Tai lieu tn hoa sinh cong nghiep bai tlc, enzyme, vtmc, chat beo
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3"2.1.
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3.2.x6co-!nh loai tipit beng sac try l6p nning ffhin-I-ayer-cliromatograpkrS"
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pha trnh hdp
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pban
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Tg
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c5'c chdt duqc phin tach trong
chuydn dich lon hon so v6i chAt c6 6i luc ldn hon- Nhd vay mli
b c6c vi td kh6c nfau. Trong sec kf I6p n6ng, pha trnh Ii dung mOi
qud rrinh cha,v sac k!
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pha dOng.
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tx-ong t-6'-p:r.(f.-c!'6t ir6p -t!g-nhd.-tin-Lr-gg
chuvdnd6.ng cham hon tro-tg.be thdlg p4q hi. Qlat
su rudng t6.c niLy mlL chft phan tich
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nhanh hop vi a@-toi:ffi-oilts sa-i-},
ph-dii tich c6 6i lu_c y6u hon vti ifitrin-rt"tr"vdn ddng
rhU.
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pha tinh.
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sic ky trdh gi5y, sac hi lqp
Tuong tu nhu mgi ky thuat sdc }cf kh6c, dic biet 1i-v6i
cdn ph5'n tfch duoc cho tuong tdc v6i hai
gr6ngd-Uq--trc-LnQ! ngltye! ticrdtdoll-g!-fu: Chdt
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qu6 trinh sac ky, c6c plian tLi
(duoc dDng. dti chudn bi lqD rn6ng h{p thu). Tuy nhien,uong
vdi.chdt mart-9 c6 dinh v) trd
dung mOi c6 cuc chuytin dOng qua chdt mang c6 thd iidn ket
thhnh pha trnh.
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b. Chdt nrang itlta tuilt
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m6t bln m6ng cfia cellulose, thuy tinh hiy nhua' drroc phri
bin gidy cellulose ding cho sac k!
rn6r lop nr6ng silicagel, nh6rn hay cellulose. c6 thd ding
duoc lim sin v6i ciLc wcb
gidy lem pha unh trong k! thuat TLC. Th6ng thudng, cdc ban TLC
1).
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li3u v)' chitiu diy.kh5-c nhau'
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vlt
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thu6c,
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chdt mang 6 day c6 thd
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qul phan t6ch mdu khi so s6nh v6i c5c mau
thfr duoc phan tich dga tron hi6u
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nen chay thri sac kf mau v,6i
pha dQng. D6i v6i mQt mAu m6i
Q4s.he
lh"lqL-{tg$-
1b9t'42q
chudn
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b6o vlr c6c axit b6o' C6c lipi
aceric cho phan tAch ckctriglycerit, cholesterol ester, ester axit
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nguyOn
c6 cuc hon nhu c6c glycerol phosphatit, sphigolipit vh glycolipit thi:gifi
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p_hgr_
higa
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tlru-ong qho -b6.n sic-
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v6-i-
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pha dQng,di til tron xu6ng, qu6
uinh sic
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mdt kh6i ban mang'
nhien s6 kh6ng holc kh6 x5c din-h git trj Rl do dung m6i c6 thd bay hoi
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Cing c6 ihd thuc hi€n sic
kf
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c{c mdu kh5
r6ch
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thudt thuc hi€n
iuong tq nhu trong sic ky' gidy hai chidu.
phni !!at kfn khi
vi
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ki Isp m6ng duoc sri dun-q rOng rdi hon sic k-f gi{y do khi nang phin
t6ch cao, thdi gian sic
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Hi€n nay, sic
kf
nhanh hcrn
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bln sic'bi duoc ldm sin v6i
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3.2.2.1. Hod chdt
- Hod chat thfch hqp cho chidt lipit
tt vit phZim (xem phdn chiel lipit) 6i,,
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chlr hdp rhu vdi
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3.2.2- Phdn thu. c
c6c
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chidu dhy mohg mu6n kh6c nhau-
- Chloroform
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duoc bdo ho1
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hoi
kf
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L'ru y, crlrig nhu trong sic l
- Hexan-diethyi ether- axit acetic (80:20:
.,',..-i,' ):1,'.'
,-,;.'--1,
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l)
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- Iod tinh rhd
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kf
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- Budng sic
lop m6ng silicagel20 x20 em
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ki
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- Bin sic
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3.2.2.2- Dlutg ctt
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- Lipit chudn
vli tinhthd iod rrong mQr binh thu!
rinh kin, dfi lon,
ruon_q
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- Budn-e hoi iod: D4t mot
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- Piprt cham mdu
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duong v6i budng di€n di.
3.2.2-3. Ti6t hdiilt
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u Ct odr bi --4g
n6i htu co-do-do pha+----P6-c-bijt--tip=ifg-r4t-p.idni.
cu. c cfia
c6c lipit thu&ng nh6 hon c6c thinh phdn khdc crla td bio vdt phd*. Tir nhtng ndm
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thOng rhudng slri dqng. c-dc he dung
1950. he dung mdi chloroforrn:methanol (2:1) duoc coi l).
hQ
dung m6i c6 kh& ndng chiet toin
hqrp
lipit.protein trong-
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bd cdc lipit c6 roirg v5.t phdm do khi nang phan tdch hicu qd6. phrlc
n'ring n-suy€n sinh chft cia c6c t€ 6eo. Tuy vfly,.hg dung m6i niy cdn hoi tari
-
hoi tan ft c6c thi.nh
phdn phi
lipit hon vi it
dQc hon,
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hexan:propanol (3:2). HC dung rn6i ni.y
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ci rnOt sd thlnh
phdn phi lipit nhu protein. MQt hg dung mOi kh6c dd duoc tim th{y, an toin hon, d6 li
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vi c{u hdc_
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c6 cDng tinh chft
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do vdy duoc sfr dun-e nhidu hon. Cho dir sri dun-s hO dung m6i chidt n).o, lipit chi€t duoc ddu
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Trong truirn-e hgo cdc vat phdm chria cric m6 Iien kdt, c6 thd cdn phii nghi€n m5u voi
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cdt tru6c khi chi6t bang dung mOi- Sau khi chidt, cdn loai U6
irtrdn chidr phi tipit beng c{ch rria
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Lipit chudn c[ng duoc chudn bi tuong tu'
b. eham m5u
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caCl, 0,5vo) trong lt'ulng hqP sr-r dL-rr:l
cjiclr chr6t Voi cjung dich muoi (KCI, NaCI lroac
sri dung hexan-plopanol' C6c xri iy nhy clng
clrio:.ofor.rn-methanol, iray Naso1 1'fivo khi
phdn dur-ig moi irol tan irong nu6c' M6u thu duoc
g6p phdn liLnr giim tird tich mau do loai b6
theo nhu cdu v) didu kien x& If miu- Giu mAu d
duoc I2Lm bay.hoi dung niOi hoac l)n-r ktrO ui
ichi'
-2OoC,r:ong tdi tr6nh sd oxy [o5 bdi oxy khOn-e
thuy iau khi tSch tt vai phdm hay
c6c mdu duoc ciem phan rich c6 thd & dan-q nguydn
dich chloroforrn (i0 mgml)' c6c mau
de dnh che n-i6t phdn. Pha mau n-ehien criu trong dung
gidY).
Cdc vdt man-{
Iot' Ndu mlu
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au1 Oi
m6p tren crla bAn sic ky'
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'Rr
di
= ------
l's-v6i
41ric-
qli4y cria dung moi:
grd-_ld Rr qhan dugc
y6i
c6c giA
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tri R, tieu- thudq' xdc dinir lo4i Iipit phan dclr
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s3qbfag
vdt mhu
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dd:u gSc
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dQn-e-t'uong -deii sg-a-lip'il-
Khoing
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ra. danh
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niru ci6 n_eay sau
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v).o rron-e budng iod uong y)'i ph6t- Lay ban sic
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bAn qaq Ki 1 9
El! -"e"h dung m61 c6ch m6p uOn
Liiybin sic xy ra,d6nh dau mfc ch4y cta dung mOi, lhm khO
Tiplr to6n
it' kh6ng nhin
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dd cho dung m6i ch.ay
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Dlt bin sac kf
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kf trong butine sdc ky
sic ky duoc nhrin-e trong h5n hqp dung m6i dDng cho sic
chim trong dung moi' Ddy kin
rirau 6 phia drr6i sao cho phdn man-e m5u kh6ng
v6i ddu nrang
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duoc phdi hiOn nhd hoi
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c. Tidn h)rnh sac kY
budng.sic
vi
thich hop khi c6c
kh6 ph6n dinh'
n€u m6u qu6 nhidu, c6c vdt m).u s6 dinh vho nhau,
nlu;
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ring
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Lhdy ckcvot
phAn t6ch 16
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miu duoc
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miu c6ch nhau4-5
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plrdn sic
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tai mot ddu cfra bln sac ky sao cho
Mau duoc dua nhitlu ldn len m6t didm clinh trrrdc
0,5 cm (xem )ai ky thu6t man-s mau trong
tai n-r6i didnr mang. dudng kinh vei n-rAu kh6ng quf
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ST
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- XEc a-inh chi sd
Chi s6 axit lh sd mg
Froc
cfta chat b6o
axii
KoH
cdn dd trung ho). c6c uxi! b.9 hr do c6 [-ong
l'gam chdr bdo.
