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TH Hoa Sinh
TH Hoa Sinh
HCM
KHOA MI TRNG & CNG NGH SINH HC
------
BI GING
THC HNH
HA SINH
MC LC
Trang
Gii thiu mn hc
sinh
1.
2.
Bi 1
nghim Ha sinh
I
L thuyt
1.
Dung dch
2.
Dung dch m
14
II
Thc hnh
20
III
Bi np
20
Bi 2
21
dinitro-salicylic (DNS)
I
L thuyt
21
1.
nh ngha
21
2.
Nguyn tc
21
3.
X l mu
21
Thc hnh
22
1.
Dng c - ha cht
22
2.
23
3.
Tnh kt qu
24
III
Bi np
24
Bi 3
25
II
Kjeldahl
I
1.
L thuyt
25
nh ngha
25
2
2.
Nguyn tc
26
Thc hnh
26
1.
Dng c - ha cht
26
2.
27
3.
Tnh kt qu
28
III
Bi np
28
Bi 4
30
L thuyt
30
1.
nh ngha
30
2.
Nguyn tc
30
Thc hnh
31
1.
Dng c - ha cht
31
2.
31
3.
Tnh kt qu
32
III
Bi np
32
Bi 5
33
L thuyt
33
1.
33
2.
34
3.
35
II
II
tnh
II
III
Thc hnh
36
1.
Dng c - ha cht
36
2.
36
3.
Tnh kt qu
38
Bi np
38
39
GII THIU MN HC
Bi ging Thc hnh sinh ho dnh cho sinh vin nm th hai khoa Mi
trng & Cng ngh Sinh hc, trng i hc K Thut Cng Ngh TP.HCM.
Vi thi lng 30 tit v tu thuc iu kin, c s vt cht ca phng th
nghim cho php, sinh vin tin hnh lm 5 bi thc hnh gm cc ni dung chnh ca
hc phn l thuyt ha sinh c s.
1. Gii thiu phng th nghim ha sinh, cch pha ch cc dung dch dung
trong th nghim ha sinh ;
2. nh lng ng kh bng phng php acid dinitro-salicylic (DNS);
3. nh lng nit tng s bng phng php Kjeldahl;
4. nh lng protein bng phng php Bradford;
5. Xc nh hot tnh enzyme amylase.
- Dng c dng ly ha cht phi tht sch v dng xong phi ra ngay,
khng dng ln np y v dng c ly ha cht.
- Khi lm vic vi cht d n, d chy khng c gn ni d bt la. Khi
cn s dng cc ha cht d bc hi, c mi,... phi a vo t ht, ch y kn np
sau khi ly ha cht xong.
- Khng ht bng pipette khi ch cn t ha cht trong l, khng ngi hay nm
th ha cht.
- Khi lm vic vi acid hay base mnh:
Bao gi cng acid hay base vo nc khi pha long (khng c nc
vo acid hay base);
Khng ht acid hay base bng ming m phi dng cc dng c ring nh ng
bp cao su.
Trng hp b bng vi acid hay base ra ngay vi nc lnh ri bi ln vt
bng NaHCO3 1% (trng hp bng acid) hoc CH3COOH 1% (nu bng base). Nu
b bn vo mt, di mnh vi nc lnh hoc NaCl 1%.
Trng hp b ha cht vo ming hay d dy, nu l acid phi sc ming v
ung nc lnh c MgO, nu l base phi sc ming v ung nc lnh c CH3COOH
1%.
Lu cc k hiu cnh bo nguy him:
Khi phi un nng dung dch ha tan, hoc qu trnh ha tan c to nhit th
phi ch nhit tr li bnh thng (nhit khng kh) ri mi thm nc ti vch
nh mc.
V d: Pha 1 lt dung dch KOH 1M
Phn t lng ca KOH: MKOH = 39 +16 +1 =56
Lng KOH pha 1 lt dung dch 1M l: 56g
Vy, cn 56g KOH, ha tan trong 1 t nc, cho vo bnh nh mc 1000. y
l phn ng ta nhit, cn lm ngui dung dch trc khi nh mc thnh 1 lt.
Nu mun pha dung dch 2M; 3M hay 0,1M; 0,05M ta cng tin hnh tng t
vi lng cn tng ng.
