Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

MC LC
CHNG I: PROTEIN.................................................................................................................................2
BI 1: PHN NG BIURE.........................................................................................................................2
BI 2: KT TA THUN NGHCH BNG MUI TRUNG TNH.........................................................4
BI 3: PHNG PHP SORENSEN.........................................................................................................6
(PHNG PHP XC NH M FORMOL)..............................................................................................................6
BI 4: NH LNGPROTEIN BNG PHNG PHP KJELDAHL..................................................7
(GING TRN M HNH).....................................................................................................................................7
CHNG II: ENZYME................................................................................................................................9
BI 1: NH HNG CA NHIT N HOT TNH CA ENZYME..........................................9
BI 2: NH HNG CA CC CHT HOT HA V KM HM N HOT TNH CA
ENZYME...................................................................................................................................................10
BI 3: XC NH HOT -AMYLASE THEO PHNG PHP WOHLGEMUTH.................11
CHNG III: GLUCIDE...........................................................................................................................13
BI 1: PHN NG VI THUC TH FEHLING..................................................................................13
BI 2: PHN NG SELIWANOFF..........................................................................................................15
BI 3: XC NH NG KH BNG PHNG PHP QUANG PH K....................................17
CHNG IV: VITAMIN............................................................................................................................19
BI 1: PHNG PHP NH TNH VITAMIN A.................................................................................19
BI 2: PHNG PHP NH TNH VITAMIN C.................................................................................20
BI 3: PHNG PHP NH LNG VITAMIN C.............................................................................21
BI 1: S NH TNG HA LIPIDE...................................................................................................23
BI 2: PHN NG X PHNG HA.....................................................................................................24
BI 3: XC NH CH S IOD CA CHT BO.................................................................................25
BI 4: XC NH CH S ACIDE CA CHT BO............................................................................27
BI 5: XC NH LIPIT TNG S.........................................................................................................28
(GING TRN M HNH)...................................................................................................................................28

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Chng I: PROTEIN
Bi 1: PHN NG BIURE
Nguyn tc:

y l phn ng c trng ca lin kt peptide (CO-NH-)

Trong mi trng kim, cc hp cht c cha t hai lin kt peptide tr ln c

th phn ng vi CuSO4 to thnh phc cht mu xanh tm, tm, tm hay .

Cng mu thay i ty thuc vo di mch peptide

Phn ng ny thng thng cng dng nh lng protein

Nguyn liu v ha cht:

Dung dch lng trng trng 1%

Ure tinh th
Dung dch NaOH 10%
Dung dch CuSO4 1%

Cch tin hnh & kt qu:

iu ch biure: cho vo ng nghim kh mt t tinh th ur, ung trn ngn la yu.
Lc u ur nng chy, n khi bt u cng li th ngng un. Qu trnh ny to ra
biure, acide cianuric v amoniac
Dng 2 ng nghim:

ng nghim

Cho vo mi ng nghim 1ml dd NaOH 10% v

1-2 git CuSO4 1%

ng 1 (ng biure)

Dung dch tm

ng 2 (3ml dd lng trng

trng 1%)

Dung dch mu tm

PTP:
(CO-NH-)+ Cu(OH)2
Ch khng cho qu nhiu CuSO4 v mu xanh ca Cu(OH)2 c to thnh
Gii thch:

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Thuc th ca phn ng mu biure l dung dch NaOH v CuSO4, i khi n cn

c gi l tc cht biure
Tuy nhin ch biure y khng phi l thuc th c cha biure m l v c biure
v protein u cho phn ng mu ging nhau vi NaOH v CuSO4, Cng mu
thay i ty thuc vo di mch peptide
Phn ng so mu Biure, lin kt peptide vi Cu2+ trong MT kim, l phn ng c
trng nhn bit protein

Nguyn tc cc phn ng mu khc ca protein:

Phn ng xantoproteic: c trng vi cc axit amin vng thm. Cc gc
amino acid Tyr,Trp,Phe trong protein tc dng vi HNO3 c to thnh mu
vng v sau khi thm kim s chuyn thnh da cam
Phn ng ninhydrin: c trng vi cc - axit amin.
Phn ng Pholia: c trng vi cc axit amin cha lu hunh
Phn ng Millon : pht hin Tyrosin. gc Tyr tc dng vi thy ngn
nitrate trong HNO3 c to thnh kt ta mu nu t
Phn ng Folin: pht hin Tyrosin, Tryptophan.
Phn ng Sakaguchi: pht hin Arginin. Gc Arg tc dng vi dung dch
kim ca -naphtol v hypobromite cho mu anh o

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 2: KT TA THUN NGHCH BNG MUI

TRUNG TNH
Nguyn tc:
Cc mui ca kim loi kim v kim th (thng dng l (NH4)2SO4 , Na2SO4,
NaCl, MgSO4) c tc dng gy kt ta thun nghch protein. Sau nu loi b nhanh
cc yu t gy kt ta ,protein tr v trng thi dung dch keo bn.
Cc protein khc nhau c th b kt ta v cc nng mui khc nhau,v vy c
th dng mui tch ring cc protein ra khi hn hp ca chng.

