Chng III

Ph khi lng
1. t vn
Mc d phng php khi kh tng i c (1910 Thomson tch c ng

v Neon 20 v 22). Song, bc ngot phng php khi ph tr thnh phng php phn tch quan trng trong Ho hu c ch t nm 1960.
Hai th mnh ng gp vo s pht trin ca phng php khi ph l :

Vi lng mu nh nht c th xc nh c khi lng tng i ca phn t v thm ch thnh phn cc nguyn t ca mt hp cht. Qua vic phn mnh trong khi ph c th suy ra cu trc hoc thng tin v phn t.
i vi cc phn t phn cc, kh bay hi, trnh phn hu c nhng phng

php ion ho nh : EI (va chm in t), ho hc (CI), t trng (field ionisation), FAB (bn ph bng nguyn t tc cao), ph khi ion th cp (sekunder ion MS), ESI (my in t).
C th xc nh khi lng tng i n 1200 v cao hn na.

2. Thit b, ghi ph : Hu c : MS, v c : ICP-MS 2.1. Nguyn l ca mt khi ph k:

24

 C 4 vng chc nng : a mu vo

To ra ion Tch khi lng Chng minh ion *) a mu vo :

Trong chn khng cao trnh va chm gia cc ion

Vn l a mu t p sut thng vo bung chn khng cao m khng cn ngt

chn khng.
C 2 phng php : a dng kh a trc tip a dng kh : dng cho mu lng hoc kh, bm qua xylanh cc cht d bay hi

c th qua h sc k kh (SKK) hoc sc k lng (GC/MS, HPLC/MS, LC/MS).

a trc tip : dng cho mu dng tinh th, sn hoc keo. Mu trong mt ci

bng nhm hoc vng, = 1mm, gn trn mt thanh t c a vo bung chn khng. Sau khi ht chn khng, ci ng mu c lm ngui v a vo bung ion ho. y n c t nng t t n khi bay hi.
Lng mu cn : 9 15

a dng kh : 0,1 1 mg ; qua SKK : 10 a trc tip : 0,001 0,1mg.

10

g.

*) To ion : T b phn a mu, mt dng phn t nh, c nh c bn vo bung ion ho. Dng phn t ny va chm vung gc vi mt dng in t (nm gia catt nng v ant). in th gia catt v ant thay i gia 0 v 300V, c ngha : nng lng ca in t l : 0 300 eV.
cc ph khi in th thp : 12 15 eV.

. ion radical M M+
Ph khi lng bnh thng : 60 100 eV

Phn ln ghi 70 eV.

Qua va chm gia in t v phn t to thnh ion phn t M + e M
+.

+ 2e

(molecular ion)
2+ hoc M + e M + 3e

25

 Nhng phn khng b ion ho s b ht ra khi bung ion qua bm chn khng. Cc

ion phn t to thnh s c tng tc v thu gn.

Vic tng tc c thc hin bi mt in th (2 10 kV), khi ra khi bung ion

ho tc t cao nht.
Vic thu gn thnh chm ion c thc hin bi mt in trng ph khi vo

phn phn tch khi l mt dng ion tp trung, ng nht.

Tc ca ion c tnh theo cng thc :

(1) (2)

z. U = v=

m. v 2 2

z = in tch ion (= n.e) m = khi lng ion v = tc ion U = in th gia tc

2. z. U m

*) Tch khi :
c thc hin thng qua t trng 1 Tesla. Nhng ion c cng in tch : ion nh hn b lch nhiu hn. Bn knh lch theo cng thc.

(3)

rm =

m. z.B

B = cng t trng T phng trnh (1) v (3) ta c phng trnh c bn ca khi ph l : (4)
2 m rm .B 2 = z 2. U

T l khi lng/in tch ph thuc vo cng t trng, bn knh lch v in

th gia tc, tc l ph thuc vo cu hnh ca thit b.