V d: Pha 500ml dung dch KCl 3M
Phn t lng ca KCl: MKCl = 39 +35,5 = 74,5
Lng KCl pha 500ml dung dch 3M l: X= (74,5 *3*500)/1000 =
111,75g
Vy, cn 111,75g KCl, ha tan trong 1 t nc, cho vo bnh nh mc 500,
nh mc n vch.
ii) Cht tan l cht rn ngm nc: khi tnh lng cht tan cn cn phi tnh
lun c khi lng cc phn t nc.
iii) Cht tan dng lng: nu cht tan l dung dch, ta phi tnh ton da vo
nng dung dch .
V d: 8) Pha 1 lt dung dch HCl 1M t HCl 37%
Phn t lng HCl: MHCl = 1+35,5 = 36,5
Lng HCl 37% pha dung dch 1M l: X = (36,5*100)/37 = 98,65g
Hay 98,65/1,19 = 83ml
Vy, ong 83ml HCl 37% cho vo bnh nh mc 1000 c sn 1 t nc.
nh m c thnh 1 lt. Tin hnh pha trong t Hotte v hi acid bay ln rt c
hi.
f) Pha nng ng lng (N)
Vic pha dung dch nng ng lng gam (N) cng tng t vi cch pha
dung dch nng phn t gam (M).
1.1.3. Hiu chnh nng dung dch
12
Khi pha ha cht, c nhiu nguyn nhn lm cho nng dung dch khng
chnh xc nh vic cn ong khng chnh xc, cc ha cht khng tinh khit hay b
ht m. Thi gian tng tr lu nn cht tan b thng hoa, b oxy ha, dung mi bay
hi, v vy phi kim tra nng thc ca cc dung dch pha sn da vo cc dung
dch c nng chnh xc c gi l dung dch chun (fixanal)
a) Dung dch chun
Dung dch chun l cc dung dch c chun b sn, m bo chnh xc v
c dng nh chun cc dung dch t pha ch khi lm th nghim. Cc cht dng
trong dung dch chun phi kh bn vng sao cho nng ca chng khng thay i
nhanh chng theo thi gian.
Pha dung dch chun, ta phi dng ng chun. ng chun l mt ng ampun
thy tinh hay nha ng kn. Bn trong cha mt lng cn chnh xc cht tan hoc
dung dch cht tan. Khi chuyn ht lng cht tan trong ng vo bnh nh mc v pha
thnh 1 lt ta c dung dch chun c nng ghi trn nhn ngoi ng.
Cch pha dung dch chun t ng chun :
- Dng inh thy tinh chc thng ampun, hng ln phu vo bnh nh mc,
dng bnh tia ra sch cht tan c trong ampun vo bnh nh mc 1 lit, va thm
nc ct va lc v a nc ct ti vch mc.
- i vi cc hp cht bn vng, c thnh phn khng thay i nh NaCl,
AgNO3, acid oxalic, ... c th pha dung dch chun trc tip bng cch cn chnh xc
cht cn pha, pha long v nh mc ti th tch ng.
- i vi cc cht nh NaOH, HCl, Na2S2O3, ... khng th pha ngay c dung
dch chun, do cc cht ny thng khng bn vng v d thay i thnh phn, v vy
sau khi pha phi hiu chnh li nng .
V d: NaOH thng nhim mt lng Na2CO3 rt d chy nc, HCl d bay
hi, Na2S2O3 d b mt nc tinh th khi ngoi khng kh.
b) Phng php hiu chnh nng dung dch
i vi cc cht d thay i thnh phn khi dng rn, nu mun pha dung
dch c nng chnh xc, ta pha dung dch c nng gn ng, sau hiu chnh
nng ca dung dch da vo phn ng vi mt dung dch chun thch hp.
13
V d: Pha dung dch NaOH 0,1 N t NaOH rn v dng dung dch chun
H2SO4 0,1N chun li.
i vi cc dung dch d thay i trong qu trnh bo qun, mi ln s dng li
phi xc nh li h s hiu chnh nng dung dch
Xt mt phn ng trung ha acid - base, 1 ion gam H+ s phn ng vi 1 ion
gam OH-. Do :
C1V1 = C2V2
Nu gi Cp l nng dung dch nh pha v Ct l nng thc ca dung dch
ta c h s hiu chnh K:
K= Cp/Ct = Vp/Vt
V d: Dung dch chun l H2SO4 0,1N, dung dch nh pha l NaOH 0,1N.
