Ky Thuat Sac Ky Trog Hoa Sinh

K thut sc k
ng dng trong nghin cu v ha sinh hc

HVTH : Hunh Cng Ti L Hong Thanh Vy Trang 1

NG DNG K THUT
SC K TRONG NGHIN
CU HA SINH

K thut sc k


MC LC
MC LC .................................................................................................................. 1
M U .................................................................................................................... 5
NI DUNG ................................................................................................................ 6
1. Tng quan v k thut sc k ................................................................................. 6
1.1. Lch s sc 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 k thut 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
K thut sc k


2.5.Sc k gel ........................................................................................................ 21
2.6. Sc k i lc ................................................................................................... 23
2.7. Sc k tng tc k nc .............................................................................. 23
2.8. Sc k kh....................................................................................................... 24
2.9. Sc k lng cao p (HPLC) ........................................................................... 27
2.9.1. Sc k phn b hiu nng cao ................................................................. 28
2.9.2. Sc k hp ph hiu 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 c SEC) ....................... 29
2.10. So Snh Gia 2 K Thut 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................................. 30
3.2. ng dng trong thu nhn v tinh sch ........................................................... 32
3.2.1. ng dng sc k ct ................................................................................ 32
3.2.1.1.Tinh ch acid 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
K thut sc k


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 t mt r ng ....................................................... 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 s ng dng c th ca 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 s dng tinh sch protease trong sc k i lc ....................... 49
4.1.4. Mt s phng php c nh ligand trn matrix ..................................... 52
4.1.4.1. C nh ligand thng qua cc tc nhn hot ha cyanogen bromide . 52
KT LUN .............................................................................................................. 56
TI LIU THAM KHO ........................................................................................ 57

K thut sc k


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 nh
cc 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.

K thut sc k


NI DUNG
1. Tng quan v k 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 trin nhanh chng vi
nhiu k thut khc nhau nh sc k giy, sc k lp mng, sc k trao i ion, sc
k 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 sc k
K thut sc k


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 v
mt 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 rn
hoc 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 s
ko 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
K thut sc k


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.
K thut sc k


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 b
bao 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).
K thut sc k


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 peak
a. 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.
K thut sc k


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 l
thuyt 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 cn bng. 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
K thut sc k


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 khin cc vt sc k to hn. Bt giy hp thu nc, nc b gi
li 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 b
theo 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.
K thut sc k


(c) Nhng cht khc nhau s c tri ra nhng im khc bit trn bng , to
thnh mt sc k .
Hin nay, sc k giy ang dn c thay th bng sc k lp mng. Do phn
ln 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 l hay 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 cc
dung 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.
K thut sc k


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 pha long 2-5%, nh mt vi
qun chm thnh mt im gn trn pha tnh, v tr phi trn cao hn mt
cht 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 cht s 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.
K thut sc k


-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 thc cn thit, tm bn phi va bnh gii ly. Dng bt ch vch nh
nt xut pht v mc tin tuyn dung mi.
-Chun b dung dch mu: Mu l cht lng, c th chm trc tip mu ln bn
mng, cn mu l dung dch qu st, c th pha long mu. Vi mu l cht rn
phi ha tan trong dung mi hu c ph hp, nng 2-5%. Nh mt vi qun
dung dch mu ln b mt tm sc k lp mng mt 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 ha dung mi,
ngi ta t tm mng 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 trong dung mi gii ly, v nh th dung
mi 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
K thut sc k


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 ny nhn ra cc hp cht c th hp
thu tia UV, cc hp cht c mu ti sm trn nn sng; n chiu tia UV 366nm nh
sng ny pht hin nhgn hp cht c pht hunh quang, cc vt sm ca cht mu
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.
K thut sc k


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 pha tnh (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 qu trnh 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
K thut sc k


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 gi li 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 tuy
c 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 thu t c chiu cao khong 2cm trong ct,
th m nh kho bn di ct cho dung mi chy ra, hng vo mt becher
trng 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 st vi 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
K thut sc k


trong ct, va bp va ry pipet dc 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 mu
c 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 ra sch 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 hp thu, 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 c
mang 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-
K thut sc k


