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NI DUNG
I.

M U T VN
1.

LCH S PHT TRIN PHNG PHP SC K Nh thc vt hc ngi Nga Mikhail Tswett (Mikhail Semyonovich Tsvet) pht minh ra k thut sc k vo nm 1903 khi ng ang nghin cu v chlorophyll. Khi cho dung dch cc sc t thc vt trong ether du ha ln mt ct c nhi bi bt mn calci carbonat, ng nhn thy sc t b hp ph trn u ct. Khi cho dung mi nguyn cht (ether du ha) ln ct, cc sc t di chuyn trong ct v tch thnh nhng vng mu xp chng ln nhau. Phng php ny sau c t tn l sc k (Chromatography: Sc k ngha l ghi mu, trong ting Hy Lp ch chroma c ngha l mu; n va l tn ca Tsvet trong ngha ting Nga va l mu ca cc sc t thc vt ng phn tch lc by gi, cn graphein l vit). Tn ny vn tip tc c dng d cc phng php ny cn c s dng tch cc cht khng mu. 1938 Izmailov v Schraiber xy dng v sau nm 1958 Stahl hon thin phng php sc k lp mng. Sc k tip tc pht trin khng ngng nh vo nhng kt qu nghin cu ca Archer John Porter Martin v Richard Laurence Millington Synge trong sut nhng nm thp nin 40 v 50 ca th k 20. Hai ng a ra nhng nguyn tc v k thut c bn ca sc k phn b lm ng lc cho s pht trin ca nhiu phng php sc k khc nhau nh sc k giy (PC), sc k kh (GC) v sc k lng cao p (HPLC). Sc k kh pht trin mnh m vo nhng nm 1960, trong khi s ra i ca sc k lng cao p vo cui thp nin 60 l mt bc ngot mi trong lch s sc k. GC v HPLC l nhng phng php sc k hu hiu v ngy nay c s dng rng ri trong cc phng th nghim hin i trn ton th gii. Bn cnh mt s phng php sc k khc vn tip tc c s dng v pht trin nh: sc k ngc dng, sc k ct

2.

VAI TR CA K THUT SC K IU CH TRONG NGHIN CU DC LIU T khi ra i v c nhng bc pht trin mnh m, sc k tr thnh mt phng php hu hiu v khng th thay th trong vic phn tch cht tinh khit ra khi mt hn hp nh tnh, nh lng, tinh khit ha cng nh phn lp cc cht mong mun. Sc k c ng dng rng ri trong nhiu lnh vc nh cng ngh sinh hc, ha tng hp, dc liu, ha phn tch, kim nghim v.v Bn cnh vic s dng nh tnh, nh lng cc cht (sc k phn tch) th sc k cn dng phn lp, iu ch, tinh ch cc cht mong mun trong mt hn hp nhiu thnh phn (sc k iu ch hay cn gi l sc k ch ha). y l mt ng dng ht sc quan trng, c bit trong ha tng hp v dc liu. Nh chng ta bit mt trong nhng yu cu c bn trong nghin cu dc liu l phi phn lp v xc nh c cc hot cht tinh khit c dc tnh trong dc liu. T bo cha hng trm loi hp cht ha hc khc nhau bao gm cc hp cht c trng lng phn t nh n cc i phn t nh protein, acid nucleic, lipid v.vDo qu trnh chit xut cc hp cht mong mun trong dc liu lun km theo mt lng ln tp cht. Trong nhiu trng hp phng php duy nht thu c hot cht tinh khit l thc hin sc k iu ch. C nhiu loi sc k iu ch c ng dng trong dc liu nh sc k lp mng iu ch, sc k lng p sut trung bnh, sc k lng cao p, sc k phn b ngc dng v c bit l sc k ct. Do cc phn t hu c trong dc liu v cng a dng vi nhiu hp cht c trng lng phn t v phn cc gn ging nhau nn khng th c mt k thut sc k chung cho cc loi hp cht khc nhau. Ty vo tng trng hp c th cc nh khoa hc s la chn phng php sc k cng nh iu kin sc k ti u iu ch hp cht mong mun. Vic tm hiu tng k thut sc k iu ch v u, nhc im, nguyn tc, iu kin sc k v cch tin hnh s gip chng ta c c s la chn ti u trong qu trnh phn tch cc hp cht t dc liu. SC K LP MNG IU CH

II.

NI DUNG CHNH
1.

Mt trong nhng k thut sc k nhanh, tin li, d thc hin v thng dng nht hin nay l sc k lp mng.
1. Nguyn tc :

Phng php sc k lp mng (SKLM) c dng nh tnh, th tinh khit v i khi bn nh lng hoc nh lng hot cht thuc. Sc k lp mng l mt k thut tch cc cht c tin hnh khi cho pha ng di chuyn qua pha tnh trn t hn hp cc cht cn tch. Pha tnh l cht hp ph c chn ph hp theo tng yu cu phn tch, c tri thnh lp mng ng nht v c c nh trn cc phin knh hoc phin kim loi. Pha ng l mt h dung mi n hoc a thnh phn c trn vi nhau theo t l quy nh trong tng chuyn lun. Trong qu trnh di chuyn qua lp hp ph, cc cu t trong hn hp mu th c di chuyn trn lp mng, theo hng pha ng, vi nhng tc khc nhau. Kt qu, ta thu c mt sc k trn lp mng. C ch ca s chia tch c th l c ch hp ph, phn b, trao i ion, sng lc phn t hay s phi hp ng thi ca nhiu c ch ty thuc vo tnh cht ca cht lm pha tnh v dung mi lm pha ng. i lng c trng cho mc di chuyn ca cht phn tch l h s di chuyn Rf c tnh bng t l gia khong dch chuyn ca cht th v khong dch chuyn ca dung mi:
trong : a l khong cch t im xut pht n tm ca vt mu th, tnh bng cm. b l khong cch t im xut pht n mc dung mi o trn cng ng i ca vt, tnh bng cm. Rf: Ch c gi tr t 0 n l.

Nguyn tc trn l nguyn tc chung cho c th tinh khit, nh tnh, nh lng, nhng i vi trng hp iu ch, c nhng khc bit nh sau:

Khc bit ln nht l SKLM iu ch l trong a s cc trng hp bn mng c s dng c kch thc 20x20, dy lp hp thu 1-5mm. Dung dch mu phi c chm thnh 1 dy bi ng mao qun.

Sau khi ly gii, trn bn s c nhiu dy, dng li lam hoc spatule co ly lp hp thu c cha dy cht ta cn. Dng dung mi thch hp chit mu cht ra khi silicagel hoc alumin. 1. u nhc im v kh nng ng dng : 1. u im : u im chung ca SKLM

K thut n gin, d thc hin, c th thc hin mi phng th nghim. Thi gian thc hin nhanh, ti a vi gi. Do cch thc hin n gin nn c th dng SKLM kho st cc h dung mi cho cc phng php sc k khc. Nhn chung SKLM l phng php c tnh ng dng rt cao, thng dng nht hin nay. u im ca sc k lp mng iu ch so vi sc k ct l : Nhanh, d tm c cc dung mi thch hp tch cc cht. Cc vng c cha cht cn thit s tp trung ti 1 v tr nht nh d dng pht hin, d dng c lp. SKLM tch tt l nh t l ln gia cht tch : cht hp thu silicagel t 1:1000 n 1:10000 trong khi t l ny sc k ct l 1:50 n 1:100 Thnh phn pha ng d thay i trong qu trnh khai trin. Cc vt sau khai trin thng b ko ui. SKLM iu ch ch dng khi hn hp cn tch c s lng t, vi trm miligam, cn nu mu nhiu, vi gam th tch bng sc k ct s kinh t hn.
1. Pha tnh :

2. Nhc im :

Trong SKLM c rt nhiu cc cht c dng lm pha tnh, trong ph bin nht l cc cht hp ph nh silicagel, Kieselguhr (celite), Alumina, bt giy, polyamid, Sephadex Pha tnh c th c trn hoc khng trn thm cht kt dnh ty i tng cn phn tch. i khi pha tnh cn c trn thm cht ch th hunh quang. Vic la chn pha tnh ph thuc rt ln vo cht cn iu ch thn du hoc thn nc. Ngoi ra cn phn bit bt SKLM

vi bt sc k ct do bt dng cho sc k ct c kch thc ln hn nhiu. Ngy nay, cc bn mng trng sn silicagel c dng ph bin nht. Bn mng dng trong SKLM iu ch thng c kch thc c5 bit, 20x20 mm, b dy 1-5mm. L cht hp ph c s dng rng ri nht hin nay, c th dng tch alkaloid hoc cc hp cht thn du. Tnh hp ph ca silicagel ph thuc ln vo cc nhm OH b mt. Kch thc ht 10-40 micromet, din tch b mt 200-400 m2/g. Din tch b mt 100 m2/g, b dy lp mng ph thuc lng cht cn iu ch. C 3 dng nhm oxyd l nhm acid, base hoc trung tnh c la chn da trn tnh cht ca cht cn iu ch. L cht cao phn t thin nhin, c dng di dng bt mn v khng cn cht kt dnh. Thng dng phn tch cc cht thn nc trong sc k phn b. Nhc im chnh l khng dng c cc thuc th mnh nh NaOH, H2SO4 nhn bit cc vt sc k.

