46 August / September 2016

occurs, positively charged at the protonated nitrogen atom of the bicyclic quinuclidine moiety and negatively charged at the sulphonic acid functional group. These CSPs separate amino acids by an electrostatic interaction between the charged species, supported (structure dependant) by hydrogen bonding, Van der Waals forces, p−p stacking and hydrophobic interactions.

A recent study [17] showed the influence of steric effects. Enantioselectivity increases with the side chain length when it is linear and increases with the bulkiness and rigidity of the side chain when it is branched. Steric effects may also be responsible for the longer retention of b-amino acids on these CSPs. There are indications that the amino group may not always be necessary for enantioselectivity, as evidenced by the separations achieved for N-protected amino acids, such that the CSP behaves in such cases as a chiral anion exchange rather than a zwitterionic CSP.

The main advantage of these phases is the ability to provide reversal of elution order [Figure 2] by switching columns, although the resolution may differ slightly mainly because the two CSPs are not exact enantiomeric versions of the same selector.

The next generation: Superficially and Fully Porous Particles

Higher performance smaller particle size CSPs have been made available over recent years utilising 3µm and sub-2µm particles; in contrast, CSPs for amino acid separations have until recently largely remained in 5 and 3 µm formats. Since their launch in 2006 for reversed phase separations [24], superficially porous particles (SPP) have continued to gain success and widespread application. A porous layer over a solid core of the SPP improves mass transfer kinetics since analytes cannot diffuse into the particle. It was realised that higher efficiencies and faster separations could be readily achieved from the resulting reduced band broadening [25]. The narrower particle size distribution provided by this technology leads to a more homogeneously packed bed and a consequential higher back pressure (albeit lower than that for sub-2µm columns,[26]), such that these phases could be used on both HPLC and the then new UHPLC instruments. Later research [27] also showed that the increased efficiencies of these particles is largely due to improvements in the A and B term of the van Deemter equation (eddy and longitudinal diffusion, respectively) resulting from homogeneously packed columns.

Recently, several studies have been carried out to incorporate this technology into chiral applications. The practical difficulties of bonding more bulky chiral selectors onto sub-2µm particles along with the tendency of the particles to aggregate were overcome in a study by Sciascera et al, creating a sub-2µm version of the brush-type CSP, Whelk-O1 [28].

The axial and radial temperature gradients that result from frictional heating in SPPs [29] can have a marked effect on CSPs made from these phases when mass transfer effects are more prominent.

A new fully porous particle (FPP) was recently developed that has an extremely high efficiency and a reduced plate height of 1.7 in narrow bore columns [31]. This was investigated as a potential particle for CSPs resulting in very fast separations in seconds rather than minutes [32,33]. Traditional 5µm and new 1.9µm TPP teicoplanin bonded phases were compared [33] and showed a 3-4-fold increase in efficiency (N/m) with a reduction in reduced plate height from 3.5 to 2.5. Methionine, for instance, exhibited a resolution of 3.0 in MeOH/ H2

O in under 40 seconds on a 5 x 4.6mm 1.9µm FPP teicoplanin column. It was noted that the increased permeability of this CSP enabled fast separations at high flow rates without excessive frictional heating. High speed peptides separations were also demonstrated; a separation of the dipeptide DL-Leu-DL-Ala was possible in less than one minute (Figure 3) [30].

Figure 3. Separation of the dipeptide LeuAla on Teicoplanin bonded SPP particles. [Data courtesy of D W Armstrong]

A further study [30] showed that in high water content mobile phases, axial temperature gradients improved mass transfer and countered any loss in efficiency due to radial temperature gradients and eddy diffusion. This resulted in a significantly increased efficiency for teicoplanin bonded SPP: high resolutions of 1.6 to 3.0 were achieved in less than one minute for a range of amino acids in MeOH/H2


Gasparrini et al [32] in contrast utilised a novel bonding chemistry on the same 1.9µm TPP that resulted in a protonated amino group on the teicoplanin structure, such that this CSP maintains a zwitterionic character, as evidenced by the separation of a range of hydrophobic, neutral and permanently charged solutes. This developmental CSP provided a high selectivity of 2.25 to 10.7 for a range of N-protected amino acids in RP with a 10 cm column length that minimises the impact of extra column effects. Reducing this to 2cm maintained relative efficiencies and resulted in extremely fast separations. BOC-D,L-Met, for example, separated in under 1 minute in RP, with a resolution of 2.20 and average efficiency of (93,575 N/m) at 2 mL/min. Ultra-fast separations using a 1cm column were also explored and

Table 1. Commonly used Chiral Stationary Phases for Amino Acid separations


CHIRAL LIGAND Macrocyclic glycopeptide Teicoplanin

Macrocyclic glycopeptide Teicoplanin Aglycone Chinchona alkaloid

Ligand Exchange

Macrocyclic glycopeptide Brush type


Quinine, quinidine bonded with ACHSA CHIRALPAK ZWIX(+) and ZWIX(-) Crown Ether Phenylalanine Amino Acid Ristocetin A

1-(3,5-dinitrobenzamido)- 1,2,3,4-tetrahydrophenanthrene

Polycyclic amine Inclusion complex

(S)-valine and 3,5-dinitroaniline urea Cyclodextrin Cyclodextrin

CROWNPAK CR-I Astec CLC-D, CLC-L Nucleosil Chiral CHIROBIOTIC R Whelk-O1

P-CAP Chirex


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