TRNG I HC BCH KHOA TPHCM KHOA K THUT HA HC NGNH CNG NGH THC PHM

inh lng axit amin bang sac k

GVHD: Phan Ngc Ha Nhm: Trn Ngc Minh Chu 61000283 Vng Bu Chu 61000286 Vng Hunh Thanh Hi 61000876 Nguyn Thin Ngn 61002085 1/11/2012

Mc lc
Mc lc.......................................................................................................................................................... 1 I. 1. 2. II. 1. 2. 3. 4. III. 1. 2. a. b. c. d. 3. T Ph Ph Ph Ph Ph X M C h th u ............................................................................................................................................. 2 h ........................................................................................................................................... 2 hi .................................................................................................................................. 2

u t.......................................................................................................................................... 3 g h g h g h g h g h t u t OPA ........................................................................................................ 3

t o d n xu t NBD-F .................................................................................................... 5 t o d n xu t PITC ....................................................................................................... 5 t o d n xu t FMOC-Cl ............................................................................................... 6 s ........................................................................................................................... 6

Gii thiu........................................................................................................................................... 6 C S S S S S s h gi g h h g h s .................................................................................................................... 8

h ....................................................................................................................................... 8 gi (Paper chromatograhhy) ........................................................................................... 11 g .......................................................................................................................... 17 HPLC ............................................................................................................ 21 g h ........................................................................................................ 31

I. X phn tch amino acid trong m u protein, m u ph i i i .

c thy phn t o thnh nh ng

Thy phn bng acid l ph g h th g th ng nh t thy phn protein tr c khi tin hnh pnn tch amino acid. Ph g h thy phn bng acid c th t o ra s phn ct khc nh u i vi mt vi amino acid- ph hy hon ton hoc mt phn. Mt s h h hi thy phn bng acid. - Trytophan b ph hy hon ton - Serine v threonine b ph hy mt phn - Methionine c th b oxy ha - Cysteine th g c pht hi n nh l cystine (nh ng cystine th ng hi n r t km bi v chng b phn hy mt phn). - Asparagine v glutamine t c pht

g ng b chuyn thnh acid aspartic v acid glutamic.

Nh vy, nu k thut thy phn khng tt c th gy ra nhiu kt qu khng chnh xc. 1. - Lm giu m u - Lo i b t p ch t. 2. - Thu thp protein d ng tinh s chs d g h g h t ta thun nghch sc k qua ct tr i ion, ry phn t , h g h sc k i l lo i b cc protein t p.

- Mt s ph
Ph g p

g h

thy phn th g

c s dng

p 1: Dng HCl 6N v phenol (t 0.1- 1 %)

Ph g h th g c s dng nh t l dng HCl c cha phenol. Thm he v g n ph n ng halogen ha tyrosine.Dung dch thy phn: HCl 6N cha t 0,1 n 1 % phenol. Ph g h tin hnh:

- Cho m u vo tube thy phn v lm kh (m u long acid).

h n c khng pha

- Thm dung dch thy phn vo m u vi t l nh t nh. - Lm l nh tube cha m u. - M u th g c thy phn 110oC, trong 24h trong chn khng hoc tr g h tr g n qu trnh oxy ha. Thi gian thy phn c th th i ty theo m h phn tch. - Sau khi thy phn lm kh m u tr Ph g p - M u g h h g lo i b acid tha.

p 2: Dng Mercaptoathanesulfonic (MESA) 2.5M.

c lm kh trong mt tube tr c khi thy phn. ha dung dch thy phn.

- Tube cha m u ny cho vo mt tube ln h

- Tube th h c ti h h tr g h h g h i dung dch o o thy phn. Tube th h c gia nhi t t 170 C n 185 C kho ng 12 pht. - Sau khi th tha. h tu e c lm kh trong ch h g lo i b acid dung dch

II. 1. g p p O-phthaladehyde hoc ortho-phthaladehyde (OPA) l cc hp ch t ha hc i cng thc C6H4(CHO)2.OPA l thuc th cho amin bc mt, nh ng cng c p dng cho phn tch amino acid.

Th g c vit tt OPA, phn t ny l mt dialdehyde bao gm hai nhm formy (CHO) gn lin k trn mt vng benzene.L ch t rn mu vng nh t c dung trong vi c tng hp cc ch t d vng v l mt thuc th trong phn tch ca cc axit amin. -ortho-phthaladehyde (OPA) c ha tan v h tr 11,5.n nh y c m vi tia c c tm v oxy khng kh. g u g i h c pH <

-o-phthaladehyde (OPA) ph n ng vi cc axit amin trong s c mt ca cc hp ch t thiol,hnh thnh cc s n phm hunh quang. Ph n g c s dng cho vi c t o d n xu t trong phn tch ca cc axit amin bng s tr i ion. C 2 phng php to dn xut OPA: t o d n xu t tin ct (precolumn derivatization) v t o d n xu t sau ct (derivatization postcolumn) 2.1.1 Dn xut tin ct: Nguyn tc chung t o precolumn dervatization l t o d n xu t ca cc axit amin vi o-phthalaldehyde (OPA) , ti the t h HPLC g c pha,cc sc k l g c trang b mt f u r etri pht hi n cc d n xu t axit amin. C g hunh quang ca phc OPA- it i c theo di vi c sng kch thch 348 nm. O-phthalaldehyde (OPA) ph n ng vi cc axit amin vi s c mt ca hp ch t thi hnh thnh cc s n phm hunh quang.N acetyl-L cystein v 2mercaptoethanol c th c s d g h thi . - OPA t n khng pht hu h qu g v h g h h g n kt qu phn tch. - h t v nh ca n trong dung d h c cng vi t ph n ng nhanh nn c th t o d n xu t t ng v phn tch bng cch s dng mt m y l y m u t ng,d n xu t nh y r t cao,gii h n pht hienj ti thiu l 50fmol(10-15mol) - Tuy nhin ,v khng c kh g h n ng vi axit amin th c p nn lm h n ch u th c h g h t o d n xu t . h g h hng phn t h c cc axit amin th c p(v d h r i e).

