Ethyl Paraben, retention

Figure 10.5. HPLC chromatogram of ethyl paraben used as a reference application for problem diagnostics. The inset shows the HPLC conditions and the typical performance parameters such as plate count, pressure, and retention time.

The earlier gradient start from %B = 45% down to 10% in steps of five causes a significant change in the separation. The two peaks for ethyl paraben and benzene even move past each other. Interesting is that the last peak is shifted toward the end only by almost a minute (differences between 0.97 and 1 are shaded gray), this means that butyl paraben in contrast to all earlier peaks remains stuck at the front of the column. The peak width of butyl paraben remains constant, although the retention times increase. The elimination concentration does not change either. 

Retention time 14-16 Internal standard ethyl paraben Limit of quantitation 7.5 pg/mL... 

With only two chromatograms at appropriate isocratic conditions, the retention times for all other isocratic conditions can be predicted provisionally. Assume that we have a 15 cm SunFire column in the drawer as well as a test mixture of methyl-, ethyl-, propyl-, and butyl-paraben. Then we can start with, for example, 55% methanol and 45% water and obtain the chromatogram in Figure 3.12. 

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