Reversed-phase chromatography applications

In SPE, choice of an appropriate solid phase is based on their possible molecular interactions with the targeted compounds. For historical reasons, reversed-phase type adsorbents have been largely utilized to extract compounds from aqueous samples, given the fact that many of them have been developed already for use in reversed-phase chromatography applications and are commercially available. Ion exchange adsorbents have been used to retain counter ions based on electrostatic interactions. Solid phase adsorbents combining hydrophobic and ionic interactions are also present, for example graphitized carbon black (GCB) obtained by... 

Recently, new approaches of sorbent construction for reversed-phase chromatography have been developed. Silicas modified with hydrocarbon chains have been investigated the most and broadly utilized for these aims. Silica-based materials possess sufficient stability only in the pH 2-8 range. Polymeric HPLC sorbents remove these limitations. Tweeten et al. [108] demonstrated the application of stroongly crosslinked styrene-divinylbenzene resins for reversed-phase chromatography of peptides. 

Compared with liquid column chromatography, in PLC there is a certain limitation with respect to the composition of the mobile phase in the case of reversed-phase chromatography. In planar chromatography the flow of the mobile phase is normally induced by capillary forces. A prerequisite for this mechanism is that the surface of the stationary phase be wetted by the mobile phase. This, however, results in a Umitation in the maximum possible amount of water applicable in the mobile phase, is dependent on the hydrophobic character of the stationary RP phase. To... 

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. 

Obviously, the monolithic material may serve its purpose only if provided with a suitable surface chemistry, which depends on the desired application. For example, hydrophobic moieties are required for reversed phase chromatography, ionizable groups must be present for separation in the ion-exchange mode, and chiral functionalities are the prerequisite for enantioselective separations. Several methods can be used to prepare monolithic columns with a wide variety of surface chemistries. 

One application in liquid chromatography which does alter the separation process is the use of a specific series of derivatives to enable the separation of chiral (optical isomers) forms of alcohols, amines and amino acids using reverse-phase separation. FLEC is available in the two chiral forms (+)-l-(9-fluorenyl) ethyl chloroformate and (—)-l-(9-fluorenyl) ethyl chlorofor-mate (Figure 3.12). Reaction of two stereoisomers of a test compound (e.g. T+ and T—) with a single isomer of the derivatizing reagent (e.g. R+) will result in the formation of two types of product, T+R+ and T—R+. It is possible to separate these two compounds by reverse-phase chromatography. 

Fnhanced-fluidity liquid reversed-phase chromatography has numerous applications including the separation of nonpolar and polar compounds. For example, EFLC and nonaqueous reversed-phase HPLC are the common means of achieving effective separations of high molecular weight homologous compounds. 

Applications of Reversed-Phase Chromatography in Peptide Isolation... 

B. Do, S. Robinet, D. Pradeau and F. Guyon, Speciation of arsenic and selenium compounds by ion-pair reversed-phase chromatography with electrothermal atomic absorption spectrometry. Application of experimental design for chromatographic optimisation, J. Chromatogr. A, 918(1), 2001, 87-98. 

Several new phases applied to maltosaccharide analysis that seem to be promising have been described in the literature, for instance, a silica-phase covered with polymeric polyamine resin, and an entirely polymeric resin containing an amide function (35). In any case, partition chromatography is restricted mainly to the utilization of polar-bonded phases, as already described. Nevertheless, there have been a certain number of applications of reversed-phase chromatography that permit relatively simple separations to be achieved. Octadecyl-bonded silica phases are the most widely used, although few applications involve carbohydrate analysis. Their interest lies rather in the analysis of derivated sugars, where the selectivity increases (36). 

WO Landen Jr, RR Eitenmiller. Application of gel permeation chromatography and nonaqueous reverse phase chromatography to high pressure liquid chromatographic determination of retinyl palmitate and /3-carotene in oil and margarine. J Assoc Off Anal Chem 62 283-289, 1979. 

Recent advances in chromatography have made it possible to employ microbore HPLC for the determination of NOC. Its main advantage is that it uses a very low mobile-phase flow (20-100 /rl/min). This might make the TEA compatible with a reversed-phase system. Massey et al. (73), in fact, have successfully used reversed-phase chromatography for the HPLC-TEA determination of V-nitroso-V, 7V -di methylpiperazinium iodide. A 500-mm X 1-mm microbore ODS column and a mobile phase consisting of 0.1 M ammonium heptane-sulfonate in methanol water (70 30) (flow rate 20 /zl/min) was used for the HPLC separation. In another study, Riihl and Reusch (74) used a microbore Spherisorb 3 SW column for HPLC-TEA determination of volatile V-nitrosamines. The mobile phase was a mixture of 2-propanol and n-hexane (2.5 97.5). Further application of such techniques for the determination of various polar NOC, especially A-nitrosamides, in foods is desirable. 

The application area of IPC and IC is overlapping, whereas IPC fills the gap between IC and reversed phase chromatography [ 3]. As one borderline case IPC can be described as IC with dynamically coated exchangers. The ion pairing reagent is simply... 

Many applications have been found for reversed-phase chromatography in HPLC. The composition of the stationary phase is more easily controlled than with the TLC methods, and thus provides more reproducible separations. The use of bonded non-polar phases enables gradient elution to be carried out in a reversed-phase system. This approach has been useful for the analysis of polar compounds and gives improved separations compared with normal-phase HPLC. These methods usually involve separation with systems consisting of Carbowax, C -polymer or similar phases bonded or physically coated on the support. 

Lamb s group has adsorbed the hydrophobic crown ethers and cryptands shown in Fig 1 onto reversed phase chromatography packings for application to ion chromatography in the analysis of cations and anions. A brief introduction to ion chromatography (IC) is in order to lay the foundation for a description of this work. 

Afton, S., K. Kubachka, B. Catron, et al. 2008. Simultaneous characterization of selenium and arsenic analytes via ion-pairing reversed phase chromatography with inductively coupled plasma and electrospray ionization ion trap mass spectrometry for detection. Applications to river water, plant extract and urine matrices. J. Chromatogr. A 1208 156-163. 

The synthesis of phosphopeptides is typically confirmed mass spectrometri-cally using either a MALDI (matrix-assisted laser desorption/ionizafion) or an ESI (electrospray ionization) source, and the peptide purity is determined by reversed-phase chromatography coupled to an UV detector (Figs. 1 and 2B). Whenever possible, phosphopeptide analyses should be complimented by mass spectra recorded in negative ion mode. For most biochemical applications it is necessary to purify the peptides by HPLC techniques (Fig. 2A). 

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