glucose blood

EXPERIMENT REPORT

“Determining The Percentage of Blood Glucose”

Submitted by

DESIANA ANGGRAENI (12030194234)

INTERNATIONAL CHEMISTRY EDUCATION 2012

THE STATE UNIVERSITY OF SURABAYA

FACULTY OF MATHEMATICS AND NATURAL SCIENCES

DEPARTMENT OF CHEMISTRY

2014

I. TITLE OF EXPERIMENT:Determining The Percentage of Blood Glucose

II. DAY/DATE START OF EXPERIMENT:Friday, October 24th 2014

III. DAY/DATE FINISH OF EXPERIMENT:Friday, October 24th 2014

IV. PURPOSE:To DetermineThe Percentage of Blood Glucose

V. BASIC THEORY:Glucose

Glucose is a simple carbohydrate that is the brain's principal source of energy.

Our bodies obtain glucose from the carbohydrates in plant products. Table sugar, for

example, is a disaccharide consisting of glucose and fructose. Starch is a mixture of

amylose, a linear polysaccharide, and amylopectin, a highly branched polysaccharide.

Starch is broken down into glucose by the enzymes in saliva and the small intestine.

This is why eating white bread or potatoes, which are rich in starch, raises the blood

sugar very quickly.

Normal Blood Sugar

The concentration of blood sugar (glucose) changes throughout the day based

on what we eat and our activity level. The normal concentration of blood sugar should

be between 70 to 130 milligrams per deciliter (mg/dL) before meals, and less than 180

mg/dL after meals, according to the National Institutes of Health. This corresponds to

3.9 to 7.2 millimoles per liter (mmol/L) before meals and less than 10 mmol/L after

meals. The Fasting Blood Sugar (FBS) is normally tested in the morning after having

abstained from food for at least 8 hours. This test is done to check for prediabetes and

diabetes. A healthy person will have FBS between 70 and 100 mg/dL because the

values can vary depending on physical activity and the method used for testing.

Laboratory testing generally uses blood drawn from a vein, whereas home testing uses

blood obtained by pricking a finger.

Energy from glucose is obtained from the oxidation reaction

C6H12O6 + 6O2 → 6CO2 + 6H2O

where a mole of glucose (about 180 grams) reacts with six moles of O2 with an energy

yield ΔG = 2870 kJ. The six moles of oxygen at STP would occupy 6 x 22.4L = 134

liters. The energy yield from glucose is often stated as the yield per liter of oxygen,

which would be 5.1 kcal per liter or 21.4 kJ per liter. This energy yield could be

measured by actually burning the glucose and measuring the energy liberated in a

calorimeter.

Estimation of Blood GlucoseThe importance of testing the blood glucose level comes from the fact that the

brain cells are very dependent on the extracellular glucose concentration for their

energy supply; hypoglycemia is likely to impair cerebral functions as well as do the

hyperglycemia especially of rapid onset, which can cause cerebral dysfunction by

affecting extracellular osmolarity.

Method:

Many methods were developed to estimate the glucose level in body fluids among

which the commonly used nowadays, the enzymatic methods. These methods can be

summarized and categorized into

A) Reduction methods : These methods depend on the reductive property of

glucose(aldose)

1-Ferriccyanide( Hoffman’s) method: using ferricyanide which is reduced by the

glucose .

Fe+++ Fe++ (color change from yellow to colorless solution that will

diminish the absorbance measured photometerically )

2-Copper sulfate methods :

Benedict: The reagent contains Na-citrate &Na carbonate with CuSO4.

It gives

color acc. To conc. of glucose (green-----yellow-----brown-----red).

Fehling : using KOH &Na/K tartrate with CuSO4

Folin- Wu : Alkaline Cu SO4 +Phosphomolybdic acid molybdenum blue

by reducing Cu2O CuO2

3-Smogi-Nelson method : using Arsenomolybdate

N.B. The reduction methods need alkaline medium &heat . These methods are

qualitative & semi-quantitative.

B) Aromatic amines method :

O-toludine +glucose (aldhyde) heat &acidity glucosamine (colored )

C) Enzymatic methods:

1-Hexokinase methods(The reference method).

With pre-deproteinization of sample or without.

Glucose +ATP +HKADP+G6P

G6P +NAD +G6PD 6 P-gluconolactone +NADH+H

(measured at 340)

Spectrophotometric Measurement of Glucose

The spectrophotometer measures absorbance. Absorbance values, by

themselves, do not describe the concentration of a substance. However, we can

determine the concentration of a substance in a solution using a standard curve. A

standard curve translates absorbance values into concentration. For an example. We

can construct a standard curve by making solutions with a known concentration of the

substance we are measuring and then measuring their absorbance. Graphing the

concentration on the x-axis and the absorbance on the y-axis, we can see that there is

a linear relationship between concentration and absorbance. Thus a standard curve is

not really a curve, but a straight line. Beers Law describes this linear relationship:

Using the standard curve, we can determine the concentration of other

solutions, by locating the absorbance of that solution on the y-axis and drawing a

horizontal line to the standard curve line. Then you can draw a vertical line from that

intersection to the x-axis to determine the concentration.

