The broad peaks split peaks and variable peak heights in HPLC may be imperfectly filled sample in the sample loop or injector, incompatibility of sample solvents with the mobile phase. It is good to dissolve and inject the sample components in the mobile phase whenever possible. Or else, ensure that the injection solvent is of lower strength as compared with the mobile phase.
Keep in mind that several autosamplers use distinct syringe wash solutions. Ensure that this solution is weaker than the mobile phase and is compatible with it. This is particularly significant while exchanging between reversed and normal phase analyses.
The guard columns and filters are preventing the strongly retain compounds and particles to accumulate on the HPLC column. The lifespan of such devices depends on the composition of the mobile phase, the pH of the mobile phase and purity of the sample, etc. As these products become plugged or contaminated with particles, hence the system pressure increases and peaks getting broad.
Some of the causes of peak tailing in HPLC chromatography are given below.
- Whenever possible prepare the sample in the same mobile phase that you use for separation.
- Don’t overload the column.
- If the retention time is too long it shows peak tailing. Modify the method thus that peaks elute earlier.
- Large amounts of extra-column cause peak tailing hence where possible, reducing tubing diameter and length to protect, especially in post-columns.
- The precipitation of the sample causes a slow dissolution of the analytes and it causes peak tailing so make sure that the molecule is properly soluble with your solvent.
- If the detector time too slow, increase the detector Hz rate.
- If the column volume too large it causes of tailing hence reduce the lengths and tubing diameters where possible, mainly post-column.
- Check the pKa value of the analyte and maintain the pH of the mobile phase as required (+/- 2 pH of pKa).
- Use of a contaminated column.
- Obstructed or dirty guard columns and filters cause tail.
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