Type V CSPS

Type V CSPs are protein phases. Because of the well established chemo- and stereospecificity of enzymes, a large number of experimentalists have adapted proteins in one form or another as stationary phases for chiral separations. The intermolecular forces responsible for analyte binding to these biopolymers are the same as for most other CSPs but the size and complexity of proteins makes them difficult to study computationally. One would think that with approximately 400 entries in the Brookhaven Protein Databank to select from, separation scientists would have used one of these proteins as a chiral selector and then use those atomic coordinates to carry out molecular modeling studies. Only one example has appeared in the literature where information from the PDB has been used to serve as a beginning point for molecular modeling of a protein CSP. In all other examples the CSP is viewed as having an unknown structure and Quantitative Structure-Enantioselective Retention Relationships (QSERRs) have been carried out. 

Because so few type V CSPs have well established molecular structures, most scientists are forced to use a series of probe molecules and some sort of regression analysis to divulge pertinent information about the mechanism of chiral recognition. For example, Norinder and Hermansson [85] separated thirty-five N-aminoalkylsuc-cinimides, 36, on an ai-acid glycoprotein (AGP) column. 

To explore the relationship between molecular structure and enantioselectivity a principal component analysis with partial least squares projection techniques allowed the authors to determine which of 50 physicochemical descriptors correlated with separation factors. A partial list of variables used in their models is given in Table 4. 

Similar descriptors along with indicator variables for the other R groups on 36 provide the other variables used. 

Four significant principal components describing 85% of the variance were found. The most important variables were associated with positions 6 and 7 on the aryl ring. Lipophilic groups containing aromatic character are especially important for enantioselectivity and the length of the aliphatic side chain was also found to be important. 

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The ability of proteins to stereoselectively bind small molecules has been used to develop a series of commerdally available protein-based CSPs (the type V HPLC CSPs), including phases that contain immobilized AGP (84), HSA (85), BSA (86), and ovomucoid (OVM) (87) (see Table 5). All these CSPs are useful in the HPLC resolution of enantiomeric compounds and appear to have an extremely wide range of applications, and the AGP CSP seems to have the broadest utility of any of the current CSPs (9-11). However, although the type V CSPs display high enantioselectivities, they also have low capadties due to the relatively small amounts of the chiral selector that can be immobilized per g silica. Thus, these CSPs are useful... 

The binding interactions between the solute and protein usually involves stereospedfic and nonstereospecific mechanisms. These mechanisms make the type V CSPs sensitive to the composition of the mobile phase, temperature, flow rate, and pH. These parameters can be adjusted to improve the chromatography and stereoselectivity of specific solutes on the AGP CSP (88,89), OVM CSP (90,91), BSA CSP (92,93), and HSA CSP (94). 

Alcohols as mobile-phase modifiers. As with the other type V CSPs, the addition of an alcohol to the mobile phase appears to reduce the hydrophobic interactions between the solute and SA CSP, which results in lower k values and reduced a s(9-ll). An example of this phenomenon are the effects of ethanol, 1-propanol, and 1-butanoI on the retention and enantioselective resolution of N-benzoyI-D,L,alanine on the BSA CSP (111). The addition to a mobile phase composed of phosphate buffer (50 mM, pH 7.0) of 2% (v/v) of ethanol reduced the k of the last eluting enantiomer by 33% and the observed a by 6%. When ethanol was replaced by 1-propanol, the observed reductions were 67 and 40%, respectively, and when 1-butanol was the modifier, the observed reductions were 80 and 77%, respectively. In practice, 1-propanol appears to be the most commonly used alcoholic modifier. 

Type V. When the CSP is a protein and the solute/CSP complexes are based on combinations of hydrophobic and polar interactions... 

Solutes containing asymmetric sulfur atomes or asymmetric phosphorous atoms can be resolved v/ith or without an aromatic moiety in the molecule. For example, the anticancer drug ifosfamide, an oxazaphos-phorane not containing an aromatic moiety, has been resolved on this type of CSP (73). In addition, these are excellent CSPs for the resolution of enantiomeric molecules with an axis of dissymmetry (atrop isomers) (9-11). 

Modifier additives also play a role in method optimization and are typically added to the modifier at concentrations less than 1 % (v/v). Additives can provide increased efficiency by minimizing undesirable interactions between the analyte and the CSP, and may be necessary to elute certain types of compounds. The type of additive (acidic or basic) that will produce the best results depends upon the functionality of the analyte [72]. Certain additives are strongly retained on the stationary phase, and their effect may persist even after they are removed from the eluent [22]. The impact of both modifiers and additives can also be affected by the proximity of the operating conditions to the critical point of the eluent [73]. 

The PO mode is a specific elution condition in HPLC enantiomer separation, which has received remarkable popularity especially for macrocyclic antibiotics CSPs and cyclodextrin-based CSPs. It is also applicable and often preferred over RP and NP modes for the separation of chiral acids on the cinchonan carbamate-type CSPs. The beneficial characteristics of the PO mode may arise from (i) the offset of nonspecific hydrophobic interactions, (ii) the faster elution speed, (iii) sometimes enhanced enan-tioselectivities, (iv) favorable peak shapes due to improved diffusive mass transfer in the intraparticulate pores, and last but not least, (v) less stress to the column, which may extend the column lifetime. Hence, it is rational to start separation attempts with such elution conditions. Typical eluents are composed of methanol, acetonitrile (ACN), or methanol-acetonitrile mixtures and to account for the ion-exchange retention mechanism the addition of a competitor acid that acts also as counterion (e.g., 0.5-2% glacial acetic acid or 0.1% formic acid) is required. A good choice for initial tests turned out to be a mobile phase being composed of methanol-glacial acetic acid-ammonium acetate (98 2 0.5 v/v/w). 

