Reversed-phase chromatography characteristics

Fig. 3.1c. Summary of characteristics of normal and reverse phase chromatography...

General Characteristics of Reversed-Phase Chromatography Separations of... 

Mobile-Phase Characteristics in the Reversed-Phase Chromatography of... 

The retention times of peptides with fewer than 20 residues in reversed-phase chromatography can be predicted with a high degree of accuracy based on their amino acid composition and the characteristics of their N-terminal and C-terminal amino acids. A number of researchers (66 -75) have studied the role of amino acids in peptide retention and have established retention coefficients for the different amino acids. The retention coefficient value of each amino acid is normally calculated by regression analysis of the retention times for peptides of known composition. 

The following are suggested supplemental experiments which will further increase your understanding of the use and characteristics of reverse-phase chromatography. They may be done under the operating conditions previously established, depending on the lab time available and course requirements. 

Kennedy 1990). This form of chromatography uses hydrophilic gel-based material as a matrix, which has been partially substituted on the surface with non-polar alkyl (e.g., methyl or octyl) or aryl (e.g., phenyl) groups (Table 4-3). Similar materials are used in reverse-phase chromatography (RPC), but the degree of substitution used for HIC chromatography (10-50 pmol ml-1 gel) is much lower than that used in RPC (100-500 pmol ml-1 gel). The solvent used for elution in HIC is low ionic strength buffer and not organic solvents which are characteristic of RPC. 

The molecular characteristics exploited for the separation of proteins and peptides include molecular size (size exclusion chromatography, SEC), net charge (ion-exchange chromatography, IEC), polarity (reverse-phase chromatography, RPC), hydrophobicity (hydrophobic interaction... 

A common characteristic of all polystyrene/divinylbenzene-based ion-exclusion phases is the high retention of aromatic carboxylic acids. This is due to 7t-7t interactions between the aromatic ring systems of the polymer and the solute. The separation of aromatic carboxylic acids, therefore, is more elegantly accomplished by the various procedures of reversed-phase chromatography. 

Taking aliphatic monocarboxylic acids as an example, the elution order, according to Fig. 4-7, is formic acid > acetic acid > propionic acid and so forth. Such separations have characteristics similar to that of reversed-phase chromatography. Van-der-Waals forces between the solute and the polymeric resin material (mainly benzene rings) as well as the decrease in solubility of the solutes in the eluent influence their distribution between the stationary and mobile phases. 

The high hydrophobicity of the surface would make the divinylbenzene particle the ideal packing for reversed-phase chromatography. However, the slow mass-transfer characteristic of polymeric packings is especially pronounced for divinylbenzene packings and has prevented it from displacing... 

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