Phn A: L THUYT

1. KHI NIM V PHNG PHP

Phng php quang ph t ngoi kh kin, hay cn gi l
phng php quang ph hp th, hay phng php o quang
da trn kh nng hp th chn lc cc bc x ri vo dung

dch ca cht nghin cu trong mt dung mi nht nh.

Cc bc sng cc i hp th c trng cho tng cht, hoc t l hp th gia cc bc sng l c s

nh tnh. hp th cc bc x ph thuc vo nng ca cht nghin cu trong dung dch cn o l c s

nh lng. Ngoi ra, vic o quang trong mt iu kin quy nh v dung mi, nng , bc sng cn c
c s cho php th tinh khit.

Phng php o quang ngoi kh nng phn tch cc cht trong dung dch n cht tinh khit, n cn gip
cc cht trong dung dch hn hp nhiu cht, nh s h tr ca phn mm x l vi tnh
2. I CNG V NH SNG V QUANG PH HP TH

2.1. nh sng: nh sng l mt loi bc x in t. nh sng va c bn cht sng va c bn cht h

tnh cht ca nh sng ph thuc vo bn cht sng, mt s tnh cht khc ph thuc vo bn cht ht.
Hnh 1: Bc x in t theo hng X
M- Vect t

E- Vect in

- Do nh sng c bn cht sng nn n c c trng bi mt trong cc i lng sau:

- bc sng, di sng (trong vng t ngoi kh kin hay dng n v l nm;

1nm = 10-9 m)

- Tn s (s dao ng trong mt giy)

C- Vn tc ng sng

C
= ------
Hnh 2: di sng

- Trong chn khng nh sng c tc 3,0 x 108 m/s

- Mt tia sng c th coi nh mt chm cc ht gi l lng t nh sng hay photon. Mi tia sng mang
lng nht nh ng vi cc photon mang nng lng E.

E = h. = h.(c/)
h: hng s Planck (h = 6,62.10-34JS)

2.2. Quang ph hp th

Phng php quang ph hp th da vo hin tng hp th nng lng ca cc bc x nh sng chiu v

nghin cu. Khi chiu nh sng vo mt cht n s lm thay i nng lng ca nguyn t, phn t.
2.2.1. Nng lng ca phn t:
Trong :

Ep = Et + Ee + E + Er

Ep- nng lng ca phn t

Ee- nng lng in t

Et- nng lng tnh tin

E- nng lng dao ng

Er- nng lng quay

Nu chiu tia t ngoi kh kin vo phn t n s lm thay i thnh phn Ee nn c gi l ph in t.

Nu chiu tia hng ngoi vo phn t n s lm thay i trng thi dao ng ca phn t (E ) nn c
dao ng.

2.2.2. S hp th nng lng ca phn t

Mi phn t ca mt cht c mt s trng thi nng lng xc nh v gin on. Trng thi nng lng th
l trng thi c bn. Khi hp th nng lng, phn t chuyn sang trng thi nng lng cao hn gi l trng
thch.
M + h

M*

Phn t trng thi kch thch th khng bn vng, c xu hng nhanh chng quay v trng thi nng l
u, gi l s phc hi hay quay tr v
M*

M + h (dng bc x)

M*

M + h (calo) (dng nhit)

Phn t thng trng thi kch thch rt ngn 10-9 n 10-6 giy.

Hnh 3: S cc mc nng lng

E0, E1, E2, l cc mc nng lng in t.
- ng vi mi mc nng lng in t c nhiu mc nng lng dao ng v0, v1, v2.
- ng vi mi mc nng lng dao ng c nhiu mc nng lng quay ro, r1, r2.

S hp th nng lng ca phn t c tnh cht c trng. Mun chuyn t mc nng lng E 0 ln mc n
E1 cn mt bc x c nng lng:

E = h = E1 E0

Ch nhng bc x c nng lng ng vi khong cch ca cc mc nng lng trong phn t mi c h

C nhng s chuyn mc nng lng d xy ra v cng c nhng s chuyn mc nng lng kh xy ra.

c ngha l c nhng bc x c hp th mnh, c nhng bc x c hp th yu v cng c nhng bc x

c hp th.
2.2.3. Ph hp th phn t

Phng php hp th phn t l phng php phn tch da vo nng lng ca nh sng b hp th b
ca cht nghin cu.
Ph chnh l mt dy cc i tng nghin cu c sp xp theo mt trt t nht nh.

Quang ph theo ngha hp l ch kho st hp th phn nh sng nhn thy, nhng theo ngha rng l k
ngoi vng nh sng nhn thy bao gm nh sng hng ngoi v t ngoi.

Mi phn t c mt s mc nng lng nht nh cn gi l c trng thi nng lng xc nh. Khi phn t

nng lng ca nh sng th trng thi nng lng ca phn t cng thay i t mc nng lng thp ln m
lng cao. S hp th nh sng l c trng v chn lc.

i vi cc phn t trong thc t c nhiu vi mc nng lng gn nhau to nn kh nng hp th cc b

bc sng gn nhau. V th ph hp th phn t khng phi l ph vch m l ph di hin di dng cc

th.

.3. Quang ph t ngoi kh kin

2.3.1. Mu sc ca nh sng: Tu theo bc sng m nh sng c chia thnh tng vng :
-Vng t ngoi 185 400 nm

-Vng kh kin 400 760 nm

Ring trong vng kh kin (mt nhn thy c) c nhiu mu, mi mu ng vi mt chm tia c bc
nhau:

760 630 nm

Cam

630 600 nm

Vng

600 570 nm

Lc

570 500 nm

Lam

500 450 n

Chm

450 430 nm

Tm

430 400 nm

Chm tia sng n sc l chm c mt bc sng duy nht.

2.3.2. S hp th nh sng v mu sc ca vt
S ph nhau ca cc tia sng:

Chiu hai tia sng (v d: tia vng v tia lam) vo mn nh (trng), iu chnh cng thch hp ca hai
lc thu c nh sng trng (khng mu). Ta ni hai tia (mu) trn ph nhau (hay b sung nhau).
Cc cp mu ph nhau:
Lc vng
Vng
Da cam

ta

Tm
Lam
Lam lc
Lc lam
Lc

Chng ta bit rng nh sng t nhin khi phn tch ra cha rt nhiu mu sc, nhng li khng mu v cc
ph nhau tng i mt.
Cc cht hp th nh sng mt cch chn lc:

C cc tia sng vi bc sng nht nh c hp th mnh hn cc tia khc. V th trong phn nh sn

phn x n mt ta th cng ca nhng tia ph vi nhng tia b hp th tr nn mnh hn v ta cm
ca nhng tia .
V d: nu mt vt c mu th hp th mnh mu xanh lc v ngc li
Tm li, mu ca vt v mu b vt hp th l hai mu ph nhau.
2.3.3. S hp th v cu trc phn t
- Cc cht hu c c dy ni n: - C H; - C - C - c cc in t trong lin kt n l in t
+ h

S chuyn dch in t t mc nng lng ln * yu cu mt nng lng tng ng vi cc bc x

sng 130nm .
Cc cht c dy ni i hoc ba C = C ; C C trong cc lin kt ny c c lin kt
+ h
S chuyn dch in t t mc nng lng

* hp th mc nng lng thp hn ng vi 18

Tm li: Gia cu trc ho hc v quang ph hp th c mi quan h cht ch. Ch c nhng phn t no

in t d b kch thch mi hp th nng lng nh sng chuyn t trng thi c bn sang trng thi b k

l cc phn t c cha nhng ni i, ni i lin hp, nhn thm hay nhng d t N, S, O. Mi cc

trn quang ph u c ngun gc l s chuyn trng thi ca in t trong phn t.
2.3.4. nh lut Lambert Beer

Chiu mt chm tia n sc c cng I0 qua dung dch c chiu dy 1. Sau khi b hp th, cng
cn li I .
truyn qua T = I / I0 . hp th A = - lg T = lg( I0 / I)

 hp th A (mt quang A ) ca dung dch t l thun vi nng C ca dung dch theo biu thc :
A = k . l .C
Trong : - k l h s hp th ph thuc vo cu to ca cht tan trong dung dch.
- l l chiu dy lp dung dch
+ Trng hp C tnh theo mol/l v l tnh bng cm, ta c k =
Do

A = .l.C

c gi l h s hp th phn t v A = khi C = 1mol/l v l = 1cm

c trng cho bn cht ca cht tan trong dung dch ch ph thuc bc sng nh sng n sc. nhn
c khi ngi ta vit:

A = .l.C

+ Trng hp C tnh theo phn trm (kl/tt) c biu th bng gam trong 100 ml dung dch, l theo cm, k
l h s hp th ring hoc h s tt ring, k hiu E .
A = E .l.C
A = E khi C = 1% v l = 1cm

h s hay dng trong m t cu trc, tnh cht quang ph ca cc cht hu c, cn trong phn tch kim n
hay dng E .
Cc iu kin p dng nh lut:

- nh sng n sc: Khi bc sng thay i cc h s hp th cng thay i. Mt chm tia cng n s
lut cng ng.

