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TRNG I HC K THUT CNG NGH TP.

HCM
KHOA CNG NGH THC PHM







Ti liu thc hnh

PHN TCH THC PHM


Ngi bin son:
ThS. Hunh Quang Phc
TS. Ng i Nghip
ThS. Trn Th Ngc Mai
KS. Trn Th Hng Hnh
KS. Nguyn Th Thu H


Ti liu lu hnh ni b
Ti liu thc hnh Phn tch thc phm
Trang 1
TRNG I HC K THUT CNG NGH TP. HCM
KHOA CNG NGH THC PHM

NI QUI PHNG TH NGHIM

1. Trang phc
Sinh vin vo PTN phi eo bng tn, mc o blouse trng v mang giy, dp c quai
hu.
2. Gi gic
Sinh vin i th nghim ng gi qui nh
Sinh vin khng c php ra khi phng th nghim khi cha c s cho php ca gio
vin hng dn.
Sinh vin khng t v PTN khi cha c s ng ca gio vin ang ging dy.
3. Nhim v
Phi c v tm hiu bi trc khi vo PTN
Nghe v thc hin ng cc ch dn c th ca gio vin, khng c lm cc th
nghim ngoi gio trnh khi cha c s ng ca gio vin hng dn.
C thi khoa hc nghim tc trong PTN nh:
- Khi lm vic vi cc ha cht c hi n thn th phi s dng cc thit b bo
h lao ng nh gng tay, mt n phng c .
- Khi lm vic vi cc kh c hay dung mi d bay hi phi lm vic trong t ht
- Cc dung dch b c lin quan n acid hay kim mnh phi pha long v thi
b t t, sau s dng vi nc xi mnh.
Thit lp qui trnh lm vic chi tit v trnh cho gio vin lin quan.
Vch k hoch lm vic trc khi vo phng th nghim.
Ton b qu trnh lm vic phi c ghi chp li cn thn di dng nht k lm vic.
Tun th qui nh phng chy cha chy.
Trc nht v rc ng ni quy nh sau mi bui hc.
Tp.H Ch Minh, thng nm 2011
Ph trch phng th nghim
Ti liu thc hnh Phn tch thc phm
Trang 2
AN TON PHNG TH NGHIM

1. Cn thn khi tin khi tin hnh th nghim, khng c s dng nhng my mc, dng c
m cha bit cch s dng. Phi hiu r tnh cht cc ha cht trnh tai nn ng tic.
2. Khng c dng cc dng c thy tinh cha ra sch, cc dng c thy tinh bn phi
ring hoc ra ngay sau khi dng.
3. Tt c cc chai l ng ha cht phi c ghi nhn. Khi dng ha cht phi c k nhn
hiu, dng xong phi li ch c. Nhn ghi ha cht bng ting nc ngai th phi xem
xt cn thn ch no cha r phi tra cu ti liu, khng c on. Ha cht cha dng
ngay phi ghi li trnh nhm ln. Trn thc t phn ln cc ha cht l cht c nn phi
ht sc cn thn.
4. Khi ht ha cht bng ng ht (pipette) phi s dng bp cao su.
5. Khi theo di dung dch ang si hoc tinh th ang chy khng c mt gn; khi
mt cht lng vo cc phi xa mt. Nn dng knh bo h lao ng.
6. Lm g nguy him phi ch c ngi ng bn cnh. un mt cht lng trong ng nghim
phi ming ng quay v pha khng c ngi. lc un khng c gi ng nghim ng
yn m phi lc u, un ton b b mt ng nghim phn cha cht lng.
7. Khi lm vic vi cht d chy th tuyt i:
- Khng dng la ngn
- Khng lm vic bn cnh la ngn
- Khng cht d chy bn cnh ngun sinh nhit.
8. Khi lm vic vi cht d n: nh nitrate, chlorate, pemanganate, bicromate, peoxyt th
phi cn thn v ng qui cch.
9. Khi lm vic vi cc acid v base mnh phi:
- Khng ra ngoi.
- Khi acid hay base vo nc khi pha long chng (khng c nc vo acid
hay base).
- Sang chai phi dng phu (khi rt phi quay nhn ln pha trn, chai kia phi trn
bn, tuyt i khng cm trn tay).
- Khng ht acid hay base khi trong chai cn qu t.
- Khi un si phi cho bt hoc bi thy tinh iu ha, trnh bn hay tro ra
ngoi.
10. Khi lm vic vi dng c in:
- Tay phi tht kh, ch lm vic cng phi kh.
Ti liu thc hnh Phn tch thc phm
Trang 3
- Cn thn khi dng in c in th 220 Vol.
11. Khi lm vic vi dng c thy tinh
- Trnh v.
- Dng c loi no dng cho vic , ch c un vi dng c thy tinh chu nhit v
dng cho chn khng nhng dng c c bit dng trong chn khng.
12. Khi c bt k s c g v ng ng dn nc trong PTN, phi nhanh chng kha van ca
ng ng dn nc.

Ti liu thc hnh Phn tch thc phm
Trang 4



MC LC



BI 1: PHA CH HA CHT V XC NH NNG DUNG DCH...................................................... 5
BI 2: NH LNG NIT TNG BNG PHNG PHP MICRO-KJELDAHL....................................... 9
BI 3: NH LNG PROTEIN THEO PHNG PHP LOWRY............................................................ 12
BI 4: NITROGEN NITRITE................................................................................................................... 14
BI 5: NH LNG NG KH BNG PHNG PHP ACID DINITROSALICYLIC (DNS) ........... 16
BI 6: SC K CT (COLUMN CHROMATOGRAPHY) LY TRCH - KHO ST SC T L CY... 19
BI 7: SC K BN MNG (THIN LAYER CHROMATOGRAPHY) PHN TCH HN HP
VANILLIN V
|
NAPHTHOL.................................................................................................... 23
BI 8: PHNG PHP SC K TRN GIY (PAPER CHROMATOGRAPHY) ...................................... 31
Ti liu thc hnh Phn tch thc phm
Trang 5
Bi 1: PHA CH HA CHT V XC NH NNG
DUNG DCH
1. L thuyt
1.1 Cch pha cc nng
a. Nng phn trm theo khi lng
Th d: Mun pha 500g dung dch sulfat ng 20% t tinh th ngm nc CuSO
4
.5H
2
O
Ta bit:
Phn t khi ca CuSO
4
khan nc: 160
Phn t khi ca CuSO
4
.5H
2
O l: 250
Mun pha 500g CuSO
4
20% th cn mt lng CuSO
4
khan nc l: (20.500)/100 = 100g.
Mun c 160g khan nc th ta phi cn 250g CuSO
4
.5H
2
O. Vy mun c 100g CuSO
4

