Electrophoresis, borates

The incubation mixtures were assayed by the double chromatographic method (33,40) or by a combination of high voltage borate electrophoresis and reverse flow chromatography (32 40 ) on Whatman 3MM paper using chloroform-methanol-H20 (50 40 10) as solvent system. The appropriate areas of each chromatogram were determined quantitatively in a toluene scintillation system with a Beckman scintillation counter (model LS-3133T). 

Amino acid analysis of a strong acid hydrolysate (2 N HCl at 100° C for 3 h) identified only glutamic acid and this was confirmed by gas chromatography-mass spectroscopy (Fig. 4). Mild acid hydrolysis (0.5 N HCl at 80°C for 1 h) followed by direct insertion mass spectroscopy gave a spectrum identical to authentic ribose-5-phosphate (Fig. 5). Borate electrophoresis of the alditol derived from the stored material and of its acid... 

Fig. 6. A Borate electrophoresis of the alkaline product with standards ( ). Ribositol-5-phosphate standard elutes at the same position. B The product of 2 N HCl hydrolysis of the alkaline NaB H4 -treated material. C The product of alkaline phosphatase treatment. (From [24] with permission)...

Weber, P. L. Buck, D. R. Capillary Electrophoresis A Past and Simple Method for the Determination of the Amino Acid Composition of Proteins, /. Chem. Educ. 1994, 71, 609-612. This experiment describes a method for determining the amino acid composition of cyctochrome c and lysozyme. The proteins are hydrolyzed in acid, and an internal standard of a-aminoadipic acid is added. Derivatization with naphthalene-2,3-dicarboxaldehyde gives derivatives that absorb at 420 nm. Separation is by MEKC using a buffer solution of 50 mM SDS in 20 mM sodium borate. 

Catechin and epicatechin are two flavanols of the catechin family. They are enantiomers. The capillary zone electrophoresis (CE) methods with UV-detection were developed for quantitative determination of this flavanols in green tea extracts. For this purpose following conditions were varied mnning buffers, pH and concentration of chiral additive (P-cyclodextrin was chosen as a chiral selector). Borate buffers improve selectivity of separation because borate can make complexes with ortho-dihydroxy groups on the flavanoid nucleus. 

Electrophoretic condition 60 cm (effective length of 50 cm)x75 p.m I.D. fused capillary column, run buffer borate buffer pH 9,0, P-cyclodextrin, electrophoresis voltage 20 kV, detection at 254 nm. 

Stellwagen, NC, Apparent Pore Size of Polyacrylamide Gels Comparison of Gels Cast and Run in Tris-acetate-EDTA and Tris-borate-EDTA Buffers, Electrophoresis 19, 1542, 1998. Stellwagen, NC Gelfi, C Righetti, PG, The Free Solution Mobility of DNA, Biopolymers 42, 687, 1997. 

FIGURE 18.2 Capillary gel electrophoresis separation of an octylphenol ethoxylate sulfate (with an ethylene oxide chain length from 1 to 8). Run conditions pH 8.3 (100 mM tris-borate, 7 M urea) 50 pm x 75 cm J W polyacrylamide gel capillary (PAGE-5, 5%T, and 5%C) run at 20 kV with a 5kV injection for 5 s UV detection at 260nm. 

Lin et al. [95] used capillary electrophoresis with dual cyclodextrin systems for the enantiomer separation of miconazole. A cyclodextrin-modified micellar capillary electrophoretic method was developed using mixture of /i-cyclodextrins and mono-3-0-phenylcarbamoyl-/j-cyclodextrin as chiral additives for the chiral separation of miconazole with the dual cyclodextrins systems. The enantiomers were resolved using a running buffer of 50 mmol/L borate pH 9.5 containing 15 mmol/L jS-cyclodextrin and 15 mmol/L mono-3-<9-phcnylcarbamoyl-/j-cyclodextrin containing 50 mmol/L sodium dodecyl sulfate and 1 mol/L urea. A study of the respective influence of the /i-cyclodcxtrin and the mono-3-(9-phenylcarbamoyl-/i-cyclodextrin concentration was performed to determine the optical conditions with respect to the resolution. Good repeatability of the method was obtained. 