- Cng mt dung dch nhng o trn cc my khc nhau c th thu c cc tr s A khc nhau. C nhi
nhn nhng trc ht l do tnh n sc ca nh sng.

- Khong nng thch hp: Do nhiu nguyn nhn vt l (s phn x, s khuch tn nh sng), ho hc

ly, nh hng ca lc ion) m nh lut Lambert Beer ch ng trong mt gii hn ca nng . V vy

dng phng php nh lng cn phi kho st k trc tm khong nng .
- Cc yu t ho hc khc: Lm th no cht hp th nh sng khng b bin i bi cc phn ng ho

dung dch. V vy, pH dung dch, s c mt cc cht l c kh nng phn ng vi cht cn o hoc gy nhi
hay tng cng) s hp th nh sng ca cc cht cn o u phi tnh n.
3. MY QUANG PH T NGOI KH KIN (UVVIS)

3.1. S cu to
My o quang ph UV-Vis l mt thit b dng o hp th hoc truyn qua ca dung dch gm

phn c bn sau:
- Ngun sng: cung cp cc bc x in t
- B phn tn sc: c nhim v chn t ngun bc x mt bc sng c trng
- B phn ng mu o
- B phn detector dng o cng tia bc x

Ngoi ra cn c cc b phn khc nh l thu knh hoc gng c nhim v chuyn tip cc tia sng qua thi

3.2. Cu to chi tit v tnh nng

3.2.1. Ngun sng

Mt ngun sng l tng l ngun sng to ra cc bc x c cng khng i trn ton b khong b

vi nhiu thp v n nh trong khong thi gian di. Trn thc t ngun sng tho mn tt c cc iu ki
khng tn ti.
C hai loi ngun sng hay c s dng trong my quang ph UV-Vis:

3.2.1.1. n h quang deuterium: Sinh ra cng lin tc trong vng UV v cung cp cng c th d
trong vng Vis (hnh 4).

n h quang deuterium hin i c nhiu thp do c mt b phn hn ch nhiu trn ton b thit b

ng. Theo thi gian, cng cc tia sng pht ra t n h quang deuterium gim dn. n loi ny c th
dng khong 1000 gi (tc l thi gian m cng ca n gim cn mt na so vi ban u).
Hnh 4: Cng tia sng ca n h quang deuterium
3.2.1.2. n halogen tungsten
Sinh ra cng tt trn mt phn vng UV v ton b vng Vis (hnh 5).
Hnh 5 : Cng tia sng ca n halogen tungsten
Loi n ny c nhiu rt thp v dao ng t v c thi gian s dng khong 10.000 gi.

a s cc my quang ph dng o trong khong UV Vis c cha hai loi n. Trong cc thit b ny ho

phn chn ngun bt gia hai loi n khi cn hoc nh sng pht ra t hai ngun c trn vi nhau t
ngun c nht.

Ngoi ra cn mt ngun sng khc l n xenon to ra cng lin tc trn ton b vng UV-Vis (hn

nhin, v nhiu t n xenon rt ln so vi hai loi n trn cho nn n xenon ch c dng trong c
phn x khuych tn, cng cao ca tia sng c quan tm hng u.

Hnh 6 : Cng tia sng ca n xenon

3.2.2. B phn tn sc

B phn tn sc to ra cc bc sng khc nhau t cc tia sng c tn sc cc gc khc nhau. Khi

hp vi mt khe ra thch hp, b phn ny c th c dng chn ra mt bc sng c trng (chnh xc

di sng hp) ca tia sng t ngun lin tc. B phn tn sc c hai loi: lng knh v cch t, thng
nhiu trong cc my quang ph UV-Vis.

- Lng knh to ra nh sng nhiu mu nh cu vng t nh sng mt tri. Nguyn tc ny c s d

my o quang ph. Lng knh cu to n gin v r tin, nhng gc tn sc khng tuyn tnh (hnh 7a). H

tn sc li ph thuc vo nhit . V vy, a s cc my quang ph hin i c b phn cch t thay th

knh. B phn ny c lm bng thu tinh. V pha trn c cc rnh rt hp, kch thc ca cc rnh ny th

nh bc sng ca nh sng c tn sc. Cui cng, mt lp nhm c ph ln trn to ra mt ngun

Tia sng chiu vo cch t c phn x li cc gc khc nhau, ph thuc vo bc sng.
- Cch t to ra gc tn x tuyn tnh khng ph thuc vo nhit . Tuy nhin, chng phn x tia sng
t khc nhau, nn c hin tng gi ln nhau (hnh 7b). Chnh v vy, ngi ta phi s dng h thng lc

rng ch cc tia sng phn x cn thit n c detector. Mt cch t lm va tn x ng thi hi t tia sn

B n sc ho gm mt khe ti, mt b phn tn sc v mt khe ra. L tng nht l tia i ra khi b

l tia n sc. Tuy nhin, trn thc t tia i ra lun l mt di sng i xng. rng ca di chiu cao
rng di ca thit b.
Hnh 7 : B phn tn sc
3.2.3. B phn ng mu o

Cc o phi hon ton trong sut tt c cc bc sng v bt k s hp th no t cc cng lm gim kh

th tuyn tnh ca mu o. V d, thit b c th cho gii hn trn ca khong tuyn tnh l 2,5 AU nhng n
hp th 1 AU th khong cn li cho mu ch l gia 1 v 2,5 AU tc l cn 1,5 AU.
Nhng cc r tin nht c lm bng plastic. Cc cc ny khng th dng vi tt c dung mi v hp

di 300nm. Cho nn khng thch hp vi vic o vng ny. c tnh ca tng loi cc ph thuc vo nh

Cc thu tinh t hn mt cht so vi cc plastic, chng bn hn. Cc thu tinh hp th mnh vng d
bi vy khng thch hp o vng ny.

Cc thch anh trong sut cho n tn di 210nm. Cc o tt nht hin nay l cc silica tng hp vi c

cao, c th trong sut n di 190nm. Hnh 8 cho thy tnh cht hp th ca cc cc o c lm bng c
liu khc nhau.

Hnh 8 : c tnh truyn qua ca nguyn liu lm cc o

B dy ca cc o c th t 0,1 n 10 cm nhng hay dng nht l cc c b dy 1 cm. i khi cc cc

cng c dng i vi nhng cht c nng thp hoc c hp th km.
3.2.4. Detector
Detector c nhim v chuyn tn hiu nh sng thnh tn hiu in
Mt detector l tng s cho p ng tuyn tnh trn mt khong rng, vi nhiu thp v nhy cao.
Cc my quang ph thng c detector l ng nhn quang hoc detector diode quang.

- ng nhn quang c chc nng t hp cc tn hiu chuyn i qua vi giai on khuych i trong thn

Bn cht ca nguyn liu lm cathode l xc nh nhy ca ph. Mt nhn quang in n to ra nh

ton b khong UV Vis. Loi detector ny sinh ra nhy cao mc nh sng thp. Tuy nhin, trong ln
dng phn tch quang ph, nhy cao thng i i vi nng thp dn n hp th thp.

pht hin chnh xc s khc nhau rt nh gia mu trng v mu th, detector phi c nhiu thp m
cao.