1. Silicagel : l dn xut ca acid silicic


2. Nhm oxyd:

3. Cellulose :

Ngoi nhng cht thng c s dng lm pha tnh nh trn, ngi ta cn dng thch cao, kieselguhr, polyamid hoc nha trao i ion.
1. Pha ng :

Vic la chn loi dung mi v t l dung mi l vic gp nhiu kh khn nht trong c qui trnh sc k. Vic la chn dung mi khng c mt qui tc c th no m ch yu do sc k s b thm d v ch yu nht vn da vo kinh nghim. H dung mi c chn phi cng n gin cng tt v phi tinh khit. C th cn c vo nhng yu t nh : C ch sc k ca pha tnh. Tnh cht ca hn hp cn phn tch.

bay hi, nht, tinh khit ca dung mi. Cn iu chnh sc ra gii ca pha ng tr s Rf nm trong khong 0,2-0,8 t phn gii cc tr.

Trc khi thc hin vic iu ch, dung mi phi c kho st k vi nhng bn mng v k thut nh SKLM nh tnh. Trong qu trnh phi tm c h dung mi ph hp nht mi thc hin iu ch.
1. Phng php pht hin v thu nhn kt qu trong SKLM

iu ch:

a s cc hp cht hu c khng c mu nn sau khi tin hnh sc k, khng th nhn thy vt trn bn, phi s dng cc phng php vt l hoc ha hc nhn thy cc dy vt. Gip bit c v tr ca vt mu trn tm bn. Phn hp cht trn bn, ch tip xc vi thuc th b h hi do xy ra phn ng, khng th s dng co bn, c lp hp cht. trnh b hao ht, dng tm knh hoc tm chn nilon che bt mt phn bn, phun xt thuc th ln phn bn khng b che. Sau khi pht hin, co bn mng phn khng b xt, chn dung mi thch hp ly cht cn iu ch. Nhanh, tin li, nhn c vt m khng gy hao ht mu. Tuy nhin, vt c th v tr x trn bn, nhng mt li nhn thy v tr biu kin ca n, ngha l mt v tr khc. H qu l s nh du sai v tr ca vt v sau khi ly gii khng thu c cht cn iu ch. Cn kim tra mt vi ln bng cch che mt phn bn v phun xt cc thuc th nhn r v tr ca cc vt. t bn trong mt bung c hi iod, khi thy vt hin ln trn bn, ly bn ra khi bung, nh du v tr ca vt, phi bn ngoi qut hi iod s bay i mt. Sau khi hi iod bay i mt, co bn, chn dung mi thch hp thu c cht cn phn tch.

1. Phun xt vi thuc th c trng :


2. Soi bn di n UV :

3. H hi iod :

2.

SC K CT
1.

SC K CT C IN
1.

Nguyn tc v yu cu ca phng php:

Nguyn l tch ca sc k ct cng nh ca cc loi sc k khc: mt mu th c np ln mt ct cha cht hp ph (thng l silica gel, nhm oxyt).Ct cha cht hp ph ny ng vai tr l mt pha tnh. Mt dung mi khai trin (pha ng) di chuyn dc theo ct s lm di chuyn cc cu t ca mu th, do cc cu t ny c phn cc khc nhau nn i lc ca chng vi pha tnh cng khc nhau, v vy chng b dung mi gii hp v b y i vi vn tc khc nhau to cc di ring bit c v tr khc nhau ra khi ct ti cc thi im khc nhau, lm c s cho vic phn tch nh tnh v nh lng. Pha tnh c nhi sn vo trong ct pha trn mt lp bng (sseer ngn nhng vt rn chy vo dch thu c) v thm mt lp mng ct Nu khng cn thn khi np mu th vo c th dn n lm xo trn pha tnh v hiu sut chit xut s thp v vy cn thm 1 lp ct vo pha trn pha tnh na n nh ct. iu quan trng nht l khng c pha tnh trng thi kh v khng c c bt kh. V vy phi lun gi cho pha tnh c bao ph bi pha ng. Bi v silica gel v nhm oxyt c th to mt lp kh dy, qu trnh chit tch c xu hng mt nhiu thi gian nu trng lc ch l 1 lc duy nht ko pha ng i. Qu trnh ny c th c tng tc bi vic tng p sut u ct hay chn khng pha di ct. Cc phng php ny s c trnh by phn sau.

1. u v nhc im ca phng php, kh nng v mc ng dng:

1. u im:

C th c s dng trong c hai lnh vc phn tch v iu ch. Sc k ct khng nhng dng xc nh s lng cc thnh phn ca mt hn hp m n cn c th c dng tch v tinh ch nhng thnh phn phn tch. iu ny tri ngc vi sc k giy, ch l mt phng php phn tch. Pha tnh v cc dng c th nghim r tin, d kim; c th trin khai vi mt lng ln mu cht. Khi chy ct sc k cn i hi s ch theo di lin tc duy tr mc dung mi v nu khng s d gy xut hin cc bt kh lm tc ct, nt ct, v bin dng ng i ca dung mi. Chy ct tn thi gian v v cng vic lp i lp li, c bit l khi chy cc mu ln. Qu trnh tch chm v hiu qu thp so vi sc k lng cao p.

1. Nhc im

1. Pha tnh v cch thc chun b ct sc k 1. Pha tnh

Loi cht s dng lm pha tnh l nhng cht hp ph, chng thay i theo phn cc ca cht cn phn lp. Hin nay ph bin l silicagel v nhm oxyt, l nhng cht hp ph phn cc (cht hp ph pha thun), chng c dng phn lp nhng cht c phn cc yu n phn cc trung bnh. C ht hp ph: C ht nh (15- 40m), c ht trung bnh (4063m), c ht th (63- 200m). Tr s Rf trn bn mng cng ln th c th dng c ht th, ngc li l c ht nh. Ht cng nh hiu qu tch cng tt nhng tc chy cng chm Cht hp ph sau khi cn s c hot ha 1 ch (nhit * thi gian) thch hp. i khi gim hot silica gel ngi ta cn thm khong 10- 15% nc vo silica gel, o u ri mi em sy trong hp kn 1050C* 2-3 gi. Sau khi hot ha hoc gim hot, cht hp ph c n tht ngui trong bnh ht m. Ct sc k thng l mt ng thy tinh c ng knh t 50mm tr ln, chiu cao t 50cm n 1m vi 1 ci vi pha di.

1. Cch thc chun b ct sc k

Chiu cao ct va phi, cng cao th kh nng tch cng hiu qu nhng nguy c mt mu cng ln. Ct c ra sch, sy kh, gn trn trn mt gi vng chc. Phng php nhi ct:

Phng php kh: Ct u tin c in y vi bt pha tnh kh, sau b sung pha ng t t cho n khi pha tnh hon ton t, v k t thi im ny khng c php ct kh. Phng php t: Mt hn dch tng i long, to t cht hp ph ha trong dung mi nn, c cn thn vo trong ct cha mt lp dung mi nm trn lp bng y ct. M kha ct ti a. Ch phi loi tr bong bng kh bng cch cho t t v thng xuyn v nh ct. C gng a ton b khi hn dch bng 1 ln rt duy nht. T thi im ny khng c khi bt kh.

n nh ct: Ct v tr thng ng, nn ct bng 2 nt cao su g nh u quanh ct n khi khng cn bt trong ct, mt thong phng v khng tt xung c na. Sau khi nn ct cho dung mi chy qua ct trong mt thi gian thch hp ty theo kch thc n nh ct. ct cng n nh th cc bng tch s cng phng v gn.

