Amino acids enantiomeric separations

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. 

Amide derivatives of d-amino acid solutes (III) were tested for resolution. An increase in the bulkiness of the N-alkyl moiety R improved the separation factors (d), i.e., the enantioselectivity. The highest d value (1.43 2(v/v) 2-propanol in n-hexane) was obtained for N-tert-butylamides. Thus, enantiomeric N-tert-butylamide derivatives of N-acyl-d-amino acids were separated with larger d values than corresponding 0-alkyl ester derivatives. 

Cyanuric chloride on monosubstitution with nucleophiles such as methanol or 4-amino-azobenzene followed by displacement of a second chlorine with alanine amide gave compounds which are used for precolumn derivatization of amino acids. The diastereoisomers formed are resolved by reverse-phase HPLC <92MI 6l2-0i>. Enantiomeric amino acids are separated by HPLC on bis[carbamoyl(alkyl)methylamino]-6-chloro-l,3,5-triazine derived stationary phases <93JC277>. 

Bhushan and Ali (1987) tested amino acid separations on silica gel layers impregnated with various metal salts. Bhushan and Reddy (1989) reported the separation of phenylthiohydantoin (PTH) amino acids on silica gel with new mobile phases. Laskar and Basak (1988) de.scribed a new ninhydrin-based procedure that produced different colors and good sensitivity for amino acid detection. Bhushan and Reddy (1987) reviewed the TLC of PTH amino acids. Gankina et al. (1989) described a unidimensional multistep silica gel HPTLC method for the separation and identification of PTH and dansylamino acids. Bhushan et al. (1987) developed numerous solvent systems for effective separations of 2,4-dini-trophenyl-(DNP) amino acids. Bhushan (1988) reviewed the TLC resolution of enantiomeric amino acids and their derivatives. Kuhn et al. (1989) reported the amino acid enantiomer separation by TLC on cellulose of d- and L-tryptophan and methyltryptophan. Guenther (1988) determined TLC-separated enantiomers by densitometry. 

A good enantiomeric resolution of a-amino acids was recently achieved by using chiral complexes of copper (II) with A, A -di-n-propyl-L-alanine (DPA) as the additive in the mobile phase. Actually, the mixture of amino acids is separated into four groups by conventional ion-exchange chromatography and then resolved by means of the chiral DPA reagent (Fig. 5). 

It was not the aim of this chapter to describe all chromatographic separations of racemic amino acids accomplished so far, but rather demonstrate applicability of these methods to separation of DL-amino acids. Chiral separations, using TLC, enables rapid and inexpensive testing both of optical enantiomers, their derivatives, peptides, and control of enantiomeric purity. 

Table 3-1. Values of enantiomeric resolution of DNP-amino acids in a running electrolyte containing the three fractions 1, 2, and 3 of the cyclo(Arg-Lys-X-Pro-X-(3 Ala) sublibrary separated by preparative HPLC.

Although these Boc derivatives underwent methylation with poor selectivity (compared to 3-amino-N-benzoyl butanoates [106] and Z-protected methyl 4-phen-yl-3-aminobutanoate [107]), epimers were succesfully separated by preparative HPLC or by flash chromatography. However, saponification of the methyl ester caused partial epimerization of the a-stereocenter and a two-step (epimerization free) procedure involving titanate-mediated transesterification to the corresponding benzyl esters and hydrogenation was used instead to recover the required Boc-y9 -amino acids in enantiomerically pure form [104, 105]. N-Boc-protected amino acids 19 and 20 for incorporation into water-soluble /9-peptides were pre-... 

Mixing the additive in the eluent used as a mobile phase can also modify the chromatographic system (dynamic modification), but the use of modified adsorbents has led to an improvement of resolution. Example works include that by Armstrong and Zhou [11], who used a macrocyclic antibiotic as the chiral selector for enantiomeric separations of acids, racemic drugs, and dansyl amino acid on biphenyl-bonded silica. 

Separation of amino acids, peptides, and proteins Amino acids are interesting molecules by themselves from an analytical point of view for two reasons. They are inherently enantiomeric and are the building blocks of peptides and proteins. The separation of amino acids is usually done through a derivatization process due to the fact that the absorbance in the UV is low. The most frequently used derivatization is done by fluorescent tagging. Sensitivity can reach the subfemtomole level.136 139 Temperature control can be used to separate conformers.140 Two conformers of Tyr-Pro-Phe-Asp-Val-Val-Gly-NH2 and four conformers of Tyr-Pro-Phe-Gly-Tyr-Pro-Ser-NH2 were separated at subzero temperatures by including glycerol as an antifreeze component of the buffer. 

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... 

With the increased popularity of LC-MS, the problem of overlapping enantiomer peaks from other amino acids has largely been resolved. The mass spectrometer can act as an additional dimension of separation (based on mass to charge ratio). Thus, only amino acids having the same mass-to-charge ratio must be separated achirally (see Desai and Armstrong, 2004). This additional dimension of separation also has implications for the applications in the matrices discussed previously. With the ability of the mass spectrometer to discriminate on the basis of mass, this lessens the need for complete achiral separation. For example, an LC-MS method was recently developed to study the pharmacokinetics of theanine enantiomers in rat plasma and urine without an achiral separation before the enantiomeric separation (Desai et al., 2005). In such matrices, proteins must still be removed by appropriate sample preparation. 

Iida, T., Matsunaga, H., Fukushina, T., Santa, T., Homma, H., Imai, K. (1997). Complete enantiomeric separation of phenylthiocarbamoylated amino acids on a tandem column of reversed and chiral stationary phases. Anal. Chem. 69, 4463-4468. 

Many compounds are less soluble as racemates than as their pure enantiomers. It thus appears probable that evaporation of an amino acid solution with a low ee should cause selective precipitation of the racemate crystals, which in turn should lead to an increase of the ee. Extremely simple manipulations, carried out in the chemistry department of Columbia University, led to a drastic increase in enantiomeric excess of phenylalanine 500 mg phenylalanine (with a 1 % ee of the L-component) was dissolved in water, and the resulting solution slowly evaporated until about 400 mg had crystallised out. The remaining solution contained a few mg of phenylalanine with 40% ee of the L-component (i.e., a 70 30 ratio of l to d). If 500 mg of such a solution (40% ee in water) is allowed to evaporate and is separated from the racemate, the result is about 100 mg, with 90% ee of the L-enantiomer (Breslow and Levine, 2006). 

Amino acid derivatives can be examined for enantiomeric purity by the same procedures after removal of the protecting groups. Another approach is to couple them directly with another derivative to give protected dipeptides whose diastereomeric forms are usually easy to separate by HPLC (see Section 4.11). An A-protected amino acid is coupled with an amino acid ester, and vice versa. Use of soluble carbodiimide as reagent (see Section 1.16), followed by aqueous washes, gives clean HPLC profiles. It is understood that the derivative that serves as reagent must have been demonstrated to be enantiomerically pure.43 84-89... 

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