A.

Tittle of Experiment : Determination of Blood Glucose

B. Date of Experiment : Wednesday, 26th of September 2018
C. Purpose of Experiment : Determining the blood glucose level in blood
D. Basic Theories :
Monosaccharides
Monosaccharides are the simplest form of carbohydrates and may be
subcatagorized as aldoses or ketoses. The sugar is an aldose if it contains
an aldehyde functional group. A ketose signifies that the sugar contains
a ketonefunctional group.

Definition of Glucose

Glucose, a monosaccharide sugar, carbohydrates are used as the most

important primary energy source in the body. Glucose is a precursor for the
synthesis of all other carbohydrates in the body as glycogen, ribose and deoxiribose
in nucleic acids, galactose in lactose milk, in glycolipids, and in glycoproteins and
proteoglycans (Murray RK et al., 2003).

Glucose Metabolism
Glucose, fructose and galactose in through the intestinal wall into the
bloodstream. Fructose and galactose are converted in the body into glucose.
Glucose is the end result of digestion and absorbed as a whole as carbohydrates.
Blood glucose levels varied with the power of absorption, will be higher after a
meal and will be dropped if there is no food intake for several hours. Glycogen can
pass freely exit and enter the cell where glucose can be used solely as a source of
energy. Glucose is stored as glycogen in liver cells by insulin (a hormone secreted
by the pancreas). Glycogen is converted back into glucose by the action of glukogen
(another hormone secreted by the pancreas) and adrenaline is a hormone secreted
by the adrenal glands.
Hyperglycemia
Hyperglycemia is a condition where blood sugar levels soaring or excessive,
which eventually will become a disease called diabetes mellitus (DM) is a disorder
caused by the body lacks insulin hormone, consequently the glucose stays
circulating in the bloodstream and difficult to penetrate the wall cell. This situation
is usually caused by stress, infection, and the consumption of certain drugs.
Hyperglycemia is characterized by polyuria, polidipsi, and poliphagia, as well as
severe fatigue and blurred vision. (Nabyl, 2009).

Hypoglycemia
Hypoglycemia or decrease in blood sugar levels is a condition where blood
glucose levels are below normal, which may occur due to an imbalance between the
food you eat, physical activity and medications used. Hypoglycemia syndrome
characterized by clinical symptoms include patients feel dizzy, weak, trembling,
sight became blurred and dark, sweating, increased heart rate and sometimes to loss
of consciousness (shock hypoglycemia). (Nabyl, 2009).

How to Measure Blood Sugar Levels

There are three ways to measure blood sugar levels:

o Random blood sugar test

These tests measure the glucose in the blood is taken at any time, without regard
to meals.

o Fasting blood sugar test

This test using samples taken on an empty stomach, after we did not eat or drink
anything (except water) for at least eight hours.

o Glucose tolerance test

This test starts with a fasting blood sugar test. Then we are given a sweet drink
containing sugar with a certain size. Then the blood sugar level was measured using
several blood samples taken at specific time intervals.
2.1.5 Determining Blood Glucose
Blood glucose can be determined in many ways by chemical
or enzymatically. In general the method of determination of blood glucose can
determined by a variety of ways:
a. chemical methods
1.) The oxidation-reduction method
This method, serum proteins and compounds of non glucose reducing
precipitated for example by adding a solution of zinc chloride and barium
hydroxide. Furthermore, glucose is oxidized under alkaline conditions and with
heating using an oxide, such as copper (II) oxide produce a copper (I) oxide which
is proportional to the concentration of glucose. Copper (I) oxide produced will
reduce the acid solution of molybdate into arseno molybdate arseno blue, a colored
compound with the color intensity is proportional to blood glucose levels. The
principle of the determination reaction by using copper (II) oxide are:

Glucose + Cu 2+ a mixture of acids, sugars + Cu2O

Cu2O + acid molybdate + 4H + 2Cu2 + + arseno blue molybdate
2.) The method of condensing
In the condensation method, glucose is condensed with ortho-toluidine by heating
in glacial acetic acid to form glukosilamin and then form a Schiff base that has a
green color. Green Schiff base of the absorption is proportional to blood glucose
levels. The principle of the reaction is as follows:

