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Phng php sc k lng hiu nng cao (HPLC)

HPLC l ch vit tt 4 ch ci u bng ting Anh ca phng php sc k lng hiu nng cao (High Performance Liquid Chromatography), trc y gi l phng php sc k lng cao p (High Pressure Liquid Chromatography).

1. Khi nim Phng php sc k lng hiu nng cao (HPLC) ra i nm 1967-1968 trn c s pht trin v ci tin t phng php sc k ct c in. HPLC l mt phng php chia tch trong pha ng l cht lng v pha tnh cha trong ct l cht rn c phn chia di dng tiu phn hoc mt cht lng ph ln mt cht mang rn, hay mt cht mang c bin bng lin kt ha hc vi cc nhm chc hu c. Phng php ny ngy cng c s dng rng ri v ph bin v nhiu l do: c nhy cao, kh nng nh lng tt, thch hp tch cc hp cht kh bay hi hoc d phn hy nhit. Phm vi ng dng ca phng php HPLC rt rng, nh phn tch cc hp cht thuc tr su, thuc khng sinh, cc cht ph gia thc phm trong lnh vc thc phm, dc phm, mi trng 2. Phn loi Da vo s khc nhau v c ch tch chit s dng trong HPLC, ngi ta chia HPLC thnh 4 loi:

Sc k hp ph hay sc k lng rn (adsorption/liquid chromatography). Sc k phn b (partition chromatography). Sc k ion (ion chromatography). Sc k ry phn t (size exclusion/gel permeation chromatography).

Trong , sc k phn b (SKPB) c ng dng nhiu nht v c th phn tch c nhng hp cht t khng phn cc n nhng hp cht rt phn cc, hp cht ion c khi lng phn t khng qu ln (<3000). SKPB c chia thnh hai loi da trn phn cc tng i gia pha tnh v pha ng: sc k pha thng SKPT (normal phase chromatography) v sc k pha o SKP (reversed phase chromatography). Trong SKPT, pha tnh s dng c phn cc cao hn pha ng. Pha tnh loi ny s c i lc vi cc hp cht phn cc. SKPT dng tch v phn tch cc hp cht c phn cc cao vi phn t lng khng ln lm.

SKP l thut ng ch mt loi sc k trong pha tnh t phn cc hn pha ng. Phng php ny dng phn tch cc hp cht t khng phn cc n phn cc. Hu ht cc hp cht hu c c mch carbon di (t phn cc) rt thch hp cho phn tch bng SKP. Dung mi s dng trong SKP l cc dung mi phn cc, trong dung mi nc ng vai tr quan trng m li r tin. Do , SKP c ng dng nhiu v ph bin hn SKPT. 3 Ph nh on ph o

Trong sc k phn b ni chung, pha tnh l nhng hp cht hu c c gn ln cht mang rn silica hoc cu thnh t silica theo hai kiu:

Pha tnh c gi li trn cht mang rn bng c ch hp ph vt l sc k lng-lng (liquid-liquid chromatography). Pha tnh lin kt ha hc vi cht nn sc k pha lin kt (bonded phase chromatography) Trong qu trnh s dng, ngi ta nhn thy sc k pha lin kt c nhiu u im hn sc k pha lng-lng v mt s nguyn nhn sau:
o

Pha tnh trong h sc k lng-lng d b ha tan bi pha ng nn d b mt mt pha tnh trong thi gian s dng v gy nhim i vi hp cht phn tch. Do pha tnh ca sc k lng-lng d tan trong pha ng nn ngi ta khng th ng dng phng php ra gii gradient dung mi.

V vy, ngi ta thng ch quan tm n loi sc k phn b pha lin kt v phn ln cc loi ct s dng hin nay trong sc k phn b u c cu trc dng ny. B mt cc ht silica SiO2 (cc ht ny c ng knh 3, 5 hoc 10 m) c x l (thy phn) bng cch un nng vi HCl 0,1M trong mt hoc hai ngy to ra nhng nhm SiOH nh sau (thng thng ch c khong 8 mol SiOH/m2 b mt):

Hnh 1. B mt silica thy phn

Sau b mt silica thy phn ny s c cho phn ng vi cc organochlorosilan to ra cc pha tnh khng phn cc, phn cc trung bnh hoc rt phn cc ty theo nhm R gn vo.