1. Chun b mu sc k, a mu ln ct 1. Mu sc k

Cn xc nh r hp cht cn chit xut. Quy trnh chit xut phi ph hp vi nhm hp cht ny. Mu th trc khi phn tch phi c loi tp bng phng php thch hp. Mu th c th dng bt kh nhng thng dng dung dch kh m c (khng c qu m c) trong 1 dung mi khng qu phn cc so vi h dung mi sp dng khai trin ct. L tng nht l dung mi nn ca h dung mi pha ng. Nu mu kh ha tan trong dung mi nn, c th dng mt dung mi khc phn cc hn dung mi nn ha tan mu. VD: Pha ng l Benzen - EtOAc th tt nht ha tan mu trong Benzen, nu mu khng tan trong Benzen th c th dng dung mi phn cc hn Benzen mt cht (CH2Cl2, CH3Cl, hn hp Benzen - EtOAc) ha tan mu.

Mu dng bt kh: trn dung dch mu vi gi ti xp va , bc hi ht dung mi thu c mt bt mu c th cht mn ti xp.

1. a mu ln ct 1. Np mu dng bt kh (t c s dng)

Rc u mu ln lp dung mi va trn nh ct. Ch : Lp dung mi cha li trn nh ct phi hi d so vi kh nng ht dung mi ca lng bt mu.

2. Np mu dng dung dch

Dng pipet khc hoc pipet Pasteur di c gn qu bp hoc bnh lng gn c u di a nh nhng dung dch mu vo lp dung mi cha trn nh ct. Np mu xong, m kha ct chm cho ton b dung dch mu thm u vo cht hp ph. Dng dung mi ha tan mu ra lng ct vi ln n khi dch ra tr nn trong sut khng mu. Trong khi ra vn m kha cho chy vi tc thch hp. T gi tr i tuyt i khng nt ct.

5. Dung mi sc k: 1. Cch la chn dung mi

Tm dung mi sc k da vo ti liu tham kho nhng thng nht vn l phi d tm h dung mi ny bng phng php SKLM trn cng chng loi pha tnh. H dung mi ny nn gm ti a 2 thnh phn v sau khi chy xong trn sc k SKLM cho kt qu:

o o o

Cc vt c di chuyn, c tch nhau, khng b ko thnh vt di. Cc vt cn tch c Rf khong 0.25 0.35 Cc vt ng l i tng cn nghin cu.

Ghi nhn h dung mi va chn, xc nh dung mi nn (l dung mi km phn cc nht trong h trn). Dung mi nn u tin ha tan cht hp ph nhi ct, ha tan mu np ct v lm dung mi u khai trin ct.

Ch : Khng nn khai trin ct sc k ngay vi h dung mi va chn bng SKLM m phi dng h dung mi cng dy nhng km phn cc hn chy ct.

1. Thay i dung mi trong qu trnh sc k (s tng dn phn cc ca dung mi):


Dung mi c thay i sao cho phn cc thay i khng t ngt. T l ca dung mi phn cc hn (B) trong h dung mi (A - B) s c tng t t. Dung mi mi s c thay i vo thi im khi lng dung mi c trn ct gn ht. Thn trng trong thay i dung mi nht l dung mi phn cc mnh.

6. Pht hin v thu nhn kt qu sc k


Theo di kh nng tch: quan st mu, chm trn SKLM bn silica gel F254 soi UV Thu phn on: Hng cng sm cng tt nht l khi phn lp cc cht khng mu. gp cc phn on: Theo di bng SKLM, gp cc ng hng c sc k ging nhau, ghi lai th tch ca phn on mi gp. Bo co kt qu: phi ghi r cc thng s v cc chi tit. Khi kim tra cc thnh phn trong phn on, lun chm km mu ton phn tin theo di. Cc thnh phn thu c sau khi sc k c th s dng cc phng php o UV, hunh quang, HPLC, GC, tin hnh kim tra, nh gi, nhn bit chnh xc hn cc thnh phn c trong mu.

7. ng dng

Chun b mu. Phn lp cc hormon tng trng steroid, hormon sinh dc v cc hp cht lin quan. Ra gii cc thuc tr su. Tch cc thuc tr su gc clo. Phn lp khng sinh.

Tch cc cht lipid. Phn lp alkaloid. Tch cc hp cht cha nito t cc hydrocarbon. Lm tinh khit dc liu. Tch hp cht vng thm t hn hp mch thng-vng thm. Phn tch cc vitamin Kh mu du, cht bo v sp bng phng php thm lc.

2. SC K CT NHANH (FC) 1.

Nguyn tc v yu cu ca phng php:

Sc k ct nhanh, cn c bit l sc k ct vi p lc trung bnh, c ng dng u tin bi Clark Still trng i hc Columbia v c m t trong J.Org Chem 43, 2923 (1978) Sc k ct nhanh khc cc k thut truyn thng khc 2 im:

S dng cc phn t silica gel nh 250-400 mesh V gii hn dng chy ca dung mi gy bi nhng phn t silica gel nh, do , kh nn (p lc t 10-15 psi) c s dng y dung mi qua ct pha tnh. Do qu trnh sc k nhanh, c phn gii cao.

c im : K thut tng t vi sc k ct truyn thng, ngoi tr vic dung mi c y qua ct nh p sut trn u ct. Tc dng ln, thi gian trin khai ngn. , tng kh nng phn tch vi cc phng php c. Dung mi c nht thp. Lng mu ln, phn on hng ln.
1. u, nhc im - kh nng ng dng

1. u im

Nh kh nn y dung mi gii ly nn thi gian trin khai nhanh hn sc k ct c in (thi gian trin khai ngn nh hn 20 pht). Cho php phn lp mu 0.01-10 g trong 10-15 pht. C th tch rt tt hn hp c khong 4 cht. Thit b n gin, r tin so vi HPLC. phn gii trung bnh (ln hn 0.15). Khng tch c hn hp phc tp hay tch ring cht cn phn lp m ch thu c cc phn on cha cht cn phn lp. thc hin cn phi c my bm nn kh v mt b phn gm nhng ct v nhng np vn kn chuyn dng, c kch thc t

2. Nhc im

nh n ln. Nhng b phn ny tng i t tin nu so vi sc k c in.


3. ng dng

Tch cc phn on n gin t hn hp phc tp. Sau a cc phn on ny ln sc k ct c in tch thnh cc phn on tinh khit.

3. Pha tnh-Cch chun b ct sc k

Lng silicagel cn nhi ct: ph thuc vo khong chnh lch v Rf ca cc hp cht cn tch v ph thuc vo lng mu. n gram mu cn 30-100n gram silicagel. i vi mu d tch th chn t l silicagel 30:1, mu kh tch th phi tng lng silicagel. S dng lng silicagel ln th s cn thi gian di hn chy ct. T trng bt silicagel khong 0.75 gram/ lt. Chn ct ty vo yu cu iu ch. Still v cng s ngh bng di y
Th tch dung mi ra gii (ml) Lng mu np (mg) Rf > 0.2 Rf >0.1 C phn on(ml)

ng knh ct (mm)

10 20 30 40 50

100 200 400 600 1000

100 400 900 1600 2500

40 160 360 600 1000

5 10 20 30 50

Nhi ct

Chn ng knh ct theo bng trn, chiu di ca ct FC thng thng khong 18 inches (46 cm). Tuy nhin, c mu v s khc

bit Rf gia cc hp cht cn phn tch nh hng n vic la chn ng knh ct. S dng mt ct bng thy tinh, dng mt t bng gn chn ngay pha trn kha ngn silica gel chy t ct qua l kha. Tip theo, thm vo mt lp ct sch dy khong inch trn lp bng. Ch s dng khi tht cn thit c c b mt bng phng, v c cng ng knh vi thn ct. Sau khi chc chn b mt bng phng, dng phu rt silicagel vo ct (chiu di ct silica gel khong 6-10 inches).

CH : lm bc ny trong t ht v Silica gel l ha cht c th gy ung th.


