Ky Thuat Sac Ky Trog Hoa Sinh

8/11/2019 Ky Thuat Sac Ky Trog Hoa Sinh

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Kthut sc kng dng trong nghin cu vha sinh hc

HVTH : Hunh Cng TiLHong Thanh Vy Trang 1

NG DNG K THUT

SC K TRONG NGHIN

CU HA SINH


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MC LC

MC LC.................................................................................................................. 1

M U.................................................................................................................... 5

NI DUNG................................................................................................................ 6

1. Tng quan v k thut sc k................................................................................. 6

1.1. Lch ssc k .................................................................................................. 6

1.2.nh ngha sc k ............................................................................................. 6

1.3. Phn loi cc phng php sc k ................................................................... 8

1.3.1. Theo bn cht vt l cc pha ..................................................................... 8

1.3.2.Theo hin tng sc k .............................................................................. 8

1.3.3.Theo kthut v phng tin sc k.......................................................... 9

2. Cc k thut sc k.............................................................................................. 11

2.1.Sc k giy ...................................................................................................... 11

2.2.Sc k lp mng ............................................................................................. 13

2.3.Sc k ct: ....................................................................................................... 17

2.4. Sc k trao i ion ......................................................................................... 19


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2.5.Sc k gel ........................................................................................................ 21

2.6. Sc k i lc ................................................................................................... 23

2.7. Sc k tng tc knc .............................................................................. 23

2.8. Sc k kh....................................................................................................... 24

2.9. Sc k lng cao p (HPLC) ........................................................................... 27

2.9.1. Sc k phn bhiu nng cao ................................................................. 28

2.9.2. Sc k hp phhiu nng cao (sc k lng - rn LSC) .......................... 28

2.9.3.Sc k trao i ion hiu nng cao (IEC) .................................................. 28

2.9.4. Sc k lng hiu nng cao trn gel (sc k loi cSEC) ....................... 29

2.10. So Snh Gia 2 KThut Sc K Lng Cao p (HPLC) v Sc K Kh

(GC) ...................................................................................................................... 29

3. ng dng .............................................................................................................. 30

3.1. ng dng trong phn tch .............................................................................. 30

3.1.1. ng dng sc k i lc trong phn tch thc phm................................. 303.2. ng dng trong thu nhn v tinh sch ........................................................... 32

3.2.1. ng dng sc k ct ................................................................................ 32

3.2.1.1.Tinh chacid amin ................................................................................ 32

3.2.1.2. ng dng trong tinh sch khng sinh................................................... 32

3.2.1.3. Phn tch glucose v fructose .............................................................. 34

3.2.1.4. Tinh sch glycerol ................................................................................ 35

3.2.1.5. Tinh sch fructose syrup....................................................................... 36

3.2.1.6. Tinh sch protein .................................................................................. 36


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3.2.1.6.Tch chit cc hp cht ru cao phn t............................................. 38

3.2.1.7.Qu trnh ti sinh nha trao i ion ....................................................... 38

3.2.1.8.Tch chit glucose tmt rng ....................................................... 38

3.3.2. ng dng sc k i lc ............................................................................ 40

3.2.2.1. Tinh sch protein .................................................................................. 41

3.2.2.2.Tinh sch interferon............................................................................... 43

3.2.2.3.Tinh sch khng th............................................................................... 44

4. Mt sng dng cthca phng php sc k ................................................ 45

4.1.ng dng sc k i lc trong tinh sch protease ............................................ 45

4.1.1.Nguyn l ................................................................................................. 45

4.1.2. Cc matrix thng dng .......................................................................... 48

4.2.3.Ligand sdng tinh sch protease trong sc k i lc ....................... 49

4.1.4. Mt sphng php cnh ligand trn matrix ..................................... 52

4.1.4.1. Cnh ligand thng qua cc tc nhn hot ha cyanogen bromide. 52

KT LUN.............................................................................................................. 56

TI LIU THAM KHO........................................................................................ 57


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M U

Cc t bo sng cha hng trm loi hp cht ha hc khc nhau. Cc hp

cht ny bao gm nhng i phn t nh protein, acid nucleic, lipid, cng nhcc hp cht c trng lng phn t nh. Nhng hp cht trn c th hin din s

lng nh, di dng vt (enzyme) hay s lng nhiu (cc protein cu to).

Ngi ta mun bit cc thnh phn ha hc ca t bo, nhm hiu r cc quy trnh

bin i cn bn ca n, qua gip cuc sng ngy cng thm tt p. Mun vy,

ngi ta phi tm cch tch cc tch ring chng xc nh cu trc ha hc.

T , k thut tch ring cc hp cht ra i vi tn gi sc k

(chromatography). Ngy nay, sc k c s dng tch tt c cc hp cht d

c mu hay khng, d trng lng phn t nh hay ln.


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NI DUNG

1. Tng quan vk thut sc k

1.1. Lch s sc k

Nm 1903, nh bc hc Nga Michael Tswett cho dung dch cc sc t thc

vt trong ete du ho ln ct nhi bt mn canxi cacbonat, ng thy cc sc t b hp

ph ln trn u ct.Khi cho ete du ho ln ct, cc sc t di chuyn trong ct t

trn xung di, mi sc t c mt tc ring, tch thnh nhng vng hay vng

mu xp chng ln nhau, hnh thnh mt h m Tvest gi l sc . ng t tn

cho phng php tch ny l sc k (Chromatography). Trong ting Hy

Lp,chroma c ngha l cht mu, graphein c ngha l vit. Tn gi ny ngy

nay vn c s dng mc d phng php ny cn c dng tch cc cht khng

mu.

n thp k 1930-1940, phng php ny c pht trinnhanh chng vi

nhiu k thut khc nhau nh sc k giy, sc k lp mng, sc k trao i ion, sck i lc.

Nm 1954, Mould D.L pht trin sc k gel tch cc hp cht mang in

tch theo trng lng phn t ca chng. n nm 1964, Moor gi l gel

permeation chromatography hay gi l sc k lc gel.

Nm 1906, sc k kh c bit n nhng n 1952, k thut ny mi pht

trin mnh m, nht l trong thp nin 1960.

Nm 1967, Horvath C. l tc gi to my sc k lng cao p.

1.2.nh ngha sck


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Sc k l mt nhm cc phng php ho l dng tch cc thnh phn ca

mt hn hp. S tch sc k c da trn s phn chia khc nhau ca cc cht

khc nhau vo hai pha lun tip xc v khng ho ln vo nhau: mt pha tnh vmt pha ng (Trong th nghim ca Tvest: pha tnh l canxi cacbonat, pha ng l

ete du ho). Pha tnh tr hon s di chuyn ca cc thnh phn trong mu. Khi cc

thnh phn ny di chuyn qua h thng sc k vi tc khc nhau, chng s c

tch khi nhau theo thi gian. Mi mt thnh phn i qua h thng trong mt

khong thi gian ring bit, gi l thi gian lu. Trong k thut sc k, hn hp

c chuyn ch trong cht lng hoc kh v cc thnh phn ca n c tch ra do

s phn b khc pha nhau ca cc cht ha tan khi chng chy qua pha tnh rnhoc lng. Nhiu k thut khc nhau c dng phn tch hp cht phc tp

da trn i tnh khc nhau ca cc cht trong mi trng ng kh hoc lng v i

vi mi trng hp ph tnh m chng di chuyn qua nh giy, gelatin hay gel

magnesium silicate,...

Sc k l phng php phn tch v tinh sch cc phn t sinh hc. Sc k

l phng php nhanh, d dng v khng nh hng n protein, y l phng

php c ngh trong nghin cu nh lng protein hay cc phn t.

Cc giai on ca qu trnh sc k:

Qu trnh sc k gm 3 giai on chnh:

a) a hn hp ln pha tnh (v d: a dung dch cc sc t ln u ct canxi

cacbonat). Cc cht c gi trn pha tnh.

b) Cho pha ng chy qua pha tnh (dung mi du ho qua ct), pha ng sko theo cc cht di chuyn trn pha tnh vi tc khc nhau, tch khi nhau v

c v tr khc nhau trn pha tnh to thnh sc k (chromatogram). Giai on

ny gi l khai trin sc k. Nu tip tc cho pha ng chy qua th cc cht c th

ln lt b ko ra ngoi pha tnh (v d: ra khi ct). l qu trnh ra gii v


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dung mi dng c v dung mi ra gii (eluent), dch hng c cui ct gi l

dch ra gii (eluate). Nu cc cht c tch trn pha tnh (sc k khai thm ta c

th ly tng phn pha tnh c mang cht (phn on bt trn ct) em chit ly cht.Nu cc cht c tch ra ngoi pha tnh (sc k ra gii) ta c th hng thu ly cc

phn on dch ra gii c cc cht cn phn tch.

c) Pht hin cc cht: Cc cht mu c th pht hin d dng, cc cht khng mu

c th pht hin bng n t ngoi hay bng cc thuc th. Trong sc k ra gii c

th pht hin cc cht khi chng i ra khi ct bng cch cho dung dch ra gii i

qua mt b phn pht hin gi l detector t sau ct.

1.3. Phn loi cc phng php sc k

1.3.1. Theo bn cht vt l cc pha

Pha ng c th l mt cht lng hay cht kh

Pha tnh c th l mt cht rn (ht xp hay bt mn) hay mt cht lng (c gi

trn mt cht mang rn).