Thit b dng sector t (magnetic sector)

26

B phn tch tnh in (E)

B phn tch t (B)

Khe vo

Khe ra Thu gom

Ngun ion

S thit b khi ph hi t kp (double focussed): (xem hnh trn)

Mun phn bit ion c cng s khi nhng thnh phn khc nhau nh: CO, C2H4, N2 phi dng khi ph phn gii cao. Thit b phn gii cao c thm mt in trng tnh t trc t trng (nh trn)
y l thit b hi t kp (double focussed), c th xc nh khi lng ion ti

chnh xc l ppm, v nh th xc nh c thnh phn nguyn t ca ion phn t.

Thit b phn gii cao i hi khe ion phi hp, do lm gim nhy.
12

16

14

CO 27,9949

C2H4 28,0313

N2 28,0061

12,000

1,00782

15,9949 14,0031

Khi mt hn hp c nhiu cht (M1+; M2+; M3+; ...) trong bung ion ho ta la chn 1 ion phn t no (v.d. M1+) cho qua khe, to s va chm ion ny phn mnh tip (fragmentation) v ghi ph ca ion ny. l k thut MS/MS. *) Chng minh ion :
Khi cho in th gia tc v cng t trng khng i ta s c phng trnh (5) :

(5)

m 2 = hng s. rm z

Khi cho in th gia tc v bn knh lch khng i ta c :

m = hng s. B2 z m m (trc y l ) mt bn knh lch cho trc th ch cn z e thay i (qut, scan) cng t trng. Trong trng hp ny ta mt dng c thu ion u ra ca b phn phn tch khi. tng dng in ion ta dng thit b nhn in t (electron multiplier).

Tc l : xc nh

27

3. S phn mnh ca cc hp cht hu c

Pic mnh nht cho cng tng i l 100% gi l pic c s (base peak). rel.%

l k hiu cng tng i. *) Ion phn t (molecular ion).

Tr mt s ngoi l, cn li pic c khi lng cao nht thng l pic ca ion phn

t.
Cc trng hp ngoi l : pic [M+1] hoc [M+H] do 1 H gn vo phn t (hay
+ +

gp c bit lp cht amin, ancol).

Thnh thong pic M
+.

khng ghi c, thay vo l pic [M R]+.

ng v (isotop) : Trong cc hp cht hu c c 3 loi sau :

Nguyn t tinh khit : 19F, 31P, 127I. Nguyn t vi 1 ng v chnh (> 98%) : H (1H), C (12C), N (14N), O (O16). Nguyn t c 2 ng v chnh : S (32S, 34S), Cl (35Cl, 37Cl), Br (79Br, 81Br).
V d : Vng pic ion phn t ca hp cht C7H6ClNO (M = 155) : m/z = 155 : + 12 C 71H635Cl114 N116 O1 m/z = 156 : +
12

(1)
100% 155

+ 12 C 71H 52 H135Cl114 N116 O1 +

12

(3) (4)

rel.Int.(%)

C 613C11H635Cl114 N116 O1

(2)

50

157 156 158 150 160 m/z

C 71H635Cl115N116 O1

+ 12 C 71H635Cl114 N117O1 m/z = 157 :

12

(5) (6) (7) (8) (9) (10)

C 513C 21H635Cl114 N116 O1

+ 12 C 71H 42 H 235Cl114 N116 O1 + 12 C 71H637Cl114 N116 O1 + 12 C 71H635Cl114 N118O1 + 12 C 613C11H 52 H135Cl114 N116 O1

28

m/z = 158 :

12

C 613C11H637Cl114 N116 O1

(11)

............................................. ............................................. Pic c khi lng cao nht v l thuyt l m/z = 173 (13 C 72 H637Cl15N18O) . Do xc sut mt s ng v l rt nh nn ch c (1), (2), (8) v (11) l c ng gp chnh cho cng pic (xem hnh bn).
Quy tc nit : Ion phn t c khi lng chn th s c s chn nguyn t nit (N0,

