DIKTAT MATA KULIAH

K I M I A AN ALI S I S

:
Drs. Adiwarna
Disusun oleh

JURUSAN TEKNIK KIMIA FAKULTAS TEKNIK

UNIVERSITAS MUHAMMADIYAH JAKARTA

2008

1. Pengantar kimia Analisis

Kimia analisis adalah suatu cabang ilmu kimia yang digunakan untuk analisa kimia
berdasarkan besaran parameter kimia dan fisika dari suatu sampel secara kualitatif dan
kuantitatif.
1.1. analisa kualitatif dan kuantitatif,
a. Analisa kimia kualitatif adalah analisa kimia untuk mengetahui jenis atom, unsure, ion,
molekul atau spesies yang terdapat dalam suatu larutan sampel. Analisa kualitatif bisa
dilakukan dengan analisa aroma, warna, warna pembakaran, rasa, warna nyala, analisa
kation, analisa anion, menggunakan pereaksi spesifik, atau dengan menggunakan alat
analisa kimia kualitatif.
- Analisa kation meliputi golongan I yakni Ag+, Hg2+2, Pb+2 ( golongan chlorida ), golongan
IIA yakni Hg+2, Cu+2, Bi+3, Cd+2 (golongan H2S), golongan IIB yakni As+3, As+5, Sb+3, Sb+5,
Sn+2, Sn+4 (golongan S=); golongan III yakni Fe+3, Cr+3, Al+3, Co+2, Ni+2, Mn+2, Zn+2
(golongan hidroksida), golongan IV yakni Ca+2, Ba+2, Sr+2 (golongan karbonat), Golongan V
yakni Na+, K+, Mg+2, NH4+, H+, Li+ (golongan sisa)
- Analisa anion meliputi golongan :
-- A1 CO3=, HCO3-, S=, S2O3=, SO3=, NO2-, OCl-, CN-, CNO- ( golongan asam encer),
-- A2 yakni F-, SiF6=, Cl-, Br-, I-, NO3-, ClO3-, ClO4-, MnO4-, CNS-, HCOO-, H3CCOO-,
C2O4=, tartarat, citrate (golongan sulfat pekat).
-- Golongan B1 yakni SO4=, S2O8=, PO4-3, PO3-3, AsO4-3, AsO3-3, CrO4=, Cr2O7=, SiO3=, SiF6=,
Salisilat, Benzoat, H4C4O4= (golongan pengendapan),
-- golongan B2 yakni MnO4-, MnO4=, CrO4=, Cr2O7= (golongan redoks)

b. Analisa kimia kuantitatif adalah analisa kimia untuk mengetahui kadar atom, unsur, ion,
molekul, spesies yang terdapat dalam suatu sampel. Analisa kuantitatif dinyatakan dalam
satuan %, ppt, molaritas, normalitas, molalitas, ppm, ppb.

1.2. Sampling
Sampling adalah cara pengambilan sampel untuk digunakan dalam analisa kimia. Pada
dasarnya cara sampling dapat dibagi menjadi :
a. Gross sampling adalah pengambilan sampel secara kasar
b. Random sampling adalah penganbilan sampel secara acak dari distribusi sampel
c. Sistematik sampling adalah pengambilan sampel secara sitematik dari distribusi
sampel
d. Industrial sampling adalah pengambilan sampel sesuai dengan sampling port yang
telah ditetapkan dalam suatu sistem industri kimia.
3.2. kesalahan analisa
3.2.1. Kesalahan konstan
a. kesalahan operasional dan personal
b. kesalahan alat dan reagen
c. kesalahan metode
d. kesalahan additive dan proporsional
3.2.2. Kesalahan aksidental
3.2.3. Minimasi kesalahan
a. kalibrasi Perlakuan dengan blanko
c. Perlakuan larutan standar
d. menggunakan berbagai cara analisa
e. Perlakuan analisa parallel
f. Standard addisi
g. standard dalam
h. Aplikasi metode
i. Dilusi isotop
3.2.4. Statistik sampling
a. Ketepatan : nilai hasil ukur analisa mendekati nilai sebenarnya
b. Ketelitian : selisih antara dua hasil ukur
c. Nilai rata-rata (: Jumlah hasil ukur (xi)/ jumlah pengukuran (n)

ml
d. Deviasi relatif rata-rata :
e. Kesalahan
- kesalahan absolut ( e )
- kesalahan random ( xi -
- bias (
e = (xi -
e. Distribusi Normal

f. Pembandingan hasil

h. Standar deviasi
--------------------------------------------------

s = (Xi Xr )2/ n - 1

VARIANSI = (Xi Xr )2/ n - 1

i. Koefisien variansi

- uji t
t = ( x - n )/s
uji t untuk pembandingan metode

t10 dari tabel = 2,23 untuk P = 0,05

- uji F
F = S2A /S2B

- uji

j. Analisa parallel

ts/n = kesalahan absolut

L = ( 100 z ) %
L = rentang data
1.3. tipe-tipe analisa kimia

Analisa kimia dapat digolongkan menjadi :

- Analisa pendahuluan adalah analisa secara awal dari suatu sampel.
- Analisa proximate dimana kadar masing unsur dalam sampel ditentukan berdasarkan
senyawa-senyawa yang ada dalam sampel .
- Analisa parsil adalah analisa menentukan konstituen tertentu dalam sampel.
- Analisa konstituen runut adalah analisa penentuan kontituen dengan kadar sangat kecil
sekali.
- Analisa komplit adalah analisa masing-masing konstituen tergantung pada kadar nya
dalam sampel
analisa kimia berdasarkan kadar sampel dapat dibagi menjadi :
- Analisa makro yakni penentuan konstituen dalam kadar lebih besar dari 0,1 gr.
- Analisa semi mikro yakni penetuan kadar konstituen berkisar antara 0,01 0,1 gr.
- Analisa mikro yakni penetuan konstituen dalam sampel kecil dari 0,001 gr,
1.4. Teknik analisa kimia
Beberapa teknik yang sering digunakan dalan analisa kimia kuantitatif adalah :
a. penentuan kadar yang cocok untuk suatu reaksi kimia baik dengan mengukur kadar reagen
yang diperlukan untuk menyempurnakan reaksi atau pun berdasarkan kadar produk yang
dihasilkan.
b. menentukan alat analisa elektrik yang tepat. ( Amperometri, voltametri, konduktometri,
potensiometri, coulombmetri )
c. Penentuan dengan menggunakan alat optik tertentu ( Spektrofotometer sinartampak-UV,
spektrofotometer inframerah, spektrofotmeter resonansi magnit inti NMR, Spketrofotometer
massa, spektrofotometer sinar X, spektrofotometer serapan atom, Spektrofotometer massa
2. Prinsip dasar kimia analisis
2.1. disosiasi elektrolitik,

Beberapa reaksi analisa kualitatif dan kuantitatif berlangsung dalam larutan encer. Oleh
karena itu Hal ini merupakan pengetahuan dasar dimana pada kondisi tersebut reaksi ini
berlangsung. Konsep tersebut merupakan teori sederhana dari disosiasi elektrolitik.
Ionisasi asam dan basa di dalam larutan.
Suatu asam bisa didefinisikan sebagai suatu senyawa yang bila dilarutkan dalam air akan
mengalami disosiasi dengan pembentukan ion hidrogen sebagai ion positif. Kenyataannya ion
hydrogen tak terdapat dalam kedaan bebas dalam larutan encer, tetapi setiap ion hydrogen
bergabung dengan satu molekul air membentuk ion hidroksonium. Ion hidroksonium adalah
proton terhidrasi sebagai berikut :
a. Asam mono proton

HCl + H2O

HNO3 + H2O

H3O+ + Cl-

H3O+ + NO3 -

Ionisasi dapat menggambarkan seberapa besar kecengerungan ion hydrogen untuk bergabung
dengan molekul air membentuk ion hidroksonium. Asam klorida dan asam nitrat diatas
terurai dengan hampir sempurna di dalam larutan encer sesuai dengan persamaan reaksi di
atas. Hal ini dapat dijelaskan dengan pengukuran titik beku dan metoda lainnnya.
b. asam poliproton terionisasi dalam beberapa tahap. Pada asam sulfat satu ion hidrogennya
dapat terdisosiasi hamper sempurna

H2SO4 + H2O

H3O+ + HSO4-

HSO4- + H2O

H3O+ + SO4=

H3PO4 + H2O

H3O+ + H2PO4-

H2PO4- + H2O

H3O+ + HPO4=

HPO4= + H2O

H3O+ + PO4-3

c. Asam lemah
HOAct + H2O

H3O+ + ActO -

c. basa
NaOH + 2H2O

Na(H2O)2+ + OH-

d. Basa poli hidroksi

Ca(OH)2 + 4 H2O

Ca(H2O)4+2 + 2 OH-

Al(OH)3 + 6H2O

Al(H2O)6+3 + 3OH-

Kesetimbangan asam dan basa

Menurut Bronsted-Lowry definisi asam dan basa adalah sebagai berikut :
Asam adalah spesis yang mendonorkan (H+).
and
bases are adalah spesis yang menerima proton

Air
Air akan mengalami reaksi kesetimbangan berikut :
H2O

H+ + OH-,

Maka konstanta kesetibangan air :

[H+] [OH-]
Keq = ---------[H2O]
Konsentrasi air dalam larutan air konstan dan ungkapan ini disederhanakan menjadi:
Kw = (55,56 M) * Keq = [H +] [OH-]
mana Kw disebut disosiasi konstan air dan sama 1.00x10-14 pada suhu kamar. Konsentrasi
[H +] dan [OH-] karena sama 1.00x10-7 M.

asam
Asam dalam air akan memisahkan, yang itu menyumbangkan proton itu. Kami menyebutnya
asam yang terdisosiasi asam benar-benar kuat dan asam yang terdisosiasi asam hanya
sebagian yang lemah.
Kuat Asam Contoh:
HNO3 (aq) + H2O NO3-(aq) + H3O + (aq)
Keq = jumlah yang sangat besar
Dalam contoh ini, HNO3 adalah asam dan H2O bertindak sebagai basis.
NO3-disebut basa konjugasi dari asam HNO3, dan H3O + adalah asam konjugasi dari basa
H2O.
basa
Basis dalam air dapat menerima sebuah proton dari air untuk menghasilkan OH-. Basa kuat
adalah garam hidroksida yang terdisosiasi sepenuhnya dalam air, jadi pernyataan ini
berlebihan. Tapi basa lemah tidak harus mengandung hidroksida sendiri, tetapi mereka
menghasilkan solusi dasar dengan bereaksi dengan air.
Lemahnya dasar contoh:
NH3 (aq) + H2O NH4 + (aq) + OH-(aq)
K = 1.8x10-5
Dalam contoh ini, NH3 adalah basa dan H2O bertindak sebagai asam. NH4 + adalah asam
konjugasi dari dasar NH3, dan OH-adalah basa konjugat dari asam H2O.
Sebuah senyawa yang dapat bertindak baik se
2.2. hukum aksi massa aktivitas larutan,
k1
A + B ==== C + D
k2
v1 = k1 [ A ] [ B ]
v2 = k2 [ C ] [ D ]
k1 [ A ] [ B ] = k2 [ C ] [ D ]
k1
K = --------K2

[C][D]
= ------------------[ A] [ B ]

Titrasi terbagi menjadi 4 bahagian :

Tirasi adalah penyetaraan antara titrat ( zat yang dititrasi) dan titran (zat pentitrasi) dengan
rumus V1N1 = V2N2
1. titrasi asam basa ( kesetimbangan asam basa)
2. titrasi pengendapan ( kesetibangan hasil kali kelarutan)
3. titrasi redoks ( kesemtibangan redoks )
4. titrasi kompleksometri (kesetimbangan komleksometri)

2.1. Titrasi asam-basa

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Titrasi setup. Buret biasanya akan dipegang oleh penjepit, tidak ditampilkan di sini. Merah
muda ini kemungkinan besar disebabkan oleh penggunaan indikator fenolftalein.
Titrasi asam-basa adalah penentuan konsentrasi asam atau basa dengan persis menetralkan
asam / basa dengan asam atau basa konsentrasi diketahui. Hal ini memungkinkan untuk
analisis kuantitatif dari konsentrasi asam diketahui atau larutan basa. Itu membuat
penggunaan reaksi netralisasi yang terjadi antara asam dan basa dan pengetahuan tentang
bagaimana asam dan basa akan bereaksi jika rumus mereka diketahui.
Asam-Base titrasi juga dapat digunakan untuk mencari kemurnian persen bahan kimia.

Methoda titrasi
Sebelum memulai suatu titrasi harus dipilih indikator yang cocok. Titik ekivalen pada suatu
reaksi adalah titik dimana jumlah ekivalen dari reakstan yang bereaksi. Yang sangat realtif
tergantung dengan asam dan basa yang digunakan. Ph pada titk equivalen dapat diperkirakan
dengan menggunakan rumus berikut :

Asam uat akan bereaksi dengan basa kuat membentuk pH netral.

Asam kuat akan bereaksi dengan asam lemah membentuk pH larutan asam (pH<7) .
Asam lemah akan bereaksi dengan basa kuat membentuk larutan basa (pH>7) .

Bila asam lemah bereaksi dengan basa lemah titik equivalen akan menjadi basa jika basanya
lebih kuat dari asamnya. Jika asam dan basanya sama kuat akan terjadi titik equivalent pada
pH netral. Akan tetapi jika asam lemah sering kali tak bisa dititrasi dengan basa lemah karena
karena perubahan warna pada indikator sering kali sangant cepat oleh karena itu sulit sekali
untuk diamati perubahan warna indikatornya.
Titik pada saat perubahan warna indikator disebut titik akhir titrasi. Pemilihan indikator
yang cocok harus dilakukan, sebaiknya agar perubahan warna indikator menunjukan titik
ekivalen dari suatu titrasi.
Pertama buret harus dibasahi dengan larutan standard, pipet larutan yang tak diketahui, dan
Erlenmeyer diisi dengan aquades.
Kedua volume yang diketahui dari larutan yang tak diketahui konsentrasinya harus dipipet
dan dimasukan kedalam erlenmeyer sambil ditambahkan beberapa tetes indikator. Buret
harus selalu terisi larutan pentiter diatas skala buret agar mudah melakukan pembacaan skala
volum pentiter yang terpakai.
Kemudian larutan pentiter dialirkan dari buret ke dalam labu erlenmeyer titrat. Dengan cara
demikian bisa diketahui banyaknya pentiter terpakai untuk netralisasi. Larutan pentiter
dialirkan melalui ujung buret sampai indikator dalam larutan titrat berubah warna untuk
menadai titrasi telah berakhir.voluume larutan pentiter yang terpakai dicatat sebagai volume
untuk perhitungan titrasi..
Tiga titrasi harus dilakukan, kali ini lebih akurat, dengan mempertimbangkan kira-kira di
mana titik akhir akan terjadi. Pembacaan pada buret pada titik akhir harus dicatat, dan ratarata untuk memberikan hasil akhir. Titik akhir tercapai ketika indikator hanya berubah warna
secara permanen. Hal ini dapat dicapai dengan mencuci penurunan tergantung dari ujung
buret ke dalam labu kanan pada akhir titrasi untuk mencapai penurunan yang lebih kecil
dalam volume dari apa yang biasanya dapat dicapai dengan hanya solusi menetes dari buret.
Asam-basa titrasi dilakukan dengan indikator fenolftalein, bila asam lemah - titrasi basa kuat,
indikator biru bromthymol dalam asam kuat - reaksi basa kuat, dan indikator metil oranye
untuk asam kuat - reaksi basa lemah. Jika dasar adalah dari skala, yaitu pH> 13,5, dan asam
memiliki pH> 5,5, maka indikator kuning Alizarie dapat digunakan. Di sisi lain, jika asam
adalah dari skala, yaitu pH <0,5, dan basis memiliki pH <8,5, maka indikator biru timol dapat
digunakan.

Titrasi asam lemah

Ketika titrasi asam lemah dengan dasar yang kuat, pH sebelum titik ekivalen dapat dihitung
dengan rumus berikut: [1]
di mana:
pKa adalah disosiasi asam konstan dari asam lemah.
[OH-] ditambahkan adalah konsentrasi basa kuat ditambahkan dalam solusi akhir (tidak

dalam larutan standar asli)

[HA] total konsentrasi menyimpulkan dari kedua asam lemah dan basa konjugasinya dalam
solusi akhir.
Dengan demikian, pada penambahan basa kuat yang merupakan setengah jumlah asam lemah
dalam larutan ([OH-] = 0,5 ditambahkan [HA] total), pH menjadi sama dengan pKa.

Rumus yang lebih umum [2] yang menggambarkan titrasi asam lemah dengan basa
kuat diberikan di bawah ini
= fraksi penyelesaian titrasi ( <1 adalah sebelum , titik ekivalen = 1 adalah titik
ekivalen, dan > 1 adalah setelah titik ekivalen)
Ca, Cb = konsentrasi asam dan basa
Va, Vb = volume asam dan basa
= fraksi asam lemah yang terionisasi
Indicator

Low pH color

Transition pH
range

High pH color

Gentian violet (Methyl violet)

yellow

0.02.0

blue-violet

Leucomalachite green (first

transition)

yellow

0.02.0

green

Leucomalachite green (second

transition)

green

11.614

colorless

Thymol blue (first transition)

red

1.22.8

yellow

Thymol blue (second transition)

yellow

8.09.6

blue

Methyl yellow

red

2.94.0

yellow

Bromophenol blue

yellow

3.04.6

purple

Congo red

blue-violet

3.05.0

red

Methyl orange

red

3.14.4

orange

Bromocresol green

yellow

3.85.4

blue

Methyl red

red

4.46.2

yellow

Methyl red

red

4.55.2

green

Azolitmin

red

4.58.3

blue

Bromocresol purple

yellow

5.26.8

purple

Bromothymol blue

yellow

6.07.6

blue

Phenol red

yellow

6.88.4

red

Neutral red

red

6.88.0

yellow

Naphtholphthalein

colorless to
reddish

7.38.7

greenish to
blue

Cresol Red

yellow

7.28.8

reddish-purple

Phenolphthalein

colorless

8.310.0

fuchsia

Thymolphthalein

colorless

9.310.5

blue

Alizarine Yellow R

yellow

10.212.0

red

Litmus

red

4.5-8.3

blue

Titik ekivalen dari tirasi asam-basa

1. Titrasi asam kuat basa kuat titik ekivalen pada pH
pH = pKw
pH = pKw + pa(asam)- pa(basa)
2. Titrasi Asam lemah basa kuat
pH = pKw + pKa pa(garam)
3. Titrasi asam kuat basa lemah
pH = pKw pKb + pa(garam)
4. Titrasi asam lemah basa lemahb
pH = pKw + pKa pKb
Kw = konstanta ionisasi air
Ca = konsentrasi asam
Cb = konsentrasi basa

[edit] References
1. ^ Medical CHEMISTRY Compendium. By Anders Overgaard Pedersen and Henning Nielsen.
Aarhus University. 2008

2. ^ Quantitative Chemical Analysis, 7Ed. by Daniel C. Harris. Freeman and Company 2007.
Retrieved from "http://en.wikipedia.org/wiki/Acid-base_titration"
Categories: Titration

Kelarutan (kesetimbangan pengendapan)

Definisi dari kelarutan :
Daftar garam larut dan mereka Ksp nilai dapat ditemukan dalam buku teks kimia yang paling
umum dan analitis. Perhatikan bahwa beberapa ion yang kita anggap ion penonton ketika
membahas kesetimbangan asam-basa akan membentuk garam larut.
Kelarutan adalah didefinisikan sebagai d mol / L, g / L, atau mg / L spesies melarutkan.
Contoh 1: Bagaimana kelarutan klorida timah,, PbCl2 terkait dengan konsentrasi larutan Pb +
dan Cl-?
Setiap unit formula PbCl2 yang larut menghasilkan satu ion timah, Pb2 +, dan dua ion
klorida, Cl-. Kelarutan molar PbCl2 adalah setara dengan konsentrasi Pb2 +, SPbCl2 = [Pb2
+].
Sejak dua Cl-s diproduksi per satuan PbCl2 formula yang larut, kelarutan molar PbCl2 sama
dengan satu-setengah konsentrasi larutan Cl-, SPbCl2 = 0,5 * [Cl-].
SPbCl2 = [Pb2 +] = 0,5 * [Cl-]. Representasi berikut menunjukkan PbCl2 padat di bagian
bawah gelas dan ion dalam larutan di atas yang solid. Dalam contoh ini terdapat ion
memimpin dua dan empat ion klorida dalam larutan, menunjukkan bahwa [Pb2 +] = 0,5 *
[Cl-].
\
/
|
|
| - - - - - - - - - - - - - - --------|
| ClPb2+
|
|
Cl|
|
Pb2+
|
|
Cl|
| Cl|
|
|
| Pb
Pb
Pb
Pb
|
|Cl Cl Cl Cl Cl Cl Cl Cl |
| Pb
Pb
Pb
Pb
|
\Cl Cl Cl Cl Cl Cl Cl Cl/
Example 2: What is the solubility of barium iodate, Ba(IO3)2, in pure water at 25 oC?
The solubility equilibrium is:
Ba(IO3)2 (s)

Ba2+(aq) + 2 IO3-(aq)

Ksp = [Ba2+][IO3-]2 = 1.5x10-9

[Ba2+] = 0.5*[IO3-]
Ksp = [Ba2+](2*[Ba2+])2 = 1.5x10-9
4*[Ba2+]3 = 1.5x10-9

[Ba2+]3 = 3.75x10-10 M
S = [Ba2+] = 7.2x10-4 M
Or in mass terms: S = (7.2x10-4 M)(487 g/mol) = 0.35 g/L
ada alasan bahwa saya mengambil garam jelas seperti barium iodat untuk contoh ini.
Menentukan kelarutan Ba (IO3) 2 sederhana dibandingkan untuk menentukan kelarutan
garam larut banyak. Ba2 + adalah ion penonton jadi kami tidak khawatir tentang hal itu
bereaksi dengan pH = 7 air. Bagaimana dengan anion iodat? Ini adalah dasar dan dapat
bereaksi dengan air:
IO3-(aq) + H2O HIO3 (aq) + OH-(aq)
Sejauh mana hasil reaksi ini diberikan oleh nilai Kb. Ka untuk asam iodic adalah 0,17 (itu
adalah asam cukup kuat).
Kb = Kw / Ka = 1x10-14/0.17 = 5.9x10-14.
Ini nilai Kb sangat kecil sehingga kita dapat menyimpulkan bahwa hidrolisis oleh iodat tidak
mempengaruhi kelarutan Ba (IO3) 2.
Contoh 3: Apa kelarutan barium iodat, Ba (IO3) 2, pada 25 oC dalam larutan 0,10 M barium
nitrat, Ba (NO3) 2?

Masalahnya sudah diatur sama seperti sebelumnya:

Ba (IO3) 2 (s) Ba2 + (aq) + 2 IO3-(aq)
Ksp = [Ba2 +] [IO3-] 2 = 1.5x10-9
Perbedaannya adalah bahwa ada dua sumber dari Ba2 +, yang 0,10 M Ba (NO3) 2 dan
pembubaran Ba (IO3) 2:
[Ba2 +] = 0,10 M + 0,5 * [IO3-]
Ksp = (0,10 M + 0,5 * [IO3-]) * [IO3-] 2 = 1.5x10-9
Kita bisa memecahkan persamaan kubik atau kita bisa mencoba mengabaikan [Ba2 +] yang
berasal dari pembubaran Ba (IO3) 2 dibandingkan dengan 0,10 M nitrat barium.

Ksp = (0.10 M)[IO3-]2 = 1.5x10-9

[IO3-]2 = 1.5x10-8
[IO3-] = 1.2x10-4 M
S = 0.5*[IO3-], so
S = 6.1x10-5 M
Kami asumsi bahwa S << 0,1 M dibenarkan.
Bandingkan hasil ini dengan kelarutan dalam air murni, S = 7.2x10-4 M. Kelarutan barium
iodat jauh lebih sedikit ketika salah satu dari ion yang terlibat dalam kesetimbangan hadir
dalam larutan. Efek ini disebut efek umum-ion, dan dijelaskan secara kualitatif dengan
prinsip LeChatelier itu.
Akankah penambahan ion lain seperti Na + dan Cl-mempengaruhi keseimbangan ini? Untuk

no pendekatan pertama. Namun, menambahkan ion penonton untuk solusi meningkatkan

kekuatan ion dan mempengaruhi koefisien aktivitas

.
Di mana dari contoh-contoh berikut ini kita harus khawatir tentang kegiatan?
kelarutan barium iodat dalam air
kelarutan barium iodat dalam larutan 0,10 M barium nitrat
kelarutan barium iodat dalam larutan 0,50 M NaCl
Contoh 4: Will bentuk endapan ketika 100,0 mL 0,2 M Fe3 + dalam 0,5 M H2SO4 dicampur
dengan 100,0 mL 1,0 M NaOH?
Sebelum memikirkan keseimbangan, melihat masalah ini dan memutuskan apakah sesuatu
akan terjadi ketika dua solusi dicampur. Larutan basa kuat dicampur dengan larutan yang
mengandung asam kuat. Asam dan basa akan bereaksi sampai salah satu dari keduanya
dikonsumsi. Dalam hal ini jumlah yang sama mol H + dan OH-dicampur sehingga solusi
yang dihasilkan akan berada di pH = 7.
The solubility equilibrium is:
Fe(OH)3 (s)

Fe3+(aq) + 3OH-(aq)

Ksp = 2x10-39 = [Fe3+] [OH-]3

Untuk menentukan apakah endapan akan terbentuk: Q menemukan dan membandingkannya
dengan KSP. (Ayat ini menyebut kuantitas produk ion, P, tapi itu adalah ekspresi yang sama
seperti Q.)
Jika Q> Ksp konsentrasi ion yang lebih besar dari konsentrasi kesetimbangan dan endapan
akan terbentuk.
Jika Q <Ksp konsentrasi ion berada di bawah konsentrasi keseimbangan mereka dan tidak
ada bentuk endapan.
Q = CFe3 + COH-3 = (0.100 M) (1x10-7 M) 3 = 1.00x10-22
1x10-22> 2x10-39
Q> Ksp
Jadi endapan akan terbentuk.
Apa yang akan menjadi [Fe3 +] dari solusi yang dihasilkan?
2x10-39 = [Fe3 +] (1x10-7 M) 3
[Fe3 +] = 2x10-18 M

Ini adalah diprediksi [Fe3 +] dalam air. Dalam sistem air nyata jumlah total zat besi
dalam larutan bisa jauh lebih tinggi karena besi yang bisa eksis dalam berbagai
bentuk seperti kompleks besi dan koloid.
Selain: Besi dan asam sulfat yang hadir di drainase tambang karena oksidasi
pirit, FeS2. Salah satu reaksi yang terjadi adalah:
FeS2 (s) + 14 Fe3 + (aq) + 8 H2O + 15 Fe2 (aq) + 2 SO42-(aq) + 16 H + (aq)
Hasil terakhir [Katrina J. Edwards, dan rekan kerja, Sains 287 (2000) 1796.]
Telah mengidentifikasi mikroorganisme yang mengoksidasi Fe2 + Fe3 + untuk
menjaga reaksi ini terjadi.
Ketika drainase tambang diencerkan secukupnya untuk menaikkan pH, besi
presipitasi sebagai hidroksida besi dan menciptakan lapisan jeruk di sungai dan
sungai
Competing Equilibria
What is the solubility of CaCO3 in water at 25 oC?
The solubility is equal to [Ca2+]. The dissolution equilibrium is:
CaCO3 (s)

Ca2+(aq) + CO32-(aq)

but there are competing equilibria:

CO32-(aq) + H2O

HCO3-(aq) + OH-(aq)

We will neglect HCO3-(aq) + H2O

H2CO3(aq) + OH-(aq). Solving the problem with this
equilibrium included follows the same procedure, just with more equations.
Ksp = [Ca2+][CO32-] = 4.5x10-9
[HCO3-][OH-]
Kb = ------------ = 2.13x10-4
[CO32-]
We can always write mass and charge balance expressions to get more equations to solve for
multiple unknowns:
[Ca2+] = [CO32-] + [HCO3-] + [H2CO3]
2[Ca2+] + [H+] = 2[CO32-] + [HCO3-] + [OH-]
We are neglecting H2CO3 and since we know the solution will be basic we can probably
neglect [H+], which will be less than 1x10-7 M. We can check this assumption when we get an
answer. We then have:
[Ca2+] = [CO32-] + [HCO3-]
2[Ca2+] = 2[CO32-] + [HCO3-] + [OH-]

If we multiply the simplified mass balance expression by two and relate it to the charge
balance expression we get:
2[CO32-] + 2[HCO3-] = 2[CO32-] + [HCO3-] + [OH-]
[HCO3-] = [OH-]
We can use this relationship to simplify the Kb expression:
[HCO3-]2
Kb = -------[CO32-]
or [HCO3-] = (Kb[CO32-])1/2
So we can eliminate [HCO3-] from [Ca2+] = [CO32-] + [HCO3-] to get:
[Ca2+] = [CO32-] + (Kb[CO32-])1/2
Now we can eliminate [CO32-] using the Ksp expression.
[Ca2+] = Ksp/[Ca2+] + (KbKsp/[Ca2+])1/2
Multiplying each side by [Ca2+] and rearranging gives:
[Ca2+]2 - (KbKsp)1/2[Ca2+]1/2 - Ksp = 0
or
[Ca2+]2 - (9.8x10-7)[Ca2+]1/2 - 4.5x10-9 = 0
The easiest way to solve this equation is to put the expression [Ca2+]2 + (9.8x10-7)[Ca2+]1/2 4.5x10-9 in a spreadsheet and try a series of values for [Ca2+] to find the one that gives a result
closest to zero.
In the absence of the second equilibrium the solubility would have been:
Ksp = 4.5x10-9 = [Ca2+][CO32-]
[Ca2+] = (4.5x10-9)1/2
S = 6.7x10-5 M
Which gives us a starting point for finding [Ca2+].
[Ca2+]

[Ca2+]2 - (9.8x10-7)[Ca2+]1/2 - 4.5x10-9

6.00x10-5

-8.49x10-9

7.00x10-5

-7.80x10-9

8.00x10-5

-6.87x10-9

9.00x10-5

-5.70x10-9

1.00x10-4

-4.30x10-9

1.20x10-4

-8.35x10-10

1.30x10-4

1.23x10-9

1.40x10-4

3.50x10-9

This set of data shows that the solution is between 1.20x10-4 and 1.30x10-4. We can do
another set of data at smaller intervals to pin down the solution:
[Ca2+]

[Ca2+]2 - (9.8x10-7)[Ca2+]1/2 - 4.5x10-9

1.22x10-4

-4.40x10-10

1.23x10-4

-2.40x10-10

1.24x10-4

-3.68x10-11

1.25x10-4

1.68x10-10

1.26x10-4

3.76x10-10

[Ca2+] = 1.2x10-4 M
The competing equilibrium for carbonate increases the calcium carbonate solubility by a
factor of approximately 2 (LeChatelier strikes again).
The assumption we made that 2[Ca2+] >> [H+] in basic solution was valid.

More Metal Hydroxide Equilibria and Another Intro to Complexation

An example where Ksp is relatively large and we can neglect competing equilibrium:
Mg(OH)2 (s)

Mg2+(aq) + 2 OH-(aq)

Ksp = [Mg2+][OH-]2 = 7.1x10-12

The solubility of Mg(OH)2 is equal to [Mg2+].
We need another equation to solve for [Mg2+]. From the stoichiometry of the reaction we
know that 2 [Mg2+] = [OH-]. Substituting into the Ksp expression to eliminate [OH-]:
Ksp = [Mg2+](2 [Mg2+])2 = 7.1x10-12
4 [Mg2+]3 = 7.1x10-12

[Mg2+]3 = 1.8x10-12
[Mg2+] = 1.2x10-4
An example where Ksp is very small and we can neglect hydroxide from the metal hydroxide
dissolution:
Al(OH)3 (s)

Al3+(aq) + 3 OH-(aq)

Ksp = [Al3+][OH-]3 = 3x10-34

The solubility of Al(OH)3 is equal to [Al3+].
Since Ksp is so small we can try solving this problem assuming that the [OH-] from the
Al(OH)3 dissolution is negligible compared to the [OH-] from the dissociation of water.
Ksp = [Al3+][OH-]3 = 3x10-34
[Al3+] = 3x10-34 / [OH-]3
pH

[OH-]

[Al3+]

1x10-11

3x10-1

1x10-10

3x10-4

1x10-9

3x10-7

1x10-8

3x10-10

1x10-7

3x10-13

At some pH the [Al3+] reaches a minimum and then increases with further increase in pH. The
solubility of Al(OH)3 increases because a soluble complex, Al(OH)4-, begins to form at high
pH.
Precipitation (Insoluble Salts)
pengantar
Banyak ion logam membentuk senyawa yang larut dalam air. Kami menyebutnya garam larut
(duhhh) atau presipitat. Umum presipitat karbonat, hidroksida, sulfat, dan sulfida. Ion yang
kita anggap ion penonton ketika membahas kesetimbangan asam-basa akan membentuk
garam larut.
Garam larut dalam kontak dengan air mempertahankan keseimbangan dengan ion. Dalam
kasus sederhana di mana tidak ada ion umum atau kesetimbangan bersaing, konsentrasi ion
bergantung hanya pada konstanta kesetimbangan untuk endapan tertentu. Ketika kita
berbicara tentang kesetimbangan kelarutan kita selalu menulis keseimbangan dengan padat di
sebelah kiri. Sebagai contoh:
Ba (IO3) 2 (s) Ba2 + (aq) + 2 IO3-(aq)
Ekspresi konstanta kesetimbangan untuk garam larut ditulis mengikuti aturan yang sama
seperti untuk keseimbangan lainnya. Konstanta kesetimbangan disebut produk kelarutan,
Ksp. Ekspresi Ksp untuk kesetimbangan di atas adalah:

Ksp = [Ba2+][IO3-]2

Ksp Values for Some Precipitates

Formula

Name

Ksp

AgCl

silver chloride

1.8x10-10

Al(OH)3 aluminum hydroxide 2x10-32

BaCO3

barium carbonate

5x10-9

Ba(IO3)2

barium iodate

1.6x10-9

BaSO4

barium sulfate

1.3x10-10

Fe(OH)2

iron(II) hydroxide

8x10-16

Fe(OH)3

iron(III) hydroxide

4x10-38

FeS

iron sulfide

6x10-18

PbCrO4

lead chromate

1.8x10-14

Pb(OH)2

lead hydroxide

2.5x10-16

PbS

lead sulfide

7x10-28

PbSO4

lead sulfate

1.6x10-8

The equilibria of insoluble salts is described in more detail in the document on solubility.

2.2.Titrasi pengendapan (Argentometry)

Dalam kimia analitik, argentometri adalah jenis titrasi yang melibatkan perak (I) ion.
Biasanya, digunakan untuk menentukan jumlah klorida hadir dalam sampel. Larutan sampel
dititrasi terhadap larutan perak nitrat konsentrasi diketahui. Ion klorida bereaksi dengan perak
(I) ion klorida untuk memberikan perak larut:
Cl- (aq) + Ag + (aq) AgCl (s) (Ksp = 1,70 10-10)
CrO4-2 (aq) + Ag + (aq) Ag2CrO4 (s) (Ksp = 1,70 10-10)
Metode Volhard
Contoh titrasi kembali, metode Volhard, dinamai Yakub Volhard, melibatkan penambahan
perak nitrat kelebihan analit, klorida perak disaring, dan perak nitrat tersisa dititrasi terhadap
tiosianat, [1] dengan besi (III) sebagai indikator yang membentuk darah-red [Fe (OH2) 5
(SCN)] 2 + pada titik akhir:

[edit] Mohr method

Dalam metode Mohr, dinamai Karl Friedrich Mohr, kalium kromat merupakan indikator,
memberikan kromat perak merah setelah semua ion klorida bereaksi:
Cl- (aq) + Ag + (aq) AgCl (s) (Ksp = 1,70 10-10)
CrO4-2 (aq) + Ag + (aq) Ag2CrO4 (s) (Ksp = 9 10-12)
Solusinya harus mendekati netral, karena bentuk hidroksida perak pada pH tinggi, sedangkan
kromat membentuk H2CrO4 pada pH rendah, mengurangi konsentrasi ion kromat, dan
menunda pembentukan endapan. Karbonat dan fosfat mengendap dengan perak, dan harus
absen untuk mencegah hasil yang tidak akurat.
Metode Mohr dapat disesuaikan untuk menentukan kandungan klorin total sampel dengan
membakar sampel dengan kalsium, maka asetat besi. Kalsium asetat "perbaikan" klorin
bebas, endapan karbonat, dan menetralkan solusi yang dihasilkan. Asetat ferri menghilangkan
fosfat. Semua klorida dilarutkan keluar dari residu, dan dititrasi. [1]
Metode Volhard
Contoh titrasi kembali, metode Volhard, dinamai Yakub Volhard, melibatkan penambahan
perak nitrat kelebihan analit, klorida perak disaring, dan perak nitrat tersisa dititrasi terhadap
tiosianat, [1] dengan besi (III) sebagai indikator yang membentuk darah-red [Fe (OH2) 5
(SCN)] 2 + pada titik akhir:
Cl- (aq) + Ag + (aq) AgCl (s) (Ksp = 1,70 10-10)
Ag+ (aq) + SCN (aq) AgSCN (s) (Ksp = 1.16 1012)
Fe(OH)(OH2)52+ (aq) + SCN (aq) [Fe(OH2)5(SCN)]2+ + OH (Kst = 3,6 x 10-14)

Fajans method
alam metode Fajans, dinamai Kazimierz Fajans, biasanya diklorofluoresein digunakan
sebagai indikator, titik akhir ditandai dengan suspensi hijau berubah merah muda. Sebelum
titik akhir titrasi, ion klorida tetap lebih. Mereka menyerap pada permukaan AgCl,
menanamkan muatan negatif pada partikel. Melewati titik akhir-perak, kelebihan (I) ion
menyerap pada permukaan AgCl, menanamkan muatan positif. Pewarna anionik seperti
diklorofluoresein tertarik pada partikel, dan mengalami perubahan warna pada adsorpsi, akili
titik akhir. Eosin (tetrabromofluorescein) cocok untuk titrasi melawan bromida, iodida, dan
anion tiosianat, memberikan tajam akhir-poin dibandingkan diklorofluoresein. Hal ini tidak
cocok untuk titrasi terhadap anion klorida karena mengikat AgCl lebih kuat daripada klorida
tidak. [2]
Cl- (aq) + Ag + (aq) AgCl (s) (Ksp = 1,70 10-10)
Ind-n (aq) + Ag + (aq) AgnInd (s) (Ksp = 6,3 10-16)

Ind = indikator

3. Titrasi redoks
Titrasi redoks adalah titrasi antara oksifator dengan reduktor. Oksidator meningkatkan
muatan hasil reduksi, dan reduktor menurunkan muatan pengoksidasi. Contoh reaksi oksidasi
reduksi adalah titrasi permanganometri antara anion permanganate dengan anion oksalat pada
suhu 50 70 oC.
MnO4 + 8H+ + 5e Mn2+ + 4H2O

Eo = +1.5119 Volt

C2O4=
CO2 + 2eEo = - 0,44 Volt
---------------------------------------------------------------------------------------------2 MnO4- + 16 H+ + 10e
2 Mn+2 + 8 H2O
Eo = + 1,5119 Volt
5 C2O4=

10 CO2 + 10e-

Eo = - 0,44

Volt

+
----------------------------------------------------------------------------------------------2 MnO4- + 16 H+ + 5 C2O4=
2 Mn+2 + 8 H2O + 10 CO2 Eo = + 1,0719 Volt
Esel

= (b Eo1 + a Eo2)/ab - RT/ab F ln K

Esel

= (b Eo1 + a Eo2)/ab - 0,05916/ab ln K

pada keadaan STP

(aMn+2)2
K = --------------------------------------(aMnO4-)2 (aC2O4=)5 (aH+)16
Contoh lain dari reaksi redok adalah reaksi iodometri
2I3- + 2e-

6I-

Eo = 0,535

Volt

2S2O3=
S4O6= + 2eEo = - 0,17 Volt
----------------------------------------------------------------------------------------------------------- +
2I3- + 2S2O3=
6I- + S4O6=
Eo = 0,365 Volt

Table of Standard reduction

potentials www.vaxasoftware.com Half
reaction

o (V)

Li+ + e Li(s)
3.0401
+

K + e K(s)
Ca2+ + 2e Ca(s)
Na+ + e Na(s)
Mg2+ + 2e Mg(s)
Al3+ + 3e Al(s)
Mn2+ + 2e Mn(s)
2H2O + 2e H2(g) + 2OH
Zn2+ + 2e Zn(s)
Cr3+ + 3e Cr(s)
Fe2+ + 2e Fe(s)
Cr3+ + e Cr2+(s)
Cd2+ + 2e Cd(s)
Ni2+ + 2e Ni(s)
Sn2+ + 2e Sn(s)
Pb2+ + 2e Pb(s)
2H+ + 2e H2(g)
Cu2+ + e Cu+(s)
SO42 + 4H+ + 2e SO2(g) + 2H2O
Cu2+ + 2e Cu(s)
O2(g) + 2H2O + 4e 4OH
Cu+ + e Cu(s)
I2(s) + 2e 2I
MnO4 + 2H2O +3e MnO2(s) + 4OH
Fe3+ + e Fe2+
Ag+ + e Ag(s)
NO3 + 4H+ + 3e NO(g) + 2H2O
Br2(l) + 2e 2Br
Br2(aq) + 2e 2Br
2IO3 + 12H+ + 10e I2(s) + 6H2O
O2(g) + 4H+ + 4e 2H2O
Cr2O72 + 14H+ + 6e 2Cr3+ + 7H2O
Cl2(g) + 2e 2Cl
-PbO2(s) + 4H+ + 2e Pb2+ + 2H2O
-PbO2(s) + 4H+ + 2e Pb2+ + 2H2O
ClO + 2H+ + 2e Cl + H2O
MnO4 + 8H+ + 5e Mn2+ + 4H2O
Au3+ + 3e Au(s)
H2O2(l) + 2H+ + 2e 2H2O
Au+ + e Au(s)
F2(g) + 2e 2F
+2.890

+REDUCING
2.931
2.868
2.7144
2.3568
1.676
1.185
0.8277
0.7628
0.74
0.440
0.42
0.40
0.236
0.13
0.1266
0.0000
+0.159
+0.17
+0.3394
+0.414
+0.5180
+0.535
+0.597
+0.769
+0.7996
+0.96
+1.066
+1.0873
+1.2093
+1.2288
+1.33
+1.3601
+1.458
+1.468
+1.46
+1.5119
+1.52
+1.78
+1.83
+OXIDIZING

Teknik pemisahan campuran dengan kromatografi terdiri dari :

a.
b.
c.
d.
e.
f.
g.

