Introduction to high performance liquid chromatography
The basic concept and separation theory of high performance liquid chromatography are consistent with classical liquid chromatography and gas chromatography, so that its plate theory and kinetic theory can be used for high performance liquid chromatography.
1. High-performance liquid chromatography The typical high-performance liquid chromatograph includes three parts: infusion system, chromatography column and detection system. The mobile phase is input using a high pressure pump.
This high pressure pump should meet the following conditions:
(1) The flow rate is constant, there is no pulsation, and there is a large adjustment range.
(2) It is resistant to solvent corrosion.
(3) There is a high output pressure, generally reaching 15~300 kg/c, and some up to 800 kg/c.
(4) The dead volume of the pump should be small.
The gradient elution device must have two high-pressure pumps, one to deliver strong solvent and one to deliver weak solvent. The two pumps are controlled by computer, and the composition of the mobile phase can be changed according to certain requirements to improve the separation effect.
It is usually injected directly with a micro-syringe, or it can be injected with a six-way valve. The detectors used in HPLC are most commonly used for UV absorption detection with sensitivity up to ng levels. In addition, there are fluorescent detectors, differential precipitation detectors, electrochemical detectors, and the like.
2. Types and applications of HPLC
(1) Liquid-solid adsorption chromatography: The stationary phase is an adsorbent having an adsorption activity, and commonly used are silica gel, alumina, high molecular organic acid or polyamide gel. The mobile phase in liquid-solid adsorption chromatography is divided into two types: “bottom†and eluent depending on the role of the mobile phase. The primer determines the separation of the basic chromatogram, and the eluent serves to adjust the sample components. The residence time is long and has a selective effect on certain components in the sample.
The combination and selection of the mobile phase intermediate and eluent components directly affect the separation of the chromatogram. Generally, the primer is a solvent with lower polarity, such as n-hexane, cyclohexane, pentane, petroleum ether, etc. The de-agent is selected according to the nature of the sample, such as ethers, esters, ketones, alcohols and acids. This method can be used to separate isomers, antioxidants and vitamins.
(2) Liquid-liquid partition chromatography: The stationary phase is composed of a monomer fixing solution. The functional group of the fixing solution is bonded to the thin shell or the porous silica gel, and is acid-washed, neutralized, dried and activated to maintain a certain silicon hydroxyl group on the surface. This liquid-liquid chromatography in which a chemically bonded phase is a stationary phase is called chemical bonded phase chromatography.
Another liquid-liquid partition chromatography using the principle of ion pairing is ion pair chromatography. Chemical bonding chromatography:
1 Polar phase chromatography: The stationary phase is a polar group, three kinds of cyano group, amino group and dihydroxy group. The mobile phase is a non-polar or less polar solvent. The component with a small polarity first peaks and the peak with a large polarity is called normal phase chromatography, which is suitable for separating polar compounds.
2 non-polar phase chromatography: the stationary phase is a non-polar group, such as octadecyl (C18), octyl (C8), methyl and phenyl, etc., the mobile phase uses a strong polar solvent, Such as water, alcohol, acetonitrile or inorganic salt buffer.
The most commonly used are mixed solvents prepared in different proportions of water and methanol. Water not only acts as an elution but also masks the silanol groups on the surface of the support to prevent smearing due to adsorption. The component with a large polarity first peaks, and the component with a small polarity has a peak, which is exactly opposite to the normal phase method, so it is called reverse phase chromatography. This method is applicable to the separation of small molecular substances such as peptides, nucleotides, sugars, and derivatives of amino acids.
Ion pair distribution chromatography:
1 Normal phase ion pair chromatography: This method is usually adsorbed on silica gel as a stationary phase by water, and the counter ion with opposite charge to the separated component is dissolved in water or buffer at a certain concentration on silica gel.
The mobile phase is a less polar organic solvent. During the chromatography, the ions to be separated form a neutral ion pair with the paired ions in the aqueous phase, and are distributed in the aqueous phase and the organic phase to achieve separation. The advantage of this method is that the mobile phase has a large choice, and the disadvantage is that the stationary phase is easy to lose.
2 reverse ion pair chromatography: the stationary phase is a hydrophobic bonded silica gel, such as a C18 bonded phase, the ions to be separated and the oppositely charged paired ions are simultaneously present in the mobile phase of strong polarity, and the resulting neutral ion pair The separation is carried out between the mobile phase and the bonded phase. The advantage of this method is that there is no loss of the stationary phase, the mobile phase contains water or the buffer is more suitable for the separation of ionized compounds.
(3) Ion exchange chromatography: The principle is the same as ordinary ion exchange. In ion exchange HPLC, the stationary phase uses an ionic bonded phase, so this method is also called ionic bonded phase chromatography. The mobile phase is mainly an aqueous solution, and the pH is preferably near the pK value of the separated acid or base.
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