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BO CO
THC HNH HA SINH
MSSV:1211100186
Ng L Hng Duyn
MSSV: 1211100062
Cao Th Nhm
MSSV:1211100142
on Ngc King
MSSV:1211100293
V Nguyn Anh Th
MSSV:1211100192
Phan Th Thy Vy
MSSV: 1211100283
L thuyt
Quy tc lm vic trong phng th nghim ha sinh
I.
Phi lm vic trong t ht bt c khi no un nng axit hoc thc hin phn ng
vi cc axit t do
-
Kim c th lm chy da
Mang gng tay , khu trang khi lm vic vi dung dch kim
Cc ha cht dng phn tch, lm tiu bn, tin hnh phn ng, trong phng th
Ha cht c bo qun trong chai l, thy tinh hoc nha ng kn c nhn ghi
tn ha cht, cng thc ha hoc, mc sch, tp cht, khi lng tnh, khi lng phn
t, ni sn xut , iu kin bo qun.
Cch s dng v bo qun ha cht:
-
Khi lm vic vi ha cht , nhn vin phng th nghim cng nh sinh vin cn ht
sc cn thn trng gy nhng tai nn ng tic cho mnh cng nh cho ngi khc.
Khng ht axit hay baz bng ming m phi dng cc dng c ring nh: ng bp
cao su.
II.
Dung dch :
-
Nng phn trm khi lng, % (w/w): l s gam cht tan c trong 100g dung
dch.
Nng trm khi lng th tch (w/v): l s g cht tan c trong 100ml dung dch.
Nng phn trm th tch th tch , % (v/v): l s ml dung cht c trong 100ml
dung dch.
Nng phn t g hay nng mol, (mol/L): l s phn t g ( hay s mol) cht tan
dung dch.
S ng lng cht tan = s mol (n) x h s ng lng (z)
-
tham gia.
Nng dung dch bo ha: l nng dung dch khi ti a cht ha tan c
mt trong dung dch.
n v nng dng trong cc php phn tch vi lng :
-
Miligam phn trm , mg% : mg cht tan trong 100g dung dch
Phn nghn ,0/00 : s gam cht ha tan trong 1000g dung dch
Phn triu , ppn : s mg cht ha tan trong 1kg hay 1 lt dung dch
Khi pha dung dch ta cn phi tnh lng kt tinh c sn ging nh phn a
-
Cht tan dng lng : mt s cht tan dng lng nh HCl, H2S04
Ngy nay a s cc dung dch th nghim c pha ch theo nng khi lng- th
tch (w/v).
i.
-
Mun pha dung dch nng 1M ca mt cht no , ta tnh khi lng phn t cht
theo n v gam. Cn chnh xc lng cht tan , qua phu cho vo bnh nh mc c
dung tch 1 lt . Cho vo tng lng nc ct nh , lc ha tan hon ton v a nc
ct ti mc . Chuyn dunh dch sang bnh cha, lc trn u ng nht.
Khi phi un dung dch ha tan, hoc qu trnh ha tan c ta nhit th phi ch
nhit tr li bnh thng (nhit khng kh) ri mi thm nc ti vch nh mc.
-
Cht tan l cht rn ngm nc : khi tnh lng cht tan cn cn phi tnh lun c
Cht tan dng lng: nu cht tan l dung dch, ta phi tnh ton da vo nng
dung dch .
Hiu chnh nng dung dch.
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.
Cch pha dung dch 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.
-
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.
-
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 .
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.
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
III.
Dung dch m
CU HI N TP
Cu 1:
Pha 1L dung dch NaOH 40% (w/v): cn X=(40x1000)/100=400(g) NaOH kh.
