MT S BI TH NGHIM THC HNH

THEO CHNG TRNH OLYMPIC

V Gio dc Trung hc B Gio dc v o to

Bi 1. Xc nh St c trong thuc vin cha St

L thuyt
St l mt thnh phn thit yu ca hng cu (hemoglobin), gip vn chuyn oxi
trong mu n mi phn ca c th. N cng gi vai tr sinh t trong nhiu phn
ng trao i cht. Thiu st c th gy bnh thiu mu l h qu ca mc hng cu
trong mu thp. Thiu st l s suy dinh dng khong cht ph bin nht trn th
gii. Mt cch gim s thiu ht st l cha tr bng vin cha st.
Hot cht trong thuc vin cha st l st(II) hin din trong thuc vin kho st
di dng st(II) fumarat. Ngoi hp cht hu c st(II) ny, thuc vin c cha
cc cht khc nh nhng tc nhn lin kt. Cu trc ca axit fumaric l:
OH
O

OH

Axit fumaric
St(II) v 1,10-phenanthroline to phc c mu vng cam/
[(C12H8N2)3Fe]2+. Mt quang (absorbance) ca phc ny, xc nh ti 510
nm trong dung dch m (pH=8) l mt php o hm lng st ca vin thuc.
Do 1,10-phenanthroline ch lin kt vi st(II) v st(II) d b oxi ha thnh
st(III), thm hidroxiamoni clorua (hydroxylammonium chloride) kh ton
b st(III) thnh st(II). Mt s phn ng n gin l:
3+

2 NH2OH + 4 Fe N2O + 4 H + H2O + 4 Fe

N
N

1,10-Phenanthroline

219

2+

Thit b v Ha cht tin hnh

THIT B

HA CHT

Cn

Vin thuc cha Fe (II)

Ci, chy s;

HCl 4M;

Cc 100 mL;

Dung dch 1,10-phenanthroline;

Thit b siu m (ultrasonic bath);

Dung dch hydroxylammonium

Bp in;

chloride;

Phu Hirsch cha mt lp nh cht

gip lc nhanh;
Bnh nh mc 250 mL v 100 mL;
Pipet;
Quang ph k;
Phng php tin hnh
Dng cn xc nh khi lng ca vin thuc cha st chnh xc n 1
mg. Vin thuc c tn cn thn thnh bt trong mt ci v chuyn nh lng
vo cc 100 mL bng mt lng nh nc ct. Thm axit clohidric (5 mL, 4
o

M). un nng cc cht trong cc n khong 60 C trn bp in. Dung dch i

sang mu vng.
t cc vo thit b siu m (ultrasonic bath) trong t nht 5 pht. Gi cc n
nh bng mp xp (styrofoam). Phu Hirsch cha mt lp nh cht gip lc
nhanh (Hi-flo filter aid) c lm m v p cht trn lc, dng phu ny lc
huyn ph bng cch ht. Ra cht gip lc nhanh (Hi-flo filter aid) bng lng
d nc ct. Nc lc c chuyn cn thn vo bnh nh mc (250 mL) v
thm nc ct, khuy lin tc iu chnh th tch cui. Dng pipet ht 10
mL dung dch ny v cho vo bnh nh mc 100 mL. Li iu chnh th tch
bng nc ct ng thi khuy u.
T dung dch ny, dng pipet ly 10 mL v cho vo bnh nh mc 100 mL.
Sau , thm dung dch 1,10-phenanthroline (10 mL) v dung dch hidroxi

220

amoni clorua (hydroxylammonium chloride) (1 mL) . K tip, iu chnh th

tch dung dch bng dung dch m (pH 8).
Mt quang ca dung dch ny c o bng my so mu (quang ph k)
ti 510 nm so vi nc trong cuvet 1,000 cm.
Hy tnh lng st trong vin thuc cha st da trn hp th mol (h s
tt, e) bit ca phc st(II) phenanthroline ti 510 nm. hp th mol ca
phc st(II) phenanthroline ti 510 nm bng 11100 M-1cm-1.
Quan trng
lai b sai lch trong mt quang khi ni vi my so mu s dng, mt h
s iu chnh c ghi trn my so mu hc sinh dng trong th nghim. Mt
quang quan st c cn phi nhn vi h s ny thu c mt quang
ng ca dung dch phc st.

Bi 2. Xc nh cc mu v c cha bit
L thuyt
C 12 mu cha bit ng trong ti bng cht do bao gm 9 dung dch
cha bit, mi dung dch c ng trong ng nh git v 3 mu cht rn ng
trong ba l ming rng. Tt c cc mu cha bit u c nh s vi 3 ch
s. Hy kim tra cn thn cc m s mu theo danh sch cc mu v c cha
bit ri vit s bo danh v tn ca mnh vo t giy. (Danh sch c km
theo cc mu cha bit ca hc sinh). Mi l ng cht rn c khong 20
miligam di dng bt hoc tinh th ca mt hp cht tinh khit. Mi ng nh
git cha khong 1,5ml dung dch ca mt hp cht tinh khit c ha tan
trong nc ct. Nng ca cc dung dch cha bit nm trong khong t 0,05
n 0,5 M(mol/lit).
Cc dung dch cha bit l nh sau:
HCl

H2O2

H2SO4

ZnCl2

NH4SCN

NaOH

Na2CO3

Na2SO3

BaCl2

K4Fe(CN)6

Ch :
221

(1) C 2 mu cha bit c lp li.

(2) H2O kt tinh trong tinh th ngm nc c b qua trong cc cng thc cho
trn.
Trn bn th nghim ca hc sinh c mt hp nha ng cc dng c, mu
cha bit v cc thuc th c s dng trong bi thc hnh ny.
Danh sch cc dng c
Dng c

Dng c

S lng.

lng.
in cc dy Pt

in cc dy Au

Hp ng pin

Pin

Bn lm trng nh

Bn mng bng nha mu

git

en

Ko ct

Tha cph

ng nh git (1 mL)

Danh sch thuc th

Thuc th

Nng .

Thuc th

Nng .

KI

0.1M

pp (phenolphtalein)

0.01

FeCl3

0.1M

Dung dch tinh bt

0.01

Mc c hi v an ton ca cc ha cht
Ha cht

Cng thc

c hi

an ton

Axit clohidric

HCl

36/37/38

26

Axit sunfuric

H2SO4

35

26-30-45

Dung dch Natri

NaOH

35

26-36/37/39-45

H2O2

22-41

26-39

Na2CO3

36

22-26

hidroxit
Dung dch
Hydroperoxit
Dung dch Natri

222

cacbonat
Dung dch Bariclorua

BaCl2

20-25

45

Dung dch Natrisunfit

Na2SO3

31-36/37/38

26-36

Dung dch Km clorua

ZnCl2

22-34-50/53

26-36/37/39-45-6061

Dung dch Kali

K4Fe(CN)6

32

22-24/25

20/21/22-32-52/53

13-61

FeCl3

22-34

26-36/37/39-45

KI

22-24/25 *

40

36/37

hexaxyanoferat (II)
Dung dch Amoni

NH4SCN

thioxyanat
St (III) clorua (rn)
Kali iotua (rn)
Dung dch tinh bt
Cht ch th
Phenolphthalein
Tin hnh
1. S dng bn thuc th c cp, cc phn ng gia cc mu cha bit vi
nhau v thit b in phn n gin nhn bit cc mu cha bit v vit
tr li ca em (di dng s vi 3 ch s - nh cch nh s mu ca cc
mu cho) vo cc trng trong t phiu tr li.
Ch : Sau khi kt thc cng vic hy cho hai dy vng (Au) v Platin(Pt) v cc pin vo cc
ti nilon ban u ca chng ri li tt c dng c v ha cht (k c cc mu cha bit) vo
hp nha ng v tr ban u.

2. Trong bi thc hnh ny hc sinh thc hin mt lot php th xc nh

(hoc khng nh) cc mu cha bit. Hc sinh cn nm c cc phn ng
ho hc lin quan n cc php th tin hnh v vit c cc phng
trnh phn ng:
A. Vit phng trnh in phn xy ra dng ion rt gn c ghi trng thi
tn ti khng nh mt mu cha bit l dung dch cha ZnCl2.

223

B. Vit mt phng trnh phn ng dng lm sch kt ta Zn trn b

mt in cc bng cc dng c v ha cht cho trong bi ny.