khan nc th phi cn mt lng CuSO
4
.5H
2
O l: (250.100)/160= 156g.
Lng nc cn thm: 500 156 = 334g.
b. Nng phn trm khi lng theo th tch
- Ta ha tan lng cht cn trong mt t nc v thm nc cho ti th tch ng.
- Th d: Cn chun b 1 lt dung dch NaCl 30%th ta cn mt lng NaCl l:
(300.1000)/100 = 300g ha tan trong 1 t nc v thm nc cho th tch 1 lt.
- Trng hp cc ha cht c ngm nc, khi cn ta phi tnh thm c lng nc trong
phn t nh trng hp trn.
- Trng hp cht ha tan l cht lng ta cng lm tng t nh trn, ngha l cn cht tan
v dung mi em trn ln vi nhau cho u l c. Nhng vic cn cht lng khng c
thun li cho lm nn cn phi a cht lng v n v th tch theo cng thc: V = P/d
- Mt khc i vi cht lng thng dng c gii hn ha tan ti a tnh theo %.
- Th d: H
2
SO
4
ha tan ti a 96%, HCl 37%, H
3
PO
4
65%... cho nn khi cn cc cht lng
ny phi tnh c s g c thc trong dung dch pha cho chnh xc.
Nu ta xem HCl l 100% th khi pha dung dch 10% ta ch vic cn 10g HCl v thm vo
90ml nc trn u l c. Nhng thc ra HCl ch c 37% nn trng lng cn phi l
(100.10)/37 = 27,02g hay 27,02/1,19 = 23ml v thm lng nc 100 27,02 = 72,98g hay
72,98ml.
c. Nng phn t gam
- Mol hoc phn t gam l khi lng ca cc cht tnh ra gam bng khi lng phn t ca
n. Dung dch phn t gam l dung dch cha 1 phn t gam cht ha tan trong 1 lt.
Ti liu thc hnh Phn tch thc phm
Trang 6
- chun b dung dch 1M ca cht no , ngi ta tnh khi lng phn t. Ly lng
cn chnh xc em ha tan trong dung mi cho thnh 1 lt dung dch (dng bnh nh mc).
Khi phi un nng dung dch hay khi phn ng ta nhit hay thu nhit phi cho nhit
v bnh thng (20
0
C) ri mi thm nc ti vch.
- Th d: Cn 0,5 lt dung dch K
2
Cr
2
O
7
0,1M; M(K
2
Cr
2
O
7
) = 294,2
chun b dung dch K
2
Cr
2
O
7
0,1M cn ly 0,1M cn ly 0,1 phn t gam, ngha l
29,42g K
2
Cr
2
O
7
. chun b 0,5 lt ta ch cn cn 29,42 . 0,5 = 14,71g pha trong bnh nh
mc 500ml.
Nu cht tan l cht lng c phn trm thp (cha c 100%) ta phi ch ti nng %
ti a ca chng tnh ton.
- Th d: Pha HCl 1M t HCl 37%
M(HCl) = 36,5
Ta phi cn: (36,5 x 100)/37 98,65g HCl
Hay: 98,65/1,19 83ml HCl
Vy ta phi ly 83ml HCl 37% pha vi nc ct thnh 1 lt th c dung dch HCl c nng
1M.
d. Nng ng lng gam (N)
Dung dch nguyn chun 1N cha 1 ng lng gam cht tan trong 1 lt.
ng lng gam s c nh ngha theo mi trng hp ring bit t phng trnh phn
ng dng trong lc nh phn.
* Trong nh phn acid hay base
ng lng gam ca mt cht acid l khi lng cht c th cho ra trong phn ng 1
ion gam H
+
.
ng lng gam ca mt cht base l khi lng cht c th cho ra trong phn ng 1
ion gam OH
-
.
- Th d:
H
+
Cl
-
+ NaOH Na
+
Cl
-
+ H
2
O
1 phn t gam HCl cho ra 1 ion gam H
+
Vy ng lng gam HCl = 1 phn t gam HCl

2H
+
SO
4
--
+ 2 Na
+
OH
-
2Na
+
SO
4
--
+ H
2
O
1 phn t gam H
2
SO
4
cho ra 2 ion gam H
+

Vy ng lng gam H
2
SO
4
= 1/2 phn t gam H
2
SO
4

Mt phn t gam NaOH cho ra 1 ion gam OH
-

Ti liu thc hnh Phn tch thc phm
Trang 7
Vy 1 ng lng gam NaOH = 1 phn t gam NaOH
Mt dung dch nguyn chun HCl cha 36,5g HCl trong 1 lt.
Mt dung dch nguyn chun H
2
SO
4
cha 98/2 = 49g H
2
SO
4
trong 1 lt.
Con s 1 hay 2 c dng chia phn t gam trong nhng th d trn c gi l h s
nguyn chun . Trong trng hp tng qut s c gi l v M/ c gi l
ng lng gam phn ng.
* Trong trng hp phn ng oxy ha - kh
Mun tm ng lng ca mt cht trong h thng oxy ha kh, ngi ta em chia
phn t gam cho s in tch trao i trong phn ng m cht tham gia.
Th d:
2Na
2
S
2
O
3
+ I
2
Na
2
S
4
O
6
+ 2NaI
S in tch trao i phn ng ny l I. Do N = M/I
Cch pha: vic pha dung dch nng ng lng gam (N) cng tng t nh pha
nng phn t gam (M) nhng thay i s phn t gam (M) bng ng lng gam
(N).
Lu : Hu ht cc ha cht rn khng tinh khit hon ton. Khi tnh ton pha dung dch t cc
ha cht rn cn phi tnh ti tinh khit ca ha cht.
1.2 Hiu chnh nng dung dch
a. Dung dch chun
Trong qu trnh pha ha cht c nhiu yu t lm sai s nng nh: vic cn o khng
chnh xc, cc cht cha tinh khit hay ht nc, lu b thng hoa hay b oxy ha. Do
ngi ta phi kim tra nng thc ca cc dch pha da vo cc cht n nh hay nng
chnh xc c coi l dung dch chun. Cc cht dng trong dung dch chun phi bn vng
nng cht phn ng ny khng thay i nhanh chng vi thi gian.
b. Cch xc nh nng
- Hai dung dch phn ng vi cng mt th tch s c cng mt chun . Khi mt th tch V
1

ca dung dch c chun C
1
(C
1
, N) tc dng trn mt th tch V
2
ca dung dch c chun
C
2
(C
2
, N), chng ta c th vit rng s ha tr gam tc dng vi nhau u bng nhau trong 2
trng hp:
C
1
V
1
= C
2
V
2

- H thc trn dng hiu chnh li nng mt s dung dch cho chnh xc.
Th d:
Ta c dung dch chun H
2
SO
4
0,1N chnh xc. Mt dung dch NaOH ta pha ly nng
nh pha l 0,1N. em chun ta thy 10ml H
2
SO
4
0,1N tc dng vi 11ml NaOH ta pha. vy
nng NaOH ta pha l: C
1
= (V
2
C
2
)/V
1
= 0,091N. H s 10/11 = 0,91 l h s hiu chnh.
Ti liu thc hnh Phn tch thc phm
Trang 8
2. Thc hnh pha ch v hiu chnh nng dung dch
2.1 Pha ch dung dch
Yu cu: Pha 100ml dung dch Na
2
S
2
O
3
0,1N t Na
2
S
2
O
3
rn (Ch quan st trn nhn chai
tm cc thng tin v ha cht rn)
2.2 Chun dung dch Na
2
S
2
O
3
bng dung dch K
2
Cr
2
O
7

a. Nguyn tc
Trong mi trng acid, vi lng tha KI v lng xc nh dung dung dch K
2
Cr
2
O
7
chun:
Cr
2
O
7
2-
+ 9I
-
+ 14H
+
= 2Cr
3+
+ 3I
3
-
+ 7H
2
O
Lng I
3
-
(phc ca dung dch I
2
) to thnh c chun bng dung dch Na
2
S
2
O
3
.
b. c im
- Chun trong mi trng c [H
+
] = 0,2 - 0,4M. Mi trng cng acid cng lm tng
tnh nh lng ca cn bng chun nhng cng lm cho I- d oxi ha bi oxi ca
khng kh.
- phn ng hon ton, cn s dng KI thch hp [I
-
] >2% v yn hn hp trong
ti khong 10 pht.
- nh im cui: s dng ch th h tinh bt.
c. Th nghim
* Ha cht
- Dung dch chun K
2
Cr
2
O
7
0,05N.
- Na
2
S
2
O
3
rn
- Dung dch KI 10%.
- Dung dch H
2
SO
4
(1:5) hoc HCl (1:1)
- Dung dch ch th h tinh bt.
* Dng c
- Buret 25ml: 1 cy/t.
- Erlen 100ml: 3 ci/t.
- Pipet 5ml: 3 ci/t.
- Bnh nh mc 100ml: 1 ci/t.
* Cch tin hnh
- Buret: Dung dch mu Na
2
S
2
O
3
.
- Erlen: ht V(ml) dung dch K
2
Cr
2
O
7
0,05N + 5ml H
2
SO
4
+ 5ml KI, yn khong 10
pht. Chun n khi dung dch c mu vng nht + vi git ch th h tinh bt. Chun
tip n mt mu xanh (dung dch c th khng mu hoc c mu xanh xm nht ca
Cr
3+
).
* Kt qu
- Xc nh
f
V t 3 ln chun .
- Xc nh h s hiu chnh nng ca Na
2
S
2
O
3
trong dung dch pha c.