Trong : R l pha tnh hay gi l nha (resin), G l nhm chc mang in
tch c c nh trn pha tnh hay cn gi l nhm chc hat ng ca nha. C l
i ion ca G. S l cht hu c c mang in tch tri du vi G.
Cc loi ht nha trao i ion: nha polystyren, silicagel, polymer
carbohydrate.
Cc bc trong sc k trao i ion:
-Nhi nha trao i ion vo ct: Gi ct thng ng trn gi, kha vi bn
di ct. Nha trao i ion c cn bng trong dung dch m, lng nha
v th tch dung mi sao cho c th rt nha d dng vo ct, khng to ra nhng
bt kh nm gi cc ht nhc. yn 5-10 pht cho cc ht nha lng xung, rt
y ct bng dung dch m, m kha cho ht nha lng xung. Khi nhi ct
hon tt, cn cn bng ct bng chch cho dung dch m chy qua ct, cui cng
kim tra pH ca dung dch chy ra khi ct.
-Np mu cht ln u ct: dung dch mu c lc trong sut trc khi np
vo ct, lc bng t giy lc hay ngang qua mt lp celite. Lng mu ty vo
lng nha cng nh kh nng trao i ca nha. iu quan trng nht l lm sao
cho tt c cc cu t quan trng ca mu cht c hp thu ht vo nha, tc l
ch s lng mu hn l th tch ca mu. np mu ln ct: m kha h
mc dung dch m xung va st mc nha trn u ct, kha li. dng pipet
ht v t dung dch mu ln u ct, m kha cho dung dch mu ht vo lp nha
trn u ct. yn 10-20 pht cho mu cht tip xc cn bng vi nha.
-Khi tt c mu c gn ln u ct nha, cho vi ml dung dch m ban
u chy qua, ni ct vi bnh cung cp dung dich gii ly.
-Gii ly bng cch tng dn nng dung dch gii ly: hn hp mu ang gn
vo nha trao i ion trong dung dch m c nng thp. c th tch mu
K thut sc k


cht ra khi nha thng, cn phi gia 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 cu
nha, 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)), tin trnh 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 phn t 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 dch
tch 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 thch ra 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
K thut sc k


phn t c trng lng phn t (TLPT) nh hn kch thc l rng, s ton phn
hay bn phn lt vo l rng, tuy rng cui cng ri cng theo dng chy i ra khi
ct nhng s chm tr hn. Dng chy pha ng khin cc phn t ln khng th
chui vo l rng ca mng gel, nhanh chng i xuyn qua ct, ra khi ct, trong khi
phn t nh ra khi ct lu hn v cn chui vo v i ra khi l rng ca gel. Nh
vy, cc thnh phn khc nhau ca hn hp mu khi i ngang qua ct sc k gel, s
ra khi ct ln lt theo trnh t TLPT ln i ra khi ct trc, phn t nh i ra
khi ct sau.
Sc k gel phi thc hin trong ct hnh ng tr bng thu tinh, gm cc bc
sau:
Nhi gel vo ct: cc ht gel trng n hin din dng huyn ph trong
dung mi ph hp cho vo ct. Huyn ph c chnh sao cho tht st nhng khi
rt vn chy vo ct thnh dng d dng. Cn bng ct bng cch cho dung mi
gii ly chy ngang qua ct, lng dung mi c th tch bng 2-3 ln th tch ct.
Kim tra s cht ch ca ct bng cch cho mt lng mu th vo u ct. mu
th thng c chn l dextran blue 2000, c mu xanh dng c th c
quan st bng mt thng s di chuyn ca mu trong ct.
t mu cn sc k ln u ct gel: dung dch mu cn phn tch phi c
lc b nhng hp cht cn dng rn, cn. Dung dch mu ho tan vo dung dch
m, t ln u ct ging nh sc k ct.
Trin khai sc k gel: trin khai gii ly rt n gin, ch s dng k thut dung
mi n nng (ngha l s dng mt loi dung mi hay hn hp hai dung mi,
khi bt u cho ti khi kt thc qu trnh gii ly ch s dng mt loi dung mi ).
gii lyc th bng trng lc nhng rt tt khi s dng bm y t u ct. hng
dung mi gii ly trong nhng l nh, mi l c mt th tch nht nh. Cc cht tan
s ra khi ct theo th t TLPT gim dn.
K thut sc k


