In method development work, it is often encountered that the method cannot be reproduced during validation, and at this time, many people will attribute the “wrong” to the column. Indeed, there are very few “hypersensitive” items that are extremely sensitive to the internal environment of the chromatographic column and cannot be reproduced. But most of the failure to reproduce is caused by other reasons. Today, we will talk about these other reasons.
Column changes during use, commonly known as column modification
- This problem often occurs when the method is developed using a chromatographic column that has been tested for other items, and then the chromatographic column is replaced for reproduction. This is because some special substances (may be trace impurities or strong retention substances in the sample) will be adsorbed on the packing materials and cannot be eluted, which will cause modification to the chromatographic column. When detecting some specific components, the retention ability of the components will be affected, resulting in the inability to reproduce the separation effect after replacing the chromatographic column. The best way to avoid this is to reproduce the results with a new column as soon as the method is established. If the new column does not reproduce the results, there is a high possibility that the old column has been modified.
- Sometimes, the method development is carried out using a chromatographic column that has not been tested for other items. When the method is reproduced later, it will also happen that the replacement of the column cannot be reproduced. This is because many samples with different conditions and different pretreatment methods are used in the method development process, which may also modify the chromatographic column. Therefore, even if the method is developed with a column that has not been used in other projects, after the method is determined, it needs to be reproduced with a brand-new column.
- The extreme conditions of the chromatographic column (such as high pH value, high temperature, low pH value, etc.) lead to damage to the chromatographic column. The method developed on this chromatographic column often fails to reproduce when the new column is replaced. Case. High pH will cause the silica gel to dissolve and the column will collapse. Low pH will cause the bonded phase to fall off easily, and high temperature will cause the packing materials to collapse and harden, which will change the retention capacity of the chromatographic column. Therefore, the method using this condition must be reproduced with a new chromatographic column after the method is determined.
- The chromatographic column has been used with ion-pairing reagents. The ion-pairing reagents will modify the chromatographic column and cannot be completely rinsed. Therefore, it is recommended that the chromatographic column that has used ion-pairing reagents be dedicated to the column.
The specific reasons, we will continue to answer for you in the next article! To be continued~