Do , da vo bn cht cc pha ta phn bit cc phng php (trong tn ca

phng php, pha ng c nu trc pha tnh):

Sc k lng - lng (liquid - liquid chromatography LLC)

Sc k lng - rn (liquid - song chromatography LSC)

Sc k kh - lng (gas - liquid chromatography GLC)

Sc k kh - rn (gas - solid chromatography GSC)

1.3.2.Theo hin tng sc k

Sc k hp ph (absorption chromatography): pha tnh l mt cht rn c kh

nng hp ph, l cc phng php sc k lng - rn v kh - rn.


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Sc k phn b (partition chromatography): pha tnh l cht lng khng ho

ln c vi pha ng, cht lng ny c bao trn b mt ca mt cht rn gi l

gi hay cht mang v phi l cht tr, khng tham gia vo sc k. Sc k phn bbao gm sc k lng - lng v sc k kh - lng.

Sc k trao i ion (ion - exchange chromatography): pha tnh l cht nha

trao i ion chp cht cao phn t c mang nhng ion c kh nng trao i vi cc

ion cng du ca dung dch hn hp sc k).

Sc k theo loi c (size - exclusion chromatography): cn gi l sc k trn

gel. Cc phn t c ln s c lot cc phn t nh hn s c tch theo kch

thc do cc phn t nh di chuyn chm hn.

1.3.3.Theo k thut v phng tin sc k

Phng php gi pha tnh:

Sc k trn ct (column chromatography: CC) pha tnh c cha trong mt

ct bng kim loi hay thu tinh.

Sc k lp mng (thin layer chromatography: TLC): pha tnh c trng u

v gi trn mt phng ca bn thu tinh, nha hay nhm. Lp mng pha tnh

thng l: silicagel, nhm oxit, cenlulose, cht nha trao i ln v c chiu dy

khong 0,25 - 0,5mm.

Sc k giy (paper chromatography - PC) pha tnh (lng) c thm trn mt

loi giy lc c bit gi l giy sc k

Theo cch cho pha ng chy:

Sc k khai trin (development chromatography) cho pha ng kp cc cht

chy v tch trn pha tnh (Sc nm trn pha tnh).


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HVTH : Hunh Cng TiLHong Thanh Vy Tr ang 10

Sc k ra gii (elution chromatoglaphy) cho pha ng chy v kp cc cht

ln lt ra ngoi pha tnh (ra khi ct, ra khi giy).

Tc di chuyn ca mt cht, peak v hnh dng peak

Tc di chuyn ca mt cht

Tc di chuyn ca mt cht c th c c trng bi h s phn b ca n

gia hai pha hoc bi cc i lng v s lu gi ca cht trn pha tnh (thi

gian lu, th tch lu)

a. Thi gian lu v th tch lu

Thi gian lu l thi gian cn mt cht di chuyn qua ct sc k, khi ra

khi ct nh thit b detector ghi nhn tn hiu v xut hin rc trn sc (tnh t

lc bm mu n khi xut hin peak).

Tm l thi gian lu ca mt cht khng b lu gi, ngha l tc di chuyn

ca n bng tc di chuyn trung bnh ca dung mi, thi gian ny c gi l

thi gian cht. tR cng ln, cht cng b lu gi mnh v tc di chuyn ca n

cng nh.b. H s phn b

Trong : Cs v Cm l nng cht tan trong pha tnh v pha ng khi cn bng

c thit lp. Khi nng nh K ph thuc vo bn cht cc pha, cht tan v nhit

K cng ln th cht phn b cng nhiu trong pha tnh v di chuyn cng

chm.

Peak v hnh dng peaka. Hnh dng peak: Hnh dng l tng ca tc l lc i xng, nhng thc t lc

sc k ch gn i xng. Chiu rng ca tc c o 1/10 chiu cao ca peak.


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b. S ko di peak: l kt qu ca s di chuyn nhanh chm khc nhau ca cc phn

t ca cng mt cht khi i qua ct sc kh. Cc lc ra chm bao gi cng t hn.

c. Hiu lc ca ct: Hiu lc ca ct thng c o bng hai thng s: s a lthuyt N v chiu cao a l thuyt H. Ta c th coi nh ct sc kh c chia thnh

N lp hay N tng mng, mi mt lp s phn b cht tan vo hai pha t trng

thi cnbng. Nhng tng mng gi nh ny c gi l a l thuyt. Nu ct sc

kh c chiu di l L th: H = N/L. Ct c N ln l ct c hiu lc cao. 6

d. phn gii: phn gii Rs l t s khong cch gia hai peak v rng

trung bnh

ca peak.

Rs = 0,75 hai pic tch khng tt, cn xen ph nhau nhiu;

Rs = 1,0 hai pic tch kh tt, cn xen ph nhau 4%;

Rs = l,5 hai pic tch hon ton (chi xen ph 0,3%).

2. Cc k thut sc k

2.1.Sc k giy

Sc k giy l mt phng php phn tch d dng cc thnh phn ca mt

hn hp (acid amin). Trong sc k giy, pha tnh l mt t giy bng cellulose ( th

d nh t giy thm, giy lc trong phng th nghim). Cc lai giy ny c tnh i

nc nn trn thc t, pha tnh l mt lp nc (H2O) tht mng c che ph

ln trn b mt ca t giy, chnh v th sc k giy l lai sc k phn chia. Pha

ng l cht lng( c th l cht lng hay mt hn hp dung mi).

Phng php ny thng c s dng tch cc cht a nc nh amino

acid, ng,.. Trong giy, cellulose c dng si, cc si ny khi nm cnh nhau s


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to thnh mng li vi cc l rng to. Khi ly gii, dung mi di chuyn dc theo b

mt si v cc l rng s c ph y dung mi. Nh th, cc cht tan b phn tn

nhiu trong l rng khincc vt sc k to hn. Bt giy hp thu nc, nc b gili trong cu trc glucopyranose bng cu ni hydrogen, v th qu trnh sc k xy

ra theo c ch phn chia.

Hnh: Sc k giy, cc cht mu di chuyn theo dung mi ln giy vi cc

im khc nhau.

(a) Mt git hn hp (drop of mixture) t mt gc mnh giy lc, mt cnh giy

nm trong dung mi.

(b) Dung mi di chuyn ln tm giy bng lc ht mao mch, cc cht s phn btheo cc t l khc nhau Mi hp cht di chuyn vi tc phn nh kch thc

ca phn t ca n v ha tan ca n trong dung mi.


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(c) Nhng cht khc nhau s c tri ra nhng im khc bit trn bng , to

thnh mt sck .

Hin nay, sc k giy ang dn c thay th bng sc k lp mng. Do phnln cc cht hu c khng c mu, nn sc k giy s khng cho thy c v tr

ca mu cht trong qu trnh dung mi di chuyn i ln giy.

Nhc im sc k giy: Trong giy, cellulose dng nhng si di nm song

song k nhau mt cch t nhin, v mt h thng mng nh th chc chn c cc l

hng. Khi dung mi gii ly di chuyn (mang theo cc cht tan, solute) dc theo b

mt ca si, cc l rng ny s c dp ph y dung mi, v cc cht tan c dp

khuch tn vo cc l rng ny, khin cc cht tan cng lc cng to dn so vi vt

chm to mc xut pht ban u. Cn nu si cellulose c nn cht qu dng

chy s rt kh di chuyn.

2.2.Sc k lp mng

Sc k lp mng lhay cn gi l sc k phng (planar chromatography), da

ch yu vo hin tng hp thu trong pha ng l dung mi hoc hn hp ccdung mi, di chuyn ngang qua mt pha tnh l mt cht tr (th d nh: silicagel

hay oxid alumin). Pha tnh c trng thnh mt lp mng, u, ph ln nn phng

nh tm king, tm nhm hay tm plastic. Do cht hp thu c trng thnh mt

lp mng nn phng php ny c gi l sc k lp mng.

Bnh sc k: Mt chu, h, l bng thy tinh, hnh dng a dng, c np y.

Pha tnh: Mt lp mng khong 0,25 nm ca mt loi hp cht hp thu(silicagel, alumin,..) c trng thnh lp mng, u, ph ln tm king, tm

nhm, hay tm plastic. Cht hp thu trn nh gi sulphat canxi khan, tinh bt

hay mt lai polymer hu c.


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Mu cn phn tch: thng l hn hp gm nhiu cht vi phn cc khc

nhau. S dng khong 1ul dung dch mu vi nng phalong 2-5%, nh mt vi

qun chm thnh mt im gn trn pha tnh, v tr phi trn cao hn mtcht so vi mt thong ca cht lng cha trong bnh.

Hnh: Bnh sc k lp mng

Pha ng: dung mi hay hn hp 2 dung mi, di chuyn chm chm dc theo

tm lp mng, v li ko mu cht i theo n. Dung mi di chuyn cng cao nh

tnh mao qun.

Mi thnh phn chts di chuyn vi vn tc khc nhau, i pha sau mc ca

dung mi.

Vn tc di chuyn ny ph thuc vo cc lc tng tc tnh in m pha tnh

mun nu gi cc mu cht li pha tnh v ty thuc vo ha tan ca mu cht

trong dung mi.

u im:

-Ch cn mt lng rt t mu phn tch

-C th phn tch ng thi mu v cht chun i chng trong cng iu kin phn

tch.


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-Tt c cc hp cht trong mu phn tch c th c nh v trn tm sc k lp

mng.

Cc bc thc hin sc k lp mng:

-Chun b ng vi qun: ng thy tinh c ng knh trong ng nh, khang 1 -

2mm, mt u c vt nhn, di 10-20cm. S dng ng vi qun chm nhiu

lai mu dung dch khc nhau,ch cn sau mi ln s dng, ra sch vi qun bng

dung mi hu c nh aceton.

