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Cellulase and pentosanase were largely separated from polygalacturonase in the crude preparation, Enzyme 19-D by paper chromatography. A partial separation of pentosanase from other enzymes of the mixture was effected by paper electrophoresis. All of the enzymes studied except cellulase were inactivated by heating at 74–75 °C.; almost 50% of the cellulase activity was retained. Polygalacturonase and pectinesterase were inactive above pH 7.0. Most of the enzymes studied appeared to have two or more components.

The paper chromatography of phenols (expecially phenolic acids), aglycones, glycosides and certain sugars is carried out with two new solvents. The authors have also examined these substances with the aid of paper electrophoresis and a borate buffer. A detection reagent for o -diphenols on paper is described.

Aliphatic monoamines, diamines, or polyamines have been separated by paper electrophoresis. The separation of a mixture of the bacterial growth factors putrescine, 1,3-propanediamine, spermidine, and spermine has been achieved by this same technique, while spermidine and spermine have also been separated and may be identified in tissue hydrolyzates by paper chromatographic and bioautographic procedures which are described.

An aqueous solution of sodium molybdate has been used as a specific reagent for the detection of phenolic compounds with o-dihydroxy groupings. The paper electrophoretic behaviour of phenolic compounds has been studied using a series of different buffers with pH values ranging from 5.2 to 10.0. o-Dihydroxy compounds can readily be detected by the use of ammonium molybdate buffer, and other structural features can be revealed by the choice of suitable electrolyte solutions. Paper partition chromatography of phenolic compounds using molybdate-treated paper in described.

The paper chromatography of vat and sulphur dyes, using reducing solvents at 80°, has been carried out, the dyes being separated as their leuco compounds. In both cases ascending paper chromatography was applied. The relation between dye constitution and chromatographic behaviour has been examined.

A number of simple chromatographic applications are described in which sheet paraffin or other plastic film is used as an adhesive vapor barrier: 1. A funnel attached to a paraffin-enclosed narrow paper wick for spotting large volumes of sample solution as small spots of controlled size. 2. A funnel for the elution of a spot and its reapplication to another paper in a single operation. 3. A method for elution or chromatography which requires no jars or other rigid saturation chambers.

A paper Chromatographic method for the analysis of phenylalanine in serum is described. The amino acids, including phenylalanine, are isolated from the deproteinised serum by means of a cation exchanger. The phenylalanine is then separated by paper chromatography at elevated temperature and estimated colorimetrically. Normal values are given. The excretion of phenylalanine and phenylpyruvic acid is correlated with the serum phenylalanine level. The method is specific and the reproducibility is good. Its application in a case of phenylketonuria is reported.

Inorganic anions were studied by paper electrophoresis and ion-exchange paper chromatography employing electrolytes and eluents with various metal ions (Mg2+, Al3+ and Cd2+). It was found that the metal ion has a strong effect on the movement of anions in paper electrophoresis both by ion pair and complex formatin In ion-exchange paper chromatography the effect is small. Data are given for twenty inorganic anions.

The behaviour is described of several barbiturates when subjected to chromatography on filter paper impregnated with olive oil. Results are given for various methanolic and aqueous inorganic solvents. The technique affords a fairly rapid separation of slow-, medium- and quick-acting barbiturates but the order of separation is different from those obtained with conventional and ion-exchange paper chromatography.

Measurement of the increase of spot areas of solutions on moistened filter paper was carried out. The results were used to calculate the feasibility of separations by paper electrophoresis, and the method is suggested as a simple technique for measuring relative diffusion rates.

An ascending chromatographic method was used to identify acidic mucopolysaccharides. On silica-gel-treated glass papers, a displacement type of chromatography with ethanol-salt solutions separated seven mucopolysaccharides. The compounds were located by charring with sulfuric acid or different staining technics. The method is convenient, reproducible, and rapid and can be used to identify small quantities of mucopolysaccharides in mixtures. Certain difficulties as and identification procedure were observed, however, and are discussed. Observations on glass-fiber papers are useful in studying different reactions of mucopolysaccharides with various salts and stains.

The design and manipulation of an apparatus for centrifugal acceleration of paper chromatography has been described. The main novel features consists of using filter paper strips instead of circular sheets and a constant flow solvent reservoir enclosed in the chromatography tank. Our arrangement is particularly suited for the quantitative analysis of materials labelled with radioactive isotopes of short half-lives and examples are given of the separation of some 82 Br- and 131 I-labelled substances. Advantages of the above method over circular paper chromatography are discussed.

