Therefore, the total number of hydrophobic groups dominates the elution process during RP-HPLC. Many proteins unfold upon contact with the hydrophobic ligands and by being dissolved in an organic solvent of low pH. This chromatography is based on hydrophobic interaction between hydrophobic ligands attached to a column support and hydrophobic patches on the protein. RP-HPLC can also be used for the purification of integral membrane proteins. Welling, Sytske Welling-Wester, in Journal of Chromatography Library, 2000 12.4.4. Recently this methodology has proved advantageous for linking LC separations directly to electrospray mass spectrometry. In the case of heparin oligosaccharides, these are first converted into organic salts, often a hydrophobic ammonium salt, for example, tetrabutylammonium by neutralizing the acidic form of the oligosaccharide with, for example, tetrabutylammonium hydroxide ( 17). This technique has only been used to separate relatively small oligosaccharides with long run times and problematic desalting making the technique laborious ( 16). The compounds are therefore separated on account of their differing hydrophobic character. Reverse-phase HPLC involves binding an organic molecule to a stationary phase, often silica derivatized with alkyl chains, in a relatively polar environment (the mobile phase), which could contain water, and then eluting the organic molecule using a gradient of a less polar organic solvent. Turnbull, in Chemistry and Biology of Heparin and Heparan Sulfate, 2005 3 Reverse-Phase Ion-Pairing High Performance Liquid Chromatography The radiosequencing procedure gives only limited information about the cleavage sites of insulin since the peptide bonds cleaved beyond the radiolabeled tyrosine (Tyr A14, Tyr A19, Tyr B16, or Tyr B26) are not identified. 6.ĭetermine the cycle at which the radioactivity (-Tyr) is released and identify subsequently the amino-terminal residue of radiolabeled intermediate. Submit resuspended samples to automated Edman degradation as described by Clot et al. 4.Ĭollect the major radioactive components in the eluates and freeze-dry the samples. Monitor eluates online for radioactivity using a γ-detector. 2.Ĭhromatograph the radioactive samples using a mixture of 0.1% trifluoroacetic acid (TFA) in water (solvent A) and 0.1% TFA in acetonitrile (solvent B) with a flow rate of 1 ml/min and an isocratic elution of 28% solvent B (70 min). Load the radioactive hydrolytic sample (diluted in 15% acetic acid) onto a RP-HPLC column (microBondapak C18 column, 0.39 × 30 cm, 10 − 5 m particle size) connected to a single pump liquid chromatograph. Monoiodinated insulins (especially Tyr A14 insulin and Tyr A19 insulin) have allowed the first identification of radioactive insulin intermediates produced in vivo within rat hepatic endosomes ( Seabright & Smith, 1996). With HPLC procedures, radiolabeled insulin may be used as the protease substrate. Reverse-phase (RP) HPLC is by far the most sensitive assay able to detect a single peptide bond cleavage. RP-HPLC allows purification of most classes of compounds, including compounds present in various herbal products, and is often the most preferable choice when analyzing and attempting to separate and identify compounds from a complex mixture. In RP-HPLC the polar molecules travel through the column more quickly. They spend less time in solution in the solvent, and this slows them down on their way through the column, which means longer retention time. They are less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules. Nonpolar compounds in the mixture tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. Therefore, polar molecules in the mixture spend most of their time moving with the solvent.
In RP-HPLC there is strong attraction between the polar solvent and polar molecules in the mixture being passed through the column, but there is not much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution.
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