Paper chromatography sample application

The place of gas chromatography (GC) in chemical analysis has been well established. Recent developments in theory and improvements in technique have made it possible to apply GC to a variety of physicochemical measurements. The advantages of GC over other techniques lie in its accuracy, convenience, specificity, versatility, speed, and ability to use only small quantities of sample. Thus, in recent years, many reviews and hundreds of papers emphasizing nonanalytical applications have appeared. 

Tn the previous papers of this series (1, 2, 3, 4) calibration and repro- ducibility of gel permeation chromatography (GPC) have been extensively examined. This paper describes the application of GPC to two selected samples of linear polyethylenes, one having a narrow molecular weight distribution (NMWD) and another a broad molecular weight distribution (BMWD). These samples were distributed by the Macro-molecular Division of IUPAC (5) for the molecular characterization of commercial polymers. The average molecular weights by GPC are compared with the data obtained from infrared spectroscopy, osmotic pressure, melt viscosity, and intrinsic viscosity. Problems associated with data interpretation are discussed. 

The procedure of paper and thin-layer chromatography. A Application of the sample. B Setting plate in solvent chamber. C Movement of solvent by capillary action. D Detection of separated components and calculation of Rf. 

Partition chromatography as described in this section may be applied to two major types of problems (1) identification of unknown samples and (2) isolation of the components of a mixture. The first application is, by far, the more widely used. Paper chromatography and TLC require only a minute sample size, the analysis is fast and inexpensive, and detection is straightforward. Unknown samples are applied to a plate along with appropriate standards, and the chromatogram is developed as a single experiment. In this way any changes in experimental conditions (temperature, humidity, etc.) affect standards and unknowns to the same extent. It is then possible to compare the Rf values directly. 

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. 

Classic paper chromatography like distribution chromatography is also carried out today in most cases in layers on a support sheet. To obtain low standard deviations extensive automatization from sample application to evaluation is necessary. The essential advantage in comparison to liquid column chromatography is the possibility of separating several samples side by side, because therefor usually no apparatus is required. 

Deacetylation was accomplished by treating 258 mg of the above product for 40 min with 5 ml of 6 N HCl at 121 C. The resulting was dried under reduced pressure and dissolved in water. After adjusting the pH to 7.0 with aqueous ammonia, the solution was evaporated to dryness again. The residue was dissolved in a little water and applied to a 0.9 X 30 cm column of resin XE-64 which had been previously equilibrated with 1 M ammonium formate pH 4.0 and washed with 250 ml of water. The column was washed with 850 ml of water after application of the sample followed by 70 ml of 0.72 M acetic acid to remove unhydrolyzed material. The e-pyridoxyl lysine was eluted with 0.81 M acetic acid, and fractions containing the product were evaporated to dryness. On recrystallization of pyridoxyl-lysine from ethanol the yield was 110 mg of pale yellow crystals, m.p. 214-214.5°C. On paper chromatography in butanol pyridine acetic acid-HjO (30 20 6 24 v/v) Rf = 0.28 (Ronchi et al. 1969). 

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. 

TLC and paper chromatography, while quite simple and inexpensive, are limited in their application to relatively simple mixtures. More sophisticated instrumental methods have been developed that allow smaller samples to be analyzed with much better resolution of different compounds. These methods also use the same principal of using stationary and mobile phases based upon a specific physical property that isolates the components of the mixture (Fig. 4.35). The most widely used methods... 

Substances separated on paper and thin-layer chromatograms are revealed or detected by derivatization to coloured or fluorescent products, and these are interpreted visually, evaluated semi-quantitatively with reference to standards run under identical conditions, or quantitated by elution of the coloured or fluorescent spots and instrumental determination by colorimetry or fluorimetry respectively. In paper chromatography the spots can be cut out with scissors or removed with a paper punch thin layers are usually scraped off the support and analysed in some appropriate way. A neat application is to analyse TLC spots directly by FAB MS [8], and this potentially provides identification as well as quantitation. It is one of the advantages of paper chromatographic and TLC methods that several samples and standards can be run under identical conditions, but the accuracy and precision of quantitation by these means are usually not nearly as good as with liquid or gas-liquid chromatographic separations. 

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