TRNG I HC BCH KHOA

THNH PH H CH MINH
KHOA K THUT HO HC
NGNH CNG NGH SINH HC

TH NGHIM HO SINH
BI 7

NH LNG NITO TNG THEO PHNG

PHP KJELDAHL

GVHD: HUNH NGC OANH

Nhm TN: Nhm 4_ chiu th 6
Ngy TN
: 26/03/2010
TRN TH KIM DUNG
60700358
NGUYN TH HNG H 60700701
NGUYN TH HI
60700678

BI 7a:
THC HIN CHNG CT M BNG B
CHNG CT KJELDAHL
1. NGUYN TC
Di tc dng ca H2SO4 c nhit cao, cc hp cht c cha nito b
phn hu v oxy ho n CO2 v H2O cn nito chuyn thnh amoniac
(NH3) v tip tc kt hp vi H2SO4 to thnh mui amoni sulfat
Qu trnh c thc hin theo cc bc sau:
Bc 1: V c ho nguyn liu
R-CH-COOH + H2SO4
NH2

CO2+H2O+(NH4)2SO4

Xc tc

Bc 2: Ct m
phn ng xy ra trong thit b ct m
(NH4)2SO4 +2NaOH
Na2SO4 +2NH3 +2H2O
Phn ng xy ra trong bnh hng
NH3 +H2SO4
(NH4)2SO4 +H2SO4 d

Bc 3: Chun lng H2SO4 cn tha trong bnh hng bng NaOH

D
P2
C

E
B

F
P1

B CHNG CT M KJELDAHL

GII THIU TERMAMYL

-Termamyl l tn th trng ca enzyme amylase
-Termamyl l mt loi alpha-amylase n nh nhit sn xut bi mt chng
Licheniformis Bacillus.
ng dng:
-Termamyl c s dng trong cc ngnh cng nghip sau y:
Tinh bt

Tinh bt l sn phm t nhin quan trng nht c nhiu ng dng trong k

thut v trong i sng con ngi. Nhiu nc trn th gii s dng ngun
tinh bt t khoai ty, la m, ng (sn), cn ring nc ta th s dng go
v khoai m l ngun tinh bt ch yu. Trong ch bin tinh bt v ng,
cng on quan trng nht l thu phn tinh bt v cc ng n gin. Sau
, ch yu trn c s ng n nh ln men, ngi ta s nhn c rt
nhiu sn phm quan trng nh: ru cn, ru vang, bia, cc loi acid hu
c, amino acid,.
Qu trnh thu phn tinh bt gm hai cng on ch yu l giai on h ho
v giai on ng ho. thc hin hai cng on cng ngh ni trn,
trong thc t sn xut ngi ta p dng hai cch: Thu phn tinh bt bng
acid v bng enzym. thu phn tinh bt t lu ngi ta s dng acid
v c nh HCl v H2SO4. Nhng kt qu cho thy, thu phn bng acid rt
kh kim sot v thng to nhiu sn phm khng mong mun v khng
p ng tiu chun an ton thc phm. Do vy, vic thay th v ng dng
enzym thu phn tinh bt l mt kt qu tt yu ca lch s pht trin.
Enzym amylase c tm ra gp phn quan trng cho nhiu ngnh
cng nghip ch bin thc phm. Enzym amylase c th tm thy nhiu
ngun khc nhau nh amylase t thc vt, ng vt v VSV. Amylase cng
ngy cng c thay th acid trong sn xut qui m cng nghip. Hin
nay, cc nh sn xut c th s dng amylase c kh nng chu nhit cao m
khng b mt hot tnh, chng hn amylase c tch chit t VSV, c th l
cc chng vi khun chu nhit c phn lp t nhng sui nc nng.
Ngoi ra, amylase cn c nhiu u im hn khi s dng acid thu phn
tinh bt: Nng lng xc tc thp, khng yu cu cao v thit b s dng,
gim chi ph cho qu trnh tinh sch dch ng.
Ngun amylase c th ly t mm thc, mm i mch ( malt), ht bp ny
mm, hay t nm mc, Nguyn liu cho sn xut l go, bp, khoai m,
y l nhng ngun nguyn liu d tm, r tin c th tm thy d dng
nc ta. Do , y l mt li th v l hng pht trin mnh lm c s cho
nhiu ngnh khc pht trin. V d: sn xut bnh ko, bia, cn, sir v lm
mm vi,
Trong cng nghip tinh bt, Termamyl c s dng cho cc ha lng lin
tc ca tinh bt nhit ln ti 105-110 C, li dng s n nh nhit cc
ca enzym ny.
Ru
Trong cng nghip ru, Termamyl c s dng cho tinh bt mng trong
chng ct mashes.
Ma ng
Trong cng nghip ng, Termamyl c s dng ph v cc mt tinh