2.1.2 Dn xut sau ct: - Nguyn tc t o d n xu t sau ct l s dng ct tr i i tch cc axit amin t do,tip theo l qu trnh oxy ha cc axit aminn bng hypochlorite natri ri th c hi n ph n ng vi OPA vi s c mt ca hp ch t thi h Nacetyl-L-cystein v 2-mercaptaoethanol. - Mc d OPA khng ph n ng vi cc axit amin th c ( h r i e) t o thnh cc ch t hu h qu g, h g qu tr h h ng hypochlorite natri cho php cc amin th c p ph n ng vi OPA. - C g hunh quang ca phc OPA- i i vi mt c sng kch thch 348 nm v pht x c sng 450 nm v vy khi tin hnh phn t h u g u d hunh quang 348 nm. - Gii h n pht hi n ti thiu kho ng vi chc pmol cho hu ht cc d n xu t axit amin, tuyn tnh trong ph m vi mt vi n vi chc nmol So vi d n xu t tin ct th d n xu t c th h t h 2. c axit amin th c p. nh y th h tu hi hi u im l

g p p o d n xu t NBD-F Nguyn tc chung ca t o d n xut NBD-F l ph n ng ca cc axit amin vi 7fluoro-4-nitrobenzo-2-a-1.3-diazole (NBD-F)ph n ng vi cc axit amin k c axit amin th c t o thnh s n phm hunh quang,ti the t h HPLC h o vi s dng u d hu h qu g s g h th h 480 v c sng pht x l 530 nm. C iu ki n tt nh t cho ph n ng t o d n xu t l nhi t 60oC v i tr ng ki tr g 5 ht, s u u g i HCL sung vo hn h h n ch h h ng ca cc ch t nn hunh quang. u i : nh y c h g h OPA h g u i h h n g Gii h n pht hi n c kho ng 10 fmol. 3. h g h gn vi h g h t o d n xu t tin ct c vi acid amin th c p. t o d n xu t ny cho mi acid amin l

g p p o d n xu t PITC Cc thuc th c kh nng t o ra cc d n xu t h p thu t ngo i cho cc amino acid gm: PITC (phenylisothiocyanate), Dansyl-Cl (5-N, N-dimethylaminonaphthalene-1-sulfonyl chloride). - Phenyl isothiocyanate (PITC) l mt thuc th s d g tr g HPLC h o g c PITC, t nh y c h OPA v h g th t ng hon ton tuy nhin PITC c th t o s n phm nh vi t t c it i , tr g it amin th c p (proline).

- PITC phn ng vi it i t o thnh phenylthiocarbamyl (PTC) h p th c c i c sng t ngo i 254 nm. - u tin lo i ht HCL v h.S u hi h sung thuc th t o d n xu t g eth :TEA : c c t: PITC=7:1:1:1, lm s ch d n xu t bng chc h p su t trong 30-45 pht. - L u tr m u d n xu t PITC sau khi s y kh,ti s dng dn xu t bng cch ha tan l i n vo dung d h m photphat pH=7,4 v acetonitrile vi t l 95:5 ch c th gi c ti t ngy nhi t phng,hay 60-70 gi hi trong t l nh. - Pht hi n cc d n xu t bng cch s dng mt my d tia c c tm 254 nm, gii h n pht hi n ti thiu c kho ng 1 pmol cho hu ht cc d n xu t acid amin. Tuy t h thu c trong kho ng 20-500 pmol. - Cc thuc th PITC l d h i, h th lo i b thuc th tha, qu gi m thiu kh g h h ng ca thuc th . - Thuc th PITC c u im l lm m t ho t tnh ca nhm amino trn amino acid do cc mi sc k t ko ui h . PITC ch c th p dng cho tr ng hp t o d n xu t tin ct v s chun b m u v t o d n xu t yu cu thi gian di. 4. g p peptide. N p o d n xu t FMOC-Cl

FMOC-Cl l thuc th dng b o v nhm amino trong qu trnh tng hp 1979, Anson Moye v Boning gii thi u FMOC nh l thuc th

t o d n xu t hunh quang cho amino acid bc mt v hai. Nh c im duy nh t ca thuc th ny l ph i tch lo i thuc th d (FMOC-Cl hay FMOC-OH) ra kh i dung dch tim tr c khi III. g p p g php sc k vo ct sc k.

1. Gii thiu im chung ca h

Ph g php s c pht tri v u tin bi Mikhail Semyonovich Tsvet (1901) trong qu trnh nghin cu v h r h . n 1952, Ar her J h P rter M rti Ri h r L ure e Mi i gt S ge t gi i Nobel ha hc v sc k phn tch. T thut s g v g c pht tri h h n ngy nay.

