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CU TO, NGUYN TC HOT NG V NG DNG CA HPLC-FPLC

M u

T khi c nh thc vt hc ngi

Nga Mikhail Semyonovich Tsvet pht minh ra n nay, k thut sc k pht trin nhanh chng trong sut th k 20. Cc nh nghin cu nhn thy nguyn tc nn tng ca sc k Tsvet c th c p dng theo nhiu cch khc nhau, t xut hin nhiu loi sc k khc nhau
18721919

M u

High-performance liquid

chromatography-HPLC (sc k lng hiu nng cao) xut hin t nhng nm 1960 nh l nhnh ca sc k lng thc hin trn ct,trong cng ngh ch to ct v cc b phn ca thit b c ci tin

M u
So vi sc k lng ct p sut thp truyn thng, HPLC c nhng u im vt tri hn nh:
Cc ng dng ca HPLC trong thi k u Tc nhanh gm c phn tch d lng thuc tr su gii acid c ci thin tt hn trong rauphn qu, cc hu c, lipid, acid amin, cht c v vitamin. Ngy nay, sc k nhy tt hn lnghiu nng cao tip tc c ng dng nhiu loi vt liu phn Kh tch nng ti s dng ctsinh hc khc.Trong c k thut phn tch protein L tng choliquid nhng ion hoc phn t ln FPLC (Fast protein chromatography)

Thu hi mu d

M u CU

TO, NGUYN TC HOT NG V NG DNG CA HPLC-FPLC


ca HPLC-FPLC nhm ng dng trong phn tch sinh hc rt cn thit cho hot ng nghin cu v sau ca sinh vin chnh l l do em chn ti

Vic nghin cu cu to, nguyn l hot ng

Chng I
1.1.1 Sc k l g?
Sc k l khi nim chung p dng cho nhiu k thut tch a dng da vo s phn on hoc phn b 1.1 Phng php sc k (pha ng) v ca mu (cht tan) gia pha di chuyn pha c nh (pha tnh) QUAN TNG

1.1.1 Sc k l g?
Trong h thng sc k ch c cc phn t pha ng mi

chuyn ng dc theo h sc k. Cc cht khc nhau s c i lc khc nhau vi pha ng v pha tnh. Trong qu trnh chuyn ng dc theo h sc k ht lp pha tnh ny n lp pha tnh khc, s lp i lp li qu trnh hp ph, phn hp ph. H qu l cc cht c i lc ln vi pha tnh s chuyn ng chm hn qua h thng sc k so vi cc cht tng tc yu hn pha ny

1.1.1 Sc k l g?
C nhiu nguyn nhn khc nhau dn n s phn b trong hai kh nng tan ccs thnh phn Kt qu lpha cc nh thnh phn cha trong mu c tch ra

trong tng 2 pha,kh nng hp ph, trao i ion, kch thc thnh di trong pha ng cc phn t nhng chnh s lp i lp li hin tng hp ph phn hp ph ca cc cht khi dng pha ng chuyn ng qua pha tnh l nguyn nhn ch yu ca vic tch sc k

1.1.1 Sc k l g?
C nhiu tiu ch phn loi cc phng php sc k, trong tiu ch c s dng nhiu nht l phn loi theo h pha, tc l cht phn tch phn b gia hai pha l g
Phng php sc k

1.1.2 Phn loi sc k


Sc k kh
k SK Sc khrn giy Sc k

Sc k lng
SK phng in di phn b
mao qun lng-lng
Sc k Sc k Sc k HPLC

in di

SK kh-lng

rn-lng

lp mng

1.1.3 Phn loi sc k


Trong h sc k, cht phn tch c th l cc ion, phn t trung ho hay cc cht phn cc; i tng tc ng ca n cng c th l ion, phn t trung ho hay cht phn cc. T chng tng tc vi nhau theo cc lc lin kt khc nhau. Ngi ta chia cc lc lin kt thnh 4 loi 1.1.4 Cc lc lin kt trong h sc k
Lc lin kt ion Lc phn cc Lc Van der Waals Lc tng tc k nc

1.2 Sc k lng trn ct

Cu to h thng HPLC:
Bnh cha pha ng.

