Qualitative method for high performance liquid chromatography analysis

There are many factors affecting the migration of solute during the analysis of high performance liquid chromatography. The retention values ​​of the same component under different chromatographic conditions are very different. Even under the same operating conditions, the retention of the same component on different columns is also It can vary widely, so HPLC is more difficult to characterize than gas chromatography.

Qualitative methods commonly used in high-performance liquid chromatograph analysis are characterized by the use of known standard samples, the selectivity of the detector, and the use of UV detectors for full-wavelength scanning.

First, use known standard samples to characterize:

Characterization of unknown compounds using standard samples is the most commonly used qualitative HPLC method.

Since each compound has a characteristic retention under specific chromatographic conditions (mobile phase composition, column and column temperature, etc.), it can be characterized using retention values. If the test compound is consistent with the retention value of the standard sample under the same chromatographic conditions, it can be preliminarily considered that the test compound is the same as the standard sample. If the mobile phase composition is changed several times and the retention value of the test compound is still consistent with the retention value of the standard sample, it can be further confirmed that the test compound is the same compound as the standard sample.

Second, the use of detector selectivity:

The same detector responds differently to different kinds of compounds, and different detectors respond differently to the same compound. Therefore, when a test compound is detected by two or more detectors, the detection sensitivity ratio of the test compound to the two detectors or several detectors is closely related to the nature of the test compound, and can be used to The test compound is characterized. This is the basic principle of dual detector characterization.

The connection of the dual detector system has a series connection and a parallel connection. When one of the two detectors is non-destructive, a simple series connection can be used by connecting the non-destructive detector in series with the destructive detector. If both detectors are destructive, they must be connected in parallel by connecting a tee to the outlet of the column and coupling it to the two detectors. The dual detection systems most commonly used for qualitative analysis in high performance liquid chromatography are ultraviolet detectors (UVD) and fluorescence detectors (FLD).

Third, the use of UV detector full-wavelength scanning function to characterize:

UV detectors are the most widely used detectors in high performance liquid chromatography. The full-wavelength scanning UV detector provides some valuable qualitative information based on the UV spectrum of the compound being tested. The traditional method is to have a maximum value of the peak of a component on the chromatogram, that is, when the concentration is maximum, the component is retained in the detection tank by stopping the pump, and then the full wavelength of the components in the detection tank is performed. Scan to obtain an ultraviolet-visible spectrum of the component, and take the possible standard samples in the same manner. Comparing the two spectra can identify whether the component is the same as the standard sample. Certain compounds with specific UV spectra can also be identified by reference to standard spectra.

In addition, the use of diode array detectors to determine the three-dimensional spectrum of the chromatographic signal, time and wavelength is more advantageous than traditional methods.

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