Sahli’s Acid Hematin Method for the Estimation of Hemoglobin

In this Acid hematin method of hemoglobin determination post we briefly summarise about: principle, reagents requirements, sahli’s acid hematin method procedure, result, application and limitations of Acid hematin method.

Acid Hematin Method Definition

The basic idea behind sahli’s acid hematin method is that when blood is mixed with N/10 Hydrochloric acid (HCl), the haemoglobin in RBCs is converted to acid hematin, a dark brown compound.

Haemoglobin (Hb) carries oxygen to cells from the lungs. If your haemoglobin levels are low, you have anaemia, a condition in which your body is not getting enough oxygen, causing fatigue and weakness. If your haemoglobin levels are high, this usually means you have too many red cells which is called polycythaemia.

Acid Hematin Method Principle

Blood is mixed with N/10 HCl resulting in the conversion of Hb to acid hematin which is brown in color in sahli’s acid hematin method. The solution is diluted till its color matches with the brown colored glass of the comparator box. The concentration of Hb is read directly using acid hematin method.

Acid Hematin Method Requirements


1. Sahli’s hemoglobinometer: This equipment consists of a comparator with a brown glass standard and Sahli’s graduated hemoglobin tube which is marked in percent and gram.

2. Hemoglobin pipette or Sahli’s pipette (marked at 0.02 ml or 20 μl).

3. Stirrer (a small glass rod).

4. Dropping pipette (dropper).

5. Blood is obtained directly by skin puncture or EDTA-anticoagulated venous blood.


1. N/10 HCl

2. Distilled Water


Acid Hematin Method of Hemoglobin Determination

Acid Hematin Method Procedure

  1. In sahli’s acid hematin method add N/10 HCl into the tube up to mark 2g%. Mix the EDTA sample by gentle inversion and fill the pipette with 0.02ml blood. Wipe the external surface of the pipette to remove any excess blood. Add the blood into the tube containing HCl.
  2. Wash out the contents of the pipette by drawing in and blowing out the acid two to three times. Mix the blood with the acid thoroughly. Allow to stand undisturbed for 10min. Place the hemoglobinometer tube in the comparator and add distilled water to the solution drop by drop stirring with the glass rod till its color matches with that of the comparator glass.
  3. While matching the color, the glass rod must be removed from the solution and held vertically in the tube. Remove the stirrer and take the reading directly by noting the height of the diluted acid hematin and express in g% in sahli’s acid hematin method.

Acid Hematin Method Advantages

  1. It is the simple and easy method and may be done at any place because apparatus can be picked up anywhere.

Acid Hematin Method Limitations

  1. Visual intensity may be different for different individuals by this method, we are not able to measure the inactive hemoglobin.
  2. This method estimates only oxy Hemoglobin. Carboxyhemoglobin and methemoglobin cannot be estimated.
  3. The endpoint disappears soon so it is difficult to know the actual endpoint and also the Proper stable standard is not available
  4. The resulting solution is not a clear solution but a suspension due to the action of hydrochloric acid on the proteins and lipids.