The solvent chosen for injection can affect the appearance of chromatographic peaks.
A recent observation in the laboratory of one of the authors (B.A.) of this column highlights a problem that has been mentioned in "LC Troubleshooting" discussions before, but its importance justifies additional discussion about the selection of the injection solvent to use with a liquid chromatography (LC) method. In this particular case, it was observed that the peak areas were constant with two different injection solvents, but the peak heights were not. Similar results were observed with methods for two different compounds (A and B). Let's see what the likely cause is for this problem.
Effect of Injection Solvent
First, consider the cases where the injection solvent is no stronger than the mobile phase [Figures 1(b)–1(d)]. In each case, the retention times of each peak are approximately the same. Although we don't have data for the peak areas in each case, we'll assume they are the same, because the same volume of the sample at the same concentration was used in each case. Note, however, that the peak heights differ. Water is the weak mobile-phase solvent in this example, and the less water that is present in the injection solvent, the shorter the peak heights are. These observations are consistent with the problem presented at the beginning, where peak areas and retention times were constant, but peak heights dropped when less water was used in the injection solvent.
Next, notice what happens when the sample of Figure 1(a) was injected in 30 μL of 100% acetonitrile — the peaks are broader, distorted and at shorter retention times. This example continues the pattern of Figures 1(b)–1(d), where the peaks are broader and shorter as less water is used in the injection solvent.