Nguyn liu v ha cht:

Dung dch lng trng trng khng pha long.

Dung dch (NH4)2SO4 bo ha .
Tinh th NaCl.
Nc ct.

Cch tin hnh & kt qu:

1) Cho vo ng nghim 3ml lng trng trng + 3ml dd (NH4)2SO4 bo ho, lc u n
khi dung dch bn bo ho v xut hin kt ta globulin. 5 pht, lc b kt tu ( thm
t giy lc bng dd (NH4)2SO4) vo mt ng nghim khc .
Cho vo dch lc 3g tinh th (NH4)2SO4 (nu dch lc l 4ml) cho n khi dung dch bo
ho, lc, albumin kt ta,lc, thu kt ta
Cho ring kt ta globulin v albumin vo 2 ng nghim tng ng, thm vo 2 ng t 23ml nc ct, lc u.
Kt qu:
ng 1: tc l ng cha kt ta globulin ,sau khi cho nc ct vo th kt ta tan
t t trong nc ct .
Do Globunlin vn dng kt ta , l do Globulin b bin tnh ,khng
quay v trng thi dung dch keo ngay c ( hin tng kt ta khng
thun nghch ).
ng 2 : tc l ng cha kt ta albumin,sau khi cho nc ct vo th kt ta tan
ngay trong nc ct.
Do Albumin tr v trng thi dung dch keo nh ban u, kt ta tan hon
ton (hin tng kt ta thun nghch).
2)Cho vo ng nghim 3ml lng trng trng , cho tip NaCl vo ,dng a thu tinh
khuy cho n khi dung dch bo ho. yn 5 pht th thy xut hin kt ta a . Lc kt
ta sau acid ho phn dch lc bng cch thm vo 1 git CH3COOH 1% ,th thy
xut hin kt ta b.
Kt qu:
Kt ta a l Globulin.
Kt ta b l Albumin.

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Lp K14S1_Nhm 2_t 5

Gii thch :
Mi loi protein kt ta cc nng khc nhau .Khi cho NaCl vo ,u tin
Globulin kt ta trc .Sau lc ht kt ta ,cho vo dch lc CH3COOH 1%, pH
gim lm xut hin kt ta Albumin.

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 3: PHNG PHP SORENSEN

(Phng php xc nh m formol)
Nguyn tc:
Cc amino acide c th phn ng vi formol trung tnh to thnh dn xut
metylenic.Kt qu l nhm amin mt tnh cht c bn ca n ,ngc li nhm
cacboxyl trong amino acide tn ti dng metylen t do c th chun vi NaOH .
Phng php c ng dng trong trng hp nhm cacboxyl v nhm amin t
do bng nhau.

Nguyn liu v ha cht:

khi dng).

Dung dch formol (pha formol : nc ct t l 1:1 v ch chun b trc

Nc mm,nc ct.
Dung dch NaOH 0,1N.
Thuc th Phenolphtalein 3%.

Cch tin hnh & kt qu:

Ly 2 bnh nn v nh du bnh 1 v bnh 2
Bnh 1 : Dng lm bnh kim tra. Cho vo 50ml dd formol v thm vo 3
git phenolphthalein 3%.Sau dng dd NaOH 0,1N hiu chnh mu trong
bnh cho n khi ch cn mu hng nht. Ta c formon trung ha
Bnh 2 : Dng lm bnh th nghim. Cho vo 10ml dch mu ( c pha long
t 5 n 20 ln ), thm 10ml dd formol trung ho v 3 git phenolphthalein
3%, lc u cho phn ng xy ra hon ton. Sau chun bng dd NaOH
0,1N cho n khi dd trong bnh 2 c mu hng (m hn mu bnh 1)
Thc hin 3 th tht v 3 th khng(thay dd m bng nc ct), ly tr s trung bnh:

Ln 1
Ln 2
Ln 3
Trung bnh

Th khng

Th tht

0,4
0,5
0,4
V1= 0,45ml = 0,00045 l

10,6
10,7
10,5
V2= 10,6ml = 0,0106 l

Lng m formol c trong 1l nguyn liu :

M(g/l) = 1,4 ( V2 V1 )/a
Trong :
V1 l th tch NaOH 0,1N trung bnh ca 3 ln th khng
V2 l th tch NaOH 0,1N trung bnh ca 3 ln th tht
a l lng mu (ml) ng vi 10ml dch mu pha long x ln (a= 1 ml =
0,001 l).
M(g/l) = 1,4 (0,0 106 0,00045 )/0.001 = 14,21

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 4: NH LNGPROTEIN BNG PHNG

PHP KJELDAHL
(Ging trn m hnh)
Nguyn tc:

Di tc dng ca H2SO4 c nhit co ,cc hp cht hu c c cha

nit b phn hy v b oxy ha n CO2 v H2O ,cn nit chuyn thnh ammoniac v
tip tc kt hp vi H2SO4 to thnh mui amoni sulphate . Qu trnh c tin hnh
theo cc bc sau :

