Reasons for Peak Tailing of HPLC Column
High performance liquid chromatography columns are frequently used in many industries and today, HAWACH will discuss 9 reasons for peak tailing of high performance liquid chromatography columns and hope we will use it more efficiently.
1. Physical damage to the HPLC column
Physical damage to the chromatographic column is the root cause of peak tailing. The solution is to replace the column with a new one.
2. Packing contamination in the column
The impurities in the mobile phase and the sample are the main sources of contamination of the column. The various solvents used in the mobile phase are at least analytically pure, and try to use chromatographically pure reagents. The water used in the mobile phase should be ultrapure water or double distilled water in all glassware. Use 0.45um solvent microporous filter (filter) to remove possible particles before use. The mobile phase is recommended to be used now. For salt-containing solutions, pay special attention to bacteria or precipitation when placed in long-term.
In addition, it is necessary to ensure that the container storing the mobile phase is clean. For complex samples, you can choose 0.45um solvent microporous filter or sample pretreatment column to pretreat the sample to ensure that the sample does not contain particulate impurities.
If the sample is inconvenient to handle, use a guard column. When the packing in the column is contaminated, the column head screw can be removed, and the contaminated packing in the front section of the column can be dug out with a special tool, and then refilled with the same packing for repair. Or flush the column with a mobile phase that can dissolve the contaminants in the opposite direction of the column used (about 20 to 30 column volumes, or depending on the specific situation; in addition, the detector cannot be connected at this time) to flush the contaminants Take out the column and use it in the direction indicated on the chromatographic column.
3. Foreign matter at the entrance of the HPLC column
When it is determined that there is a foreign body at the entrance of the column, you can remove the column head screw, take out the filter cap and place it in a 20% nitric acid solution, clean it with ultrasonic for about 20 minutes, then place it in ultrapure water and clean with ultrasonic for about 20 minutes. After 10 minutes, reload it into the chromatographic column.
4. The sample concentration is too high and the column is overloaded
Sample overload on the column can cause peak broadening and tailing (or tongue sticking). Appropriately reduce the injection volume or sample concentration (if necessary, increase the sensitivity of the detector) until the peak shape and retention time no longer change, this adverse effect can be eliminated. Reducing the injection volume can also improve its resolution. Under normal circumstances, the injection volume of each compound in the sample in a 150×4.6mm column is kept within the range of 3～50ug, which will not cause obvious overload.
5. The sample solvent is wrong
Choose a suitable sample solvent to eliminate unnecessary interference. Dissolve the sample with mobile phase.
6. Extra-column effect
Extra-column effects (that is, the tubing between the injection valve, the HPLC column and the detector is too long, the diameter is too thick, the tubing joints are not matched, and there is dead volume) is one of the main factors that affect the efficiency of the chromatographic column. Therefore, under possible conditions, the connecting pipes at both ends of the HPLC column should be as short as possible, the inner diameter of the connecting pipe should be as small as possible, the cut must be flat and smooth, and the dead volume should be reduced as much as possible to prevent the HPLC column from not reflecting due to sample diffusion. Real column efficiency and other situations occur.
7. Insufficient or inappropriate cushioning
In a separation with poor buffering or low ionic strength, poor retention time reproducibility and peak tailing may also occur. Increasing the buffer concentration (to match the sample size) can improve this situation.
8. Silanol group function
Careful analysis of the surface properties of the packing in the HPLC column shows that the surface of the packing in the reversed-phase chromatography column is about half covered by the bonded phase, and the rest are unbonded silanol groups. The so-called retention refers to the result of the interaction between the sample molecule and the bonded phase. However, the interaction between acidic or basic compounds and silanol groups or metal impurities remaining on the surface of the silica gel causes a double retention mechanism, so peak tailing occurs.
In order to reduce peak tailing, 25mM triethylamine (inhibitor) can usually be added to the mobile phase. Triethylamine can interact strongly with the silanol group, thereby reducing or blocking the interaction between the sample molecule and the silanol group to ensure the normal progress of the retention mechanism and greatly alleviate peak tailing. The use of long-chain silanol-based inhibitors has a slower onset, but has a longer duration than triethylamine.
Because in the inhibitor molecule, in addition to the polar interaction between the amine group and the silanol group, the non-polar part of the macromolecule and the stationary phase will also have a reverse interaction to produce a retention phenomenon. If the inhibitor is added to the sample, the effect will be significant.
9. Metal contamination in the column
Metal contamination (Fe, Ni, etc.) in the column can cause peak tailing of certain compounds. Metal can be brought in by the packing itself, or the metal filter at the column inlet can be slowly dissolved by the corrosive mobile phase and deposited into the column packing along with the mobile phase. For example, after the C18 column packing is contaminated with metal, the acid/alkaline sample is tailed, and the packing that can be bonded after alkaline washing/acid washing can get sharp and symmetrical peaks.