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Analyte Retention in Reversed Phase HPLC

The hydrophobicity of an analyte molecule will be the primary indicator as to the retentivity in reversed phase HPLC. 

Hydrophobicity is often expressed as Log P which is a measure of the way an analyte (in its neutral form) partitions between two immiscible solvents (usually octanol and water) under standard conditions
(Equation 1). 

The higher the value of Log P (between –1 and +1) the more hydrophobic the molecule.

Polar analytes interacting with silica surface silanol groups undergo an adsorption type interaction as well as their partitioning behavior – this can lead to detrimental peak shape effects along with an increase in retention time.

The structure of the sample molecules will also give clues as to their elution order.  The elution order is governed by the water solubility of the molecule and the carbon content for an analogous series.  Some observations governing sample elution order include (again, these assume compounds are of analogous series):

  • The less water soluble a sample is, the more retention
  • Retention time increases as the number of carbon atoms increase
  • Branched-chain compounds elute more rapidly than normal isomers
  • Unsaturation decreases retention
  • Neutral polar and charged species typically show the least retention followed by acid, then basic compounds all eluting early

The general order of elution is:

Aliphatics > induced dipoles (e.g. CCl4) > permanent dipoles (e.g. CHCl3) > weak Lewis bases (esters, aldehydes, ketones) > strong Lewis bases (amines) > weak Lewis acids (alcohols, phenols) > strong Lewis acids (carboxylic acids)

Ionic compounds generally elute with the void volume of the column.

Retention order
Figure 1: Retention order in reversed phase HPLC.

Retention order:

1.   Straight chain hydrocarbon – most hydrophobic (C11)
2.   Straight chain hydrocarbon – less hydrophobic (C10)
3.   Branched n-hydrocarbon (C10)
4.   Unsaturated C10 – (more polar due to π electron dipole in the double bond)
5-7.   Analytes with functional groups.  Ionized analytes will elute fastest of all
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