Electrophoresis acid preparation

A wide range of nucleic acids including RNAs, DNA fragments, plasmids, and oligonucleotides can be separated effectively by SEC on the basis of molecular size. Accordingly, it is possible to adopt SEC as an alternative to gel electrophoresis for analytical purposes. Furthermore, because the separated components in samples can be recovered easily and yet almost quantitatively by collection of column effluent, SEC should be superior to gel electrophoresis for preparative purposes. Consequently, SEC seems to be a useful technique for the separation and purification of nucleic acids. 

The thiazolecarboxylic acid structure (40) was also guessed in a similar way, from tracer experiments. The unknown compound was converted into the thiamine thiazole by heating at 100°C and pH 2. On paper electrophoresis, it migrated as an anion at pH 4. Tracer experiments indicated that it incorporated C-l and C-2 of L-tyrosine, and the sulfur of sulfate. The synthetic acid was prepared by carboxylation of the lithium derivative of the thiamine thiazole, and the derivatives shown in Scheme 19 were obtained by conventional methods. Again, the radioactivity of the unknown, labeled with 35S could not be separated from structure 40, added as carrier, and the molar radioactivity remained constant through several recrystallizations and the derivatizations of Scheme 17. 

Gotti et al. [42] reported an analytical study of penicillamine in pharmaceuticals by capillary zone electrophoresis. Dispersions of the drug (0.4 mg/mL for the determination of (/q-penicillaminc in water containing 0.03% of the internal standard, S -met hy I - r-cystei ne, were injected at 5 kPa for 10 seconds into the capillary (48.5 cm x 50 pm i.d., 40 cm to detector). Electrophoresis was carried out at 15 °C and 30 kV, with a pH 2.5 buffer of 50 mM potassium phosphate and detection at 200 rnn. Calibration graphs were linear for 0.2-0.6 pg/mL (detection limit = 90 pM). For a more sensitive determination of penicillamine, or for the separation of its enantiomers, a derivative was prepared. Solutions (0.5 mL, final concentration 20 pg/mL) in 10 mM phosphate buffer (pH 8) were mixed with 1 mL of methanolic 0.015% 1,1 -[ethylidenebis-(sulfonyl)]bis-benzene and, after 2 min, with 0.5 mL of pH 2.5 phosphate buffer. An internal standard (0.03% tryptophan, 0.15 mL) was added and aliquots were injected. With the same pH 2.5 buffer and detection at 220 nm, calibration graphs were linear for 9.3-37.2 pg/mL, with a detection limit of 2.5 pM. For the determination of small amounts of (L)-penicillamine impurity, the final analyte concentration was 75 pg/mL, the pH 2.5 buffer contained 5 mM beta-cyclodextrin and 30 mM (+)-camphor-10-sulfonic acid, with a voltage of 20 kV, and detection at 220 nm. Calibration graphs were linear for 0.5-2% of the toxic (L)-enantiomer, with a detection limit of 0.3%. 

In the purification of pectinesterase from the fruits of Citrus nat-sudaidai,61 fractional salting-out with ammonium sulfate was followed by chromatography on a column of DEAE-cellulose and by separation of the active fraction on Sephadex G-100. A preparation (purified solution) having a specific activity 460-fold greater than that of the original extract was obtained. Its homogeneity was checked by disc electrophoresis, and its amino acid content was determined and fundamental, kinetic data were obtained. 

By use of a crude preparation obtained after the cultivation of Aspergillus niger,104 pectinesterase was purified by repeated chromatography on DEAE-cellulose, using gradient elution. The homogeneity of the product was checked by free electrophoresis, sedimentation analysis, and determination of the N-terminal amino acid (phenylalanine). 

Several research groups used another interesting column technology as an alternative to the modification of the capillary surface. This method is inherited from the field of electrophoresis of nucleic acids and involves capillaries filled with solutions of linear polymers. In contrast to the monolithic columns that will be discussed later in this review, the preparation of these pseudostationary phases need not be performed within the confines of the capillary. These materials, typically specifically designed copolymers [85-88] and modified den-drimers [89], exist as physically entangled polymer chains that effectively resemble highly swollen, chemically crosslinked gels. 

The formation of DNP or dansyl amino acid derivatives followed by chromatography or electrophoresis is a useful technique in certain circumstances. The preparation of DNP derivatives may be indicated when the sample for analysis contains a variety of other substances, removal of which would be complicated, leading possibly to considerable analytical errors. However, the derivative formation and extraction is time consuming and itself can introduce inaccuracies into the analysis and should be used only when it offers an advantage over the separation of untreated amino acids. 

After completion of electrophoresis, the gel was stained with Coomassie blue. To prepare the staining solution, 40 mg Coomassie blue R-250 was dissolved in 25 ml isopropanol and 10 ml glacial acetic acid. The solution was filtered and the volume was increased to 100 ml with distilled water. The gel was placed in staining solution for 1 h, followed by washing with 10% acetic acid/2.5% isopropanol. 

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