Solvat ha silicagel:

S dng qu bp cao su g nh v u n vo thnh ct nh v silicagel. Cho mt lng dung mi ra gii thch hp vo silicagel. Dng kh nn y dung mi qua silica. Khi c khong vi trm mililit, lp silicagel trn b mt s ng nht hn v cc bt kh b by trong silicagel c y ra ngoi. Tip tc cho dung mi i qua silicagel n khi nhn ton ct ng nht. Lm vi ln tt c silicagel c solvat ha. Khng u ct b kh.
3. Yu cu-K

thut chun b mu sc k, a mu ln ct

1. Np mu

Cho tt c cc dung mi cn trn b mt chy xung n khi ngang bng b mt ca silica. Nu mt trn ca silica gel khng bng phng, g nh vo thnh bn n khi bng. Ha tan mu vo mt lng ti thiu dung mi. Np mu ln ct, trnh khng lm xo trn b mt ca silicagel, ra thnh ct vi ln vi lng ti thiu dung mi chy sc k.Sau mi ln thm, phi lng dung mi chy xung gn b mt ca silicagel. Sau thm mt t ct trn b mt silicagel silicagel khng b xo trn khi thm dung mi. Sau , lm y phn cn trng cn li ca ct bng dung mi chy sc k v ni vi h thng p sut. Ra gii vi tc khong 2 inches/ pht l ti u. M my ht chn khng v kha.. Trong khi my ht chn khng vn cn hot ng, rt dung mi ( c trn trc, nh hexane v ethylacetate). Cho dung mi qua ct n ht lng dung mi d kin. Lc ny, ng kha v tho my ht chn khng.

2. Chy ct sc k

Cc phn on ca ct c hng trong nhng ng test, c kch c ph hp vi kiu ct v phn cc. ta s dng ng nghim di khong 13mm cho mu nh( 5-50mg), v ng ln hn cho mu ln hn. ti u ha hiu qu sc k, nn thu cc phn on sao cho tng th tch ca chng khng qu 1/10 ca ct. Ta bt u thu cc phn on ngay sau khi np mu; s khng lu cho nhng hp cht km phn cc ra gii khi ct. Mt khi ta chy ct, tt nht l khng nn dng li bt c lc no. ng ct kh hay ra gii ht dung mi cho n khi tt c cc hot cht c ra gii ht.

3. V sinh ct

Sau khi hon thnh, ra gii cc dung mi ra khi ct bng cch s dng kh nn. Khi tt c cht lng chy ra khi ct th dng chn khng (bm) y ht dung mi cn li trong ct ra ngoi. Cho tt c silicagel vo thng cha silicagel thi.

3. Cch thc la chn dung mi

Hp cht cn tch nn c TLC Rf t khong 0.15 n 0.20 vi h dung mi c chn. H dung mi kt hp mt dung mi c phn cc cao hn vi mt dung mi c phn cc thp hn l la chn thch hp nht v ta c th iu chnh phn cc trung bnh mt cch d dng. T l ca cc dung mi quyt nh phn cc ca h dung mi v tc phn gii cc hp cht cn c tch ra. Dung mi c phn cc cao s gia tng tc ra gii cc cht. Cc h dung mi kt hp thng thng nhm tng phn cc l dichloromethane/hexane, ether/hexane, hexane/ethyl acetate, v dichloromethane/methanol. Nu Rf khong 0.2, ta s cn mt lng dung mi khong 5 ln lng silicagel kh chy ct.

3. Phng php pht hin v thu nhn kt qu sc k

Dng sc k lp mng xc nh phn on cha cht cn phn lp. Nn chm 10 phn on trn 1 bn mng v trin khai sc k cng 1 lc. Nhng phn on c cng hp cht cn tch c gp li trong 1 ng thy tinh khc, sau c c mu bng cch c quay di p sut gim. CHROMATOGRAPHY VLC)
1.

3. SC K CT CHN KHNG (VACUUM LIQUID

Nguyn tc v yu cu ca phng php:

Sc k ct chn khng (VLC) l 1 dng sc k ct ci tin. H thng hot ng nh p sut ht di vi ct, dung mi chy xuyn qua h thng vi lu lng ln. Mc ch l tch 1 mu phc tp thnh vi phn on c phn cc khc nhau. y l 1 dng sc k hp ph, c kh nng tch trung bnh. M t dng c: tng t nh sc k ct c in nhng thay i 1 s k thut

Ct sc k: ct bng thu tinh hay s, thnh dy, c li xp, ng knh ln (5-10 cm), ngn (20-40 cm) hay dng phu lc Buchner. Khng c kho. Hng phn on bng cc erlen c khc vch thay v bng ng nghim. Erlen c vi ni vi b to p sut gim, c iu chnh bng 1 van x.
1. u, nhc im, ng dng: 1. u im: So vi sc k ct c in:

Thi gian thc hin nhanh (vi gi), np c nhiu mu t tn dung mi. D thu hi dung mi bng c quay do th tch mi phn on ln. Tch tt v d theo di bng sc k lp mng

So vi sc k ct nhanh:

H thng n gin hn, d dng thay i dung mi hn. Thao tc d dng hn.
1. Nhc im

Khng tch ngay thnh cht tinh khit c (tr nhng hn hp n gin hay cc vt cch xa nhau) Phi c dng c thch hp (ct c thnh dy, c li xp; h thng ht)
1. ng dng:

Tch hn hp thnh vi phn on c phn cc khc nhau, mi phn on gm t cht hn d dng phn lp tip bng SKC c in. Hot ng n gin nn c ng dng rng trong lnh vc hp cht thin nhin. Thch hp i vi dch chit dc liu th. Gip nh hng trong th nghim sinh hc 1 cch tp trung v t lng ph.
1. Chun b cho SKC chn khng 1. Pha tnh

Thng s dng silicagel 60 vi c ht nh (10-40 m), hay trung bnh (40- 63 m), i khi dng c ht mn hoc th Ngoi ra c th s dng nhm oxit, Celite
1. Chun b mu: tng t SKC c in 2. Pha ng

Dung mi c chn tin hnh VLC phi c kh nng di chuyn cc thnh phn trong hn hp cn tch bng sc k lp mng n Rf >0.30 (sau 1 ln khai trin) Thay i phn cc h dung mi khai trin theo nguyn tc tng t SKC c in nhng nhanh hn ( do ch tch thnh vi phn on).
1. Tin hnh sc k 1. Nhi ct v n nh ct

Nhi ct bng phng php bt kh. G nh vo thnh ct, sau nn cht khi bt bng cch cho p sut ht bn di, kt hp vi nn bng nt cao su bn trn. n nh ct bng cch cho dung mi chy qua ct bng p sut ht vi th tch bng 2-3 th tch cht hp ph Khi lp dung mi cui cng cn cch b mt cht hp ph khong 1- 2 cm th tin hnh np mu
1. Np mu

Mu dng dung dch hay hn dch vi th tch khng vt qua 1/5 th tch cht hp ph. Ho tan mu theo nguyn tc ging SKC c in

Rt thng dung dch mu ln lp dung mi cha trn nh ct. Sau dng dung mi ra thnh ct ri np dung mi y ct. Trong qu trnh ny, p sut ht vn hot ng.
1. Khai trin

Tin hnh khai trin v hng cc phn on bng erlen. Hng ht dung mi trong ton b khi cht hp ph ri ngng p sut v thay erlen khc. Np tip dung mi vi lng va . tch cc phn an phn cc hn, dng h dung mi tng dn (tng t SKC c in)
1. ng dng c th

Sc k ct chn khng c th p dng i vi nhiu loi hp cht. Bng vic vn dng vic thay i dung mi v cht hp ph c th tch thnh nhng cht tinh khit 1 cch tit kim hn so vi sc k ct c in. VD1: Chit alkaloid t Aconitum columbianum Ranunculaceae (Pelletier 1985) Dch chit (1 g) pH 9 v 12 chy qua ct VLC vi cht hp ph l 65 g nhm oxid 60 (type E, Merck) Vi h dung mi toluene- chloroform (1-4) chit c 265 mg talatizamine (a) Vi chloroform- methanol (19-1) chit c 247 mg cammaconine (b)

(a)
(b)

So vi sc k lp mng, vic tinh ch aconitin nhanh hn, r hn, thun li hn, khng cn phi co lp cht hp ph em gii hp ph. VD2: Chit gingerols t Zingiber officinale Zingiberaceae (Zarate 1992) Dch chit acetone ca Zingiber officinale Zingiberaceae (300 mg) c trn vi silicagel 60 (40-63 m, Merck 9385, 9.5 g). lm bay hi v tri bng ln trn phu Buchner 100 ml, ng knh 3.5 cm, t 1 ming giy lc chn pha trn mu v thm t t 25 ml hexan . Chy sc k vi h dung mi c phn cc tng dn (hexan- dietyl eter, dietyleter- methanol) Bng cch ny, ta tch c hu nh hon ton gingerols t shogaols.

