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Polyamide-layer chromatography of nucleic acid bases and nucleosides
... Reaction products after assays were mixed with 20 nmol uracil, deoxyuridine and deoxyuridinemonophosphate . Thin layer chromatography was performed according to the method by Wang and Wang (1967), using polyamide layer plates (BDH), and tetrachloromethane, acetic acid and acetone (4:1:4, by volume) as solvent. Spots detected under UV-light were cut out of the plate and radioactivity measured in a liquid scintillation counter. ...
Uracil-DNA glycosylase (UDG; UNG) has been purified 17000-fold from Atlantic cod liver (Gadus morhua). The enzyme has an apparent molecular mass of 25 kDa, as determined by gel filtration, and an isoelectric point above 9.0. Atlantic cUNG is inhibited by the specific UNG inhibitor (Ugi) from the Bacillus subtilis bacteriophage (PBS2), and has a 2-fold higher activity for single-stranded DNA than for double-stranded DNA. cUNG has an optimum activity between pH 7.0-9.0 and 25-50 mM NaCl, and a temperature optimum of 41 degrees C. Cod UNG was compared with the recombinant human UNG (rhUNG), and was found to have slightly higher relative activity at low temperatures compared with their respective optimum temperatures. Cod UNG is also more pH- and temperature labile than rhUNG. At pH 10.0, the recombinant human UNG had 66% residual activity compared with only 0.4% for the Atlantic cUNG. At 50 degrees C, cUNG had a half-life of 0.5 min compared with 8 min for the rhUNG. These activity and stability experiments reveal cold-adapted features in cUNG.
The methodology for the detection of picogram quantities of nucleotides directly from TLC plates without the use of radioactive
labeling has been developed. The method couples thin-layer chromatography (TLC) separation with matrix-assisted laser desorption/ionization
mass spectrometry (MALDI MS) detection. The TLC/MALDI coupling protocol was studied and optimized for the separation and detection
of deoxyribonucleotides. Several ammonia based solvents were examined as potential extraction solvents for the TLC/MALDI coupling
protocol. It was found that in order to obtain maximum MALDI signal intensity and minimal analyte spreading, the extraction
solvent should produce Rf-values for the analytes in the range of 0.3–0.4. Rf-values above this range led to extensive analyte spreading and those below this range resulted in poor extraction. Various
MALDI matrices and co-matrices were investigated, the best results were obtained using 2′,4′,6′-trihydroxyacetophenone (THA)
as a matrix. The extraction solvent chosen was an ammonium hydroxide/methanol (100 mM/30%, Rf = 0.28–0.38) solvent system which was found to provide the best sensitivity, minimal lateral spreading and excellent matrix
transfer. Using the optimized coupling protocol, the detection limits for the deoxyribonucleotide monophosphates were established
at or better than 10 picograms.
The separation of nineteen organophosphorus pesticides by one-dimensional and two-dimensional chromatography and their detection on polyamide layers were investigated. A comparison was made between the detection limits on polyamide and silica gel layers, and this showed the advantages of polyamide layer chromatography.Some correlations between the chemical structure of the samples and their RF values on polyamide layers and their connection with the type of solvent system used were discussed.
Compounds separated on polyamide thin-layers and located in an appropriate manner can be introduced directly into the ion source of the mass spectrometer together with the chromatographic polyamide adsorbent. In spite of the large excess of polyamide, excellent mass spectra are obtained exhibiting low backgrounds. In this way, organic compounds can be rationally identified on a nanogram scale not requiring reference substances. The application of the procedure is described for selected compounds of different classes of substances, e.g., phenols, steroids, nucleosides, biogenic amines, and amino acids.
Poly(ethylene terephthalate) film was used as supporter for preparation of polyamide layer. The most convenient method of preparation was described. This layer can be cut into any size and its sensitivity is higher than the glass plate supported layer. Twenty aromatic nitro compounds were used to show the behavior of polyester film based polyamide layer.
AbstractA non‐protein fraction from crude Formosan cobra venom was found to be a nucleoside mixture. The components were identified as guanosine, adenosine and inosine by paper chromatographic and polyamide thin‐layer chromatographic correlations and their spectroscopic data. The mole ratio of guanosine/adenosine/inosine was found to be 7:2.5:1.
Paper chromatography using the polyamide impregnated paper were reviewed. The method for preparing plates coated with a thin‐layer of polyamide for chromatographical work was devised. Circular polyamide plates for horizontal chromatography was also prepared. These plates were applied to the chromatography of the phenols, phenolic carboxylic acids and tropolones. The reproducibility of the chromatogram were excellent. It was found that polyamide thin‐layer chromatography is the most convenient method for the separation of phenolic substances ever found. The R f values of phenols, phenolic carboxylic acids and tropolones are tabulated and the relations between solvents, R f values and structure of phenols are discussed.
The separation of seventeen DNP derivatives of amino acids, 2,4-dinitroaniline and 2,4-dinitrophenol by polyamide layer chromatography is investigated. Convenient procedure for the preparation of durable polyamide layers are described. A table of RF values in ten solvent systems and ultraviolet contract photographs of three one-dimensional chromatograms and three two-dimensional chromatograms are given. The present method is better than paper on thin-layer chromatography in speed and efficiency.
- Wang