Usually we encounter various strange problems in the process of doing experiments. Among them, the double peaks of the chromatographic peaks can be said to be a type of problems that are often encountered.

In HPLC analysis, when the chromatographic column is normal, the sample sensitivity is sufficient, the analysis method is suitable, and the chromatographic peak is under the condition of short peak time (excluding gradient), the peak shape should be symmetrical and sharp. However, when the understanding of the sample is insufficient, the method is not appropriate, and the sample processing method and injection method are unreasonable, various unexpected problems will occur, and it is difficult to make a reasonable explanation for the chromatographic peaks, especially for novices. Chromatographic double peaks refer to a substance that has double peaks in the chromatogram. There are four reasons for this situation.

1. Chromatographic blockage or contamination

If you find that each chromatographic peak appears double peaks when analyzing the sample (the faster the peak appears, the less likely it is to appear double peaks), especially when using a single pure substance, you can judge that the chromatographic column has a problem (the column head is damaged or the fixed phase transition of the column head is dirty or lost). If the sample injection volume is small, the original chromatographic column is normal, and the shape of chromatographic peaks is mostly one large peak with one small peak, which is not necessarily trailing. Generally, this should be due to the blockage of the column head end, and the reverse connection of the chromatographic column for washing and maintenance can be solved under normal circumstances. If the peaks are tailing and the strength of the two peaks is not much different, the column head filler is more likely to be contaminated or the bonded phase is lost. At this time, the chromatographic column can be repaired or a new chromatographic column can be used. It is recommended that the maintenance be handled by the manufacturer.

2. Inappropriate solvent polarity and sample amount

General books and literatures do not mention this content, and this is indeed a very important reason for the emergence of bimodal. At present, the HPLC analysis is mostly reversed-phase chromatography, and the mobile phases are mostly methanol, acetonitrile, water and various additives to improve the separation performance. The sample is generally dissolved in a solvent soluble in the mobile phase. The best dissolution method is to use the mobile phase, but many cases are inconsistent. When reagents with high polarity strength are used as solvents, such as pure methanol, pure acetonitrile and pure ethanol, while the analytical system is mainly composed of water, the sample injection volume is large, such as 20ul. A single pure substance has two peaks, and the second peak is smaller than the first peak (each time is different), and it is trailing. The retention time will be advanced (relative to the small injection volume). Reduce the injection volume by more than half, and the peak type will become normal. This is because the polarity of the sample solvent is too different from that of the mobile phase, and the mobile phase has no time to dilute it to equilibrium.


Another reason is that the injection volume is not necessarily large, but the concentration is very large. The two peaks on the chromatogram are close together, and are basically the same height without trailing (if the peak appears quickly, it may also be a chromatographic column problem). It is OK to dilute the sample and inject it again. This is caused by excessive injection volume and overload of chromatographic column.

3. The characteristics and H value of the sample are not known

Some samples have tautomerism due to the characteristics of their chemical structures, and the tautomers cannot be separated, but exist in a dynamic equilibrium. In chromatographic analysis, under a specific condition, a substance will appear double or even triple peaks. At this time, the double peaks are generally close to each other, and the height is basically the same, and there is no tailing. If the conditions are slightly changed, especially if the pH is changed, the double peak phenomenon will disappear.

The effect of pH on peak shape is evident during buffer mobile phase equilibration, and this double-peak situation is often encountered due to continuous changes in pH when injecting continuously. In addition, during sample analysis, the pH of the mobile phase should be kept away from the isoelectric point of the analyte as far as possible, otherwise it is easy to cause double peaks. When ion-pairing reagents are used for analysis, poor selection of conditions can easily lead to the generation of double peaks.

4. Unreasonable instrument parameter setting

The reference wavelength is set incorrectly, such as setting the analysis wavelength to 254nm and the reference wavelength to 400nm, which may not affect most compounds. But if the tested compound also has strong UV absorption at 400nm, higher than 254nm. In this way, when the peak is out, due to the offset effect of the background, the original peak becomes two symmetrical peaks, and if the peak valley between the two peaks is reversed by 180 degrees, it happens to be a complete peak. In this case, the reference wavelength should be set larger, or canceled.

The above are the reasons for the double peaks and the corresponding solutions that the editor has sorted out for you. High performance liquid chromatography is a very precise analysis system. Once abnormal peak shape occurs, you need to carefully investigate the cause and find a suitable solution.

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