V c ha mu :
R-CHNH2 COOH + H2SO4 CO2 + H2O + (NH4)2SO4

Ct m :
(NH4)2SO4 + 2 NaOH Na2SO4 + 2NH3 + 2H2O

Sau lng NH3 c hi nc li cun bng mt dng c l my

Parnas Wargner (my chng ct m )v c dn n mt bnh tam gic c cha
mt lng thi H2SO4.T y cho php chng ta xc nh c lng NH3 phng
thch ra ,c ngha l xc nh c lng m c trong mu nguyn liu :
NH3 + H2SO4 (NH4)2SO4 + H2SO4d

Chun H2SO4 d bnh hng bng dung dch NaOH 0,01N

Nguyn liu v ha cht:

Vt phm c cha Nit ( nc mm ,u h ,cc loi thc phm )

H2SO4m c.

K2SO4 tinh khit.

CuSO4 tinh khit.

Se.

NaOH 40%.

H2SO4 0,1N : 2,8ml H2SO4 m c (d=1,84/lt).

NaOH 0,1N : 4g NaOH tinh th/lt.

Phenolphtalein 1% trong cn .

HCl long .
(Nn dng ng chun chun b H2SO4 v NaOH 0,1N)

Cch tin hnh & kt qu:

u tin ht 1ml (i vi nguyn liu lng ) hay cn chnh xc 1g (i vi

nguyn liu kh nghin nhuyn ) cho vo bnh Kjendanl ,cho thm 10ml H 2SO4
m c (d=1,84). tng qu trnh v c ha (t chy) cn cho thm cht xc
tc.Tt nht l dng 0,5g hn hp K2SO4 : CuSO4 : Se (100:10 :1). C th dng mt
mnh Se kim loi 0,05g hoc dng hn hp CuSO4 v K2SO4 hoc acid perchloric.
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Lp K14S1_Nhm 2_t 5

Hn hp xc tc c tc dng lm tng nhit si,do lm tng vn tc ca qu

tnh phn ng. Sau khi thm cc cht xc tc un nh hp cho n khi dunh dch hon
ton mt mu. Ch un mnh khi hn hp hon ton chuyn sang dch lng. Trong
qu trnh un thnh thong lc nh,trng kho lo sau cho khng cn mt vt en no
ca mu nguyn liu th nghim cha b phn hy st li trn thnh bnh .un cho ti
khi dung dch hon ton trng.

Sau chuyn ton

b dung dch sau khi v c
ha xong bnh Kjendahl vo
bnh nh mc 100ml ,thm
nc ct cho n ngn chia,lc
u .Dng c ct trc khi s
dng phi ra sch : ly vo
bnh tam gic 10ml H2SO4
0,1N ,thm vi git ch th
phenolphthalein v lp vo
my ct nh m hnh . un si
nc trong bnh cu to hi
nc ,m nc v ng sinh hn
.Sau qua phu cho vo bnh
ct 10ml dd th nghim t bnh
nh mc v tip 8-10ml
dung dch NaOH 40% .Trng phu bng mt lng nh nc ct ,ri ng kha .Hi
nc t bnh cu sc qua bnh phn ng v ko theo NH3 sang bnh hp ph . Qu
trnh ct kt thc sau 15 pht .nh phn lng H2SO4 d bng dung dch NaOH
0,1N.

Sau khi ly bnh hp ph t bnh nc ct vo ,ng kha,ng thi

m kha bnh ra .Dung dch chuyn t bnh ct sang bnh ra ,tho b dung dch
bn i .Sau khi th nghim xong phi ra sch my ct vi lng 1 ln bng HCl long
v nhiu ln bng nc ct .
Tnh kt qu :
Hm lng nit tnh theo cng thc sau :
X =

( a b). 0,0014 .10000 .100

10 .V

X: hm lng nit (g/l)

a: s mol H2SO4 0,1N em hp th NH3
b: s mol NaOH 0,1N tiu xchun tiu tn cho chun s
V: s ml phm vt em v c ha
0,0014: lng g nit ng vi 1ml H2SO4 0,1N

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Chng II: ENZYME

Bi 1: NH HNG CA NHIT N HOT
TNH CA ENZYME
Nguyn tc:
Nhit c nh hng quan trng n tc phn ng ca enzyme. S tng tc
phn ng ca enzyme bng cch nn nhit ch c hiu qu trong mt khong
nhit tng i hp
Nhit ti thch ca phn ln cc enzyme l trong khong 40-60 0C. nhit
trn 800C hu ht cc enzyme b bin tnh khng thun nghch

Cch tin hnh & kt qu:

Chun b 4 ng nghim, cho vo mi ng 2ml dd tinh bt 1%
ng 1 : gia nhit 85-900C
ng 2 : cho vo nc
ng 3 : t m 45-500C
ng 4 : nhit phng

