I believe that many people have encountered such a situation. After a period of use of the column, the resolution of adjacent chromatographic peaks becomes worse, and the peak shape also becomes wider, or when the method is developed, the resolution of the target compound is not good. What are the reasons for this situation? How to troubleshoot after encountering a problem? Discuss with you today.
In liquid chromatography, the degree of resolution (Rs) is the most important part of chromatographic separation. Regardless of whether the ultimate purpose of chromatography is quantitative and qualitative analysis or sample preparation, it is achieved on the basis of a certain degree of resolution. The formula for calculating the degree of separation is as follows:
Among them, Rs represents the degree of resolution, tR2 represents the retention time of the next peak in the adjacent peaks, tR1 represents the retention time of the previous peak in the adjacent peaks, and W1 and W2 are the peak widths W of the two peaks at the baseline.
The greater the difference in retention time between chromatographic peaks, or the narrower the peak shape, the better the separation (increased resolution). For chromatographic separations, baseline separation helps to obtain accurate quantitative results. Two chromatographic peaks of similar size, with baseline separation corresponding to Rs>1.5.
What are the main reasons for the decrease in resolution?
It can be seen from the calculation formula of the resolution that the most intuitive reasons for the decrease of the resolution are: peak shape broadening, tailing or forward extension, and retention time changes. Therefore, the main reasons for the decrease in resolution are:
- The column has been used for too long, and the column efficiency has decreased
- The column or guard column or system is contaminated
- The sample is contaminated and deteriorated, resulting in peak shape broadening, tailing or front delay, or interference of spurious peaks
- The dead volume outside the instrument column is too large (the pipeline is too long, the inner diameter of the pipeline is too large, the volume of the detector flow cell is too large, etc.), resulting in a decrease in the column efficiency and a widening of the peak shape
- Influence of column temperature
The degree of separation is reduced, how to solve it?
If it is in the daily experiment, if the resolution is reduced, first check whether the chromatographic column (including the guard column) is contaminated or invalid, and clean or replace it. Secondly, check for the problem of sample contamination and re-prepare the sample Perform analysis. Finally, troubleshoot system contamination and mobile phase contamination, clean instrument tubing, replace in-line filters, and change mobile phase. By eliminating the above-mentioned possibilities, the problem of reduced resolution can be basically solved.
If you encounter low resolution during method development, there are many influencing factors, such as column type, particle size of packing material, type and ratio of organic phase, type and concentration of buffer salt, column temperature and column length etc. The specific impact will be discussed with you one by one later.