Enantiomers stationary phase

Figure 1. Plots of capacity factors k vs /5-CD concentration for methylphenobarbital enantiomers. Stationary phase 10 nm LiChrosorb RP-18 mobile phase 20% ethanol-buffer solution of pH=2.0 containing various /9-CD concentrations temperature 25 "C. (Reprinted with permission from ref. 26. Copyright 1986 Marcel Dekker.)...

Twenty-eight chiral compounds were separated from their enantiomers by HPLC on a teicoplanin chiral stationary phase. Figure 8-12 shows some of the structures contained in the data set. This is a very complex stationary phase and modeling of the possible interactions with the analytes is impracticable. In such a situation, learning from known examples seemed more appropriate, and the chirality code looked quite appealing for representing such data. 

Figure 8-12. Examples of las eluted enantiomers In a chromatographic separation on chiral HPLC with teicoplanin stationary-phase.

A few GLC stationary phases rely on chemical selectivity. The most notable are stationary phases containing chiral functional groups, which can be used for separating enantiomers. ... 

An alternative model has been proposed in which the chiral mobile-phase additive is thought to modify the conventional, achiral stationary phase in situ thus, dynamically generating a chiral stationary phase. In this case, the enantioseparation is governed by the differences in the association between the enantiomers and the chiral selector in the stationary phase. 

Most chiral chromatographic separations are accompHshed using chromatographic stationary phases that incorporate a chiral selector. The chiral separation mechanisms are generally thought to involve the formation of transient diastereomeric complexes between the enantiomers and the stationary phase chiral ligand. Differences in the stabiHties of these complexes account for the differences in the retention observed for the two enantiomers. Often, the use of a... 

Chiral separations present special problems for vaUdation. Typically, in the absence of spectroscopic confirmation (eg, mass spectral or infrared data), conventional separations are vaUdated by analysing "pure" samples under identical chromatographic conditions. Often, two or more chromatographic stationary phases, which are known to interact with the analyte through different retention mechanisms, are used. If the pure sample and the unknown have identical retention times under each set of conditions, the identity of the unknown is assumed to be the same as the pure sample. However, often the chiral separation that is obtained with one type of column may not be achievable with any other type of chiral stationary phase. In addition, "pure" enantiomers are generally not available. 

Three general methods exist for the resolution of enantiomers by Hquid chromatography (qv) (47,48). Conversion of the enantiomers to diastereomers and subsequent column chromatography on an achiral stationary phase with an achiral eluant represents a classical method of resolution (49). Diastereomeric derivatization is problematic in that conversion back to the desired enantiomers can result in partial racemization. For example, (lR,23, 5R)-menthol (R)-mandelate (31) is readily separated from its diastereomer but ester hydrolysis under numerous reaction conditions produces (R)-(-)-mandehc acid (32) which is contaminated with (3)-(+)-mandehc acid (33). 

Chiral Chromatography. Chiral chromatography is used for the analysis of enantiomers, most useful for separations of pharmaceuticals and biochemical compounds (see Biopolymers, analytical techniques). There are several types of chiral stationary phases those that use attractive interactions, metal ligands, inclusion complexes, and protein complexes. The separation of optical isomers has important ramifications, especially in biochemistry and pharmaceutical chemistry, where one form of a compound may be bioactive and the other inactive, inhibitory, or toxic. 

Separation of enantiomers by physical or chemical methods requires the use of a chiral material, reagent, or catalyst. Both natural materials, such as polysaccharides and proteins, and solids that have been synthetically modified to incorporate chiral structures have been developed for use in separation of enantiomers by HPLC. The use of a chiral stationary phase makes the interactions between the two enantiomers with the adsorbent nonidentical and thus establishes a different rate of elution through the column. The interactions typically include hydrogen bonding, dipolar interactions, and n-n interactions. These attractive interactions may be disturbed by steric repulsions, and frequently the basis of enantioselectivity is a better steric fit for one of the two enantiomers. ... 

The potential for use of chiral natural materials such as cellulose for separation of enantiomers has long been recognized, but development of efficient materials occurred relatively recently. Several acylated derivatives of cellulose are effective chiral stationary phases. Benzoate esters and aryl carbamates are particularly useful. These materials are commercially available on a silica support and imder the trademark Chiralcel. Figure 2.4 shows the resolution of y-phenyl-y-butyrolactone with the use of acetylated cellulose as the adsorbent material. 

Synthetic chiral adsorbents are usually prepared by tethering a chiral molecule to a silica surface. The attachment to the silica is through alkylsiloxy bonds. A study which demonstrates the technique reports the resolution of a number of aromatic compoimds on a 1- to 8-g scale. The adsorbent is a silica that has been derivatized with a chiral reagent. Specifically, hydroxyl groups on the silica surface are covalently boimd to a derivative of f -phenylglycine. A medium-pressure chromatography apparatus is used. The racemic mixture is passed through the column, and, when resolution is successful, the separated enantiomers are isolated as completely resolved fiactions. Scheme 2.5 shows some other examples of chiral stationary phases. 

Scheme 2.5. Chiral Stationary Phases for HPLC Separation of Enantiomers...

