High performance liquid chromatography is the diagnostic method of choice for haemoglobin variants in a large number of laboratories worldwide. It is able to detect and resolve a large number of haemoglobin variants, especially when used in conjunction with another method of screening / identification (e.g. haemoglobin electrophoresis). In addition, it is able to quantify the amount of variant haemoglobin present, which itself is a useful parameter in identification.

Method:

  • Exchange column containing small sphreres of silica which are modified to be weakly cationic
  • Anticoagulated specimen is lysed and diluted in buffer, then injected into column
  • Hb is absorbed into column and eluted by means of a gradient of increasing ionic strength
  • Eluate passes through a photometer which measures changes in absorbance

Reasons to use:

  • Major discriminatory value:
    • Separates haemoglobin S from Hb Lepore, Hb D-Punjab and Hb G-Philadelphia
    • Separates haemoglobin E from Hb C, Hb O-Arab
  • Pitfalls: cannot discriminate between HbA2 and HbE

Typical Retention Times:

Window / PeakRetention Time (min)Causes / Variant Haemoglobin
P10.63 – 0.85Bilirubin, haemoglobin H, haemoglobin Bart’s
F0.98 – 1.2Haemoglobin F
P21.24 – 1.4Glycated haemoglobin, haemoglobin Hope
P31.4 – 1.9Post-translationally modified haemoglobin A, haemoglobin J-Oxford
A1.3 – 3.1Haemoglobin A, glycated S, Köln
A23.3 – 3.9Haemoglobin A2, E, Lepore
D3.9 – 4.3Haemoglobin D-Punjab, G-Philadelphia
S4.3 – 4.7Haemoglobin S, Q-Thailand
C4.9 – 5.3Haemoglobin C, O-Arab
Typical retention times of normal and variant haemoglobins on HPLC