Hydrocarbonaceous bonded phases

It is likely that small molecules such as short oligopeptides have almost equal access to the hydrocarbonaceous sublayer at the surface of the bonded phases and thus their retention behavior is not affected significantly by the size of the hydrophilic polyether moieties. 

Horvath, C.S. and Melander, W.R., Liquid chromatography with hydrocarbonaceous bonded phases theory and practice of reversed phase chromatography, J. Chromatogr. ScL, 15, 393, 1977. 

Besides hydrocarbonaceous bonded phases, other nonpolar stationary phases can also be employed in RPC. Although some of them will be... 

The reagents cfin be conveniently classified according to (i) the chemical nature of tfie lorganic moiety of interest, (fi) the number of reactive substitumits in th mblecule, and (liO the nature of the reactive fimctions. TaUe n shows cfa orosilane reagents that can be used for the preparation of hydrocarbonac us bonded phases. The first commercial stationary idiases were prq ared with tridilorooctadecylsilane. In recent years, however, the enq loyment of octadecyl and octyldimethylchlorosflanes has been on the increase. 

Hydrocarbonaceous bonded phases described in the literature have at vialues ranging frolm 2 to 4. It is believed that among monomeric phases, those having the highest ol values are the best for use in RPC. In Table III the pertinent clttihtcteristics of some monomeric bonded phases are listed. 

Quite generally a hydrocarbonaceous bonded stationary phase is expected to bind preferentially eluites having the same nonpolar moity as that at the surfsce. Thus, selective stationary phases can be prepared by using, for instance, adamantyl or cholestanyl functions. According to preliminary experiments in our laboratory cholestanyl bonded phases preferentially retain steroids in RPC. At the present stage of development it ap-... 

As discussed by Minors in the first volume of this series (57), modern liipiid chromnlography employs bonded stationary phases not only with hydrocarbonaceous, but also with polar ligates, me bonded phases of intermediate polarity (7/5) can be used with either polar or nonpolar mobile phases. With a sufficiently polar eluent the technique falls into the category of RPC as this chromatographic method by defiiiition employs a mobile phase more polar than the stationary phase so that retention increases with decreasing polarity of eluites having similar molecular dimensions. 

The present popularity of RPC, however, is due to the deyelopment of hydrocarbonaceous bonded phases in microparticulate fond which yield columns particularly suitable for use in sophisticated HPL uipment. Although the column constitutes only a small part of an an ytical liquid chromatograph, both in dimension and price, it occupies a supremely important position. First of all, the chromatographic process tjdces place in... 

The column should permit the modulation of retention behavior over a very wide range of conditions. This requirement in fact means that the stationary phase is inert, that is it does not facilitate specific interactions with certain molecular functions of solute molecules with the concomitant advantage of a relatively clean and rapid adsorption-desorption kinetics. Preferably then the stationary phase has no functional groups such as fixed charges that would have strong affinity to counterionic solutes and exclude solutes of co-ionic nature. In this regard the properties of well-prepared hydrocarbonaceous bonded phases indeed approach those that we would expect from an ideal phase. 

In view of the foregoing discussion of the properties of hydrocarbonaceous bonded phases on silica support it is readily appreciated that well-prepared stationary phases presently used in RPC approach the ideal with the exception of their relatively poor stability in contact with aqueous eluents, particularly at high pH, and the fact that surface silanol groups cannot be completely eliminated. The latter may interact with polar solutes, particularly when the dielectric constant of the eluent is rrlHlively low. Neveritieirss, residual surface silanolii can be masked bs alkylamines in the eluent with the result that peak tailing, when it is due to... 

It is seen that the trichlorosilane reacts with the silanol groups to form siloxane bridges. Subsequently the residual chlorines are hydrolyzed. Under carefiiUy controlled reaction conditions it is possible to obtain a product in which the hydrocarbonaceous layer at the surface is similar to that in a corresponding monomeric bonded phase. However, the hydrolysis of chlorines that did not react with surface silanbis may result in a silanol concentration at the surface that is higher than that in the silica gel proper used as the starting material for the reaction with alkyltri-chlorosilanes. 

Factors that influence the retentive powers and selectivity of such bonded phases include the surface concentrations of hydrodartenaceous ligates and free silanol groups. The thermodynamic aspectitm solute interactions with the hydrocarbonaceous ligates at the surface, which are hydrophobic interactions in the case of aqueous eluents, are discussed later in this chapter within the framework of the solvophobic theory. In practice, however, solute interactions with surface silanol which may be termed silanophilic interactions can also contribute ]to retention (71, 75, 93), particularly in the case of amino compounds. Consequently the retention mechanism may be different from that which would be ol served with an ideal nonpolar phase. Therefore, increasing attention is paid to the estimation of the concentration of accessible sianols and to their elimination from the surface of bonded phases. 

In Table IV various specifications of commercial hydrocarbonaceous bonded phases are listed. It is seen that the conversion of surface sila-... 