- tng kh nng , diode quang c s dng trong cc my o quang ph. Trc kia, diode qua

nhy thp trong khong UV thp, nhng vn ny c hiu chnh trong cc detector hin i. Gii hn

xp x 170-1100 nm i vi detector s dng silicon. Cc my quang ph hin i c mt dy cc detec

quang thay v mt detector.

- H quang hc : Cc thu knh hoc cc gng lm c dng chuyn tip v hi t tia sng qua thi

thu knh n gin th r tin nhng b sai lch v mu, l v cc tia sng c bc sng khc nhau khng

t cng ng mt im trong khng gian. Tuy nhin, vi thit k cn thn, s sai lch v h mu ca cc th
th trong h quang hc c th c dng loi tr ln nhau v mt h quang hc c hiu qu c th c

vi cc hp phn n gin v r tin ny. Cc gng lm khng t bng thu knh v hon ton khng c s
v h mu. Tuy nhin, b mt bng nhm th d dng b n mn, do vy t hiu qu

3.3. Phn loi my o quang ph

3.3.1. My quang ph mt chm tia
C hai loi: quang ph thng thng v diode array
3.3.1.1. My o quang ph thng thng

Tia sng a sc t ngun c hi t vo khe ti ca b phn n sc ho, n s truyn mt cch chn l

hp ca tia sng. Tia sng ny sau i qua b phn o mu n detector.

 hp th ca mu c xc nh bng cch o cng nh sng n detector ca mu trng v so s

cng nh sng n detector sau khi i qua mu th
Hnh 9 : S ca my o quang ph mt chm tia thng thng

Nh cp trn, a s cc my o quang ph c hai ngun n, mt n deuterium v mt n tun

dng hoc l ng nhn quang hoc gn y hay dng hn l diode quang lm detector.
Thit k ny l thch hp vi vic o hp th mt im ca ph. N khng thch hp trong vic o

phn khc nhau cc bc sng khc nhau hoc trong vic thu ph ca mu. thc hin qu trnh ny c

quang thng thng, cc phn ca b phn n sc ho phi quay cho nn ny sinh vn l s khng lp l
c hc i vi thit b. Hn na qu trnh thu s liu rt chm.
3.3.1.2. My o quang ph diode array
- nh sng a sc t ngun c i qua mu o v c hi t vo mt khe ti ca b a sc ho.

- B phn a sc ho tn sc nh sng vo chui diode, trn mi diode o mt di hp ca ph. rn

tia sng c pht hin bi mt diode lin quan n kch c ca khe ti ca b a sc ho v kch c ca d
diode c chc nng nh l mt khe ra ca b n sc ho.
Hnh 10 : S ca my quang ph diode array
- V tr tng i ca mu v b phn tn sc l ngc so vi my quang ph thng thng, cu hnh ny
ra l ngc v h quang hc.

- hn ch cc phn ng quang ho c th xy ra, mt b phn ng c dng ngn tia sng

pht ra cho ti lc o. Khi qu trnh o c bt u, b phn ng s t ng m v tia sng i qua mu
diode. S khc nhau v cng ca nh sng n detector khi o mu trng v mu th c ni n
quang ph thng thng.

- My quang ph diode array bi vy o rt nhanh do cc s liu c thu ng thi, lp li ca b

v c tin cy cao.
Hnh 11 ch ra h quang hc ca my o quang diode arry hin i HP 8453.
Hnh 11: S my HP 8453

Cu hnh mt chm tia ny c s ti thiu ca phn h thng quang thu c hiu qu cao nht v c
diode c th o t 190-1100 nm vi phn gii cao.

- My mt chm tia thng r v c h quang hc n gin v bi vy nhy cao.

- My quang ph diode array c bit thch hp vi mt chm tia v ph thu c rt nhanh v v c th

gia mu trng v mu th c hn ch. Hn na cht ni chun c th c dng loi s dao ng ca
- Trong my quang ph mt chm tia, mu trng v mu th c o lin tip nhau, vi khong thi gian
nhau vi giy khi o im v mt vi pht nu ta o c di ph. S dao ng ca n c th xy ra v gy ra
ln trong qu trnh o.
3.3.2. My quang ph hai chm tia

- My hai chm tia c pht trin loi s thay i ca cng n gia cc ln o mu trng v

loi ny mt b phn ng ngt nh sng c t trn ng i ca h quang hc gn ngun sng. B p

ngt ny chuyn ng ca tia sng gia ng n mu trng v mu th ri n detector. N s quay m

sao cho vic o lin tip gia mu trng v mu th s xy ra vi ln trong mt giy, bi vy s hiu chn
thay i ca cng n trong thi gian o (hnh 12).

- So snh vi my mt chm tia, my hai chm tia c h quang hc phc tp hn, nn nhy gim.
nhy cao phi o trong thi gian di.

- Theo l thng, n nh cao ca my hai chm tia l l do chnh trong vic thit k ca my quang
nng cao. Tuy nhin, vi nhng tin b gn y v n v thit k in m tng c n nh ca loi
tia v dn n s quay tr li ca my mt chm tia. My mt chm tia c nhy cao v d s dng
4. NG DNG TRONG PHN TCH, KIM NGHIM

4.1. nh tnh
4.1.1. S dng cc cc i hp th
Cc cc i hp th l c trng nh tnh ca cc cht nn hay c dng nh tnh.
V d: Dung dch trong nc
Vitamin B12 c cc cc i 278 nm, 361 nm v 550 nm
Chloramphenicol c cc i 278 nm
Dexamethanson c cc i 240 nm
4.1.2. S dng cc cc i hp th kt hp vi dng ph
Cho bit mc tinh khit ca mt cht :
V d: Vitamin B12 (theo USP 24)

A361/A278 t 1,70 n 1,90

A361/A550 t 3,15 n 3,40

Penixilin V (theo BP 2003) A306 0,36

A274 0.56

4.2. nh lng

Nhng vn trnh by y nhm gip cho vic tm hiu, nhn xt v nhng qui trnh nh lng c sn
t xy dng nhng qui trnh kim nghim.
4.2.1. Chn cc iu kin nh lng
4.2.1.1. Chn bc sng
Ta thng chn bc sng ng vi cc i hp th ln nht.
- Khi ng chun c dc ln nht. Cng mt sai s A sai s C nh nht.
- Ti max, sai s bc sng t nh hng.
4.2.1.2. Chn khong nng thch hp
Khong nng trong quan h gia hp th v nng l tuyn tnh.

Mt vn khc c lin quan, c ghi trong mt s dc in l nng phi c chn sao cho h
c ri vo khong vng ti u l 0,2 0,8 v cng gn 0,43 cng tt.
iu ny c rt ra khi nghin cu s ph thuc gia sai s tng i dA/A v gi tr hp th.

Nu c iu kin ta vn nn tun theo yu cu ny, mc d c th vi my o hin i vi khuch

ch th hin s th cc vn va nu thay i
4.2.1.3. Chn cc iu kin lm vic khc
c kt qu ng, cht cn nh lng phi hp th nh sng ti vng my c th o c v khng
bi cc cht khc. Nhng trng hp nh dung dch tim B 12, thuc nh mt Chloramphenicol 0,4%, cc
va nu tho mn d dng. Tuy nhin, nhiu trng hp phi p dng cc bin php khc.

Chit cht cn kim nghim khi tp ri mi nh lng c (v d: chit Vitamin B 1 bng dung dch
khi cc t dc trong vin).

Lm cc phn ng mu (i khi kh phc tp)

V d: Dexamethason hp th cc i ti 240 nm, nhng bc sng ny nhiu cht hu c khc cn

(nht l trong cc ch phm). V vy, ngi ta dng phn ng vi Xanh Tetrazolium trong mi trng Te
amoni hydroxyd, sn phm mu o bc sng 485 nm.

nh hng ca pH

Trong dung dch nc, pH c nh hng rt ln n bc sng hp th cc i (max) cng nh hp th

ca dung dch (Amax). (Hnh 13)

Khi pH thay i th max ca dung dch s chuyn dch v Amax cng thay i. Nu max chuyn v bc s

gi l s chuyn (bathocromic shift). Nu max chuyn v bc sng ngn th gi l s chuyn xanh (hyps

 nh hng ca dung mi

shift).