Ti liu thc hnh Phn tch thc phm
Trang 9


Bi 2: NH LNG NIT TNG BNG PHNG PHP MICRO -
KJELDAHL

1. Mc ch
Phng php Micro - Kjeldahl thng c dng xc nh tng lng nit trong
nguyn liu

2. Nguyn tc
Di tc dng ca H
2
SO
4
m c nhit cao, cc hp cht cha hu c b phn
hy v b oxy ha n CO
2
v H
2
O cn nit chuyn thnh NH
3
v tip tc kt hp vi
H
2
SO
4
to thnh mui amoni sulfat.

3. Nguyn liu, thit b, dng c, ha cht
* Nguyn liu
- Mu bt u phng sy kh n mt m hon ton
* Thit b
- H thng ct m
- Bp in
- T ht
* Dng c
- Bnh Kjeldahl
- Pipette 10ml
- Becher
- Erlen
- Mung inox
- Da nha
- Bnh nh mc 250ml
- Phu thy tinh
- Burette 25ml
- Gi burette
- Kp burette
- Bnh xt nc ct
- ng bp cao su
* Ha cht
- H
2
SO
4

- NaOH 45%
- Hn hp CuSO
4
: K
2
SO
4
theo t l 1:3
- H
2
SO
4
0,1N
- Ch th methyl red (hay phenolphtalein)
Ti liu thc hnh Phn tch thc phm
Trang 10
4. Tin hnh
* V c ha mu
- Cn chnh xc khong 3g mu rn tch m hon ton (sy 100 105
0
C n khi
lng khng i).
- Gi mu bng giy lc khng tro.
- Chuyn mu vo bnh v c ha (bnh Kjeldahl).
- Thm vo bnh v c ha 10ml H
2
SO
4
m c v 0,2g hn hp CuSO
4
: K
2
SO
4
theo t
l 1:3.
- t bnh v c ha ln bp un v un n khi dung dch trong sut v c mu xanh
ngc (khng cn) th dng qu trnh v c mu. ngui.
- Tin hnh nh mc ln 250ml

* Lp h thng ct m theo s


A: Bnh un to hi nc
B: Bnh thu dch thi
C: Bnh ct (cha dung dch v c pha long v NaOH m c)
D: Phu dn cht th v NaOH vo bnh ct C
E: H thng sinh hn
F: Bnh thu NH
3

P, P
1
, P
2
: Cc kha dn

* Chun b vn hnh h thng ct m
- Cho nc vo bnh un (1/2 2/3V) th tch ca h thng sinh hn (m van nc).
- ng cc kha ca h thng.
- S dng n cn un nng bnh un.
- Khi nc ngng t u ra ca h thng sinh hn th tin hnh quy trnh ra.
Ti liu thc hnh Phn tch thc phm
Trang 11
- Cho nc ct vo phu, np mu v m kha nc vo bnh ct.
- Tt n cn, nc bnh ct s chuyn vo bnh x, m kha bnh x x nc ra
ngoi.
- Lp li quy trnh ra t nht hai ln.
* Ct m
- Chun b bnh thu NH
3
cha 10ml dung dch H
2
SO
4
0,1N, 2 git ch th methyl red (hay
phenolphtalein) v nhng ngp u ra ca ng sinh hn.
- M n cn un nng bnh un.
- Mu v c ha v pha long c cho vo phu np mu (khong 10ml).
- M kha phu np mu t t a mu vo bnh ct.
- Ra phu np mu 3 ln bng nc ct v m.
- Cho 10ml NaOH 45% vo phu np mu, m kha t t lu khng x ht.
- Ra phu np mu 3 ln (cho t t vo khng x ht).
- Thc hin qu trnh ct trong 10 pht.
* Chun , tnh kt qu
H bnh thu NH
3
khi u ra ng sinh hn v thc hin trong 5 pht. Ly bnh thu NH
3

ra v tin hnh chun .
Hm lng m tng X (%) c tnh theo cng thc:
(%)
.
100 . . .( 0014 , 0
) 1 , 0 1 , 0
m v
V f v V
m
m Nth NaOH Ntr NaOH


f : h s hiu chnh nng NaOH
V
NaOH 0,1 tr
(trng) : th tch NaOH 0,1N chun mu trng (ml)
v
NaOH 0,1N th
(tht) : th tch NaOH 0,1N chun mu tht (ml)
v
m
: th tch mu a vo ct (10 ml)
V
m
: th tch mu nh mc (250 ml)
m : khi lng mu a vo v c ha (g)
* Xc nh h s hiu chnh nng f
Ly 10ml H
2
SO
4
0,1N vo erlen, thm 2 git ch th phenolphtalein em chun
bng NaOH 0,1N n khi xut hin mu hng nht th ngng. Tnh nng thc t ca
NaOH, s dng h thc hiu chnh nng :
C
1
.V
1
= C
2
.V
2

f: l t s gia nng thc t v nng theo l thuyt ca NaOH
Ti liu thc hnh Phn tch thc phm
Trang 12
Bi 3: NH LNG PROTEIN THEO PHNG PHP LOWRY

1. Nguyn tc
Hu ht cc protein u cha Tyrosin v Tryptophan. Hm lng ca nhng acid amin ny
ty thuc vo loi protein. V vy, nhng protein cng mt loi vi nhau c hm lng cc
acid amin ny ging nhau.
Khi cho protein tc dng vi thuc th Folin s to thnh mt phc cht c mu. Cng
mu ca phc ny t l vi hm lng Tyrosin v Tryptophan (cng l hm lng protein). V
th, ta c th dng phng php so mu xc nh hm lng protein.
2. Dng c v ha cht
* Dng c
- Buret 25ml
- Pipet 1ml, 2ml, 5ml, 10ml chnh xc
- Bnh nh mc 50ml
- Erlen 250ml
* Ha cht
- Dung dch albumin 0,1% : cn chnh xc 0,1g albumin pha vi nc ct thnh 100ml.
- Dung dch A: cn 2g Na
2
CO
3
ha tan trong NaOH N/10 thnh 100ml
- Dung dch B: cn 0,5g CuSO
4
.5H
2
O ha tan trong dung dch citrat natri 1% to thnh
100ml.
- Dung dch C: (ch pha dng trong ngy) l hn hp ca hai dung dch A v B c
pha theo t l 49:1
- Thuc th Folin : thuc th ny pha sn, cch u ch nh sau:
o Cn: 100g Natri tungstat v 25g Natri molybdate tht tinh khit. Thm vo 700ml
nc ct v 50ml acid orto phosphoric 85%. Khuy cho ha tan v thm vo
100ml acid clohydric m c. Tip tc khuy ri cho vo mt bnh cu nt nhm
2 lt. un hon lu trong 10 gi.
o Sau khi un cho vo 150g Lithium sulfat. Khuy cho n khi tan hon ton ri
vo thm 5 10ml brom 1/3 (hoc 2 3ml Brom lng). Khuy u v un si
trong t ht kh c trong 15 pht. Lm lnh nhit thng. Cho vo bnh nh
mc 1 lt v thm nc ct cho . Lc k v lc (nu dung dch khng trong). Dung
dch c mu vng chanh, nu chuyn sang mu xanh l khng dng c. Bo qun
trong chai thy tinh mu.
3. Thc hnh
* Dng ng chun
Ta thc hin ng chun vi mt loi protein tinh khit c sn, thng l albumin ca b
ng kh theo bng sau c c cc dung dch albumin chun c nng protein t 0
n 250 /ml:
Ti liu thc hnh Phn tch thc phm
Trang 13
ng nghim s 0 1 2 3 4 5
Nng protein g/ml 0 50 100 150 200 250
Dung dch albumin 0,1%(ml) 0 0,5 1,0 1,5 2,0 2,5
Nc ct (ml) 10 9,5 9,0 8,5 8,0 7,5

