Welcome to Scribd, the world's digital library. Read, publish, and share books and documents. See more
Download
Standard view
Full view
of .
Look up keyword
Like this
95Activity
0 of .
Results for:
No results containing your search query
P. 1
Estimation of blood glucose using glucose oxidase (Biomedical practical for biochemistry II)

Estimation of blood glucose using glucose oxidase (Biomedical practical for biochemistry II)

Ratings:

4.38

(8)
|Views: 18,968 |Likes:
Published by kiedd_04
enzymatic method yields maximum specificity for glucose estimation
enzymatic method yields maximum specificity for glucose estimation

More info:

Published by: kiedd_04 on Feb 01, 2008
Copyright:Attribution Non-commercial

Availability:

Read on Scribd mobile: iPhone, iPad and Android.
download as DOC, PDF, TXT or read online from Scribd
See more
See less

03/25/2013

pdf

text

original

 
TITLE:
Estimation of Blood Glucose Using Glucose Oxidase
AIMS AND OBJECTIVES:
To understand the importance of measuring blood glucose level.
To understand the principles of enzymatic estimation of glucose.
INTRODUCTION
Enzymatic method yields maximum specificity for glucose estimation. Glucose can bemeasured by its reaction with glucose oxidase, in which gluconic acid and hydrogen peroxideare formed. Hydrogen peroxide than reacts with an oxygen acceptor, such as ortho-dianisidine,phenylamine-phenazone or any other chromogenic oxygen acceptors, in a reaction catalysed byperoxidase to form a colour.One of the advantages of enzymatic method is its specificity. This enables the estimationof glucose even in a complex mixture. Thus, this method is widely used in the field ofclinical chemistry and for food analysis. In clinical chemistry, the enzymatic analysis ofglucose in blood and urine has been modified as dipstick test. However in the presentpractical, the conventional enzymatic method shall be used.
METHOD:
1.SAMPLES TEST:
a.
The following substances are added in the tube.
i.
1.8 ml sodium sulphate-zink sulphate
ii.
0.1 ml bloodiii.0.1 ml NaOH (0.1M)b.Mix carefully.
c.
Centrifuge at 3000 RPM for 5 min.
d.
Transfer the supernatant into new test tube.
Note: The glucose concentration in the supernatant is 1/20 of the concentration in the bloodsample.
1
 
2.
BLANK & STANDARD:
a.
Glucose standards 12 mg/dl are provided.
 b.
Preparing concentration glucose standard point.
TUBESCONCENTRATION(mg/dL)GLUCOSESTANDARD (mL) WATER (mL)TOTAL mLPER TUBE
13.0M
1
V
1
: M
2
V
2
(12)V
1
: 3.0 (2.0 )V
1
: 0.5 mLH
2
O = 2.0 ml -0.5 mL= 1.5 mL2.026.0M
1
V
1
: M
2
V
2
(12)V
1
: 6.0 (2.0 )V
1
: 1.0 mLH
2
O = 2.0 ml –1.0 mL= 1.0 mL2.039.0M
1
V
1
: M
2
V
2
(12)V
1
: 9.0 (2.0 )V
1
: 1.5 mLH
2
O = 2.0 ml –1.5 mL= 0.5 mL2.0412.0M
1
V
1
: M
2
V
2
(12)V
1
: 12.0 (2.0 )V
1
: 2.0 mLH
2
O = 2.0 ml –2.0 mL= 0 mL2.0
BLANK-
-2.0 mL2.0
c.
Then, add 5 ml ortho-tolidine mixture where consisting;
i.
1%
 
ortho-tolidineii.4mg (100 units) peroxidase
iii.
12.5 mg (500 units) glucose oxidase in 100
ml
phosphate buffer, pH 7.0.
TUBESMINUTE ADDEDORTHO-TOLIDINEORTHO-TOLIDINE mLTOTAL mL PER TUBEBLANK
25.07.0145.07.0265.07.0385.07.0
4
105.07.0
SAMPLE
125.07.0iv.Mix quickly.
v.
The time is staggered every 2 minutes when adding the ortho-tolidine solutionas shown table above.
vi.
Incubate the tubes at room temperature for exactly 10 min.
vii.
Then, measure at absorbance 625nm.Notes:
2
 
Ensure that each tubes absorbance is read exactly after 10 min.
If the absorbance reading for the sample is too high, dilute thesupernatant which was obtained earlier, (2x) with water and repeat thesubsequent steps.
RESULTS:GLUCOSE CONCENTRATION (mg/dL)ABSORBANCE READING (nm)
00.0003.00.1816.00.1209.00.00612.00.005SAMPLE0.045
DISCUSSION:
On this experiment, blood glucose is estimated by using enzymes glucose oxidase.Where Ortho-toluidine is use as chemical for exploited to quantitative carbohydrates molecules to formSchiff bases with aromatic amines. Ortho-tolidine in a hot acidic solution will yield a colored compoundwith absorbance maxima at 625 nm.
GLUCOSE OXIDASEGlucose + O
2
+ H
2
O
Glucose Oxidase
Gluconic Acid + H
2
O
2
H
2
O
2
+ Reduced chromogen
Peroxidase
Oxidized chromogen + H
2
OGlucose oxidase is the most specific enzyme reacting with only β–D-glucose and glucoseoxidase converts β–D-glucose to gluconic acid. Added Mutarose to the reaction can facilitate theconversion of α– D-glucose to β–D-Glucose. Oxygen is consumed and hydrogen peroxide isproduced. The reaction is measured based on rate of disappearance of oxygen. Spectrophotometer isused for read absorbance, where it proportional to the amount of glucose presents in the sample.However, on this experiment, the absorbance reading for concentration reading is not expectedas well and it fully incorrect. Because the absorbance reading supposedly from the lower thru highvalue. But, on another hand for comparison with other group shown that the results reading arecorrect and accordingly as table below.
GLUCOSE CONCENTRATION (mg/dL)ABSORBANCE READING (nm)
00.0003.00.0566.00.1609.00.22512.00.225The problems that can be come across are shown in table below;
3

Activity (95)

You've already reviewed this. Edit your review.
1 hundred reads
1 thousand reads
Monir Chowdhury added this note
Thanks for this topics.
Anil Jha added this note
Theoriticaly this method is fine but practically this method has not give perfect result, due to chemicals preparation is not given briefly and time duration also not given in this methods, Overall this method is fine.
nice i luv t thank u so much..................
Sanchit Saluja liked this
Zhi Yan liked this
Mariah Lee liked this

You're Reading a Free Preview

Download
scribd
/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->