Separation voltage

Accordingly, the ionic conductivity in an electrolyte with negligible electronic conduction (/jon jtolal) may be determined by Ohm s law, provided that unpolarizable electrodes are employed. To overcome this limitation, separate voltage probes in the shape of identical electronic leads connected to the electrolyte at positions separated by a distance L may be employed (four-probe technique [38]). Under these... 

FIGURE 16.2 Representative base peak electropherograms from CZE runs of RPLC fractions, (a) Fraction 15 (5 peptide identifications) and (b) fraction 20 (19 peptide identifications). Column, bare fused silica capillary, 60 cm x 180 pm ODx30pm i.d. separation voltage, 15 kV observed CZE current, 1.91 p.A running electrolyte, 200 mm acetic acid + 10% isopropanol temperature, 22°C injection time, 10 s at 2 psi ( 4 nL total injection volume) supplementary pressure, 2 psi flow rate, 25nL/min spray voltage, 1.5 kV (reprinted with permission from Electrophoresis). 

Figure 11 Electropherogram of a mixture of five amino acids using indirect CL detection. Conditions 21-kV separation voltage, and 2 s at 21 kV for sample injection sample concentration 0.5 mM of each amino acid. Peak identities (1) arginine (2) leucine (3) serine (4) cysteine (5) aspartic acid. (From Ref. 86, with permission.)...

CZE has been employed for the analysis of another set of dyes in foodstuffs. The chemical structures, numbers and names of the dyes included in the investigation are listed in Fig. 3.142. A fused-silica capillary column of 57 cm length (50 cm effective length 75 jum i.d.) was employed for the separations. The capillary was conditioned by 1.0 M NaOH for 20 min followed by 10 min wash with water and 10 min wash with the running buffer. The buffer was prepared by adding NaOH to 10 mM phosphoric acid to reach pH 11.0. The capillary was thermostated at 25°C and the separation voltage was 20 kV. A hydrodynamic injection mode was applied (0.5 psi, 4 s, 21 nl) and spectra of... 

CZE using cyclodextrins (CD) as buffer additives has also been employed for the determination of synthetic dyes in various food products. The synthetic dyes New coccine, Erythrosine, Allura red AC, Tartrazine, Sunset yellow FCF, Brilliant blue FCF, Indigo carmine and Fast green FCF were included in the experiments. Measurements were carried out in a used-silica capillary (47 cm length, 40 cm to the detector, 50 pm i.d.). Capillary temperature was 25°C and separation voltage was 20 kV. Pressure injection was performed... 

Another study employed CE for the determination of the stoichiometry of the conjugation reaction between immonuglobulin and Lissamine rhodamine-B sulphonyl chloride (LRSC). The chemical structure of the dye is shown in Fig. 3.162. Separation of the unconjugated dye from the conjugated end product was performed by CE using an uncoated fused-silica capillary column (60 cm X 75 //m i.d.). The running buffer consisted of 10 rnM borate and 0.5 mM sodium dodecyl sulphate. The separation voltage was 20 kV and analytes were detected by a fluorescence detector. It was concluded from the results that the CE method combined with... 

The synthesis of phtalocyanine dyes was followed by CE and the purity and composition of the end product was investigated by the same method. The chemical structure of the newly synthetized zinc phtalocyanine tetrasulphonic acid is shown in Fig. 3.163. An uncoated fused silica capillary (65cm X 75 /.an i.d.) was employed for the separation of dye components. Running buffers were 10 mM ammonium acetate (pH adjusted to 9.4 with ammonia) and 10 mM potassium dihydrogenphosphate (KH2P04) (pH = 9.0). Samples were injected hydrodynamically (50 mm for 10 s). Separation was realized in the constant current mode at 30 pA, which required about 28 kV separation voltage. Analytes... 

The synthesis of a new near-infrared cyanine dye was monitored by CE and fluorescence detection. The chemicals structure of the dye and its synthetic precursor are depicted in Fig. 3.165. The analysis of the dye was realized in fused-silica capillaries of 75 and 100 /an i.d. The total and effective lengths of capillaries were 75 and 60 cm, respectively. The separation voltage was 30 kV and separations were carried out at ambient temperature. The running buffer was 2.5 mM Na2B407 (pH = 9.2). A near-infrared laser-induced fluorescence detector was applied. Electropherograms illustrating the separation of the dye are shown in Fig. 3.166. 

