A question from Yahoo! Answers:

Can anyone explain how does HPLC measure HbA1c? What exactly is HPLC?

Let’s start with the last part of the question – “What exactly is HPLC?”

HPLC or High Performance Liquid Chromatography in simple terms is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate components of a mixture followed by identification and quantitation of each component. In case of HPLC analysis of glycosylated hemoglobin (HbA1c) , the “mixture” is a blood plasma sample.

“how does HPLC measure HbA1c?”
The blood plasma components, including HbA1c, get separated due to a variety of chemical interactions between plasma molecules and the column of the HPLC instrument. The levels of detected substances are plotted in the form of a series of peaks . The area under the peak is proportional to the amount of analyte present and by calculating the area of the peak using the mathematical function of integration.

The levels of HbA1c in the original blood plasma sample is determined by comparing the areas of the HbA1c and HbA (hemoglobin alpha-chain) peaks.

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