HAWACH To Talk About The 30 FAQs About HPLC Column (Chapter 5)

Q21: Why is there peak broadening?
1. The sample volume is too large: use the mobile phase to match the sample, the total sample volume is less than 15% of the first peak;
2. Cause peak expansion in the injection valve: discharge bubbles before and after injection to reduce diffusion;
3. The sampling rate of the data system is too slow: the set rate should be greater than 10 points per peak;
4. The time constant of the detector is too large: set the time constant to 10% of the half-width of the first peak of interest;
5. The viscosity of the mobile phase is too high: increase the HPLC column temperature and use a low-viscosity mobile phase;
6. The volume of the detection pool is too large: use a small volume pool to remove the heat exchanger;
7. Retention time is too long: increase the content of strong solvent during isocratic elution, gradient elution can also be used;
8. The external volume of the HPLC column is too large: reduce the connecting pipe diameter and connecting pipe length to very small;
9. Sample overload: enter small concentration and small volume samples.

Q22: Why do peaks sometimes appear during the experiment?
The mobile phase used has absorption at the detection wavelength, and if the solution has no absorption at this wavelength or absorbs lower than the mobile phase, caves will appear in the mobile phase and peaks will appear after passing through the HPLC column.

Q23: Why are there “fat” peaks and “flat” peaks? How to avoid it?
Injecting a large volume of sample with a strength greater than that of the mobile phase usually damages the quality of the chromatogram and results in “fat” and flat peaks. The following rules should be followed to choose a solvent to dissolve the sample:
A. Dissolve the sample with the mobile phase and inject it.
B. Use a large volume of weak solvent to dissolve the sample. For example, reverse phase chromatography uses water to dissolve the sample for injection. The main disadvantage is that after each injection, a large negative peak appears at the beginning of the chromatogram, and sometimes the sample peak is also affected.
C. Dissolve the sample with a strong solvent when needed.

HAWACH To Talk About The 30 FAQs About HPLC Column (Chapter 5)