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to an electrophoretic separation chip is one possible answer. However, there is no reason why reactions cannot be carried out on such devices. Since it does not require the investment of a large chemical plant, the reactions can be performed where required, thus reducing the need to transport hazardous chemicals across countries. Since many reactors can be con- structed on a single chip, and many chips located in the same area, it is evident that this technology will provide hazardous or chemically unstable chemicals where they are required. Further Reading Altria KD (ed.) (1996) Capillary Electrophoresis Guide- book,Principles,Operation, and Applications. New Jer- sey: Humana Press. Harrison DJ and Van den Berg A (eds) (1998) Micro Total Analysis Systems ’98. Dordrecht: Kluwer Academic Publishers. Haswell SJ (1997) Developments and operating character- istics of microSow injection analysis systems based on electroosmotic Sow. Analyst 122: 1Rd1OR. Manz A and Becker H (1997) Microsystem Technology in Chemical and Life Sciences. Berlin: Springer. Madou M (1996) Fundamentals of Microfabrication. Boca Raton: CRC. Martin AJP (1962) Opening lecture. In: Van Swaay M (ed.) Fourth International Symposium on Gas Chromatogra- phy. London: Butterworths. Oefner PJ, Bonn GK and Chiesa C (1995) Encyclopaedia of Analytical Chemistry, pp. 1041}1152. London: Aca- demic Press. Pethig R and Markx GH (1997) Applications of dielec- trophoresis in biotechnology, Trends in Biochemistry 15: 426}432. Regnier F (1999) The evolution of analysis in life science research and molecular medicine: the potential role for separations. Chromatographia 49: S56dS64. Tsuda T (ed.) (1995)Electric Field Applications, pp. 47}73. Weinheim: VCH. Nonaqueous Capillary Electrophoresis S. H. Hansen, I. Bj[rnsdottir and J. Tj[rnelund, Royal Danish School of Pharmacy, Copenhagen, Denmark Copyright^ 2000 Academic Press Electrophoresis is a separation technique that is nor- mally performed in an aqueous environment. This is due to the fact that the separationmechanism is based on the difference in migration rate of charged species in an electric Reld. Species (ions/molecules or par- ticles) with a difference in their charge over size ratio will exhibit a difference in migration rate. Most charged species are fairly soluble in aqueous media and thus water is the most obvious solvent for elec- trophoresis. However, in a number of nonaqueous solvent systems, it is possible to obtain sufRcient conductivity to perform electrophoresis. If such sys- tems are utilized with the technique of capillary elec- trophoresis, a number of advantages compared to aqueous systems are obtained in the separation of small molecules. Nonaqueous electrophoresis of bi- opolymers like polysaccharides, nucleic acids and proteins is not of practical use due to lack of solubility of such molecules in organic solvents. NonaqueousCapillary Electrophoresis Only a few attempts to perform nonaqueous paper electrophoresis have been described and these articles were reviewed in 1978. In 1984 nonaqueous capillary electrophoresis (NACE) was brieSy mentioned in a single publication, but not utilized further. How- ever, since 1993 the use of nonaqueous media for capillary electrophoresis has seen renewed interest in the separation of drug substances due to the high separation selectivity obtained in these systems. The electrophoretic migration of the solutes is in- Suenced by the nature of the solvent or solvent mix- ture used for the electrophoresis medium in three main ways: 1. The mobility may change due to changes in the size of the solvated ion. 2. The dielectric constant of the organic solvent may inSuence the equilibrium of the protolytic dis- sociation. The higher the value of the dielectric constant, the higher the degree of ionization of acids and bases. 