B phn kh kh (degasse) Bm cao p B phn np mu Ct sc k (pha tnh)


u d ( detector) In d liu

H thng my tnh c phn mm ghi nhn tn hiu, x l d liu v iu khin h thng

Bnh cha pha ng

Thng thng my HPLC c 4 bnh cha pha ng. Mc ch: To nng pha ng theo gradient thc hin ra gii trong qu trnh sc k. Khng s dng ht 4 bnh khi dng my.

B phn kh kh

Vai tr: loi tr cc bt nh c trong dng pha ng. Cc hin tng khi khng kh sch bt kh : + T l pha ng cu cc ng dung mi ly khng ng s lm cho pic khng chnh xc. + Nu lng bt kh qu ln, bm s khng bm c pha ng, my ngng lm vic.

Bm cao p

Cu to: Bm p sut khng i c thit k dng pittong Mc ch: vn chuyn pha ng qua h thng sc k. hoc dng bm tim. Dng tim trc to chy ra dng chy ng, khngv c xung nhng Dng pittong tovt dng c xung vy phi c b th tch bm hn ch. phn kh xung c hc hocchy in trong t bm loi tr dao ng ca c im: Lu lng dng thng l 1ml/pht p sut. B phnkim kh xung c hc gm b phn c th thay c sot m bo tnh1 ng v chnh xc i th tchHai tng thay i p sut ( v d nh kim loi ng bms thng c s dng vn hnh loi ch d dng, ng i cha yth cht lng d nn) :bin p sut khng hoc tch khng i.

B phn np mu
Nguyn tc hot ng ca van np mu: Cu to ca van np mu Vai tr: a mu vo dng pha ng np vo ct sc Khi Van van np np mu v ltr van LOAD: 6 cng, mu trong c np c 1 qua cng bm k. tim npvo mu vng vonay, ngoi, vng np lc mu. pha ng chy trc tip Ngy hu nh tt c h thng HPLC u s vo Vng ct np sc mu(sample k. mu. Bm loops) c a th tch c nh np p dng van np tim s mu n van Khi sut van thng. np Th n tch v vng tr INJECT: np thng vng cha dng l mu 10tr mu npxoay mu vo ct thnh 100 microlit 1 b phn trong dng chy ca pha ng v mu s Van theo cpha th ng iu chnh vo ct. xoay theo cc v tr LOAD v INJECT

Ct sc k
Vai tr: L cng c chnh tch ring cc cu t c trong mu. Cu to: Gm 2 phn chnh: +Phn cng +Vt liu nhi ct

Ct sc k
Phn cng
Dng ng bng thp khng r, thy tinh silica nung chy, titan, nha PEEK (polyether ether ketone); c 1 u ni vi b np mu, 1 u ni vi detector ca h thng. phn tch: L cc ct ph kin t trc ct phn tch Ct precolumn: HPLC. 2 loi ct ph bin nht ct precolumn ha v Kch thc ct thng dng c l ng knh trongbo 4, 5, 6 mm; ct bo v: 15, 25cm. chiu di ct l 10, Ct precolumn bocc ha: cha nhng ht silica trncos Ct c nhi bng ht 3, 5, 10 mm, vn hnh kch iu kin thc ln. Ct c t gia bm v b np mu bo ha lu lng 1-2ml/pht.
pha ng vi silica, n gip lm chm qu trnh ha tan cc vt liu nhi trn nn silica trong qu trnh s dng pha ng cha nc. Ct bo v: c ph bn trong bng lp mng pha lin kt hoc vt liu nhi vi ht ging ct phn tch. Ct c t gia b np mu v ct phn tch bo v ct phn tch khi b nhim bn bi nhng thnh phn c tnh hp th mnh. Ct bo v cn c nhi li hoc thay th khi nng lc lin kt b gim ng k v tp cht bt u xm nhp vo ct phn tch.

