13

CHNG 3: TNG QUAN V SC K LNG11 3.1 PHNG PHP SC K LNG HIU NNG CAO (HPLC) HPLC l mt phng php tch v phn tch cc hp cht c s dng rng ri

v ph bin nht hin nay v nhiu l do: c nhy tng i cao, c kh nng nh lng tt, thch hp cho vic tch cc hp cht kh bay hi hoc d b phn hy nhit, c phm vi ng dng tri rng trong nhiu lnh vc t nghin cu khoa hc trong cc phng th nghim n cng nghip v mt s lnh vc khc.

Hp cht c th phn tch bng sc k lng nh acid amin, protein, acid nucleic, hydrocacbon, carbohydrate, thuc khng sinh, thuc tr su, cc hp cht v c Da vo s khc nhau v c ch chit tch s dng trong sc k lng hiu nng cao m ngi ta c th phn chia n ra lm cc loi: sc k hp ph, sc k phn b, sc k ion, sc k ry phn t trong sc k phn b c ng dng rng ri v ph bin. Ty theo phn cc pha tnh v dung mi pha ng, ngi ta phn bit: sc k lng pha thng v sc k lng pha o. Sc k lng pha thng: pha tnh c phn cc cao hn phn cc ca dung mi pha ng, dng tch v phn tch cc hp cht c phn cc cao vi phn t lng khng ln lm. Sc k lng pha o: ngc vi sc k pha thng, pha tnh c phn cc thp, pha ng c phn cc cao hn. Phng php ny dng phn tch cc hp cht t khng phn cc n phn cc va. Dung mi s dng l dung mi phn cc, trong nc ng vai tr quan trng m li r tin, do sc k lng pha o c s dng nhiu nht.

3.2 CC THNG S CA PHNG PHP SC K LNG 12

3.2.1 H s phn b Cn bng ca mt cu t trong h sc k c th c m t bng phng trnh n

gin sau: A pha ng A pha tnh

14

H s cn bng K cho cn bng ny c gi l t l phn b v c tnh nh sau:

Cs: nng cu t trong pha tnh Cm: nng cu t trong pha ng S: Pha tnh M: Pha ng K ty thuc bn cht pha tnh, pha ng v cht ha tan. K nh: cht di chuyn

nhanh; K ln: cht di chuyn chm. Thng thng K 1 cht c i lc vi pha tnh nu khng th n s di chuyn

nhanh qu lm cho kh tch. Hai cht mun tch ra khi nhau th phi c K khc nhau. 3.2.2 Thi gian lu tR

L thi gian cht phn tch sau khi tim vo ct n u d c tnh theo cng thc:

tR = tR + tM

tM: thi gian lu cht ca cu t khng b gi li trn ct, c tnh gn ng theo cng thc:

2

M0,5 ( )t L dc

V

trong : L: chiu di ct, cm

time

M

S

CCK

15

dc: ng knh ct, cm V: tc dng pha ng, mL/pht C th nhn danh cht thng qua thi gian lu v trong iu kin th nghim trn

ct thit b sc k lng nht nh, thi gian lu ca cht l mt i lng xc nh. Thi gian lu cng ln th h s phn b cng ln.

3.2.3 H s dung lng m t tc lu ca cu t phn tch trong ct ngi ta s dng mt h s

quan trng gi l h s dung lng k. Cs.Vs'

C .VM M M

Vsk KV

Vs: th tch pha tnh Vm: th tch pha ng k' c th tnh theo cng thc

k phi > 1 peak ca cht tan tch khi peak ca dung mi nhng khng qu ln v lc thi gian phn tch s ko di v mi b t do cht trong ct qu lu. Khong k l tng l t 2 n 5 nhng khi phn tch mt hn hp phc tp chng ta c th chp nhn k trong khong t 0,5 n 20.

k tng hoc gim ty thuc mnh ca dung mi pha ng. Trong sc k phn b pha o ngc, mnh ca dung mi tng theo phn trm cht hu c.

Thng thng trong sc k phn b pha o ngc, khi gim dung mi hu c trong pha ng 10% th k s tng ln gp 2 n 3 ln.

3.2.4 Hiu nng Khi ni n hiu nng ca ct l ni n kh nng tch mi sc k ca mt cu t

trn ct. hiu nng N c biu din nh s a l thuyt ca ct. N cng ln hiu nng tch ca ct cng ln.