Cu 2:
Nng mol ca H2SO4 l:
CM = (10.d.C%)/M = (10x97x1.84)/98 = 18.2 (mol)
Th tch H2SO4 cn s dng l:
VH2SO4 = (VH2SO4 2M. CMH2SO4)/CMH2SO4 = (2x500)/18.2 = 54.91(ml)
Lng nc cn b sung l: 500 54.91 = 445.09 (ml)
Cu 3:
Pha 250ml dung dch CuSO4 1M, cn m = n.M = 0.25 x 250 = 62.5 (g)
CuSO4.5H2O
Cu 4:H2SO4 2M = H2SO4 4N
C1.V1 = C2.V2 V2 = (1000 x 0.1)/4 = 25 (ml)
Cu 5:
-
Cu 6:
-
Lng dung dch HCl 37% cn dng l: Y = (100 * 10)/37 = 27.03 (g)
=> 27.03 /1.19 = 22.71 (ml)
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 t l thun vi nng
ng kh trong mt phm vi nht nh.
THC HNH
I.
1. Dng c
-
My o mu
Bp in
2. Ha cht
-
-Thuc th DNS
II.
Tin hnh
Gi nh
Vt ly nc cho vo bnh nh mc
Thm nc ct 100ml
ng1
ng2
ng3
ng ng5
ng6
ng7
4
Glucose 1mg/ml
0.2
0.4
0.6
0.8
Nc ct(ml)
0.8
0.6
0.4
0.2
Mu pha long(ml)
0.2
0.4
0.6
0.8
DNS (mol)
OD540nm
0.333
0.712
1.233
1.469
1.623 0.179
o bc sng 540nm
1,6
OD540nm
1,4
1,2
1
y = 1,7206x + 0,0347
R = 0,9756
0,8
0,6
0,4
0,2
0
0
0,2
0,4
0,6
C
0,8
1,2
10
Tnh kt qu:
T phng trnh th ng cong chun tnh c mg/ml ng kh trong dung dch
ng pha long.
Nhn vi h s pha long n c lng ng trong 1 ml dung dch gc
Chn h s pha long n sao cho OD nm trong gii hn ng chun
Da vo phng trnh ng tuyn tnh gia nng ng kh (mg/ml) vi mt
quang OD540 trn ta tnh c hm lng ng kh P trong mu phn tch X.
y = 1,7206x + 0,0347 x =
0,0347
1,7206
0,1790,0347
1,7206
= 0.084
Tnh hm lng ng trong nguyn liu (mg/g) = X * n * V/m = 0.084 * 25 * 100/20 = 10.5
11
CU HI N TP
Cu 1: ng kh l cc ng cha nhm aldehyde (-CHO) hoc ketone (-CO) nh
glucose , fructose ,arabinose , maltose , lactose (saccharose , trehalose khng phi ng
kh.
Cu 2: p dng phng php o ng kh trong trng hp phn tch nng
ng kh trong dung dch.
Cu 3 : Sai s h thng do nhng l do:
- Ha cht chun b cho th nghim khng c chun.
- Do vn chuyn mu khng ng cch
- Thao tc o khng chnh xc
- Ghi khng chnh xc ch s
- dng c cuvete khi o OD
- Ht ha cht khng chnh xc
Cu 4 : Khi o OD my bo over l do mu qu m c cn phi pha long li.
Ga tr OD nm trong khong t 1 n 5 .V nu cao qu hoc thp qu ng chun s
b lch.
Cu 5: Trong bi thc hnh chn ng glucose dng ng chun .iu ny
khng lun ng .v ng kh c nhiu loi nh fructose, maltose..
12
H S CHUYN I
6.25
5.30
5.46
5.71
5.3
5.3
5.3
6.25
5.95
5.83
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 2H20 + 2SO2 + O2
13
14
5ml
dd trong sut
Chng ct m
35ml
Chun H2SO4 d
Dd trc chun
Dd sau chun
15
CU HI N TP
Cu 1) Gii thch ngha cc bc trong th nghim
a) V c ha mu.
- Oxi to thnh trong phn ng li oxi ha cc nguyn t khc. Cc phn t cha
nito di tc dng ca H2SO4 to thnh NH3.NH3 kt hp vi H2SO4 d to thnh
(NH4)2SO4 tan trong dung dch.