Bi 3. Xc nh cacbonat v hiro photphat trong mt mu lm cht mi

L thuyt
Na2CO3, CaCO3 v Na2HPO4 l cc thnh phn chnh ca cc bt mi. Trong
bi th nghim ny, phi xc nh cc ion cacbonat v hiro photphat trong mt
mu mi bng hai chun axit-baz.
u tin, thm mt lng chnh xc axit clohiric (c ly vi lng d) vo
mu th. Phn ng xy ra, hiro photphat chuyn thnh H3PO4, cn cc ion
cabonat chuyn thnh CO2 sau thot ra ht khi b un si. Cc ion canxi c ban
u trong mu chuyn vo dung dch. V cc ion ny c th nh hng n kt
qu phn tch nn chng c kt ta trong CaC2O4 v lc b trc khi chun .
Tip n, axit photphoric va to thnh c chun bng dung dch NaOH c
nng chnh xc vi hai cht ch th khc nhau l: Bromcrezon xanh
(Bromocresol Green, BCG) v Thymolphthalein (TP). Bc th nht ca chun
ny l: H3PO4 (v lng d HCl) c chun ti ion H2PO4-, im kt thc
bc chun ny c mi trng hi axit (pH khong ~4.5). im ny lm cho
BCG chuyn t mu vng sang mu xanh. Bc th hai ca chun ny: tip tc
bc trn cho ti khi to ra HPO42-. im kt thc bc hai ny xy ra khi TP
khng mu chuyn sang mu xanh (mi trng c tnh kim, pH vo khong 10).
Lng ion CO32- trong mu c tm ra khi da vo lng khc nhau gia:
a) Lng cht chun ng vi lng ban u ca HCl ( dng ha tan mu)
b) Lng cng ca cht chun ng vi im kt thc chun th hai (ch
th TP).
Lng ion HPO42- c tm ra khi da vo lng khc nhau gia lng cht
chun dng t ti hai im kt thc chun (ch th TP v BCG).

224

Thit b v Ha cht tin hnh

THIT B

HA CHT

Cn

Bt mi cha Na2CO3, CaCO3 v

Phu lc;

Na2HPO4

Cc 100 mL;

HCl 1M;

ng ong;

Nc ct

Bp in;

Dung dch K2C2O4 15%;

Bnh hnh nn (Erlenmeyer);

Dung dch NaOH;

Bnh nh mc 100 mL;

Dung dch Thymolphthalein (TP)

Pipet;

Dung dch BromoCresol Green

(BCG)

Qui trnh tin hnh

Bc 1. Ha tan mu v ui CO2
Thm ng 10,00 mL dung dch HCl nng khong 1 mol/L (xem tr s chnh
xc ny c ghi trn nhn ca l) vo mu bt mi c trong cc c y bng
mt knh ng h thy tinh (mi thao tc phi chnh xc: ly dung dch bng
mt pipet! Khng c b np y cc ra trnh tht thot ha cht!). Sau
giai on thot kh mnh kt thc, dng bp in cn thn un nng dung dch
trong cc (vn phi y cc bng mt knh ng h) ti khi ht kh thot ra. Tip
n, un cn thn dung dch cn li trong cc cho si trong khong 2-3 pht.
Bc 2. Kt ta canxi
Nhc cc khi bp in. Dng nc ct ra phn hi nc ngng t mt knh
ng h cho chy vo cc. Dng ng ong ly 1-2 mL dung dch K2C2O4 15%. Cho
t t lng ny theo thnh vo cc (mt khong 10 n 20 pht) cho ti lc kt ta
hon ton. Dng khong thi gian ch i ny xc nh nng chnh xc ca
dung dch NaOH (theo phng php di y).
Bc 3. Xc nh nng chnh xc ca dung dch NaOH

225

Dng mt pipet ly 10,00 mL dung dch HCl ri cho vo bnh nh mc 100 mL,
thm nc ct cho n vch, lc bnh trn u. Rt dung dch NaOH y buret.
Dng pipet ly 10,00 mL dung dch HCl trong bnh nh mc cho vo mt bnh
hnh nn (Erlenmeyer). Thm vo erlenmeyer ny 1-2 git dung dch
Thymolphthalein ri chun bng dung dch NaOH ti khi mu xanh xut hin
trn vng xoy ca dung dch v bn ch trong khong 5 - 10 giy.
Ti y v phn sau: Hy lp s chun mc cn thit. Cn lu rng tr
s th tch dung dch cn dng nhiu nht v t nht ch cch nhau c 0,10 mL. S
liu th tch dung dch bo co c chnh xc ti 0,01 mL.
3.1a Hy in y vo bng trong Phiu tr li.
3.1b Hy tnh nng ca dung dch NaOH (theo mol/L).
Bc 4. Lc b canxi oxalat
Sau khi kt ta c hu ht CaC2O4, dng phu lc dung dch vo bnh nh mc
100 mL. Nc lc ny hi b c do c mt lng nh canxi oxalat nhng khng
gy nh hng ti vic chun . Dng nc ct ra sch kt ta ri b giy lc c
kt ta vo thng ng rc. Thm nc ct vo bnh ng nc lc cho ti vch v
lc u.
Bc 5. Chun mu dng Bromocresol Green
Dng pipet ly 10,00 mL nc lc thu c sau bc 4 cho vo mt Erlenmeyer
ri thm tip vo 3 git dung dch BCG. Hy chun b mt Erlenmeyer khc c
dung dch i chng gm 3 git dung dch NaH2PO4 15%, 3 git dung dch BCG
v 15-20 mL nc ct. Dng dung dch NaOH chun dung dch nc lc ti khi
mu ca dung dch ny trng vi mu ca dung dch i chng th dng.
3.2 Hy in y vo bng trong Phiu tr li.
Bc 6. Chun mu dng Thymolphthalein
Dng pipet ly 10,00 mL nc lc thu c sau bc 4 trn cho vo mt
Erlenmeyer ri thm tip vo 2 git dung dch TP. Dng dung dch NaOH
chun dung dch nc lc ti khi mu xanh xut hin v bn trong khong 5
10 giy th dng.
226

3.3 Hy in y vo bng trong Phiu tr li.

Bc 7. Cc tnh ton
3.4. Hy tnh khi lng ca CO32- trong mu dng.
3.5. Hy tnh khi lng ca HPO42- trong mu .
Bc 8. Cc cu hi thm cho bi th nghim ny
Hy tr li cc cu hi sau y vo Phiu tr li.
3.6a. Hy ch ra mt phn ng (vit phng trnh) lm cn tr s phn tch mu khi
c mt Ca2+.
3.6b. Danh mc cc li c th phm phi cc bc khc nhau c nu ra trong
bng ca Phiu tr li. Hy ch ra trong s nhng li no dn n sai s khi xc
nh hm lng ca CO32- v/hoc HPO42-. Hy dng cc k hiu sau y: 0 nu
khng c sai s nh c d on, + hoc nu c sai lch nhiu hn (sai
s dng) hoc sai lch t hn (sai s m) so vi thc t.

Bi 4. Chun complexon;
V d ca s xc nh ion kim loi dng php o complexon.
L thuyt
Nng ion Ni2+ c th c xc nh bng s to phc vi EDTA
(etylendiamin tetraaxetat).
EDTA l mt ligand nhiu rng to phc 1: 1 vi ion Ni2+. Cht ch th l
murexide cng c th to phc vi ion Ni2+ nhng phc ny khng bn bng
EDTA. Mc ch ca th nghim ny l xc nh lng nc kt tinh trong
niken sunfat.
Ha cht cn thit:
Niken sunfat (300 mg)

M an ton:
R 20/21/22, 42/43, 45, 46 S 26, 27, 28, 36/37/39, 45

Dung dch EDTA tiu chun

Cht ch th murexide
Ammoni clorua (3 g)

R 22 S 36

R - S 22, 24/25
R 22, 36 S 22

227

Ammoniac m c (20 mL)

Dng c cn thit:
Cn
Cc ong 100 mL
Bnh tam gic
B thit b chun
Tin hnh
Cn chnh xc khong 300 mg niken sunfat v ha tan vo nc. Dng
cc ong 100 mL.
iu ch dung dch m bng cch ha tan 2,7 g ammoni clorua v 17,5
mL ammoniac m c trong 50 mL nc. y dung dch EDTA tiu chun
0,01 M vo mt buret. Dng pipet ly 10,00 mL dung dch niken sunfat cho vo
cc hnh nn 200 mL v pha long vi khong 90 mL nc. Va thm va
khuy u 10 mL dung dch m vo cc hnh nn. Thm mt t cht ch th
murexide rn v m bo tan ht. Chun vi dung dch EDTA n khi i
mu t vng sang tm. Khi mu i chm, thm mt t ammoniac m c lc
cui chun . Th nghim ny cn c thc hin hai ln.
Ghi li cc s liu sau:
1. Lng dung dch EDTA theo mL. Cng ghi li chnh xc chun ca dung dch.
2. Khi lng niken sunfat.xH2O.
3. Tnh nng Ni2+ trong dung dch.
4. Tnh s mol nc kt tinh trong mt mol niken sunfat.

