Electrochromatographic separations

Lin, C.-C., and Liu, C.-Y. (2004). Proline-coated column for the capillary electrochromatographic separation of amino acids by in-column derlvatlzatlon. Electrophoresis 25, 3216-3223. 

Bedalr, M., and El Rassl, Z. (2003). Capillary electrochromatography with monolithic stationary phases III. Evaluation of the electrochromatographic retention of neutral and charged solutes on cationic stearyl-acrylate monoliths and the separation of water-soluble proteins and membrane proteins. /. Chromatogr. A 1013, 47-56. 

More complex is the separation of charged analytes in CEC, which is the result of the interplay of chromatographic and electrophoretic processes that is considered in the definition of the electrochromatographic retention factor, or overall retention factor, k introduced by Rathore and Horvath [140] ... 

The selectivity coefficient or separation factor, a, in CEC is given by the ratio of the electrochromatographic retention factors of the two analytes (1 and 2) migrating as adjacent peaks ... 

The limits of the electrochromatographic retention factor defined by Equation 6.54 are that both k and must be separately evaluated under specific experimental conditions used in CEC. These limitations can be avoided describing the retention of analytes in CEC by a peak locator that can be evaluated directly from the electrochromatogram as follows [256] ... 

Figure 8.8—Electrochromatographic separation of aromatic hydrocarbons. The movement of the mobile phase is strictly due to the electro-osmotic flow. In contrast to HPLC, no pressure is exerted at the head of the column. Separations can be carried out with a very high efficiency.

Zeng used a silica monolith modified with the liquid crystalline crown ether 29 as a column material in capillary electrochromatography (Scheme 17) [50]. Polycyclic aromatic compounds, benzenediols, pesticides, and steroids were successfully separated on the column. Introduction of the liquid crystalline crown ether led to a significant improve of the electrochromatographic performance. 

Figure 5.14 Electrochromatographic separation of neutral molecules on a 230 mm x 50 mm I.D. capillary packed with 3 mm Hypersil ODS. (A) Capillary electrochromatography, applied voltage 23 kV, (B) micro-HPLC, pressure 130 bar. Peaks, in order of elution, represent thiourea, benzylalcohol, benzaldehyde, benzene, and naphthalene. (Reprinted from Ref. 38 with permission.)...

Fig. 6.2. Electrochromatographic separation of benzyl alcohol (1), resorcinol (2), methylparaben (3), and p-naphthol (4) using a soft gel column (Reprinted with permission from [27], Copyright 1998 Wiley-VCH). Conditions Column 48.5 cm (24 cm active) x 75 pm i.d., stationary phase 4.1% T, 9.7% C, 0.7% S poly(2-acrylamido-2-methyl-l-propanesulfonic acid-co-N-isopropyl acrylamide-co-methylene bisacrylamide) mobile phase 20 80 acetonitrile and 2.5 mol/L phosphate buffer pH 6.8 16 kV.

Fig. 6.21. Electrochromatographic separation of benzene derivatives on monolithic capillary column prepared by UV initiated polymerization. Conditions capillary column, 100 pm i.d. x 25 cm active length stationary phase poly(butyl methacrylate-co-ethylene dimethaciylate) with 0.3 wt. % 2-acrylamido-2-methyl-l-propanesulfonic acid pore size, 296 nm mobile phase, 75 25 vol./vol mixture of acetonitrile and 5 mmol/L phosphate buffer pH 7 UV detection at 215 nm 25 kV pressure in vials, 0.2 MPa injection, 5 kV for 3 s. Peaks thiourea (1), benzyl alcohol (2), benzaldehyde (3), benzene (4), toluene (5), ethylbenzene (6), propylbenzene (7), butylbenzene (8), and amylbenzene (9).

Fig. 6.22. Electrochromatographic separation of Gly-Tyr (1), Val-Tyr-Val (2), methionine enkephalin (3), and leucine enkephalin (4) on monolithic methacrylate capillary column with a pore size of 492 nm. (Reprinted with permission from [55]. Copyright 1999 Wiley-VCH). Conditions Mobile phase 10% of aqueous 10 mmol/L sodium 1-octanesulfonate and 90% of a 2 8 mixture of 5 mmol/L phosphate buffer pH=7.0 and acetonitrile. UV detection at 215 nm. Total sample concentration 1 mg/mL.

Fig. 7.9. Electrochromatographic separation of a mixture serotonin and its metabolites on an etched liquid crystal modified capillary.

Fig. 7.12. Electrochromatographic separation of the optical isomers of D,L-chlorodiazepoxide on an etched cyclodextrin modified capillary. pH = 3.0, V = 20 kV, L = 45 cm, 1 = 25 cm. Peaks 1 = impurity 2,3 = D,L isomers of chlorodiazepoxide (individual isomers not identified).

The first generation of research groups working in the field of enantiomer separation by CEC did not pressurize the flow system [38-41]. The result were long elution times associated with broad tailing peaks. Now, most of the recently published papers deal with electrochromatographic enantiomer separation under elevated pressure by pressurizing both ends of the capillary [48-52, 54,55,57,58,62]. In some cases pressurization of the inlet vial alone was described [42-45,59,60]. 

Comparing the two methods p-LC and p-CEC, the electrochromatographic method always shows higher theoretical plate numbers and resolutions at comparable elution times [39-41] (see Fig. 9.8). The Chirasil-Dex-monolith [44] consists of a single piece of a chiral modified porous solid, thus frits are no longer required and the risk of air bubble formation decreases. Pressurization is not insisted. As shown in Fig. 9.9 the enantiomer separation of mephobarbital on a Chirasil-Dex-monolith can be performed... 

Peters et al. reported on rod-CEC on a chiral monolith [50] which was prepared by copolymerization of the chiral monomer 2-hydroxyethyl methacrylate (A -L-valine-3,5-dimethylanilide) carbamate with ethylene dimethylacrylate, 2-acrylamido-2-methyl-l-propanesulfonic acid and butyl or glycidyl methacrylate in the presence of a porogenic solvent. The electrochromatographic enantiomer separation of 7V-(3,5-dinitrobenzoyl)leucine diallylamide was feasible at 25 kV the inlet and outlet buffer vials were both pressurized. 

Capillaries packed with poly-A-acryloyl-L-phenylalanine ethyl ester (Chiraspher) modified silica were used for electrochromatographic enantiomer separation of ben-droflumethiazide. To suppress bubble formation, the inlet buffer vial was pressurized to 12 bar and the outlet buffer vial to 4 bar [42], Acetonitrile or methanol were used as organic modifier whereby acetonitrile was superior to methanol. Non-aqueous p-CEC was performed on helical poly(diphenyl-2-pyridylmethylmethacrylate) coated on wide-pore aminopropyl silica [56]. With this chiral stationary phase, the enantiomer separation of Trogers base, benzoin acetate, methylbenzoin and trans-stilbene oxide was achieved by pressure-supported CEC, applying a higher pressure to the inlet than to the outlet buffer vial. 

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