M

MR'

tttk

2

21

R

2

R

wt5,55

wt16

HLN

16

Vi W: b rng mi sc k W1/2: b rng ca mi phn na chiu cao S a l thuyt cng ln, b rng W cng nh, mi cng nhn. 3.2.5 chn lc

2 2 2

1 1 1

''

R M R

R M R

K t t tK t t t

chn lc l mt thng s rt quan trng, lin quan ti kh nng tch ca cc cu t cn phn tch, n ty thuc bn cht ca pha tnh, pha ng.

Hai cht tch c khi # 1 v cng ln, kh nng tch cng cao. C nhiu yu t nh hng n chn lc nh thnh phn dung mi pha ng,

pH ca mi trng, bn cht ca pha tnh, nhit . Trong s thay i pha ng t dung mi ny sang dung mi khc s lm thay i ng k.

3.2.6 phn gii phn gii (Rs) ty thuc vo iu kin pha ng, pha tnh v c tnh ca hp

cht cn phn tch m ta c c sc k vi nhng mi ph ln nhau hoc tch hn nhau. phn gii Rs l mt thng s dng nh gi mc tch ph, kh nng tch ca cc mi sc k s c ngha quan trng trong vic phn tch mt cch nh lng.

Phng trnh trn thch hp cho vic tnh Rs khi 2 mi tch hn nhau (Rs 1,5). Trong trng hp Rs nh hn, tc mi b trng lp th vic xc nh W1 v W2 s

kh khn hn l W1/2(1) v W1/2(2) lc ny Rs s c tnh theo: 2 1

1/2(1) 1/2(2)

1,18 R Rt tRsW W

'A

'B

A

B

KK

KK

2wwtt

R21

RRS

12

17

2

2

'14 1 ' 1s

kR Nk

Khi k1= k2, 1: 1 '14 ' 1skR N

k

c th phn tch mt cch nh lng th 2 mi k nhau phi tch hn nhau, tc Rs 1,3. Khi Rs 1 cn phi thay cc thng s thc nghim lm tng Rs.

Tm li: khi tin hnh mt quy trnh phn tch bng sc k lng hiu nng cao, th cn ch ti kh nng phn tch ca cc mi trn sc k . C nhiu yu t nh hng ln php xc nh v ty theo yu cu cn phn tch nh tnh, nh lng hay bn nh lng m chng ta s ti u ha cc thng s thc nghim ca pha tnh cng nh pha ng cho hp cht cn nghin cu.

3.3 GII THIU CC B PHN CA THIT B SC K LNG HIU NNG CAO

Thit b sc k lng hiu nng cao bao gm cc b phn chnh: bm, b phn tim mu, ct sc k phn tch, u d, b phn iu khin v x l s liu.

Hnh 10: M hnh c bn ca HPLC. Nguyn l hot ng: mu sau khi c tim vo ct s c pha ng li ko qua

ct. Da vo kh nng tng tc khc nhau gia cc cht c trong nn mu vi pha tnh v pha ng m chng c tch ra khi nhau v sau khi ra khi ct s c ghi nhn bi b d c th.

18

Sc k lng c th ghp vi nhiu loi u d khc nhau nh u d ph t ngoi kh kin UV-Vis, u d hunh quang FLD, u d ch s khc x RID, u d in ha ECD, u d khi ph MS Hin nay u d khi ph c ng dng rng ri trong phn tch vt v cc hp cht nhn danh chnh xc.

Ty theo tnh cht ca cht cn kho st m ta c th chn la pha tnh, pha ng (bng 1).

Loi ct Pha tnh Dung mi Cht cn phn tch C18 Octyldecyl ACN, MeOH, H2O Khng phn cc C8 Octyl ACN, MeOH, H2O Khng phn cc Phenyl Styryl ACN, MeOH, H2O Axit bo, cht c lin kt i Cyano Cyanopropyl ACN,MeOH,H2O, THF Xeton, aldehyd

Amino Aminopropyl ACN, MeOH, H2O, THF,

CHCl3, CH2Cl2

ng, anion

Diol Dihydroxyhexyl ACN, MeOH, H2O, THF Protein

SAX Aromatic Quaterany m Anion Aromatic m SCX Acid sulfonic ACN, MeOH, H2O

Cation

Alkyl ether m Diethyl aminoethyl

DEAE Diethylamine ACN, MeOH, H2O

Protein, anion

Alkyl ether m CM Acid acetic ACN, MeOH, H2O

Protein, cation

Si Hexane Silica

Silanol chloroform

Hp cht hu c phn cc, ng phn

Bng 1: Loi ct, pha tnh, pha ng v hp cht phn tch thng dng 13.