3. The acid}base property of the solute, expressed by its pKa value, may change due to the differenti- ating effect of many organic solvents. The latter effect of the three is the most signiRcant, as the dissociation constant, Ka, may change many orders of magnitude for different solvents. The increased selectivity of separation in organic solvents compared to aqueous systems is due to the fact that the levelling effect of water is eliminated. If II /ELECTROPHORESIS /Nonaqueous Capillary Electrophoresis 1293 Table 1 Classification of organic solvents according to their Br+nsted acid}base behaviour Solvent designation Relative acidity Relative basicity Examples Neutral # # MeOH, glycerol, phenol, tert, butyl alcohol Amphiprotic Protogenic # ! Sulfonic acid, formic acid, acetic acid Protophilic ! # Liquid ammonia, FA, NMF Dipolar protophilic ! # DMSO, DMF, tetrahydrofurane, 1,4-dioxan, pyridine Aprotic Dipolar protophobic ! ! MeCN, acetone, nitrobenzene, sulfolane, PC Inert ! ! Aliphatic hydrocarbons, benzene, 1,2-dichlorethane, tetrachloromethane ! indicates weaker and# indicates stronger acid or base than water. DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide: FA, formamide; MeCN, acetonitrile; MeOH, methanol; NMF, N-methylformamide; PC, propylene carbonate. Solvents in italic are the ones that are preferred for NACE. Reproduced with permission from TjCrnelund J and Hansen SH (1999) Journal of Biochemistry and Biophysical Methods 38: 139}153. Table 2 Physicochemical parameters of selected solvents Solvent Viscosity, � (cP) Dielectric constant, � �/� pKauto Tboil (3C) UV cutoff (nm) (1 cm cuvette) Water 0.89 78.4 89.9 14 100 (200 FA 3.3 111 33.6 16.8 210 275 NMF 1.65 182 110.3 10.7 182 275 DMF 0.8 36.7 45.9 29.4 153 260 DMSO 1.99 46.7 23.4 33.3 189 260 MeOH 0.544 32.7 60.6 17.2 65 205 PC 2.5 64.4 25.7 Not detected Protolysis 242 200}230 MeCN 0.34 37.5 110.3 Not detected Protolysis 82 200}230 Glycerol 945 42.5 0.045 * 290 205 Acetic acid 1.0430 6.152 5.91 14.45 118 * All values are at 253C unless otherwise stated in subscript. For abbreviations, see Table 1. Reproduced with permission from TjCrnelund J and Hansen SH (1999) Journal of Biochemistry and Biophysical Methods 38: 139}153. strong acids or bases are dissolved in water, they all show up with about the same acid or base strength. If the same acids or bases are dissolved in organic sol- vents they will exhibit very different protolytic behav- iour depending on the degree of dissociation, which again depends on the solvent in question. Important factors inSuencing the choice of organic solvent or solvent mixture for a given separation are volatility, the dissolving power towards suitable elec- trolytes, viscosity and dielectric constant, UV trans- parency and, last but not least, the effect on the separation selectivity of the system. Information on the viscosity and volatility, the auto protolysis con- stant, the dielectric constant at standard conditions and the UV transparency of the neat solvents may be found in the literature. In contrast, data on solvent mixtures and systematic studies of how to choose solvents and electrolytes in order to control the selectivity of the electrophoretic system are limited and thus the choice of separation media is still a mat- ter of trial and error. Solvents may be classiRed ac- cording to their Br+nsted acid}base behaviour; a sim- pliRed version of this classiRcation is shown in Table 1. Practical Considerations Choice of Organic Solvent The physical chemical properties of the organic sol- vents preferred for NACE are given in Table 2 and, as mentioned above, the physical constants have a major impact on the choice of solvent or solvent mixture for a given electrophoretic separation. Some of the more practical considerations are the chemical resistance of parts in the CE instrument towards the solvent, the volatility of the solvent, the solvating power of the solvent towards electrolytes, the UV transparency and the viscosity of the solvent. 