228

Bi 5. Phn tch nh tnh cc hp cht hu c

th nghim ny bn phi nhn bit 7 cht rn cha bit ghi trong danh sch
cc cht trang 7, chng l cc thuc ph bin trong cuc sng hng ngy v l
cc tc nhn hu ch trong ha hu c. t iu ny, phi tin hnh cc phn
ng ho hc theo qui trnh sau v phn tch kt qu thu c.
-

Cc l dn nhn cht cha bit nh:

L U-1, L U-2, L U-3, L U-4, L U-5, L U-6, L U-7

Phn ng th 1: Th tnh tan

Lc ng nghim vi CH3CN, 1M HCl, nc v 1M NaOH.
Phn ng th 2: th vi 2,4-DNPH
Ha tan mt cht cha bit vi 95% EtOH v th vi dung dch ca 2,4dinitrophenylhydrazin trong axit sunfuric c v 95% ethanol (2,4-DNPH).
Phn ng th 3: th vi CAN
Trn dung dch Xeri(IV) ammoni nitrat trong HNO3 long (k hiu nhn l
CAN) vi CH3CN c hn hp. Cho cht cha bit vo dung dch hn
hp. Nu c s i mu dung dch, th dung dch ny c th cha ancol,
phenol hoc andehit.
Phn ng th 4: Php th Bai (Baeyer)
Trong ng nghim, ha tan cht cha bit vi CH3CN. Va lc va cho t
t vo dung dch th 5 git dung dch 0.5% KMnO4.
Phn ng th 5: Th pH
Trong ng nghim, ho tan cht cha bit vi 2 ml 95% EtOH. Dng giy
pH o pH ca dung dch.
Phn ng th 6: th vi st (III) clorua
Ly dung dch thu c t Phn ng th 5 v cho 5 git dung dch 2.5%
FeCl3.

229

Thit b v Ha cht tin hnh

THIT B

HA CHT

Cn

7 cht cha bit

Phu lc;

HCl 1M;

Cc 100 mL;

NaOH 1M;

ng nghim;

Nc ct

Tha;

Dung dch KMnO4;

CH3CN;
Dung dch CAN
2,4 DNPH
Dung dch FeCl3 2,5%
C2H5OH
Giy pH

Qui trnh tin hnh

Cc li khuyn hu ch
a) Trng lng ca tha (spatula) ly y cht khong 15~20 mg.
b) Lau k tha bng giy lau sau khi dng ly cht.
c) Sau khi cho bt k tc nhn no miu t di y vo dung dch ca mu
cha bit, phi trn k v quan st thn trng hn hp thu c.
d) nhn im ti a, phi tin hnh tt c cc phn ng th v ghi vo bng.
Phn ng th 1: Th tnh tan
Cho vo ng nghim mt tha y cht (15~20 mg) cha bit v1 ml of
CH3CN. Lc ng nghim v ghi li tnh tan. Lp li th nghim vi 1M HCl,
nc v1M NaOH.
Phn ng th 2: th vi 2,4-DNPH
Cho khong 15~20 mg mt cht cha bit vo ng nghim v ha tan vi 2
ml 95% EtOH (i vi cc cht tan c trong nc, th ly khong15~20

230

mg ho vo trong 1 ml nc). Cho vo 5 git dung dch ca 2,4dinitrophenylhydrazin trong axit sunfuric c v 95% ethanol (k hiu nhn
l 2,4-DNPH).
Phn ng th 3: th viCAN
Trn 3 ml dung dch Xeri(IV) ammoni nitrat trong HNO3 long (k hiu
nhn l CAN) vi 3 ml CH3CN trong ng nghim. ng nghim khc cho
khong 15~20 mg cht cha bit vo 1 ml dung dch hn hp. (i vi cht
tan trong nc, th u tin ho khong 15~20 mg mu trong 1 ml nc, v
sau cho thm 1 ml thuc th CAN. Nu c s i mu dung dch, th
dung dch ny c th cha ancol, phenol hoc andehit.
Phn ng th 4: Php th Bai (Baeyer)
Trong ng nghim, ha tan khong 15~20 mg cht cha bit vi 2 ml
CH3CN (i vi cht tan c trong nc, th ho khong 15~20 mg cht
vi 1 ml nc). Va lc va cho t t vo dung dch th 5 git dung dch
0.5% KMnO4.
Phn ng th 5: Th pH
Trong ng nghim, ho khong 15~20 mg cht cha bit vi 2 ml 95%
C2H5OH (i vi cht tan c trong nc, th ho khong 15~20 mg cht
vi 1 ml nc. Dng giy pH o pH ca dung dch
Phn ng th 6: th vi st (III) clorua
Ly dung dch thu c t Phn ng th 5 v cho 5 git dung dch 2.5%
FeCl3.
Ghi kt qu
1. Ghi cc kt qu th vo t Phiu Tr li. Vit O nu tan, cn X nu khng tan
i vi phn ng th tnh tan. Vit (+) i vi phn ng dng tnh, cn () cho
phn ng m tnh i vi cc phn ng th 2 ~ 4 v 6. Vit a, b v n tng ng
vi dung dch c tnh axit, baz hoc trung tnh, cn pH vi phn ng th 5.
2. Da trn kt qu th, hy cho bit cu to ph hp ca cc hp cht cha
bit, suy t cc cht cho trong danh sch. Vit cht ny vo thch hp.
231

Cc hp cht cha bit c th l

COOH

HO

OH
HO

HO
NH2

HO

OCH3

(A)

NH2

OCH3

OH

(F)

(G)

(E)

OH
HCl

H
CH3
O

CH3

HO
OH

COOH
H3C

HCl

H3CO

CH3
N

(K)

(M)

H
N

CHO

CH3
O

HO

(Q)

OH
OCH3

HO
OCH3

(T)

O
(W)

(V)

Bi 6. Sc k trao i ion cc aminoaxit

Trao di ion l mt phng php phn tch v iu ch quan trng cho php
phn tch cc cht mang in. S tng tc gia cc nhm ion ca cht vi cc
gc gn trn nha l c s ca phng php ny. bi ny, phi phn tch mt
hn hp cc aminoaxit, tip theo th nh tnh tng loi aminoaxit c tch ra
t ct bng phn ng mu c trng. Do th sinh phi sp hng o ph nn
chng ti ngh bn hy bt u vi bi thc hnh s 1.
O

NH2

N
OH
NH2

N
H

His

HS

OH

HN

NH

OH

NH2

Cys

NH2

Arg

Cho mt hn hp gm ba aminoaxit: histidin, cystein v arginin (xem cu trc

trn). Polistyren lin kt cho bi gc sunfat l nha trao i cation (xem s

232

di y). Trc khi th nghim ct sc k trao i ion c nhi sn v cn

bng vi Dung dch ra gii 1 (pH 4,9).
Qui trnh tin hnh
Tin hnh sc k. Bc 1.
a dung dch cc aminoaxit ln ct sc k.
u tin, m kha cho dung mi trong ct chy xung bnh tam gic
(Erlenmeyer) c ghi Cht thi sao cho dung mi vn cn nm trn b mt
cht nhi v trnh khng cho n b kh. ng kha li v thn trng dng
mt syranh cho dung dch phn tch ln ct. M kha v cho dung dch ny
ngm vo cht nhi (x dung mi vo bnh Cht thi). ng kha ct v cn
thn m (nh) t t kp ng cho chy vo khong 1 mL Dung dch ra gii 1
(ng vi ~ 1 cm ca cht lng trn ct). Dng hai tay ni cht u ni c nhm
trong (nhm ci) u ct vo u ni c nhm ngoi (nhm c) u ng
dn dung dch ra gii 1 (xem k vic ni cht u thy tinh vi ct). B bnh
Cht thi ra v thay vo cc ng nghim trn gi. M t t kp ng v m
kha dung dch ra gii chy xung qua ct. Bt u qu trnh ra gii (lun
m kha ct khi bt u ra gii v ng kha li khi ngng ra).
Thu gom cc phn on vo ng nghim, ly khong 2,5 mL (xem mi tn s
). Nu thy cn th dng bt d nh du. Sau khi gom c t 4 n 8 ng,
ngng ra gii v sau phn tch nh tnh cc mu (phn on) thu c.

233

ng
kp ng
u ni nhm
ngoi

u ni
nhm trong

dung dch ra gii

lp dung
mi
nha trao i
ion
Kha ct

nh tnh cc mu thu c
nh tnh cc aminoaxit da trn phn ng ca nhm -amino vi natri 2,4,6trinitrobenzen sunfonat (TNBS):
O 2N

NO 2
NH2

HOOC
R

+
O
Na
O S

HOOC

NH

NO 2
NO2

NaHSO 3

O 2N

NO 2

nh tnh c thc hin trong cc l ca tm nha polistyren, mi l tng

ng vi mi ng nghim xc nh. Trc khi th, u tin hy trn 1 mL dung
dch TNBS vi 10 mL dung dch m cacbonat v sau cho 0,1 ml hn hp
thu c vo mt na cc l trn tm nha (t A1 n H5). Tip theo, cho 0,1
mL ca phn on cn phn tch vo l. Bt u th vi A1, v tip tc vi B1,
C1, v.v. (di chuyn t trn xung v t tri sang phi). Nu aminoaxit c mt
trong phn on phn tch th mu vng m s xut hin trong l trong khong
234