19

nh lng anthocyanin trong cc dch trch bng phng php HPLC ngi ta cn kt hp u d UV/Vis vi u d ECD. Khng ging HPLC bn nh lng ch lm sch, HPLC- UV/Vis- ECD dng phn tch. Cht phn tch c tim t ng v cng vi pha ng ti ct. Cc hp phn khc tng tc khc nhau vi pha tnh v pha ng. Cht phn tch sau khi c ra gii i n detector mt cch ring bit. Phng php HPLC-UV/Vis-ECD c mt s c im sau: Pha ng:

Pha ng trong HPLC c a lin tc vo h thng. Pha ng c th theo chng trnh gradient dng hoc ng dng, nhng pha ng c nh thng mt thi gian lu v kh tch cc mi. Hin nay thng dng nht l dng gradient. Khi ra gii trong sc k pha o, lng dung mi hu c tng dn ln. Ti lc tim mu, mt lng pha ng yu hn i vo h (t hp phn hu c hn). Sau li tng t l hu c trong pha ng ln ra gii tt c nhng cht b gi li pha tnh. Ct 10:

C nhiu loi pha tnh khc nhau, dn n c ch ra gii cng khc nhau. Trong nghin cu ny s dng ct pha o vi pha tnh khng phn cc v pha ng phn cc. Pha tnh cha cc hp phn ging RMe2SiCl vi R l cc nhm alkyl (C18H37 hoc C8H17). Hp phn khng phn cc c i lc vi pha tnh nn thi gian lu di hn. Thi gian lu c th iu chnh bng cch thay i phn cc ca pha ng. ng knh trong ca ct ln to sc cha ln, c dng trong HPLC bn iu ch lm sch anthocyanin. Ct phn tch dng trong HPLC-UV/Vis-ECD c ng knh trong ID nh hn (C18 250 x 4.6mm).

3.3.1. u d UV-Vis u d c gn n UV (thng l n deuterim 190-360nm) v n Vis (n

tungsten, 360-800nm). V vy cc hp phn hp thu nh sng t khong 180- 800nm s c u d pht hin.

Cht phn tch sau khi ra gii ra khi ct chuyn qua 1 t bo cm bin hnh tr. nh sng trong vng UV/Vis truyn qua t bo ti mng quang in. Nu cht phn tch hp thu c nh sng 1 bc sng no , mng quang in s pht hin ra s thay

20

i. Tn hiu pht ra t mng quang in s chuyn ti b khuch i n b ghi o v h thng thu d liu.

Cng dng sng truyn qua cell o (I) t l vi nng ca cht phn tch theo nh lut Lamber- Beer (- h s hp thu phn t, c- nng (mol/L), l- chiu di ng truyn quang (l lun l 1 cm), Io l cng ca tia sng ti cell):

A= l c

100

A log

tiem

II

DientichECDCF V nDientichUVC

l

Anthocyanin c 2 bc sng hp thu 530nm v 280nm, v vy c th s dng u d UV/Vis. Trong HPLC nh lng, c th dng UV/Vis kt hp vi ECD hoc vi MS.

3.3.2. u d ECD: 3.3.2.1 Tnh cht in ha ca anthocyanin: Hot tnh chng oxi ha ca cc hp cht thin nhin ph thuc vo s lng cc

phn t hin din trong mu v cu trc ca chng, v d v tr nhm -OH hay -OCH3. Anthocyanin c c tnh chng oxi ha do cc nhm -OH trn vng thm. Cht chng oxi ha l mt cht kh khi tng tc vi cc gc t do, ngha l chng b oxi ha trn b mt in cc. Cht chng oxi ha ngn s hnh thnh v hot ng ca oxi v nit.

Ta c th thy hnh 11, nhm catechol vng B v cc nhm resorcinol vng A ca catechin c th b oxi ha. Vng A v B khng lin hp nhau nn s oxi ha cc nhm -OH ca vng ny khng nh hng ti cc nhm -OH trn vng kia. Cu trc catechin c cc nhm -OH gn vo vng thm c th b oxi ha. Vng B d b oxi ha hn vng A, v vy khi p th 100 800mV th cc -OH trn vng B s b oxi ha, trn 1500mV vng A s b oxi ha.

21

Hnh 11: S oxi ha ca cc nhm catechol. Mt s nghin cu cho thy cc nhm -OH v tr meta (trn vng A) kh b oxi

ha hn v tr para hoc ortho (trn vng B). Kt qu l, anthocyanin s b oxi ha nhiu bc cc th p khc nhau, ty vo th t oxi ha ca cc nhm -OH khc nhau. So vi u d UV/Vis, u d ECD nhy hn v chn lc hn i vi anthocyanin c hm lng nh.

