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Publisher Summary This chapter focuses on the separation of histidine degradation products and related imidazoles. The separation of imidazoles by paper chromatography has been well documented. The data presented here include details of R f values for enzymatically and nonenzymatically formed degradation products of histidine as well as for the more commonly occurring related imidazoles. Electrophoretic mobilities at two different pH values are also given for these compounds. Paper chromatograms were run overnight on Whatman No. 1 paper, in the ascending direction, and the solvent front was allowed to reach the edge of the paper, 27 cm from the baseline. A number of detection methods are listed as footnotes to the table. Routinely, it has been found convenient to include urocanic acid as an internal marker with all samples because this compound can be readily detected by viewing the papers under ultraviolet light prior to treatment with a specific spray reagent. Papers with both imidazoles and ninhydrin-positive compounds present can be treated successively with ninhydrin and diazotized p-chloroaniline, in that order.

Publisher Summary This chapter provides an overview of paper chromatography of carbohydrates and related compounds. The chapter surveys the application of filter-paper chromatography to carbohydrates and certain of their derivatives. It discusses the different methods of arranging the filter paper in the chromatographic chamber— descending, ascending, and circular or horizontal development. The filter paper most commonly used is Whatman No. 1. Many different solvent developers are used in the separation of sugars and related compounds. Three of these, phenol–water, collidine–water, and l-butanol–acetic acid–water, also commonly employed in the resolution of amino acid mixtures, are widely used. On theoretical grounds, low retention factor ( R F ) values, in the range of 0.2 to 0.3, are most desirable for maximum separations. The size of the areas occupied by the spots results in overlapping at low R F values, even though the partition coefficients are favorable for separation.

Publisher Summary This chapter discusses analytical and semipreparative separations of nucleic acid components by paper electrophoresis technique. The decision whether to use paper electrophoresis depends on the amount of material to be run and the type of separation. The smallest amount of nucleotide easily detected on paper by its ultraviolet absorption is about 5μg, and the largest amount of nucleotide mixtures conveniently handled in a single electrophoretic run is about 10 mg. Because of their insolubility, except at extreme acid or alkaline pH, the purines and pyrimidines are better separated by paper chromatography, as also are many of the methylated bases and nucleosides. Only a few of the isomeric polynucleotides can be separated by paper electrophoresis. However, many of these are resolved by the technique of electrophoresis on Diethylaminoethyl cellulose (DEAE) paper. The criteria for selecting a paper are that it should (1) give a low ultraviolet absorbing background after elution of the substances, (2) have a reasonable tensile strength when wet, and (3) give compact spots with minimum trailing. For analytical work Whatman 54, and for separation of larger amounts, Whatman 3MM are used.

Publisher Summary This chapter discusses the paper chromatography of sugars. In paper chromatography, a sheet of filter paper serves as the support for an immobile aqueous phase over which a partially miscible organic solvent is permitted to flow. A solute mixture introduced into such a system can be separated into its components provided that they possess significantly different partition coefficients between the mobile phase and water-saturated cellulose. When the partition favors the aqueous phase, the solute will tend to remain near the point of application. Conversely, when the organic phase is favored, the solute will move with the solvent. The application of this principle by Partridge resulted in a method for the qualitative analysis of a mixture of sugars. In qualitative procedure satisfactory chromatograms can be obtained from a sugar solution containing relatively large amounts of protein and salt. To verify the identification of unknown sugars, it is desirable to have milligram quantities of the individual components. Such quantities can be acquired by isolations from band chromatograms.

Publisher Summary Paper chromatography is gradual extension of the principles to simplified systems wherein solid supports were employed to hold one of the phases. This chapter describes the application of filter paper chromatography to the qualitative analysis of amino acids. It also describes the equipment, materials, and manipulations useful to the beginner confronted with the average biochemical problems. In practice, qualitative paper chromatography consists of a sequence of basic operations: (1) preparation of the sample, (2) determination of optimal aliquot, (3) application of the sample to the paper sheet, (4) development of the chromatogram with selected solvents, (5) detection of the separated compounds, and (6) identification of the spots. Special requirements frequently may add steps to the basic operations. The full power of paper chromatography usually can be realized most readily with equipment which permits the running of duplicate and control chromatograms simultaneously in the same chamber.

Publisher Summary This chapter presents the results of a number of studies that have been carried out at the Research Department of The Graphic College of Denmark and are relevant to the complicated processes involved in the absorption of printing ink into and its drying on paper. The studies have been made particularly on coated paper so as to discover the influence of the paper properties on the change of hue of colored printing inks and on phenomena like chalking and ghosting. The report is in three parts: (1) studies of the absorption of inks into paper, (2) studies of the change of hue of printing inks as a function of various paper properties, and (3) studies of the drying of printing inks on coated paper in relation to chalking and ghosting. Many experiments failed to establish a generally valid relationship between laboratory measurements of ink properties and practical printings because the properties of the ink change with time after printing, and in a way depending on the properties of the paper and their printing conditions.