bt trong nc tri cy ma. Qua ni dung tinh bt ng th gim v lc

ti nh my lc to iu kin.
Dt may
Trong ngnh cng nghip dt may, Termamyl c s dng cho hng dt
trc khi nhum nhit cao.
HOT NG
-Enzyme termamyl l mt endoamylase b gy mi lin kt hydrolyses 1,4alpha glycosidic trong amylose v Amylopectin, hai thnh phn ca tinh bt.
Do ,tinh bt b ph v nhanh chng to dextrins v oligosaccharides.
-Termamyl hot ng ti u pH 7 v 90 C.
-Termamyl l mt amylase c s dng loi b vt bn tinh bt .
-Termamyl hiu qu loi b vt bn tinh bt v cung cp trng. Termamyl
l c hiu qu ti mi trng kim tng i cao v nhit cao.

Tng quan v enzym amylase

Amylase l g?
Amylase l mt h enzyme rt ph bin trong th gii sinh vt . Cc enzyme
ny thuc nhm enzyme thy phn, xc tc phn gii lin kt ni phn t
trong nhm polysaccharide vi s tham gia ca nc.
C 6 loi enzyme c xp vo 2 nhm: Endoamylase ( enzyme ni bo ) v
exoamylase ( enzyme ngoi bo ).
Endoamylase gm c -amylase v nhm enzyme kh nhnh. Nhm
enzyme kh nhnh ny c chia thnh 2 loi: Kh trc tip l Pullulanase (
hay -dextrin 6 glucosidase); kh gin tip l Transglucosylase (hay oligo1,6-glucosidase) v maylo-1,6-glucosidase. Cc enzyme ny thy phn cc
lin kt bn trong ca chui polysaccharide.
Exoamylase gm c -amylase v -amylase. y l nhng enzyme thy
phn tinh bt t u khng kh ca chui polysaccharide.
C cht tc dng ca amylase l tinh bt v glycogen:
Tinh bt: l nhm Carbohydrate thc vt, c ch yu trong cc loi c
nh khoai lang, khoai ty, khoai m , trong cc ht ng cc, cc loi ht v
c cng thc tng qut l (C6H12O6)n.Tinh bt t mi ngun khc nhau
u c cu to t amylase v amylopectin ( Meyer, 1940 ). Cc loi tinh bt
u c 20-30% amylase v 70-80% amylopectin. Trong thc vt, tinh bt
c xem l cht d tr nng lng quan trng.
-Amylase c trng lng phn t 50.000 160.000 Da, c cu to t 2001000 phn t D-glucose ni vi nhau bi lin kt -1,4-glucoside to thnh

mt mch xon di khng phn nhnh.

-Amylopectin c trng lng phn t 400.000 n hng chc triu Da, c
cu to t 600-6000 phn t D-glucose, ni vi nhau bi lin kt -1,4glucoside v -1,6-glucoside to thnh mch c nhiu nhnh. Tinh bt
khng tan trong nc lnh nhng khi hn dch tinh bt b un nng ( 60850C ) th tinh bt s b h ha v c gi l h tinh bt . Di tc dng
ca enzyme amylase, tinh bt b thy phn do cc lin kt glucoside b phn
ct. S thy phn tinh bt bi enzyme amylase xy ra theo 2 mc : Dch
ha v ng ha. Kt qu ca s dch ha l to ra sn phm trung gian
dextrin v khi dextrin tip tc b ng ha th sn phm l maltose v
glucose.
-Cabohydrate trong thc phm l ngun cung cp nng lng quan trng
cho c th con ngi. Rau v qu cng l ngun cung cp tinh bt v tinh
bt ny mt phn c chuyn ha thnh disaccharide v glucose.
Carbohydrate c mt trong hu ht cc loi thc phm nhng ngun cung
cp ch yu l ng v tinh bt.
Glycogen: l mt loi Cabohydrate d tr. ng vt c d tr trong c
th ng vt v c c th chuyn ha s dng t t. Amylase c vai tr
quan trng trong s chuyn ha glucid t bo ng vt, VSV, glucogen
c cu to t cc glucose lin kt vi nhau bi lin kt -1,4-glycoside
cc v tr phn nhnh, glucose ni vi nhau bng lin kt -1,6-glycoside.
Glycogen c s mch nhiu hn tinh bt. Phn t lng trong khong 2
triu-3 triu Da. Glycogen d tan trong nc, nu nh chng ta n qu nhiu
Carbohydrate th c th chng ta s chuyn ha chng thnh cht bo d tr.
ng vt v ngi, glycogen tp trung ch yu trong gan.
c tnh v c ch tc dng ca enzym -amylas
c tnh ca enzym -amylase
-amylase t cc ngun khc nhau c thnh phn amino acid khc nhau, mi
loi -amylase c mt t hp amino acid c hiu ring. -amylase l mt
protein giu tyrosine, tryptophan, acid glutamic v aspartic. Cc glutamic
acid v aspartic acid chim khong tng lng amino acid cu thnh nn
phn t enzyme:
- -amylase c t methionine v c khong 7-10 gc cysteine.
- Trng lng phn t ca -amylase nm mc: 45.000-50.000 Da ( Knir
1956;Fisher,Stein, 1960 ).
- Amylase d tan trong nc, trong dung dch mui v ru long.
- Protein ca cc -amylase c tnh acid yu v c tnh cht ca globuline.
-im ng in nm trong vng pH=4,2-5,7 ( Bernfeld P, 1951 ).
--amylase l mt metaloenzyme. Mi phn t -amylase u c cha 1-30

