Optically active isomers, separation

Faolini and Divizia have succeeded in partially resolving inactive linalol into its optically active isomers hut only to the extent of optical rotations of + 1° 70 and — 1° 60 respectively. Linalol was converted into its acid phthalate, and an alcoholic solution of this compound was treated with the equivalent quantity of strychnine. By fractional crystallisation the laevo-rotatory salt, yielding dextro-rotatory linalol, separates first, leaving the more soluble dextro-rotatory strychnine salt, which yielded laevo-rotatory linalol in the mother liquor. 

Chiral stationary phases for the separation of enantiomers (optically active isomers) are becoming increasingly important. Among the first types to be synthesized were chiral amino acids ionically or covalently bound to amino-propyl silica and named Pirkle phases after their originator. The ionic form is susceptable to hydrolysis and can be used only in normal phase HPLC whereas the more stable covalent type can be used in reverse phase separations but is less stereoselective. Polymeric phases based on chiral peptides such as bovine serum albumin or a -acid glycoproteins bonded to... 

Three methods are known, all due to Pasteur, by which an inactive mixture can be separated into its optically active isomers —... 

Alanine, aspartic acid, glutamic acid, tyrosine, leucine, phenylalanine and also a-amino-n-caproic acid and a-aminobutyric acid have in this way been separated by Fischer into their optically active isomers. To these must be added ornithine which was synthesised by Sorensen in 1903, and separated into d- and 1-ornithine in 1905. 

This is the first stereospecific cleavage of a Si—Si bond by an alkali metal. Functional optically active disilanes were first prepared in 1967 by Sommer et al.528, 529, and the optically active isomers were successfully separated. 

Separation of optically active isomers is one of the most important areas of HPLC apphcation in the pharmaceutical industry. Since most of biological systems are predominantly homochiral, different enantiomers of the same drug could have different effect and potency, and the development of enan-tioselective analytical (and preparative) separation methods is very important. Detailed description of chiral HPLC separation is given in Chapter 22 of this book here we only briefly review the specifics of distinctive types of chiral stationary phases (CSP). 

One of the first chromatographic separation of enantiomers was by Gil-Av, who used gas chromatography to separate the trifluoroacetyl derivatives of some amino acids on an optically active stationary phase consisting of N-trifluoroacetyl-L-phenylalanine cyclohexyl ester. It was noted that the L isomers eluted last on the L stationary phase but first on the D stationary phase. Gas chromatography has not proved the ideal technique for separating optically active isomers as all solutes... 

Sulfonium ions differ substantially in structure. The inversion barrier is much higher (AH = 20 + 30 kcal mole ) and in son cases optically active isomers having a high stability were separated (e.g. Me, Et, n-Bu—The bond angles are much smaller and much closer to 90°. With increasing size of the electronic cloud around the heteroatom, the hybridization of the free electron pair with those involved in chemical bonding is of less importance and bond angles approach 90° e.g. ... 

The optically active isomers of endo-dicyclopentadiene have been obtained (460) by reaction of a-phenylethylamine with (methoxydi-cyclopentadiene)chloroplatinum(II) dimer, separation of the diastereo-isomcrs of the monomeric (CioHi20CH3)Pt(amine)Cl, and treatment of these with KCN. 

Verapamil is a synthetic compound possessing slight structural similarity to papaverine. It can be separated into its optically active isomers, of which the levorotatory enantiomer is the most potent. It is absorbed rapidly after oral administration. The drug is metaboli zed quickly and. as a result, has low bioavailability. The liver is the main site of first-pass metaboli.sm. forming several products. The preferential metabolic step involves N-dealkylation. followed by O-demethylation. and subsequent conjugation of the product before elimination. The metabolites have no significant biological activity. Verapamil has an elimination half-life of approximately 5 hours. 

Stereoisomers can be classified into two types enantiomers and dia-stereomers. Enantiomers (mirror images) have identical physical and chemical properties and therefore are not separated on the conventional reversed-phase stationary phases. Their separation will not be discussed. Diastereomers are isomers which are not mirror images of the parent. They have slightly different physical and chemical properties and can often be separated on conventional stationary phases. There are two classes of diastereomers optically active isomers when the API has two or more stereocenters and non-optically active geometric isomers, such as cis-trans, syn-anti, etc. Stereoisomers of chiral molecules must be included in the peak set. 