The great interest in the possibility of replacing silici based bonded phases by carbon in RPC is understandable because the carbon is expected to be more stable toward aqueous eluents than the iflica-suppoited hydrocarbonaceous phases that are used almost exclusively today. Even if a carbonaceous sorbent with uniform surface and fovdrable porosity would be avaflable its stability may not live up to this expectation, however. The carbon surfoce is readily oxidized and can undergo other chemical transformations with concomitant changes in its retention properties. 

In spite of the preceding observation that eluite retention in RPC with hydrocarbonaceous bonded phases may not occur by partitio ng of the eluite between two liquid phases, theoretical considerations based on the solvophobic treatment of solvent effects shows that it might be possible to relate the observed retention factors to partition coefficients between water and an organic solverit. Such a relationship would be quite useful in light of the scale developed by Hansch and his co-workers (2/12, 283) to characterize hydrophobic properties of drugs and other biologically active... 

In RPC separation of peptides, the fundamental structural properties of the amino adds within the sequence and the relative accessibility of the nonpolar amino add residues to a large measure determine the overall selectivity that can be achieved with a defined RPC systemJ20-23 As a consequence, peptides typically elute from RPC sorbents in the order of their relative hydrophobicities, for a pre-selected mobile-phase composition, pH, and temperature. However, the relative hydrophobicities of different peptides are also conditional on the solvation environment in which they are placed. The exposure or greater accessibility of previously sequestered polar or hydrophobic amino acid side chains in polypeptides with well-developed secondary structures will thus significantly affect the relative binding affinities of these peptides to hydrocarbonaceous-bonded phase surfaces. 

The reasons causing denaturation in RPC are obvious low pH, low ionic strength, organic eluents, and, last but not least, the contact with the rigid and hydrophobic surface. The question how to suppress the unfolding is of practical importance. Low temperature and short contact time with the hydrocarbonaceous layer are recommended. Bonded phases with moderate hydrophobicity may be advantageous, e g., phenyl-... 

As discussed briefly earlier, the stationary phases commonly encountered in high performance liquid chromatography are either polar adsorbents (silica, alumina) or those which contain bonded phases (Yost et al. 1980, Braithwaite and Smith 1985). The latter are most likely to be silica coated with either polar (—NH2, —CN, or diol) or nonpolar (hydrocarbonaceous) ligands covalently attached via siloxane bonds. 

Determinations of the adsorption isotherms for a number of organic solvent-water systems in contact with hydrocarbonaceous stationary phases have shown that a layer of solvent molecules forms on the bonded-phase surface and that the extent of the layer increases with the concentration of the solvent in the mobile phase. For example, methanol shows a Langmuir-type isotherm when distributed between water and Partisil ODS (56). This effect can be exploited to enhance the resolution and the recoveries of hydrophobic peptides by the use of low concentrations, i.e., <5% v/v, of medium-chain alkyl alcohols such as tm-butanol or tert-pentanol or other polar, but nonionic solvents added to aquo-methanol or acetonitrile eluents. It also highlights the cautionary requirement that adequate equilibration of a reversed-phase system is mandatory if reproducible chromatography is to be obtained with surface-active components in the mobile phase. 

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. 

One approach to solving the problem of residual silanol interactions has involved improvements in the synthetic procedures for the production of hydrocarbonaceous stationary phases. One synthetic approach for the elimination of residual silanol groups involves the reaction of the bonded phase with a small silylating reagent such as trimethylchlorosilane which is presumed to have easier access to silanol groups than bulkier, long-chained chlorosilanes. An alternative, synthetic approach involves surface polymerization of the stationary phase, which is believed to reduce the accessibility of surface silanol groups to polar analytes in the mobile phase. Stationary phases produced by the former method are often referred to as end-capped and stationary phases produced by the latter method are sometimes called base-deactivated. ... 

Hennion, M.C., Picaud, C. and Caude, M. (1978) Influence of the number and the length of alkyl chains on the chromatographic properties of hydrocarbonaceous bonded phases. J. Chromatogr. 166, 21-35. 

The rapid development of hydrocarbonaceous bonded phases (nonpolar stationary phases with hydrocarbon chains covalently attached to the silica support) in the 1970s and 1980s for the popular re-versed-phase chromatographic (RPC) technique has, however, eclipsed this technique somewhat, lately. Nevertheless, LLPC has become one of the most powerful separation techniques for the isolation of natural products and biopolymers. 

RPIP chromatography uses a hydrocarbonaceous stationary phase and either an aqueous or aqueous-organic mobile phase which also contains the counter-ion. The stationary phase is usually an octadecyl bonded phase and the mobile phase is usually an aqueous buffer with either methanol or acetonitrile as an organic modifier. The choice of counter-ions depends on the solutes to be separated, but generally for the separation of acids a hydrophobic organic base is added to the mobile phase, while for the separation of bases a hydrophobic organic acid is added. Separations of other compounds are similarly obtained by the addition of an appropriate counter-ion. 

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