nguyn t gam Ca/mol, nhng khng t hn 1-6 nguyn t gam/mol Ca tham

gia vo s hnh thnh v n nh cu trc bc 3 ca enzyme, duy tr hot
ng ca enzyme ( Modolova, 1965 ). Do , Ca cn c vai tr duy tr s
tn ti ca enzyme khi b tc ng bi cc tc nhn gy bin tnh v tc ng
ca cc enzyme phn gii protein. Nu phn t -amylase b loi b ht Ca
th n s hon ton b mt ht kh nng thy phn c cht. -amylase bn
vi nhit hn cc enzyme khc. c tnh ny c l lin quan n hm
lng Ca trong phn t v nng Mg2+. Tt c cc amylase u b kim
hm bi cc kim loi nng nh Cu2+, Ag+, Hg2+. Mt s kim loi nh :Li+,
Na+, Cr3+, Mn2+, Zn2+, Co2+, Sn2+, Cr3+, khng c nh hng my n
-amylase.
Thnh phn amino acid ca -amylase nm mc Aspergillus nh sau
( g/100 g protein ): alamine=6,8 ; glycine= 6,6; valine= 6,9, leucine= 8,3;
Isoleucine= 5,2; prolin= 4,2, phenylalanine= 4,2; tyrosine=9,5; trytophan=
4,0; xetin= 6,5; trionin= 10,7, cystein + cystine= 1,6; glutamic acid= 6,9;
amide= 1,5 ( Akabori et amiloza, 1954 ). Khng ging cc -amylase khc,
amylase ca Asp.oryzae c cha phn phi protein l polysaccharide. Polyose
ny bao gm 8 mol maltose, 1 mol glucose, 2 mol hexozamin trn 1 mol
enzyme ( Akabori et amiloza, 1965 ). Vai tr ca polyose ny vn cha r,
song bit c rng n khng tham gia vo thnh phn ca trung tm
hot ng v nm pha trong phn t enzyme.
-amylase ca nm mc hu nh ch tn cng nhng ht tinh bt b thng
tn. Sn phm cui cng ca thy phn amylase l glucose v maltose. i
vi nm si t l l 1:3,79 ( Hanrahan, Caldwell, 1953 ) .-amylase ca nm
si khng tn cng lin kt -1,6 glucoside ca amylopectin, nn khi thy
phn n s to thnh cc dextrin ti hn phn nhnh. y l mt cu trc
phn t tinh bt do enzym -amylase phn ct to thnh dextrin ti hn phn
nhnh.
Sn phm thy phn cui cng ca tinh bt di tc dng ca amylase nm
si ch yu l maltose, th n l maltotriose. Khi dng nng -amylase
VSV tng i ln c th chuyn ha 70-85% tinh bt thnh ng ln
men. Cn cc -amylase ca nm mc th mc ng ha n glucose v
maltose c th ln ti 84-87%.
iu kin hot ng ca -amylase t cc ngun khc nhau thng khng
ging nhau. pH ti thch cho hot ng ca -amylase t nm si l 4,0-4,8 (
c th hot ng tt trong vng pH t 4,5-5,8 ). Theo s liu ca Liphis, pH
ti thch cho hot ng dextrin ha v ng ha ca ch phm amylase t
Asp.oryzae trong vng 5,6-6,2. Cn theo s liu ca Fenixova th pH ti
thch cho hot ng dextrin ha ca n l 6,0-7,0.