Gi cc ng trong 15 pht dd trong ng t n cc nhit

tng ng

Tip tc cho vo mi ng 0,5ml dd amylase cng t cc

nhit trn v t cc ng nghim cc nhit c

Sau 1, 2, 4, 6, 8 v 12 pht ly t mi ng 1 git, nh vo cc

git thuc th Lugol chun b sn trn bn knh. Quan st mu to thnh

1 pht
2 pht
4 pht
6 pht
8 pht
12 pht

ng 1

ng 2

ng 3

ng 4

Xanh en
Xanh en
Xanh en
Xanh en
Xanh en
Xanh en

Tm en
en nht
Nu en
Nu en
Nu t
Nu en

en
Tm en
Nu nht
Nu nht nht
Nu nht hn
Nu rt nht

en m
Nu t
Nu t nht
Nu t nht
Nu t nht hn
Mu ca lugol

Qua bng trn ta thy : trong ng 1 v ng 2 th mu dd tinh bt thay i khng ng

k, trong ng 3 v ng 4 mu ca dd tinh bt thay i nh enzyme thy phn
Gii thch :
Nhit c nh hng quan trng n tc phn ng ca enzyme. Nhit ti
thch ca phn ng enzyme nm trong phm vi 40-500C v c th bin i ph thuc vo
nhiu yu t nh sch ca enzyme, thi gian phn ngNhit m enzyme b mt
hon ton hot tnh xc tc gi l nhit ti hn, thng vo khong trn 700C. nhit
ti hn, enzyme b bin tnh, t khi c kh nng c hi phc li hot . Ngc li,

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

di 00C, hot enzyme tuy b gim nhng li c th tng ln khi a v nhit bnh
thng

Bi 2: NH HNG CA CC CHT HOT HA

V KM HM N HOT TNH CA ENZYME
Nguyn tc:
Cht hot ha c kh nng lm tng cng tc dng ca enzyme. Cht km hm l
gim i lc ca enzyme vi c cht hay lm enyme mt kh nng kt hp vi c cht

Cch tin hnh & kt qu:

Chun b 3 ng nghim:

ng nghim

Sau cho vo mi ng 1ml nc bt v 1ml dd tinh

bt, lc u. Sau vi pht, thm vo mi ng vi git
thuc th Lugol, lc u

ng 1

1ml nc ct

Mu vng hi m

ng 2

1ml dd NaCl 1%

Mu vng nht hn

ng 3

1ml dd CuSO4

Mu xanh en

Kt qu:
Ta thy enzyme ha tan tt trong nc, trong dung dch mui long nhng khng tan
trong dung mi khng phn cc

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Lp K14S1_Nhm 2_t 5

Bi 3: XC NH HOT -AMYLASE THEO

PHNG PHP WOHLGEMUTH
Nguyn tc:

Cc amylase l cc enzyme thy phn tinh bt, c 2 loi l -amylase v

-amylase. S tc dng ca hn hp -amylase v -amylase trn tinh bt cho ta
hn hp cng lc cng nhiu glucose, maltose v dextrin n gin

xc nh hot ca enzyme, ta xc nh nng enzyme thp nht c

kh nng gy thy phn hon ton tinh bt cho cc sn phm khng i mu dd iode.
l nguyn tc ca phng php Wohlgemouth

n v Wohlgemouth l lng enzyme cn thit thy phn hon ton

1mg tinh bt 37oC sau 30 pht c Cl- lm cht hot ha

Nguyn liu v ha cht:

Dung dch amylase (dd nc bt pha long 10 ln) => nc bt ca bn n

Dung dch tnh bt 0,1%
Dung dch NaCl 0,9%
Dung dch Iode 0,02N
Nc ct

Cch tin hnh & kt qu:

Chun b 10 ng nghim, nh s t 1-10,cho vo mi ng 1ml dd NaCl 0,9%. Trong
ng nghim 1 cho vo 1ml dch enzyme, lc u. Sau ly 1ml t ng nghim 1 cho
vo ng nghim 2, lc k v lp li cho ti ng nghim 10 th ht 1ml v b i. Trong
mi ng nghim cho vo 2ml dd tinh bt 0,1%, lc u v vo t m n nhit 37 oC
trong 30pht. Khi thi gian ht, lm ngui cc ng n nhit phng v cho vo mi
ng 2 git dd Iode 0,02N v lc u

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Lp K14S1_Nhm 2_t 5

Kt qu:
Ta thy 6 ng nghim u thy phn hon ton. ng 7 phn hy c , cn 3 ng
cn li hon ton khng b phn hy
Hot enzyme:
X= 2n *2
(trong n l s th t ng nghim c nng enzyme thp nht v c thy phn
hon ton)

X= 26 *2=128
Gii thch:
Khi chng ta pha long nc bt 10 ln v dch enzyme trong nc bt li c pha
long n 10 ln tip theo (qua 10 ng nghim) th n ng nghim 7, lng tinh bt cn
rt t nn ch thy phn c . n 3 ng nghim cui (ng nghim 8,9,10), lng
enzyme khng cn nn khng th thy phn c tinh bt
Vai tr ca NaCl trong th nghim: Cl- trong NaCl lm cht hot ha
enzyme