Trong khi cng lng cht hp ph tin hnh vi FC, chy bng h dung mi toluene- methanol (80-5) khng tch c gingerols t shogaols iu ny cho thy kh nng tch cao ca VLC vi 1 lng nh cht hp ph.

3. SC K CT KH (DDC) 1.

Nguyn tc:

K thut ny tng ng vi k thut sc k lp mng iu ch


DDC l mt phng php sc k " khng ra gii ", ngha l, nhng cht cn tch s nm li trong ct cui qu trnh sc k. Vic s dng ng nilon p mng cho php d dng ly ra nhng cht phn tch bng cch ct ng thnh tng on. Trong TLC, dng dung mi chy nh lc mao dn chng li trong lc trong khi phng php DCC, dung mi chy nh trng lc. Pha tnh dng trong DDC l pha tnh s dng trong sc k lp mng. 1. u nhc im ca phng php :

2.1 u im: Phng php sc k ct kh l phng php bt cu gia phng php sc k lp mng (TLC) v sc k ct iu ch c in. p lc chi ph v thit b t hn so vi phng php sc k lng iu ch. Cng nh cc phng php sc k ct ni chung, DCC dng c c trong phn tch v iu ch. DDC nhanh, d dng v hiu qu cao trong vic tch v/hoc tinh ch cc cht vi s lng ln. Ging nh TLC, DDC l mt phng php "khng ra gii" s dng ng nilon nh l vt h tr v c th dng UV-254 nhn bit. Cng nh cc loi sc k ct, cc ct vi hu ht mi kch c cha y pha tnh trong sc k ct kh c th dng tch, hoc lm sch mt lng rt ln cc cht. 2.2 Nhc im:

thit lp ct i hi k thut, s kho lo v cn thi gian. i hi s ch lin tc trong sut qu trnh tin hnh. 1. Kh nng v mc ng dng

Cng nh cc phng pho sc k ct ni chung, DCC dng c c trong phn tch v iu ch. DCC c dng xc nh s lng thnh phn cht trong hn hp, phn tch v lm tinh khit nhng thnh phn ny dng cho nhng phn tch sau . Sc k ct kh p dng thnh cng cho nhng cht nh thuc nhum, alkaloids, v nhng hp cht d vng khc, mc d c th tch

bng cc loi ct sc k khc nhng s gp nhiu kh khn ng k. Lipid cng c th c tch bng sc k ct kh. 4. Cch thc tin hnh 4.1 Chun b ct:

ng nylon p mng di khong 20m c dng gi silicagel trong sut qu trnh chy sc k v c bn di dng cun c chiu rng thay i. Ct ng thnh nhng an c chiu di mong mun. Cho php tha mt on 10cm bt phn cui ng. ng mt u ng bng cch hn trn la ga, gp v kp , buc li hay bt c cch no ph hp. D dng loi b nhng np gp trn ng p mng, bng cch thi mt dng kh nng qua ng sau khi lc ng trong acetone. Cho mt ming m cotton hoc len thy tinh vo u hn kn ca ng v dng kim m thng nhiu ln cho php khng kh thot ra khi dung mi di chuyn trong ct.

4.2 Lm y ct:

tch 1g cht th cn dng khong 300g pha tnh trong ng c kch thc 40mmx1m. Cn thn thm tng lng nh pha tnh n 3/4 ng. Sau mi lng nh c thm vo, nn g nh ct vo mt b mt cng hoc nn cht bng my lc rung. Khi vic lm y pha tnh hon tt, kp c nh ct trn gi.

4.3 Chun b mu sc k:

Chun b mu sc k: thm khong 1g pha tnh vo mt t ml mu th. Lm bay hi dung mi bng my c quay, n khi mu th v pha tnh thnh dng bt chy t do (free flowing powder). Mu th nn c trn vi mt lng pha tnh bng t nht 10 ln khi lng ca n trong mt ng cuvet.

4.4 Np mu ln ct: C 2 phng php: np mu kh v np mu t. Np mu kh: mu th ht bm trong pha tnh ( c chun b nh trn ) s c cho ln trn u ct thnh mt lp mng, phng. Ph ln trn lp mng ny khong 2cm pha tnh tinh khit.

Np mu t: Nu mu th np vo ct l dung dch m c, cn ch thu c vng bt u cng hp cng tt. Dng mu th dng lng c th gy ra nhng vng g gh hn l dng mu th dng ht bm vo mt lng pha tnh ( mu kh ). Sau khi dung dch thm nhp vo phn u ct, thm khong 2cm pha tnh tinh khit. 4.5 Khai trin ct sc k: Sau khi np mu, bt u cn thn cho dung mi vo trn u ct bng bnh lng. Dung mi nn di chuyn t t v chc chn trong ct. Mc dung mi trn b mt ct nn c nh l 1-2 cm l tt nht, t c bng cch chn phu bnh lng ( ng np y trn bnh ) trn u ct khong 1 cm. Khi dung mi tin ti y ct th dng li. Ty theo chiu di ct, nht ca hn hp dung mi, v chiu cao ca mc cht lng trn u ct, thi gian khai trin ct sc k s ko di trong khong 15-30 pht. Cng nh vi sc k lp mng, thnh phn ca mu th ban u s c tch khi nhau trong ct. Th tch dung mi cho vo trn u ct khng nn vt qu lng cn thit lm y cc ch trng gia cc cht hp ph. 4.6 Hi phc cc cht tch: Sau khi kt thc khai trin ct, ct c t nm ngang trn mt b mt phng thch hp ct. Nhng vng phn tch c nh du trn ct bng mt thng hoc chiu tia UV nu cn thit. Mt cch khc l ct ng theo chiu dc, xt thuc th hin mu vo mt ct ca mt na ct v i chiu vi na ct cn li xc nh cc vng phn tch. Sau ct s c ct thnh tng phn bng dao. C th ct ct thnh nhng lt mng m khng lm v, vn pha tnh. Cc lt ct s c cho vo phu Buchner v tin hnh chit cc cht bng nhng phng php thch hp. 5. Pha tnh trong DCC Pha tnh dng trong DCC l nhng cht nh Silica DCC, Alumina DCC ... c xp l 60 Ao, phn b 63-200um, pht hunh quang UV 254.
Sn phm T trng

pH

Hot

Cht pht

Din tch b

tnh Silica for DCC Alumina for DCC 7 7 III III

quang 0.5 % 0.5 %

mt 0.5 g/ml 500-600 m2/g 0.8 g/ml 200 m2/g

6. Cch thc la chn v thay i dung mi trong qu trnh sc k Hn hp dung mi s dng l dung mi cho kt qu tt nht trong sc k lp mng s b. 2. SC K LNG P SUT TRUNG BNH (MPLC) 1. Nguyn tc L k thut sc k lng c tin hnh trong iu kin p sut trung bnh t 5-30 bar 1. S cu to ca 1 h thng MPLC

1. Dung mi:

MPLC thng chim 1 lng ng k dung mi do chi ph ca cc dung mi khc nhau l mt yu t quan trng. Hu ht cc dung mi ph bin nh n-hexan, ethylacetat, methanol, chloroform, diclorometan hon ton ph hp MPLC sau khi chng ct n gin. Dung mi khng cn thit phi kh kh hoc lc qua mng lc trc khi s dng. Cc my bm rt vng chc v c thit k chu ng 1 lng nh ht trong dung mi. Cn lu l chloroform, dung mi ny thng c cha vt ca HCl, c th gy thit hi ng k cho trang thit b v cc cht nhy cm vi acid. Hu ht HCl c th c loi b bng cch lc qua natri cacbonat trc khi s dng. L mt k thut m 2 hay nhiu dung mi c iu chnh thay i t l trong qu trnh chy theo mt chng trnh xc nh trc. Cc chi tit k thut ca vic chy gradient l lp trnh khc nhau cho cc h thng MPLC khc nhau nhng ni chung nguyn tc hot ng ca mt my trn gradient khng khc my so vi mt my trn gradient HPLC thng thng. Chng trnh c xy dng t thnh phn mu v kch thc ct. Ni chung dung mi phn cc nn c tng ln t t phn u ca chng trnh v tng nhanh phn cui ca chng trnh. Vic trn gradient cng rt hu ch cho vic ra v ti sinh ct sau khi s dng. Mt bm sc k c th chu c p lc ln n 30 bar v cung cp mc lu lng dung mi n 100ml/min l ph hp cho MPLC. V d vi silicagel v lu lng dung mi 20-50 ml/min p lc thng l 5-15 bar. p lc trn 15 bar c th xy ra v d nh sau khi ti mu nng, rt kh x l v h thng ng phn no d b r r ti nhng ch p lc cao. ng knh ct v di c sn t cc nh sn sut khc nhau. Cc ct Buchi c ng knh t 1.5- 10cm, di l 23 cm hoc 46 cm. Ct thch hp nht cho cc gradient nhanh chng l 23 cm. Cc ct c th c trang b tin ct, iu ny ko di thi gian