 bn i vi tc dng ca acid cng khc khc nhau. -amylase ca

Asp.oryzae bn vng i vi acid tt hn l -amylase ca malt v vi khun
Bac.subtilis. pH= 3,6 v 0oC, -amylase ca malt b v hot hon ton
sau 15-30 pht; -amylase vi khun b bt hot n 50%, trong khi hot
lc ca -amylase ca nm si hnh nh khng gim bao nhiu ( Fenilxova,
Rmoshinoi 1989 ). Trong dung dch -amylase nm si bo qun tt pH=
5,0-5,5; -amylase dextrin ha ca nm si en c th chu c pH t 2,52,8. 0oC v pH= 2,5, n ch b bt hot hon ton sau 1 gi.
Nhit ti thch cho hot ng xc tc ca -amylase t cc ngun khc
nhau cng khng ng nht, -amylase ca nm si rt nhy cm i vi tc
ng nhit. . Nhit ti thch ca n l 500C v b v hot
70OC( Kozmina, 1991 ).
Trong dung dch m pH= 4,7, -amylase ca Asp.oryzae rt nhy vi tc
ng ca nhit cao, thm ch 400C trong 3 gi hot lc dextrin ha ca
n ch cn 22-29%, hot lc ng ha cn 27-85%. 500C trong 2 gi, amylase ca nm si ny b v hot hon ton ( Miller v cng s ).

C ch tc dng ca enzym -amylase

-amylase ( 1,4--glucan-glucanhydrolase ). -amylase t cc ngun khc
nhau c nhiu im rt ging nhau. -amylase c kh nng phn cch cc
lin kt -1,4-glucoside nm pha bn trong phn t c cht ( tinh bt hoc
glycogen ) mt cch ngu nhin, khng theo mt trt t no c. -amylase
khng ch thy phn h tinh bt m n thy phn c ht tinh bt nguyn
song vi tc t rt chm.
Qu trnh thy phn tinh bt bi -amylase l qu trnh a giai on.
+ giai on u ( giai on dextrin ha ):Ch mt s phn t c cht b
thy phn to thnh mt lng ln dextrin phn t thp (-dextrin ), nht
ca h tinh bt gim nhanh ( cc amylose v amylopectin u b dch ha
nhanh ).
+ Sang giai on 2 ( giai on ng ha ): Cc dextrin phn t thp to
thnh b thy phn tip tc to ra cc tetra-trimaltose khng cho mu vi
iodine. Cc cht ny b thy phn rt chm bi -amylase cho ti
disaccharide v monosaccharide. Di tc dng ca -amylase, amylose b
phn gii kh nhanh thnh oligosaccharide gm 6-7 gc glucose( v vy,
ngi ta cho rng -amylase lun phn ct amylose thnh tng on 6-7 gc
glucopiranose 1 ).
+ Sau , cc poliglucose ny b phn cch tip tc to nn cc mch
polyglucose colagen c ngn dn v b phn gii chm n maltotetrose v

maltotriose v maltose. Qua mt thi gian tc dng di, sn phm thy phn
ca amylose cha 13% glucose v 87% maltose. Tc dng ca -amylase
ln amylopectin cng xy ra tng t nhng v khng phn ct c lin kt
-1,6-glycoside ch mch nhnh trong phn t amylopectin nn d c chu
tc dng lu th sn phm cui cng, ngoi cc ng ni trn ( 72%
maltose v 19% glucose ) cn c dextrin phn t thp v isomaltose 8%.
Tm li, di tc dng ca -amylase, tinh bt c th chuyn thnh
maltotetrose, maltose, glucose v dextrin phn t thp. Tuy nhin, thng
thng -amylase ch thy phn tinh bt thnh ch yu l dextrin phn t
thp khng cho mu vi Iodine v mt t maltose. Kh nng dextrin ha cao
ca -amylase l tnh cht c trng ca n. V vy, ngi ta thng gi loi
amylase ny l amylase dextrin ha hay amylase dch ha.
Cc giai on ca qu trnh thy phn tinh bt ca -amylase:
+ Giai on dextrin ha:
Tinh bt dextrin phn t lng thp
+ Giai on ng ha:
Dextrin tetra v trimaltose di & monosaccharide
Amylase oligosacharide poliglucose
Maltose maltotriose maltotetrose
2.NGUYN LIU, HO CHT , THIT B:
NGUYN LIU: Mu termamyl v c ho v pha long
HO CHT :
H2SO4 N/100
NaOH N/100
Metyl
CCH TIN HNH:
Bc 1: V c ho mu _ termamyl dng c v c ho mu sn.
Bc 2 : Ct m
a )Ra my :
- un si bnh A v m nc vo ng lm lnh E. Kho P 1 v P2 u ng ,
sau cho vo phu D t nc ct (khong 10ml) v cho chy xung bnh C
bng P2.
Hi nc ca bnh A s t t qua erlen F, c nh vy cho n khi erlen F
c khong 5ml nc th ngng un bnh A, lm ngui bnh A , p sut trong

