INTRODUCTION:

Hemoglobin (Hb), the main component of red blood cells, is a protein that carries oxygen from

the lungs to the body’s 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 CO2 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 lung’s 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. Colorimetric Methods

ii. Gasometric Methods

iii. Specific Gravity Methods

iv. 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. Measures all forms of hemoglobin except sulfhemoglobin

ii. Can be easily standardized

iii. Cyanmethemoglobin reagent (also called Drabkin’s 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 (Drabkin’s 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 µl 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

Hb (g/dL)

Volume of Stock

(µl)

Volume of

Distilled water

(µl)

Volume of

Drabkin’s

Reagent (µl)

Absorbance

2 20 80 3000 0.589

4 40 60 3000 1.164

6 60 40 3000 1.766

8 80 20 3000 2.258

10 100 - 3000 2.650

unknown 100 - 3000 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.

Drabkin’s reagent contains iron, potassium cyanide, and sodium bicarbonate as well as

potassium ferricyanide. It is a pale yellow solution, odorless and an alkaline solution. Drabkin’s

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 (CuSO4) 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 Drabkin’s solution

must wrap with aluminum foil to prevent light contact with Drabkin’s 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. Drabkin’s Reagent [pdf]. [Online] Available at:

http://www.sigmaaldrich.com/content/dam/sigmaaldrich/docs/Sigma/Datasheet/3/

d5941dat.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].