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e- t
ho|
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3.3- X1,c tiiah c6c clei so Iy
phin ring.
+ KOH _+
+ HrO
Dg" .rio luong KOH dr)ng dd trung hoi c6c axit, tinh chi sd axit
3.3-1.2 Ho6, chdt
.,4,1f L{ (^.
Ete etylic
RCOOK
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Rugu etyltc 96Vo
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KoH v) c6c axit b6o
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RCOOH
chudn do, khi d6 girla
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ry do c6 trong chdt b6o xiry ra
KoH
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bang dung dich
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hoi chft b6o
Trun-e
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33-I-1. Nguyin tdc
KOH 0,lN trong rugu
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Dung dich phenolphtalein |o/o trong ruou
TA
3.3.1-3. Cdch tidn hnnh
lim
lic
ddu.
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chua tan heL c6 thd
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vin
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thdn' chdt b6o
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Ldy viro binh cdu hoic binh n6n sach, kho; dung tich 250 rnl chilh x6c m6r
Iuong m5u cdn ch{t b6o (cdn rr6n can phan fch)
rg-khqitg 3ffi!ftlr^vio d6
jQ- 5Q mI h5n hop ete - ruou g6vo tilf=]:1) dd hoi ran chdr b69... Ndu sau
khi l6c cdn
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nguoi, 4-iqh.pban h6n-hq-p--b-arg-d-u-rg
ciqb $oH 0,1N trong ruqq (dr)ng
dung dich KoH trong rucru dd fdnh nhfrng sai s6t c6 thd
xdy ra do srr thuj phan cria
xi phbng, trong rruimg hqp,khi h5n hqrp chria qud nhidu nu6c tir zov_o rr& ren) y_f,c_lii
th!-p-henolp_bl{e_4_.1i5 giot dung dich tVo rrong ruou) .Iq
ddn !!U_{"d! tign m4u hOlg
nroi, tru-dng-bqp__gtra't b6o c6_mhuJ.tr4pr,ttriding
si_li.thi timotpiJalein (1ml dun_e dich
7Vo trong ruou) dinh p_t$_p.!,q*
xa_nh,_hodc dr)ng ankati xanh 68 (tml dung dich
-d{9
lVo r,rong flrou)-
:
5.611b-f
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b - Sd ml dung dlch KOH 0,1N dDng d-inh
m . Lugng mdu th( nghiQm (g)
phan
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tinh theo c6ng thric
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Chi s6 axit
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3.3-I-1-- Tinlt kdt qud
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f - He s6 hieu chinh'n6ng d6 cria dung crich KoH 0,rN dem dr)ng
5,511 - Sd *g KOH c6 rrong lml KOH 0,lN
50
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f:.S.2.
X5c clinh chi sd cira xh phdng
Chis6xiphbngl}'tucrrgmgKoHcdnddtrurrghoh..#c..axiib6otudocdng
mg KoH cdn dd xi phdng
c5 trong 1 g ch{t b6o. N6i cd'ch kh{c l} luong
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nhr: Iien ket
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ho6ckcg}ixerircflngnhudiitnrngho}'axitb6otudoc6trortglgbh{tb6o.
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3-3-2-1, NguY€n tdc
ST
dich KOH chudn dQ thi chdt b6o bi thui
Dun s6i chdt b6o v6i mot lucrng drr dung
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- ----
cHzocoRl
-I
CHOCOR2
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phdn.
+ R,COOH +
+ 3H"O
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+ RCOOH
C6c axit b6o duoc
gili
ph6ng ra sd phin tmg vdi KOH
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R-COOH
+ KOH -) RCOOK +
F{tO
TA
theo so dd-treri 'C6c axit b6o tu do cfrng phln rlng v6i KOH
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xi Phbng'
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tinh duo.c chi s6
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I-ugngkidndu,kh6ngthamgiaphinrlngvotctcaxitb6oduo.cdinhphinbang
b6o'
dd Fung hoi clc loai ax'it b6o trong chdt
HCI chudn- Drr.a vio lugng kidni tiOu t6n
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cH.'ocoR3
R2COOH
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3.3.2.2. EIod chdt
U
KOH tinh khidt hoi ta-n trong 1 ljt nrot
Dung dich KOH 0,5N trong nJgu: 30g
H
:
Dung dich HCI0'5N
Dung. dich phenol phtalein
'l'Vo
r.ong nrgu hodc timol'phtaletrt lVo
$gr[
ST
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:*:, ;"
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etylictinhkhi6tdun;r5n!c6tipS.nesinhhlnthuh6i'dungdichrJdy€nmot
trong binh dlrng miLu nAu'
. ngiy dOm, gau d6 lgc vi biq qu5'n
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,,ll';tTlil,*","*,
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viobinh20mldungdichKoH0,5Ntrongrugll,daybinhbangnritc66ngsinh|ein
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khfvidunsdic6chthufh6nhqptronglgib.N6uxlphdngho6cilcchftkh6tanc6
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thcm 5 gigt dung dich phenol
phbng hori xong; lhm nguQi h6n hqrp,
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phtaieinhodctimolphtalein llotoalrugrrvidinhphanbingdungdichHCl0;5N.
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sau khi
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propiJie, butYlic hodC amilic-
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m6i c6 nhi6t do s6i cao nhu toluen' flrou
thd th€m khoing 2oml rnQt trong cdc dung
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SongsonghmthfnghiQmkidmchrtrrg,thaychdtb6obangmQtlugrrgnu6c
vi n6ng d6 cfia kidm c6 thd bi bi6n ddi)'
ruong rlng (cdn ldm thi nghiQm ki6m chrfirg
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kit
qud
Chi sd xh phong (X-,;) rfnh rheo cOng rhuc
(n
- b)"f,.28,os. f"
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Xp=
:
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3-3-2-4- Tinlz
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i6rr%
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rrl HCI 0,5N dDng dinh phan nl6u thi
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b - Sd
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nghiQm
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ST
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ding
- He s6 higu chinh ndng dd crja HCl dem
fz - H9 sd hieu chinh ndng dQ clra KOH dem dr)ng
28,05 - Luong mg KOH c6 trong 1 rnl KOH 0,5N
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ml HCI 0,5N ding dinh phAn mAu kidm chrlng
f,
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a - 56
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Trong d6:
m - Luong mdu cdn ch{t b6o (g)
fe LL
>3
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Chi
sde*E'sdmg KOH cdn dd xlphbng
hod ciic glyxerit (este) c6 trong 1g chdt b€o
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33-3-- Xic dinh dri s6 este
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Chi s6 ese (Es) bing hieu crja chi s6 xi phbng'vi chi sd axit
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Es=Xp-A*
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Trong thr,tc td sin xuft, chi sd este cho ph€p tinh duoc luong glyxerin c6 thd thu
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Him luong glyx.titt
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3.3.4. X6c tfinh
Chi s6 iod
r
3.3.4.1.
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Ili
li
dri
GVo
sd iod
:
0-0547
-
Es
blng dung dich Giublo
rd g*r, iod kdt
hgrp
:
Ngttendic
du. a
tr€n nguy€n ticiod kdt hqrp v6i cdc axit
k6p- Nhmg.d nhiet dQ thudng, iodphan rlng v6i axit b6o c6 trong thinh phdn cfra
f
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b6o
r{t ch{m,
cdn khi dun n6ng thi' iod. ker hqp
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chit
:cdc ,n6i .k6p kh6ng ddng d6u,
'khong lim no hoin toin duo.c ciic n6i k6p- C{c halogen khr{.c (clo-brom) phin rfug v6i
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axit b6o r{t m4nh;cho n6n ngubi ta thay iq6 bAng cric
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kh{c-
hqrp chdt cria
n6 vqr cdc halogen
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vfi
nhau
vI kdt
hgp
vlo
cdc n6i k6p cira
ST
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axit b€o chua no c6 uong chft b6o.
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Hgqz + 21, + HgIz +
M
iod duo.c tao thinh khi cdc ch{t d6 tl{'c dgng
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Giublo dtng dung dich ruou cria iod vdi HgCl2 dd,xdc dinh chi s6 iod- Clorua
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v.b.o
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$6p
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Phrrung
'ftY
v6i 100 gam chdt b6o.
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crja chdt b6o tinh theo cdng thrlc
TA
U
khi xi phdog hoi chft b6o.
