Reversed phase hplc reversed phase hplc principle

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Reversed-Phase HPLC Reversed-Phase HPLC Principle: Partition of analytes between mobile phase and stagnant phase inside the pore space + adsorption on the surface of bonded phase . Stationary Phase: Hydrophobic surfaces of moieties bonded on silica (C18, C8, C5, Phenyl, CN) Mobile phase: Methanol or Acetonitrile and Water. Applications: ~80% of all separations done on RP HPLC.
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REVERSED PHASE REVERSED PHASE 80% Octadecylsilica (ODS, C18) 10% Octylsilica (C8) 5% Butylsilica (C4) 3% Phenyl 2% Cyano (CN)
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REVERSED PHASE SEPARATION PRINCIPLE SEPARATION PRINCIPLE Nonpolar (nonspecific) interactions of analyte with hydrophobic adsorbent surface (-C18, C8, Phenyl, C4) Different sorption affinities between analytes results in their separation More polar analytes retained less Analytes with larger hydrophobic part are retained longer Almost no separation of structural isomers REVERSED PHASE SEPARATION PRINCIPLE (2) Nonspecific (hydrophobic) interactions are at least ten times weaker than polar small differences in component molecular structure could have a significant effect in their retention Reversed Phase Normal Phase Octane, k’ = 4.3 Octanol, k’ = 3.5 Nonane, k’ = 7.8 Nonanol, k’ = 3.3
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REVERSED-PHASE REVERSED-PHASE INTERACTIONS INTERACTIONS Influence of the eluent hydrophobicity
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