N2, N4, ... ), khi lng l s l nguyn t nit (N1, N3, N5, ...).
Nng lng ion ho mt cht hu c thu ion phn t thng l t 7 14 eV (1 1 1

eV = 23,04 kcal mol

= 96,3 kJ.mol ). 10,17 eV 9,88 eV 10,48 eV

V d : n-hexan cyclohexan etanol

ax-acetic metyl amin triflormetan

10,35 8,97 13,84

Xc nh chnh xc cng thc cng ca ion phn t : cn dng ph khi phn gii

cao. phn gii A ca mt khi ph k c tnh nh sau:

A= m m

Hai pic k nhau c cho l phn gii nu chng khng trng ln nhau qu

10%. tch vch c m/z = 950 khi vch m/z = 951 th my cn c phn gii l :

950 = 950 1
Khi ph k c phn gii thp thng c A = 1000 2000.
V phn gii cao :

Trong cc cng thc (2) (5) trang trc ta c khi lng nh sau : 156,017147 156,020069 156,010827 156,018008 (2) (3) (4) (5)

29

 tch c cc vch ny cn c phn gii nh sau : A(2)/(3) =

156 = 53388 0,002922 156 = 24684 0,006320

A(2)/(4) = A(2)/(5) =

156 = 180765 0,000863

Phn gii

Khng phn gii Mun ghi c c 4 pic th thit b khi ph cn phn gii 181.000. Thit b khi ph phn gii cao cn thm mt in trng trc t trng, nh vy cc ion c thu gom (focusse) 2 ln (double focussed - hi t kp) .
Phng php ghi ph

*) Chiu sng cc knh nh : Trong mt ph khi phn gii cao cht cn nghin cu v mt cht so snh thng l perfluokerosen cng c bay hi trong mt t trng v in th gia tc khng i. Ph c ghi trn mt tm knh nh. o sng ti (cng ) ca vt en (vch) v so snh vi ph cht chun, ta thu c s khi lng phn gii cao.Tin li ca phng php ny l cng mt lc c th ghi c tt c cc pic vi lng mu rt nh (khi nghin cu trao i cht, vt liu sinh hc). Bt tin l phi c thm thit b o sng ti v khong cch cc vt en. *) Ghi theo qut dng (qut t trng):
y ph c ghi bng cch: 3 i lng l dng ion, dng ion tng, bin thin t trng c x l theo thi gian v biu din thng qua my tnh.

Ngy nay : Cc tn hiu in c tch trong mt my tnh trong qu trnh ghi, sau s tnh ton v in ra ph. Ph ny s cho thng tin v khi lng v cng pic ( m/z v rel.%).
30

4. Nhng phn ng ct mnh chnh ca cc phn t hu c 4.1. Phn ct v tr

Nhng lin kt v tr i vi d nguyn t (N, O, S...) d b phn ct, v in tch sinh ra khi phn ct c cc d nguyn t lm bn vng.

Ph khi ca 2-Butanon

S phn mnh ca 2-Butanon S phn mnh rt gn ca 2Butanon

Ph khi ca 5- Androstan-3-on-etylen-acetal

31

S phn mnh ca 5- Androstan-3-on-etylen-acetal

32

Ph khi ca cyclo-hexanon

m/z = 55

S phn mnh ca cyclohexanon

Ph khi ca cyclohexanol
4.2. Phn ct v tr benzyl v allyl

Ph khi ca 2 aminoetanol

*) Phn ct v tr benzyl

Ph khi ca butylbenzen

S phn mnh ca butylbenzen

33

S phn mnh ca o-clortoluen

Ph khi ca o-clortoluen

Cp ion m/z 91/65 v m/z 77/51 l c trng cho cc hp cht thm th mt ln Ta thy phn ct benzyl mnh hn phn ct phenyl.

alkyl

m/z = 91 l pic c s ca cc hp cht n-ankyl benzen nh toluen, etylbenzen, propylbenzen, pentylbenzen, o-, m-, p-xylen.
*) Phn ct v tr allyl

S phn mnh ca 4 metyl-1hexen

Ph khi ca 4 metyl-1- hexen

t th hin hn phn ct benzyl

4.3. Phn ct cc lin kt khng hot ho

Ph khi ca nhng cht khng cha d nguyn t (phn ct ), khng cha nhm

phenyl (phn ct benzyl) v lin kt C=C(phn ct allyl) th tun theo quy lut phn ct cc lin kt khng hot ho.