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Lp K14S1_Nhm 2_t 5

Chng III: GLUCIDE

Bi 1: PHN NG VI THUC TH FEHLING
Nguyn tc:
Do c cha chc aldehyde hoc ceton cho nn cc monosaccharide c tnh
kh v c gi l ng kh. Nu 2 monosaccharid kt hp vi nhau
bng 2 hydroxyl glucoside th disacccharide to thnh b mt tnh kh.
Cn nu nhm OH glucoside ca nhm ny lin kt vi nhm OH alcol
ca monosaccharid kia th disaccharid to thnh vn cn tnh kh
Khi un ng kh vi dd thuc th Fehling th kt ta ca Cu2O hnh
thnh ( do ng kh kh Cu(OH)2 c trong Fehling thnh CuO2)

Nguyn liu v ha cht:

Dung dch Glucose 1%

Dung dch Mantose 1%
Dung dch Saccharide 1%
Thuc th Fehling A (CuSO4)
Thuc th Fehling B (Seignet + NaOH)

Cch tin hnh & kt qu:

Ly 3 ng nghim, nh s t 1-3 nh sau :

ng 1 : cho 2ml glucose 1% + 1ml Fehling A + 1ml Fehling B + un

trong 5 pht
- Kt qu: c kt ta gch ti ( Cu2O )
- PTP:

ng 2 : cho 2ml maltose 1% + 1ml Fehling A + 1ml Fehling B + un

trong 5 pht
- Kt qu: c kt ta gch ti ( Cu2O )
- PTP:

ng 3 : cho 2ml saccharide 1% + 1ml Fehling A + 1ml Fehling B + un

trong 5 pht
- Kt qu: khng hin tng
- PTP:
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Lp K14S1_Nhm 2_t 5
Gii thch:
Thuc th Fehling l hn hp 2dd : dd
CuSO4 v dd mui Seignet vi NaOH. Khi trn
2 dd trn vi nhau th xy ra phn ng :
CuSO4 + 2 NaOH Cu(OH)2 + Na2SO4
Sau dd CuSO4 tc dng vi mui seignet
to mui phc ha tan, dd c mu xanh thm
Mui phc trn l hp cht khng bn
Trong mi trng kim, cc mono v 1 s
dixacarit kh Cu2+ di dng alcolat ng thnh
Cu2+, chc aldehit b oxi ha thnh axit hoc
mui tng ng
Do glucose v maltose c tnh kh nn khi
un vi dd thuc th Fehling th kt ta ca
Cu2O hnh thnh ( do ng kh Cu(OH )2
c trong Fehling thnh CuO2 )

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Bi 2: PHN NG SELIWANOFF
Nguyn tc:

Khi trn dd acid ascorbic vi dd xanh metylen th dd s mt mu. Phn

ng xy ra do acid ascorbic b oxi ha thnh acid dehidro ascorbic v xanh metylen
tr thnh dng khng mu

Nguyn liu v ha cht:

Dung dch fructose 1%

Dung dch glutose 1%
Dung dch NaCl 1%
Thuc th Seliwanoff

Cch tin hnh & kt qu:

Cho vo mi ng nghim 2ml thuc th Seliwanoff v thm vo :
ng 1 : 2ml dd fructose 1%
ng 2 : 2ml dd glucose 1%
ng 3 : 2ml nc ct

Tt c un cch thy trong 5 pht

Kt qu

ng 1

C mu anh o ti

ng 2

Mu vng nht

ng 3

Khng hin tng

Gii thch :
Phn ng seliwanoff c trng i vi cc fructose v cc cetohexose nhng khng
nhy i vi cc aldohexoz

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Bi 3: XC NH NG KH BNG PHNG
PHP QUANG PH K
Nguyn tc:

Cc loi ng kh c kh nng kh Ferricyanur (Fe3+) thnh Ferrocyanur

(Fe2+) ion ny c mu xanh m (xanh prusse). Do ta c th dng phng php so
mu nh lng ng

Chun b mu:

1g nguyn liu thm + H2SO4 10% (ngp mu), un cch thy khong 1h,
ly ra cho NaOH vo trung ha n pH=7, lc nu c cn ta c dung dch mu

Ly 1ml dd mu pha long thnh 30-100ml dd dng xc nh ng

kh

Chun b mu:

1g nguyn liu thm + H2SO4 10% (ngp mu), un cch thy khong 1h,
ly ra cho NaOH vo trung ha n pH=7, lc nu c cn ta c dung dch mu

Ly 1ml dd mu pha long thnh 30-100ml dd dng xc nh ng

kh

Cch tin hnh & kt qu:

Thc hin 9 ng nghim theo mu sau:
S ng
ml glucose 0,02mg/ml

1
0

2
0

ml nc ct

ml ng nh nng
Nng trong mi ng
chun ( g/ml)