Publisher Summary This chapter discusses the use of filter paper supports for plant cultures. The use of ashless filter paper supports has been recommended for the culture of isolated plant tissues of the callus type. These tissues are cultured according to Gautheret's technique, which consists in culturing explants, 2–3 mm side measurement, weighing about 150 mg, in vertical tubes containing about 25 ml of nutrient medium generally solidified with agar, as these tissues cannot tolerate immersion in a liquid. These paper supports can be made very quickly and placed in position and 100 tubes can be equipped with them in one-quarter of an hour. It is observed that if the support is lowered too far into the liquid, a bubble of air remains enclosed under the support. Growth in a liquid medium surmounted by a filter paper support is less abundant than on agar. Culture on ashless filter paper is suitable not only for tissue cultures, but also for the culture of embryos, seedlings and any other plant material not too large in size, and which it is required to culture aseptically on a nutrient medium of strictly calculated composition.

Publisher Summary The chapter presents a survey of techniques that help one to understand the principles on which electrophoretic separation on paper is based, enables one to appreciate the possibilities, and makes one aware of the limits and errors of the method. In this survey, both the 1D and the 2D methods are reviewed. The unidimensional type of paper electrophoresis is an extension of free boundary electrophoresis. There are several differences between the two systems. These differences simplify the qualitative and quantitative appraisal of separation after the run on paper. These two methods are described and for each of them a description of the available apparatus with special regard to its structure in promoting and controlling the key function of migration is followed by a study of the anticonvectional medium, the buffer, and the electrical field. The quantitation of results is surveyed. Finally, the chapter considers the new possibilities to describe the trend of electrophoretic methods and indicates their probable development in the future. The chapter concludes that if the electrophoretic tool is called upon to bring a definite advance to medical science, only accurate techniques and reproducible results under standardized conditions can warrant an important future.

Publisher Summary This chapter presents the paper chromatography of glutathione and its hydrolysis products. Paper chromatography offers possibilities for overcoming difficulties encountered when trying to distinguish among sulfhydryl compounds present simultaneously in solution. Sulfhydryl peptides can be chromatographed successfully if first stabilized by coupling with N-ethylmaleimide that eliminates a major obstacle, the tendency of SH compounds to streak on the paper. Several methods of detecting maleimide derivatives of sulfhydryl compounds in quantities of the order of 2 γ or less are theoretically possible. First is the synthesis of C14 labeled N-ethylmaleimide of sufficiently high specific activity to permit ready detection of sulfhydryl imides by radioautography or Geiger–Mueller tube. Second is preparations of other maleimide derivatives containing groups having large extinction coefficients in the ultraviolet region offer another possible solution. Finally, a maleimide derivative with a functional group that couple with a chromophore, such as an azo dye, forming an intense color, serves to detect small amounts of sulfhydryl containing compounds in a more specific manner than the ninhydrin reaction.

Publisher Summary This chapter discusses the ion-exchange paper chromatography method for the separation of nucleoside diphosphate sugars and related nucleotides. By combining the rapidity and discrimination of ionexchange resins with the sensitivity of filter paper as a supporting medium, a rapid and efficient separation of small quantities of the nucleotides is acheived. The method is capable of discriminating between derivatives of the various naturally occurring purine and pyrimidine bases; and conditions may readily be selected for separation of the base homologous nucleotides with varying numbers of phosphate substituents. Since the method is based in part on ion-exchange principles, substituents either on the base or phosphate component of the nucleotide predictably alters the mobility of the product on polyethyleneimine paper. Nucleoside diphosphate sugars can be separated from mono-, di-, and triphosphates of the same nucleoside in about 40 minutes. Nucleoside diphosphate sugars differing in the base moiety separate in 3–4 hours. Separations of the common ribo- and deoxyribonucleoside monophosphates are also obtained in 3–4 hours. It is a convenient procedure to check reaction products in the intereonversions of the nucleotides.

Publisher Summary Nucleic acid components on paper chromatograms are photographed by making a contact print by filtered ultraviolet light onto sensitized paper through the dried paper chromatograms. In the procedure, the source of ultraviolet light is a Hanovia Homesun lamp, a 450-watt, 200-volt, mercury arc lamp. This is used in conjunction with a specially constructed filter. Various sensitized papers used for making contact copies of printed pages are suitable for the present purpose, for example, Kodagraph Contact Standard. Time of exposure and distance of paper from the light source are determined with known standards. For making the prints, it is quite satisfactory to pin the chromatogram paper over the photographic paper on a planoconvex wooden surface. A piece of plywood is fastened to a board with a small strip of wood under the center to bow the plywood out a little. After development, the paper is fixed and washed in the usual way.