Three types of macrocyclic antibiotic CSPs have been commercialized by Astec (Whippany, New York) and show complementary enantioselec-tivity Chirobiotic V (selector vancomycin), Chirobiotic T (teicoplanin)... 

Many of the chiral molecules containing amide groups were bonded to a solid support for the preparation of CSPs [16-19]. The racemic compounds resolved on these CSPs include a-hydroxycarbonyls, /i-hydroxycarbonyls, amino acids, amino alcohols, amine, and derivatized and underivatized diols. The preliminary chiral diamide phase [(/V-foriuyl-L-valyl)aminopropyl)silica gel] has sufficient separability for racemic /Y-acylatcd a-amino acid esters but not in other types of enantiomer [16]. Most of the eluents used with these CSPs are of normal phase mode, including w-hcxanc, 2-propanol, chlorinated organic solvents, and acetonitrile. 

Abou-Basha and Aboul-Enein [22] presented an isocratic and simple HPLC method for the direct resolution of the clenbuterol enantiomers. The method involved the use of a urea-type CSP made of hS )-indoline-2-carboxylic acid and (R)-1 -(naphthyl) ethylamine known as the Chirex 3022 column. The separation factor (a) obtained was 1.27 and the resolution factor (Rs) was 4.2 when using a mobile phase composed of hexane-1,2-dichloroethane-ethanol (80 10 10, v/v/v). The (+)-enantiomer eluted first with a capacity factor (k) of 2.67 followed by a (—)-enantiomer with a k of 3.38. Biesel et al. [23] resolved 1-benzylcyclohexane-1,2-diamine hydrochloride on a Chirex D-penicillamine column. Gasparrini et al. [24] synthesized a series of the chiral selectors based on /ra s -1,2 - d i a m i n o eye I o hexane. The developed CSPs were used for the chiral resolution of arylacetic acids, alcohols, sulfoxides, selenoxides, phosphinates, tertiary phosphine oxides, and benzodiazepines. In another study, the same authors [25] described the chiral resolution of /i-aminocstcrs enantiomers on synthetic CSPs based on a re-acidic derivatives of trans- 1,2-diaminocyclohexane... 

Fig. 9.12. Enantiomer separation of Fmoc-Leu on a quinine carbamate-based WAX-type CSPs by CEC under a slight overpressure (8 bar). Conditions capillary, 100 pm I.D. x 25 cm (overall length, 33.5 cm) acetonitrile-methanol (80 20, v/v) 200 mM acetic acid, 10 mM triethylamine -25 kV UVdetection, 254 nm. Reproduced from [58], with permission.

Fig. 9.36. Enantioscparation of a-aryl propionic acids on 1-allyl-tcrguride-ba.scd WAX type CSP (a) fenoprofen. (b) flobufen. (c) naproxen, and (d) ketoprofen. Exp. cond., 20 mM potassium acetate (pH 3.6)-ttcetonitrile (50 50, v/v( How rate. I.O ml/min detectum. 255 nm (reprinted with permission from Ref. 1.3971).

Fig. 9.38. Loadability of different CSPs under bateh-ehromatography arnditions. (a) Triigcr base on Chi-ralpak AD methanol vs. aeetonitrilc (

Figure 13.19 HPLC enantioseparation of racemic dichloroprop 43 on the (DHQD)2PHAL-type CSP (61). Column (150 mm X 4 mm i.d.) mobile phase methanol/acetic acid/ ammonium acetate (92 2 0.5, v/v/w) flow rate 1 ml/min UV detection 254 nm column temperature 25 °C [108].

Fig. 7.7 Effect of various types of additives on the reversed-phase enantiomer separation of neutral, acidic and basic analytes on a polysaccharide-type CSP. Column CH I RALCEL AD-RH (150 x 4.6 mm i.d.), mobile phase aqueous mobile phase containing modifier indicated in the figure/acetonitrile (60/40 v/v), flow rate 0.5 mb min" , temp. 25 °C, detection UV 254 nm. (Reprinted with permission from [116]).

Fig. 7.12 En antiomer separation of racemic analytes achieved on silica-supported poly(frons-l, 2-diaminocyclohexane-diacryl-amide)-type CSPs prepared by surface-initiated graft polymerization. (A) temazepam, n-hexane/ethanol (50/50 v/v) (B) binaphthol, dichloromethane/methanol (97/3 v/v) ...

Fig. 7.17 Complementary chiral recognition profiles of glycopeptide-type CSPs. (A) N-CBZ-norvaline on vancomycin (left) and teicoplanin (right). Mobile phase methanol/1 % triethy-lammonium acetate (20/80 v/v) pH 4.1.

The large number of CSP s developed, tested and marketed present somewhat of a problem for how best to categorize them. Wainer has suggested a classification scheme for HPLC CSP s based on the mode of formation of the solute-CSP complex [16]. There are five categories, labeled Type l-V, and molecular modeling has been done on most of these. The categories and modes of association are ... 

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