Question

How can you combine standard addition and internal standard techniques in an HPLC experiment?

Answer 1

An internal standard in analytical chemistry is a chemical substance that is added in a constant amount to samples, the blank and calibration standards in a chemical analysis. This substance can be used for calibration by plotting the ratio of the analyte signal to the internal standard signal as a function of the analyte standard concentration. This is done to correct analyte losses during sample preparation. The internal standard is a compound that must be show similar behaviour to the analyte.

This ratio for the samples is then used to obtain their analyte concentrations from a calibration curve. The internal standard used needs to provide a signal that is similar to the analyte signal in most ways but sufficiently different so that the two signals are readily distinguishable by the instrument.

An external standard is like the internal standard (known behaviour), but is not added to the unknown. Rather it is run alone, as a sample, and usually at different concentrations, so you can generate a standard curve. Again, the peak areas are related to the known amounts of external standard run. External standards do not correct for losses that may occur during preparation of the sample, such as extraction, centrifugation, evaporation, etc. Internal standards would correct for this if added to the unknown at the beginning of the sample preparation.

The measurement uncertainty based on the Guide of Uncertainty Measurement (GUM) depends on the proper combination of various sources of uncertainty and therefore the mathematical model used to calculate the appropriate analyte in question, from a calibration curve with external or internal standard.

Internal standardization is a more effective technique than external standardization, because lower measurement uncertainty values were obtained, once that the internal standardization corrects more effectively the greater uncertainty source - bias correction factor.