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Very large molecules, however, can result in an incomplete interaction between the large analyte surface and the ligands alkyl chains and can have problems entering the pores of the stationary phase.Retention time increases with hydrophobic surface area.Branched chain compounds elute more <a href="http://www.targetmol.com/compound/Daptomycin">Targetmol's Daptomycin</a> rapidly than their corresponding linear isomers because the overall surface area is decreased.Aside from mobile phase surface tension, other mobile phase modifiers can affect analyte retention.For example, the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions, and because the entropy of the analytesolvent interface is controlled by surface tension, the addition of salts tend to increase the retention time.This technique is used for mild separation and recovery of proteins and protection of their biological activity in protein analysis. Another important component is the influence of the pH since this can change the hydrophobicity of the analyte.For this reason most methods use a buffering agent, such as sodium phosphate, to control the pH.The buffers serve multiple purposes: they control pH, neutralize the charge on any residual exposed silica on the stationary phase and act as ion pairing agents to neutralize charge on the analyte.Ammonium formate is commonly added in mass spectrometry to improve detection of certain analytes by the formation of ammonium adducts.A volatile organic acid such as acetic acid, or most commonly formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column eluent.Trifluoroacetic acid is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving retention of analytes such as carboxylic acids in applications utilizing other detectors, as it is one of the strongest organic acids.The effects of acids and buffers vary by application but generally improve the chromatography.Reversed phase columns are quite difficult to damage compared with normal silica columns; however, many reversed phase columns consist of alkyl derivatized silica particles and should never be used with aqueous bases as these will destroy the underlying silica particle.They can be used with aqueous acid, but the column should not be exposed to the acid for too long, as it can   corrode the metal parts of the HPLC equipment.RPHPLC columns should be flushed with clean solvent after use to remove residual acids or buffers, and stored in an appropriate composition of solvent.The metal content of HPLC columns must be kept low if the best possible ability to separate substances is to be retained.A good test for the metal content of a column is to inject a sample which is a mixture of, and, bipyridine.Because the, bipy can chelate the metal, the shape of the peak for the, bipy will be distorted when metal ions are present on the surface of the silica.It is generally a low resolution chromatography and thus it is often reserved for the final, polishing step of purification.It is also useful for determining the tertiary structure and quaternary structure of purified proteins.SEC is used primarily for the analysis of large molecules such as proteins or polysaccharides.

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