Ly 10ml H2SO4 0,1N cho vo erlen, thm ba git ch th mu phenolphtalien,
dng burette chun bng NaOH c 11 ml NaOH
Vy nng thc t NaOH pha l : Ct = (10*0,1)/11 = 0,091 N
H s iu chnh K: K= 0,091/0,1 = 10/11 = 0,91
1.2. Dung dch m:
1.2.1. nh ngha dung dch m
pH mi trng lm thay i cu trc khng gian protein v mt s amino acid
c mch nhnh phn ly (COO- v NH4+ to lin kt ion). Hat ng ti u ca protein
ph thuc vo cu trc khng gian nht nh trong mi trng, ngha l ph thuc vo
t l phn ly ca mch nhnh thnh ion, hay ni tm li l ph thuc vo pH mi
trng.
Dung dch m l dung dch c pH khng thay i nhiu lm khi mt lng
nh acid (H+) hoc base (OH-) c thm vo. Nh vy, dung dch m bao gm mt
cp acid base lin hp (acid yu v mui ca acid yu ny hoc base yu v mui ca
base ny) v t l ca chng s quyt nh pH ca dung dch.
Vic pha dung dch m tun theo nguyn tc chn cp acid base c hng s
phn ly pK A/B gn vi pH m mun pha v phi trn chng vi s mol bng nhau.
Th d, c m pH gi tr 4.75, chn cp acid - base CH3COOH
(0,1M)/CH3COONa (0,1M). Nu thm vo dung dch 0.001 mol HCl th pH ca h
vn ch mc 4.748 gn bng 4.75. Ngha l pH thay i rt t.
14
---------->
NH4+
OH-
pH
15
pH
9,80
0,2
6,60
6,50
3,50
8,20
9,70
0,6
6,77
6,00
4,00
8,31
9,40
0,6
7,09
5,50
4,50
8,41
9,00
1,0
7,36
5,00
5,00
8,51
8,75
1,25
7,50
4,50
5,50
8,60
8,50
1,50
7,60
4,00
6,00
8,69
8,00
2,0
7,78
3,00
7,00
8,84
7,70
2,30
7,88
2,00
8,00
8,98
7,50
2,50
7,94
1,00
9,00
9,11
7,00
3,00
8,08
0,00
10,0
9,24
pH
pH
46,5
3,50
3,0
23,0
27,0
4,8
43,8
6,20
3,2
20,5
29,5
5,0
40,0
10,0
3,4
18,0
32,0
5,2
37,0
13,0
3,6
16,0
34,0
5,4
35,0
15,0
3,8
13,7
36,3
5,6
33,0
17,0
4,0
11,8
38,2
5,8
31,0
19,0
4,2
9,5
40,5
6,0
28,0
22,0
4,4
7,2
42,8
6,2
25,5
24,5
4,6
16
pH
pH
93,5
6,50
5,6
45,0
55,0
6,9
92,0
8,00
5,8
39,0
61,0
7,0
90,0
10,0
5,9
33,0
67,0
7,1
87,7
12,3
6,0
28,0
72,0
7,2
85,0
15,0
6,1
23,0
77,0
7,3
81,5
18,5
6,2
19,0
81,0
7,4
77,5
22,5
6,3
16,0
84,0
7,5
73,5
26,5
6,4
13,0
87,0
7,6
68,5
31,5
6,5
10,5
89,5
7,7
62,5
37,5
6,6
8,50
91,5
7,8
56,5
53,5
6,7
7,00
93,0
7,9
51,0
49,0
6,8
5,30
94,7
8,0
17
pH
pH
10
990
5,0
372
628
6,6
18
982
5,2
492
508
6,8
30
970
5,4
612
388
7,0
49
951
5,6
726
274
7,2
79
921
5,8
818
182
7,4
121
879
6,0
885
115
7,6
184
816
6,2
936
64
7,8
264
736
6,4
969
31
8,0
pH
pH
5,0
3,6
16,8
2,8
6,4
3,4
24,2
2,6
8,2
3,2
32,4
2,4
11,4
3,0
44,0
2,2
18
pH
pH
4,0
8,6
22,4
9,6
6,0
8,8
72,2
9,8
8,8
9,0
32,0
10,0
12,0
9,2
38,6
10,4
16,8
9,4
45,5
10,6
pH
pH
46,3
3,7
3,6
20,0
30,0
4,8
44,0
6,0
3,8
14,8
35,2
5,0
41,0
9,0
4,0
10,5
39,5
5,2
36,8
13,2
4,2
8,8
41,2
5,4
30,5
19,5
4,4
4,8
45,2
5,6
25,5
24,5
4,6
19
20
BI 2. NH LNG NG KH BNG
PHNG PHP ACID DINITRO - SALICYLIC (DNS)
I. L thuyt
1. nh ngha
ng kh l cc ng cha nhm aldehyde (-CHO) hoc ketone (-CO) nh
glucose, fructose, arabinose, maltose, lactose; trong khi saccharose, trehalose
khng phi ng kh.