The separations of steroid oestrogens achieved by paper chromatography have been reviewed with special reference to studied involving epimeric oestrogens and to oestrogens which have been isolated recently. Methods of detection of these compounds on paper chromatograms have also been discussed. The mobilities of thirty-four different oestrogens and their derivatives in many solvent systems have been tabulated. It is hoped that this review will be found useful in providing a collection of data from which separations of mixtures of oestrogens can be planned.

1. (1) The binding of acid dyes by denatured protein on filter paper has been investigated with special reference to Light Green. 2. (2) The uptake of dye, measured by elution, has been shown to vary according to the method of denaturation, the nature of the dye solvent (particularly the salt and and ethanol concentration) and finally with the duration and temperature of the subsequent washing procedure. 3. (3) Albumin and globulin do not behave in the same manner with different denaturing agents so that different methods give different albumin/globulin dye uptake ratios. 4. (4) The stoichiometric relationship between dye and protein is consistently nonlinear even at lower concentrations of protein on the paper and the same pattern can be demonstrated with protein on cellulose acetate membrane. It is suggested that this non-linear relationship at low protein densities is due to interference by the cellulose supporting medium and other anions. 5. (5) Intrinsic constants and the number of binding sites at infinite dye concentration have been measured under standardized conditions for Light Green, Brom-Cresol Green and Lissamine Green. The results have been compared and differences discussed. 6. (6) In the quantitative measurement of protein based on dye uptake, the importance of restricting the protein density on the paper is stressed.

1. 1. An established method of chromatography and polychromatic staining of phospholipids on untreated paper has been more fully investigated with respect to sulphatides. 2. 2. A rapid staining method for sulpholipids on paper chromatograms is described. 3. 3. The optical properties of spherical aggregates of the sulphatides under crossed polarizers are noted. 4. 4. Sulphatides have been shown, by chromatography, to occur in many tissues including those of rat, mouse, ox, reptile, certain marine animals, Aplysia, Archidoris, Coryphella, in some microsomal fractions and in lipovitellin. Sulpholipids also occur in fungi.

A convenient and relatively efficient technique was developed for the routine measurement of radioactivity distribution on one-dimensional paper chromatograms containing low amounts of H3- and C14-labeled compounds which are insoluble in the scintillation solvent toluene. A pilot strip, 2 cm wide, was cut out from the chromatogram and divided into 3 4 cm bands along the length of the chromatostrip. Each band was placed in a standard 20-ml screw-top glass vial. Methanol (0.2 ml) was added and the contents were shaken occasionally for 2 or 3 min. Both the methanol and filter paper band were flooded with 10 ml scintillation fluid and the contents were counted. This technique was found to be more satisfactory in terms of efficiency and reproducibility than several other methods tested.

The reaction on paper between reducing sugars and ammonia has been studied. The reaction products were separated by developing the paper chromatogram with an ammonia-containing solvent. In all cases there were obtained three ninhydrin-, as well as p-anisidine-, reactive spots, the relative intensities of which depended on the reaction time, on the concentration of ammonia, and on the presence or absence of catalysts. These spots are provisionally identified as hexose-ammonia, hexose-1-amine, and dihexosamine. The value of paper chromatography in study reactions involving labile intermediates is discussed.

A simple procedure for obtaining good infrared spectra of compounds eluted from paper chromatograms has been described. As little as 25 μg of many phenolic acids may be isolated, characterized, and identified from paper chromatograms.

Summary A paper chromatographic procedure is described for detecting dithioacetals of sugars. A 1% aqueous solution of potassium permanganate was used as the spraying reagent, by which the sugar dithioacetals were detected as the brown spots on paper chromatogram. A series of developing solvents was investigated, and some of them were found useful for separating 2-amino-2-deoxy- d -glucose diethyl dithioacetal from 2-amino-2-deoxy- d -galactose diethyl dithioacetal, and both the diethyl dithioacetals from uronic acid diethyl dithioacetals.