3 pht. Ly mu trong l u lm chun i chiu. nh gi ng mu,

bn nn tm nha ln t giy trng.
Lu : Dng pipet my ly tt c cc cht lng m c th tch 0,1 mL. Bn
nn dng mt u ht nha cho tt c cc phn on c mt cht (nh).
6.1a nh du m t s lc cng mu (nh tnh) trn tm nha (c l ) vo
Phiu Tr li. Dng cc k hiu sau: (-)- khng mu, 1-mu yu, 2- mu va phi v
3- mu mnh. Tip tc nh du s m t ny trong qu trnh sc k.
Tip tc ra gii thu cc phn on v phn tch chng cho n khi bn nhn
c t nht 2 l c mu nh l A1, iu ny ch ra rng aminoaxit th nht
hon ton ra ht khi ct (kt thc nh (peak) th nht).
Tin hnh sc k. Bc 2.
Ngay sau khi kt thc thu gom nh (peak) th nht, bn phi thay Dung dch
ra gii th 2. lm iu ny, hy ng kha ct, ng (vn cht) kp ng
dn (Quan trng !), tho ng dn ang ni vi chai ng Dung dch ra gii
th 1 v ni n vi chai ng Dung dch ra gii th 2. Gi cht u ni nhm
u ct.
6.1b. Khi cc Dung dch ra gii c thay i, hy nh du bng cch v
cc ng thng nm gia cc l tng ng tm nha.
Tip tc ra gii, thu cc phn on v phn tch nh tnh chng nh miu
t trn.
Tin hnh sc k. Bc 3.
Ngay sau khi kt thc thu gom nh (peak) th 2, bn phi thay Dung dch ra
gii th 3 nh miu t bc 2. Tip tc sc k cho n khi aminoaxit th 3
hon ton ra khi ct.
Dng qu trnh sc k bng cch ng kha ct v vn cht kp ng.
Da vo kt qu phn tch nh tnh, hy chn nhng phn on c cha cc
aminoaxit.
6.1.c Hy in vo Phiu Tr li nhn ghi (s th t) ca cc l ng vi cc
phn on chn trn.
235

6.2 Gp li cc phn on c cng mt nh v dng ng ong o th tch

ca tng phn on gp. Bo co th tch ca cc phn on gp ngoi tr
lng dng cho phn tch nh tnh. Ghi cc kt qu thu c vo Phiu
Tr li.
Rt cc phn on gp vo l thy tinh nu c ghi nhn Peak 1, Peak 2
Peak 3. Chun b cc mu phn tch nh lng trn my quang ph nh
m t di y.
Khi kt thc bi thi thc hnh, hy nt cc l v chng trn bn. Cc
phn on gom sau s c nhn vin phng th nghim phn tch kim
tra li.
Phn tch quang ph
i vi mi mu, bn cn phi a 2 cuvet cho ngi o mu. Chun b mu
nh sau.
Quan trng! Khi bo qun, lun cuvet trong hp! Tt c cc cuvet c 2
mt hng v 2 mt trn nm thng ng dng o. Khi dng cuvet, khng
c chm vo mt dng o, nu khng bn s thu c gi tr mt
quang sai.
Php th s 1(nh 1). Nng cystein c xc nh bng phn ng Ellman:
NO 2

+
NH3

+
O

SH

OH

H3N

-H 2O

S
-

+
NO 2

NO 2

NO 2

ng nghim A1 (ng i chiu). Cho 0,1 mL Dung dch ra gii 1 ly t ng

nha nh vo mt ng nghim v cho thm vo 2.9 mL tc nhn Ellmann.

236

ng nghim B1 (ng mu phn tch). Cho 0,1 ml dung dch phn tch vo mt
ng nghim v cho thm vo 2.9 mL tc nhn Ellmann.
Trn u cc ng nghim v chuyn mi hn hp sang cc cuvet tng ng c
ghi A1 (cho mu i chiu) v B1 (cho mu phn tch).

Mu th s 2 (nh 2). Xc nh nng histidin da trn kh nng ca gc

imidazol phn ng vi cc hp cht diazo (phn ng Pauli).
ng nghim A2 (ng i chiu). Cho 2,8 mL dung dch m Tris-HCl vo mt
ng nghim, cho thm vo 0,1 mL Dung dch ra gii 2 ly t ng nha nh v
0,1 mL tc nhn Pauli.
ng nghim B2 (ng mu phn tch). Cho 2,8 mL dung dch m Tris-HCl vo
mt ng nghim, tip theo cho thm 0,1 mL dung dch cn phn tch v 0,1 mL
tc nhn Pauli.
Trn u cc ng nghim v chuyn mi hn hp sang cc cuvet tng ng c
ghi A2 (cho mu i chiu) v B2 (cho mu phn tch).
Mu th s 3 (nh 3). Xc nh nng ca arginin da trn kh nng ca
gc guanidin phn ng vi mt s phenol trong iu kin kim v cht oxi ha
(phn ng Sakaguchi).
ng nghim A3 (ng i chiu). Cho 0,1 mL Dung dch ra gii 3 vo mt ng
nghim, tip theo cho thm 1,5 mL dung dch NaOH 10%, 1 mL dung dch 8hydroxiquinolin v 0,5 mL dung dch natri hypobromua.
ng nghim B3 (ng mu phn tch). Cho 0,1 mL dung dch phn tch vo mt
ng nghim, tip theo cho thm 1,5 mL dung dch NaOH 10%, 1 mL dung dch
8-hydroxiquinolin v 0,5 mL dung dch natri hypobromua.
Lc mnh cc ng nghim trong 2 pht (Quan trng!) v quan st s to thnh
mu vng cam. Cho 0,2 mL dung dch ur 8 M vo mi ng, trn k v ly
khong 3 mL ca mi hn hp cho vo cc cuvet tng ng ghi A3 (cho mu
i chiu) v B3 (cho mu phn tch).

237

Tt c cc hn hp cn phi phn tch bng quang ph khng c sm hn 10

pht v khng c mun hn 2 gi sau khi chun b xong mu. a 6 cuvet
cho nhn vin o mu. Trong trng hp phi sp hng ch, hy ngh nhn
vin o mu ghi m s th sinh ca bn ln bng nh du sp hng. Bn s
c gi khi n lt mnh. Trong thi gian ny bn c th bt u lm bi th
hnh s 2.
Trong trng hp cc mu ca bn khng kp kho st trong khong thi gian
ph hp nu trn (hon ton khng th xy ra), bn phi chun b li mu
mi.
Nhn bn in ph cc mu ca bn v kim tra li. K nhn vo bn ph v
xin ch k ca nhn vin o mu.
6.3 Hy xc nh hp ph cc bc sng tng ng v tnh hm lng
(theo mg) ca mi aminoaxit trong hn hp ca bn. di quang l 1,0 cm.
in vo Phiu Tr li, nh rng 1 mol ca mi aminoaxit cho 1 mol sn phm
tng mg.
D liu i chiu:
Gi tr ca cc h s tt:

Khi lng phn t ca

Sn phm ca phn ng Ellmann: 13600 M-1 cm-1 aminoaxit:

410 nm

Cystein 121 g/mol

Sn phm ca phn ng Pauli: 6400 M-1 cm-1 470 Histidin 155 g/mol
nm

Arginin 174 g/mol

Sn phm ca phn ng Sakaguchi: 7700 M-1 cm-1

500 nm

6.4 V 3 cu trc cng hng ca cc gc phn t tham gia vo s to thnh

hn hp mu trong phn ng Ellmann

238

Bi 7. Phn tch nh lng Axit Ascorbic trong vin Vitamin C

L thuyt
Thnh phn chnh trong vitamin C thng mi l axit ascorbic (H2C6H6O27,
FW = 176,12). Axit ascorbic va l mt axit, va l mt cht kh, do , c
chun axit-baz v chun oxi ha kh u c th s dng xc nh
lng axit ascorbic trong nhng vin vitamin C thng mi.
Vitamin C l 1 cht chng oxy ha cn thit i vi dinh dng ca con
ngi. Thiu vitamin C c th dn n bnh scurvy (scobat) c trng khin cho
xng v rng khng bnh thng v mt s bnh khc.
Vitamin C l tn thng gi ca axit L-ascorbic (AsA), c danh php
quc t l 2-oxo-L-threo-hexono-1,4-lactone-2,3-enediol, CTPT C6H6O6 (M=
176,1g/mol), CTCT nh sau:

Trong cng thc cu to ca ascorbic ta nhn thy c C4 v C5 l 2

cacbon bt i xng, v vy ascorbic c 4 ng phn quang hc l axit Lascorbic, axit izo L-ascorbic, axit D-ascorbic v axit izo D-ascorbic. Trong s
cc ng phn ny ch c axit L-ascorbic v izo L-ascorbic l c tc dng cha
bnh cn 2 ng phn cn li l cc khng vitamin, tc l c ch tc dng ca
vitamin. Trong t nhin ch tn ti dng axit L-ascorbic, cc ng phn cn li
c to ra theo con ng tng hp.
Axit ascorbic tn ti dng tinh th hoc bt kt tinh trng hoc hi ng vng,
khng mi, c v chua, tan nhiu trong nc (300g/lt), t tan hn trong ru v
khng tan trong chloroform, benzene hay cc dung mi h c khng phn cc.
Axit ascorbic rt d b phn hy di tc dng ca nh sng v nhit .
Dung dchaxit ascorbic khng bn, rt d b oxy ha di tc dng ca oxy