Fig. 3.172. Non-aqueous capillary electrophoresis with electrochemical detection of a dye mixture containing (a) 1.7 jUg/ml malachite green, (b) 0.70 jug/ml crystal violet, (c) 4.3 /ig/ml rhodamine B, and (d) 9.1 X 10-6 M ferrocene. Experimental conditions capillary dimensions, 95 cm X 75 pm i.d. running electrolyte, acetonitrile containing 1 M HAc and 10 mM NaAc electrokinetic injection, 20 s 5 kV separation voltage 20 kV applied detection potential, 1.55 V. Reprinted with permission from F.-M. Matysik [206].

Separation conditions can be optimized with respect to the capillary length (L), capillary ID, separation voltage (V), and separation temperature (T). 

FIGURE 3 Effect of separation voltage on CE-SDS separation of a protein. 

The separation conditions were subsequently optimized using the same antibody molecule. Focusing times of 1, 5, 10, 15, and 20 min were studied using a separation voltage of 25 kV. The optimized focusing time for sufficient separation was determined to be 10 min (Figure 19). 

Separation voltages of 5, 10, 15, 20, 25, and 30 kV were evaluated for the same molecule using a 10 min focusing time. The results are shown in Figure 20. The data demonstrate that 25 kV is an appropriate separation voltage for this molecule. 

Capillary length Capillary inner diameter (ID) Separation voltage Separation temperature Focusing time... 

Separation conditions in general refer to the entire system, including sample, instrument, detection, capillary, buffer, separation voltage, time, and polarity. Again, due to the flexibility in development that CZE methods afford, special attention to each of these variables may be necessary. Once the method has been optimized, qualification testing... 

Figure 13.9 Microchip-based micellar electrokinetic chromatography (MEKC) electro-pherogram of a mixture of nitroaromatics and nitramines. Analytes 20 ppm of each (1) TNB, (2) DNB, (3) NB, (4) TNT, (5) tetryl, (6) 2,4-DNT, (7) 2,6-DNT, (8) 2-, 3-, and 4-NT, (9) 2-Am-4,6-DNT, (10) 4-Am-2,6-DNT. Conditions MEKC buffer, 50 mM borate, pH 8.5, 50 mM SDS, 5 M Cy7, separation voltage 4 kV, separation distance 65 mm. (Reprinted in part with permission from [37]. Copyright 2000 American Chemical Society.)...

Due to the low conductivity in organic solvents, the currents are found to be orders of magnitude smaller than in aqueous CE. Hence, Joule heating virtually does not occur, even at high background electrolyte concentrations up to 0.1 mol/L. This allows us to work at a very high separation voltage. Nevertheless, the ability of a solvent to dissolve ionic species limits the number of solvents that can be used in CE. For instance, common buffer ions used in aqueous CE, such as phosphate and borate, cannot be employed... 

Fig. 6 Free-solution CE separation of PNA/DNA hybrid from excess PNA probe. M13 mpl8 ssDNA 4.2 X 10-8 M, and PNA probe 1.3 X 10-7 M. Detection LIF 488/520 nm. Buffer TBE, 7 M urea (pH 8.0). CE conditions 50-mm-i.d. polyacrylamide-coated capillary (27 cm in length and 20 cm to detector), 10 s gravity injection, separation voltage — 10 kV. Laser-induced fluorescence detection with excitation at 488 nm and emission at 520 nm. The buffer contained Triszborate (pH 8.0) with 7 M urea buffer. (From Ref. 37.)...

Fig. 9 Electropherograms showing 3 nM fluorescently labeled 11-mer in the absence (A) and in the presence (B) of 0.7 /j,M SSB protein in the running buffer. The conditions used were as follows separation capillary, 35 cm, 20-/xm i.d. running buffer, 25 mM disodium tetraborate (pH 9.1) separation voltage, 25 kV excitation wavelength, 488 nm emission wavelength, 515 nm and temperature, 25 (1°C. Approximately 1 nL of sample solution was injected electrokinetically. The asterisk indicates the migration time of the solvent, The traces Iv and Ih, corresponding to vertically and horizontally polarized fluorescence intensities, respectively, are shown separated for clarity. (From Ref. 48.)...

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