Ht 0,4ml dung dch protein c nng khc nhau t cc ng nghim va pha trn theo
th t t 0 n 5 vo by ng nghim sch khc nhau (gm hai ng th khng v nm ng t 1
n 5). Thm vo 2ml dung dch C. Lc u v yn nhit phng trong 5 pht. Sau
thm vo 0,2ml thuc th Folin, lc u trong 5 10 pht, thm nc ct cho 5ml. em
o mt quang bc sng 750nm hay 500nm.
X l s liu thu c theo phng php bnh phng cc tiu.V biu biu din s
bin thin ca mt quang (
A
OD) theo nng protein chun ( /ml).
* Xc nh hm lng protein trong mu
Ht 0,4ml dung dch protein cn xc nh cho vo mt ng nghim sch v sy kh. Thm
vo 2ml dung dch C. Lc u v yn nhit phng trong 5 pht. Sau thm vo
0,2ml thuc th Folin, lc u trong 5 10 pht, thm nc ct cho 5ml. em o mt
quang bc sng 750nm hay 500nm. (Nn lm 3 ng nghim ly trung bnh).
* Tnh ton
T ng chun so snh mt quang ca ng nghim cha mu protein. T suy ra hm
lng protein ca nguyn liu l a ( /ml).
Lng protein (M) c trong 1g nguyn liu c tnh theo cng thc:

m
n a
M
. 10 .
3
= mg protein/1g
Vi
a: l hm lng protein ( /ml dung dch)
n: l h s pha long
m: khi lng nguyn liu ly phn tch (g)
Ti liu thc hnh Phn tch thc phm
Trang 14
Bi 4: NITROGEN NITRITE

1. Nguyn tc
- Nitrite c xc nh bng phng php so mu, mu do phn ng t cc dung dch
tham chiu v mu sau khi tc dng vi acid sulfanilic v naphthylamine mi
trng pH bng 2 2,5 to thnh hp cht tm ca acid azobenzol naphthylamine
sulfonic.
- Phng php diazo thch hp khi hm lng N NO
2
t 1 25mg/l.


















Hnh 4.1: C ch phn ng to mu
2. Dng c, thit b v ha cht
* Dng c, thit b
- Bnh nh mc 10ml
- Pipet 1ml, 5ml, 10ml.
- Bu ht an ton
- Spectrophotometer.
* Ha cht
- Dung dch chun: dung dch lu tr NO
2
- 1000ppm: ha tan 1,5 g NaNO
2
khan
trong nc ct v nh mc thnh 1 lt.
- Dung dch NO
2
- chun 10ppm: ly 10ml dung dch chun 1000ppm + nc ct nh
mc thnh 1 lt.
Ti liu thc hnh Phn tch thc phm
Trang 15
- Dung dch sulfanilic: cn 0,6g acid sulfanilic + 70ml nc nng ngui + 20ml
HCl m c, pha long thnh 100ml vi nc ct.
- Dung dch naphthylamine chlohydrate: cn 0,6g naphthylamine chlohydrate + 50ml
nc ct + 1ml HCl m c + nc ct thnh 100ml. Pha dng ngay hoc gi
nhit thp.
3. Thc hnh
- Chun b mu v dung dch tham chiu ng thi
STT ng nghim 1 2 3 4 5 M
1
M
2

Dd NO
2
chun (10ppm) (ml) 0 1 2 3 4
Mu (ml) 8 8
Dung dch sulfanilic (ml) 0,5
i 10 pht
Dung dch naphthylamine 0,5
i 20 pht
H
2
O (ml) 9 8 7 6 5 1 1
C (mg/l) 0 Cx Cx
- Thm vo mu cc dung dch theo ng th t trong bng.
- o hp thu A bc sng 520nm.
4. Cch tnh
- T ng chun o hp thu.
- S dng phng php bnh phng cc tiu lp phng trnh y = ax + b, v gin
A = f(C). T tr s hp thu ca mu A
m
suy ra nng C
m
.
C
m
(NO
2
-
) =
8 . 1000
. 1000 . 10 . f C
x
(ppm)
f: pha long ca mu trc khi hin mu
5. Cu hi chun b
1.1. Phn tch mt mu nc ngm, kt qu hm lng nitrite cao, c th kt lun g?
1.2. Nitrogen thng tn ti dng no trong nc mt, nc ngm?
Ti liu thc hnh Phn tch thc phm
Trang 16
BI 5: NH LNG NG KH BNG PHNG PHP ACID
DINITROSALICYLIC (DNS)

1. Nguyn tc
Phng php da trn c s phn ng to mu gia ng kh vi thuc th acid
dinitrosalicylic.Phn ng xy ra trong mi trng kim v c gia nhit. Cng mu ca
hn hp phn ng t l thun vi nng ng kh trong mt phm vi nht nh. Da
theo th ng chun ca glucose tinh khit vi thuc th acid dinitrosalicylic s tnh
c hm lng ng kh ca mu nghin cu.
2. Nguyn liu, thit b, ha cht
* Nguyn liu
- Chui s chn
* Thit b
- My quang ph
- Bp in hoc b iu nhit
* Dng c
- ng nghim
- Gi ng nghim
- Becher 500 ml
- Becher 250 ml
- Becher 100 ml
- Kp ng nghim
- Bnh xt nc ct
- Bp cao su
- Ci chy s
- Da nha
- Dao
- Mung Inox
- Pipette 5 ml
- Pipette 1 ml
- Phu thu tinh
- Bnh nh mc 100 ml
- Cuvet nha
- Giy lc
- Erlen 250 ml
- Erlen 100 ml
- a khuy
* Ha cht
- Thuc th acid dinitrosalicylic (DNS):
Nc ct : 1416 ml
Acid dinitrosalicylic ( C
7
H
4
N
2
O
7
) : 10,6 g
Ti liu thc hnh Phn tch thc phm
Trang 17
NaOH : 19,8 g
Sau khi ha tan thm vo dung dch gm:
Mui seignette (KNaC
4
H
4
O
6
.4H
2
O) : 306 g
Phenol (C
6
H
5
OH) : 7,6 ml
Natri metabisulfit (Na
2
S
2
O
5
) : 8,3 g
- Glucose 0,1%
- Acetat ch 10 %
- Na
2
HPO
4
bo ha
- Acid trichloroacetic 10%,
- NaOH 5% ,
- Ch th methyl red
- Ch th phenolphtalein
- Na
2
CO
3
bo ha.
- Cn 70 80
o
.
- Giy lc
3. Tin hnh
* X l mu
Ty vo i tng nghin cu m cch chun b dung dch dng nh lng ng c
khc nhau:
- Nguyn liu khng cha nhiu tinh bt
Dng nc nng trch ly ng. Cn 12g mu nu l nguyn liu kh (cy, l hoc qu
kh) hoc 5 10g nu l nguyn liu ti c hm m cao (rau, qu ti). Cho vo ci s
nghin tht nh (c th nghin vi bt thy tinh hay ct sch). Trch ly nhiu ln bng 30ml
nc ct nng 70 80
o
C. Chuyn lng dch vo bnh nh mc, b phn b.
- Nguyn liu cha protein (da,)
Trch ly dung dch (V<50ml), em kt ta protein v cc tp cht bng dung dch acid
trichloroacetic 10% (hoc Acetat ch 10 %), sau trung ha bng dung dch NaOH 5% vi ch
th methyl red (mu chuyn sang vng) hoc Na
2
HPO
4
bo ha.
- Nguyn liu cha nhiu acid hu c (da, chanh, kh,)
Trong qu trnh trch ly ng, sacarose c th b thy phn mt phn do s c mt ca acid
hu c c sn trong nguyn liu, do khi xc nh ng kh phi trung ha acid hu c
bng dung dch NaOH 5% hay Na
2
CO
3
bo ha.
Nghin nhuyn nguyn liu trong ci s vi mt t nc ct. Nh 3 git ch thi methyl
red hoc phenolphtalein v cho t t tng git NaOH 5% n khi xut hin mu hng nht.
Tip tc trch ly bng nc m nh trn
- Nguyn liu cha nhiu tinh bt (chui, khoai lang,..)
Trch ly ng bng 30ml ru 70 80
o
. Trong trng hp ny khng cn kt ta protein
v lng protein chuyn vo dung dch khng ng k.
Mu qua x l thm nc ct ti vch nh mc 100ml, lc qua giy lc vo cc. Nc
qua lc l dung dch mu cn phn tch.
Ti liu thc hnh Phn tch thc phm
Trang 18
* Dng th chun Glucose
ng s
ng i
chng
1 2 3 4 5 Mu
Dd glucose chun 0,1% (ml) - 0,2 0,4 0,6 0,8 1,0 -
Dd mu (ml) - - - - - - 1,0
Nc ct (ml) 3,0 2,8 2,6 2,4 2,2 2,0 2,0
Thuc th DNS (ml) 1,0 3,0 3,0 3,0 3,0 3,0 3,0
OD
540nm