D-glucose + ortho-toluidine glukosilamin Schiff

base (-H2O green)

b. enzymatic method
1.) glucose oxidase method
In the method of glucose oxidase, glucose in the presence of oxygen to be oxidized
by the enzyme glucose oxidase to form glucuronic acid and hydrogen peroxide.
Hydrogen peroxide will oxidize the chromogen formed catalyzed by peroxidase to
form a colored oxidized chromogen. The amount of colored product formed
according to blood glucose levels. The principle of the reaction is as follows:

Glucose + O2 + H2O → glucuronic acid + H2O2

H2O2 + chromogen → oxidized chromogen + H2O
Chromogen that is often used is ortho-toluidine are on blue. 2.) The method of
hexokinase

In the hexokinase method, the glucose in the presence of ATP by the enzyme
hexokinase difoforilasi produce glucose-6-phosphate and ADP. Furthermore,
glucose-6-phosphate and NADP by the enzyme glucose-6-phosphate
dehydrogenase is converted into 6-phosphogluconate and NADPH. NADPH
formed can be measured absorbance and is proportional to blood glucose levels.

Glucose + ATP → Glucose-6-phosphate + ADP

glucose-6-phosphate + NADP → 6-
phosphogluconate + NADPH

Specthtofotometry Uv-Vis

UV-Visible spectrophotometry is one of the most frequently employed

technique in pharmaceutical analysis. It involves measuring the amount of
ultraviolet or visible radiation absorbed by a substance in solution. Instrument
which measure the ratio, or function of ratio, of the intensity of two beams of light
in the U.V-Visible region are called Ultraviolet-Visible spectrophotometers. In
qualitative analysis, organic compounds can be identified by use of
spectrophotometer, if any recorded data is available, and quantitative
spectrophotometric analysis is used to ascertain the quantity of molecular species
absorbing the radiation. Spectrophotometric technique is simple, rapid, moderately
specific and applicable to small quantities of compounds.

The fundamental law that governs the quantitative spectrophotometric

analysis is the Beer -Lambert law. Beer’s law: It states that the intensity of a beam
of parallel monochromatic radiation decreases exponentially with the number of
absorbing molecules. In other words, absorbance is proportional to the
concentration. Lambert’s law: It states that the intensity of a beam of parallel
monochromatic radiation decreases exponentially as it passes through a medium of
homogeneous thickness. A combination of these two laws yields the Beer-Lambert
law. (Siladitya Behera,2012)
Blood Glucose Test

A blood sugar test is a procedure that measures the amount of sugar, or

glucose, in your blood. Your doctor may order this test to help diagnose diabetes.
People with diabetes can also use this test to manage their condition.

Blood sugar tests provide instant results and let you know the following:

 your diet or exercise routine needs to change

 how your diabetes medications or treatment is working

 if your blood sugar levels are high or low

 your overall treatment goals for diabetes are manageable

Your doctor may also order a blood sugar test as part of a routine checkup.
They may also be looking to see if you have diabetes or prediabetes, a condition
where your blood sugar levels are higher than normal.

Lambert Beer Law

The Beer-Lambert law relates the attenuation of light to the properties of the
material through which the light is traveling. This page takes a brief look at the
Beer-Lambert Law and explains the use of the terms absorbance and molar
absorptivity relating to UV-visible absorption spectrometry.

The Absorbance of a Solution

For each wavelength of light passing through the spectrometer, the intensity
of the light passing through the reference cell is measured. This is usually referred
to as IoIo - that's II for Intensity.
 Figure 1.11.1: Light absorbed by sample in a cuvetter

The intensity of the light passing through the sample cell is also measured for
that wavelength - given the symbol, II. If II is less than IoIo, then the sample has
absorbed some of the light (neglecting reflection of light off the cuvetter surface).
A simple bit of math is then done in the computer to convert this into something
called the absorbance of the sample - given the symbol, AA. The absorbance of a
transition depends on two external assumptions.

