Reversed-Phase Chromatography RPC

In reversed-phase chromatography (RPC), the mobile phase modulator is typically a water-miscible organic solvent, and the stationary phase is a hydrophobic adsorbent. In this case, the logarithm of solute retention factor is commonly found to be linearly related to the volume fraction of the organic solvent. 

Hydrophobic interaction chromatography (HIC) occupies a unique niche in the field of analytical chromatography. A particular advantage of HIC is its unique selectivity. Whereas ion-exchange chromatography (IEC) principally reveals differences based on the surface charge of native proteins, HIC reveals differences based principally on their surface hydrophobicity. HIC is complementary to reversed-phase chromatography (RPC) in a different sense. Whereas HIC discriminates primarily on the basis of surface hydrophobicity, RPC principally reveals differences based on total hydrophobicity of all the hydrophobic residues of denatured proteins. 

Several researchers have used liquid chromatography in its different modes, such as reversed phase chromatography (RPC), hydrophobic interaction chromatography (HIC), lEC, SEC, and AC, to analyze and fractionate food proteins and peptides. 

This chapter commences with Section 7.1 which deals with reversed-phase chromatography (RPC) and related techniques as applied to synthetic peptides 1-3 A detailed discussion on RPC is presented. Basic considerations are covered as are issues of fundamental physical chemistry. Many examples of chromatography and quantitative relationships are described for peptides and peptide derivatives. There is also an extensive table of naturally occurring peptides that have been isolated and purified by RPC techniques. The section includes many examples of RPC and HPLC profiles of peptidic systems. 

Reverse phase chromatography (RPC) hydrophobicity-based separation... 

The surfaces of proteins are mostly hydrophilic. Although the majority of the hydrophobic residues tend to be buried in the interior of the protein, some hydrophobic regions are also found on the surface (Voet and Voet, 1995 Ladisch, 2001). The level of surface hydrophobicity differs from one protein to another, mainly as a consequence of the amino acid composition and sequence. Difference in surface hydrophobicity is the property exploited in hydrophobic interaction chromatography (HIC) and reverse phase chromatography (RPC). 

Since the commencement of this serial publication high-performance liquid chromatography (HPLC) has continued its meteoric growth, and HPLC is now safely entrenched as the premier analytical technique for mixtures of nonvolatile substances. During the past three years the acceptance of HPLC in the life sciences and the expansion of its scope to the rapid separation of biopolymers has been perhaps the most momentous event. The exploitation of the potential of reversed-phase chromatography (RPC) with hydrocarbonaceous bonded phases as a versatile, efficient, and convenient technique is particularly noteworthy in this regard. As it stands now, HPLC has become an indispensable tool in the armamentarium of life scientists and has found wide use on a quotidian basis. 

The differences in sizes and locations of hydrophobic pockets or patches on proteins can be exploited in hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC) discrimination is based on interactions between the exposed hydro-... 

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. 

Reversed-phase chromatography (RPC) is a method in which molecules are bound hydrophobically to nonpolar ligands in the presence of a polar solvent. Solutes are generally bound in an acidic mobile phase with elution occurring during a gradient to an organic solvent. 

Reversed Phase Chromatography (RPC), Principles and Standard Conditions... 

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... 

Figure 1.3. Schematic diagrams depicting separation modes of (a) normal-phase chromatography (NPC) and (b) reversed-phase chromatography (RPC).

FIPLC columns can be categorized into four major modes (NPC, RPC, IEC, and SEC) in addition to other specialized modes (e.g., affinity, chiral, or specified applications).1,3,9 Since reversed-phase chromatography (RPC) is used in 70-80% of all FIPLC applications, RPC columns for small molecules are the focus of this discussion. 

Table 8.3 lists some common chromatographic modes based on the analyte s molecular weight and polarity. All case studies will focus on reversed-phase chromatography (RPC), the most common mode for small organic molecules. Note that ionizable compounds (acids and bases) are often separated by RPC with buffered mobile phases (to keep the analytes in a non-ionized state) or with ion-pairing reagents. 

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