HAWACH To Talk About The 30 FAQs About HPLC Column (Chapter 4)

 Q15:  In addition to the flow rate, what other factors can cause pressure changes?

There are four factors that will bring a change of pressure. Change the composition and temperature of the mobile phase; change the column length, the HPLC column inner diameter, and the particle size of the column; the column’s sudden blocking pressure increases (normally, the column pressure increases gradually with other conditions unchanged).

Q16: What are strong solvents and weak solvents?
The peak capacity factor and retention time can be changed by changing the composition of the mobile phase or the strength of the solvent. Under certain conditions, the solvent that reduces the retention time or shortens the analysis time is a strong solvent, and the solvent that increases the retention time or extends the analysis time is a weak solvent.

Q17: How can the peak position be rearranged?
When analyzing multi-component samples, only the strength (composition percentage) of the mobile phase is changed without changing its composition. Generally, only the retention time of all components is changed, and the rearrangement of peak positions does not occur.

The rearrangement of the peak position may occur under the following conditions: the mobile phase is replaced with a strong solvent; the pH value is changed; the column packing is changed; the column temperature is changed; the composition of the mobile phase is changed (such as the addition of ion pair reagent triethylamine, etc.).

Q18: In addition to online degassing, what are the commonly used laboratory degassing methods?
Heating and reflux degassing are degassing effect are ideal, but it cannot be maintained; helium degassing has a good degassing effect and can remove more than 90% of the air, but helium is too expensive, so it is not used much; vacuum degassing’s effect is second only to helium degassing, but it is easy to cause volatile loss of sample solution during degassing; ultrasonic degassing can only remove about 30% of the air, but it is generally used in the laboratory. At present, we still strive to use online degassing, which is convenient and effective.

Q19: Occurrence and treatment of chromatographic double peaks
In HPLC analysis, when the chromatographic column is normal, the sample sensitivity is sufficient, the analysis method is appropriate, and the peak shape should be symmetrical and sharp under the condition that the peak time is short (excluding gradient).

However, when the sample is not well understood, the method is improper, the sample processing method and the injection method are unreasonable, various unexpected problems will occur, and it is difficult to make a reasonable interpretation of the chromatographic peak, especially for novices. The chromatographic double peak refers to a substance that clearly appears, but a double peak appears in the chromatogram, indicating that it contains two substances. There are four reasons for this situation.

1. Chromatography column
If you analyze the sample and find that there are double peaks in each chromatographic peak (the faster the peak, the possibility of double peaks will be reduced), especially when using a single pure substance, you can be sure that the column has a problem: the column head is damaged or the column head stationary phase dirty or lost.

If the injection volume is small, the original chromatographic column is normal, and the shape of the chromatographic peak is mostly a large peak with a small peak, not necessarily tailing, which should generally be the column head is blocked. Connect the column in reverse and rinse or pickle with a mobile phase or other solvents to wash away the residue that is stuck in the column head, and then reverse. In general, it will do.

And sometimes the recoil is normal. If the peak is tailed, the difference between the strength of the two peaks is not large, the column head stationary phase is dirty or more likely to be lost. At this time, the injection head can be unscrewed, the microporous filter can be sonicated, and the column header can be scraped off a part of the packing. Fill it with new packing and tighten, but this kind of work requires a certain technique, and it cannot do frequently. Otherwise, it will be scrapped due to low efficiency.

2. Solvent polarity and injection volume
The general HPLC books and literature will not mention this content, and this is indeed a very important reason for the occurrence of double peaks. At present, HPLC analysis is mostly reversed-phase chromatography, and the mobile phase is mostly methanol, acetonitrile, water, and various additives which are added to improve separation performance. The sample is generally dissolved in a solvent that is compatible with the mobile phase. The recommended dissolution method is to dissolve with the mobile phase, but in many cases it is inconsistent.

When using reagents with strong polar strength of solvents, such as pure methanol, pure acetonitrile, and pure ethanol, and the analysis system is mainly water, the sample injection volume is large, such as the 20ul quantitative tube. Under this condition, it is completely certain pure materials produce double peaks, the second peak is smaller than the first peak (not the same every time), and tail. The retention time will be advanced (relative to the small injection volume), reducing the injection volume by more than half, the peak will become normal.

This is caused by the polar difference between the solvent of the sample and the mobile phase, and the mobile phase is too late to dilute it to equilibrium. One of the reasons mentioned above is that the injection volume causes double peaks. The other reason is that the injection volume is not necessarily large, but the absolute volume is large. The double peaks on the chromatogram are close together, basically at the same height, without tailing (If the peak appears quickly, it may be a column problem). It is sufficient to dilute the sample and re-inject. This is due to the excessive injection volume and the overload of the column.

3. Characteristics of the sample
Some samples have tautomerism due to the chemical structure, and this tautomer cannot be separated but exists in a dynamic equilibrium. In the chromatographic analysis, under a specific condition, a substance will have double peaks, which are close together, basically high, no tailing. The conditions change slightly, especially pH, the double peak phenomenon will disappear, such as Erythromycin, etc. Some samples can not see double peaks on the ultraviolet chromatogram, but under LC-MS, using a mass spectrometer, the total ion current of the mass spectrum is more obvious, for example, the pesticide acetamiprid ).

4. Parameters
The recorded parameters are generally internal and do not need to be modified, but the parameters of GC and HPLC are not completely consistent. For example, the general recording time interval on the C-R3A data recorder is 2ms for GC and 5ms for HPLC column. One peak will become two or more peaks.

Q20:  What are the basic indicators for evaluating a column?
The basic indicators for evaluating a chromatographic column are the number of plates, peak asymmetry factor, column pressure drop, applicable range and concentration of bonded phase, and peak capacity.

(To be continued)

HAWACH To Talk About The 30 FAQs About HPLC Column (Chapter 4)