3
0,0
5
0,9
5

4
0,1

5
0,2

6
0,4

7
0,6

0,9

0,8

0,6

0,4

12

1
17,994

2
20,337

Cho vo mi ng 1ml thuc th A (Na2CO3 + KCN/H2Oct) v 1ml thuc th

B(K3Fe(CN)6/ H2Oct), lc u, un si cch thy 15 pht
Lm lnh, thm 5ml thuc th C (Fe[NH4(SO4)2] + Lauryl sulfat/H2SO4), lc u,
yn 15pht
o Mode Absorbance vi di sng 690nm

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Lp K14S1_Nhm 2_t 5

th theo nng ng v hp thu

1.6
1.4

hp thu

1.2
1
0.8
0.6
0.4
0.2
0
0

10

15

20

25

Nng

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Chng IV: VITAMIN

Bi 1: PHNG PHP NH TNH VITAMIN A
Nguyn tc:
Di tc dng ca H2SO4 m c , Vitamin A b mt nc v to thnh sn phm
ngng t c mu khng bn .Sau bin i nhanh thnh mu c trng ca liporom
(nu en).

Cch tin hnh & kt qu:

Nh 1 git du c ha tan trong 25

git Cloroform, sau cho 1 git H2SO4 vo
ri lc u

Do khi tc dng vi H2SO4, vitamin

A trong du c b mt nc to thnh sn phm
ngng t c mu hng khng bn, sau bin
i nhanh thnh mu nu en

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Bi 2: PHNG PHP NH TNH VITAMIN C

Nguyn tc:
Khi trn dd acid ascorbic vi dd xanh metylen th dd s mt mu .Phn ng xy ra do
acid ascorbic b oxy ho thnh acid dehydro ascorbic v xanh metylen tr thnh dng
khng mu

Nguyn liu v ha cht:

Dd Vitamin C.
Xanh metylen 0,01%,
Dd NaOH 5%.

Cch tin hnh & kt qu:

Chun b 2 ng nghim:
ng 1: 1ml dd Vitamin C

Thm vo 2 git xanh

T mu xanh trong sut
metylen, lc u
ng 2: 1ml nc ct
Thm vo 3 git NaOH 5%, Vn gi nguyn mu trong
lc u
sut
Gii thch: Do Vitamin C lm mt mu dd xanh metylen

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Bi 3: PHNG PHP NH LNG VITAMIN C

Nguyn tc:
xc nh hm lng Vitamin C (Acid ascorbic) trong nguyn liu bng phng
php chun iod ngi ta cho tt c acid ascorbic b oxy ho bi iod , phn iod cn tha
s cho mu xanh vi dung dch tinh bt . iu ni ln phn ng kt thc

Nguyn liu v ha cht:

dd HCl 5%.
dd I2 0,01N.
dd tinh bt 1% .
Chanh tri ,bi ,cam.

Cch tin hnh & kt qu:

Ly 10g tht tri chanh v 20ml HCl 5% vo ci s gi ,nghin nh cho n
khi dung dch c dng ng th, dng nc ct chuyn ton b dch chit ng th
vo trong bnh nh mc 100ml, thm nc ct n vch 100ml. Lc u cho lng
axit ascorbic c trong nguyn liu c ho tan hon ton, lc cn vo trong bnh
tam gic 250ml, ta c dung dch mu hay dd nguyn liu
Ht 20ml dd nguyn liu c lc cn vo trong bnh nn, thm5-10 git h
tinh bt 1%
Dng I2 0,01N chun cho n khi dung dch bt u xut hin mu xanh lam
nht l c
Lp li chun 3 ln, ly kt qu trung bnh ta c V(ml) dd I 2 0,01N dng
chun Vitamin C

Chun bng dd I2 0,01N

Ln 1
Ln 2
Ln 3
Trung bnh

Trong :

X(%) =

0,5
0,5
0,6
V= 0,533ml

Hm lng Vitamin C : X(%) =

V .V1 .0,00088 .100

V2 .m

m : lng mu th nghim (5gr)

0,00088 : s gram acid ascorbic ng vi 1ml dd I2 0,01N.
V1 : th tch dd mu (50ml).
V2 : th tch dch mu ly xc nh ( 20ml).
V: s ml Iode 0,01N dng chun
0,533 .50 .0,00088 .100
= 0,023452 %
20 .5

Phn ng oxy ho acide ascorbic :

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Lp K14S1_Nhm 2_t 5

It tng i khng tan trong nc, nhng iu ny c th ci

thin bng cch pha trn it vi ioua v hnh thnh triioua: I 2 +
I

I3

Triioua oxy ha vitamin C to acid dehydroascorbic:

C6H8O6 + I3 + H2O C6H6O6 + 3I - + 2H +

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Chng V: LIPIDE
Bi 1: S NH TNG HA LIPIDE
Nguyn tc:

S nh tng ho lipide trong c th l giai on u ca s hp th

chung.

gi s cn bng gia mi trng phn ho ( H2O ) vi pha phn tn

(lipide) th phi cn c cht gy nh tng ( natri carbonat , protein, x phng , mui
mt,.)