un (un cch thy) cc ng nghim ng 5 pht
Lm ngui n nhit phng, o mt quang (OD) bc sng 540nm
* Lu
- Phn ng to thnh trong mi trng kim v vy nhng mu c cha acid phi c
trung ha trc khi em phn tch.
- Phng php ny c hiu cho tt c cc ng kh.
- Cc mu un c th c mt thi gian (20 pht) trc khi o. Cc mu khng
un s b thy phn dn dn.
- Nhng dung dch ng m c phi c pha long sao cho mu dung dch mm lt
trong khong dy mu ng 1 n 5
4. X l kt qu
- Xy dng ng chun glucose y = f(x) vi trc tung (y) l mt quang, trc honh
(x) l hm lng glucose (mg) bng phng php bnh phng cc tiu.
- Da vo ng chun, tnh nng X (mg/ml) glucose trong dung dch mu
5. Tnh ton
Hm lng glucose trong nguyn liu c tnh theo cng thc:
. .100
1000.
X V
G
m
=
f
mu
(%)
Vi:
m : lng mu em th nghim (g)
V : th tch nh mc dung dch th nghim (100ml)
X : nng ng kh trong dung dch mu tnh theo glucose (mg/ml)
f
mu
: h s pha long mu (nu c)
Ti liu thc hnh Phn tch thc phm
Trang 19
Bi 6: SC K CT (COLUMN CHROMATOGRAPHY)
LY TRCH V KHO ST SC T L CY
1. Nguyn tc
Trong cc loi l cy thng c cha sc t gm chlorophyll, carotenoid Nhng loi sc
t ny c dng rng ri trong thc phm cng nh y dc. bi thc hnh ny, chng ta
dng phng php sc k ngoi hp (hp ph) tch chng ra khi hn hp.
Trc ht cc sc t c cho hp ph vo ct cellulose (cellulose c xem nh l phase
c nh), sau dung ly chng nh cc loi dung mi khc nhau (phase di ng) tch chng
ra khi hn hp. Nh ta bit cc loi sc t c trong l cy, nu ng v mt phn cc tng
dn th s nh sau: carotene, xanthophyl, chlorophyll b, chlorophyll a. Nh vy ta s cho dung
ly chng bng cc loi dung mi phn cc tng dn: ether du ha, ether du ha 2% acetone,
ether du ha 5% acetone v cui cng l ether du ha 20% acetone. Cui cng ta s tch
c cc sc t ring bit.
Sau khi c c nhng sc t, ta phi nh tnh v nh lng chng. V nh tnh, chng ta
kho st ph hp thu ca n. Mi sc t s c ph hp thu ring, nh nhng mi hp thu cc
i cho php chng ta xc nh chng. Mun nh lng ring tng sc t, thng thng ngi
ta dng phng php thc nghim (gn ng) v chng ta tuy c lp c chng nhng nhng
sc t ny d b phn hu cho nn kh m c c cht chun.


2. Thit b, dng c, ha cht, nguyn liu
* Thit b
- Cn phn tch
- Spectrophotometer
- Thit b lc chn khng
Hnh 6.1: Trnh t thao tc trn sc k ct
Ti liu thc hnh Phn tch thc phm
Trang 20
* Dng c
- Ct sc k
- Becher
- Pipette
- ng ong 50ml
- a thy tinh
- ng nghim
- Bnh tia
- Ci s
- Giy lc 8 |
* Ha cht
- Bt cellulose (dng cho sc k hp ph)
- Petrlium ether
- Acetone
* Nguyn liu
- L rau m
3. Tin hnh
* Ly trch sc t t l cy
Nghin nhuyn trong ci s 20g l cy ti (thng dng l l rau m). Thm vo 30ml
acetone, trn nhiu ln trong 10 pht, lc qua phu Buchner v hng phn acetone.
cht trch bng acetone vo trong 1 phu chit, ta trch ra ln th 2 vi ether du ha.
Mun vy, ta vo phu chit 10ml ether du ha, lc nh, yn s tch thnh 2 phase. B
phase di, thm 50ml nc ct, lc nh (c th c nh tng, thi gian lc khong 5 10
pht), b phn di (phase nc) v c lm nh th khong 10 ln cho n khi ht mi acetone
(c th ngi xc nh, nu dung dch cn cha acetone th khng th tch carotene ra c).
Mc ch l c mt hn hp cha cc sc t trong dung mi v cc. Nu dung dch cn nhiu
(>2ml), un cch thu n khi th tch cn khong 2ml (ong th tch chnh xc ca dung dch
ny). Cc sc t ha tan trong ether du ha s c tch ra nh php sc k ngoi hp trn ct
cellulose.
* S phn ly cc sc t trn ct cellulose
vo di ct 1 ming bng gn nh. Ngm trong ether du ha 1 lng va bt
cellulose trong 15 pht v vo ct, va , va g nh vo ct bt cellulose lng u v
nh ng cho cellulose kh. B cao ca ct cellulose khong 12 15cm. yn trong
khong 15 pht cho ct n nh, m kha cho dung mi chy xung cho n khi mc ether du
Ti liu thc hnh Phn tch thc phm
Trang 21
ha va xung n u ct cellulose, cho vo mt cch thn trng 1ml dung dch sc t trong
ether du ha.
M kha cho dung mi chy xung, khi dung mi cn li trn ct va chm n u ct, ta
cho 1ml ether du ha, lm nh th 3 ln cho hn hp sc t nm hon ton trong ct, lc
ny sc t c ngoi hp (hp ph) trn ct cellulose.
S phn ly c thc hin vi cc dung mi ether du ha c nng acetone cao dn.
1. Ether du ha
c dng dung ly carotene. Khi phn ether du ha dng ra h xung u ct, ta
1 lng va ether du ha dung ly (khong 5 10ml), ta hng phn dung dch cha
carotene. Ta c cho chy n khi no phn di ct ra ht phn carotene.
2. Hn hp ether du ha 2% acetone
vo ct hn hp ether du ha + 2% acetone, ta thy xut hin 3 lp: xanthophyl v
chlorophyll b, chlorophyll a (xanthophyl c mu vng, chlorophyll b c mu xanh l cy,
chlorophyll a c mu xanh v u). C tip tc dung ly s thu c xanthophyl. Phn
xanthophyl thng kh nhiu, v vy khi dung ly gn ht xanthophyl, ta bt u cho ether du
ha 5% acetone vo, c 2 sc t xanthophyl v chlorophyll b u di chuyn nhng xanthophyl
di chuyn nhanh hn.
3. Hn hp ether du ha 5% aceton
Hn hp ny dng dung ly chlorophyll a
4. Hn hp ether du ha 20% aceton

