What causes the tailing of HPLC column?
High performance liquid chromatography column is composed of high pressure liquid pump separation system, detector and liquid chromatography column (including column temperature control system). The performance of liquid chromatography column is directly related to the accuracy and applicability of separation and analysis.What causes the tailing of HPLC column?
1. Physical damage of column
The physical damage of chromatographic column is the root cause of peak tailing. The solution is to replace the new column.
2. Packing contamination in the column
Impurities in mobile phase and sample are the main pollution sources of chromatographic column.
At least pure solvents should be used for analysis. The water used in the mobile phase should be ultra pure water or double distilled water from all glassware. Before use, filter with 0.45um solvent microporous filter (filter) to remove the possible particles. The mobile phase is recommended to be used and prepared at present. For the solution containing salt, special attention should be paid to bacteria or precipitation. In addition, it is necessary to ensure that the container for storing mobile phase is clean.
For complex samples, 0.45um solvent microporous filter (filter) or sample pretreatment column can be selected to pretreat the sample to ensure that there are no particulate impurities in the sample. If the sample is inconvenient to handle, a protective column should be used. When the packing in the column is polluted, the screw at the head of the column can be removed, and the contaminated filler in the front section of the column can be dug out with special tools, and then filled with the same filler again to repair. Or wash the chromatographic column (about 20 to 30 times the volume of the column, or as the case may be; in addition, the detector can not be connected at this time), wash the pollutant out of the column, and then use it according to the direction indicated by the column.
3. There are foreign matters at the column inlet
When it is determined that there is foreign matter at the column inlet, remove the screw on the column head, take out the filter cap and put it in 20% nitric acid solution, clean it with ultrasonic wave for about 20 minutes, then put it in ultra pure water, and clean it with ultrasonic wave for about 10 minutes, and then put it into the chromatographic column again.
4. The sample concentration is too high to cause column overload
The overload of the sample on the column can cause the peak broadening and tailing (or tongue stretching).
The adverse effect can be eliminated by appropriately reducing the injection volume or sample concentration (the sensitivity of the detector can be increased if necessary) until the peak shape and retention time are no longer changed. The resolution can also be improved by reducing the injection volume. Under normal conditions, the injection volume of each compound in the sample is maintained in the range of 3-50ug in a 150 × 4.6mm column, which will not cause obvious overload.
5. The sample solvent is not correct
Choose the appropriate sample solvent to eliminate unnecessary interference. The mobile phase should be used to dissolve the sample.
6. Out of column effect
The out of column effect (i.e., the pipeline between injection valve, chromatographic column and detector is too long, the diameter is too large, the pipe joint does not match, and there is dead volume) is one of the main factors affecting the column efficiency. Therefore, under possible conditions, the connecting pipeline at both ends of the column should be as short as possible, the inner diameter of the connecting tube should be as small as possible, the incision must be smooth and smooth, and the dead volume should be reduced as much as possible, so as to prevent the situation that the real column efficiency can not be reflected due to the sample diffusion.
7. Insufficient or inappropriate buffer
In the separation with poor buffer or low ionic strength, the retention time reproducibility is poor and the peak shape is tailed. Increasing the buffer concentration (matching the sample size) can improve this situation.
8. Silanol group action
A careful analysis of the surface properties of the packing in the column shows that the surface of the packing material is about half covered by the bonding phase, and the rest is the unconjugated silanol group. The so-called retention is the result of the interaction between the sample molecules and the bonding phase. However, the interaction between acidic or basic compounds and the residual silanol groups or metal impurities on the surface of silica gel leads to double retention mechanism, thus leading to peak tailing.
In order to reduce the peak tailing, 25mm triethylamine (inhibitor) can be added into the mobile phase. Triethylamine has strong interaction with silanol group, which can reduce or block the interaction between sample molecule and silanol group, so as to ensure the normal retention mechanism and greatly alleviate the peak shape tailing. Using long chain silyl alcohol group inhibitors, although the effect is slow, but the persistence is longer than triethylamine. In addition to the polar interaction between the amino group and the silanol group, the nonpolar part of the macromolecule and the stationary phase will also react in reverse phase, resulting in the 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 the tailing of some compounds. The metal can be brought in by the packing itself, or the metal filter at the inlet of the column can be slowly dissolved by the corrosive mobile phase and deposited in the column packing with the mobile phase. For example, after the C18 column packing is contaminated by metal, the acid / alkaline sample tailing can be caused. The packing can be bonded after alkaline washing / pickling, and the peak obtained is sharp and symmetrical.
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