34

Ph khi ca 1-florheptan

Ph khi ca 1-clorheptan

Ph khi ca 1-bromheptan
4.4. Phn ng Retro - Diels - Alder (RDA)

Ph khi ca 1-iodmheptan

Cc h vng 6 cha mt ni i c th b ph vng to thnh hai mnh : cu t "En" v "Dien". Thng thy cu t "dien" mang in tch trong ph ; cu t "en" cng thy. Vng 6 c th khng cha, cha 1 hoc nhiu d nguyn t, n cng c th l mt phn ca h vng ln. Phn ng RDA c th xy ra ion phn t, cng c th xy ra phn mnh m ni i c hnh thnh do mt phn ct khc (v d phn ct ). Phn ng RDA l mt qu trnh trung ho in.

35

C ch I:

C ch II: Ph khi ca 1,2,3,4-tetrahydrocarbazol

(m/z = 143)

Phn ng RDA cc hp cht khc :

Ph khi ca 5,7-dihydroxy-4'metoxyisoflavanon

S phn mnh ca 5,7-dihydroxy-4'-metoxyisoflavanon

Nhiu hp cht thin nhin nh indol ancaloit, tetrahydroisochinolin ancaloit, flavon, isoflavon, rotenoit c vng trung tm c th to phn ng RDA trong khi ph k. Cc mnh to thnh do phn ng RDA s cho cc thng tin rt hu ch gii cu trc cc cht ny.
4.5. Chuyn v Mc Lafferty
Chuyn v Mc Lafferty tng t phn ng Norrish II trong quang ho, trong nhit phn ester, trong phn ng Tschugaev,v trong phn ng "En".

36

 Cn gi l phn ct

c dch chuyn H.

Nhng nhm c th tham gia chuyn v Mc Lafferty : C=O (axit carboxylic, ester, andehyt, keton, amit, lactam, lacton) ; C=N (azometin, base Schiff, hydrazon, oxim, semicarbazon) ; S=O (sulfonester) ; C=C (alkyl aren, alkylheterocyclen, benzyleter, olefin). C ch I :

C ch II :

V d : CH3 CH 2 CH 2 COOCH3

Qua phn ct

Chuyn v Mc. Lafferty

Ph khi ca metyl butanoat S phn ct mnh ca metyl butanoat

37

Ph khi ca butyl benzoat

S phn ct mnh ca butyl benzoat c trng cho cc metylester l pic m/z = 74 (Mc Laffesrty) v m/z = 59 (phn ct ), cho etyl ester l 88 v 73.
4.6. Phn ng onium

quan st thy nhng mnh ion c d nguyn t tch in dng nh oxonium, amonium, phosphonium, sulphonium.

Thng

Qua phn ct

Qua phn ct

phn ng onium

Chuyn v Mc. Lafferty

phn ng onium

Ph khi ca N-isopropyl- Nmetylbutylamin

S phn mnh ca N-isopropyl- Nmetylbutylamin

38

in tch O Qua phn ct

Qua phn ct

phn ng onium

Ph khi ca N-butylacetamit

S phn mnh ca N-butylacetamit

4.7. Mt CO
Nhng

cht hoc nhng mnh ion c cha nhm C=O d b mt 28 n v khi

(CO).