Vt liu nhi ct HPLC


Cc yu cu chung ca vt liu nhi ct + Vt liu nhi ct c chc nng chnh l lm cht mang ca h thng sc k. Trong sc k lng-lng c in, vt liu nhi ch c chc nng lm cht mang cho pha tnh. Trong sc k loi tr kch thc, s tng tc gia cht tan v vt liu nhi ct l khng cn thit ( do qu trnh tch ch da trn s khc bit v kch thc phn t). Trong sc k hp th ( sc k trao i ion, sc k i lc) vt liu nhi thc hin ng thi c chc nng l cht mang v chc nng ca pha tnh. Do , c th thy, vt liu nhi c th c lin quan n qu trnh tch tht s hoc khng. + Vt liu nhi phi l sn c vi kch thc ht chnh xc, ng u, t thay i. + bn c hc ln chng li p sut to ra trong qu trnh nhi ct v s dng. + bn ha hc cao, trnh b n mn nhanh chng.

Vt liu nhi
Cht nhi ct da trn nn silica
1. rng: +p ng tt cc ca vt ct. 2. Silica Pha lin kt: c tokh ra nh linyu kt cu ng ha trliu canhi nhm Cc ht silica c nhiu hnh dng v cc kchnhm thc khc nhau mc hydrocacbon vo b mt silica qua silanol ti bty mt ch s dng scca k m tanhi chn loi ht silica cho thch hp. silica Nhc imct chnh cht trn nn : b khung +Cc htdch silica ht cha nhiu l nh, c thnh phn ha tan dn trong dung c nc khi pH>8. ch yu SiO2 vi mi nguyn t nm tm t din u.titania, Ti b mt Mt s l vt liu c s dung thay th: alumina, zirconia, ht, mt ha tr cn li hydroxyapatite. thng c nhm OH chim, v gi l cacbon graphit rng, silanol, c tcnhi dng bin tnh b mt ca ht cch silicaph do tnh hot 3. Vt liu c mng mng: To bng lp cht mng ln ng caca n. vi ht tr, thng khng rng. Vt liu lm li c th c b mt bn cht nh ht silica hoc polymer, latex. Cc nhm chc nh cc v tr trao i ion ch c b mt, li ch m nhn lm tng bn c hc. Nhc im chnh: lp ph b mt lm mt i s lng tm tng tc lm hiu qu lin kt gim.

Cht nhi ct polymer:


- Bn - C th thay i cc c tnh tng tc qua bin i ha hc trc tip.

C 2 dng vt liu polymer:


1. Vi l: cc ht tn ti dng gel, mi loi ht c 1 xp nht nh, chng c th trng n hoc co li khi c s thay i pha ng, tuy nhin c th gy v ht, truyn khi km, tr lc dng chy ln 2. L ln: C nhiu lin kt ngang(>50%), gm 1 mng cc ht gel tiu cu lin kt nhau to thnh 1 ht ln. Thng c dng trong HPLC hn dng vi l.

Detector
Vai tr: Nhn bit s thay i nng trong dung dch ra khi ct v biu hin dng tn hiu in. La chn detector da vo: bn cht, nng cht tan, nhy, phm vi tuyn tnh ca detector, tnh tng thch vi dung mi v ch chy ca pha ng ( ng dng hay theo gradient) s dng, v c tnh kinh t, chi ph vn hnh Cc loi detector c s dng rng ri nht : detector quang ph kh kin-t ngoi, ph hunh quang o ch s khc x phn tch in ha

Detector o d hp th UV-VIS
S dng khi hp cht c cha nhm mang mu: cht khng no nh ketone, hp cht c ni i lin hp, vng thm, mt s ion v c v cc phc cht khc. ln ca tn hiu hp th t l thun vi nng cht phn tch theo nh lut Beer: A= e x d x C Detector UV-VIS c 3 kiu thit b khc nhau: + Bc sng o c nh + Bc sng o thay i c + S dng mng diode

Detector c bc sng o c nh: L kiu thit k n gin nht, vn hnh bc sng c nh. S dng 1 knh lc tch vch bc x n t ngun bc x (nh n thy ngn) D vn hnh, khng t tin Kh nng ng dng b hn ch. Detector c bc sng thay i c: c s dng ph bin nht n gin, vn hnh cc bc sng c th iu chnh c. Ngun bc x l n tungsten hoc deuterium, vic la chn bc x c thc hin nh my n sc. Detector s dng mng diode: L phng php hin i, em li nhiu thng tin hn v thnh phn mu. Cho php nhn bit cc hp cht c trong 1 hn hp v c th dng nh gi tinh khit. Chi ph ln. thit b ny, ton b nh sng t n deuterium c tri thnh 1 ph trn mt mng cc photodiode gn trn 1 con chip silic. Thit b o ng thi hp th ti tt c cc bc sng v my tnh x l s liu.

