Proteomics/Protein Separations - Chromatography/Reversed Phase

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Reversed Phase
Immobilized Metal Ion Affinity Chromatography (IMAC) Hydrophobic Interaction Chromatography (HIC)

Chapter written by: Laura Grell and Alexander Butarbutar
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Chapter modified by Kai Burnett and Dalia Ghoneim
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Reversed PhaseEdit

Reversed Phase Chromatography - Proteins with exposed hydrophobic region (red) will be able to bind to the immobilized hydrocarbon stationary phase; the rest simply wash off

   Reverse Phase chromatography is a separation based on the solubility of the protein. The term ‘reverse’ was derived from its predecessor named ‘normal’ phase chromatography, which utilized a polar stationary phase such as silica. In reverse phase, the stationary phase is packed with silica modified with silyl ethers containing non-polar alkyl groups typically C8 or C18. This creates a hydrophobic stationary phase. The mobile phase on the other hand, contains relatively polar organic solvents such as methanol, butanol, isopropanol, acetonitrile (Ref1).

   Utilization of these polar solvents introduces very harsh conditions for the protein, thus the method will generally work well for smaller and more stable proteins. All peptides and proteins carry a mix of hydrophilic and hydrophobic amino acids, but those with high net hydrophobicity will be able to participate in hydrophobic interactions with the stationary phase. As mixtures of proteins are applied to the column, polar proteins will elute first while non-polar proteins will bind to the column. Elution of the bound hydrophobic protein can be accomplished by increasing the concentration of organic solvent, which increases the retention time of a particular component. Reverse phase chromatography is commonly coupled with mass spectrometry in an effort to quantify the protein that is eluted from the column (Res1).

Stages in Reversed Phase ChromatographyEdit

The stages in Reversed Phase Chromatography are very similar to the stages found in Ion exchange Chromatography. Once again, the process can be summarized in four steps(Res2):

  1. Equilibration
  2. Sample Application
  3. Elution
  4. Regeneration


In this step the hydrophobic column is primed by applying the specific sample buffer. Because the column is hydrophobic, water molecules tend to be ordered at the junction between the column and the buffer.

Application of the SampleEdit

In this step, the sample protein is injected into the system. Proteins in the mixture that have a high percentage of exposed hydrophobic amino acid residues will be adsorbed to the hydrophobic stationary phase. Other proteins in the mixture will be washed out.


This stage in the process involves changing the buffer conditions to elute the bound hydrophobic protein. The most common way to do this is to use a gradient that slowly increases the hydrophobicity using an organic buffer. The order in which proteins are desorbed is usually relative to the number of external hydrophobic residues each protein has (Res 3).


This stage involves washing off any remaining protein from the stationary phase and returning the conditions back to the way they were at the start of the process. This involves creating a less hydrophobic environment.


Here is a sample diagram of a reversed phase separation run for 80 minutes, reaching a 70% organic solvent level with three elution peaks. ReversePhase1.jpg

Note the gradient delay in this diagram. The delay is common to all liquid chromatography gradients, and is caused by a delay in the software telling the gradient to start pumping, and the solvent reaching the column. Also note the organic solvent concentration doesn't start at 0%. This is because the bonded hydrocarbons are much less efficient at binding to other hydrophobic molecules without a small amount of organic solvent present.


In order to see greater peak separation, you can run the same proteins again for a shorter period of time, or lower the maximum amount of organic solvent. Using the above diagram, it appears that the last protein elutes at about 50% organic solvent concentration. After taking into account the delay, the last peak elutes and an adjusted 40% organic solvent concentration. A new separation can then be done, using a 40% maximum organic solvent concentration, which would generate much more widely separated peaks.

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  1. University of Adelaide Department of Chemistry Determination of caffeine in soft drinks by HPLC
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  3. Andrew Guzzetta Reverse Phase HPLC Basics for LC/MS
  4. Reversed Phase Chromatography (RPC)


  1. Eschelbach JW, Jorgenson JW. Improved protein recovery in reversed-phase liquid chromatography by the use of ultrahigh pressures. Anal Chem. 2006 Mar 1;78(5):1697-706.