C ch ca qu trnh oxi ha xy ra theo th t sau: 1. S oxi ha cc nhm -OH trn vng B th dng b 2. S oxi ha cc nhm -OH trn vng A th cao hn

Th oxi ha ty thuc vo pH, khi pH tng ln s lm gim th oxi ha. Ni chung, cc nhm -OH v tr ortho trn vng B b oxi ha 450mV. Nhm

methoxyl v ng lm tng kh nng chng oxi ha ca anthocyanin. Vng A v B khng lin hp nhau nn s oxi ha ca cc nhm -OH trn vng A khng nh hng ng k ti s oxi ha cc nhm -OH vng B v ngc li. V vy t phng php quang ph c th kt lun rng vng A khng nh hng trn tnh cht ph ca cc gc t do t vng B.

3.3.2.2 Nguyn l ca HPLC/ECD6,14,15,16

22

Thit k my HPLC - ECD v mt c bn khng khc g HPLC truyn thng, ngoi tr u d quang hc c thay bng u d ECD. u d ECD c dng t 1974. Lc v c ch m t h thng HPLC - ECD hnh 14. Thit b gm bm y dung mi ng bng p sut h thng. Ct phn tch cha vt liu nhi ct (thng l lp nn tr c ph vi b mt k nc, chng hn cc dn xut ca hydrocacbon a vng) phn tch cht da trn thi gian lu. u d in ha ni trc tip vi ct. tng hiu sut v lp li, mu c tim bng h thng tim mu t ng, b to gradient iu khin thnh phn pha ng, tt c cc h thng ny u phc v cho my tnh v my tnh thu ph sc k . lm phong ph phn d liu, u d PDA ni trc tip v t trc u d ECD. Thnh phn pha ng cha nng cht in ly cao thun tin cho dng i qua u d ECD. Thng pha ng mi trng acid cha Li3PO4 hoc Na3PO4. Li3PO4 d tan trong dung mi hu c (MeOH hoc ACN) v vi khun khng pht trin khi c mt Li nn Li3PO4 thng a dng trong dung mi ng. Cn lm sch dung mi ng vi kh tr He loi oxi v oxi cn tr vic pht hin cht phn tch siu nhy.

Khi cht phn tch i qua in cc lm vic (in hnh l in cc Cacbon nhng thng l in cc kim loi), phn ng ha hc xy ra nh cht phn tch mt mt (hoc nhiu) in t. Nhng in t ny c pht hin bng mt dng chy qua in cc lm vic. V th, tn hiu ha hc chuyn thnh tn hiu in t m khng cn cht mang t tnh trung gian. Dng chy qua in cc lm vic vo mt thi im no l mt hm tuyn tnh vi s phn t b oxi ha ti b mt in cc. Th in ha ca in cc lm vic c th bin i xc nh mt cch chn lc cht phn tch; bt k cht no ra khi ct sc k m khng b oxi ha di th p vo th u d s khng pht hin c. Do ECD c mt chc nng chn lc ni ti m cc h thng u d khc khng c. Thm vo , ECD cc k nhy, gii hn pht hin nh gp 10 1000 ln nhy ca u d UV/Vis v gp 10 ln u d hunh quang.

23

Hnh 12: H thng LC-UV-ECD ti phng th nghim

Hnh 13: Nguyn l hot ng ca u d in ha. A: ECD loi ampe gm u d gn vo thnh ca rnh hp, cht phn tch c chuyn qua. Phn ng in ha chuyn cht X thnh sn phm b oxi ha Y. B: ECD dng coulom gm mt in cc xp m cht phn tch s chuyn qua, phn ng in ha hiu qu hn v ci thin c s truyn khi.

u d in ha hnh 13A l loi u d ampe. y l dng c bn nht ca ECD. Khi cht phn tch chy qua in cc lm vic (thng l Cacbon nhng cng c

