Reversed-phase chromatography salts

Reversed-phase chromatography is widely used as an analytical tool for protein chromatography, but it is not as commonly found on a process scale for protein purification because the solvents which make up the mobile phase, ie, acetonitrile, isopropanol, methanol, and ethanol, reversibly or irreversibly denature proteins. Hydrophobic interaction chromatography appears to be the least common process chromatography tool, possibly owing to the relatively high costs of the salts used to make up the mobile phases. 

Hydrophobic interaction chromatography (HIC) can be considered to be a variant of reversed phase chromatography, in which the polarity of the mobile phase is modulated by adjusting the concentration of a salt such as ammonium sulfate. The analyte, which is initially adsorbed to a hydrophobic phase, desorbs as the ionic strength is decreased. One application demonstrating extraordinary selectivity was the separation of isoforms of a monoclonal antibody differing only in the inclusion of a particular aspartic acid residue in the normal, cyclic, or iso forms.27 The uses and limitations of hydrophobic interaction chromatography in process-scale purifications are discussed in Chapter 3. 

In reversed-phase chromatography, the addition of buffers or salts to the mobile phase reduced the separation of a mixture of ILs with different cations and anions to a separation based on the hydrophobicity of the cation [25]. Plots... 

Common surfactants that have been used in MEKC, are listed in Table 3.1 with the respective critical micelle concentrations the most popular are SDS, bile salts, and hydrophobic chain quaternary ammonium salts. Selectivity can also be modulated by the addition to the aqueous buffer of organic solvents (methanol, isopropanol, acetonitrile, tetrahydrofuran, up to a concentration of 50%). These agents will reduce the hydrophobic interactions between analytes and micelles in a way similar to reversed-phase chromatography. Organic modifiers also reduce the cohesion of the hydrophobic core of the micelles, increasing the mass transfer kinetics and, consequently, efficiency. Nonionic... 

A solution of potassium nitrosodisulfonate (Fremy s salt) (0.56 mmol) dissolved in 5 ml water was added to a solution of the product from Step 7 (0.18 mmol) dissolved in 5 ml dioxane, the mixture stirred at ambient temperature 2 hours, and concentrated. The material was purified by reverse phase chromatography and the product isolated in 54% yield having a HPLC retention time of 1.75 minutes. 

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