-Chun b tm bn mng: tm bn mng thng mi 20x20cm, dng ko ct

bn vi kich thccn thit, tm bn phi va bnh gii ly. Dng bt ch vch nhnt xut pht v mc tin tuyndung mi.

-Chun b dung dch mu: Mu l cht lng, c th chm trc tip mu ln bn

mng, cn mu ldung dch qu st, c th pha long mu. Vi mu l cht rn

phi ha tan trong dung mi huc ph hp, nng 2-5%. Nh mt vi qun

dung dch mu ln b mt tm sc k lp mngmt cch thn trng, trnh khng

cho lm lng b mt ca lp mng. Mi vt chm trn bn khng cha nhiu hn

12ug (10ug l ti u) mu cht.

-Sy nh dung mi bay i khi vt chm, ri nhng bn vo dung dch gii

ly

-Gii ly dung mi gii ly di chuyn ln: Sau khi bnh bo h a dung mi,

ngi ta t tmmng vo bnh khai trin cho cc vt chm mu b cnh pha

di y bnh. Cnh y ca tm lp mng ngp trong dung mi gii ly khang

0.5-1cm. Cc vt mu khng c ngp trongdung mi gii ly, v nh th dungmi s khuch tn vo trong dung mi.

-Hin hnh cc vt sau khi gii ly: Cc hp cht c mu s c nhn thy

bng mt thng, nhng phn ln cc hp cht hu c khng c mu, nn nu


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mun nhn thy cc vt, cn s dng phung php vt l (Pht hin bng tia t

ngoi UV: n chiu tia UV 254nm nh sng nynhn ra cc hp cht c th hp

thu tia UV, cc hp cht c mu ti sm trn nn sng; n chiutia UV 366nm nhsng ny pht hin nhgn hp cht c pht hunhquang, cc vt sm ca chtmu

c mu sng trn nn bn mng sm mu) hay dng phng php ha hc (Bng

cch dung thuc th hin hnh nh hi iod, 2,7-fluorescein pht hin a s hp cht

hu c, ninhydrin pht hin aminoacid hay amin, 2,4-dinitrophenylhydrazin pht

hin aldehyde hay caton, clorur antimony pht hin steroid hay vitamin hay

carotenoid,)

- 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.


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Hnh: Cc bc ca qu trnh sc k lp mng

2.3.Sc k ct:

Sc k ct c tin hnh iu kin p sut kh quyn. Pha tnh l nhng ht

c kch thc tng i ln (50-150um), c np trong ct thy tinh. Mu cht

cn phn tch c t trn u ct, pha trn phatnh (c mt lp thy tinh che ch

lp mt khng b xo trn), bnh cha dung mi gii ly c t phi trn cao.

Dung mi gii ly ra khi ct phn bn di ct c hng vo nhng l nh

ngay ng dn ra ca ct. Phng php ny thng lm cho qutrnh tch b chm,

hiu qu thp so vi sc k lng cao p (HPLC). Tuy vy, sc k ct cng c u

im l pha tnh v cc dng c r tin, d kim, c th trin khai vi mt lng

mu tng i ln.

Cc bc thc hin sc k ct:

-La chn cht hp thu :pha tnh l silicagel loi thng, hp cht khng phn

cc c gii ly khi ct trc, hp cht phn cc c gii ly sau. V 2 phn t


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khng phn cc, phn t na c trng lung phn t ln s c tnh phn cc mnh

hn phn t kia, n b pha tnh gili trong ct nn di chuyn ra khi ct chm hn

so vi cc phn t nh, v cng c khi n li lu hn trong ct so vi phn t tuyc tnh phn cc.

- La chn dung mi:Mu cn sc k uc ho tan hon ton trong dung mi

ph hp vi nng 10mg/ml, gi l dung dch mu (A). Chun b 4-6 tm bn

mng 2,5x10cm. Chm ln nhng tm bn ny mi tm khong 2 -5ul dd(A). Mi

bn mng c trin khai vi mt loi dung mi gii ly khc nhau, k pht hin

bng n UV hay thuc th. Vi n dung mi s d dng thy c dung mi no

ph hp. T kt qu , c gng tm mt hn hp dung mi, trong mt dung mi

phn cc v mt dung mi km phn cc th d nh ete du ha: etyl acetate.

-Np cht hp thu dng kh vo ct: dng kp gi cho ct thng ng trn gi,

cho dung mi loi km phn cc nht vo khong 2/3 chiu cao ct. Cho cht hp

thu dng kh vo thng trong ct, u n, mi ln mt lng nh, va cho va kh

nh vo thnh ct. Khi lp cht hp thut c chiu cao khong 2cm trong ct,

th m nhkho bn di ct cho dung mi chy ra, hng vo mt bechertrng bn di ct, dung mi ny d c rt li ln u ct. Sau khi np xong,

cho dung mi chy qua cht hp thu vi ln n khi cht hp thu trong ct ng

nht.

-Np mu: Mu dng lng cho trc tip ln u ct sc k. Nu mu dng

rn th ho tan mu cht vo trong mt lng nh dung mi, loi dung mi cho khi

u sc k. Thc hin np mu ln ct nh sau:

+M kho cho dung mi chy ra khi ct h mc dung mi trong ct

xung sao cho va stvi mt thong ca cht hp thu trong ct.

+ng kho li, np dung dch mu vo u ct. Mun np mu, s dng mt

pipet ht dung dch mu cht, t u pipet gn st mt thong cu cht hp thu


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trong ct, va bp va ry pipetdc quanh thnh ct cho dung dch chy ra theo

thnh trong ca ct, chm xung b mt cht hp thu.

+M kho bn di cho dung mi chy ra khi ct, lm cho dung dch muc thm ht vo cht hp thu trn u ct, cn canh chng khng cho cht hp

thu u ct b kh.

+Dng pipet cho mt lung nh dung mi mi ln u ct, ng thi dng

dung mi ny rasch ng m dung dch dnh trn thnh ct.

+M kho cho dung mi chy ra. Lp la vi ln gip cho dung dch mu thm

su vo cht hpthu, dung mi trong sut khng ly mu ca cht mu.+S dng bng thy tinh, bng gn, ct hay giy lc t nh ln mt thong

cht hp thu bo v mt ct.

+Cho dung mi vo y ct tin hnh gii ly trn ct.

2.4. Sc k trao i ion

S trao i ion l s gn kt c tnh cht thun nghch gia cc phn t cmang in tch. Trong sc k ct nhi, pha tnh R c gn thm nhm chc G mang

in tch. Gi s cho i ngang qua ct mt hn hp mu cht ban u c cha nhiu

lai cht tan (solute) khc nhau, cht tan no c mang in tch ngc du vi in

tch ca nhm chc, th d cht tan S, cht tan d ui i ion C ra th ch vo,

gn vo pha tnh, v nh th cht tan s b gi li trong ct, trong khi nhng cht

khc ca hn hp mu cht ban u s khng b gi li nn i ra khi ct. k thut

ny tch ring c hp cht S ra khi hn hp ban u.

RG- + S+ ------ RG-S+ + C+

Hoc RG+ +S- --- RG+S- + C-


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cht ra khi nha thng, cn phigia tng lc ion ca dung dch m bng cch

cho thm NaCl. Khi c thm NaCl trong dung dch m, ion ca dung dch m s

cnh tranh vi hn hp mu cht, ginh ly nhng nhm chc hat ng cunha, y hp cht ra khi nha, ri theo dng chy i ra khi ct.

Trong phng php sc k trao i ion, pha tnh l nhng ht mang sn mt

in tch nht nh, nhng ht ny s tng tc vi cc phn t (protein) mang

in tch tri du vi chng. C th, nu ht mang in m (nh ct

carboxymethyl-cellulose (CM-cellulose)), tintrnh c gi l sc k trao i ion

dng, th s tng tc vi nhng phn t mang in tch dng. Ngc li, nu

ht mang in tch dng (nh ct diethylaminoethyl-cellulose (DEAE-cellulose)),

gi l sc k trao i ion m, th tng tc vi phn t mang in tch m. V th,

nhng protein cng du vi ct s chy ra khi ct trong khi nhng protein tri du

b gi li ct.

phng thch nhng protein ny, ta tng nng ion ca pha ng, nhng

ion ny s th phnt protein tng tc vi cc ht mang in tch. V d, trong sc

k trao i ion dng, ta thm mui natri clorua hay mui khc trong dung dchtch gii bi v ion natri s tranh bm vo ct vi cc protein c in tch dng, do

, nhng protein mang in tch dng c phng thchra ngoi ct ln lt theo

ln v in tch.

2.5.Sc k gel

Trong sc k gel, pha tnh l mng polymer c l rng v cc l rng ny

c ph y dung mi dng lm pha ng. Mt hn hp gm nhiu hp cht c

trng lng phn t khc nhau c th tch ring c hay khng l tu vo kch

thc l rng. Cc phn t c kch thc ln hn l rng, khng th chui lt vo

bn trong l rng, nhanh chng theo dng chy ca pha ng i ra khi ct. cc


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ng dng: k thut ny dng tch cc i phn t c ngun gc sinh hc

nh: protein, polysaccharide, acid nuleic, enzyme,.. Ngi ta ng dng vo vic

an TLPT ca mt hp cht cha bit.

