General Principles: RPLC separates on the basis of hydrophobicity. As with other HPLC techniques there is a polymeric stationary phase, of for example polystyrene/divinylbenzene. The mobile phase is usually a combination of a weak aqueous buffer or a dilute acid and a water miscible organic solvent. For effective separation of proteins the mobile phase is generally a gradient system required to achieve separation and is preferably linear for convenience. RP-HPLC can efficiently separate molecular species that are exceptionally similar to one another in terms of structure or weight. 

RP-HPLC procedures currently represent the majority of applications for peptide analysis and purification adn over 80% of all analytical sutides with proteins. The dominant effectin refersed-phase chromatography is a hydrophobic interaction between the nonpolar amino acid residues of peptides or proteins and the nonpolar ligands, typically immobilized onto the surface of a spherical porous silica partcile, although nonpolar polymeric sorbents derived, elg., from cross-liniked polystyrene-divinylbenzene can also be employed. (Analysis of Proteins, unit 10:13:1, Current Protocls in Molecular Biology “”HPLC of peptides and proteins: standard operating conditions”). 

A sample is dissolved in mobile phage (1) column: the sample is pumped under high pressure onto a column. Need to consider the mode of separation here (ie., adsorption, reversed phase, ion exchange). (2)elution: the sample is eluted through the column (3) detection: the eluant is monitored with a detector. Detector possibilities include UV, radiochemical, fluorescent, Mass Spec. A new method is evaporative light scattering where there is 1) nebulization (the column effluent passes through a needle, mixes with nitrogen gas and forms a dispersion of droplets, 2) mobile phage evaporation (the droplets pass through a heated drift tube where the mobile phase evaporates, leaving a fine mist of dried sample particles and 3) detection (these sample particles pass through a flow cell where they intercept a laser light beam which scatters the light generating an electrical signal). (4)analysis: Data from the detectors is fed into a computer.

Comparison with HIC: RPLC is closely related to HIC as both are based upon interactions between solvent accessible non-polar groups on the surface of biomlecules and hydrophobic ligands of the matrix. However, ligands used in RPLC are more highly substituted with hdryophobic ligands than HIC ligands. While the degree of substitution of HIC absorbents may be in the range of 10-50 umoles/mL of matrix of C2-C8 aryl ligands, several hudred umoles/mL of matrix of C4-C8 alkyl ligands are used for RPLC. RPLC is also distinct in that HIC is performed in aqueous solvent conditions and changes in ionic strenght are used to elute the column. The protein typically binds in the native state via hydrophobic groups located on the surface of the protein, and the native state is retained during the elution conditions. In contrast, RPLC utilizes a hydrophobic solvent (typically acetonitrile) and the binding of a ligand is a function of the phase partition between the hydrophobic nature of the solvent and column functional group. Proteins are typically denatured to some extent in such solvents and bind due to the hydrophobic nature of the entire polypeptide sequence. Since the majority of hydrophobic groups are located in the core of globular proteins, the binding is related to the extent of denaturation of the protein and accessbility of these groups to the column functional groups.

The Source 30RPC column is a polymeric reverse phase matrix. It is based on rigid, monosized 30 micron diameter polystyrene/divinyl benzemne beads. 

Parameters/Conditions

Mobile Phase:

The organic solvents acetonitrile and methanol are often used in the mobile phase in reversed phase chromatography (“Tips for practical HPLC analysis –Separation Know-how – Shimadzu LC World Talk Special Issue Volume 2.)

In RP_HPLC iscratic eltuion, step elution or gradient elution modes can be utilized to purify peptides and prtoeins. Besides the requirement for an organic solvent to be used as a surface tension modified, ion-pair reagents are utilized at low pH (e.g., pH 2.1) to suppress silanophilic interactions between free silano groups on the silica surface and basic amino acid reisudes.  (Analysis of Proteins, unit 10:13:1, Current Protocls in Molecular Biology “”HPLC of peptides and proteins: standard operating conditions”).