2. Nguyn tc
Phng php ny da trn c s phn ng to mu gia ng kh vi thuc
th acid dinitrosalicylic (DNS). Cng mu ca hn hp phn ng t l thun vi
nng ng kh trong mt phm vi nht nh. So mu tin hnh bc sng
540nm. Da theo th ng chun ca glucose tinh khit vi thuc th DNS s tnh
c hm lng ng kh ca mu nghin cu.
Phng trnh phn ng to mu gia ng kh v DNS acid:
Acid dinitrosalicylic
3. X l mu
Ty vo i tng nghin cu (ht, qu,...) m cch chun b dung dch nghin
cu dng nh lng ng c khc nhau i cht, nhng nguyn tc chung nh
sau:
- Trng hp nguyn liu th nghim khng cha qu nhiu tinh bt hoc
inulin, c th chit ng t nguyn liu bng nc. Cn v cho vo ci s 1 g
nguyn liu ht hay cc mu th nghim thc vt kh nh cy, l hoc qu kh,...
c nghin nh (v sy kh n khi lng khng i). Nu l nguyn liu ti (nh
hoa, qu ti) th cn 5 10 g. Nghin cn thn vi bt thy tinh hay ct sch v 30
21
22
- Dung dch glucose chun: pha sn dung dch glucose chun 10 mg/ ml (bo qun t
lnh vi ngy).
2. Tin hnh
a) Chit xut ng trong thc vt
- 5g rau ci ngt nghin nh cho vo becher 100ml + 10ml cn 900 un cch
thy (si 3 ln) khuy u bng a thy tinh ngui lc (ch gn ly phn
trong).
- Cho tip 10ml cn 800 vo cc ng b, khuy u, un si 2 ln trn ni un cch
thy ngui lc (lp li 2 ln)
- a phn b ln lc v ra bng ru nng (800)
- Ru qua lc c cho bay hi trong phng hoc trn ni cch thy un nh. Sau
khi cho bay hi ru, mu c th lu trong bnh ht m.
- Cn kh trong cc c pha long thnh th tch V = 50ml vi nc ct ta c
dung dch ng gc. Pha lang dung dch ng gc vi mt h s pha lang n thch
hp.
b) Dng ng chun v xc nh hm lng ng
- Cho vo 6 ng nghim sch vi cc cht c th tch nh bng sau:
Ho cht
ng
ng 1 ng 2 ng 3 ng 4
ng 5
C
Glucose 10
0.2
0.4
0.6
0.8
1.0
10
9.8
9.6
9.4
9.2
9.0
mg/ml (ml)
Nc ct (ml)
Nng
(mg/ml)
OD540nm
24
Mu
H s chuyn i
Ngun gc ng vt
6.25
Ht bng
5.30
u phng
5.46
u nnh
5.71
Ht hng dng
5.3
Cm da
5.3
Ht m
5.3
Bp
6.25
Go
5.95
La m
5.83
25
2. Nguyn tc
a) V c ha mu
- Trc tin mu c v c ha bng H2SO4 c nhit cao v c cht xc
tc. Cc phn ng ca qu trnh v c ha xy ra nh sau:
2H2SO4 2H2O +2SO2+ O2
- Oxi to thnh trong phn ng li oxi ha cc nguyn t khc. Cc phn t
cha nit di tc dng ca H2SO4 to thnh NH3. V d cc protein b thy phn
thnh acid amine; carbon v hidro ca acid amine to thnh CO2 v H2O; cn nit
c gii phng di dng NH3 kt hp vi H2SO4 d to thnh (NH4)2SO4 tan trong
dung dch
2NH3 + H2SO4 (NH4)2SO4
Cc nguyn t P, K, Ca, Mg,... chuyn thnh dng oxide: P2O5, K2O, CaO,
MgO,...
