Principles of liquid-liquid distribution chromatography

Principle of liquid-liquid distribution chromatography: Separation is achieved based on the different solubility of substances in two immiscible (or partially immiscible) liquids. Components with larger distribution coefficients also have larger retention values.

Liquid liquid distribution chromatography can be divided into normal phase chromatography (NPC) and reverse phase chromatography (RPC) based on the polarity of the stationary and mobile phases.

Normal phase chromatography: using polar stationary phases (such as polyethylene glycol, amino and nitrile bonded phases); The mobile phase is a relatively non-polar hydrophobic solvent (alkanes such as n-hexane and cyclohexane), and ethanol is often added to regulate the retention time of the components. Commonly used for separating compounds with moderate and strong polarity, such as phenols, amines, carbonyls, and amino acids.

Reverse phase chromatography: Non polar stationary phases (such as C18, C8) are generally used; The mobile phase is water or buffer, and organic solvents such as methanol, acetonitrile, isopropanol, acetone, and tetrahydrofuran that are miscible with water are often added to adjust the retention time. Suitable for separating non-polar and weakly polar compounds. RPC is widely used in modern liquid chromatography, and according to statistics, it accounts for about 80% of the entire HPLC application.

With the rapid development of column fillers, the application scope of reverse phase chromatography has gradually expanded and has now been applied to the analysis of certain inorganic or easily dissociated samples. To control the dissociation of the sample during the analysis process, buffer solutions are commonly used to control the pH value of the mobile phase. However, it should be noted that the pH values used for C18 and C8 are usually 2.5-7.5 (2-8). A pH too high will cause the silica gel to dissolve, while a pH too low will cause the bonded alkyl groups to detach. There are reports that new product columns can be operated within the pH range of 1.5~10.