Hn hp ny dng dung ly chlorophyll b.
* V quang ph hp ph ca cc sc t
cc phn dung ly hng c vo trong quang ph k, o di song t 350 -700nm
v tng dn tng 20nm, ta s c s thay i mt quang ca cc sc t ny. (Chn dung dch
khng l ether du ha khi o carotene, hn hp ether du ha + 2% acetone khi o
xanthophyl)
Xanthophyl
(vng)
Chlorophyll a
(xanh l cy)
Hnh 6.2 Qu trnh tch sc t trong ct
Chlorophyll b
(xanh v u)
Ti liu thc hnh Phn tch thc phm
Trang 22
V quang ph hp thu ca cc sc t c phn ly (trc tung l mt quang, trc honh l
di sng)
Ch : Trong khi o quang ph hp thu, nh o mt quang ca cc dung dch:
- Chlorophyl a, chlorophyll b cc di sng 661,6 v 644,8nm.
- Vi carotene v xanthophyl th trn ln ri o mt quang di sng 470nm.
4. nh lng
Cc sc t c nh lng theo cc cng thc thc nghim sau:
C
a
= 11,24A
661,6
2,04A
644,8
C
b
= 20,13A
644,8
4,19A
661,6

C
a+b
= 7,05A
661,6
+ 18,09A
644,8


C
x+c
=

Trong : C
a
: nng chlorophyll a (g/ml)
C
b
: nng chlorophyll b (g/ml)
C
a+b
: nng tng cng ca chlorophyll (g/ml)
C
x+c
: nng tng cng ca carotenoid (carotene v xanthphyl) (g/ml)
A
z
: mt quang o di sng z.
Lu : khi tnh nng cc sc t, mt quang ca cc sc t c tnh trn cng mt n v
th tch c nh. Mun vy, chng ti ngh lc u ong th tch ca carotene gi s V ml,
sau vi cc dung dch xanthophyl, chlorophyll a v chlorophyll b chng ta c trn bp cch
thu loi bt dung mi sao cho cc loi sc t u c th tch thu c l V ml.
1000A
470
1,90C
a
63,14C
b

214
Ti liu thc hnh Phn tch thc phm
Trang 23
BI 7: SC K BN MNG (THIN LAYER
CHROMATOGRAPHY)
PHN TCH HN HP VANILLIN V
|
NAPHTHOL
PHN TCH CC SC T T L CY

1. S lc v l thuyt
1.1 nh ngha
- Sc k l qu trnh tch cc cu t ca mt hn hp da vo vic cc cu t ny s phn b
khc nhau gia pha tnh v pha ng. Pha tnh c th l ct nhi (sc k ct) v pha ng l
dung mi hu c s di chuyn ngang qua; pha tnh c th l mt lp mng cht hp ph, lc
pha ng s c ht thm ln lp mng nh lc ht mao dn.
- Sc k bn mng l k thut phn b rn - lng, trong pha lng di ng c cho i trn
mt lp mng cht hp ph c trng ln mt nn phng bng vt liu nh thy tinh, nhm
hoc plastic. Do cht hp ph c trng trn mt lp mng nn c tn gi chung l tm bng
mng.
1.2 Cht hp ph
- C 2 loi cht hp ph thng dng l alumin G v silicagel G. Ch G do ch Gypsum (sulfat
calcium). Trong cht hp ph dng cho bn mng thng c 10 13% cht gypsum lm
cht kt dnh. Mun c mt dung dch st cht hp ph trng bn, c th dng dung mi
clorur methylen to dung dch st, mn, ng nht v khng lm cho gypsum kt dnh li
nn c th dnh dung dch st ny trong nhiu ngy nu cha kp trng bn. C th s dng
nc lm dung mi, bn trng ri s kh trc hn nhng nn dng ngay v lu s c hin
tng vn cc.
1.3 Trng bn
1.3.1 Trng bn kh nh (2-3x6-8)
- Cc tm king c ra sch bng cc cht ty ra thng dng loi ht cc vt du m, cc
vt bn; nu khng th ra bng dung dch acid cromic; trng li bng nc ct; dng ng cho
ro nc v sy kh.
- Chun b dung dch st: trong mt bnh hnh khi tr hoc khi ch nht c np y cha sn
100ml CH
2
Cl
2
hoc 100ml nc ct, va cho t t va khuy u 30g cht silicagel G hoc
alumin G.
- Nhng bn: ghp 2 bn king st kht vo nhau ri nhng ngp vo dung dch st ni trn, rt
bn ln t t ra khi dung dch, yn 1 pht cho dung dch chy tr vo chai. Tch ring 2 bn
v t nm ngang trong 10 pht, dng dao lam co b phn bt d tha 4 cnh, sy bn trong
30 pht 110
0
C. Ly ra, ngui, bc bn trong t giy thm bo qun trong bnh ht m hoc
ni kh ro.
Ti liu thc hnh Phn tch thc phm
Trang 24
1.3.2 Trng bn kh ln (4x12, 5x10, 10x20)
- Chun b dung dch st: theo ch dn nh sn xut hoc tnh ton lng silicagel v lng
nc cho mt bn nh sau: bn 10x20, dng 3g silicagel trong 10ml nc. Cho bt kh vo
mt becher nh c sn lng nc cn thit, dng a khuy u, nh hn hp trong 1 -2 pht
to ra mt dung dch st, ng nht, khng c bt kh.
- Trng bn: tay tri cm bn, bn nm trn lng bn tay, dng 3 ngn tay ci, tr v gia
gi 2 bn cnh tm bn, tay phi cm becher cha dung dch st, va rt va ko ph dung dch
ln mt u cnh ca bn, va nghing nh bn dung dch chy u xung v ph ht tm
bn; c th rt thm vi git gel ln tm king ch cha c trng hoc dng m nhn ca
becher chm b cc bt kh trn bn trng. yn nhit phng cho ro mt ri sy kh
110
0
C trong 30 pht, bo qun k dng dn.
1.4 La chn dung mi
- Vic chn dung mi trin khai ty thuc vo mu cn tch ly. Vi mu cha bit thnh phn,
cha bit ti liu tham kho, cn th nghim vi nhiu loi dung mi khc nhau, t loi khng
phn cc n loi phn cc.
- Cch xc nh nhanh loi dung mi ph hp vi mu:
Chm dung dch mu thnh nhiu chm bng u nhau ln trn cng mt bn mng, cc vt
chm cch nhau 1 cm. Dng cc ng vi qun a cc dung mi c phn cc khc nhau,
thm nh ln vt chm mu, mi vt chm mu mt git dung mi loi khc nhau. Sau khi
chm, dung mi s lan ta to thnh vng trn. Dng vit ch khoanh trn vt lan xa nht ca
dung mi. Quan st cc vng trn ng tm: dung mi no lm cho mu lan ra ngoi cng lc
vi tin tuyn dung mi th dung mi qu phn cc; dung mi no m mu nm ti ch l
khng phn cc.
Hoc d quan st hn nn thit lp mt lot th nghim vi nhng bnh trin khai sc k
bn mng trong mi bnh cha mt trong cc dung mi vi phn cc tng dn: hexan,
benzene, chloroform, eter diethyl, acetone, methanol. Chun b cc tm bng mng c cha cc
mu cht nh nhau ri nhng mi tm vo mt bnh nh chun b. Ghi nhn di ng ca
cc cu t trong mu:
o Nu dung mi no khin cho tt c cc cu t nm ti ch mc xut pht th dung mi
cha phn cc: dung mi khng ph hp.
o Nu dung mi no khin tt c cc cu t di chuyn ht ln mc tin tuyn dung mi th
dung mi qu phn cc: dung mi khng ph hp.
o Nu dung mi no c th lm cho cht mu ban u tch thnh nhiu vt khc nhau mt
cch gn, r, sc nt v v tr cc vt nm khang 1/3 n 2/3 chiu di bn sc k th
dung mi ph hp.
o Nu qua qu trnh trin khai m nhn thy h thng n dung mi khng cho nhng vt
gn, r, sc nt th cn th trin khai vi h thng hn hp dung mi.
Dung mi no tch tt cht trn sc k bn mng s thch hp cho sc k ct.
Ti liu thc hnh Phn tch thc phm
Trang 25