C nhiu dung mi trong sut thch hp cho vng kh kin v phn ln vng t ngoi. Cc dung mi

khit. Phn ln cc cht mch thng no u l nhng dung mi thch hp cho vng t ngoi. Khi chn dung

c s ho tan th dung mi khng phn cc tt hn dung mi phn cc v cc dung mi khng phn c

nh hp th nhn hn.

Bnh thng nc v cc alcol u c th l dung mi cho vng t ngoi, nhng nu cn etylic c l

th s b hp th rt mnh cho nn phi lu v tinh khit ca dung mi.
Hnh 13: nh hng ca pH i vi ph t ngoi ca Phenobacbitone
A: trong HCl 0,1M ; B: trong m borat 0,05M (pH = 9,2) ;
C: trong NaOH 0,5M (pH = 13)
4.2.2. Cc phng php nh lng
4.2.2.1. Phng php s dng E
l phng php s dng trc tip nh lut Lamber Beer, ch yu di dng:
A = E .l . C

khi l = 1cm

C=

Mun p dng phng php ny cn phi c my quang ph hp th (vi b n sc ho dng lng knh
t), ng thi my phi c chun c v bc sng ln hp th.

chun ho v bc sng c th dng nhng knh chun nh knh Holmium hay Dydimium vi dng p
tr s cc i xc nh.

chun ho v hp th cng c th dng cc knh lc chun km theo my hay dng cc dung dch
th chun c c bc sng ln hp th. Hay dng hn l dung dch K2Cr2O7 trong H2SO4 0,01N.
4.2.2.2. Cc phng php s dng cht chun

Nh phng php ng chun, phng php so snh v phng php thm u i hi phi c cht chu

mt s dc in lun lun qui nh phi lm so snh vi chun. Nhng chun ny phi c sn xut v
bi nhng c quan c trch nhim.
4.2.2.3. Chun o quang

S thay i v hp th ca dung dch c th c dng xt v s thay i nng ca mt thnh p

qu trnh nh lng. hp th t l trc tip i vi thnh phn hp th. th biu din s ph thuc gi

th v th tch dung dch chun (trong trng hp phn ng l hon ton) bao gm 2 ng thng m giao

ng ny chnh l im tng ng.

Hnh 14 : Cc dng ca chun o quang.

(a) ng biu din ca s chun m ch c dung dch chun c s hp th, nh trong trng
lng As3+ bng dung dch Bromat Bromid.

(b) ng biu din ca s chun m sn phm to ra c s hp th nh trong trng hp

Cu2+ bng EDTA.
(c) ng biu din ca s chun khi dung dch chuyn thnh sn phm khng hp th

(d) ng biu din ca s chun mt dung dch c mu to ra sn phm khng mu bi mt dung d

c mu.

(e),(f) ng biu din ca s chun khi ta thm lin tip cc dung dch chun to ra lin tip hai ph
hp th khc nhau.
u im: C th dng nh lng o th tch cc dung dch c mu m khng tm c ch th mu thch
4.2.2.4. Phng php chit o quang (hay phng php acid mu)

+ Nguyn tc: Chn mt cht acid mu thch hp cho tc dng vi cht base hu c trong nhng iu
thch hp, theo c ch:
xA-m +

yB(H+)n

(acid)

(base)

Ax-mBy(H+)n
(cp ion)

+ Cc yu t nh hng:
-pH: mi acid mu to cp ion vi base hu c mt pH nht nh
V d:

+ Helianthin thng pH 4,4 4,6

+ Xanh bromothymol thng pH 6,6 6,8
pH c nh hng rt ln ti s chit cp ion vo pha hu c.

- Dung mi: Cn la chn dung mi thch hp chit trit cp ion to thnh m khng cho cht acid m

Thng thng s dng cc dung mi: dichloroethan, chloroform, dichloromethan Trong mt s trng h
cho thm mt s cht c kh nng phn cc cao vo dung mi solvat ho cp ion to thnh d
vic chuyn chng sang pha hu c.

- Ngoi ra, nng thuc th, s ln chit, thi gian chit, khong tuyn tnh, s bn vng ca cp m
cng rt cn c lu .
+ Cc v d:
nh lng

Chlorpheniramin

Phenylpropanolamin

Atropin sulfat

Acid mu
pH
Dung mi

maleat
Helianthin
4,5
Chloroform

hydroclorid
Xanh bromothymol
7,0
Chloroform

o A

422 2nm

416 2nm

Lc bromocresol
5,6
Chloroform
416 nm

4.2.2.5. nh lng hn hp
a) Phng php ton ph: Do mt quang c s cng tnh
nh lng ng thi hai thnh phn c ph hp th chng ln nhau.

Ta c th o hp th ca dung dch hn hp ti t nht hai bc sng khc nhau trong s chnh lch
hp th ca hai thnh phn cng ln cng tt. (Hnh 15)
Hnh 15 : Bc sng c chn nh lng hai thnh phn
( bc sng c nh du bng mi tn).
(k1/k2)1 = max

(k1/k2)2 = max

Nu gi A1 v A2 l hp th ca dung dch c cha hai thnh phn 1 v 2 ti cc bc sng 1 v 2 ta

A1 = C1 + C2

A2 = C1 + C2

Cc h s , , v c th xc nh c bng cc php o ring bit cc dung dch ca cc thnh ph

cht. Cn hai nng C1 v C2 cha bit, ta c th tnh c bng cch gii h 2 phng trnh trn.

Phng php ny v nguyn tc c th em p dng cho cc hn hp n thnh phn. o mt qua

bc sng v gii h phng trnh c dng:
A1 = C1 + C2 + .......+ Cn
A2 = C1 + C2 +.........+ Cn
..........................................................
An = C1 + C2 +.........+ Cn
Gii h phng trnh ny ta c cc nng phi tm C1, C2, ..., Cn.
Thc t ch p dng tho mn cho cc hn hp 2 hoc 3 thnh phn v hai l do chnh sau: Tng tc

t c th lm sai lch nh lut Beer, lm cho h s k khng cn l h s trung thc na. Hn na cc sai s c

c v cc s liu mt quang s tng ln gp bi khi gii h phng trnh, lm cho sai s ln. Gn y, ng

d kin khc phc tr ngi ny bng phng trnh phc tp hn, trong cc php o mt quang cc b
ln hn n nhiu ln v cch tt nht l gii h phng trnh bng my tnh.
b) Phng php ph o hm bc hai

Phng php ph o hm l s chuyn dng ca ph hp th sang ph o hm bc mt, bc hai ho

hn. Ph o hm bc mt l th ca gradient ng cong hp th (tc ca s thay i hp th vi

dA/d). i vi di sng ph o hm bc hai l th ca cong ca ph hp th (d 2A/ d2). Nu

tun theo nh lut Lambert Beer, o hm bc hai ti bt k di sng no () cng lin quan ti n
phng trnh sau:
d2A

d2A(1%, 1 cm)
=

d2

Trong :

d2

cd

A: hp th bc sng

A(1%, 1cm): hp th ring bc sng

c: Nng ca cht hp th biu th di dng % (kl/tt)
d: B dy ca lp cht hp th tnh bng cm.

4.3. Mt s ch trong thc nghim

- Truc khi o mu, phi bt my trc 15 pht cho my n nh
- Mu em o phi bo m trong
- Cc o phi tt, thng cho php sai s v dy ca cc l 0,01 mm.
- thu c kt qu chnh xc, nn s dng cng cc o vi mu chun v mu th

- t cc o phi thng ng trong khoang o mu, nn lun lun hng cng mt mt ca cc o v

sng.
- Cc o phi oc trng ra t nht 3 ln bng dung dch nh o.

