Knowledge about HPLC
High performance liquid chromatography has the characteristics of “four high and one wide”:
1. High pressure: The mobile phase is a liquid. When flowing through the column, the resistance is large. In order to pass the column quickly, high pressure must be applied to the carrier liquid.
2. High speed: fast analysis speed, fast liquid carrier flow rate, much faster than classical liquid chromatography. Usually one sample is analyzed for 15 to 30 minutes, and some samples can be completed within 5 minutes, usually less than 1 hour.
3. High efficiency: high separation efficiency. The stationary phase and mobile phase can be chosen for excellent separation and many times higher separation efficiency than industrial rectification columns and gas chromatography.
4. High sensitivity: UV detector up to 0.01 ng, injection volume in the order of μL.
5. Wide range of applications: More than 70% of organic compounds can be analyzed by high performance liquid chromatography, especially for the separation and analysis of high boiling point, macromolecule, strong polarity, poor thermal stability compounds, showing advantages.
6. Columns can be used repeatedly: separate columns can be used to separate different compounds
7. Samples are small and easy to recycle: the sample is not destroyed after passing through the column, and a single component can be collected or prepared.
In addition, high-performance liquid chromatography has the advantages that the column can be used repeatedly, the sample is not destroyed, and it is easy to recycle, but it also has disadvantages. Compared with gas chromatography, it has its own advantages and complements each other. A disadvantage of high performance liquid chromatography is the “extra-column effect”. Between the injection and the detector, in any dead space other than the column (injector, column fitting, connecting tube and detection cell, etc.), if the flow pattern of the mobile phase changes, any diffusion of the separated material and Retention will significantly lead to a broadening of the chromatographic peak and a decrease in column efficiency. High performance liquid chromatography detectors are less sensitive than gas chromatography.
1. Adsorption Chromatography
2. Partition Chromatography
3. Ion Chromatography
4. Size Exclusion Chromatography
5. Bonded-phase chromatography
6. Affinity Chromatography