Choice of Organic Solvent

The physical chemical properties of the organic solvents 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.

Table 2 Physicochemical parameters of selected solvents

Solvent Viscosity, j (cP) Dielectricconstant, s s/t pKauto Tboll(°C) UVcutoff

(1 cm cuvette)

Solvent Viscosity, j (cP) Dielectricconstant, s s/t pKauto Tboll(°C) UVcutoff

(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

242

200-230

Protolysis

MeCN

0.34

37.5

110.3

Not detected

82

200-230

Protolysis

Glycerol

945

42.5

0.045

290

205

Acetic acid

1.0430

6.152

5.91

14.45

118

All values are at 25°C unless otherwise stated in subscript. For abbreviations, see Table 1. Reproduced with permission from Tjcrnelund J and Hansen SH (1999) Journal of BiochemistryandBiophysical Methods 38: 139-153.

All values are at 25°C unless otherwise stated in subscript. For abbreviations, see Table 1. Reproduced with permission from Tjcrnelund J and Hansen SH (1999) Journal of BiochemistryandBiophysical Methods 38: 139-153.

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-aminocaproicacid pH 4.0with 25 mmol L~1 of3-(A/,W-dimethylmyristylammonium)propanesulfonateand 15 mmol L~1 ofTween® 20 added. Apparatus: Quanta 4000. Conditions: 64 cm (56 cm to the detector) x 75 i.d. capillary, hydrostatic (10 cm) injection for 15 s, ambient (27-30°C), 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: 64cm (55.5 cm to the detector) x50 ^m i.d. capillary, injection of 3 sat 5 kPa (50 mbar), 25°C, 25 kV (7 ^A) and UV detection at 214 nm. Adapted with permission from Bjornsdottir I, Tjornelund J and Hansen SH (1996) Selectivity enhancement in capillary electrophoresis using nonaqueous media. Journal of Capillary Electrophoresis 3: 83-87.

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-aminocaproicacid pH 4.0with 25 mmol L~1 of3-(A/,W-dimethylmyristylammonium)propanesulfonateand 15 mmol L~1 ofTween® 20 added. Apparatus: Quanta 4000. Conditions: 64 cm (56 cm to the detector) x 75 i.d. capillary, hydrostatic (10 cm) injection for 15 s, ambient (27-30°C), 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: 64cm (55.5 cm to the detector) x50 ^m i.d. capillary, injection of 3 sat 5 kPa (50 mbar), 25°C, 25 kV (7 ^A) and UV detection at 214 nm. Adapted with permission from Bjornsdottir I, Tjornelund 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 acetonitrile (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

Figure 2 Electropherogramsof five basic drugs with equal or very similar mass overcharge 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: 83-87.

Figure 2 Electropherogramsof five basic drugs with equal or very similar mass overcharge 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: 83-87.

vents have a UV cutoff at 214 nm or above (Table 2). Nevertheless, solvents like MeCN and MeOH may 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 sufficient of the application.

On the positive side, organic solvents often intensify the fluorescence 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.

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