239

khng kh, c bit l khi c mt mt s kim loi nng: Fe, Cu V vy cn

phi bo qun vitamin C trong bng ti v nhit thp.
Ha tnh ca vitamin C l ha tnh ca nhm chc lacton, ca cc nhm
hydroxyl, ca lin kt i, song quan trng nht l nhm chc endiol. Chnh
nhm ny quyt nh tnh axit v tnh kh ca axit ascorbic.
Nguyn tc phng php
1. Phng php axit baz
Trong dung mi nc, axit ascorbic l axit phn ly hai nc vi cc gi tr
pKa ln lt bng 4,2 v 11,6 tng ng vi s phn ly H+ ca nhm OH nh
vo C3 v C2 .
Axit ascorbic d dng phn ng vi cc dung dch kim to mui.
CH2OH
H

OH

O
O

H
HO

HOH2C

NaOH

(CHOH)3

COONa

H2O

OH

nh lng axit ascorbic c th dng phn ng chun nc 1 vi NaOH,

ch th phenolphatalein.
1. Phng php oxi ha kh:
Axit L- ascorbic b oxi ha thnh axit L- dehydroascorbic theo bn phn ng
oxiha sau y ( E0= 0,127V pH=5)
CH2OH

CH2OH
H

OH

OH

O
O

O
H
HO

H
O

OH

Axit ascorbic

2H+

2e-

Axit dehidroascorbic

Qu trnh oxy ha ascorbic xy ra hai mc khc nhau:

- S oxy ha thun nghch vitamin C thnh axit dehydroascorbic: tnh
cht ny v cng quan trng i vi tc dng sinh hc ca axit ascorbic l tham
gia xc tc cc qu trnh oxy ha kh xy ra trong c th.
240

- S oxy ha bt thun nghch bin vitamin C thnh cc sn phm khc

khng c hot tnh v bin mu. Phn ng ny tng nhanh theo pH v nhit
ca dung dch.
Cc cht oxy ha thng dng oxi ha axit ascorbic l: brom, iot,
thuc th Fehling, dung dch KMnO4, dung dch AgNO3, 2,6diclorophenolindophenol. Phng php chun c tin hnh bng cch
nh t t dung dch thuc th t buret vo dung dch c cha axit ascorbic
trong mi trng thch hp. im tng ng c nhn nh s chuyn mu
ca dung dch khi c cht ch th thch hp.
Phng php ny c th p dng xc nh trc tip vitamin C trong
cc mu thc phm. Trong cc i tng khc nh rau qu, thc phm, nc
gii kht c thnh phn tng i phc tp, cha nhiu cht kh khc nhau,
dung dch c v c mu, gy kh khn trong vic xc nh im cui ca qu
trnh chun .
Trong th nghim ny hm lng vitmain C trong vin nn c xc nh
bng phng php chun axit- baz hoc chun oxi ha kh. Axit
ascorbic c xc nh da trn phn ng oxi ha n bng iot (trong KI d)
theo phng php chun trc tip vi cht ch th h tinh bt.
Th nghim ny gm hai phn, phn u dng chun axit-baz xc
nh lng axit ascorbic trong mt vin vitamin C. Phn th hai dng chun
oxi ha kh thc hin xc nh tng t.
S nh gi c da trn s chnh xc ca mi php chun . Tnh 30%
cho chun axit-baz, tnh 60% cho chun oxi ha kh v 10% cho s so
snh hai phng php.
KIM TRA THUC TH V THIT B TRC KHI TH NGHIM
Thuc th
Dung dch NaOH

Thit b
ng ong

(trn nhn c ghi nng )

10 mL

x1

100 mL

x1

Dung dch Thiosunfat (Na2S2O3) Cc thy tinh

241

(trn nhn c ghi nng )

100 mL

x2

Dung dch Iod (0.01 M)

250 mL

x2

Cht ch th

Bnh Erlenmeyer

Dung dch Phenonphtalein

Dung dch metyl

Dung dch h tinh bt

125 mL

x4

250 mL

x2

Giy lc

x 10

Giy cn

x 10

Mold and Pastel

1 b

Buret (1 rack)

x2

Buret Brush

x1

Bnh nh mc, 100 mL

x1

Spatula

x1

Phu x 1
Pipet (20 mL) / Bm an ton

1 b

Pipet Pasteur (ng nh git) x 6

Bn chi

x1

Tin hnh:
Ha tan vin vitamin C trong nc, lc nu cn thit. Th tch cui cng ca
dung dch nn l 100 mL.
Chun b cc dung dch:
* Dung dch vitamin C chun:
Cn chnh xc lng c 0,1 gam axit ascorbic trn cn phn tch v chuyn
nh lng vo bnh nh mc dung tch 250 ml. Thm khong 2 gam axit
oxalic vo bnh nh mc, thm nh mc n 2/3 th tch bnh v lc u cho
cht rn tan ht sau nh mc n vch mc bng nc ct. Nt kn bnh

242

trnh s oxi ha ca oxi khng kh. Dung dch ny c dng chun dung
dch iot.
* Dung dch it:
Ha tan 5 g KI v 0,268 g KIO3 trong 200 ml nc ct, thm 30 ml axit
sunfuric 3 M v chuyn vo bnh nh mc 500 ml, nh mc n vch mc, ta
c dung dch KI3
Phn 1: Chun axit-baz.
1-1 Dng pipet pipet 10 mL ht dung dch trn cho vo mt bnh Erlenmeyer.
Chn cht ch th thch hp thc hin s chun .
1-2 Lp li 3 ln bc th 2.
Phn 2: Chun oxi ha kh
2-1 S dng dung dch thiosunfat chun xc nh nng dung dch iod
cho.
2-1-1

Dng pipet 20 mL a dung dch iodin vo bnh Erlenmeyer, ri chun

bng cch s dng dung dch Na2S2O3 chun. Dng tinh bt lm
cht ch th.

2-1-2 Lp li 3 ln bc th 4.
2-2 Xc nh lng axit ascorbic.
2-2-1

Dng Pipet 10 mL a dung dch t bc 1 vo bnh Erlenmeyer.

Thm vo vi git tinh bt lm cht ch th v chun vi dung dch
iod.

2-2-2

Lp li 3 ln bc th 6.

Bng d liu 3
3-1

3-2
3-2-1

Chun axit - baz

Chun ln 1

Dung dch Vitamin C

mL; Dung dch NaOH dng

mL

Chun ln 2

Dung dch Vitamin C

mL; Dung dch NaOH dng

mL

Chun ln 3

Dung dch Vitamin C

mL; Dung dch NaOH dng

mL

Chun oxi ha kh
Xc nh nng iot

243

Chun ln 1

Dung dch Iod

mL; Dung dch Na2S2O3 dng

mL

Chun ln 2

Dung dch Iod

mL; Dung dch Na2S2O3 dng

mL

Chun ln 3

Dung dch Iod

mL; Dung dch Na2S2O3 dng

mL

3-2-2 Xc nh axit ascorbic

Chun ln 1

Dung dch Vitamin C

mL; Dung dch Iod dng

mL.

Chun ln 2

Dung dch Vitamin C

mL; Dung dch Iod dng

mL.

Chun ln 3

Dung dch Vitamin C

mL; Dung dch Iod dng

mL.

Cu hi
7-1

Gi s axit ascorbic l mt n axit, dng d liu t chun axit-baz

tnh lng axit ascorbic trong c vin vitamin C.

7-2

Phn ng ca I2 vi Na2S2O3 nh sau:

2 S2O3

2-

+ I2 S4O6

2-

+ 2I

Tnh nng dung dch iod.

7-3

Phn ng ca axit ascorbic vi I2 l:

-

H2C6H6O6 + I2 C6H6O6 + 2 I + 2H

Tnh lng axit ascorbic trong c vin vitamin C

7-4

So snh u im v khuyt im ca hai phng php chun .