1294 II /ELECTROPHORESIS /Nonaqueous Capillary Electrophoresis Figure 1 Electropherograms of imipramine and four derivatives. (A) 50 mmol L�1 6-aminocaproic acid pH 4.0; (B) 50 mmol L�1 6-amino caproic acid pH 4.0 with 25 mmol L�1 of 3-(N,N-dimethylmyristylammonium)propanesulfonate and 15 mmol L�1 of Tween� 20 added. Apparatus: Quanta 4000. Conditions: 64 cm (56 cm to the detector)�75 �m i.d. capillary, hydrostatic (10 cm) injection for 15 s, ambient (27}303C), 20 kV (62 �A) and UV detection at 214 nm. (C) 25 mmol L�1 ammonium acetate and 1 mol L�1 acetic acid in acetonitrile. Apparatus: HP3DCE instrument. Conditions: 64 cm (55.5 cm to the detector)�50 �m i.d. capillary, injection of 3 s at 5 kPa (50 mbar), 253C, 25 kV (7 �A) and UV detection at 214 nm. Adapted with permission from BjCrnsdottir I, TjCrnelund J and Hansen SH (1996) Selectivity enhancement in capillary electrophoresis using nonaqueous media. Journal of Capillary Electrophoresis 3: 83}87. Solvents with a high vapour pressure and thus a high volatility (e.g. methanol (MeOH) and aceto- nitrile (MeCN)) may be inconvenient for automated analysis in some instruments due to problems with evaporation of the electrophoresis medium from the run buffer vials as well from the sample vials. In CE the detection is often performed by measuring the UV absorbance of the analyte at a relatively short wavelength (e.g. at 214 nm or below) in order to increase the sensitivity. However, many organic sol- II /ELECTROPHORESIS /Nonaqueous Capillary Electrophoresis 1295 Figure 2 Electropherograms of five basic drugs with equal or very similar mass over charge ratio. (A) 50 mmol L�1 6-aminocaproic acid pH 4.0; (B) 50 mmol L�1 6-amino caproic acid pH 4.0 with 25 mmol L�1 of Tween� 20 added. Apparatus and conditions as in Figure 1A. (C) 25 mmol L�1 ammonium acetate and 100 mmol L�1 sodium acetate in methanol#acetonitrile (1 : 1 v/v) and 25 kV (23 �A). Apparatus and other conditions as in Figure 1C. Adapted with permission from BjCrnsdottir I, TjCrnelund J and Hansen SH (1996) Selectivity enhancement in capillary electrophoresis using nonaqueous media. Journal of Capillary Electrophoresis 3: 83I87. vents have a UV cutoff at 214 nm or above (Table 2). Nevertheless, solvents likeMeCN andMeOHmay be used for measurements performed at a wavelength as low as 200 nm as the light path through the capillary is very short compared to the 1 cm cuvette used for the determination of the UV cutoff wavelength. The amides and dimethylsulfoxide can only be used when detection at wavelengths above c. 245 nm is sufRcient of the application. On the positive side, organic solvents often inten- sify the Suorescence relative to what is observed for given solutes in aqueous media. This has been used to decrease detection limits in NACE for analysis of tetracyclines in biological matrices. Choice of Electrolyte The choice of electrolyte is important and will inSu- ence the separation. However, due to the low solubil- ity of many electrolytes in organic solvents, it can be difRcult to Rnd a suitable electrolyte. The more polar solvents, like MeOH, DMSO, formamide, N-methyl- formamide and N,N-dimethylformamide, possess 1296 II /ELECTROPHORESIS /Nonaqueous Capillary Electrophoresis Figure 3 Electropherograms of cis-trans- and diastereo-isomers. (A) Separation of cis- and trans-flupenthixol decanoate using 50 mmol L�1 ammonium acetate and 1 mol L�1 acetic acid in methanol#acetonitrile (1 : 1, v/v), above: cis-flupenthixol decanoate with 0.5% trans-isomer added; below: trans-flupenthixol decanoate. Conditions: 64 cm (55.5 cm to the detector)�50 �m i.d. capillary, injection for 3 s at 5 kPa (50 mbar), 253C, 30 kV (9 �A) and UV detection at 230 nm. Test solution: 5.0 mg mL�1 of the sample in methanol#acetonitrile (1 : 1 v/v). Peak identity: 1, cis-flupenthixol decanoate; 2, trans-flupenthixol decanoate; U, unknown. (B) Separation of dipeptides (diastereomers); (C) separ- ation of quinine and quinidine (diastereomers). Conditions as in (A) with a detection