24

khi l kim loi), phn ng ha hc xy ra lc ny cht phn tch mt i mt hoc nhiu in t (tc l cht phn tch b oxi ha). Nhng in t ny c pht hin di hnh thc dng in chy qua in cc lm vic. Do thng tin ha hc v cht phn tch c chuyn thnh tn hiu in m khng cn cht mang t tnh hoc quang hc trung gian. Dng chy qua pin in ha bt k thi im no u tuyn tnh vi s mol c oxi ha trn b mt pin ti thi im . Th in ha ca in cc lm vic c th bin i cho php xc nh cht phn tch chn lc hn da trn th oxi ha; bt k hp cht no i ra khi ct m khng oxi ha di th p vo cell o th u d s khng nhn ra. Do vy u d ECD c mt chc nng chn lc ni ti m cc u d quang hc khng c. Tuy nhin, loi u d ny t nhiu khng hiu qu v ch c 5 - 10% cht phn tch chuyn qua in cc lm vic s khuch tn n b mt in cc v thc hin qu trnh in ha. Do dng hnh hc ca u d nn ch cho php s oxi ha ti lp phn cch (boundary), ch mt phn cht phn tch truyn qua u d c oxi ha. Loi u d coulom tt hn (hnh 13B), loi ny c matrix xp, thng l Cacbon. in cc so snh v in cc b tr (counter) dng kim loi (thng l Paladium). i vi dung dch long, u d coulom gn nh y ht cht phn tch chuyn ha trn in cc di th p ca u d. Dng c hng trc tip vo u d ch khng phi truyn qua. Din tch b mt in cc tng ng k, dng chuyn ng mnh nn pha trn lp dung dch phn bin gip cc iu kin c tha mn. V vy, u d nhy hn 10 - 20 ln u d ampe v ci thin c s truyn khi. Trong thc hnh u d ampe v u d coulom khng khc nhau nhiu v nhy nhng nhn chung u d culong c a chung hn.

3.3.2.3 C ch Cc cht c th kh hoc oxi ha tri qua phn ng oxi ha trn b mt in cc,

to s thay i dng v c u d ECD ghi nhn. Hnh 14 m t c ch ca pin in ha: 1 cp in cc c t vo trong pin v p th dc theo in cc. Khi th ln, phn ng din ra v xut hin dng in.

Cng dng I c ghi o theo thi gian, din tch peak ng vi tng in tch Q trao i gia in cc v cht c hot tnh in ha, c tnh theo cng thc sau:

Din tch peak = Q = i . dt

25

nh lut in phn Faraday lin kt Q vi nng cht hot ng in cc qua phng trnh:

Q = n F ntim Vi ntim = [C] Vtim

Q = n [C] VtimF Trong my Dionex chng ti biu din theo hiu in th V thay v cng I,

cng dng ny c bin i thnh hiu th bng cch nhn vi in tr R Din tch peak = R i . dt = n [C] Vtim F

Vi: R: l in tr ca thit b (c th tnh ton hoc bit c t nh sn xut), cn gi

l h s i: dng o c bi u d in ha. n: s electron trao i lin quan ti qu trnh in cc. C: nng ca cht hot ng in cc. Vtim: th tch tim (L) F: hng s Faraday = 96500 (C/mol). ntim l s mol cht phn tch c tim vo my. Nu bit c s electron trao i n trong hp cht c tnh in ha, s bit c

nng cht phn tch t din tch peak sc k HPLC- ECD.

26

Hnh 14: S ca h HPLC- ECD. ng mi tn m l pha ng. ng t nt ch mi lin h gia cc thit b.

Hnh 15: (a) m phng phn ng trn pin in ha (2 in cc t trong dung dch cha hp cht c hot tnh in ha); (b) pin in ha tht- in cc b tr (A) p th c nh bi Amp.1, th c chn lc t my in th (P) kt ni vi ngun in. Dng chy qua in cc lm vic c iu khin bng b ampe th 2 (Amp.2) v d liu i ra hng vo u c hoc h thng qun l d liu; (c) h in cc culong s dng in cc carbon graphite l xp, trn mi n v in cc c 1 in cc carbon l xp, trn c 2 mt c gn in cc so snh v in cc b tr. Khi p sut dc theo in cc kh nh, cc n v in cc c th kt ni li thnh chui, to mng array.

Mt pin in ha c bn cn 3 in cc: in cc lm vic ( y s din ra qu trnh oxi ha hoc kh), in cc b tr v in cc so snh (dng in chy qua rt b, dung ha bt k s thay i trong dn nn ca pha ng). Pin in ha c s dng c nhy cao, b mt rng m bo 100% cht phn tch hot tnh in cc s b oxi ha hoc kh. Dng o u ra c n v mV (U = I R, U c n v l mV, I c n v l mA, R c n v l ).

Vic trao i in t xy ra ti ranh gii ca in cc lm vic v cht hot ng trong dung dch in dn. T l trao i in t, tc ln ca dng chuyn qua, chnh l tc phn ng ti th p. T l ny cng ty thuc vo tc ca cht kho st n b mt in cc bng khuch tn v i lu.