Bi 8. Tng hp v phn tch Aspirin

L thuyt
Aspirin, acid acetylsalicylic l mt axit hu c c cha c este hu c. N c
s dng rng ri trong y hc nh mt loi thuc gim au v nh mt loi thuc
gim st. N thng c iu ch bng phn ng ca axit salixylic vi
anhydrit axetic theo phn ng sau:

axit Salixylic

anhydrit axetic

axit axetyl salixylic

244

axit axetic

Mt lng axit axetylsalixylic c th c xc nh bi s chun vi mt baz

mnh nh Natri hyroxit
CH3COO-C6H4COOH(aq) + OH-(aq) CH3COOC6H4COO-(aq) + H2O(l)
Tuy nhin, axit axetylsalixylic cng l mt este nn d dng b thy phn khi
chun vi mt baz mnh, do trong mi trng kim n b phn hy dn
n sai st trong s phn tch. Nh vy, khi p dng phng php chun th
tt c axit c mt trong dung dch s thy phn hon ton trong NaOH d. Mt
mol axit trong aspirin phn ng va vi mt mol NaOH, mt mol este trong
aspirin phn ng va vi mt mol NaOH. Nh vy s mol NaOH phn ng
s gp i s mol aspirin, sau lng NaOH tha s c chun vi dung
dch axit chun. Trong th nghim ny, axit acetylsalicylic s c chun b,
tng lng acid c mt s c xc nh bng cch s dng mt phng php
chun li.
Ha cht v Thuc th
axit Salixylic HOC6H4COOH
anhydrit axetic (CH3CO)2
axit photphoric H3PO4 v axit sunfuric m c
Etanol C2H5OH
Natri hyroxit NaOH 0,50 mol.L1.
axit clohyric HCl 0,30 mol.L1.
Cht ch th phenolphtalein.
Cht
CH3CO2C6H4C
CH3C2O3CH3
H3PO4
H2SO4
C2H5OH
NaOH(aq)
HCl(aq)

Trng thi
Cht rn
Cht lng
m c
m c
Cht lng
0.50 molL0.30 molL-

K hiu R
22 36 37 38 41 61
10 20 22 34
23 24 25 35 36 37 38
23 24 25 35 36 37 38
11 20 21 22 36 37 38
35
23 25 34 38

245

K hiu S
22 26 36 37 39
26 36 37 39 45
23 30 36 37 39 45
23 30 36 37 39 45
7 16 24 25 36 37 39
26 37 39 45
26 36 37 39 45

Thit b v thy tinh

phu Buchner

cc, 100 ml,

Bnh tam gic (Erlenmeyer) 250 mL giy lc

Pipet, 5 ml v 10 ml

bnh lc chn khng

Xi lanh c chia , 50 ml

ng mao dn im nng chy

buret, 50 ml

Nhit k, 110 C

Thanh khuy

Nhit nng chy b my

Ming knh ng h

Bnh ra

A. Tng hp Aspirin, axit axetylsalixylic

1. Cn chnh xc 3,00 gam axit salixylic trong bnh tam gic nh mc 100 mL
2. Thm 6,00 mL anhyrit axetic v 4 8 git axit photphoric vo bnh v
khuy, trn k
3. un nng dung dch n khong 80 100oC bng cch t bnh trong nc
nng khong 15 pht.
4. Thm tng git 2,0 ml nc lnh cho n khi anhydrit axetic phn hy hon
ton v sau thm 40 mL nc v lm mt dung dch trong bnh nc .
Nu tinh th khng xut hin, dng thanh khuy c vo thnh bnh to ra
kt tinh.
5. Dng giy lc s dng lc. Lc cht rn bng cch lc ht thng qua phu
Buchner v ra cc tinh th vi vi ml nc lnh vo khong 5oC.
6. kt tinh li, bng cch ha tan cc tinh th vo cc v thm 10 ml etanol,
sau thm 25 ml nc m.
7. y cc bi ming knh ng h v khi s kt tinh bt u th t cc
trong bnh nc hon tt s kt tinh li.
8. p dng ht lc nh m t trong bc 5.
9. t cc sn phm vo giy lc vi ming knh ng h v sy kh 100 C
trong khong 1 gi ri xem xt sn phm.

246

10. Xc nh im nng chy (135 C) xc minh tinh khit.

B. Xc nh lng axit acetylsalicylic
1. Ha tan 0,5 g aspirin vo 15 ml etanol trong mt bnh tam gic 250 ml.
2. Thm 20 mL dung dch NaOH 0.50 molL-1.
3. tng tc phn ng thy phn, un nng cc mu trong mt cc nc
khong 15 pht sau khi b sung hai hoc ba chip si vo bnh xoy trn bnh
thnh thong.
Ch : Trnh un si, bi v cc mu c th b phn hy.
4. Lm lnh mu n nhit phng v thm 2- 4 git ch th phenolphtalein
vo bnh. Mu sc ca dung dch l mu hng nht. Nu dung dch khng
mu th thm 5 ml dung dch NaOH 0,50 mol. L-1 ri lp li cc bc 3 v 4.
5. Ghi li tng khi lng dung dch NaOH 0,50 mol.L-1 c thm vo.
6. Chun baz tha trong dung dch bng dung dch HCl 0,30 mol.L-1 cho
n khi mu hng bin mt v dung dch tr nn c.
7. Ghi li khi lng dung dch HCl 0,30 mol.L-1 c thm vo.
8. Lp li cc bc ca s chun hai ln na bng cch s dng hai mu
mi.
X l d liu
1. Tnh hm lng ca Aspirin chun b:

Axit salixylic

Anhyrit axetic

Axit axetylsalixylic

Axit axetic

Khi lng aspirin theo l thuyt:

n(salicylic acid) =

3, 00
138,12

= 0,0217 mol, n(aspirin) = 0,0217 mol

Khi lng aspirin = 0.0217180,2 = 3.906 g

247

Thc nghim:
Khi lng sn phm kh (aspirin) thu c theo thc nghim = 3,03 g
n(aspirin) hay 3,03/180,2 = 0,01682 mol
Hm lng ca aspirin =

3,03
100% =
3,91

77%

Hm lng thp do tan ca aspirin trong nc lnh

2. Tnh lng axit axetylsalixylic c trong mu asppirin
n(axit axetylsalixylic) theo l thuyt=

1, 00 g
180, 0 g / mol

= 5.55 mmol

40 ml NaOH 0,5 M cha 20 mmol

Chun 1,00 g mu vi HCl 0.30 M, thc nghim dng trung bnh 27.0
ml
27 ml HCl 0,3M cha 8,10 mmol
n(NaOH dng phn ng vi axit axetylsalixylic) = 20.0 8.10 = 11.9
mmol
1,0 mol axit axetylsalixylic phn ng vi 2,0 mol NaOH nn
n (axit axetylsalixylic) =

11, 9
=
2

5.95 mmol

loi b axit axetic to ra sau phn ng, qu trnh kt tinh li (ti kt tinh)
c lp i lp li
v ra sch mu bng nc d. Do lng mu gim t 1,50 g n 1,05 g.
Trong s chun ti kt tinh 1,00 g mu vi HCl 0.30 M, thc nghim
dng trung bnh 28.9 ml.
28,9 ml HCl 0,30M cha 8,67 mmol
n(NaOH dng phn ng vi axit axetylsalixylic) = 20,0 8,67 = 11,3 mmol
1,0 mol axit axetylsalixylic phn ng vi 2,0 mol NaOH nn
n (axit axetylsalixylic) =

11, 3
2

= 5,67 mmol

3. Tnh tinh khit ca aspirin v biu th bng t l phn trm khi lng
im nng chy ca mu l 132oC ch ra rng mu khng tinh khit. Khi

248

lng axit axetylsalixylic tm thy trong (b) nhiu hn so vi l thuyt ch ra

rng phng php tnh cho thy khng ch axit axetylsalixylic m cn axit
salixylic v sn phm ph axit axetic cng khng phn ng. V vy n s
nh hng n tinh khit ca mu aspirin.

Bi 9. Thy phn N-axetyl-alanin bng enzim;

v d ca qu trnh ha hc thn thin vi mi trng
L thuyt:
Phn ng sinh hc c xc tc bi enzim. Nhiu enzim biu l kh nng
chn lc cao v thng c th xc tc chn lc cho mt ng phn i quang
ca hn hp trit quang. Trong ha hc hin i enzim c dng cho nhiu
qu trnh tin hnh trong ng nghim, c bit trong s tng hp cc ng phn
quang hc tinh khit. Th nghim ny xem xt s thy phn N-axetyl-alanin vi
enzim axylaza I (acylase I).
S phn ng c nu di y:

Hn hp raxemic N-axetyl-alanin

N-axetyl-alanin

alanin

C th theo di din tin phn ng da trn s to thnh alanin nh phn

ng vi ninhidrin nh di y.

ninhidrin

indan-1,2,3-trion

alanin

anion mu tm lmax = 592 nm

249

Ha cht cn thit:

M an ton:

N-axetyl-alanin raxemic (265 mg)

R-S-

Acylase I (10 mg)

R-S-

Liti hidroxit (85 mg)

R 23-34 S 45-26-27-36/37/39

Dung dch ninhidrin (Sigma N1632) (2 mL) R 11-20/21/22-34 S 16-26-2736/37/39

Dimetyl sunfoxit (khang 70 mL)
m liti axetat pH 5,2

R 36/37/38 S 26-36-23
R 20/21/22-63 S 22-36

Tin hnh:
Ha tan 262 mg (2,0 mmol) hn hp raxemic N-axetyl-alanin trong 10
mL nc. Sau thm t t v khuy nh dung dch cha 84 mg (2,0 mmol) liti
hidroxit monohidrat trong 4 mL nc. Theo di pH bng giy pH n khi t
pH = 7. Thm dung dch acylase I (10 mg) trong nc ng thi khuy mnh
trong 2 pht. [ iu ch dung dch enzim ny bng cch thm enzim vo 5 mL
nc v lc bng lc thy tinh nh c ph vi diatomit]. K tip, thm nc
vo th tch tng cng t chnh xc 20,0 mL. Hn hp phn ng c gi
ti nhit 37C (chng cch thy) trong 60 pht. Dng ng tim hoc pipet
nh ly chnh xc 0,25 mL dung dch enzim cho vo ng nghim ri thm 1,25
mL ninhidrin (Sigma N 1632), (1.25 mL). un nng hn hp ny trong nc
si khong 20 pht, lc y s c mu tm thm. Sau khi ngui, cho hn hp
ny vo dung dch m c cha dung dch liti axetat 4 M (pH = 5,2) v dimetyl
sulfoxit theo t l 1 : 3 trong ng ong 250 mL. iu chnh th tch n 250 mL.
Sau o s hp th bng ph k ti l = 592 nm. Dng ninhidrin trong cng
dung dch m (liti axetat trong dimetyl sulfoxit) tham chiu. epurple
complex = 13350 L mol1 cm1.
Ghi li cc s liu sau:
1. Nng ban u ca hn hp raxemic N-axetyl-alanin.