wavelength of 214 nm. Adapted with permission from Han- sen SH, BjCrnsdottir I and TjCrnelund J (1997) Separation of cationic cis-trans (Z-E) isomers and diastereomers using nonaqueous capillary electrophoresis. Journal of Chromatography A 792: 49}55. a good solvating power towards the electrolytes com- monly used in NACE. So far, ammonium acetate has been the most frequently used electrolyte in NACE systems and acetic acid or sodium acetate have often been used in combination with ammonium acetate in order to adjust the acid}base properties of the electrophoresis medium. Quaternary ammonium salts have also been used a number of times with success, e.g. in the separation of phenols and car- boxylic acids. More rarely, Tris, magnesium acetate, citric acid, formic acid, triSuoroacetic acid and methanesulfonic acid have been used. When coupling CE to mass spectrometry (MS), it is an advantage to choose a volatile electrolyte, e.g. ammonium acetate, in order to limit background noise or cluster ion formation. Other Additives A number of polyalcohols and surfactants such as the Tweens� have been used as additives. Their primary function is to decrease the electroosmotic Sow (EOF) and thus prolong the time for elec- trophoretic separation. Also chiral separations are possible in NACE using either cyclodextrines or chiral counter ions as additives. Reversal of EOF The separation of anionic solutes in CE may lead to extended time of analysis due to their migration in the direction opposite to EOF. One method of decreasing the analysis time is to reverse the EOF, thus making the anions migrate in the same direction as the EOF. In aqueous CE, the addition of long alkyl chain trimethylammonium ions is used for this purpose, e.g. in the analysis of inorganic anions and phenols. This principle may also be used in NACE. However, the long alkyl chain trimethylammonium ions are not able to form hemimicelles at the inner capillary sur- face when using nonaqueous solvents and thus the EOF is not reversed. Addition of the polycation hexa- dimethrine bromide to the nonaqueous electrophor- esis medium may result in suitable and stable systems with reversed EOF, even when used at fairly low concentrations (0.001}0.05%). Applicability of NACE In CE the separation of solutes is due to differences in the charge over size ratios and thus very similar substances may be difRcult to separate in aqueous CE unless special mechanisms like micellar elec- trokinetic chromatography (MEKC) are involved. Of course this involves addition of one or more sur- factants. II /ELECTROPHORESIS /Nonaqueous Capillary Electrophoresis 1297 Table 3 Applications of NACE in analysis of food, pharmaceuticals and biological fluids Solvents Electrolytes Analytes Applications within food NMF-dioxane (1 : 1 v/v) 40 mmol L�1 Tris, 2.5 mmol L�1 anthraquinone-2-carboxylic acid Free saturated long chain fatty acids (n-C14-n-C26). Separation of dimeric and trimeric acids and hydrogenated fish oil NMF 500 mmol L�1 magnesium acetate tetrahydrate Tetracycline (TC), oxytetracycline (OTC), chlorotetracycline (CTC), demeclocycline, 4-epitetracycline, anhydrotetracycline, 4-epianhydrotetracycline, and desmethyltetracycline TC, OTC and CTC in milk and plasma Propylene carbonate Tetraalkylammonium ions, long chain trimethylammonium ions 20 mmol L�1 tetradecylammonium bromide (vitamin K1 and preservatives) Phenanthrene, �-naphthol, preservatives: methylparaben, ethylparaben and propylparaben, thiourea (EOF marker) and vitamin K1 Applications within pharmaceuticals 10}100% MeOH Ammonium acetate, acetic acid Haloperidol and synthetic putative metabolites, pyrazoloacridine and mifentidine Mixture of MeOH and H2O Ammonium acetate, acetic acid Haloperidol, cimetropium and mifentidine MeOH 5 mmol L�1 ammonium acetate, 100 mmol L�1 acetic acid Haloperidol and its synthetic putative metabolites, pyrazoloacridine and its synthetic putative metabolites, mifentidine and its synthetic