Sc k l ph g h h t h, h i, h t h h t d a vo s phn b khc nhau ca chng gi h i h g v h t h. Khi tip xc vi h t h, ut ca hn hp s phn b gi h t h v h ng t g ng vi tnh ch t ca chng (tnh h p ph, tnh tan). Trong cc h thng sc k ch c cc phn t h ng mi chuy ng dc theo h sc k. Cc ch t khc nhau s c i l c khc nhau vi h t h v h ng. Trong qu trnh pha ng chuy ng dc theo h sc k ht l h t h n lp h t h h , s l i p l i qu trnh h p ph, ph n h p ph. H qu l cc ch t c i l c ln vi pha tnh s chuy ng ch h qu h thng sc k so vi cc ch t t g tc yu h vi pha ny. Nh vy m g i ta c th tch cc ch t qua qu trnh sc k . C s c h g h sc k

Ph g php sc k d a vo s phn b khc nhau ca cc ch t gi h ng v h t h. C hiu nguyn nhn n s phn b khc nhau gi a nhau ca cc ch t, h ng chnh s l i p l i hi t ng h p ph - ph n h p ph ca cc ch t khi dng h ng chuy g qu h t h gu h h yu ca vi c tch sc k. Hu ht cc ph g h s u c d a theo nguyn l trn, v hi u qu ca t g h g php ph vo s chn l a gi h g h t h [8]. Phn lo i qu trnh sc k Tr g h g h s h ng ph i tr ng thi l ng hoc rn. D a vo tr ng thi tp hp c thnh hai nhm sc k kh v sc k l ng. D v h g v h t h g i ta l i chia cc ph g h . Ty theo tr ng thi tp hp ca cc pha, lo i t ta phn bi t cc lo i s h b ng gt u th h t h h t h g, g i ta chia sc k h tr i ca cc ch t gi a h sc k thnh nhm nh v s hnh thnh s g i

Mt s ph

g h

sc k trong phn tch amino acid

Phn tch amino acid h g h nh thnh ph i i g h h ng chng trong m u protein hay peptide. Phn tch amino acid c th s dng sc k gi y (paper chromatography), sc k lp m ng (thin layer chomatography- TLC), sc k kh (gas chromatography), s tr i ion (ion exchange chromatography), sc k l ng cao p (high performance liquid hr t gr h ), i n di mao qu n (capillary electrophoresis) . 2. p g p a. h gh Sc k kh l mt h g h h ng l g c thay bng 1 dng kh lin tc ch qu h t h. Cc ch t c tch ra kh i hn hp bi t gt h h u a chng vi h t h. Do kh g h t m ca ch t h , g h h g g v i tr ca mt h ng th c s trong 1 h thng sc k. N ch lm nhi m v li cun cc ch t h h u tr g h h i h y d the h t h chng c th t g t vi pha t h. V th dng kh ch y trong ct sc k ch c gi h g. g v i tr y cc ch t ra kh i h t h tr g h t kh chnh l nhi t . Cc ch t c nhi t si khc p

nhau s b u gi hay b li cun bi cc dng kh mang khc nhau.T h t kh i nhau.Do ph i h h i c th c li cu i i dng kh mang, ch nh ng ch t h i i c th c phn tch. V vy sc k kh ch p d g c cho cc ch t c kh g h i hi t tin hnh sc k. C u t o ca mt h thng sc k kh hi i bao gm: Kh mang v h th g iu chnh kh:c 2 lai kh mang thng dng nh t l it v he i.Kh g h i h g c c cc kh l khc h h ng ti phn t h h t g t vi cc ch t phn tch vi h t h h t o nn cc peak l trong s . tinh khit ca kh th g th ng ph i l h 99,95%. c c t dng ti u, t h th g v iu chnh p su t u ng kh c s dng. C g u v thit b u:L i u i d ng l g v h thng.C g u c gia nhi t cho t t c cc thnh phn ca m u l g h h i i v h thng.Vi sc k kh mao qu ng m u th ng h g v t qu 10ml.Vi sc k kh ct nhi ng m u c th l h . Ct sc k:D ng c in l ct nhi vi mt ct thy tinh hay thp khng r h h i u ng knh trong 2-4mm, di 1-4m, bn trong nhi h t h. Tuy nhin ngy nay phn ln cc ct sc k kh l mao qu n.Ct mao qu n th g c lm bng silica nung ch ng knh trong 0,1-0,53 mm vi chiu di 10-100 h h . t th g th ng c ng knh trong 0,2mm, 0,25 mm hay 0,32 mm vi chiu di 25-60m. Thnh torng ca ct mao qu n c th trng ln 1 l h t h ng vi b dy 0,2-0,5 (WCOT) h c ph mt lp h t c mt l h t h ng bao quanh (SCOT) hoc mt lp ph xp ca ch t h p ph hay ry phn t (PLOT) ai ct mao qu n thng dng nh t hi n nay l WCOT. So vi ct nhi ct mao qu n c hi u g t h h r t nhiu. Mt ct mao qu n 60 m c th c 180000-300000 thu t so vi 4000 thu t ca ct nhi 2m. Lai ct nhi ng knh nh h (0,75 ) e h ng trung gian gii a ct nhi v mao qu n. Ph t h tr g s h g.C h t h rn vi h h p ph (silicagel, nhm oxit) ry phn t hay gp trong ct nhi. Tu hi h t h ph bin nh t h t h g the h phn b.Ph t h ng c th ph ln mt gi mang trong ct nhi hay trng ln thnh trong ca ct mao qu n. L g h t h tr g t cng ln , kh g t h v ng m u c th phn tch ca ct cng cao.Trong ct nhi g h t h th chim ti 25% khi ng ca vt li u nhi ct.

