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Separation Mode Description: Popular Phases Used:
Reversed Phase Being the most common principle HPLC/UHPLC separation mode, reversed phase chromatography offers dynamic retention of compounds with hydrophobic and organic functionality. Retention of these compounds by reversed phase involves a combination of hydrophobic and van der Waals type interactions between each target compound and both the stationary phase and mobile phase.
  • C18 HPLC columns are the most preferred as they offer an excellent range of hydrophobic separation power along with high surface area coverage
  • C8 HPLC columns are used when less retention compared to a C18 is needed
  • C4 and C5 HPLC columns are commonly used to separate large macromolecules such as proteins
Normal Phase Normal phase chromatography (NPC) is used to separate hydrophobic compounds and matrices that are retained too strongly by reversed phase and have minimal solubility in aqueous mobile phases. Normal phase stationary phases typically include polar functional groups that interact with analytes primarily via dipole-dipole and hydrogen bonding interactions.
  • Silica HPLC columns mainly retain analytes by polar interactions
  • Cyano (CN) HPLC columns offer unique polar selectivity for analytes
  • Amino (NH2) HPLC colimns offer retain analytes through polar and hydrpgen boning interactions
HILIC HILIC is a particularly useful separation mode for polar organic compounds that are poorly retained by reversed phase. Retention of these polar compounds using reversed phase methods is often difficult because of co-elutions with the solvent front or elutions within the chromatographic region where ion suppression is the greatest.
  • Unbonded Silica HPLC columns draw and retain a water-enriched layer onto the surface of the silica which facilitates the interaction of polar compounds with the stationary phase for increased retention.
Ion Exchange Ion-exchange (IEX) chromatography involves interactions between a charged stationary phase and the oppositely charged mobile analytes. In cation-exchange chromatography positively charged molecules are attracted to a negatively charged stationary phase. Likewise, in anion-exchange chromatography negatively charged molecules are attracted to a positively charged stationary phase.
  • SCX for positively charged compounds
  • SAX for negatively charged compounds
  • WCX for compounds such as carboxylic acids
  • WAX for comounds that contain weakly charged amino groups
Ion Exclusion Ion exclusion (IEC) chromatography is a process of separating components in a mixture by means of an ion-exchange resin that excludes highly ionized particles and retains slightly ionized or non-ionized particles. Ion exclusion (IEC) chromatography applies to the retention of organic acids, carbohydrates, sugars, starches, and oligosaccharides, using a sulfonated stationary phase.
  • cross-linked sulfonated styrene-divinylbenzene (SDVB) and multiple ionic forms (Ag+ , K+, etc.)