52
TA
ST
.C
U
H
M
TA
IL
IE
U
F.r-Cli=CH-R,-COOH+ IC'l --> Rt
T ?*'*'-COOH
ici
O
M
LuqnrglCldu,khdngthamgiavlophirr'fngkdthWy6icdcndikSp'dinhphdnbing
dungdichtiosunfatnatrichudndQsaukhidlth€mdungdichKlvirnu6cvb'oh6nhqp
+
NarSnOu
H
oA t&u,
C[
kh6ng nhtrng
.hi-;; ,hdJi*
IL
IE
U
*,
Uao
hoi cAc n6i k6p mh cbn c6 thd thay
phii tuin th&.nghiom ngdt c{c didu
hydro & c6c nh6m no, vi vly cdn
phuoneph6p x6c dinh chi s6 iod cho
ki0n tidn hinh criaphucnig ph6p' Trong c6c
tisu chudn'
Giubio vln dugc xem lh phuong ph6p
rhd nguy€n
ft
TA
ST
.C
O
U
EU
H
ST
zNaI
2NarS2O3
M
Iz *
12
U
KCI +
CII+KJ
.C
pb6.n ung.
dOn
nay phuong ph6p
IL
I
3.3-4.2. EIod chdt
U
tr0n ldn hai dung dich sau
:
H
IE
.C
O
- Hod Zilgiodtrong 500 ml ruou 96oC
vi lgc' Sau khi trdn cho
- Hoii 30 gam HgCl, trong 500 ml ruou 960
U
cA.ch
M
TA
*DungdichGiublo:.h6nhqrpGiubloduocpha4Sgibtru6ci<}ridtngbang
IL
ST
dQ bdn cla
= i,lajnham lim tang th€m
trong binh miudung dich' Bio quin dung dich Giublo
gan. Na'$O3 trong ntr6c idt dun s6i
+ Dung dich Na2SrO3 0,1N : hol tan 24'8
dd nguOikh6ng c6 CO2 cho't6i 1 lit.
l
H
U
TA
rhem vho h5n hqp 25mI HCI (d
+ Dune dich KI\'OVo
O
M
'
+ Clorofocm
ST
.C
-.-
TA
I
LI
E
U
.
3-3.4-3" Cdch tidn hdnh
U
sau d6 cho vio binh
vio binh n5n 10 ml clorofocm, Iilc nhg cdn th{n'
D4y kin binir ng.y bang n6t mii c6 th{m
dung 25 ml dung rtich Giublo bang bur€r
y€n trong t6i & nhiet d6 thrrdng trong
u6t dung dich KI- Lic cdn than binh vh dd
U
H
IL
no ttri cimg ad tau' Tidp d6" them
Ddu c6 chng nhidu a,xit b6o khdng
10mI KI107o dd tao
thinlt ket tira Flglj
M
U
kholng lL-llgib.
TA
ST
IE
.C
O
M
chdt b6o- Them
.C
O
vio dd hoi
thi phii cho th€m dung dich KI
Ndu sau khi tic mi vin c6 kdr rira d6 HgI2
IL
IE
U
H
U
IL
IE
TA
H
U
ST
.C
O
M
U
ST
tan kdt tria-
TA
ST
.C
U
TA
IL
IE
U
H
M
Thom
vio b)nh loOml FI,o vi dinh phdn iod
giii
ciuoc
ph6ng ra bdng dun-e
ui.:! Na"$QrO,lN cho d6n khi xuft hiOn mdu v).ng
rom. Th6m vig binh imr
dich hd tinh bot ivo vir dinh phan tidp cho
den mat m)r.u xanh. Trong 1jc churin
M
can
.C
ki€n nh*
EOn
IL
IE
U
(b
:
- a).f .0.01269100
U
TA
I_
H
M
Chi s6 iod Ct) tinh theo c6ne rhfc
ST
.C
O
lic
U
3.3.4.4. Tinh kft qud
EU
H
di€-u
ST
r} clorofocm vio I6p nu6c.
song song ridn hinh rhi nghiQm kidm chring r.rong
cing
(khong c6 chft b6o).
O
manh dd iod chuydn
.
dung
Trong d6
:
IL
I
a.- 56 ml dung dich Na"SrO.0,lN dr)ng dinh
phAn mAu rhi nghiQm_
U
M
H
dDng.
U
chft b6o, gam
IE
.C
m_- Lugng m6u cAn
O
TA
b'- sd ml dung dich Narsro3 0,rN dDng dlnh phan mdu
kitim chring_
f-- He sd hieu chinJr ndng d6 cfra NarSrO30,1N dem
I
IL
Xic ttinh chisd iod bdng iod clorua
U
3.3.5.
LI
E
X
0,lN
H
U
0,01269.- 56 gam iod nrcrng ring v6i 1 ml dung
dich Na,$O3
TA
ST
100.- Hg sd qui chudn cho 100 gam chdt b6o.
_ i
(Kh6ng dring HgCl, dQc)
.
":
:t
;;'1r
--,
O
M
TA
I
3.3-5-I- Nguy€n tdc
-i
Zzu + KO,
+ 6HCI
= 3ClI + 3KCl +
Iod crorua
3HrO
M
U
.
ST
.C
Phuong phSp n).y nhanh v). crlng.ri m6t phuong
ph6p ticu chudn.
-:
IL
IE
U
H
U
IL
IE
TA
H
U
ST
.C
O
M
U
M
O
ST
Dung dich iod clorua trong HCI 0,2N: cho
vio binh nrit mli tl,r gam Kr, 7,0
gam iodat ket; (KI03),50 mI nu6c
cdt,jO
HCI dam d4c.(d = l,l9) vi lic
"rf
cho hoi tan hoan toln iod' sau d6 them
20 mr clcirofocm- V&a l6c that manh
.C
U
TA
IL
ST
IE
.C
O
U
H
lod clorua kgt hqrp vio cdc ndi k6p cira oit b6o c6
trong chdt b€o- Luong c[
du d;nh ph'' beng dung dlch tiosunfat nhu hong phuong
phdp dtng dung dich
Giublo' Tu d6 dE ding bi6t duo.c lugng clr
dn kdt hqp v6i ch{r b6o.vi tinh chi sd iod3.3-5.2. Hod. chdt
5A-
TA
ST
.C
U
TA
IL
IE
U
H
M
dng
vira cho rhem
giot KJa3lTo cho cicn
kli miu tirrr cita clorofocm
bi mat oi-
ch6 t6i'
1074
ST
- Dung dich KI
mbLu &
O
trong b)nh thuli tinh
.C
I
M
-Chuydndungdichsangph€uchiStddyOn,b6lopnu6cdivid'Llngdichcdniai
cho t6i I ljt- 86'o quAn thu6c thri
chuydn sang blnh dinh mfc rdi them nu6c cat
TA
IL
IE
U
ST
.C
O
i
H
M
i
U
I
U
ch{t b6o v}- sau d'6,25 ml dung dich clorqa iod
15 phrit- Tidp d6, cho th€n i0 ml
trong HCI 0,2N. L6c ddu, d4y kin binh iai vh dd yen
gili pn6ng ra bang dung dlch
dung dich KJ, IO/o,50 mI nu6c cft. Dfnh phaq iod
rnl dung dich hd tmh bot
natri g"trN cho ddn mlu vlng rom. Sau d6, th€m 1
IL
I
EU
H
5 ml ete etylic
vdio binh dd holi tan
U
TA
riosunfat
H
U
d
e ph6p
Giubto.
IL
hrlc
$S ^r,
TA
ST
IE
.C
U
O
mL
H
M
m{t mdu hc}Ln
clorofocm v-i dinh phan tidp cho ddn khi dung dich bf
"cin'
manh
y
llc
Chf
b€o"
chft
trOn
thu
hdp
bi
Cho rh€m clorofocm vho d€.giAi ph6ng iod
kiiim chring- cni s6 iod tfnh
trong hic dinh phan- Ddng thdi ti6n hbnh l)m thf nghigrn
\Vo,3
,, ,
i;Y5r
dlnh
,
,.\l
LI
E
TA
I
dr+ng 911a geq
p
iod
pgrgxit c6 trong
clft
ci
b€o_:
*4
,
U
KI thii ra iod theo Phan rlng
sau
U
H
M
fty'
(t4o
chi s6 peroxit dua tren nguyen tiic.- c6c peroxit cfra chzft bio
phin r?ng v6i
uinh 6i cria chdt b€o) trong m6i trudng axit c6 kha q+g
x.a'c dlnh
thhnh trgng qu6
0
ST
.C
3.3.6-1. Ngwy|rc tdq
O
M
U
ttd phin ring vdi hydro hoat dQng ciia cdc
peroxit lil s6 gryr iod duoc
prroxit chrla trong 100 gam chdt b6o" n6i c'{ch khe", "hi's5
v6i 100 gam ch{t b6o hhb t5'c
gii.,i ph6ng ra khi cho dung dich iodua kali trlc dung
s6
:
M
U
TA
IL
ST
IE
.C
O
Rr-cfl $H Rz
R-CH-CH-Rz
to'* 2CH3cooK'+ H'o+I'
5 5 +2w + 2cH3cooH -+
.C
zNaI +
Na2Sn06
s6 peroxit-
U
vio lugng riosunfat ti€u t6n khi dinh Phen iod tinh chi
IL
IE
U
H
U
H
U
IL
IE
ST
I
TA
.C
O
M
Dlra
+ Iz
ST
2Na2S20.