Cc ch t l ng s d g h t h g ng v ngun g v phn c c. Hai l i h t h th g ng nh t l d n ch t ca polisiloxan v polyethylen glycol.Mi h t h gii h n nhi t c h g v t qua gii h n ny s lm nhi t h t h u h h ng. Bung ct: Nhi t l mt y t gh qu trng trong vi c tch cc ch t qu trnh sc k kh. Trong sut qu trnh phn tch nhi t ct ph i g u trn tan b ct. Nhi t ct ph i h, h h v th i theo yu cu phn tch. Phn tch sc k kh c th c th c hi the iu ki ng nhi t h hit t g the h g tr h hi t. t iu ny tan b ct s t trong mt khoang kn gi l bung ct. Bung ct c cch nhi t v c cc thit b gia nhi t, theo di nhi t v phn phi nhi t, gi cho ct h t h g u, nh v tun theo yu cu phn tch. C nhiu l i ete t r h h u c s dng trong sc k kh.Ph bin nh t l detector ion ha ngn l (FID). ai ph thng pht hi c hu ht cc ch t phn tch, nh y v c kh ang tuy t h ng hc kh rng, thch hp cho vi h ng. Lai ny c thit k gi v g t g i r tin.Detector MS vi nhiu thut h h u g g h bi h v tnh hi u dng ca n.N pht hi c r t nhiu ch t g t detector r t chn lc khi s dng k thut pht hi n chn lc ion.Ph khi nh y cao v kh ang tuy t h ng hc ln thch hp cho vi h ng.Detector Ms cn cung c p nh ng thng tin h u ch v khi u g h t h v h m h nh c u trc cc ch t. nh mt ch t g i thi gi u (tr) g i ta cn s dng ch s K v ts. S vi tr ch s u Kovats hh tr nh ng sai l ch do thit b v iu ki n phn tch. u h im ca sc k kh S h h g h hi u g t h r t , h hiu so vi sc k HPLC. N c th phn tch nh ng hn hp r t phc t p m HPLC khng phn tch ni. Nh i h g h h c th s d g h h g h h t h ch t h i. C h t h g h i nhi t phn tch mu h t h c cn ph i bi i d n ch t h i ng cch ethyl ha.D v n c th s dng trong sc iu ch h g tr th c t sc k kh g h h g tr g h t h h t h v nh g.Tr g h t h c li u sc k kh thch hp cho phn tch tinh du v cc ch t h i. Mt ng dng khc hi g qu t h t h ng cc ch t b o v th c vt tr c li u, dioxin v ch t c h i trong th c ph ,.C h t ny th ng tn t i ng r t nh trong hn hp phc t p r t kh pht hi , h h g h h ng chng b g h g h sc k khc.

b. g (Paper chromatograhhy) Gii thi u v sc k gi y i. h Ngh Sc k gi y (SKG) l lo i sc k dng gi thut ha h h t h g t h v hn hp ca cc ch t u, c bi t l sc t. gi g h t h. t k nh cc thnh phn trong cng mt

ii. ng Dng Th g g th nghi tinh khit, h t h, h ng hay phn tch cc ch t. t k thut h u ch bi v n th c hi t g i nhanh chng v ch cn mt ng nh vt li u m u. Th g th ng sc k gi g nh tnh s n ph h h ng ch t. iii. Nguyn l ho t ng Trong sc k gi , h t h v h g u l ch t l g. Ph t h c h p th trong cc l xp ca gi y (ch t mang). S phn ly trong sc k gi i qu n cc nguyn tc gi g h tr g sc k lp m ng. Ch t c phn b gi a mt h t h v h g. Ph t h th g c hoc thnh ph th c t hn hp dung mi ca h g c ht chn lc vo gi . Ph ng l mt h dung mi di chuy i h t h, g the u t c trong m u cng vi n. Cc ch t c trong thnh phn ca m u s tch theo s h p thu m nh hay yu ty thuc vo kh g t gt a cc ch t vi h t h. Nh vy SKG l sc k phn b l ng -l ng, d a vo h s phn b khc

nhau ca cc c u t trong hn hp 2 ch t l c u t c trong thnh phn m u. iv. H S Di Chuyn

g h g h t

h u phn tch cc

t tr g h di chuyn ca mt ch t g i t th g g h s di chuy Rf v c tnh bng kho ng cch di chuyn ca ch t so vi s di chuyn ca dung mi. Gi tr ca Rf bao gi g h h 1.

Tr

g : : H s di chuyn

a l kho ng cch di chuyn ca ch t phn tch b l kho ng cch di chuyn ca dung mi. Tr g tr ng hp sc k lin t h g h c gii tuyn ca dung i, g i ta dng h s di chuyn Rr. Rr l t s gi a kho ng cch di chuyn ca ch t phn tch v kho ng cch di chuyn ca ch t dng lm chun so snh. Gi tr Rr c th nh h h h 1. TRANG THIT B i. Gi y sc k: Ch t m g tr g SKG th ng l cellulose tinh khit d ng gi c bi t v ht khc nhau nn c hi u ng tch khc nhau, dng lm ch t cho pha t h. Ng i r , SKG h g h s tr i ion, s h o Wh CM 50,Wh P 20, Wh t 30, Wh s, i gi y lm t s i thy tinh WhGF/A, WhGF/B, WhGF/C,.C th g s k thut c SKG: dy (mm), tr g ng (g/m2), chiu cao th m dung mi trong mt thi gi (h), * iu ki n l a chn gi y sc k: + C sc c n v

+ ng nh t v c h t v t trng + Tinh khit ha hc + t h p ph.

ii. Dung mi Sc k gi th ng l sc k phn b u g i h t h th ng dng l cc dung d h , h ng l nh g u g i phn c c th h h t h. * iu ki n dung mi + Tinh khit cao +C u g i g h ng ca c u t ph i khc nahu trong hai pha. h g c bi i.