bnh A gim xung ng thi p sut trong bnh B cng gim , do nc

trong bnhC s rt qua B .
- Khi nc rt ht ta li thm nc vo phu D, v li m kp cho nc
chy qua C, k nc s rt qua B.
Ta lm nh vy chng vi ln.
- Nuc bnh B y ta m kp P1 cho nc chy ra.
Kho kp P1 v P2 li.
b)Chun b dung dch bnh hng NH3 (bnh F):
Cho vo bnh hng F (erlen 250 ml) 15ml H2SO4 N/100 v 5 git thuc th
metyl , dung dc c mu tm .
t bnh hng sao cho u mt ca ng sinh hn E ngp trong dung dch ca
bnh hng.
c)Thc hin chung ct li cun hi nc:
- Lc ny bnh A ang c un si, kp P1 v P2 ng. vo phu D 10
ml dung dch termamyl, m kp P2 sao cho dung dch chy t t xung bnh
C, ng P2 li. nc trng vo D v m kp cho nc chy t t xung C
cho n khi mc nc trong D xung gn st kp P2 th kho kp P2 li. Sau
trng D bng nc ct vi ln vi thao tc nh trn.
- Thm vo phu D 10 ml NaOH m c . M kho P2 t t cho NaOH
chy t t xung C tng git mt. Nu nc trong C tro ln mnh qu th
kho P2 li, ri sau li tip tc cho NaOH chy gn ht xung C.
Trng phu D 2 ln bng nc ct , ch cho nuc chy gn st ti P2 m thi.
- Tip tc chng ct li cun hi nc trong 5 pht, sau h erlen xung
sao cho u mt ca ng sinh hn trong khng kh , un tip 3 pht. Ra u
mt ca sinh hn E nm trong khng kh, un tip 3 pht na. Ra u mt
li bng nc ct, ri ly erlen ra nh lng H2SO4 tha.
- Ngng un bnh A , ra my vi ln.
*Ta thc hin li mt ln th khng thay v dng termamyl ta dng 10 ml
nc ct.
Bc 3: Chun
Lng H2SO4 N/100 cn tha trong bnh F c chun bng NaOH
N/100. Qu trnh chun kt thc khi dung dch chuyn t tm sang
mu vng nht.

RA MY CHNG
CT M3.S KHI
RA MY CHNG CT M 2
N 3 LN
CHUN B DUNG DCH BNH
HNG F : 15 ml dung dich H2SO4
N/100, 5 git metyl

V C HO
MU
TERMAMYL

UN SI BNH A

CHO VO PHU D, TRNG LI

PHU BNG NC CT

CHO 10 ml DUNG DCH NaOH ,

TRNG LI BNG NC CT

-CHNG CT LI CUN HI NC TRONG 5 PHT

-SAU H ERLEN XUNG SAO CHO U MT
CA NG SINH HN TRONG KHNG KH, UN TIP
3 PHT.
-RA U MT CA SINH HN E NM TRONG
KHNG KH, UN TIP 3 PHT NA.
-RA U MT LI BNG NC CT

LY DUNG DCH TRONG BNH F

CHUN VI NaOH N/100 N KHI
DUNG DCH CHUYN SANG VNG
NHT

MU
TERMAMYL
10 ML

NaOH
N/100
10 ml

XC NH H S HIU CHUN :
Ly 2 erlen , cho vo mi erlen 20 ml H2SO4 N/100 v vi git metyl .
Sau chun bng dung dch NaOH N/100 .
4. KT QU
Mu trng (V0)
Mu chun ( V1)
Xc nh h s hiu chnh (ln 1) (V
NaOH1)
Xc nh h s hiu chnh (ln 2) (V
NaOH2)

Th tch NaOH N/100 (ml)

12.8
7.9
16.8
17

a) H s hiu chun
V NaOH1 = 16,8 ml
V NaOH2 = 17,0 ml
vy V NaOH = 16,9 ml
H s hiu chun
X

20 ml NaOH N / 100
V NaOH

N / 100

20
1.1834
16.9

b) Tnh lng m c trong mu

Gi Vo l th tch NaOH ca ln th khng , Vo=12,8ml
V1 l th tch NaOH ca ln th tht , V1=7,9 ml
Lng NaOH tng ng vi NH3 phng thch bi 10 ml dung dch
termamyl v c ho v pha long
V=V0-V1 = 12.8 7.9 =4.9
S mol NH3 phng thch tng ng vi s mol NaOH. Do , s mol
NH3 phng thch bi 10ml dung dch termamyl l :

V . X
V . X .10 5 4.9 1.1834 10 5 5.798 10 5 ( mol )
100.1000

S gram m c trong 10ml dung dich termamyl v c ho v pha long

l:
14.V.X.10-5 =14*5.798*10-5 =8.118*10-4 (g)
S gram m c trong 100ml dung dch m v c ho l
14.V . X .10 5.

100
14.V . X .10 4 8.118 10 3 ( g )
10

S gram m c trong 1 lit mu ban u

14.V . X .10 4.