Publisher Summary The complete separation of all nucleotides obtainable from nucleic acids is a matter for the employment of a number of techniques, only one of which is paper chromatography. Spots can be detected by their ultraviolet absorption visually 1,2 or by photography. Nucleotides may also be detected by spraying with dilute FeC1 3 , which complexes with the phosphate groups. The spots are then detected by spraying the paper to detect free ferric ions. For separation Descending chromatography is almost imperative because many solvents require to be run off the end of the paper. Very large tanks are to be avoided if possible. Appropriate marker spots should be used as RF values tend to be variable. It is advisable to use solvents which do not absorb ultraviolet light. Strongly acid solvents will hydrolyze purine deoxyribonucleotides, cyclic mononucleotides, and ribo-oligonucleotides. Mixtures separated by chromatography can be separated further or resolved by by 0.1 N HC1 for 4 hours at 20 °, pancreatic ribonuclease, and prostatic phosphomonoesterase..

Publisher Summary This chapter describes the role of paper electrophoresis of proteins in investigating the chemical nature of physiological and pathological conditions; and the individual patient. The value of paper electrophoresis in determining the diagnosis and prognosis, in the assessment of progress, and in the control of therapy has been evaluated in the chapter. Paper electrophoresis of proteins is a rapidly developing technique, and conclusions drawn at present are scarcely more than those of a preliminary report. It has been very widely used in elucidating the changes in protein metabolism, which occur in different conditions, amplifying and extending the information already obtained by the more laborious technique of boundary electrophoresis. The use of paper electrophoresis as a diagnostic procedure is thus less assured than its use in other fields, and it would appear that techniques that give better resolution of individual protein components, such as electrophoresis in starch gel or electrophoresis in agar combined with immunological reactions (G18), could profitably be developed further as aids to diagnosis.

Publisher Summary This chapter discusses the development of surface smoothness of a lightweight publication paper under increased loading of the finishing supercalender. A 28 lb/ream basestock consisting of approximately 70 percent hardwood and 30 percent softwood chemical pulp has been coated on a 12 inch trailing blade pilot coater with a single coat on the wire side under various coating conditions. Supercalendering and blade coating exert, therefore, a combined beneficial influence on the printability of the finished paper. Blade coating fills the deep regions of the basestock and applies only a thin coating layer to the highest portions, thus, equalizing the sheet caliper to some extent. The coating thickness of a blade coated sheet varies from a small fraction of the final paper caliper to 50 percent and more according to the void depth of the basestock before coating. Supercalendering reduces the coated paper caliper at the thickest areas, thus, again equalizing the sheet caliper in the same direction. It effects this by increasing density at these high surface locations and throughout the sheet under these surface areas.

Publisher Summary This chapter discusses paper electrophoresis. Paper electrophoresis, introduced by Koenig in 1937, is several years older than modern paper chromatography. Paper electrophoresis was first used in conjunction with paper chromatography. The inhibiting influence of colloid size is compensated by the number of charged sites on the particle. The size and shape of the colloidal particles are relatively without influence. Other carriers such as powdered glass, gelatin, agar, cotton, asbestos, silica gel, starch, ion-exchange resins and other materials can be used as the carrier medium. The medium should serve primarily as a support and consequently should not influence the migration by absorption, etc. However, if absorption or ion exchange is desired, starch or columns of ion-exchange resins can be employed. Although excellent separation of high molecular weight substances can be achieved at voltages of 80-400 V, the greater rates of diffusion of amino acids and peptides tend to vitiate the separation achieved by low voltage paper electrophoresis. This adverse effect of diffusion can be prevented by greatly increasing the voltage applied which correspondingly increases the rate of migration.

Publisher Summary This chapter presents experiments on deformations of paper in stacks, in which sheets were conditioned at a fixed humidity and then printed with a screen line plate by means of a Vandercook proof press. The prints were stacked progressively during printing. The stack was exposed to another humidity for a time and then, a second color was printed upon the first one, from the same plate. From the interference of the two line patterns, it was possible to measure deformation of the paper. Every sheet was measured and the results for an offset paper, a conventional coated paper, and a machine-coated paper are presented in the chapter. The change in dimension is given on the abscissa in units of 0.1 mm/m. The trials were made on three papers: (1) an offset paper, (2) a conventional coated paper, and (3) a machine-coated paper. The results show that the deformation was greater in the transverse direction than in the longitudinal direction and the deformations were not the same whether the transverse direction or the longitudinal direction was parallel to the cylinder axis; this effect shows that the press has a laminating action.

