SPECTROSCOPIC EXAMINATION OF BLOOD FOR THE DETECTION OF ABNORMAL HAEMOGLOBIN PIGMENTS

SPECTROSCOPIC EXAMINATION OF BLOOD FOR THE DETECTION OF ABNORMAL HAEMOGLOBIN PIGMENTS 

Certain chemicals and drugs may react with haemoglobin to form abnormal pigments which affect its capacity to bring about gaseous exchange. 

Three such pigments,methaemoglobin,sulphhaemoglobin and carboxyhemoglobin,can be differentiated from each other with the help of a spectroscope.

The spectroscope splits white light into its component colours with the help of a prism or a diffraction grating. Some substances absorb light at various wavelengths. 

When viewed through the eyepiece of the spectroscope, this absorbed light appears as a dark band at that particular wavelength. Such absorption bands are specific for each compound and is called an absorption spectrum of that substance. 

Technique Dilute the sample of blood 1:10 or 1:20 with distilled water and view it through a spectroscope. Absorption bands appear either in the green or the red part of the spectrum, depending on the type of the pigment present (Fig. 6.5)

Oxyhaemoglobin (normal Hb) shows a narrow band at 575 nm (yellow region) and a wider band at 540 nm (green region).

Methaemoglobin shows three bands which may not be very well defined, at 630 nm, 575 nm and 538 nm.

Sulphhaemoglobin also shows three absorption bands at 618 nm, 575 nm and 538 nm.

Carboxyhaemoglobin has an absorption spectrum very similar to that of oxyhaemoglobin, with bands at 570 nm, 534 nm and a weak band at 418 nm.

Note

Simple chemical tests help in the differentiation of hemoglobin pigments if the absorption bands are not very clear. For example, 

Dilute the blood sample 1:500. 

The normal oxyhaemoglobin will show a yellowish-red color. If, however, carboxyhaemoglobin is present in it, a purplish tint will be observed. 

To one drop of blood on a slide, add two drops of sodium hydroxide (NaOH, 250g/L) solution.

A normal specimen of blood will change to a brownish red colour whereas that containing carboxyhaemoglobin will remain red.

iii. The absorption bands of methaemoglobin and sulphhaemo-globin are very similar. 

To differentiate between them, add 2-3 drops of sodium cyanide solution (50g/L) to a solution of haemoglobin. The cyanide will convert methaemoglobin into cyanmethaemoglobin which does not show any absorption band at the red region (630 nm). 

Absorption spectrum of sulphaemoglobin remains unchanged.