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 tnh vi 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.
K thut sc k


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 nc khc c trn b mt protein. Khi cho
mu protein vo trong iu kin nng mui cao p cc phn k nc li gn
nhau 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) cc phn k nc trn
phn t tng tc cc nhm c nh. Cc phn t gn kt c ra gii 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<500.
Sc k kh rn (GSC - gas solid chromatography): pha tnh rn l mt cht hp
ph, y l sc k kh hp ph v sc k kh lng (GLC - gas liquid
chromatography): pha tnh lng c bao hay gn trn mt cht mang rn (solid
support) y l sc k kh phn b.
S dng phng php sc k kh c kh nng tch c hon ton nhng cht
hu c tng t, v d nh o-; m-; p-xilen khng th tch c bng phng php
K thut sc k


chng ct phn on nhng tch c kh n gin bng sc k kh, cng nh 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: sc
k 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
K thut sc k


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 trong ct
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 in
ca 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.
K thut sc k


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 o
c 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 my sc 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.
K thut sc k


Ct sc k: Kiu ct thng ph thuc vo phng php dng tch, nhng
thng l nhng ct bng thp khng g, c c l chnh xc ng knh t 3 n 4
tm v di t 10 n 30cm (nhng ct dng cho SEC ng rng hn v di n
100cm). Loi ct ny c hiu lc rt cao, c s (ra l thuyt ln n 100.000 a cho
1m chiu di ct)
Detector : L b phn pht hin cc cht khi chng ra khi ct v ghi nhn cc tn
hiu ghi trn sc k . Hin ang s dng cc loi detector sau: Detector t ngoi
(UV): Dng n thu ngn cho vch 254nm. Nhiu cht hp th bc sng ny.
Detector t ngoi v kh kin (UV-VIS): Dng ph quang k la chn bc sng t
195 n 750 nm. Loi ny hin nay c dng nhiu nht. Detector in ho v
detector hunh quang c nhy v chn lc cao dng trong phn tch vt.
Dng detector in ho c th pht hin c nhng lng picrogam (10-12g)
Cc phng php sc k hiu nng cao
2.9.1. Sc k phn b hiu nng cao
Gm hai loi: Sc kh lng - lng v sc k pha lin kt.
- Sc k lng - lng (LLC): Pha tnh l cht lng c bao trn b mt ca cc ht
cht mang, tc l c hp ph trn cht mang.
- Sc k pha lin kt (BPC): Pha tnh c gn ho hc vi cht mang to ra lin
kt siloxan ni nhm c - silic vi silicagen.
2.9.2. Sc k hp ph hiu nng cao (sc k lng - rn LSC)
Pha tnh l cht rn m trn b mt c cha cc nhm hiroxil phn cc, pha ng
l mt dung mi khng phn cc.
2.9.3.Sc k trao i ion hiu nng cao (IEC)
K thut sc k


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 SEC l 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 c t 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 tch
ca ct v bung tim
mu)
K thut sc k


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 cht phn 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
Detecter u d Thng dng nht l UV-
VIS
Thng dng l FID, dng
cho phn tch cc cht hu
c
K thut sc k ct c s dng 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 s nhng
ng dng ca k thut sc k ct v ang c p dng quy m cng nghip
vi nng sut cao nh: trch ly ng t mt 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 tch thc phm
K thut sc k


Phn tch c t
Sc k lp mng: (Thin Layer Chromatography TCL).
Phng php ny da trn s hp ph khc nhau ca cc ochratoxin trn lp
silicagel (pha tnh) v kh nng ha tan ca chng trong h dung mi (pha ng).
Cc ochratoxin ln lt c tch trong qu trnh h dung 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 s c 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 333
nm. S hin din ca ochratoxin trong mu c xc nh bng cch so snh da
vo v tr so vi vt hunh quang chun. S nh tnh bng mt cho kt qu tng
i. Dng my o mt pht hunh quang (fluorodensimeter) thay mt thng
gim sai s.
Sc k lng cao p: (High Performance Liquid Chromatography HPLC)
K thut HPLC nhanh, nhy, cho kt qu phn 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 t C8 n C18). Pha ng
l hn hp cc dung mi c phn cc khc nhau, qua ct vi vn tc dng 1 10
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 b thch 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 x bc 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
K thut sc k


