Experiment 7: Estimation of Hemoglobin in Blood Objectives 1. To study t e structure as !ell as t e range of Hemoglobin and its effect of abnormality in blood.

". To measure t e concentration of sample emoglobin and find t e concentration for un#no!n sample by using spectrop otometer.

$ntroduction Blood is a type of connective tissues. Hemoglobin consists of 1 molecule of globin and % molecules of eme &eac containing 1 molecule of iron in t e ferrous state'. (lobin consists of " pairs of polypeptide c ains. $n t e emoglobin molecule) eac polypeptide c ain is associated !it 1 eme group* eac molecule of oxygen or +O" eme group can combine !it 1

Hemoglobin carries oxygen from places of ig oxygen pressure &lungs' to places of lo! oxygen pressure &tissues') ! ere it readily releases t e oxygen. Hemoglobin also returns +O" from t e tissues to t e lungs. Bot ig and lo! emoglobin counts indicate defects in t e balance of red blood cells in t e blood) and may indicate disease. ,t a pressure of 1-- mmHg in t e lung.s capillaries) /01/23 of t e Hb is combined !it oxygen. $n t e perip eral tissues) ! ere t e pressure may be as lo! as "-mmHg) less t an 4-3 of t e oxygen remains combined !it Hb. T e normal emoglobin content in

uman varies !it altitude. 5ormal emoglobin content for male is in t e range of 14.2 to 17." g6d7) ! ile female falls in t e range of 1".1 to 10.1 g6d7. Hig er1t an1normal emoglobin may indicate: +ongenital eart disease &in erited disease ! ic present ! en birt ' +or pulmonale 8ulmonary fibrosis 8olycyt emia vera $ncreased 9B+ formation associated !it excess ert ropoietin

7o!er1t an1normal emoglobin may indicate: ,nemia &various types' Eryt ropoietin deficiency &from #idney disease' 9ed blood cell destruction associated !it transfusion reaction Bleeding 7ead poisoning :alnutrition 5utritional deficiencies of iron) folate) vitamin B11") vitamin B1; Over1 ydration T e emoglobin concentration in blood decreased as caused t e anemia

symptom and increased in concentration !ill causes t e blood viscosity increase. ,nemia is affected by eit er less1t an1optimal production of red blood cells or emoglobin) or increased destruction or s ortened life span of red blood cells. $ron deficiency is t e main cause of significant anemia during pregnancy t at eventually causes t e c ild birt to ave anemia. 8olycyt emia is one of t e conse<uence diseases of increasing in blood

viscosity ! ic due to a combination of ig altitude residency and poor pulmonary function. T e abnormal emoglobin concentration in a pregnant mot er !ill also cause some effect on t e fetus suc as anemia and iron poisoning.

:et ods for emoglobinometry can be grouped into % main classes depending on t e basic tec ni<ue employed !it variants !it in eac class: &i' &ii' &iii' &iv' +olorimetric met ods&colour c anges indicate reaction' (asometric met ods =pecific gravity met ods + emical met ods emoglobin determination is t e cyanmet emoglobin

T e met od of c oice for

met od ! ic is a type of colorimetric met od. T e principle of t is met od is t at ! en t e blood is mixed !it a solution containing potassium ferricyanide and potassium cyanide) t e potassium ferricyanide oxidi>es iron to form met emoglobin &unable to transport oxygen'. T e potassium cyanide t en combines !it met emoglobin to form cyanmet emoglobin) ! ic is a stable colour pigment read p otometrically at a !ave lengt of 0%-nm by spectrop otometer. =everal advantages of using cyanmet emoglobin met od are: i' ii' iii' :easures all forms of emoglobin except sulp emoglobin +an be easily standardi>ed +yanmet emoglobin reagent &also called ?rab#in.s solution' is very stable.

:aterials 1. Test tubes ". +entrifuge tubes 4. +uvette %. :icropipettes 0. 8ipette ;. ,luminum foil 7. Hemoglobin standard &Hgb' standard 2. +yanmet emoglobin reagent &?rab#in.s solution' /. ?istilled !ater 8rocedure 1. , serial dilution of emoglobin standard is prepared from t e range 1-g6d7 @ "-g6d7. T e dilution is done follo!ing t e formula :1A1 B :"A".

:1 B :olarity of stoc# reagent :" B Cinal molarity needed A1 B Aolume of stoc# reagent A" B Cinal volume needed 12mg6d7: &"-'&A1' B &12'&1m7' B -./m7 of Hemoglobin "-g6d7 D -.1m7 of distilled !ater 1;mg6d7: &12'&A1' B &1;'&1m7' B -.2//m7 of Hemoglobin 12g6d7 D -.111m7 of distilled !ater 1%mg6d7: &1;'&A1' B &1%'&1m7' B -.270m7 of Hemoglobin 12g6d7 D -.1"0m7 of distilled !ater 1"mg6d7: &1%'&A1' B &1"'&1m7' B -.207m7 of Hemoglobin 12g6d7 D -.1%4m7 of distilled !ater 1-mg6d7: &1"'&A1' B &1-'&1m7' B -.244m7 of Hemoglobin 12g6d7 D -.1;7m7 of distilled !ater ". 0m7 of +yanmet emoglobin reagent is pipetted into eac tube. 8rior to t at) t e tubes are !rapped !it used as a blan#. 4. Tube is left to stand for 1- minutes. %. T e absorbance &,' in t e spectrop otometer is read at 0%- nm for eac sample) >eroing t e spectrop otometer !it t e blan# solution. 0. ,n ,bsorbance versus Hemoglobin concentration in grams 3 grap is plotted on a linear grap paper. ;. T e result is interpreted and t e un#no!n concentration of t e determined from t e grap . emoglobin is aluminum foil. "-E of t e appropriate sample including t e un#no!n concentration sample is added into respective tube. +yanmet emoglobin is