+Thnh phn dung mi trong qu trnh s + Dung mi d kh i lo i kh i gi y. iii. Bnh sc k

Th ng bnh hnh tr hay hnh hp ch nht v ch t li u th ng lm bng thy tinh, c np kn. Nn dng np thy tinh c mp mi nhm, khng nn dng nt cao su. QU TRNH V K THUT SC K i. Chun b Chn bnh s h th c thch hp. Bnh ph i h th c thch hp, c n y kn. Np c l gi lp bnh g h g u g i. Tr g h gi g ng dung mi v treo gi y s c thit k thch hp cho kiu khai trin sc k. Chun b dung mi: Trn cc dung mi theo t l . Thnh phn v t l cc h dung i qu nh trong cc chuyn lun. Vi c trn cc thnh ph c tin hnh trong bnh g n, sau khi l u, yn. Nu c tch lp th g n l y l h th , th ng l l i, u g i h t h bo ho gi y s s u hi h m cc ch t phn tch, cn l tr g u g i h ng. Nu dung mi khng tch lp th dng h h u g i bo ho gi y sc k. (vd: h butanol-acid acetic- c 50:1:50 sau khi trn xong s phn thnh hai lp (2 pha), mt lp s c acid bo ha butanol ( h t h), t lp s ut h ( h ng)) Chun b gi y: Gi y sc k l lo i gi c bi t v ph hp vi yu cu cho sc k, c b dy thch hp. Tu the h th c ca bnh v s ng cc vt cn ch m m ct nh ng kh gi y hnh ch nht thch hp, chiu di dc theo th gi y. Chiu rng ph i nh h hiu di ca g u g i h g h g c nh h 2,5 . B di ph i tnh sao cho khi treo gi v h h g u i ch m vo mng dung mi trong khi bo ho dung

i. Tr ng hp bnh sc k c hnh chung p th gi y ph i cun trn v c nh bng mc thy tinh hoc khu bng ch. ng knh ca cun gi y ph i nh h ng knh c gi (s u hi h h s rt u g i v ) hm trnh gi y ch v th h v s qu tr h h t h i. ii. S a so n ch t th Ch t th ph i c lo i t p ch t h t v c ha tan vi mt ng dung mi ti thiu. Du g i h c s dng trong phng th nghi m l: chloroform, methanol, acetol. iii. Ch m dung dch ln gi y K mt ng ch m nh song song vi mp gi ( ng v h ch m sc k) theo chiu rng v cch mp gi 3 i vi s i sao cho cc vt ch m khng b ngp v u g i, 6 i vi s i u g v ch ch m khng trng vi thy tinh gi y m th h 2 hi t gi y treo vo mng phn trn ca bnh.Dng ng mao qu n hay micropipet 1-10 l ch m ln nh g i h d u trn gi y, vt ch m ph i g , ng knh khng qu 5mm. Mi ln ch m ph i s y kh mi ch m tip ln 2. L ng ch m v n g dung dch ch t th qu nh trong chuyn lun. Khi ch m nhiu vt trn mt d i gi y th vt ch m n ph i cch vt ch m kia t nh t l 30 mm iv. Khai trin sc k Gi c ch m dung d h v c b h h i u g i s c nhng vo dung i h ng. Sau khi trin khai xong, lm kh gi y bng nhi t phng hoc s y 100oC trong 10-15 pht. C nhiu h g h u g khai trin sc k gi y ty thuc vo ch t cn phn tch m dng cho thch hp. Ph g Ph Kh i Trin S i : u gi y c ch t phn tch nhng vo dung mi h. Ph ng ch y ln nh l c ht mao qu n. S i u g: u gi y c ch m ch t phn tch nhng vo m ng dung mi. Ph ng ch y t trn u im Dng c gi n Thao tc nhanh C th h ng v tch cc ch t c Rf khc xa nhau Nh im Trng l c tc d g g c chiu vi l c ht mao qu n nn tc ht gi m nhanh khi ch y ln. Dng c phc t h h g h h .

C th tch cc ch t c Rf khc nhau. C th h ng

xung nh trng l c. S i g g: Gi y ct hnh vung hoc hnh ch nht t nm ngang trn thy tinh. u gi y ch m ch t phn tch nhng vo chu dung mi. Sc k hnh trn: Gi y ct hnh trn c mt b , c nhng vo chn dung mi. Ph g i t tm ra theo ngoi vng trn. Sc k hai ln: s dng hai dung mi v quay 90 gi y gi . u ti t u gi h ng xung t g t h s i xu g, n khi ch t phn tch gi a t gi y th xoay gc 90o gi a t gi y v chuyn sang h dung mi khc. v. Pht hi n cc vt

Dng c gi n c th t trong t m hoc t l nh. h g h h ng, acid amin, ch t bo.

Thao tc nhanh Vng tch nh , chnh xc Hi u g t h h h g h tr . Hi u ng tch tt C th tch cc ch t c khc nhau. Tch hn hp phc t p ca cc hp ch t; acid amin, g,

Ch g nh tnh Vi c so snh vi ch t khc m u h h . Dng c phc t h h g h h .

