High performance liquid chromatography (HPLC) is an important method for separating and analyzing mixtures of complex organics, drugs and biological macromolecules. As a modern analytical technique, the application of high performance liquid chromatography has become more and more extensive. When the sample composition is relatively simple, usually using a chromatographic column and selecting a separation mode can get good sample separation results. But for some more complex components, no matter how to optimize the chromatographic conditions and parameters, some of them cannot be separated well. In this case, chromatography-chromatographic hyphenated technology can be used. Chromatography-chromatography hyphenated technology refers to the hyphenation of a variety of different types of chromatographic methods, also known as multi-dimensional chromatography, and its main function is to improve the resolution and peak capacity of the chromatogram.
Chromatography-chromatography hyphenated technology combines chromatographic columns with the same flow, the same separation mechanism but different selectivity in series, or combines chromatographic columns with different mobile phases, different separation mechanisms, and different selectivities. Common hyphenated methods include gas chromatography-gas chromatography (GC-GC), high-performance liquid chromatography-gas chromatography (HPLC-GC), and high-performance liquid chromatography-high-performance liquid chromatography (HPLC-HPLC).
The key to process technologies such as multidimensional chromatography, automatic high-performance liquid chromatography, and online pre-processing is column switching (CS). As early as the 1970s, HPLC column switching technology was applied with the development of dead volume switching valves. However, it was not until the 1990s that with the increasing emphasis on the analysis of complex mixtures, the application of HPLC column switching technology increased dramatically.
HPLC column switching technology is the use of multi-channel switching valves to change the direction of the mobile phase or the mobile phase system, so that the eluent enters the secondary column (analysis column) from the primary column (pretreatment column) at a specific time. Because the chromatographic columns that achieve different separation goals rely on switching valves to connect, it is called column switching technology. The basic principle of online pre-purification is to complete the separation of the measured components and interfering impurities in the pretreatment column to achieve the purpose of purification and enrichment. Then use the column switch to transfer the tested component to the analytical column to complete the determination of the tested component.
In the analysis of biological samples, with the widespread application of HPLC, sample pretreatment is particularly important. However, pretreatment is generally time-consuming, consumes reagents, and requires a sufficient amount of samples, which often brings large errors to the results. The HPLC column switching technology can solve these problems. HPLC column switching technology can realize online separation of samples. Without reducing the sensitivity, HPLC column switching technology can enhance the selectivity of the chromatogram, carry out online enrichment of trace samples, and prevent samples from being contaminated during the pretreatment process. In addition, HPLC column switching technology can separate complex components in a variety of switching methods within a column switching system to achieve multiple separation goals. There are multiple pumps, multiple analytical columns or pretreatment columns, and multiple elution systems in a column switching system.
After continuous development, HPLC column switching technology has been valued by more and more researchers, and is widely used in many fields such as environmental protection, food inspection, biochemical analysis, medicine and so on. Among them, HPLC column switching technology is the most widely used in in vivo drug analysis.
Biological samples (such as plasma, serum, etc.) often contain large amounts of protein. The protein can react with the organic solvent in the mobile phase to produce precipitation, which can block and deteriorate the chromatographic column. Therefore, the sample cannot be directly injected in the traditional GPLC method. However, the HPLC column switching technique can be used for direct sample injection and analysis. A large number of biological samples are directly introduced into the pretreatment column. On the pretreatment column with water as the pretreatment mobile phase, the protein and some other endogenous impurities in the sample are washed and removed by water. The tested component is retained on the pretreatment column, and is purified and concentrated. Then the column is switched, and the measured component is flushed onto the analytical column by the analytical mobile phase for separation and analysis.
Biological samples are usually low due to the measured component concentration and often contain protein. The pre-treatment process is complicated and time-consuming. The use of HPLC column switching technology can simplify the sample preparation process. Usually add an appropriate protein precipitation agent (such as methanol, acetonitrile, etc.) to the sample. After removing the protein, the supernatant was injected in large quantities. In the pretreatment mobile phase dominated by water, the tested components are retained on the pretreatment column and the impurities are washed away. Then the switching valve is automatically switched, and the components are separated after being flushed into the analytical column by all or part of the analytical mobile phase.
In vivo drug analysis, in order to improve the detection sensitivity of certain compounds (such as amino acids, hormones, fatty acids, etc.), derivatization is often required. Using HPLC column switching technology can automatically complete the derivatization process online, and obtain good precision.