Sorption reversed phase

Some authors have suggested the use of fluorene polymers for this kind of chromatography. Fluorinated polymers have attracted attention due to their unique adsorption properties. Polytetrafluoroethylene (PTFE) is antiadhesive, thus adsorption of hydrophobic as well as hydrophilic molecules is low. Such adsorbents possess extremely low adsorption activity and nonspecific sorption towards many compounds [109 111]. Fluorene polymers as sorbents were first suggested by Hjerten [112] in 1978 and were tested by desalting and concentration of tRN A [113]. Recently Williams et al. [114] presented a new fluorocarbon sorbent (Poly F Column, Du Pont, USA) for reversed-phase HPLC of peptides and proteins. The sorbent has 20 pm in diameter particles (pore size 30 nm, specific surface area 5 m2/g) and withstands pressure of eluent up to 135 bar. There is no limitation of pH range, however, low specific area and capacity (1.1 mg tRNA/g) and relatively low limits of working pressure do not allow the use of this sorbent for preparative chromatography. 

Solutes will interact with the reverse phase surface in much the same way as they do with the silica gel surface. There will be basically two forms of interaction, by sorption and by displacement. Sorption interaction has been experimentally confirmed by Scott and Kucera (10) by measuring the adsorption isotherm of acetophenone on the reverse phase RP18 from a 40%w/v acetonitrile mixture in water. The authors noted that there was no change in the acetonitrile concentration, as the solute was adsorbed. Displacement interactions, although certain to occur, do not appear to have been experimentally demonstrated to date. 

Several theoretical models, such as the ion-pair model [342,360,361,363,380], the dyneuaic ion-exchange model [342,362,363,375] and the electrostatic model [342,369,381-386] have been proposed to describe retention in reversed-phase IPC. The electrostatic model is the most versatile and enjoys the most support but is mathematically complex euid not very intuitive. The ion-pair model emd dynamic ion-exchange model are easier to manipulate and more instructive but are restricted to a narrow range of experimental conditions for trtilch they might reasonably be applied. The ion-pair model assumes that an ion pair is formed in the mobile phase prior to the sorption of the ion-pair complex into the stationary phase. The solute capacity factor is governed by the equilibrium constants for ion-pair formation in the mobile phase, extraction of the ion-pair complex into the stationary phase, and the dissociation of th p ion-pair complex in the... 

Lochmiiller, C.H. and Wilder, D.R., The sorption behavior of aUcyl bonded phases in reversed-phase, high performance liquid chromatography, J. Chromatogr. Sci., 17, 574, 1979. 

To determine the effect of mobile-phase composition on the sorption behavior of TGs on reverse-phase columns, two mixtures were employed acetonitrile/ethanol (80 20) and aceto-nitrile/methanol (80 20). A very rapid analysis resulted, with excellent peak shape and adequate resolution, when ethanol was used as the secondary solvent. Substituting an equal amount of methanol for ethanol resulted in increased solute retention, poor detector response, and asymmetrical peaks. Methanol forms a monomolecular layer on octadecyl-derived silica, which may explain the increase in solute retention caused by methanol. Also, the use of methanol would... 

Donati, L., Keizer, J., Bottoni, P., Scenati, R., Funar, E. (1994) Koc estimation of diethylatrazine, diisopropylatrazine, hexazionone, and terbuthylazine by reversed phase chromatography and sorption isotherms. Toxicol. Environ. Chem. 44(1-2), 1-10. 

On the other hand, the lack of internal pore structure with micropellicular sorbents is of distinct advantage in the analytical HPLC of biological macromolecules because undesirable steric effects can significantly reduce the efficiency of columns packed with porous sorbents and also result in poor recovery. Furthermore, the micropellicular stationary phases which have a solid, fluid-impervious core, are generally more stable at elevated temperature than conventional porous supports. At elevated column temperature the viscosity of the mobile phase decreases with concomitant increase in solute diffusivity and improvement of sorption kinetics. From these considerations, it follows that columns packed with micropellicular stationary phases offer the possibility of significant improvements in the speed and column efficiency in the analysis of proteins, peptides and other biopolymers over those obtained with conventional porous stationary phases. In this paper, we describe selected examples for the use of micropellicular reversed phase... 

The concept behind the invention is shown in Figure 9.36. It operates with two mechanisms—size exclusion and reverse phase bonded sorption. The purpose is to facilitate the injection of plasma samples without prior clean up to remove proteins that normally clog a reverse phase column. The pore size of the stationary phase is small enough that the large protein molecules cannot enter and the outer surfaces to which they are exposed do not retain them at all, so they are eluted off the column... 

Solute retention in reversed-phase HPLC is dependent on the different distribution coefficients established between a polar mobile and a nonpolar stationary phase by the peptidic components of a mixture. Although there are many similarities between reversed-phase HPLC separations of peptides and the classical liquid-liquid partition chromatographic methods, it is debatable whether the sorption process in reversed-phase HPLC arises due to partition or adsorption events, i.e., whether the nonpolar stationary phase functions as a bulk liquid or as an adsorptive monolayer. These aspects and the theoretical models for reversed-phase HPLC are discussed in a subsequent section. 

The basic model for the separation of peptidic solutes on nonpolar stationary phases assumes that reversible interactions of the solute molecules S, S2,. . . , S occur with the hydrocarbonaceous ligand L and that the interactions are due to hydrophobic associations and not to electrostatic or hydrogen bonding effects. Conceptually, the sorption of peptides to alkyl-bonded reversed phases under these conditions can be based either on partition or on adsorption processes. In a partition pro-... 