2.6. Sc k i lc

Sc k i lc hp th pht hin cc i lc sinh hc m mt phn t sinh hc c

vi mt phn t khc c c nh trn mt pha n nh. C nhiu phng php

ra gii cc phn t i lc v c nhiu loi sc k i lc khc nhau, cc bc theo

gradient vn thng c s dng. Thng s dng trn cc h thng c p sut

thp. Cc gi th (thng l agarose) thng chu c p sut di 50psi.

Nhc im: t tin, kh ty ra,chn lc ligand (phi t), gn kt ligand

ln gi th hot ha thng gp nhiu kh khn.

u im: pht trin phng php tinh sch trn protein A (protein A l

antigen cho IgG), bc bt gi tt, cho php phn tch protein dng nguyn thy

cn hat tnhvi dng bin tnh.

S dng sc k i lc cho tng tc gia khng nguyn khng th: ta c th

tinh sch mt khng nguyn bng cch cho mu i qua ct c cha gi th lin kt

vi khng th thu nhn protein. Ngc li, mt gi th c lin kt vi mt khng

nguyn chuyn bit s bt gi khng th chuyn bit cho khng nguyn . ng

dng nhiu nht l gn kt protein A vo gel gn kt vi cc globulin min dch.

2.7. Sc k tng tc k nc

Sc k tng tc k nc l phung php b sung kh tt cho cc k thut

phn tch khc. Trong nhng nm gn y, phng php ny tr nn thng dng

nh l bc u tin trong qu trnh sc k.


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Loi ht Macro-Prep c dn xut l nhm k nc, c th l gc methyl hay t-

butyl. Cc gc ny hp dn cc gc k nckhc c trn b mt protein. Khi cho

mu protein vo trong iu kin nng mui cao p cc phn k nc li gnnhau v nh vi nhau. Qu trnh ra gii c thc hin bng cch gim dn nng

mui cho n khi cc phn k nc i li vo trong dung dch.

c im: Phn tch cc phn t theo tnh k nc ca phn t . Cc nhm k

nc khc nhau c c inh trn b mt gi th. Di iu kin nng mui cao

(thp) ccphn k nc trn

phn t tng tc cc nhm c nh. Cc phn t gn kt c ragii bng

cch gim p lc ion v v vy ha tan cc phn t ra khi gi th.

u im: L bc cho php c mu, cng sut cao, nhanh, mu c thu nhn

trong nng mui thp.

Nhc im: mt s protein c th b ta nng mui cao, kh nng cp

quy m ln.

2.8. Sc k kh

Sc k kh l phng php c dng tch cc cht th kh bay hi, vi

pha ng l cht kh, gi l kh mang (carrier gas). Sc k kh cn p dng cho cc

cht kh, lng, rn d bay hi v bn nhit cao, c TLPT M


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chng ct phn on nhng tch c kh n gin bng sc k kh, cngnh s

dng tch nhng hn hp rt phc tp nh kh thi t cha trn 300 hp cht.

Vi vic ra i ca nhiu loi detector nhiu phng php mi xut hin nh: sck kh - khi ph (GS - MS), sc k kh - hng ngoi (GC - IR)... lm tng kh

nng phn tch ca sc k kh.

Hnh: S ca mt my sc k kh

1. Kh mang

Kh mang l mt kh tr nh nh, hen, ngon, hidro... trong hai l kh mang

ph bin nht. Kh c cha trong bom kh c gn van gim p v iu chnh.Kh

mang cn c tinh khit cao v phi khng tng tc vi mu, ch mang mu i

qua ct: Tn hiu etect c ph thuc vo s khc nhau v tnh cht gia kh mang

vcht cn phn tch.

2. B bm mu

Thng dng bm tim bm trc tip mu qua mt vch polime silicon

chu nhit cao vo ct hoc vo phng un nng mu mu lng c th bay hi


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nhanh chng. Yu cu cn 15 thit l mu phi c p sut hi c th c lm

bay hi trong phng mu. Nhit phng mu c th cao hn nhit trongct

mt cht qu trnh bay hi c thc hin d dng.3. Ct sc k

Ct c t trong l c th iu chnh nhit trong qu trnh thc hin. Ct

thng dng nht trong phn tch l nhng ng bng thp khng r (ng, thp) hoc

bng thu tinh di t 1 n 10 m v c ng knh t 2 n 4 mm. Chng c un

cun trn cho khp vi phng l. Ct c nhi bng nhng phn t rn hot ng

nh mt pha tnh (GSC): l nhng ht nh xp gn trn mt trong ct hoc pha

tnh c tm trn cc ht nh . Trong sc k kh lng (GLC), pha tnh c gi

trn cht mang, l nhng ht cht rn nh, bn nhit, tr v mt ho hc, c l c

1 - 5 m, b mt ring ln t 1 - 10 m2/g. Pha tnh phi chu nhit, ho lng nhit

phn tch, tr v ho hc. Thng hay s dng cc polyme xp hoc gel

aluminosilicat kh nc.

4. Detector

Yu cu v detector rt nghim ngt: Mi hp phn c trong 0,1 l mu phi

c pht hin mc 1% cho nn phi nhanh chng pht hin c 0,002 l (c

khi lng c 10-6g) mu. Cc detector hin nay o c nhng lng nh hn

n my bc. Hin nay trong phng php GC ngi ta s dng nhng loi detector

sau:

Detector dn nhit (TCD): Vic vn hnh ca detector da trn s cn bng

nhit ca mt dy dn nung nng. Khi c cht cn phn tch i qua th dn inca hn hp kh s thp i, dy s nng ln, xut hin mt tn hiu in. Detector

dn nhit thch hp vi nhiu cht cn phn tch.


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Detector ion ho ngn la (FID): Khi t chy cc hp cht hu c trong ngn

la, chng s to ra cc ion. Cc in cc gn ngn la s pht hin ra s c mt

ca cc ion bng s xut hin mt dng in nh chy qua mch in. C th oc nhng dng ngay c khi chng ch xp xi bng 10-12

2.9. Sc k lng cao p(HPLC)

- p dng cho phn tch mu dng lng, rn c TLPT M>2000. - Cc thnh phn

chnh ca mysc k lng hiu nng cao

Bnh cha dung mi

Thng lm bng thu tinh, i khi lm bng thp khng r, trong phng php ra

gii thng thng ch cn mt bnh cha dung mi, trong phng php ra gii

gradient thng dng 2, 3, 4 bnh cha cc dung mi khc nhau v h dung mi ra

gii l hn hp ca cc loi dung mi trn trn ln vi nhau theo ti l c xc

nh. Cn loi cc ht v cc kh ho tan trong dung mi.

H thng bm

Bm dng trong phng php phi to c p sut cao (3000 - 6000 psi hay

khong 250 - 500at), lu lng bm khong 0,1 n 10 ml/ph, phi tr vi cc

dung mi. H thng bm ny phi c kh nng bm pha ng qua ct p sut cao

m khng b ngt qung hoc to nhp sng c th lm sai lch cc lc thu c. C

nhiu kiu bm dng trong cc my HPLC; nhng kiu phng xoay chiu hin

nay c dng ph bin nht.

H bm mu (h tim mu)

Mu c bm vo ct nh h thng van mu. Ngi ta bm mu vo vng mu

vi th tch thng l t 10 n 20 l.


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Pha tnh rn cha cc nha trao i ln di dng bt mn, pha ng thng l

dung dch nc.

2.9.4. Sc k lng hiu nng cao trn gel (sc k loi c SEC)

Phng php ny c ng dng ch yu cho cc cht c phn t lng ln. Cht

nhi cho SECl nhng ht xp ca silicagent hay cc polyme c kch thc nh (

10 m). Pha tnh l dung mi nm trong cc l xp ca ht, pha ng l dung mi

chy gia cc ht. Ch nhng phn t nh mi khuch tn vo lp xp, khi ra gii

cc phn t s ln lt ra theo ct ln n nh.

2.10. So Snh Gia 2 K Thut Sc K Lng Cao p (HPLC) v Sc K Kh

(GC)

HPLC GC

bay hi Khng yu cu bay hi,

mu phi tan c trong

pha ng

Mu phi bay hi c

phn cc Tch c c 2 loi hp

cht phn cc v khng

phn cc

Mu phn cc v khng

phn cc

bn nhit Php phn tch c thc

hin ti nhit thp(nhit phng hay thp

hn)

Mu buc phi tn ti

nhit cao (nhit tchca ct v bung tim

mu)


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Khi lng phn t Khng c gii hn trn v

mt l thuyt, trn thc t

ho tan l gii hn

c trng < 500 amu

Chun b mu Mu buc phi lc, mu

nn c dung mi ho tan

nh pha ng

Dung mi phi bay hi v

c nhit si thp hn

cc chtphn tch.

Lng mu Lng mu ph thuc vo

ng knh (trong) ca

ct

Thng t 1 5 l

C ch tch Thc hin c 2 pha

ng v tnh

Ch c pha ng l mang

mu

Detecteru d Thng dng nht l UV-

VIS

Thng dng l FID, dng

cho phn tch cc cht hu

cKthut sc k ct c sdng quy m thng mi cho vic tinh sch cc

sn phm ln men, nguyn liu sinh hc v ha hc hu c. Mt vi trong snhng

ng dng ca kthut sc k ct v ang c p dng quy m cng nghip

vi nng sut cao nh: trch ly ng tmt r, phn tch glucose v fructose, phn

tch cc ru bc cao, phn tch cc acid amin.

3. ng dng

3.1. ng dng trong phn tch

3.1.1.ng dng sc k i lc trong phn tchthc phm


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Phn tch c t

Sc k lp mng: (Thin Layer ChromatographyTCL).