VI. TOOLS AND MATERIALS:

Tools Materials Spectronic-20 Cu alkalis solution Centrifuge ZnSO4.7H2O 5% solution Test tube Ba(OH)2 0.3 N solution Water bath Glucose standard primary 0.01 mg/L Measured glass Arsenmolibdat reagent Cup glass Sample blood

2 drops of blood “oxalated”

-Added 1.9ml of aquadest-Centrifuged-Added 1.5ml Ba(OH)2 0.3N-Stirred-Added 1.5ml ZnSO4.7H2O 5%-Let It for 5 minutes-Centrifuge for 10 minutes

Residue Filtrate

-put in tube 1ml filtrateAdded 3-5 drops of Biuret

Filtrate of free protein blood

1 ml of filtrate of free protein blood

-Added into test tube-Added 1ml of Cu alkalis reagent-Evaporated into water bath for 20 minutes-Cooled with tap water-Stirred-Added 1ml of Arsenomolibdat reagent-Absorbed by spec-20 with wavelength 660nm

Absorbance value

VII. PROCEDURE1. Deproteination of blood filtrate

2. Determining the percentage of blood glucose

0.01 mg/ml 0.02 mg/ml 0.03 mg/ml 0.04 mg/ml 0.05 mg/ml


Absorbance value

1ml of aquadest


Absorbance value

3. Standard curve preparation

4. Blanco solution

2 drops of blood “oxalated”

-Added 1.9ml of aquadest-Added 1.5ml Ba(OH)2 0.3N-Stirred-Added 1.5ml ZnSO4.7H2O 5%-Let It for 5 minutes-Centrifuge for 10 minutes

Residue Filtrate

-put in tube 1ml filtrateAdded 3-5 drops of Biuret

Filtrate of free protein blood

VIII. RESULT OF EXPERIMENT

No. Procedure Observation Hypothesis/Reaction Conclusion1. Deproteination of blood filtrate Before:

Desi blood=Red +++Jannah blood=Red ++++Biuret=colorlessBa(OH)2=colorlessZnSO4.7H2O=colorlessAfter :-Added aquadestDesi blood=red ++Jannah blood=red ++++-Added Ba(OH)2 0.3N:Desi blood=red +Jannah blood=red +++All of sample form ppt-Added ZnSO4.7H2O:Desi blood=red+Jannnah blood=+++All of sample form ppt-cebtrifuged:2 layers Upper layer=filtrate, (colorless)Lower layer=residue coagulated blood (red)-Filtrate+BiuretDesi blood=colorlessJannah blood=colorless

Blood containe glucose and protein

Biuret test to showing there aren’t protein by colourless solution

Blood filtrate free from protein, its shown by colourless solution

1 ml of filtrate of free protein blood


Absorbance value

2. Determining the percentage of blood glucose Before:Blood filtrate=colourlessCu alkalis=blueArenomolibdat=yellowAfter-Added Cu alkalis=Desi blood filtrate=turbid (++) in blue solution Jannah blood filtrate= turbid (+) in blue solution-boiled waterDesiana blood filtrate=blue and form precipitateJannah blood filtrate=green and form precipitate-Added aresomolibdat=Desi blood filtrate=light green + appears bubblesJannah blood filtrate=dark green and appears bubbles

Glucose will reduce Cu2+ ion in base condition, result of the reduction will oxidation reaction by arsenomolybdate resulting blue color. Blue color that will be measured to determine the absorbancex

Desi blood :Conc=0.066WL 660nm=0.331Absorbance=-0.075The percentage of blood glucose = 0.066Jannah blood:Conc=0.596WL 660nm=2.609Absorbance=2.203The percentage of blood glucose= 0.598

3. Standard curve preparation BeforeGlucose filtrate= colorlessCu alkalis=blueArenomolibdat=yellowAfter-diluted all concentration=colorless-Added Cu alkalis= light blue-Boiled water= light blue-Added arsenomolibdat= green and appears bubbles

When the concentration of solution is bigger, the absorbance will be bigger too, so curve will increase linearly

Std 1= 0.097 (conc 0.01)Std 2=0.121 (conc 0.02)Std 3=0.181 (conc 0.03)Std 4=0.215 (concb0.04)Std 5=0.264 (conc 0.05)

