Paper chromatography sample preparation

Paper chromatography has been used successfully for many years and is still a useful tool despite the fact that thin-layer techniques, especially with readily available commercially prepared plastic or foil-backed plates, offer advantages of speed, resolution and easier handling. Larger volumes of sample can be applied to paper, permitting the subsequent elution of a particular amino... 

We have carried out an extensive literature search on sample preparation technologies and found many papers on conventional chromatography and capillary electrophoresis methods but few on NLC and NCE. It is important to mention here that sample preparation methodologies used in conventional chromatography and capillary electrophoresis can be used in NLC and NCE. The interested reader can consult our earlier books for details [20,21], However, attempts have been made to describe sample preparation protocols required in NLC and NCE techniques. Some of the important requirements and preparations are discussed below. 

Paper partition chromatographic methods have been widely applied to the analysis of tetracyclines (128, 129). Pharmaceutical aqueous suspensions for oral use are acidified with HC1 and diluted with methanol. Crystalline formulations are dissolved only in methanol. A paper chromatographic method for TC determination in pharmaceutical preparations is based on the complexation of the antibiotic with a mixture of urea and disodium edetate on paper at pH 7.4. Urea helped in the separation of degradation products and led to the formation of well defined spots (130). Samples from fermentations must be acidified with oxalic acid to liberate TC from the mycelium. TC in filtrates may be precipitated in saturated solution of sodium tetraphenyl borate, precipitate dissolved in ethyl or butyl acetate and applied for paper chromatography. Various solvent systems and hRp values for paper chromatography are given in Table 4. 

Prepared a sample of pure B chain with paper chromatography. 

Acid Hydrolysis. Twenty-five milliliters of 6N hydrochloric acid was added to 250 mg. of dry sample and the suspension was refluxed for 16 hours. The excess hydrochloric acid was removed by evaporation on a steam bath or on a rotary vacuum evaporator. The amino acids were then dissolved in 20 ml. of 10% 2-propanol. Samples so prepared were used directly for analysis by paper chromatography. Those samples to be analyzed by ion exchange chromatography were decolorized with a small amount of charcoal (Darco G-60) and filtered through paper. A 2-ml. aliquot of the filtrate was dried and redissolved in sodium citrate buffer at the pH and molarity required for application to the resin. 

Zirconium and hafnium This separation must rank as one of the major achievements of inorganic paper chromatography. The metals are present as zirconyl and hafnyl nitrates basic nitrates must be absent since these are immobile. The mixture is best prepared by digesting the sample at 80°C with concentrated nitric acid and evaporating the excess acid at the same temperature under reduced pressure the product gives a clear solution when dissolved in water. 

As a rule, chemical methods used in the examination of writing materials require initial preparation of a sample for study. Paper chromatography, thin-layer chromatography and capillary electrophoresis are experimental techniques often applied. These methods lead primarily to separation of the dyes contained in the ink under examination and to the discrimination of ink samples. The techniques are simple to use, require a small amount of sample for examination, are selective and give reproducible results. Their basic disadvantage, however, is the necessity to isolate the ink from the substrate (e.g. paper) on which the examined document has been prepared. Solvent extraction of the ink often leads to partial damage of the document. 

Amino-l-0 -o-ribofuranosyl)imidazole-4-carboxamide (5.16 g, 20 mmol) and hexachloroethane (40 g, 100 mmol) were added to a solution of raethanolic NaOMe (prepared from 7.8 g of metallic Na and 150 mL of MeOH), and the solution was heated to reflux for 3 h with stirring. Paper chromatography of this solution showed two spots which, on elution, corresponded spectrophotometrically to the starting AICA-riboside and inosine (I). HjO (100 mL) was added, the hexachloroethane was removed by filtration and the filtrate was neutralized to pH 7 by adding portionwise Amberlite IR-120 (IH form). The resin was removed by filtration and washed with ILO. The combined filtrate and washings were concentrated in vacuo to give a crystalline mass, which was rccrystallizcd (HjO) to afford a pure sample yield 3 g (51 %). 

The carbohydrate analyses reported by Sundman, Saarnio and Gustafs-son for sulfate pulps are listed in Table XVII. These results were obtained by quantitative, paper chromatography on completely hydrolyzed samples. In another study,"" the uronic acid anhydride content of some wood pulps was determined. It was noted " that sulfate pulps prepared from hardwoods retain a considerable proportion of uronic acid, whereas sulfate pulps from softv oods may be entirely free from polyuronides. 

The residue then was dissolved in 30 ml of 0.04 M aqueous triethyl-ammonium bicarbonate buffer (pH 7.5), (resulting pH 5.6). It was chromatographed on a DEAE cellulose column (HCOJ form) (3.4 X 15.0 cm), with elution first with 500 ml of water and then with a linear gradient of trimethylammonium bicarbonate at pH 7.2 (0-0.07 M). XXVII was completely resolved from some of XXVIII which was formed under the above reaction conditions. However, it was still contaminated by small amounts of cAMP and ethyl 2-diazo-malonic acid. These impurities were apparently generated from the hydrolysis of some XXVII when the triethylammonium bicarbonate was removed. Pure samples of XXVII were obtained by preparative paper chromatography on either Whatman 40 or 3 MM paper with overnight development with ethanol 0.5 M ammonium acetate, pH 7.0 (5 2, v/v). 

This sample preparation technique is widely used in the determination of halogens, sulphur, phosphorous in organic compounds as well as for the determination of Hg, Zn, Mn, Ni, Co, Fe, Cu, etc. An excellent application is the separation of compounds by paper chromatography in which the spot of interest is cut out of the paper and burned, as described above. 

Materials. The following Epon prepolymers (Shell Chemical Company) were used Epons 825, 828, 834 (all liquid to semisolid) and 1001, 1002, and 1004 (all solids). Approximate compositions (degree of polymerization) were determined by gel permeation chromatography. Bimodal blends were made by mixing Epon 825 with Epon 1004, as shown in Table 1. Sample preparation and the method of curing have been described in the preceding paper (29). 

CE, with its high resolving power, rapid method development, easy sample preparation, and low operational cost, is reported to be an excellent technique for resolving caseins (including different genetic variants), peptides derived from them, and whey proteins (16-19). Peptide profiles obtained by CE supplement the information obtained by reversed-phase high performance liquid chromatography (RP-HPLC) (17, 20). The application of CE to the assessment of proteolysis in milk and different cheese types has acquired an enormous importance in recent years. Reviews on the application of CE to this field can be found in papers by Otte et al. (21) and Redo et al. (22). 

Occurrence and preparation. The major heptose of the LPS of Vibrio cholera Inaba 569B was proved to be D-glycero-h-manno-heptose (47), by isolating the sugar through repeated purification on paper chromatography, and was confirmed by comparison with an authentic sample (synthesized via condensation of nitromethane with D-galactose).246... 

F values are not exactly reproducible from laboratory to laboratory or even on different runs in the same laboratory, so that they should be considered mainly as guides to relative migration distances and sequences. Factors causing Rp values to vary include dimensions of the apparatus nature and size of the layer or paper direction of mobile phase flow the volume and composition of the mobile phase equilibration conditions temperature humidity and sample preparation preceding chromatography. 

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