1000
1000
14 * 4.9 *1.1834 *
8.118 ( g / l )
Vm
1

5.NHN XT
Phng php cho kt qu kh chnh xc, thc hin kh n gin, sai s
khng cao.
Lng m c trong mu termamyl tng i thp.

6. M RNG

B CHNG CT KJELDAHL
LN

Johan Gustav Christoffer Thorsager

Kjeldahl
(1849-1900), nh ho hc ngi an Mch

nh lng nit tng s theo phng php Microkjeldahl

1. Nguyn tc: Trong phn t protein, hm lng nit chim t l 15- 18%
khi lng phn t. Do nu xc nh c hm lng % nit trong
nguyn liu, ty theo mi loi protein m ta nhn vi h s khc nhau.
Hm lng protein ng vt: % protein = % N protein x 6,25
Hm lng protein thc vt: % protein = % N protein x 6,25 i vi ht
ng cc. % protein = % N protein x 5,75 i vi ht u .
Di tc dng ca H2SO4 c, nhit cao nit c trong thnh phn cc
hp cht hu c to thnh (NH4)2 SO4. Dng kim c, y nit ra khi
mui ca n di dng NH3 kt hp vi hi nc to thnh NH4OH. Lng
nit c trong thnh phn NH4OH c xc nh bng h chun H2SO4NaOH hoc H3BO3- H2SO4 0,01N.
2. Ha cht v nguyn liu:

Thuc th hn hp: xanh metylen 0,1% v metylen 0,1% trong ethanol

960 vi t l ; H2SO4 c; hn hp xc tc K2SO4/ CuSO4 vi t l 3/1;
H2SO4 0,01N; NaOH 0,01N; H3BO3 2%; ethanol 960; axit trichloacetic
20%. Cc nguyn liu c ngun gc ng vt nh tht, c, ngun gc thc
vt nh go, ng, u , sy kh tuyt i 1050C
3. Dng c:
ng ong 100ml, 50ml, 10ml; bnh nh mc 100ml (3); cc 250ml, 100ml,
50ml(3), a thy tinh (3); l ng ha cht(7); my Microkjeldahl bn t
ng hoc t ng; bp un; ng sinh hn
4. Tin hnh:
Cn 200mg nguyn liu dng bt sy kh tuyt i cho vo bnh kjeldahl,
dng ng ong 10ml H2SO4 c vo bnh kjeldahl s thy xut hin mu
nu en do nguyn liu b oxy ha. Cho thm 200mg hn hp xc tc, lc
nh, y kn bnh, mt thi gian cho H2SO4 tc dng vi nguyn liu s
gim bt c thi gian un. Sau t bnh kjeldahl ln bp un (t hi
nghing), y ming bnh bng phu thy tinh, t bp vo trong phng ht
kh. Khi si, gi nhit bp un khng qu cao trnh ha cht tro
ra, va trnh mt amoniac.
Trong khi un theo di s mt mu en ca dung dch trong bnh un, khi
thy dung dch c mu trong sut th c th lc nh bnh ko ht cc phn
t trn thnh bnh cha b oxy ha vo trong dung dch. Tip tc un cho
n khi dung dch trong hon ton. ngui bnh, chuyn dung dch sang
bnh nh mc 100ml, dng nc ct trng li bnh kjeldahl v dn ti vch.
- Giai on ct m theo phng php dng h chun H2SO4- NaOH:
Cho nc ct 1 ln ti 2/3 th tch ca bnh, cm in un si nc trong
bnh. Hi nc v nhit ca bnh 1 gip cho nit trong (NH4)2SO4 ca
dch nghin cu b NaOH 30% y ra di dng NH3. NH3 c ngng t

nh h thng lm lnh v hi nc to thnh NH4OH. Sau khi nc trong

bnh si, cho vo bnh hng 10ml H2SO4 0,01N v 3 git thuc th hn
hp, dung dch trong bnh nn s c mu tm hng chng t mi trng axit.
t bnh hng vo gi sao cho u mt ca ng sinh hn ngp trong dung
dch ca bnh hng. Nu lng axit trong bnh hng khng ngp, c th
cho thm vo mt t nc ct v m.