Kromatografi kertas (PC)

Kromatografi lapisan tipis (TLC)
Kolom kromatografi (CC)
Kromatografi ion (IC)
Kromatografi cair bertekanan tinggi (HPLC)
Kromatografi gas (GC)
Kromatografi elektroforesis

4.3. Kromatografi kertas,

Chromatography jar

Kertas kromatografi adalah teknik kimia analitik untuk memisahkan dan mengidentifikasi
campuran atau yang bisa berwarna, terutama pigmen. Hal ini juga dapat digunakan dalam
warna sekunder atau primer dalam percobaan tinta. Metode ini telah digantikan dengan
kromatografi lapis tipis, namun masih merupakan alat pengajaran yang kuat. Dua-cara kertas
kromatografi, juga disebut dua dimensi kromatografi, melibatkan menggunakan dua pelarut
dan putar kertas 90 di antara. Hal ini berguna untuk memisahkan campuran kompleks dari
senyawa yang sama, misalnya, asam amino

Technique of Paper Chromatography

Tempat terkonsentrasi kecil solusi yang berisi sampel zat terlarut diterapkan pada secarik
kertas kromatografi sekitar dua cm dari dasar piring, biasanya menggunakan tabung kapiler
untuk presisi maksimum. Sampel ini diserap ke kertas dan dapat membentuk interaksi dengan
itu. Setiap zat yang bereaksi atau obligasi dengan kertas tidak dapat diukur dengan
menggunakan teknik ini. Makalah ini kemudian dicelupkan ke dalam perawatan pelarut,
seperti etanol atau air, mengambil tempat cocok yang berada di atas permukaan pelarut, dan
ditempatkan dalam wadah tertutup.
Bergerak pelarut kertas dengan aksi kapiler, yang terjadi sebagai akibat dari daya tarik
molekul pelarut untuk kertas, hal ini juga dapat dijelaskan sebagai diferensial adsorpsi dari
komponen zat terlarut ke dalam pelarut. Sebagai pelarut naik melalui kertas itu bertemu dan
melarutkan campuran sampel, yang kemudian akan melakukan perjalanan ke kertas dengan
sampel zat terlarut pelarut. Berbeda senyawa dalam campuran sampel pada tingkat yang
berbeda karena perbedaan dalam kelarutan dalam pelarut, dan karena perbedaan ketertarikan
mereka pada serat di koran. Komponen lebih mudah larut yang lebih lanjut ia pergi.
Kromatografi kertas mengambil mana saja dari beberapa menit sampai beberapa jam.
Dalam beberapa kasus, kromatografi kertas tidak pigmen terpisah sepenuhnya, ini terjadi
ketika dua zat tampaknya memiliki nilai yang sama dalam pelarut tertentu. Dalam kasus ini,

dua arah kromatografi digunakan untuk memisahkan beberapa tempat-pigmen.

[Sunting] kromatografi Ascending
Dalam metode ini, pelarut adalah di kolam renang di bagian bawah kapal di mana kertas yang
supported.The cromatogram menaik dilipat di atas batang gelas yang setengah lainnya
menjadi teknik chromatogram.This turun memberikan pemisahan cepat seperti yang dari
teknik individu .
[Sunting] kromatografi Descending
Dalam metode ini, pelarut disimpan dalam sebuah palung di bagian atas ruangan dan
dibiarkan mengalir ke bawah kertas. Cairan bergerak turun oleh aksi kapiler serta oleh gaya
gravitasi, sehingga metode ini juga dikenal sebagai metode gravitasi. [Rujukan?] Dalam hal
ini, aliran lebih cepat dibandingkan dengan metode ascending, dan kromatografi adalah
menyelesaikan lebih cepat. Aparat dibutuhkan untuk kasus ini lebih canggih. Pelarut
berkembang ditempatkan di palungan di bagian atas yang biasanya terdiri dari bahan inert.
Makalah ini kemudian ditangguhkan dalam pelarut. Zat yang tidak dapat dipisahkan dengan
metode naik kadang-kadang dapat dipisahkan dengan metode menurun. [Rujukan?]
[Sunting] Kromatografi sebagai seni
Kromatografi dapat digunakan sebagai alternatif seni karena warna tertentu yang
menciptakan, tapi kromatografi kertas jarang menghasilkan warna dipahami jika dijalankan
dengan benar. Kadang-kadang, hasil kromatografi dapat memulai dengan yang tidak
diinginkan "berdaun" warna, tetapi jika dilakukan dengan benar dan lengkap, warna megah
dapat diproduksi.
[Sunting] R nilai
Nilai R dapat didefinisikan sebagai rasio dari jarak yang ditempuh oleh substansi ke jarak
yang ditempuh oleh pelarut. Nilai R biasanya dinyatakan sebagai pecahan dari dua tempat
desimal tetapi disarankan oleh Smith bahwa angka persentase harus digunakan sebagai
gantinya. Jika R nilai solusi adalah nol, zat terlarut tetap dalam fase diam dan dengan
demikian itu bergerak. Jika R value = 1 maka zat terlarut tidak memiliki afinitas untuk fase
diam dan perjalanan dengan pelarut depan.
Analisa kualitatif adalah nilai Rf = hC/hS, hC tinggi naik komponen, hS = tinggi naik pelarut
Analisa kuantitatif :
a. Perbandingan luas puncak noda
b. Perbandingan berat noda
c. Perbadinga noda setelah dilarutkan
d. planimetri

Displacement chromatography
Tempat terkonsentrasi kecil solusi yang berisi sampel zat terlarut diterapkan pada secarik
kertas kromatografi sekitar dua cm dari dasar piring, biasanya menggunakan tabung kapiler
untuk presisi maksimum. Sampel ini diserap ke kertas dan dapat membentuk interaksi dengan
itu. Setiap zat yang bereaksi atau obligasi dengan kertas tidak dapat diukur dengan
menggunakan teknik ini. Makalah ini kemudian dicelupkan ke dalam perawatan pelarut,
seperti etanol atau air, mengambil tempat cocok yang berada di atas permukaan pelarut, dan
ditempatkan dalam wadah tertutup.
Bergerak pelarut kertas dengan aksi kapiler, yang terjadi sebagai akibat dari daya tarik

molekul pelarut untuk kertas, hal ini juga dapat dijelaskan sebagai diferensial adsorpsi dari
komponen zat terlarut ke dalam pelarut. Sebagai pelarut naik melalui kertas itu bertemu dan
melarutkan campuran sampel, yang kemudian akan melakukan perjalanan ke kertas dengan
sampel zat terlarut pelarut. Berbeda senyawa dalam campuran sampel pada tingkat yang
berbeda karena perbedaan dalam kelarutan dalam pelarut, dan karena perbedaan ketertarikan
mereka pada serat di koran. Komponen lebih mudah larut yang lebih lanjut ia pergi.
Kromatografi kertas mengambil mana saja dari beberapa menit sampai beberapa jam.
Dalam beberapa kasus, kromatografi kertas tidak pigmen terpisah sepenuhnya, ini terjadi
ketika dua zat tampaknya memiliki nilai yang sama dalam pelarut tertentu. Dalam kasus ini,
dua arah kromatografi digunakan untuk memisahkan beberapa tempat-pigmen.
[Sunting] kromatografi Ascending
Dalam metode ini, pelarut adalah di kolam renang di bagian bawah kapal di mana kertas yang
supported.The cromatogram menaik dilipat di atas batang gelas yang setengah lainnya
menjadi teknik chromatogram.This turun memberikan pemisahan cepat seperti yang dari
teknik individu .
[Sunting] kromatografi Descending
Dalam metode ini, pelarut disimpan dalam sebuah palung di bagian atas ruangan dan
dibiarkan mengalir ke bawah kertas. Cairan bergerak turun oleh aksi kapiler serta oleh gaya
gravitasi, sehingga metode ini juga dikenal sebagai metode gravitasi. [Rujukan?] Dalam hal
ini, aliran lebih cepat dibandingkan dengan metode ascending, dan kromatografi adalah
menyelesaikan lebih cepat. Aparat dibutuhkan untuk kasus ini lebih canggih. Pelarut
berkembang ditempatkan di palungan di bagian atas yang biasanya terdiri dari bahan inert.
Makalah ini kemudian ditangguhkan dalam pelarut. Zat yang tidak dapat dipisahkan dengan
metode naik kadang-kadang dapat dipisahkan dengan metode menurun. [Rujukan?]
[Sunting] Kromatografi sebagai seni
Kromatografi dapat digunakan sebagai alternatif seni karena warna tertentu yang
menciptakan, tapi kromatografi kertas jarang menghasilkan warna dipahami jika dijalankan
dengan benar. Kadang-kadang, hasil kromatografi dapat memulai dengan yang tidak
diinginkan "berdaun" warna, tetapi jika dilakukan dengan benar dan lengkap, warna megah
dapat diproduksi.
[Sunting] R nilai
Nilai R dapat didefinisikan sebagai rasio dari jarak yang ditempuh oleh substansi ke jarak
yang ditempuh oleh pelarut. Nilai R biasanya dinyatakan sebagai pecahan dari dua tempat
desimal tetapi disarankan oleh Smith bahwa angka persentase harus digunakan sebagai
gantinya. Jika R nilai solusi adalah nol, zat terlarut tetap dalam fase diam dan dengan
demikian itu bergerak. Jika R value = 1 maka zat terlarut tidak memiliki afinitas untuk fase
diam dan perjalanan dengan pelarut depan.
[Sunting] Penemuan
Munculnya kromatografi perpindahan dapat dikaitkan dengan Tiselius [1], yang pada tahun
1943 pertama kali diklasifikasikan mode kromatografi yang frontal, elusi, dan perpindahan.
Kromatografi Pemindahan sejak menemukan berbagai aplikasi dari isolasi unsur transuranic
[2] untuk entitas biokimia [3]. Pemindahan kromatografi adalah metode kromatografi di
mana komponen diselesaikan dalam berturut-turut "persegi panjang" zona zat murni sangat
terkonsentrasi daripada pelarut dipisahkan "puncak". Molekul-molekul dipaksa untuk
bermigrasi ke kolom oleh gelombang maju dari molekul displacer yang memiliki afinitas
yang lebih tinggi untuk fase diam daripada larutan umpan. [4] Karena migrasi paksa,
konsentrasi produk yang lebih tinggi dan kemurnian dapat diperoleh dibandingkan dengan
moda lain kromatografi. Teknik ini ditemukan kembali oleh Horvath [5] dan telah sejak
menemukan banyak aplikasi, terutama di bidang pemurnian makromolekul biologi.
[Sunting] Prinsip

Prinsip dasar kromatografi perpindahan adalah: Sebuah molekul dengan afinitas tinggi untuk
matriks kromatografi (displacer) akan bersaing secara efektif untuk situs mengikat, dan
dengan demikian menggantikan seluruh molekul dengan afinitas yang lebih rendah [6] Ada
perbedaan jelas antara perpindahan dan kromatografi elusi. . Dalam modus elusi, zat biasanya
muncul dari sebuah kolom di sempit, puncak Gaussian. Pemisahan yang luas dari puncak,
sebaiknya ke baseline, yang diinginkan untuk mencapai pemurnian maksimal. Kecepatan di
mana setiap komponen campuran perjalanan ke kolom dalam mode elusi tergantung pada
banyak faktor. Tapi untuk dua zat untuk melakukan perjalanan dengan kecepatan yang
berbeda, dan dengan demikian dapat diselesaikan, harus ada perbedaan substansial dalam
beberapa interaksi antara biomolekul dan matriks kromatografi. Parameter operasi yang
disesuaikan untuk memaksimalkan efek dari perbedaan ini. Dalam banyak kasus, dasar
pemisahan puncak dapat dicapai hanya dengan elusi gradien dan beban kolom yang rendah.
Dengan demikian, dua kelemahan kromatografi elusi modus, terutama pada skala preparatif,
adalah kompleksitas operasional, karena gradien pelarut memompa, dan throughput rendah,
karena beban kolom yang rendah. Pemindahan kromatografi memiliki keunggulan
dibandingkan kromatografi elusi dalam komponen diselesaikan menjadi zona berturut-turut
zat murni daripada "puncak". Karena proses mengambil keuntungan dari nonlinier dari
isoterm, feed kolom yang lebih besar dapat dipisahkan pada kolom tertentu dengan
komponen dimurnikan pulih pada konsentrasi lebih tinggi secara signifikan.
[Sunting] Teori dan Metode
Seperti ditunjukkan di atas, proses kromatografi perpindahan dapat dipecah menjadi tiga
tahap yang berbeda: loading, perpindahan, dan regenerasi. Demi kesederhanaan, contoh
pemurnian biomolecule diberikan di sini akan dibatasi untuk protein. Pada prinsipnya, semua
biomolekul kepentingan komersial saat ini - termasuk oligonukleotida dan antibodi - dapat
dimurnikan dalam mode perpindahan, dengan pilihan yang tepat matriks kolom dan displacer.
Memuat fase
Pemurnian dimulai dengan kolom disetimbangkan dengan buffer pemuatan mirip dengan
metode kromatografi elusi. Campuran pakan yang mengandung protein murni dalam buffer
dimuat ke kolom pada tingkat yang cukup lambat, dalam kondisi di mana bahan baik
dipertahankan. Tujuan dari langkah ini adalah untuk memulai pengikatan biomolekul dalam
keadaan semiequilibrium. Hal ini ditunjukkan dalam grafik sebagai campuran protein dimulai
pemisahan awal ke komponen kuning dan ungu.
Perpindahan fase
Setelah sampel telah dimuat, kolom diberi makan larutan displacer pada konsentrasi yang
cukup rendah (biasanya 5 mM) dalam buffer yang sama yang digunakan untuk memuat
sampel. Displacer ini dirancang untuk mengikat lebih erat dengan matriks dari salah satu
biomolekul dan dengan demikian "mendorong" semua komponen campuran dari matriks
depan itu. Selama masing-masing komponen sampel dan displacer tersebut tidak ireversibel
teradsorpsi pada matriks kolom, ada beberapa dari masing-masing terus menyerap ke, dan
dari desorbing, matriks.
Dalam modus elusi, hasil yang optimal diperoleh ketika konsentrasi sangat rendah sehingga
komponen individu bertindak independen dan tidak bersaing untuk situs mengikat matriks.
Dalam modus perpindahan, komponen sampel diperkenalkan dalam bentuk yang jauh lebih
terkonsentrasi, dan sehingga memungkinkan untuk komponen yang mengikat kuat - baik
displacer atau salah satu protein dalam campuran sampel - bersaing untuk situs mengikat.
Komponen mengikat kuat (awalnya, displacer sendiri) kemudian menggantikan dengan
berhasil bersaing untuk situs mengikat. Berdasarkan kekuatan masing-masing mengikat,
setiap komponen dalam sampel asli kemudian menjadi "displacer" untuk komponen kurang
terikat erat berikutnya. Jadi kereta perpindahan didirikan sebagai berdekatan, band terfokus

dengan sedikit tumpang tindih (kuning dan ungu dalam grafik). Dalam menjalankan sukses,
kereta sepenuhnya dibentuk sebelum komponen bunga tiba di bagian bawah kolom. Pecahan
dapat dikumpulkan dan pemotongan produk yang diinginkan dibuat.
regenerasi fase
Ketika displacer yang menerobos, yaitu, mulai muncul dari kolom, jalankan selesai dan
regenerasi kolom dapat dimulai. Regenerasi dilakukan dengan menggunakan buffer yang
dapat menghapus displacer dari matriks, diikuti oleh equilibrium dengan memuat penyangga
dalam persiapan untuk menjalankan berikutnya. Karena displacers harus mengikat lebih kuat
dari protein apapun yang dimurnikan, maka diharapkan bahwa penghapusan mereka dari
kolom harus memerlukan beberapa kondisi khusus. Bagian non-sepele dari desain yang
displacer baik, maka, adalah penggabungan dari beberapa fitur struktural yang
memungkinkan untuk penghapusan lengkap dari matriks di bawah beberapa kondisi.

Keuntungan dari kromatografi perpindahan

Sebuah perbedaan penting antara perpindahan dan kromatografi langkah gradien adalah
bahwa bagian depan displacer selalu tetap di belakang zona pakan yang berdekatan dalam
kereta perpindahan sedangkan desorbents (misalnya, garam dalam pertukaran ion, pengubah
organik dalam fase terbalik kromatografi) bergerak melalui zona pakan. Modus perpindahan
kromatografi mengambil keuntungan dari karakteristik termodinamika dari sistem
kromatografi untuk mengatasi banyak kekurangan elusi kromatografi preparatif dan gradien.
Ini karakteristik perpindahan membuatnya kurang peka terhadap beban pakan daripada mode
elusi operasi, sehingga memungkinkan untuk memberikan throughputs proses yang lebih
tinggi. Keuntungan lain adalah resolusi tinggi yang dapat memberikan perpindahan

dibandingkan dengan proses elusi. Pemindahan kromatografi memanfaatkan kompetisi,

nonlinear multikomponen antara komponen yang akan dipisahkan, sehingga dalam resolusi
yang lebih tinggi, khususnya di kalangan spesies yang terkait erat. Sebaliknya, dalam proses
elusi (misalnya, linear gradien, gradien langkah), pemisahan terjadi di bawah kondisi yang
mengikat relatif lemah (yang penting untuk mendapatkan dari zat terlarut kolom). Faktor
pemisahan antara zat terlarut dengan demikian lebih rendah dalam elusi daripada di
pengungsian, menyebabkan resolusi miskin dalam mode elusi operasi. Keuntungan lain dari
perpindahan termasuk kontrol yang lebih baik atas konsentrasi produk dan munculnya produk
dalam konsentrasi yang relatif rendah pengubah fase gerak. Kombinasi throughputs tinggi
dan resolusi tinggi dalam suatu proses tunggal membuat perpindahan modus operasi yang
menarik untuk pemurnian protein.

[edit] Applications
Pemurnian kromatografi protein dari campuran kompleks bisa sangat menantang, terutama
ketika campuran mengandung protein yang sama dipertahankan atau bila diinginkan untuk
memperkaya komponen jejak dalam feed. Selanjutnya, beban kolom sering terbatas ketika
resolusi tinggi diperlukan menggunakan mode tradisional kromatografi (misalnya linear
gradien, isokratik kromatografi). Dalam kasus ini, kromatografi perpindahan merupakan
teknik yang efisien untuk pemurnian protein dari campuran kompleks pada beban tinggi
kolom dalam berbagai aplikasi. Sebuah kemajuan penting dalam keadaan seni kromatografi
pemindahan adalah pengembangan rendah displacers massa molekul untuk pemurnian
protein dalam sistem pertukaran ion [7]. [8] [9]. Penelitian ini adalah signifikan dalam bahwa
itu mewakili keberangkatan utama dari kebijaksanaan konvensional bahwa polimer
polyelectrolyte besar dibutuhkan untuk menggantikan protein dalam sistem pertukaran ion.
Rendah displacers massa molekul memiliki keuntungan operasional yang signifikan
dibandingkan dengan displacers polyelectrolyte besar. Misalnya, jika ada tumpang tindih
antara displacer dan protein yang menarik, bahan-bahan massa molekul rendah dapat dengan
mudah dipisahkan dari protein dimurnikan selama pasca-perpindahan pengolahan
menggunakan ukuran standar berbasis metode pemurnian (misalnya ukuran kromatografi
eksklusi, ultrafiltrasi) . Selain itu, perilaku garam bergantung adsorpsi ini displacers MW
rendah sangat memudahkan regenerasi kolom. Ini displacers telah digunakan untuk berbagai
macam pemisahan resolusi tinggi di sistem pertukaran ion [10] [11] [12] [13] [14] [15] [16].
Selain itu, kegunaan kromatografi perpindahan untuk pemurnian faktor pertumbuhan
rekombinan [17], antigen protein vaksin [18] dan oligonukleotida antisense [19] juga telah
ditunjukkan. Ada beberapa contoh di mana perpindahan kromatografi telah diterapkan pada
pemurnian protein menggunakan pertukaran ion, interaksi hidrofobik, serta terbalik
kromatografi fase [20]. Kromatografi Pemindahan cocok untuk memperoleh jumlah mg
protein dimurnikan dari campuran kompleks dengan menggunakan kolom kromatografi
standar analitis pada skala bangku. Hal ini juga sangat cocok untuk memperkaya komponen
jejak dalam feed. Kromatografi Pemindahan dapat dengan mudah dilakukan dengan
menggunakan berbagai sistem resin termasuk, pertukaran ion, HIC dan RPLC [21], [22] Duadimensi kromatografi merupakan pendekatan yang paling menyeluruh dan ketat untuk
evaluasi proteome. Sementara pendekatan diterima sebelumnya telah digunakan pendekatan
modus elusi kromatografi seperti pertukaran kation HPLC fase terbalik, hasil biasanya sangat
rendah membutuhkan kepekaan analitis dalam picomolar berkisar femptomolar [23]. Sebagai
kromatografi Pemindahan menawarkan keuntungan dari konsentrasi komponen jejak, dua
dimensi kromatografi memanfaatkan perpindahan daripada modus elusi pada langkah
kromatografi hulu merupakan alat yang berpotensi kuat untuk analisis komponen jejak,
modifikasi, dan identifikasi komponen dinyatakan minor proteome. [ 24]

[edit] References
1. ^ A. Tiselius. Displacement development in adsorption analysis. Ark. Kemi. Mineral Geol.
16A: 118 (1943).
2. ^ G. T. Seaborg. The Transuranium Elements. Science 104(2704):379-386 (1946).
3. ^ J. Frenz and C.S. Horvath. High performance displacement chromatography. pp 212-314 in
C. Horvath (Ed.) High Performance Liquid Chromatography-advances and perspectives. Vol.
5, Academic Press, San Diego, CA.
4. ^ N. Tugcu . Purification of proteins using displacement chromatography. pp 71-89 in M.
Zachariou (Ed.) Methods in Molecular Biology: Vol 421 Affinity Chromatography: Methods
and Protocols. 2nd edition. Humana Press, Totowa NJ.
5. ^ C.S. Horvath, A. Nahum, and J. Frenz. High performance displacement chromatography. J.
Chromatogr. 218, 365393(1981).
6. ^ Displacement Chromatography 101. [1] Sachem, Inc. Austin, TX 78737
7. ^ S. M. Cramer and G. Jayaraman, Current Opinions in Biotechnology 4: 217-225, (1993)
8. ^ G. Jayaraman, S. Gadam, and S. M. Cramer. J. Chromatogr. A 630:53-68. (1993)
9. ^ G. Jayaraman, Y. Li, J. A. Moore, and S. M. Cramer. J. Chromatogr. A 702:143-155. (1995)
10. ^ A. Kundu, S. Vunnum, G. Jayaraman, and S. M. Cramer. Biotech. and Bioeng. 48: 452-460.
(1995)
11. ^ A. Kundu, S. Vunnum, and S. M. Cramer. J. Chromatogr. A, 707:57-67. (1995)
12. ^ A. Kundu, S. Vunnum, and S. M. Cramer. Adsorption 4:3-4. (1998)
13. ^ A. Kundu, K. Barnthouse, and S. M. Cramer. Biotech. and Bioeng., 56:119-129. (1997)
14. ^ KA. Kundu, A. A. Shukla, K. A. Barnthouse, J. Mooreand S. M. Cramer. BioPharm 10 :64.
(1997)
15. ^ A. Kundu, and S. M. Cramer. Anal. Biochem., 248:111-116. ( 1997)
16. ^ A. A. Shukla, K. A. Barnthouse, S. S. Bae, J. A. Moore, and S. M. Cramer.. J. Chromatogr.
A 814:1-2. (1998)
17. ^ K. A. Barnthouse, W. Trompeter, R. Jone, P. Inampudi, R.Rupp, and S. M. Cramer. J.
Biotechnol. 66:125-136 (1998)
18. ^ A. A. Shukla, R. L. Hopfer, D. N. Chakravarti, E. Bortell, and S. M. Cramer. Biotechnol.
Prog. 14: 91-101(1998)
19. ^ N. Tugcu, R. R. Deshmukh, Y. S. Sangvic, J. A. Moored, and S. M. Cramer J. Chromatogr.
A 923:65-73(2001)
20. ^ R. Freitag and J. Breier. J. Chromatogr. A 691, 101112 (1995).
21. ^ Trace Component Amplification. [2] Sachem, Inc. Austin, TX 78737
22. ^ N. Tugcu, R. R. Deshmukh, Y. S. Sanghvi, and S. M. Cramer. Reactive and Functional
Polymers 54, 3747(2003).
23. ^ . E. Nagele, M. Vollmer, P. Horth, and C. Vad. 2D-LC/MS techniques for the identification
of proteins in highly complex mixtures. Expert Reviews in Proteomics. Vol. 1, No. 1, Pages
37-46 (2004).
24. ^ 2D-HPLC Course SACHEM, Inc. Austin, TX, 78737[3]

Retrieved from "http://en.wikipedia.org/wiki/Displacement_chromatography"

4.4. TLC, kolom dan

Ion chromatography
Pertukaran ion kromatografi (atau kromatografi ion) adalah proses yang memungkinkan
pemisahan ion dan molekul polar berdasarkan muatan mereka. Dionex Corp

memperkenalkan sistem komersial pada awal tahun 1970 yang menggunakan teknologi
revolusioner penekanan untuk mengurangi konduktivitas latar belakang, sehingga
meningkatkan kemampuan deteksi ion. Hal ini dapat digunakan untuk hampir semua jenis
molekul dibebankan termasuk protein yang besar, nukleotida kecil dan asam amino. Solusi
harus disuntikkan biasanya disebut sampel, dan komponen individual dipisahkan disebut
analit. Hal ini sering digunakan dalam pemurnian protein, analisis air, dan kontrol kualitas

Sejarah
Ion methods have been in use since 1850, when H. Thompson and J. T. Way, researchers in
England, treated various clays with ammonium sulfate or carbonate in solution to extract the
amonia dan kalsium rilis. Pada tahun 1927, kolom pertama mineral zeolit digunakan untuk
menghilangkan kalsium dan ion magnesium mengganggu dari solusi untuk menentukan isi
sulfat air. Versi modern dari IEC dikembangkan selama Proyek Manhattan perang. Suatu
teknik yang diperlukan untuk memisahkan dan berkonsentrasi pada unsur-unsur radioaktif
yang diperlukan untuk membuat bom atom. Para peneliti memilih adsorben yang akan latch
ke unsur transuranium dibebankan, yang kemudian dapat dielusi diferensial. Pada akhirnya,
sekali declassified, teknik ini akan menggunakan resin IE baru untuk mengembangkan sistem
yang sering digunakan saat ini untuk pemurnian tertentu biologi dan inorganics. Pada awal
1970-an, kromatografi ion dikembangkan oleh Hamish Kecil dan rekan kerja di Dow
Chemical Company sebagai metode baru yang dapat digunakan dalam analisis IEC otomatis.
Ini kemudian mengarah pada pembentukan Dionex Corp (Dow-Ion Exchange) yang
memimpin pasar di IC peralatan dan perkembangan. IC menggunakan resin ion lemah untuk
fase stasioner dan suatu stripper menetralkan tambahan, atau penekan, kolom untuk
menghilangkan ion latar belakang eluen. Ini adalah teknik yang kuat untuk menentukan
konsentrasi rendah ion dan sangat berguna dalam studi kualitas lingkungan dan air, antara
aplikasi lain

Prinsip dasar
ion Kromatogram
Pertukaran ion kromatografi mempertahankan molekul analit pada kolom berdasarkan
coulombic (ion) interaksi. Permukaan fase diam menampilkan kelompok fungsional ion (RX)
yang berinteraksi dengan ion analit muatan yang berlawanan. Jenis kromatografi dibagi lagi
menjadi kromatografi penukar kation dan anion kromatografi pertukaran. Senyawa ionik
yang terdiri dari spesies kationik M + dan spesies anionik B-dapat dipertahankan oleh fase
diam.
Kation kromatografi pertukaran mempertahankan kation bermuatan positif karena fase diam
menampilkan kelompok fungsional bermuatan negatif:
Anion kromatografi penukar anion mempertahankan menggunakan gugus fungsional
bermuatan positif:
Perhatikan bahwa kekuatan ion baik C + atau A-dalam fase gerak dapat disesuaikan untuk
menggeser posisi kesetimbangan dan dengan demikian waktu retensi.
Kromatogram ion menunjukkan kromatogram yang diperoleh dengan kolom penukar anion.

[edit] Typical technique

Metrohm 850 Ion chromatography system

Sampel diperkenalkan, baik secara manual atau dengan autosampler, menjadi sebuah loop
sampel volume yang diketahui. Sebuah larutan buffered dikenal sebagai fase gerak membawa
sampel dari lingkaran ke kolom yang berisi beberapa bentuk materi fase diam. Ini biasanya
resin atau matriks gel yang terdiri dari agarosa atau manik-manik selulosa dengan kovalen
terikat kelompok fungsional yang bertugas. Analit target (anion atau kation) dipertahankan
pada fase diam tetapi dapat dielusi dengan meningkatkan konsentrasi dari spesies yang sama
menuduh bahwa akan menggantikan ion analit dari fase diam. Misalnya, dalam kromatografi
penukar kation, analit bermuatan positif bisa digusur oleh penambahan ion natrium
bermuatan positif. Analit kepentingan maka harus dideteksi dengan beberapa cara, biasanya
oleh konduktivitas atau UV / Visible absorbansi cahaya.
Dalam rangka untuk mengontrol sistem IC, kromatografi sistem data (CDS) biasanya
diperlukan. Selain sistem IC, beberapa CDSs juga dapat mengontrol kromatografi gas (GC)
dan HPLC sistem.

[edit] Separating proteins

Preparative-scale ion exchange column used for protein purification.

Protein memiliki kelompok fungsional banyak yang dapat memiliki baik muatan positif dan
negatif. Pertukaran ion kromatografi memisahkan protein sesuai dengan muatan bersih
mereka, yang tergantung pada komposisi fase gerak. Dengan menyesuaikan pH atau
konsentrasi ion dari fase gerak, molekul protein yang berbeda dapat dipisahkan. Misalnya,
jika protein memiliki muatan positif bersih pada pH 7, maka akan mengikat ke kolom manikmanik bermuatan negatif, sedangkan protein bermuatan negatif tidak akan. Dengan
mengubah pH sehingga muatan total pada protein negatif, juga akan dielusi.

Elution by changing the ionic strength of the mobile phase is a more subtle effect - it works
as ions from the mobile phase will interact with the immobilized ions in preference over

those on the stationary phase. This "shields" the stationary phase from the protein, (and vice
versa) and allows the protein to elute.

[edit] References

Ion Chromatography by Hamish Small ISBN 0-306-43290-0

Handbook of Ion Chromatography by Joachim Weiss, Dionex Corporation, ISBN
978-3527287017
Ion Chromatography by James S. Fritz and Douglas T. Gjerde, ISBN 3527299149
Ion Chromatography (Journal of Chromatography Library) by P.R. Haddad and
P.E. Jackson, ISBN 0444882324
Sample Preparation Techniques for Ion Chromatography by A. Seubert et al.,
Metrohm AG, Order Number 8.025.5003
Air Monitoring by Ion Chromatography by M. Lubli, D. Parab, K. Viehweger,
Metrohm AG, Order Number 8.035.5003

Thin layer chromatography

From Wikipedia, the free encyclopedia
Jump to: navigation, search

Thin layer chromatography

Separation of black ink on a TLC plate

Acronym

TLC

Classification

Chromatography
Other Techniques

Related

Agarose gel electrophoresis

SDS-PAGE

This box: view talk

Kromatografi lapis tipis (TLC) adalah teknik kromatografi yang digunakan untuk
memisahkan campuran [1] kromatografi lapis tipis dilakukan pada selembar kaca, plastik,
atau aluminium foil, yang dilapisi dengan lapisan tipis bahan adsorben, biasanya silika gel,.
aluminium oksida, atau selulosa. Ini lapisan adsorben dikenal sebagai fase diam.
Setelah sampel telah diterapkan di piring, campuran pelarut atau pelarut (dikenal sebagai fase
gerak) ditarik piring melalui kapiler. Karena analit yang berbeda naik pelat TLC pada tingkat
yang berbeda, pemisahan dicapai [2]..
Kromatografi lapis tipis menemukan banyak aplikasi, termasuk:
penentuan komponen tanaman mengandung
pemantauan reaksi organik.
menganalisis ceramides dan asam lemak
deteksi pestisida atau insektisida dalam makanan dan air
menganalisis komposisi zat warna dari serat dalam forensik, atau
mengidentifikasi senyawa hadir dalam substansi tertentu
pengujian kemurnian radiokimia radiofarmasi
Sejumlah perangkat tambahan dapat dibuat dengan metode asli untuk mengotomatisasi
langkah-langkah yang berbeda, untuk meningkatkan resolusi dicapai dengan TLC dan
memungkinkan kuantisasi lebih akurat. Metode ini disebut sebagai HPTLC, atau "high
performance TLC".

[sunting] persiapan pelat

Pelat TLC biasanya tersedia secara komersial, dengan ukuran partikel berkisar standar untuk
meningkatkan reproduktifitas. Mereka dibuat dengan cara mencampurkan adsorben, seperti
gel silika, dengan sejumlah kecil pengikat lembam seperti kalsium sulfat (gipsum) dan air.
Campuran ini menyebar sebagai bubur tebal pada lembar pembawa tidak aktif, biasanya kaca,
aluminium foil tebal, atau plastik. Pelat yang dihasilkan dikeringkan dan diaktifkan dengan
pemanasan dalam oven selama tiga puluh menit pada 110 C. Ketebalan lapisan adsorben
biasanya sekitar 0,1-0,25 mm untuk tujuan analitis dan sekitar 0,5 -. 2,0 mm untuk KLT
preparatif [3]

[edit] Technique

Development of a TLC plate, a purple spot separates into a red and blue spot.

Kromatogram dari 10 minyak esensial diwarnai dengan pereaksi vanillin.

Proses ini mirip dengan kromatografi kertas dengan keuntungan dari berjalan lebih cepat,
pemisahan yang lebih baik, dan pilihan antara fasa diam yang berbeda. Karena kesederhanaan
dan kecepatan TLC sering digunakan untuk reaksi kimia pemantauan dan analisis kualitatif
produk reaksi.
Tempat kecil larutan yang mengandung sampel diterapkan ke piring, sekitar satu sentimeter
dari dasar. Plat tersebut kemudian dicelupkan ke dalam pelarut yang cocok, seperti heksana
atau etil asetat, dan ditempatkan dalam wadah tertutup. Pelarut bergerak piring dengan aksi
kapiler dan memenuhi campuran sampel, yang dibubarkan dan dilakukan atas piring dengan
pelarut. Berbeda senyawa dalam campuran sampel pada tingkat yang berbeda karena
perbedaan dalam daya tarik mereka ke fase diam, dan karena perbedaan kelarutan dalam
pelarut. Dengan mengubah pelarut, atau mungkin menggunakan campuran, pemisahan
komponen (diukur dengan nilai Rf) dapat disesuaikan. Juga, pemisahan dicapai dengan pelat
KLT dapat digunakan untuk memperkirakan pemisahan kolom kromatografi. [4]
Pemisahan senyawa didasarkan pada persaingan zat terlarut dan fase gerak untuk tempat
mengikat fase diam. Misalnya, jika fase silika gel biasa digunakan sebagai fase diam dapat
dianggap polar. Mengingat dua senyawa yang berbeda dalam polaritas, senyawa polar yang
lebih memiliki interaksi kuat dengan silika dan karena itu lebih mampu untuk menghilangkan
fase gerak dari tempat mengikat. Akibatnya, senyawa kurang polar bergerak lebih tinggi
piring (menghasilkan nilai Rf yang lebih tinggi). Jika fase mobile berubah menjadi lebih
polar pelarut atau campuran pelarut, itu lebih mampu menghilangkan zat terlarut dari tempat
silika mengikat dan semua senyawa pada pelat TLC akan bergerak lebih tinggi piring. Praktis
hal ini berarti bahwa jika Anda menggunakan campuran etil asetat dan heptana sebagai fase
gerak, menambahkan hasil asetat lebih etil nilai Rf yang lebih tinggi untuk semua senyawa
pada pelat TLC. Mengubah polaritas fase gerak biasanya tidak akan menghasilkan urutan
terbalik dari menjalankan senyawa pada pelat TLC. Sebuah seri eluotropic dapat digunakan
sebagai panduan dalam memilih fase gerak. Jika urutan terbalik dari menjalankan senyawa
yang diinginkan, fase stasioner apolar harus digunakan, seperti C18-difungsikan silika.
[Sunting] preparatif TLC
TLC juga dapat digunakan pada skala semi-preparatif kecil untuk memisahkan campuran
hingga beberapa ratus miligram beberapa. Campuran ini tidak "terlihat" pada pelat TLC
sebagai titik, melainkan diterapkan ke piring sebagai lapisan tipis bahkan horizontal ke dan
tepat di atas tingkat pelarut. Bila dikembangkan dengan pelarut senyawa terpisah di band
horisontal daripada tempat horizontal dipisahkan. Setiap band (atau band yang diinginkan)
yang dikerok bahan backing. Bahan backing kemudian diekstraksi dengan pelarut yang sesuai
(misalnya DCM) dan disaring untuk memberikan materi terisolasi setelah penghapusan
pelarut. Untuk skala kecil reaksi dengan produk mudah dipisahkan, preparatif TLC dapat

menjadi jauh lebih efisien dari segi waktu dan biaya daripada melakukan kromatografi
kolom. Jelas, seluruh piring tidak dapat dikembangkan atau produk kimia akan dihancurkan
kimia. Jadi teknik ini paling baik digunakan dengan senyawa yang berwarna, atau terlihat di
bawah sinar UV. Atau, bagian kecil dari piring dapat dikembangkan misalnya kimia
memotong bagian keluar dan kimia berkembang, atau masking sebagian besar dari piring dan
mengekspos bagian kecil untuk pengembang kimia seperti yodium.
[Sunting] Analisis
Sebagai bahan kimia yang terpisah mungkin tidak berwarna, beberapa metode yang ada
untuk memvisualisasikan tempat:
Seringkali sejumlah kecil senyawa neon, biasanya mangan-diaktifkan seng silikat,
ditambahkan ke adsorben yang memungkinkan visualisasi dari tempat bawah blacklight
(UV254). Lapisan adsorben sehingga akan berpendar hijau muda dengan sendirinya, tapi
tempat analit memuaskan fluoresensi ini.
uap Yodium adalah reagen warna umum tidak spesifik
reagen warna khusus yang ada di mana lempeng TLC adalah dicelupkan atau yang
disemprotkan ke piring [5]
Dalam kasus lipid, kromatogram dapat ditransfer ke membran PVDF dan kemudian
mengalami analisis lebih lanjut, misalnya spektrometri massa, teknik yang dikenal sebagai
Far-Timur blotting.
Setelah terlihat, nilai Rf, atau faktor retensi, dari tempat masing-masing dapat ditentukan
dengan membagi jarak yang ditempuh oleh produk dengan total jarak yang ditempuh oleh
pelarut (solvent depan). Nilai-nilai ini tergantung pada pelarut yang digunakan, dan jenis
pelat KLT, dan tidak konstanta fisik. Eluen pada lapisan tipis diletakkan di atas piring
[Sunting] Aplikasi
Dalam kimia organik, reaksi yang kualitatif dipantau dengan TLC. Tempat sampel dengan
tabung kapiler ditempatkan di piring: tempat dari bahan awal, tempat dari campuran reaksi,
dan "co-spot" dengan baik. Sebuah piring (3 oleh 7 cm) kecil TLC berlangsung beberapa
menit untuk menjalankan. Analisis kualitatif, dan akan menunjukkan apakah bahan awal telah
menghilang, yaitu reaksi selesai, jika produk apapun telah muncul, dan berapa banyak produk
yang dihasilkan (meskipun ini mungkin berada di bawah-diperkirakan karena co-elusi).
Sayangnya, TLC dari suhu rendah reaksi dapat memberikan hasil yang menyesatkan, karena
sampel dipanaskan sampai suhu kamar dalam kapiler, yang dapat mengubah reaksi-sampel
hangat dianalisis dengan TLC yang tidak sama dengan apa yang ada dalam labu suhu rendah .
Salah satu reaksi tersebut adalah pengurangan DIBALH dari ester untuk aldehida.
Sebagai contoh kromatografi dari ekstrak daun hijau (bayam misalnya untuk) dalam 7 tahap
pembangunan. Karoten elutes cepat dan hanya terlihat sampai langkah 2. Klorofil A dan B
berada setengah jalan di langkah terakhir dan lutein pewarnaan senyawa pertama kuning.

Step 1

Step 2

Step 3

Step 4

Step 7
Step 6
Step 5
Dalam satu studi TLC telah diterapkan dalam pemutaran reaksi organik [6] misalnya dalam
fine tuning-sintesis BINAP dari 2-naftol. Dalam metode ini solusi alkohol dan katalis
(misalnya (III) klorida besi) adalah tempat yang terpisah pada garis dasar, kemudian bereaksi
dan kemudian langsung dianalisis.

[edit] References
1. ^ Laurence M. Harwood, Christopher J. Moody. Experimental organic chemistry: Principles
and Practice (Illustrated edition ed.). pp. 159-173.
2. ^ Vogel's Textbook of Practical Organic Chemistry (5th Edition) (Hardcover) by A.I. Vogel
(Author), A.R. Tatchell (Author), B.S. Furnis (Author), A.J. Hannaford (Author), P.W.G.
Smith ISBN 0582462363
3. ^ Tables showing the thickness value of commercial regular and preparative Thin Layer
Chromatography plates
4. ^ Fair, J. D.; Kormos, C. M. J. Chromatogr. A 2008, 1211(1-2), 49-54.
(doi:10.1016/j.chroma.2008.09.085)
5. ^ Stains for Developing TLC Plates
http://www.ux1.eiu.edu/~cfthb/research/handbook/TLCstains.htm
6. ^ TLC plates as a convenient platform for solvent-free reactions Jonathan M. Stoddard, Lien
Nguyen, Hector Mata-Chavez and Kelly Nguyen Chem. Commun., 2007, 1240 - 1241,
doi:10.1039/b616311d

Column chromatography

Typical set up for manual column chromatography.

