Making an HPLC Calibration work (Part 1)

Calibration of an HPLC method is necessary to give quantitative results. However there is more than type of calibration available, and each has its merits and limitations. The aim is to select the most appropriate one, and
then to avoid the myriad of small pitfalls, because no-one wants almost the right answer!

FIVE TYPES OF CALIBRATION

1. External Standard Calibration
This involves analysing a series of standards covering the concentration range of interest. For example:
Level 1 - 40mg/l
Level 2 - 70mg/l
Level 3 - 100mg/l
Level 4 - 130mg/l
Level 5 - 160mg/l

The peak for each component is integrated and identified and the peak area is plotted against concentration to give a calibration curve (see below). Each component should be present in the standard mix, and hence a series of calibration curves result from a single set of injections, one curve for each component. When an unknown sample is run, the peaks are integrated and identified, and the peak areas are related to a concentration from the calibration graph. If the calibration curve is a straight line, and it goes through zero, it is valid to use a single point calibration. For example, only the highest standard is run, and the line passes through this point and zero. If the points do not give a straight line, or the curve does not pass through zero, a multi-point calibration should be used, and normally five concentration levels are required. Samples are run after the calibration standards, and hence it is recommended that known standards (QC Standards) are run periodically as unknowns to check the integrity of the calibration. An external standard calibration works provided that there is no variation in injection volume, and that the HPLC conditions do not change during the run. It is frequently asked if it is acceptable to use a single standard and inject a series of different volumes to construct the calibration. This is generally not acceptable. It is unusual for the accuracy and precision of the autosampler to be good enough, and since a constant volume of sample will be injected, the system should be calibrated in the same way.