The term 'electrochromatography' is used to denote the specific cases where chromatography and ionophoresis operate simultaneously. The electric field may be applied either in the direction of flow of the solvent or at right angles to it. The filter paper is previously treated with the relevant buffer solution and the developing solvent too is saturated with it. Colloidal graphite painted on the two sides of the paper serves as electrodes. A detailed study has been made of a number of amino acids at different pH's with phenol-buffer as the developing solvent. On ordinary paper acidic amino acids move towards the anode, basic ones towards the cathode, neutral amino acids do not move at all. Using buffer-treated papers, however, the movement depends upon the pH of the buffer and the isoelectric points of the amino acids. By choice of suitable pH and application of electric field in proper direction amino acids having very close RF values (except isomers) can be separated from one another.

In order to effectively control slime production in paper plants and other major water consuming industries, the efficacy of selected chemical compounds as inhibitors of growth of Sphaerotilus natans was investigated. It was determined that a few products were effective against Sp. natans in paper mill process water (80°F). These are listed in the following order of decreasing effectiveness: chlorine, Busan 90, chlorine dioxide and Slimacide V-10. In vitro studies can suggest those chemicals which may be useful; however, the choice of inhibitors depends largely on the individual plant, i.e., the mill processes' capacity to tolerate the concentration, contact time or cost of the specific chemical.

1. 1. A method for the detection of Jaffe-positive substances in human blood serum using the technique of paper partition chromatography is described. 2. 2. A partial solution of the problem of apparent creatinine has been furnished by identification of pyruvate as a second Jaffe-positive compound beside creatinine in a water-soluble fraction of acetone-filtrate of sera; this finding was confirmed polarographically as well as spectrophotometrically . Up till now the quantitative significance of this substance has not been considered in connection with this problem. 3. 3. After a few preliminary experiments, paper chromatography of the insoluble residue of acetone-filtrate of serum has been left open for further investigation. It seems likely that the final solution of the problem of apparent creatinine will cover both possibilities which were discussed in the introduction, irrespectively of whether the chromatographic investigation of the insoluble fraction might show the presence of some new unknown Jaffe-positive component.

The conditions for the quantitative determination of melibiose (or raffinose after invertase hydrolysis) on paper chromatograms by either the use of “Genochrome” or N,N -diethyl- p -phenylenediamine as dip reagents were determined. The maximum density of the spots produced by the reaction of the chromogenic reagent with the sugar was measured by reflected light of 515 mμ wavelength. A linear relationship between the logarithm of the sugar concentration and the reflection density reading was observed with sugar concentrations between 20 and 75μg./5 μl. and, therefore, was the most favorable range in which to make the measurements. When the samples and the sugar standards are measured on the same paper chromatogram, the replicates agree within ±5%; between several chromatograms prepared the same day, replication values will be within ±10%.

A simplified method for the quantitative determination of keto-acids by means of paper chromatography of their 2,4-dinitrophenylhydrazones is reported. 1. 1. The identification of these compounds on the filter paper is discussed, especially of those compounds which give more than one spot. 2. 2. The method is described in detail, espicially with regard to the pyruvic and α-ketoglutaric acid content in biological material. The recovery of these two keto-acids, starting from known aqueous solutions, averages 94%; on the basis of these results a standard curve can be drawn. According to this curve, recovery from deproteinized extracts of liver and blood averages 91.6% for pyruvic acid and 96.7% for α-ketoglutaric acid. 3. 3. Using this method the pyruvic and ketoglutaric acid content of rat blood, muscle, liver and brain is estimated.

A method is described for rapid analysis of phenylalanine and tyrosine using eluates of whole blood that has been impregnated into filter paper. This method employs semi-automated ion-exchange column chromatography (amino acid analyzer) and will yield reasonably accurate and consistent quantitative values for phenylalanine and tyrosine concentrations within two hours. The technique has significant value in rapidly confirming the presence of increased phenylalanine with or without a concomitant increase in tyrosine concentration in autoclaved or unautoclaved blood filter paper specimens. Thus phenylketonuria can be differentiated from hyperphenylalaninemia due to tyrosinemia within hours after the initial identification of an elevated phenylalanine concentration by a screening method such as the Guthrie test. In addition, further evaluations of phenylalanine and tyrosine concentrations can be facilitated in infants on whom only blood filter paper specimens are obtainable.

The new paper chromatographic system, isopropyl ether-ethylene glycol system, provides an improved and faster separation of steroids, particularly estrogens in paper chromatograms. The mobilities of a number of steroids are listed.

