Calibration with an Internal Standard

Some of the factors mentioned earlier, which introduce error when calibrating with external standards only, can be compensated for by introducing a constant amount of an IS in both the unknown and the standard calibration samples. The IS should be a compound with a chanical nature similar to that of the analytes, so that it will pass through the sample extraction and preparation procednre similarly. In general, it should elute in the chromatographic system close to the other peaks in the system, bnt separated from all of them, so it can be identified and measured accurately. One calcnlates the ratio of the peaks responses to their concentrations. Then the ratio for each analyte peak (A) is divided by the ratio of the IS peak, to give the relative response factor (RRF), for the analyte to the IS, as summarized in Equation 11.13  

Ratio A Area Ax Cone. IS Ratio IS Area ISxCone. A 

For a given constant concentration of IS added, if we plot the ratio of the analyte peak area to the IS peak area against the analyte concentration A, we should get a standard curve whose slope is the RRF. In a fashion analogous to Equation 11,12 for external standard calibration, we can determine the ratio U of analyte of unknown concentration to IS peak area and calculate the unknown concentration C using Equation 11.14  

The criteria for acceptable linearity of least squares fit and zero intercept when plotting ratios of analyte to IS areas versus concentration are similar to the case for external standard calibrations described earlier. More than one IS can be used, both for calculating RRTs to compensate for retention time variations as well as the RRFs for improving quantitation. The variations that a quantitation IS can compensate for depend upon the point at which it is introduced in the analysis. If it is put into the final extract prior to injection on the chromatograph, it can correct for concentration variations due to evaporative volume changes, variations in injection volume, and variations in detector response. This is called an injection IS. If the IS is put into the initial sample, and into calibration standards prepared in an equivalent matrix, it can additionally correct for variations in recovery during the sample preparation process. This is called a method IS. Combined use of separate compounds for each purpose can aid in determining the causes of peak area variability. 

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