(m/z = 65)

S phn mnh ca phenol

Ph khi ca phenol

39

Ph khi ca Dispiro[4.1.4.1]-dodecan-6,12dion

(m/z = 65)

S phn mnh ca Dispiro[4.1.4.1]dodecan-6,12-dion

5. Cc phn ng nhit trong my khi ph 5.1. Nhng loi quan trng nht ca phn ng nhit Tch loi nhng mnh nh :

Decacboxyl ho (CO2) Decacbonyl ho (CO) CH3COOH HX (H2O, HCl...)

*) Phn ng retro :
Retroaldol :

40

 Retro-Diels-Alder

*) Isomer ho *) Dehydro ho, hydro ho

*) Nhit phn cc hp cht cha nit bc 4 * Dealkyl ho 6. Cc phng php v khi nim khc 6.1. Ion ho va chm in t (EI) - Dng cho nhng cht d bay hi phn t nh hoc t phn cc.
EI : Bt li l hay mt ion phn t hoc ion phn t cng yu. 10 20% hp cht c PTL < 300 khng thy ion phn t.

Li : c nhiu mnh ion, c th so snh ph vi ph ca cht bit, s dng ngn hng ph.
6.2. Ion ho ho hc (CI) - Phng php CI dng cho cc cht kh bay hi nh:

Nhng cht 200 Dalton nhng nhiu nhm phn cc Cht t 800 25.000 Dalton

CI : Dng mt cht kh (v d CH4, isobutan, NH3) vi mt p sut khong 102 Pa a vo bung ion ho. Cht kh ny va chm vi cc in t bn ra t mt catt nng vi nng lng 300 eV v b ion ho.

V d :
+ . + 2e CH 4 + e CH 4

Tip theo n b phn mnh mt phn :

+ . + CH CH 4 CH 5 + CH. 4 3
+ + CH3 + CH 4 C 2 H5 + H2

41

Khi ta cho bc hi cht cn nghin cu M vo hn hp ion ny, ta s c :

+ M + CH 5 MH + + CH 4

Phng php CI c th o c cc ion phn t v do xc nh c trng lng phn t. u tin, mt cht kh (v d hydrocacbon, H2, H2O, NH3, alcol, kh tr) qua va chm vi electron s b ion ho (p sut kh 1 kPa).

Trong trng hp l kh metan s xut hin [CH4]+, cn dng kh isobutan hoc

NH3

s c [C4H9]+, [NH4]+

Nu s dng cht kh khc (v d kh tr, CO2, N2) th khng xy ra proton ho m xy ra trao i in tch (charge exchange CE) : [He]+ + M [M]+ + He
Cc phn ng khc nh cng i in t :

M + [X]+ [MX]+

Bt anion :

AB + [X]+ [B]+ + AX

Ion ho hc l phng php m du b sung cho phng php EI.

Ph khi CI ca lysin metyl ester (Kh metan) Ph khi CI ca lysin metyl ester (Kh isopropan)
6.3. Ion ho bi in t (electrospray ionization ESI)

Dung dch cht cn o c phun qua mt capila vi tc 1 20 l/pht vo mt bung trong thit b.

42

S xut hin nhng git nh tch in, khi dung mi (MeOH/H2O ; CH3CN/H2O) bc hi, cc git s nh hn na v c a vo b phn phn tch trong mt capila.

S ion ho bi in t (my in t , ESI) 6.4. Bn ph bng dng nguyn t nhanh (Fast-Atom-Bombardment, FAB)

Dng cho nhng phn t kh hoc khng bay hi.

Nguyn l : Mt dng nguyn t trung tnh c tc nhanh c dng bn vo mt lp cht cn nghin cu mng trong bung ion ho. Qua xut hin ion ca cht cn phn tch.

Dng nguyn t khng tch in, c tc cao, thng dng l argon (Ar), hn

hu cng dng xenon (Xe) v c to ra bng mt sng lc, ion Ar+ c gia tc (5 10 keV) trong mt bung va chm, khi va chm vi nguyn t Ar trung tnh s to ra Aro c tc cao.

