Common Terms And Matters Needing Attention Of HPLC Column
Short for High-performance liquid chromatography, HPLC is an analytical chemistry technique used to separate, quantify and identify the components which are in a mixture. In the HPLC technique, the pumps push some liquid solvents which contain a mixture of the through a HPLC column which contains solid absorbent materials.
The principle of HPLC is easy to understand. Each of the components in the sample will interact with the absorbent material in a different way, which will cause different flow rates in the various components, and the components will be separated as they flow out of the HPLC column.
The HPLC column is called the stationary phase, which is made from different substances. You can find that silica is popular in the absorbent material, as it’s highly compact in nature. The full lines of Hawach HPLC column products will help you to get faster separation for similar samples. The wonderful performance of the HPLC columns will get your HPLC method greatly improved.
Common terms for HPLC
1. Chromatogram: a graph of the response signal generated by the HPLC column effluent when passing through the detector system versus time or mobile phase effluent volume, or a paper chromatogram (ch or paper layer spot observed by appropriate methods), The distribution map of the band.
2. Chromatographic peak: The differential curve of the response signal generated when the effluent from the HPLC column passes through the detector system.
3. Peak base: The straight line connecting the start and end of the peak (CD in Figure 1).
4. Peak height (h): The distance from the maximum point of the chromatographic peak to the peak bottom (BE in Figure 1).
5. Peak width (W): The distance between the two points where the tangent line and the bottom of the peak intersect at the inflection points on both sides of the peak (F, G in Figure 1) (KL in Figure 1).
6. Half-peak width (W h / 2, the peak with a half-height): Make a straight line parallel to the bottom of the peak through the midpoint of the peak height. The distance between this straight line and the two points that intersect at both sides of the peak (Figure 1 HJ).
7. Peak area (A): the area between the peak and the peak bottom (CHEJDC in Figure 1).
8. Tailing peak (tailing peak): the trailing edge is the gentler asymmetric peak.
9. Leading peak: the leading edge is gentler than the trailing edge of the asymmetric peak. (Also known as tongue extension peak, forward extension peak).
10. Ghost peak: In addition to the chromatographic peaks normally generated by the components, the chromatographic peaks appearing on the spectrum due to changes in instrument conditions and other reasons, that is, peaks not generated by the sample. This chromatographic peak does not represent a specific component, and it is easy to give errors in qualitative and quantitative results. (Also known as Ghost Peak).
11. Distorted peak: Asymmetrically shaped chromatographic peaks. The peaks and trailing peaks belong to this category.
12. Negative peak: also known as inverted peak, negative peak, that is, the direction of the peak is opposite to the chromatographic peak in the usual direction.
Matters needing attention during the use of HPLC column
1. The HPLC column bed must always be kept dry, especially during the injection test and HPLC column flushing process, and the mobile phase should not be left empty for over 30 minutes. If not, the HPLC column will dry up and bring a large amount of non-ejectable bubbles, leading to partial collapse or central cracking.
2. It is strictly forbidden to use force to throw it and the accidental impact or falling at high places should be prohibited no matter in use or storage. Or, it will easily cause mechanical damage to the column bed, resulting from no regeneration ability.
3. The valve or pipe must be cleaned, when the HPLC column and the chromatograph are coupled.
4. Avoid using high viscosity solvents as the mobile phase.
5. The sample to be injected should be completely dissolved.
6. Most reversed-phase HPLC columns have a pH stability range of 2-7.5, which does not exceed the pH range of the column.
7. After the end of the analytical work, the sample remaining in the injection valve should be cleaned and the HPLC column should be cleaned with a suitable solvent.
8. If the analytical HPLC column is not used for a long time, it should be stored and sealed with a suitable organic solvent.