Reversed-phase high-performance liquid chromatography of human haemoglobin chains

Area of Science:

• Biochemistry
• Analytical Chemistry
• Hematology

Background:

• Human hemoglobin is composed of various globin chains.
• Accurate separation and identification of these chains are crucial for diagnosing hemoglobinopathies.

Purpose of the Study:

• To develop and validate a reversed-phase high-performance liquid chromatographic (RP-HPLC) method for separating human hemoglobin chains.
• To assess the method's resolution, reproducibility, and potential for quantitation.

Main Methods:

• Utilized a LiChrospher 100 CH-8/2 column with a ternary eluent (acetonitrile-methanol-0.155 M NaCl, pH 2.7).
• Employed a 60-min linear gradient for chain separation, with adjustments for specific variants like A gamma T.
• Investigated the role of silanophilic interactions and NaCl concentration in retention.

Main Results:

• Achieved improved resolution between multiple human hemoglobin chains, including (delta beta) Lepore, beta A, beta S, alpha, G gamma, and A gamma.
• Demonstrated good reproducibility of retention times (+/- 1.5%) and a recovery rate of 82%.
• Identified that NaCl addition is critical for suppressing silanol adsorption, and methanol concentration affects solvation.

Conclusions:

• The developed RP-HPLC method provides sensitive and reproducible separation of human hemoglobin chains.
• This technique shows significant potential for the diagnosis of hemoglobinopathies and research on hemoglobin variants.