John W. Dolan

Calibration

Most LC methods are used for quantitative analysis — that is, the methods that answer the "how much is there?" question. Nearly all of these methods rely on comparison of the peak area (or less often, peak height) of a sample with that of a reference standard. To do this, we use a calibration curve (also referred to as a standard curve or sometimes "line"). The two most popular methods of calibration are external and internal standardization. In external standardization, the calibration is based upon comparing the response of the reference standard with its concentration. For internal standardization, a constant concentration of an internal standard is added to each sample and the ratio of the response of the analyte to that of the internal standard is compared with the concentration. The external-standard method is simpler and will be used for all the examples discussed here; the details of these two methods will be discussed in a future article.

where y is the response (area), x is the concentration, m is the slope of the curve, and b is the y-intercept. (Even though the relationship is linear, we still call it a curve.) When the curve goes through the origin (x = 0, y = 0), b = 0, and the curve can be expressed as