Chromatographic bimodal refers to the same substance, which shows bimodal in the chromatogram, which makes us mistake it for two substances. Why does this happen and how to avoid it during the experiment? Today, Xiaobian will talk with you.

In HPLC analysis, when the chromatographic column is normal, the sample concentration is appropriate, the analysis method is appropriate, and the peak time is short, the peak shape should be a Gaussian peak, symmetrical and sharp. However, in practice, if the sample properties are not understood, the pretreatment is inappropriate or the analysis method is unreasonable, the peak shape will be abnormal, and the double peak phenomenon is one of the common problems of liquid chromatography. The causes of chromatographic double peaks are generally as follows:

Chromatographic column

If it is found that each chromatographic peak is bimodal when analyzing the sample, especially when analyzing a single pure substance, it can be determined that there is a problem with the chromatographic column, which is generally caused by the damage of the column head. If the injection volume is small, the original chromatographic peak is normal, and the peak shape is mostly one large peak with one small peak. When it is not necessary to drag the tail, it should be considered that the column head end is blocked, and the chromatographic column can be reversed (Welch chromatographic column can be reversed), washed with mobile phase or acid or other organic solvents, washed away the substances blocked at the column head end, and then connected to the test, which will usually be improved. Of course, there can be no recoil, and the positive impact sometimes has an effect.

If there is little difference between the strength of the two peaks, the possibility of dirty packing at the column head end or column loss is greater. At this time, the column head can be unscrewed, the sieve plate can be removed for ultrasound, the contaminated packing at the column head end can be scraped off, and then tightened after filling with new packing. However, this is best operated by professionals and can not be done often, otherwise the chromatographic column will be scrapped due to the reduction of column efficiency after a few times. If the above operation still cannot solve the problem, it may be caused by column collapse, and the chromatographic column needs to be replaced.


At present, HPLC analysis is mostly reversephase chromatography, mobile phase is mostly methanol, acetonitrile, water, adding a variety of additives to improve the separation performance. The sample is generally dissolved in a solvent that dissolves with the mobile phase, and the best solution is to dissolve with the mobile phase. In practical analysis, sometimes a certain buffer is added for the solubility or stability of the sample, but the acidity and alkalinity of the buffer may lead to sample transformation, resulting in bimodal phenomenon. Change the buffer, adjust the pH of the solvent, or configure the sample with the mobile phase.

In addition, the sample should be prepared and used now to avoid the solvent effect caused by the change of the organic phase ratio and pH value of the sample solution.

Injection volume

When dissolved in solvent polarity strength of reagent samples, such as methanol, acetonitrile, such as ethanol, and analysis system is given priority to with water, if the sample sample quantity is big, such as into 20 mu L, target at this time there will be a bimodal, smaller than in the second peak (is different every time), and the tail, the retention time in advance () relative to sample quantity is little, will halve sample quantity, The peak pattern will return to normal. This is caused by the fact that the polarity of the solvent of the sample is too different from that of the mobile phase, and the mobile phase has no time to dilute it to reach equilibrium. In this case, the sample amount needs to be reduced or the mobile phase needs to dissolve the sample.

Another reason is that the sample quantity is not necessarily large, but the absolute amount is very large. The double peaks on the chromatogram are close together, basically the same height, without trailing (if the peak is very fast, it may also be the column problem). The sample is diluted and then injected, which is caused by the overload of the column due to the large amount of injection.

PH value

The influence of pH value of the system on chromatographic bimodal appears in all links, especially in the process of buffer mobile phase equilibrium. This kind of double peak is often encountered when continuous injection is affected by the continuous change of pH. In addition, during sample analysis, the pH of the mobile phase should be as far away from the isoelectric point of the analyte as possible, otherwise it is easy to cause double peaks. In the analysis of ion pair reagents, double peaks can also be caused if the liquid phase conditions are not selected well.

Sample characteristics

Some samples have isomers due to the characteristics of their chemical structure. Some samples can not see double peaks on the UV chromatogram, but under LC-MS, it is obvious on the total ion flow diagram (TIC) of the mass spectrum, such as acetamiprid.

Instrument parameter setting

The reference wavelength is set incorrectly, for example, the analysis wavelength is set at 254 nm and the reference wavelength is set at 400 nm, which may not affect most compounds. However, if the tested compound also has strong UV absorption at 400 nm, which is higher than 254 nm. In this way, when it comes out of the peak, due to the deduction of the background, one summit has become two symmetrical peaks, and if the peak valley between the two peaks is reversed by 180 degrees, it happens to be a complete peak. At this time, set the reference wavelength higher or cancel.

Of course, if the instrument has a large dead volume, it may also lead to double peaks.

In the case of insufficient understanding of samples, improper analysis methods, unreasonable sample processing methods and injection methods, various unexpected problems will occur, and it is difficult to make a reasonable explanation for chromatographic peaks, especially for novices. Therefore, understanding the causes of each abnormality and taking reasonable improvement measures according to the causes are the most effective way to avoid the occurrence of abnormal peaks.

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