1.5 Chm bn
- Trc khi chm mu ln bn phi vch ln "mc xut pht" cch y bn 1cm; v ln "mc
tin tuyn dung mi" cch u bn 0,5cm.
- Mu l cht lng th s dng trc tip. Nu mu l cht rn, ly 1mg mu t ln mt king
ng h, ha tan mu vi vi git dung mi d bay hi nh acetone. Dng vi qun nhng nh
phn u nhn vo dung dch mu, lc mao dn s ht dung dch mu vo vi qun, chm nh
phn u nhn c cha mu ln trn bn mng ti mt im cch y bn 1cm. Cn thn, nh
nhng u nhn ca vi qun chm nh, khng lm thng l b mt. Chm vo v ly ra tht
nhanh dung dch mu thm vo bn to thnh mt im trn nh. Thi nh ln vt chm
dung mi bay hi mau khng lan thnh vt chm to. C th chm thm ln ngay vt c vi ln
na c vt m, r, ng knh khng qu 2mm. Nn chm nhiu ln, mi ln mt lng
nh dung dch mu hn l chm mt ln vi lng mu ln. Nu cn phi chm cng lc nhiu
vt chm trn cng mt bn th cc vt chm phi cch u nhau 1cm v cch y bn 1cm v
cch 2 cnh bn 1cm.
1.6 Chun b bnh trin khai
Bnh hnh khi tr hoc khi ch nht c ng knh ln hn b ngang bn mt t. t mt
t giy lc bao ph mt trong ca bnh nhng vn cha mt khong c th quan st bn
trong. Tnh ton lng dung mi gii ly sao cho khi vo bnh sao cho khi vo bnh, lp dung
mi s dy khang 0,5-0,7cm. Cho dung mi gii ly vo bnh, yn 5-10 pht bo ha hi
dung mi trong bnh. Bn mng c cm thng ng v c nhng vo dung mi trong bnh,
khi nhng vo phi cn thn hai cnh bn ca bn khng chm vo thnh bnh; lc v tr
ca cc vt chm mu nm trn cao cch mt thong ca dung mi khong 0,5cm. y np
bnh, dung mi s c ht ln nh lc ht mao dn. Theo di khi mc dung mi ln n vch
tin tuyn dung mi th ly bn ra khi bnh. Sy nh bng my sy. Quan st bng mt v dng
vit ch khoanh nh cc vt thy c. Cn nu khng thy g, c th nhn bn di n t
ngoi, bng hi iod hoc phun bn vi cc thuc hin hnh thch hp.
Dung mi khng
phn cc
Dung mi ph
hp
Dung mi qu
phn cc
Vt chm mu
Tin tuyn dung
mi
Hnh 7.1: Cch la chn dung mi
Ti liu thc hnh Phn tch thc phm
Trang 26



1.7 Hin hnh cc vt chm sau khi trin khai
- Thuc hin hnh thng dng nht l Iod. Mun hin hnh, cho bn mng trin khai, sy kh
vo mt l ming rng c np y, trong l c mt l nh cha vi tinh th iod. y np li,
hi iod ta ra chim ht l v cc vt s hin hnh r trn bng mng. Ly bn mng ra khi l,
dng vit ch khoanh du cc vt v theo thi gian iod s thng hoa v cc vt s bin mt.
- Phng php thng dng k tip l nhn bn di n t ngoi. Cc vt cht s thy nh
nhng vt sng trn nn bn.
- Mt phng php khc l trn thm mt cht pht hunh quang vo cht hp ph trng bn.
Lc di n t ngoi c bn mng s pht sng cn cc vt l nhng im ti.
- a s cc nhm chc hu c c th nhn thy c khi phun dung dch acid sulfuric m c
ln bn ri t bn vo l sy 110
0
C.
- Vi hp cht aldehyd, ceton th phun thuc th 2,4-dinitrophenylhydrazin ln bn s thy xut
hin cc vt mu vng, vng cam hoc .
- Vi hp cht phenol th dng FeCl
3
.
- pht hin cc acid carboxylic th dng bromocresol lc.
- Cc hp cht d b oxid ha th dng permanganate kali, dicromat kali.
- pht hin amin th dng paradimethylaminobenzaldehyd.
- Cc amino acid th dng dung dch ninhydrin.
1.8 Gi tr Rf
- Trong qu trnh gii ly, mt hp cht no phi lun lun di chuyn mt an di nht nh
so vi on di m dung mi di chuyn. T l ny l Rf

Rf =
Hnh 7.3: Tin trnh sc k
Hnh 7.2: Bnh trin khai sc k
on ng di chuyn ca dung mi
on ng di chuyn ca cht
Ti liu thc hnh Phn tch thc phm
Trang 27
- Trong iu kin nht nh, tr s Rf l hng s cho bt k mt cht no. C th dng Rf xc
nh mt hp cht cha bit nhng phi c s kt hp vi nhiu d kin khc.
- o Rf: cc vt chm c ng knh nh, chn ngay tm ca vt. Vi cc vt lan rng, nn
chy li bn mng mi v chm cc mu thnh im nh. Cc vt ko di c ui nn chn
trng tm ca vt.
2. Thc hnh
2.1 Phn tch hn hp vanillin v | naphthol
* Dng c v ha cht
Dng c Ha cht
ng nghim c np 1 Silicagel 0,8g
King thy tinh 4x12cm 2 Chloroform 13ml
King thy tinh 10x10cm 2 Acetat ethyl 7ml
Ly thy tinh y bng 2 Natri sulfat
ng ong 10ml 2 Dd vanillin
Becher 50ml 1 Dd -naphthol
ng vi qun 3 Iod
n cn 1 Dd FeCl
3