Valproic acid has been determined in human serum using capillary electrophoresis and indirect laser induced fluorescence detection [26], The extract is injected at 75 mbar for 0.05 min onto a capillary column (74.4 cm x 50 pm i.d., effective length 56.2 cm). The optimized buffer 2.5 mM borate/phosphate of pH 8.4 with 6 pL fluorescein to generate the background signal. Separation was carried out at 30 kV and indirect fluorescence detection was achieved at 488/529 nm. A linear calibration was found in the range 4.5 144 pg/mL (0 = 0.9947) and detection and quantitation limits were 0.9 and 3.0 pg/mL. Polonski et al. [27] described a capillary isotache-phoresis method for sodium valproate in blood. The sample was injected into a column of an EKI 02 instrument for separation. The instrument incorporated a conductimetric detector. The mobile phase was 0.01 M histidine containing 0.1% methylhydroxycellulose at pH 5.5. The detection limit was 2 pg/mL. 

Figure 3.12 Metabolic profiling by capillary electrophoresis, (a) Comparative carbohydrate profiles of M. truncatula tissue obtained using 4-aminobenzonitrile derivatization, capillary electrophoresis with a 150 mM borate buffer, pH = 9, and on-column UV detection at 214 nm. (b) Anion profile from M. truncatula using capillary electrophoresis and indirect UV detection. The separation buffer was 5 mM K2C1O4, 1% Waters OFM-Anion BT, pH 8.0.

Budtz-Olsen (B13) has shown that Hp grouping is possible on vertical paper electrophoresis if Consden and Powell s (C3) borate-barbiturate buffer is used, since differences between the migration rate of the different Hp s is then great enough. It is, however, doubtful whether this is possible when the concentration of the Hp is low. 

Fig. 17.11. Bottom CGE separation of components of poly U (sigma) in 25% pluronic F127. Top Note the resolution of two contaminants between each of the oligonucleotides from about 15 to 27 nucleotides long in this expanded section of the bottom electropherogram. Electrophoresis was performed in 25% pluronic F127 in tris-borate-EDTA buffer (90 mM tris, 90 mM boric acid, 2 mM Na EDTA, pH 8.3.) (25°C, 500 V cm-1, effective column length 30 cm). Reprinted with permission from Ref. [82],...

Fig. 3.161. (A) Zone electrophoresis patterns of FITC-labelled transferrin samples by fluorescence detection. The unbound dye (providing a main peak and several minor ones) was not removed from the samples. Experimental conditions background electrolyte, 100 mM borate buffer, pH 8.3 voltage, 20 kV capillary 59 cm (effective length 41 cm) X 75 pm i.d. injection of samples 100 mbar x s 20°C detection with fluorescence detector (240 - 400 nm, broadband excitation filter and a 495 nm cut-off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 13 pm (1 mg/ml) Tf and (a) 0.01 mM FITC, (b) 0.1 mM FITC, and 1 mM FITC. (B) Zone electrophoresis patterns of an FITC-labelled transferrin sample by simultaneous fluorescence (upper trace, left axis) and UV detection (lower trace, right axis). The unbound dye shows several peaks with both detections. Experimental conditions background electrolyte, 100 mM borate buffer, pH 8.3 voltage, 20 kV capillary 59 cm (effective length fluorescence 41 cm, UV 50.5 cm) X 75 pm i.d. injection of samples 100 mbar X s 20°C detection with fluorescence detector (240 - 400 nm, broadband excitation filter and a 495 nm cut off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 6.5 pm (0.5 mg/ml) Tf and 0.1 mM FITC. Reprinted with permission from T. Konecsni et al. [199].

Other buffers that have been used for continuous, native electrophoresis are Tris-glycine (pH range 8.3 to 9.5),19 Tris-borate (pH range 8.3 to 9.3),26 and Tris-acetate (pH range 7.2 to 8.5).27 Borate ions26 can form complexes with some sugars and can therefore influence resolution of some glycoproteins. 

Tris-sulfate/Tris-borate, Tris-formate/Tris-borate, and Tris-citrate/Tris-borate have been advocated as electrophoresis buffers.5 For basic proteins, a low-pH alanine-acetate system28 is often used. 

Figure 1.9 Use of capillary electrophoresis for the analysis of amoxicillin from four suppliers (a-d). Reprinted from [18], copyright 1994, with permission from Elsevier. (AMOX is amoxicillin, AMP is ampicillin, PENV is penicillin V, DG is a degradant, and 1 and 2 are unspecified impurities. Capillary 80 cm X 50 (tm i.d. [75 cm to detector] [Polymicro Technologies] running buffer 100 mM pH 8 Na2HP04 containing 50 mM sodium dodecyl sulfate and 50 mM sodium borate voltage 18 kV injection at 100 mm for 15 s detector UV 205 nm.)...

