Reverse-Phase HPLC

Reversed-phase chromatography (RP-HPLC) separates molecules on the basis of differences in their hydrophobicity. The components of the analyte mixture pass over stationary-phase particles bearing pores large enough for them to enter, where interactions with the hydrophobic surface removes them from the flowing mobile-phase stream. The strength and nature of the interaction between the sample particles and the stationary phase depends on both hydrophobic interactions and polar interactions. As the concentration of organic solvent in the eluant increases, it reaches a critical value for each analyte which desorbs it from the hydrophobic stationary-phase surface and allows it to elute from the column in the flowing mobile phase. Since this elution depends on the precise distribution of hydrophobic residues in each species, each analyte elutes from the column at a characteristic time, and the resulting peak can be used to confirm its identity and quantify it.

Varying types of RP-HPLC columns are available with different pore sizes and hydrophobic coatings, allowing the investigator to manipulate the strength of the analyte-stationary phase interaction. Typically, longer alkane chains are used for peptides and small molecules to maximize the strength of the interaction, while shorter alkane chains are used for larger analytes such as proteins. Additionally the steepness of the organic solvent gradient in the mobile phase can be varied to alter the sample binding characteristics and analyte retention times.

At Analytical Ventura, we manipulate the parameters of RP-HPLC to maximize the separation of your analyte while optimizing the resolution, column capacity, and run time, among other parameters. Additionally, with access to all other types of chromatography, we are capable of developing a comprehensive purification and analysis strategy for your product. We also offer ESI-MS services coupled to RP-HPLC to assist us in elucidating its identity and structure.

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