Bnh phun sc k 1
Bnh hi iod 1
a thy tinh 1
Qu bp cao su 1
Giy lc 1
Bt ch, thc k 1
My sy 1
* Cch tin hnh
Trng bn mng
- V sinh bn thy tinh.
Ti liu thc hnh Phn tch thc phm
Trang 28
- Cho 0,8g silicagel vo ng nghim c np, thm 3ml nc ct, y np li v lc tht mnh
to mt hn hp huyn ph. Nhanh chng m np ng nghim v trng u silicagel ln bn
thy tinh 4x12cm. Sau khi trng silicagel xong, bn sc k kh t nhin trn mt bn ri sy
110
0
C trong vng 30 pht.
- Tin hnh trng 2 bn sc k.
Chun b trin khai
- Lau tht sch v sy kh bnh trin khai. Ct giy lc v p vo mt trong ca bnh. Cho 10ml
h dung ly chloroform acetate ethyl (t l 8:2 hoc 5:5) vo bnh.
- y np bnh li v yn cho h dung mi thm t ht giy lc.
- Thc hin 2 bnh trin khai vi h dung ly nh trn.
Trin khai sc k bn mng
- Cc dung dch chm bn gm 3 loi: Dung dch vanillin, dung dch -naphthol, dung dch hn
hp ca vanillin v -naphthol.
- Dng ng vi qun chm u 3 dung dch trn ln cng mt bn mng v cch mp di
1cm. nh du mp trn xc nh mc h dung ly thm t (khong 0,5cm). yn cho cc
vt chm kh, ri cho bn mng vo bnh trin khai, y np bnh li. Khi h dung ly thm t
n mc nh du mc trn th ly bn mng ra. yn cho bn kh t nhin ri mi em
hin mu.
- Tin hnh trin khai 2 bn mng.
Hin mu bn mng v tnh gi tr Rf
- Sau khi trin khai v kh t nhin, mt bn mng c hin hnh bng cch cho vo bnh
hi iod , mt bn mng c hin hnh bng cch phun dung dch FeCl
3
ri sy kh.
- Quan st 2 bn mng v tnh gi tr Rf ca vanillin v -naphtol ca bn hin mu bng hi
iod v bng dung dch FeCl
3
.
Ti liu thc hnh Phn tch thc phm
Trang 29
2.2 Phn tch cc sc t t l cy
* Dng c v ha cht
Dng c, thit b Nguyn liu, ha cht
ng nghim c np 1 Rau ngt 20g
Bn mng 1 Acetone 50ml
Bnh trin khai 1 Petroleum ether 50ml
Cc nung 1
ng ong 50ml 1
Becher 50ml 1
ng vi qun 3
Phu chit
Bnh phun sc k 1
Bnh hi iod 1
Ci chy s 1
a thy tinh 1
Qu bp cao su 1
Giy lc 1
Vit ch, thc k 1
My sy 1
Thit b lc chn khng 1
* Cch tin hnh (Mu chun b chung cho 1 nhm ln)
Ly trch sc t t l cy
- Nghin nhuyn trong ci s 20g l cy ti (thng dng l l rau ngt). Thm vo 30ml
acetone, trn nhiu ln trong 10 pht, lc qua phu Buchner v hng phn acetone.
- cht trch bng acetone vo trong 1 phu chit, ta trch ra ln th 2 vi ether du ha.
Mun vy, ta vo phu chit 10ml ether du ha, lc nh, yn s tch thnh 2 phase. B
phase di, thm 50ml nc ct, lc nh (c th c nh tng, thi gian lc khong 5 10
pht), b phn di (phase nc) v c lm nh th khong 10 ln cho n khi ht mi acetone
(c th ngi xc nh, nu dung dch cn cha acetone th khng th tch carotene ra c).
Mc ch l c mt hn hp cha cc sc t trong dung mi v cc. Nu dung dch cn nhiu
Ti liu thc hnh Phn tch thc phm
Trang 30
(>2ml), un cch thu n khi th tch cn khong 2ml (ong th tch chnh xc ca dung dch
ny). Cc sc t ha tan trong ether du ha s c tch ra nh php sc k bn mng.
Chun b bn mng (mi t 1 bn mng)
- Dng vit ch lm du v k mt ng theo chiu ngang ca bn mng (cch y khong 1
cm).
- Lm du mc trn (cch mt trn khong 2cm)
Trin khai sc k bn mng
- Dng ng vi qun chm u dung dch sc t ly trch c phn trc trn ln cng mt
bn mng (ngay trn ng k sn). Dng my sy sy nh cho kh. Thc hin lp li thao tc
trn t nht 3 ln.
- Cho bn mng vo bnh trin khai c cha sn h dung mi (chiu cao mc dung mi khong
0,5 cm):
- T 1: petroleum ether.
- T 2: hn hp petroleum ether v 2% acetone.
- T 3: hn hp petroleum ether v 5% acetone.
- T 4: hn hp petroleum ether v 10% acetone.
- T 5: hn hp petroleum ether v 15% acetone.
- T 6: hn hp petroleum ether v 20% acetone.
- y np bnh li.
- Khi h dung ly thm t n mc nh du mc trn th ly bn mng ra, yn cho bn
kh t nhin.
Tnh gi tr Rf
- Tnh gi tr Rf i vi cc sc t.
- Cho bit trong l cy c hn hp sc t: chlorophyll a (mu xanh v u), chlorophyll b (mu
xanh l cy), xanthophyl (mu vng), carotene (mu vng cam).
3. Cu hi
1. Tm hiu cc sc t t nhin?
2. Mun thu c cc sc t ta phi lm nh th no?
3. Tm hiu cng thc nh lng cc sc t sau khi thu?
Ti liu thc hnh Phn tch thc phm
Trang 31
Bi 8: PHNG PHP SC K TRN GIY
(PAPER CHROMATOGRAPHY)
1. Nguyn l
- Nguyn tc ca sc k trn giy l da vo tnh cht c h s phn chiakhc nhau trong hai
dung mi khng ho ln nhau nn sc k trn giy cn gi l sc k phn chia trn giy.
- i vi mi dung mi c hiu, s di chuyn ca mt cht c c trng bi h s Rf.



2. Dng c, ho cht
- My sy
- Bt ch, compass, ko
- Cc nung
- Giy sc k Whatman s 1 hoc FN s 4.
- Dung dch acid glutamic chun (acid glutamic 1mg/ml H
2
O).
- Cc acid amin Arg, Leu, Cys v Pro
- Bt ngt
- Dung mi Butanol: Acid acetic: H
2
O t l 5: 2: 3 theo th tch. Lc u trong bnh gn,
yn 1-2 ngy cho tch thnh hai lp r rt.lp di l lp nc bo ho butanol, lp
trn l butanol bo ho nc, cho thm 1-2% butanol lm dung mi chy sc k.
- Thuc th hin mu: 0,2g ninhydrin pha trong aceton cho va 100ml.
- Bnh hoc chung thu tinh kn.
- Micropipet hoc ng mao dn nh chm sc k.
- Bnh phun thuc th hin mu.



Hnh 8.1: Sc k giy
on ng di chuyn ca dung mi
on ng di chuyn ca cht
Rf =
Ti liu thc hnh Phn tch thc phm
Trang 32
3. K thut tin hnh
- Sc k trn giy mt chiu i c trang b n gin hn c. C th t trang b bng cch dng 1
bnh hoc chung thu tinh c np y kn.
- K 1 ng thng cch mp di t giy chy sc k khong 2cm. Trn ng k ny, dng
micropipet chm vt acid glutamic chun cnh cc vt dung dch bt ngt cn th. Khong
cch cc vt chm t 2-3cm. Th tch cc vt t 5-10l. Chm nhiu ln, ch kh li chm. C
th dng my sy tc sy nh cho mau kh. ng knh mi vt cng gn cng tt, thng
khng qu 0,5cm. Sau khi chm sc k, cun giy thnh hnh tr c vo trong bnh sc k
cha dung mi butanol bo ho nc v y kn bnh.
- Khi dung mi trin khai gn ht t giy chy sc k, ly ra kh nhit phng.
- Phun dung dch Ninhydrin 0,2% hin mu.
- C th kh t nhin hoc sy nh t sy (60
o
trong 30 pht).
4. Tnh ton v yu cu
- So snh Rf ca vt mu hin ln dung dch bt ngt cn th vi dung dch acid glutamic
chun. Nu dung dch bt ngt sau trin khai trn giy sc k c mt vt duy nht v c cng
Rf vi vt acid glutamic chun l bt ngt tinh khit khng c ln tp cht khc.
- So snh Rf ca hn hp acid amin v cc acid amin chun.