During a study to determine the importance of the glycone moiety in the mechanism of separation of tecoplanin bonded phases [20], it was expected that enantioselectivity would disappear one it was removed. In fact, selectivity remained but changed such that the aglycone version enabled alternative separations and mobile phases for different amino acids. It was made available commercially as CHIROBIOTIC TAG in 2000. In contrast to teicoplanin, this CSP contains a single primary amine that remains charged under usual HPLC conditions.

Figure 1. Separation of peptide isoforms of Luteinising hormone-releasing factor, LHRH [Data courtesy of D W Armstrong]

Mobile phases

Most mobile phases in use for the macrocyclic glycopeptide and chinchona alkaloid based CSPs are of either polar organic or aqueous organic composition. Polar organic mobile phases are principally a non-aqueous methanol / acetonitrile mix, with the addition of acid and base where control of the ionisable moieties of the glycopeptide CSP and solutes is required. The polar organic was first developed by Armstrong in 1993 for use with cyclodextrin CSPs (commercially, CYCLOBOND, [15] and was then extended to the macrocyclic glycopeptide phases when they were launched in 1994. In recent years, polar organic has also been adopted by several other CSPs including brush type CSPs for general chiral separations [16]. For amino acid separations on the chinchona alkaloid based CSPs [17], formic acid, diethlyamine and water are usually added in small quantities to control the comparative strengths of anionic and cationic interactions. Simple alcohol water mixes are also used with the macrocyclic glycopeptide phases, adding buffer when the amino acid has ionisable functional groups additional to the alpha zwitterion moiety.

Macrocyclic glycopeptides CSPs

The variety of functional groups of macrocyclic glycopeptide phases (known commercially as CHIROBIOTIC) provides high selectivity for ionisable and zwitterionic compounds, including amino acids. Since just after their launch in 1994, the use of the macrocyclic glycopeptide, teicoplanin has been successfully used for the separation of a wide range of amino acid types and small peptides [18]. The range of enantioselectivity

of teicoplanin is based on its ability to offer multiple mechanisms that includes hydrogen bonding, dipole-dipole, π−π, van der Waals, hydrophobic, ionic and steric interactions. NMR and HPLC Investigations by Gasparrini et al [19] indicated that free carboxylic groups can strongly interact by hydrogen bonding with the CSP peptide amide moieties, providing high selectivity for a wide range of free and N-protected amino acids and peptides. The amine group of the amino acid can be free or blocked with N-bonded functional groups such as 6-aminoquinolyl-N- hydroxysuccinimidyl carbamate (AQC, trade mark Waters Corporation), benzoyl, N-tert- butoxycarbonyl (tBOC), carboxybenzyl (CBZ), 5-(dimethylamino)naphthalene-1-sulphonyl (dansyl) and 9-fluorenylmethoxycarbonyl (FMOC), making this method suitable for the monitoring of peptide synthesis. In each case for all amino acids, the elution order is L before D and the mobile phase alcohol / water, adding formic acid for acidic amino acids and ammonium acetate buffer for the basic ones.

An interest in the therapeutic effects of changing the conformation of one or more amino acids in a peptide led to the use of teicoplanin for the separation of peptide isoforms, using a simple mobile phase of acetonitrile/formic acid (Figure 1). This method enabled selectivity between peptides with just one amino acid difference, even if not in the terminal position [7].

Zwitterionic Ion Exchange Type CSPs

By combining quinine with (S,S)-trans- 2-aminocyclohexane sulphonic acid (ACHSA), or quinidine with the (R,R) form, and bonding at the C-9 position via a carbamate linkage, two new 3 and 5µm brush type chinchona alkaloid CSPs (commercially, CHIRALPAK ZWIX(+) and ZWIX(-), respectively) were commercially introduced in 2012 [21,22,23]. The phases incorporate a weak anionic and a strong cationic interaction site. By selecting particular mobile phase conditions, both the amphoteric amino acid analyte and the zwitterionic CSP can become charged; polar organic solvents in combination with added acid and base ensure that ionisation

Figure 2. Reversal of elution order of L-enriched trans 4-hydroxy proline on CHIRALPAK ZWIX(+)/ZWIX(-) columns.

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