Seorang ahli kimia pada tahun 1950 dengan menggunakan kromatografi kolom. Wadah
Erlenmeyer berada di lantai.
Kromatografi kolom dalam kimia adalah metode yang digunakan untuk memurnikan
senyawa kimia individu dari campuran senyawa. Hal ini sering digunakan untuk aplikasi
pada skala preparatif dari mikrogram sampai kilogram.
Kolom preparatif kromatografi klasik, adalah tabung gelas dengan diameter dari 50 mm dan
tinggi 50 cm sampai 1 m dengan keran di bagian bawah. Dua metode yang umumnya
digunakan untuk menyiapkan kolom, metode kering, dan metode basah. Untuk metode
kering, kolom pertama diisi dengan bubuk kering fase stasioner, diikuti dengan penambahan
fase gerak, yang memerah melalui kolom sampai benar-benar basah, dan dari titik ini tidak
pernah diperbolehkan untuk kering. Untuk metode basah, bubur dibuat dari eluen dengan
bubuk fase diam dan kemudian dengan hati-hati dituangkan ke dalam kolom. Perawatan
harus diambil untuk menghindari gelembung udara. Sebuah solusi dari bahan organik dipipet
di atas fase diam. Lapisan ini biasanya atasnya dengan lapisan pasir kecil atau dengan kapas
atau wol kaca untuk melindungi bentuk lapisan organik dari kecepatan eluen baru
ditambahkan. Eluen secara perlahan melewati kolom untuk memajukan bahan organik.
Seringkali reservoir eluen bola atau corong pisah eluen-penuh dan tutup diletakkan di atas
kolom.
Komponen individu dipertahankan oleh fase diam berbeda dan terpisah dari satu sama lain
ketika mereka sedang berjalan pada kecepatan yang berbeda melalui kolom dengan eluen.
Pada akhir kolom mereka mengelusi satu per satu. Selama proses kromatografi seluruh eluen
dikumpulkan dalam serangkaian fraksi. Komposisi aliran eluen dapat dipantau dan setiap
fraksi dianalisis untuk senyawa terlarut, misalnya dengan kromatografi analitis, serapan UV,
atau fluoresensi. Senyawa berwarna (atau senyawa neon dengan bantuan lampu UV) dapat
dilihat melalui dinding kaca sebagai band bergerak.

Proses pemisahan lapisan komponen pada kolom kromatografi

Column chromatography proceeds by a series of steps.

[edit] Stationary phase (adsorbent)

The stationary phase or adsorbent in column chromatography is a solid. The most common
stationary phase for column chromatography is silica gel, followed by alumina. Cellulose
powder has often been used in the past. Also possible are ion exchange chromatography,
reversed-phase chromatography (RP), affinity chromatography or expanded bed

adsorption (EBA). The stationary phases are usually finely ground powders or gels and/or
are microporous for an increased surface, though in EBA a fluidized bed is used.

Mobile phase (eluent)

Fase diam atau adsorben dalam kromatografi kolom solid. Fase diam yang paling umum
untuk kromatografi kolom silika gel, diikuti oleh alumina. Bubuk selulosa telah sering
digunakan di masa lalu. Juga mungkin adalah pertukaran ion kromatografi, terbalik-fase
kromatografi (RP), kromatografi afinitas atau diperluas tidur adsorpsi (EBA). Para fasa diam
biasanya bubuk ditumbuk halus atau gel dan / atau mikroporous untuk permukaan meningkat,
meskipun dalam EBA tempat tidur terfluidisasi digunakan.
Fase gerak (eluen)
Fase gerak atau eluen adalah salah pelarut murni atau campuran pelarut yang berbeda. Hal ini
dipilih sehingga faktor retensi nilai senyawa bunga kira-kira sekitar 0,2-0,3 untuk
meminimalkan waktu dan jumlah eluen untuk menjalankan kromatografi. Eluen juga telah
dipilih sehingga senyawa yang berbeda dapat dipisahkan secara efektif. Eluen dioptimalkan
dalam pretest skala kecil, sering menggunakan kromatografi lapis tipis (KLT) dengan fase
diam yang sama.
Tingkat aliran lebih cepat dari eluen meminimalkan waktu yang dibutuhkan untuk
menjalankan kolom dan dengan demikian meminimalkan difusi, menghasilkan pemisahan
yang lebih baik, lihat persamaan Van Deemter itu. Sebuah kolom laboratorium sederhana
berjalan oleh aliran gravitasi. Laju aliran seperti kolom dapat ditingkatkan dengan
memperpanjang kolom diisi segar eluen di atas puncak fase diam atau dikurangi dengan
kontrol sentuh. Laju aliran yang lebih baik dapat dicapai dengan menggunakan pompa atau
dengan menggunakan gas terkompresi (misalnya udara, nitrogen, atau argon) untuk
mendorong pelarut melalui kolom (flash kromatografi kolom) [1] [2].
Ukuran partikel dari fase diam umumnya halus di kromatografi kolom flash daripada dalam
kromatografi kolom gravitasi. Sebagai contoh, salah satu nilai yang paling banyak digunakan
silika gel dalam teknik pertama adalah jala 230-400 (40-63 pM), sedangkan teknik yang
kedua biasanya membutuhkan jala 70-230 (63-200 pM) silika gel [3].
Sebuah spreadsheet yang membantu dalam keberhasilan pengembangan kolom lampu kilat
telah dikembangkan. Spreadsheet memperkirakan volume retensi dan volume band analit,
nomor fraksi diharapkan mengandung analit masing-masing, dan resolusi antara puncak yang
berdekatan. Informasi ini memungkinkan pengguna untuk memilih parameter yang optimal
untuk preparatif skala pemisahan sebelum kolom flash itu sendiri yang dicoba. [4]

Automated Systems

Sebuah sistem kromatografi ion otomatis.

Kromatografi kolom merupakan tahap yang sangat memakan waktu di lab apapun dan

dengan cepat dapat menjadi hambatan untuk setiap laboratorium proses. Oleh karena
itu, beberapa produsen telah mengembangkan sistem otomatis flash kromatografi
(biasanya disebut sebagai LPLC, kromatografi cairan tekanan rendah, sekitar 50-75
psi) yang meminimalkan keterlibatan manusia dalam proses pemurnian. Sistem
otomatis akan mencakup komponen biasanya ditemukan pada lebih sistem HPLC
mahal seperti pompa gradien, port sampel injeksi, detektor UV dan kolektor untuk
mengumpulkan fraksi eluen. Biasanya sistem ini otomatis dapat memisahkan sampel
dari beberapa miligram hingga skala kg industri dan menawarkan solusi yang jauh
lebih murah dan lebih cepat untuk melakukan beberapa suntikan pada persiapanHPLC sistem.
Resolusi (atau kemampuan untuk memisahkan campuran) pada sistem LPLC akan
selalu lebih rendah dibandingkan dengan HPLC, sebagai bahan kemasan dalam kolom
HPLC bisa jauh lebih kecil, biasanya hanya 5 micrometre sehingga luas permukaan
meningkat fase stasioner, meningkatkan interaksi permukaan dan memberikan
pemisahan yang lebih baik. Namun, penggunaan media ini kemasan kecil
menyebabkan tekanan balik tinggi dan mengapa itu disebut kromatografi cair tekanan
tinggi. Kolom LPLC biasanya dikemas dengan silika sekitar 50 mikrometer, sehingga
mengurangi tekanan balik dan resolusi, tetapi juga menghilangkan kebutuhan untuk
mahal pompa tekanan tinggi. Produsen kini mulai pindah ke yang lebih tinggi tekanan
sistem flash kromatografi dan telah disebut ini kromatografi cair tekanan sebagai
media (MPLC) sistem yang beroperasi di atas 150 psi.
Perangkat lunak mengendalikan sistem otomatis akan mengkoordinasikan komponen,
memungkinkan pengguna untuk hanya mengumpulkan faksi-faksi yang mengandung
senyawa target mereka (dengan asumsi mereka terdeteksi pada detektor sistem) dan
membantu pengguna untuk menemukan bahan murni yang dihasilkan dalam kolektor
fraksi. Perangkat lunak ini juga akan menyelamatkan kromatografi yang dihasilkan
dari proses untuk keperluan penarikan kembali arsip dan / atau lambat.
Sebuah contoh wakil dari kromatografi kolom sebagai bagian dari latihan
laboratorium sarjana adalah pemisahan tiga komponen (dari 28) dalam minyak
spearmint: carvone, limonene dan dehydrocarveol [5]. Sebuah setup mikro yang
terdiri dari pipet Pasteur sebagai kolom dengan fase diam silika gel dapat cukup. The
eluen awalnya adalah heksana dan polaritas pelarut meningkat selama proses dengan
menambahkan etil asetat.
[Sunting] Kolom Kromatogram Perhitungan Resolusi
Biasanya, kromatografi kolom diatur dengan pompa peristaltik mengalir buffer dan
sampel solusi melalui bagian atas kolom. Solusi dan buffer melewati kolom di mana
seorang kolektor fraksi pada akhir setup kolom mengumpulkan sampel dielusi.
Sebelum koleksi fraksi, sampel yang dielusi dari kolom lulus melalui detektor seperti
spektrofotometer atau spektrometer massa sehingga konsentrasi sampel dipisahkan
dalam campuran larutan sampel dapat ditentukan.
Misalnya, jika Anda adalah untuk memisahkan dua protein yang berbeda dengan
kapasitas mengikat yang berbeda untuk kolom dari sampel solusi, jenis yang baik
detektor akan menjadi spektrofotometer menggunakan panjang gelombang 280 nm.
Semakin tinggi konsentrasi protein yang melewati solusi dielusi melalui kolom,
semakin tinggi absorbansi panjang gelombang itu.
Karena kromatografi kolom memiliki aliran konstan solusi dielusi melewati detektor
pada konsentrasi yang berbeda-beda, detektor harus plot konsentrasi sampel dielusi
selama perjalanan waktu. Ini plot konsentrasi sampel terhadap waktu disebut
kromatogram.

Tujuan utama dari kromatografi adalah untuk memisahkan komponen yang berbeda
dari campuran solusi. Resolusi itu menyatakan tingkat pemisahan antara komponenkomponen dari campuran. Semakin tinggi resolusi dari kromatogram, semakin baik
tingkat pemisahan sampel kolom memberikan. Data ini adalah cara yang baik untuk
menentukan pemisahan kolom sifat bahwa sampel tertentu. Resolusi dapat dihitung
dari kromatogram.
Kurva terpisah dalam diagram mewakili profil yang berbeda konsentrasi sampel elusi
dari waktu ke waktu berdasarkan afinitas mereka ke kolom resin. Untuk menghitung
resolusi, waktu retensi dan lebar kurva diperlukan.
Waktu Retensi: Waktu dari awal deteksi sinyal oleh detektor dengan tinggi puncak
profil konsentrasi elusi dari masing-masing sampel yang berbeda.
Lebar Curve: Lebar kurva profil konsentrasi sampel yang berbeda dalam
kromatogram dalam satuan waktu.
Sebuah metode sederhana dari menghitung resolusi kromatogram adalah dengan
menggunakan model plat [6]. Model piring mengasumsikan bahwa kolom dapat
dibagi menjadi sejumlah bagian, atau piring dan keseimbangan massa dapat dihitung
untuk setiap piring individu. Pendekatan ini mendekati kurva kromatogram yang khas
sebagai kurva distribusi Gaussian. Dengan melakukan ini, lebar kurva diperkirakan
sebagai 4 kali standar deviasi dari kurva, 4. Waktu retensi adalah waktu dari awal
deteksi sinyal dengan waktu ketinggian puncak kurva Gaussian.
Dari variabel pada gambar di atas, resolusi, nomor plat, dan tinggi piring model plat
kolom dapat dihitung dengan menggunakan persamaan:
Resolusi (Rs):
Rs = 2 (TRB - tra) / (WB + WA)
Dimana:
TRB = waktu retensi dari B terlarut
TRA = waktu retensi zat terlarut A
wb = Gaussian kurva lebar B terlarut
Wa = Gaussian kurva lebar zat terlarut A
Plat Nomor (N):
N = (TR2) / (w / 4) 2
Plat Tinggi (H):
H=L/N
Dimana L adalah panjang kolom [6].
[Sunting] Column Adsorpsi Equilibrium
Untuk kolom adsorpsi, resin kolom (fase diam) terdiri dari microbeads. Bahkan
partikel yang lebih kecil seperti protein, karbohidrat, ion logam, atau senyawa kimia
lainnya yang terkonjugasi ke microbeads. Setiap partikel mengikat yang melekat pada
microbead dapat diasumsikan untuk mengikat dalam rasio 1:1 dengan sampel zat
terlarut dikirim melalui kolom yang perlu dimurnikan atau dipisahkan.
Mengikat antara molekul target untuk dipisahkan dan molekul mengikat manik-manik
kolom dapat dimodelkan menggunakan reaksi kesetimbangan yang sederhana Keq =
[CS] / ([C] [S]) di mana Keq adalah konstanta kesetimbangan, [C] dan [ S] adalah
konsentrasi molekul target dan molekul mengikat resin kolom, masing-masing. [CS]

adalah konsentrasi kompleks dari molekul target terikat pada resin kolom. [6]
Menggunakan ini sebagai dasar, tiga isoterm yang berbeda dapat digunakan untuk
menggambarkan dinamika mengikat kromatografi kolom: linear, Langmuir, dan
Freundlich.
Isoterm linear terjadi ketika konsentrasi zat terlarut perlu dimurnikan adalah relatif
sangat kecil untuk molekul pengikatan. Dengan demikian, ekuilibrium dapat
didefinisikan sebagai:
[CS] = Keq [C].
Untuk menggunakan skala industri, molekul yang mengikat total pada manik-manik
kolom resin harus diperhitungkan karena situs kosong harus diperhitungkan.
Langmuir isoterm Freundlich dan isoterm berguna dalam menggambarkan
keseimbangan ini. Langmuir isoterm:
[CS] = (KeqStot [C]) / (1 + Keq [C]), di mana Stot adalah molekul mengikat total
pada manik-manik.
Freundlich isoterm:
[CS] = Keq [C] 1 / n
Isoterm Freundlich digunakan ketika kolom dapat mengikat sampel yang berbeda
dalam larutan yang perlu dimurnikan. Karena sampel yang berbeda memiliki
konstanta mengikat yang berbeda dengan manik-manik, ada banyak yang berbeda
Keq ini. Oleh karena itu, Langmuir isoterm bukan model yang baik untuk mengikat
dalam hal ini. [6]
[Sunting] Lihat pula
Kromatografi, sebuah artikel gambaran yang mencakup semua teknik kromatografi.
kromatografi cair kinerja tinggi (HPLC) untuk kromatografi kolom menggunakan
tekanan tinggi.
protein Cepat kromatografi cair (FPLC) untuk pemisahan protein menggunakan
kromatografi kolom.

[edit] References
1. ^ Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43(14), 2923-2925.
(doi:10.1021/jo00408a041)
2. ^ Laurence M. Harwood, Christopher J. Moody. Experimental organic chemistry: Principles
and Practice (Illustrated edition ed.). pp. 180-185. ISBN 978-0632020171.
3. ^ Normal phase column chromatography, Material Harvest
4. ^ Fair, J. D.; Kormos, C. M. J. Chromatogr. A 2008, 1211(1-2), 49-54.
(doi:10.1016/j.chroma.2008.09.085)
5. ^ Isolation of Three Components from Spearmint Oil: An Exercise in Column and Thin-Layer
Chromatography Davies, Don R.; Johnson, Todd M. J. Chem. Educ. 2007 84Abstract
6. ^ a b c d Harrison et al. Bioseparations Science and Engineering. Oxford University Press.
New York, New York. 2003

[edit] External links

High performance liquid chromatography

High performance liquid chromatography

Sebuah HPLC. Dari kiri ke kanan: Sebuah perangkat memompa menghasilkan

gradien dari dua pelarut yang berbeda, kolom baja ditegakkan dan aparatus untuk
mengukur absorbansi

HPLC apparatus.
Kromatografi cair kinerja tinggi (atau kromatografi cair tekanan tinggi, HPLC) merupakan
bentuk kromatografi kolom sering digunakan dalam kimia biokimia dan analitis untuk
memisahkan, mengidentifikasi, dan menghitung senyawa berdasarkan polaritas istimewa
mereka dan interaksi dengan fase diam kolom ini. HPLC menggunakan berbagai jenis fase
diam (biasanya, hidrofobik rantai karbon jenuh), sebuah pompa yang menggerakkan fase
gerak (s) dan analit melalui kolom, dan detektor yang memberikan waktu retensi charateristic
untuk analit. Detektor juga dapat memberikan informasi characterisitc lainnya (yaitu UV / Vis
data spektroskopi untuk analit jika demikian dilengkapi). Analit waktu retensi bervariasi
tergantung pada kekuatan interaksi dengan fase diam, rasio / komposisi pelarut (s) yang
digunakan, dan laju aliran fase gerak.
Dengan HPLC, pompa (bukan gravitasi) memberikan tekanan yang lebih tinggi diperlukan
untuk mendorong fase mobile dan analit melalui kolom padat. Kepadatan meningkat muncul
dari ukuran partikel yang lebih kecil. Hal ini memungkinkan untuk pemisahan yang lebih
baik pada kolom panjang pendek bila dibandingkan dengan kromatografi kolom biasa.
[Sunting] Operasi
Sampel yang akan dianalisis diperkenalkan dalam volume kecil dengan aliran fase gerak.
Gerak analit melalui kolom diperlambat oleh bahan kimia tertentu atau interaksi fisik dengan
fase diam seperti melintasi panjang kolom. Berapa banyak analit diperlambat tergantung pada
sifat analit dan komposisi fase stasioner dan mobile. Waktu di mana mengelusi analit tertentu
(keluar dari ujung kolom) disebut waktu retensi, waktu retensi dalam kondisi tertentu
dianggap sebagai karakteristik mengidentifikasi cukup unik dari analit tertentu. Penggunaan
ukuran partikel yang lebih kecil kemasan kolom (yang menciptakan backpressure lebih
tinggi) meningkatkan kecepatan linear memberikan komponen lebih sedikit waktu untuk
menyebar dalam kolom, yang mengarah ke resolusi ditingkatkan dalam kromatogram yang

dihasilkan. Pelarut umum digunakan termasuk kombinasi bercampur air atau cairan organik
berbagai (yang paling umum adalah metanol dan asetonitril). Air mungkin berisi buffer atau
garam untuk membantu dalam pemisahan komponen analit, atau senyawa seperti asam
trifluoroasetat yang bertindak sebagai agen pasangan ion.
Sebuah perbaikan lebih lanjut untuk HPLC telah bervariasi komposisi fase gerak selama
analisis, hal ini dikenal sebagai elusi gradien. Sebuah gradien normal untuk kromatografi fase
terbalik mungkin mulai metanol 5% dan kemajuan linear untuk metanol 50% lebih dari 25
menit, gradien yang dipilih tergantung pada seberapa hidrofobik analit tersebut. Gradien
memisahkan campuran analit sebagai fungsi dari afinitas analit untuk komposisi fase saat
bergerak relatif terhadap fase diam. Proses partisi ini mirip dengan apa yang terjadi selama
ekstraksi cair-cair tetapi terus menerus, tidak bertahap. Dalam contoh ini, menggunakan
gradien air / metanol, komponen lebih hidrofobik akan mengelusi (datang dari kolom) ketika
fase gerak sebagian besar terdiri dari metanol (memberikan fase gerak relatif hidrofobik).
Senyawa-senyawa yang lebih hidrofilik akan terelusi dalam kondisi metanol relatif rendah /
air yang tinggi.
Pemilihan pelarut, aditif dan gradien tergantung pada sifat fase diam dan analit. Seringkali
serangkaian tes yang dilakukan pada analit dan sejumlah berjalan sidang dapat diproses
dalam rangka untuk menemukan metode HPLC yang memberikan pemisahan terbaik dari
puncak.

[edit] Types
[edit] Partition chromatography

HPLC moderen
Seorang diri modern yang terkandung HPLC. Dalam gambar ini, sebuah Agilent 1.200
terhubung ke PC
Partisi kromatografi adalah jenis pertama dari kromatografi yang dikembangkan kimiawan.
Prinsip Koefisien partisi telah diterapkan dalam kromatografi kertas, kromatografi lapis tipis,
fase gas dan cairan-cairan aplikasi. 1952 Nobel Prize di bidang kimia itu diperoleh oleh
Archer John Porter Martin dan Richard Laurence Millington Synge bagi perkembangan
mereka dari teknik, yang digunakan untuk pemisahan mereka dari asam amino. Partisi
kromatografi menggunakan pelarut dipertahankan, di permukaan atau di dalam biji-bijian
atau serat dari matriks "lembam" pendukung yang solid seperti kromatografi kertas, atau
mengambil keuntungan dari beberapa coulombic tambahan dan / atau hidrogen donor

interaksi dengan dukungan yang solid. Molekul menyeimbangkan (partisi) antara fase diam
cair dan eluen. Dikenal sebagai Kromatografi Interaksi Hidrofilik (HILIC) di HPLC, metode
ini memisahkan analit didasarkan pada perbedaan kutub. HILIC paling sering menggunakan
fase terikat diam polar dan non-polar, air larut, fase gerak. Partisi HPLC telah digunakan
historis pada silika tak terikat atau mendukung alumina. Masing-masing bekerja secara
efektif untuk memisahkan analit oleh perbedaan kutub relatif, bagaimanapun, HILIC
memiliki keuntungan memisahkan asam, larutan basa dan netral dalam kromatogram tunggal.
Analit polar berdifusi ke dalam lapisan air stasioner terkait dengan fase diam polar dan
dengan demikian dipertahankan. Kekuatan retensi meningkat dengan polaritas analit
meningkat, dan interaksi antara analit polar dan fase diam polar (relatif terhadap fase gerak)
meningkatkan waktu elusi. Kekuatan interaksi tergantung pada kelompok fungsional dalam
molekul analit yang mempromosikan partisi tetapi juga dapat mencakup coulombic
(elektrostatik) interaksi dan kemampuan hidrogen donor.
Penggunaan pelarut polar lebih dalam fase gerak akan mengurangi waktu retensi analit,
sedangkan pelarut hidrofobik lebih cenderung meningkatkan waktu retensi.
Partisi dan NP-HPLC telah jatuh dari kasih karunia di tahun 1970-an dengan perkembangan
fase terbalik HPLC karena kurangnya reproduksibilitas waktu retensi sebagai air atau pelarut
organik protik mengubah keadaan hidrasi dari silika atau media kromatografi alumina. Barubaru ini telah menjadi berguna lagi dengan perkembangan fase terikat HILIC yang
meningkatkan reproduktifitas.
[Sunting] kromatografi fase normal
Untuk detail lebih lanjut tentang topik ini, lihat kromatografi fasa air normal.
Juga dikenal sebagai HPLC fase normal (NP-HPLC), atau adsorpsi kromatografi, metode ini
memisahkan analit didasarkan pada adsorpsi dengan kimia permukaan stasioner dan dengan
polaritas. Itu adalah salah satu jenis pertama HPLC yang dikembangkan kimiawan. NPHPLC menggunakan fase diam polar dan non-polar, non-berair fase gerak, dan bekerja
efektif untuk memisahkan analit mudah larut dalam pelarut non-polar. Rekan analit dengan
dan dipertahankan oleh fase diam polar. Kekuatan adsorpsi meningkat dengan polaritas analit
meningkat, dan interaksi antara analit polar dan fase diam polar (relatif terhadap fase gerak)
meningkatkan waktu elusi. Kekuatan interaksi tidak hanya tergantung pada kelompok
fungsional dalam molekul analit, tetapi juga pada faktor sterik. Pengaruh sterics pada
kekuatan interaksi memungkinkan metode ini untuk menyelesaikan (terpisah) isomer
struktural.
Penggunaan pelarut polar lebih dalam fase gerak akan mengurangi waktu retensi analit,
sedangkan pelarut hidrofobik lebih cenderung meningkatkan waktu retensi. Pelarut polar
yang sangat dalam campuran cenderung menonaktifkan fase diam dengan menciptakan
lapisan air stasioner terikat pada permukaan fase diam. Perilaku ini agak aneh bagi fase
normal karena hal ini sangat murni mekanisme adsorptif (interaksi adalah dengan permukaan
yang keras daripada lapisan lembut pada permukaan).
NP-HPLC jatuh dari kasih karunia di tahun 1970-an dengan perkembangan fase terbalik
HPLC karena kurangnya reproduksibilitas waktu retensi sebagai air atau pelarut organik
protik mengubah keadaan hidrasi dari silika atau media kromatografi alumina. Baru-baru ini
telah menjadi berguna lagi dengan perkembangan fase terikat HILIC yang meningkatkan
reproduktifitas.
[Sunting] Pemindahan kromatografi
Prinsip dasar kromatografi perpindahan adalah: Sebuah molekul dengan afinitas tinggi untuk
matriks kromatografi (displacer) akan bersaing secara efektif untuk situs mengikat, dan
dengan demikian menggantikan seluruh molekul dengan afinitas yang lebih rendah [1] Ada
perbedaan jelas antara perpindahan dan kromatografi elusi. . Dalam modus elusi, zat biasanya
muncul dari sebuah kolom di sempit, puncak Gaussian. Pemisahan yang luas dari puncak,

sebaiknya ke baseline, yang diinginkan untuk mencapai pemurnian maksimal. Kecepatan di

mana setiap komponen campuran perjalanan ke kolom dalam mode elusi tergantung pada
banyak faktor. Tapi untuk dua zat untuk melakukan perjalanan dengan kecepatan yang
berbeda, dan dengan demikian dapat diselesaikan, harus ada perbedaan substansial dalam
beberapa interaksi antara biomolekul dan matriks kromatografi. Parameter operasi yang
disesuaikan untuk memaksimalkan efek dari perbedaan ini. Dalam banyak kasus, dasar
pemisahan puncak dapat dicapai hanya dengan elusi gradien dan beban kolom yang rendah.
Dengan demikian, dua kelemahan kromatografi elusi modus, terutama pada skala preparatif,
adalah kompleksitas operasional, karena gradien pelarut memompa, dan throughput rendah,
karena beban kolom yang rendah. Pemindahan kromatografi memiliki keunggulan
dibandingkan kromatografi elusi dalam komponen diselesaikan menjadi zona berturut-turut
zat murni daripada "puncak". Karena proses mengambil keuntungan dari nonlinier dari
isoterm, feed kolom yang lebih besar dapat dipisahkan pada kolom tertentu dengan
komponen dimurnikan pulih pada konsentrasi lebih tinggi secara signifikan.

Reversed phase chromatography (RPC)

Sebuah kromatogram campuran kompleks (air parfum) diperoleh terbalik fase HPLC
Untuk detail lebih lanjut tentang topik ini, lihat Terbalik-fase kromatografi.
Fase balik HPLC (RP-HPLC atau RPC) memiliki fase non-polar stasioner dan Aqueous, fase
gerak cukup polar. Satu fase diam umum adalah silika yang telah diobati dengan RMe2SiCl,
di mana R adalah gugus alkil rantai lurus seperti C18H37 atau C8H17. Dengan fase stasioner,
waktu retensi lebih lama untuk molekul yang lebih non-polar, sedangkan molekul polar
mengelusi lebih mudah. Penyidik dapat meningkatkan waktu retensi dengan menambahkan
lebih banyak air ke fase gerak, sehingga membuat afinitas analit hidrofobik untuk fase relatif
hidrofobik stasioner kuat ke fase gerak sekarang lebih hidrofilik. Demikian pula, penyidik
dapat mengurangi waktu retensi dengan menambahkan lebih pelarut organik untuk eluen.
RPC begitu umum digunakan yang sering salah disebut sebagai "HPLC" tanpa spesifikasi
lebih lanjut. Industri farmasi secara rutin menggunakan RPC untuk memenuhi syarat obat
sebelum pembebasan mereka.
RPC beroperasi pada prinsip kekuatan hidrofobik, yang berasal dari simetri tinggi dalam
struktur air dipole dan memainkan peran paling penting dalam semua proses dalam ilmu
kehidupan. RPC memungkinkan pengukuran kekuatan interaktif. Pengikatan analit ke fase
diam sebanding dengan luas permukaan kontak sekitar segmen non-polar dari molekul analit
pada hubungan dengan ligan dalam eluen air. Efek solvophobic didominasi oleh kekuatan air
untuk "pengurangan rongga-" sekitar analit dan rantai-C18 versus kompleks keduanya.
Energi yang dilepaskan dalam proses ini sebanding dengan tegangan permukaan eluen (air:

7.3 10-6 J / cm , metanol: 2,2 10-6 J / cm ) dan permukaan hidrofobik analit dan ligan
masing . Retensi dapat dikurangi dengan menambahkan pelarut kurang polar (metanol,
asetonitril) ke dalam fase gerak untuk mengurangi tegangan permukaan air. Elusi gradien
menggunakan efek ini dengan secara otomatis mengurangi polaritas dan tegangan permukaan
dari fase gerak berair selama analisis.
Sifat struktur molekul analit memainkan peran penting dalam karakteristik retensi. Secara
umum, suatu analit dengan luas hidrofobik lebih besar permukaan (CH, CC, dan umumnya
non-polar ikatan atom, seperti SS dan lain-lain) hasil dalam waktu retensi lebih lama karena
meningkatkan non-polar permukaan molekul daerah, yang non -berinteraksi dengan struktur
air. Di sisi lain, gugus polar, seperti-OH,-NH2, COO-atau-NH3 + mengurangi retensi karena
mereka terintegrasi dengan baik ke dalam air. Molekul yang sangat besar, namun, dapat
mengakibatkan interaksi yang tidak lengkap antara permukaan analit besar dan rantai alkil
ligan dan dapat memiliki masalah memasuki pori-pori fase diam.
Waktu retensi meningkat dengan luas permukaan hidrofobik (non-polar). Senyawa rantai
bercabang mengelusi lebih cepat dari isomer berhubungan linear karena luas permukaan
keseluruhan menurun. Senyawa organik Similarly with ikatan CC single mengelusi lambat
dibandingkan dengan C = C or CC-triple Bond sebagaimana ikatan ganda atau triple lebih
pendek daripada single CC-bond.
Selain fase tegangan permukaan ponsel (kekuatan organisasi dalam struktur eluen), lainnya
pengubah fase gerak dapat mempengaruhi retensi analit. Misalnya, penambahan garam
anorganik menyebabkan peningkatan linear moderat dalam tegangan permukaan larutan
berair (1,5 10-7 ca. J / cm Mol per untuk NaCl, 2,5 10-7 J / cm Mol per untuk (NH4)
2SO4), dan karena entropi dari interface analit-pelarut dikendalikan oleh tegangan
permukaan, penambahan garam cenderung meningkatkan waktu retensi. Teknik ini
digunakan untuk pemisahan ringan dan pemulihan protein dan perlindungan dari aktivitas
biologis mereka dalam analisis protein (kromatografi interaksi hidrofobik, HIC).
Komponen penting lainnya adalah pengaruh pH karena ini dapat mengubah hidrofobisitas
analit. Untuk alasan ini metode yang paling menggunakan agen buffering, seperti natrium
fosfat, untuk mengontrol pH. Buffer melayani beberapa tujuan: mereka mengontrol pH,
menetralisir muatan pada setiap silika terkena residu pada fase diam dan bertindak sebagai
agen pasangan ion untuk menetralkan muatan pada analit. Formate Amonium biasanya
ditambahkan dalam spektrometri massa untuk meningkatkan deteksi analit tertentu dengan
pembentukan adduct amonium. Suatu asam organik yang mudah menguap seperti asam
asetat, atau asam formiat paling sering, sering ditambahkan ke fase gerak jika spektrometri
massa digunakan untuk menganalisis kolom eluen. Asam trifluoroasetat jarang digunakan
dalam aplikasi spektrometri massa karena kegigihan dalam detektor dan sistem pengiriman
pelarut, tetapi bisa efektif dalam meningkatkan retensi analit seperti asam karboksilat dalam
aplikasi menggunakan detektor lainnya, karena merupakan salah satu asam organik terkuat.
Efek dari asam dan buffer bervariasi oleh aplikasi tetapi umumnya meningkatkan
kromatografi.
Kolom fase terbalik cukup sulit untuk merusak dibandingkan dengan kolom silika normal,
namun, banyak kolom fase terbalik terdiri dari alkil partikel silika diderivatisasi dan tidak
boleh digunakan dengan dasar air karena ini akan menghancurkan partikel silika yang
mendasarinya. Mereka dapat digunakan dengan asam encer, tetapi kolom tidak boleh terkena
asam terlalu lama, karena bisa menimbulkan korosi bagian logam dari peralatan HPLC. RPHPLC kolom harus memerah dengan pelarut bersih setelah digunakan untuk menghapus sisa
asam atau buffer, dan disimpan dalam sebuah komposisi yang tepat dari pelarut. Kandungan
logam dari kolom HPLC harus tetap rendah jika kemampuan terbaik untuk zat terpisah
dipertahankan. Sebuah tes yang baik untuk kandungan logam dari kolom adalah untuk
menyuntikkan sampel yang merupakan campuran dari 2,2 '- dan 4,4' - bipiridin. Karena 2,2 '-

bipy dapat khelat logam, bentuk puncak untuk 2,2'-bipy akan terdistorsi (ekor) ketika ion
logam yang hadir pada permukaan silika. [Rujukan?] ..
[Sunting] Ukuran kromatografi eksklusi
Untuk detail lebih lanjut tentang topik ini, lihat kromatografi eksklusi ukuran.
Ukuran kromatografi eksklusi (SEC), juga dikenal sebagai kromatografi permeasi gel atau
kromatografi gel filtrasi, memisahkan partikel berdasarkan ukuran. Ini umumnya merupakan
kromatografi resolusi rendah sehingga sering dicadangkan untuk langkah final, "polishing"
pemurnian a. Hal ini juga berguna untuk menentukan struktur tersier dan struktur kuaterner
protein dimurnikan. SEC digunakan terutama untuk analisis molekul besar seperti protein
atau polimer. SEC bekerja dengan menjebak molekul-molekul yang lebih kecil dalam poripori partikel. Molekul-molekul yang lebih besar hanya melewati pori-pori karena mereka
terlalu besar untuk masuk ke pori-pori. Oleh karena itu molekul yang lebih besar mengalir
melalui kolom lebih cepat daripada molekul yang lebih kecil, yaitu lebih kecil molekul,
semakin lama waktu retensi.
Teknik ini banyak digunakan untuk penentuan berat molekul polisakarida. SEC adalah teknik
resmi (suggested by European Pharmacopeia) untuk perbandingan berat molekul yang
berbeda tersedia secara komersial molekul rendah heparins berat.
[Sunting] Ion kromatografi penukar
Untuk detail lebih lanjut tentang topik ini, lihat pertukaran ion kromatografi.
Dalam pertukaran ion kromatografi, retensi didasarkan pada daya tarik antara ion terlarut dan
situs dibebankan terikat pada fase diam. Ion muatan yang sama dikecualikan. Jenis penukar
ion meliputi:
Polistirena resin - Ini memungkinkan hubungan lintas yang meningkatkan stabilitas rantai.
Tinggi linkage lintas Mengurangi meliuk, yang meningkatkan waktu ekuilibrasi dan akhirnya
meningkatkan selektivitas.
Selulosa dan ion dekstran penukar (gel) - Ini memiliki ukuran pori yang lebih besar dan
kepadatan muatan yang rendah membuat mereka cocok untuk pemisahan protein.
Controlled-pori kaca atau silika berpori
Secara umum, penukar ion mendukung pengikatan ion biaya tinggi dan jari-jari yang lebih
kecil.
Peningkatan ion tanding (berkenaan dengan kelompok-kelompok fungsional dalam resin)
konsentrasi mengurangi waktu retensi. Peningkatan pH mengurangi waktu retensi dalam
pertukaran kation sementara penurunan pH mengurangi waktu retensi dalam pertukaran
anion.
Bentuk kromatografi secara luas digunakan dalam aplikasi berikut: pemurnian air,
prekonsentrasi komponen jejak, ligan-kromatografi penukar, pertukaran ion kromatografi
protein, tinggi-pH anion-kromatografi penukar karbohidrat dan oligosakarida, dan lain-lain.
[Sunting] Bioaffinity kromatografi
Proses kromatografi bergantung pada properti dari zat biologis aktif untuk membentuk stabil,
kompleks yang spesifik, dan reversibel. Pembentukan kompleks ini melibatkan partisipasi
pasukan molekul umum seperti Van der Waals interaksi, interaksi elektrostatik, dipol-dipol
interaksi, interaksi hidrofobik, dan ikatan hidrogen. Ikatan, efisien biospecific dibentuk oleh
aksi simultan dan terpadu dari beberapa kekuatan di situs mengikat pelengkap.
[Sunting] kromatografi fase berair yang normal
Aqueous fase kromatografi normal (ANP) adalah teknik kromatografi yang meliputi wilayah
fase gerak antara fase terbalik kromatografi (RP) dan organik kromatografi fase normal
(ONP). Teknik ini digunakan untuk mencapai selektivitas unik untuk senyawa hidrofilik,
menunjukkan elusi fase normal yang menggunakan reverse-fase pelarut. [Rujukan?]
[Sunting] isokratik aliran dan elusi gradien
Sebuah pemisahan di mana komposisi fase gerak tetap konstan di seluruh prosedur disebut

isokratik (berarti komposisi konstan). Kata ini diciptakan oleh Csaba Horvath dari
Universitas Yale [rujukan?], Yang merupakan salah satu pelopor dari HPLC.
Komposisi fase gerak tidak harus tetap konstan. Sebuah pemisahan di mana komposisi fase
gerak berubah selama proses pemisahan digambarkan sebagai elusi gradien. [2] Salah satu
contoh adalah gradien mulai metanol 10% dan berakhir pada metanol 90% setelah 20 menit.
Kedua komponen dari fase gerak biasanya disebut "A" dan "B", A adalah "lemah" pelarut
yang memungkinkan zat terlarut untuk mengelusi hanya perlahan-lahan, sedangkan B adalah
"kuat" pelarut yang cepat elutes zat terlarut dari kolom . Sebuah pelarut sering air, sedangkan
B adalah pelarut organik bercampur dengan air, seperti asetonitril, metanol, THF, atau
isopropanol.
Dalam elusi isokratik, lebar puncak meningkat dengan waktu retensi linear menurut
persamaan untuk N, jumlah pelat teoritis. Hal ini menyebabkan kerugian yang terlambateluting puncak menjadi sangat datar dan luas. Bentuk dan lebar dapat menjaga mereka dari
yang diakui sebagai puncak.
Elusi gradien mengurangi retensi nanti-eluting komponen sehingga mereka mengelusi lebih
cepat, memberikan sempit (dan lebih tinggi) untuk komponen yang paling puncak. Ini juga
meningkatkan bentuk puncak untuk puncak ekor, sebagai meningkatnya konsentrasi eluen
organik mendorong bagian tailing dari puncak ke depan. Ini juga meningkatkan tinggi puncak
(peak terlihat "tajam"), yang penting dalam analisis jejak. Program gradien mungkin
termasuk mendadak "langkah" peningkatan persentase komponen organik, atau lereng yang
berbeda pada waktu yang berbeda - semua sesuai dengan keinginan untuk pemisahan
optimum dalam waktu yang minimal.
Dalam elusi isokratik, selektivitas tidak berubah jika dimensi kolom (panjang dan diameter
dalam) perubahan - yaitu, elusi puncak dalam urutan yang sama. Dalam elusi gradien, urutan
elusi dapat berubah sebagai dimensi atau perubahan laju alir. [Rujukan?]
Kekuatan pendorong yang berasal dari kromatografi fase terbalik dalam urutan tinggi struktur
air. Peran fase gerak organik adalah untuk mengurangi urutan tinggi dengan mengurangi
kekuatan penghambat dari komponen berair.
[Sunting] Parameter
[Sunting] diameter internal
Diameter internal (ID) dari kolom HPLC merupakan parameter penting yang mempengaruhi
sensitivitas deteksi dan selektivitas pemisahan elusi gradien. Hal ini juga menentukan
kuantitas analit yang dapat dimuat ke kolom. Kolom besar biasanya terlihat dalam aplikasi
industri, seperti pemurnian produk obat untuk digunakan nanti. Rendah-ID kolom telah
meningkatkan sensitivitas dan konsumsi pelarut lebih rendah dengan mengorbankan
kapasitas loading.
kolom ID yang lebih besar (lebih dari 10 mm) yang digunakan untuk memurnikan jumlah
bahan yang dapat digunakan karena kapasitas beban yang besar.
kolom skala Analytical (4.6 mm) telah menjadi jenis yang paling umum dari kolom, kolom
meskipun kecil dengan cepat mendapatkan popularitas. Mereka digunakan dalam analisis
kuantitatif tradisional sampel dan sering menggunakan detektor absorbansi UV-Vis.
Narrow-bore kolom (1-2 mm) yang digunakan untuk aplikasi saat sensitivitas lebih
diinginkan baik dengan khusus UV-vis detektor, deteksi fluoresensi atau dengan metode
deteksi lainnya seperti kromatografi cair-spektrometri massa
Kolom kapiler (di bawah 0,3 mm) yang digunakan hampir secara eksklusif dengan deteksi
alternatif berarti seperti spektrometri massa. Mereka biasanya terbuat dari kapiler leburan
silika, daripada tabung stainless steel yang lebih besar mempekerjakan kolom.
[Sunting] Ukuran partikel
HPLC paling tradisional dilakukan dengan fase diam melekat pada bagian luar kecil partikel
silika bulat (manik-manik yang sangat kecil). Partikel ini datang dalam berbagai ukuran

dengan 5 pM manik yang paling umum. Partikel yang lebih kecil umumnya memberikan luas
permukaan lebih banyak dan pemisahan yang lebih baik, tetapi tekanan yang dibutuhkan
untuk meningkatkan kecepatan optimal linear dengan kebalikan dari kuadrat diameter
partikel [3] [4] [5].
Ini berarti bahwa perubahan partikel yang setengah besar, menjaga ukuran kolom yang sama,
akan menggandakan kinerja, tetapi meningkatkan tekanan yang dibutuhkan dengan faktor
empat. Partikel yang lebih besar yang digunakan dalam HPLC preparatif (diameter kolom 5
cm sampai dengan> 30 cm) dan untuk non-HPLC aplikasi seperti solid-fase ekstraksi.
[Sunting] ukuran pori
Fasa diam banyak berpori untuk memberikan luas permukaan yang lebih besar. Pori-pori
kecil memberikan luas permukaan yang lebih besar sedangkan ukuran pori yang lebih besar
memiliki kinetika yang lebih baik, terutama untuk analit yang lebih besar. Sebagai contoh,
suatu protein yang hanya sedikit lebih kecil dari pori bisa memasuki pori-pori tetapi tidak
mudah meninggalkan sekali di dalam.
[Sunting] Tekanan Pompa
Pompa bervariasi dalam kapasitas tekanan, namun kinerja mereka diukur pada kemampuan
mereka untuk menghasilkan laju aliran yang konsisten dan direproduksi. Tekanan dapat
mencapai setinggi 40 MPa (6000 lbf/in2), atau sekitar 400 atmosfer. Modern HPLC sistem
telah ditingkatkan untuk bekerja pada tekanan yang lebih tinggi, dan karena itu dapat
menggunakan ukuran partikel jauh lebih kecil dalam kolom (<2 m). Ini "Ultra Cair Kinerja
Tinggi Kromatografi" sistem atau RSLC / UHPLCs dapat bekerja sampai dengan 100 MPa
(15.000 lbf / in ), atau sekitar 1000 atmosfer. Istilah "UPLC" adalah merek dagang dari
Corporation Waters, namun kadang-kadang digunakan untuk merujuk ke teknik yang lebih
umum.

