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.
We can't share the complete details of the methods because they are proprietary, but the key elements follow. A 10 ppm concentration
of sample is prepared in distilled water or in 100% methanol, and 20 μL of this solution is injected. A 250 mm × 4.6 mm C18
column packed with 5-μm diameter particles is used with UV absorbance detection at 210 nm. For compound A (method A), a mobile
phase of 60:40 methanol–water is used at a flow rate of 1.1 mL/min; for compound B (method B), the mobile phase is 70:30 methanol–water
with a flow rate of 1.4 mL/min. The data are summarized in Table I. Both compounds (A and B) are well retained with k-values of approximately 2.6 in each case. You can see that the peak area differences between injection in methanol and water
are ≤0.1% for both methods. However, the peak heights are approximately 30% larger for the water injections.
Table I: Chromatographic results for two methods A and B
Effect of Injection Solvent
The chromatograms of Figure 1 illustrate the influence of the injection solvent on the appearance of peaks in the chromatogram
for another sample (1). In each case, the reversed-phase column was operated with an 18% acetonitrile–buffer mobile phase,
where the buffer was 81:1 water–acetic acid. Injection volumes of 30 μL were made in various injection solvent compositions.
Figure 1: Chromatograms for caffeine (first peak of each chromatogram) and salicylamide (second peak). Injections of 30 μL
of ~1 mg/mL of each analyte dissolved in (a) acetonitrile, (b) mobile phase, (c) 81:1 water–acetic acid, and (d) water. Column:
250 mm × 4 mm, 10-μm dp; mobile phase: 18:81:1 acetonitrile–water–acetic acid; flow rate: 1 mL/min. Adapted from reference 1.
First, consider the cases where the injection solvent is no stronger than the mobile phase (Figures 1b–1d). 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 1a 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 1b–1d, where the peaks are broader
and shorter as less water is used in the injection solvent.