2NH3 + H2SO4 (NH4)2SO4
b) Chng ct m:
- ui NH3 ra khi dung dch bng NaOH
- (NH4)2SO4 + 2NaOH 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
Cu 2. Tho lun cc yu t dn n sai s.
Ht khng chnh xc mu
Chun khng chnh xc
Sai s ca my chng ct
Cu 3. V sao phi bo m NaOH trong bnh chng ct m d?
V NaOH d th s chuyn tt c (NH4)2SO4 thnh NH3
Cu 4. Nu thay H2SO4 trong bnh hng bng acid boric (H3BO3) th kt qu c thay
i g khng?
Ta c: 0.1N H2SO4 = 0.05M H2SO4
Ta chuyn qua phng trnh v 1.42 l h s nito, c 1ml H2SO4 dng trung ha
NH4OH th tng ng vi 1.42mg nito. V ly 1ml nn nhn thm cho 10-3
2NH4OH
0.1*10-3
H2SO4
0.05*10-3
mN=0.1*10-3 * 14.2
16
3NH4OH
0.15*10-3*14.2
0.05*10-3
L thuyt
1. Nguyn tc
Phng php ny da trn s thay i bc sng hp thu cc i ca thuc nhum
Coomassie Brilliant Blue khi to phc hp vi protein. Trong dung dch mang tnh acid,
khi cha kt ni vi protein th thuc nhum c bc sng hp thu cc i 465 nm; khi
kt hp vi protein th thuc nhum hp thu cc i bc sng 595 nm. hp th
bc sng 595 nm c lin h mt cch trc tip ti nng protein.
xc nh protein trong mu, u tin ta xy dng mt ng chun vi dung
dch protein chun bit trc nng . Dung dch protein chun thng l bovine
serum albumin (BSA) . Sau khi cho dung dch protein vo dung dch thuc nhum, mu
s xut hin sau 2 pht v bn ti 1 gi. Tin hnh o dung dch bng my quang ph k
ta c ODx, hp th s t l vi lng trong mu. Thc hin mt i chng vi nc
ct (OD0). Ly gi tr OD = ODx - OD0. Lng protein mu trong dung dch o c xc
nh bng cch chm trn ng chun theo OD, ly gi tr ca im trn trc honh.
chnh l lng protein mu trong dung dch o.
17
THC HNH
1. Dng c - ha cht
1. Dng c
- My o quang ph
- ng nghim (14)
- Pipette 5ml, 1ml (c th thay bng pipetteman)
- ng h bm giy
- Giy thm
- Gi ng nghim
- Bnh tia ng cn
2. Ha cht
- Cn 900
- Dung dch protein chun: cn 10 mg albumin bng cn phn tch, pha trong 1 ml
nc ct, lc u cho tan. Gi -200C. Khi dng pha long ra 100 ln, c dung dch c
nng 0,1 mg/ml.
- Dung dch thuc th Bradford: dd thuc th c thnh phn trong 100ml nh sau:
Coomassie Brilliant Blue: 0,005g
18
Methanol: 4,7g
Phosphoric acid 85%: 8,5g
Phm mu Coomasie Brilliant Blue c lm tan trong ethanol trong mt chai
ng mu ti c np. B sung phosphoric acid v chnh ti 100ml bng nc ct. Lc
u, gi 40C
- Dung dch cn xc nh hm lng protein (phng th nghim cung cp)
2. Tin hnh
- Thu dch trch ly protein nh trong bi enzyme (5 g malt trch ly bng nc thu
c V = 100 ml). Pha long mu vi h s pha long n ln c mu phn tch (mu
X).
- Lp mt lot 6 ng theo s th t 0, 1, 2, 3, 4, 5 v 1 ng nghim cha mu cn
phn tch X
- Dng ng h bm giy, canh thi gian 0 pht cho 5ml thuc th vo ng nghim
0, lc u yn. thi im 1 pht cho 5ml thuc th vo ng nghim 1, lc u
yn,... c tip tc cho n ht.