[edit] References
1. ^ Displacement Chromatography 101. [1] Sachem, Inc. Austin, TX 78737
2. ^ A recent book provides a comprehensive treatment of the theory of high-performance
gradient chromatography: Lloyd R. Snyder and John W. Dolan (2006). High-Performance
Gradient Elution: The Practical Application of the Linear-Solvent-Strength Model. Wiley
Interscience. ISBN 0471706469..
3. ^ Fast and Ultrafast HPLC on sub-2 m Porous Particles Where Do We Go From
Here? - LC-GC Europe
4. ^ Xiang, Y.; Liu Y. and Lee M.L. (2006). "Ultrahigh pressure liquid chromatography using
elevated temperature". Journal of Chromatography A 1104 (1-2): 198202.
doi:10.1016/j.chroma.2005.11.118.
5. ^ Horvth, Cs.; Preiss B.A. and Lipsky S.R. (1967). "Fast liquid chromatography.
Investigation of operating parameters and the separation of nucleotides on pellicular ion
exchangers". Analytical Chemistry 39: 14221428. doi:10.1021/ac60256a003.

[edit] External links

Brief Guide to HPLC Instruments A video that describes the different parts of a
HPLC instrument and the role that each performs
HPLC Primer Beginners Guide to Liquid Chromatography, J.C. Arsenault and P.D.
McDonald [2009] - A detailed primer with history, glossary, and technology sections
A History of Chromatography - P.D. McDonald, The Quest for Ultra Performance
in Liquid Chromatography - Origins of UPLC Technology, P.D. McDonald [2009] - a
history of the 100 years of chromatography, with glossary and detailed notes

Beginners Guide to UPLC Technology - Beginners Guide to UPLC (UltraPerformance Liquid Chromatography), E.S. Grumbach, J.C. Arsenault, and D.R.
McCabe [2009] - a good introduction to UPLC Technology
Scientific Calculator for HPLC

4.5. kromatografi gas

Gas chromatography-mass spectrometry

Example of a GC-MS
Kromatografi gas-spektrometri massa (GC-MS) adalah sebuah metode yang menggabungkan
fitur dari gas-cair kromatografi dan spektrometri massa untuk mengidentifikasi zat yang
berbeda dalam sampel uji. Aplikasi GC-MS termasuk deteksi narkoba, kebakaran investigasi,
analisis lingkungan, investigasi bahan peledak, dan identifikasi sampel tidak diketahui. GC /
MS juga dapat digunakan dalam keamanan bandara untuk mendeteksi zat dalam bagasi atau
pada manusia. Selain itu, dapat mengidentifikasi elemen dalam bahan yang sebelumnya
dianggap telah hancur di luar identifikasi.
GC-MS telah banyak digembar-gemborkan sebagai "standar emas" untuk identifikasi zat
forensik karena digunakan untuk melakukan tes tertentu. Sebuah tes yang spesifik positif
mengidentifikasi kehadiran sebenarnya zat tertentu dalam sampel yang diberikan. Sebuah tes
non-spesifik hanya menunjukkan bahwa zat yang jatuh ke dalam kategori zat. Meskipun tes
non-spesifik statistik bisa menyarankan identitas substansi, ini bisa menyebabkan identifikasi
positif palsu.
[sunting] Sejarah
Penggunaan spektrometer massa sebagai detektor dalam kromatografi gas dikembangkan
selama tahun 1950-an oleh Roland Gohlke dan Fred McLafferty. [1] [2] Perangkat ini sensitif
yang besar, rapuh, dan awalnya terbatas pada pengaturan laboratorium. Perkembangan
komputer terjangkau dan miniatur telah membantu dalam penyederhanaan penggunaan
instrumen ini, serta perbaikan besar diperbolehkan dalam jumlah waktu yang diperlukan
untuk menganalisis sampel. Pada tahun 1996 yang top-of-the-line kecepatan tinggi GC-MS
unit menyelesaikan analisis dari accelerants api dalam waktu kurang dari 90 detik, sedangkan
generasi pertama GC / MS akan diperlukan setidaknya 16 menit [rujukan?]. Hal ini telah
menyebabkan untuk diadopsi secara luas dalam sejumlah bidang.

[edit] Instrumentation
Main articles: gas chromatograph and mass spectrometer

The insides of the GC-MS, with the column of the gas chromatograph in the oven on the
right.
Bagian dalam GC-MS, dengan kolom kromatografi gas di oven di sebelah kanan.
GC-MS terdiri dari dua blok bangunan utama: kromatografi gas dan spektrometer massa. The
kromatografi gas menggunakan kolom kapiler yang tergantung pada dimensi kolom ini
(panjang, diameter, ketebalan film) serta sifat fase (misalnya 5% fenil polisiloksan).
Perbedaan sifat kimia antara molekul yang berbeda dalam campuran akan memisahkan
molekul sebagai sampel perjalanan panjang dari kolom. Molekul-molekul mengambil jumlah
waktu yang berbeda (disebut waktu retensi) untuk keluar dari (elusi dari) kromatografi gas,
dan ini memungkinkan hilir spektrometer massa untuk menangkap, mengionisasi,
mempercepat, menangkis, dan mendeteksi molekul terionisasi secara terpisah. Spektrometer
massa melakukan hal ini dengan memecah molekul masing-masing menjadi fragmen
terionisasi dan mendeteksi fragmen menggunakan massa mereka dengan perbandingan massa
dan muatan.

GC-MS schematic
Kedua komponen, digunakan bersama-sama, memungkinkan tingkat yang jauh lebih halus
identifikasi substansi dari salah satu unit yang digunakan secara terpisah. Hal ini tidak
mungkin untuk membuat identifikasi yang akurat dari suatu molekul tertentu dengan
kromatografi gas atau spektrometri massa saja. Proses spektrometri massa biasanya
membutuhkan sampel yang sangat murni sedangkan kromatografi gas menggunakan detektor
tradisional (misalnya Flame Detector Ionisasi) mendeteksi beberapa molekul yang terjadi
untuk mengambil jumlah waktu yang sama untuk melakukan perjalanan melalui kolom (yaitu
memiliki waktu retensi yang sama) yang mengakibatkan dalam dua atau lebih molekul koelusi. Kadang-kadang dua molekul yang berbeda juga dapat memiliki pola yang sama dari
fragmen terionisasi dalam spektrometer massa (spektrum massa). Menggabungkan dua proses
membuatnya sangat tidak mungkin bahwa dua molekul yang berbeda akan berperilaku

dengan cara yang sama di kedua kromatografi gas dan spektrometer massa. Oleh karena itu
ketika sebuah spektrum massa mengidentifikasi muncul pada waktu retensi karakteristik
dalam analisis GC-MS, biasanya meminjamkan untuk kepastian meningkat bahwa analit dari
bunga dalam sampel.
[Sunting] Split / splitless GC-MS inlet
Sampel diperkenalkan ke kolom melalui inlet. Inlet ini biasanya injeksi melalui septum.
Setelah di inlet, ruang dipanaskan bertindak untuk menguapkan sampel. Dalam sistem split,
aliran konstan gas pembawa bergerak melalui inlet. Sebagian dari aliran gas pembawa
bertindak untuk mengangkut sampel ke dalam kolom. Bagian lain dari aliran gas pembawa
akan diarahkan untuk membersihkan inlet dari setiap injeksi berikut sampel (pembersihan
septum). Namun bagian lain dari aliran diarahkan melalui ventilasi perpecahan dalam rasio
set yang dikenal sebagai rasio split. Dalam sistem splitless, keuntungan adalah bahwa jumlah
yang lebih besar dari sampel diperkenalkan ke kolom. Namun, sistem split lebih disukai bila
detektor sensitif untuk melacak jumlah analit dan ada kekhawatiran tentang overloading
kolom.
[Sunting] Purge dan perangkap GC-MS
Untuk analisis senyawa volatil sistem konsentrator Purge dan Perangkap (P & T) dapat
digunakan untuk memperkenalkan sampel. Analit target diekstrak dan dicampur dengan air
dan dimasukkan ke dalam ruang kedap udara. Gas inert seperti Nitrogen (N2) ditiupkan
melalui air, hal ini dikenal sebagai pembersihan. Senyawa-senyawa volatil pindah ke ruang
atas di atas air dan ditarik sepanjang gradien tekanan (yang disebabkan oleh pengenalan gas
pembersihan) keluar dari ruangan. Senyawa-senyawa yang mudah menguap diambil
sepanjang garis dipanaskan ke 'perangkap'. perangkap adalah kolom bahan adsorben pada
suhu ambien yang memegang senyawa dengan mengembalikan mereka ke fase cair.
Perangkap tersebut kemudian dipanaskan dan senyawa sampel diperkenalkan ke kolom GCMS melalui antarmuka volatil, yang merupakan sistem inlet perpecahan. P & T GC-MS
sangat cocok untuk senyawa organik volatil (VOC) dan senyawa BTEX (senyawa aromatik
terkait dengan minyak bumi) [3].
[Sunting] Jenis Detektor Spektrometer Massa
Jenis yang paling umum dari spektrometer massa (MS) terkait dengan kromatografi gas (GC)
merupakan spektrometer massa quadrupole, kadang-kadang disebut dengan nama HewlettPackard (sekarang Agilent) perdagangan "Mass Detector Selektif" (MSD). Lain detektor
relatif umum adalah spektrometer massa perangkap ion. Selain itu orang dapat menemukan
spektrometer massa sektor magnetik, namun instrumen tertentu yang mahal dan besar dan
tidak biasanya ditemukan di high-throughput laboratorium layanan. Detektor lainnya dapat
ditemui seperti waktu penerbangan (TOF), tandem quadrupoles (MS-MS) (lihat di bawah),
atau dalam kasus sebuah perangkap MSN ion dimana n menunjukkan tahap nomor massa
spektrometri.
[Sunting] Analisis
Sebuah spektrometer massa biasanya digunakan dalam salah satu dari dua cara: Full Scan
atau Pemantauan Ion Selektif (SIM). Instrumen GC / MS khas mampu melakukan kedua
fungsi baik secara individu atau bersamaan, tergantung pada konfigurasi instrumen tertentu.
[Sunting] Full Scan MS
Ketika mengumpulkan data dalam modus scan penuh, berbagai target fragmen massa
ditentukan dan dimasukkan ke dalam metode instrumen. Sebuah contoh dari berbagai khas
fragmen massa untuk memantau akan m / z 50 sampai m / z 400. Penentuan apa yang
berkisar untuk menggunakan sebagian besar ditentukan oleh apa yang salah mengantisipasi
berada di sampel sementara yang sadar akan kemungkinan gangguan pelarut dan lainnya.
Sebuah MS tidak harus ditetapkan untuk mencari fragmen massa terlalu rendah atau pun
seseorang bisa mendeteksi udara (ditemukan sebagai m / z 28 karena nitrogen), karbon

dioksida (m / z 44) atau gangguan lain yang mungkin. Selain itu jika satu adalah dengan
menggunakan berbagai besar scan maka sensitivitas instrumen yang menurun karena
melakukan scan lebih sedikit per detik karena setiap scan harus mendeteksi berbagai fragmen
massa.
Full scan berguna dalam menentukan senyawa yang tidak diketahui dalam sampel. Ini
menyediakan informasi lebih dari SIM ketika datang untuk konfirmasi atau menyelesaikan
senyawa dalam sampel. Selama pengembangan instrumen metode mungkin umum untuk
pertama menganalisa larutan uji dalam modus scan penuh untuk menentukan waktu retensi
dan sidik jari fragmen massa sebelum pindah ke metode instrumen SIM.
[Sunting] pemantauan ion terpilih
Dalam pemantauan ion terpilih (SIM) fragmen ion tertentu dimasukkan ke dalam metode
instrumen dan hanya fragmen massa yang terdeteksi oleh spektrometer massa. Keuntungan
dari SIM adalah bahwa batas deteksi yang lebih rendah karena instrumen yang hanya melihat
sejumlah kecil fragmen (misalnya tiga fragmen) selama setiap scan. Lebih scan dapat terjadi
setiap detik. Karena hanya fragmen massa beberapa bunga sedang dipantau, gangguan
matriks biasanya lebih rendah. Untuk tambahan mengkonfirmasi kemungkinan hasil positif
potensial, relatif penting untuk memastikan bahwa rasio ion fragmen massa berbagai
sebanding dengan standar referensi yang dikenal.
[Sunting] Jenis Ionisasi
Setelah molekul perjalanan panjang kolom, melewati jalur transfer dan masuk ke dalam
spektrometer massa mereka terionisasi dengan berbagai metode dengan metode yang
biasanya hanya satu yang digunakan pada waktu tertentu. Setelah sampel terfragmentasi
maka akan terdeteksi, biasanya oleh dioda multiplier elektron, yang pada dasarnya mengubah
massa fragmen terionisasi menjadi sinyal listrik yang kemudian terdeteksi.
Teknik ionisasi yang dipilih adalah independen menggunakan Full Scan atau SIM.
[Sunting] Elektron Ionisasi
Sejauh ini merupakan bentuk paling umum dan mungkin standar ionisasi adalah ionisasi
elektron (EI). Molekul-molekul masuk ke dalam MS (sumber adalah quadrupole atau
perangkap ion sendiri dalam perangkap ion MS) di mana mereka dibombardir dengan
elektron bebas yang dipancarkan dari filamen, tidak banyak berbeda dengan filamen yang
akan menemukan di bola lampu standar. The membombardir elektron molekul, menyebabkan
molekul untuk fragmen dengan cara yang khas dan direproduksi. Ini "keras ionisasi" hasil
teknik dalam penciptaan fragmen lebih banyak massa rendah untuk mengisi rasio (m / z) dan
sedikit, jika ada, molekul mendekati unit massa molekul. Ionisasi keras dianggap oleh
spectroscopists massa sebagai mempekerjakan pemboman elektron molekul, sedangkan
"ionisasi lunak" adalah biaya oleh tabrakan molekul dengan gas diperkenalkan. Pola
fragmentasi molekul tergantung pada energi elektron diterapkan pada sistem, biasanya 70eV
(elektron Volt). Penggunaan 70eV memfasilitasi perbandingan spektrum yang dihasilkan
dengan National Institute of Standar (NIST-USA) perpustakaan spektrum menerapkan
program pencocokan algoritma dan penggunaan metode analisis yang ditulis oleh lembaga
standardisasi metode banyak.
[Sunting] Ionisasi Kimia
Artikel utama: ionisasi kimia
Dalam ionisasi kimia gas pereaksi, biasanya metana atau amonia dimasukkan ke dalam
spektrometer massa. Tergantung pada teknik (CI positif atau negatif CI) yang dipilih, ini gas
reagen akan berinteraksi dengan elektron dan analit dan menyebabkan 'soft' ionisasi molekul
bunga. Sebuah ionisasi lembut fragmen molekul ke tingkat yang lebih rendah daripada
ionisasi keras EI. Salah satu manfaat utama menggunakan ionisasi kimia adalah bahwa
sebuah fragmen massa erat sesuai dengan berat molekul analit kepentingan diproduksi.
Positif Kimia Ionisasi

Dalam Ionisasi Kimia Positif (PCI) gas reagen berinteraksi dengan molekul target, paling
sering dengan pertukaran proton. Ini menghasilkan spesies dalam jumlah yang relatif tinggi.
Negatif Kimia Ionisasi
Dalam Ionisasi Kimia Negatif (NCI) gas pereaksi mengurangi dampak elektron bebas pada
analit target. Ini energi menurun biasanya meninggalkan fragmen pasokan yang besar.
Bagian ini membutuhkan ekspansi dengan:
Memperbarui. Informasi berikut ini dalam proses sedang diperbarui:.
Tujuan utama dari analisis instrumen untuk mengukur jumlah zat. Hal ini dilakukan dengan
membandingkan konsentrasi relatif antara massa atom dalam spektrum yang dihasilkan. Dua
jenis analisis yang mungkin, komparatif dan asli. Analisis Perbandingan dasarnya
membandingkan spektrum yang diberikan ke perpustakaan untuk melihat apakah spektrum
karakteristiknya hadir untuk beberapa sampel di perpustakaan. Hal ini paling baik dilakukan
oleh komputer karena ada segudang distorsi visual yang dapat terjadi karena variasi dalam
skala. Komputer juga dapat secara bersamaan berkorelasi lebih banyak data (seperti waktu
retensi diidentifikasi oleh GC), untuk lebih akurat berhubungan data tertentu.
Metode lain dari analisis mengukur puncak dalam hubungannya dengan satu sama lain.
Dalam metode ini, puncak tertinggi diberikan 100% dari nilai, dan puncak lainnya yang
ditugaskan nilai proporsional. Semua nilai di atas 3% ditugaskan. Massa total senyawa yang
tidak diketahui biasanya ditandai dengan puncak orangtua. Nilai ini puncak orangtua dapat
digunakan agar sesuai dengan rumus kimia yang mengandung berbagai unsur yang diyakini
berada di kompleks. Pola isotop dalam spektrum, yang unik untuk elemen yang memiliki
isotop banyak, juga dapat digunakan untuk mengidentifikasi berbagai elemen hadir. Setelah
rumus kimia telah disesuaikan dengan spektrum, struktur molekul dan ikatan dapat
diidentifikasi, dan harus konsisten dengan karakteristik direkam oleh GC / MS. Biasanya,
identifikasi dilakukan secara otomatis oleh program yang datang dengan instrumen, diberi
daftar unsur-unsur yang bisa hadir dalam sampel.
Sebuah "spektrum penuh" analisis menganggap semua "puncak" dalam spektrum.
Sebaliknya, ion pemantauan selektif (SIM) hanya memonitor puncak yang dipilih terkait
dengan zat tertentu. Hal ini dilakukan dengan asumsi bahwa pada waktu retensi yang
diberikan, satu set ion adalah karakteristik dari senyawa tertentu. Ini adalah analisis cepat dan
efisien, terutama jika analis memiliki informasi sebelumnya tentang sampel atau hanya
mencari beberapa zat-zat tertentu. Ketika jumlah informasi yang dikumpulkan tentang ion
dalam kromatografi menurun diberikan puncak gas, sensitivitas meningkat analisis. Jadi,
analisis SIM memungkinkan untuk jumlah yang lebih kecil dari suatu senyawa untuk
dideteksi dan diukur, tetapi tingkat kepastian tentang identitas senyawa yang berkurang.
[Sunting] GC-MS/MS
Ketika fase kedua fragmentasi massa ditambahkan, misalnya menggunakan quadrupole kedua
dalam instrumen quadrupole, hal itu disebut MS / MS atau Tandem MS. Tandem spektrometri
massa (MS / MS) adalah teknik yang lebih kuat untuk quantitate tingkat rendah senyawa
target di hadapan latar belakang matriks sampel tinggi.
The kuadrupol pertama (Q1) terhubung dengan sel tabrakan (q2) dan quadrupole lain (Q3).
Kedua quadrupoles dapat digunakan dalam pemindaian atau mode statis, tergantung pada
jenis MS / MS analisis yang dilakukan. Jenis analisis meliputi produk ion scan, prekursor ion
scan, Pemantauan Reaksi Tunggal (SRM) dan Pemantauan Reaksi Beberapa (MRM) dan
Scan Rugi Netral. Sebagai contoh: Ketika Q1 berada dalam mode statis (melihat satu massa
hanya seperti di SIM), dan Q3 berada dalam mode pemindaian, satu memperoleh apa yang
disebut produk spektrum ion (juga disebut "putri spektrum"). Dari spektrum ini, seseorang
dapat memilih ion produk terkemuka yang dapat menjadi ion produk untuk ion prekursor
yang dipilih. Pasangan ini disebut "transisi" dan membentuk dasar untuk SRM (MRM

kadang-kadang digunakan sebagai istilah). SRM sangat spesifik dan hampir menghilangkan
latar belakang matriks.
[Sunting] Aplikasi
[Sunting] Pemantauan Lingkungan dan Pembersihan
GC-MS menjadi alat pilihan untuk melacak polutan organik di lingkungan. Biaya GC-MS
peralatan telah menurun secara signifikan, dan kehandalan telah meningkat pada saat yang
sama, yang telah memberikan kontribusi terhadap peningkatan adopsi dalam studi
lingkungan. Ada beberapa senyawa yang GC-MS tidak cukup sensitif, termasuk pestisida dan
herbisida tertentu, tetapi untuk analisis yang paling organik dari sampel lingkungan, termasuk
kelas utama banyak pestisida, sangat sensitif dan efektif.
[Sunting] Forensik Pidana
GC-MS dapat menganalisis partikel dari tubuh manusia dalam rangka untuk membantu
menghubungkan seorang kriminal untuk kejahatan. Analisis puing-puing kebakaran
menggunakan GC-MS mapan, dan bahkan ada American Society didirikan untuk Bahan
Pengujian (ASTM) standar untuk analisis kebakaran puing-puing. GCMS / MS sangat
berguna di sini sebagai sampel sering mengandung matriks yang sangat kompleks dan hasil,
yang digunakan di pengadilan, harus sangat akurat.
[Sunting] Penegakan Hukum
GC-MS semakin digunakan untuk mendeteksi narkotika ilegal, dan akhirnya bisa
menggantikan obat-mengendus anjing [1]. Hal ini juga sering digunakan dalam toksikologi
forensik untuk menemukan obat dan / atau racun dalam spesimen biologi dari tersangka,
korban, atau almarhum .
[Sunting] Keamanan
A pasca-11 September pengembangan, sistem deteksi bahan peledak telah menjadi bagian
dari semua bandara AS. Sistem ini berjalan pada sejumlah teknologi, banyak dari mereka
didasarkan pada GC-MS. Hanya ada tiga produsen disertifikasi oleh FAA untuk menyediakan
sistem ini, [rujukan?] Salah satunya adalah Thermo Detection (sebelumnya Thermedics),
yang menghasilkan Egis, garis GC-MS berbasis detektor bahan peledak. Dua lainnya
produsen Teknologi Barringer, kini dimiliki oleh Sistem Deteksi Smith dan Instrumen Jalur
Ion, bagian dari Infrastruktur Umum Sistem Keamanan Listrik.
[Sunting] Makanan, Minuman dan Parfum Analisis
Makanan dan minuman mengandung senyawa aromatik banyak, sebagian alami terdapat
dalam bahan baku dan beberapa membentuk selama pemrosesan. GC-MS secara luas
digunakan untuk analisis senyawa yang meliputi ester, asam lemak, alkohol, aldehida, terpene
dll Hal ini juga digunakan untuk mendeteksi dan mengukur kontaminan dari pembusukan
atau pemalsuan yang mungkin berbahaya dan yang sering dikendalikan oleh pemerintah
lembaga, untuk pestisida misalnya.
[Sunting] Astrochemistry
Beberapa GC-MS telah meninggalkan bumi. Dua dibawa ke Mars oleh program Viking. [4]
Venera 11 dan 12 dan Pioneer Venus menganalisis atmosfer Venus dengan GC-MS. [5] Probe
Huygens dari misi Cassini-Huygens mendarat satu GC-MS pada Saturnus terbesar bulan,
Titan [6]. Materi di 67P/Churyumov-Gerasimenko komet akan dianalisa oleh misi Rosetta
dengan kiral GC-MS pada tahun 2014. [7]
[Sunting] Kedokteran
Dalam kombinasi dengan pelabelan isotop senyawa metabolik, GC-MS digunakan untuk
menentukan aktivitas metabolik. Sebagian besar aplikasi didasarkan pada penggunaan 13C
sebagai label dan pengukuran rasio 13C/12C dengan spektrometer massa rasio isotop
(IRMS), sebuah MS dengan detektor yang dirancang untuk mengukur ion pilih beberapa dan
kembali nilai-nilai sebagai rasio.

[edit] See also

Liquid chromatography-mass spectrometry

Ion mobility spectrometry-mass spectrometry
Prolate trochoidal mass spectrometer

[edit] References
1. ^ Gohlke, R. S. (1959). "Time-of-Flight Mass Spectrometry and Gas-Liquid Partition
2.
3.
4.
5.
6.

7.

Chromatography". Analytical Chemistry 31: 535. doi:10.1021/ac50164a024.

^ Gohlke, R (1993). "Early gas chromatography/mass spectrometry". Journal of the
American Society for Mass Spectrometry 4: 367. doi:10.1016/1044-0305(93)85001-E.
^ "Optimizing the Analysis of Volatile Organic Compounds - Technical Guide" Restek
Corporation, Lit. Cat. 59887A
^ The Development of the Viking GCMS
^ V. A. Krasnopolsky, V. A. Parshev (1981). "Chemical composition of the atmosphere of
Venus". Nature 292: 610613. doi:10.1038/292610a0.
^ H. B. Niemann, S. K. Atreya, S. J. Bauer, G. R. Carignan, J. E. Demick, R. L. Frost, D.
Gautier, J. A. Haberman, D. N. Harpold, D. M. Hunten, G. Israel, J. I. Lunine, W. T.
Kasprzak, T. C. Owen, M. Paulkovich, F. Raulin, E. Raaen, S. H. Way (2005). "The
abundances of constituents of Titans atmosphere from the GCMS instrument on the Huygens
probe". Nature 438: 779784. doi:10.1038/nature04122.
^ Goesmann F, Rosenbauer H, Roll R, Bohnhardt H (2005). "COSAC onboard Rosetta: A
bioastronomy experiment for the short-period comet 67P/Churyumov-Gerasimenko".
Astrobiology 5 (5): 622631. doi:10.1089/ast.2005.5.622.

[edit] Bibliography

Robert P., Dr Adams (2007). Identification of Essential Oil Components By Gas

Chromatography/Mass Spectrometry. Allured Pub Corp. ISBN 1-932633-21-9.
Adlard, E. R.; Handley, Alan J. (2001). Gas chromatographic techniques and applications.
London: Sheffield Academic. ISBN 0-8493-0521-7.
Eugene F. Barry; Grob, Robert Lee (2004). Modern practice of gas chromatography. New
York: Wiley-Interscience. ISBN 0-471-22983-0.
Eiceman, G.A. (2000). Gas Chromatography. In R.A. Meyers (Ed.), Encyclopedia of
Analytical Chemistry: Applications, Theory, and Instrumentation, pp. 10627. Chichester:
Wiley. ISBN 0-471-97670-9
Giannelli, Paul C. and Imwinkelried, Edward J. (1999). Drug Identification: Gas
Chromatography. In Scientific Evidence 2, pp. 362. Charlottesville: Lexis Law Publishing.
ISBN 0-327-04985-5.
McEwen, Charles N.; Kitson, Fulton G.; Larsen, Barbara Seliger (1996). Gas
chromatography and mass spectrometry: a practical guide. Boston: Academic Press. ISBN 012-483385-3.
McMaster, Christopher; McMaster, Marvin C. (1998). GC/MS: a practical user's guide. New
York: Wiley. ISBN 0-471-24826-6.
Message, Gordon M. (1984). Practical aspects of gas chromatography/mass spectrometry.
New York: Wiley. ISBN 0-471-06277-4.
Niessen, W. M. A. (2001). Current practice of gas chromatography--mass spectrometry. New
York, N.Y: Marcel Dekker. ISBN 0-8247-0473-8.
Weber, Armin; Maurer, Hans W.; Pfleger, Karl (2007). Mass Spectral and GC Data of Drugs,
Poisons, Pesticides, Pollutants and Their Metabolites. Weinheim: Wiley-VCH. ISBN 3-52731538-1.

[edit] External links

GCMS - How does it work?

MeSH Gas+chromatography-mass+spectrometry
GCMS Tutorial
Gas Chromatography-Mass Spectroscopy Background
Introduction to Mass Spectrometry

1. Spektrofotometer single beam

1. Spektrofotometer single beam

Gambar : Bagan spektrofotometer single beam

2. Spektrofotometer double beam

Gambar bagan spektrofotometer double beam

No
1
2
3
4
5
6
7
8
9

No

C (ppm)
A
1,02
0,1
2,21
0,2
3,23
0,3
4,51
0,4
5,12
0,5
5,79
0,6
7,11
0,7
8,35
0,8
9,27
0,9

A-Ar

C-Cr

(A-Ar)2

(C-Cr)2

1,02

0,1

-0,4

4,16

0,16

17,30

2,21

0,2

-0,3

2,97

0,09

8,81

3,23

0,3

-0,2

1,95

0,04

3,80

4
5

4,51
5,12

0,4
0,5

-0,1
0

0,67
0,06

0,01
0

0,45
0,00

5,79

0,6

0,1

0,61

0,01

0,37

7,11

0,7

0,2

1,93

0,04

3,73

8,35

0,8

0,3

3,17

0,09

10,06

9,27

0,9

0,4

4,09

0,16

Rata
2

46,61

4,5

16,74
61,255
49

5,18
0,0111
6
0,0987
01
0,9972
87

0,5

k
m
r

0,6

(A-Ar)(CCr)
1,6635555
56
0,8906666
67
0,3897777
78
0,0668888
89
0
0,0611111
11
0,3862222
22
0,9513333
33
1,6364444
44
6,046

Infrared spectroscopy
From Wikipedia, the free encyclopedia
(Redirected from Infrared Spectrometer)
Jump to: navigation, search
For a table of IR spectroscopy data, see infrared spectroscopy correlation table.
Infrared spectroscopy (IR spectroscopy) is the subset of spectroscopy that deals with the
infrared region of the electromagnetic spectrum. It covers a range of techniques, the most
common being a form of absorption spectroscopy. As with all spectroscopic
techniques, it can be used to identify compounds or investigate sample composition.
Infrared spectroscopy correlation tables are tabulated in the literature. A common
laboratory instrument that uses this technique is an infrared spectrophotometer.

[edit] Background and theory

The infrared portion of the electromagnetic spectrum is divided into three regions; the near-,
mid- and far- infrared, named for their relation to the visible spectrum. The far-infrared,
approximately 40010 cm1 (100030 m), lying adjacent to the microwave region, has low
energy and may be used for rotational spectroscopy. The mid-infrared, approximately
4000400 cm1 (302.5 m) may be used to study the fundamental vibrations and associated
rotational-vibrational structure. The higher energy near-IR, approximately 140004000 cm1
(2.50.8 m) can excite overtone or harmonic vibrations. The names and classifications of
these subregions are merely conventions. They are neither strict divisions nor based on exact
molecular or electromagnetic properties.
Infrared spectroscopy exploits the fact that molecules have specific frequencies at which they
rotate or vibrate corresponding to discrete energy levels (vibrational modes). These
resonant frequencies are determined by the shape of the molecular potential energy
surfaces, the masses of the atoms and, by the associated vibronic coupling. In order for a
vibrational mode in a molecule to be IR active, it must be associated with changes in the
permanent dipole. In particular, in the BornOppenheimer and harmonic approximations,
i.e. when the molecular Hamiltonian corresponding to the electronic ground state can be
approximated by a harmonic oscillator in the neighborhood of the equilibrium molecular
geometry, the resonant frequencies are determined by the normal modes corresponding to
the molecular electronic ground state potential energy surface. Nevertheless, the resonant
frequencies can be in a first approach related to the strength of the bond, and the mass of the
atoms at either end of it. Thus, the frequency of the vibrations can be associated with a
particular bond type.
Simple diatomic molecules have only one bond, which may stretch. More complex molecules
have many bonds, and vibrations can be conjugated, leading to infrared absorptions at
characteristic frequencies that may be related to chemical groups. For example, the atoms in a
CH2 group, commonly found in organic compounds can vibrate in six different ways:
symmetrical and antisymmetrical stretching, scissoring, rocking, wagging and twisting:

Symmetrical
stretching

Antisymmet
rical
stretching

Scissoring

Rocking

Wagging

Twisting

The infrared spectrum of a sample is collected by passing a beam of infrared light through the
sample. Examination of the transmitted light reveals how much energy was absorbed at each
wavelength. This can be done with a monochromatic beam, which changes in wavelength
over time, or by using a Fourier transform instrument to measure all wavelengths at once.
From this, a transmittance or absorbance spectrum can be produced, showing at which IR
wavelengths the sample absorbs. Analysis of these absorption characteristics reveals details
about the molecular structure of the sample. When the frequency of the IR is the same as the
vibrational frequency of a bond, absorption occurs.
This technique works almost exclusively on samples with covalent bonds. Simple spectra
are obtained from samples with few IR active bonds and high levels of purity. More complex
molecular structures lead to more absorption bands and more complex spectra. The technique
has been used for the characterization of very complex mixtures.

[edit] Sample preparation

Gaseous samples require little preparation beyond purification, but a sample cell with a long
pathlength (typically 510 cm) is normally needed, as gases show relatively weak
absorbances.
Liquid samples can be sandwiched between two plates of a high purity salt (commonly
sodium chloride, or common salt, although a number of other salts such as potassium
bromide or calcium fluoride are also used).[1] The plates are transparent to the infrared light
and will not introduce any lines onto the spectra. Some salt plates are highly soluble in water,
so the sample and washing reagents must be anhydrous (without water).
Solid samples can be prepared in four major ways. The first is to crush the sample with a
mulling agent (usually Nujol) in a marble or agate mortar, with a pestle. A thin film of the
mull is applied onto salt plates and measured.[2]

The second method is to grind a quantity of the sample with a specially purified salt (usually
potassium bromide) finely (to remove scattering effects from large crystals). This powder
mixture is then crushed in a mechanical die press to form a translucent pellet through which
the beam of the spectrometer can pass.[3]
The third technique is the "cast film" technique, which is used mainly for polymeric
materials. The sample is first dissolved in a suitable, non hygroscopic solvent. A drop of this
solution is deposited on surface of KBr or NaCl cell. The solution is then evaporated to
dryness and the film formed on the cell is analysed directly. Care is important to ensure that
the film is not too thick otherwise light cannot pass through. This technique is suitable for
qualitative analysis.
The final method is to use microtomy to cut a thin (20100 micrometre) film from a solid
sample. This is one of the most important ways of analysing failed plastic products for
example because the integrity of the solid is preserved.
It is important to note that spectra obtained from different sample preparation methods will
look slightly different from each other due to differences in the samples' physical states.

[edit] Conventional method

Typical apparatus
A beam of infrared light is produced and split into two separate beams. One is passed through
the sample, the other passed through a reference which is often the substance the sample is
dissolved in. The beams are both reflected back towards a detector, however first they pass
through a splitter which quickly alternates which of the two beams enters the detector. The
two signals are then compared and a printout is obtained.
A reference is used for two reasons:

This prevents fluctuations in the output of the source affecting the data
This allows the effects of the solvent to be cancelled out (the reference is usually a
pure form of the solvent the sample is in)

[edit] FTIR
Main article: Fourier transform spectroscopy
Fourier transform infrared (FTIR) spectroscopy is a measurement technique for
collecting infrared spectra. Instead of recording the amount of energy absorbed when the
frequency of the infra-red light is varied (monochromator), the IR light is guided through an
interferometer. After passing through the sample, the measured signal is the interferogram.
Performing a Fourier transform on this signal data results in a spectrum identical to that
from conventional (dispersive) infrared spectroscopy.
FTIR spectrometers are cheaper than conventional spectrometers because building an
interferometer is easier than the fabrication of a monochromator. In addition, measurement of
a single spectrum is faster for the FTIR technique because the information at all frequencies
is collected simultaneously. This allows multiple samples to be collected and averaged
together resulting in an improvement in sensitivity. Virtually all modern infrared
spectrometers are FTIR instruments.

[edit] Absorptions bands

Main article: Infrared Spectroscopy Correlation Table

Wavenumbers listed in cm1.

[edit] Uses and applications

Infrared spectroscopy is widely used in both research and industry as a simple and reliable
technique for measurement, quality control and dynamic measurement. It is of especial use in
forensic analysis in both criminal and civil cases, enabling identification of polymer
degradation for example. It is perhaps the most widely used method of applied
spectroscopy.[citation needed]
The instruments are now small, and can be transported, even for use in field trials. With
increasing technology in computer filtering and manipulation of the results, samples in
solution can now be measured accurately (water produces a broad absorbance across the
range of interest, and thus renders the spectra unreadable without this computer treatment).
Some instruments will also automatically tell you what substance is being measured from a
store of thousands of reference spectra held in storage.
By measuring at a specific frequency over time, changes in the character or quantity of a
particular bond can be measured. This is especially useful in measuring the degree of
polymerization in polymer manufacture. Modern research instruments can take infrared
measurements across the whole range of interest as frequently as 32 times a second. This can
be done whilst simultaneous measurements are made using other techniques. This makes the
observations of chemical reactions and processes quicker and more accurate.
Techniques have been developed to assess the quality of tea-leaves using infrared
spectroscopy. This will mean that highly trained experts (also called 'noses') can be used more
sparingly, at a significant cost saving.[4]
Infrared spectroscopy has been highly successful for applications in both organic and
inorganic chemistry. Infrared spectroscopy has also been successfully utilized in the field of
semiconductor microelectronics[5]: for example, infrared spectroscopy can be applied to
semiconductors like silicon, gallium arsenide, gallium nitride, zinc selenide, amorphous
silicon, silicon nitride, etc.

[edit] Isotope effects

The different isotopes in a particular species may give fine detail in infrared spectroscopy.
For example, the O-O stretching frequency (in reciprocal centimeters) of oxyhemocyanin is
experimentally determined to be 832 and 788 cm1 for (16O-16O) and (18O-18O) respectively.
By considering the O-O as a spring, the wavenumber of absorbance, can be calculated:

where k is the spring constant for the bond, c is the speed of light, and is the reduced
mass of the A-B system:

(mi is the mass of atom i).

The reduced masses for 16O-16O and 18O-18O can be approximated as 8 and 9 respectively.
Thus

Where is the wavenumber [wavenumber = frequency/(speed of light)]

The effect of isotopes, both on the vibration and the decay dynamics, has been found to be
stronger than previously thought. In some systems, such as silicon and germanium, the decay
of the anti-symmetric stretch mode of interstitial oxygen involves the symmetric stretch mode
with a strong isotope dependence. For example, it was shown that for a natural silicon
sample, the lifetime of the anti-symmetric vibration is 11.4 ps. When the isotope of one of the
silicon atoms is increased to 29Si, the lifetime increases to 19 ps, similarly, when the silicon
atom is changed to 30Si, the lifetime becomes 27 ps.[6]

[edit] Two-dimensional IR
Two-dimensional infrared correlation spectroscopy analysis is the application of 2D
correlation analysis on infrared spectra. By extending the spectral information of a
perturbed sample, spectral analysis is simplified and resolution is enhanced. The 2D
synchronous and 2D asynchronous spectra represent a graphical overview of the spectral
changes due to a perturbation (such as a changing concentration or changing temperature) as
well as the relationship between the spectral changes at two different wavenumbers.
Main article: Two-dimensional infrared spectroscopy

Pulse Sequence used to obtain a two-dimensional Fourier transform infrared spectrum. The
time period 1 is usually referred to as the coherence time and the second time period 2 is
known as the waiting time. The excitation frequency is obtained by Fourier transforming
along the 1 axis.
Nonlinear two-dimensional infrared spectroscopy[7][8] is the infrared version of correlation
spectroscopy. Nonlinear two-dimensional infrared spectroscopy is a technique that has
become available with the development of femtosecond infrared laser pulses. In this
experiment first a set of pump pulses are applied to the sample. This is followed by a waiting
time, where the system is allowed to relax. The waiting time typically lasts from zero to
several picoseconds and the duration can be controlled with a resolution of tens of
femtoseconds. A probe pulse is then applied resulting in the emission of a signal from the

sample. The nonlinear two-dimensional infrared spectrum is a two-dimensional correlation

plot of the frequency 1 that was excited by the initial pump pulses and the frequency 3
excited by the probe pulse after the waiting time. This allows the observation of coupling
between different vibrational modes; because of its extremely high time resolution it can be
used to monitor molecular dynamics on a picosecond timescale. It is still a largely unexplored
technique and is becoming increasingly popular for fundamental research.
Like in two-dimensional nuclear magnetic resonance (2DNMR) spectroscopy this technique
spreads the spectrum in two dimensions and allow for the observation of cross peaks that
contain information on the coupling between different modes. In contrast to 2DNMR
nonlinear two-dimensional infrared spectroscopy also involve the excitation to overtones.
These excitations result in excited state absorption peaks located below the diagonal and
cross peaks. In 2DNMR two distinct techniques, COSY and NOESY, are frequently used.
The cross peaks in the first are related to the scalar coupling, while in the later they are
related to the spin transfer between different nuclei. In nonlinear two-dimensional infrared
spectroscopy analogs have been drawn to these 2DNMR techniques. Nonlinear twodimensional infrared spectroscopy with zero waiting time corresponds to COSY and
nonlinear two-dimensional infrared spectroscopy with finite waiting time allowing
vibrational population transfer corresponds to NOESY. The COSY variant of nonlinear twodimensional infrared spectroscopy has been used for determination of the secondary structure
content proteins.[9]

[edit] References
1. ^ Laurence M. Harwood, Christopher J. Moody. Experimental organic chemistry:
Principles and Practice (Illustrated edition ed.). pp. 289-292.
2. ^ Laurence M. Harwood, Christopher J. Moody. Experimental organic chemistry:
Principles and Practice (Illustrated edition ed.). pp. 292.
3. ^ Laurence M. Harwood, Christopher J. Moody. Experimental organic chemistry:
Principles and Practice (Illustrated edition ed.). pp. 295-296.
4. ^ Luypaert, J.; Zhang, M.H.; Massart, D.L. (2003), "Feasibility study for the use of
near infrared spectroscopy in the qualitative and quantitative analysis of green tea,
Camellia sinensis (L.)", Analytica Chimica Acta, 478(2), Elsevier, pp. 303312
5. ^ Lau, W.S. (1999). Infrared characterization for microelectronics. World Scientific.
6. ^ Isotope Dependence of the Lifetime of the 1136-cm[sup -1] Vibration of Oxygen in
Silicon K. K. Kohli, Gordon Davies, N. Q. Vinh, D. West, S. K. Estreicher, T.
Gregorkiewicz, I. Izeddin, and K. M. Itoh, Phys. Rev. Lett. 96, 225503 (2006),
DOI:10.1103/PhysRevLett.96.225503
7. ^ P. Hamm, M. H. Lim, R. M. Hochstrasser (1998). "Structure of the amide I band of
peptides measured by femtosecond nonlinear-infrared spectroscopy". J. Phys. Chem.
B 102: 6123. doi:10.1021/jp9813286.
8. ^ S. Mukamel (2000). "Multidimensional Fentosecond Correlation Spectroscopies of
Electronic and Vibrational Excitations". Annual Review of Physics and Chemistry 51:
691. doi:10.1146/annurev.physchem.51.1.691.
9. ^ N. Demirdven, C. M. Cheatum, H. S. Chung, M. Khalil, J. Knoester, A. Tokmakoff
(2004). "Two-dimensional infrared spectroscopy of antiparallel beta-sheet secondary
structure". Journal of the American Chemical Society 126: 7981.
doi:10.1021/ja049811j.