250

2. Hp th ti l = 592 nm.
3. Tnh s mmol alanin to thnh trong phn ng enzim. Dng nh lut
Lambert-Beer.
4. Tnh phn trm chuyn ha.
Cu hi:
9-1

Alanin to thnh c quang hot khng?

9-2

N-axetyl-alanin vn khng quang hot, quang hot hn hay tinh khit v

mt quang hc khi s chuyn ha nh hn 50%?

9-3

Cng cu hi nh trn khi s chuyn ha t chnh xc 50%?

9-4

C th chuyn ha trn 50% hay khng?

Ghi ch quan trng:

Nu thi gian cho php, c th dng phn ng sau 10, 25, 40 v 60 pht
xc nh nng alanin trong mi trng hp. T , v th biu din
nng alanin theo thi gian, v c lng thi gian phn ng ti u.

Bi 10 - Phn ng ngng t andol thn thin vi mi trng.

L thuyt
c gng thn thin hn vi mi trng, ngi ta ngy cng ch nhiu n
vic gim thiu mt lng ln dung mi dng trong cc phn ng ha hc.
Trong th nghim sau, mt phn ng andol c thc hin khi khng c dung
mi.
O
MeO

CHO

NaOH
MeO

A
C18H18O4
or C18H16O3

251

Thit b v Ha cht s dng

Thit b

Ha cht

Cc thy tnh 25 cm3

3,4-dimetoxibenzandehit (DMBA)

Bnh tam gic 100 cm3

1- indanon (C9H7ON)

Tha kim loi

NaOH

a thy tinh

HCl 3M

pH k

C2H5OH (ethanol)

Phu Hirsch

(C2H5)2O (diethyl ethe)

Phu Buchner

C7H14 (Heptane)

Bm ht chn khng

CH3COOC2H5 (ethyl ethanoat)

Thit b phn tch sc k lp mng

TLC
n t ngoi (UV)
Tin hnh
1. Cho 3,4-dimetoxibenzandehit (DMBA 0,50 g; 3,0 mmol) v 1-indanon
(0,40 g; 3,0 mmol) vo mt cc thy tinh dung tch 25 cm3. Dng mt tha
kim loi nghin nh hai cht rn v c xt cho n khi chng to ra mt
cht du trong.
2. Cho NaOH (0,1 g; 2,5 mmol) vo hn hp phn ng, nu c sinh ra bt
k s vn cc no th phi nghin nh v tip tc c xt cho n khi hn
hp chuyn thnh cht rn.
3. yn hn hp trong 20 pht. Sau thm 4 cm3 dung dch HCl 3M v
c xt quanh cc y xung tt c sn phm bm trn thnh cc. Dng
a thy tinh u bng nghin nh bt k s vn cc no xut hin.
a) o v ghi li pH ca dung dch.

252

4. Lc chn khng qua phu Hirsch tch ly sn phm th. Trng cc

vi 2 cm3 dung dch HCl 3M v dng n ra sn phm th trn phu
Hirsch, tip tc ht chn khng thm 10 pht lm kh sn phm.
b) Ghi li khi lng sn phm th (c th n vn cn hi t) v cho vo l
dn nhn CPA.
5. Chy sc k lp mng (TLC) vi h dung mi Et2O:heptane (1:1) kim
tra phn ng kt thc hay cha. Cho sn hai cht u pha trong etyl
etanoat. Pha sn phm th vo etyl etanoat. [Lu : c th s dng tt c 3
bn mng cho, nhng ch cn np 1 bn cho vo ti nilon c kha (Ziploc
bag), ghi tn v s bo danh ca mnh. y chnh l bn mng m th sinh s
v vo phiu tr li.]
c) Dng n t ngoi (UV) pht hin cc vt cht; ly bt ch khoanh trn v
tr ca cc vt; sao chp (copy) bn mng vo phiu tr li, v sau cho
bn mng vo ti nilon c kha (Ziploc bag), ghi s bo danh ca mnh.
Xc nh RF v ghi li gi tr tm c.
6. Dng mt bnh tam gic 100 cm3 c thanh khuy t kt tinh sn
phm th trong h dung mi 9:1 EtOH:H2O (Lu : dng phu thy tinh
lc nng, cn phi c qu trnh ny loi i mt lng nh tp cht khng
tan). Dng a thy tinh u bng nghin cht vn cc nu c. ngui
bnh nn cha dung dch lc n nhit phng v sau lm lnh
trong chu (dng khay xp polistyren lm chu ng ) trong mt
gi, sau lc qua phu Busn (Buchner) thu sn phm sch, ri ht
thm 10 pht cho kh sn phm. Cho sn phm vo l dn nhn RPA
v ghi s bo danh ca mnh.
d) Ghi li khi lng ca sn phm tinh ch.
e) V cc cu trc c th c i vi sn phm A, da trn cc thng tin cho
trong phiu tr li.

253

f) Ph

13

C NMR ca A c dn ra trang sau. Cc pic do dung mi CDCl3,

c nh du sao (*). Da vo ph ny kt lun cng thc no l ng

i vi A. nh du cu tr li trong phiu tr li.
g) Tnh hiu sut phn trm sn phm tinh ch, da trn cng thc ng vi
cu trc m bn nu trn.

Bi 11. Tng hp Benzylhydantoin

L thuyt
-Amino axit l nhng vin gch xy dng cc peptit v protein. Chng
cng thng c dng lm vt liu ban u trong tng hp dc phm. Trong
th nghim ny S-phenylalanine A thin nhin c chuyn theo hai bc thnh
benzylhydantoin C, l cht trung gian hu ch iu ch cc dn xut c hot
tnh sinh l khc nhau.
BC 1

BC 2
O

O
H2N

OH

1) NaOCN, NaOH (aq)

HN

2) HCl (aq), 25 oC

Khoi lng phan t = 165,19

OH
NH2

O
HCl (aq), 80 oC

Khoi lng phan t = 208,22

Phng php tin hnh

BC 1
S dng phng php phn tch sc k lp mng TLC
S dng phng php chng cch ct
BC 2
S dng phng php phn tch sc k lp mng TLC
S dng phng php chng cch ct
S dng n cc tm (UV)

254

HN
C

NH
O

Khoi lng phan t = 190,20

BC 1
Gi li mt lng rt nh cht ban u A phn tch sc k lp mng TLC
(xem di y). Cho (S)-phenylalanin A (500 mg, 3 mmol, lng chnh xc
c ghi trn nhn ca l nh) vo mt bnh cu c di, thm natri xianat
(300mg; 4,6 mmol), nc (3 mL) v mt c t (stirring bar). Thm hai git
dung dch natri hidroxit (1M) trong nc vo huyn ph khuy. Bnh cu
c ni vi b ngng t v un nng hn hp phn ng n 80 oC bng chng
cch ct va ng thi s dng khuy t.
Quan trng
t n nhit thch hp kp thi v khng mt qu nhiu thi gian, hy
m in nung ct ngay khi bt u th nghim ny. Dng nhit k kim tra
thng xuyn v cn thn nhit ca ct.
Sau khi un nng hn hp phn ng ti 80 oC trong t nht 30 pht, ngui
dung dch trong sut thu c v nhit phng v vo mt cc nh hnh
nn. Ra bnh cu vi mt t nc. Axit ha dung dch bng cch thm tng
git axit clohidric (4 M) n pH < 3 ng thi khuy t. Thm t nc vo
huyn ph trng thu c d khuy.
Lc kt ta trng bng cch ht, ra kt ta bng nc ly d (trn lc) ri
ra hai ln vi lng nh di-isopropyl ete loi hu ht nc cn bm vo.
Dn xut ca ure B c trn lc v ht trong t nht 3 pht loi b cng
nhiu cng tt dung mi cn st li.
Mt lng nh dn xut ca ure B to thnh c gi li phn tch sc k lp
mng TLC sau ny.
BC 2
Dn xut ca ure B nay c chuyn sang bnh cu c di v thm axit clohidric
(4 M, 3 mL). Cho c t vo, khuy k huyn ph v un nng n 80 oC bng
chng cch ct. Thu c dung dch trong sut. Sau thi gian phn ng khong
30 pht, hn hp phn ng (lc ny cng c th c cht t kt ta) c