Capillary zone electrophoresis is a powerful tool for the separation of water-soluble vitamins, such as nicotinic acid and vitamin C, with high-pH borate or phosphate buffers. Most simultaneous separations have been performed for fat-soluble vitamins, such as vitamins A and E, by MEKC. Here, organic... 

Gel Electrophoresis This method was used in the determination of the purity of native lysozyme and identification of ozonized products. Different gel concentrations (7,8,9,10%) and buffer solutions (0.25M borate, pH 8.7 0.025M phosphate, pH 7.1) were tried and the best results were obtained with 7% gel in pH 8.7 buffer. 

Qi, S., Li, Y., Deng, Y., Cheng, Y, Chen, X., and Hu, Z., Simultaneous determination of bioactive flavone derivatives in Chinese herb extraction by capillary electrophoresis used different electrolyte systems—borate and ionic liquids, /. Chromatogr. A, 1109, 300-306,2006. 

Standard research-grade agarose is usually sufficient, but special agaroses may be used for specific applications (e.g. high-resolution gels). The electrophoresis buffer is usually prepared and stored as a 10 x concentrated stock. The most commonly used buffer is Tris-borate-EDTA (TBE), or alternatively, one may use Tris-acetate-EDTA (TAE) buffer ... 

Capillary electrophoresis has been applied by Chen and Gu (281) for simultaneous determination of oxytetracycline, tetracycline, chlortetracycline, and doxycycline residues in bovine milk. Separation was performed on a noncoated capillary column, 57 cm total length with 50 cm effective length, 75 m internal diameter (i.d.) and 375 m outside diameter (o.d.), using a mobile phase containing 10 mM sodium dodecyl sulfate, 50 mM borate, and 50 mM phosphate, pH 8.5. Under these conditions, concentrations below 10 ppb could be determined in milk using an ultraviolet spectrophotometer set at 370 nm. 

Dissolve in H2O and acidify with 3N HCl to pH 3.5. Collect the solid and wash with H2O. The air-dried ppte is extracted with 70% aqueous EtOH, filtered hot and cooled slowly. Fine yellow needles of the acid crystallise out, are filtered and dissolved in the minimum quantity of 0.0IN NaOH and reppted with N HCl to pH 3.5. It is then recrystd from 70% aqueous EtOH (3x). The final product (acid) is dried at 80° in a vacuum for 24h, m >300°dec. It contains one mol of water per mol of acid (C30H36N4O13.H2O). The product is pure as revealed by electrophoresis at pH 5.6 and 8.6, and by TLC in isoBuOH-isoPrOH-AcOH-H2O (60 60 5 5 by vol) or isoPrOH or pH 8.0 borate buffer. [AC 31 456 7959]. 

This mode of electrophoresis, in which the electrolyte migrates through the capillary, is the most widely used. The electrolyte can be an acidic buffer (phosphate, citrate, etc.) or basic buffer (borate) or an amphoteric substance (a molecule that possesses both an acidic and an alkaline function). The electro-osmotic flow increases with the pH of the liquid phase, or can be rendered non-existent. 

Figure 26-31 Separation of natural isotopes of 0.56 mM Cl by capillary electrophoresis with indirect spectrophotometrlc detection at 254 nm. Background electrolyte contains 5 mM CrOJ to provide absorbance at 254 nm and 2 mM borate buffer, pH 9.2. The capillary had a diameter of 75 m, a total length of 47 cm (length to detector = 40 cm), and an applied voltage of 20 kV. The difference in electrophoretic mobility of 36C and 37CI is just 0.12%. Conditions were adjusted so that electroosmotlc flow was nearly equal to and opposite electrophoretic flow. The resulting near-zero net velocity gave the two isotopes maximum time to be separated by their slightly different mobilties. [From C. A Lucy and T. L McDonald, "Separation of Chloride Isotopes by Capillary 35 40 45 Electrophoresis Based on the Isotope Effect on Ion Mobility"Anal.

Buffers appropriate for electrophoresis gels include Tris-glycine, Tris-acetate, Tris-phosphate, and Tris-borate at concentrations of about 0.05 M. 

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