- Cc phi c dng cn thn trnh xc, trnh li vt tay trn b mt ca cc o, v du t vt t

gy ra s hp th ln.

- Khi o mu c chun b trong dung mi d bay hi th phi dng np y trong qu trnh o mu,
s v nng .

- i vi nhng mu yu cu nghim ngt v nhit th phi c b phn iu nhit trong sut qu trnh

Bi thc hnh:
nh lng Paracetamol trong vin nn bng phng php o quang
(theo DVN III)
1. Nguyn tc

Trong cng thc cu to ca paracetamol c cc lin kt i lin hp, do vy, n c kh nng hp th n

ngoi. Ngoi ra, paracetamol cn ho tan trong kim. V vy, s dng dung dch trong kim

paracetamol bng o quang ti bc sng cc i hp th 257nm.

Tnh kt qu theo A(1%, 1cm); ly 715 l gi tr A(1%, 1cm) .

2. Tin hnh

Cn 20 vin, tnh khi lng trung bnh vin, nghin thnh bt mn. Cn chnh xc mt lng bt vin t

khong 0,15 g paracetamol, cho vo bnh nh mc 200 ml, thm 50 ml dung dch natri hydroxyd 0,1N (TT
nc, lc 15 pht thm nc n vch. Lc u, lc qua giy lc kh, b 20 30 ml dch lc u.
Ly chnh xc 10 ml dch lc cho vo bnh nh mc 100 ml. Thm nc va n vch, lc u.

Ly chnh xc 10 ml dung dch trn cho vo bnh nh mc 100 ml, thm 10 ml dung dch natri hydroxyd

thm nc n vch, lc u. o hp th ca dung dch bc sng 257 nm, dng cc dy 1cm. M

dung dch natri hydroxyd 0,01N. Tnh hm lng paracetamol (C 8H9NO2) theo A(1%, 1cm); ly 715 l gi
1cm) bc sng 257 nm

3. Tnh kt qu
Lng paracetamol c trong vin tnh theo khi lng trung bnh vin c xc nh theo cng thc
X(gam) = At x 200 x m /715 x mt
Trong :
At: hp th ca mu th
715: Gi tr A(1%, 1cm) bc sng 257 nm
m: Khi lng trung bnh ca vin (gam)
mt: Khi lng bt vin dng th (gam).

PHNG PHP QUANG PH

Phng php quang ph ng dng trong ngnh kim nghim thuc l mt s phng php phn tch s d
quang hc: Ph hp th t ngoi, kh kin, hng ngoi; Ph hp th nguyn t; Ph hunh quangthuc v
phng php phn tch quang hc.

Cc phng php phn tch quang hc da trn vic nghin cu s tng tc ca bc x nh sng trn ch
hoc s pht ra cc bc x nh sng di mt tc ng ha l no .
V d: Khi chiu nh sng vo mt dung dch, c hin tng khc x hoc quay mt phng nh sng
hoc c th c s hp th quang hoc pht hunh quang.
V vy kt qu nghin cu cho php ngi ta a ra cc phng php phn tch quang hc khc nhau.

1 - Phng php quang ph hp th UV-VIS ( phng php o quang).

Da trn kh nng hp th chn lc cc bc x ri vo dung dch cht nghin cu, hp th ny ph
nng cht tan.
2 -Phng php quang ph hp th IR (quang ph hng ngoi).

Khi phn t cht nghin cu b chiu tia hng ngoi, cc nhm nguyn t ca n s hp th nng lng v

trng thi dao ng, to nn mt nh hp th c trng trn ph IR, bc sng xut hin nh v tp hp c
c s ca vic nh tnh.
3 - Phng php quang ph hunh quang.

Da trn kh nng pht ra cc bc x nhn thy c bc sng xc nh v c cng ph thuc vo h

ca cht nghin cu , khi n b mt chm tia t ngoi chiu vo.
4 - Phng php quang ph hp th nguyn t.

Da trn kh nng hp th cc bc x c trng chiu vo khi cht nghin cu c nguyn t ha tr

la hoc trong l. Da vo phn nng lng bc x b hp th c th nh lng c cc nguyn t.
5 - Phng php o phn cc.
Da trn s o gc quay ca mt phng nh sng phn cc i qua dung dch nghin cu.
6 - Phng php o khc x
Da trn s o chit sut ( gc khc x) ca nh sng i qua cht nghin cu.

Ngoi ra cn cc phng php phn tch quang hc khc nh o c, o quang ngn la, o ph
nguyn t, o ph pht x Plasma
Trong chng ny chng ti ch gii thiu cc phng php quang ph c ng dng nhiu trong kim
thuc, l : Phng php quang ph UV-VI S;
Phng php quang ph hunh quang.

PHN TCH, KIM NGHIM THUC BNG QUANG PH HUNH QUA

1. KHI NIM V PHNG PHP
Phng php quang ph hunh quang (Fluorescence spectrophotometry) l mt trong cc phng php

quang hc. Cc phng php phn tch quang hc da trn vic nghin cu s tng tc ca bc x nh sng
kho st hoc s pht ra cc bc x nh sng di mt tc ng ha l no .
V d: Khi chiu nh sng vo mt dung dch, c hin tng khc x hoc quay mt phng nh sng
hoc c th c s hp th hoc pht hunh quang.
V vy kt qu nghin cu cho php ngi ta a ra cc phng php phn tch quang hc khc nhau.

- Phng php quang ph hp th ( phng php o quang).

- Phng php quang ph hunh quang.
- Phng php quang ph hp th nguyn t.
- Phng php o phn cc.
- Phng php o khc x...

Phng php hunh quang da trn nguyn tc: Khi chiu mt chm tia t ngoi (bc x kch thch) v
cht, cc cht ny pht ra bc x nhn thy c bc sng xc nh ty thuc tng cht v c cng ph
hm lng ca chng. Khi ngng chiu bc x kch thch, nu bc x pht quang tt ngay th s pht quang

hunh quang, nu bc x pht quang cn tn ti mt lc th gi l ln quang. Trong bi ny chng ti ch

hunh quang.

Trong kim nghim thuc, quang ph hunh quang c ng dng nh tnh, nh lng cc dc ch

nng pht hunh quang hoc to c dn cht pht hunh quang. Tuy khng ph bin bng quang ph

nhng quang ph hunh quang c nhy cao hn hng trm ln v c th phn tch chn lc cc cht trong
2. I CNG V QUANG PH HUNH QUANG

2.1. S hnh thnh quang ph hunh quang

2.1.1. Khi nim v hin tng pht quang

Hin tng pht quang ni chung l tnh cht ca cc cht c kh nng pht quang di cc tc nhn k

khc nhau. Cc tc nhn c th do ma st, do nhit, do hiu ng cc phn ng ha hc, do tc nhn chi
cc bc x in t... (c gi l: nhit pht quang, in pht quang, ha pht quang, sinh pht quang...

nhn kch thch l cc tia t ngoi hoc phn c bc sng ngn ca nh sng kh kin ta c hin tng ph
quang hc hay cn gi l hunh quang.
2.1.2. S to thnh ph hunh quang - C ch ca s pht quang

Theo thuyt lng t, mi ht s cp (nguyn t, ion, phn t) c mt h thng duy nht cc trng thi n

Trng thi nng lng thp nht gi l trng thi c bn (S 0). Khi b kch thch, nh c hp th photon n
nng lng ca photon s c truyn sang ht v ht s c chuyn sang mt nng lng cao hn gi l
kch thch S1; S2 ... ( mi trng thi phn t c th tn ti cc phn mc nng lng khc nhau 0; 1; 2; 3...
thi gian rt ngn (10-6 10-9 giy ) cc ht trng thi kch thch s tr v trng thi c bn ban u theo
cc hin tng sau y:

- Hin tng bt hot (Deactivation; Vibrational relaxation): Cc phn t mc nng lng cao ca trn

tr v mc nng lng thp nht ca trng thi v gii phng ra nng lng di dng nhit nng do va chm

phn t lm tng nhit mi trng, gi l hin tng phc hi khng bc x hay l s "th gin" ph
ng sau khi b kch thch (thi gian phc hi < 10-12 giy).