putative metabolites MeOH and mixture of MeOH and MeCN Ammonium acetate, tetrabutylammonium bromide, tetrabutylammonium hydrogen sulfate and tetrapentylammonium bromide Tamoxifen and four phase I metabolites MeOH, MeCN, mixture of MeOH and MeCN, formamide, NMF, DMF, DMA, DMSO 25 mmol L�1 ammonium acetate, 0}1 mol L�1 acetic acid or 100 mmol L�1 sodium acetate Application: 25 mmol L�1 ammonium acetate, 1 mol L�1 acetic acid in MeCN Imipramine, di-desmethylimipramine, desmethylimipramine, methylimipramine and imipramine-N-oxide. Maprotiline, amitriptyline, litracene, protriptyline and nortriptyline. Application: imipramine N-oxide and impurities MeOH : MeCN : DMF (45 : 49 : 6 v/v/v) 25 mmol L�1 ammonium acetate, 10 mmol L�1 citric acid and 118 mmol L�1 methanesulfonic acid Tetracycline and three degradation products. Tetracycline, oxytetracycline, doxycycline, desmethyltetracycline and chlortetracycline MeOH : MeCN (1 : 1 v/v) 20 mmol L�1 ammonium acetate, 1 mol L�1 acetic acid Morphine analogues, antihistamines, antipsychotics and stimulants Formamide, NMF or DMF Citric acid or acetic acid mixed with Tris. Chiral selectors: �-CD, �-CD and derivatized �-CD. Addition of long chain alkyl ammonium salts investigated Racemic mixtures of chlorphedianol, chlorcyclizine, ethopropazine, mianserin, nefopam, primaquine, propiomezine, trihexyphenidyl, trimeprazine, trimipramine and thioridazine NMF, formamide and mixtures of both 25}200 mmol L�1 �-CD, 10 mmol L�1 NaCl Dansylated amino acids NMF 5}100 mmol L�1 �-CD and 10 mmol L�1 NaCl Dansylated amino acids MeOH Ammonium acetate, acetic acid, quinine N-3,5-dinitrobenzylated amino acids, ($)-1,1�-binaphthyl-2,2�-diyl hydrogen phosphate and N-[1-(1-naphthyl)ethyl]phthalomic acid MeCN ($)-Camphorsulfonic acid potassium or sodium salt, 1 mol L�1 acetic acid 0.2 mol L�1 Tween 20 Atenolol, bisoprolol, bunitrolol, metroprolol, pindolol, propranolol, salbutamol, ephedrine, epinephrine, cisapride and synthetic impurities Formamide Tetra-n-butylammonium perchlorate. Chiral selector: (#)-18-crown-6-tetracarboxylic acid 1-Naphthylethylamine, 1-phenylethylamine, phenylalanine, DOPA, tryptophan, norephedrine, noradrenaline and 2-amino-1,2-diphenylethanol 1298 II /ELECTROPHORESIS /Nonaqueous Capillary Electrophoresis Table 3 Continued Solvents Electrolytes Analytes Formamide, NMF, DMF, DMA, DMSO, MeOH, MeCN and mixtures of MeOH and MeCN 25 mmol L�1 ammonium acetate, 1 mol L�1 acetic acid Morphine, codeine, normorphine, thebaine, noscapine and papaverine. Application: morphine in opium tincture MeOH : MeCN (75 : 25) 25 mmol L�1 ammonium acetate, 1 mol L�1 acetic acid Morphine NMF 500 mmol L�1 magnesium acetate tetrahydrate Oxytetracycline in an ointment Mixtures of MeOH and MeCN Ammonium acetate, ammonium chloride, acetic acid, trifluoroacetic acid, formic acid, methane sulfonic acid Cis-trans (Z-E) isomers of chlorprothixene, thiothixene, clopenthixol, flupenthixol, flupenthixol decanoate, clomiphene and diastereomers: L-Ala-L-Phe, L-Ala-D-Phe; quinine, quinidine, cinchonine and cinchonidine Mixtures of MeOH and MeCN Sodium acetate A range of penicillins, cephalosporins and nonsteroidal anti-inflammatory drugs MeOH 20 mmol L�1 CAPS and 0}40 mmol L�1 Brij 35 Mesoporphyrin, coporphyrin, pentaporphyrin, hexacarboxylporphyrin, heptacarboxylporphyrin and uroporphyrin Applications within biological fluids 10}100% MeOH in H2O 20 mmol L�1 ammonium acetate, 1% acetic acid Pyrazoloacridine, two metabolites and a synthetic degradation product in urine NMF 500 mmol L�1 magnesium acetate tetrahydrate Tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), demeclocycline, 4-epitetracycline, anhydrotetracycline, 4-epianhydrotetracycline and desmethyltetracycline. TC, OTC and CTC in cow milk and human plasma MeOH 5 mmol L�1 ammonium acetate, 100 mmol L�1 acetic acid Mifentidine and three metabolites in rat liver homogenate MeOH : MeCN (1 : 1 v/v) 50 mmol L�1 ammonium acetate, 159 mmol L�1 sodium acetat