255

ngui n nhit phng. Lc huyn ph thu c bng cch ht, ra k vi

nc v sau cng ra hai ln vi lng nh di-isopropyl ete. Sn phm c
li trn lc t ht trong t nht 3 pht. Sau thu ly trn giy lc v kh
trong khng kh trong t nht 30 pht.
Sn phm cui C, cht B v cht ban u A (xem trn) c em phn tch sc
k lp mng TLC. phn tch, cc lng nh mi cht c ha tan trong
lng rt t axeton tinh khit. Cc mu nh ca cc dung dch ny c cho ln
bn TLC bng cch dng cc ng mao dn c cp. S phn tch c tin
hnh mi ln vi hai bn TLC. Cc bn TLC c x l vi cht ra gii l
dung dch axit fomic 2% trong etyl axetat. Sau khi ra gii, phn tch cc bn
TLC bng n cc tm (UV-lamp). nh du r vch u (starting line), vch
cui (front line) v cc im kch hot cc tm (UV-active spots) bng bt ch.
Sao chp s vo trong T Bi lm. Cc tr s Rf c xc nh. Cui
cng, gi bn TLC vi kt qu phn tch tt nht trong parafilm vo trong ti
nha (plastic bag) dng bng dnh dn li.
Sn phm cui C c chuyn vo mt l nh xc nh khi lng rng
trc (khi lng c ghi trn nhn). Cn l cha sn phm v tnh hiu
sut ca sn phm C.
Gio vin s kim tra cht lng ca benzylhydantoin do hc sinh iu ch v
xc nh im nng chy bng thit b o im nng chy t ng.

256

CC CNH BO V CC NGUY HIM C TH GP

V KHUYN CO V AN TON
TRONG KHI LM TH NGHIM
1. Cnh bo cc nguy c c bit (K hiu R - Risk)
R 1. Gy n khi dng kh.
R 2. Nguy c n khi va p, ma st, c la hoc ngun gy chy khc.
R 3. Nguy c gy n rt cao khi va p, ma st, c la hoc ngun gy chy khc.
R 4. To ra cc hp cht n kim loi rt nhy.
R 5. un nng c th gy n.
R 6. Gy n khi tip xc hoc khng tip xc vi khng kh.
R 7. C th gy chy.
R 8. Tip xc vi vt liu chy c th gy chy.
R 9. Gy n khi trn vi cht d chy
R 10. C th chy.
R 11. D chy.
R 12. Rt d chy.
R 13. Kh ha lng rt d chy.
R 14. Phn ng mnh lit vi nc.
R 15. Tip xc vi nc gii phng kh d bc chy.
R 16. Gy n khi trn vi cc cht oxi ha.
R 17. T bc chy trong khng kh.
R 18. Khi s dng, c th to ra hn hp hi vi khng kh gy chy hoc n.
R 19. C th to ra cc peoxit gy n.
R 20. Nguy him khi ht vo.
R 21. Nguy him khi tip xc vi da.
R 22. Nguy him nu nut vo.
R 23. Ng c khi ht vo.
R 24. Ng c khi tip xc vi da.
R 25. Ng c nu nut vo.
R 26. Rt c khi ht vo.
R 27. Rt c khi tip xc vi da.
R 28. Rt c nu nut vo.

257

R 29. Tip xc vi nc gii phng kh c.

R 30. Khi s dng, c th rt d chy.
R 31. Tip xc vi axit gii phng kh c.
R 32. Tip xc vi axit gii phng kh rt c.
R 33. Gy nguy him do cc tc ng tch ly.
R 34. Gy bng.
R 35. Gy bng nng.
R 36. Gy cay mt.
R 37. Kch thch h h hp.
R 38. Gy mn nga da.
R 39. Nguy him do cc tc ng nghim trng khng th loi b.
R 40. C th nguy him do cc tc ng nghim trng khng th loi b.
R 41. Nguy him do gy hng mt nng.
R 42. C th gy s mi khi ht vo.
R 43. C th gy mn nga khi tip xc vi da.
R 44. Nguy c gy n nu un nng trong bnh kn
R 45. C th gy ung th.
R 46. C th gy tn hi gen.
R 47. C th gy tn hi phi.
R 48. Nguy him do b tn hi ko di.

2. Khuyn co v an ton (K hiu S - Safety)

S 1. Nt kn bnh cha.
S 2. cch xa tm vi ca tr con.
S 3. Gi ni thong mt.
S 4. Bo qun cch xa khu dn c.
S 5. Bo qun bnh cha di cc iu kin....(cht lng c nh sn xut a ra
ch dn ring).
S 6. Bo qun di cc iu kin... (kh tr c nh sn xut ch dn ring).
S 7. Bo qun bnh cha dng ng kn.
S 8. Bo qun bnh cha kh ro.
S 9. Bo qun bnh cha ni thng gi.
S 10. Bo qun bnh cha cht trong bnh dng t.
S 11. Trnh tip xc vi khng kh.

258

S 12. Khng bo qun bnh cha dng kn.

S 13. Bo qun cch xa thc phm, nc ung v thc phm cho gia sc.
S 14. Bo qun cch xa ..... ( cc cht k nhau phi c nh sn xut ch nh).
S 15. Bo qun cch xa nhit.
S 16. Bo qun cch xa ngun pht la. Cm ht thuc.
S 17. Bo qun cch xa cc cht d chy.
S 18. Tip xc v m bnh cha ho cht cn thn.
S 19 Khi s dng ha cht khng n hoc ung ng thi.
S 20. Khi s dng ha cht khng ht thuc.
S 21. Khng ht bi ha cht.
S 22. Khng ht kh/khi/ hi/ kh phun sng.
S 23. Trnh tip xc ha cht vi da.
S 24. Trnh ha cht bn vo mt.
S 25. Trong trng hp b bn vo mt, phi ra ngay vi nhiu nc v n c
quan y t.
S 26. Ci b ngay o qun b nhim bn ha cht.
S 27. Khi b dnh vo da, ra ngay vi mt lng nhiu.....(do nh sn xut ch nh).
S 28. Khng lm kh kit bnh cha.
S 29. Khng bao gi c cho nc vo sn phm ny.
S 30. Bo qun cch xa cc cht gy chy.
S 31. Cn c cc bin php phng s phng in.
S 32. Trnh va p v ma st.
S 33. Cht ny v bnh cha n phi c loi b theo cch an ton thch hp.
S 34. Mc qun o bo v thch hp.
S 35. eo gng tay thch hp.
S 36. Trong trng hp khng thng thong, phi eo thit b tr h hp.
S 37. eo phng tin bo v mt/ mt.
S 38. v sinh sn v cc vt dng b nhim bn ha cht ny, cn s dng ... (do
nh sn xut ch nh).
S 39. Trong trng hp chy v/ hoc n khng c ht khi.
S 40. Trong thi gian phun khi / phun sng phi eo thit b tr h hp thch hp.
S 41. Trong trng hp chy, s dng ....(ch r chnh xc dng loi dng c cu
ha no. Nu nc lm tng nguy c th KHNG bao gi c dng nc)
S 42. Nu cm thy ngi khng khe, n c quan y t ngay (c bin ch dn)
259

S 43. Trong trng hp tai nn hoc nu ngi khng c khe n c quan y t

ngay (c bin ch dn).

3. V d v ngha R v S
Di-isopropyl ete (Di-isopropyl ether)
Cng thc

C6H14O

Khi lng phn t

im nng chy

-85 oC im si

Khi lng ring

0,72 g/cm3

102,17

68 oC

R11

Rt d chy

R19

C th to peoxit d n

R66

Lm cho da kh nt nu b dy nhiu ln

R67

Ha cht dng hi gy chng mt v bun nn

S9

Gi ni thng gi

S16

Trnh tia la - Khng ht thuc

S29

Khng c vo h thng thot nc

S33

Cn c bin php chng phng tnh in

Axit clohidric (Hydrochloric acid)

Cng thc

HCl

Khi lng ring

R11

Khi lng phn t 36,46

0,909

Rt d chy

R37/38 Gy nga mt, h thng h hp v da

S16

Trnh tia la - Khng ht thuc

S26

Nu ri vo mt, ra ngay vi nhiu nc v xin tr gip y t

S45

Khi b tai nn hoc khi thy khng khe, xin tr gip y t ngay (nu c th,
mang theo nhn ha cht)

S7

Cn gi bnh tht kn

Metanol (Methanol)
Cng thc

CH4O

Khi lng phn t

im nng chy

-98 oC

Khi lng ring

0,79 g/cm3

R11

im si

32,04
65 oC

Rt d chy

R23-25

Rt c khi ngi, tip xc vi da hoc ung

R39/23

Rt c v gy h qu nghim trng khng th hi phc khi ngi, tip

24/25

xc vi da hoc ung

260

S7

Cn gi bnh tht kn

S16

Trnh tia la Khng ht thuc

S36/37
S45

Mang bao tay v mc y phc bo h

Khi gp tai nn hoc cm thy khng khe, xin tr gip y t ngay (nu c th,
mang theo nhn ha cht)

261