9esult +oncentration Tube 5o 1 " 4 % 0 ; 7 of Hemoglobin &g6d7' 11" 1% 1; 12 "Aolume of Hemoglobin &m7' -.244 -.207 -.270 -.22/ -./ 1 Aolume of distilled !ater &m7' -.1;7 -.1%4 -.1"0 -.111 -.1 ,bsorbance &,0%-' -.1;; -."-7 -.";-.";0 -.4-% -.44/ -.";7

?iscussion

F en t e blood is added !it al#aline ?rab#in.s solution) cyanmet aemoglobin is formed. +yanmet aemoglobin is a stable pigment ! ere t e emoglobin inside t e blood ! ic is present as oxidised to and bound to a cyanide radicle.

?rab#in.s 9eagent is used for t e <uantitative) colorimetric determination of emoglobin concentration in ! ole blood at 0%- nm. +lassic tec ni<ues for determining blood emoglobin !ere based on estimation of oxygen) carbon monoxide capacity) or iron

content. T e assays proved unreliable because of t e

eterogeneous nature of

emoglobin. , colorimetric cyanmet emoglobin met od !as proposed ! ere total emoglobin at al#aline pH is rapidly converted to t e cyanoderivative. T e absorbance of t e cyanoderivative is determined at 0%- nm. T e met od !as simplified by combining t e separate reactants) al#aline ferricyanide and cyanide) into a single reagent.

?rab#in.s solution reacts !it all forms of emoglobin except sulf emoglobin) a pigment t at normallu occurs in only minute concentrations in blood. T e broad absorption pea# of cyanmet emoglobin permits its measurement using bot !ide and narro! band!idt instruments &04- @ 00- nm'. =igma provides a stable) dry ?rab#in.s 9eagent) ! ic is combined !it a surfactant minimi>es turbidity sometimes caused by t e presence of eryt rocyte stroma.

T e result of t e experiment is <uite accurate and consistent. T e result is ig ly accurate due to t e good pipetting s#ill of t e experimenter. , linear grap is obtained from t e result. T e grap indicates t e relations ip bet!een t e absorbance and emoglobin concentration. , greater absorbance !ill be obtained if t ere is a ig er emoglobin concentration. T e principle be ind it is t at ! en t e concentration of emoglobin increases) t e lig t intensity increases too) causing t e absorbance to increase at t e same time. T e un#no!n concentration of t e sample of t e experiment ! ic is obtained from t e grap is 10.2 g6d7. T e accuracy and consistency of t e result is due to t e good and accurate pipetting s#ill of t e experimenters.

T ere are some precautions in t e use of t e spectrop otometer. T e lamp and t e electronics of t e spectrop otometer ave to !arm up before t e experiment. T e experiment starts !it measure t e !avelengt of t e blan#. T e reason for set t e Gblan#H is t at all errors of measurement t at may be introduced into absorption spectrum from t e cuvette material) solvent) temperature fluctuations) gases in t e atmosp ere are

ta#en into account. Besides) t e fingerprints and !ater or li<uids outside t e cuvette must be !iped off before t e cuvette is put into t e spectrop otometer. T is is to prevent t e fingerprint from absorb some of t e radiant energy t at may lead to t e incorrect of result before measuring. Besides) t e micropipettes s ould be turned into t e correct volume. T ere is a minimum volume stated at t e top of micropipettes. T e volume turned must not be lo!er t an t e minimum volume stated o!ever it able to be turned. T is !ill cause t e inaccurate of volume obtained from t e micropipette. T e ?rab#in.s reagent s ould be #ept a!ay from reac ing of t e lig t by covering it !it aluminium foil to prevent t e un!anted reaction. T e sample blood s ould be #eep refrigerated or placed in an ice box to preserve fres ness.

+onclusion $n t e end of t e experiment !e !ere able to understand t e structure as !ell as t e range of emoglobin and its effect of abnormality in blood. T e emoglobin range and concentration is determined by t e cyanmet emoglobin met od ! ic is a type of colorimetric met od. T roug t is met od !e are able to determine t e un#no!n sample concentration. T e un#no!n concentration of t e sample of t e experiment ! ic is obtained from t e grap is 10.2 g6d7.

9eference 1. Drabkins Direction. 9etrived from) ttp:66!!!.unm.edu6Irrobergs604;?rab#ins?irections.pdf

". Haemoglobin. 9etrived from) ttp:66!!!.liv.ac.u#6Igd!ill6medic6 aem. tml 4. 7ab manual version -11-2. Measurement of Protein Solutions.