Sources of variation in the column-chromatographic determination of glycohemoglobin (HbA1)

We studied the influence of many variables on the cation-exchange chromatographic determination of glycohemoglobin was minicolumns (HbA1) and with macrocolumns (HbA1a+b and HbA1c). Minicolumns are most sensitive to buffer composition. The best conditions are pH 6.78 and sodium, potassium, and phosphate concentrations of 54.8, and 36 mmol/L, respectively. In fact, there is no clear cut-off point for elution. A cyanide concentration of 1.5 mmol/L has no negative effects. For macrocolumns, the influence of temperature is demonstrated; 20 degrees C is optimal. A higher temperature caused results for HbA1 to be higher. For minicolumns, the temperature effect corresponds to 1% HbA1 per 1 degrees C, necessitating rigorous temperature control. The amount of sample applied also influences the results. Therefore a constant column load, chosen between 1.3 and 2.0 mg of Hb permilliliter of resin, is advised. Heparinized blood can be kept for three days at 4 degrees C; hemolyzed samples are best stored at -70 degrees C.