Food preservatives—sorbic, benzoic, p-hydroxybenzoic acid and p-hydroxybenzoates—have been extracted from foods directly on to chromatographic paper with ethyl ether. Paper chromatographic separation of the extracted compounds was effected on the same paper. Methyl, ethyl and propyl p-hydroxybenzoates have been separated from each other during ether extraction and from the critical pair, the butyl and benzyl p-hydroxybenzoates, on a paper impregnated with sodium hydroxide. An examination was made of the reactions which distinguish sorbic and benzoic acids when both are present.

A technique has been developed by which complex mixtures of amines can be sepated by column chromatography on the ion exchange resins Amberlite CG-50 and Amberlite CG-120. Identification of amines is made on the basis of the speed of their elution from the resin column, as well as by subsequent paper chromatography of effluent fractions. The method can be applied to amines derived from various physiological fluids, and offers the possibility of isolating unidentified compounds for chemical characterization. Using the combined technique of ion exchange column chromatography followed by paper chromatography, 26 amines were identified in human urine. these were: methylamine, dimethylamine, ethylamine, ethanolamine, β-hydroxypropylamine, pyrrolidine, piperidine, putrescine, cadaverine, 2,2'-dithiobis(ethylamine), histamine, N-acetylhistamine, i -methylhistamine, p -hydroxybenzylamine, 3-methoxy-4-hydroxybenzylamine, p -tyramine, m -tyramine, octopamine, synephrine, 3-methoxytyramine, normetanephrine, metanephrine, tryptamine, serotonin, bufotenin, and kynuramine. An additional 20 unidentified bases, presumably amines, were found to be regularly excreted in urine. Evidence is presented supporting the identification of kynuramine, β-hydroxy-propylamine, putrescine, cadaverine, and 2,2'-dithiobis-(ethylamine). These 5 amines have not previously been considered to be constituents of normal human urine. It is suggested that study of the urinary excretion of amines may provide useful clues as to the mechanisms involved in various metabolic disorders, including those characterized by mental dysfunction.

Three products formed during the paper chromatography of glycine with water-saturated phenol as a solvent have been investigaetd. These unknowns were stable condensation products whose formation was dependent upon the presence of glycine ad formaldehyde. They were ninhydrin sensitive. Alkaline hydrolysis yielded glycine. For chromatographic practices, the amount of glycine or formaldehyde which may be converted into the condensation products will depend upon the amount of the other component in the mixture. Addition of 1% formaldehyde normally complexes all of the glycine. In usual chromatographic experiments part of the glycine may be complexed by residual formaldehyde present in chromatographic phenol. Glycine condensation may be minimized by redistilling the phenol solvent with aluminum turnings and sodium bicarbonate and by washing the filter paper with 1% oxalic acid.

A paper chromatographic system was developed for the resolution of mixtures of sulfated mucopolysaccharides. The effect of varying the ammonium formate buffer/isopropanol ratio in the developing solution on the RF values of heparin and chondroitin sulfate is presented. A complete separation of heparin from chondroitin sulfate was obtained with three solvent mixtures containing different proportions of buffer and isopropanol. One of these solvent mixtures, ammonium formate buffer-isopropanol (65:35) v/v, resolved commercial beef heparin into two principal components, on “fresh” paper. This system was used to compare beef heparin with mouse mast cell tumor heparin, rat heparin, sheep lung heparin, chondroitin sulfate, and β-heparin. A common metachromatic component with an RF of approximately 0.5 was0found in all of the heparin preparations chromatographed with this solvent mixture. “Aging” of the filter paper resulted in the loss of the ability of the buffer-isopropanol mixture (65:35) v/v to resolve the beef heparin. Increase of the buffer-isopropanol ratio to (70:30) v/v restored the resolution of the beef heparin on “aged” paper. Heparitin sulfate and α-heparin monosulfate were compared to beef heparin using the latter system.

A survey of various buffers as agents for the paper ionophoresis of hexose phosphate esters and inorganic phosphate has revealed 0.2 M borate buffer at pH 9.5 as the most suitable for separation of the phosphate compounds into four distinct groups. Variations in molarity and pH of the borate can be utilized to separate the three reducing aldohexoses not resolved by the above conditions. These procedures have been used to identify tentatively a previously unknown compound in potatoes as fructose 1-phosphate. The advantages of paper electrophoresis as well as correlation between movement and relative charge density are discussed.