AbsorbanceA1= -0.309A2=-0.285A3=-0.225A4=-0.191A5=-0.142

y=4.28x-0.358R2=0.987

0.01 mg/ml

0.02 mg/ml

0.03 mg/ml

0.04 mg/ml

Absorbance

0.05 mg/ml

1ml of aquadest


Absorbance value

4. Blanco solution Beforeaquadest= colorlessCu alkalis=blueArenomolibdat=yellowAfter-Added Cu alkaline= light blue-Bolied= light blue-Added Arsenomolibdat= green ++ and appears bubbles from bottom tube

Blanco solution is to ensure that the standard solutions

Absorbance Blanco =0.406

IX. DATA ANALYSIS

1. Deproteination of blood filtrate

First, preparation of sample blood. In our experiment we used two samples,

they are Desi blood and Jannah blood. Each of samples placed in centrifuge

tube, and dissolved it with water and stirred to make it soluble, in desi blood

the color is red +++ and jannah blood is red ++++. After the samples of blood

was diluted, the samples mixed with Ba(OH)2, this adding to give base

condition and also to precipitate iron ion in haemoglobin, this ions changes to

Fe(OH)2 molecule as red precipitate, the changes of desi blood sample are red

+ color and there are red ppt while jannah blood sample are red +++ color and

there are red ppt. And then the adding ZnSO4 after Ba(OH)2, Protein is

removed as Zinc proteinate, Sulphydryl compounds as Zinc salts and the

remaining zinc and barium ions as Zinc hydroxide and Barium sulphate.

ZnSO4 + Ba(OH)2 Zn(OH)2 + BaSO4

helps to precipitate it and also denaturation the protein perfectly, but because

of the density of protein is higher than glucose, the samples that will be

analyzed have to centrifuge to separate protein from glucose, cause protein can

disturb the measuring of blood glucose percentage. The samples after

centrifuged, desi blood sample from 2 layers, at upper layer(filtrate) is

colorless and lower layer(residue) is coagulated blood(red) and jannah blood

sample also form 2 layers too, at upper layer(filtrate) is colorless and lower

layer(residue) is coagulated blood(red) . The filtrate is taken into different tube

and added 3 drops of biuret reagent. Biuret reagent to identify protein, if the

filtrate contain protein, the filtrate solution will change to blue solution, but in

our samples are still colourless, it indicates that our filtrate sample free of

protein.

2. Determining the percentage of blood glucose

1 ml of filtrate of free protein blood is added into test tube, 1ml of Cu

alkalis reagent is added into the filtrate, desi blood filtrate become turbid (++)

in blue solution and jannah blood filtrate turbid(+) in blue solution. In hot

alkaline solution, glucose reduces cupric ion to cuprous ion with formation of cuprous oxide. Desi blood filtrate is blue and form precipitate and jannah blood is green and form precipitate.

The reaction :

Cu +2 + glucose Cu2O + oxidation products of glucose

Added Phosphomolybdic (or Arsenomolybdic) acid (MO+6) is reduced by the

cuprous ion to form compounds with lower oxidation states of molybdenum,

which have a blue color and suitable for photometric measurement, but i our

samples are green.

Continued with the absorbance with specnometer-20 wavelength 660nm. This

measurement show that the absorbance of desi blood is 0.075 and jannah

blood is 2.205

3. Standard curve preparation

In objective of this step experiment to make a curve that have regression as

determining the percentage of blood glucose. To begin of this experiment, we

have to prepare the standard solution by continues diluting, that the first from

0.01 mg/ml to 0.02 mg/ml, 0.03 mg/ml, 0.04 mg/ml and 0.05 mg/ml by

formula :

M1.V1=M2.V2.

In each of standard solution are added 1 ml of alkaline copper reagent and

evaporated for 20 minutes, each of tube are resulting light blue solution. The

adding of alkaline Cu in each tube caused Cu2+ will be reducted by Cu+ in base

condition, and after the evaporation, the standard solution still light blue. This

function of evaporation to increase reaction rate of alkaline Cu. Continue

before by adding arsenomolybdate, we have to stabilize by cooling the

standard solution. The change after added by arsenomolybdate is green and

appreas bubbles. The function of adding arsenomolybdate to redissolve Cu2O.

Continued with the absorbance with specnometer-20 wavelength 660nm. This

measurement show that

Std 1= 0.097 (conc 0.01) A1= -0.309

Std 2=0.121 (conc 0.02) A2=-0.285

Std 3=0.181 (conc 0.03) A3=-0.225

Std 4=0.215 (concb0.04) A4=-0.191

Std 5=0.264 (conc 0.05) A5=-0.142

So we can make the curve, and we got the equation y=4.28x-0.358 R2=0.987

0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05 0.055

-0.35

-0.25

-0.15

-0.05

f(x) = 4.28000000000002 x − 0.358800000000001R² = 0.987238078813487

Absorbance Vs Concentration

Concentration

Abso

rban

ce

And from the curve we can calculate the percentage blood glucose of sample, by imagine “y” as absorbance of sample, so we got the percentage of blood glucose in desi blood = 0.066 mg/ml and The percentage of blood glucose in jannah blood = 0.598 mg/ml.