Publisher Summary This chapter discusses the filter paper disk techniques for assaying radioactive macromolecules. The method consists of numbering a series of Whatman No. 1 (or No. 3) cellulose disks and then applying the impure radioactive sample, such as an aliquot of a reaction mixture, to the properly numbered disk. The enzyme reaction is stopped by placing the filter paper disk in a large beaker of trichloroacetic acid. A large number of disks, for example, all the time points in a multitube kinetic experiment or density gradient run, can be accumulated and subsequently processed simultaneously through several reagents necessary for extraction of extraneous material. After radioactive precursors have been removed from the radioactive product, the washing reagents that may interfere with the analysis of the radioactive product are removed by suitable solvents and residual water is removed by drying either with solvents, air, or heat. The radioactivity of the sample can then be analyzed for the various types of isotopes that it may contain, either by scintillation techniques or by a straightforward Geiger counting procedure, under standard counting conditions. Descriptions of several specific modifications of this disk procedure are outlined in the chapter, which are useful for a number of enzymatic polymerizations and depolymerizations that use radioactive substrates to measure the radioactive product.

Publisher Summary Using histochemical techniques as well as ascending paper chromatography, it could be shown that the pineal organ of the rat contains phospholipids, triglycerides, and fatty acids. This chapter discusses of lipochondria that are present in the pineal parenchymal cells and contain triglycerides and phospholipids. They are evidently homologous to the “Golgi bodies” in the cells of the other organs. Under experimental conditions, they produce “myelin figures.” It appears that the pineal lipid level may change under physiological and experimental conditions. Probably, at least some of these quantitative variations are caused, by the function of the adenohypophysis, while also direct orthosympathetic neural regulation may play a certain part. The exact role of the pineal lipids in the functioning of the organ, may be in elaborating one or more specific secretory products, is still unknown.

The article describes the separation of gamma-aminobutyric acid (Gaba) from mouse-brain extract using one-dimensional paper chromatography. (Author)

A computer program has been written which calculates DPM for single and dual labeled samples after correction for quenching. The program in ALGOL is executed from a remote terminal and uses punched paper tape input logged directly from the liquid scintillation counter in the proper format. Multiple quench indexes are used to calculate the best estimate of efficiencies for each isotope; channel ratios, AES ratios and AES counts are all used for single isotope samples and the two AES indexes only for dual labeled samples are used in the estimate computed in either case from a single sample counting. A separate counting produces data for the channels ratio computation for dual label samples using either the screening method or the general method where appropriate. Divergence of efficiencies computed from the different indexes indicates non-identity with the standards. The algorithm has been tested with serum, urine and aqueous biological samples.

Publisher Summary The development of procedures for peptide mapping requires a technique that is capable of giving highly valuable information on the primary structure of protein molecules. This chapter discusses the procedure of peptide mapping by using two major techniques, namely––paper chromatography and electrophoresis. An enzymatic digest of a protein to chromatography is subjected, followed by high-voltage electrophoresis in the second dimension. The relative mobility of each peptide in the two dimensions depends on its composition and to a lesser extent its sequence. The procedure is designed to afford a rough identification of differences in amino acid sequence between two closely related molecules, such as residue replacements due to genetic mutation. Several solvent systems are used for peptide mapping and vary in applicability to different proteolytic digests. A variety of amino acid-specific reagents used to stain a peptide map and enhance its usefulness are focused. Many specific staining techniques found in standard texts dealing with chromatography and are helpful in technique of peptide mapping such as Ehrlich stain for indoles are discussed. Interpretation of the map, peptide mapping for preparative purposes, peptide mapping to evaluate digestion by exopeptidases, and technique of diagonal peptide mapping are also discussed in the chapter.

Publisher Summary Most of the methods for the separation of deoxyoligonueleotides are directly applicable to the separation of ribooligonucleotides, but not vice versa. Methods based on the presence of two adjacent hydroxyl groups are obviously not applicable to the deoxyribose series. There are methods that are more convenient for the deoxy series, for example, the method of Felix et al., because d-T and d-C and their derivatives are widely separated whereas the positions of the derivatives of C and U are rather close, and experienced hands are required to obtain reliable results with the ribo derivatives. Paper chromatography is used to identify a compound by comparison of its location with that of an authentic sample. Both the experimental and the “authentic” spots are then eluted and read against controls obtained by eluting the closely located blank spots of identical dimensions.

Publisher Summary This chapter describes the behavior of carbohydrates during electrophoresis in the various electrolytes and the modes and positions of the attachment of the complex agents to the polyhydroxy compounds and indicates the use of these methods in current problems. The facts described in Foster's are repeated, except where completeness seems desirable. Molecules that possess a net charge, or which may be given by simply controlling the pH of their environment, migrate in an applied electrical field as anions or cations according to the properties of their functional groups. The lower affinity of sulfonated benzeneboronic acid for diols, together with this entropy effect explains the observation in contrast to borate solution; no contribution to mobility arises from such a diol grouping as that on C-4 and C-6 in D-glucopyranose.