High-efficiency liquid phase layering (HPLC)

the basic concept and separation theory of high-efficiency liquid phase layering method are consistent with the classical liquid phase
chromatography
method and gas chromatography, so its tower plate theory and dynamic theory can be used for high-efficiency liquid phase chromatography.. 1. High-efficiency liquid phase analyzer typical high-efficiency liquid phase
including infusion system, layering column and detection system three parts. The flow phase is entered with a high-pressure pump.
this high-pressure pump should meet the following conditions:
(1) flow is constant, no pulsation, and has a large adjustment range.
(2) is resistant to solvent corrosion.
(3) has a high output pressure, generally to reach 15 to 300 kg/c, and some up to 800 kg/c
(4) pump dead volume is small.gradient escape device must have two high-pressure pumps, one transport strong solvent, one transport weak solvent, two pumps operating speed with computer control, and according to certain requirements to change the composition of the flow phase, to improve the separation effect.generally with trace syringes directly into the sample, can also be used six-way valve sample. The detectors used in HPLC are mostly UV absorption detection with sensitivity up to ng levels. In addition, there are fluorescent detectors, oscillostic light detectors,
electrochemical
detectors, etc.. 2. HPLC type and application
(1) liquid-solid adsorption layering: fixed phase is an adsorption agent with adsorption activity, commonly used silicone, alumina, polymer
organic
acid or polyamide gel. The flow phase in liquid-solid adsorption layering depends on its role, divided into "substrate" and elsalizer two categories, the substrate determines the separation of the basic chromatography, the elvenomy agent regulates the length of stay of the sample part, and has a selective effect on a few parts of the sample.the combination and selection of the components of theflow phase midseed and the washesic directly affects the separation of chromatography, and the general substrate is a less polar solvent, such as positiveane, cyclane, pulcone, petroleum ether, etc., and the washetic agent selects targeted solvents according to the nature of the sample, such as ether, ester, ketone, alcohol and acid. This method can be used to isolate isomers,
antioxidants,
vitamins, etc.(2) liquid-liquid distribution layering: the fixed phase is made up of monosomal fixed liquid. The hydrogenic group of the fixing fluid is combined with thin shell or porous silicone, which is picked, mesothized,
dryed
and the surface is maintained with a certain amount of silicone hydroxyl. This liquid-liquid layering with chemical bonding phase as the fixed phase is called chemical bonding phase layering.another liquid-liquid distribution layering using the principle of ion pairs is ion-to-layer analysis. Chemical bonding layering:
(1) polar bonding phase layering: fixed phase for polar groups, cyanide, amino and dihydroxy. The flow phase is a non-polar or less polar solvent. The small polarity of the first part out of the peak, the polarity of the second out of the peak, which is called positive phase layering method, suitable for the separation of polar
compounds
.(2) Non-polar bonding phase layering: fixed phase is a non-polar group, such as octane (C18), octane (C8), methyl and benzene, etc., flow with strong polar solvents, such as water, alcohol, ethyl or inorgesal salt buffer.most commonly used is different proportions of water and methanol preparation of mixed solvents, water not only elusive action can also cover the surface of the carrier of silicon hydroxyl, to prevent adsorption to the tail phenomenon. Polar large parts first out of the peak, polarity of small parts out of the peak, exactly the opposite of the positive phase method, so called inverse stratific analysis. This method is applicable to the separation of small molecular substances, such as peptides,
nucleotides
, sugars,
amino acids
derivatives.ion-to-distribution stratography:
(1) positive-phase ion-to-layer analysis: This method often adsorption of water on silicone as a fixed phase, the separation of the part with the opposite charge of the paired ions in a certain concentration of water or buffer stained on the silicone.flow phase is a low-polar organic solvent. In the process of stratography, the ions to be separated are paired with the water phase to form a neutral ion pair, which is distributed in the water phase and the organic phase, and separation is achieved. The advantage of this method is that the flow phase has a large choice, the disadvantage is that the fixed phase is easy to lose.(2) Reverse ion-to-layer analysis: fixed phase is hydrophobic bonded silicone, such as C18 bonded phase, to be separated ions and paired ions with opposite charges are present in the strong polar flow phase at the same time, the resulting neutral ion pairs are distributed between the flow phase and bonding phase, and are separated. The advantage of this method is that there is no loss problem in the fixed phase, the flow phase contains water or buffer is more suitable for the separation of ionist compounds. (3) ion exchange: the principle is the same as ordinary ion exchange. In ion exchange HPLC, fixed phase multi-use ion bonding phase, so this method is also known as ion bonding phase layering. The flow phase is mainly an aqueous solution, and the pH value is preferably near the pK value of the separated acid and alkali.
.