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 vt
Sc 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 t qu 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 ph ln trn nha trao i ion.
Nhng loi nha trao i ion tiu chun thng c s dng l Dowex HGR
hay Duolite C-25. Tuy nhin, hiu qu phn tch acid amin ca cc loi nha trao
i ion truyn thng s c 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 dch ng
carboxylate.
Do s khc nhau v tnh nng sinh hc hay gi tr dinh 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 ngh sn 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 th c thc hin vi dn xut ng ca nha trao i ion iminodiacetate.
3.2.1.2. ng dng trong tinh sch khng sinh
K thut sc k


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 s
dng trong tinh sch khng sinh bng sc k ct nh: Nha anion ho nc
Sephadex A25, nha base yu Amberlite 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
Aureomycin mt loi khng sinh chng li cc khi u. Trong qu trnh ny, t 64
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 ph s dng silicagel cng c s dng trong tinh
sch carminomycin cng 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 t l 17 3. Sn phm thu c c tinh sch > 90%.
Mt loi khng sinh khc l Cephalosporin c sinh ra t qu trnh ln men
bi vi khun Streptomyces sp 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 t 10 g saponin th c th thu c 403
mg saikosaponin c, 1210 mg saikosaponin a v 1604 mg saikosaponin d
K thut sc k


3.2.1.3. Phn tch glucose v fructose
Trong cng nghip sn xut ng, k thut sc k cng gi mt vai tr quan
trng trong vic tch chit thu sn phm ng n t hn hp syrup glucose
fructose (thu c t qu 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 M trong ngnh sn xut ng glucose v fructose t HFCS (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 s ln lt thu c l glucose, higher saccharide, hn hp glucose fructose 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 qu nghin cu thu c l trnh t thu nhn sn phm cui ct sc k l khc
nhau khi s dng cc loi pha tnh khc nhau, c th l s thu c fructose trc
ri sau ti glucose khi s dng nha trao i ion m v ngc li.
K thut sc k


iu cng cho thy l thc t trong vic phn tch fructose ra khi glucose th
ta c th s dng c 2 loi nha trao i ion dng ln ion m, tuy nhin trong cng
nghip vn thin v s dng nha ion dng hn l do v c bn th n bn hn v
r tin 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 80
o
C nhm y nhanh qu trnh phn tch glycerol ra
khi mui v dung dch glrcerol ngm nc c nht cao s gy 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
K thut sc k


3.2.1.5. Tinh sch fructose syrup
Qu trnh loi b 3 5% oligosachharide ra khi fructose syrup l cn thit v s
c mt ca oligosaccharide ny s lm gim ngt ca syrup hoc cn tr enzyme
ng phn ha glucose thnh fructose. Qu trnh loi b oligosaccharide ny c
din t bi 2 nh nghin cu l Hirota v Shioda. H s dng 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 79 89% cha 9 20%
l oligosaccharide, v dng oligosaccharide cha 64 76% oligosaccharide ha tan.
3.2.1.6. Tinh sch protein
K thut sc k


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 s dng cc loi gel mm chu
nn nh Saphadex G-200 v Ultrogel AcA34. 3 mu tip theo s dng cc loi gel
chu nn km hn nh: Sepharose 4B, Sephadex G-75 v G-50. Kt qu cho thy l
nng sut tinh sch trong trng hp 2 thu c cao hn 4 ln so vi trng hp
th nht. Qu trnh tinh sch cc loi protein ti t hp bng k thut sc k cho
thy tnh u vit trong vic bo ton cu trc b 3 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 nng
cng cho thy kh nng ng dng trong vic tch chit v tinh sch cc
polypeptide (133). Cc nh nghin cu xc nh c th nhng loi acid v base
ra gii thu c hiu qu cao trong qu trnh tinh sch cc di- v tripeptide vi
tinh khit cao nht.
Mt v d v qu trnh phn tch hn hp enkephain peptide (mt loi peptide
chc nng iu ha s gim au ca c th):

