Internal standards are commonly used in chromatography to calculate the concentration of an analyte. For any particular detector, the relative response factor for the analyte compared to the internal standard must be determined first. Calibrating the linearity of the response factor for an analyte compared to the internal standard requires making a series of solutions with the same concentration of standard, and a varying concentration of analyte. Plotting the response of the analyte relative to the standard (peak area of analyte/peak area of standard) versus the concentration of the analyte relative to the standard ([analyte]/[standard]) should produce a straight-line graph whose slope is the relative response factor, RRF. area of analyte signal/area of standard signal = RRF(concentration of analyte/concentration of standard)
The table at the right gives some data for a chromatographic analysis of an analyte, using an internal standard. (Peak areas of each are measured by a mass spectrometer). The volume injected for each sample is similar, but not exactly the same.
Sample Analyte Conc. Standard Conc. Anaalyte peak area Standard peak area
1 1.0 10.0 307 2995
2 5.0 10.0 3649 6201
3 10.0 10.0 3047 2813
A. Calculate the peak area ratio (analyte/standard) and the concentration ratio [analyte]/[standard] for each of the samples, and calculate RRF for each sample (peak area ratio/concentration ratio).
B. Using Excel, plot the area ratio on the y-axis and the concentration ratio on the x-axis and use the SLOPE function or the trendline to get a slope for the line, which should be the weighted average of RRF. Compare this value to the simple average of the three values calculated above.