Dr.

Rasha Nassra
Lecturer of
Medical Biochemistry
Introduction

 Haemoglobin is a protein with a prosthetic

group haem united to the protein globin.
 Its molecular weight is 68000.

 Haem is an iron-prophyrin compound.

Porphyrin is formed of four pyrrol rings united
together by four methane bridges.
Introduction

Normal Value:

 Adult male: 14-18 g/dl blood.

 Adult female: 12-16 g/dl blood

Introduction

 When haemglobin level decreases below

the normal value, this denotes the presence
of anaemia.

 While an increase in its level is found in

polycythemia.
Determination of haemoglobin in blood by
colorimetry

 Principle:
Haemoglobin is oxidized by potassium
ferricyanide into methaemoglobin which is
converted into cyanomethaemoglobin by
potassium cyanide.

The intensity of absorbance of

cyanomethaemoglobin is proportional to
haemoglobin concentration.
Reagents:

1- Ferricyanide-cyanide reagent
2-Cyanomethaemoglobin standard: 15 g/dl.
 Blank Standard Sample

Distilled water 10 μl - -

Cyanomet -Hb standard - 10 μl -

Blood sample - - 10 μl

Ferricyanide-cyanide reagent 2.5 ml 2.5 ml 2.5 ml

 Incubate for 10 minutes, then read the
absorbance of standard
(A. standard) and sample (A. sample)
against blank at wave length
540 nm
using a spectrophotometer.
Calculation:

Conc. Sample

= A.sample x conc. standard

A standard

= ….. g/dL