Commonly Used Quantitative Methods in Chromatography

As we all know, chromatographic qualitative analysis is to determine the compounds represented by each chromatographic peak. Since various substances have definite retention values under certain chromatographic conditions, the retention values can be used as a qualitative indicator. Various chromatographic qualitative methods are based on retention values. However, different substances may have similar or identical retention values under the same chromatographic conditions, that is, retention values are not exclusive. It is therefore difficult to characterize a completely unknown sample based on retention values alone. If you make a preliminary judgment on the composition of the sample on the basis of understanding the source, nature, and analysis purpose of the sample, and then combine the following methods, you can determine the compounds represented by the chromatographic peaks.

There are various qualitative methods in chromatography. Today, I will briefly share several qualitative methods commonly used in chromatography.

Standard sample qualitative

The use of standard samples to characterize unknown compounds is a commonly used HPLC qualitative method. Since each compound has a characteristic retention value under specific chromatographic conditions (mobile phase composition, chromatographic column and column temperature, etc.), the retention value can be used for characterization. If the retention values of the tested compound and the standard sample are consistent under the same chromatographic conditions, it can be preliminarily considered that the tested compound is the same as the standard sample. If the retention value of the tested compound is still consistent with the retention value of the standard sample after changing the composition of the mobile phase for many times, it can be further confirmed that the tested compound and the standard sample are the same compound.

Characterization of full wavelength scanning function of UV detector

UV detectors are the most widely used detectors in high performance liquid chromatography. A full wavelength scanning UV detector can provide some valuable qualitative information based on the UV spectrum of the detected compound. The traditional method is that when the chromatographic peak of a certain component on the chromatogram has a maximum value, that is, when the concentration is the largest, the component is retained in the detection cell by means of stopping the pump, and then the components in the detection cell are subjected to full wavelength analysis. Scan to obtain the UV-Vis spectrum of the component, and then take possible standard samples and process them in the same way. 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 comparison with standard spectra.

Selective characterization of detectors

The response of the same detector to different kinds of compounds is different, and the response of different detectors to the same compound is also different. Therefore, when a test compound is detected by two or more detectors at the same time, the ratio of the detection sensitivity of the two detectors or several detectors to the test compound is closely related to the properties of the test compound, which can be used to detect The tested compounds were identified. This is the rationale for dual detector characterization.

The connection of the dual detector system includes series connection and 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 before the destructive detector. If both detectors are destructive, they should be connected in parallel by connecting a tee at the outlet end of the chromatographic column and connecting them to the two detectors respectively. The dual detection systems most commonly used for qualitative analysis in HPLC are the ultraviolet detector (UVD) and the fluorescence detector (FLD).

Offline Analysis Qualitative

If semi-preparative or preparative mode is used, a fraction of the liquid sample from the HPLC effluent can be collected so that a certain amount of pure analyte can be collected for off-line structure determination experiments. Because of the large amount of sample available, the time frame limitations of on-line analysis do not exist, and off-line structural analysis can often provide conclusive structural information for a collected chromatographic peak. Offline analysis includes traditional analysis methods such as FTR, NMR and mass spectrometry. As long as there are enough samples, we can also use wet chemical tests, X-ray crystallography or other analytical techniques for offline analysis of samples.