O
Iod duo.c i+o ihinh diruh phdn bdng dung dich tiosunfat
TA
ST
.C
U
TA
IL
IE
U
H
M
'
- axit axetic bens
U
M
TA
IL
I
EU
H
U
TA
IL
IE
U
ST
.C
O
H
M
U
ST
.C
O
M
- Du,rg dich Kj bdo hoa,r6i oha
- Dung dich NarSrO, 0,01N.(phurnr6c khi drihg
fir dung dich Na.sro3',1N
bing nu6e cfr dun s0i dd nguQi kh6ng c6 chria
COr)
-Dung dich hd titlhbltlzo
r\t'v"I-[L"d' [cJ . iLA,
--a \
(jaof cdch tiin hdnh
!"..r/"o
^ |y.Jv
\---l tAv vdo binh n6n '250,
/'5 gam cntt a;{m€rn vio ,#; _ 10 mr
'clorofocrn
ad hoa hnichd'r b50 vr sau d6, r0 - 2o/r
cH3cooH Aar(g-Qranhqir axit ,"{
axetic bdng vi. clorofocm 2: r),1
ml dung dich KI bdo hoi m6i pha (hoac
mQr n
-i '
thd KD- Lic h6n hqp that cdn rh&n
trong 5 -a(toni Th6m vho h6n hqp 25
rnl nudc
c{t vi dinh phan iod tao thinh bang aung
aichfisro3 0,01N vfi chi rhi hd rinr bor
(0,5 mI dung dich hd rini bQt
tVd.
Ddng rhdi tien henh rhi nghiQm kidm ch6ng
(thay ch{t b6o bing 3 - 5 mI nu6c cdt).
33-6-4-Tinh kdt.qud.
U
(a -b).f..o,ootz6g-too
IE
=
m
U
H
U
TA
ST
p
IL
.C
O
H
Chi s6peFdxft (p) tinh theo c6ng thric
sau:
Tmng d6:
a
- 56 mI hiposunfit ding duJh pfan miu tl:f nghiQm_
ST
.C
f - He sd hieu chinh
O
M
TA
I
LI
E
:m
no'ng d6 cria hiposunfiL
m - Trgng luo.ng m6u thf nghiem (g)
Sd qam iod trrcng Ung
U
-.
viri lml Na"SrO, O,OIN-
U
H
M
0;0O1269
IL
IE
U
M
.C
H
U
ST
t;
IL
IE
U
:=:*'
TA
H
U
ST
.C
O
M
O
U
TA
IL
ST
IE
.C
O
100 - Hg sd qui chuydn rheo 100
56
TA
ST
.C
U
H
TA
IL
IE
U
cAc
PflAN
NV
FF{IJoNG PHAF xAq; rllzuel
vrrAMiN
M
M
-
I !54J
U
H
I
HOCH-.O-\
-rH
-;-o .-:i
(
\/'4n
OFI
:
-O.():o
g\
axit ciehydroascachic
axit ascochic
'
I
HOCH
(dang khri)
U
O
H
M
TA
Axitascocbicbiph6hu}r{tnhanhdu6it6cdun.4cirac6cc|rdtoxyhtldv)rbdrr
axit ascocbicitrong mdu trdng cdc
trong moi trudng axit- Do vty ngudi ta lhudnq-chigt
U
EU
H
U
F
OH
IL
IE
U
cHpH
'" -' cHpH
IL
I
2
TA
M
-*
hai nh6m enol v}
Axit ascocbic (vitamin C) li m6t hqp ch{t chua no'c6
tai clu6i hai dang oxi ho6v) dang kh&nh6m hydroxin, ho) tan trong nu6c vi tdn
*tr.!:
ST
.C
O
v'E
ST
.C
O
C (axit ascocbic)
IE
.C
ZVo"'
dung dich axit nhu axetic 5To. axil meta photphonc
TA
IL
ST
iPhuongphdpctuatr0nrrguyOntscl}axitascochicc6khindngox;vhoikhu
U
%->r
H
U
thuf,nnghiclrch6t"hiJ!id4.j,.JL@iclophenoIindophenol(DCIP).
thi
t6n ta tinh dtrgc
Dua vho
LI
E
rr8hi€m-Socidph6ntmggifraaxitascocbicv6ich{tchithiDCTPnhusau:
cF[rol-f
'I
HOCI{
cl
OH
M
U
-o(}
TA
IL
ST
IE
.C
O
U
H
M
U
ST
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O
M
TA
I
I
O
ch{t chi rhi nny.llmQt hsp ch{t c6 khi "u."e 99i T}Y,f?-T:-'H|f:i:
*i."1"
IE
U
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ST
U
IL
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TA
H
U
ST
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t
H
O
M
i
IL
"o
cdn dang khri khong mru (DCIP c6
*a". 'i+:'l'n:-"l-'.1i,'"1":-d:l1i"J5f::
U
ili.iE-ft
".'i
"," -oit'lili'r',iii'ud,
ilir#',H'rx'ui*;:;;";;i:-;cd
5'l
TA
ST
.C
U
TA
IL
IE
U
H
M
r-ir-oi tr-rrimg
")4.1.2. Hof chat
vi
mOi rnrdnrg axir yeu" c6
h'il:-e 6 mOi truhrg c6 pH <
rnlu iim trorrg hho6'rrg pI{
4'0)'
M
;5 niu
trl 4 d3n 5
kidm
O
l,)-i '::-'lh iron=
.{rit
lr:eta - photphoric ZVo hay axit axetic lVo
ST
-
.C
- HCI i% haY CH3COOH5%
U
H
M
- Du"g iilch oxalat arnOn bdo ny
IL
IE
U
ST
.C
O
- Kl irr-h lhd
il2fr.2Vo
t6i)
- .i.,ji ascocbic (vitamin c) tinh kidt (bao quln kin trong I9 mdu
- h:,.g 'ich hd tinh bOt 17o
v)o binl dinh
- Dune dich thudc chi thi DCIP 0,001N: cho 0,15'gam DCIP
tidp d6 dung dich d6rn phobphat
:i.uic cunE tich 500 ml thern 350 rni nu6c cdt vi
IL
I
EU
H
U
TA
-
U
IE
.C
U
O
7 ng)'y)'
suan trcng binh c6 mlu & noi t6i (kh6ng qu6'
H
M
TA
PH6,9-T,Ochot6iv4chmrlc.Vltrongdungdichnu6c'thudcth&niybiph6
ruyr{tnlanhnOnduo.cphav1idungdichc[em.ThuScthftDCIPduocb6'o
TA
IL
ST
-Dung dich d6m PhotPhat PH 6,
KII.PO. trong 1 lit nu6c c{t) v}
H
U
2.HrO :rong 1 lit nrrdc c{t)va nhau ru6c khi dem ding-
U
4.1.3. Cich ti6n h).Lnh
O
M
TA
I
LI
E
5 ml dich chidt dDng
C:n l{y l-io c6i srl lugng mlu thi nghiQm thd q)'o <19 oong
gidu vitamin C nhu 6t''
dinh phan chrla fd 0,15 ddn 0,20 mg tittft tg C lcic nguyen liQu
hroi khdc nhu rau qui*" 10
khoai tay, cha]lh.-. cdn3 -5 garq- c6c nguy€n liQu thuc vlt
ST
.C
-50gam,cicsinPhdm,d6nghQP5-10gam"'nrlthgoh)mluqnrgvitamiilCtrong
tlng loai nguyOn 1i0u mlL ldy luong mlu cln cho thf-ch hqp)-
U
M
Ngaysaukhildymauxong,thOmHCllVo(hodcCH3-COOHIVg)v>rccdcv6i
dich arit (khoang
dd rna.u thf nghiqm drro.c ngam kin hoin toin trong dung
U
H
O
lugng dn
O
dd tn{ng c6i v}r thom ddn vqch nrfc, hodc dDng
dd kdt tta protein, Ioqi trd ch{t ktrri vi sai t6-
C6 thd anng axit oxalic
LVo
IL
IE
U
H
U
IL
IE
TA
H
U
ST
.C
O
M
axerat ch\.SVo (5rn11
lVo cho q'dn vlch mrlc-
.C
tinh loc dd dnng) rdi them HA
ST
cho qu6.
