EXPERIMENT REPORT “Determining The Percentage of Blood Glucose” Submitted by DESIANA ANGGRAENI (12030194234) INTERNATIONAL CHEMISTRY EDUCATION 2012 THE STATE UNIVERSITY OF SURABAYA
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
-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
1ml of aquadest
-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
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
-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
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
-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
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