Thay i t l dung mi:

2. My bm:

3. Ct :

sng ca ct tch c th s dng li nhiu ln hn. Silicagel ht mn th kh nng phn tch tt hn ht th. Mi quan h gia kch thc mu v ng knh ct ngh cho MPLC (cn lu rng s lng mu cng ph thuc vo mc kh ca qu trnh phn tch)

Mu (g) < 0.5 0.5-5 5-10 10-20 20-50

ng knh ct (cm) 1.5 2.6 hoc 3.6 4.9 7 10

4. Chun b mu sc k a mu ln ct:

Mu c th lng tim hoc mu c ha tan sau bm ln nhng vt liu khc trc khi ti ln ct. Mu lng nn pha long mu vi t l cao dung mi (ln n 1:5) trnh qu ti u ct v tch kt qu gp kh khn. Tt nht l nn ha tan mu trong hn hp dung mi ban u ca chng trnh gradient. Mu rn c nhiu im thun li hn mu lng tim. S dng tt trong h thng MPLC t ng. Sau khi cho mu vo, ch cn nhn nt cho my bt u chy. H thng s t cn bng ct, ti mu, nhn tn hiu v ra sc k , ti cn bng li ct m khng cn can thip su hn. iu ny cho php cc hot cht km tan trong pha ng c th d dng ti ln h thng MPLC m khng cn ha tan trong dung mi mnh hn, iu c th lm gim hiu lc ca ct. C nhiu cht hp ph c th la chn cho mu rn. V d cht ti trn ct C18 c th hp ph ln Celite, cht ny c s tng tc yu vi a s hp cht nn d dng tch ra vo ct. Cn trong pha thun c th chn silica, Celite, hoc cc vt liu khc.

5. Pht hin v thu nhn kt qu:

S dng detector UV l , ghi nhn s k v cc phn on.


1. u nhc im ca phng php, kh nng v mc ng

dng:
1. u im:

Gi c phi chng v nhanh chng. Khng i hi thit b tn km (nh trong HPLC kch thc ht nh, dung mi khi chy qua pha tnh xut hin mt p sut ngc i hi phi cn c mt bm cao p duy tr p sut thng c p sut ngc ). Mt khc kch thc ht nh c th gii hn mc lu lng v lm gim phn gii. Kh nng ti c lng mu ln. Ct c th ti s dng. Ch cn detector UV l . Khng qu tn km dung mi. phn gii ch t mc trung bnh khng cao nh HPLC. Ch ph hp phn tch cc hn hp thnh phn on n gin. Kh nng phn tch cc thnh phn phc tp km hn HPLC. Thanh lc cc sn phm tng hp, c lp cc hp cht t nhin, n gin ha hn hp trc khi tin hnh sc k vi k thut cao khc, chia hn hp phc tp thnh cc phn on n gin phn tch.

2. Nhc im:

3. Kh nng ng dng:

4. ng dng c th:

Phn lp cc cht tinh khit t cy curcuma xanthorrhiza Roxb.(Zingiberaceae) bng hai k thut MPLC v sc k ct nhanh. 2. SC K LNG CAO P IU CH (PHPLC) 1. Nguyn tc v yu cu ca phng php

1. Nguyn tc:

HPLC iu ch l k thut phn lp v tinh khit ha mt lng ln cc hp cht. Trong HPLC iu ch, quan trng nht l lng mu tinh khit c phn lp, khc hon ton HPLC phn tch. Do HPLC iu ch cao vai tr ca hiu sut chit tch.

1. Yu cu:

H thng HPLC iu ch cn phi c kho st trc cc iu kin ti u bng h thng HPLC phn tch:

Chng trnh gradient khng cn s dng do tn km v chi ph. Mu th c th ch cn c x l trc theo phng php thch hp. phn gii cc cht khi kho st bng HPLC phn tch cng cao th cng d dng p dng k thut qu ti ct trong HPLC iu ch. (K thut qu ti ct ph thuc vo pha tnh, tnh cht ca cht phn tch, v tnh tan ca cht phn tch trong dung mi pha ng)

Ct s dng thng c ng knh trong > 30 mm (ct c ng knh trong t 8-30 mm c gi l HPLC bn iu ch). Kch thc ht pha tnh trong HPLC iu ch rt quan trng, nhng do ct lun c s dng tnh trng qu ti, nn cc loi ht pha tnh c kch thc nh v mc tin nh 1.8, 3.0 v 3.5 m dng trong HPLC phn tch th khng cn thit.

Nu mu phc tp vi phn gii thp gia cc cht v k thut qu ti ct kh c p dng th c th s dng pha tnh vi ht c ng knh 5 m. Nu mu tch tt, pha tnh vi cc ht ng knh 7-10 m thng c chn. i khi c th chn ht pha tnh c ng knh ln hn. Tuy nhin vic la chn pha tnh vi cc ht c ng knh thch hp cn phi cn bng gia hiu sut phn lp mu, tinh khit v lu lng mu.

Kch thc ht pha tnh trong HPLC iu ch ln nn p sut cn thit thp hn so vi HPLC phn tch, do cho php tc dng cao hn v lng mu np vo nhiu hn. u d phi nhy, thng s dng nht l u d khc x k vi sai hay u d UV. Lng mu np vo phi tnh ton ph hp vi ng knh trong ca ct. Thng thng, ct ng knh trong 4.6 mm thng dng trong HPLC iu ch vi c mu nh, ct ng knh trong 7.8 mm dng trong HPLC bn iu ch, ct ng knh 21.2 mm thng dng trong HPLC iu ch lng mu ln. Ct vi ng knh trong 30, 50 mm v ln hn c th c s dng trong trng hp m rng c mu. Dung mi pha ng phi hon ton tinh khit v d bay hi thu c cc cht tinh khit Qu ti ct tan ca cht phn tch trong dung mi > 20 g/l iu kin ng dng

Tm li: iu kin l tng ca mt h thng HPLC iu ch l:


Kch thc ht pha tnh t 10 -15 m Tc dng cao 2. u im, nhc im, kh nng v mc ng dng:

1. u im:

HPLC iu ch l mt phng php hu hiu tch, tinh ch v kim tra tinh khit ca cc hp cht t nhin. Cho php phn lp hng gam mu th. Trong quy m cng nghip c th tch lng mu th hng kg. Tn km Kh khn trong vn hnh Vic xc nh iu kin sc k phi c tin hnh theo quy m ln dn HPLC iu ch c ng dng rng ri trong phn lp cc hp cht t nhin, p dng trong cc lnh vc:

1. Nhc im:

1. Mc ng dng:

nh tnh: s b hay khng nh Dng lm cht chun, cht trung gian tng hp, nguyn liu th nghim sinh hc,.. Dng lm nguyn liu lm thuc

3. Pha tnh v cch thc chun b pha tnh

Khi chn pha tnh, quan trng l chn ht vi kch c l ph hp vi iu kin phn tch. B mt tip xc cng ln th kh nng phn tch cng tt, tuy nhin cn lu vi kch thc l nht nh, b mt cng ln th tnh khng bn ca tiu phn pha tnh cng tng. Trn thc t, pha tnh thng dng nht trong HPLC iu ch l pha lin kt gia silica v cc gc phn cc gn nhm silanol, silyl ete. Ngoi ra cn c th do lin kt gia silica vi nhm amino v cyano. C ch gn kt l do cc ni hydrogen trong phn t. Tuy nhin do silica c tnh hi acid nn trn b mt silica cng c s trao i cation. Pha o c to thnh do s lin kt gia nhm silanol v cc nhm khng phn cc C8 C18 nh dimethyloctadecyl,Trn

Pha thun:

Pha o:

thc t khng th lin kt hon ton cc nhm silanol b mt do cc nhm silanol khng phn ng s gn cc phn t phn cc lm qu trnh sc k cc pic b ko ui. V vy cn p dng k thut kha ui bng cch cho nhm silanol khng phn ng tc dng vi mt cht silyl ha chui ngn, nh cloroalkylsilan (trimethylclorosilan, dimethylclorosilan,). 4. Yu cn v k thut chun b mu sc k, a mu ln ct
1. Yu cu mu sc k:

tan trong pha ng hp l (c bit trong HPLC pha o)

Nu mu tan t trong pha ng => th tch tim ln => hiu qu phn tch gim. Nu mu tan nhiu => dung mi chy qua ct m c v ha tan nhiu c th dn n ta trn b mt ct => hng ct.