S electron trao i n ca cc cht in hot c th c xc nh bng phng php vol-ampe. Nguyn tc ca phng php c m t nh sau:

27

Cch xc nh s electron trao i bng vol-ampe 19,20,21: Cc phng php in ha s dng trong h sinh hc m phng bn cht chng

oxi ha da trn mi lin h cht ch gia kh nng oxi ha kh ca ha cht trong dung dch v hot tnh in ha ti b mt in cc, cng ca cht chng oxi ha trong mi trng v th p chng b kh trn b mt in cc (cht kh cng mnh th p cng t dng). Thc t mt cht chng oxi ha l cht kh trong tng tc vi gc t do, lc ny chng s b oxi ha, tuy nhin khng phi lc no cng vy. Nhng cht chng oxi ha khc nhau phn ng vi cc gc t do v cc cht oxi ha khc nhau nhiu mc , cng nh chng s hot ng trn cc b mt in cc khc nhau vi mc khc nhau. D bin i nh vy nhng chng cng cho php nh danh v nh lng. Mc khc, cht chng oxi ha ngn s hnh thnh v kh nng hot ng ca nitrogen v oxygen t nhng dng hot ng khc.

Trong phng php vol-ampe, th p ca in cc lm vic thay i tuyn tnh vi thi gian, bt u t th E m khng xy ra phn ng in ha n gi tr E xy ra s kh/oxi ha cht nghin cu. Sau khi i ngang qua vng th c mt hoc hai phn ng in ha xy ra, hng qut tuyn tnh o li, cc sn phm cng nh sn phm trung gian to ra t ln qut u s thc hin phn ng in ha v c ghi nhn. Khong thi gian th nghim (chnh l vn tc qut v th E nghin cu) c th bin i 102-10-3s mc d cc phn ng nh lng thng gii hn 10-10-3s. Cht in ly b tr c mt km hm s di chuyn ca cc in tch.

Cc th nghim c thc hin trn cell 3 in cc kn kh ni vi dng argon, dung dch c sc kh argon 5-10 pht ui oxi trc mi ln o, th tch cell l 10mL. Qut th t -200 ti 1200mV v -200 ti 600mV. in cc so snh l calomen bo ha cch bit vi dung dch bi cu ni, bn trong cu ni c cng dung mi v cht in ly b tr. in cc b tr c dng xon din tch b mt 1cm2, l 1 si platium di khong 5cm ng knh 1mm. in cc lm vic gm nhng a thu c t mt ct ngang ca si dy bng vng c cc ng knh khc nhau t bn trong vt liu thy tinh. Gia cc ln chy, in cc lm vic c nh bng bng bt alumina trong 3 pht loi b cc sn phm oxi ha hp th mnh trn b mt in cc, nhng sn phm ny s cn tr b mt in cc, nh hng ti ln th th 2 d l pH no. in cc lm vic

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cng c th lm bng Cacbon ng knh 3mm hoc c dng a quay ng knh 2mm nm gia li Teflon, hot ng vn tc gc = 105rad/s, qut th 20mV/s m bo trng thi n nh. S electron trong phn ng in ha c th c xc nh bng nhiu cch. Phng php ph bin nht dng php o in lng coulometry, xc nh s lng ca cht in phn gii phng trong thi gian in phn bng cch o s culong s dng. Tuy nhin khi s dng gi tr n t phng php culong gii thch ph CV thng gp tr ngi, v khong thi gian v cc iu kin phn ng tng i khc nhau. Trong php o in lng coulometry thi gian l 1 gi v lp phn ng l dung dch gc ca cell o, cn trong phng php CV thi gian ngn hn 1 giy v lp phn ng khong mt vi mm tnh t b mt in cc.

Cc bi bo trc y thng dng mt phng php rt ph bin xc nh n trong CV l: cht chun, thng l ferrocene (n = 1), c thm vo cng vi mu trong cell o CV, ri so snh trc tip cng peak ca chun v cht nghin cu. Tuy nhin vic so snh ny da trn gi thit h s khuch tn ca chun v mu l nh nhau (thng h s khuch tn rt kh bit trc). Ngoi ra cng phi tnh n cc phn ng ha hc ng th v d th c th nh hng ti hnh dng v chiu cao ca sng in th. Do phng php ny lun thiu chnh xc. Tuy nhin, nu o lng t k thut tc thi:

Itransient = n D1/2 F A co ( Tc)-1/2 itransient t l vi n D1/2 Vi Tc: thi gian kho st ring. A: din tch b mt in cc. F: hng s Faraday = 96500. D: h s khuch tn b mt. co: nng ca dung dch. Kt hp vi php o in lng voltametry trng thi tnh (o dng Faraday i

qua dung dch in phn khi p th thch hp vo in cc lm vic)

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isteady state = n D 4F co ro isteady state t l vi n D. D: h s khuch tn cu. ro: bn knh.

transientsteadystate

i ni

, lc ny h s khuch tn D c gin lc.