S u hi h i trin cc ch t c phn b trn gi y cc v tr khc nhau hnh thnh s . C th php hi n cc ch t bng mt (ch t mu), hay bng cch chiu tia UV, hay phun thuc th u h : i h ri , i i e, U IM v KHUYT IM i. u im K thut v dng c L h g h gi n,d th c hi n. ng m u phn tch nh . h t h, h g, nh t p ch t

vi h t h v

C th ng d g i qu ,.

nh nhiu ch tiu h

Sc k gi y hi h nguyn t phng x . ii. Khuyt im

g h

gi n nh t tch v pht hi n cc ch t c cha

Khng lm sc k ch ha bng sc k gi y. Khng dng cc ha ch t t h tr g h t hi n vt.

Thi gian khai trin lu t 4-5 gi (mt m u sc k gi y c th khai trin t 2-3 ngy). Gi y d h p ph cc ha ch t h tht k. tr g i tr ng nn ph i b o qu n gi y sc k

NG DNG CA SC K GIY TRONG K THUT NH TNH i. nh tnh v th tinh khit T g t h tr g sc k lp m g, th ng sc k so snh vi ch t chu nh tnh (d a vo gi tr Rf ). nh tnh mt ht g i chc chn cn tin hnh sc k vi t nh t vi h dung mi khc nhau. Ch t th c coi l tinh khit khi trn s khng c vt l . ii. Mt s v d nh tnh xylose bng s c k gi y H dung mi: BuOH-pyrimidinGi y sc k: Wh No 1 hay gi t g g.Ch m ch t chun v mu th cch ba gi y 6cm g tr t trong 100ml BuOH g , ng ch m .Vt ch m c c (6:4:2)

Thuc th : Ha 1,66 g acid phtalic v 0.93g aniline mi h Ch t chun: Ha tan xylose chun thnh dung d h 100 g/ 2 M u th : C 1 g X se h tr ng knh khng qu 3mm. g th h 10

, ng ch m 2

Khai trin: Theo k thut trn xung. Cho dung mi vo h h h i dung mi trong thi gi tr 16h. t gi v h h 4h. S u h i trin, l y ra s y kh,phun thuc th , s y 105oC/5=10 pht, c th soi UV. Tnh .

c.

Ch t h p ph p a

p a ng

d. Khi ni m

g a

HPLC l vit tc ca 4 ch i u bng ting anh c h g h sc k l ng cao p (high pressure liquid chromatography). HPLC t h g h hi t h tr g h g h t g v h t h h tr g t h t r h hi i g tiu h h t h t g h t h t g r , h t h t g i g i t h h vi h h h u . Ph g h g g s g r g ri v h i v hiu : h , h g h g tt, thch h t h h h t h h i h h h hi t. Ph g h sc k l ng cao p (HPLC): HPLC l mt sc k ct ( u hr t gr h ) i vi mt detector nh c th pht hi c cc ch t tch ra trong qu trnh ch y sc k. Vi nh ng tin b k thut v ct, ete t r hu n sc k ct th h h g h h tch c t nhanh v hi u su t cao. Lo i ny cn ph i c h th g y h ng vi p su t n kho ng 30Mpa (300 atm) nhm t o dng ch y vi u g vi / ht. S ng mu phn tch bng HPLC ch cn kho ng 20microlit. Phn lo i D v s h HPLC th h 4 i:

h u v

h t h hit s

g tr

g HPLC, g i t

hi

S S S S

h h h s g r ( s r ti / iqui hr t gr h ). h ( rtiti hr t gr h ). i (i hr t gr h ). r h t (size e usi /ge er e ti hr t gr h ).

Tr g , sc k phn b (SKPB) c ng dng nhiu nh t v c th phn tch c nh ng hp ch t t khng phn c n nh ng hp ch t r t phn c c, hp

ch t ion c khi ng phn t khng qu l (<3000). SKPB c chia thnh hai lo i d tr phn c t g i gi h t h v h ng: s h th ng SKPT (normal phase chromatography) v s h o SKP (reverse h se chromatography). Tr g SKPT, h t h s d g phn c h h g. Ph t h i ny s c i l c vi cc hp ch t phn c . SKPT g tch v phn tch cc hp ch t phn c c cao vi phn t ng khng ln lm. SKP thut ng ch mt lo i s tr g h t h t h h h g. Ph g h g h t h hp ch t t khng phn c n phn c c. Hu ht cc hp ch t h u ch carbon di (t phn c c) r t thch hp cho phn tch b g SKP. Du g i s d g tr g SKP u g i h , tr g u g i g v i tr qu trng m l i r ti . D , SKP c ng dng nhiu v ph bi h SKPT Nguyn tc c h g h :

HPLC h g h s c pht trin d tr h g h ghi sc k ct, cc ch t phn tch khi qua ct sc k do c i l c khc nhau vi h ng( dung mi) v h t h ( h t nhi trong ct) m chng ra kh i ct vi thi gian khc nhau,ch t no c i l c vi h t h h s gi trong ct u h , h t c i l c nh vi h t h s ra kh i ct cng vi h ng s h . Th v dung mi nh git qua mt ct ghi s i tc dng ca trng l c, g i t t ln dung mi p su t kho g 400 t s dch chuyn x r h h h . Ph g h h h h g t s dng cc h t h th c nh trong ct h p ph v t g mt tip xc gi h t h v h t ch t h t h i qu . iu ny s t g ng kh g h t h h t c trong hn hp. Ph t h t yu t quan trng quyt nh b n ch t ca qu trnh sc k v lai sc k . Nu h t h h t h p ph th ta c Sc k h p ph pha thun ho h o. Nu h t h h t tra i Ion th ta c S tr i ion. Nu h t h h t L ng th ta c Sc k phn b hay sc k chit . Nu h t h Ge th t Sc k Gel hay Ry phn t . Song d d ng no th h t h tr g HPLC g h i tho mn nh ng yu cu s u : Ph i tr v n v ng vi iu ki n c i tr ng sc k, khng c ph n ng ho hc ph vi dung mi r a gi i hay vi ch t h t h, m b ch ca s t h g the n ch t chnh c h t h, h h g t ch t phn tch cn tch.

