Amino acid chiral separation

Miniaturized columns have provided a decisive advantage in speed. Uracil, phenol, and benzyl alcohol were separated in 20 seconds by CEC in an 18 mm column with a propyl reversed phase.29 A19 cm electrophoretic channel was etched into a glass wafer, filled with a y-cyclodextrin buffer, and used to resolve chiral amino acids from a meteorite in 4 minutes.30 A 6 cm channel equipped with a syringe pump to automate sample derivatization was used to separate amino acids modified with fluorescein isothiocyanate.31 Nanovials have been used to perform tryptic digests on the 15 nL scale for subsequent separation on capillary Electrophoresis.32 A microcolumn has also been used to generate fractions representing time-points of digestion from a 40 pL sample.33 A disposable nanoelectrospray emitter has been... 

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... 

The alkylated 3,6-dialkoxy-2,5-dihydropyrazines are hydrolyzed by treatment with 0.25 N hydrochloric acid (2 equivalents of H+) at room temperature to give the hydrochlorides of the corresponding alkylglycine methyl ester 3 and the chiral auxiliary amino acid methyl ester 2. Hydrolysis of the dihydropyrazines 1 under more drastic conditions (10-30 equivalents of 6 N hydrochloric acid) yields the corresponding diketopiperazines which are very stable to further hydrolysis. Basification of the hydrochlorides with aqueous ammonia liberates the free a-amino esters. In general, the chiral auxiliary amino ester is separated by distillation. 

Since Pasteur separated crystalline sodium ammonium tartrate manually in 1848, many researchers have worked on the subject of enantiomeric separation. In 1939 Henderson and Rule fully separated derivatives of camphor by column chromatography using lactose as a stationary phase material [1]. Gil-Av et al. [2] were able to separate amino acid derivatives on a polysiloxane-based stationary phase by gas chromatography (GC) in 1966. Since then many approaches for a successful distinction between enantiomers have been developed for capillary GC and liquid chromatography [3]. It is still a current topic for researchers searching for chiral separation with SciFinder [4] results in 812 hits and searching for chiral recognition leads to 285 hits for the year 2003 only. 

Similarly, enantiopure 3-substitutcd-/V-rnc thy I benzyl /3-sultams have been converted into A -methylbenzyl-a-amino acid thioesters via sulfenylation and Pummerer rearrangement with high or complete retention of configuration. Chiral sulfoxides were prepared by sulfenylation followed by oxidation of trans-isomers as two separable A and B stereoisomers. Treatment with TFAA gave chiral cr-amino acid thioesters in high yields with a de > 90%. Slight epimerization of the cr-chiral center of the cr-phenyl thioesters has been observed under the reaction conditions whereas no epimerization was observed in the case of -/-butyl thioesters (Scheme 28) <1998JOC8355>. 

Type IV includes chiral phases that usually interact with the enantiomeric analytes through the formation of metal complexes. There are usually used to separate amino acid enantiomers. These types of phases are also called ligand exchange phases. The transient diastereomeric complexes are ternary metal complexes between a transitional metal (usually Cu +), an amino acid enantiomeric analyte, and another compound immobilized on the CSP which is able to undergo complexation with the transitional metal (see also the ligand exchange section. Section 22.5). The two enantiomers are separated based on the difference in the stability constant of the two diastereomeric species. The mobile phases used to separate such enantiomeric analytes are usually aqueous solutions of copper (II) salts such as copper sulfate or copper acetate. To modulate the retention, several parameters—such as the pH of the mobile phase, the concentration of the copper ion, or the addition of an organic modifier such as acetonitrile or methanol in the mobile phase—can be varied. 

A second strategy used to separate amino acids is based on the fact that two enantiomers react differentiy with chiral reagents. An enzyme is typically used as the chiral reagent. 

Most of amino acids are chiral and as discussed earlier, the chiral separation is a challenging problem. Another possibility to separate amino acids is to use enzymes that can recognize and selectively separate them. As enzymes also catalyze chemical reactions on substrate molecules that they bind to, this creates an unwanted problem because the reaction products must be separated in the following step. This problem can be circumvented by using apoenzymes as molecular-recognition transporter. Apoenzymes are enzymes that lack in their cofactors they cannot transform substrates into products. 

Ghosh, S., Hui, T., Uddin, M. S., Hidajat, K. (2013). Enantioselective separation of chiral aromatic amino acids with surface functionalized magnetic nanoparticles, 105, 267-277. 

Most of amino acids are chiral and as discussed earlier, the chiral separation is a challenging problem. Another possibility to separate amino acids is to use enzymes that can recognize and selective separate them. Because enzymes... 

Another elegant example of the imitation of the properties of biopolymers by synthetic polymers comes from the school of E. Bayer of Tubingen (172). They have prepared chiral polysiloxane polymers for resolution of optical antipodes. The prochiral polymeric backbone was a copolymer of poly [(2-carboxypropyl)methylsiloxane], octamethylcyclotetrasiloxane, and hexa-methyldisiloxane. Amino acids or small peptides were covalently linked to this polymer in order to introduce a chiral surface. For this, the free carboxyl function of the polymer was reacted with the L-amino acid in the presence of DCC (see Chapter 2). The individual chiral centers (amino acids) on the polymer surface were separated by siloxane chains of specified length in order to achieve optimum interaction with the substrate and polymer viscosity. An example of great value for optical resolution is the polymer designated chirasil-Val, containing 0.86 mmole of iV-tert-butyl-L-valin-amide per gram of polymer (Fig. 5.14). 

Proteias, amino acids bonded through peptide linkages to form macromolecular biopolymers, used as chiral stationary phases for hplc iaclude bovine and human semm albumin, a -acid glycoproteia, ovomucoid, avidin, and ceUobiohydrolase. The bovine semm albumin column is marketed under the name Resolvosil and can be obtained from Phenomenex. The human semm albumin column can be obtained from Alltech Associates, Advanced Separation Technologies, Inc., and J. T. Baker. The a -acid glycoproteia and ceUobiohydrolase can be obtained from Advanced Separation Technologies, Inc. or J. T. Baker, Inc. 

Diamide Chiral Separations. The first chiral stationary phase for gas chromatography was reported by GH-Av and co-workers in 1966 (113) and was based on A/-trifluoroacetyl (A/-TFA) L-isoleucine lauryl ester coated on an inert packing material. It was used to resolve the tritiuoroacetylated derivatives of amino acids. Related chiral selectors used by other workers included -dodecanoyl-L-valine-/-butylamide and... 

Among various types of chiral stationary phases, the host-guest type of chiral crown ether is able to separate most amino acids completely (58). 

Achiral Columns Together with Chiral Mobile Phases. Ligand-exchange chromatography for chiral separation has been introduced (59), and has been appHed to the resolution of several a-amino acids. Prior derivatization is sometimes necessary. Preparative resolutions are possible, but the method is sensitive to small variations in the mobile phase and sometimes gives poor reproducibiUty. 

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]. 

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