- Hin tng chuyn ni (Internal conversion): Cc nguyn t t mc thp nht ca trng thi kch thch

thi c bn gii phng ra nng lng bng cch bc x cc photon nh sng, gi l s phc hi c bc x,
bc x ny chnh l hunh quang (ko di 10-9 10-6 giy).

- Hin tng vt ni h (Intersystem crossing): Cc phn t trng thi kch thch S 1khng chuyn v tr

bn S0 m chuyn sang trng thi siu bn T1, ri mi t T1chuyn v S0 v bc x cc photon nh sng lm

trnh pht quang xy ra chm gi l hunh quang chm hoc ln quang (10-3 10 giy)
Cc hin tng ny c biu din s hnh 1.
Hnh
. Cc hin tng quang hc xy ra sau khi cc ht s cp b kch thch

Trong hin tng pht quang, mt phn nng lng ca nh sng kch thch b tiu tn chuyn thnh n

do hin tng bt hot v vy nng lng nh sng kch thch bao gi cng ln hn nng lng nh sng b

ni cch khc, bc sng nh sng bc x lun di hn bc sng ca nh sng kch thch, to ra s dch ch

i pht quang v pha bc sng di hn so vi cc i hp th, gi l s dch chuyn Stock. Cht no c

chuyn Stock cng ln th php o hunh quang cng chnh xc (v t b nh hng ca ph hp th). V d:
2.

Hnh 2: Ph kch thch EX v ph bc x y EM ca Griseophunvin.

Cc i EX = 297 nm. Cc i EM = 416 nm.

2.2. Cc i lng c trng ca ph hunh quang

Hiu sut hunh quang (K hiu l F): L c trng vt l ca mt phn t, cho bit kh nng hunh q
mt cht

S photon pht x
F

0 < F < 1

S photon hp th

Cng hunh quang (K hiu l F): L s liu thc nghim biu th hot tnh hunh quang ca m
n c kch thch do hp th mt bc x thch hp.
= F .IA . k (1)

F
Trong :

IA :

l cng nh sng kch thch

l h s hp th

F :

l hiu sut hunh quang.

p dng nh lut Lambert - Beer thay gi tr ca IA vo (1) v thc hin bin i ton hc ta c:
F = k . F . I0 . 2,3 lC
Trong :

I0

l cng nh sng ti

l h s hp th mol

l chiu dy ca lp dung dch (cm)

C :

l nng cht pht quang trong dung dch (mol/l)

Trong iu kin xc nh th k, F, I0, , l l nhng hng s, ta thay bng mt hng s k', ta c:

F = k' . C
Ngha l trong mt iu kin xc nh th F t l thun vi C.
Tuy nhin cc nghin cu nu ra rng im ny ch ng khi dung dch long:
4

(mol/l);

A < 0,002

Nu dung dch c nng ln hn hoc c tp cht th s xy ra s tt hunh quang, khng cn s t

gia cng hunh quang v nng
V d: Xem hnh 3

Hnh 3: Mt v d v s ph thuc gia cng hunh quang v nng

2.3. nh hng ca cu trc phn t n hiu sut hunh quang.

T c ch ca s pht quang ta thy nhng cht c kh nng hp th t ngoi u c kh nng pht hun

Trn thc t li khng phi nh vy , c nhiu l do khin chng khng pht quang c. n nay vn ch
qui lut chnh thc d on mt cht c pht quang hay khng. Ngi ta mi ch a ra mt s nhn xt
sau:
2.3.1. V cu to phn t

Cc cht c cu to dy ni i lin hp th c kh nng pht quang, v cng nhiu dy ni i lin h

nng pht quang (hiu sut hunh quang) cng cao.
Cc nhm chc c kh nng cho in t th lm tng hiu sut hunh quang.
V d: -OH, -NH2 , -OCH3 ...
Cc nhm chc c kh nng nhn in t th lm gim hiu sut hunh quang:
V d: -NO2, -COOH, - CHO, Br-, I- ...
V tr cc nhm th trn nhn thm c th lm tng hoc gim hiu sut hunhquang:

V d: V tr Para v Ortho lm tng s khng nh v ca in t trong nhn thm cng lm tng hiu s
quang, ngc li v tr Meta th lm gim.
ng phn Cis lm tng pht quang v Trans lm gim pht quang ca mt hp cht.
2.3.2. V rn chc ca cu trc phn t
Cc cht c cu to phn t rn chc th kh nng pht quang cao, v ngc li.
V d: Fluorene, Rosamine, Fluorescein: Pht quang mnh
Biphenyl, Malachite, phenolphtalein: Khng pht quang

S khc nhau c bn ca cc hp cht trn l: Fluorene c cu ni methylen, Rosamine, Fluorescein c

oxygen to cho cc hp cht trn mt cu trc bng phng, rn chc nn lm cho cc nhn thm khng

ng mt cch t do na do kh nhng nng lng di dng nhit nng do va chm m phi gii ph
lng bng con ng pht quang.
2.3.3. V cc dn cht ha hc

C nhng hp cht bnh thng th khng pht quang hoc pht quang yu, nhng khi chuyn sang cc
khc th pht quang rt mnh.
V d:
Khng pht quang

Pht quang mnh

Tetracyclin

Tetracyclin + Ca2+ + Barbiturat

Hydrocortison

Hydrocortison +H2SO4 c + ethanol

Adrenalin

Adrenochrom

Vitamin B1

Thiocrom

2.4. Cc yu t mi trng nh hng n ph hunh quang

2.4.1. S c mt ca cc cht tan khc
C th gy ra cc hin tng sau:

Hiu ng lc ni: Cc cht khc c th khng pht quang nhng li hp th nh sng ca cht pht q
gim cng hunh quang.

S lm tt ha hc: Cc cht khc lm tt mt phn hay ton b cng pht quang do va chm
bin i ha hc ca cht pht quang.
C ch:

F + h F *
F * F + h'

do va chm nn

F*+Q F+Q*

Kt qu l Q ng vai tr ca mt cht lm tt ha hc ( Quencher).

V d:
- Cl- l Q ca Quinin cho nn trong mi trng HCl th Quinin khng pht quang.

- NaHSO3 l Q ca Vitamin B2 nn ta nh lng B2 bng phng php o tt hunh quang ca B2 b

hydrosulfit.

- ADN l Q ca Acridin nn trong xt nghim sinh ha ngi ta s dng Acridin trong vic nh lng
2.4.2. Nng ca ion H+, vai tr ca pH
pH c vai tr quan trng. Mt s cht c tnh acid yu hay base yu th cng pht quang ph thuc
pH. V d: Ta c acid AH, trong nc AH phn ly theo phng trnh :
AH

A- + H +

Nu dng pht quang l AH th khi H+ tng th phn ng theo chiu nghch F s tng

Nu dng pht quang l A- th ngc li F s gim trong mi trng acid v tng trong mi trng kim
2.4.3. nh hng ca dung mi

Cng pht quang v bc sng cc i thng thay i theo dung mi v nhng tc ng khc nhau:

S lm tt ho hc do dung mi : Quinin sulfat pht quang mnh trong mi trng H 2SO4nhng hu n

pht quang trong mi trng HCl ; oxy ho tan trong dung mi lm bin i ho hc ( oxy ho ) ca cht ph
nht tng lm tng cng pht quang.
tinh khit ca dung mi: dung mi c tp hunh quang s lm sai lch kt qu, lm gim nhy ca
2.4.4. nh hng ca nhit

Nhit tng thng lm gim cng pht quang do lm tng tn s va chm ca phn t lm cho h
bt hot tng ln.

