INTRODUCTION:

Hemoglobin (Hb), the main component of red blood cells, is a protein that carries oxygen from
the lungs to the bodys tissues, and carbon dioxide from the tissues to the lungs to be exhaled.
Hemoglobin consists of 1 molecule of globin and 4 molecules of heme (each containing 1
molecule of iron in the ferrous state). Globin consists of 2 pairs of polypeptide chains. In the
hemoglobin molecule, each polypeptide chain is associated with 1 heme group; each heme group
can combine with 1 molecule of oxygen or CO2.

Hemoglobin carries oxygen from places of high oxygen pressure (lungs) to places of low oxygen
pressure (tissues), where it readily releases the oxygen. Hemoglobin also returns CO 2 from the
tissues to the lungs.

Both high and low hemoglobin counts indicate defects in the balance of red blood cells in the
blood, and may indicate disease.

At a pressure of 100 mmHg in the lungs capillaries, 95-98% of the Hb is combined with oxygen.
In the peripheral tissues, where the pressure may be as low as 20 mmHg, less than 30% of the
oxygen remains combined with Hb.

The normal hemoglobin content in human varies with altitude. Normal hemoglobin content for
male is in the range of 13.8 to 17.2 g/dL, while female falls in the range of 12.1 to 15.1 g/dL.

Methods for hemoglobinometry can be grouped into 4 main classes depending on the basic
technique employed with variants within each class:
i.
ii.
iii.
iv.

Colorimetric Methods
Gasometric Methods
Specific Gravity Methods
Chemical Methods

The method of choice for hemoglobin determination is the cyanmethemoglobin method (This is
a type of colorimetric method). The principle of this method is that when blood is mixed with a
solution containing potassium ferricyanide and potassium cyanide, the potassium ferricyanide
oxidises iron to form methemoglobin, which is a stable color pigment read photometrically at a
wavelength of 540nm.

Three advantages of the cyanmethemoglobin method are:

i.
ii.
iii.

Measures all forms of hemoglobin except sulfhemoglobin

Can be easily standardized
Cyanmethemoglobin reagent (also called Drabkins solution) is very stable (UTAR,
2015).

OBJECTIVES:
1. To study the principle of hemoglobin.
2. To determine the absorbance of hemoglobin at different concentration by using
spectrophotometer.
3. To study the use of cyanmethemoglobin reagent.
4. To determine the unknown sample concentration from the graph.

MATERIALS:
Test tubes, test tube rack, micropipette, pipette tips, hemoglobin standard (Hgb) standard,
cyanmethemoglobin reagent (Drabkins solution), distilled water, spectrophotometer, Eppendorf
tubes, Eppendorf tube rack, pipette, pump, vortex, aluminum foil, cuvette, beaker

PROCEDURE:

1. A serial dilutions of Hemoglobin standard had been prepared from the range 2 g/dL 10
g/dL.
2. About 3 mL of Cyanmethemoglobin reagent had been pipetted in to each tube. 100 l of
the

appropriate

sample

were

added

into

each

tube. Anything

other

than

Cyanmethemoglobin reagent was not added to the reagent BLANK.

3. Tubes were allowed to stand for 10 minutes.
4. Absorbance in the spectrophotometer was read at 540nm, the spectrophotometer was set
to zero with the BLANK solution.
5. A graph of absorbance vs. Hemoglobin concentration in grams/dL was plotted on graph
paper.

RESULT:
Concentration of

Volume of Stock

Hb (g/dL)

(l)

2
4
6
8
10
unknown

20
40
60
80
100
100

Volume of

Volume of

Distilled water

Drabkins

Absorbance

(l)
80
60
40
20
-

Reagent (l)
3000
3000
3000
3000
3000
3000

0.589
1.164
1.766
2.258
2.650
2.039

DISCUSSION:
The graph obtained shows a straight line. These indicate that the absorbance is directly
proportional to the hemoglobin concentration. When the concentration of hemoglobin increase,
the absorbance increase. The higher the concentration of hemoglobin, the more the molecules in
the solution, more light is being absorbed and thus, concentration increase.
Drabkins reagent contains iron, potassium cyanide, and sodium bicarbonate as well as potassium
ferricyanide. It is a pale yellow solution, odorless and an alkaline solution. Drabkins reagent is
used for the quantitative, colorimetric determination of hemoglobin concentration in whole blood
at 540 nm (Sigma-Aldrich, n.d.).

