Estimation of Hydrocortisone by Reversed-Phase Chromatography (RP-HPLC/RP-UPLC) (CAT#: STEM-CT-2469-CJ)

Reversed-phase chromatography is a technique using hydrophobic molecules covalently bonded to the stationary phase particles in order to create a hydrophobic stationary phase, which has a stronger affinity for hydrophobic or less polar compounds. The use of a hydrophobic stationary phase is essentially the reverse of normal phase chromatography, since the polarity of the mobile and stationary phases have been inverted – hence the term reversed-phase chromatography. Reversed-phase chromatography employs a polar (aqueous) mobile phase. As a result, hydrophobic molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase, and hydrophilic molecules in the mobile phase will pass through the column and are eluted first. Hydrophobic molecules can be eluted from the column by decreasing the polarity of the mobile phase using an organic (non-polar) solvent, which reduces hydrophobic interactions. The more hydrophobic the molecule, the more strongly it will bind to the stationary phase, and the higher the concentration of organic solvent that will be required to elute the molecule.

Immunology/Inflammation; Biopharmaceuticals; Pharmaceuticals

1. Sample preparation.
2. Select the appropriate HPLC/UHPLC columns.
3. Set the operating conditions of the instrument and run the equipment.
4. Collect and analyse data.

• Sample: Blood; Urine; Plasma; Tissues; Drugs; Proteins; Peptides; Nucleic acids; Nucleotides & More.
• Equipment: HPLC machine; Reversed phase HPLC/UHPLC columns; HPLC/UHPLC systems.
• (Optional) Chromatographic solvents; Eppendorf vials; HPLC autosampler vials; Hypercarb Porous Graphitic Carbon HPLC Columns.

1. Reversed-phase chromatography is a commonly used, high-throughput analytical technique that allows the separation of analytes based on differences in hydrophobicity.
2. The reverse phase HPLC uses a nonpolar stationary phase and a polar mobile phase whereas the normal phase HPLC uses a polar stationary phase and a less polar mobile phase.
3. Reversed phase high performance liquid chromatography has been used successfully in the analysis and purification of polypeptides, small polypeptides and drugs.