1. The absorbance is directly proportional to the concentration (cc) of the

solution of the the sample used in the experiment.
2. The absorbance is directly proportional to the length of the light path
(ll), which is equal to the width of the cuvette.

Assumption one relates the absorbance to concentration and can be expressed

as

A∝c(1.1)(1.1)A∝c

The absorbance (AA) is defined via the incident intensity IoIo and transmitted
intensity II by

A=log10(IoI)(1.2)(1.2)A=log10⁡(IoI)

Assumption two can be expressed as

A∝l(1.3)(1.3)A∝l

Combining Equations 1.11.1 and 1.31.3:

A∝cl(1.4)(1.4)A∝cl

This proportionality can be converted into an equality by including a

proportionality constant (ϵϵ).

A=ϵcl(1.5)(1.5)A=ϵcl
 This formula is the common form of the Beer-Lambert Law, although it can
be also written in terms of intensities:

A=log10(IoI)=ϵlc(1.6)(1.6)A=log10⁡(IoI)=ϵlc

The constant ϵϵ is called molar absorptivity or molar extinction coefficient

and is a measure of the probability of the electronic transition. On most of the
diagrams you will come across, the absorbance ranges from 0 to 1, but it can go
higher than that. An absorbance of 0 at some wavelength means that no light of that
particular wavelength has been absorbed. The intensities of the sample and
reference beam are both the same, so the ratio Io/IIo/I is 1 and the log10log10 of 1
is zero. (Jim, Clark 2017)

E. Tools and Materials

Tools:
Spectronic 20 Uv-Vis 1
Sentrifuge 1
Reaction Tube 8
Water Bath 1
Electric stove 1
Measuring glass 1
Champin glass 1
Materials:
Cu alkali Solution sufficiently
Ba(OH)2 solution sufficiently
ZnSO4 solution sufficiently
Arsenomolybdate sufficiently
(NH4)2SO4 sufficiently
1. Deproteinasi of blood filtrate

3 ml of aquadest + 2 drops Na-sitrat

- Entered into centrifuge tube

- Enetered 1 ml of blood stirred
- Added 1 ml of Ba()OH2 0,3N and stirred
- Added 2 ml of Zn (SO4)7.H2O 5% and stirred
- Added 2 ml of (NH4)2SO4 0,5N and stirred
- Let it stand for 15 minutes
- Sentrifuge for 15 minutes at speed of 3500 rpm
- Decanted

Residue Filtrate

- taken 1 ml then tested

by 3 drops of biuret

Free protein sample

2. Determinaton of blood glucose content

1 ml of free blood filtrate

- Entered into test tube

- Aadded 3 ml of cu alkalis reagent
- Pu in boiling point for 15 minutes
- Put in cold water for 10 minutes
- Added 2 drops of arsnomolibdat reagnt
- Stirred well
- Read the absorbance with a spectronic 20 at
weavelength 660nm

Absorbance
3. Making a standart curve

1 ml of glucose solution 0,1mg/ml ; 0,3mg/ml ; 0,5mg/ml ; 0,7mg/ml ; 0,9mg/ml ;

- Entered into test tube

- Aadded 3 ml of cu alkalis reagent
- Pu in boiling point for 15 minutes
- Put in cold water for 10 minutes
- Added 2 drops of arsnomolibdat reagnt
- Stirred well
- Read the absorbance with a spectronic 20 at
weavelenth 660nm

Absorbance

4. Blanko solution

1 ml of aquadest

- Entered into test tube

- Aadded 3 ml of cu alkalis reagent
- Pu in boiling point for 15 minutes
- Put in cold water for 10 minutes
- Added 2 drops of arsnomolibdat reagnt
- Stirred well
- Read the absorbance with a spectronic 20 at
weavelenth 660nm