Nu s dng cc in cc khc nhau cho php o tc thi v trng thi tnh, cn iu chnh dng o c v c hiu ng din tch in cc. Tuy vy chng ta c th trnh vic chun ha ny nu cc php o ging nhau thc hin trn cng in cc v cng mt cht chun bit n v D ging nh ferrocene.

3.3.3 Gii thiu v LC/MS/MS12

Sc k lng ghp khi ph l mt thit b l tng cho cc phng th nghim. LC/MS l phng php c dng trong phn tch vt v cc hp cht cn nhn danh chnh xc v trong nhng iu kin vn hnh nht nh ngoi thi gian lu c trng, ha cht cn c nhn danh bng khi ph ca n. nhy LC/MS ty thuc vo hp cht cn phn tch v giao din s dng. Trong sc k lng ghp khi ph, hn hp cht phn tch sau khi tch ra khi ct s i qua mt ng truyn n u d khi ph. Ct sc k lng cho php tch cht cn quan tm, khi ph cho php tch ion cn quan tm v cho bit phn t lng ca cht phn tch. Mt h thng LC/MS/MS s phn mnh ion m thnh cc ion con v tch cc ion con nh danh v nh lng.

Mt h LC/MS c bn gm h thng bm sc k lng, b phn tim mu, ct sc k, u d khi ph.

Nhim v ca u d khi ph: Nhn danh ha cht:

Mt mi trn sc k vi thi gian lu xc nh c trng cho ha cht, tR ny chnh l thi gian t lc ha cht bt u vo ct v ra khi ct vo u d khi ph.

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Mi ny biu din tng cng cc ion sinh ra t phn t ha cht thng qua k thut ion ha xc nh. Tng cc mi ny trn sc hp thnh sc tng ion (total ion chromatogram).

Mi ny cng c th biu din cng ca mt loi ion sinh ra t s phn mnh ca phn t ha cht hoc t s phn mnh ca mt loi ion ca ha cht.

Khi ph tng ng ca ha cht hoc khi ph biu din cng cc ion sinh ra t mt ion nht nh. Nh th vic nhn danh thng qua va thi gian lu va khi ph s chnh xc hn l ch da vo thi gian lu. nh lng nng ca ha cht trong mu. Cc b phn chnh ca khi ph gm: b to ion, b tch ion, b d ion.

So snh 2 k thut ion ha APCI v ESI: Ging nhau: - u thuc loi ion ha mm, t to s phn mnh, trong nhiu trng hp cn thy c phn t ban u. - Nu c s phn mnh ion th cch phn mnh cng n gin, gip on nhn cu trc cht ban u d dng hn. - C hai k thut u c th to ion m hoc dng ty theo cu trc hp cht kho st.

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Khc nhau:

K thut phun ion (ESI: electrospray ionization)

- Ion to trong pha dung dch - Thch hp cho cht khng bn nhit - C th to s phn mnh t hn APCI - Thch hp cho cht kh phn cc - Thch hp cho cht c phn t khi

ln

K thut ion ha ha hc p sut thng (APCI: atmospheric pressure chemical

ionization)

- Ion to trong pha hi - Thch hp cho cht khng bn nhit - C th to s phn mnh nhiu hn ESI - Thch hp cho cht t phn cc - Thch hp cho cht c phn t khi nh

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K thut APCI: khi phn t cht phn tch v dung mi i qua mt l sy s ha hi, cc phn t hi c kh N2 phun ra v i vo vng phng in (corona discharge). Hin tng ion ha xy ra:

C ch to ion dng: N2 + e- N2 + 2e- H2O + e- H3O+ +2e- H2O + H3O+ H3O+ + OH M + H3O+ MH+ + H2O C ch to ion m: vi nhng phn t c H linh ng, trong vng phng

in, c s ion ha ca phn t bng cch mt i H+, phn cn li l mt anion: OH + M (M-H)- + H2O

K thut phun ion ESI: - Cc phn t ha cht v dung mi ra khi ct sc k c a vo mt ng mao qun bng kim loi cao th v sau c phun mn bng kh N2 thot ra chung quanh ng mao qun. - Di tc dng ca in th cao, c s to thnh nhng ht nhuyn mang in tch thot ra t u ng mao qun. - Sau cc phn t dung mi t t bc hi. Cc ht mang in c th tch nh dn v do s y nhau gia cc in tch cng du, chng s b ra thnh nhng ht nh mang in tch. - Sau cc ion dng hoc m to thnh c a vo b phn tch ion qua mt ca rt nh. Dung mi v kh N2 b bm ht ra ngoi.