Horse hemoglobin is a globular protein which carry oxygen from lungs to other parts of the
body. Each hemoglobin protein structure consists of four polypeptide subunits, which are held
together by noncovalent interactions such as ionic bonds, hydrogen bonds, hydrophobic
interactions, and van der Waals forces, as well as four heme pigments, one in each of the subunits
(Sadava et al., 2008). These heme groups contain positively-charged iron (Fe2+) molecules
which can reversibly bind to oxygen molecules and transport them to various areas of the body
(Sadava et al., 2008). As the heme groups bind or release their oxygen loads, the overall
hemoglobin undergoes conformational changes which alters their affinity for oxygen (Sadava et
al., 2008).

Figure 2: Structure of horse hemoglobin.

The specific gravity method is one of the method used to measure the concentration of
hemoglobin without any special instrument. The principle of this method is by dropping a drop
of blood into copper sulfate (CuSO 4) solution and observe the position of the blood in the
solution. If the drop float on the surface of solution, the specific gravity of blood is lighter than
the specific gravity of the solution which mean the concentration of hemoglobin is low. If the
drop sink to the bottom of solution, the specific gravity of blood is heavier than the specific
gravity of the solution which mean the concentration of hemoglobin is high. Blood with normal
concentration of hemoglobin will sink rapidly in copper sulfate solution whereas blood with

lower concentration of hemoglobin will float on the surface of copper sulfate solution. Recently,
these method is used to test for potential blood donors and hematocrit detection in a faster way
(Estridge, Reynolds and Walters, 2000).

There are some precautions needed when carry out this experiment. Changes the pipette tips after
transfer a solution. This is because different concentration of solution transfer by the same tips
will cause contamination and thus the result obtained is incorrect. When transfer the sample into
cuvette, make sure there is no bubbles as it will affect the reading of absorbance. Besides, make
sure the test tube is clean and the cuvette is also clean. Vortex the mixture for few seconds so that
the mixture can mix well and evenly. Insert the blank cuvette into the spectrophotometer first
before insert the cuvette contain the sample. All the test tube that contain Drabkins solution must
wrap with aluminum foil to prevent light contact with Drabkins solution as the solution are light
sensitive. It will react quickly once it exposed to light.

CONCLUSION:
When the concentration of hemoglobin increase the absorbance increase. The absorbance is
directly proportional to the concentration of hemoglobin. The concentration of unknown is 6.8
g/dL.

REFERENCES:
1. Estridge, B.H., Reynolds, A.P. and Walters, N.J., 2000. Basic medical Laboratory
Techniques. 4th ed. United States of America:Thomson Learning.
2. Sadava et.al, 2008. The Science of Biology. 8th ed. Massachusetts and Virginia: Sinauer
Associates Inc. and W.H. Freeman and Company.
3. Sigma Aldrich, n.d. Drabkins Reagent

[pdf].

[Online]

Available

at:

http://www.sigmaaldrich.com/content/dam/sigmaaldrich/docs/Sigma/Datasheet/3/d5941d
at.pdf [Assessed on 25 February 2015].
4. UTAR, 2015. Lab Manual: Estimation of hemoglobin in blood. Perak: UTAR.

5. Zoological Science, 2003. Significance of Affinity and Cooperativity in Oxygen Binding

to Hemoglobin of Horse Fetal and Maternal Blood. [Online]. Available at:
http://www.bioone.org/doi/pdf/10.2108/zsj.20.1087 [Assessed on 25 February 2015].