Absorbance
No. Observation Result
Experiment Procedure Reaction Conclusion
Exp Before After
H OH
1. Deproteination of Blood Filtrate - Aquadest: - Aquadest+ C
Experiment 1:
colorless blood + based on the
H OH
- Blood: dark NaSitrate: light experiment,
red (++) red solution HO H O filtrate does
- Ba(OH)2: - +Ba(OH)2 : H OH not contain
white dark red H C
protein.
solution solution Indicated by
- Zn(SO)- - +ZnSO4.7H2O: CH OH
2 (aq) + Cu2+ (aq) + negative
OH
4.7H2O: dark red result from
O
C
colorless precipitate biuret test.
H OH
- Na.sitrate: - After stand for
dark red(+) 15 minutes: HO H

- (NH4)2SO4: dark red H OH

colorless - After
H C OH
centrifuge:
there are two 5OH- (aq)  CH OH +
2

layers colorless Cu2O (s) + 3H2O (l)

at the top and
dark red  2Cu2O (s) + [AsMo12O40]3-
precipitate at (aq)  Cu2+ (aq) +
the bottom [AsMo12O40]4 (aq)
 Determination of Blood Glucose Content - Protein free - Filtrate + Cu- The level of glucose based on Based on the
blood alkali: light theory is: 70-90 mg/100mL experiment,
filtrate: blue solution the level of
colorless - After boiled for glucose in
solution 15 minutes: blood is
- Cu alkali: green solution
2. blue - After colded in
solution water +
- Arseno arsenomolybda
Molybdate: te: green
light blue solution
solution - Absorbance

3. Making standard curve - Glucose - Nelson A+B: Experiment 1:

solution: pale blue Based on the
- Nelson A: solution experiment, it
colorless - 1 mL of can be
solution glucose + Cu conclude that
- Nelson B: alkali: pale the curve of
pale blue blue standard
- Arsenomol - 0.3 mg/ml of solution is
ybdate: glucose + Cu linear.
colorless alkali: pale
solution blue solution
- 0.5 mg/ml of
glucose + Cu
alkali: pale
blue solution
 - 0.7 mg/ml of
1 ml of glucose solution 0,1mg/ml ; glucose + Cu
0,3mg/ml ; 0,5mg/ml ; 0,7mg/ml ; alkali: pale
0,9mg/ml ; blue
- 0.9 mg/ml of
- Entered into glucose + Cu
test tube alkali: pale
- Added 3 ml of blue solution
cu alkalis - 0.3 mg/ml of
reagent glucose + Cu
alkali + heated:
- Put in boiling
orange+blue
point for 15
- + cooling
minutes water: form
- Put in cold precipitate
water for 10 - + arseno
minutes molybdate:
- Added 2 drops precipitate
of dilute
arsnomolibdat - 0.7 mg/ml
reagnt glucose + Cu
- Stirred well alkali + heated:
- Read the orange (+)
- + cooling
water:
Absorbance precipitate
- + arseno
molybdate:
precipitate
dilute
4. Blanco Solution - - 0.9 mg/ml + Experiment 4:
glucose + Cu based on the
alkali + heated: experiment, it
orange (++) can be
- + cooling conclude that
water: form blanco
precipitate absorbance
- + value is used
arsenomolybda to
te: precipitate comparasion.
dilute
- 0.1 mg/ml
glucose + Cu
alkali + heated:
orange-blue
- +cooling: form
precipitate
- +
arsenomolybda
te: precipitate
dilute
- A0.1: 0.48
- A0.3: 0.606
- A0.5: 0.809
- A0.7: 0.973
- A0.9: 1.443
- Ablanco: 1.514
H. Analysis and Explanation
Determining the blood glucose can be experimentally done by Cu alkali
test. As we know that glucose can react with Cu that can form precipitate of
glucose that indicated by the form of red precipitate. The principle is redox
reaction, where the glucose is oxidized by Cu alkali solution then the Cu alkali
will reduced by glucose. Testing of glucose contain in blood, firstly the protein
inside blood should be precipitate or filtrate by doing decantation and spinning
process. But, before that the blood should be added with several reagent like
Ba(OH)2, Zn(SO)4.7H2O, Na sitrate and (NH4)2SO4. All the reactant having their
own function to differentiate between glucose and protein inside blood.