The circulins—As early as 1949, Peterson and Reineke characterized circulin as its sulphate. Total hydrolysis yielded D-leucine, L-threonine and L-K,y-diaminobutyric acid together with an optically active isomer of pelargonic acid. The existence of two components, found by Peterson and Reineke was later confirmed by the chromatographic separation of crude circulin into two major components, named circulin A and circulin B. In addition there was evidence for at least three other ninhydrin-positive, biologically active entities. In the hydrolysate of circulin A, L-isoleucine was found besides the amino acids previously reported . Quantitative amino acid analysis showed circulin A and B to be composed of L-a,y-diamino-butyric acid, L-threonine, D-leucine, L-isoleucine and ( + )-6-methyloctanoic acid in the molar ratio 6 2 1 1 1. After partial acid hydrolysis, fractionation and structure determination of the resulting peptides, circulin A and circulin B were formulated as cyclodecapeptides . Very recently, however, Japanese workers have revised the structure of circulin A. According to them, circulin A differs from colistin A only by a replacement of L-leucine in the latter by L-isoleucine Figure 1.7). 

A second approach to Isomer separation by HPLC Is to use a non-optlcally active stationary phase and an optically active solvent. If the amino acids can Interact with both the stationary and mobile phases, but one of the Isomers Interacts more strongly with the mobile, optically active phase, separation of the Isomers Is possible (49). In 1979, several laboratories reported methods Involving the use of chiral mobile-phases containing zlnc(II) or copper (II) complexed to an L-amlno acid (51-53). A distinct advantage of these methods Is that they do not require derlvatlza-tlon of the sample prior to analysis. However, separation of a complete mixture of amino acids (such as that obtained from a protein hydrolysate) has not been reported. 

The relationship between cardiac depressant, local anesthetic and 6-adrenergic blockade actions has evoked considerable interest. A common mechanism could be responsible for all actions. However, the separation of racemic alprenolol and propranolol into their optically active isomers resulted in only the levo isomers being potent 6-blockers while both isomers possessed local anesthetic and cardiac depressant actions . The separation of bunolol into its optical isomers resulted in a further separation of the actions of 6-blockers. Bunolol is devoid of significant antiarrhythmic and local anesthetic activities. Dextro-bunolol is inactive as a 6-blocker but did possess a cardio-depressant action equal to that of the leyo isomer. Thus, no correlation between local anesthetic, 6-blockade or toxic cardiac depression actions exist. Separate mechanisms for the three activities probably are involved. 

Until the discoveries of Alfred Werner, it was thought that carbon had to be present in a compound for it to be optically active. Werner prepared the following compound containing OH ions as bridging groups and separated the optical isomers, a. Draw structures of the two optically active isomers of this compound. 

A new class of membrane materials, the molecularly imprinted membranes, probably has found the highest number of applications in the pharmaceutical field, due to the great advantage of being able to discriminate enantiomers from an optically active isomer complex mixture. These types of materials, which are based on the molecular recognition from an active site of the membrane and imprinted molecule (template), are particularly used in chromatography for the separation of the enantiomers and the controlled release of pharmaceutical optically active substance. 

Capillary columns having a chiral stationary phase are used for the separation of optically active isomers or enantiomers, namely, species that have the same... 

As noted in Section 2 the d- and fi-isomers of optically active molecules exhibit somewhat different order parameters in liquid crystals possessing a local screw sense (such as cholesteryl derivatives). Accordingly d- and C-isomers should separate on cholesteric substrates. For 3,3,3-trichloropropylene oxide the difference in the order parameter is of the two isomers is AS z = 0.00015 (cf Section 2) for a compensated nematic mixture of cholesteryl derivatives. On the basis of Equ. (42) one would expect a separation factor of a 1.002. Up to the present the separation of optically active isomers on cholesteric substrates has not been achieved. 

A similar situation is encountered in ansa-type compounds (e.g., benzene derivatives in which para or meta positions are linked via heteroatoms containing bridge). In these compounds, as in cyclophanes, when a bridge is sufficiently short, the benzene ring rotation can be hindered, making feasible separation of individual optically active isomers. Figure 2.23 shows schematic representation of isomers of an ansa-type compound. 

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