19
ng nghim
Nc ct (ml)
0.9
0.9
0.7
0.6
0.5
0.1
0.2
0.3
0.4
0.5
TT Bradford
10
20
30
40
50
4.14
OD595nm
0,5
OD 595nm
0,4
0,3
y = 10,514x + 0,0015
R = 0,9675
0,2
0,1
0
0
10
20
30
40
50
C (g /ml)
60
20
= 10,514 + 0.0015 =
= 0,1035
100
10
= 1.035
CU HI N TP:
1. Gii thch ngha cc bc trong th nghim.
- Pha long mu vi h s pha long n ln c mu phn tch
Phi gim bt nng protein trong mu, khi o nng quang khng b vt
qu gii hn cho php.
- Lp mt lot 6 ng theo s th t 0, 1, 2, 3, 4, 5 v 1 ng nghim cha mu
cn phn tch X.
Phi pha cc nng khc nhau lp ng chun. xc nh protein trong
mu.
- Dng ng h bm giy, canh thi gian 0 pht cho 5ml thuc th vo ng
nghim 0, lc u yn. thi im 1 pht cho 5ml thuc th vo ng
nghim 1, lc u yn,... c tip tc cho n ht.
Phng php ny da trn s thay i bc sng hp thu cc i ca thuc nhum
Coomassie Brilliant Blue khi to phc hp vi protein.
- Tnh thi gian ng 0 c 20 pht, tin hnh o hp thu ca dung dch
bc sng 595 nm. Ta c gi tr OD0, thi im 21 pht o ng 1 (OD1),...
tng t c cch mt pht o cho ht cc ng. Ghi li gi tr OD
Trong 20 pht thuc nhum kt hp vi protein khi thuc nhum hp thu cc
i bc sng 595 nm.
21
22
L THUYT:
I.
a. nh ngha
Enzyme l cht xc tc sinh hc c bn cht l protein v c tnh c hiu cao. Mi
enzyme c kh nng xc tc cho mt hoc mt s phn ng nht nh. Hot ng hay
hot tnh ca enzyme cng mnh th lng c cht c chuyn ha hoc lng sn
phm to thnh trn mt n v thi gian cng ln. V vy c th nh gi hot tnh xc
tc ca enzyme bng cch xc nh tc chuyn ha c cht hoc tc tch ly sn
phm phn ng.
23
II.
Hot tnh ring ca mt ch phm enzyme c trng cho tinh sch ca ch phm
enzyme. Hot tnh ring c biu th bng s n v enzyme/mg protein ( U/mg
protein) hoc (Kat/kg protein), trong hm lng protein c xc dnh bi phng
php Lowry hoc Bradford.
III.
Ch :
-
Cc thng s nhit , pH, nng ion v thnh phn dung dch m nh hng
ln hot tnh enzyme. Th hot tnh enzyme phi c tin hnh trong iu kin thch
hp nh iu kin sinh l, k tn tr thc phm hoc k m hot lc c th t ti u.
-
24
Khi xc nh hot tnh phi lm mu i chng( enzyme b bt hot trc khi tip
IV.
ton 10 mg tinh bt sau thi gian phn ng 30 pht trong iu kin th nghim.
THC HNH:
1. Dng c - ha cht:
a. Dng c.
-
My lc.
B iu khin.
Bnh nh mc.
ng nghim.
ng h bm giy.
Giy thm.
Gi ng nghim.
b. Ha cht:
25
Dung dch m.
-
Dung dch m
Dung dch m acetate pH= 4,7 (xc nh hot tnh enzyme nm mc): trn 1 th
Dung dch m phosphate pH= 4,9 (xc nh hot tnh enzyme ca malt): trn 10
ml dung dch Na 2HPO 4 1/15 M vi 990 ml dung dch KH 2PO 4 1/15 M nhn c 1 l
dung dch m phosphate pH = 4,94. Kim tra pH.