 C kh g t h hn lc mt hn hp ch t tan nh t h tr g iu ki n sc k nh t nh. Tnh ch t b mt ph i h, c bi t tr g xp ca n v khng b th i hay bin d ng trong qu trnh sc k, khng b phn r i p su t cao ca qu trnh sc k. Cn b g ng ca qu trnh sc k ph i x y ra nhanh v lp l i tt. C h th mi m b thu c kt qu tch tt. C h t ph i t g i ng nh t, gh l ng knh ca cc h t ln nh t so vi cc h t nh nh t khng chnh l ch nhau qu 10% v trn 85% s h t ph i nm tr g v g h th tru g h. C h th ct tch mi c hi u qu cao. u h im c h t h:

Cng vi h t h r a r i ch t phn tch ra kh i ct ,chng ta cn c mt pha g . Nh vy nu chng ta n p m u phn tch gm hn hp ch t phn tch A,B,C .. Vo ct phn tch ,kt qu cc ch t A,B,C.. S c tch ra kh i h u s u hi i qu t .Quyt nh hi u qu ca s tch sc k tng h t gt .

Ch t khuch tn F1 F2 F3

Pha tnh

Pha

ng

Tng c 03 t gt s quyt nh ch t c r a r i ra kh i ct tr c tin khi l u gi trn ct l nh nh t ( F1) .v g c l i .i vi mi ch t ,s u gi qui nh bi 03 l F1,F2,F3 .Tr g F1 v F2 gi vai tr quyt nh .cn F3 l yu t h h ng khng ln . F1 c gi ch t phn tch trn ct .F2 l l c ko c h g i vi ch t phn tch ra kh i ct . Nh vy vi cc ch t khc nhau th F1 v F2 l khc nhau ,Kt qu l cc ch t khc nhau s di chuyn trong ct vi t khc nhau v tch ra kh i nhau khi ra kh i ct ( h h h i )

Phn minh ho qu trnh tch cc cht A v B trong ct tch sc k

Ph

ng

S g t u g i qu t i h qu . iu h vt i u tr g t v t v h t hu h .

h h i

t h h th i ti s t. Th v , h g qu t i 400 su t s g t h th h t h h r t hiu h t h t h hiu h t g t gi h qu . iu h h t h i t th h h h

thit b

2 2

3
4

Detector 8 7

H thng sc k lng cao p (HPLC)

Tr 1234567-

g :

Bnh ch u g i h ng B phn kh kh B B phn tim m u (bng tay hay autosample) Ct s ( h t h) ( g i i tr ng hay trong b iu nhi t) Detector (nhn tn hi u) H thng my tnh gn phn mm nhn tn hi u v x l d li u v iu khin h thng HPLC. 8- In d li u.

i. Bnh ng dung mi: Hi n t i HPLC th g 4 g u g i v u .Ch h chng ta s dng 4 bnh cha dung mi cng 1 l r a gi i theo t l mong mun v tng t l dung mi c 4 u g 100%. Tuy nhin theo kinh nghi m th chng ta t khi s dng ti cho h h g u c pha tr ng nh t h ,h h g trnh r a gi i nh. : - T tc u g i u g h HCLP iu ph i l dung mi tinh khit v c ghi r trn nhn l dung cho HPLC hay dung mi tinh khit phn tch. - T t c cc ha ch t u g pha m u v pha h m ph i c s dng la ha ch t tinh khit phn tch. Nhm m h tr h h ng ct sc k hay nhiu ng nn,t o ra cc oeak t p trong qu trnh phn tch. ii. B kh Degasse: - M h a b kh nhm lo i tr bt nh cn st l i tr g u g i h ng. - Nu h tr g qu tr h h t h u g i h ng cn st cc bt kh th mt s hi t g s u s x y ra: - T l h ng c ng dung mi l h g g s lm cho thi gi u c e th i. - Tr g tr ng hp bt qu nhiu b kh kh khng th lo i tr ht c th c th pump s h g ht u g i hi su t khng ln v my sc k s ngng ho t ng. Trong b t c tr ng hp no nu tr g h t qu phn tch sai. 3 v 2 g gi h qu

iii. Pump cao p: M h h ng vo ct th c hi n qu trnh chia tch sc k. Pump ph i t c p su t cao kho ng 3000-6000 PSI ho 250 t n 500 at (1at=0,98 bar) v pump ph i t o dng lien t . u ng t 0,1- 9,999 ml/pht. T th hi h g nh theo thong s y dung mi lin tc. i t.hi n t i 2 ist e

iv. B phn tim mu (injection): M h: u vo ct h t h the h g h h g gng dng ch y.C 2 cch l y m u vo trong ct; bng m u th cng (tim bng tay) v tim m u t ng (autosample). v. Ct sc k:

Th g th ng: L = 10 30 cm v c th ni tip 2 ct hoc nhiu hn ID = 4 10 mm, kch thc ht nhi: 3, 5 v 10m 40.000 60.000 a/m ct Ct tc cao v hiu qu hn L = 3 - 7 cm v c th ni tip 2 ct hoc nhiu hn ID = 1 4,6 mm, kch thc ht nhi: 3 hoc 5 m 100.000 a/m ct

Ct ch ,

h t h

c coi l tri tim ca h th g s

ng cao p.

