Those with experimental experience know that in HPLC analysis, if the performance of the chromatographic column is normal, the sample concentration is appropriate, the analysis method is appropriate, and the chromatographic peak should be symmetrical and sharp under the condition that the peak time is short, as shown in Figure 1 below. shown.
Generally, when there are double peaks in chromatography, we can check for the following reasons. However, in actual operation, if the sample is not understood, the pretreatment is not appropriate, or the analysis method is unreasonable, the peak shape will be abnormal. Peak phenomenon is one of the common problems in liquid chromatography, as shown in Figure 2 below. Chromatographic double-peak refers to the fact that it is obviously the same substance, but there are double peaks in the chromatogram, which makes the experimenter mistakenly think that it contains two substances, thus affecting the judgment.
When chromatographic double peaks generally appear, we can check for the following reasons–
- Incorrect connector connection
If you find problems with the shape of each peak during use of the column, first check for connection problems. The line may be too long or too short, which can lead to leaks or peak bifurcation/tailing. Leaks will occur if the line is too long and the seals are not positioned properly. If the tubing is not pushed out far enough, a dead space will be created, creating a mixing chamber, resulting in extra-column volume and poor peak shape.
If each chromatographic peak is found to have double peaks during sample analysis, especially when a single pure substance is used, and all connectors are connected normally, it can be determined that there is a problem with the chromatographic column, usually the column head is damaged or fixed. caused by contamination.
If the injection volume is small, the original chromatographic column is normal, and the shape of the chromatographic peaks is mostly a large peak with a small peak, not necessarily tailing. This is generally because the column head is blocked. Connect the chromatographic column in reverse, rinse or acid wash with mobile phase Or other solvents, flush out the residue blocking the head of the column, and then reverse it, usually it will do. Of course there is no recoil, and forward rushing is sometimes normal.
If the peak is tailing, the intensity of the double peaks is not much different, and the stationary phase at the head of the column is more likely to be dirty or lost. It is recommended to perform daily or abnormal regeneration flushing according to the instructions. If flushing does not improve peak output, it is recommended to contact the column manufacturer for troubleshooting or repair.
- Guard column failure
If the sample matrix is dirty, after using for a period of time, the problem of peak splitting or tailing is likely to be caused by the failure of the guard column. Generally, if the number of plates, pressure or resolution changes by more than 10%, the guard column needs to be replaced. The guard column is a consumable item, and it is recommended to replace it directly without regeneration.
- Injection volume
When using reagents with high solvent polarity, such as pure methanol, pure acetonitrile, and pure ethanol, and the analysis system is dominated by water, if the sample injection volume is large, such as the quantitative tube is 20 μL, then a single pure substance will Double peaks appear, the second peak is smaller than the first peak (not the same every time), and tailing, the retention time will be advanced (relative to the small injection volume), the injection volume will be reduced by more than half, and the peak shape will be becomes normal. This is a problem of solvent effect caused by the polarity of the solvent of the sample and the mobile phase being too different, and the mobile phase is too late to dilute it to reach equilibrium. In this case, it is recommended to use the mobile phase as a diluent or reduce the injection volume to eliminate the solvent effect.
Another reason is that the injection volume is not necessarily large, but the absolute volume is large, and the double peaks on the chromatogram are close to each other, basically the same height, and no tailing (if the peaks are very fast, it may also be a problem with the chromatographic column. ). It is enough to dilute the sample before injecting it, which is caused by overloading the column due to the excessive injection volume.
- Solvent problems
At present, HPLC analysis is mostly reversed-phase chromatography, and the mobile phases are mostly methanol, acetonitrile, and water, and various additives are added to improve the separation performance. The sample is generally dissolved in a solvent that is compatible with the mobile phase, and the best dissolution method is to dissolve in the mobile phase. In actual analysis, sometimes a little buffer is added for the solubility or stability of the sample, and the acidity of the buffer may lead to the conversion of the sample, resulting in a double peak phenomenon. At this point, you need to change the buffer, adjust the pH of the solvent, or configure the sample with the mobile phase.
In addition, the samples should be prepared and used immediately to avoid solvent effects caused by changes in the organic phase ratio and pH value of the sample solution.
- Sample Characteristics
Some samples have tautomerism due to the characteristics of their chemical structures, and the tautomers cannot be separated, but exist in a dynamic equilibrium. During chromatographic analysis, under a specific condition (such as pH value, mobile phase polarity, temperature, etc.), a substance will appear double peaks, the double peaks are very close, basically the same height, no tailing, and the conditions are slightly With a change, especially in pH, the bimodal phenomenon will disappear.
The influence of the pH value of the system on the chromatographic double peaks appears in each link (mentioned above), especially in the process of buffer mobile phase equilibrium. Such double peaks are 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.