My Microkjendahl
Cc b phn ca my:
1. Bnh un
2. Bnh ct
3. H thng sinh hn
4. Bnh hng
5. Bnh ng dung dch thi

Cho vo bnh ct 10ml dung dch nghin cu v 3 git thuc th hn hp,

sau trng phu bng nc ct v m. Cho tip vo bnh ct 15ml NaOH
30% ri ng cht van li. Dung dch trong bnh ct chuyn t mu tm
hng sang mu xanh l m th hin mi trng kim. Sau 8-10 pht ct
m, kim tra xem NH4OH cn c to ra khng, dng giy qu th
dung dch u ng sinh hn. Nu giy qu cn mu xanh th phi ct m
tip, nu giy qu khng i mu th qu trnh ct m xong, ly bnh
hng em chun .
- Chun : Dng NaOH 0,01N chun cho ti khi dung dch trong bnh
hng chuyn t mu tm hng sang mu xanh l m. Lng NaOH 0,01N
dng chun bng H2SO4 0,01N cn d trong bnh hng.
- Tnh kt qu: Hm lng % nit tng s hay nit ton phn c tnh theo
cng thc N% = 1,42 x( V1 V2) x 100 / a
V1: S ml H2SO4 0,01N cho vo bnh hng.
V2: S ml NaOH 0,01N chun .
( V1 V2): S ml H2SO4 0,01N trong bnh hng b trung ha bi
NH4OH c to ra do qu trnh chng ct m
1,42 l h s: c 1ml H2SO4 0,01N dng trung ha NH4OH th tng
ng vi 1,42 mg nit.
a l s nguyn liu tnh bng gam ca mu ct m
Sau khi tnh c nit tng s, em kt qu nhn vi h s tng ng s
cho ra hm lng protein trong nguyn liu..

BI 7b:
XC NH NITO TNG VI MU M TIU NH
BNG MY KJELDAHL T NG
1. NGUYN TC
Trc tin mu c v c ho bng H2SO4 m c nhit cao v c
cht xc tc . Cc phn ng ca qu trnh v c ho xy ra nh sau:
2 H2SO4
2H2O +2SO2 +O2
Oxy to thnh li oxy ho cc nguyn t khc : cacbon to thnh CO2,
hydro to thnh H2O , cn nito gii phng ra di dng NH3 kt hp vi
H2SO4 d to thnh (NH4)2SO4 tan trong dung dch.
NH3 + H2SO4
(NH4)2SO4
QU TRNH CHNG CT M BNG H THNG T NG
Cht m c v c ho nm di dng amoni sulfat khi c tc dng
vi cht kim mnh nh NaOH s phng thch ra amoniac.
(NH4)2SO4 +2NaOH
Na2SO4 +H2O +2NH3
Sau , NH3 c hi nc li cun v a vo dung dch H3BO3. Trong
dung dch ny, NH3 tn ti di dng ion NH4+ v gii phng ra ion H2BO3Chun lng ion nu bng HCl chun s xc nh c lng amoniac
sinh ra. T , suy ra c lng m trong mu nguyn liu ban u.
CC THIT B T NG CHNG CT KJELDAHL

THIT B V C
HO MU

THIT B CHNG
CT M

2.THIT B V DNG C
B chng ct m Kjeldahl t ng
Erlen 250 ml
Burret 25 ml
3. HO CHT
Dung dch H2SO4 m c
Dung dch NaOH 40%
Hn hp xc tc K2SO4 v CuSO4 (t l 3:1)
Dung dch H3BO3%
Thuc th hn hp metyl 0.1 % pha trong cn
4.CCH TIN HNH
- V c ho mu : cho 5 (g) m tiu nh vo ng Kjeldahl , thm vo 5ml
dung dch H2SO4 m c v hn hp xc tc K2SO4 v CuSO4. a ng
vo my v c ho mu.

- Dnh m bng my Kjeldahl :

Mu sau khi v c ho c a vo my Kjeldahl nh m. ng thi ,
a vo mt erlen c cha thuc metyl thu mu .
Mu ny sau em i chun bng dung dch HCl 0.1 N n khi xut
hin mu hng nht.
S KHI
5g M TIU NH
DUNG DCH H2SO4
HN HP K2SO4 V
CuSO4
V C HO MU

NH M BNG MY KJELDAHL T NG

ERLEN CHA
METYL

THU MU

CHUN MU BNG DUNG DCH HCl 0.1 N

KT QU
Mu trng
Mu tht

th tch HCl 0.1 N (ml)

0.01
5.2

Hm lng Nito tng s ( Nt) trong nguyn liu l:

Nt

(V1 Vt ) 0.0014 1000 (5.2 0.01) 0.0014 *1000

7.266( g / l )
V
1

V1: th tch HCl 0.1 N chun mu tht

Vt: th tch HCl 0.1 N chun mu trng
V:th tch dch mu ly ban u
0.0014 : s gram nito ng vi 1ml HCl 0.1N
6.NHN XT
Lng nit c trong mu ban u kh cao.
Phng php ny kh chnh xc v n gin.
7.M RNG
Ngoi ra cn c mt s my chng ct t ng Kjeldahl c thit k khc

nh lng N - Protein bng my Kjeldahl

Nguyn tc ca qu trnh phn tch
Mu c v c ho bng H2SO4 nhit cao v c cht xc tc.
Cc phn ng ca qu trnh v c ho mu xy ra nh sau:
2H2SO4