Phng php ny da trn s hp ph khc nhau ca cc ochratoxin trn lpsilicagel (pha tnh) v khnng ha tan ca chng trong hdung mi (pha ng).

Cc ochratoxin ln lt c tch trong qu trnh hdung mi di chuyn theo lc

mao dn. trn bn silicagel. Silicagel c pht ln mt bn mng (nhm hoc

nha) c kch thc thng l 20 cm x 20 cm, dch chit sau khi c c sc ha

vo hn hp dung mi benzen: acetonitril. Chm khong 10 50 l dung dch

ochratoxin ha tan ln bn silicagel, tin hnh song song vi chun bit trc

nng . Sau khi sc k, sy kh bn, soi di n hunh quang bc sng 333nm. Shin din ca ochratoxin trong mu c xc nh bng cch so snh da

vo vtr so vi vt hunh quang chun. Snh tnh bng mt cho kt qutng

i. Dng my o mt pht hunh quang (fluorodensimeter) thay mt thng

gim sai s.

Sc k lng cao p: (High Performance Liquid ChromatographyHPLC)

Kthut HPLC nhanh, nhy, cho kt quphn tch chnh xc hn TCL. Gm 2 giai

on: Giai on tch cc cht phn tch: gm pha tnh v pha ng. Pha tnh thng

dng ct pha o, cc cht dng nhi trong ct sc k c tnh phn cc thp

(thng dng l hp cht silicagel c gn cc chui alkyl tC8 n C18). Pha ng

l hn hp cc dung mi c phn cc khc nhau, qua ct vi vn tc dng 110

ml/pht nh h thng bm cao p. Thng s dng h dung mi gm nc,

methanol, acetonitril. Giai on phn tch cc cht sau khi ra khi ct: Da vo tnh

cht ca cc cht cn phn tch, mt thit bthch hp gi l u d (detector) s

c ni trc tip vo u ra ca ct sc k pht hin tng cht phn gii ra khi

ct. u d hunh quang thng c s dng bc sng kch thch 333 nm,

pht xbc sng 460 nm.Vic nh tnh hon ton da vo thi gian lu ca cc

cht phn tch v chun. Sau khi xc nh thi gian ra khi ct cc cht cn phn


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tch l ochratoxin, da vo din tch ca mu so vi din tch ca chun bit

trc nng nh lng ochratoxin c trong mu cn phn tch.

Phn tch vi sinh vtSc k i lc min dch

3.2. ng dng trong thu nhn v tinh sch

3.2.1. ng dng sc k ct

3.2.1.1.Tinh ch acid amin

Ngy nay, nhng hn hp acid amin phc tp thu c tqu trnh ln men hay

thy phn protein c tch chit v tinh ch bng nhng phng php ra gii

chuyn dng sau khi acid amin c hp phln trn nha trao i ion.

Nhng loi nha trao i ion tiu chun thng c sdng l Dowex HGR

hay Duolite C-25. Tuy nhin, hiu quphn tch acid amin ca cc loi nha trao

i ion truyn thng sc tng cng khi kt hp vi cc loi nha acrylic (69).

V dung dch to ra ng lc tt nht cho qu trnh phn tch l dung d ch ngcarboxylate.

Do skhc nhau vtnh nng sinh hc hay gi trdinh dng khc nhau m yu

cu cn phi phn tch 2 dng ng phn D v L ca cc acid amin l rt cn thit,

c bit l trong cng nghsn xut 2 loi acid amin thit yu l L-phenylalanin v

L-tryptophan th qu trnh phn tch chng ra khi dng ng phn D l rt quan

trng. Qu trnh phn tch hai dng ng phn dng D v dng L ca cc loi acid

amin c thc thc hin vi dn xut ng ca nha trao i ion iminodiacetate.

3.2.1.2. ng dng trong tinh sch khng sinh


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Mt trong nhng ng dng quan trng ca k thut sc k ct l vo khu tinh

sch trong cng nghip sn xut khng sinh. Mt trong s nh phn tch khng

sinh neomycin B ra khi neomycin C bng nhm -xt (72). Cc loi nha c sdng trong tinh sch khng sinh bng sc k ct nh: Nha anion ho nc

Sephadex A25, nha base yuAmberlite IRA 68.

Sc k ct trao i ion vi pha tnh l Amberlite IRA 93 v sc k ct lc gel

Sephadex G-50 c p dng thnh cng trong vic phn tch khng sinh

Aureomycinmt loi khng sinh chng li cc khi u. Trong qu trnh ny, t64

lt canh trng sau ln men s tinh sch v thu c khong 430 mg ch phm

khng sinh ng kh.Ngoi ra, sc k ct hp phsdng silicagel cng c sdng trong tinh

sch carminomycincng l mt trong cc loi khng sinh chng li khi u. pha

ng c s dng trong trng hp ny l hn hp dung dch chloroform v

methanol vi tl173. Sn phm thu c c tinh sch > 90%.

Mt loi khng sinh khc l Cephalosporin c sinh ra tqu trnh ln men

bi vi khun Streptomycessp cng c tinh sch bng sc k ct s dng pha

ng l dung dch chloride form chy qua pha tnh l nha QAE Sephadex.

Mt ng dng quan trng khc khc ca k thut sc k ct trong cng nghip

khng sinh l tch chit cc loi khng sinh chng vim saikosaponin a, c, v d ra

khi hn hp th. vi phng php ny th t10 g saponin th c ththu c 403

mg saikosaponin c, 1210 mg saikosaponin a v 1604 mg saikosaponin d


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3.2.1.3. Phn tch glucose v fructose

Trong cng nghip sn xut ng, k thut sc k cng gimt vai tr quan

trng trong vic tch chit thu sn phm ng n thn hp syrup glucose

fructose (thu c tqu trnh nghch o ng sucrose hay thy phn tinh bt).

Quc gia ng dng sc k quy m cng nghip trong vic phn tch glucose

v fructose l Mtrong ngnh sn xut ng glucose v fructose tHFCS (high

fructose corn syrup).

Nhn li lch s th qu trnh phn tch fructose ra khi glucose c khi

xng bi LeFevre v Serbia nm 1962, lc ny pha tnh c s dng trong h

thng sc k l nha ion dng vi 4% lin kt ngang ca Ca, Ba, Sr, v Ag.

Nguyn tc l do glucose v cc oligosacharide khng to c lin kt mnh vi

nha ion trong ct sc k nh fructose nn khi cho hn hp chy qua ct sc k th

ta sln lt thu c l glucose, higher saccharide, hn hp glucosefructose v

cui cng l fructose tinh khit.

Trong mt nghin cu ca mnh, Ghim v Chang (81) tin hnh kho st nh

hng ca in tch nha trao i ion n qu trnh tch chit glucose v fructose.

Kt qunghin cu thu c l trnh tthu nhn sn phm cui ct sc k l khc

nhau khi sdng cc loipha tnh khc nhau, cthl sthu c fructose trc

ri sau ti glucose khi sdng nha trao i ion m v ngc li.


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iu cng cho thy l thc ttrong vic phn tch fructose ra khi glucose th

ta c thsdng c2 loi nha trao i ion dng ln ion m, tuy nhin trong cng

nghip vn thin vsdng nha ion dng hn l do vc bn th n bn hn vrtin hn so vi nha ion m.

3.2.1.4. Tinh sch glycerol

Mt trong nhng qu trnh phn tch sc k c ng dng quy m cng

nghip u tin l qu trnh tinh sch glycerol. Trong qu trnh ny, nhit

dung dch mu c nng ln 80oC nhm y nhanh qu trnh phn tch glycerol ra

khi mui v dung dch glrcerol ngm nc c nht cao sgy kh khn cho qu

trnh chy sc k.

V d: biu phn tch ca dung dch 10% NaCl v 36,5% glycerine vi nha

Dowex 50x8


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3.2.1.5. Tinh sch fructose syrup

Qu trnh loi b35% oligosachharide ra khi fructose syrup l cn thit v s

c mt ca oligosaccharide ny slm gim ngt ca syrup hoc cn trenzyme

ng phn ha glucose thnh fructose. Qu trnh loi boligosaccharide ny c

din tbi 2 nh nghin cu l Hirota v Shioda. H sdng nha cation mnh

di dng Ca thu c dng fructose cha n 97% fructose ha tan v cha

n 2% oligosaccharide, v dng glucose c tinh khit 7989% cha 9 20%

l oligosaccharide, v dng oligosaccharide cha 6476% oligosaccharide ha tan.

3.2.1.6. Tinh sch protein


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Kelley v cng s (121) kho st ng dng ca qu trnh sc k gel trong

cng nghip tinh sch protein. Trong nghin th nghim ny, Kelly ch s dng

nhng ct sc k c dung tch >4L. 7 mu u tin sdng cc loi gel mm chunn nh Saphadex G-200 v Ultrogel AcA34. 3 mu tip theo sdng cc loi gel

chu nn km hn nh: Sepharose 4B, Sephadex G-75 v G-50. Kt qucho thy l

nng sut tinh sch trong trng hp 2 thu c cao hn 4 ln so vi trng hp

thnht. Qu trnh tinh sch cc loi protein ti thp bng kthut sc k cho

thy tnh u vit trong vic bo ton cu trc b3 cng nh hot tnh sinh hc ca

cc loi protein.