U
U
TA
tich "100" rnl' Thim vAo
Tidp d6, chuydn hoin toin h6n hqrp vio Uinf, C"1 -rlc dung
b6 protein v)'o gitip
binh dinh mrlc dung dich axit meta-photphaic 2% (25 mi dd Ioar
M
IL
ST
IE
.C
+5mI]Ha'|%).Khil{y'rnluchriiffinh{ingdrlngcubdngs6tho4cbsrrgd6ng.
tr€n 10 ph6t)'
Nghiiin czin than m6u nguy€n liQu (kh6n-g nen nghidn qui lau
TA
ST
.C
U
H
M
TA
IL
IE
U
Kiii thi nglri€ni vA c6c pf,fun v1t c6 agudn gdc tiqag
tncloaxetic 2a7o sao cho nong
, |,"- ^
uulrS
,,,3;
^-.t+
4.\_LL
ciia nd dat 5vo trotg ciung dich (25mi)-
v}.o biruh n6n dDng rich 50
rnl (hay c6c) 5ml,1g6c !g9__c_9__chrla
O
l{y
.C
Dr)ng pipet
M
Truong hqrp kh6ng c6 axit metaphoQhoric hod,c oxaljc cd rr-ij chi dr)ng ri6ng
mQtminhaxiiHCl ld/ohay cH3cooH svo.Dd,yen l0phrir,sau.i.iccdnihd:rv).loc.
Chrft
song song
j
H
ST
.C
O
lq9llu"-
lim thi nghiQm $_d1q_chgmg., dinn phan
IL
IE
U
M
U
ST
vitarnin c, th€m 5mI dung dich oxalat am6n bio h.oi, 10ml uro va
dung dich chi thi DCIP tD rnicroburet cho tdi khi xudt hiQn rnlu hdng bdn (kh6ng
m{t
miu trong m6t phtit). Trudng hgrp dr)ng axit oxalic thi khorig.cd'n cho th6rn oxalat
c6c rhu6c thii dem dr)ng.
EU
H
U
TA
- Khi x6c dinh vitamin C rrong mQt phdrn vAr nio d6 phAi ridn hlnh pha_n tich ir n-hfr
ln
2 mdu thi nghiom, d6i vdi rn6i rnAu - }ldn x6c dinh, kdt qui dinh phan 2 lAn khong
dtoc
sai i€ch nhau qu6 0,03
ml DCTP 0,001N.
,_-"-T->-_aa_
rJ."
U
tfrih theo c6ng thrlc :
"--
X=
,\
H
U
i-!r:r1--
IE
if.--'i.'-
! mI hoac fr hon lml).
IL
iuong
ST
FIa.m
.C
4"tr-4"Tfnh kdt qlla
?i\"-
lucmg DCIP dilns dd ciinh ph6n n6n
H
tong khoang L-2 m1(kh6ng qud
vi
LI
E
U
Trsng d5
U
khOng quri 2 phrit
M
&
dil
O
TA
- Thdi gian din.h phel k6o
TA
IL
I
- Phai dr)ng riricroburer dd dinh phan-
thi
b - 56 ml'ciri thr
a
i
- sd ml chi
:_:'j
-i
r';rir-t:.
0,001N dirng dinh ph6n dich chi6t vi'raml'r
r:
i,:.::
-r--.\
i -:i"
l'i
!.j...
:;
-::.
-
-
c
0,001N dilng dlrh phAn m5u kidm chrlng
O
M
f - Sr5 mg axit ascocbic rlng v6i 1mI duug dich dem'dDng_
_-Vtdng ttidm - Luong mdu ttif nghiQm (g)
U
Xdc dinh hQ sdf
M
. t\r
ST
.C
TA
I
:
IE
.C
O
U
H
Hoe tan 1-1,5 mg @dy u6c lucnrg khdng cdn can)'axit ascocbis tinll khidt uong 50rnl
H2Wa2Va L{y chlnh xdc 5mI dung d.ich d6 vh chudn dO bdng dung dich chi thj DCIp
IL
IE
U
H
U
IL
IE
TA
H
U
ST
.C
O
M
U
O
ST
KIO30,001N cho ddn khi xu{t hi€n miu xanh- HQ s6 f duoc tinh theo c6ng thfc :
.C
U
TA
Lfy vio m6t binh lfir{c 5nrl dung dich.axit ascocbic tinir k\idt ren, rhem m6r it
tiiiii thd Kr (3=4.rng) vi 5 gigt dung dich hd rinh botlvo,rOi aior, phan bang dung dich
M
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0,001N cho ddn miu hdng bdn (khOng m{r miu sau 1 phrit).':'
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sd mg axit ascocbic ring
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0,088
0,088.
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a
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rlich chi thi DCIP 0,00iN
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4-LXiLc atinh vitamin B, (thiamin) bins phucng ph:{p do miu hu}nh quang
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- Chd phdm photrataza
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- Doog dich vitamin B1 ti€u
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v4y, c-6 thd ddnh gid hoat
hoi cc ch{t ho4c
chuydn
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gian clng ldn-
phin rlirg tao thinh-tBn rnQt don vi thdi
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phuong phdp 6p k6, phuong phrip ph6 quang kd, phuong ph6p chudn dq Uen hic, phucrng ph6p
sic hf vi.phuong phdp ho6 hoc-
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rrrg ho6 ho.c khdc nhau dd dinh luong cAc.co c
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didu ki€n x:{c dinh, t6c
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thinh trong dung dich phan rmg du6i t6c dung
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enzirn
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dugc bidu di6n bang don
(I).
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enzim trong h5n hqp phin rlng- Luong enzim
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Theo qui u6c qudc te, don vi enzim ti6u chud
,
citit
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mit philt
&
nkfrn
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hod lmicromot'co
ngubi ta tidn hanh x6c dinh hoat dQ enzim &
TA
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v6i firng enzim mQL
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don
vi ti6u
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theo
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micro duong luong cria c5.c li€n kdt (peptit, glu
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micromol ccch{t A (honc B), n6u A
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ln luong enzim x.iic tdc chuydn ho6 rnQt rni
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du mili don vi (mU) hey kilo don vi (KLI)-
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vi enzim tr€n lmg
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protein-
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Niu bidt rrgn-q lugng phtn tri cua erzim c6 thd tinh duoc hoat
du6i tic dgng cfia I phen -tfr enzirn sau 1 phf t-
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Neu bidt 16 trung tlm hoat dOng cria phen t& enzim c6 $d tinh dugc hoat dd cria brung
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thich .3ng r,ri.i iri.,lng clurrg riltli ,-:'f,p \Q - 2(J lein ithur}n3i 1! 10 ldrr) ciOi troil3il'ong suot'
Sar-r cl6 dcn-i k-rc hoac ly tim di c5 cirri-/c ch.ing Llich enzirn
tlung
N€u Ih cic ciro ph.im k/ rlruai iray thurln khier phai hoi chj phdm vh.o nu6c hoac
sau ci5
clich clgni thich fnrg r6i dirng quc thuy tinh iri cdn thA.n r'6i mOt luong nh6 dung n-rOi'
clicli ti3nr c5 pI,.
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tlii phrii loc lrof,c 11' tim-
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X6E;jfi5 hoat
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.
riO proteaza.
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GLiilIr) iiF-o phuong phirp Anson cei t:dn
tic
phuong phip dua
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sirOt
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'rrong
thgm nuric hoic ciung clich rigrn clro.clen m6t thd tich nhdt ciirh. Neu dung clich khOng
phlLn protein.caz-einal natri (co chdt c6 thti
i)
cazein
EU
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bing che phdm enzinr c6 trong dung dich ngfiiOn cfr.r r6i tidp ci6 ldLr-n vd
phiim
hpat erzim vi kct trla protein chrra bi thuy prhAn bang axit tricloaxetic. Dinh lugng sin
vlo cld
cluqc rqo rhiph trong pliin ring ihuy phln bing phin fng. miu v6i thr-rdc thu foiin- Dua
U
irLh luo
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trOn lImg Drotetn cuia che ohdm.
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int
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ciinh tai cdc tri s6 pH sau
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r:gc
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| 1<- -Ir
- < A t0,7 ddi v6i proteinaza axit
Yrr-J'Y
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I pH =_1.,2 ! Q,2 d6i v6i Protern az-a trung tinh
' pH = 9,5 + 0,2 d6ivdi proteinaza kiiim
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do
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riOns c&a
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ax-it amin cria cdc enzim v
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5.7.2Hol' chdt vir dung dich enzim
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ml a-rit axetic bang trong nudc cat t6i 1 lit dung dich.
