In the process of method development, we often encounter situations where the method cannot be reproduced during verification. At this time, many people will attribute this “error” to the chromatographic column. Indeed, there are very few “hypersensitivity” items that are extremely sensitive to the internal environment of the chromatographic column and cannot be reproduced. But most of them are caused by other reasons. Today, let’s talk about these other reasons.
The change of chromatographic column during use is commonly known as column modification
- This problem often occurs when the chromatographic column that has tested other items is used for method development, and then the chromatographic column is replaced for reproduction. This is because some special substances (possibly trace impurities or strong retention substances in the sample) will be adsorbed on the filler and cannot be eluted, which will modify the chromatographic column. When testing some specific components, it will affect the retention capacity of components, resulting in the inability to reproduce the separation effect after replacing the chromatographic column. The best way to avoid this situation is to use a new chromatographic column to reproduce the results immediately after the method is determined. If the new column cannot reproduce the results, it is likely that the old column is modified.
- Sometimes, chromatographic columns that have not tested other items are used for method development. Later, when reproducing, it will also occur that the replacement of columns cannot be reproduced. This is because many samples are treated under different conditions and different pretreatment methods will be used in the process of method development. In this case, the chromatographic column may also be modified. Therefore, even if the method is developed with a chromatographic column that has not been done in other projects, after the method is determined, it also needs a new chromatographic column for reproduction.
- The extreme conditions of the chromatographic column (such as high pH value, high temperature, low pH value, etc.) lead to the damage of the chromatographic column. The method developed on this chromatographic column often can not be reproduced when replacing a new column. High pH value will cause dissolution of silica gel and collapse of chromatographic column; Low pH value will cause the bonded phase to fall off easily, and high temperature will lead to the collapse and hardening of the filler, which will change the retention capacity of the chromatographic column. Therefore, using this conditional method, after the method is determined, it must be reproduced with a new chromatographic column.
- If the chromatographic column has used the conditions containing ion pair reagent, the ion pair reagent will modify the chromatographic column and cannot be completely washed. Therefore, the chromatographic column that has used ion pair reagent is recommended to be used for a special purposes.
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