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Chromatographic double peaks refer to the phenomenon where the same substance appears as two peaks in the chromatogram, leading analysts to mistakenly believe it contains two substances. So, what are the main reasons for the occurrence of double peaks? Today, Sister Xiao Xi will discuss with you the reasons behind "double peaks" in liquid chromatography.

In HPLC analysis, under conditions where the column is normal, sample concentration is appropriate, the analytical method is suitable, and the chromatographic peak elutes in a relatively short time, the peak shape should be symmetrical and sharp. However, in practice, if the sample is not well understood, sample pretreatment is improper, or the analytical method is unreasonable, abnormal peak shapes may occur, with double peaks being one of the common issues in liquid chromatography.

The reasons for chromatographic double peaks generally include the following:

Chromatographic Column

If double peaks appear for every chromatographic peak during sample analysis, especially when using a single pure substance, it can be concluded that the column is problematic, usually due to column head damage or contamination of the stationary phase at the column head.

If the injection volume is small and the column was previously normal, the peak shape often appears as one large peak with a small peak, not necessarily tailing. This is generally caused by a clogged column head. Reversing the column, flushing with mobile phase, acid washing, or using other solvents to remove residues clogging the column head, and then reversing it back usually resolves the issue. Of course, sometimes normal flushing without reversal may also work. If the peak tails and the double peaks are of similar intensity, it is more likely due to contamination or loss of the stationary phase at the column head. In this case, the injector head can be unscrewed, the frit ultrasonicated, a portion of the packing material at the column head scraped off, and new packing material added before tightening. However, this requires high technical skill and should not be done frequently, as it may reduce column efficiency and render the column unusable after a few attempts. If the above methods do not resolve the issue, it may be due to column collapse, necessitating column replacement.

Solvent Issues

Most HPLC analyses today use reversed-phase chromatography, with mobile phases typically consisting of methanol, acetonitrile, water, and various additives to improve separation. Samples are usually dissolved in a solvent compatible with the mobile phase, ideally using the mobile phase for dissolution. In practice, small amounts of buffer may be added for sample solubility or stability, but the acidity or alkalinity of the buffer may cause sample transformation, leading to double peaks. In such cases, the buffer should be replaced, the solvent pH adjusted, or the sample prepared using the mobile phase.

Additionally, samples should be prepared and used immediately to avoid changes in the organic phase ratio or pH of the solvent, which can cause solvent effects.

Injection Volume

When using solvents with high polarity strength, such as pure methanol, pure acetonitrile, or pure ethanol, in an analysis system where water is the main component, a large injection volume (e.g., 20 µl in a quantitative loop) may cause a single pure substance to exhibit double peaks. The second peak is usually smaller than the first (varies each time) and tails, with retention time shifting earlier (relative to smaller injection volumes). Reducing the injection volume by more than half will normalize the peak shape. This occurs because the solvent polarity of the sample differs significantly from the mobile phase, and the mobile phase cannot dilute it quickly enough to achieve equilibrium. In such cases, reducing the injection volume is necessary.

Another reason is that the injection volume may not be large, but the absolute amount is significant. The double peaks on the chromatogram are close together, nearly equal in height, and without tailing (if elution is rapid, it could also be a column issue). Diluting the sample before injection resolves this issue, as it is caused by column overloading due to excessive sample amount.

Sample Characteristics

Some samples, due to their chemical structure, exhibit tautomerism, where tautomers cannot be separated and exist in a dynamic equilibrium. During chromatographic analysis, under specific conditions (e.g., pH, mobile phase polarity, temperature), a substance may show double peaks that are close together, nearly equal in height, and without tailing. Slight changes in conditions, especially pH, can make the double peaks disappear, as seen with erythromycin, for example.

Some samples may not show double peaks in UV chromatograms, but in LC-MS with mass spectrometric detection, the total ion chromatogram may clearly display them, as with the pesticide acetamiprid.

pH Value

The pH of the system affects chromatographic double peaks at various stages (as mentioned earlier), particularly during the equilibration process of buffer mobile phases. During continuous injections, double peaks often occur due to continuous pH changes. Additionally, during sample analysis, the pH of the mobile phase should be kept away from the isoelectric point of the analyte to avoid double peaks. When using ion-pair reagents, improper condition selection can also easily lead to double peaks.

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