0,livl
:
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+ Pha dung dich (B) axit photphoric
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+ Pha aung ai"n (A) axit a-retic O,1N{
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:,1:;r,i axit phophoric trong
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arit octo-boric 0,llvl
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pii:..:.lti3rrvhohSnhqpdOmnlyc6clugrrgNaoHlNkhd.cnhau,taduocciic
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cic: :::-. .zr ring 0,5M
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L{r' =- - - :::-z ho6 chft dd pha c5c dung dich A' B' C tren gfp 5 IAn so
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Tl,-i:t .:_-. :1: ,hri tich bang nhau cria cric dung dich A, B, c,
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+ Fha:::z :ich @) nahi hYdroxit 1N :
dd
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;r.. j: --. -.:l!1 ii:c 1dy 2l,5m:axir bdng; 26,25mlaxit photphoric v)' 30,99 ant boric)'
d€rn 0, lM- Dung ::::. _3_, -. -; :ang O,Oilv[ tr6n 9 thd tich nu6c c{t v6i m6t thd tfch
- Dung ci--'. --i- --:-::-xe[ic 0,3M
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felin : Chudn bi nhu trong phAn l'5-2
lit dung dich-.-). :ha icing 16,45m1 Ho dam dac bing il16c c{t't6i' rn6t
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::r:: -- '-: -.:= :rudc c{t t6i vach mric, 15c ddu v} dem loclft dung dich'
- Dune :::.- --.-: :-::;'c-dc 1N pha !o:?ulg82,2LIHCI bang nu6c c{tt6i m6t
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nghi€nc,1:=.=-::.1,;n:5cdrrngdichd0rncfing.nhukhoingpH-uf9bheP_ddq!i€trritenzim'
di.m bio <:r,::-. --.-- --!thoac ldm giirn hoaf tinh xric t6c cria n6'
+
croreinaza axit (pHcira h6n hqp phin ri'rg I) 2'5 0'2):
a ),,': .; ;;-!
'in
ciru trOn cAn phan
nif ii:ra
Dol .;oi :ra:z!ns= axit (pH crta h6n hqp pfuin ilng
td
5
5
M
t0'2)
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ticl- Chi. cdn than chO Phdt bing que
nl6 vdi m6tit dung d.ich d€m van ndng 0,1M,pH2'5' Sau;d6;
thuy
=rh -ns :dc
dich dQm niy td
chuvSn .:i:: --- vio bin}l dinh mrlc dring tfch 1o0m] vi th€m dung
rtich dem v4n
v?ch a.ruc. 55: nuSn pha lodng dung dich erzim thi cfrn'g ding dung
Car
74,'
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Can 01,-19 chdphdrn nghi€n c'Jru. Chi cdn thin chO phdm bang que thuy tinh tiong cdc
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Ddi voi proreinaza rnmg nnh
Lty 0,1 -
1g chd phdm erzim
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vi
H crta hdn hgp phdn trng ld
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i
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95'iA2)
,2)
nhu cde chdphdm enzim tr0n, thay pH cria ddm
- Dung dich co ch{t (cazeinat natri)
:9,5-
:
cazeirat:natri kh6 vh hoi. tan trong 90 mi
TA
q Ddi voi proteirutza axit (pH 25) cln29
chudn bi nrong ru
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vi fdn hhn]r nhu trSn dr)ng dung dlch dQm v3n ndng
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nly t6i vach mrlc-
thOm dung dich Citn-i
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.
inric dung tich 1O0ml
O
c
ihh
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dich vdo binh
vlo 3 - 3,5m1F{Cl lN.
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dung dich cazeinlt na'ui bang axil clohydric r.udng thfp hon 5,1 thi dung
xu6ng
vihSn hq bi phan thenh 2
M
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dich bi vdn duc chrit it (tao $enh
ciLc vdm
pha
nh6), cbn & pH 4,5
- k€rtia vl dung -Oi,
,
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c6 c6c vdn l6n llng
.
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th€m
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dung dich d€m 0,01M, pF{ 5,5. Sau ci6, dua pH cria dung dich xu6ng 2,5 bang cdch
,
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Khi axit hod tidp tuc dung dich cazeinat natri bang HCI cho tdi pH 3, 1 - 3 ,2 thi c6c vfir,
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lhfp hon ta v5n duoc
H
dung dich ddng
nhdt.
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ihCm axit clohydric thAt nhadr gho tdi pH 3
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th€m duag rlich dQm 0,1M, pH 5,5, cho t6i vach mric'
Ddi vdi proteinaza trung tinh (pH.7A hoi t4n 29 cazeinat natri khd vio 90 ml dung
IE
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--', . , -,
["tt"" rg "o"i""t n'iri
cria dung dlch co ch{t vd 5,5- Sau d6" chuydn toan bQ du_ng dlch sang binh din}r
vi
,
th€m dung dlch dpm va-n narrg 0'1M,
ning 0,1M, pH 5,5. Thern *Ol y;i gig! {ung dph HCI lN
dung tich 10qrd
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manh liOn tgc dung
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ltic-'
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vl
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b-
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chu3dn dung dich sang bintr dinh mrlc 100m1
.p!?:5
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tr€p clugg d!ch'co chft bang axit clohydric cho t6i pH 2,5 ho/c
IL
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dich dOrr van ndng 0,1M; p}l7,2-
lN cho tdi klti'
pHT,2- chuydn toan bQ dung dich sang binh dinh mric dung tfch 100mf vi th€m dung
M
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cho tdi va-ch rnfc-
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dich d€.m 0,1lvl pH7
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Ndu pH cria dung dich co chft th{p lnon'l;2 thi thein nrng gigt.NlOH
TA
ST
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vdi Drote,inozc lcidm (pH
dOm van
nang 0,1M, pii
vd 9,5. Chuydn todn
bQ
9i)
9,-5. ThOm
hon tan 29 cazeinat natri khO v)o 90 ml dung dich
it giot NaOH lN
dd dua pH cria dung
dung dich sang binh dinh mric dung
fch
100mI v), thOm dung
cazeinat natri, ngud ta pha dung dicn *.r ch{t & TO}C
.C
Lan cria
ST
nit ngiin thdi gian hol
O
dich dOrn 0,IM, pH 9,5 cho t6i Yach mfcDd
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5.1.3. Di6u kicn
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Thdi han bio qgan dung dich cazeirat nati2Vo trong t& ianh khdng qu62 ngey-
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tOn m6y khuaf tn-
=
thuf phan & ndng dQ protein l% (n6ng dd co chft trong dung dich,
EU
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thu.c hiQn phan rlng
vi
sau khi
nhi€t dO 30oC- Trudc
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trQn lAn dung dich co ch{t v6i dung dich enzim) trong,vbng 10 phrit
khi
dich co ch{t
M
d Dii
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thuj phan, dung diqh co chat lAn dung dich enzim ddu phii dua
v€
nhiOt d6 30oC-
vi kdt r&a phdn co_chft kh6ng bi thui pha$P eruint, ngudi
3M bang thd tfcn cria h5n
1 luqnF d"ng di.h ufi
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IL
kidm tinh-
U
tffi,
vi 0,07 - O,41d6i v6i proteinazavh.0,2 - 0,6 ddi v6i
ST
dung dich dem scj rniu ndm hong pham
proteinaza trung
H
lic trQn ddu v) gifi & 300C bong 20 phrit- Sau d6 dern lgccdn l{y phii tinh to6n sao cho iugng co chdt cbn du nhi€u vd mAt dO quang crla
rdi
hqP phan rlng,
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*g
M
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ta thOm vio h5n hCP phen
H
Tuy v-fly, trong mQt s6 wong hqp
t"i
thu6c
vio
dang chdphdm,
TA
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Dd dinh chi su thuj phdn
chft lucrng'cria chd
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phdm (mrlc dQ thudn khid| enzim nghi€n criu rnh ph+m vi m{t dQ quang do d.uo-c c6 thd c6
cri.c tr1 sd
khdc
il.-.,
.
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TA
I
Citng mot c.hd plldm enzim nhtrng pH cria hdn lqp phan ring kh:d.c nh^o lhi phem vi
mat dO quang do duo-c cfrng cgllq_biqqddi- V-L thg,. khi tiOn Mnh x{c dinh ho+.t dQ proteaza
x{c dinh hoa.t dQ cria ri6 2- 3 lln b cic chd dQ pha lodng khdc
nhau (5- 10 didm) t4r cdng mQt g! qd pH cho Eudc- Sau di, [im x9m t4r 9d.c gi6 ql nio cria
mAt dO quang thi thu duo.c hoat dQ cria chd phdm nhu nhau- TU d6 mgi den hinh xdc dinh hoat
tim dugc.