V vy trong my quang ph hunh quang n ngun c cng sut ln nn phi c knh cch nhit hoc c
lm mt n.
3. MY QUANG PH HUNH QUANG

3.1. S cu to (Hnh 4)
- Ngun sng (Source)
- B phn to nh sng n sc (Monochromator offilter)
- Cc ng mu o (Sample cell)
- My hin tnh hiu (Detector)
- B ghi tn hiu (Electronis and recorder)

(a)

(b)

Hnh 4 . S cu to ca my quang ph hunh quang: (a) hoc (b)

3.2. Cu to chi tit

3.2.1. Ngun sng ( Source):

Mt trong cc loi n sau:

- n thy ngn (Hg) cho cc vch kch thch ch yu:

253,7; 313; 365; 404,7; 407,8; 435,8; 546,1; 577,0; 579,1 nm.
- n xenon cho ph kch thch lin tc vng UV v mt phn vng VIS.
- n Deuteri cho ph kch thch lin tc vng 200 n 300 nm.
- Ngun Laser cho bc x kch thch cng cao.
3.2.2. B phn to nh sng n sc ( Monochromator or filter)
(Cho c nh sng kch thch v nh sng bc x)
Mt trong cc loi sau:
- Knh lc (Filter)
- Cch t (Grating)
- Lng knh (Prism)
- My pht tia n sc.
Ring lng knh hin nay t dng
3.2.3. Cc ng mu o ( Sample cell; cuvette)
- Hnh khi ch nht, bn mt trong sut, cnh v y c lm bng; hoc hnh ng tr trong sut,
c lm bng.

- Trong my, cuvette c iu chnh cc gc khc nhau so vi phng ca tia sng kch thch
vi hp th ca mu th (gc 900 vi a s mu th, gc 370 vi cc mu c hp th ln...). (Hnh 5)
Hinh 5. S v tr cuvette
3.2.4. Pht hin tn hiu ( Detector)
nh sng pht quang c thu nhn bng nhn quang in t, c hai loi:
+ Photocell ;

+ Photomultiplier

3.2.5. B ghi tn hiu ( Electronics & recorder)

Dng quang in c khuych i v o bng cc my o thch hp, c 3 loi :
+ My o ch th kim ;

+ My t ghi ;

+ My vi tnh

4. PHP O PH HUNH QUANG

Ph hunh quang bao gm ph kch thch v ph bc x

4.1. Ph kch thch - Excitation spectrum (EX)

L ng biu din s ph thuc ca cng pht quang tng i (relative intensity) theo bc sn
sng kch thch.

Bc sng cc i ca ph kch thch cng chnh l bc sng cc i hp th ca cht c kch th

hot ha) v vy ph kch thch cng chnh l ng biu din mi quan h gia cng hp th ca cht
sng bc x hp th.

4.2. Ph bc x (Ph hunh quang) : Emission spectrum (EM)

Fluoreseence spectrum (FL)

L ng biu din s ph thuc ca cng pht quang tng i (cng hunh quang ca ch
hat ho) theo bc sng ca nh sng pht ra.
V d ph hunh quang ca mt s cht (hnh 6,7):

Hnh 6: Ph kch thch v ph bc x ca Vitamin B1

(Dung dch 1 g/ml , cc i EX = 370 nm, cc i EM = 425 nm)

Hnh 7: Ph kch thch v ph bc x ca Histamin

(Dung dch 1 g/ml , cc i EX = 350 nm, cc i EM = 440 nm)

Ghi ch : Khi ghi ph hunh quang ca mt s cht ta thng thu c ngoi ph ph hunh quang

s ph khc nh ca nh sng tn x, ca gii Raman, ca dung mi v cc o, ca nh sng tn x th cp

sng phn x, nhng ph hunh quang ca mu vn th hin r rt nht v da vo ph ny ta c th xc

cc i bc x (bc sng pht quang mnh nht) ca cht .
V d: Natri salicylat c cc i bc x EM = 435 nm (Hnh 8)

Hnh 8: Ph hunh quang ca Natri salicylat

4.3. Ph hunh quang 3 chiu (Ph pht quang ton phn)

L ph biu din s ph thuc ca cng pht quang tng i theo c bc sng ca nh sng kch

bc sng ca nh sng bc x. Trn cc my quang ph hunh quang th h mi c km theo my vi tnh ch

ghi c ph hunh quang ca mu o tng bc sng kch thch. Kt qu ta ghi c mt ph kh

chiu.
V d: Mt ph khng gian 3 chiu (Hnh 9)
a)

b)

Hnh 9: a. Ph pht quang ton phn pha trc bc x t 270 - 415 nm

b. Ph pht quang ton phn pha sau bc x t 415 - 270 nm
Trong :
- Trc tung biu din bc sng kch thch EX.
- Trc honh biu din bc sng bc x EM.
- Cc ng ln sng (ng mc: Contour) biu din cng pht quang.

Vi ph hunh quang ton phn cc thng s u c lu tr trong my tnh cho php ta thc hin

chng trnh x l ton hc khc nhau. Trong trng hp phi xc nh mt thnh phn trong hn hp ta c

chn cp bc sng EX v EM ti u xc nh thnh phn ny m cc thnh phn khc nh hng t nht.

V d: Trn my HITACHI F - 4500 ta ghi c ph hunh quang 3 chiu theo chng trnh 3D-Scan
dch Morphin cho php ta chn cc i kch thch EX = 280 nm v cc i bc x EM = 345 nm (Hnh 10).

Hnh 10: Ph hunh quang 3 chiu ca Morphin.HCl

4.4. Mt vi nt so snh php o ph hunh quang vi php o ph UV-VIS

4.4.1. nhy
Php o ph hunh quang c nhy cao hn so vi php o UV - VIS. V d Vitamin B 1 nng
quang l 10-1 g/ml; nng o UV - VIS l 10 g/ml.
4.4.2. V tnh c hiu

Php o ph hunh quang c tnh c hiu cao hn php o UV - VIS v ch c nhng cht pht qu
chuyn c thnh dn cht pht quang mi xc nh c bng php o ph hunh quang v vi mt cht

th ch c mt cp cc i EX v EM nht nh nn ta c th la chn nh lng cht dng n

trong mt hn hp.

V d trong USP XXI v XXII qui nh nh lng Vitamin B 1 v Vitamin B2 trong vin Decavitamin b
o ph hunh quang.
4.4.3. V s hn ch

Php o ph hunh quang do tnh c hiu cao nn kh nng p dng hn ch, khng ph bin nh

UV - VIS. Mt khc do nhy cao nn nng dung dch em o rt thp lm cho sai s tng i ca p

cao hn, c th nh DVN I qui nh sai s ca php o UV VIS l 1% th ca hunh quang l 3% - 5

4. NG DNG TRONG KIM NGHIM THUC, PHN TCH

Trong ngnh y t php o ph hunh quang c ng dng nh tnh, nh lng cc cht trong thu
dch sinh ho.

4.1. nh tnh

Da vo cp bc sng cc i EX/EM nh tnh v vi mt cht nht nh c kch thch bng m

bc sng no th s cho mt tia c bc sng hunh quang nht nh. Mt s v d (Bng 1):
Bng 1. Mt s v d v cp bc sng cc i EX/EM nh tnh.

Cht phn tch

Retinol
Vitamin K1
Cholesterol
Estron
Hydrocortison
Testosteron
Serotonin
Serotonin
Noradrenalin
Phenylamin
L - Tyrosin
Tryptophan
Dansyl-arginin
Dasyl - Clorid
Chlortetracyclin
Chlortetracyclin
Griseofulvin
Pyrimidin
Indol
Adenin
Chlorophyll

Mi trng
Cyclohexan
Ethanol
Dn cht
H2SO4
H2SO4/Ethanol
H2SO4/Ethanol
HCl 0,1N
Acid acetic 0,1N
Dn cht
Nc
Nc
Dd NaOH pH 11
Nc pH 8,5
Nc pH 8,5
Nc
KOH 0,01N
Methanol
Ethanol
Nc
Dd H2SO4 pH 1
Ethanol

Nng o

Gii hn pht hin

EX max

(g/ml)

(g/ml)

(nm)

4,05
1
1
1
0,1
0,1
1
1
1
100
10
0,1
1
0,1
1
1
1
10
0,1
10
0,1

0,003
0,001

0,001
0,01
1
0,01
0,001
0,001
0,001

0,0001
0,5
0,001
0,1

350
348
470
470
470
470
301
301
396
263
282
311
338
324
386
531
297
279
254
254
430

E
475
420
519
535
525
520
338
338
405
284
305
350
536
500
506
416
416
344
440
367
674

4.2. nh lng

Da vo s ph thuc tuyn tnh gia cng hunh quang vi nng cht pht quang m phng p
p dng nh lng.
Phng php hunh quang c nhy cao v c hiu cao. My quang ph hunh quang c ci

cng hin i nn phng php ny c nhiu ng dng. Trong kim nghim thuc, phng php hunh
c a vo nhiu dc in: USP, BP, EC, JP.....
Trong xt nghim sinh ho c nhiu ti liu, gio trnh, thng tin khoa hc.