The main material that be used in this experiment is blood. The reagents
that used are Ba(OH)2, Zn(SO)4.7H2O, Na sitrate, Cu alkali, arsenomolybdate
and (NH4)2SO4. The tools that used in this experiment are sentrifuge tube,
reaction tube, measuring glass, and spectronic 20. The sentrifuge is used to
separate between filtrate and residue of sample. While, spectonic 20 is used to
observe the absorbance data of sample and aquadest.

Firstly, the preparation of deproteination of blood. Blood color

is dark red color with thick concentration. The blood is taken as
much as 1 mL and added with Na Sitrate solution. After that the
solution become dark red solution. The function of adding the Na
citric solution is to prevent the blood precipitate or coagulate. The
blood become more soluble than before. Then, adding the several
reagents such as 1 ml of Ba(OH)2 0,3N and stirred, 2 ml of Zn
(SO4)7.H2O 5% and stirred, 2 ml of (NH4)2SO4 0,5N and stirred.
The mixture color become dark red solution (Figure H.1).
The second experiment is done to know the blood glucose
content. Firstly, 1 mL of free protein blood sample is entered into
test tube. Added with 3 mL of Cu alkali reagent. The addition of Cu alkali is used
Figure H.1 to oxidator of glucose. So the Cu2+ ion will reduce become Cu+
Blood after
Then boiled up for 15 minutes. Then, cool down for 10 minutes.
adding the
reagent Then, added 2 mL of arseno molybdate reagent. The boiling process
is used to denaturate the rest of protein and also cut the glucose bound. So, when
it’s react with Cu alkali, it will produce Cu-glucose complex. After that, measure
the absorbency data with spectrofotometry 20 at 660 nm wave length.
The third experiment is purposed to make the standard curve. Firstly
prepared 5 kinds of glucose solution with different composition. They are 1 ml
of glucose solution 0,1mg/ml ; 0,3mg/ml ; 0,5mg/ml ; 0,7mg/ml ; 0,9mg/ml. each
sample is entered into 5 different reaction tube. Then, each of tube is added with
Cu alkali solution to oxidate the glucose solution. Then, the solution is boiled for
15 minutes to cut the bond of glucose and also to accelerate the reaction process.
Then, after 15 minutes the solution is put into cool water for 10 minutes. After
that, added arsenomolybdate solution to activate the reaction with Cuprooxide.
Then, measure the absorbency data at 660 nm wavelength. The result of
measurements are:

Concentration Absorbency
0.1 0.48
0.3 0.606
0.5 0.809
0.7 0.973
0.9 1.443

The fourth experiment, is done to prepare blanco solution. The first thing to
do is 1 mL of aquadest is entered into test tube. Then, added with Cu alkali
solution into tube. Put into boiling water for 15 minutes. After that, cooled for
10 minutes. The boiling process is used to accelerate the reaction aquadest and
Cu alkali. Then, added with arseno molybdate to react cuprooxide. The solution
then readed the absorbancy at 660 nm with spectronic 20. The result of
measurement is
Na sitrate solution is used to make the blood oxalated. Because, if the blood
is stay opened in open air without given any, the blood be denaturated, and the
blood will precipitate and difficult to do the experiment. Ba(OH)2 is used to
created base condition of blood. So the protein will be denaturized because of
base condition. As we know that one of the factors that can cause the
denaturation of protein is acid or base condition. Zn(SO)4 is used to catalyst of
 deproteination of blood. (NH4)2SO4 is used to be the salt that will bound the
protein. The sentrifuge process is used to separate the glucose and protein of
blood. So it will form red precipitate that contain protein and another mineral
inside blood. The sentrifuge process is the process of spinning the blood tube so
it will separate the blood into filtrate and residue.
The work principle of spectrofotometri Uv-Vis is firstly, the light is shooted
into the monochromator that contain dispersion glass that will pass through the
monochromator and then pass through the sample and cached by screen inside
spectrophotometry and readed by the tools the absorbency data. The process of
standardization is firstly diluted 10 mL of sample then, taken 1 mL of it then,
diluted become 0.7 mL.
Based on the experiment, the level of glucose is 106.86 mg/100mL. This
result contains into hyperglycemia. Because the normal level of glucose inside
blood is 70-90 mg/100mL. this condition can be influenced by the activity of
volunteer that lack of sport activity. So, the glucose can not be converted into
energy to body. Beside that, another factor that influencing the glucose level is
the ability of heart to change glucose over number into glucagon. Another factor
is the food that consumed by volunteer maybe contain high glucose level.
I. Conclusion
The indicator of blood that free of protein is it has no result when tested by
biuret solution. Usually if the protein tested by biuret it will change become blue
solution.
Based on the experiment result, the level of glucose is 106.86 mg/100 mL.
it is included into hyperglycemia that caused by several factors. Maybe the main
factor is food that consumed by volunteer. But another factor are the lack of sport
activity, stress, hormone working and so on.