K thut sc k ct vi pha tnh l cc loi nha khng hot ng chc nngcng cho thy kh nng ng dng trong vic tch chit v tinh sch cc

polypeptide (133). Cc nh nghin cu xc nh cthnhng loi acid v base

ra gii thu c hiu qucao trong qu trnh tinh sch cc di- v tripeptide vi

tinh khit cao nht.

Mt v dvqu trnh phn tch hn hp enkephain peptide (mt loi peptide

chc nng iu ha sgim au ca c th):


38/57



3.2.1.6.Tch chit cc hp cht ru cao phn t

Kthut sc k ct cng c p dng quy m cng nghip trong vic tch

chit, tinh sch ng n v ru cao phn ttnguyn liu lignin cellulose nh

g(147,148). in hnh l qu trnh phn tch xylitol v sorbitol ra khi galactitol,

mannitol v arabinitol sdng nha trao i ion di dng Calci, v sau mt

loi nha cation Nhm sc sdng phn tch xylitol ra khi sorbitol.

3.2.1.7.Qu trnh ti sinh nha trao i ion

Qu trnh ti sinh nha trao i ion sthi ra dung dch c cha mui bra tritct sc k v cc loi acid hay base c sdng cho vic ra gii. Sau cc

loi mui ny li sc tch ra khi hn hp trn bng phng php ra gii ion.

Mt ct sc k nhi vi mt loi nha trao i ion c bit sc sdng cho qu

trnh ny. Qu trnh phc hi cht ti sinh ny i hi ngun chi ph u t cho ct

sc k, loi nha sdng v qu trnh vn hnh, kim sot. Qu trnh i hi lp

t mt hthng quy m ln, cho nng sut ra gii cao nhng phi hp l vmt

kinh t.

3.2.1.8.Tch chit glucose t mt r ng

Mt rl mt phphm trong qu trnh sn xut ng tinh luyn tma hay c

ci ng. Hm lng ng cn li trong mt rthng nm trong khong t30

60% (tnh theo tng cht rn ha tan). Qu trnh xl sc k ct trao i ion loi

b cc loi mui trc qu trnh kt tinh c th gip gim s tht thot ng

sucrose vo mt rny.

Khi mt rc cho chy qua ct sc k, cc ht trng thi ion ha cao c

gii phng ra khi cc ht nha trong khi cc phn t ng khng tch in s

c hp ph chn lc ln b mt hot ng ca cc ht nha trao i ion. Qu


39/57



trnh ny sgip phn tch ng ra khi cc hp chp khng phi ng c ngun

gc v c ln hu c c trong mt r. Mt rtma v tcci ng c cha cc

tp cht hu c khng phi ng khc nhau, mt rtma cha t cc acid amin vc nhiu cc cu tmu sinh ra t s thoi bin ng khhn mt r t cci

ng.

Chai loi mt rc ngun gc tma hay cci ng u c thc tinh

sch vi cng hthng sc k ct sdng Dowex 50WX4 di dng ion K+. Trong

trng hp sdng h thng sc k ny th 50% sucrose trong mt cci ng

c thu hi vi tinh sch 80%, v 65% sucrose trong mt rtma c thu hi

v tinh sch 68%. Vi tinh sch ny th ng sucrose c thc ngcquay li giai on trung gian cho qu trnh kt tinh. Nghin cu ca Takahashi v

Takikawa (153) chra rng c thkt hp qu trnh ra gii ion v kthut hon

lu trong nhng iu kin ti thch c ththu c hiu sut tinh sch 90% cho mt

rtcci ng.

Nhng phn tch kthut (155) vqu trnh ui ion thu hi sucrose tmt r

cci ng chra rng, vi cng mt chi ph u t cho vic thu hi sucrose t

mt rth qu trnh sc k ny mang tnh hiu qukinh tcao nht.

Ngoi ra , kthut sc k cn c sdng tch cc sn phm phc trong

mt rnh: ng betaine, inositol, raffinose, v adenosine. C th sdng nha

cation Ca thu hi 70% inositol, sdng nha cation Na tch chit raffinose,

v c ththu hi 66% - 86% ng raffinose c tinh sch 91% - 93% ty thuc

vo tlsucrose/raffinose c trong mt.

Mt trong nhng nghin cu gn y nht vng dng ca kthut sc k trong

cng nghip xl mt rl ca Neuzil v Fergin (162), h p dng kthut sc

k lin tc UOP thu hi sucrose t mt r. Trong phng php ny, h s

dng cht hp phkhng hot ng chc nng v dung mi ra tch l alcohol. T


40/57



dung dch rc tl30% sucrose, 10% KCl v 10% betaine, khi p dng kthut

ny c ththu c 90% sucrose vi tinh khit ln n 99%.

3.3.2. ng dng sc k i lc

Ban u, kthut sc k i lc c pht minh nhm mc ch tch chit v tinh

sch protein. V c ng dng kh thnh cng trong lnh vc ny (Bng 1).

Tuy nhin, ng dng ca kthut sc k ny khng bgii hn chtrong lnh vc

tinh sch protein, m n cn mang li hiu qu tt trong qu trnh tinh sch

carbohydrate v glycoprotein vi s sdng lectin cnh. (Bng 2). Nhng th

thhormone dng trong tinh sch hormone hay nhng khng thn dng c s

dng trong tinh sch khng nguyn. Nhng c quan t ca t bo v thm ch l

ton btbo cng c tinh sch nhvo kthut sc k i lc.

Bng 1: Proteins Purified Using Affinity Chromatography (2)

Proteins Purified Ligand

Succinic thiokinase Coenzyme A

Phosphofructokinase Adenosine monophosphate

Transcobalamin I and II Coenzyme B12

Methylmalonyl - CoA mutase Coenzyme B12

Luciferase Flavin

Dihydrofolate reductase Folate

Tyrosine aminotransferase Pyridoxal phosphate

Aspnrtate aminotransferase Pyridoxal phosphate

Glutamate oxaloacetate transaminase Pyridoxal phosphate

Aldolase Nucleotide phosphates

Citrate synthetase Nucleotide phosphates

ribunuclease reductase Nucleotide phosphates


41/57



Sialyltransferase Nucleotide phosphates

Galactosyltransferase Nucleotide phosphates

Glycogen synthetase Nucleotide phosphates

Bng 2: Lectin-Agarose Affinity Chromatography Purifications of Glycoproteins

(2)

Lectin Ligand Glycoprotein Purified

Concanavalin A - AntitrypsinHerpes-specific membrane glycoproteins

Horseradish peroxidase

Human alkaline phosphatase

Interferon

Immunoglobulin A

Receptors for insulin

Receptors for epidermal growth factor

Porcine enteropeptidase

Rat brain glycopeptides

Thyrotropin

Ricinus communis - Fetoprotein

Wheat germ agglutin Erythropoietin

Glycophorin A

Receptors for insulin

Receptors for somatomed

3.2.2.1.Tinh sch protein


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Wickerhauser v cng sca ng (70) sdng heparin Sepharose tin

hnh tinh sch antithrombin III thuyt thanh ngi. Chui qu trnh tinh sch ny

c th hin trong hnh sau: (5.36)Plasma Cohn Fraction IV - 1

Cryosupernatant +20% polyethylen glycol

(final concentration)

+20% polyethylen glycol Batch adsorption with

(final concentration) heparin-Sepharose(5-8 g Fraction IV-1 to 1 ml gel)

Batch adsorption with

heparin-Sepharose

(45 ml plasma to 1 ml gel)

Wash with 12.5 liters of

0.15 and 0.5 M NaCl

Elute with 12.5 liters of 2 M NaCl

Concentrate on Pellicon cassette system to 500 ml

Pasteurize with addition of citrate to 0.5 M pH 7.55, 10 h 60oC

Desalt on Sephadex G-50 2.5 liter column

750ml solution

Sterile filter

Lyophilize

Fisher v Newsholme (72) pht trin mt quy trnh kt hp gia sra tch

enzyme adenosine kinase ththng sc k lc gel vo shp phenzyme ln trn

5 AMPSepharose 4B trong ct sc k i lc.


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Dung dch m c s dng trong sut qu trnh tinh sch bao gm 4mM

Na2H2PO4, 2mM EDTA v 5% glycerol pH 7.0 v nhit 4oC. Enzyme

adenosine kinase sc ra tch ra khi ct sc k v thu c dung dch mcha 0.6mM enzyme.

Qu trnh sc k i lc trn calmodulin cnh c xem l phng php tinh

sch rt nhanh seminalplasmin ca b n tinh sch 99%. Qu trnh ra gii

seminalplasmin khi calmodulin cnh i hi cn phi c mt ca EDTA v 4M

urea trong dung dch m.

Hai loi enzyme malate dehydrogenase v 3-hydroxybutyrate dehydrogenase thu

c tcht chitRhodopseudomonas spheroides cng c tinh sch bi 2 ct sck i lc phi tmu lin tip. Enzyme 3-hydroxybutyrate dehydrogenase c ra

tch bi dung dch KCl 1M v enzyme malate dehydrogenase c tch ra sau

bi dung dch m c cha thm 2mM NADH. Vi kthut sc k i lc c ththu

c 3-hydroxybutyrate dehydrogenase vi hiu sut 80% trong khi nu dng

nhng phng php khc th qu trnh tinh sch ny phi tri qua 9 giai on ring

bit nhng hiu sut t c chl 9%.

3.2.2.2.Tinh sch interferon

Knight v Fahey (75) sdng Blue Sepharose Cl-4B trong qu trnh tinh sch

i lc interferon tnguyn bo si ca con ngi. Interferon shp phln vt liu

i lc trong dung dch NaCl 1 hoc 2M trong khi cc protein tp khc th khng.

