I.

II.
III.
IV.

Title of Experiment
: Determination of blood glucose levels
Date before Experiment
: Monday, September 26, 2016
Date after Experiment
: Monday, September 26, 2016
Objective Experimenting :
To determine the blood glucose levels
V. Basic Theory :
a. Glucose
Blood glucose is a monosaccharide carbohydrates in the form contained in
the blood. Organs that influence the glucose metabolism including liver and
pancreas. Blood glucose are in balance and regulate hormonal teroid the
hormone, insulin, the hormone efineprin and growth hormones (Ganong, 1990).
Beside that, glucose which is one of the most important carbohydrates are used as
a source of energy for animals and plants. Glucose is one of the main results of
photosynthesis and respiration early for. Natural form (D-glucose) is also called
dextrose. Glucose is a aldohexoses and is often called dextrose because it has the
properties can rotate polarized light to the right.
Sugar contained in two enantiomers (mirror isomers), D-glucose and Lglucose, but in organisms, which are found only D-isomer.Suatu isomer D or L
shaped carbohydrates associated with conformational isomeric on carbon 5. If
you're in the right projection Fischer , then the shape of the ring is the enantiomer
D, if to the left, then it becomes enantiomer L. Very catchy, referring to D for
"dextro", which is the Latin root for "right", while L for "levo" which is the root
word " left ". the structure of the ring itself can be formed through two different
ways, which produces glucose- (alpha) and (beta). Structurally, glucose- and
- different in hydroxyl groups attached to the first carbon on cincinnya.Bentuk
has a hydroxyl group "below" the hydrogen (as the molecule is commonly
described, as shown in the picture above), while the form of hydroxyl group is
"above" the hydrogen. the two forms are formed alternately at all times in a
solution of water, until it reaches the ratio stable : 36:64, in a process called
mutarotation can be accelerated (Lehninger, 1982).

Changes

the

Fischer

projection

to

Haworth

projection

-D-

glucopyranose
b. Blood Glucose
Blood glucose is the sugar found in the blood that form of carbohydrates in
the diet and are stored as glycogen in the liver and skeletal muscle. Energy for
most of the functions of cells and tissues derived from glucose.
Blood glucose levels that are known to help predict the metabolism that
may occur in a cell with a sugar content provided. If the content of glucose in the
body greatly exaggerated the glucose catabolism will experience a reaction
enzymatically to produce energy. However, if the glucose content is below the
minimum threshold, then the pyruvic acid produced from catabolism could
undergo enzymatic processes in anabolism through glukoneogenesi to synthesize
glucose and meet normal levels of glucose in the blood (blood plasma) that is 65110 mg / dl (3, 6 to 6.1 mmol / L) (Murray, 2003).
In medicine, blood sugar is a term that refers to glucose levels in the
blood. Blood glucose levels are regulated in the body. Glucose is passed through
the blood is the main source of energy for cells - the cells of the body. Generally,
blood glucose levels are at levels of 70-110 mg / dl. Abnormal glucose
metabolism may cause hyperglycemia (when blood sugar levels are at high levels
(> 110 mg / dl)) and hypoglycemia (when blood glucose levels are too low (<70
mg / dl)).
Blood glucose will reduce Cu2+ in alkaline conditions, the result of the
reduction will react with arseno molybdate produces a blue color. glucose can
reduce Cu2+for glucose is a reducing sugar which can reduce an electron acceptor

compounds. In addition Ba(OH)2 little clot of blood samples. Ba(OH) 2 This

function gives alkaline and precipitate iron ions in the blood. One of the
precipitated iron ions are iron ions in hemoglobin, the iron ion is converted into a
molecule Fe(OH)2 in the form of red sludge. In ZnSO 4 then red precipitate out of
the sample more and terakoagulasi become pale. The addition of ZnSO 4 serves to
precipitate and denature protein completely. Because the density greater than the
density of glucose to be analyzed, then the separation is then performed with a
centrifuge to precipitate the protein. This precipitation aims to separate the
protein from glucose because it would interfere with the measurement.
Determination will be done by means of spectrophotometry. To measure
arbsorbansinya then added Cu alkalis and also arsenomolibdat. The addition of
Cu alkalis under alkaline conditions will reduce Cu2+ into Cu compound which
beereaksi with arsenomolbdat menghailkan blue.
c. Method of measuring glucose levels:
1. Chemical methods. The principle of this examination, the process of
condensation of glucose with amines and acids achromatic glacial heated
atmosphere, thus forming a green colored compound is then measured by
2.