An interesting and practical example of the use of thermodynamic analysis is to explain and predict certain features that arise in the application of chromatography to chiral separations. The separation of enantiomers is achieved by making one or both phases chirally active so that different enantiomers will interact slightly differently with the one or both phases. In practice, it is usual to make the stationary phase comprise one specific isomer so that it offers specific selectivity to one enantiomer of the chiral solute pair. The basis of the selectivity is thought to be spatial, in that one enantiomer can approach the stationary phase closer than the other. If there is no chiral selectivity in the stationary phase, both enantiomers (being chemically identical) will coelute and will provide identical log(Vr ) against 1/T curve. If, however, one... 

The column was 25 cm long, 4.6 mm I.D. and packed with Partisil 10. It is seen that linear curves were obtained for three different solutes and two different moderators in n-heptane. Scott and Beesley [14] obtained retention data for the two enantiomers, (S) and (R) 4-benzyl-2-oxazolidinone. The column chosen was 25 cm long, 4.6 mm I.D. packed with 5 mm silica particles bonded with the stationary phase Vancomycin (Chirobiotic V provided by Advanced Separations Technology Inc., Whippany, New Jersey). This stationary phase is a macrocyclic glycopeptide Vancomycin that has a molecular weight of 1449.22, and an elemental composition of 54.69% carbon. 

It is seen from equation (22) that there will, indeed, be a temperature at which the separation ratio of the two solutes will be independent of the solvent composition. The temperature is determined by the relative values of the standard free enthalpies of the two solutes between each solvent and the stationary phase, together with their standard free entropies. If the separation ratio is very large, there will be a considerable difference between the respective standard enthalpies and entropies of the two solutes. As a consequence, the temperature at which the separation ratio becomes independent of solvent composition may well be outside the practical chromatography range. However, if the solutes are similar in nature and are eluted with relatively small separation ratios (for example in the separation of enantiomers) then the standard enthalpies and entropies will be comparable, and the temperature/solvent-composition independence is likely be in a range that can be experimentally observed. 

Erank H, G. J. Nicholson and E. Bayer, Rapid gas cliromatographic separation of amino acid enantiomers with a novel cliiral stationary phase , J. Chromatogr. Sci. 15 174-176(1977). 

HPLC separations are one of the most important fields in the preparative resolution of enantiomers. The instrumentation improvements and the increasing choice of commercially available chiral stationary phases (CSPs) are some of the main reasons for the present significance of chromatographic resolutions at large-scale by HPLC. Proof of this interest can be seen in several reviews, and many chapters have in the past few years dealt with preparative applications of HPLC in the resolution of chiral compounds [19-23]. However, liquid chromatography has the attribute of being a batch technique and therefore is not totally convenient for production-scale, where continuous techniques are preferred by far. 

The first partial chiral resolution reported in CCC dates from 1982 [120]. The separation of the two enantiomers of norephedrine was partially achieved, in almost 4 days, using (/ ,/ )-di-5-nonyltartrate as a chiral selector in the organic stationary phase. In 1984, the complete resolution of d,l-isoleucine was described, with N-dodecyl-L-proline as a selector in a two-phase buffered n-butanol/water system containing a copper (II) salt, in approximately 2 days [121]. A few partial resolutions of amino acids and dmg enantiomers with proteic selectors were also published [122, 123]. 

W. H. Pirkle and B. C. Hamper, The direct preparative resolution of enantiomers by liquid chromatography on chiral stationary phases in Preparative Liquid Chromatography, B. A. Bidling-meyer (Ed.), Journal Chromatography Library Vol. 38, 3 Edition, Elsevier Science Publishers B. V, Amsterdam (1991) Chapter 7. 

A. M. Stalcup, Cyclodextrin bonded chiral stationary phases in enantiomer separations in A practical approach to chiral separations by liquid chromatogra.phy, G. Subramanian, VCH, Weinheim (1994) Chapter 5. 

Enantiomeric separations have become increasingly important, especially in the pharmaceutical and agricultural industries as optical isomers often possess different biological properties. The analysis and preparation of a pure enantiomer usually involves its resolution from the antipode. Among all the chiral separation techniques, HPLC has proven to be the most convenient, reproducible and widely applicable method. Most of the HPLC methods employ a chiral selector as the chiral stationary phase (CSP). 

Fig. 3-1. Separation of racemic 3,5-dinitrobenzamido leucine Al.A -diallylamide on silica and polymer-based chiral stationary phases. Conditions column size 150 x 4.6 mm i.d. mobile phase 20 % hexane in dichloromethane flowrate 1 mL min injection 7 pg. Peaks shown are l,3,5-tri-rert.-butylbenzene (1), R-enantiomer (2) 5-enantiomer (2 ). (Reprinted with permission from ref. [8]. Copyright 1997 American Chemical Society.)...

Proteins. A chiral stationary phase with immobilized a -acid glycoprotein on silica beads was introduced by Hermansson in 1983 [18, 19]. Several other proteins such as chicken egg albumin (ovalbumin), human serum albumin, and cellohy-drolase were also used later for the preparation of commercial CSPs. Their selectivity is believed to occur as a result of excess of dispersive forces acting on the more retained enantiomer [17]. These separation media often exhibit only modest loading capacity. 

Despite the difficulties caused by the rapidly expanding literature, the use of chiral stationary phases (CSPs) as the method of choice for analysis or preparation of enantiomers is today well established and has become almost routine. It results from the development of chiral chromatographic methods that more than 1000 chiral stationary phases exemplified by several thousands of enantiomer separations have been described for high-performance liquid chromatography (HPLC). 

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