b) Chng ct m:
- ui NH3 ra khi dung dch bng NaOH
(NH4)2SO4 + 2 NaOH = Na2SO4 +H2O + 2NH3
- NH3 bay ra c lm lnh bin i thnh NH4OH ri vo bnh hng, bnh
hng cha H2SO4 0.1N
2NH4OH + H2SO4 (NH4)2SO4 + 2H2O + H2SO4 d
c) Chun H2SO4 d
- Chun H2SO4 d bng NaOH 0.1N
H2SO4 d + NaOH Na2SO4 + H2O
II. Thc hnh
1. Dng c - ha cht
a) Dng c
- Bnh ph mu
- Bp un
- B chng ct Kjeldahl nh lng Nit gm:
Bnh ct m (bnh Kjeldahl)
ng dn kh
ng sinh hn
26
B chng ct Kjeldahl
b) Ha cht + nguyn liu
- Bt u nnh (0.1g), nc tng 1-2 ml
- H2SO4 m c
- NaOH 40%
- H2SO4 0.1N chun
- NaOH 0.1N chun
- Thuc th Tashiro
- Cht xc tc: hn hp K2SO4 : CuSO4 (3:1)
2. Tin hnh
a) V c ha mu
- Cn 100mg bt u nnh sy kh tuyt i cho vo bnh Kjeldahl, cho tip
5ml H2SO4 m c s thy xut hin mu nu en (do nguyn liu b oxi ha).
Cho thm vo 200mg cht xc tc, lc nh, y kn khong 3 pht. t bnh
Kjeldahl ln bp un, y ming bnh bng mt phu thy tinh.
Lu giai on ny phi thc hin trong t Hotte, t bnh hi nghing trn
bp, trnh trng hp khi si mnh ha cht bn ra ngoi, khi si gi nhit bp
un va phi trnh ha cht tro ra ngoi v khng b bay mt ammoniac.
27
- Trong khi un, theo di s mt mu en ca dung dch trong bnh un, khi
thy dung dch gn nh trong sut th c th lc nh bnh ko ht cc phn t trn
thnh bnh cn cha b oxi ho vo trong dung dch. Tip tc un cho n khi dung
dch trong hon ton. ngui bnh ri chuyn ton b dung dch sang bnh nh mc
100 ml, dng nc ct v m trng li bnh Kjeldahl v nh mc n vch.
b) Ct m
- Chuyn 50ml dung dch trong bnh nh mc trn vo bnh ct m c sn
50ml nc ct v 3 git thuc th Tashiro lc ny trong bnh c mu tm hng. Tip
tc cho vo bnh ct 20ml NaOH 40% cho n khi ton b dung dch chuyn sang
mu xanh l m (thm 5ml NaOH 40% nu dung dch trong bnh cha chuyn ht
sang mu xanh l m).
- Tin hnh lp h thng ct m, cho vo bnh hng 20ml H2SO4 0.1N v 3
git thuc th Tashiro (dung dch c mu tm hng). t bnh hng sao cho ngp u
ng sinh hn. Bt cng tc ct m. Kim tra xem hi ra t u ng sinh hn c lm
xanh giy qu khng, nu khng thm NaOH 40%.
- Sau khi ct m 20-25 pht kim tra xem NH4OH cn c to ra khng,
dng giy qy th u ng sinh hn. Nu giy qy khng i sang mu xanh l
c. Ngng ct m, i h thng ngui mi tho h thng em i ra.
c) Chun :
- Chun H2SO4 d trong bnh hng bng NaOH 0.1N cho n khi mt mu
tm hng v chuyn sang mu xanh l m. Ghi nhn th tch NaOH 0.1 N s dng.
