There is a special mode of reversed-phase chromatography, non-aqueous reversed-phase chromatography (NARP), in which the column is non-polar (such as C18) and the mobile phase, as the name implies, consists entirely of organic phases. Non-aqueous reverse phase chromatography (NARP) is mainly used to separate samples with strong hydrophobicity, such as lipids, synthetic polymers and so on. The mobile phase in non-water reversed-phase chromatography is composed of organic solvent A with strong polarity and organic solvent B with weak polarity. Usually A solvent commonly used is acetonitrile or methanol, B solvent is tetrahydrofuran, isopropyl alcohol, dichloromethane, methyl tert-butyl ether or other weak polarity of organic solvents. Sample retention is controlled by changing the polarity of the %B or B solvent.

The retention mechanism of reversed-phase chromatography has long been the focus of research. The localization of solute molecules in stationary phase may exist in several forms, such as solphobicity, adsorption, and distribution. The hydrophobic interaction assumes that the solute molecule is aligned with and attached to the complex. Adsorption means that solute molecules do not penetrate into the stationary phase but remain between the stationary phase and the mobile phase liquid. The distributive action is similar to that of a liquid in which solute molecules dissolve. See below:

Among them, the role of hydrophobic solvent is widely accepted theory: relatively speaking, hydrophobic solute molecules prefer to adsorb on hydrophobic alkyl groups, so it is also called hydrophobic retention. In reversed-phase chromatography, alkyl and other bonded non-polar stationary phase, mobile phase for water, organic solvent, buffer and other polar solvents. The longer the bond chain, the stronger the hydrophobicity, the larger the solute retention value; The larger the mobile phase surface tension, dielectric constant and polarity are, the stronger the interaction between solute and bonded phase is, the worse the elution ability of mobile phase is, and the larger the solute retention value is. The weaker the polarity of the solute, the stronger the hydrophobicity and the greater the retention value.

For non-aqueous reversed-phase chromatography, the principle is consistent with that of reversed-phase chromatography. The stationary phase is non-polar stationary phase. Due to its strong hydrophobicity and large retention, the sample does not use strong polar aqueous solution as the mobile phase, but is entirely composed of organic solvents.

The optimization of non-aqueous reversed-phase chromatography is the same as that of reversed-phase chromatography. It is mainly adjusted by changing the polarity of% B or B solvent. Both isosceles and gradients can be used. At the same time, the column temperature also has an impact on the separation of samples. Generally, the mixed solvent of ACN (a) and THF (b) is preferred as the initial mobile phase. If the retention of 100% THF sample is still too strong, it can be replaced by B solvent with weak polarity (such as dichloromethane or chloroform), but the detection wavelength of dichloromethane or chloroform should be considered.

Nonaqueous reversed-phase chromatography is a special mode of reversed-phase chromatography. The chromatographic column is the same as the non-polar chromatographic column commonly used in reversed-phase chromatography. The mobile phase is all organic phase, and the sample retention is controlled by changing the polarity of solvents with weak polarity; It mainly separates samples with strong hydrophobicity and insoluble in water. In normal work, non-aqueous reversed-phase chromatography can be considered when similar substances are encountered.

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