-
amylase kim.
-
hn hp ha tan hon ton, sau chuyn dung dch sang b nh nh mc 1000 ml, b
sung nc ct n vch mc. Bo qun dung dch trong b nh mu nu ch ti.
-
Trch ly enzyme:
Enzyme c trch ly t ch phm hay t bo, m ng thc vt bng dd m c pH
thch hp nh
-
26
Th nghim
i chng
Mu trng
Dd Tinh bt 1%
2 ml
2 ml
Nc ct
2ml
1ml
1ml
1ml
Dd NaCl 3%
0,5ml
0,5ml
0,5ml
1ml
40 , 30 pht xy ra phn ng
HCl 1N
1ml
1ml
1ml
Dd enzyme
1ml
1ml
n vch
n vch
n vch
Iod
0,5ml
0,5ml
0,5ml
27
Tnh kt qu:
00 :
0: .
=
00 0
20
00
1,0720,106
20
1,072
= 18,022
18,022 50 100
=
= 901,1
)=
10
10 10
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 em trch ly, g
28
CU HI N TP
Cu 1: Phn bit cc dng chnh ca enzyme amylase v ngun thu chng.
C 3 dng chnh:
--amylase (Endo -1,4-glucanase; EC 3.2.1.1) c trong nc bt, ht ha tho ny
mm, ty tng, nm mc, vi khun
--amylase (Exo -1,4-glucanase; EC 3.2.1.2) c nhiu thc vt (ht c)
-Glucoamylase
Cu 2: Bn cht ca hot tnh enzyme l g? Nu cc phng php o hot tnh ca
enzyme
Bn cht: hot tnh ca enzyme cng mnh th lng c cht c chuyn ha hoc
lng sn phm to thnh trn mt n v thi gian cng ln.
Hai nhm phng php chnh sau:
- o lng c cht b mt i hay lng sn phm c to thnh trong mt thi gian
nht nh ng vi mt nng enzyme xc nh.
- o thi gian cn thit thu c mt lng bin thin nht nh ca c cht hay sn
phm tng ng vi mt nng enzyme xc nh.
Cu 3: C cht v sn phm ca enzyme -amylase l g? Da trn pp no ngi ta
chn pp xc nh hot tnh nh trong th nghim?
C cht ca enzyme -amylase l tinh bt. Enzyme ny phn gii lin kt 1,4glycoside gia chui polysaccharide (hot tnh endoamylase) to thnh maltose, dextrin
phn t thp.
Nguyn tc chung da trn c s thy phn tinh bt bi enzyme trong dung dch
enzyme nghin cu thnh cc dextrin c phn t lng khc nhau. o cng mu to
thnh gia tinh bt v cc sn phm thy phn ca n vi iode bng my so mu s tnh
c hot tnh enzyme.
Cu 4: C th s dng pp o ng kh bng DNS acid o hot tnh enzyme amylase khng? Ti sao?
29
L THUYT
NGUYN TC
Dng c .
Phu lc.
Giy lc .
30
Ethanol 96 %.
Acetone.
Ether du ha .
Cn 95%.
II.
TIN HNH.
Lc.
nh du cc vt sc t bng bt ch.
31
TNH KT QU
Rf=7,5/8
TR LI CU HI
Cu1 : phn cc ca cc dng sc t quang hp trong hnh 6.1 lcholorophyll A v
cholorophyll b c phn cc ln hn b-carotene , b-carotene c phn cc thp , cn
zeaxanthin c phn cc ln hn b-carotenen nhng nh hn cholorophyll A v
cholorophyll B
Cu2 :Phi dng ethan 95% trch ly sc t m khng dng nc v cc sc t ch tan
trong dung mi hu c , khng tan trongnc
Cu3 :Gi tr Rf ca sc t l 7,5/8 . Tn sc t c gi tr Rf = 7,5/8 l zeaxanthin