K thut sc k


3.2.1.6.Tch chit cc hp cht ru cao phn t
K thut sc k ct cng c p dng quy m cng nghip trong vic tch
chit, tinh sch ng n v ru cao phn t t nguyn liu lignin cellulose nh
g (147,148). in hnh l qu trnh phn tch xylitol v sorbitol ra khi galactitol,
mannitol v arabinitol s dng nha trao i ion di dng Calci, v sau mt
loi nha cation Nhm s c s dng 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 s thi ra dung dch c cha mui b ra tri
t ct sc k v cc loi acid hay base c s dng cho vic ra gii. Sau cc
loi mui ny li s c 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 s c s dng 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 s dng v qu trnh vn hnh, kim sot. Qu trnh i hi lp
t mt h thng quy m ln, cho nng sut ra gii cao nhng phi hp l v mt
kinh t.
3.2.1.8.Tch chit glucose t mt r ng
Mt r l mt ph phm trong qu trnh sn xut ng tinh luyn t ma hay c
ci ng. Hm lng ng cn li trong mt r thng nm trong khong t 30
60% (tnh theo tng cht rn ha tan). Qu trnh x l 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 r ny.
Khi mt r c 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
K thut sc k


trnh ny s gip phn tch ng ra khi cc hp chp khng phi ng c ngun
gc v c ln hu c c trong mt r. Mt r t ma v t c ci ng c cha cc
tp cht hu c khng phi ng khc nhau, mt r t ma cha t cc acid amin v
c nhiu cc cu t mu sinh ra t s thoi bin ng kh hn mt r t c ci
ng.
C hai loi mt r c ngun gc t ma hay c ci ng u c th c tinh
sch vi cng h thng sc k ct s dng Dowex 50WX4 di dng ion K
+
. Trong
trng hp s dng h thng sc k ny th 50% sucrose trong mt c ci ng
c thu hi vi tinh sch 80%, v 65% sucrose trong mt r t ma c thu hi
v tinh sch 68%. Vi tinh sch ny th ng sucrose c th c ngc
quay li giai on trung gian cho qu trnh kt tinh. Nghin cu ca Takahashi v
Takikawa (153) ch ra rng c th kt hp qu trnh ra gii ion v k thut hon
lu trong nhng iu kin ti thch c th thu c hiu sut tinh sch 90% cho mt
r t c ci ng.
Nhng phn tch k thut (155) v qu trnh ui ion thu hi sucrose t mt r
c ci ng ch ra rng, vi cng mt chi ph u t cho vic thu hi sucrose t
mt r th qu trnh sc k ny mang tnh hiu qu kinh t cao nht.
Ngoi ra , k thut sc k cn c s dng tch cc sn phm ph c trong
mt r nh: ng betaine, inositol, raffinose, v adenosine. C th s dng nha
cation Ca thu hi 70% inositol, s dng nha cation Na tch chit raffinose,
v c th thu hi 66% - 86% ng raffinose c tinh sch 91% - 93% ty thuc
vo t l sucrose/raffinose c trong mt.
Mt trong nhng nghin cu gn y nht v ng dng ca k thut sc k trong
cng nghip x l mt r l ca Neuzil v Fergin (162), h p dng k thut sc
k lin tc UOP thu hi sucrose t mt r. Trong phng php ny, h s
dng cht hp ph khng hot ng chc nng v dung mi ra tch l alcohol. T
K thut sc k


dung dch r c t l 30% sucrose, 10% KCl v 10% betaine, khi p dng k thut
ny c th thu c 90% sucrose vi tinh khit ln n 99%.
3.3.2. ng dng sc k i lc
Ban u, k thut 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 k thut sc k ny khng b gii hn ch trong lnh vc
tinh sch protein, m n cn mang li hiu qu tt trong qu trnh tinh sch
carbohydrate v glycoprotein vi s s dng lectin c nh. (Bng 2). Nhng th
th hormone dng trong tinh sch hormone hay nhng khng th n dng c s
dng trong tinh sch khng nguyn. Nhng c quan t ca t bo v thm ch l
ton b t bo cng c tinh sch nh vo k thut 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
K thut sc k


