Stationary derivatization

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). 

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. 

Other examples of irreversible derivatization on treatment with iodine have been described for phenohc steroids (estrone derivatives [256]), morphine [257] and 23 other pharmaceuticals [258]. These reactions are probably favored by the presence of silica gel as stationary phase and by the influence of light. 

Recently, multidimensional GC has been employed in enantioselective analysis by placing a chiral stationary phase such as a cyclodextrin in the second column. Typically, switching valves are used to heart-cut the appropriate portion of the separation from a non-chiral column into a chiral column. Heil et al. used a dual column system consisting of a non-chiral pre-column (30 m X 0.25 mm X 0.38 p.m, PS-268) and a chiral (30 m X 0.32 mm X 0.64 p.m, heptakis(2,3-di-(9-methyl-6-(9-tert-butyldimethylsilyl)-(3-cyclodextrin) (TBDM-CD) analytical column to separate derivatized urinary organic acids that are indicative of metabolic diseases such as short bowel syndrome, phenylketonuria, tyrosinaemia, and others. They used a FID following the pre-column and an ion trap mass-selective detector following the... 

The educt, a racemate, is derivatized before the separation with an agent which might be achiral or unichiral (Fig. 7-1), and afterwards is passed through a chromatographic system which is equipped with a stationary phase. This stationary phase may also be achiral or unichiral in nature. 

Type Eduet Derivatizing Agent number type Stationary phase type of binding or reaetion... 

This strategy is the one most commonly used for the analytical determination of ena-tiopurity. A given racemate is reacted with a unichiral derivatizing agent, and the resulting pair of diastereomers is separated on an achiral stationary phase, in most of the cases on a reversed-phase type (Fig. 7-2). 

Derivatization of a racemic compound with an achiral group may play an important role in the analysis of a chiral compound (Fig. 7-15). In the case of substances with low or no UV-activity, the compounds can be rendered detectable by introducing an UV-absorbing or fluorescent group. If the racemate itself shows selectivity on a chiral stationary phase (CSP), this method can be applied to reduce the limit of detection. Examples have been reported in the literature, especially for the derivatization of amino acids which are difficult to detect using UV detection. Different derivatization strategies can be applied (Fig. 7-16). 

Mourier s report was quickly followed by successful enantiomeric resolutions on stationary phases bearing other types of chiral selectors, including native and deriva-tized cyclodextrins and derivatized polysaccharides. Many chiral compounds of pharmaceutical interest have now been resolved by packed column SFC, including antimalarials, (3-blockers, and antivirals. A summary is provided in Table 12-2. Most of the applications have utilized modified CO, as the eluent. 

With the development of HPLC, a new dimension was added to the tools available for the study of natural products. HPLC is ideally suited to the analysis of non-volatile, sensitive compounds frequently found in biological systems. Unlike other available separation techniques such as TLC and electrophoresis, HPLC methods provide both qualitative and quantitative data and can be easily automated. The basis for the HPLC method for the PSP toxins was established in the late 1970 s when Buckley et al. (2) reported the post-column derivatization of the PSP toxins based on an alkaline oxidation reaction described by Bates and Rapoport (3). Based on this foundation, a series of investigations were conducted to develop a rapid, efficient HPLC method to detect the multiple toxins involved in PSP. Originally, a variety of silica-based, bonded stationary phases were utilized with a low-pressure post-column reaction system (PCRS) (4,5), Later, with improvements in toxin separation mechanisms and the utilization of a high efficiency PCRS, a... 

The most common technique used for agrochemicals is reversed-phase SPE. Here, the bonded stationary phase is silica gel derivatized with a long-chain hydrocarbon (e.g. C4-C18) or styrene-divinylbenzene copolymer. This technique operates in the reverse of normal-phase chromatography since the mobile phase is polar in nature (e.g., water or aqueous buffers serve as one of the solvents), while the stationary phase has nonpolar properties. 

Membranes offer a format for interaction of an analyte with a stationary phase alternative to the familiar column. For certain kinds of separations, particularly preparative separations involving strong adsorption, the membrane format is extremely useful. A 5 x 4 mm hollow-fiber membrane layered with the protein bovine serum albumin was used for the chiral separation of the amino acid tryptophan, with a separation factor of up to 6.6.62 Diethey-laminoethyl-derivatized membrane disks were used for high-speed ion exchange separations of oligonucleotides.63 Sulfonated membranes were used for peptide separations, and reversed-phase separations of peptides, steroids, and aromatic hydrocarbons were accomplished on C18-derivatized membranes. 

Many times an analyte must be derivatized to improve detection. When this derivatization takes place is incredibly important, especially in regards to chiral separations. Papers cited in this chapter employ both precolumn and postcolumn derivatization. Since postcolumn derivatization takes place after the enantiomeric separation it does not change the way the analyte separates on the chiral stationary phase. This prevents the need for development of a new chiral separation method for the derivatized analyte. A chiral analyte that has been derivatized before the enantiomeric separation may not interact with the chiral stationary phase in the same manner as the underivatized analyte. This change in interactions can cause a decrease or increase in the enantioselectivity. A decrease in enantioselectivity can result when precolumn derivatization modifies the same functional groups that contribute to enantioselectivity. For example, chiral crown ethers can no longer separate amino acids that have a derivatized amine group because the protonated primary amine is... 

One of the most powerful methods for determining enantiomer composition is gas or liquid chromatography, as it allows direct separation of the enantiomers of a chiral substance. Early chromatographic methods required the conversion of an enantiomeric mixture to a diastereomeric mixture, followed by analysis of the mixture by either GC or HPLC. A more convenient chromatographic approach for determining enantiomer compositions involves the application of a chiral environment without derivatization of the enantiomer mixture. Such a separation may be achieved using a chiral solvent as the mobile phase, but applications are limited because the method consumes large quantities of costly chiral solvents. The direct separation of enantiomers on a chiral stationary phase has been used extensively for the determination of enantiomer composition. Materials for the chiral stationary phase are commercially available for both GC and HPLC. 

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