Peak Shape Changes with Increased Injection Volume

As discussed in this series, changes in peak shape are a common problem in HPLC analyses. Ideally, peaks should be symmetrical, with a Gaussian shape [D. R. Stoll, LC-GC N. Am. 39 (2021), pp. 353–362]. The symmetry of a peak may be quantified by calculating the USP tailing factor (T), as illustrated in Figure 1. A tailing factor of 1 indicates perfect symmetry, while values less than 1 are referred to as fronting and values greater than 1 as tailing. Many methods require that the tailing factors for all peaks must be within a specified range. Tailing factors that deviate significantly from 1 may decrease the resolution of peaks that elute close together, making integration more difficult [D. R. Stoll, LC-GC N. Am. 39 (2021), pp. 353–362]. Also, when the peak symmetry is poor the peak is often wider than it should be, which decreases the peak height. In applications involving the detection, and quantification of analytes present at low concentrations, this may decrease the precision of the results as well as the limits of quantitation, and detection.

An important step when developing methods for quantifying analytes that are present in low concentrations is to optimize the injection volume. Ideally, peak heights and peak areas increase linearly with the injection volume for a fixed sample composition until mass, volume or detector overload begins to occur [U. D. Neue, HPLC Columns: Theory, Technology, and Practice, Wiley-VCH, New York, 1997, pp. 355–356]. In the example shown in Figure 2A a mixture of six analytes each at a concentration of 0.2 µg/mL was separated using a 5–95% acetonitrile gradient. The injection volume was 2 µL and a 2.1 x 50 mm column was used, so the injection volume was 1.1% of the column volume. Since the general guidance is that the injection volume should be 1–10% of the column volume [Waters Knowledge Base 48961], there appeared to be room to increase the injection volume to increase the signal-to-noise ratios. When the injection volume was increased to 4 µL the chromatogram shown in Figure 2B was obtained. While the areas of all six peaks increased by a factor of two, as expected, the first two peaks didn’t increase in height, but became broader with pronounced fronting.

As discussed in the first two parts, there are several possible causes of changes in peak symmetry, including issues with the HPLC system, the mobile phase, the sample, and the column [J. W. Dolan and L. R. Snyder, Troubleshooting LC Systems, Springer Science+Business Media, New York, 1989, pp. 385–420]. As previously discussed, a good starting point for troubleshooting is to carefully analyze the chromatograms to observe whether the change in peak shape is seen for all the peaks, or only some of them. When only some of the peaks in a chromatogram show fronting peaks, as in Figure 2B, possible causes include coelution of an interfering compound, mass overload, and the use of a sample solvent that is too strong. Because the issue was observed after increasing the injection volume and the analyte concentrations are low, the latter cause seems the most likely. The sample solvent used for the chromatograms shown in Figure 2A and 2B was 50/50 v/v acetonitrile/water, chosen because some of the analytes have limited solubility in water. Since the gradient starts with an acetonitrile concentration of only 5%, the sample solvent is considerably stronger than the initial mobile phase. Because the first analytes to elute are the least hydrophobic, they are most affected by the strong sample solvent. To test this hypothesis, a series of samples was prepared with the same analyte concentrations, but with different acetonitrile/water ratios. The tailing factor results for the first two peaks are shown in Figure 3. The tailing factors for both analytes decrease as the acetonitrile concentration is increased, demonstrating that the use of a strong sample solvent is the cause of the fronting peaks seen in Figure 2B. To avoid this issue, while ensuring that the most hydrophobic analytes are dissolved, an acetonitrile concentration of 10% was chosen. The chromatogram resulting from the injection of 4 µL of a sample dissolved in 10/90 v/v acetonitrile/water is shown in Figure 2C. Now all the peaks show the expected two-fold increase in height compared to the 2 µL injection, with good peak symmetry. When optimizing the injection volume the strength of the sample solvent relative to the initial mobile phase composition should always be considered.