2 H2O + 2SO2

+ O2

Oxy to thnh trong phn ng li oxy ho cc nguyn t khc. Cc phn

t cha nit di tc dng ca H2SO4 to thnh NH3. Cc protein b thu
phn thnh axit amin. Cacbon v hydro ca axit amin to thnh CO2 v H2O,
cn nit c gii phng di dng NH3 kt hp vi axit H2SO4 d to
thnh (NH4)2SO4 tan trong dung dch.
2NH3 + H2SO4

(NH4)2SO4

Cc nguyn t khong khc nh P, K, Ca, Mg,chuyn thnh dng oxit:

P2O5, K2O, CaO, MgOtuy tn ti trong dung dch mu nhng khng nh
hng n kt qu phn tch.
ui amoniac ra khi dung dch bng NaOH.
(NH4)2SO4 + 2NaOH = Na2SO4 + H2O + 2NH3

NH3 bay ra cng vi nc sang bnh hng. Bnh hng c cha H3BO3 do
NH3 bay ra s tc dng ngay vi H3BO3 theo phn ng:
2NH3 + 2H2O + 4H3BO3 = (NH4)2B4O7 + 7H2O
Lng (NH4)2B4O7 c xc nh thng qua vic chun bng HCl
0,25N cho n khi dung dch chuyn sang mu hng nht v khng b mt
mu.
Nhng yu t c th nh hng n kt qu phn tch
+ Nng HCl 0,25N khng m bo chnh xc
+ Ly mu khng i din
+ Cn mu khng chnh xc
+ Ngun nc b nhim N
Kh nng ng dng ca phng php Kjeldahl
Phng php ny c kh nng ng dng cho hu ht cc loi mu:
+ Mu thc phm: thu hi sn, nc mm, cc loi u, thc n nui
tm c, rau qu
+ t, nc thi
+ B men bia, bnh du u nnh, bnh du cao su
Nguyn tc hot ng ca my Kjeldahl
Mu c a vo bn t (h thng v c ho mu) thng qua cc ng
Kjeldahl Sau khi v c ho mu xong, ton b mu + ng Kjeldahl c
a qua h thng chng ct NH3 ton b sn phm c a qua thit b
chun Kt Qu.
Thc Hnh
Qui trnh
Phn tch mu u phng:
Bc 1: Chun b mu
-Mu ht c nghin mn

-Sy mu 80oC cho n khi t trng lng khng i

Bc 2: V c ho mu
-Cn 0,3 1 g mu kh c nghin mn.
-Cho mu vo tn y ca ng Kjeldahl (chu khng dnh trn thnh
ng)
-Cho 5g hn hp xc tc K2SO4 v CuSO4 theo t l 10:1
-Dng pipet ht 15ml H2SO4 m c (d=1,84) cho vo ng v c ho
mu c cha hn hp trn.
-Lp ng trn vo h thng v c ho mu.
-Vic v c ho mu hon ton khi ton b dung dch trong ng v c
ho mu c mu xanh trong sut.
Bc 3: Chng ct mu
-Lp ng v c ho mu vo b chng ct, lp bnh hng cha 10-60ml
H3BO3 4% v 7 git dung dch ch th mu.
-Khi ng my ct.
-Qu trnh chng ct tin hnh khong 390 giy th ton b kh NH3
chuyn sang bnh hng.
-Khi NH3 c ct hon ton, h bnh hng xung, dng tia nc ct nh
trng sch axit dnh u ng lm lnh.
Bc 4: Chun
Bc 5: Tnh kt qu
Ta c 1ml HCl 0,25N tng ng vi 0,0035g N.
Thng qua s ml HCl 0,25N, ngi ta bit c s lng axit boric kt
hp vi NH3, t bit lng NH3 gii phng t mu.
N tng (%) = (VHCl * 0,35)/ P mu
Trong : V tnh bng ml
P tnh bng g

Protein tng (%) = (VHCl * 0,35* 6,25)/ P mu

Mt s thit b v ho cht s dng
Thit b:
- T sy mu
- My nghin mu
- Cn phn tch 4 s
- My phn tch m t ng Kjeldahl
- Thit b chun
- My ct nc 2 ln
Ho cht:
- Hn hp K2SO4 v CuSO4 theo t l 10:1
- Nc ct 2 ln
- NaOH 32%
- H2SO4 m c
- HCl 0,24N
- Acid Boric 4%
- Cht ch th mu: cch pha:
+ Ho tan 0,264g methyl trong 250 ml C2H5OH (cn tuyt i)
+ Ho tan 1,28g Bromocresol xanh l cy trong 50ml C2H5OH (cn tuyt
i)
+Trn u 2 dung dch trn, b sung 96ml HCl 0,25N. nh mc n
1000ml bng C2H5OH.