Dng Aro nguyn t tc cao c hng vo lp cht cn phn tch gn vo mt

matrix (thng dng glycerin, thioglycerin hoc 3-nitrobenzyl ancol) [1-2 g cht trong 1-2 l glycerin) trn mt mi nhn bng ng.

Vic chun b mu cng phc tp, cn s kho lo v kinh nghim. Ph FAB/MS c dng cho cc axit hu c (COOH, SO3H, OPO3H) v mui,

polypeptit, oligosaclarit, nucleotit, ph thng cho ion phn t dng (positive ion) : MH+ ; hoc ion phn t m (negative ion) : [M+H] [M+( glycerin)]+

. Thnh thong cng thy ion [M+Na]+,

43

6.5. Gii hp t trng (Field-Desorption FD) :

Mt si kim loi mnh (5 m) s c cy vi mt kim nh. Mt lng nh cht (~ 1 g) c ho trong dung mi v a ln si kim loi. Si kim loi t trong in k 8 kV v b t nng. V kim nh c cy vo l rt nhn nn u kim s c mt in th ln n 108 Vcm 1, iu ny dn ti : 1 e ca cht cn o c chuyn vo orbital d trng ca kim loi lm kim trn si kim loi xut hin ion phn t dng ca cht. Qua lc y Columb, ion phn t ny s c gii hp. Trong ph s c ion M+ v c th MH+. Cc phng php ci tin : Laser-Desorption, ph khi ion th cp (sekunder ion mass spectrometry SIMS) v californium plasma desorption.

V cc ion phn t cht nghin cu i khi mi trng lng hoc rn trong thi gian
12

ngn (10

s) nn hu nh khng c phn hu do nhit thu c ion phn t.

Katt

+ 8kV Si kim loi O+ O+ O+ O+ Kim nh ion phn t

+ 8kV

Si kim loi

S ca phng php gii hp t trng (FD)

*/ Californium (252Cf)-Plasma desorption :

ng v phng x 252Cf phn r thnh 2 phn chy ngc chiu nhau vi ng nngl 79 v 104 MeV, khi gp cht nghin cu trn tm Ni s lm tng nhit 10.000oK, phn t cht dng plasma s c gii hp dng + v v tng tc vo bung phn tch. Thit b MS ny dng k thut thi gian bay (time of flight TOF). Phng php ny c th xc nh c cht c phn t lng t 10.000 20.000 Dalton vi chnh xc 10 20 n v khi.

44

Cc phng php ion ho hay c dng

ion ho Phng php (EI) Ht ion ho eLoi ion M+v cc mng ion (CI) CH5 , NH4 , Ar
+ + +

Tn hiu ph

khong PTL 3,,500

Phn ng phn hu nhit c

- C th

V d: NH4: [M+H]
+

Sn phm cng hp

3,500

[M+H4]+ M, Cluster (FAB) z.B.Aro , nng lng cao V d: [M+H]+ [M+Na] [M+K]
+

Cng cht mang V d: [2glycerin+H]

3,500

Rt him

Cluster V d: [M+H]+

(ESI)

Khng c ( tnh in)

100,000

Khng

(TSI)Va chm nhit

Thng xuyn v/d CH3CO2NH4

[M+H]+ [M+H4]+

Thnh thong cng dung mi

3,500

Khng

45

6.6. Kt ni cc thit b khc vi my khi ph

GC/MS
Ion ho v thu gom ion trong chn khng Phn tch H thng khi detect

Kh mang

Bm mu

SKK

ngun ion FID- detect v my ghi

MS My tnh

ts

MS bn cu t

SKK cho bn cu t v pic dung mi (LM)

ts

T ph khi ca hn hp 4 ng phn ca dn xut pyrrolizidin tch ph ca A, B (sn phm chnh 95%, M = 223 v C, D so snh

Cu trc ca ng phn chnh

Cng tng i

Ph khi ca tng cu t

46

T l ng v phng x ca Clor v Brom

47

T l ng v phng x ca Clor v Brom

48