[edit] External links

"
7.4. NMR,
Analisa spektrofotometri inframerah (IR) adalah suatu cara analisa kimia berdasarkan
spektrum vibrasi dalam suatu molekul senyawa kimia pada daerah panjang gelombang 2,5
25 m. Sebenarnya spektrum IR terbagi menjadi 3 bagian seperti tabel dibawah ini :
Tabel : pembagian daerah spektrum inframerah
No Daerah spektrum
1
2
3

Tipe translasi tingkat energi Panjang gelombang

m
Inframerah dekat
overtone
0,75 2,5
Inframerah sedang Vibrasi, rotasi
2,5 - 25
Inframerah jauh
Vibrasi rangka, rotasi
25 - 1000

Frequency
cm-1
13.300 4000
4000 400
400 10

Spketrum inframerah dekat adalah merupakan pita spektrum akibat adanya overtone
dari vibrasi stretching hidrogen, yang dapat digunakan untuk analisa qualitatif bermacammacamgugus fungsi. Alat analisa spektrum inframerah dekat serupa dengan spektrofotometer
sinar tampak berperan memberikan interpretasi semua senyawa kecuali monoatomik dan
homopolar seperti Ne, He, O2, N2, dan H2.
Daerah spektrum inframerah sedang akan memberikan informasi kualitatif dan
kuantitatif dari gugus fungsi dan struktur molekul suatu senyawa. Alat analisa spektrum
inframerah sedang ini berbeda dengan spektrofotometer sinar tampak. Daerah spektrum
inframereh sedang ini terbegi menjadi daerah fundamental frequensi 900 4000 m dan
daerah finger print 400 900 m yang berperan menunjang interpretasi dari spektrum
fundamental frekuenai.
Daerah spektrum inframerah jauh memberikan informasi terutamasekali mengenai
daerah spektrum translasi rotasi, vibrasi kisi kristal, dan vibrasi rangka dari molekul besar.
Ada 2 proses absorpsi radiasi elektromagnetik oleh senyawa
a. Energi radiasi yang diekspos harus cocok benaar dengan energi yang diperlukan molekul.
b. Akan terjadi coupling antara radiasi dan senyawa.
Macammacam vibrasi spektrum inframerah :
a. vibrasi stretching ( vibrasi ulur-tarik) adalah vibrasi yang terjadi menjauh dan mendekat ke
pusat vibrasi. Vibrasi stretching ini terbagi menjadi vibrasi stretching
- simetri ( serentak ), gugus serentak bergerak mendekati dan menjauhi pusat vibrasi.
- asimetri (selang-seling), gugus yangterikat ke pusat vibrasi bergerak berselang seling
mendekati dan menjauhi pusat vibrasi.
b. Vibrasi bending ( vibrasi ayun) adalah vibrasi yang terjadi berayun dalam suatu gugus
dengan jarak tetap dari pusat vibrasi. Vibrasi bending ini terbagi menjadi :
- scissoring (gunting) gerakan pada vibrasi ini seperti gerakan gunting pada sumbu axis.
- rocking (sapu) gerakan pada vibrasi ini seperti gerakan menyapu pada sumbu axis dimana
masing-masing gugus terikat ke pusat vibrasi akan bergerak pada arah yang sama.

7.4.

Nuclear magnetic resonance

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This article is about the physical phenomenon. For its use as a method in spectroscopy, see
Nuclear magnetic resonance spectroscopy.
"NMR" redirects here. For other uses, see NMR (disambiguation).

900 MHz, 21.2 T NMR Magnet at HWB-NMR, Birmingham, UK

Nuclear magnetic resonance (NMR) is a physical phenomenon in which magnetic nuclei
in a magnetic field absorb and re-emit electromagnetic radiation. This energy is at a
specific resonance frequency which depends on the strength of the magnetic field and the
magnetic properties of the isotope of the atoms; in practical applications, the frequency is
similar to VHF and UHF television broadcasts (601000 MHz). NMR allows the
observation of specific quantum mechanical magnetic properties of the atomic nucleus.
Many scientific techniques exploit NMR phenomena to study molecular physics, crystals,
and non-crystalline materials through NMR spectroscopy. NMR is also routinely used in
advanced medical imaging techniques, such as in magnetic resonance imaging (MRI).
All isotopes that contain an odd number of protons and/or of neutrons (see Isotope) have
an intrinsic magnetic moment and angular momentum, in other words a nonzero spin,
while all nuclides with even numbers of both have a total spin of zero. The most commonly
studied nuclei are 1H and 13C, although nuclei from isotopes of many other elements (e.g.
2H, 6Li, 10B, 11B, 14N, 15N, 17O, 19F, 23Na, 29Si, 31P, 35Cl, 113Cd, 129Xe, 195Pt) have
been studied by high-field NMR spectroscopy as well.
A key feature of NMR is that the resonance frequency of a particular substance is directly
proportional to the strength of the applied magnetic field. It is this feature that is exploited in
imaging techniques; if a sample is placed in a non-uniform magnetic field then the resonance
frequencies of the sample's nuclei depend on where in the field they are located. Since the
resolution of the imaging technique depends on the magnitude of magnetic field gradient,
many efforts are made to develop increased field strength, often using superconductors.
The effectiveness of NMR can also be improved using hyperpolarization, and/or using twodimensional, three-dimensional and higher-dimensional multi-frequency techniques.
The principle of NMR usually involves two sequential steps:

The alignment (polarization) of the magnetic nuclear spins in an applied, constant

magnetic field H0.

The perturbation of this alignment of the nuclear spins by employing an electromagnetic, usually radio frequency (RF) pulse. The required perturbing frequency is
dependent upon the static magnetic field (H0) and the nuclei of observation.

The two fields are usually chosen to be perpendicular to each other as this maximizes the
NMR signal strength. The resulting response by the total magnetization (M) of the nuclear
spins is the phenomenon that is exploited in NMR spectroscopy and magnetic resonance
imaging. Both use intense applied magnetic fields (H0) in order to achieve dispersion and
very high stability to deliver spectral resolution, the details of which are described by
chemical shifts, the Zeeman effect, and Knight shifts (in metals).
NMR phenomena are also utilized in low-field NMR, NMR spectroscopy and MRI in the
Earth's magnetic field (referred to as Earth's field NMR), and in several types of
magnetometers.

Contents
[hide]

1 History
2 Theory of nuclear magnetic resonance
o 2.1 Nuclear spin and magnets
o 2.2 Values of spin angular momentum
2.2.1 Spin behavior in a magnetic field
2.2.2 Magnetic resonance by nuclei
2.2.3 Nuclear shielding
o 2.3 Relaxation
3 NMR spectroscopy
o 3.1 Continuous wave (CW) spectroscopy
o 3.2 Fourier transform spectroscopy
o 3.3 Multi-dimensional NMR Spectroscopy
o 3.4 Solid-state NMR spectroscopy
o 3.5 Sensitivity
o 3.6 Isotopes
4 Applications
o 4.1 Medicine
o 4.2 Chemistry
o 4.3 Non-destructive testing
o 4.4 Acquisition of dynamic information
o 4.5 Data acquisition in the petroleum industry
o 4.6 Flow probes for NMR spectroscopy
o 4.7 Process control
o 4.8 Earth's field NMR
o 4.9 Quantum computing
o 4.10 Magnetometers
5 Makers of NMR equipment
6 See also
7 Notes
8 References
9 External links
o 9.1 Tutorial
o 9.2 Animations and Simulations
o

9.3 Video

[edit] History
Nuclear magnetic resonance was first described and measured in molecular beams by Isidor
Rabi in 1938,[1] and in 1944, Rabi was awarded the Nobel Prize in physics for this work.[2]
In 1946, Felix Bloch and Edward Mills Purcell expanded the technique for use on liquids
and solids, for which they shared the Nobel Prize in Physics in 1952.[3][4]
Purcell had worked on the development of radar during World War II at the Massachusetts
Institute of Technology's Radiation Laboratory. His work during that project on the

production and detection of radio frequency power and on the absorption of such RF power
by matter laid the background for Rabi's discovery of NMR.
Rabi, Bloch, and Purcell noticed that magnetic nuclei, like 1H and 31P, could absorb RF
energy when placed in a magnetic field and when the RF was of a frequency specific to the
identity of the nuclei. When this absorption occurs, the nucleus is described as being in
resonance. Different atomic nuclei within a molecule resonate at different (radio) frequencies
for the same magnetic field strength. The observation of such magnetic resonance frequencies
of the nuclei present in a molecule allows any trained user to discover essential, chemical and
structural information about the molecule.
The development of NMR as a technique in analytical chemistry and biochemistry
parallels the development of electromagnetic technology and advanced electronics and their
introduction into civilian use.

[edit] Theory of nuclear magnetic resonance

[edit] Nuclear spin and magnets
All nucleons, that is neutrons and protons, composing any atomic nucleus, have the
intrinsic quantum property of spin. The overall spin of the nucleus is determined by the spin
quantum number S. If the number of both the protons and neutrons in a given nuclide are
even then S = 0, i.e. there is no overall spin. Then, just as electrons pair up in atomic
orbitals, so do even numbers of protons or even numbers of neutrons (which are also spin-12
particles and hence fermions) pair up giving zero overall spin.
However, a proton and neutron will have lower energy when their spins are parallel, not antiparallel, since this parallel spin alignment does not infringe upon the Pauli Exclusion
Principle, but instead it has to do with the quark structure of these two nucleons. Therefore,
the spin ground state for the deuteron (the deuterium nucleus, or the 2H isotope of hydrogen)
that has only a proton and a neutroncorresponds to a spin value of 1, not of zero. The
single, isolated deuteron therefore exhibits an NMR absorption spectrum characteristic of a
quadrupolar nucleus of spin 1, which in the "rigid" state at very low temperatures is a
characteristic ('Pake') doublet, (not a singlet as for a single, isolated 1H, or any other isolated
fermion or dipolar nucleus of spin 1/2). On the other hand, because of the Pauli Exclusion
Principle, the tritium isotope of hydrogen must have a pair of anti-parallel spin neutrons (of
total spin zero for the neutron-spin pair), plus a proton of spin 1/2. Therefore, the character of
the tritium nucleus is again magnetic dipolar, not quadrupolarlike its non-radioactive
deuteron neighborand the tritium nucleus total spin value is again 1/2, just like for the
simpler, abundant hydrogen isotope, 1H nucleus (the proton). The NMR absorption (radio)
frequency for tritium is however slightly higher than that of 1H because the tritium nucleus
has a slightly higher gyromagnetic ratio than 1H. In many other cases of non-radioactive
nuclei, the overall spin is also non-zero. For example, the 27Al nucleus has an overall spin
value S = 52.
A non-zero spin is thus always associated with a non-zero magnetic moment () via the
relation = S, where is the gyromagnetic ratio. It is this magnetic moment that allows the
observation of NMR absorption spectra caused by transitions between nuclear spin levels.
Most nuclides (with some rare exceptions) that have both even numbers of protons and even
numbers of neutrons, also have zero nuclear magnetic moments, and they also have zero

magnetic dipole and quadrupole moments. Hence, such nuclides do not exhibit any NMR
absorption spectra. Thus, 18O is an example of a nuclide that has no NMR absorption,
whereas 13C, 31P, 35Cl and 37Cl are nuclides that do exhibit NMR absorption spectra. The
last two nuclei are quadrupolar nuclei whereas the preceding two nuclei (13C and 31P) are
dipolar ones.
Electron spin resonance (ESR) is a related technique in which transitions between
electronic spin levels are detected rather than nuclear ones. The basic principles are similar
but the instrumentation, data analysis, and detailed theory are significantly different.
Moreover, there is a much smaller number of molecules and materials with unpaired electron
spins that exhibit ESR (or electron paramagnetic resonance (EPR)) absorption than those
that have NMR absorption spectra. ESR has much higher sensitivity than NMR does.

[edit] Values of spin angular momentum

The angular momentum associated with nuclear spin is quantized. This means both that the
magnitude of angular momentum is quantized (i.e. S can only take on a restricted range of
values), and also that the orientation of the associated angular momentum is quantized. The
associated quantum number is known as the magnetic quantum number, m, and can take
values from +S to S, in integer steps. Hence for any given nucleus, there is a total of 2S + 1
angular momentum states.
The z-component of the angular momentum vector (S) is therefore Sz = m, where is the
reduced Planck constant. The z-component of the magnetic moment is simply:

[edit] Spin behavior in a magnetic field

Splitting of nuclei spin states in an external magnetic field

An intuitive model. Nuclei behave like they had own magnetic moments (spin magnetic
moments). By itself, there is no energetic difference for any particular orientation (only one
energy state, on the left), but in external magnetic field there is a high-energy state and a lowenergy state depending on the relative orientations of the magnet to the external field, and the
orientation of the magnetic moment can precess relative to it. The external field can be
supplied by a large magnet and also by other nuclei in the vicinity.
Consider nuclei which have a spin of one-half, like 1H, 13C or 19F. The nucleus has two
possible spin states: m = 12 or m = 12 (also referred to as spin-up and spin-down, or
sometimes and spin states, respectively). These states are degenerate, that is they have the
same energy. Hence the number of atoms in these two states will be approximately equal at
thermal equilibrium.
If a nucleus is placed in a magnetic field, however, the interaction between the nuclear
magnetic moment and the external magnetic field mean the two states no longer have the
same energy. The energy of a magnetic moment when in a magnetic field B0 is given by:

Usually the z axis is chosen to be along B0, and the above expression reduces to:

or alternatively:

As a result the different nuclear spin states have different energies in a non-zero magnetic
field. In less formal language, we can talk about the two spin states of a spin 12 as being
aligned either with or against the magnetic field. If is positive (true for most isotopes) then
m = 12 is the lower energy state.
The energy difference between the two states is:

and this difference results in a small population bias toward the lower energy state.

[edit] Magnetic resonance by nuclei

Resonant absorption by nuclear spins will occur only when electromagnetic radiation of
the correct frequency (e.g., equaling the Larmor precession rate) is being applied to match
the energy difference between the nuclear spin levels in a constant magnetic field of the
appropriate strength. The energy of an absorbed photon is then E = h0, where 0 is the
resonance radiofrequency that has to match (that is, it has to be equal to the Larmor
precession frequency L of the nuclear magnetization in the constant magnetic field B0).
Hence, a magnetic resonance absorption will only occur when E = h0, which is when 0 =
B0/(2). Such magnetic resonance frequencies typically correspond to the radio frequency
(or RF) range of the electromagnetic spectrum for magnetic fields up to roughly 20 T. It is
this magnetic resonant absorption which is detected in NMR.[citation needed]

[edit] Nuclear shielding

It might appear from the above that all nuclei of the same nuclide (and hence the same )
would resonate at the same frequency. This is not the case. The most important perturbation
of the NMR frequency for applications of NMR is the "shielding" effect of the surrounding
shells of electrons.[5] Electrons, similar to the nucleus, are also charged and rotate with a spin
to produce a magnetic field opposite to the magnetic field produced by the nucleus. In
general, this electronic shielding reduces the magnetic field at the nucleus (which is what
determines the NMR frequency).
As a result the energy gap is reduced, and the frequency required to achieve resonance is also
reduced. This shift in the NMR frequency due to the electronic molecular orbital coupling to
the external magnetic field is called chemical shift, and it explains why NMR is able to
probe the chemical structure of molecules, which depends on the electron density distribution
in the corresponding molecular orbitals. If a nucleus in a specific chemical group is shielded
to a higher degree by a higher electron density of its surrounding molecular orbital, then its
NMR frequency will be shifted "upfield" (that is, a lower chemical shift), whereas if it is less
shielded by such surrounding electron density, then its NMR frequency will be shifted
"downfield" (that is, a higher chemical shift).
Unless the local symmetry of such molecular orbitals is very high (leading to "isotropic"
shift), the shielding effect will depend on the orientation of the molecule with respect to the
external field (B0). In solid-state NMR spectroscopy, magic angle spinning is required to
average out this orientation dependence in order to obtain values close to the average
chemical shifts. This is unnecessary in conventional NMR investigations of molecules, since
rapid "molecular tumbling" averages out the chemical shift anisotropy (CSA). In this case,
the term "average" chemical shift (ACS) is used.

[edit] Relaxation
For more details on this topic, see Relaxation (NMR).
The process called population relaxation refers to nuclei that return to the thermodynamic
state in the magnet. This process is also called T1, "spin-lattice" or "longitudinal magnetic"
relaxation, where T1 refers to the mean time for an individual nucleus to return to its thermal
equilibrium state of the spins. Once the nuclear spin population is relaxed, it can be probed
again, since it is in the initial, equilibrium (mixed) state.
The precessing nuclei can also fall out of alignment with each other (returning the net
magnetization vector to a non-precessing field) and stop producing a signal. This is called T2
or transverse relaxation. Because of the difference in the actual relaxation mechanisms
involved (for example, inter-molecular vs. intra-molecular magnetic dipole-dipole
interactions ), T1 is usually (except in rare cases) longer than T2 (that is, slower spin-lattice
relaxation, for example because of smaller dipole-dipole interaction effects). In practice, the
value of
which is the actually observed decay time of the observed NMR signal, or free
induction decay, (to 1/e of the initial amplitude immediately after the resonant RF pulse)-also depends on the static magnetic field inhomogeneity, which is quite significant. (There is
also a smaller but significant contribution to the observed FID shortening from the RF
inhomogeneity of the resonant pulse). In the corresponding FT-NMR spectrummeaning the
Fourier transform of the free induction decay--the

time is inversely related to the width

of the NMR signal in frequency units. Thus, a nucleus with a long T2 relaxation time gives
rise to a very sharp NMR peak in the FT-NMR spectrum for a very homogeneous ("wellshimmed") static magnetic field, whereas nuclei with shorter T2 values give rise to broad FTNMR peaks even when the magnet is shimmed well. Both T1 and T2 depend on the rate of
molecular motions as well as the gyromagnetic ratios of both the resonating and their strongly
interacting, next-neighbor nuclei that are not at resonance.
A Hahn echo decay experiment can be used to measure the dephasing time, as shown in the
animation below. The size of the echo is recorded for different spacings of the two pulses.
This reveals the decoherence which is not refocused by the pulse. In simple cases, an
exponential decay is measured which is described by the time.

[edit] NMR spectroscopy

Main article: NMR spectroscopy

Bruker 700 MHz. Nuclear Magnetic Resonance (NMR) spectrometer

NMR spectroscopy is one of the principal techniques used to obtain physical, chemical,
electronic and structural information about molecules due to either the chemical shift,
Zeeman effect, or the Knight shift effect, or a combination of both, on the resonant
frequencies of the nuclei present in the sample. It is a powerful technique that can provide
detailed information on the topology, dynamics and three-dimensional structure of molecules
in solution and the solid state. Thus, structural and dynamic information is obtainable (with or
without "magic angle" spinning (MAS)) from NMR studies of quadrupolar nuclei (that is,
those nuclei with spin S > 12) even in the presence of magnetic "dipole-dipole" interaction
broadening (or simply, dipolar broadening) which is always much smaller than the
quadrupolar interaction strength because it is a magnetic vs. an electric interaction effect.
Additional structural and chemical information may be obtained by performing doublequantum NMR experiments for quadrupolar nuclei such as 2H. Also, nuclear magnetic
resonance is one of the techniques that has been used to design quantum automata, and also
build elementary quantum computers.[6][7]

[edit] Continuous wave (CW) spectroscopy

In its first few decades, nuclear magnetic resonance spectrometers used a technique known as
continuous-wave spectroscopy (CW spectroscopy). Although NMR spectra could be, and
have been, obtained using a fixed magnetic field and sweeping the frequency of the
electromagnetic radiation, this more typically involved using a fixed frequency source and
varying the current (and hence magnetic field) in an electromagnet to observe the resonant
absorption signals. This is the origin of the counterintuitive, but still common, "high field"
and "low field" terminology for low frequency and high frequency regions respectively of the
NMR spectrum.
CW spectroscopy is inefficient in comparison with Fourier analysis techniques (see below)
since it probes the NMR response at individual frequencies in succession. Since the NMR
signal is intrinsically weak, the observed spectrum suffers from a poor signal-to-noise ratio.
This can be mitigated by signal averaging i.e. adding the spectra from repeated

measurements. While the NMR signal is constant between scans and so adds linearly, the
random noise adds more slowly - proportional to the square-root of the number of spectra
(see random walk). Hence the overall signal-to-noise ratio increases as the square-root of the
number of spectra measured.

[edit] Fourier transform spectroscopy

Most applications of NMR involve full NMR spectra, that is, the intensity of the NMR signal
as a function of frequency. Early attempts to acquire the NMR spectrum more efficiently than
simple CW methods involved illuminating the target simultaneously with more than one
frequency. A revolution in NMR occurred when short pulses of radio-frequency radiation
began to be used -- centered at the middle of the NMR spectrum. In simple terms, a short
square pulse of a given "carrier" frequency "contains" a range of frequencies centered about
the carrier frequency, with the range of excitation (bandwidth) being inversely proportional
to the pulse duration. The Fourier transform of an approximately square wave contains
contributions from all the frequencies in the neighborhood of the principal frequency. The
restricted range of the NMR frequencies made it relatively easy to use short (millisecond to
microsecond) radio frequency pulses to excite the entire NMR spectrum.[citation needed]
Applying such a pulse to a set of nuclear spins simultaneously excites all the single-quantum
NMR transitions. In terms of the net magnetization vector, this corresponds to tilting the
magnetization vector away from its equilibrium position (aligned along the external magnetic
field). The out-of-equilibrium magnetization vector precesses about the external magnetic
field vector at the NMR frequency of the spins. This oscillating magnetization vector
induces a current in a nearby pickup coil, creating an electrical signal oscillating at the NMR
frequency. This signal is known as the free induction decay (FID), and it contains the vector
sum of the NMR responses from all the excited spins. In order to obtain the frequencydomain NMR spectrum (NMR absorption intensity vs. NMR frequency) this time-domain
signal (intensity vs. time) must be Fourier transformed. Fortunately the development of
Fourier Transform NMR coincided with the development of digital computers and the
digital Fast Fourier Transform. Fourier methods can be applied to many types of
spectroscopy. (See the full article on Fourier transform spectroscopy.)
Richard R. Ernst was one of the pioneers of pulse NMR, and he won a Nobel Prize in
chemistry in 1991 for his work on Fourier Transform NMR and his development of multidimensional NMR (see below).

[edit] Multi-dimensional NMR Spectroscopy

The use of pulses of different shapes, frequencies and durations in specifically designed
patterns or pulse sequences allows the spectroscopist to extract many different types of
information about the molecule. Multi-dimensional nuclear magnetic resonance spectroscopy
is a kind of FT NMR in which there are at least two pulses and, as the experiment is repeated,
the pulse sequence is systematically varied. In multidimensional nuclear magnetic resonance
there will be a sequence of pulses and, at least, one variable time period. In three dimensions,
two time sequences will be varied. In four dimensions, three will be varied.
There are many such experiments. In one, these time intervals allow (amongst other things)
magnetization transfer between nuclei and, therefore, the detection of the kinds of nuclearnuclear interactions that allowed for the magnetization transfer. Interactions that can be

detected are usually classified into two kinds. There are through-bond interactions and
through-space interactions, the latter usually being a consequence of the nuclear
Overhauser effect. Experiments of the nuclear Overhauser variety may be employed to
establish distances between atoms, as for example by 2D-FT NMR of molecules in solution.
Although the fundamental concept of 2D-FT NMR was proposed by Jean Jeener from the
Free University of Brussels at an International Conference, this idea was largely developed
by Richard Ernst who won the 1991 Nobel prize in Chemistry for his work in FT NMR,
including multi-dimensional FT NMR, and especially 2D-FT NMR of small molecules. [8]
Multi-dimensional FT NMR experiments were then further developed into powerful
methodologies for studying biomolecules in solution, in particular for the determination of
the structure of biopolymers such as proteins or even small nucleic acids.[9]
In 2002 Kurt Wthrich shared the Nobel Prize in Chemistry (with John Bennett Fenn
and Koichi Tanaka) for his work with protein FT NMR in solution.

[edit] Solid-state NMR spectroscopy

Main article: Solid-state nuclear magnetic resonance
This technique complements X-ray crystallography in that it is frequently applicable to
molecules in a liquid or liquid crystal phase, whereas crystallography, as the name implies, is
performed on molecules in a solid phase. Though nuclear magnetic resonance is used to
study solids, extensive atomic-level molecular structural detail is especially challenging to
obtain in the solid state. There is little signal averaging by thermal motion in the solid state,
where most molecules can only undergo restricted vibrations and rotations at room
temperature, each in a slightly different electronic environment, therefore exhibiting a
different NMR absorption peak. Such a variation in the electronic environment of the
resonating nuclei results in a blurring of the observed spectrawhich is often only a broad
Gaussian band for non-quadrupolar spins in a solid- thus making the interpretation of such
"dipolar" and "chemical shift anisotropy" (CSA) broadened spectra either very difficult or
impossible.
Professor Raymond Andrew at Nottingham University in the UK pioneered the development
of high-resolution solid-state nuclear magnetic resonance. He was the first to report the
introduction of the MAS (magic angle sample spinning; MASS) technique that allowed him
to achieve spectral resolution in solids sufficient to distinguish between chemical groups with
either different chemical shifts or distinct Knight shifts. In MASS, the sample is spun at
several kilohertz around an axis that makes the so-called magic angle m (which is ~54.74,
where cos2m = 1/3) with respect to the direction of the static magnetic field B0; as a result of
such magic angle sample spinning, the chemical shift anisotropy bands are averaged to their
corresponding average (isotropic) chemical shift values. The above expression involving
cos2m has its origin in a calculation that predicts the magnetic dipolar interaction effects to
cancel out for the specific value of m called the magic angle. One notes that correct
alignment of the sample rotation axis as close as possible to m is essential for cancelling out
the dipolar interactions whose strength for angles sufficiently far from m is usually greater
than ~10 kHz for C-H bonds in solids, for example, and it is thus greater than their CSA
values.

There are different angles for the sample spinning relative to the applied field for the
averaging of quadrupole interactions and paramagnetic interactions, correspondingly ~30.6
and ~70.1
A concept developed by Sven Hartmann and Erwin Hahn was utilized in transferring
magnetization from protons to less sensitive nuclei (popularly known as cross-polarization)
by M.G. Gibby, Alex Pines and John S. Waugh. Then, Jake Schaefer and Ed Stejskal
demonstrated also the powerful use of cross-polarization under MASS conditions which is
now routinely employed to detect low-abundance and low-sensitivity nuclei.

[edit] Sensitivity
Because the intensity of nuclear magnetic resonance signals and, hence, the sensitivity of the
technique depends on the strength of the magnetic field the technique has also advanced over
the decades with the development of more powerful magnets. Advances made in audio-visual
technology have also improved the signal-generation and processing capabilities of newer
instruments.
As noted above, the sensitivity of nuclear magnetic resonance signals is also dependent on
the presence of a magnetically susceptible nuclide and, therefore, either on the natural
abundance of such nuclides or on the ability of the experimentalist to artificially enrich the
molecules, under study, with such nuclides. The most abundant naturally occurring isotopes
of hydrogen and phosphorus (for example) are both magnetically susceptible and readily
useful for nuclear magnetic resonance spectroscopy. In contrast, carbon and nitrogen have
useful isotopes but which occur only in very low natural abundance.
Other limitations on sensitivity arise from the quantum-mechanical nature of the
phenomenon. For quantum states separated by energy equivalent to radio frequencies,
thermal energy from the environment causes the populations of the states to be close to equal.
Since incoming radiation is equally likely to cause stimulated emission (a transition from the
upper to the lower state) as absorption, the NMR effect depends on an excess of nuclei in the
lower states. Several factors can reduce sensitivity, including

Increasing temperature, which evens out the population of states. Conversely, low
temperature NMR can sometimes yield better results than room-temperature NMR,
providing the sample remains liquid.
Saturation of the sample with energy applied at the resonant radiofrequency. This
manifests in both CW and pulsed NMR; in the first case (CW) this happens by using
too much continuous power that keeps the upper spin levels completely populated; in
the second case (pulsed), each pulse (that is at least a 90 pulse) leaves the sample
saturated, and four to five times the (longitudinal) relaxation time (5 T1) must pass
before the next pulse or pulse sequence can be applied. For single pulse experiments,
shorter RF pulses that tip the magnetization by less than 90 can be used, which loses
some intensity of the signal, but allows for shorter recycle delays. The optimum there
is called an Ernst angle, after the Nobel laureate. Especially in solid state NMR, or
in samples with very few nuclei with spins > 0, (diamond with the natural 1% of
Carbon-13 is especially troublesome here) the longitudinal relaxation times can be on
the range of hours, while for proton-NMR they are more on the range of one second.
Non-magnetic effects, such as electric-quadrupole coupling of spin-1 and spin-32
nuclei with their local environment, which broaden and weaken absorption peaks.

14N,

an abundant spin-1 nucleus, is difficult to study for this reason. High resolution
NMR instead probes molecules using the rarer 15N isotope, which has spin-12.

[edit] Isotopes
Many chemical elements can be used for NMR analysis.[10]
Commonly used nuclei:

1H,

the most commonly used spin nucleus in NMR investigation, has been studied
using many forms of NMR. Hydrogen is highly abundant, especially in biological
systems. It is the nucleus most sensitive to NMR signal (apart from 3H which is not
commonly used due to its instability and radioactivity). Proton NMR produces narrow
chemical shift with sharp signals. Fast acquisition of quantitative results (peak
integrals in stoichiometric ratio) is possible due to short relaxation time. The 1H
signal has been the sole diagnostic nucleus used for clinical magnetic resonance
imaging.
2H, a spin 1 nucleus commonly utilized as signal-free medium in the form of
deuterated solvents during proton NMR, to avoid signal interference from
hydrogen-containing solvents in measurement of 1H solutes. Also used in determining
the behavior of lipids in lipid membranes and other solids or liquid crystals as it is a
relatively non-perturbing label which can selectively replace 1H. Alternatively, 2H
can be detected in media specially labeled with 2H. Deuterium resonance is
commonly used in high-resolution NMR spectroscopy to monitor drifts in the
magnetic field strength (lock) and to improve the homogeneity of the external
magnetic field.
3He, is very sensitive to NMR. There is a very low percentage in natural helium, and
subsequently has to be purified from 4He. It is used mainly in studies of endohedral
fullerenes, where its chemical inertness is beneficial to ascertaining the structure of
the entrapping fullerene.
11B, more sensitive than 10B, yields sharper signals. Quartz tubes must be used as
borosilicate glass interferes with measurement.
13C spin-1/2, is widely used, despite its relative paucity in naturally occurring carbon
(approximately 1%). It is stable to nuclear decay. Since there is a low percentage in
natural carbon, spectrum acquisition on samples which have not been experimentally
enriched in 13C takes a long time. Frequently used for labeling of compounds in
synthetic and metabolic studies. Has low sensitivity and wide chemical shift, yields
sharp signals. Low percentage makes it useful by preventing spin-spin couplings and
makes the spectrum appear less crowded. Slow relaxation means that spectra are not
integrable unless long acquisition times are used.
14N, spin-1, medium sensitivity nucleus with wide chemical shift. Its large
quadrupole moment interferes in acquisition of high resolution spectra, limiting
usefulness to smaller molecules and functional groups with a high degree of
symmetry such as the headgroups of lipids.
15N, spin-1/2, relatively commonly used. Can be used for labeling compounds.
Nucleus very insensitive but yields sharp signals. Low percentage in natural nitrogen
together with low sensitivity requires high concentrations or expensive isotope
enrichment.
17O, spin-5/2, low sensitivity and very low natural abundance (0.037%), wide
chemical shifts range (up to 2000 ppm). Quadrupole moment causing a line

broadening. Used in metabolic and biochemical studies in studies of chemical

equilibria.
19F, spin-1/2, relatively commonly measured. Sensitive, yields sharp signals, has
wide chemical shift.
31P, spin-1/2, 100% of natural phosphorus. Medium sensitivity, wide chemical shifts
range, yields sharp lines. Spectra tend to have a moderate amount of noise. Used in
biochemical studies and in coordination chemistry where phosphorus containing
ligands are involved.
35Cl and 37Cl, broad signal. 35Cl significantly more sensitive, preferred over 37Cl
despite its slightly broader signal. Organic chlorides yield very broad signals, its use
is limited to inorganic and ionic chlorides and very small organic molecules.
43Ca, used in biochemistry to study calcium binding to DNA, proteins, etc.
Moderately sensitive, very low natural abundance.
195Pt, used in studies of catalysts and complexes.

Other nuclei (usually used in the studies of their complexes and chemical binding, or to
detect presence of the element):

6Li, 7Li
9Be
19F
21Ne
23Na
25Mg
27Al
29Si
31P
33S
39K, 40K, 41K
45Sc
47Ti, 49Ti
50V, 51V
53Cr
55Mn
57Fe
59Co
61Ni
63Cu, 65Cu
67Zn
69Ga, 71Ga
73Ge
75As
77Se
81Br
87Rb
87Sr
95Mo
109Ag
113Cd

119Sn
125Te
127I
133Cs
135Ba, 137Ba
139La
183W
199Hg

[edit] Applications
[edit] Medicine

Medical MRI
See also: Magnetic resonance imaging
The application of nuclear magnetic resonance best known to the general public is magnetic
resonance imaging for medical diagnosis and magnetic resonance microscopy in
research settings, however, it is also widely used in chemical studies, notably in NMR
spectroscopy such as proton NMR, carbon-13 NMR, deuterium NMR and phosphorus-31
NMR. Biochemical information can also be obtained from living tissue (e.g. human brain
tumors) with the technique known as in vivo magnetic resonance spectroscopy or
chemical shift NMR Microscopy.
These studies are possible because nuclei are surrounded by orbiting electrons, which are
charged particles that generate small, local magnetic fields that add to or subtract from the
external magnetic field, and so will partially shield the nuclei. The amount of shielding
depends on the exact local environment. For example, a hydrogen bonded to an oxygen will
be shielded differently than a hydrogen bonded to a carbon atom. In addition, two hydrogen
nuclei can interact via a process known as spin-spin coupling, if they are on the same
molecule, which will split the lines of the spectra in a recognizable way.
As one of the two major spectroscopic techniques used in metabolomics, NMR is used to
generate metabolic fingerprints from biological fluids to obtain information about disease
states or toxic insults.

[edit] Chemistry
By studying the peaks of nuclear magnetic resonance spectra, chemists can determine the
structure of many compounds. It can be a very selective technique, distinguishing among
many atoms within a molecule or collection of molecules of the same type but which differ
only in terms of their local chemical environment. NMR spectroscopy is used to
unambiguously identify known and novel compounds, and as such, is usually required by
scientific journals for identity confirmation of synthesized new compounds. See the articles
on carbon-13 NMR and proton NMR for detailed discussions.
By studying T2 information, a chemist can determine the identity of a compound by
comparing the observed nuclear precession frequencies to known frequencies. Further
structural data can be elucidated by observing spin-spin coupling, a process by which the
precession frequency of a nucleus can be influenced by the magnetization transfer from
nearby chemically bound nuclei. Spin-spin coupling is observed in NMR of hydrogen-1 (1H
NMR), since its natural abundance is nearly 100%; isotope enrichment is required for most
other elements.
Because the nuclear magnetic resonance timescale is rather slow, compared to other
spectroscopic methods, changing the temperature of a T2*experiment can also give
information about fast reactions, such as the Cope rearrangement or about structural
dynamics, such as ring-flipping in cyclohexane. At low enough temperatures, a distinction
can be made between the axial and equatorial hydrogens in cyclohexane.
An example of nuclear magnetic resonance being used in the determination of a structure is
that of buckminsterfullerene (often called "buckyballs", composition C60). This now famous
form of carbon has 60 carbon atoms forming a sphere. The carbon atoms are all in identical
environments and so should see the same internal H field. Unfortunately,
buckminsterfullerene contains no hydrogen and so 13C nuclear magnetic resonance has to be
used. 13C spectra require longer acquisition times since carbon-13 is not the common isotope
of carbon (unlike hydrogen, where 1H is the common isotope). However, in 1990 the
spectrum was obtained by R. Taylor and co-workers at the University of Sussex and was
found to contain a single peak, confirming the unusual structure of buckminsterfullerene.[11]

[edit] Non-destructive testing

Nuclear magnetic resonance is extremely useful for analyzing samples non-destructively.
Radio waves and static magnetic fields easily penetrate many types of matter and anything
that is not inherently ferromagnetic. For example, various expensive biological samples,
such as nucleic acids, including RNA and DNA, or proteins, can be studied using nuclear
magnetic resonance for weeks or months before using destructive biochemical experiments.
This also makes nuclear magnetic resonance a good choice for analyzing dangerous samples.

[edit] Acquisition of dynamic information

In addition to providing static information on molecules by determining their 3D structures in
solution, one of the remarkable advantages of NMR over X-ray crystallography is that it can
be used to obtain important dynamic information including the low-frequency collective
motion in proteins and DNA, for example in the Ca2+-calmodulin system.[12] The low-

frequency internal motion in biomacromolecules and its biological functions have been
discussed by Chou.[13]

[edit] Data acquisition in the petroleum industry

Main article: NMR in porous media
Another use for nuclear magnetic resonance is data acquisition in the petroleum industry
for petroleum and natural gas exploration and recovery. A borehole is drilled into rock and
sedimentary strata into which nuclear magnetic resonance logging equipment is lowered.
Nuclear magnetic resonance analysis of these boreholes is used to measure rock porosity,
estimate permeability from pore size distribution and identify pore fluids (water, oil and gas).
These instruments are typically low field NMR spectrometers.

[edit] Flow probes for NMR spectroscopy

Recently, real-time applications of NMR in liquid media have been developed using
specifically designed flow probes (flow cell assemblies) which can replace standard tube
probes. This has enabled techniques that can incorporate the use of high performance liquid
chromatography (HPLC) or other continuous flow sample introduction devices.[14]

[edit] Process control

NMR has now entered the arena of real-time process control and process optimization in
oil refineries and petrochemical plants. Two different types of NMR analysis are utilized to
provide real time analysis of feeds and products in order to control and optimize unit
operations. Time-domain NMR (TD-NMR) spectrometers operating at low field (220 MHz
for 1H) yield free induction decay data that can be used to determine absolute hydrogen
content values, rheological information, and component composition. These spectrometers
are used in mining, polymer production, cosmetics and food manufacturing as well as coal
analysis. High resolution FT-NMR spectrometers operating in the 60 MHz range with
shielded permanent magnet systems yield high resolution 1H NMR spectra of refinery and
petrochemical streams. The variation observed in these spectra with changing physical and
chemical properties is modeled using chemometrics to yield predictions on unknown
samples. The prediction results are provided to control systems via analogue or digital
outputs from the spectrometer.

[edit] Earth's field NMR

Main article: Earth's field NMR
In the Earth's magnetic field, NMR frequencies are in the audio frequency range, or the
very low frequency and ultra low frequency bands of the radio frequency spectrum.
Earth's field NMR (EFNMR) is typically stimulated by applying a relatively strong dc
magnetic field pulse to the sample and, after the end of the pulse, analyzing the resulting low
frequency alternating magnetic field that occurs in the Earth's magnetic field due to free
induction decay (FID). These effects are exploited in some types of magnetometers,
EFNMR spectrometers, and MRI imagers. Their inexpensive portable nature makes these
instruments valuable for field use and for teaching the principles of NMR and MRI.

An important feature of EFNMR spectrometry compared with high-field NMR is that some
aspects of molecular structure can be observed more clearly at low fields and low
frequencies, whereas other aspects observable at high fields are not observable at low fields.
This is because:

Electron-mediated heteronuclear J-couplings (spin-spin couplings) are field

independent, producing clusters of two or more frequencies separated by several Hz,
which are more easily observed in a fundamental resonance of about 2 kHz. "Indeed it
appears that enhanced resolution is possible due to the long spin relaxation times and
high field homogeneity which prevail in EFNMR."[15]
Chemical shifts of several ppm are clearly separated in high field NMR spectra, but
have separations of only a few millihertz at proton EFNMR frequencies, so are
usually lost in noise etc.

[edit] Quantum computing

Main article: Nuclear magnetic resonance quantum computer
NMR quantum computing uses the spin states of molecules as qubits. NMR differs from
other implementations of quantum computers in that it uses an ensemble of systems, in this
case molecules.

[edit] Magnetometers
Main article: Magnetometer
Various magnetometers use NMR effects to measure magnetic fields, including proton
precession magnetometers (PPM) (also known as proton magnetometers), and
Overhauser magnetometers. See also Earth's field NMR.

[edit] Makers of NMR equipment

Major NMR instrument makers include Oxford Instruments, Bruker, Spinlock SRL,
General Electric, JEOL, Kimble Chase, Philips, Siemens AG, Varian, Inc. and Agilent
Technologies, Inc..

[edit] See also

Carbon-13 NMR
Chemical shift
Dynamic nuclear polarisation (DNP)
Earth's field NMR (EFNMR)
Free induction decay (FID)
In vivo magnetic resonance spectroscopy (MRS)
J-coupling
Larmor equation (Not to be confused with Larmor formula).
Larmor precession
Low field NMR
Magic angle spinning
Magnetometer
Magnetic resonance imaging (MRI)

NMR crystallography
NMR spectra database
NMR spectroscopy
NMR Microscopy
Nuclear magnetic resonance in porous media
Nuclear quadrupole resonance (NQR)
Protein dynamics
Protein NMR
Proton NMR
Rabi cycle
Relaxometry
Relaxation (NMR)
Sow-Hsin Chen, MIT
Spin echo
Solid-state NMR
Zero field NMR

[edit] Notes
1.
2.
3.
4.
5.
6.
7.

8.
9.
10.
11.
12.
13.
14.
15.