Interferon tinh khit sau sc ra tch ra khi Blue Sepharose bng dung dch

ethylen glycol 50%. Dung dch ra khi ct sc k c thu nhn vo mt dung dch

m sao cho to c dung dch c cha 37% ethylen glycol v interferon bn hn

khi trong dung dch ethylen glycol 37%. Thm mt ct sc k sc sdng

nhm thu c dung dch c nng interferon cao hn. Sn phm thu c c

hot tnh l 5x108n v/mg.


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Bng 3: Purification of Human Fibroblast Interferon (75)

3.2.2.3.Tinh sch khng th

Sc k i lc c s dng cho vic tinh sch khng th vi cc khng

nguyn l cht mang. Nhng khng th cnh cng c sdng trong tinh

sch protein v enzyme. Trong iu kin l tng, nhng ct sc k i lc ny gip

phn tch 1 khng thra khi hn hp th chqua 1 bc thc hin.

Proteins Purified on Immuno Affinity Columns (38)


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4. Mt sng dng cthca phng php sc k

4.1.ng dng sc k i lc trong tinh sch protease

4.1.1.Nguyn l

u tin l cc ligand lin kt ng ha trvi cc matrix khng ha tan, nh cc

ht agarose, sau matrix c cho vo ct sc kHn hp dung dch cha

protein cn tinh sch sc cho vo ct khi sxy ra stng tc gia protein

vi cc ligand c c nh trn matrix, do protein s b gi li trn matrix,


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nhng cht khng tng tc vi matrix schy qua ct. Stng ny c tnh c

th v thun nghch. Protein c tch ra khi matrix bng cch lm yu lin kt

gia chng bng phng php gia gii khc nhau. S tng tc sinh hc gialigand v phn ttinh sch c thl kt quca lin kt tnh in hoc tng tc k

nc, lc van der Waals hoc l lin kt hydro. ra gii c thsdng ligand

cnh tranh c trng, hoc l khng cnh tranh bng cch thay i pH, nng ion

hoc l sphn cc ph vlin kt.

Qui trnh thc hin

Hnh 1: Cc bc trong sc k i lc

Bc 1: Chn mi trng i lcChn mi trng c sn (ligand lin kt sn vi matrix). Nu khng c th phi

chn ligand thch hp to mi trng i lc c hiu. n nh mi trung i lc

trong dung dch m lin kt (binding buffer).

Bc 2: Chun bmi trng vdung dch m


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Vic ti s dng mi trng i lc cn phi ph thuc vo bn cht ca mu.

Lu trnhnhng cht bo.

Bc 3: Chun bmu vchy muChun b mu: Mu cn c lm sch trc khi chy sc k.. Nu c th nn

kim tra i lc ca ligand vi protease cn tch. Sau , hiu chnh pH mu v loi

bcc thnh phn gy gin on lin kt. Chy mu: Chy mu di iu kin ti

u cho qu tr.nh lin kt cc phn tcn tch vi ligand. Cn phi kim sot tc

d.ng chy, thtch mu khng nh hng n hiu qulin kt.

Bc 4: Ra gii

Thu hi protease mc tiu bng cch thay i cc iu kin thch hp cho qutrnh ra gii. C nhiu phng php ra gii, khng c phng php chung nht

nh. C th tin hnh qu tr.nh ra gii c hiu bng cch s dng mt ligand

cnh tranh hoc qu tr.nh ra gii khng c hiu bng cch thay i pH, lc ion

hoc phn cc. Kt quthu c protease dng tinh sch hoc c c.

Phng php 1: Thay i dung dch m

Phng php 2: Thay i pH hoc nng tc nhn cn cho qu trnh ra

gii. Phng php ny c thgy hi cho ligand.

Phng php 3: Thm mt cht cnh tranh lin kt. Phng php ny c th

ci thin tnh c hiu ca mi trng m sdng cc nhm ligand c hiu.

Cc phng php ra gii c tnh chn lc c ng dng kt hp vi cc

nhm ligand c hiu.

- Ra gii pH

Sthay i pH sc nh hng n mc ion ho ca cac nhm trn ligand v

protease lin kt. Sthay i ny c thtc ng trc tip n vtr lin kt, hoc

tc ng gin tip n sthay i i lc do thay i cu h.nh. Gim pH l phng

php thng sdng nht ra gii. Tuy nhin vic thu cc phn on phi c

tin hnh iu kin pH trung tnh (v dTris-HCl 1M, pH 9).


48/57



- Ra gii lc ion

Thng sdng NaCl (dung dch m) lm tng lc ion. Cc enzym thng

c ra gii vi nng thp hn hoc bng 1M NaCl.- Ra gii cnh tranh

Thng sdng tch nhng cht lin kt trn mi trng c hiu nhm hoc

khi i lc lin kt gia protein mc tiu vi ligand tng i cao. Cht ra gii c

thcnh tranh lin kt vi protein mc tiu hoc ligand. C thsdng c cht vi

nng tng theo gradient hoc dng xung. Thng sdng nng xp xvi

nng ca ligand. Nu c cht lin kt yu hn th sphi sdng vi nng

cao hn.- Ra gii vi cht ra gii c phn cc thp:

Thng sdng dioxane (10%) hoc ethylen glycol (50%).

- Ra gii chaotroptic:

Chsdng khi khng dng c phng php khc. Phng php ny lm thay

i cu trc ca protein c ra gii. Cc tc nhn thng dng l guanidin

hydrocloride, ure.

Bc 5: n nh li mi trng i lc trong binding buf fer

4.1.2. Cc matrix thng dng

Matrix i lc thng c chun bbng lin kt ng ha trca mt ligand vi

cht rn htr. Agarose hay Sepharose, Sephadex v tinh bt l nhng cht rn h

trtt bi v. chng dto dn xut vi cc ligand khc nhau. Tuy nhin gel agarose

l c sdng nhiu nht v. c bn cao v cho php cc i phn txm nhp

d dng. chun b ct i lc, matrix phi c hot ha c th gn ligand

bng cc lin kt ng ha tr. Trong mt strng hp, mt spacer arm c dng

trong qu tr.nh hot ha, v sau ligand c thlin kt ng ha tr. Lin kt ny

c thc thc hin bng nhiuphng php. Bng 4.10 a ra nhng tc nhn


49/57



thng dng hot ha matrix v cnh ligand. Tuy nhin, cc matrix hot ha

. c thng mi ha v c th s dng rt tin li ph hp vi nhng ligand

c chn. Sn phm thng mi thng gp nht l: Sepharose 4B, 6B: chagarose c lin kt ngang 4%, 6% tng ng.

AH Sepharose: Sepharose c nhm amine tdo.

CH Sepharose: Sepharose c nhm carboxyl tdo.

CNBr-activated Sepharose: Sephaose c hot ho bng CNBr

Bng 2: Mt stc nhn hot ha matrix v cnh ligand phbin

4.2.3.Ligand sdng tinh sch protease trong sc k i lc

Vi mi protease khc nhau sc mt ligand tng ng. Trong sc k. i lc

tinh sch, phn tch protease tcc ngun khc nhau, ligand thng sdng nht l

cc polypeptide antibiotics gramicidin S hoc cc bacitracin. Ngoi ra ngi ta cn

nghin cu tng hp cc ligand i lc c hiu mi tcc dn xut ca polypeptide.

Cc peptide ny c cu trc ging cu trc cc c cht ca enzym, ng thi cha

cc lin kt khng li sthuphn protein.


50/57



Bng 3: Mt sligand thng sdng trong sc k. i lc

Mt sc cht tng hp tng ng vi cc protease khc nhau:

Subtilisin v thermitase: Z-Ala-AIa-Leu-pNA (p-nitroanilide).

Subtilisin-like serine protease: Ala-Ala-Leu-pNA

Enzyme trcy bcng anh kininase X: Glp-Ala-Ala-Leu-pNA.

-Chymotrypsin: Suc-Phe-pNA.

Kallikrein: Z-D-Ala-Leu-Arg-pNA.

Leu-aminopeptidase: Leu-pNA.

Trypsin ca b. v cua: Bz-D,L-Arg-pNA.

Protease PC v papain: Glp-Phe-Ala-pNA.

Carboxypeptidase PC: DNP-Ala-Ala-Leu.

Tng hp mt sligand:

* Boc-Leu-N(CH2 CH2)2O

HOBt (1-hydroxybenzotriasol) (0.69 g, 5.16 mmol) c ho tan trong 5ml hn

hp THF (tetrahydrofuran)-DMF (1:10) (1) Boc-Leu-OH (1 g, 4.3 mmol) ho tan

trong 5ml THF (2) Ho trn (1) vi (2). Hn hp thu c em lm lnh n 0oC.

Sau thm DCC c ho tan trong 3ml THF vo. Cui cng thm mopholine

(473 L, 5.4 mmol) vo v khuy 15 pht 20oC. Dicyclohexylurea kt ta c


51/57



lc ra, tch ly dung mi. Phn cn du c.n li c ho tan trong 120ml

ethylacetate. Dung dch ny sau c ra bng nc, b.o ho NaHCO3 v nc,

lm kh vi MgSO4. Cui cng bc hi dung mi, thu c 0.91g Boc-Leu-N(CH2CH2)2O, hiu sut 81%.