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 - Antitrypsin
Herpes-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
K thut sc k


Wickerhauser v cng s ca ng (70) s dng heparin Sepharose tin
hnh tinh sch antithrombin III t huyt 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 60
o
C
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 s ra tch
enzyme adenosine kinase t h thng sc k lc gel vo s hp ph enzyme ln trn
5 AMP Sepharose 4B trong ct sc k i lc.
K thut sc k


Dung dch m c s dng trong sut qu trnh tinh sch bao gm 4mM
Na
2
H
2
PO
4
, 2mM EDTA v 5% glycerol pH 7.0 v nhit 4
o
C. Enzyme
adenosine kinase s c ra tch ra khi ct sc k v thu c dung dch m
cha 0.6mM enzyme.
Qu trnh sc k i lc trn calmodulin c nh c xem l phng php tinh
sch rt nhanh seminalplasmin ca b n tinh sch 99%. Qu trnh ra gii
seminalplasmin khi calmodulin c nh 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 t cht chit Rhodopseudomonas spheroides cng c tinh sch bi 2 ct sc
k i lc phi t mu 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 k thut sc k i lc c th thu
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 ch l 9%.
3.2.2.2.Tinh sch interferon
Knight v Fahey (75) s dng Blue Sepharose Cl-4B trong qu trnh tinh sch
i lc interferon t nguyn bo si ca con ngi. Interferon s hp ph ln vt liu
i lc trong dung dch NaCl 1 hoc 2M trong khi cc protein tp khc th khng.
Interferon tinh khit sau s c 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 s c s dng
nhm thu c dung dch c nng interferon cao hn. Sn phm thu c c
hot tnh l 5x10
8
n v/mg.
K thut sc k


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 c nh cng c s dng trong tinh
sch protein v enzyme. Trong iu kin l tng, nhng ct sc k i lc ny gip
phn tch 1 khng th ra khi hn hp th ch qua 1 bc thc hin.
Proteins Purified on Immuno Affinity Columns (38)
K thut sc k


4. Mt s ng dng c th ca 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 tr vi 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 s c cho vo ct khi s xy ra s tng tc gia protein
vi cc ligand c c nh trn matrix, do protein s b gi li trn matrix,
K thut sc k


nhng cht khng tng tc vi matrix s chy qua ct. S tng 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 gia
ligand v phn t tinh sch c th l kt qu ca lin kt tnh in hoc tng tc k
nc, lc van der Waals hoc l lin kt hydro. ra gii c th s dng ligand
cnh tranh c trng, hoc l khng cnh tranh bng cch thay i pH, nng ion
hoc l s phn cc ph v lin kt.
Qui trnh thc hin

Hnh 1: Cc bc trong sc k i lc
Bc 1: Chn mi trng i lc
Chn 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 b mi trng v dung dch m
K thut sc k


Vic ti s dng mi trng i lc cn phi ph thuc vo bn cht ca mu.
Lu trnh nhng cht bo.
Bc 3: Chun b mu v chy mu
Chun 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
b cc thnh phn gy gin on lin kt. Chy mu: Chy mu di iu kin ti
u cho qu tr.nh lin kt cc phn t cn tch vi ligand. Cn phi kim sot tc
d.ng chy, th tch mu khng nh hng n hiu qu lin kt.
Bc 4: Ra gii
Thu hi protease mc tiu bng cch thay i cc iu kin thch hp cho qu
trnh 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 qu thu 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 th gy 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 s dng 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
S thay i pH s c nh hng n mc ion ho ca cac nhm trn ligand v
protease lin kt. S thay i ny c th tc ng trc tip n v tr lin kt, hoc
tc ng gin tip n s thay i i lc do thay i cu h.nh. Gim pH l phng
php thng s dng nht ra gii. Tuy nhin vic thu cc phn on phi c
tin hnh iu kin pH trung tnh (v d Tris-HCl 1M, pH 9).
K thut sc k