IE
.C
1.1.4. Cdch ti6n hanh
IL
ST
a-Ti6n hnnh zrtc ilinh
fingnghiQm, mdi 6ng c6 Zmt
H
U
IL
IE
TA
.C
ST
U
H
U
IE
IL
hl sd bi kdt
ST
d6 protein du cfing nhq san phdr*;thuf phdn cao phan
U
ltf
O
M
axit ticloraxetic 0,3M,
.C
O
U
2
TA
co ch{t, r6i ddt vio mfy didu nhiet & 30oCSau 10 phri! cho vio m6i 6ag2mldung dich enz-im,lac trQn ddu vi dd cho thui phan dring 10
m6i 6ng 4mI
phrfit 6 30oC- Dd dinh chi phan rlng enzim b'dng.cdch th0m vio cLZ 6ag
]ghiem,
Cho vi.o
M
O
dO trong vDng mAt dQ quang vda
ST
.C
phi.ri
U
mfi
U
H
M
cria mdt chd pbdm
'76
TA
ST
.C
U
TA
IL
IE
U
H
M
*, nghi€m .i -?OoC
"U"
th6m 20 phrit n[a. Sau dfy loc h6n hop qua gi{y !9c -k}e- tr5n phdu nh6 vi.o cic dng nghiOm
hra;
(Jruaf trQn nhanh h6n hgp dd kdt
r&a hohn tohn
proetil rdi git
L{y Trrn dich loc cho v}o 6ng nghi€m vi thcm vio d6 5mI
dung dich NarCOrO,5M, i,iruai ddu li0n tuc vi th€m lmi thudc thri folin phxn tich- Dd yen
h6n hez Eilan l:rng 20 phrit- Sau phdn rlng dung dich c6 miu xanh da trbi- Do cudng d6 miu
.C
O
M
kh6 i
ST
cria dung dich tr6n mdy so mhu iluang dien d6i nguo.c v6i mAu kidm chrirrg-
H
M
U
Ti6n hanh lem thi nghi€m kidm chring bing cr{.ch th€m cdc hod.ch{t khdc theo th'1 tJ
IL
IE
U
ST
.C
O
_ - !s'Iq_g_le!. lhem vlo 2ml dung dich ernim Qqgg$f _lodng nhu & mdu rtr "eEeor)_ 4{Tt.
git
triclorexetic Q3M,
h6n hqp bong rndy didu nhiet 10 phrit & :00C, sau d6 mdi them vio
TA
EU
H
U
2ml dung dich co chft vi. gft2} phrit & nhidt do nly- Qua 20 phrtt, dern loc dung dich . Lfy
. 1ml dung dich cho vio dng ngtriQm kh6, thCm 5ml Na2CO3 0,5M, khuaf
lcm vh 6 6u6c s6ng
L = 656 ; 67O nm-
Dq"S dudng chudn theo tyrorin
U
b.
II
:'-
M
TA
hfp thu:6nh sdng
IE
IL
chudn.
U
lpM ryrozin trong 1ml dung dich ryrozin
H
Phai xay dqng dudng chudn theo tyrozin,d6i v6i m6i mdy so m).u quang dien
LI
E
U
Idn pha thu6c thii
mOL
v) tinrg
:
dich g6c tyrozin c6 ndn! dQ
1}.
: tOrVtitpmoVlml). Cen 181,19 mg ryrozin
TA
I
Pha dung
folin
du.a
m). tinh duong luqrrg Fyrozin, hlc Jd tinh mAt d6 quzrng c6 duo.c cria
TA
niy
.C
drrdrng chudn
ST
heo
U
O
Mudn tinh ho4t dQ proteaza phii xay dqng dubng chudn theo ryroztn, rdi sau d6
H
IL
I
Do cudng dQ miu cria cric dung dich thf nghiQm bang mdy so miu v6i cu-vet c6 chidu dny l@
hoi tan rong'dung dich HCI O;zN'idi cfiuydn vao bintr dilh mrlc 1 Iit
- --- 0;2N cho t6i vach mfc.
vA them FICI
O
M
tinh khidq
iiU
-
2ml dich gdc
+ 48 mt
-
4ml dich
+46m1HCI0,2N dugc ndng
-
5ml
aicn gdc + 49ml HCl0,2N duoc ndng d6 0,2:104M (0,02 pmol/ml)
U
U
H
HCl02N
dQ 0,8-
l04M (0,08 pnnoVml)
IE
ctich g6c + 45ml
dus_c ndng dQ 1,0- 10-4M
(0,10 prmoUml)
tQ,t,rnl Hcl 0,2N dugc n6ng dQ 1,5- 104M
(0,t5 pmol/ml)
O
g6c + 35ml HCI0,2N duo-c ndng dQ 3,0-
l04M (0,30 gmoUrnl)
IE
IL
\r
U
t'
H
U
IL
IE
TA
H
U
ST
.C
O
M
U
ST
. 15mI dich
M
dich g6c + 40ml HCl0,2N duoc no'ng ftQZ,O- 10a4 (0,20 pmoVnrf)
.C
- - 10ml
Hq 0,2N duoc ndng a$ O,+.f O4Vt (0,04 [moVrnl) -
IL
g6c
- t':-r dich g6c
U
ST
.C
-
TA
ST
.C
O
M
Til dung dich g6c niy ta pha ddy dung dich nhu sau:
TA
ST
.C
U
H
TA
IL
IE
U
M
L{y N;6c
dung dich dd pha lodng tr€n
hrc vi. cho'r).o cdc
lrnl, cho v}.o c6c dng ngtriQni kho, khuaf Iien
'i;,o nghiQm m5i 6ng 5mI NarCC3 O,5Vi vd 1m-l-thu6c thi- foi.n phan tfchL)Lrn mdt miu kidm ci,-3-.g-.thay lml dich chudn ryrozh bang lml nu6c c{t_
O
M
Dd yen c{c h5n hgrp phan rhg 20 phrit- Do cudng d6 mdu cfia c6c dung dich lytozin
dly lcm).
di
mO
ti
2 mdu cdn khi{c
n-hau
vL tldn hdnh hai thi ngiriQm
tren iiay.
IL
IE
U
ST
.C
O
song song nhu
tt
H
M
Phii pha clic dung dich ryrozin
ST
.'
=656-
U
670 nrn vi. cuvet
.C
v6i thu6c th& fotin bang mr{y so miu quang die,\ ildi ngugc v6i mflu kidm ch6ng (i,
Theo s6liOu tung binh thu duoc fd hai thf nghi€m dung duhng chudn tyrozr,^- T166
TA
truc ho)nh ghi hnm lugng Lyroztn (C) ( pmoUrnl), tnlc hrng ghi mat dQ quang nrong rlng (D)-
Gia sri, hen dulng chudn O,lpmol tyrozin trong
lpmol tyrozia
IL
I
5.1.4-
sE
Tinh k6t
iml
cho mAt dQ quang la 0,15i, thi
cho 1,51- Nhu vfly duong luong ryrozin s6 beng 1,51.
qui
tffi
TA
Hoat dO prote:za (.gdP)
bang dvlgHay dvlmt duoc x6c dinh theo c6ng thtic
:
U
EU
H
U
.
H
M
D'4 .tooo
HdP:
'
IE
.C
thd tich cria h6n hqrp phan
zmt(S)+znt(E)+ 4mt(TCA) _
zmt(E)
LI
E
U
H
xung axit tricloraxetic :
fng v). thd tich cria dung dich enzim
TE. Duong luqng tyrozin
xic dinh
sau
IL
gita
khi bd
TA
lO
do dugc cria m6u-
U
4.Ty
dQ quang
ST
b day : D. Mat
U
O
'7E.IO.m
^
thed duong chudn (duong
.tyro4n tri mAt dQ
quang mi lFmol fyrozin c6
lrnl dung, dich.chuCn khi ph;in foe odi thudc thri
-tr9ng
folin c5 thd c6 duo. c)-
O
M
TA
I
-Iugng
phan protein co ch{t
(tinh bang mg c6 trong
IE
M
t6i cfic
.C
Phuong ph6p dua trpn sr5 thuli phan tinh b6t b6i c6c enzim cria hO amjlaza
O
U
5.2-1. Nguydn td'c
a amilaza tIIdA) theo Rukhliadeva - Geriacheva.
IL
ST
S-Z-Xfic dinh hoat.dO
TA
.C
O
U
H
M
U
phri"o-nft,
m- Lugng chd phdm enzim (l{y dd thuj
lml dung dich enzim)
ST
.C
m- rtrAi gi"n ttr"l; phan
IL
IE
U
H
U
IL
IE
TA
H
U
ST
.C
O
M
U
ST
dextrin c6 phdn tri luqtng khdc nhau. Do cuirng dQ mhu t4o thinh giea tinh bQt v). c6c sin
78
TA
ST
.C
U
H
M
'
TA
IL
IE
U
L{y ft.:6c dung dich da pha Iodng tr€n lrnl,
hrc v). cho .ri.o c5.c
d;r_q
cho v),o c6c dng nghi€m khO, khuay lien
nghiQm m5i 6ng 5mI NarCO3 0,5VI
vl
1m_}thudc th& foL,n phen rfch_
O
M
L)m m6t mau kitlm ci^-3-.g..thay lmL dich chudn tyroztn bang lml nu6c q{tDd y6n c6c h6n hqrp phan rlng 20 ph(t- Do cuimg d6 m).u cia c6c dung dich tyrozin
cr{.c
dung dich ty.rozin
fi
2 rndu can kh5.c nhau
M
Phii pha
H
IL
IE
U
truc hoinh ghi hlm lugng tyrozin (C) ( prmoVrnl), tn+c hrng ghi m{t dQ quang nrong rrng @)-
TA
EU
H
Ib ti6" henh hai thi nehiem
Theo sdliOu trung binh-thu duoc r& hai'thi nghiQm drrng duhng chudn ryrszh^- Treri
Gil sri, Een duhng chudn O,lprmol tyrozin trong iml cho mit
U
ST
.C
O
song song nhu dd mO ti-tren day.