3. Tnh kt qu:
Hm lng % nit tng s c tnh theo cng thc
N (mg%) = {1,42 * (V1-V2)*100/a}*2
V1: s ml H2SO4 cho vo bnh hng
V2: s ml NaOH 0.1N chun
a s miligam nguyn liu
1,42: h s, c 1 ml H2SO4 dng trung ha NH4OH th tng ng vi
1,42 mg Nit
III. Bi np
1. Gii thch ngha cc bc trong th nghim.
28
29
30
31
ng nghim
Nc ct, ml
Albumin chun (0,1mg/ml),
ml
TT Bradford, ml
5
?
OD595nm
(Sinh vin in nng albumin (g/ml) vo bng trn)
32
33
V i n h n g en z y m e cn c ch t h o t h a h o c ch t n n h th p h i ch o cc ch t
n y v o en z y m e tr c k h i ch o c ch t v o h n h p p h n n g .
-
N n g c ch t tro n g p h n n g en z y m e p h i tro n g g i i h n th c h h p ,
th a b o en z y m e, n h n g k h n g q u cao k m h m en z y m e. S au k h i d n g p h n
n g l n g c ch t c ch u y n h a 2 0 -3 0 % .
-
T h i g ian x c n h h o t tn h th n g 5 -3 0 p h t. T t n h t l x c n h t c b an
u c a p h n n g (3 0 -6 0 g iy ), v g iai o n n y t c p h n n g l n n h t, sau b t
u g im (H n h 1 ).
34
K h i x c n h h o t tn h p h i l m m u i ch n g so n g so n g v i m u th n g h im .
T ro n g m u i c h n g en z y m e p h i b b t h o t tr c k h i ti p x c v i c ch t.
n c ct tro n g b n h n h m c 1 0 0 m l, lc u. t v o b p cch th y an g s i, lc li n
t c ch o n k h i tin h b t tan h o n to n. S au lm n g u i v b su n g 1 0 m l m acetate
p H = 4,7 (h o c 1 0 m l d u n g d ch m p h o sp h ate p H = 4,9 ) b su n g n c ct n v ch
m c, lc u . D u n g d ch c ch u n b tro n g n g y s d n g .
-
D u n g d c h en z y m e g c
2. T in h nh:
a) T rch ly en zym e: en zy m e c trc h ly t ch p h m h a y t b o , m n g th c
v t b n g d u n g d c h m c p H th c h h p .
i) Trch ly enzyme t vi khun: C n m = 0 ,1 g ch p h m n g h i n c u , d n g a
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M u trn g
2 ml
2 ml
2 ml
1 ml
1 ml
1m l
0 ,5 m l
0 ,5 m l
0 ,5 m l
4 0 o C , 1 0 p h t t n h it
D u n g d c h en z y m e
1 ml
4 0 o C , 3 0 p h t x y ra p h n n g
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H Cl 1N
1 ml
1 ml
1 ml
E nzym e
1m l
1 ml
C h u y n san g b n h n h m c 1 0 0 m l
N c ct
n v c h
n v c h
n v c h
0 ,5 m l
0 ,5 m 1
0 ,5 m 1
Io d e
D u n g d c h i ch n g c m u x a n h
D u n g d c h th n g h i m c m u tm v i c n g k h c n h a u t y v o l n g tin h
b t ch a b th y p h n .
o m t q u a n g (O D ) c a cc d u n g d c h b c s n g = 6 2 0 n m so v i m u
trn g .
3. T nh k t qu:
S m g tin h b t b th y p h n S =
x 20
S n V
10 m
Hat tnh ring (n v hat tnh/mg protein) = hat tnh ton phn/mg protein
(Bradford).
trong :
n = h s pha long
V = th tch dch trch ly, ml
m = khi lng enzyme, g
III. Bi np
1. n v hat tnh enzyme amylase trong bi l g ?
2. So snh cc h s pha lang, chn h s pha lang no ?
3. C th s dng phng php o ng kh bng DNS acid o hat tnh enzyme
amylase khng ? Ti sao ?
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