J. Refferences

Jim Clark. 2017. The Absorbance of a Solution. Cornwall: Chemistry School.

Lehninger al. 1982. Fundamentals of biochemistry vol 1. Suhartono mt,

translator.Jakarta: Erlangga.
Matthews. K. 2012. Biochemistry. Oregon: Oregon University.
 MaryAnn DePietro. 2018. Blood Sugar Test. Online: healtline.com .

Murray and robert K., et al. 2003. Biochemical harper. Jakarta: medical
book publishers EGC.
Siladitya Behera; Subhajit Ghanty; AF. UV-Visible Spectrophotometric Method
Development and Validation of Assay of Paracetamol Tablet Formulation.
West Bengal: Journal of Analytical and Bioanalytical Technique.

K. Answer of Qustions

1. the level of glucose inside blood is 106.85 mg/100mL

2. To accelerate the reaction process and to cut the glucose bound.

3. Insulin roles is used to change the over number of glucose into glucagon and
saved inside heart.

M. Calculation

ATTACHEMNT

1. CALCULATION
Known :
- Solution = 2 mg/mL
Ask :
- Volume for solution ?
Answer :
- For 0.9 mg/ml
M1.V1 = M2.V2
0.9 mg/ml . 10 ml = 2 mg/ml . V2
9 ml = 2V2
V2 = 4.2 ml
- For 0.7 mg/ml
M1.V1 = M2.V2
0.7 mg/ml . 10 ml = 0.9 mg/ml . V2
7 ml = 0.9V2
V2 = 7.77 ml
- For 0.5 mg/ml
M1.V1 = M2.V2
0.5 mg/ml . 10 ml = 0.7 mg/ml . V2
5 ml = 0.7 V2
V2 = 7.1 ml
- For 0.3 mg/ml
M1.V1 = M2.V2
0.3 mg/ml . 10 ml = 0.5 mg/ml . V2
3 ml = 0.5V2
V2 = 6 ml
- For 0.1 mg/ml
M1.V1 = M2.V2
0.1 mg/ml . 10 ml = 0.3 mg/ml . V2
9 ml = 0.3V2
V2 = 3.33 ml
- Calculation the blood glucose
y = 1.1465x + 0.2889
1.514= 1.1465x + 0.2889

1.1465x = 1.514 – 0.2889

1.1465x = 1.2251

X = 1.2251/1.1465

X = 1.0685 mg/ml

= 106.85 mg/100 ml
L. Documentary

Figure L.2 Apparatus to

Figure L.1 Materials to do the experiment Figure L.3 The blood
do experiment after adding the Na
Citric

Figure L.5 Aquades as

Figure L.4 The blood stabilizer of sentrifuge Figure L.6 The
before sentrifuge process sentrifuge tool

Figure L.9 Glucose test

Figure L.7 Blood after Figure L.8 Cu alkali before boiling process
sentrifuge process solution

Figure L.11 the result of

boiling proces
 Figure L.10 boiling Figure L.12 Aquadest
process of sample solution as blanco

Figure L.13. the addition

Figure L.14 the addition Figure L.15 the process
of Nelson A solution
of Nelson B solution of absorbency
measurement