Getting Started in HPLC

Section 2F. HPLC Data Systems

Before an LC system can provide quantitative results, it must be calibrated for the analytes of interest. Calibration consists of running a series of chromatograms at different analyte concentrations that bracket the expected concentrations of analyte in the samples.

In the early days of HPLC, quantitation was done graphically on the basis of a calibration curve or line as shown on the right. The best-fit straight line was drawn through the calibration points. When the actual sample was run, the area of the analyte peak was measured and the corresponding concentration (or amount) of analyte was read directly from the graph.

Most data systems today calculate a response factor from an appropriate least-squares fit to the calibration points. This response factor is essentially the slope of the calibration line. Dividing the response factor into the area of the analyte peak obtained from the sample generates the concentration or mass of analyte in the sample. The procedure is exactly equivalent to the old manual graph technique.

In its simplest form, a calibration curve is a plot of peak size (area or height) as a function of the amount (mass or concentration) of analyte injected. When a sample is run and the peak size is measured, the amount of analyte can be read directly from the graph or computed by dividing the peak size by the response factor.




A calibration curve is only   valid if the samples and calibrators are run under identical   conditions. If anything changes -- column, mobile phase, flow, temperature, injection volume, whatever -- then the calibration must   be rerun before samples are analyzed.



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Last revised: April 06, 2001.