Do You Really Know About SPE Normal Phase and Reverse Phase Extraction?

Solid phase extraction is a pretreatment technology based on liquid-solid chromatographic separation, which can selectively adsorb and selectively elute target compounds. According to its retention mechanism, the force is mainly divided into non-polar interaction, polar interaction, and ion exchange (electrostatic attraction). Ion exchange cartridges are aimed at dissociable charged substances. In the analysis of pesticide residues and animal residues, normal phase extraction and reverse phase extraction cartridges are mostly used. Today we will discuss normal phase extraction and reverse phase extraction.

Reversed phase mechanism

Reversed phase mode refers to the separation mode in which the polarity of the stationary phase of the SPE cartridge is less than the polarity of the mobile phase. It mainly uses the non-polar force between the carbon-hydrogen bonds of the stationary phase functional groups and the carbon-hydrogen bonds of the target compound, which is suitable for extracting and separating non-polar to medium-polar targets from the polar matrix.

For the target that is adsorbed on the reversed-phase SPE cartridge by non-polar force, it can be eluted with a weakly polar solvent, such as chloroform, cyclohexane, ethyl acetate, etc. As long as the elution strength of the solvent is sufficient to destroy the van der Waals force between the target and the adsorbent, the target can be eluted from the SPE cartridge. Even for the more polar methanol, it has sufficient non-polar force to elute many compounds. Sometimes a single solvent cannot completely elute the highly hydrophobic target, you can consider using dichloromethane: ethyl acetate (1:1, volume ratio).

In reversed-phase mode, the polarity of the solvent system should gradually decrease in the order of sample solvent, rinsing solvent, and elution solvent, while their elution intensity gradually increases. It must be ensured that the selected sample solvent cannot elute the target. The selected eluent should elute the interference to the maximum without eluting the target. The selected eluent should be able to completely elute the target.

Normal phase mechanism

The normal phase mode refers to the separation mode in which the polarity of the stationary phase of the SPE cartridge is greater than the polarity of the mobile phase. It mainly uses the polar interaction between the polar surface of the solid phase extraction material and the polar functional group of the target. The strength of the polar force is greater than the non-polar force but smaller than the ionic force. Common polar functional groups include hydroxyl, amine, and sulfhydryl groups. The weakly polar matrix environment is conducive to the formation of polar forces because weakly polar solvents do not have functional groups that can form hydrogen bonds with the polar stationary phase.

Therefore, in the normal phase extraction of SPE, the sample matrix is mostly weakly polar, such as n-hexane, dichloromethane, rape oil, etc., and most of the targets contain polar functional groups. Common polar stationary phases are silica gel, alumina, Florisil, and bonded silica gel containing cyano (CN), amino (NH2), and glycol (2OH) groups.

In the normal phase mode, the polarity of the solvent system should gradually increase in the order of sample solvent, rinsing solvent, an elution solvent, and their elution intensity should also gradually increase. It must be ensured that the selected sample solvent cannot elute the target. The selected eluent should elute the interference to the maximum without eluting the target, so the eluent should be able to completely elute the target.

Choose reversed phase or normal phase?

So in the face of reversed-phase and normal-phase solid-phase extraction, which one should we choose in practical applications? The HAWACH description in the following is for your reference.

Most compounds have multiple functional groups. In actual operation, which extraction mechanism is mainly considered based on the properties of the target and interference, for example, 2-naphthylamine is a weakly basic compound (pKa=4.16), which can be in a cationic state under certain pH conditions. This compound has both hydrophobic and hydrophilic groups. At this time, the extraction mechanism that is conducive to separating the target and the interference can be selected according to the sample matrix. When the target is in a weakly polar sample matrix, the polar interaction can be used to select the normal phase extraction mode; if it is in a polar sample environment, the non-polar interaction can be used to select the reverse phase extraction mode.

In addition, the pH of the extraction environment can be adjusted to two pH units lower than the target pKa, that is, pH=2.16, using cation exchange mechanism. The following summarizes the main factors affecting the choice of retention mechanism: The first influencing factor of retention mechanism: the nature of the target functional group. The second influencing factor of the retention mechanism: the nature of the sample matrix.

HAWACH has successively launched silica-based reversed-phase, normal-phase, and ion exchange cartridges for different testing items, as well as SPE products with polymer matrix, inorganic matrix, and hybrid adsorption mechanism. Welcome to contact HAWACH for more.