There are many kinds of HPLC classification, which can be classified according to different bases.
1. According to the polarity of the mobile phase and stationary phase, HPLC can be divided into normal phase chromatography and reversed-phase chromatography.
Normal-phase high-performance liquid chromatography: The stationary phase in the column is composed of polar compounds such as silica gel and alumina. When the chromatography is run, the non-polar compounds are eluted first because the polar compounds in the sample have a stronger affinity for the stationary phase, so their retention time in the column is longer than that of the non-polar compounds. Many substances can be analyzed by normal-phase chromatography, but because drugs, foods, and other biological products are mostly non-polar, they are not as widely used in everyday life as reverse-phase chromatography.
Reversed-phase high performance liquid chromatography: The stationary phase is composed of non-polar compounds, such as octadecylsilane, Reversed Phase C4 HPLC Columns, C8, C18, and other organic compounds. The mobile phase is polar. Therefore, compounds with high polarity are eluted first, and compounds with low or no polarity are eluted last.
HPLC mostly analyzes substances such as drugs, food, and biochemical molecules, and they are all polar substances (water-soluble) in nature. Therefore, reversed-phase high performance liquid chromatography is more widely used.
2. According to the different separation principles, HPLC can be divided into the following six types.
Affinity chromatography: mainly utilizes the affinity between the sample and immobilization to achieve separation.
Ion exchange chromatography: based on the reversible exchange of charged groups between the stationary phase and the mobile phase to achieve separation. Ion exchange resins are used to separate samples containing charged ions. For anions, an anion exchange resin is used; for cations, a cation exchange resin is used. Mainly used to separate acidic and basic compounds.
Ion-pair chromatography: An ion-pairing agent is added to the mobile phase or stationary phase of reversed-phase chromatography to form “counter ions” with ionizable components in the sample. Commonly used ion pairing agents are pentane, hexane, heptane, or octane sulfonate.
Adsorption chromatography: It uses the principle of different adsorption, and separates according to the different affinity of the components in the sample to the stationary phase.
Size Exclusion Chromatography: Separation of components in a sample in order of molecular size. The chromatographic column is made of soft gels such as agarose, dextran, polyacrylamide, etc., and semi-rigid gels such as alkyl dextran and polystyrene can also be used.
Chiral chromatography: used to separate optically active isomers in a sample. The stationary phase is chemically bonded silica gel. Often used in medicine, biology, and other fields.
3. According to different analysis purposes, HPLC can be divided into analytical liquid chromatography and preparative liquid chromatography.
Analytical liquid chromatography: The qualitative and quantitative analysis of the components in the mixture is mainly carried out by using the detector.
Preparative liquid chromatography: mainly to separate and purify the components in the mixture. Preparative columns and injection volumes are larger than analytical ones.
4. As we all know, the separation of the mixture is accomplished by the mobile phase flowing through the HPLC column, where the mobile phase can be a single solvent or a mixed solvent, so HPLC can be divided into isocratic elution and gradient elution. Take off two types.
Isocratic elution: There is only one solvent and all operations are performed in one solvent.
Gradient elution: The mobile phase consists of two or more solvents, and their concentration ratios are constantly changed.
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