4. Blanco solution

In preparation of blanco solution to indentify zero point of standard solution,

so we can use it as standard comparison of sample that will be tested. Blanco

solution preparation we can make by 1 ml of aquadest reacted with alkaline

solution and then cooled it. The solution is light blue, and evaporated it for 20

minutes, the solution still light blue and after that we cooled it for about 5

minutes and added arsenomolybdate, the solution change to green color and

appears bubbles from the bottom tube. Continued by measured the absorbance

with spec-20 wavelenght 660nm, Absorbance Blanco is 0.406.

X. DISCUSION

In our experiment we find some error, its can see from the normal percentage of glucose. The normal concentration of blood sugar should be between 70 to 130 milligrams per deciliter (mg/dL) before meals, and less than 180 mg/dL after meals, according to the National Institutes of Health, and in our experiment far from that data. From the The absorbance of desi sample is minus value, this is caused the blood that we got just 1 drop and the preparation of standard solution, the concentration is too low. This concentration make the calculation of volume is decimal value, like concentration 0.03 mg/ml we got volume 33.33 ml. So when we take the solution with 33.33 ml, we can’t make the exact measurement of the solution, and as the theoretically if the standard preparation there are mistake on the dilution, it can make error for another experiment. Also, from blanco solution too green for normal blanco, cause of measurement when addition of alkaline copper.

XI. CONLUSION

From the data experiment that we have

1. Blood filtrate free from protein, its shown by colourless solution.

2. Preparation of standard solution resulting equation y=4.28x-0.358, R2=0.987

3. The percentage of blood glucose in Desi blood = 0.066 mg/ml and The

percentage of blood glucose in Jannah blood = 0.598 mg/ml.

XII. QUESTIONS AND ANSWERS1. Determine the percentage of blood glucose/100ml blood!

Answer:From the regression that we have got

y=4.28x-0.358 we imagine that “y” is absorbance of sample, soThe percentage of desi blood glucose :Yd =4.28x-0.358-0.075 = 4.28x-0.358x =0.066The percentage of jannah blood glucose :Yj =4.28x-0.3582.203 = 4.28x-0.358x =0.598

2. What the function the boiling process?Answer: To speed up the rate of reaction by alkaline Cu

3. Explain what the role of insulin in the regulation of glucose levels?Answer:Insulin is a hormone produced by beta cells in a part of the pancreas known as the islets of Langerhans. Glucose is the fuel that provides energy for cells throughout our body. Insulin controls how much glucose the liver produces and also helps to move glucose from the bloodstream into our cells, where it is needed as a source of energy.

REFERENCES

FDA.2014.Blood Glucose Monitoring Devices.U.S:Silver Spring

Macvol.2014. Spectrophotometric Measurement of Glucose. http://employees.csbsju.edu/mcampos/bio114/labmaterials/lab.2.writeup.03.pdf (acessed on 30/10/2014)

Nelson, David L.1982. Lehninger Principles of Biochemistry, Fourth Edition.Jakarta:Erlangga.

Tim Dosen.2014.Petunjuk Praktikum Biokimia.Surabaya:Unesa

Somogyi,Michael.1945.Determination of Blood Sugar.St.Louis:American Society For Biochemistry

ATTACHMENT

No. Procedure Picture1. Deportenination of glucose filtrate

Taken 0.1 ml of sample A and B blood “oxalated”

0.1 ml of blood + 1.9 ml of aquadest

0.1 ml of blood + 1.9 ml of aquadest + 1.5 ml of Ba(OH)2

0.1 ml of blood A and B+ 1.9 ml of aquadest + 1.5 ml of Ba(OH)2 + 1.5 ml of ZnSO4.7H2O

Centrifuged

Separated filtrate from the residue

Fitrate ResidueTested with biuret

2. Determining the percentage of Blood Glucose A and B Blood filtrate free of protein

Blood filtrate + Cu Alkalis

Heated and cooled

Heated Cooled+ 1 ml arsenomolibdat

3. Preparation of Standard Curve

1 ml of glucose with concentration 0.01mg/ml ; 0.02 mg/ml ; 0.03 mg/ml ; 0.04 mg/ml ; 0.05 mg/ml

Added by Cu alkalis

Heated and cooled

Added by 1 ml of arsenomolibdat reagent

4. Blanco Solution

1 ml of aquadest

1 ml of aquadest + 1 ml of Cu alkalis

Heated and cooled

glucose blood

Documents