^ I.I. Rabi, J.R. Zacharias, S. Millman, P. Kusch (1938). "A New Method of Measuring
Nuclear Magnetic Moment". Physical Review 53 (4): 318327. Bibcode 1938PhRv...53..318R.
doi:10.1103/PhysRev.53.318. PMID 9981980.
^ Biography of I. Rabi at Nobelprize.org
^ Filler, Aaron (2009). "The History, Development and Impact of Computed Imaging in
Neurological Diagnosis and Neurosurgery: CT, MRI, and DTI". Nature Precedings.
doi:10.1038/npre.2009.3267.5.
^ 1952 Nobel Prize for Physics at Nobelprize.org
^ Principle of Shielding and Deshielding | NMRCentral.com
^ Quantum automaton and quantum computation (see also references therein)
^ Lieven M. K. Vandersypen; Steffen, Matthias; Breyta, Gregory; Yannoni, Costantino S.;
Sherwood, Mark H.; Chuang, Isaac L. (2001). "Experimental realization of Shor's quantum factoring
algorithm using nuclear magnetic resonance". Nature 414 (6866): 883887. arXiv:quant-ph/0112176.
Bibcode 2001Natur.414..883V. doi:10.1038/414883a. PMID 11780055.
^ "Nuclear Magnetic Resonance Fourier Transform Spectroscopy" Ernst's Nobel
lecture. (Includes mention of Jeener's suggestion.)
^ I.C. Baianu. "Two-dimensional Fourier transforms". 2D-FT NMR and MRI. PlanetMath.
http://planetmath.org/encyclopedia/TwoDimensionalFourierTransforms.html. Retrieved 200902-22.
^ Multinuclear NMR
^ R. Taylor, J.P. Hare, A.K. Abdul-Sada, H.W. Kroto (1990). "Isolation, separation and
characterization of the fullerenes C60 and C70: the third form of carbon". Journal of the Chemical
Society, Chemical Communications 20 (20): 14231425. doi:10.1039/c39900001423.
^ Chou, J. J.; Li, S.; Klee, C. B.; Bax, A. (2001). "Solution structure of Ca2+-calmodulin
reveals flexible hand-like properties of its domains". Nature Structural Biology 8 (11): 990997.
doi:10.1038/nsb1101-990. PMID 11685248.
^ Kuo-Chen Chou (1988). "Low-frequency collective motion in biomacromolecules and its
biological functions". Biophys Chem 30 (1): 348. doi:10.1016/0301-4622(88)85002-6.
PMID 3046672.
^ R.L Haner and P.A, Keifer (2009). "Flow Probes for NMR Spectroscopy". Encyclopedia of
Magnetic Resonance. doi:10.1002/9780470034590.emrstm1085. ISBN 0470034599.
^ Robinson J. N. et al. (2006). "Two-dimensional NMR spectroscopy in Earth's magnetic
field". Journal of Magnetic Resonance 182 (2): 343347. Bibcode 2006JMagR.182..343R.

doi:10.1016/j.jmr.2006.06.027. PMID 16860581.

http://www.sfu.ca/~simonw/phys431/references/nmr/robinson_nmr_imaging_JMR2006.pdf.

[edit] References

Gary E. Martin, A. S. Zektzer (1988). Two-Dimensional NMR Methods for

Establishing Molecular Connectivity. New York: Wiley-VCH. p. 59. ISBN 0-47118707-0. http://books.google.com/books?
id=9ysYrpe_NoEC&printsec=frontcover.
J.W. Akitt, B.E. Mann (2000). NMR and Chemistry. Cheltenham, UK: Stanley
Thornes. pp. 273, 287. ISBN 0-7487-4344-8.
J.P. Hornak. "The Basics of NMR". http://www.cis.rit.edu/htbooks/nmr/. Retrieved
2009-02-23.
J. Keeler (2005). Understanding NMR Spectroscopy. John Wiley & Sons. ISBN 0470-01786-4.
Kurt Wthrich (1986). NMR of Proteins and Nucleic Acids. New York (NY), USA:
Wiley-Interscience. ISBN 0-471-11917-2.
J.M Tyszka, S.E Fraser, R.E Jacobs (2005). "Magnetic resonance microscopy: recent
advances and applications". Current Opinion in Biotechnology 16 (1): 9399.
doi:10.1016/j.copbio.2004.11.004. PMID 15722021.
J.C. Edwards. "Principles of NMR". Process NMR Associates. http://www.processnmr.com/pdfs/NMR%20Overview.pdf. Retrieved 2009-02-23.
R.L Haner, P.A. Keifer (2009). Encyclopedia of Magnetic Resonance. John Wiley.
doi:10.1002/9780470034590.emrstm1085.

[edit] External links

7.5. massa, Mass spectrometry

From Wikipedia, the free encyclopedia

Jump to: navigation, search

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio
of charged particles.[1] It is used for determining masses of particles, for determining the
elemental composition of a sample or molecule, and for elucidating the chemical structures
of molecules, such as peptides and other chemical compounds. The MS principle consists
of ionizing chemical compounds to generate charged molecules or molecule fragments and
measuring their mass-to-charge ratios.[1] In a typical MS procedure:
1. A sample is loaded onto the MS instrument and undergoes vaporization
2. The components of the sample are ionized by one of a variety of methods (e.g., by
impacting them with an electron beam), which results in the formation of charged
particles (ions)
3. The ions are separated according to their mass-to-charge ratio in an analyzer by
electromagnetic fields
4. The ions are detected, usually by a quantitative method
5. The ion signal is processed into mass spectra
MS instruments consist of three modules:

An ion source, which can convert gas phase sample molecules into ions (or, in the
case of electrospray ionization, move ions that exist in solution into the gas phase)
A mass analyzer, which sorts the ions by their masses by applying electromagnetic
fields
A detector, which measures the value of an indicator quantity and thus provides data
for calculating the abundances of each ion present

The technique has both qualitative and quantitative uses. These include identifying
unknown compounds, determining the isotopic composition of elements in a molecule, and
determining the structure of a compound by observing its fragmentation. Other uses include
quantifying the amount of a compound in a sample or studying the fundamentals of gas
phase ion chemistry (the chemistry of ions and neutrals in a vacuum). MS is now in very
common use in analytical laboratories that study physical, chemical, or biological properties
of a great variety of compounds.

Contents
[hide]

1 Etymology
2 History
3 Simplified example
4 Creating ions
o
4.1 Inductively coupled plasma
o
4.2 Other ionization techniques
5 Mass selection
o
5.1 Sector instruments
o
5.2 Time-of-flight
o
5.3 Quadrupole mass filter
o
5.4 Ion traps

5.4.1 Three-dimensional quadrupole ion trap

5.4.2 Linear quadrupole ion trap

5.4.3 Orbitrap
o
5.5 Fourier transform ion cyclotron resonance
6 Detectors
7 Tandem mass spectrometry
8 Common mass spectrometer configurations and techniques
9 Chromatographic techniques combined with mass
spectrometry
o
9.1 Gas chromatography
o
9.2 Liquid chromatography
o
9.3 Ion mobility
10 Data and analysis
o
10.1 Data representations
o
10.2 Data analysis
11 Applications
o
11.1 Isotope ratio MS: isotope dating and tracking
o
11.2 Trace gas analysis
o
11.3 Atom probe
o
11.4 Pharmacokinetics
o
11.5 Protein characterization
o
11.6 Glycan analysis
o
11.7 Space exploration
o
11.8 Respired gas monitor
12 See also
13 References
14 Bibliography
15 External links

[edit] Etymology
The word spectrograph had become part of the international scientific vocabulary by 1884.
[2][3]
The linguistic roots are a combination and removal of bound morphemes and free

morphemes which relate to the terms spectr-um and phot-ograph-ic plate.[4] Early
spectrometry devices that measured the mass-to-charge ratio of ions were called mass
spectrographs which consisted of instruments that recorded a spectrum of mass values on
a photographic plate.[5][6] A mass spectroscope is similar to a mass spectrograph except that
the beam of ions is directed onto a phosphor screen.[7] A mass spectroscope configuration
was used in early instruments when it was desired that the effects of adjustments be quickly
observed. Once the instrument was properly adjusted, a photographic plate was inserted and
exposed. The term mass spectroscope continued to be used even though the direct
illumination of a phosphor screen was replaced by indirect measurements with an
oscilloscope.[8] The use of the term mass spectroscopy is now discouraged due to the
possibility of confusion with light spectroscopy.[1][9] Mass spectrometry is often abbreviated
as mass-spec or simply as MS.[1]

[edit] History
For more details on this topic, see History of mass spectrometry.

Replica of an early mass spectrometer

In 1886, Eugen Goldstein observed rays in gas discharges under low pressure that
traveled away from the anode and through channels in a perforated cathode, opposite to the
direction of negatively charged cathode rays (which travel from cathode to anode).
Goldstein called these positively charged anode rays "Kanalstrahlen"; the standard
translation of this term into English is "canal rays". Wilhelm Wien found that strong
electric or magnetic fields deflected the canal rays and, in 1899, constructed a device with
parallel electric and magnetic fields that separated the positive rays according to their chargeto-mass ratio (Q/m). Wien found that the charge-to-mass ratio depended on the nature of the
gas in the discharge tube. English scientist J.J. Thomson later improved on the work of
Wien by reducing the pressure to create a mass spectrograph.
The first application of mass spectrometry to the analysis of amino acids and peptides was
reported in 1958.[10] Carl-Ove Andersson highlighted the main fragment ions observed in the
ionization of methyl esters.[11]
Some of the modern techniques of mass spectrometry were devised by Arthur Jeffrey
Dempster and F.W. Aston in 1918 and 1919 respectively. In 1989, half of the Nobel Prize
in Physics was awarded to Hans Dehmelt and Wolfgang Paul for the development of the
ion trap technique in the 1950s and 1960s. In 2002, the Nobel Prize in Chemistry was
awarded to John Bennett Fenn for the development of electrospray ionization (ESI) and
Koichi Tanaka for the development of soft laser desorption (SLD) and their application to
the ionization of biological macromolecules, especially proteins.[12]

[edit] Simplified example

Schematics of a simple mass spectrometer with sector type mass analyzer. This one is for the
measurement of carbon dioxide isotope ratios (IRMS) as in the carbon-13 urea breath test

The following example describes the operation of a spectrometer mass analyzer, which is of
the sector type. (Other analyzer types are treated below.) Consider a sample of sodium
chloride (table salt). In the ion source, the sample is vaporized (turned into gas) and ionized
(transformed into electrically charged particles) into sodium (Na+) and chloride (Cl-) ions.
Sodium atoms and ions are monoisotopic, with a mass of about 23 amu. Chloride atoms and
ions come in two isotopes with masses of approximately 35 amu (at a natural abundance of
about 75 percent) and approximately 37 amu (at a natural abundance of about 25 percent).
The analyzer part of the spectrometer contains electric and magnetic fields, which exert
forces on ions traveling through these fields. The speed of a charged particle may be
increased or decreased while passing through the electric field, and its direction may be
altered by the magnetic field. The magnitude of the deflection of the moving ion's trajectory
depends on its mass-to-charge ratio. Lighter ions get deflected by the magnetic force more
than heavier ions (based on Newton's second law of motion, F = ma). The streams of
sorted ions pass from the analyzer to the detector, which records the relative abundance of
each ion type. This information is used to determine the chemical element composition of the
original sample (i.e. that both sodium and chlorine are present in the sample) and the isotopic
composition of its constituents (the ratio of 35Cl to 37Cl).

[edit] Creating ions

Main article: Ion source
The ion source is the part of the mass spectrometer that ionizes the material under analysis
(the analyte). The ions are then transported by magnetic or electric fields to the mass
analyzer.
Techniques for ionization have been key to determining what types of samples can be
analyzed by mass spectrometry. Electron ionization and chemical ionization are used for
gases and vapors. In chemical ionization sources, the analyte is ionized by chemical
ion-molecule reactions during collisions in the source. Two techniques often used with liquid
and solid biological samples include electrospray ionization (invented by John Fenn[13])
and matrix-assisted laser desorption/ionization (MALDI, initially developed as a similar
technique "Soft Laser Desorption (SLD)" by K. Tanaka[14] for which a Nobel Prize was
awarded and as MALDI by M. Karas and F. Hillenkamp[15]).

[edit] Inductively coupled plasma

Inductively coupled plasma (ICP) sources are used primarily for cation analysis of a wide
array of sample types. In this type of Ion Source Technology, a 'flame' of plasma that is
electrically neutral overall, but that has had a substantial fraction of its atoms ionized by high
temperature, is used to atomize introduced sample molecules and to further strip the outer
electrons from those atoms. The plasma is usually generated from argon gas, since the first
ionization energy of argon atoms is higher than the first of any other elements except He, O,
F and Ne, but lower than the second ionization energy of all except the most electropositive
metals. The heating is achieved by a radio-frequency current passed through a coil
surrounding the plasma.

[edit] Other ionization techniques

Others include glow discharge, field desorption (FD), fast atom bombardment (FAB),
thermospray, desorption/ionization on silicon (DIOS), Direct Analysis in Real Time
(DART), atmospheric pressure chemical ionization (APCI), secondary ion mass
spectrometry (SIMS), spark ionization and thermal ionization (TIMS).[16] Ion
attachment ionization is an ionization technique that allows for fragmentation free analysis.

[edit] Mass selection

Mass analyzers separate the ions according to their mass-to-charge ratio. The following
two laws govern the dynamics of charged particles in electric and magnetic fields in vacuum:
(Lorentz force law);
(Newton's second law of motion in non-relativistic case, i.e. valid only at ion
velocity much lower than the speed of light).
Here F is the force applied to the ion, m is the mass of the ion, a is the acceleration,
Q is the ion charge, E is the electric field, and v B is the vector cross product of
the ion velocity and the magnetic field
Equating the above expressions for the force applied to the ion yields:
This differential equation is the classic equation of motion for charged
particles. Together with the particle's initial conditions, it completely determines
the particle's motion in space and time in terms of m/Q. Thus mass spectrometers
could be thought of as "mass-to-charge spectrometers". When presenting data, it
is common to use the (officially) dimensionless m/z, where z is the number of
elementary charges (e) on the ion (z=Q/e). This quantity, although it is
informally called the mass-to-charge ratio, more accurately speaking represents
the ratio of the mass number and the charge number, z.
There are many types of mass analyzers, using either static or dynamic fields, and
magnetic or electric fields, but all operate according to the above differential
equation. Each analyzer type has its strengths and weaknesses. Many mass
spectrometers use two or more mass analyzers for tandem mass spectrometry
(MS/MS). In addition to the more common mass analyzers listed below, there are
others designed for special situations.
There are several important analyser characteristics. The mass resolving power
is the measure of the ability to distinguish two peaks of slightly different m/z. The
mass accuracy is the ratio of the m/z measurement error to the true m/z. Mass
accuracy is usually measured in ppm or milli mass units. The mass range is the
range of m/z amenable to analysis by a given analyzer. The linear dynamic range
is the range over which ion signal is linear with analyte concentration. Speed
refers to the time frame of the experiment and ultimately is used to determine the
number of spectra per unit time that can be generated.

[edit] Sector instruments

For more details on this topic, see sector instrument.
A sector field mass analyzer uses an electric and/or magnetic field to affect the
path and/or velocity of the charged particles in some way. As shown above,
sector instruments bend the trajectories of the ions as they pass through the
mass analyzer, according to their mass-to-charge ratios, deflecting the more
charged and faster-moving, lighter ions more. The analyzer can be used to select
a narrow range of m/z or to scan through a range of m/z to catalog the ions
present.[17]

[edit] Time-of-flight
For more details on this topic, see time-of-flight mass spectrometry.
The time-of-flight (TOF) analyzer uses an electric field to accelerate the ions
through the same potential, and then measures the time they take to reach the
detector. If the particles all have the same charge, the kinetic energies will be
identical, and their velocities will depend only on their masses. Lighter ions
will reach the detector first.[18]

[edit] Quadrupole mass filter

For more details on this topic, see Quadrupole mass analyzer.
Quadrupole mass analyzers use oscillating electrical fields to selectively
stabilize or destabilize the paths of ions passing through a radio frequency (RF)
quadrupole field created between 4 parallel rods. Only the ions in a certain
range of mass/charge ratio are passed through the system at any time, but changes
to the potentials on the rods allow a wide range of m/z values to be swept rapidly,
either continuously or in a succession of discrete hops. A quadrupole mass
analyzer acts as a mass-selective filter and is closely related to the quadrupole
ion trap, particularly the linear quadrupole ion trap except that it is designed to
pass the untrapped ions rather than collect the trapped ones, and is for that reason
referred to as a transmission quadrupole. A common variation of the transmission
quadrupole is the triple quadrupole mass spectrometer. The triple quad has
three consecutive quadrupole stages, the first acting as a mass filter to transmit a
particular incoming ion to the second quadrupole, a collision chamber, wherein
that ion can be broken into fragments. The third quadrupole also acts as a mass
filter, to transmit a particular fragment ion to the detector. If a quadrupole is made
to rapidly and repetitively cycle through a range of mass filter settings, full
spectra can be reported. Likewise, a triple quad can be made to perform various
scan types characteristic of tandem mass spectrometry.

[edit] Ion traps

[edit] Three-dimensional quadrupole ion trap

For more details on this topic, see quadrupole ion trap.
The quadrupole ion trap works on the same physical principles as the
quadrupole mass analyzer, but the ions are trapped and sequentially ejected. Ions

are trapped in a mainly quadrupole RF field, in a space defined by a ring

electrode (usually connected to the main RF potential) between two endcap
electrodes (typically connected to DC or auxiliary AC potentials). The sample is
ionized either internally (e.g. with an electron or laser beam), or externally, in
which case the ions are often introduced through an aperture in an endcap
electrode.
There are many mass/charge separation and isolation methods but the most
commonly used is the mass instability mode in which the RF potential is ramped
so that the orbit of ions with a mass a > b are stable while ions with mass b
become unstable and are ejected on the z-axis onto a detector. There are also nondestructive analysis methods.
Ions may also be ejected by the resonance excitation method, whereby a
supplemental oscillatory excitation voltage is applied to the endcap electrodes,
and the trapping voltage amplitude and/or excitation voltage frequency is varied
to bring ions into a resonance condition in order of their mass/charge ratio.[19][20]
The cylindrical ion trap mass spectrometer is a derivative of the quadrupole
ion trap mass spectrometer.

[edit] Linear quadrupole ion trap

A linear quadrupole ion trap is similar to a quadrupole ion trap, but it traps ions
in a two dimensional quadrupole field, instead of a three-dimensional quadrupole
field as in a 3D quadrupole ion trap. Thermo Fisher's LTQ ("linear trap
quadrupole") is an example of the linear ion trap.[21]
A toroidal ion trap can be visualized as a linear quadrupole curved around and
connected at the ends or as a cross section of a 3D ion trap rotated on edge to
form the toroid, donut shaped trap. The trap can store large volumes of ions by
distributing them throughout the ring-like trap structure. This toroidal shaped trap
is a configuration that allows the increased miniaturization of an ion trap mass
analyzer. Additionally all ions are stored in the same trapping field and ejected
together simplifying detection that can be complicated with array configurations
due to variations in detector alignment and machining of the arrays.[22]

[edit] Orbitrap
For more details on this topic, see Orbitrap.
These are similar to Fourier transform ion cyclotron resonance mass
spectrometers (see text below). Ions are electrostatically trapped in an orbit
around a central, spindle shaped electrode. The electrode confines the ions so that
they both orbit around the central electrode and oscillate back and forth along the
central electrode's long axis. This oscillation generates an image current in the
detector plates which is recorded by the instrument. The frequencies of these
image currents depend on the mass to charge ratios of the ions. Mass spectra are
obtained by Fourier transformation of the recorded image currents.

Orbitraps have a high mass accuracy, high sensitivity and a good dynamic range.
[23]

[edit] Fourier transform ion cyclotron resonance

For more details on this topic, see Fourier transform mass spectrometry.
Fourier transform mass spectrometry (FTMS), or more precisely Fourier
transform ion cyclotron resonance MS, measures mass by detecting the
image current produced by ions cyclotroning in the presence of a magnetic
field. Instead of measuring the deflection of ions with a detector such as an
electron multiplier, the ions are injected into a Penning trap (a static
electric/magnetic ion trap) where they effectively form part of a circuit.
Detectors at fixed positions in space measure the electrical signal of ions which
pass near them over time, producing a periodic signal. Since the frequency of an
ion's cycling is determined by its mass to charge ratio, this can be deconvoluted
by performing a Fourier transform on the signal. FTMS has the advantage of
high sensitivity (since each ion is "counted" more than once) and much higher
resolution and thus precision.[24][25]
Ion cyclotron resonance (ICR) is an older mass analysis technique similar to
FTMS except that ions are detected with a traditional detector. Ions trapped in a
Penning trap are excited by an RF electric field until they impact the wall of the
trap, where the detector is located. Ions of different mass are resolved according
to impact time.

[edit] Detectors

A continuous dynode particle multiplier detector.

The final element of the mass spectrometer is the detector. The detector records
either the charge induced or the current produced when an ion passes by or hits a
surface. In a scanning instrument, the signal produced in the detector during the
course of the scan versus where the instrument is in the scan (at what m/Q) will
produce a mass spectrum, a record of ions as a function of m/Q.
Typically, some type of electron multiplier is used, though other detectors
including Faraday cups and ion-to-photon detectors are also used. Because
the number of ions leaving the mass analyzer at a particular instant is typically

quite small, considerable amplification is often necessary to get a signal.

Microchannel plate detectors are commonly used in modern commercial
instruments.[26] In FTMS and Orbitraps, the detector consists of a pair of metal
surfaces within the mass analyzer/ion trap region which the ions only pass near as
they oscillate. No DC current is produced, only a weak AC image current is
produced in a circuit between the electrodes. Other inductive detectors have also
been used.[27]

[edit] Tandem mass spectrometry

Main article: Tandem mass spectrometry
A tandem mass spectrometer is one capable of multiple rounds of mass
spectrometry, usually separated by some form of molecule fragmentation. For
example, one mass analyzer can isolate one peptide from many entering a mass
spectrometer. A second mass analyzer then stabilizes the peptide ions while they
collide with a gas, causing them to fragment by collision-induced dissociation
(CID). A third mass analyzer then sorts the fragments produced from the
peptides. Tandem MS can also be done in a single mass analyzer over time, as in
a quadrupole ion trap. There are various methods for fragmenting molecules
for tandem MS, including collision-induced dissociation (CID), electron
capture dissociation (ECD), electron transfer dissociation (ETD), infrared
multiphoton dissociation (IRMPD), blackbody infrared radiative
dissociation (BIRD), electron-detachment dissociation (EDD) and surfaceinduced dissociation (SID). An important application using tandem mass
spectrometry is in protein identification.[28]
Tandem mass spectrometry enables a variety of experimental sequences. Many
commercial mass spectrometers are designed to expedite the execution of such
routine sequences as selected reaction monitoring (SRM) and precursor ion
scanning. In SRM, the first analyzer allows only a single mass through and the
second analyzer monitors for multiple user-defined fragment ions. SRM is most
often used with scanning instruments where the second mass analysis event is
duty cycle limited. These experiments are used to increase specificity of
detection of known molecules, notably in pharmacokinetic studies. Precursor ion
scanning refers to monitoring for a specific loss from the precursor ion. The first
and second mass analyzers scan across the spectrum as partitioned by a userdefined m/z value. This experiment is used to detect specific motifs within
unknown molecules.
Another type of tandem mass spectrometry used for radiocarbon dating is
Accelerator Mass Spectrometry (AMS), which uses very high voltages,
usually in the mega-volt range, to accelerate negative ions into a type of tandem
mass spectrometer.

[edit] Common mass spectrometer configurations and

techniques
When a specific configuration of source, analyzer, and detector becomes
conventional in practice, often a compound acronym arises to designate it, and

the compound acronym may be better known among nonspectrometrists than the
component acronyms. The epitome of this is MALDI-TOF, which simply refers
to combining a matrix-assisted laser desorption/ionization source with a
time-of-flight mass analyzer. The MALDI-TOF moniker is more widely
recognized by the non-mass spectrometrists than MALDI or TOF individually.
Other examples include inductively coupled plasma-mass spectrometry
(ICP-MS), accelerator mass spectrometry (AMS), Thermal ionizationmass spectrometry (TIMS) and spark source mass spectrometry (SSMS).
Sometimes the use of the generic "MS" actually connotes a very specific mass
analyzer and detection system, as is the case with AMS, which is always sector
based.
Certain applications of mass spectrometry have developed monikers that although
strictly speaking would seem to refer to a broad application, in practice have
come instead to connote a specific or a limited number of instrument
configurations. An example of this is isotope ratio mass spectrometry
(IRMS), which refers in practice to the use of a limited number of sector based
mass analyzers; this name is used to refer to both the application and the
instrument used for the application.

[edit] Chromatographic techniques combined with mass

spectrometry
An important enhancement to the mass resolving and mass determining
capabilities of mass spectrometry is using it in tandem with chromatographic
separation techniques.

[edit] Gas chromatography

A gas chromatograph (right) directly coupled to a mass spectrometer (left)

See also: Gas chromatography-mass spectrometry
A common combination is gas chromatography-mass spectrometry (GC/MS or
GC-MS). In this technique, a gas chromatograph is used to separate different
compounds. This stream of separated compounds is fed online into the ion
source, a metallic filament to which voltage is applied. This filament emits
electrons which ionize the compounds. The ions can then further fragment,
yielding predictable patterns. Intact ions and fragments pass into the mass
spectrometer's analyzer and are eventually detected.[29]

[edit] Liquid chromatography

See also: Liquid chromatography-mass spectrometry
Similar to gas chromatography MS (GC/MS), liquid chromatography mass
spectrometry (LC/MS or LC-MS) separates compounds chromatographically
before they are introduced to the ion source and mass spectrometer. It differs
from GC/MS in that the mobile phase is liquid, usually a mixture of water and
organic solvents, instead of gas and the ions fragments cannot yield predictable
patterns. Most commonly, an electrospray ionization source is used in LC/MS.
There are also some newly developed ionization techniques like laser spray.

[edit] Ion mobility

See also: Ion mobility spectrometry-mass spectrometry
Ion mobility spectrometry/mass spectrometry (IMS/MS or IMMS) is a
technique where ions are first separated by drift time through some neutral gas
under an applied electrical potential gradient before being introduced into a mass
spectrometer.[30] Drift time is a measure of the radius relative to the charge of the
ion. The duty cycle of IMS (the time over which the experiment takes place) is
longer than most mass spectrometric techniques, such that the mass spectrometer
can sample along the course of the IMS separation. This produces data about the
IMS separation and the mass-to-charge ratio of the ions in a manner similar to
LC/MS.[31]
The duty cycle of IMS is short relative to liquid chromatography or gas
chromatography separations and can thus be coupled to such techniques,
producing triple modalities such as LC/IMS/MS.[32]

[edit] Data and analysis

Mass spectrum of a peptide showing the isotopic distribution

[edit] Data representations

See also: Mass spectrometry data format
Mass spectrometry produces various types of data. The most common data
representation is the mass spectrum.
Certain types of mass spectrometry data are best represented as a mass
chromatogram. Types of chromatograms include selected ion monitoring (SIM),
total ion current (TIC), and selected reaction monitoring chromatogram (SRM),
among many others.
Other types of mass spectrometry data are well represented as a threedimensional contour map. In this form, the mass-to-charge, m/z is on the x-axis,
intensity the y-axis, and an additional experimental parameter, such as time, is
recorded on the z-axis.

[edit] Data analysis

Basics
Mass spectrometry data analysis is a complicated subject that is very specific to
the type of experiment producing the data. There are general subdivisions of data
that are fundamental to understanding any data.
Many mass spectrometers work in either negative ion mode or positive ion mode.
It is very important to know whether the observed ions are negatively or
positively charged. This is often important in determining the neutral mass but it
also indicates something about the nature of the molecules.
Different types of ion source result in different arrays of fragments produced
from the original molecules. An electron ionization source produces many
fragments and mostly single-charged (1-) radicals (odd number of electrons),
whereas an electrospray source usually produces non-radical quasimolecular ions
that are frequently multiply charged. Tandem mass spectrometry purposely
produces fragment ions post-source and can drastically change the sort of data
achieved by an experiment.
By understanding the origin of a sample, certain expectations can be assumed as
to the component molecules of the sample and their fragmentations. A sample
from a synthesis/manufacturing process will probably contain impurities
chemically related to the target component. A relatively crudely prepared
biological sample will probably contain a certain amount of salt, which may form
adducts with the analyte molecules in certain analyses.
Results can also depend heavily on how the sample was prepared and how it was
run/introduced. An important example is the issue of which matrix is used for
MALDI spotting, since much of the energetics of the desorption/ionization event
is controlled by the matrix rather than the laser power. Sometimes samples are

spiked with sodium or another ion-carrying species to produce adducts rather

than a protonated species.
The greatest source of trouble when non-mass spectrometrists try to conduct mass
spectrometry on their own or collaborate with a mass spectrometrist is inadequate
definition of the research goal of the experiment. Adequate definition of the
experimental goal is a prerequisite for collecting the proper data and successfully
interpreting it. Among the determinations that can be achieved with mass
spectrometry are molecular mass, molecular structure, and sample purity. Each of
these questions requires a different experimental procedure. Simply asking for a
"mass spec" will most likely not answer the real question at hand.
Interpretation of mass spectra
Main article: Mass spectrum analysis
Since the precise structure or peptide sequence of a molecule is deciphered
through the set of fragment masses, the interpretation of mass spectra requires
combined use of various techniques. Usually the first strategy for identifying an
unknown compound is to compare its experimental mass spectrum against a
library of mass spectra. If the search comes up empty, then manual
interpretation[33] or software assisted interpretation of mass spectra are
performed. Computer simulation of ionization and fragmentation processes
occurring in mass spectrometer is the primary tool for assigning structure or
peptide sequence to a molecule. An a priori structural information is fragmented
in silico and the resulting pattern is compared with observed spectrum. Such
simulation is often supported by a fragmentation library[34] that contains published
patterns of known decomposition reactions. Software taking advantage of this
idea has been developed for both small molecules and proteins.
Another way of interpreting mass spectra involves spectra with accurate mass.
A mass-to-charge ratio value (m/z) with only integer precision can represent an
immense number of theoretically possible ion structures. More precise mass
figures significantly reduce the number of candidate molecular formulas, albeit
each can still represent large number of structurally diverse compounds. A
computer algorithm called formula generator calculates all molecular formulas
that theoretically fit a given mass with specified tolerance.
A recent technique for structure elucidation in mass spectrometry, called
precursor ion fingerprinting identifies individual pieces of structural
information by conducting a search of the tandem spectra of the molecule
under investigation against a library of the product-ion spectra of structurally
characterized precursor ions.

[edit] Applications
[edit] Isotope ratio MS: isotope dating and tracking

Mass spectrometer to determine the 16O/18O and 12C/13C isotope ratio on

biogenous carbonate
Main article: Isotope ratio mass spectrometry
Mass spectrometry is also used to determine the isotopic composition of
elements within a sample. Differences in mass among isotopes of an element are
very small, and the less abundant isotopes of an element are typically very rare,
so a very sensitive instrument is required. These instruments, sometimes referred
to as isotope ratio mass spectrometers (IR-MS), usually use a single magnet to
bend a beam of ionized particles towards a series of Faraday cups which
convert particle impacts to electric current. A fast on-line analysis of deuterium
content of water can be done using Flowing afterglow mass spectrometry, FAMS. Probably the most sensitive and accurate mass spectrometer for this purpose
is the accelerator mass spectrometer (AMS). Isotope ratios are important
markers of a variety of processes. Some isotope ratios are used to determine the
age of materials for example as in carbon dating. Labeling with stable isotopes
is also used for protein quantification. (see protein characterization below)

[edit] Trace gas analysis

Several techniques use ions created in a dedicated ion source injected into a flow
tube or a drift tube: selected ion flow tube (SIFT-MS), and proton transfer
reaction (PTR-MS), are variants of chemical ionization dedicated for trace gas
analysis of air, breath or liquid headspace using well defined reaction time
allowing calculations of analyte concentrations from the known reaction kinetics
without the need for internal standard or calibration.

[edit] Atom probe

Main article: Atom probe
An atom probe is an instrument that combines time-of-flight mass spectrometry
and field ion microscopy (FIM) to map the location of individual atoms.

[edit] Pharmacokinetics
Main article: Pharmacokinetics

Pharmacokinetics is often studied using mass spectrometry because of the

complex nature of the matrix (often blood or urine) and the need for high
sensitivity to observe low dose and long time point data. The most common
instrumentation used in this application is LC-MS with a triple quadrupole
mass spectrometer. Tandem mass spectrometry is usually employed for added
specificity. Standard curves and internal standards are used for quantitation of
usually a single pharmaceutical in the samples. The samples represent different
time points as a pharmaceutical is administered and then metabolized or cleared
from the body. Blank or t=0 samples taken before administration are important in
determining background and ensuring data integrity with such complex sample
matrices. Much attention is paid to the linearity of the standard curve; however it
is not uncommon to use curve fitting with more complex functions such as
quadratics since the response of most mass spectrometers is less than linear
across large concentration ranges.[35][36][37]
There is currently considerable interest in the use of very high sensitivity mass
spectrometry for microdosing studies, which are seen as a promising alternative
to animal experimentation.

[edit] Protein characterization

Main article: Protein mass spectrometry
Mass spectrometry is an important emerging method for the characterization and
sequencing of proteins. The two primary methods for ionization of whole
proteins are electrospray ionization (ESI) and matrix-assisted laser
desorption/ionization (MALDI). In keeping with the performance and mass
range of available mass spectrometers, two approaches are used for
characterizing proteins. In the first, intact proteins are ionized by either of the two
techniques described above, and then introduced to a mass analyzer. This
approach is referred to as "top-down" strategy of protein analysis. In the second,
proteins are enzymatically digested into smaller peptides using proteases such
as trypsin or pepsin, either in solution or in gel after electrophoretic
separation. Other proteolytic agents are also used. The collection of peptide
products are then introduced to the mass analyzer. When the characteristic pattern
of peptides is used for the identification of the protein the method is called
peptide mass fingerprinting (PMF), if the identification is performed using the
sequence data determined in tandem MS analysis it is called de novo
sequencing. These procedures of protein analysis are also referred to as the
"bottom-up" approach.

[edit] Glycan analysis

Mass spectrometry (MS), with its low sample requirement and high sensitivity,
has been predominantly used in glycobiology for characterization and
elucidation of glycan structures.[38] Mass spectrometry provides a
complementary method to HPLC for the analysis of glycans. Intact glycans may
be detected directly as singly charged ions by matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-MS) or, following
permethylation or peracetylation, by fast atom bombardment mass
spectrometry (FAB-MS).[39] Electrospray ionization mass spectrometry

(ESI-MS) also gives good signals for the smaller glycans.[40] Various free and
commercial software are now available which interpret MS data and aid in
Glycan structure characterization.

[edit] Space exploration

As a standard method for analysis, mass spectrometers have reached other planets
and moons. Two were taken to Mars by the Viking program. In early 2005 the
CassiniHuygens mission delivered a specialized GC-MS instrument aboard
the Huygens probe through the atmosphere of Titan, the largest moon of the
planet Saturn. This instrument analyzed atmospheric samples along its descent
trajectory and was able to vaporize and analyze samples of Titan's frozen,
hydrocarbon covered surface once the probe had landed. These measurements
compare the abundance of isotope(s) of each particle comparatively to earth's
natural abundance.[41] Also onboard the CassiniHuygens spacecraft is an ion
and neutral mass spectrometer which has been taking measurements of Titan's
atmospheric composition as well as the composition of Enceladus' plumes. A
Thermal and Evolved Gas Analyzer mass spectrometer was carried by the
Mars Phoenix Lander launched in 2007.[42]
Mass spectrometers are also widely used in space missions to measure the
composition of plasmas. For example, the Cassini spacecraft carries the Cassini
Plasma Spectrometer (CAPS),[43] which measures the mass of ions in Saturn's
magnetosphere.

[edit] Respired gas monitor

Mass spectrometers were used in hospitals for respiratory gas analysis beginning
around 1975 through the end of the century. Some are probably still in use but
none are currently being manufactured.[44]
Found mostly in the operating room, they were a part of a complex system, in
which respired gas samples from patients undergoing anesthesia were drawn
into the instrument through a valve mechanism designed to sequentially connect
up to 32 rooms to the mass spectrometer. A computer directed all operations of
the system. The data collected from the mass spectrometer was delivered to the
individual rooms for the anesthesiologist to use.
The uniqueness of this magnetic sector mass spectrometer may have been the fact
that a plane of detectors, each purposely positioned to collect all of the ion
species expected to be in the samples, allowed the instrument to simultaneously
report all of the gases respired by the patient. Although the mass range was
limited to slightly over 120 u, fragmentation of some of the heavier molecules
negated the need for a higher detection limit.[45]

[edit] See also

Mass spectrometry software

Calutron
Helium mass spectrometer

Mass spectrometry imaging

Reflectron

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^ Riker JB, Haberman B (1976). "Expired gas monitoring by mass spectrometry in a
respiratory intensive care unit". Crit. Care Med. 4 (5): 2239. doi:10.1097/00003246197609000-00002. PMID 975846.
^ J. W. W. Gothard, C. M. Busst, M. A. Branthwaite, N. J. H. Davies and D. M. Denison
(1980). "Applications of respiratory mass spectrometry to intensive care". Anaesthesia
35 (9): 890895. doi:10.1111/j.1365-2044.1980.tb03950.x. PMID 6778243.

[edit] Bibliography

Tureek, Frantiek; McLafferty, Fred W. (1993). Interpretation of mass spectra.

Sausalito, Calif: University Science Books. ISBN 0-935702-25-3.
http://books.google.com/?id=xQWk5WQfMQAC&printsec=frontcover.
Edmond de Hoffman; Vincent Stroobant (2001). Mass Spectrometry: Principles and
Applications (2nd ed.). John Wiley and Sons. ISBN 0-471-48566-7.
Downard, Kevin (2004). Mass Spectrometry A Foundation Course.
Cambridge UK: Royal Society of Chemistry. ISBN 0854046097.
http://books.google.com/?id=-8LtzxKrSwkC.
Siuzdak, Gary (1996). Mass spectrometry for biotechnology. Boston: Academic
Press. ISBN 0-12-647471-0.
Dass, Chhabil (2001). Principles and practice of biological mass spectrometry. New
York: John Wiley. ISBN 0-471-33053-1.
Jnrgen H. Gross (2006). Mass Spectrometry: A Textbook. Berlin: SpringerVerlag. ISBN 3-540-40739-1. http://books.google.com/?
id=e10yKTODUzoC&printsec=frontcover.
Muzikar, P., et al. (2003). "Accelerator Mass Spectrometry in Geologic Research".
Geological Society of America Bulletin 115: 643654. doi:10.1130/00167606(2003)115<0643:AMSIGR>2.0.CO;2. ISSN 0016-7606.

O. David Sparkman (2006). Mass Spectrometry Desk Reference. Pittsburgh: Global

View Pub. ISBN 0-9660813-9-0.
Tuniz, C. (1998). Accelerator mass spectrometry: ultrasensitive

7.7. Atomic absorption spectroscopy

From Wikipedia, the free encyclopedia
(Redirected from Atomic absorption spectrometer)
Jump to: navigation, search

Atomic absorption spectrometer

In analytical chemistry, atomic absorption spectroscopy is a technique for determining
the concentration of a particular metal element in a sample.[1] The technique can be used to
analyze the concentration of over 70 different metals in a solution.
Although atomic absorption spectroscopy dates to the nineteenth century, the modern form
was largely developed during the 1950s by a team of Australian chemists. They were led by
Alan Walsh and worked at the CSIRO (Commonwealth Science and Industry Research
Organisation) Division of Chemical Physics in Melbourne, Australia.[2]

[edit] Principles
The technique makes use of absorption spectrometry to assess the concentration of an
analyte in a sample. It relies therefore heavily on Beer-Lambert law.
In short, the electrons of the atoms in the atomizer can be promoted to higher orbitals for a
short amount of time by absorbing a set quantity of energy (i.e. light of a given wavelength).
This amount of energy (or wavelength) is specific to a particular electron transition in a
particular element, and in general, each wavelength corresponds to only one element. This
gives the technique its elemental selectivity.

As the quantity of energy (the power) put into the flame is known, and the quantity remaining
at the other side (at the detector) can be measured, it is possible, from Beer-Lambert law, to
calculate how many of these transitions took place, and thus get a signal that is proportional
to the concentration of the element being measured.

[edit] Instrumentation

Atomic absorption spectrometer block diagram

In order to analyze a sample for its atomic constituents, it has to be atomized. The sample
should then be illuminated by light. The light transmitted is finally measured by a detector. In
order to reduce the effect of emission from the atomizer (e.g. the black body radiation) or
the environment, a spectrometer is normally used between the atomizer and the detector.

[edit] Types of Atomizer

The technique typically makes use of a flame to atomize the sample,[3] but other atomizers
such as a graphite furnace[4] or plasmas, primarily inductively coupled plasmas, are also
used.[5]
When a flame is used it is laterally long (usually 10 cm) and not deep. The height of the
flame above the burner head can be controlled by adjusting the flow of the fuel mixture. A
beam of light passes through this flame at its longest axis (the lateral axis) and hits a detector.

[edit] Analysis of liquids

A liquid sample is normally turned into an atomic gas in three steps:
1. Desolvation (Drying) the liquid solvent is evaporated, and the dry sample remains
2. Vaporization (Ashing) the solid sample vaporises to a gas
3. Atomization the compounds making up the sample are broken into free atoms.

[edit] Radiation Sources

The radiation source chosen has a spectral width narrower than that of the atomic transitions.

[edit] Hollow cathode lamps

Hollow cathode lamps are the most common radiation source in atomic absorption
spectroscopy. Inside the lamp, filled with argon or neon gas, is a cylindrical metal cathode
containing the metal for excitation, and an anode. When a high voltage is applied across the
anode and cathode, gas particles are ionized. As voltage is increased, gaseous ions acquire
enough energy to eject metal atoms from the cathode. Some of these atoms are in an excited
states and emit light with the frequency characteristic to the metal[6]. Many modern hollow
cathode lamps are selective for several metals.

[edit] Diode lasers

Atomic absorption spectroscopy can also be performed by lasers, primarily diode lasers
because of their good properties for laser absorption spectrometry.[7] The technique is then
either referred to as diode laser atomic absorption spectrometry (DLAAS or DLAS),[8] or,
since wavelength modulation most often is employed, wavelength modulation absorption
spectrometry.

[edit] Background Correction methods

The narrow bandwidth of hollow cathode lamps make spectral overlap rare. That is, it is
unlikely that an absorption line from one element will overlap with another. Molecular
emission is much broader, so it is more likely that some molecular absorption band will
overlap with an atomic line. This can result in artificially high absorption and an improperly
high calculation for the concentration in the solution. Three methods are typically used to
correct for this:

Zeeman correction - A magnetic field is used to split the atomic line into two
sidebands (see Zeeman effect). These sidebands are close enough to the original
wavelength to still overlap with molecular bands, but are far enough not to overlap
with the atomic bands. The absorption in the presence and absence of a magnetic field
can be compared, the difference being the atomic absorption of interest.

