Reverse phase chromatography fundamentals

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. 

The best operative conditions to separate the 20 natural amino acids by using a wide variety of commercially available stationary phases used both in normal and in reversed-phase chromatography and by two-dimensional (2D) chromatography technique are described. Resolution of amino acids derivatives, which play a fundamental role in the peptide and protein sequence structures, is also reported. 

A variety of micropellicular packing materials has been developed for the analysis of both small and large molecules by various HPLC modes, including ion exchange (lEC), metal interaction (MIC), reversed phase (RPC) [4], and affinity chromatography (AC) [5]. Besides analytical applications, other possible utilization of micropellicular stationary phases includes fundamental kinetic and thermodynamic studies of the retention mechanisms on a well-defined surface. Nevertheless, a relatively limited variety of micropellicular... 

Deoxyribonucleic acid and ribonucleic acid, as well as their nucleotides, nucleosides, and base constituents play an important role in many vital biochemical processes of medical interest. To better understand these processes, fundamental investigations into the structure, occurrence, search for modifications, and biochemical impact of structural variation are required. Thus, reliable high-resolution analytical methods for the separation and identification of the nucleic acid constituents (often at extremely low concentration levels) had to be developed. Chromatography (including reversed-phase hquid chromatography), ion exchange chromatography, dHPLC, and electrophoresis... 

In reversed-phase liquid chromatography (RPLC) and electromigration techniques (CEC, CZE), aqueous solutions of methanol, acetonitrile, tetrahydrofu-ran, and dioxane are used as eluents. In this mode of LC, a fundamental concern is the pruity of water, which can contain phenols, hydrocarbons, etc. Tetra-hydrofuran is used frequently as the solvent in gel permeation chromatography. It must be stabilized by butylated hydroxytoluene used as antioxidant. Similarly, when halogenated solvents (e.g., trichloroethylene) are used triethanolamine is added as a... 

The fundamental components of any modern-day HPLC system are a solvent delivery system, a sample injector, a column, a detector, and a computer with the appropriate data acquisition and processing software. There are numerous HPLC methods described in the literature for isoflavones [13-25] and for the common anthocyanins, each method invokes different combinations of solvent systems, columns, and detectors. HPLC has been interfaced with a variety of detection methods such as ultraviolet/visible (UV/vis) spectrocopy and hquid chromatography-mass spectrometry (LC-MS) [21,22]. In this chapter, however, discussion is restricted to the most commonly used pairing in flavonoid analysis, that of a reverse-phase (RP-18) column and a UV/visible detector. 

The fundamental characteristic of ion-pair chromatography is that the addition of the counter ion enhances the retention of the solute, without which the solute would either move with the solvent, in the case of reversed-phase support, or be completely retained, in the case of normal-phase support (or at least experience severe tailing and poor resolution). The enhanced retention is a consequence of the partitioning of the ion pair into the stationary phase subsequent to partitioning of the ions into the stationary phase. Thus, the equilibrium constants defined in Fig. 2.22, which are unique for each particular solute, counter ion, and stationary and mobile phase, are the factors that define the retention of a particular solute, the column efficiency, and hence the efficiency of the separation. 

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