Detetor hunh quang:


S dng cho cc hp cht hu c c kh nng tr li 1 phn bc x

UV-VIS ti 1 bc sng di hn: hin tng pht hunh quang, v d nh: vitamin trong thc phm, thuc; cc hydrocacbon thm trong nc thi i vi nhng hp cht c kh nng pht hunh quang th vic o d dng. i vi cc hp cht khng c kh nng pht hunh quang th ta phi bin i hp cht ban u thnh cc dn xut c kh nng pht hunh quang. y l phng php c tnh chn lc v nhy rt cao, l detector l tng phn tch lng vt. C gii hn nhn bit i vi cng 1 hp cht nh hn 100-1000 ln so vi ph hp th, d b nhiu nn do bc x bn ngoi.

Detector o ch s khc x:
S dng cho hu ht cc cht phn tch ha tan trong dung mi v

khng cha tm hp th UV-VIS nh cacbohydrate, lipid t nhy hn so vi cc detector khc do dung mi pha ng chim th tch ln, cn phi c mt nng cht phn tch ln lm thay i ch s khc x. Khng th dng cho ra gii gradient.

Detector in ha:
S dng ch yu nhn bit cc ion v c, cc acid hu c khi ra

khi ct trao i ion yu; phn tch cc catechoamine, cc hp cht phenol, cacbohydrate(detector ampe k xung in), nh lng ancol gy mi, nht l terpenol Nguyn l: da vo qu trnh oxi ha kh in ha ca cc cht phn tch lm thay i dn in ca pha ng ra khi ct. C chn lc rt cao, rt nhy, thng gp 10^14 ln so vi phng php phn tch UV thng thng.

Cc detector HPLC khc:


a. b.

c.

Detector tn x nh sng Detector phng x Detector nit pht quang ha

H thng ghi, x l s liu


Vai tr: hin th kt qu v nh lng cc peak trong sc k My phn tch in t xc nh thi gian lu, din tch, chiu rng ca

cc peak bng cch theo di tn hiu detector nhn bit nhng im khi u, kt thc, cc i ca mi peak. cui mi ln chy, mt bn bo co c in ra nh my in trong lit k y nhng d liu cn thit. nu h thng c chun ha( bng cht ngoi hoc ni chun) th nng cht phn tch c xc nh. Thit b phn tch c th ng ring hoc kt ni vi my vi tnh. H thng ny c th lin kt vi h thng qun tr thng tin ca phng th nghim to thun tin cho vic thu thp, phn tch, lu tr d liu. Mc d my phn tch c th lm n gin ng k vic x l s liu nhng iu quan trng l ngi phn tch phi hiu r v nng lc, hn ch ca thit b v a ra nh gi.

Sinh vin thc hin

L Hu Lm Dng Th Quyn
Gio vin hng dn

Nguyn Th Minh Xun

Cc ch tch trong HPLC

Cc ch tch trong HPLC

Sc k pha chun (NP-HPLC) Sc k pha o (RP-HPLC) Sc k trao i ion (IC) Sc k loi tr kch thc

Sc k i lc

Cc ch tch trong HPLC


Pha chun (NP-HPLC)

Pha ng

Cc vch sc t c phn tch

Mu CaCO3

Cc ch tch trong HPLC


Pha chun (NP-HPLC)

c im: Cht lng ca pha ng khng phn cc, cht lng ca pha tnh phn cc mnh. Cu to: Pha tnh: l cht hp th phn cc nh ht silica trn hoc ht silica c gn cc pha lin kt.
Si Si

Pha ng: thng l cc dung mi khng phn cc (nh hexane ),trong c b sung tc nhn phn cc hn (nh methylene choloride) kim sot lc dung mi v chn lc.