2. K thut chun b:

Mu th c chun b theo ng chuyn lun ring. Qui trnh chun b theo nguyn tc:

Dung mi ha tan hot cht phi ha tan trong pha ng, trong nhiu trng hp dng chnh dung mi pha ng ha tan mu. Loi b cc cht khng tan trong pha ng hoc cc cht khng ra gii c bng cch lc hay chit (chit li nhiu ln, lc qua mng lc 0.2 0.45 micromet)

i vi HPLC iu ch, mu phi qua x l tng i tinh khit ch khng dng cao chit nguyn lm mu, mu em chit tch bng HPLC c t cht hn.

3. a mu ln ct o Yu cu:

To ra phn b tt mu th trn u ct gip qu trnh phn tch v ra gii ng u, thun li, nhanh chng.

b. K thut:

Tim mu bng ng tim. Tim khi ngng chy. Tim qua cc van (c hay khng c loop). Tim qua bm chnh. Tim qua bm ph. a cht rn vo. 5. Cch thc la chn v thay i dung mi
o

Qui tc chung:

Ty bn cht ca ct: ct pha o, pha thun (trong HPLC iu ch hay dng ct pha o nn dung mi ra gii thng l h tng i phn cc isocratic hoc h c phn cc t thp ln cao gradient). Ty vo phn cc ca mu th: tnh tan ca cht cn phn lp, tinh khit. H cng n gin, cng trung tnh cng tt ( bo tr ct). Khi h ra gii khng t yu cu, thay i t l, thnh phn dung mi. Dung mi s dng trong HPLC iu ch thng c kho st trc bng sc k lp mng (TLC). Trong HPLC iu ch (thng l sc k pha o) , nc c s dng nh l dung mi yu so vi cc dung mi hu c c dng ra gi cc hp cht gn vo ct. Ba dung mi hu c thng c s dng nht l acetonitrile, metanol v tetrahydrofuran, cc dung mi ny u cho tia UV truyn qua tt v cn khc nhau v kh nng tch. C ngha l nu mt dung mi khng th p ng vi yu cu chit tch th c th c thay th bng dung mi cn li. Trong nhiu trng hp th s kt hp gia hai trong ba dung mi trn l cho vic chit tch, nhng i khi vn phi cn n h ba bn dung mi. Thay i dung mi trong qu trnh sc k (ra gii gradient): qu trnh iu ch cng c th c thc hin bng cch s dng h dung mi ra gii hn hp ty vo hp cht mun chit tch tc lm thay i phn cc ca pha ng (dung mi ra gii). Vic ra gii bng gradient dung mi l cn thit khi mu th tim vo ct c lng nc cao (v d: 5% MeCN trong H2O), hay phn cc ca dung mi c tng t t theo thi gian nhm ra gii tt c cc cht c trong mu th. Mt khc i vi ct thng thng (4,5mm v di 150 mm), c tc dng bnh thng l 1 ml/ pht th qu trnh ra gii gradient ch cn n 30 pht chc chn ra gii tt c cc cht trong ct. Ra gii gradient c dng tch mt hn hp cht phc tp trong mt phn on nhng rt tn thi gian (v cn thi gian ct bt u cc iu kin ra gii mi) T ra gii gradient n ra gii ng dng (isocratic)

C th:

L tng nht l vic ra gii ch cn thc hin bng mt h dung mi, khi h dung mi c gi n nh v cn bng.

Tuy vy vic ra gii gradient c th c dng nh cng c d tm h dung mi ng dng thch hp nht, bng cch dng dung mi vi t l thay i, mt h dung mi ph hp nht c th c tm thy khi tt c cc hp cht cn chit tch u c ra gii trong vng 25 30% thay i dung mi hu c ca phn tch gradient.

6. K thut thng p dng

Trong HPLC iu ch, k thut qu ti c s dng ph bin. C 3 phng php:

Qu ti th tch Qu ti khi (hay qu ti nng )

Trong sc k phn tch, thng lng mu tim rt nh nn chiu rng pic khng b nh hng. Hn na, nng mu cng rt thp nn khng b qu ti. Qu ti th tch: th tch tim mu ln lm pic dn rng (iu kin nng mu thp khng nh hng chiu rng pic). Tuy nhin, cao ca pic khng thay i, chng t pic khng b gim i mc d gi tr k tng. Pic c xu hng b ko thnh hnh ch nht. V nguyn tc, qu ti th tch c th tng cho n khi cc pic dnh vo nhau, th tch tim mu ti a n 50 l/g pha tnh. Trn thc t, con s ny thng l 5 10 i vi sc k pha o, cn vi sc k pha thun con s ny ch bng 10% so vi pha o.

Qu ti khi: mu c ha tan ti a trong dung mi, do mu tim vo ct c nng cao. Pic trong trng hp ny c xu hng ko thnh hnh tam gic. Ti a qu ti khi l 100 lg/g pha tnh. Thc t, gi tr ny c th vt hn 5 10 ln trong sc k pha o, sc k pha thun ch bng 10% so vi pha o. Qu ti khi thng c s dng trong k thut qu ti i vi HPLC iu ch v cht phn tch ra khi ct sm vi tinh khit cao. Trong trng hp ny, ct vi cc ht ng knh ln c th c s dng m khng lm gim hiu sut phn lp cht. (Trong khi nu s dng k thut qu ti th tch th kch thc ht nhi ct rt qua trng. Nu tiu phn ht pha tnh nh ch thu c cc di cht hp). 7. Phng php pht hin v thu nhn kt qu

u d trong HPLC iu ch phi thch hp vi tc dng cao ng thi phi c bin php ngn s qu ti u d. Cc u d thng dng:

u d quang ph t ngoi kh kin (UV Vis): nhy, khng ph hy mu th, ph bin nht nhng khng pht hin c cc hp cht khng hp thu trong vng 190 900 nm u d khc x k (RI): phi c trang b b phn iu nhit gi nhit h thng n nh khong 0,5 0C, khng th p dng ra gii gradient c nhng c th pht hin tt c cc cht (vn nng) v rt bn. u d tn x nh sng (ELS): mi c s dng gn y v ha hn l u d thay th cho c u d UV Vis v RI, nhy cao, gii hn pht hin thp, khng phc tp, c th chy gradient, n nh tt tuy vy ch s dng c vi cc pha ng d bay hi v mu phn tch phi c s bay hi thp hn s bay hi ca pha ng. B phn thu nhn tn hiu: trong nhiu trng hp, b phn thu nhn tn hiu c xu hng gn lin vi h thng v quyt nh thu thp thng tin no v cc thnh phn c ra gii. Nhng thc t s can thip l khng cn thit v b phn thu thp tn hiu c lp trnh chy hon ton t ng. B phn ny l cc phn mm c ci t trong my tnh kt ni vi h thng sc k.

8. ng dng c th

Phn lp hp cht xanthin: cafein v theophyllin: sc k pha o C18, ng dng tc 0.6 mL/pht, pha ng nc:acetonitril, u d UV Phn lp khng sinh: dicloxacillan, cloxacillan, oxacillan: sc k pha o ht ng knh 5 m dp Zorbax Eclipse XDB-C18, pha ng nc:acetonitril (65:35), tc Phn lp cc cht t nhin, anthocyanin: ct 250 x 21.2 mm, pha o7 m dp PrepHT Zorbax StableBond-C18, gradient dung mi

A: 0.1% acid trifluoroacetic trong nc B: 0.1% acid trifluoroacetic trong methanol gradient: 2325% B trong 35 pht, 2553.5% B trong 50 tc dng 21.2 mL/pht, u d UV, mu tim 46.1 mg/mL tng s cht tan rn (~5 mg/mL anthocyanin).

5.