Smith-Hieftje correction (invented by Stanley B. Smith and Gary M. Hieftje) - The

hollow cathode lamp is pulsed with high current, causing a larger atom population and
self-absorption during the pulses. This self-absorption causes a broadening of the line
and a reduction of the line intensity at the original wavelength.[9]

Deuterium lamp correction - In this case, a separate source (a deuterium lamp) with
broad emission is used to measure the background emission. The use of a separate
lamp makes this method the least accurate, but its relative simplicity (and the fact that
it is the oldest of the three) makes it the most commonly used method.

[edit] References
1. ^ Sperling, Michael B.; Welz, Bernhard (1999). Atomic Absorption Spectrometry. Weinheim:
Wiley-VCH. ISBN 3-527-28571-7.
2. ^ Lvov, B. V. (2005), "Fifty years of atomic absorption spectrometry", Journal of Analytical
Chemistry 60: 382, doi:10.1007/s10809-005-0103-0

3. ^ C. T. J. Alkemade, T. Hollander, W. Snelleman and P. J. T. Zeegers, Metal Vapours in

Flames, Pergamon Press, Oxford (1982).

4. ^ B. V. L'vov, Forty years of electrothermal atomic absorption spectrometry. Advances and

5.
6.
7.
8.
9.

problems in theory, Spectrochim. Acta B 52 1239-1245 (1997).

^ J. A. C. Broekaert, Analytical Atomic Spectrometry with Flames and Plasmas, Third
Edition, Wiley-VCH, Weinheim, Germany (1998).
^ Skoog, D.; Holler, J.; Crouch, S. Principles of Instrumental Analysis, 6th ed.; Thomson
Books/Cole, 2007; pp 238.
^ O. Axner, Laser Spectrometric Techniques in Analytical Atomic Spectrometry. In: Meyers
RA ed. Encyclopedia of Analytical Chemistry, New York, John Wiley & Sons, Inc., (2000), p.
9506-9595.
^ A. Zybin, J. Koch, H. D. Wizemann, J. Franzke and K. Niemax, Diode laser atomic
absorption spectrometry, Spectrochimica Acta B 60 1-11 (2005).
^ S. B. Smith, Jr and G. M. Hieftje (1983). "A New Background-correction Method for
Atomic Absorption Spectrometry". Applied Spectroscopy 37 (5): 419424.
doi:10.1366/0003702834634893.

8. Metode analisa termal

metoda analisa termal untuk analisa kualitatif dan kuantitatif

9. X-ray fluorescence
From Wikipedia, the free encyclopedia
Jump to: navigation, search

A Philips PW1606 X-ray fluorescence spectrometer with automated sample feed in a cement
plant quality control laboratory
X-ray fluorescence (XRF) is the emission of characteristic "secondary" (or fluorescent) Xrays from a material that has been excited by bombarding with high-energy X-rays or
gamma rays. The phenomenon is widely used for elemental analysis and chemical
analysis, particularly in the investigation of metals, glass, ceramics and building
materials, and for research in geochemistry, forensic science and archaeology.

[edit] Underlying physics

Physics of X-ray fluorescence, in a schematic representation.

When materials are exposed to short-wavelength X-rays or to gamma rays, ionisation of
their component atoms may take place. Ionisation consists of the ejection of one or more
electrons from the atom, and may take place if the atom is exposed to radiation with an
energy greater than its ionisation potential. X-rays and gamma rays can be energetic enough
to expel tightly held electrons from the inner orbitals of the atom. The removal of an electron
in this way renders the electronic structure of the atom unstable, and electrons in higher
orbitals "fall" into the lower orbital to fill the hole left behind. In falling, energy is released in
the form of a photon, the energy of which is equal to the energy difference of the two orbitals
involved. Thus, the material emits radiation, which has energy characteristic of the atoms
present. The term fluorescence is applied to phenomena in which the absorption of radiation
of a specific energy results in the re-emission of radiation of a different energy (generally
lower).

Figure 1: Electronic transitions in a calcium atom. Remember, when electrons are jumping
down, one of the electrons in the lower orbital is missing.

Figure 2: Typical energy dispersive XRF spectrum

Figure 3: Spectrum of a rhodium target tube operated at 60 kV, showing continuous spectrum
and K lines

[edit] Characteristic radiation

Each element has electronic orbitals of characteristic energy. Following removal of an inner
electron by an energetic photon provided by a primary radiation source, an electron from an
outer shell drops into its place. There are a limited number of ways in which this can happen,
as shown in figure 1. The main transitions are given names: an LK transition is
traditionally called K, an MK transition is called K, an ML transition is called L, and
so on. Each of these transitions yields a fluorescent photon with a characteristic energy equal
to the difference in energy of the initial and final orbital. The wavelength of this fluorescent
radiation can be calculated from Planck's Law:

The fluorescent radiation can be analysed either by sorting the energies of the photons
(energy-dispersive analysis) or by separating the wavelengths of the radiation (wavelengthdispersive analysis). Once sorted, the intensity of each characteristic radiation is directly
related to the amount of each element in the material. This is the basis of a powerful
technique in analytical chemistry. Figure 2 shows the typical form of the sharp fluorescent
spectral lines obtained in the wavelength-dispersive method (see Moseley's law).

[edit] Primary radiation

In order to excite the atoms, a source of radiation is required, with sufficient energy to expel
tightly held inner electrons. Conventional X-ray generators are most commonly used,
because their output can readily be "tuned" for the application, and because higher power can
be deployed relative to other techniques. However, gamma ray sources can be used without
the need for an elaborate power supply, allowing an easier use in small portable instruments.
When the energy source is a synchrotron or the X-ray are focussed by an optic like a
polycapillary, the X-ray beam can be very small and very intense. As a result, atomic
information on the sub-micrometer scale can be obtained. X-ray generators in the range 20
60 kV in order to the K line, which allows excitation of a broad range of atoms. The
continuous spectrum consists of "bremsstrahlung" radiation: radiation produced when high
energy electrons passing through the tube are progressively decelerated by the material of the
tube anode (the "target"). A typical tube output spectrum is shown in figure 3.

[edit] Dispersion
In energy dispersive analysis, the fluorescent X-rays emitted by the material sample are
directed into a solid-state detector which produces a "continuous" distribution of pulses, the
voltages of which are proportional to the incoming photon energies. This signal is processed
by a multichannel analyser (MCA) which produces an accumulating digital spectrum that can
be processed to obtain analytical data. In wavelength dispersive analysis, the fluorescent Xrays emitted by the material sample are directed into a diffraction grating monochromator.
The diffraction grating used is usually a single crystal. By varying the angle of incidence and
take-off on the crystal, a single X-ray wavelength can be selected. The wavelength obtained
is given by the Bragg Equation:

where d is the spacing of atomic layers parallel to the crystal surface.

[edit] Detection
In energy dispersive analysis, dispersion and detection are a single operation, as already
mentioned above. Proportional counters or various types of solid state detectors (PIN
diode, Si(Li), Ge(Li), Silicon Drift Detector SDD) are used. They all share the same
detection principle: An incoming X-ray photon ionises a large number of detector atoms with
the amount of charge produced being proportional to the energy of the incoming photon. The
charge is then collected and the process repeats itself for the next photon. Detector speed is
obviously critical, as all charge carriers measured have to come from the same photon to
measure the photon energy correctly (peak length discrimination is used to eliminate events
that seem to have been produced by two X-ray photons arriving almost simultaneously). The
spectrum is then built up by dividing the energy spectrum into discrete bins and counting the
number of pulses registered within each energy bin. EDXRF detector types vary in
resolution, speed and the means of cooling (a low number of free charge carriers is critical in
the solid state detectors): proportional counters with resolutions of several hundred eV cover
the low end of the performance spectrum, followed by PIN diode detectors, while the Si(Li),
Ge(Li) and Silicon Drift Detectors (SDD) occupy the high end of the performance scale.
In wavelength dispersive analysis, the single-wavelength radiation produced by the
monochromator is passed into a photomultiplier, a detector similar to a Geiger counter,
which counts individual photons as they pass through. The counter is a chamber containing a
gas that is ionised by X-ray photons. A central electrode is charged at (typically) +1700 V
with respect to the conducting chamber walls, and each photon triggers a pulse-like cascade
of current across this field. The signal is amplified and transformed into an accumulating
digital count. These counts are then processed to obtain analytical data.

[edit] X-ray intensity

The fluorescence process is inefficient, and the secondary radiation is much weaker than the
primary beam. Furthermore, the secondary radiation from lighter elements is of relatively low
energy (long wavelength) and has low penetrating power, and is severely attenuated if the
beam passes through air for any distance. Because of this, for high-performance analysis, the
path from tube to sample to detector is maintained under high vacuum (around 10 Pa residual
pressure). This means in practice that most of the working parts of the instrument have to be

located in a large vacuum chamber. The problems of maintaining moving parts in vacuo, and
of rapidly introducing and withdrawing the sample without losing vacuum, pose major
challenges for the design of the instrument. For less demanding applications, or when the
sample is damaged by a vacuum (e.g. a volatile sample), a helium-swept X-ray chamber can
be substituted, with some loss of low-Z (Z = atomic number) intensities.

[edit] Chemical analysis

The use of a primary X-ray beam to excite fluorescent radiation from the sample was first
proposed by Glocker and Schreiber in 1928[1]. Today, the method is used as a nondestructive analytical technique, and as a process control tool in many extractive and
processing industries. In principle, the lightest element that can be analysed is beryllium (Z =
4), but due to instrumental limitations and low X-ray yields for the light elements, it is often
difficult to quantify elements lighter than sodium (Z = 11), unless background corrections
and very comprehensive interelement corrections are made.

Figure 4: Schematic arrangement of EDX spectrometer

[edit] Energy dispersive spectrometry

In energy dispersive spectrometers (EDX or EDS), the detector allows the determination of
the energy of the photon when it is detected. Detectors historically have been based on silicon
semiconductors, in the form of lithium-drifted silicon crystals, or high-purity silicon wafers.

Figure 5: Schematic form of a Si(Li) detector

[edit] Si(Li) detectors

These consist essentially of a 35 mm thick silicon junction type p-i-n diode (same as PIN
diode) with a bias of -1000 V across it. The lithium-drifted centre part forms the nonconducting i-layer, where Li compensates the residual acceptors which would otherwise
make the layer p-type. When an X-ray photon passes through, it causes a swarm of electronhole pairs to form, and this causes a voltage pulse. To obtain sufficiently low conductivity, the
detector must be maintained at low temperature, and liquid-nitrogen must be used for the best
resolution. With some loss of resolution, the much more convenient Peltier cooling can be
employed.[2]

[edit] Wafer detectors

More recently, high-purity silicon wafers with low conductivity have become routinely
available. Cooled by the Peltier effect, this provides a cheap and convenient detector,
although the liquid nitrogen cooled Si(Li) detector still has the best resolution (i.e. ability to
distinguish different photon energies).

[edit] Amplifiers
The pulses generated by the detector are processed by pulse-shaping amplifiers. It takes
time for the amplifier to shape the pulse for optimum resolution, and there is therefore a
trade-off between resolution and count-rate: long processing time for good resolution results
in "pulse pile-up" in which the pulses from successive photons overlap. Multi-photon events
are, however, typically more drawn out in time (photons did not arrive exactly at the same
time) than single photon events and pulse-length discrimination can thus be used to filter
most of these out. Even so, a small number of pile-up peaks will remain and pile-up
correction should be built into the software in applications that require trace analysis. To
make the most efficient use of the detector, the tube current should be reduced to keep multiphoton events (before discrimination) at a reasonable level, e.g. 520%.

[edit] Processing
Considerable computer power is dedicated to correcting for pulse-pile up and for extraction
of data from poorly resolved spectra. These elaborate correction processes tend to be based
on empirical relationships that may change with time, so that continuous vigilance is required
in order to obtain chemical data of adequate precision.

[edit] Usage
EDX spectrometers are superior to WDX spectrometers in that they are smaller, simpler in
design and have fewer engineered parts. They can also use miniature X-ray tubes or gamma
sources. This makes them cheaper and allows miniaturization and portability. This type of
instrument is commonly used for portable quality control screening applications, such as
testing toys for Lead (Pb) content, sorting scrap metals, and measuring the lead content of
residential paint. On the other hand, the low resolution and problems with low count rate and
long dead-time makes them inferior for high-precision analysis. They are, however, very

effective for high-speed, multi-elemental analysis. Field Portable XRF analysers currently on
the market weigh less than 2 kg, and have limits of detection on the order of 2 parts per
million of Lead (Pb) in pure sand.

Figure 6: Schematic arrangement of wavelength dispersive spectrometer

Chemist operates a goniometer used for X-ray fluorescence analysis of individual grains of
mineral specimens, U.S. Geological Survey, 1958.

[edit] Wavelength dispersive spectrometry

In wavelength dispersive spectrometers (WDX or WDS), the photons are separated by
diffraction on a single crystal before being detected. Although wavelength dispersive
spectrometers are occasionally used to scan a wide range of wavelengths, producing a
spectrum plot as in EDS, they are usually set up to make measurements only at the
wavelength of the emission lines of the elements of interest. This is achieved in two different
ways:

"Simultaneous" spectrometers have a number of "channels" dedicated to analysis of

a single element, each consisting of a fixed-geometry crystal monochromator, a
detector, and processing electronics. This allows a number of elements to be measured
simultaneously, and in the case of high-powered instruments, complete high-precision
analyses can be obtained in under 30 s. Another advantage of this arrangement is that
the fixed-geometry monochromators have no continuously moving parts, and so are
very reliable. Reliability is important in production environments where instruments
are expected to work without interruption for months at a time. Disadvantages of
simultaneous spectrometers include relatively high cost for complex analyses, since
each channel used is expensive. The number of elements that can be measured is
limited to 1520, because of space limitations on the number of monochromators that
can be crowded around the fluorescing sample. The need to accommodate multiple

monochromators means that a rather open arrangement around the sample is required,
leading to relatively long tube-sample-crystal distances, which leads to lower detected
intensities and more scattering. The instrument is inflexible, because if a new element
is to be measured, a new measurement channel has to be bought and installed.

"Sequential" spectrometers have a single variable-geometry monochromator (but

usually with an arrangement for selecting from a choice of crystals), a single detector
assembly (but usually with more than one detector arranged in tandem), and a single
electronic pack. The instrument is programmed to move through a sequence of
wavelengths, in each case selecting the appropriate X-ray tube power, the appropriate
crystal, and the appropriate detector arrangement. The length of the measurement
program is essentially unlimited, so this arrangement is very flexible. Because there is
only one monochromator, the tube-sample-crystal distances can be kept very short,
resulting in minimal loss of detected intensity. The obvious disadvantage is relatively
long analysis time, particularly when many elements are being analysed, not only
because the elements are measured in sequence, but also because a certain amount of
time is taken in readjusting the monochromator geometry between measurements.
Furthermore, the frenzied activity of the monochromator during an analysis program
is a challenge for mechanical reliability. However, modern sequential instruments can
achieve reliability almost as good as that of simultaneous instruments, even in
continuous-usage applications.

[edit] Sample presentation

In order to keep the geometry of the tube-sample-detector assembly constant, the sample is
normally prepared as a flat disc, typically of diameter 2050 mm. This is located at a
standardized, small distance from the tube window. Because the X-ray intensity follows an
inverse-square law, the tolerances for this placement and for the flatness of the surface must
be very tight in order to maintain a repeatable X-ray flux. Ways of obtaining sample discs
vary: metals may be machined to shape, minerals may be finely ground and pressed into a
tablet, and glasses may be cast to the required shape. A further reason for obtaining a flat and
representative sample surface is that the secondary X-rays from lighter elements often only
emit from the top few micrometers of the sample. In order to further reduce the effect of
surface irregularities, the sample is usually spun at 520 rpm. It is necessary to ensure that the
sample is sufficiently thick to absorb the entire primary beam. For higher-Z materials, a few
millimetres thickness is adequate, but for a light-element matrix such as coal, a thickness of
3040 mm is needed.

Figure 7: Bragg diffraction condition

[edit] Monochromators
The common feature of monochromators is the maintenance of a symmetrical geometry
between the sample, the crystal and the detector. In this geometry the Bragg diffraction
condition is obtained.
The X-ray emission lines are very narrow (see figure 2), so the angles must be defined with
considerable precision. This is achieved in two ways:

Flat crystal with Soller collimators

The Soller collimator is a stack of parallel metal plates, spaced a few tenths of a millimetre
apart. To improve angle resolution, one must lengthen the collimator, and/or reduce the plate
spacing. This arrangement has the advantage of simplicity and relatively low cost, but the
collimators reduce intensity and increase scattering, and reduce the area of sample and crystal
that can be "seen". The simplicity of the geometry is especially useful for variable-geometry
monochromators.

Figure 8: Flat crystal with Soller collimators

Figure 9: Curved crystal with slits

Curved crystal with slits

The Rowland circle geometry ensures that the slits are both in focus, but in order for the
Bragg condition to be met at all points, the crystal must first be bent to a radius of 2R (where
R is the radius of the Rowland circle), then ground to a radius of R. This arrangement allows
higher intensities (typically 8-fold) with higher resolution (typically 4-fold) and lower
background. However, the mechanics of keeping Rowland circle geometry in a variable-angle
monochromator is extremely difficult. In the case of fixed-angle monochromators (for use in
simultaneous spectrometers), crystals bent to a logarithmic spiral shape give the best focusing

performance. The manufacture of curved crystals to acceptable tolerances increases their

price considerably.

[edit] Analysis Lines

The spectral lines used for chemical analysis are selected on the basis of intensity,
accessibility by the instrument, and lack of line overlaps. Typical lines used, and their
wavelengths, are as follows:

elemen
line
t
Li
K
Be
K
B
K
C
K
N
K
O
K
K1,
F

waveleng
th (nm)
22.8
11.4
6.76
4.47
3.16
2.362

eleme
nt
Ni
Cu
Zn
Ga
Ge
As

waveleng
th (nm)
K1 0.1658
K1 0.1541
K1 0.1435
K1 0.1340
K1 0.1254
K1 0.1176

eleme
nt
I
Xe
Cs
Ba
La
Ce

lin waveleng
e th (nm)
L1 0.3149
L1 0.3016
L1 0.2892
L1 0.2776
L1 0.2666
L1 0.2562

eleme
nt
Pt
Au
Hg
Tl
Pb
Bi

lin wavelengt
e h (nm)
L1 0.1313
L1 0.1276
L1 0.1241
L1 0.1207
L1 0.1175
L1 0.1144

1.832

Se

K1 0.1105

Pr

L1 0.2463

Po

L1 0.1114

1.461

Br

K1 0.1040

Nd

L1 0.2370

At

L1 0.1085

1.191

Kr

K1 0.09801

Pm

L1 0.2282

Rn

L1 0.1057

0.989

Rb

K1 0.09256

Sm

L1 0.2200

Fr

L1 0.1031

0.834

Sr

K1 0.08753

Eu

L1 0.2121

Ra

L1 0.1005

0.7126

K1 0.08288

Gd

L1 0.2047

Ac

L1 0.0980

0.6158

Zr

K1 0.07859

Tb

L1 0.1977

Th

L1 0.0956

0.5373

Nb

K1 0.07462

Dy

L1 0.1909

Pa

L1 0.0933

0.4729

Mo

K1 0.07094

Ho

L1 0.1845

L1 0.0911

0.4193

Tc

K1 0.06751

Er

L1 0.1784

Np

L1 0.0888

0.3742

Ru

K1 0.06433

Tm

L1 0.1727

Pu

L1 0.0868

0.3359

Rh

K1 0.06136

Yb

L1 0.1672

Am

L1 0.0847

0.3032

Pd

K1 0.05859

Lu

L1 0.1620

Cm

L1 0.0828

K1, 0.2749

Ag

K1 0.05599

Hf

L1 0.1570

Bk

L1 0.0809

Ne
Na
Mg
Al
Si
P
S
Cl
Ar
K
Ca
Sc
Ti

K1,
2

K1,
2

K1,
2

K1,
2

K1,
2

K1,
2

K1,
2

K1,
2

K1,
2

K1,
2

K1,
2

K1,
2

line

V
Cr
Mn
Fe
Co

K1
K1
K1
K1
K1

0.2504
0.2290
0.2102
0.1936
0.1789

Cd
In
Sn
Sb
Te

K1 0.05357
L1 0.3772
L1 0.3600
L1 0.3439
L1 0.3289

Ta
W
Re
Os
Ir

L1 0.1522
L1 0.1476
L1 0.1433
L1 0.1391
L1 0.1351

Cf
Es
Fm
Md
No

L1 0.0791
L1 0.0773
L1 0.0756
L1 0.0740
L1 0.0724

Other lines are often used, depending on the type of sample and equipment available.

[edit] Crystals
The desirable characteristics of a diffraction crystal are:

High diffraction intensity

High dispersion
Narrow diffracted peak width
High peak-to-background
Absence of interfering elements
Low thermal coefficient of expansion
Stability in air and on exposure to X-rays
Ready availability
Low cost

Crystals with simple structure tend to give the best diffraction performance. Crystals
containing heavy atoms can diffract well, but also fluoresce themselves, causing interference.
Crystals that are water-soluble, volatile or organic tend to give poor stability.
Commonly used crystal materials include LiF (lithium fluoride), ADP (ammonium
dihydrogen phosphate), Ge (germanium), graphite, InSb (indium antimonide), PE (tetrakis(hydroxymethyl)-methane: penta-erythritol), KAP (potassium hydrogen phthalate), RbAP
(rubidium hydrogen phthalate) and TlAP (thallium(I) hydrogen phthalate). In addition, there
is an increasing use of "layered synthetic microstructures", which are "sandwich" structured
materials comprising successive thick layers of low atomic number matrix, and monoatomic
layers of a heavy element. These can in principle be custom-manufactured to diffract any
desired long wavelength, and are used extensively for elements in the range Li to Mg.
Properties of commonly used crystals
material

plane

d (nm)

min
(nm)

max
(nm)

intensity

thermal
durability
expansion

LiF

200

0.2014

0.053

0.379

+++++

+++

+++

LiF

220

0.1424

0.037

0.268

+++

++

+++

LiF

420

0.0901

0.024

0.169

++

++

+++

ADP

101

0.5320

0.139

1.000

++

++

Ge

111

0.3266

0.085

0.614

+++

+++

graphite

001

0.3354

0.088

0.630

++++

+++

InSb

111

0.3740

0.098

0.703

++++

+++

PE

002

0.4371

0.114

0.821

+++

+++++

KAP

1010

1.325

0.346

2.490

++

++

++

RbAP

1010

1.305

0.341

2.453

++

++

++

Si

111

0.3135

0.082

0.589

++

+++

TlAP

1010

1.295

0.338

2.434

+++

++

++

YB66

400

0.586

6 nm LSM

6.00

1.566

11.276

+++

++

[edit] Detectors
Detectors used for wavelength dispersive spectrometry need to have high pulse processing
speeds in order to cope with the very high photon count rates that can be obtained. In
addition, they need sufficient energy resolution to allow filtering-out of background noise and
spurious photons from the primary beam or from crystal fluorescence. There are four
common types of detector:

gas flow proportional counters

sealed gas detectors
scintillation counters
semiconductor detectors

Figure 10: Arrangement of gas flow proportional counter

Gas flow proportional counters are used mainly for detection of longer wavelengths. Gas
flows through it continuously. Where there are multiple detectors, the gas is passed through
them in series, then led to waste. The gas is usually 90% argon, 10% methane ("P10"),
although the argon may be replaced with neon or helium where very long wavelengths (over
5 nm) are to be detected. The argon is ionised by incoming X-ray photons, and the electric
field multiplies this charge into a measurable pulse. The methane suppresses the formation of
fluorescent photons caused by recombination of the argon ions with stray electrons. The
anode wire is typically tungsten or nichrome of 2060 m diameter. Since the pulse strength
obtained is essentially proportional to the ratio of the detector chamber diameter to the wire
diameter, a fine wire is needed, but it must also be strong enough to be maintained under
tension so that it remains precisely straight and concentric with the detector. The window
needs to be conductive, thin enough to transmit the X-rays effectively, but thick and strong
enough to minimize diffusion of the detector gas into the high vacuum of the monochromator
chamber. Materials often used are beryllium metal, aluminised PET film and aluminised
polypropylene. Ultra-thin windows (down to 1 m) for use with low-penetration long
wavelengths are very expensive. The pulses are sorted electronically by "pulse height
selection" in order to isolate those pulses deriving from the secondary X-ray photons being
counted.
Sealed gas detectors are similar to the gas flow proportional counter, except that the gas
does not flow though it. The gas is usually krypton or xenon at a few atmospheres pressure.
They are applied usually to wavelengths in the 0.150.6 nm range. They are applicable in
principle to longer wavelengths, but are limited by the problem of manufacturing a thin
window capable of withstanding the high pressure difference.
Scintillation counters consist of a scintillating crystal (typically of sodium iodide doped with
thallium) attached to a photomultiplier. The crystal produces a group of scintillations for each
photon absorbed, the number being proportional to the photon energy. This translates into a
pulse from the photomultiplier of voltage proportional to the photon energy. The crystal must
be protected with a relatively thick aluminium/beryllium foil window, which limits the use of
the detector to wavelengths below 0.25 nm. Scintillation counters are often connected in
series with a gas flow proportional counter: the latter is provided with an outlet window
opposite the inlet, to which the scintillation counter is attached. This arrangement is
particularly used in sequential spectrometers.
Semiconductor detectors can be used in theory, and their applications are increasing as their
technology improves, but historically their use for WDX has been restricted by their slow
response (see EDX).

A glass "bead" specimen for XRF analysis being cast at around 1100 C in a Herzog
automated fusion machine in a cement plant quality control laboratory. 1 (top): fusing, 2:
preheating the mould, 3: pouring the melt, 4: cooling the "bead"

[edit] Extracting analytical results

At first sight, the translation of X-ray photon count-rates into elemental concentrations would
appear to be straightforward: WDX separates the X-ray lines efficiently, and the rate of
generation of secondary photons is proportional to the element concentration. However, the
number of photons leaving the sample is also affected by the physical properties of the
sample: so-called "matrix effects". These fall broadly into three categories:

X-ray absorption

X-ray enhancement
sample macroscopic effects

All elements absorb X-rays to some extent. Each element has a characteristic absorption
spectrum which consists of a "saw-tooth" succession of fringes, each step-change of which
has wavelength close to an emission line of the element. Absorption attenuates the secondary
X-rays leaving the sample. For example, the mass absorption coefficient of silicon at the
wavelength of the aluminium K line is 50 m/kg, whereas that of iron is 377 m/kg. This
means that a given concentration of aluminium in a matrix of iron gives only one seventh of
the count rate compared with the same concentration of aluminium in a silicon matrix.
Fortunately, mass absorption coefficients are well known and can be calculated. However, to
calculate the absorption for a multi-element sample, the composition must be known. For
analysis of an unknown sample, an iterative procedure is therefore used. It will be noted that,
to derive the mass absorption accurately, data for the concentration of elements not measured
by XRF may be needed, and various strategies are employed to estimate these. As an
example, in cement analysis, the concentration of oxygen (which is not measured) is
calculated by assuming that all other elements are present as standard oxides.
Enhancement occurs where the secondary X-rays emitted by a heavier element are
sufficiently energetic to stimulate additional secondary emission from a lighter element. This
phenomenon can also be modelled, and corrections can be made provided that the full matrix
composition can be deduced.
Sample macroscopic effects consist of effects of inhomogeneities of the sample, and
unrepresentative conditions at its surface. Samples are ideally homogeneous and isotropic,
but they often deviate from this ideal. Mixtures of multiple crystalline components in mineral
powders can result in absorption effects that deviate from those calculable from theory. When
a powder is pressed into a tablet, the finer minerals concentrate at the surface. Spherical
grains tend to migrate to the surface more than do angular grains. In machined metals, the
softer components of an alloy tend to smear across the surface. Considerable care and
ingenuity are required to minimize these effects. Because they are artifacts of the method of
sample preparation, these effects can not be compensated by theoretical corrections, and must
be "calibrated in". This means that the calibration materials and the unknowns must be
compositionally and mechanically similar, and a given calibration is applicable only to a
limited range of materials. Glasses most closely approach the ideal of homogeneity and
isotropy, and for accurate work, minerals are usually prepared by dissolving them in a borate
glass, and casting them into a flat disc or "bead". Prepared in this form, a virtually universal
calibration is applicable.
Further corrections that are often employed include background correction and line overlap
correction. The background signal in an XRF spectrum derives primarily from scattering of
primary beam photons by the sample surface. Scattering varies with the sample mass
absorption, being greatest when mean atomic number is low. When measuring trace amounts
of an element, or when measuring on a variable light matrix, background correction becomes
necessary. This is really only feasible on a sequential spectrometer. Line overlap is a common
problem, bearing in mind that the spectrum of a complex mineral can contain several hundred
measurable lines. Sometimes it can be overcome by measuring a less-intense, but overlapfree line, but in certain instances a correction is inevitable. For instance, the K is the only
usable line for measuring sodium, and it overlaps the zinc L (L2-M4) line. Thus zinc, if
present, must be analysed in order to properly correct the sodium value.

[edit] Other spectroscopic methods using the same principle

It is also possible to create a characteristic secondary X-ray emission using other incident
radiation to excite the sample:

electron beam: electron microprobe (or Castaing microprobe);

ion beam: particle induced X-ray emission (PIXE).

When radiated by an X-ray beam, the sample also emits other radiations that can be used for
analysis:

electrons ejected by the photoelectric effect: X-ray photoelectron spectroscopy

(XPS), also called electron spectroscopy for chemical analysis (ESCA)

The de-excitation also ejects Auger electrons, but Auger electron spectroscopy (AES)
normally uses an electron beam as the probe.
Confocal microscopy X-ray fluorescence imaging is a newer technique that allow control
over depth, in addition to horizontal and vertical aiming, for example, when analyzing buried
layers in a painting.[3]

[edit] Instrument qualification

A 2001 review,[4] addresses the application of portable instrumentation from QA/QC
perspectives. It provides a guide to the development of a set of SOPs if regulatory
compliance guidelines are not available.
Further information: Verification and Validation

[edit] See also

Emission spectroscopy
List of materials analysis methods
Micro-X-ray fluorescence
X-ray fluorescence holography

[edit] Notes
1. ^ Glocker, R., and Schreiber, H., Ann. Physik., 85 (1928), p. 1089
2. ^ David Bernard Williams, C. Barry Carter (1996). Transmission electron
microscopy: a textbook for materials science, Volume 2. Springer. p. 559.
ISBN 030645324X. http://books.google.com/?id=667SJf95AFAC&pg=PA559.
3. ^ L. Vincze (2005). "Confocal X-ray Fluorescence Imaging and XRF
Tomography for Three-Dimensional Trace Element Microanalysis". Microscopy
and Microanalysis 11: 682. doi:10.1017/S1431927605503167.
http://journals.cambridge.org/action/displayFulltext?
type=1&fid=326128&jid=MAM&volumeId=11&issueId=S02&aid=326127.
4. ^ Kalnickya, Dennis J.; Raj Singhvi (2001). "Field portable XRF analysis of
environmental samples". Journal of Hazardous Materials 83: 93.
doi:10.1016/S0304-3894(00)00330-7.

[edit] References

Beckhoff, B., Kanngieer, B., Langhoff, N., Wedell, R., Wolff, H., Handbook of
Practical X-Ray Fluorescence Analysis, Springer, 2006, ISBN 3-540-28603-9
Bertin, E. P., Principles and Practice of X-ray Spectrometric Analysis, Kluwer
Academic / Plenum Publishers, ISBN 0-3063-0809-6
Buhrke, V. E., Jenkins, R., Smith, D. K., A Practical Guide for the Preparation of
Specimens for XRF and XRD Analysis, Wiley, 1998, ISBN 0-471-19458-1
Jenkins, R., X-ray Fluorescence Spectrometry, Wiley, ISBN 0-4712-9942-1
Jenkins, R., De Vries, J. L., Practical X-ray Spectrometry, Springer-Verlag, 1973,
ISBN 0387910298
Jenkins, R., R.W. Gould, R. W., Gedcke, D., Quantitative X-ray Spectrometry,
Marcel Dekker, ISBN 0-8247-9554-7
Van Grieken, R. E., Markowicz, A. A., Handbook of X-Ray Spectrometry 2nd ed.;
Marcel Dekker Inc: New York, 2002; Vol. 29; ISBN 0-8247-0600-5

10.

X-ray crystallography
From Wikipedia, the free encyclopedia
(Redirected from X ray diffraction)
Jump to: navigation, search

X-ray crystallography can locate every atom in a zeolite, an aluminosilicate with many
important applications, such as water purification.
X-ray crystallography is a method of determining the arrangement of atoms within a
crystal, in which a beam of X-rays strikes a crystal and diffracts into many specific
directions. From the angles and intensities of these diffracted beams, a crystallographer can
produce a three-dimensional picture of the density of electrons within the crystal. From this
electron density, the mean positions of the atoms in the crystal can be determined, as well as
their chemical bonds, their disorder and various other information.
Since many materials can form crystals such as salts, metals, minerals,
semiconductors, as well as various inorganic, organic and biological molecules X-ray

crystallography has been fundamental in the development of many scientific fields. In its first
decades of use, this method determined the size of atoms, the lengths and types of chemical
bonds, and the atomic-scale differences among various materials, especially minerals and
alloys. The method also revealed the structure and functioning of many biological molecules,
including vitamins, drugs, proteins and nucleic acids such as DNA. X-ray crystallography is
still the chief method for characterizing the atomic structure of new materials and in
discerning materials that appear similar by other experiments. X-ray crystal structures can
also account for unusual electronic or elastic properties of a material, shed light on chemical
interactions and processes, or serve as the basis for designing pharmaceuticals against
diseases.
In an X-ray diffraction measurement, a crystal is mounted on a goniometer and gradually
rotated while being bombarded with X-rays, producing a diffraction pattern of regularly
spaced spots known as reflections. The two-dimensional images taken at different rotations
are converted into a three-dimensional model of the density of electrons within the crystal
using the mathematical method of Fourier transforms, combined with chemical data known
for the sample. Poor resolution (fuzziness) or even errors may result if the crystals are too
small, or not uniform enough in their internal makeup.
X-ray crystallography is related to several other methods for determining atomic structures.
Similar diffraction patterns can be produced by scattering electrons or neutrons, which are
likewise interpreted as a Fourier transform. If single crystals of sufficient size cannot be
obtained, various other X-ray methods can be applied to obtain less detailed information;
such methods include fiber diffraction, powder diffraction and small-angle X-ray
scattering (SAXS). If the material under investigation is only available in the form of
nanocrystalline powders or suffers from poor crystallinity, the methods of electron
crystallography can be applied for determining the atomic structure.
For all above mentioned X-ray diffraction methods, the scattering is elastic; the scattered Xrays have the same wavelength as the incoming X-ray. By contrast, inelastic X-ray
scattering methods are useful in studying excitations of the sample, rather than the
distribution of its atoms.
o

[edit] History
[edit] Early scientific history of crystals and X-rays

Drawing of square (Figure A, above) and hexagonal (Figure B, below) packing from
Kepler's work, Strena seu de Nive Sexangula.
Crystals have long been admired for their regularity and symmetry, but they were not
investigated scientifically until the 17th century. Johannes Kepler hypothesized in his work
Strena seu de Nive Sexangula (1611) that the hexagonal symmetry of snowflake crystals
was due to a regular packing of spherical water particles.[1]

As shown by X-ray crystallography, the hexagonal symmetry of snowflakes results from the
tetrahedral arrangement of hydrogen bonds about each water molecule. The water
molecules are arranged similarly to the silicon atoms in the tridymite polymorph of SiO2.
The resulting crystal structure has hexagonal symmetry when viewed along a principal axis.
Crystal symmetry was first investigated experimentally by Nicolas Steno (1669), who
showed that the angles between the faces are the same in every exemplar of a particular type
of crystal,[2] and by Ren Just Hay (1784), who discovered that every face of a crystal can
be described by simple stacking patterns of blocks of the same shape and size. Hence,
William Hallowes Miller in 1839 was able to give each face a unique label of three small
integers, the Miller indices which are still used today for identifying crystal faces. Hay's
study led to the correct idea that crystals are a regular three-dimensional array (a Bravais
lattice) of atoms and molecules; a single unit cell is repeated indefinitely along three
principal directions that are not necessarily perpendicular. In the 19th century, a complete
catalog of the possible symmetries of a crystal was worked out by Johann Hessel,[3]
Auguste Bravais,[4] Yevgraf Fyodorov,[5] Arthur Schnflies[6] and (belatedly) William
Barlow. From the available data and physical reasoning, Barlow proposed several crystal
structures in the 1880s that were validated later by X-ray crystallography; [7] however, the
available data were too scarce in the 1880s to accept his models as conclusive.

X-ray crystallography shows the arrangement of water molecules in ice, revealing the
hydrogen bonds that hold the solid together. Few other methods can determine the structure
of matter with such sub-atomic precision (resolution).
X-rays were discovered by Wilhelm Conrad Rntgen in 1895, just as the studies of crystal
symmetry were being concluded. Physicists were initially uncertain of the nature of X-rays,
although it was soon suspected (correctly) that they were waves of electromagnetic
radiation, in other words, another form of light. At that time, the wave model of light
specifically, the Maxwell theory of electromagnetic radiation was well accepted among
scientists, and experiments by Charles Glover Barkla showed that X-rays exhibited
phenomena associated with electromagnetic waves, including transverse polarization and
spectral lines akin to those observed in the visible wavelengths. Single-slit experiments in
the laboratory of Arnold Sommerfeld suggested the wavelength of X-rays was about 1
Angstrm. However, X-rays are composed of photons, and thus are not only waves of
electromagnetic radiation but also exhibit particle-like properties. The photon concept was
introduced by Albert Einstein in 1905,[8] but it was not broadly accepted until 1922,[9][10]
when Arthur Compton confirmed it by the scattering of X-rays from electrons.[11] Therefore,
these particle-like properties of X-rays, such as their ionization of gases, caused William
Henry Bragg to argue in 1907 that X-rays were not electromagnetic radiation.[12][13][14][15]
Nevertheless, Bragg's view was not broadly accepted and the observation of X-ray
diffraction in 1912[16] confirmed for most scientists that X-rays were a form of
electromagnetic radiation.

[edit] X-ray analysis of crystals

The incoming beam (coming from upper left) causes each scatterer to re-radiate a small
portion of its intensity as a spherical wave. If scatterers are arranged symmetrically with a
separation d, these spherical waves will be in sync (add constructively) only in directions
where their path-length difference 2d sin equals an integer multiple of the wavelength . In

that case, part of the incoming beam is deflected by an angle 2, producing a reflection spot
in the diffraction pattern.
Crystals are regular arrays of atoms, and X-rays can be considered waves of electromagnetic
radiation. Atoms scatter X-ray waves, primarily through the atoms' electrons. Just as an ocean
wave striking a lighthouse produces secondary circular waves emanating from the lighthouse,
so an X-ray striking an electron produces secondary spherical waves emanating from the
electron. This phenomenon is known as elastic scattering, and the electron (or lighthouse) is
known as the scatterer. A regular array of scatterers produces a regular array of spherical
waves. Although these waves cancel one another out in most directions through destructive
interference, they add constructively in a few specific directions, determined by Bragg's
law:

Here d is the spacing between diffracting planes, is the incident angle, n is any integer, and
is the wavelength of the beam. These specific directions appear as spots on the diffraction
pattern called reflections. Thus, X-ray diffraction results from an electromagnetic wave (the
X-ray) impinging on a regular array of scatterers (the repeating arrangement of atoms within
the crystal).
X-rays are used to produce the diffraction pattern because their wavelength is typically the
same order of magnitude (1-100 ngstrms) as the spacing d between planes in the crystal.
In principle, any wave impinging on a regular array of scatterers produces diffraction, as
predicted first by Francesco Maria Grimaldi in 1665. To produce significant diffraction, the
spacing between the scatterers and the wavelength of the impinging wave should be similar in
size. For illustration, the diffraction of sunlight through a bird's feather was first reported by
James Gregory in the later 17th century. The first artificial diffraction gratings for visible
light were constructed by David Rittenhouse in 1787, and Joseph von Fraunhofer in
1821. However, visible light has too long a wavelength (typically, 5500 ngstrms) to
observe diffraction from crystals. Prior to the first X-ray diffraction experiments, the spacings
between lattice planes in a crystal were not known with certainty.
The idea that crystals could be used as a diffraction grating for X-rays arose in 1912 in a
conversation between Paul Peter Ewald and Max von Laue in the English Garden in
Munich. Ewald had proposed a resonator model of crystals for his thesis, but this model
could not be validated using visible light, since the wavelength was much larger than the
spacing between the resonators. Von Laue realized that electromagnetic radiation of a shorter
wavelength was needed to observe such small spacings, and suggested that X-rays might
have a wavelength comparable to the unit-cell spacing in crystals. Von Laue worked with two
technicians, Walter Friedrich and his assistant Paul Knipping, to shine a beam of X-rays
through a copper sulfate crystal and record its diffraction on a photographic plate. After
being developed, the plate showed a large number of well-defined spots arranged in a pattern
of intersecting circles around the spot produced by the central beam.[16][17] Von Laue
developed a law that connects the scattering angles and the size and orientation of the unitcell spacings in the crystal, for which he was awarded the Nobel Prize in Physics in 1914.
[18]

As described in the mathematical derivation below, the X-ray scattering is determined by

the density of electrons within the crystal. Since the energy of an X-ray is much greater than

that of a valence electron, the scattering may be modeled as Thomson scattering, the
interaction of an electromagnetic ray with a free electron. This model is generally adopted to
describe the polarization of the scattered radiation. The intensity of Thomson scattering
declines as 1/m with the mass m of the charged particle that is scattering the radiation;
hence, the atomic nuclei, which are thousands of times heavier than an electron, contribute
negligibly to the scattered X-rays.