Cc ch tch trong HPLC


Pha chun (NP-HPLC)
Nguyn tc tch :

rt yu

OH

Cc ch tch trong HPLC


Pha chun (NP-HPLC)
Cc dung mi pha ng s dng: Dung mi ch yu ( khng phn cc): Hydrocarbons (Pentane, Hexane, Heptane, Octane) Aromatic Hydrocarbons (Benzene, Toluene, Xylene) Methylene chloride Chloroform Carbon tetrachloride Dung mi ph(phn cc hoc hi phn cc) Methyl-t-butyl ether (MTBE), Diethyl ether, Tetrahydrofuran (THF),Dioxane, Pyridine, Ethyl acetate, Acetonitrile, Acetone, 2-propaol,ethanol, methanol -Dung mi ch yu c s dng chnh lm pha ng -Dung mi ph c s dng thm vo dung mi chnh vi t l nht nh nhm thay i thi gian lu.

Cc ch tch trong HPLC


Pha chun (NP-HPLC)
ng dng: Tch cc hp cht tan tt trong dung mi hu c V d: vitamin tan trong cht bo; phosphorlipid; cc hp cht, ng phn, cc carbohydrate tan tt trong nccng c th c ra gii bng sc k pha chun.

Cc ch tch trong HPLC


Pha o (RP-HPLC):
c im: Pha tnh l khng phn cc, pha ng l phn cc. Cu to: Pha tnh: ph bin nht l cc pha lin kt c chun b bng cc phn ng trn b mt cc ht silica: nhm silanol lin kt vi cc gc hydrocabon (nh phn ng vi cc chlorosilane) Da vo nhm R3 ngi ta phn ra cc loi ct: Ct C18 (ODS) : Octadecylsilane [-(CH2)17CH3] l mt trong nhng vt liu nhi ct pha o ph bin nht. Ct C8 (octyl) Ct C4 (butyl) Ct Phenyl

Cc ch tch trong HPLC


Pha o (RP-HPLC):
Cu to: Cc yu t nh hng n cht lng ca ct: Kiu nhm hu c c lin kt vi nhm silica Chiu di mch ca cht hu c Lng cht hu c c trn mt n v th tch cht nhi Kch thc v hnh dng ca cc ht mang pha tnh Din tch b mt v rng ca cu trc a hnh b mt pha lin kt Nng silanol t do

Cc ch tch trong HPLC


Pha o (RP-HPLC):
Cu to: Pha ng: thng l nc ha trn vi methanol, acetonitril hoc tetrahydrofuran. Cc cht tan c gi li do tng tc k nc vi pha tnh v c ra gii dn theo th t tng dn ca k nc Cc cht ph gia khc cng c thm vo pha ng lm v hot cc nhm silanol d. Dung dch m c thm vo nhm lm gim hoc c ch, kim sot cc qu trnh ion ha cc thnh phn c trong mu.

Cc ch tch trong HPLC


Pha o (RP-HPLC):
Nguyn tc tch :

OH

C18 OH

Cc loi dung mi s dng trong pha o: Methanol (MeOH), acetonitrile (ACN) hay THF l cc dung mi thng s dng nht cho sc k pha o.

ng dng: s dng nhiu nht trong phn tch protein thc vt. C vitamin tan trong nc hoc cht Ngoi ra cn dng phn tch cc thnh phn ca nc gii kht ( nh caffein, aspartame..); cc thnh phn phm mu, cc sc t (chlorophil, carotenoid); cc hp cht phenol c mi
Phn tch vitamin B1 mu bng HPLC pha o.

Cc ch tch trong HPLC


HPLC trao i ion

Nguyn tc: Trong : pha tnh cha cc nhm chc mang in tch dng hoc m; pha ng cha mu c bn cht mang in tri du vi in tch ca pha tnh.

Cc ch tch trong HPLC


HPLC trao i ion

Gm 2 loi sc k : trao i ion dng v trao i ion m C th, nu pha tnh mang in m (nh ct carboxymethylcellulose (CM-cellulose)), , th pha ng s l nhng phn t mang in tch dng v tin trnh c gi l sc k trao i ion dng. Ngc li, nu pha tnh mang in tch dng (nh ct diethylaminoethyl-cellulose (DEAE-cellulose)), gi l sc k trao i ion m, th tng tc vi pha ng mang in tch m.