Although the side-chain groups R, R, R have only a weak influence on the acid dissociation constants and the reverse situation, namely a change in polarity due to an ionization event in the vicinity of R, R, R, can have a significant influence on retention. The capacity factor equation describing the sorption to a reversed phase of the above simple nonpolar peptide as a function of pH is given by... 

In a series of studies we recently demonstrated (29, 30, 63-67) that the resolution of peptides on reversed phase can be profoundly influenced by the addition of appropriate counterionic reagents to a mobile phase of deflned pH, ionic strength, and water content. Retention under these conditions can be discussed in terms of ion-air associations between the ionized peptide and a counterion in the mobile phase and subsequent sorption of the complex onto the stationary phase. Alternatively, adsorption of the counterion, particularly if it is lipophilic, onto the nonpolar stationary phase may occur, and peptide retention would then be mediated by dynamic liquid-liquid ion-exchange effects. Arguments in favor of the participation of one, the other, or both of these alternative pairing-ion phenomena in ion-pair chromatography have been extensively reviewed (16, 28b, 62, 68, 68a). It can be shown (62, 68) that retention behavior in ion-pair systems can be described by... 

McCormick, R.M. and Karger, B.L. (1980) Role of organic modifier sorption on retention phenomena in reversed-phase liquid chromatography. J. Chromatogr. IW, 259-273. 

Solid-state Si and CPMAS NMR have been used to characterize three different reversed-phase materials for high-performance liquid chromatography, obtained by sorption of poly(methyloctylsiloxane) onto bare silica, titanized silica and zirconized silica pores and to study the coupling of y-methacryloxypropyltrimethoxysilane on experimental nano-porous silica fillers. 

Solid-phase extraction (SPE) is a method of sample preparation that concen-Irates and purifies analytes from solution by sorption onto a disposable solid-phase cartridge, followed by elution of the analyte with a solvent appropriate for instrumental analysis. The mechanisms of retention include reversed phase, normal phase, and ion exchange. Traditionally, sample preparation consisted of sample dissolution, purification, and extraction that was carried out with liquid-liquid extraction. The disadvantages with liquid-liquid extraction include the use of large volumes of organic solvent, cumbersome glassware, and cost. Furthermore, liquid-liquid extraction often creates emulsions with aqueous samples that are difficult to extract, and liquid-liquid extraction is not easily automated. These difficulties are overcome with solid-phase extraction. Thus, solid-phase extraction was invented in the mid-1970s as an alternative approach to liquid-liquid extraction. 

Some SPE packing materials do not receive endcapping thus, it is important to check for endcapping when choosing an SPE sorbent for a specific analyte. For example, in the case when the R group is C-18, the solute would experience reversed-phase sorption to the C-18 and in some cases sorption to the hydroxyl groups. When the interaction between the two groups is intentional, this is called a mixed-mode application of SPE. More examples of mixed-mode SPE will be shown later in this chapter. 

Figure 2.10. Reversed-phase mechanism of sorption of dioctylphthalate in SPE. [Reproduced in modified form from Zief and Kiser (1987) and published with permission.]...

Occasionally, one inadvertently may use a mixed-mode mechanism in an SPE method. A good example is the sorption of triazine herbicides onto C-18 bonded phases that are monofunctional and are not endcapped. In this case, the basic compound has the potential for hydrogen bonding to the silanol sites of the silica gel, as well as reversed-phase sorption into the C-18 bonded phase. 

The elution of analytes from reversed-phase sorbents is a rather simple process and consists of choosing a nonpolar solvent to disrupt the van der Waals forces that retain the analyte. Because the sorption process is a partitioning process, it is usually only necessary to allow the eluting solvent to have intimate contact with the bonded phase (e.g., C-18) in order to elute the analytes from the sorbent. Because the bonded phases consist of a silica matrix, they have an increased polarity compared to the original hydrophobicity of the C-18 alkane. Thus, the elution solvent must be capable of mutual solubility with the silica surface, as well as with the C-18 or other bonded phase. 

Change the form of the analyte. If the molecule is ionic, change the pH of the sample so that the molecule is nonionic this step will increase the sorption of the solute for a reversed-phase sorbent. 

Try salting out for reversed phase. Add 5 to 10% sodium chloride to the matrix, if aqueous. Hydration of the salt increases the polarity of the solvent and drives the analyte onto the reversed-phase sorbent. Change the mechanism of sorption. For example, if reversed phase is being used, and the molecule can be made ionic, change the mechanism of isolation to ion exchange. 

Change the elution solvent so that the interferences remain on the solid phase and the analyte is eluted. An example is the sorption of a herbicide from water and its elution from a reversed-phase sorbent, C-18, using ethyl acetate rather than methanol. Ethyl acetate does not remove the majority of the natural organic substances (humic substances)" from the sorbent, while methanol does. Thus, the chromatogram is considerably cleaner with the ethyl-acetate eluent. 

The fundamental step in SPE is recognizing that the hydrophobicity versus the polarity of an analyte controls the mechanism of sorption that may be selected. Thus, step one is to select a mechanism of sorption, and Figure 3.8 is a guide to this process. Part A shows the process for aqueous samples and part B for nonaqueous samples. With aqueous samples, the decision for the sorbent is based on the polarity and ionic character of the analyte. If the analyte is polar and ionic, then ion exchange is the preferred sorbent. If the analyte is polar but nonionic, then reversed phase by either C-18 or a polymeric phase is the best sorbent. If the analyte is nonpolar, then a reversed-phase sorbent such as C-8 or C-18 is chosen. 

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