Hnh 2: To nhnh trn b mt silica

Thng ch khong 50% nhm OH mt H+ to ra HCl (tc < 4mol/m2 b mt b silan ha) v s kt hp s dn dn b nh hng bi hiu ng lp th. Ngoi nhm Cl ngi ta cn s dng OCH3. Hp cht cn phn tch khi i qua pha tnh s b gi li bi nhng lc lng tng tc khc nhau ty thuc tnh cht, c im ca cht tan v pha tnh. Trong SKP, nhm th R trong hp cht siloxan hu nh khng phn cc hoc t phn cc. l cc ankyl dy di nh C8 (n-octyl), C18 (n-octadecyl) cn gi l ODS (octadecylsilan) hoc cc nhm alkyl ngn hn nh C2; ngoi ra cn c cyclohexyl, phenyl trong nhm phenyl c phn cc cao hn nhm alkyl. Ngi ta nhn thy cc alkyl dy di cho kt qu tch n nh hn cc loi khc nn y l loi c s dng nhiu nht.

Hnh 3: Cu trc ca ct ODS

Tuy nhin do hiu ng lp th nn trong cu trc ca pha tnh cn nhm OH cha phn ng, gy nh hng xu n qu trnh tch sc k ty mi trng pH phn tch. Trong mi trng qu acid (pH < 2) th c s phn ly cc nhm ether (-O-Si-C18) ra khi nn. Lc ny ct s mt hot tnh dn n cht cn phn tch khng cn tng tc tt vi pha tnh na. Trong mi trng baz (pH > 7), chnh nn silic mang pha tnh c th b ha tan (SiO2 thnh silicat), h qu l N gim v s nhnh ghp cng gim, mi rng ra v thi gian lu cng c th gim. Kt qu phn tch nh vy s mt i chnh xc. Mt trong nhng cch khc phc hin tng ny l dng cc cht nh trimethylchlorosilan ClSi(CH3)3 hoc hexamethyldisilazan (t s dng hn) tng

tc vi nhm OH ny (gi l hin tng end-capping). Lc ny ta s c loi ct t tng tc vi cht phn tch c tnh baz (ct LC-DB ca hng SUPELCO).

Hnh 4. Cu trc ct LC-DB

C mt cch khc loi tr bt nh hng ca nhm OH m khng cn tng tc vi n l thay nhng nhm methyl ca Si(CH3)2-C18 bng nhng nhm th ln hn nh isopropyl nhng nhm ny s che i nhng nhm OH, cn tr tng tc ca nhm OH vi cht cn phn tch.

Hnh 5: Cu trc ct c gc isopropyl

Ngi ta cn ghp ln dy C18 mt s nhm phn cc tng thm phn cc ca dy C18, lm ct c kh nng tch chn lc hn i vi nhng hp cht phn cc mnh (ct EPS Expended Polar Selectivity). Ngoi sn silica, thi gian gn y ngi ta c s dng n nn nha polystyren (Polystyren Reversed Phase PRP) cho php phn tch trong mi trng pH t 1 13. Ct ny d s dng trong mi trng acid v baz mnh. 4 Ph n on ph o

Pha ng trong sc k lng ni chung phi t nhng yu cu sau:


Ha tan mu phn tch. Ph hp vi u d.

Khng ha tan hay lm mn pha tnh. C nht thp trnh p sut di li cao. Tinh khit dng cho sc k (HPLC grade).

Trong sc k pha o, dung mi pha ng c phn cc cao. Trn l thuyt chng ta c th s dng kh nhiu dung mi nhng kinh nghim thc t cho thy methanol (MeOH), acetonitrile (ACN) v tetrahydrofuran (THF) l t yu cu nht. Nc l mt dung mi c cho vo cc dung mi hu c gim kh nng ra gii. Mi dung mi u c trng bi cc hng s vt l nh ch s khc x (refractive index), nht (viscocity), nhit si (boiling point), phn cc (polarity index), ra gii (eluent strength) Trong phn cc v ra gii c tc ng ln ln kh nng phn tch ca cc mi sc k.

