Normal Phase vs Reversed Phase Chromatography

Solid Phase Extraction (SPE)

• Analyte molecules are enriched on the adsorbent, while interfering components and solvent molecules (matrix) are not retained
• Remaining interfering components are washed from the adsorbent, and the analyte is removed from the adsorbent by elution with a suitable solvent

Retention of Interfering Components

• Analyte molecules show no interaction with the adsorbent, whereas interfering components and solvent molecules (matrix) are retained
• Analyte molecules are "washed" from the adsorbent, and the solid phase is used to "filter" the sample

Normal Phase SPE

• Involves a polar analyte, a mid- to nonpolar matrix, and a polar stationary phase
• Typically uses sorbents such as polar-functionalized bonded silicas (e.g. LC-CN, LCNH2, and LC-Diol) and polar adsorption media (e.g. LCSi, LC-Florisil, ENVI-Florisil, and LC-Alumina)
• Retention of an analyte is due to interactions between polar functional groups of the analyte and polar groups on the sorbent surface, including hydrogen bonding, pi-pi interactions, dipole-dipole interactions, and dipole-induced dipole interactions
• Conditioning the cartridge with a nonpolar solvent, usually the sample solvent, is required

Steps in SPE

• Column conditioning: to wash the extraction system and remove potential interferents, and to activate the solid sorbent
• Sample loading: sample is forced through the sorbent material, and the analyte of interest is retained by the sorbent
• Washing the packing: unwanted materials are washed off using the same solution
• Eluting the compound of interest: rinsing the packing with a small volume of a solution that removes compounds of interest, but leaves behind impurities

Normal Phase Elution Protocol

• Elute unwanted components with a nonpolar solvent
• Elute the first component with a polar solvent
• Elute remaining components of interest with progressively more polar solvents

Reversed Phase SPE

• Involves a mid- to nonpolar analyte, a polar (usually aqueous) or moderately polar sample matrix, and a nonpolar stationary phase
• Typically uses sorbents such as alkyl- or aryl-bonded silicas (e.g. LC-18, ENVi-18, LC8, ENVI-8, LC-4, and LC-Ph)
• Retention of organic analytes from polar solutions is due to attractive forces between the carbon-hydrogen bonds in the analyte and the functional groups on the silica surface (Van der Waals forces or dispersion forces)
• Conditioning the cartridge with methanol or acetonitrile followed by deionized water or buffer is required, and the cartridge should not be allowed to dry
• Loading the sample (dissolved in a strongly polar solvent) into the cartridge is required

Reversed Phase Elution Protocol

• Elute unwanted components with a strongly polar solvent
• Elute weakly held components of interest with a less polar solvent
• Elute more tightly bound components of interest with progressively more non-polar solvents

Ion Exchange SPE

• Used for compounds that are charged when in a solution (usually aqueous, but sometimes organic)

Learn about the general elution protocols for normal phase and reversed phase chromatography, including the steps to separate components based on polarity. Understand the use of nonpolar and polar solvents in these chromatographic techniques.