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.
Q24: Why does the front extension peak occur and how to solve it?
The HPLC column temperature is easy to cause the front extension peak. Some samples show the front extension peak at room temperature, and the phenomenon of the front extension peak disappears after increasing the temperature. In ion-pair chromatography, another reason for the extension of the peak is the use of the non-mobile phase as the sample solvent.
Therefore, in ion-pair chromatography, only the mobile phase is required to dissolve the sample, and the injection volume should not be too large, otherwise, it will cause pre-posted peaks or other problems. In RP-HPLC, the strength of the sample solution is greater than that of the mobile phase, which causes a prolonged peak. Increasing the strength of the mobile phase, reducing the strength of the sample solution, and increasing the ionic strength in the ion-pair chromatography can overcome the effect of prolonged peaks. In addition, using a mobile phase to dissolve samples is a simple and practical method.
Q25: How to simply judge whether the proportional valve is leaking?
Set the pump to use a separate channel, open the purge valve at a flow rate of 5ml/min, lift the solvent filter head in other solvent bottles until it leaves the liquid level, and observe whether the solvent in these channels is flowing, and should not flow under normal conditions
Q26: What are the reason for the peak broadening?
A: The HPLC column itself degrades during use, gradually reducing HPLC column efficiency;
B: Peak width effect outside the HPLC column. A good dedicated HPLC column used in another liquid chromatography system causes a decrease in the number of plates, indicating that the new system has a great effect of peak width outside the HPLC column;
C: The chemical effect is mostly caused by the interaction between the mobile phase and the stationary phase. Changing the mobile phase can improve the broad peak.
Q27: What are the common reasons for the slow HPLC column equilibrium?
The common reason for slow HPLC column equilibration is that the components strongly adsorb to the column in the old or new mobile phase, or the concentration in the new mobile phase is small or even zero.
A: mobile phase contains amine modifier;
B: mobile phase contains ion pair reagent;
C: silica gel HPLC column;
D: tetrahydrofuran in mobile phase.
A special HPLC column can be considered for special methods. When not in use, the HPLC column should be folded down, filled with an appropriate solvent or mobile phase, sealed and stored, and no other analysis is required.
Q28: What are the requirements for internal standards?
A: The structure or physical and chemical properties of the internal standard should be similar or similar to the analyzed component;
B: The retention value of the internal standard should be slightly larger or smaller than the retention of the analyte, and the difference should not be too large;
C: The peak of the internal standard should have a good resolution with the peaks of all analytes (R is greater than 1.5), and the internal standard must not be an interference;
D: There is no internal standard with similar structure, and similar internal standards can be retained;
E: The response of the instrument to the analyte is basically the same as the internal standard, and the size of the peak area cannot be very different.
Q29: What is the “infinite diameter effect” of HPLC?
Due to the use of high-efficiency particulate stationary phase and high-pressure mobile phase in HPLC analysis, after the sample is injected into the chromatographic HPLC column with a plunger, the molecular diffusion of the sample molecule in the HPLC column is very small due to the large resistance of the column until it flows out from the HPLC chromatographic column. It is not in contact with the inner wall of the HPLC column, so the peak shape expansion caused by the chromatogram is very small, which can maintain high column efficiency.
Q30: How to evaluate a detector?
A. Noise: Generally, noise refers to the high-frequency noise and irregular fluctuations of the baseline caused by the electrical components of the instrument, temperature fluctuations, linear pulses of voltage, and other non-solute effects;
B. Baseline drift: Drift is a slow upward or downward movement of the baseline, which can be observed for a long time (0.5~1h). It can mask noise and small peaks. Drift is related to the entire liquid chromatography system, not only caused by the detector;
C. Sensitivity (smaller detection concentration or smaller detection amount): In a particular separation work, whether the detector has sufficient sensitivity is very important. When comparing detectors, the performance index of sensitivity is often used. Sensitivity refers to the concentration or quality of the solute that enters the detector within a unit time when the signal-to-noise ratio (signal-to-noise ratio) is equal to 2;
D. Linear range: When performing quantitative analysis, the detector is expected to have a wide linear range, so that the main components and trace components can be detected simultaneously in one analysis;
E. Cell volume of the detector: it should be less than 1/10 of the elution volume of the dead-time chromatographic peak that eluted earlier, otherwise severe off-column band expansion will occur.