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- in th dng u kim to ion dng, in th m th to ion m v c quyt nh ty theo cht kho st. - Cc cht c tnh acid to ion m pH cao, cn cht c tnh baz to ion dng pH thp.

3.3.4 Gii thiu thit b khi ph FINNIGAN LCQ deca17 (hng Thermo Quest Finnigan).

Thit b HPLC (TPS Spectra system) bao gm b ly mu t ng (spectra system AS3000), bm P4000 gradient pump (spectra system P4000) v b loi kh chn khng, u d UV 6000 (spectra system SN4000) LP flow cell 5cm ni vi u d MS (LCQ Deca). Th tch anthocyanin khong 10L tim vo ct Phenomenex (Torrance, California, USA) Luna C18 (150mm 2.0mm, kch thc ht 3m), chng trnh ra gii bng 3.

Hnh 16: H thng LC-UV- MS ti phng th nghim u d MS l mt by ion LCQ-Deca (ThermoFinnigan) vi giao din ion ha phun

in t chy dng ion dng. p th dng +3 +4kV vo kim silica, dung mi to git bay ln, kh Helium y cc ht kh v gip phn nh, cc ht kh ny va chm nhau, dng in 80mA, th mao qun 23mV, nhit mao qun 225oC, thu knh (tube lens) ci

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t hiu th -5V, sheath gas (He) 1.2 L/pht v kh b tr (He) 6 L/pht. Ph hp thu DAD t bc sng 200 ti 800nm. By ion chy dng qut scan MS/MS ty thuc d liu, ln qut u ci t khong m/z= 200-1200. Ln qut th hai khai bo ion c cng cao nht t ln qut u v thu ph MS ln hai, tng t ln qut 3 MS3 khai bo ion cng cao ln 2. Nng lng va chm 28%, thi gian kch hot 30ms. Sau khi c tch nh, ion con c in tch ln hn in tch ca phn t m, ESI l k thut ion ha mm, ch cn vi giy thc hin s phn mnh.

Hnh 17: S ca giao din ESI 14

Nhn chung, 1 cht i vo my MS tri qua 3 bc: u tin, phn t c bn ph bi dng electron nng lng cao, chuyn phn t thnh ion. Ion phn t tng tc n trng tnh in. Tip theo, trong trng tnh in hay nam chm in, cc ion gia tc c phn tch ty theo t l khi lng/in tch. Cui cng, cc ion c t l khi lng/in tch c trng c pht hin. Dng ra ca u d c khuch i v ghi o.

Cht phn tch t sc k lng hoc bm xy lanh chuyn qua kim phun, to cc git tch in cui u kim. Dng He khng tch in trung ha cht lng v gip dung mi bay hi thnh git. Khi dung mi bay hi, cc phn t cht phn tch b y gn nhau hn,

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chng y ln nhau v cc git b v ra. Qu trnh ny do lc khng culong gia cc ion gy ra. Qu trnh ny s dng li khi cc phn t khng cn lin kt vi dung mi v tr thnh cc ion ring r. Sau chng c pht hin. Anthocyanin dng ion dng, v vy chng khng cn ion ha m trc tip i vo my MS sau khi bay hi dung mi. Qu trnh nh sau:

Th nghim khi tandem mass chnh l cc bc la chn khi ph vi cc dng v xut hin gia cc qu trnh. Trong th nghim tandem mass, thng tin v cu trc c tm thy qua cc mnh v ln 2 v 3 (MS2, MS3). Vi anthocyanin, khi cc nhm ng mt i, ty vo khi lng phn t b mt, c th xc nh l ng hexose hay pentose. Sau khi mt gc ng, s nhn bit nhm aglycone. Loi aglycone c nhn danh khi so snh vi nhng nhm bit.

Hnh 18: M t phng php tandem mass to ra cc mnh v ion t MS2 trong thit b triple quadropole.

Trong ion trap, cc ion b loi ra khi by ngoi tr ion cn c bn ph ln 2. Thit b p mt tn s sng radio svi tn s cng hng bng vi vn tc gc ca ion trong by. Nh tn s sng ny, nng lng ng lc c chuyn vo ion v ion bt u lun chuyn trong ion trap mt bn knh ln hn.

Ekinetic = m r 2

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Ion c ngun nng lng ng hc ny s va chm vi mt trong cc nguyn t He vn sn trong ion trap, lc ny qu trnh bn ph xut hin. Nng lng ng hc ny ty vo khuch i ca tn hiu sng radio v c hiu chnh bi s % do b vn hnh MS, trong my LC-UV-MS ti trng Roskidle khong 25-50%.