Nguyn liu v ha cht:

Du lc ( du u phng ).
Gelatin 2%.
R ng

Cch tin hnh & kt qu:

Chun b 2 ng nghim sch ,, lau kh , v tin hnh nh du ng nghim thnh
ng 1 v ng 2.
Tip theo cho vo c 2 ng 0,5ml du lc .
Sau cho vo mi ng :
ng 1 : 0,5ml gelatin 2% .
ng 2 : 0,5ml nc mt r ng .
Lc mnh c 2 ng .
* Kt qu :

ng 1

ng 2

Kt qu

Gii thch

To thnh 2 lp
,lp du ni ln
trn.(l nhng
ht du khng
nhp vo nhau
ni ln trn).
Chuyn thnh
trng c ,
mt thi gian s
phn lp .

Bn cht l protein
nh tng mnh hn r
ng

- 2010 -

Cho mt r ng vo th
do trong r ng do c
mui mt l cht gy nh
tng nn xy ra hin
tng nh tng nhng
yu hn ng 1

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Bi 2: PHN NG X PHNG HA
Nguyn tc:

Ch s savon ha l lng mg KOH cn trung ha acide bo t do cng

nh lin kt c trong 1g cht bo

Di tc dng ca kim, glyxerit b thy phn to thnh glyxerin v x

phng ( mui Natri hoc Kali ca acid bo )

Cch tin hnh & kt qu:

Cho vo cc s 0,5ml du lc + 10 ml KOH 30%

Khy u v un cch thy cho n khi qunh li th ta c x phng,

nu cho nc vo lc ln s thy xut hin bt x phng

Gii thch:
Di tc dng ca enzyme, axit hoc kim, un nng, m b thy phn to thnh
glixerin v axit bo hoc mui ca axit bo bc cao gi l x phng
Phn ng nh sau :

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Bi 3: XC NH CH S IOD CA CHT BO
Nguyn tc:

Ch s iod ca cht bo l s gram iod kt hp vi 100gr cht bo

Xc nh ch s iod l da trn kh nng ca iod kt hp c vi acide

bo ch nhng lin kt i, v mi ni i ca lipit s cho phn ng cng vi 2
nguyn t ca nhm halogen. Do , nu ta cho cht bo tc dng vi mt lng tha
iod v xc nh lng tha y gip ta suy ra ch s iod

Nh vy ch s iod xc nh tng qut cc acid bo khng no trong cht

bo

Nguyn liu v ha cht:

Du n
Cn 96o
Dd I2 0,1N
Dd Na2S2O3 0,1N dng chun
Dd tinh bt 1%

Cch tin hnh & kt qu:

Ly 6 bnh tam gic:
Bnh 1,2,3 (mu trng):1ml nc ct
Bnh 4,5,6 (mu nguyn liu): 12g du n
Thm vo mi bnh 10ml cn 96o lc u, thm 10ml I2 0,1N. Sau dng giy
bt kn ming bnh tam gic trnh bay hi I2

1ml H2O ct
Bnh

(1)

(2)

2g Du n
(3)

(4)

(5)

(6)

yn trong ti t 10 15 pht, ri sau chun bng Na2S2O3 0,1N n khi

dung dch c mu vng nht
Na2S2O3 0,1N dng chun
(ml)

9,7

9,7

9,8

9,7

9,6

9,7

Thm vo mi bnh 1ml dung dch tinh bt 1%, v tip tc chun n khi mt
mu xanh
Na2S2O3 0,1N dng chun
(ml)

0,4

0,3

0,4

0,5

0,3

0,4

Tnh ch s iod:
a: s ml Na2S2O3 0,1N dng chun mu trng
9,7 + 9,7 + 9,8 0,4 + 0,3 + 0,4
+
= 10 ,1 (ml)
3
3

a =

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

b: s ml Na2S2O3 0,1N dng chun mu nguyn liu

9,7 + 9,6 + 9,6 0,5 + 0,3 + 0,4
+
= 10,06667 (ml)
3
3

b =

m: trng lng mu tnh bng gram

0,0127: s gram iod ng vi 1ml Na2S2O3 0,1N
Ch s I2 =

( a b). 0,0127 .100

(10 ,1 10 .06667 ). 0,0127 .100
=
= 0.020955
m
2

Gii thch :

Ch s iod cho ta bit mc cha no ca cc acid bo c trong thnh

phn ca cht bo

Da vo phng php tha tr, tc l cho 1 lng d iod phn ng cng

vi du bo, ri i chun iod cn li nh vo Na2S2O3, suy ra lng iod cng
vo du bo.

Ch s iod cng cao chng t cht bo cng lng v kh nng b oxi ha

cng nhanh

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Lp K14S1_Nhm 2_t 5

Bi 4: XC NH CH S ACIDE CA CHT BO
Nguyn tc:

Ch s acide ca cht bo l s milligram KOH cn thit trung ho ht

nhng acid bo t do cha trong 1g cht bo.