Ct h t h th g th ng lm bng thp khng g, chiu di ct kho ng t 10-30 ng knh trong 1-10 mm,h t ch t nhi c =5-10 .( g i r t s tr g

h i t v h th v h h t)vi ch t nhi ct c =1,8-5 th g t g 3-10 v h i t hi u g t h .Ch t hi t t the i t v iu s .Th g th g h t hi t si i ge ( h thu ) h si i ge t gh u ( h ).Ng i r g i t g i h h : h t, e , h t tr i i . i vi t s h g h h t h i h i hi t h th h hi t h g th t t tr g h iu hi t (oven column). vi. Detector : Dete t r ghi tr s h t h g i t t v g g h h h t hi h t hi h g r h i t v h t hi u th h t h v h g.T the t h h t h t s g i ete t r th h h v h i th iu i tr g t h h t h t h.

A=k.C
Tr g : A t hi u . h t h ete t r h , g i th,

C: N g h t h g s th T ghi

hi u th h th qu g;C g h t i , hi t; hit su t vii. B ghi nhn tn hiu: gi h ghi h t hi u h t hi ghi

Dete t r tru s g. gi th v s , thi

Tr g th h th s g u, i t h e , hiu , Hi

, th h i iu g h h tr t h, th ut t th g s, h v th g s e h t h i g, s h gi i,..tr g qu tr h h t h g thi ,t h t th g s the u u g i s g h : g , SRD, viii. In kt qu:

S u hi h t h g u t s i t qu h h thi h s . Mu t qu t h t tt h t h i t tt h t h t h h u; iu i g : +Ph t h : L i h t h K h th +Ph C t. g, hi t

t ht r gi iu i s

g: th h h v t , H,t

ti h h i. Chun b dung dch axit amin chun:

- Dng pipet t ng rt 3,5,7,10 l dung dch axit amin chun cho vo ng nghi m v lm d n xu t h i vi dung dch m u - Ch S y kh m u t o d n xu t lo i b nh ng vt cui cng ca PITC - Cn chun b t o d n xu t mi ln phn tch - nhi t phng,cc m u c pha long ch cho kt qu nh trong vng 10 gi.Cn cc m u h h g th th gi c trong 60 gi,V vy khng u tr cc m u h h g. Nh ng m u kh c th nhiu tun trong t g. ii. X l mu (nh trnh by trn) iii. Dng c ha cht - Dung dch HCL 6M c b su g he 1% (10 he h v 1 HCL) - B h h it - Ch t t o d n xu t h Phe is thi te ( PITC) , - Triethylamine (TEA) - Ethanol lo i dng cho HPLC - N c c t ln 2 - Dng c v thit b h tr cho sc k. iv. Cch tin hnh: - Sau khi chun b xong m u, h u g i h ng vo bnh ch h ng. - u vo thit b kim tim m u - iu chnh cc thng s cho my sc k: nhi t , vn t g, c sng ca u d. - Khi ng my v tin hnh ch y s ,s u l s li u v tnh ton kt qa. v. X l kt qu: - Xem v so snh s ca chun v m u - Thng s thi gian - Di n tch peak - Chiu cao peak trn s - Nhn d ng peak v th t cc axit amin trn s - N g chun trong dung dch hn hp chun

- L ng chun t cc n g chun - Cng thc tnh kt qu : H ng mi axit amin trong m u h t h bng s mg c trong 1g m u bng cng thc: A=

c tnh

S M CV 1000 SC M

Vi: A n g axit amin i trong m u, mg/g -SM di n tch peak axit amin i trn s -SC di n tch peak axit amin i trn s -C n g axit amin chu i t h the - V tng th t h nh mc m u -M Khi 3. ng m u th c ph g phn tch axit amin sc k: ca m u ca chun ng chun

g a p g p p u v h im c h g h

u im: - C th h t h ng thi nhiu hp ch t - Khng c h i u - phn gi i cao nh qu trnh tch trn ct - nh y cao nh u d tch nh (1-100- Th tch m u phn L) Nh im: -Ph g h t hn lc do khng lo i tr ht c h h ng ca nn m u. u h i s gi : u i : -Ph g h th hi h h h g v th t gi . -C th h t h h h s g u t tr g u. -L g u s g t. -Thit h g ti . Nh i : -Mt s u t r t h h tr s gi .

u h i s g: u im chung ca SKLM -K thut gi n, d th c hi n, c th th c hi n mi phng th nghi m. -Thi gian th c hi n nhanh, ti vi gi. -Do cch th c hi gi n nn c th g SKLM kh o st cc h u g i h h g h sc k khc. -Nh hu g SKLM h ng php c tnh ng dng r t cao, thng dng nh t hi n nay. Nh im : -Thnh ph h ng d th i trong qu trnh khai trin. -Cc vt sau khai tri th ng b ui. -SKLM iu ch ch dng khi hn hp cn tch c s g t, vi tr miligam, cn nu m u nhiu, vi gam th tch bng sc k ct s kinh t h . u h i s h: u i : -L g u s g t. -Kh g t h h h th h t g u t h r t tt h t qu h h. - h h , h vi h h t h i g th . -Thit h g h t . Nh im : -M u h i hi t . -Thit ti .