HPLCSeparationModes 3• Separation is based on the partition equilibrium between stationary phase and mobile phase.• The polarity of the stationary phase is lower than that of the mobile phase.• Typically the mobile phase contains a mixture of organic solvents (methanol, acetonitrile, or THF) and aqueous solvents (water or buffer).• Use of lower polarity mobile phases fasten the elution.• Separation is based on hydrophilic interaction.• A high polarity stationary phase is used.• Typically the mobile phase contains a mixture of organic solvents such as acetonitrile and aqueous solvents (water or buffer).• Using the higher polarity mobile phase causes a faster elution.• Applicable for the analysis of high polar substances.• Separation is based on the partition equilibrium between the stationary phase and the mobile phase.• The polarity of the stationary phase is higher than that of the mobile phase.• Typically the mobile phase contains a mixture of organic solvents with different polarities such as hexane and isopropanol.• Using the higher polarity mobile phase causes a faster elution.• Separation is based on differences in analytes' coordination complex.• Stationary phase modified with metal sulfonate complex ion.• Works in combination with size exclusion or HILIC modes.• Separation is based on electrostatic interaction (repulsion) between the ion exchanger and ionic solutes.• Dissociated ionic molecules elute faster than non-dissociated forms.• Used mainly for the analysis of organic acids.• Separation is based on electrostatic interaction (bonding) between the ion exchanger and ionic solutes.• Electrical conductivity detector can be used with a mobile phase with low-salt concentration.• Used mainly for the analysis of inorganic compounds.• Network or pores on the surface of the packing material works as molecular sieve to separate molecules based on their sizes.• To separate molecules solely based on their sizes, it requires an analytical condition without any compounds and packing gel interaction.• The bigger the molecule size, the faster the elution sequence.• Used for molecular weight or molecular distribution determination of macromolecules and qualification of oligomers.• Separation is based on electrostatic interactions between the ion exchanger and ionic solutes.• The mobile phase of choice should have a sufficient buffering capacity at the pH that produces the largest charge differences between the analyte of interest.• The elution position is optimized by varying the pH, salt concentration, and/or ionic strength of the mobile phase.• Separation is based on hydrophobic interaction.• Hydrophobic functional group is modified on the stationary phase.• Adsorption of analytes generally occurs at a high salt concentration and they are released by lowering the salt concentration.• Used mainly for the analysis of proteins.• Separation is based on adsorption of the analyte to the specific biologically derived ligand pair.• Highly selective.• A buffer solution with the appropriate pH and ionic strength is selected based on the type of ligand, analytes, and their interaction.• Used mainly for the purification and concentration of biologically active substances.• Separation of optical isomers using chiral selectors.• Highly selective.• Separation is based on the combination of different modes.Liquid chromatography (LC) uses liquid as mobile phase (eluent). It is an analytical method that separates a mixture of compounds based on their physical and chemical differences. High performance liquid chromatography (HPLC) is a method that introduces the mobile phase under high-pressure conditions resulting in rapid and high-performance separations. The various interactions between the analyte, stationary phase (packing material), and mobile phase are the key factors for the separation. A wide variety of separation modes can be achieved by using particular combinations of stationary and mobile phases.Separation modeReversed Phase Chromatography(RPC)Hydrophilic Interaction Chromatography(HILIC)Normal Phase Chromatography(NPC)Ligand Exchange Chromatography(LEX)Ion Exclusion Chromatography(IEX)Ion Chromatography(IC)Size Exclusion Chromatography(SEC)Ion Exchange Chromatography(IEC)Hydrophobic Interaction Chromatography(HIC)Affinity Chromatography(AFC)Chiral Separation Chromatography(CS)Multimode ChromatographyCharacteristicsHPLC Separation Modes
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