* H-Leu-N(CH2CH2)2O *HCl

Boc-Leu-N(CH2 CH2)2O (0.91 g, 3 mmol) c ho tan trong 15ml HCl 3.4M

trong dioxane. Hn hp c khuy 3 gi20oC. Sau , dung mi v HCl c

bc hi p sut thp. HCl c tch ra bng cch ra li v bc hi vi methanol.

Phn c.n li em sy kh chn khng, thu c 0.642g H-Leu-N(CH2 CH2) 2O

*HCl, hiu sut 90%.* Z-Ala-Ala-Leu-N(CH2CH2) 2O

0.425g (3.15mmol) HBOt c ho tan trc trong 5ml hn hp THF-DMF

(5:1), ri c thm vo ( 0oC) 0.926g (3.15 mmol) Z-Ala-Ala-OH trong 20ml

THF kh. Tip tc cho vo hn hp trn ln lt c 0.779 g (3.78 mmol) DCC

(. ho tan trong 20ml THF), 0.749 g (3.15 mmol) H-Leu-N(CH2 CH2) 2O *HCl

vi 484 L (3.46 mmol) triethylamine. Khuy hn hp trn 20oC trong 15 gi,

sau lc dicyclohexylurea ra. Dch lc bc hi p sut thp, v ho tan phn cn

du c.n li trong 130ml ethyl acetate.

Dung dch c ra vi nc, b.o ho vi NaHCO3 v c lm kh vi

MgSO4. Cui cng bc hi p sut thp, thu c 0.83g Z-Ala-Ala-Leu-

N(CH2CH2) 2O, hiu sut 56%.

* Ala-Ala-Leu-N(CH2CH2) 2O

508g Z-Ala-Ala-Leu-N(CH2CH2) 2O c ho tan trong 7ml methanol, sau

c bi kh vi gon v thm 50mg ch. vo. Dn H2 qua dung dch trong 48 gi

20oC. Ri thm HCOOH hpH xung c.n 2-3. Lc hn hp v bc hi dung

mi di p sut thp, thu c 0.33g Ala-Ala-Leu-N(CH2CH2) 2O, hiu sut

95%.


52/57



4.1.4. Mt sphng php cnh ligand trn matrix

4.1.4.1. Cnh ligand thng qua cc tc nhn hot ha cyanogen bromide

Tc nhn ph bin nht lin kt ligand vi matrix l cyanogen bromide

(CNBr). N phn ng vi nhm hydroxyl ca Sepharose pH kim.

Phn ng:

Phn ng ca CNBr rt phc tp. H.nh 4.9 th hin c ch ca vic hot ha

Agarose cng nh cc matrix cha hydroxyl khc bng CNBr. pH cao (khong

11), CNBr phn ng vi nhm hydroxyl ca matrix to ra thnh phn hot ha

chnh, cyanate ester, v cc thnh phn ph khc, imidocarbonate (cyclic hoc

acyclic), carbamate v carbonate. Tuy nhin, cc v.ng imidocarbonate chim u th

hn trong vic hot ha cc dextran lin kt ngang v cellulose. Trong iu kin

kim nh (pH 9-10), cyanate ester v v.ng imidocarbonate phn ng ddng vi

cc nhm amine c bn ca ligand, kt qu to ra cc dn xut isourease v

imidocarbonate thay thtng ng (h.nh 2). Cc nhm amino phn ng nh trung

tm ca phn ng ny, v. vy cn thit phi tin hnh phn ng pH 810. pH

ny cc nhm amino vn khng nhn thm proton. Phn ng cp i ny khng nn

tin hnh trong mt dung dch m cha cc amine c bn, nh tris, glycine hoc

ethanol amine. V. nhng tc nhn ny scnh tranh vi cc ligand cnh. Dung

dch m c chn thung l sodium carbonate hoc bicarbonate, v iu kin cp

i ti u l pH 8.3. Sau khi ligand c lin kt v loi bcc ligand tha, cc

nhm hot ha c.n lai trong matrix c thbkha li bng cch thm vo mt lng

d cc amine c bn nhnh tris, glycine, ethanolamine

u im:

Vic hot ha bng CNBr th n gin v n h.a cho vic lin kt cc phn t

sinh hc nhy cm nh enzym, lectin v khng th. Hot ha CNBr c thc

ng dng khng ch vi agarose, dextran hay Sephadex, m c.n c th ng dng


53/57



cho cc polymer tng hp cha nhm hydroxyl. Mt khi c hot ha cc ligand

nhcng nh cc ligand c khi lng phn tln cha cc amine c bn c th

c lin kt.Nhc im:

Mt nhc im chnh ca cc matrix i lc hot ha bng CNBr l cc ligand .

cnh bri ra lin tc. Lin kt yu l nguyn nhn chnh ca skhng n nh

ca lin kt isoureas gia matrix hot ha v ligand. Mt nhc im khc ca

matrix ny l chng c thhot ng nh mt cht trao i anion yu, lm thc y

cc lin kt khng c hiu. iu ny lm cho dn xut isourease tch in dng

pH trung h.a.


54/57



Hnh 2: C chhot ha agarose (hoc cc polymer c nhm hydroxyl) bng

CNBr.

Quy trnh lm vic:Cc tc nhn:

- Cyanogen bromide (Aldrich, Milwaukee, WI)

- Sepharose 4B hoc 6B (Pharmacia)

Shot ha:

(Lu: CNBr c c tnh rt cao, phi tin hnh hot ha CNBr trong thit bkn c

van thng hi tt)

B1. Ra 100ml Sepharose 4B vi 1 lt nc . c de-ion ha trong phuthy tinh v lm kh.

B2. To huyn ph gel trong 100ml sodium carbonate 2M trong becher.

B3. H.a tan 10g CNBr trong 5ml acetonitrile v thm dung dch CNBr vo

huyn ph gel v khuy lin tc, sdng cnh khuy overhead paddle.

(Ch .: khng dng cnh khuy ttrnh lm vht gel).

B4. phn ng hot ha tip tc trong ng 2 pht nhit ph.ng.

B5. Ra nhanh cc ht gel . c hot ha vi 1 lt nc lnh, sau

ra vi dung dch m lin kt lnh (sodium bicarbonate 0.1M, cha NaCl

0.5M, pH 8.5).

B6. Tin hnh cnh ligand ngay.

Cnh ligand:

B7. To huyn ph gel . c hot ha trong dung dch m lin kt (sodium

bicarbonate 0.1M, cha NaCl 0.5M, pH 8.5) cha protein (5-10mg/ml gel)

Tip tc to phn ng lin kt trong 2 ginhit ph.ng (hoc qua m

4oC), khuy lin tc, sdng cnh khuy paddle.

B8. Ra gel . lin kt vi dung dch m lin kt, loi bcc ligand khng lin

kt.


55/57



B9. Kha cc nhm hot ng c.n li trong gel bng cch to huyn ph trong 100

ml ethanolaminr 1M hoc glycerine 0.2M, pH 8.0 trong 2 ginhit phng.

B10. Ra gel vi dung dch m lin kt, sau ra vi dung dch m acetate0.1M (pH 4.0) cha NaCl 0.5M. V ra li vi dung dch m lin kt.

B11.Cui cng ra gel vi nc. Ra tip vi dung dch m c chn cho qu

trnh sc k i lc lectron, bqua N-hydroxy-succinimide.


56/57



KT LUN

Sc k l mt k thut c ng dng rng ri trong nhiu lnh vc trn th

gii. Vit Nam tip cn k thut ny vo nhng nm ca thp nin 80. V iu kincuc sng ngy cng cao, nn i hi phi c nhng cng ngh k thut hin i

p ng nhu cu. Bn cnh cng c mt s t ngi, v li nhun c nhn m bt

chp tt c. Ngy nay, kim sot kim tra tnh hnh cht lng hng ho p

ng nhu cu cuc sng nh: thc phm, dc phm, ho cht V th k thut sc

k cng c vai tr ln trong cng tc kim tra, gim st cht lng m k thut ny

hon ton m ng c nhng yu cu trn. V d nh: kim tra d lng

khng sinh (nitrofuran, tetracycline) trong thu sn, kim tra d lng hoocmon

(clenbuterol, salbutamol) kch thch tng trng, to nc cho gia sc, kim tra d

lng mu b cm c trong thc phm v m phm nh: Sudan c trong trng

gia cm v trong son mi, 3MCPD (3_monoclo_propan_1,2_diol) c trong nc

tng, formone c trong bnh ph, xc nh mt s phng php ch bin bo qun

nhm loi tr s pht trin ca cc nm mc gy c (c c t Aflatoxin) trong

cc l hng nng sn d tr v xut khu c bit u tng v lc Trong cng

nghip dc phm, sn: Kim tra hm lng hot cht chnh trong dc phm, d

lung cht c th gy c hi Ngoi ra cn ng dng trong nhiu lnh vc khc

nh quan trc, mi trng V vy, vi tng mc ch s dng m ta c th chn

mt phng php sc k ph hp phn tch mu tng ng.


57/57


TI LIU THAM KHO

[1] Separation and Purification technique in Biotechnology, Frederick Dechow,

1989[2] Doury-Berthod, M., Poitrenaud, C., Tremillon, B., J Chromatography, 179:37

(1979)

[3] Dutcher, J.D., Hosansky, N., Donin, M.H., et al., J Am Chem Sot, 73:1384

(1951)

[4] Ghim, Y.S., Chang, H.N., Ind Eng Chem Fundam, 21:369 (1982)

[5] Pietrzyk, D.J., Cahill, W.J., Jr., Stodola, J.D., J Liquid Chromatography,

5(3):443 (1982)

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