Php o hunh quang c p dng nh lng cc cht c kh nng pht hunh quang t nhin ho
c th to ra dn cht thch hp c kh nng pht hunh quang.
* V d : xem cc v d trong bng 1 trn v cc v d sau:

nh lng Vitamin B1 bng phng php Thiochrom: Chuyn Thiamin thnh Thiochrom ri o hu
ca Thiochrom trong isobutanol bc sng EX = 365 nm v EM = 550 nm.
nh lng Vitamin B6 bng cch cho phn ng vi KCN to ra dn cht Lactonepyridoxinat ri
quang bc sng EX = 356 m v EM = 432 nm.

 nh lng Vitamin C bng cch oxy ha thnh Dehydro ascorbic ri cho phn ng vi O-phenyle
to dn cht Quinoxalin o hunh quang bc sng EX = 350 nm v EM = 425 nm.

nh lng Penicillin bng cch cho phn ng vi 2 - methoxy - 6 chloro - 9 ( - aminoethyl) aminoa
dn cht pht hunh quang bc sng EX = 420 nm v EM = 500 nm.

nh lng aminoacid bng cch cho phn ng vi phenyl acetaldehyd v ninhydrin to dn cht pht
bc sng EX = 390 nm v EM = 475 nm.

nh lng Histamin bng cch cho phn ng vi O-phthalaldehyd to thnh dn cht pht quang
sng EX = 360 nm v EM = 450 nm.

4.3. Detector hunh quang cho my sc k lng hiu nng cao (HPLC)

Quang ph hunh quang ngoi vic p dng nh tnh nh lng cc cht trn my o quang ph hu

th n cn c p dng ch to b phn pht hin ca my sc k lng hiu nng cao: Detector hunh q
cu to ging nh mt my quang ph hunh quang, xem hnh 11).

Hnh 11. S cu to ca mt detector hunh quang

Detector hunh quang ng vai tr nh mt my quang ph hunh quang kt ni vi sc k lng ph

nh tnh v nh lng cc cht sau khi ra khi ct sc k. N c p dng pht hin tt nhiu cht h

hunh quang t nhin cng nh cc dn cht c hunh quang. Hn th, nh cc b phn ly mu t ng v

dn cht ha hc vi cc thuc th m n c th pht hin trc tip trn my ngay trc khi cc cht i vo

Vi chc nng qut ph ca Detector hunh quang ta c th tm c cp bc sng EX/EM ti u cho php

Hin nay, sau Detector UV l loi ph bin nht th Detector hunh quang c nhy v chn lc cao
nhy ca Detector hunh quang ln hn hng trm ln so vi Detector UV (pht hin c picrogam).

V d: - nh lng Vitamin B 2 trong sa l mt hn hp nhiu thnh phn vi hm lng Vitamin B

Vi Detector UV bc sng cc i 265 nm th sc c nhiu thnh phn khc cn tr, nhng vi Detec
quang cho mt pic ca B2 cao v p v cc thnh phn khc khng nh hng ti s pht hin bc sng
265 nm v bc sng pht x 520 nm ( hnh 12).

- nh lng cc acid amin trong thuc b tng hp a thnh phn bng cch to dn cht tin ct (d

th o-Phthalaldehyd cho cc acid amin bc 1 v Fluoryl methyl cho cc aid amin bc 2) ri pht hin bng
hunh quang ( hnh 13).

Hnh 12: So snh Detector UV v Detector hunh quang

tch B2 trong sa.

i v

Hnh 13: Phn tch cc acid amin bc 1 v bc 2 s dng dn cht ha

tin ct vi Detector hunh quang.

4.4. K thut nh lng bng phng php o quang ph hunh quang

Ty theo cc cht cn nh lng c kh nng pht quang hay khng ngi ta tin hnh nh lng

hay gin tip thng qua dn cht cu n. S khc nhau ch giai on chun b mu o, cn cc giai on
thc hin ging nhau nh sau:
4.4.1. Chun b my mc, dng c

- My o phi c chun ha thng xuyn bng cht chun hunh quang n nh m bo ng

kt qu. Tin hnh chun my theo ch dn c ghi r rng trong ti liu hng dn s dng my ca
xut.
- Dng c phi sch, khng c dnh cht pht quang, khng c ra bng cc cht ty ra tng hp.
4.4.2. Chun b thuc th

- Cc dung mi ha cht, thuc th dng cho php o quang ph hunh quang phi ng chng loi v
khit qui nh dng cho hunh quang. Pha ch chnh xc .
4.4.3. Chun b mu o
- Mu th : x l thu c dung dch c nng o theo qui nh ca chuyn lun dc in hoc ti

- Mu trng : l dung mi pha mu th nu nh lng trc tip, cn nh lng gin tip th mu trng p
chun b ging nh mu th.
- Mu chun : pha dung dch chun theo qui nh ging nh mu th.

Ni chung nn la chn nng dung dch chun sao cho t l cng hunh quang ca th so v

khng nn nh hn 0,40 v khng ln hn 2,50. iu ny l cn thit nng dung dch o nm tron

tuyn tnh. Khi cng hunh quang khng tht s t l vi nng th bt buc phi thit lp ng ch

mt dy cc dung dch chun khc nhau c o trong cng iu kin vi mu th. Trong mt s trng

chun c th l cht i chiu c bn cht ha hc khc vi mu th, trng hp ny bt buc phi dng
chun.
4.4.4. Cch o hunh quang

Vn hnh my o trn cc my khc nhau theo hng dn ca nh sn xut my. Cch o phi tun th
nh v th t nh sau:

+ Phi chun my v im 0 vi mu trng trc khi o mu chun v th. Trong qu trnh o nu c

chnh thay i khe chiu sng th phi chnh li im 0.

+ o mu chun iu chnh nhy c c kt qu ti u, o dy mu chun phi cng mt i

im 0 v nhy.
+ o mu th cng iu kin nh mu chun.

Chun b dung dch nh m t trong chuyn lun, chuyn dung dch vo cc o ca my quang p

quang v chiu vo n tia sng kch thch vi bc sng n sc c ghi trong chuyn lun. o c
hunh quang ca dung dch bc sng pht hunh quang c ghi trong chuyn lun.

nh lng, a vo my cc ng dung mi hoc hn hp dung mi dng ho tan mu th v

my v im "0". a dung dch chun c chun b nh m t trong chuyn lun vo my v iu chn

ca my kt qu c trn thang o ln hn 50. Nu thay i khe sng iu chnh ln th hai, mt i

c t li th cng hunh quang ca chun cng phi c o li. Cui cng, a mu th vo my,
hunh quang.

Khi cng hunh quang khng tht s t l thun vi nng , vic nh lng c th c thc hin d
th chun.

Trong mt s trng hp, php nh lng c th c so snh vi mt mu chun i chiu c bn cht ho

mu th (V d: thy tinh pht hunh quang hoc dung dch mt cht pht hunh quang khc vi mu th

trng hp ny, nng ca cht th phi c xc nh bng th chun o trong cng iu kin vi mu

4.4.5. Tnh kt qu
Nng ca dung dch th (trng hp dng mt chun) c tnh theo cng thc :
Cx
Trong

(Ix x Cc ) / Ic

Ix v Ic : l cng hunh quang ca th v chun.

Cx v Cc : l nng ca dung dch th v chun.