[edit] Development from 1912 to 1920

Although diamonds (top left) and graphite (top right) are identical in chemical composition
being both pure carbon X-ray crystallography revealed the arrangement of their atoms
(bottom) accounts for their different properties. In diamond, the carbon atoms are arranged
tetrahedrally and held together by single covalent bonds, making it strong in all directions.
By contrast, graphite is composed of stacked sheets. Within the sheet, the bonding is covalent
and has hexagonal symmetry, but there are no covalent bonds between the sheets, making
graphite easy to cleave into flakes.
After Von Laue's pioneering research, the field developed rapidly, most notably by physicists
William Lawrence Bragg and his father William Henry Bragg. In 1912-1913, the younger
Bragg developed Bragg's law, which connects the observed scattering with reflections from
evenly spaced planes within the crystal.[19][20][21] The Braggs, father and son, shared the 1915
Nobel Prize in Physics for their work in crystallography. The earliest structures were
generally simple and marked by one-dimensional symmetry. However, as computational and
experimental methods improved over the next decades, it became feasible to deduce reliable
atomic positions for more complicated two- and three-dimensional arrangements of atoms in
the unit-cell.
The potential of X-ray crystallography for determining the structure of molecules and
minerals then only known vaguely from chemical and hydrodynamic experiments was
realized immediately. The earliest structures were simple inorganic crystals and minerals, but
even these revealed fundamental laws of physics and chemistry. The first atomic-resolution
structure to be "solved" (i.e. determined) in 1914 was that of table salt.[22][23][24] The
distribution of electrons in the table-salt structure showed that crystals are not necessarily
composed of covalently bonded molecules, and proved the existence of ionic compounds.
[25]
The structure of diamond was solved in the same year,[26][27] proving the tetrahedral
arrangement of its chemical bonds and showing that the length of CC single bond was 1.52
ngstrms. Other early structures included copper,[28] calcium fluoride (CaF2, also known
as fluorite), calcite (CaCO3) and pyrite (FeS2)[29] in 1914; spinel (MgAl2O4) in 1915;[30][31] the
rutile and anatase forms of titanium dioxide (TiO2) in 1916;[32] pyrochroite Mn(OH)2 and,
by extension, brucite Mg(OH)2 in 1919;[33][34]. Also in 1919 sodium nitrate (NaNO3) and

cesium dichloroiodide (CsICl2) were determined by Ralph Walter Graystone Wyckoff, and
the wurtzite (hexagonal ZnS) structure became known in 1920.[35]
The structure of graphite was solved in 1916[36] by the related method of powder diffraction,
[37]
which was developed by Peter Debye and Paul Scherrer and, independently, by Albert
Hull in 1917.[38] The structure of graphite was determined from single-crystal diffraction in
1924 by two groups independently.[39][40] Hull also used the powder method to determine the
structures of various metals, such as iron[41] and magnesium.[42]

[edit] Contributions to chemistry and material science

X-ray crystallography has led to a better understanding of chemical bonds and noncovalent interactions. The initial studies revealed the typical radii of atoms, and confirmed
many theoretical models of chemical bonding, such as the tetrahedral bonding of carbon in
the diamond structure,[26] the octahedral bonding of metals observed in ammonium
hexachloroplatinate (IV),[43] and the resonance observed in the planar carbonate group[29] and
in aromatic molecules.[44] Kathleen Lonsdale's 1928 structure of hexamethylbenzene[45]
established the hexagonal symmetry of benzene and showed a clear difference in bond
length between the aliphatic CC bonds and aromatic CC bonds; this finding led to the idea
of resonance between chemical bonds, which had profound consequences for the
development of chemistry.[46] Her conclusions were anticipated by William Henry Bragg,
who published models of naphthalene and anthracene in 1921 based on other molecules, an
early form of molecular replacement.[44][47]
Also in the 1920s, Victor Moritz Goldschmidt and later Linus Pauling developed rules for
eliminating chemically unlikely structures and for determining the relative sizes of atoms.
These rules led to the structure of brookite (1928) and an understanding of the relative
stability of the rutile, brookite and anatase forms of titanium dioxide.
The distance between two bonded atoms is a sensitive measure of the bond strength and its
bond order; thus, X-ray crystallographic studies have led to the discovery of even more
exotic types of bonding in inorganic chemistry, such as metal-metal double bonds,[48][49][50]
metal-metal quadruple bonds,[51][52][53] and three-center, two-electron bonds.[54] X-ray
crystallography or, strictly speaking, an inelastic Compton scattering experiment has
also provided evidence for the partly covalent character of hydrogen bonds.[55] In the field
of organometallic chemistry, the X-ray structure of ferrocene initiated scientific studies of
sandwich compounds,[56][57] while that of Zeise's salt stimulated research into "back
bonding" and metal-pi complexes.[58][59][60][61] Finally, X-ray crystallography had a pioneering
role in the development of supramolecular chemistry, particularly in clarifying the
structures of the crown ethers and the principles of host-guest chemistry.
In material sciences, many complicated inorganic and organometallic systems have been
analyzed using single-crystal methods, such as fullerenes, metalloporphyrins, and other
complicated compounds. Single-crystal diffraction is also used in the pharmaceutical
industry, due to recent problems with polymorphs. The major factors affecting the quality of
single-crystal structures are the crystal's size and regularity; recrystallization is a commonly
used technique to improve these factors in small-molecule crystals. The Cambridge
Structural Database contains over 500,000 structures; over 99% of these structures were
determined by X-ray diffraction.

[edit] Mineralogy and metallurgy

Since the 1920s, X-ray diffraction has been the principal method for determining the
arrangement of atoms in minerals and metals. The application of X-ray crystallography to
mineralogy began with the structure of garnet, which was determined in 1924 by Menzer. A
systematic X-ray crystallographic study of the silicates was undertaken in the 1920s. This
study showed that, as the Si/O ratio is altered, the silicate crystals exhibit significant changes
in their atomic arrangements. Machatschki extended these insights to minerals in which
aluminium substitutes for the silicon atoms of the silicates. The first application of X-ray
crystallography to metallurgy likewise occurred in the mid-1920s.[62][63][64][65][66][67] Most
notably, Linus Pauling's structure of the alloy Mg2Sn[68] led to his theory of the stability and
structure of complex ionic crystals.[69]

[edit] Early organic and small biological molecules

The three-dimensional structure of penicillin, for which Dorothy Crowfoot Hodgkin was
awarded the Nobel Prize in Chemistry in 1964. The green, white, red, yellow and blue
spheres represent atoms of carbon, hydrogen, oxygen, sulfur and nitrogen, respectively.
The first structure of an organic compound, hexamethylenetetramine, was solved in 1923.
[70]
This was followed by several studies of long-chain fatty acids, which are an important
component of biological membranes.[71][72][73][74][75][76][77][78][79] In the 1930s, the structures of
much larger molecules with two-dimensional complexity began to be solved. A significant
advance was the structure of phthalocyanine,[80] a large planar molecule that is closely
related to porphyrin molecules important in biology, such as heme, corrin and chlorophyll.
X-ray crystallography of biological molecules took off with Dorothy Crowfoot Hodgkin,
who solved the structures of cholesterol (1937), vitamin B12 (1945) and penicillin (1954),
for which she was awarded the Nobel Prize in Chemistry in 1964. In 1969, she succeeded
in solving the structure of insulin, on which she worked for over thirty years.[81]

Ribbon diagram of the structure of myoglobin, showing colored alpha helices. Such
proteins are long, linear molecules with thousands of atoms; yet the relative position of
each atom has been determined with sub-atomic resolution by X-ray crystallography. Since it
is difficult to visualize all the atoms at once, the ribbon shows the rough path of the protein
polymer from its N-terminus (blue) to its C-terminus (red).

[edit] Biological macromolecular crystallography

Crystal structures of proteins (which are irregular and hundreds of times larger than
cholesterol) began to be solved in the late 1950s, beginning with the structure of sperm
whale myoglobin by Max Perutz and Sir John Cowdery Kendrew, for which they were
awarded the Nobel Prize in Chemistry in 1962.[82] Since that success, over 48970 X-ray
crystal structures of proteins, nucleic acids and other biological molecules have been
determined.[83] For comparison, the nearest competing method in terms of structures analyzed
is nuclear magnetic resonance (NMR) spectroscopy, which has resolved 7806 chemical
structures.[84] Moreover, crystallography can solve structures of arbitrarily large molecules,
whereas solution-state NMR is restricted to relatively small ones (less than 70 kDa). X-ray
crystallography is now used routinely by scientists to determine how a pharmaceutical drug
interacts with its protein target and what changes might improve it.[85] However, intrinsic
membrane proteins remain challenging to crystallize because they require detergents or other
means to solubilize them in isolation, and such detergents often interfere with crystallization.
Such membrane proteins are a large component of the genome and include many proteins of
great physiological importance, such as ion channels and receptors.[86][87]

[edit] Relationship to other scattering techniques

Further information: X-ray scattering techniques

[edit] Elastic vs. inelastic scattering

X-ray crystallography is a form of elastic scattering; the outgoing X-rays have the same
energy, and thus same wavelength, as the incoming X-rays, only with altered direction. By
contrast, inelastic scattering occurs when energy is transferred from the incoming X-ray to
the crystal, e.g., by exciting an inner-shell electron to a higher energy level. Such inelastic
scattering reduces the energy (or increases the wavelength) of the outgoing beam. Inelastic

scattering is useful for probing such excitations of matter, but not in determining the
distribution of scatterers within the matter, which is the goal of X-ray crystallography.
X-rays range in wavelength from 10 to 0.01 nanometers; a typical wavelength used for
crystallography is 1 (0.1 nm), which is on the scale of covalent chemical bonds and the
radius of a single atom. Longer-wavelength photons (such as ultraviolet radiation) would not
have sufficient resolution to determine the atomic positions. At the other extreme, shorterwavelength photons such as gamma rays are difficult to produce in large numbers, difficult
to focus, and interact too strongly with matter, producing particle-antiparticle pairs.
Therefore, X-rays are the "sweetspot" for wavelength when determining atomic-resolution
structures from the scattering of electromagnetic radiation.

[edit] Other X-ray techniques

Other forms of elastic X-ray scattering include powder diffraction, SAXS and several types
of X-ray fiber diffraction, which was used by Rosalind Franklin in determining the doublehelix structure of DNA. In general, single-crystal X-ray diffraction offers more structural
information than these other techniques; however, it requires a sufficiently large and regular
crystal, which is not always available.
These scattering methods generally use monochromatic X-rays, which are restricted to a
single wavelength with minor deviations. A broad spectrum of X-rays (that is, a blend of Xrays with different wavelengths) can also be used to carry out X-ray diffraction, a technique
known as the Laue method. This is the method used in the original discovery of X-ray
diffraction. Laue scattering provides much structural information with only a short exposure
to the X-ray beam, and is therefore used in structural studies of very rapid events (Time
resolved crystallography). However, it is not as well-suited as monochromatic scattering
for determining the full atomic structure of a crystal and therefore works better with crystals
with relatively simple atomic arrangements.
The Laue back reflection mode records X-rays scattered backwards from a broad spectrum
source. This is useful if the sample is too thick for X-rays to transmit through it. The
diffracting planes in the crystal are determined by knowing that the normal to the diffracting
plane bisects the angle between the incident beam and the diffracted beam. A Greninger chart
can be used [88] to interpret the back reflection Laue photograph.

[edit] Electron and neutron diffraction

Other particles, such as electrons and neutrons, may be used to produce a diffraction
pattern. Although electron, neutron, and X-ray scattering are based on different physical
processes, the resulting diffraction patterns are analyzed using the same coherent diffraction
imaging techniques.
As derived below, the electron density within the crystal and the diffraction patterns are
related by a simple mathematical method, the Fourier transform, which allows the density to
be calculated relatively easily from the patterns. However, this works only if the scattering is
weak, i.e., if the scattered beams are much less intense than the incoming beam. Weakly
scattered beams pass through the remainder of the crystal without undergoing a second
scattering event. Such re-scattered waves are called "secondary scattering" and hinder the
analysis. Any sufficiently thick crystal will produce secondary scattering, but since X-rays

interact relatively weakly with the electrons, this is

generally not a significant concern. By contrast, electron
beams may produce strong secondary scattering even for
relatively thin crystals (>100 nm). Since this thickness
corresponds to the diameter of many viruses, a
promising direction is the electron diffraction of isolated
macromolecular assemblies, such as viral capsids and
molecular machines, which may be carried out with a
cryo-electron microscope. Moreover the strong
interaction of electrons with matter (about 1000 times
stronger than for X-rays) allows determination of the
atomic structure of extremely small volumes. The field of
applications for electron crystallography ranges from
bio molecules like membrane proteins over organic thin
films to the complex structures of (nanocrystalline)
intermetallic compounds and zeolites.
Neutron diffraction is an excellent method for structure determination, although it has been
difficult to obtain intense, monochromatic beams of neutrons in sufficient quantities.
Traditionally, nuclear reactors have been used, although the new Spallation Neutron
Source holds much promise in the near future. Being uncharged, neutrons scatter much more
readily from the atomic nuclei rather than from the electrons. Therefore, neutron scattering is
very useful for observing the positions of light atoms with few electrons, especially
hydrogen, which is essentially invisible in the X-ray diffraction. Neutron scattering also has
the remarkable property that the solvent can be made invisible by adjusting the ratio of
normal water, H2O, and heavy water, D2O.

[edit] Methods
[edit] Overview of single-crystal X-ray diffraction
Workflow for solving the structure of a molecule by X-ray crystallography.
The oldest and most precise method of X-ray crystallography is single-crystal X-ray
diffraction, in which a beam of X-rays strikes a single crystal, producing scattered beams.
When they land on a piece of film or other detector, these beams make a diffraction pattern of
spots; the strengths and angles of these beams are recorded as the crystal is gradually rotated.
[89]
Each spot is called a reflection, since it corresponds to the reflection of the X-rays from
one set of evenly spaced planes within the crystal. For single crystals of sufficient purity and
regularity, X-ray diffraction data can determine the mean chemical bond lengths and angles to
within a few thousandths of an ngstrm and to within a few tenths of a degree,
respectively. The atoms in a crystal are not static, but oscillate about their mean positions,
usually by less than a few tenths of an ngstrm. X-ray crystallography allows measuring the
size of these oscillations.

[edit] Procedure
The technique of single-crystal X-ray crystallography has three basic steps. The first and
often most difficult step is to obtain an adequate crystal of the material under study. The
crystal should be sufficiently large (typically larger than 0.1 mm in all dimensions), pure in
composition and regular in structure, with no significant internal imperfections such as
cracks or twinning.
In the second step, the crystal is placed in an intense beam of X-rays, usually of a single
wavelength (monochromatic X-rays), producing the regular pattern of reflections. As the
crystal is gradually rotated, previous reflections disappear and new ones appear; the intensity
of every spot is recorded at every orientation of the crystal. Multiple data sets may have to be
collected, with each set covering slightly more than half a full rotation of the crystal and
typically containing tens of thousands of reflections.
In the third step, these data are combined computationally with complementary chemical
information to produce and refine a model of the arrangement of atoms within the crystal.
The final, refined model of the atomic arrangement now called a crystal structure is
usually stored in a public database.

[edit] Limitations
See also: Resolution (electron density)
As the crystal's repeating unit, its unit cell, becomes larger and more complex, the atomiclevel picture provided by X-ray crystallography becomes less well-resolved (more "fuzzy")
for a given number of observed reflections. Two limiting cases of X-ray crystallography
"small-molecule" and "macromolecular" crystallographyare often discerned. Smallmolecule crystallography typically involves crystals with fewer than 100 atoms in their
asymmetric unit; such crystal structures are usually so well resolved that the atoms can be
discerned as isolated "blobs" of electron density. By contrast, macromolecular
crystallography often involves tens of thousands of atoms in the unit cell. Such crystal
structures are generally less well-resolved (more "smeared out"); the atoms and chemical
bonds appear as tubes of electron density, rather than as isolated atoms. In general, small
molecules are also easier to crystallize than macromolecules; however, X-ray crystallography
has proven possible even for viruses with hundreds of thousands of atoms.

[edit] Crystallization
Further information: crystallization, recrystallization, and Protein Crystallization

A protein crystal seen under a microscope. Crystals used in X-ray crystallography may be
smaller than a millimeter across.

Although crystallography can be used to characterize the disorder in an impure or irregular

crystal, crystallography generally requires a pure crystal of high regularity to solve the
structure of a complicated arrangement of atoms. Pure, regular crystals can sometimes be
obtained from natural or synthetic materials, such as samples of metals, minerals or other
macroscopic materials. The regularity of such crystals can sometimes be improved with
macromolecular crystal annealing [90][91][92] and other methods. However, in many cases,
obtaining a diffraction-quality crystal is the chief barrier to solving its atomic-resolution
structure.[93]
Small-molecule and macromolecular crystallography differ in the range of possible
techniques used to produce diffraction-quality crystals. Small molecules generally have few
degrees of conformational freedom, and may be crystallized by a wide range of methods,
such as chemical vapor deposition and recrystallization. By contrast, macromolecules
generally have many degrees of freedom and their crystallization must be carried out to
maintain a stable structure. For example, proteins and larger RNA molecules cannot be
crystallized if their tertiary structure has been unfolded; therefore, the range of crystallization
conditions is restricted to solution conditions in which such molecules remain folded.

Three methods of preparing crystals, A: Hanging drop. B: Sitting drop. C: Microdialysis

Protein crystals are almost always grown in solution. The most common approach is to lower
the solubility of its component molecules very gradually; if this is done too quickly, the
molecules will precipitate from solution, forming a useless dust or amorphous gel on the
bottom of the container. Crystal growth in solution is characterized by two steps: nucleation
of a microscopic crystallite (possibly having only 100 molecules), followed by growth of that
crystallite, ideally to a diffraction-quality crystal.[94] The solution conditions that favor the

first step (nucleation) are not always the same conditions that favor the second step
(subsequent growth). The crystallographer's goal is to identify solution conditions that favor
the development of a single, large crystal, since larger crystals offer improved resolution of
the molecule. Consequently, the solution conditions should disfavor the first step (nucleation)
but favor the second (growth), so that only one large crystal forms per droplet. If nucleation is
favored too much, a shower of small crystallites will form in the droplet, rather than one large
crystal; if favored too little, no crystal will form whatsoever.
It is extremely difficult to predict good conditions for nucleation or growth of well-ordered
crystals.[95] In practice, favorable conditions are identified by screening; a very large batch of
the molecules is prepared, and a wide variety of crystallization solutions are tested.[96]
Hundreds, even thousands, of solution conditions are generally tried before finding the
successful one. The various conditions can use one or more physical mechanisms to lower the
solubility of the molecule; for example, some may change the pH, some contain salts of the
Hofmeister series or chemicals that lower the dielectric constant of the solution, and still
others contain large polymers such as polyethylene glycol that drive the molecule out of
solution by entropic effects. It is also common to try several temperatures for encouraging
crystallization, or to gradually lower the temperature so that the solution becomes
supersaturated. These methods require large amounts of the target molecule, as they use high
concentration of the molecule(s) to be crystallized. Due to the difficulty in obtaining such
large quantities (milligrams) of crystallization grade protein, robots have been developed that
are capable of accurately dispensing crystallization trial drops that are in the order of 100
nanoliters in volume. This means that 10-fold less protein is used per-experiment when
compared to crystallization trials setup by hand (in the order of 1 microliter).[97]
Several factors are known to inhibit or mar crystallization. The growing crystals are generally
held at a constant temperature and protected from shocks or vibrations that might disturb their
crystallization. Impurities in the molecules or in the crystallization solutions are often
inimical to crystallization. Conformational flexibility in the molecule also tends to make
crystallization less likely, due to entropy. Ironically, molecules that tend to self-assemble into
regular helices are often unwilling to assemble into crystals. Crystals can be marred by
twinning, which can occur when a unit cell can pack equally favorably in multiple
orientations; although recent advances in computational methods may allow solving the
structure of some twinned crystals. Having failed to crystallize a target molecule, a
crystallographer may try again with a slightly modified version of the molecule; even small
changes in molecular properties can lead to large differences in crystallization behavior.

[edit] Data collection

[edit] Mounting the crystal

Animation showing the five motions possible with a four-circle kappa goniometer. The
rotations about each of the four angles , , and 2 leave the crystal within the X-ray beam,
but change the crystal orientation. The detector (red box) can be slid closer or further away
from the crystal, allowing higher resolution data to be taken (if closer) or better discernment
of the Bragg peaks (if further away).
The crystal is mounted for measurements so that it may be held in the X-ray beam and
rotated. There are several methods of mounting. Although crystals were once loaded into
glass capillaries with the crystallization solution (the mother liquor), a modern approach is to
scoop the crystal up in a tiny loop, made of nylon or plastic and attached to a solid rod, that is
then flash-frozen with liquid nitrogen.[98] This freezing reduces the radiation damage of the
X-rays, as well as the noise in the Bragg peaks due to thermal motion (the Debye-Waller
effect). However, untreated crystals often crack if flash-frozen; therefore, they are generally
pre-soaked in a cryoprotectant solution before freezing.[99] Unfortunately, this pre-soak may
itself cause the crystal to crack, ruining it for crystallography. Generally, successful cryoconditions are identified by trial and error.
The capillary or loop is mounted on a goniometer, which allows it to be positioned
accurately within the X-ray beam and rotated. Since both the crystal and the beam are often
very small, the crystal must be centered within the beam to within ~25 micrometers accuracy,
which is aided by a camera focused on the crystal. The most common type of goniometer is
the "kappa goniometer", which offers three angles of rotation: the angle, which rotates
about an axis perpendicular to the beam; the angle, about an axis at ~50 to the axis; and,
finally, the angle about the loop/capillary axis. When the angle is zero, the and axes
are aligned. The rotation allows for convenient mounting of the crystal, since the arm in
which the crystal is mounted may be swung out towards the crystallographer. The oscillations
carried out during data collection (mentioned below) involve the axis only. An older type of
goniometer is the four-circle goniometer, and its relatives such as the six-circle goniometer.

[edit] X-ray sources

Further information: Diffractometer and Synchrotron
The mounted crystal is then irradiated with a beam of monochromatic X-rays. The brightest
and most useful X-ray sources are synchrotrons; their much higher luminosity allows for
better resolution. They also make it convenient to tune the wavelength of the radiation, which
is useful for multi-wavelength anomalous dispersion (MAD) phasing, described below.
Synchrotrons are generally national facilities, each with several dedicated beamlines where
data is collected around the clock, seven days a week.

A diffractometer
Smaller, X-ray generators are often used in laboratories to check the quality of crystals
before bringing them to a synchrotron and sometimes to solve a crystal structure. In such
systems, electrons are boiled off of a cathode and accelerated through a strong electric
potential of ~50 kV; having reached a high speed, the electrons collide with a metal plate,
emitting bremsstrahlung and some strong spectral lines corresponding to the excitation of
inner-shell electrons of the metal. The most common metal used is copper, which can be
kept cool easily, due to its high thermal conductivity, and which produces strong K and K
lines. The K line is sometimes suppressed with a thin (~10 m) nickel foil. The simplest and
cheapest variety of sealed X-ray tube has a stationary anode (the Crookes tube) and
produces ~2 kW of X-ray radiation. The more expensive variety has a rotating-anode type
source that produces ~14 kW of X-ray radiation.
X-rays are generally filtered (by use of X-Ray Filters) to a single wavelength (made
monochromatic) and collimated to a single direction before they are allowed to strike the
crystal. The filtering not only simplifies the data analysis, but also removes radiation that
degrades the crystal without contributing useful information. Collimation is done either with
a collimator (basically, a long tube) or with a clever arrangement of gently curved mirrors.
Mirror systems are preferred for small crystals (under 0.3 mm) or with large unit cells (over
150 )

[edit] Recording the reflections

An X-ray diffraction pattern of a crystallized enzyme. The pattern of spots (called reflections)
can be used to determine the structure of the enzyme.

When a crystal is mounted and exposed to an intense beam of X-rays, it scatters the X-rays
into a pattern of spots or reflections that can be observed on a screen behind the crystal. A
similar pattern may be seen by shining a laser pointer at a compact disc. The relative
intensities of these spots provide the information to determine the arrangement of molecules
within the crystal in atomic detail. The intensities of these reflections may be recorded with
photographic film, an area detector or with a charge-coupled device (CCD) image sensor.
The peaks at small angles correspond to low-resolution data, whereas those at high angles
represent high-resolution data; thus, an upper limit on the eventual resolution of the structure
can be determined from the first few images. Some measures of diffraction quality can be
determined at this point, such as the mosaicity of the crystal and its overall disorder, as
observed in the peak widths. Some pathologies of the crystal that would render it unfit for
solving the structure can also be diagnosed quickly at this point.
One image of spots is insufficient to reconstruct the whole crystal; it represents only a small
slice of the full Fourier transform. To collect all the necessary information, the crystal must
be rotated step-by-step through 180, with an image recorded at every step; actually, slightly
more than 180 is required to cover reciprocal space, due to the curvature of the Ewald
sphere. However, if the crystal has a higher symmetry, a smaller angular range such as 90
or 45 may be recorded. The rotation axis should be changed at least once, to avoid
developing a "blind spot" in reciprocal space close to the rotation axis. It is customary to rock
the crystal slightly (by 0.5-2) to catch a broader region of reciprocal space.
Multiple data sets may be necessary for certain phasing methods. For example, MAD phasing
requires that the scattering be recorded at least three (and usually four, for redundancy)
wavelengths of the incoming X-ray radiation. A single crystal may degrade too much during
the collection of one data set, owing to radiation damage; in such cases, data sets on multiple
crystals must be taken.[100]

[edit] Data analysis

[edit] Crystal symmetry, unit cell, and image scaling

Further information: Space group
The recorded series of two-dimensional diffraction patterns, each corresponding to a different
crystal orientation, is converted into a three-dimensional model of the electron density; the
conversion uses the mathematical technique of Fourier transforms, which is explained below.
Each spot corresponds to a different type of variation in the electron density; the
crystallographer must determine which variation corresponds to which spot (indexing), the
relative strengths of the spots in different images (merging and scaling) and how the
variations should be combined to yield the total electron density (phasing).
Data processing begins with indexing the reflections. This means identifying the dimensions
of the unit cell and which image peak corresponds to which position in reciprocal space. A
byproduct of indexing is to determine the symmetry of the crystal, i.e., its space group.
Some space groups can be eliminated from the beginning. For example, reflection
symmetries cannot be observed in chiral molecules; thus, only 65 space groups of 243
possible are allowed for protein molecules which are almost always chiral. Indexing is
generally accomplished using an autoindexing routine.[101] Having assigned symmetry, the
data is then integrated. This converts the hundreds of images containing the thousands of
reflections into a single file, consisting of (at the very least) records of the Miller index of

each reflection, and an intensity for each reflection (at this state the file often also includes
error estimates and measures of partiality (what part of a given reflection was recorded on
that image)).
A full data set may consist of hundreds of separate images taken at different orientations of
the crystal. The first step is to merge and scale these various images, that is, to identify which
peaks appear in two or more images (merging) and to scale the relative images so that they
have a consistent intensity scale. Optimizing the intensity scale is critical because the relative
intensity of the peaks is the key information from which the structure is determined. The
repetitive technique of crystallographic data collection and the often high symmetry of
crystalline materials cause the diffractometer to record many symmetry-equivalent reflections
multiple times. This allows calculating the symmetry related R-factor based upon how
similar are the measured intensities of symmetry equivalent reflections, thus assessing the
quality of the data.

[edit] Initial phasing

Further information: Phase problem
The data collected from a diffraction experiment is a reciprocal space representation of the
crystal lattice. The position of each diffraction 'spot' is governed by the size and shape of the
unit cell, and the inherent symmetry within the crystal. The intensity of each diffraction 'spot'
is recorded, and this intensity is proportional to the square of the structure factor amplitude.
The structure factor is a complex number containing information relating to both the
amplitude and phase of a wave. In order to obtain an interpretable electron density map,
both amplitude and phase must be known (an electron density map allows a crystallographer
to build a starting model of the molecule). The phase cannot be directly recorded during a
diffraction experiment: this is known as the phase problem. Initial phase estimates can be
obtained in a variety of ways:

Ab initio phasing or direct methods - This is usually the method of choice for
small molecules (<1000 non-hydrogen atoms), and has been used successfully to
solve the phase problems for small proteins. If the resolution of the data is better than
1.4 (140 pm), direct methods can be used to obtain phase information, by
exploiting known phase relationships between certain groups of reflections.[102][103]

Molecular replacement - if a related structure is known, it can be used as a search

model in molecular replacement to determine the orientation and position of the
molecules within the unit cell. The phases obtained this way can be used to generate
electron density maps.[104]

Anomalous X-ray scattering (MAD or SAD phasing) - the X-ray wavelength may
be scanned past an absorption edge of an atom, which changes the scattering in a
known way. By recording full sets of reflections at three different wavelengths (far
below, far above and in the middle of the absorption edge) one can solve for the
substructure of the anomalously diffracting atoms and thence the structure of the
whole molecule. The most popular method of incorporating anomalous scattering
atoms into proteins is to express the protein in a methionine auxotroph (a host
incapable of synthesizing methionine) in a media rich in seleno-methionine, which
contains selenium atoms. A MAD experiment can then be conducted around the

absorption edge, which should then yield the position of any methionine residues
within the protein, providing initial phases.[105]

Heavy atom methods (multiple isomorphous replacement) - If electron-dense

metal atoms can be introduced into the crystal, direct methods or Patterson-space
methods can be used to determine their location and to obtain initial phases. Such
heavy atoms can be introduced either by soaking the crystal in a heavy atomcontaining solution, or by co-crystallization (growing the crystals in the presence of a
heavy atom). As in MAD phasing, the changes in the scattering amplitudes can be
interpreted to yield the phases. Although this is the original method by which protein
crystal structures were solved, it has largely been superseded by MAD phasing with
selenomethionine.[104]

[edit] Model building and phase refinement

A protein crystal structure at 2.7 resolution. The mesh encloses the region in which the
electron density exceeds a given threshold. The straight segments represent chemical bonds
between the non-hydrogen atoms of an arginine (upper left), a tyrosine (lower left), a
disulfide bond (upper right, in yellow), and some peptide groups (running left-right in the
middle). The two curved green tubes represent spline fits to the polypeptide backbone.
Further information: Molecular modeling
Having obtained initial phases, an initial model can be built. This model can be used to refine
the phases, leading to an improved model, and so on. Given a model of some atomic
positions, these positions and their respective Debye-Waller factors (or B-factors,
accounting for the thermal motion of the atom) can be refined to fit the observed diffraction
data, ideally yielding a better set of phases. A new model can then be fit to the new electron
density map and a further round of refinement is carried out. This continues until the
correlation between the diffraction data and the model is maximized. The agreement is
measured by an R-factor defined as

A similar quality criterion is Rfree, which is calculated from a subset (~10%) of reflections that
were not included in the structure refinement. Both R factors depend on the resolution of the
data. As a rule of thumb, Rfree should be approximately the resolution in ngstrms divided
by 10; thus, a data-set with 2 resolution should yield a final Rfree ~ 0.2. Chemical bonding
features such as stereochemistry, hydrogen bonding and distribution of bond lengths and
angles are complementary measures of the model quality. Phase bias is a serious problem in
such iterative model building. Omit maps are a common technique used to check for this.
[clarification needed]

It may not be possible to observe every atom of the crystallized molecule - it must be
remembered that the resulting electron density is an average of all the molecules within the
crystal. In some cases, there is too much residual disorder in those atoms, and the resulting
electron density for atoms existing in many conformations is smeared to such an extent that it
is no longer detectable in the electron density map. Weakly scattering atoms such as hydrogen
are routinely invisible. It is also possible for a single atom to appear multiple times in an
electron density map, e.g., if a protein sidechain has multiple (<4) allowed conformations. In
still other cases, the crystallographer may detect that the covalent structure deduced for the
molecule was incorrect, or changed. For example, proteins may be cleaved or undergo posttranslational modifications that were not detected prior to the crystallization.

[edit] Deposition of the structure

Once the model of a molecule's structure has been finalized, it is often deposited in a
crystallographic database such as the Cambridge Structural Database (for small
molecules) or the Protein Data Bank (for protein structures). Many structures obtained in
private commercial ventures to crystallize medicinally relevant proteins, are not deposited in
public crystallographic databases.

[edit] Diffraction theory

Further information: Dynamical theory of diffraction and Bragg diffraction
The main goal of X-ray crystallography is to determine the density of electrons f(r)
throughout the crystal, where r represents the three-dimensional position vector within the
crystal. To do this, X-ray scattering is used to collect data about its Fourier transform F(q),
which is inverted mathematically to obtain the density defined in real space, using the
formula

where the integral is taken over all values of q. The three-dimensional real vector q
represents a point in reciprocal space, that is, to a particular oscillation in the electron
density as one moves in the direction in which q points. The length of q corresponds to 2
divided by the wavelength of the oscillation. The corresponding formula for a Fourier
transform will be used below

where the integral is summed over all possible values of the position vector r within the
crystal.
The Fourier transform F(q) is generally a complex number, and therefore has a magnitude
|F(q)| and a phase (q) related by the equation

The intensities of the reflections observed in X-ray diffraction give us the magnitudes |F(q)|
but not the phases (q). To obtain the phases, full sets of reflections are collected with known
alterations to the scattering, either by modulating the wavelength past a certain absorption
edge or by adding strongly scattering (i.e., electron-dense) metal atoms such as mercury.
Combining the magnitudes and phases yields the full Fourier transform F(q), which may be
inverted to obtain the electron density f(r).
Crystals are often idealized as being perfectly periodic. In that ideal case, the atoms are
positioned on a perfect lattice, the electron density is perfectly periodic, and the Fourier
transform F(q) is zero except when q belongs to the reciprocal lattice (the so-called Bragg
peaks). In reality, however, crystals are not perfectly periodic; atoms vibrate about their mean
position, and there may be disorder of various types, such as mosaicity, dislocations, various
point defects, and heterogeneity in the conformation of crystallized molecules. Therefore, the
Bragg peaks have a finite width and there may be significant diffuse scattering, a continuum
of scattered X-rays that fall between the Bragg peaks.

[edit] Intuitive understanding by Bragg's law

An intuitive understanding of X-ray diffraction can be obtained from the Bragg model of
diffraction. In this model, a given reflection is associated with a set of evenly spaced sheets
running through the crystal, usually passing through the centers of the atoms of the crystal
lattice. The orientation of a particular set of sheets is identified by its three Miller indices (h,
k, l), and let their spacing be noted by d. William Lawrence Bragg proposed a model in which
the incoming X-rays are scattered specularly (mirror-like) from each plane; from that
assumption, X-rays scattered from adjacent planes will combine constructively (constructive
interference) when the angle between the plane and the X-ray results in a path-length
difference that is an integer multiple n of the X-ray wavelength .

A reflection is said to be indexed when its Miller indices (or, more correctly, its reciprocal
lattice vector components) have been identified from the known wavelength and the
scattering angle 2. Such indexing gives the unit-cell parameters, the lengths and angles of
the unit-cell, as well as its space group. Since Bragg's law does not interpret the relative
intensities of the reflections, however, it is generally inadequate to solve for the arrangement
of atoms within the unit-cell; for that, a Fourier transform method must be carried out.

[edit] Scattering as a Fourier transform

The incoming X-ray beam has a polarization and should be represented as a vector wave;
however, for simplicity, let it be represented here as a scalar wave. We also ignore the

complication of the time dependence of the wave and just focus on the wave's spatial
dependence. Plane waves can be represented by a wave vector kin, and so the strength of the
incoming wave at time t=0 is given by

At position r within the sample, let there be a density of scatterers f(r); these scatterers should
produce a scattered spherical wave of amplitude proportional to the local amplitude of the
incoming wave times the number of scatterers in a small volume dV about r

where S is the proportionality constant.

Let's consider the fraction of scattered waves that leave with an outgoing wave-vector of kout
and strike the screen at rscreen. Since no energy is lost (elastic, not inelastic scattering), the
wavelengths are the same as are the magnitudes of the wave-vectors |kin|=|kout|. From the time
that the photon is scattered at r until it is absorbed at rscreen, the photon undergoes a change in
phase

The net radiation arriving at rscreen is the sum of all the scattered waves throughout the crystal

which may be written as a Fourier transform

where q = kout - kin. The measured intensity of the reflection will be square of this amplitude

[edit] Friedel and Bijvoet mates

For every reflection corresponding to a point q in the reciprocal space, there is another
reflection of the same intensity at the opposite point -q. This opposite reflection is known as
the Friedel mate of the original reflection. This symmetry results from the mathematical fact
that the density of electrons f(r) at a position r is always a real number. As noted above, f(r)
is the inverse transform of its Fourier transform F(q); however, such an inverse transform is a
complex number in general. To ensure that f(r) is real, the Fourier transform F(q) must be
such that the Friedel mates F(q) and F(q) are complex conjugates of one another. Thus,
F(q) has the same magnitude as F(q) but they have the opposite phase, i.e., (q) = (q)

The equality of their magnitudes ensures that the Friedel mates have the same intensity |F|2.
This symmetry allows one to measure the full Fourier transform from only half the reciprocal
space, e.g., by rotating the crystal slightly more than a 180, instead of a full turn. In crystals
with significant symmetry, even more reflections may have the same intensity (Bijvoet
mates); in such cases, even less of the reciprocal space may need to be measured, e.g.,
slightly more than 90.
The Friedel-mate constraint can be derived from the definition of the inverse Fourier
transform

Since Euler's formula states that eix = cos(x) + i sin(x), the inverse Fourier transform can be
separated into a sum of a purely real part and a purely imaginary part

The function f(r) is real if and only if the second integral Isin is zero for all values of r. In turn,
this is true if and only if the above constraint is satisfied

since Isin = Isin implies that Isin=0.

Each X-ray diffraction image represents only a slice, a spherical slice of reciprocal space, as
may be seen by the Ewald sphere construction. Both kout and kin have the same length, due to
the elastic scattering, since the wavelength has not changed. Therefore, they may be
represented as two radial vectors in a sphere in reciprocal space, which shows the values of
q that are sampled in a given diffraction image. Since there is a slight spread in the incoming
wavelengths of the incoming X-ray beam, the values of|F(q)|can be measured only for q
vectors located between the two spheres corresponding to those radii. Therefore, to obtain a
full set of Fourier transform data, it is necessary to rotate the crystal through slightly more
than 180, or sometimes less if sufficient symmetry is present. A full 360 rotation is not
needed because of a symmetry intrinsic to the Fourier transforms of real functions (such as
the electron density), but "slightly more" than 180 is needed to cover all of reciprocal space
within a given resolution because of the curvature of the Ewald sphere. In practice, the
crystal is rocked by a small amount (0.25-1) to incorporate reflections near the boundaries of
the spherical Ewald shells.
A well-known result of Fourier transforms is the autocorrelation theorem, which states that
the autocorrelation c(r) of a function f(r)

has a Fourier transform C(q) that is the squared magnitude of F(q)

Therefore, the autocorrelation function c(r) of the electron density (also known as the
Patterson function[106]) can be computed directly from the reflection intensities, without
computing the phases. In principle, this could be used to determine the crystal structure
directly; however, it is difficult to realize in practice. The autocorrelation function
corresponds to the distribution of vectors between atoms in the crystal; thus, a crystal of N
atoms in its unit cell may have N(N-1) peaks in its Patterson function. Given the inevitable
errors in measuring the intensities, and the mathematical difficulties of reconstructing atomic
positions from the interatomic vectors, this technique is rarely used to solve structures, except
for the simplest crystals.

[edit] Advantages of a crystal

In principle, an atomic structure could be determined from applying X-ray scattering to noncrystalline samples, even to a single molecule. However, crystals offer a much stronger signal
due to their periodicity. A crystalline sample is by definition periodic; a crystal is composed
of many unit cells repeated indefinitely in three independent directions. Such periodic
systems have a Fourier transform that is concentrated at periodically repeating points in
reciprocal space known as Bragg peaks; the Bragg peaks correspond to the reflection spots
observed in the diffraction image. Since the amplitude at these reflections grows linearly with
the number N of scatterers, the observed intensity of these spots should grow quadratically,
like N. In other words, using a crystal concentrates the weak scattering of the individual unit
cells into a much more powerful, coherent reflection that can be observed above the noise.
This is an example of constructive interference.
In a liquid, powder or amorphous sample, molecules within that sample are in random
orientations. Such samples have a continuous Fourier spectrum that uniformly spreads its
amplitude thereby reducing the measured signal intensity, as is observed in SAXS. More
importantly, the orientational information is lost. Although theoretically possible, it is
experimentally difficult to obtain atomic-resolution structures of complicated, asymmetric
molecules from such rotationally averaged data. An intermediate case is fiber diffraction in
which the subunits are arranged periodically in at least one dimension.

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[edit] Further reading

[edit] International Tables for Crystallography

Theo Hahn, ed (2002). International Tables for Crystallography. Volume A, Spacegroup Symmetry (5 ed.). Dordrecht: Kluwer Academic Publishers, for the
International Union of Crystallography. ISBN 0792365909.
Michael G. Rossmann and Eddy Arnold, ed (2001). International Tables for
Crystallography. Volume F, Crystallography of biological molecules. Dordrecht:
Kluwer Academic Publishers, for the International Union of Crystallography.
ISBN 0792368576.
Theo Hahn, ed (1996). International Tables for Crystallography. Brief Teaching
Edition of Volume A, Space-group Symmetry (4 ed.). Dordrecht: Kluwer Academic
Publishers, for the International Union of Crystallography. ISBN 0792342526.

[edit] Bound collections of articles

Charles W. Carter and Robert M. Sweet., ed (1997). Macromolecular

Crystallography, Part A (Methods in Enzymology, v. 276). San Diego: Academic
Press. ISBN 0121821773.
Charles W. Carter Jr., Robert M. Sweet., ed (1997). Macromolecular Crystallography,
Part B (Methods in Enzymology, v. 277). San Diego: Academic Press.
ISBN 0121821781.
A. Ducruix and R. Gieg, ed (1999). Crystallization of Nucleic Acids and Proteins: A
Practical Approach (2 ed.). Oxford: Oxford University Press. ISBN 0199636788.

[edit] Textbooks

Blow D (2002). Outline of Crystallography for Biologists. Oxford: Oxford University

Press. ISBN 0198510519.
Burns G., Glazer A M (1990). Space Groups for Scientists and Engineers (2nd ed.).
Boston: Academic Press, Inc. ISBN 0121457613.
Clegg W (1998). Crystal Structure Determination (Oxford Chemistry Primer).
Oxford: Oxford University Press. ISBN 0198559011.
Cullity B.D. (1978). Elements of X-Ray Diffraction (2nd ed.). Reading,
Massachusetts: Addison-Wesley Publishing Company. ISBN 0534553966.
Drenth J (1999). Principles of Protein X-Ray Crystallography. New York: SpringerVerlag. ISBN 0387985875.
Giacovazzo C et al. (1992). Fundamentals of Crystallography. Oxford: Oxford
University Press. ISBN 0198555784.
Glusker JP, Lewis M, Rossi M (1994). Crystal Structure Analysis for Chemists and
Biologists. New York: VCH Publishers. ISBN 0471185434.
Massa W (2004). Crystal Structure Determination. Berlin: Springer.
ISBN 3540206442.
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5. http://en.wikipedia.org/wiki/Acid-base_titration"
Categories: Titration