SC K PHN B NGC DNG 1. Nguyn tc:

Sc k phn b ngc dng c tin hnh theo c ch phn b l hin tng cht tan c phn b ng thi vo 2 pha lng khng ng tan trong nhau.
1. Pha tnh :

L cht lng c cha trong ct Cc lai ct pha tnh: Sanki,J type,cross axis
1.

Pha ng

cht lng (thng l dung mi phn cc ): mu th l hp cht phn cc Vd:hn hp gm 2 cht a v b:

H s phn b:

Ka

Kb H s K cng ln=>cht a,b cng tp trung trong pha tnh =>pha ng cng kh tch cc cht ra khi pha tnh T s gia Ka v Kb cng ln cng d tch ring a,b Gm 3 giai an chnh: Trn ln (to s tip xc lin b mt gia 2 pha ng ,pha tnh ,mu th) n nh Phn tch
4. Mc ch ca sc k phn b ngc dng:

Tch nhng cht c M<2000 Tch cht phn cc nh polysaccaride,acid amin ,acid cacboxylic,acid nucleic,phosphate v 1 s polyme phenol (do nhng cht ny kh b tch bi c ch hp ph)
4. u im so vi sc k khc:

Ga tin r D trin khai quy m cng nghip(GC,HPLC lm khi lng ln s gim phn gii v tc dng chy khin kt qu khng chnh xc) D thc hin vi cc cht c nht cao
4. Tin hnh:

Chun b ct

Nhi ct Np mu: :mu th di dng m c (ha tan lai bt kh v rt vo ct c sn y) H dung mi khai trin: phi hp hoc dng ring

Vd:hn hp acetonitril,methanol v nc Kho st h dung mi theo trnh t gim dn phn cc(H2O MeO)=>MeOH=>(MeOH acetonitril)

4. Mt s phng

php sc k phn b ngc dng: 1. DCCC (Droplet counter current


chromatography)

L hnh thc sc k c xa.N ch s dng trng lc gip pha ng di chuyn qua pha tnh v ko cc cht cn phn tch ra theo h s phn b tng phn ca cc cht vi pha ng.

2. CPC: sc k phn b ly tm

c pht hin vo nhng nm 80 bi ngi Nht H thng gm nhng rotor ng nht vi cnh qut c quay quanh trc trung tm CPC t to rung ng v ting n ,tc quay rng ln hn HSCCC (t 500-2000rpm) N cho php s lng gn v tch lp gia h thng 2 pha khng n nh (biphasic):H2O-H2O/Butanol-H2O ng c CPC c to bi nhng a nh c ni vi nhau bi ng u ui.Rotor c b y pha tnhv cho pha ng bm qua.Nh s quay ,tc chy t b nh hng bi p sut thp CPC thch hp cho c l trong phng th nghim (vi ml) n quy m ln hn(vi lt)

3. HSCCC:

Th h mi ca CCC c pht trin bi gio s Yoichiro Ito .cu to bi nhng ng nh hnh xon c t trong tubin c nh xoay quanh trc hnh tinh v quay lch tm vi cc trc mt tri khc to s pha trn vi tc chng mt My ny ging CPC nhng tc quay cao hn.N l 1 my ly tm hnh tinh c h tr vi nhiu ct hnh hc xon c. N s dng ng mao mch thay th cho nhng ng ln trong CPC d dng trn ln cc cht vi nhau v thu nh c kch c ca my.

My HSCC

VD: Phn tch dc liu Trung Quc bng my HSCCC

HSCCC (TBE-300) t Shenzhen Tauto Biotech,China, vi 3 h thng ct c ni lin tip vi nhau (ng knh ng = 2.6 mm, tng th tch = 300 ml). Dung mi h thng cho HSCCC:

H thng dung mi A: n-hexane-ethanol-nc (10:5.5:4.5, v/v). v / v). H thng dung mi B: n-hexane-ethanol-nc (10:7:3, v/v). Cc r bt kh (20 g) ca S. miltiorrhiza Bunge miltiorrhiza Bunge t H Bc, Sn ng v tnh Giang T c thm vo to 50 ml of n -hexaneethanol (1:1, v/v). n 50 ml n-hexane-ethanol (1:1, v / v). Hn hp c rung ng trong 45 pht. N c sau ly tm 10.000 g trong 10 pht, v ni trn mt c lu. Dch chit c ra sch bng nc v etanol n khng mu. C n cn.dng h dung mi A ha tan cn u tin ct c lp y bi h dung mi A u trn,quay 900rpm trong 10 pht. Tip tc np dung mi A vo u di tc 2ml/pht. n nh ct. Sau bm phn dung mi ha tan mu th vo. Dng u d UV-Vis pht hin cc nh 280 nm. Sau 470 pht thay h thng dung mi A bng h thng dung mi B tng hiu qu phn tch. Pha tnh: h dung mi A u trn. Pha ng: 0-470 pht :h dung mi A cho u di.

T 470 pht tr i:h dung mi B cho u di.

Hn hp c tch ra thnh 12 nh.

CI TIN :

Tng hiu qu tch :dng dung dch m iu chnh pH La chn dung mi da vo 3 yu t: t trng ,sc cng lin b mt , nht ca mu th

III.

KT LUN

Sc k iu ch c nhng ng gp to ln trong qu trnh phn lp cc dc cht t dc liu. Qua chng minh c dc tnh v gip bo ch ra cc thuc c hot cht t dc liu vi tinh khit cao gp phn lm tng hiu qu iu tr cng nh gim cc tc dng ph. Trong tng lai, sc k iu ch s vn tip tc l phng php ch o phn lp cc hp cht t nhin cng nh tng hp. Tuy nhin hin nay vn cn nhiu c ch sc k vn cha c sng t v cha cho thy mi tng quan cht ch gia l thuyt v thc nghim. a s cc iu kin sc k c p dng thnh cng u phi tri qua mt thi gian nghin cu v th nghim tng i di. Bn cnh hiu sut, chi ph v quy m iu ch cn phi c ci thin, do khuynh hng sp ti trong pht trin sc k iu ch s l: Tip tc hon thin l thuyt v sc k nhm kt hp cht ch gia l thuyt v thc nghim, qua cc nh khoa hc c th d on v xc nh cc iu kin sc k ti u mt cch chnh xc hn v nhanh chng hn. t c hiu sut cao v gim chi ph iu ch cc k thut v quy trnh sc k cn c ti u ha. Qu trnh ti u ha quy trnh lun l mt vn phc tp, i hi mt thi gian th nghim lu di v c phng php ph hp. Vic nng cp quy m iu ch cng l mt trong nhng yu cu cp thit. thc hin iu ny cn c s nghin cu v ci tin v k thut sc k cng nh cc iu kin sc k nh ct sc k, pha tnh v.v

IV.

TI LIU THAM KHO


Gio trnh phng php nghin cu dc liu - i hc y dc thnh ph H Ch Minh - Ch bin PGS.TS.Trn Hng Ha phn tch tp 2-Nh xut bn y hc - Ch bin: PSG.TS.V Th Bch Hu - TS. Vnh nh Phng php phn tch cc hp cht hu c - NXB i hc quc gia TPHCM - Tc gi Nguyn Kim Phi Phng Encyclopedia of Chromatography Jack Cazes.pdf A Practical Handbook of Preparative HPLC.pdf Practical-High-Performance-Liquid-Chromatography-2005-1.pdf Preparative chromatography_wiley_2005.pdf

http://caythuocvn.com/Dlieuhoc/Tools/Phuongphap/SKLM.htm http://www.chromatography-online.org/TLC/contents.html http://www.chromatography-online.org/TLC/Phase-Selection-in-TLC/MobilePhase.html http://kerouac.pharm.uky.edu/ASRG/HPLC/theory_of_operation.html http://www.pharm.uky.edu/asrg/hplc/applications.html http://www.lcgceurope.com/lcgceurope/data/articlestandard//lcgceurope/202002/1910 3/article.pdf http://www.lcgceurope.com/lcgceurope/data/articlestandard/lcgceurope/422004/12846 9/article.pdf http://www.chem.uidaho.edu/faculty/ifcheng/Chem 253/Lecture Notes/2005/HPLC Chapter 25.pdf http://www.chromguide.com/pdf/K1_HPLC_in_Laboratory_Practice.pdf
http://siggy.chem.ucla.edu/VOH/136/flash_chromatography http://orgchem.colorado.edu/hndbksupport/colchrommienflash.html http://www. Saidsorbents.com/flash.html

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