photometry.
Enzymatic methods.
- glucose oxidase method. The principle of this examination is the
enzyme catalyzes the oxidation of glucose oxidation of glucose to
gluconic acid and hydrogen peroxide formed reacts with phenol and 4 amino phenazone with the aid of peroxidase enzymes produce
quinoneimine pink and can be measured with a photometer at = 546
-

nm.
The method hexokinase. (Down, 2000)

d. UV-Vis spectrophotometry
UV-Vis spectrophotometry is one spectroscopic analysis techniques that use
radiation sources eleltromagnetik near ultraviolet (190-380) and visible light
(380-780) using a spectrophotometer instrument. It is used for chemical analysis
of quantitative and qualitative sometimes.
Spectrophotometer consists of

spectrometers

and

photometer.

Spectrophotometer produce beams of a specific wavelength spectrum and the

photometer is measuring device ditranmisikan light intensity or absorbed.
Spectrophotometer

composed

of

continuous

spectrum

source,

monochromator, absorbing cells for the sample solution or a blank and a tool to
measure the average difference between the absorption of the sample and blank
or comparison UV-Vis spectrophotometer can make determinations on samples
in solution, gas, or steam (J.R. Day and Underwood, 1992).
e. Lambert-Beer Law
Law of Lambert-Beer (Beer's law) is the relationship between the
absorbance linearity with the concentration of the analyte solution. According to
the law of Lambert, absorption (A) is proportional to the thickness of the layer
(b) are exposed:
A = k. B
Lambert-Beer Law is used to determine the blood glucose levels where
Absorbance proportional to the glucose concentration to be measured. This
solution was measured at a wavelength of 660 nm maximum aitu. Digitally using
the Lambert-Beer law:
A=kxc xl
Where :
A
k
c
l

: Absorbance (light absorption)

: koefisisen molar solution ekstinksi
: concentration of the sample
: table cuvette

by law lambert-beer, light absorption is directly proportional to the

concentration.
VI. Equipments and Materials
a. Equipments
UV-VIS Spectrophotometer
1 piece
Centrifuge tube
1 piece
Test tube
8 pieces
Incubator
1 piece
Graduated cylinder
10 mL
1 piece
Test tube rack
1 piece
Beaker glass 100 mL
1 piece

b.

Beaker glass 50 mL

1 piece

Pipette

10 pieces

Spatula

1 piece

Propipette

1 piece

Materials

1.
2.
3.
4.
5.

Cu alkalis solution
Ba(OH)2 0,3N solution
ZnSO4.7H2O 5% solution
Standar glucose solution 2 mg/mL
Arsenomolibdat reagent

VII. Procedure
1. Deproteinase of Blood Filtrate
1 ml oxalated blood
Enetred into centrifuge tube which already contains 3 mL of aquades
Mixed well using spatula
Added 1 mL of Ba(OH)2 0,3 N
Mixed until blended
added 2 mL of ZnSO4.7H2O 5%
mixed well
added 2 mL of (NH4)2SO4
allowed to stand for 5 minutes
centrifuge for 15 minutes
decanted

residue

filtrate
Tested using biuret 3-5 drops
Observation result

2. Determine the blood glucose levels

1 mL of bood filtrate that free of protein
Entered into test tube
Added 3 mL of Cu alkalis reagent
Entered into boiling water for 15 minutes
Entered into cold water
Addeddrop by drop arsenmolibdat reagent
Mixed until blended
Read the absorbance with UV-VIS Spectrophotometer at a wavelength of 660 nm

Observation Result

3. Standar Curves Manufacture

1 mL of

1 mL of

1 mL of

1 mL of

glucose 0,3

glucose 0,5

glucose 0,7

glucose 0,9

mg/ml

mg/ml

mg/ml

mg/ml

- Poured into test tube

- Added 0,5 mL of Cu alkalis
- Entered to the boiling water for
20 minutes
- Entered into cold water
- Added drop by drop of
arsenmolibdat reagent
- Mixed until blended
- Read the absorbance with UVVIS spectrophotometer at a
wavelength of 660 nm
- observed

Observation result

4.

Blanko Solution
1 ml of aqueades
- Poured into test tube
- Added 1 mL of Cu alkalis reagent
- Entered into boiling water for 20
minutes
- Entered into cold water
- Added 1 ml of arsenmolibdat
reagent
- Mixed until blended
- Read the absorbance with UV-VIS
spectrophotometer at a
wavelength of 660 nm

Observation Result

A.
B.
C.