Chiral Derivatization Chromatography

This chapter will focus on topic 3, which is normally regarded to be chiral derivatization chromatography, but will also cover other topics that might be considered when applying derivatization techniques. The goal for the separation of the race-mates may be their analysis or their preparation. Both topics will be covered in this chapter. 

One of the most useful applications of chiral derivatization chromatography is the quantification of free amino acid enantiomers. Using this indirect method, it is possible to quantify very small amounts of enantiomeric amino acids in parallel and in highly complex natural matrices. While direct determination of free amino acids is in itself not trivial, direct methods often fail completely when the enantiomeric ratio of amino acid from protein hydrolysis must be monitored in complex matrices. 

Fig. 7-1. General reaction scheme of chiral derivatization chromatography.

Schulte, M. Chiral derivatization chromatography, in Chiral Separation Techniques, Subramanian, G. (Ed.), Wiley-VCH, Weinheim, 2001... 

Tao QF, Zeng S. 2002. Analysis of enantiomers of chiral phenethylamine drugs by capillary gas chromatography/ mass spectrometry/fLame-ionization detection and precolumn chiral derivatization. J Biochem Biophys Methods 54 103. 

Toyo oka T. 2002. Resolution of chiral drugs by liquid chromatography based upon diastereomer formation with chiral derivatization reagents. J Biochem Biophys Methods 54 25-56. 

As discussed earlier, the concepts of chiral chromatography can be divided into two groups, the indirect and the direct mode. The indirect technique is based on the formation of covalently bonded diastereomers using an optically pure chiral derivatizing agent (CDA) and reacting it with the pair of enantiomers of the chiral analyte. The method of direct enantioseparation relies on the formation of reversible quasi diastereomeric transient molecule associates between the chiral selector, e.g., i /t)-SO, and the enantiomers of the chiral selectands, [R,S)-SAs [(Ry SA + (S)-SA] (Scheme 1). 

M. Wilson and T. Walle, Silica gel high performance liquid chromatography for the simultaneous determination of propranolol and 4-hydroxypropranolol enantiomers after chiral derivatization, J. Chromatogr., 370 424-430 (1984). 

Sun, X. X., Sun, L. Z., Aboul-Enein, H. Y. Chiral derivatization reagents for dmg enantioseparation by high-performance liquid chromatography based upon precolumn derivatization and formation of diastereomers enantioselectivity and related stmcture. Biomed. Chromatogr., 2001,15,116-132. 

Wilson, M.J. Ballard, K.D. Walle, T. Preparative resolution of the enantiomers of the beta-blocking drug atenolol by chiral derivatization and high performance liquid chromatography. J.Chromatogr, 1988, 431, 222-227... 

Potts, B.D. Parli, C.J. Analysis of the enantiomers of fluoxetine and norfluoxetine in plasma and tissue using chiral derivatization and normal-phase liquid chromatography. J.Liq.Chromatogn, 1992, 15, 665-681... 

The first effective chiral stationary phase, as already referred to in chapter 1, utilized derivatized amino acids to provide chiral selectivity [7] and this was achieved as early as 1966. These types of stationary phases however, had very limited temperature stability and the optimum temperature for separation was often in excess of that at which the stationary phase was stable. The first relatively stable stationary phase, as already mentioned, was introduced by Bayer [6], who combined the derivatized optically active component of the stationary phase in a polysiloxane gum. Nevertheless, a number of small molecular weight materials were used as stationary phase in the early days of chiral gas chromatography. The first type introduced by Gil-Av are shown below. 

Anal3des derivatized with a chiral derivatization reagent have been separated by reversed-phase liquid chromatography " the reversed-phase liquid chromatography of derivatized amino acid enantiomers was analyzed using... 

The nature of the chiral derivatization reagent is critical in achieving optimum resolution of the diastereomers, and for simple carboxylic add reagents containing a phenyl or other 7t-donating group close to the chiral centre are recommended to improve the resolution. This effect was particularly well demonstrated by Kaneda [59], who showed that the S(+)-2-butanol (20) or R(—)-2-octanol (21) esters of 2-methylbutyric add were unresolved by gas chromatography but the esters with R- -methylbenzyl alcohol (19) were readily separated. (See Section 4.2.2 for the numbered structures). 

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