€ cho 1;51- Nhu v6.y ducrng luong tyrozin
5.1.4- Tinh k6t qui
lpmol tyrozin
IL
I
ST
nn.vi. cuvet dIy icrn)-
=u:u-
(tr,
U
670
.C
v6i thu6c thri folin bdng m6y so mlu quang die,r ddi nguoc v6i mlu kidm chring
sd
dQ quang la
bing 1,51-
HoatdQ yrotraza. (HdP) tinh bimg dulghay dvlml duoc x6c dinh theo c6ng thrlc
TA
0,15i, thi
:
IE
.C
U
O
H
M
U
D'4
' -'TE.lo.m'.tooo
nap
IL
ST
d day : D. M4tdQ quang do duoc cria m6u-
- 2m.I(s)+Lml(E)+ 4nl(TCA) zmt(E)
LI
E
U
H
xnng axit rriclorax 'c
TE
?rgq
TA
I
qu.ang
mi lpnol
lI
mat dQ
tyrozin c6 trong lml dung,.dich.,ch"dr',l*ri phan ftng v&i thudc thri
m- Lugng chd phdm enzim Q{y dd
O
M
ST
.C
Thdi gian thuf phan co ch{t, phrit
thuf phen protein co ch{t (tinh bang mg c6 trong
di-ch enzim)
U
lml dung
4
t"qnq tFozitr xic dinh theci duong,chudn (duong lrqng tyrozin
'folin c6 thd c6 duoc)10-
TA
U
' +-Tj, te grta ttrd tictr cria h6n hqp phan rrng v). thd tich cria dung. dich enzim sau khi bd
IE
.C
O
U
H
M
1000- HQ sdchuyCn s:rng gam chdphdm-
M
t6i cic
vi
cac san
IL
IE
U
H
U
IL
IE
TA
H
U
ST
.C
O
M
U
ST
dextiin c6 phfln t& luqng l*r6c nhau- Do cuirng dQ miu t4o thAnh gifra tinh bQt
O
Phuong phnp dua hOn su thuf phan d4h bQt b6i cic enzim cria hQ amtlaza
.C
U
s.z-l.Nguy6n fiic
TA
IL
ST
i2-Xfrrc dinh hoat.dQ aami|aiza (IIdA) theo Rukhliadeva - Geriacheva.
78
TA
ST
.C
U
H
M
o,l .15-5.i
=
100,100. t5
=
hoa-r
dd' i'{guiii ta xfc dinh ,i-ioc lr:r;ng
(.'.
n^nnns
U,VU\JUJ
M
-fi,
TA
IL
IE
U
lolng v) ding ciich efi-7rn dd pha icang ci6 dd' xdc dfnh
enz-im hong dich thuy phdn (b) le: (tinh bang gam)
g--ti
ng v or
87 1t'g glucoza-'
ST
nhy nron
.C
O
chudn thi dai lugng E
K,\i so m)u dung dich d6, nhdnduoc E=0'173' Thec dulng
U
- //-\
H
Nguyen tdc
U
.
EU
H
IL
IE
U
5.4.1--
Dga ken cd s& x6c.
TA
ST
.C
O
M
phdp vr luong cfra
IrS.+)Xec dinh hoat dQ glucoamilaza. bang'phucrog
r\--l
---:--Rod-z 6.F- Korenbiakina.
di'h ,g tdng ndng dQ glllcoza trong qu6 trinh thuy phln co chft
din d&ng
U
IE
tEo
J
IL
ST
Hd tinh bQt:
.C
Dich chidt enzim
H
M
5"4"2. E{oA chdt
''
O
TA
glucoza'
SdC va pH.4,7 trong rhdi gian I gid ldm gidi ph6ng duoc Img
U
IL
I
vi lugng cfia Redzevichtinh bOL l-ugng gLttcozaduoc x6c dinh bang phuong ph6p
tan Enh bQt tan d nhi6t d6
M6t ilon vi hnat dQ glucoarnilaza Id lttong enzim t6c dung
U
H
U
TA
Felin I :34,64 g CuSOa-5H2O trong 500 rnl
Felin Ii: 173 g KNatactrat + 50g NaOH trong 500 mI'
LI
E
H2SO4:.25Vo
z
30Va
TA
I
KI
Cfch tifu hlmh
ST
.C
5"4.3.
O
M
Nat$Or 0,lN-
a" Ckudn bi di{IE cki6t enzim
3
gam canh trubng nghidn nh6 chuydn
toin
U
M
Can
bQ
vio binh tam gi6c
25O
ml'
thern
O
dQm axetat pH'= 4,7 " Gifr h6n
O
dith hoat.il6 glucoaniiliza
IL
IE
U
H
U
IL
IE
TA
H
U
ST
.C
O
M
U
ST
.C
b. XAc
M
U
TA
IL
ST
IE
.C
U
H
hsp 6 nhiet d6 30oc tong m6y
niy li dung dich enzim g6c'
didu nhi6t I gid, thinh thoang khudy vi sau d6 derri lgc, nu6elgc
c6,thd bio quan dch chi6t enz'imtrong 1 ngiy 6 nhier dQ +zocaen++t;
vio d6 90 mr nu6c cdt vh, 10 ml
TA
ST
.C
U
TA
IL
IE
U
H
M
.
i
mI H,SO* 25Vo
H
khi l).m ngu6i cho vdo
hoi tan CurO vi
IL
IE
U
Sau
dd,
Chudn d6 bang dung dich tioiulfat
natri e.{ar$O3) 0,1N.
thOm
2 rnl Kl3OVo-
TA
song song tidn hinh thi nghi€m kidm
chring trong cDng didu ki6n nhu tr€n (cho
v)o
0,5 ml dich chidt gnzim d6 dun s6i
dd v6 hoar enzim).
Tinh k6t qui
'3'3.6o
m.l0
0,lN
crja m5u kidm chrlng
v) thj nghiQm
O
a: Hi€u sd rnl Nq$O3
U
TA
IL
I
HdG = af
H
5-4.4.
M
EU
H
U
ST
.C
O
M
U
ST
.C
O
M
Cho 1 ml tinh b6r'lVovio binh ram gidc
-50 rnl, rh6m l,_5 mI nu6c cft vh O,_5 m_l dich
chiet ( enzim vi tinn bot phai ad:oor
bong lo phdt cho dn dinh nhiet co tru6c
khi cho vdo,
ddng thdi luqng nu6c v). enzim c6
thd thay deii iu;i 15-o hoar dO enzim).
Od t0ptrut d nhiet d6
3O0c' Dinh chi phin rlng beng
1 mI fetin i vi 1 ml r.ri" u. or"
gui?j1rft (kd rt hic xuft hi€n
bot s6i ddu ti€n), iim ngudi (n€u
kh6n-e c6 rop curo mau d6 & tir{y
binh phai rang iuong
enzim' neu chi c6 curo, mi khdng
c6 mhu xanh cria dung dich felin &
tron chrlng t6 thidu
fetin phdi pha lodng dich enzim g6c)-
U
IE
.C
'
H
i0: Thdi gian
phan rfug,
IL
TA
U
ST
.
K: HQ s6 hi6u chinh n6ng d6 NarSrOr
O,lN3,3: HQ s6 chuydn rhinh dudng
m:.Lugng enzim cho vio phin rfng (gum)-
phft
LI
E
U
60: H€ s6 chuydn thlnh gid-
O
M
TA
I
5'5' x:ic dinh hoat do dudng hori
firdDh) theo phu.ong phrfp iod
5.5.1. Nguy€n tdc
Phuong phrip
ST
.C
ron su thuf phan tirr, uet t"" r"Glia
nan isiii tien
ltei [Eit
rt glu-oti
b&i enzim
x6c
dlnh
lucrng
dubnlltao
thlnh
1i
bdng phuong phr{p iod:
-"a"
' ' Hoht
dQ dudng hor{'ddc u.,m; chd khi,
ning cira
^^rt*u(thuf phan) duirng hod
tinfr bqt roi
duong khr? mantoza
U
H
M
U
du. a
U
ST
dung"l aurrgdich enzim
IL
IE
U
H
U
IL
IE
TA
H
U
ST
.C
O
M
5-5.2.Ho6 ch{t cdn
.C
M
O
U
TA
IL
ST
IE
.C
O
hodc h6n hqp manroza vi glucoza96c
Don vi hoqt d6 &tdng hod td
lrrqng enztm iong nhfrng didu ki€n nhdt dinh d 300c,
sau
I phz'it ntc tdc thui ptuin du.oc tinh bot ddn
9dc abng khir cila I micro duong luqng c6c liEn
kdt glucozit vd bidu rhi bdng
don vil gam-
88
