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 from the tissues
to the lings to be exhaled. Hemoglobin consists of one molecule of globin and four
molecules of heme (each containing one molecule of iron in the ferrous state). Globin
consists of two pairs of polypeptide chains. In the hemoglobin molecule, each
polypeptide chain is associated with one heme group each heme group can combine with
one molecule of oxygen or !"#.
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
carbon dioxide from the tissues to the lungs.
$oth high and low hemoglobin counts indicate defects in the balance of red blood cells in
the blood, and may indicate disease.
%t a pressure of &'' mm Hg in the lung's capillaries, () * (+, of the Hb is combined
with oxygen. In the peripheral tissues, where the pressure may be as low as #' mm Hg,
less than -', of the oxygen remains combined with Hb.
.he normal hemoglobin content in human /aries with altitude. 0ormal hemoglobin
content for male is in the range of &-.+ to &1.# g2d3, while female falls in the range of
&#.& to &).& g2d3.
4ethods for hemoglobinometry can be grouped into four main classes depending on the
basic techni5ue employed with /ariants within each class:
i. !olorimetric methods
ii. Gasometric methods
iii. 6pecific Gra/ity methods
i/. !hemical methods
.he method of choice for hemoglobin determination is the cyanmethemoglobin method
(this is a type of colorimetric method). .he principles of this method is that when blood is
mixed with a solution containing potassium ferricyanide and potassium cyanide, the
potassium ferricyanide oxidi7es iron to form methemoglobin. .he potassium cyanide then
combines with methemoglobin to form cyanmethemoglobin, which is a stable color
pigment read photometrically at a wa/e length of )8' nm.
.hree ad/antages of the cyanmethemoglobin method are:
&. 4easures all forms of hemoglobin except sulfhemoglobin.
#. !an be easily standardi7ed.
-. !yanmethemoglobin reagent(also called 9rab:in's solution) is /ery stable (;tar, #'&8)
"b<ecti/e(s):
&. .o study the absorbance of hemoglobin by using spectrophotometer.
#. .o study the difference of bo/ine hemoglobin and human hemoglobin.
-. .o understand and learn the method to determine the hemoglobin in the blood.
4aterials:
.est tubes, test tube rac:, micropipette (& m3), blue tip, hemoglobin standard (Hgb)
standard, cyanmethemoglobin reagent (9rab:in=s solution), distilled water,
spectrophotometer, >ppendorf tube, pipette, pump, /ortex, aluminum foil, cu/ette
?rocedure:
&. Indi/idual dilution of Hemoglobin standard had been prepared from the range of #
g2d3 @ #'g2d3.
#. %pproximately )m3 of !yanmethemoglobin reagent had been pipetted in to each
tube. & m3 of the appropriate sample had been added into each tube. %nything
other than the !yanmethemoglobin reagent was not allowed to add to the reagent
$3%0A.
-. Bortex the content in the test tube then allow the tubes to incubated for &' minutes at
room temperature.
8. .he absorbance in the spectrophotometer was read at )8'nm, the spectrophotometer
has been set to 7ero with the $3%0A solution.
). % graph of absorbance /s. Hemoglobin concentration in g2d3 was plotted on linear
graph paper.
Cesult:
!oncentration
of hemoglobin
(g2d3)
6toc: /olume
(Dl)
Bolume of
distilled water
(Dl)
%bsorbance
# &'' (''
8 #'' +''
E -'' 1''
+ 8'' E''
&' )'' )''
#' &''' *
!olour obser/ed
9iscussion:
From the graph, we can :now that the absorbance is directly proportional to the
concentration of the hemoglobin. It is a straight line graph. Ghen the concentration of
hemoglobin increases, the absorbance will also increase too as more light had been
absorb when the light pass through cu/ette. Howe/er, when the concentration of
hemoglobin is low, the amount of light absorb is low. So, the absorbance will
decrease too.
In this experiment, bo/ine hemoglobin is tested. $o/ine hemoglobin did not ha/e
huge different from the human hemoglobin. .he H* and I* subunit of human hemoglobin
and bo/ine hemoglobin ha/e ++, and +8, almost the same. .he difference between
bo/ine hemoglobin and human hemoglobin is the deletion of I*His # in bo/ine
hemoglobin which reduce the I* subunit to &8) amino acid. only the presence of &1
amino acid in the hemoglobin H chain and #8 amino acid in the hemoglobin I chain in
bo/ine hemoglobin. $o/ine hemoglobin is also one of an alternati/e source material to
use in hemoglobin-based oxygen carrier. Bovine hemoglobin afnity is
regulated by chloride ion, Cl
-
.(Safo and Abraham, !!"#
S$eci%c gravity method is one of the way to determine hemoglobin in the
blood based on oxygen combining ca$acity of hemoglobin. &he $rinci$le of
the method is blood is dro$$ed into a co$$er sulfate solution. &his method
does not needed any s$ecial instrument to estimate the hemoglobin
concentration in the dro$ of blood. 'f the dro$ is lighter than the s$eci%c
gravity of the solution which mean has low concentration of hemoglobin in
ther blood, the dro$ will (oat on the surface of the solution) if not it will sin*
to the bottom of the solution. Blood with normal amount of hemoglobin
$resent in it will falls +uic*ly whereas blood with low amount of hemoglobin
will falls slowly or not (oat on the surface. &he amount of hemoglobin $resent
in the blood will a,ect after the &his is the faster way to test for anemia. this
method is also used for hematocrit detection. &he density of the dro$ is
directly $ro$ortional to the amount of hemoblogin $resent in that dro$
(-stridge , .eynolds and /alters, !!!#
.here are some precautionary steps needed to follow throughout the experiment.
Ghen pipette solution always remember to press the tip of the pipette to the wall of
cu/ette. .his is to pre/ent bubble forming.Always remember to change the ti$ of
the $i$ette after every single $i$etting if not the will a,ect the concentration.
All the test tube that are used to contain the 0rab*in1s solution must be wra$
with aluminum foil because 0rab*in1s solution are light sensitive it will react
+uic*ly once it ex$osed to light. /hen micro$i$ette hemoglobin in the tube,
try to go in directly and release it slowly. 4a:e sure there is no water droplet
outside the cu/ette before place in the spectrophotometer to test for absorbance. %lways
set the wa/elength on the spectrophotometry before operating the spectrophotometer. %s
the light path runs from the front to bac:, ma:e sure the cu/ette is oriented correctly.
%lways remember to insert the blan: cu/ette before insert the cu/ette with the sample.
Conclusion2
From this experiment, we :now that the absorbance is directly proportional to the
concentration of the hemoglobin. .he absorbance is increasing with the hemoglobin
concentration.
.eference2
". 3tar, !"4. 5ab manual2-stimation of hemoglobin in blood. 6era*2 3&A..
. Safo, 7.8. and Abraham, 0.9., !!".The X-ray structure determination of
bovine carbonmonoxy hemoglobin at 2.1 resoultion and its relationship to
the quaternary structures of other hemoglobin crystal forms. :;nline<.
Available at2 htt$2==www.ncbi.nlm.nih.gov=$mc=articles=67C>?4!=
:Accessed2 @ Auly !"4<.
>. -stridge, B.B., .eynolds, A.6. and /alters, C.9., !!!. Basic medical
aboratory
Techniques. 4
th
ed. 3nited States of America2&homson 5earning.