Dng KOH 0,1N trung ho cc acide bo t do trong nguyn liu vi

phenolphtlein lm cht ch th mu .

Nguyn liu v ha cht:

Du n tinh luyn ,
Cn tuyt i 980 .
Dd KOH 0,1N chun .
Ch th phenolphthalein 1%

Cch tin hnh & kt qu:

Trong bnh tam gic 250ml, cn khong 5g du n v thm vo 50ml cn 980
ho tan du n .Sau em i un nh 600C
Chun nhanh vi dd KOH 0,1N khi dd trong bnh tam gic cn hi m , thm
3 git ch th phenolphthalein, chun n khi dd trong bnh tam gic c mu hng
nht bn trong 30 giy
Lm lp li th nghim 3 ln ly th tch trung bnh

Chun bng dd KOH 0,1N

Ln 1
Ln 2
Ln 3
Trung bnh

34,5
35
34
V= 34,5ml

Ta c: 5,61 l s mg KOH ng vi 1ml KOH 0,1N

V : s ml KOH 0,1N dng chun .
m : trng lng cht bo cn (5000mg)
Ch s Acid =

5,61 .V
5,61 .34 ,5
=
= 0,38709
m
5000

Gii thch :

Ch s acid: l s mg KOH cn thit trung ha cc acid bo t do c

trong 1g cht bo.

Ch s acid cho bit c cht lng cht bo, ch s acid cng cao chng
t cht bo khng ti, b phn hy v b oxi ha mt phn

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Bi 5: XC NH LIPIT TNG S
(Ging trn m hnh)
Nguyn tc:

Dng dung mi k nc trch li hon ton lipide t nguyn liu c

nghin nh. Mt s thnh phn ha tan trong cht bo cng c trch li theo, bao
gm sc t, cc vitamin tan trong cht bo, cc cht mi tuy nhin hm lng ca
chng thp. do c ln tp cht, phn trch li c gi l lipide

C 2 phng php xc nh :

Phng php xc nh trc tip : chit xut lipide ra khi nguyn liu v
cn trc tip

Phng php xc nh gin tip : chit xut lipide ra khi nguyn liu v
cn li nguyn liu

Nguyn liu v ha cht:

Dung mi chit xut lipide thng dng l ether etylic

Nguyn liu c nghin nh ,sy kh n khi lng khng i.

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Lp K14S1_Nhm 2_t 5

Thit b Soxhlet :

Cch tin hnh & kt qu:

Chun b ti bng giy lc ng nguyn liu hoc dng ng hnh tr

ng mu c sn ( ti giy lc c ct hnh ch nht, chiu di gp 2,5 ln chiu
rng, gp thnh ti tr c ng knh b hn tr chit). Ti c sy kh n khi
lng khng i v c cn trn cn phn tch ( nu xc nh theo phng php
gin tip )

Cn chnh xc 2-5 g ri cho mu vo ti giy, gp kn mp ti, t ti c

mu phn tch vo tr chit

Phng php xc nh trc tip

+

Trc khi chit, bnh cu c sy kh n kh lng khng i

t bnh cu ln ni cch thy v cho ete vo th tch bnh

Cho ti nguyn liu vo tr chit

Lp tr chit vo bnh cu

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+ Cho dung mi vo tr chit n ngp ti nguyn liu. Mc dung mi n

phn trn ng xifon tr chit
+

Lp ng sinh hn, ngm nguyn liu trong dung mi mt vi gi

t my Soxhlet vo ni cch thy sao cho s ln dung mi rt t tr

chit xung bnh cu khong 10-15 ln trong 1 gi

Th lipide chit ht cha bng cch ly mt vi git ete t u cui tr

chit cho ln a knh ng h sch, cho bay hi ht ete, nu khng c
lipide trn a knh, xem nh lipide c chit hon ton

Khi chit xong, ly bnh cu ra, lp ng sinh hn vo v ct ly ete

Sy bnh cu c cha lipide ( 60-700C trong 30 pht ) n khi lng

khng i ri em cn

Tnh kt qu

Hm lng lipide c trong 100g mu nguyn liu:

X : hm lng lipide tnh theo %
a : khi lng bnh v lipide (g)
b : khi lng bnh (g)
c : khi lng mu tch lipide (g)
Phng php xc nh gin tip :
(a b). 100
X =
c

Sau khi kt thc th nghim nh trn, ly ti mu nguyn liu ra khi bnh

chit, cho bay hi ht dung mi, sy kh n trng lng khng i

Tnh kt qu

Hm lng lipide c trong 100g mu nguyn liu :

X =

(a b). 100
c

X : hm lng lipide tnh theo %

a : khi lng ti mu nguyn liu trc khi chit (g)
b : khi lng ti mu nguyn liu sau khi chit (g)
c : khi lng nguyn liu ly xc nh cc ch s ca cht bo

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