Bng 1: Tnh cht ca mt s pha ng trong sc k lng

C ba thng s gy nh hng ln n tch cc mi sc k: s a l thuyt N, h s dung lng K, chn lc . Khi s thay i thnh phn pha ng khng em li kt qu tch mi theo yu cu th chng ta phi thay i bn cht pha ng (s dng dung mi khc), tc thay i . i khi c th phi thay i c pha tnh. Trong qu trnh tch ca SKP, s tng tc gia hp cht cn phn tch v pha ng ph thuc rt nhiu vo moment lng cc, tnh acid (cho proton) hoc tnh baz (nhn proton) ca dung mi. Thng thng pha ng trong SKP bao gm mt hn hp nc hoc dung dch m vi mt hoc nhiu dung mi hu c phn cc tan c trong nc. Nc l mt

dung mi rt phn cc nn n khng tng tc vi nhng nhm alkyl khng phn cc trong pha tnh, do n c coi nh pha ng yu nht v c tc ra gii chm nht trong tt c cc dung mi ng ca SKP. Hn hp nc v dung mi hu c thng lm gia tng nht dn n vic tng p sut ct.

Hnh 6: nht ca hn hp nc v dung mi hu c 25oC

Vic la chn dung mi v thnh phn dung mi trong pha ng c ti u ha cho nhng hp cht cn phn tch. Thng thng, ngi ta s dng hn hp dung mi MeOH/nc trc, ri ACN/nc hay THF/nc. Vi mt hn hp cht phn tch phc tp th s c s trn ln ca cc dung mi hu c vi nc. Khi la chn th phi chn cc hn hp MeOH, ACN v THF vi nc c ra gii tng ng. Thnh phn pha ng c th c nh trong sut qu trnh chy sc k (ch isocratic) hoc c thay i theo mt chng trnh nh sn (chng trnh gradien dung mi) c hiu qu tch tt hn. 5 i l phn n n on phn h

5 1 H

Cn bng ca mt cu t X trong h sc k c th c m t bng phng trnh nh sau:

Hng s cn bng K cho cn bng ny c gi l t l phn b hay hng s phn b (partition coefficient) v c tnh nh sau:

Vi CS : nng cu t trong pha tnh. CM : nng cu t trong pha ng. H s K ty thuc vo bn cht pha tnh, pha ng v cht phn tch. 5 2 Thi i n l u: tR : thi gian lu ca mt cu t t khi vo ct n khi tch ra ngi ct. tO : thi gian cho cht no khng c i lc vi pha tnh i qua ct; cng l thi gian pha ng i t u ct n cui ct v cn gi l thi gian lu cht. tR' : thi gian lu tht ca mt cu t.

Hnh 7. Thi gian lu ca cu t phn tch

5 3 H

dun l

k c nh ngha theo cng thc sau:

Vi VS : th tch pha tnh VM : th tch pha ng Nu k~ 0, tR~ tO: cht ra rt nhanh, ct khng c kh nng gi cht li.

Nu k cng ln (tR cng ln): cht trong ct cng lu, thi gian phn tch cng lu, mi c kh nng b t. Khong k l tng l 2-5, nhng khi phn tch mt hn hp phc tp, k c th chp nhn trong khong rng 1-20. 5 4 Hiu nn Hiu nng hay s a l thuyt N ca ct c trng cho kh nng tch mi sc k ca cc cu t trn ct. N cng ln, hiu nng tch cng cao. S a l thuyt c th o trn sc k . Ngi ta chng minh c:

Hay

Vi W1/2 l chiu rng mi sc k v tr chiu cao mi (pht) W l chiu rng mi sc k v tr y mi (pht) 5 5 hn l c trng cho kh nng tch hai cht ca ct.

5 6 phn i i: y l i lng biu th r c ba kh nng ca ct sc k: s gii hp, s chn lc v hiu qu tch. N c xc nh qua phng trnh sau:

Hay

Vi N c xc nh t phng trnh (2.5) hoc (2.6).


Ngun: Phng Phn tch Sc K (CASE)

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