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

As a consequence of nonideal chromatographic conditions, values for stable glycated hemoglobin (HbA1c) 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 HbA1c peak is not collected in the HbA1c fraction. Increasing the ionic strength of the eluting buffer by increasing the operating temperature to 28 degrees 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.