Hemoglobin A1C Detection

Automated cation-exchange High-Performance Liquid Chromatography (HPLC) is being used increasingly as the initial diagnostic method in many laboratories. HPLC depends on the interchange of charged groups on the ion exchange material with charged groups on the hemoglobin molecule. When a hemolysate containing a mixture of hemoglobins is adsorbed onto the resin, the rate of elution of different hemoglobins is determined by the pH and ionic strength of any buffer applied to the column. Various fractions are detected as they pass through an ultraviolet/visible light detector and are recorded on an integrating computer system. Analysis of the area under these absorption peaks gives the percentage of the fraction detected.

- The analysers are automated and permit processing of large batches

- Rapid assay time

- Very small samples (5 µl) are sufficient for analysis; this is especially useful in pediatric work

- Quantification of normal and variant haemoglobins is available on every sample

- Provisional identification of a larger proportion of variant haemoglobins can be made

- Applicable for the quantification of HbA1c (Glycated hemoglobin)

Glycation is the nonenzymatic addition of sugar residue to amino groups of proteins. Chromatographic analysis of HbA identifies several minor hemoglobins, namely, HbA1a, HbA1b and HbA1c, which are collectively referred to as HbA1. HbA1c is formed by the condensation of glucose with the N-terminal valine residue of each β-chain of HbA to form an unstable Schiff base. The Schiff base may dissociate or may undergo an Amadori rearrangement to form a stable ketoamine, HbA1c. 

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