In the chromatographic identification test, it is a common chromatographic qualitative method to identify the color latent separation with the reference substance. The principle is based on the fact that the same platform has exactly the same specific chromatographic retention behavior under the same chromatographic conditions (mobile phase composition, chromatographic column and column temperature, etc.). Generally, under the same chromatographic conditions, if the tested chemical platform and the reference substance have the same retention value, it can be preliminarily considered that the tested chemical platform and the reference substance are the same substance. The chromatographic identification methods in Pharmacopoeia and other drug quality standards are sufficient to be based on this. In the inter assay, the chromatographic retention time of the control sample and the test sample should be measured respectively. There may be many situations in banquet practice. Here are some examples:

Note: when it is preliminarily found that the retention time of the test sample and the control sample is slightly different in the experiment, it should first be determined that this difference is not caused by the difference in composition and concentration between the test sample and the control sample. The simple elimination method is to mix the test sample and the control sample in equal proportion, and observe whether two chromatographic peaks can be separated under the same chromatographic conditions. If there are two chromatographic peaks, it can be confirmed that the retention time of the test sample and the control sample is different. On the contrary, it can not be confirmed that the retention time of the test sample and the control sample is different.

Strictly speaking, according to the same retention time of the test sample and the control sample under a specific chromatographic condition, it is judged that they are the same substance, which still has a certain risk. If it is necessary to further determine that the test sample and the reference sample are the same, different chromatographic conditions (usually different mobile phases) shall be selected according to the characteristics of the reference sample to further compare whether the changes of retention values of the test sample and the reference sample under different chromatographic conditions are the same. For example, for organic acid-base compounds, the change of retention value when the pH value of mobile phase changes around its curve should be compared; For nonpolar compounds, the change law of retention value with the change of mobile solvent should be compared. If the composition of the mobile phase is changed many times and the retention values of the test sample and the control sample are completely consistent, it can be further confirmed that the test sample and the control sample are the same substance.