Cc ch tch trong HPLC


HPLC trao i ion

Cu to: Pha tnh: ty vo tng loi ct cho tng mc ch s dng khc nhau m ta c cc loi ct sau: Ct trao i cation: ct trao i cation mnh: SCX :cha nhm SO3- ( sufopropyl) v d sodium sunfate. Ct trao i cation yu: WCX :COO-( carboxyl), v d nh carboxylmethyl-cellulose CMC. Ct trao i anion: Ct trao i anion mnh: SAX: R4N+ (amine) Ct trao i anion yu: WAX : hp cht DEAE-diethyl aminoethyl.

Cc ch tch trong HPLC


HPLC trao i ion

Pha ng: Pha ng s dng trong HPLC thng l dung dch m c cha nc. Tng lc ion ca pha ng lm gim thi gian lu ca cht tan. S thay i pH ca pha ng cng lm nh hng n pha tnh v c mu. Ch ra gii thng s dng l ng dng pha ng , gradient nng , gradient pH.

Cc ch tch trong HPLC


HPLC trao i ion

ng dng: HPLC trao i ion c nhiu ng dng, t vic nhn bit cc ion v c n gin n vic phn tch cc hp cht carbohydrate v acide amine hoc tinh sch cc protein. ng dng ln nht ca HPLC trao i ion l tinh sch cc protein v phn tch cc thnh phn carbohydrate c trong cc mu thc phm, mu sinh hc

Cc ch tch trong HPLC


HPLC trao i ion

Cc ch tch trong HPLC


HPLC loi tr kch thc
Nguyn l: Cc cht trong mu c tch nh s sai khc nhau v kch thc, qu trnh khng c s tng tc v mt ha hc gia pha ng v pha tnh. Trong , pha tnh l cc vt liu c mt xp nht nh, cht tan c kch thc ln khng lt qua c cc l trong s di chuyn bn ngoi cc khong trng cc ht ca pha tnh v c ra gii ra khi ct u tin. Cc cht tan c kch thc nh hn s ln lt ra khi ct.

Cc ch tch trong HPLC


HPLC loi tr kch thc

Cc ch tch trong HPLC


HPLC loi tr kch thc

Cu to: -Pha tnh: vt liu nhi ct c chn sao cho kch thc l ca cu trc ph hp vi phm vi khi lng phn t cht ra gii. -Pha ng: chn theo tan ca mu, tnh tng thch vi ct, tnh tng tc gia cht tan v pha chun. Dung dch m b sung vo pha ng ( dng cho cc polymer sinh hc nh protein, axit nucleic) gi hot tnh sinh hc v ngn nga cc tng tc hp ph

Cc ch tch trong HPLC


HPLC loi tr kch thc

ng dng Ct sc k loi tr kch thc s dng polymer a nc c dng xc nh nhanh khi lng phn t trung bnh ca poly saccarit nh amylose, amylopeptin, cc dn xut xenlulose tan trong nc. Trong cng nghip HPLC c s dng nh gi ging u nnh nh vo hm lng protein Gn y phng php sc k ny c p dng xc nh polymer triacylglycerol trong du

Xy dng quy trnh tch

Xy dng quy trnh tch

TNG KT

TNG KT

Mt h thng HPLC c bn gm c Bm y pha ng i qua h thng. B np mu cho php mu c a vo pha ng dn vo ct.

Ct HPLC, ni gia b np mu v detector, gm c phn cng bng thp khng r c lm y bng cht nhi ct.
Detector dng trong HPLC gm c hp th UV-VIS, hunh quang, ch s khc x, in ha v mt s dng khc H thng ghi/ x l s liu c chc nng ghi li kt qu c bn ca qu trnh sc k

TNG KT

Vt liu nhi ct: Polymer (vi l hoc l to) Nn silica (silica rng,c pha lin kt, ph thnh trong ct) S thnh cng ca silica c pha lin kt m rng ng dng ch tch pha chun v pha o ca HPLC Qu trnh tch:

Theo nguyn l trao i ion, loi tr kch thc v sc k i lc

TNG KT

ng dng Phn tch cc phn t nh v ion nh ng, vitamin v axit amin Tch v tinh sch cc i phn t nh protein, axit nucleic v polysacarit

MT S HNH NH HPLC TRNG

MT S HNH NH HPLC TRNG

MT S HNH NH HPLC TRNG

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