- Ra gii lc ion
Thng s dng 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 s dng 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
th cnh tranh lin kt vi protein mc tiu hoc ligand. C th s dng c cht vi
nng tng theo gradient hoc dng xung. Thng s dng nng xp x vi
nng ca ligand. Nu c cht lin kt yu hn th s phi s dng vi nng
cao hn.
- Ra gii vi cht ra gii c phn cc thp:
Thng s dng dioxane (10%) hoc ethylen glycol (50%).
- Ra gii chaotroptic:
Ch s dng 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 buffer
4.1.2. Cc matrix thng dng
Matrix i lc thng c chun b bng lin kt ng ha tr ca mt ligand vi
cht rn h tr. Agarose hay Sepharose, Sephadex v tinh bt l nhng cht rn h
tr tt bi v. chng d to dn xut vi cc ligand khc nhau. Tuy nhin gel agarose
l c s dng nhiu nht v. c bn cao v cho php cc i phn t xm 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 s trng hp, mt spacer arm c dng
trong qu tr.nh hot ha, v sau ligand c th lin kt ng ha tr. Lin kt ny
c th c thc hin bng nhiu phng php. Bng 4.10 a ra nhng tc nhn
K thut sc k


thng dng hot ha matrix v c nh 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: ch agarose c lin kt ngang 4%, 6% tng ng.
AH Sepharose: Sepharose c nhm amine t do.
CH Sepharose: Sepharose c nhm carboxyl t do.
CNBr-activated Sepharose: Sephaose c hot ho bng CNBr

Bng 2: Mt s tc nhn hot ha matrix v c nh ligand ph bin
4.2.3. Ligand s dng tinh sch protease trong sc k i lc
Vi mi protease khc nhau s c mt ligand tng ng. Trong sc k. i lc
tinh sch, phn tch protease t cc ngun khc nhau, ligand thng s dng 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 t cc 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 s thu phn protein.
K thut sc k


Bng 3: Mt s ligand thng s dng trong sc k. i lc

Mt s c 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 t r cy b cng 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 s ligand:
* Boc-Leu-N(CH2 CH2)
2
O
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 0
o
C.
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
K thut sc k


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(CH2
CH2)2O, hiu sut 81%.
* H-Leu-N(CH
2
CH2)
2
O *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 gi 20oC. 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 h pH 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%.
K thut sc k


4.1.4. Mt s phng php c nh ligand trn matrix
4.1.4.1. C nh 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 d dng vi
cc nhm amine c bn ca ligand, kt qu to ra cc dn xut isourease v
imidocarbonate thay th tng 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 8 10. 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 s cnh tranh vi cc ligand c nh. 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 b cc ligand tha, cc
nhm hot ha c.n lai trong matrix c th b kha li bng cch thm vo mt lng
d cc amine c bn nh nh 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 th c
ng dng khng ch vi agarose, dextran hay Sephadex, m c.n c th ng dng
K thut sc k


cho cc polymer tng hp cha nhm hydroxyl. Mt khi c hot ha cc ligand
nh cng nh cc ligand c khi lng phn t ln 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 .
c nh b ri ra lin tc. Lin kt yu l nguyn nhn chnh ca s khng n nh
ca lin kt isoureas gia matrix hot ha v ligand. Mt nhc im khc ca
matrix ny l chng c th hot 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.

K thut sc k


Hnh 2: C ch hot 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)
S hot ha:
(Lu: CNBr c c tnh rt cao, phi tin hnh hot ha CNBr trong thit b kn c
van thng hi tt)
B1. Ra 100ml Sepharose 4B vi 1 lt nc . c de-ion ha trong phu
thy 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, s dng cnh khuy overhead paddle.
(Ch .: khng dng cnh khuy t trnh lm v ht 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 c nh ligand ngay.
C nh 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 gi nhit ph.ng (hoc qua m
4oC), khuy lin tc, s dng cnh khuy paddle.
B8. Ra gel . lin kt vi dung dch m lin kt, loi b cc ligand khng lin
kt.
K thut sc k


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 gi nhit phng.
B10. Ra gel vi dung dch m lin kt, sau ra vi dung dch m acetate
0.1M (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, b qua N-hydroxy-succinimide.

K thut sc k


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 kin
cuc 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, 3 MCPD (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.

K thut sc k


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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