Effect of temperature on quantifying glycated (glycosylated) hemoglobin by cation-exchange chromatography

Abstract

As a consequence of nonideal chromatographic conditions, values for glycated hemoglobin (HbA(1c)) determined by cation-exchange chromatography in a commercial minicolumn system (y) or by 'high-performance' liquid chromatography (x) differ markedly, yielding the regression line y = 0.82x + 0.6. With use of the protocol specified by the manufacturer, 20% of the HbA(1c) peak is not collected in the HbA(1c) fraction. Increasing the ionic strength of th eluting buffer by increasing the operating temperature of 28°C increases the rate of elution from the minicolumn, making results of the two methods more closely comparable (y = 0.98x - 0.22). Because at a given pH the elution volume is determined primarily by the ionic strength, close limits on the composition of the eluting buffer are set by the temperature dependence of its ionic strength. At a specified temperature and pH the position of a peak can be judged to within a volume of 1 mL if the conductivity of the eluent does not vary by more than ± 0.05 mS.