A wide range of Δ 4 - and Δ 1 , 4 -3-ketosteroids, from weakly polar to strongly polar, may be chromatographed and detected on cellulose triaacetate paper, with benzene—methanol—water mixtures as developing solvents. Three systems have been worked out. In the first a one-phase mixture of benzene, methanol and water is used as the mobile phase and the paper is not accommodated. A “reversed phase” type of chromatogram is obtained, i.e. the more polar compounds have higher R F values than the less polar ones, as is usual for chromatography on acetylated paper. If, howerver, two-phase mixtures of benzene, methanol and water are used, as was the case in the other two methods, “reversed phases”, as well as “convetional” type chomatograms may be obtained. This depeds upon whether the lower or the upper layer is used as the mobile phase, the other layer being used for accommodation of the paper. Perhaps the first-mentioned method should, in contrast to the other two, be regarded as adsorption chromatography, rather than as partition chromatography. Well-reproducible R F values are obtained and, especially with the two-phase mixtures, compact spots are found. Detection methods and R F dta are given for various Δ 4 - and Δ 1 , 4 -3- ketosteroids, ranging from C 18 to C 27 compounds and including sex hormones and adrenoccortical hormones.

The use of activation analysis in conjunction with paper chromatography, and particularly activation after chromatography, is reviewed. Irradiation facilities, the use of markers, internal standards and general activation conditions are discussed, and detailed attention is given to the behaviour of various types of paper on activation, with especial reference to stability and impurities. Practical applications dealing with phosphorus, oxygen, iodine, arsenic and various metals are reviewed.

Publisher Summary This chapter discusses the principles of paper chromatography of antibiotics. It includes all steps of analysis beginning with the preparation of samples and papers, development techniques, detection methods, and means of documentation. Paper chromatography of the best-known groups of antibiotics is also presented in the chapter. General theoretical aspects of paper chromatography are not dealt with except for those that directly emerged from studies of antibiotics. The chapter concludes with a discussion of paper chromatographic classification and systematic analysis of antibiotics.

1. Low voltage paper electrophoresis in 0.05 M citrate buffer of pH 4.8, at a voltage of 200 V, for 312 hours, is a suitable method for determining adenine nucleotides in erythrocytes. 2. Before applying the sample to the paper it is necessary to remove the excess of deproteinisation agent. 3. By means of the method described 42.6 ± 14.5 mg% ATP and 10.7 ± 9.5 mg % ADP were determined in the erythrocytes.

A critical review of the paper chromatography of Strychnos alkaloids has led to the following conclusions: 1. Paper chromatography is one of the most important methods of approach in elucidating the chemistry of Strychnos alkaloids. 2. Paper chromatography and electrophoresis, in the case of Strychnos tertiary alkaloids, are of great value for the rapid analytical identification of the various compounds. In the case of quaternary alkaloids these methods can give general information, but no absolute conclusions, about a great number of alkaloids so far known of this group, and facilitate the identification of some of the more common bases. 3. No absolute relation between RM value and structure can so far be derived from the RC values of Strychnos alkaloids. This is due to the extremely complicated structures of these alkaloids, which belong to several chemical groups, and to the difficulty of obtaining standard RC values for many quaternary alkaloids. 4. Paper electrophoresis of Strychnos tertiary alkaloids is of great importance for the identification of the different strychnine derivatives. 5. Paper chromatography and electrophoresis must be considered as the best methods hitherto used for the preparative purification of small quantities of Strychnos quaternary alkaloids.

The separation of the mixtures (a) magnesium(II), calcium(II), strontium(II), and barium(II); (b) nickel(II), cobalt(II), zinc(II), and copper(II); (c) gallium(III), indium(III) and thallium(III) has been achieved by ascending filter paper chromatography, and the sectors containing the separated cations have been cut into small pieces. The ions from the pieces have been transported to filter paper on the ring oven and the determinations of the constituents have been made by ring colorimetry.

Tritium labelled compounds on paper chromatograms are detected, localized, and the ratios of their activities determined precisely with a windowless scanner. Filter paper strip is pulled, by a wheel driven by a synchronous motor, between two windowless, flow, Geiger-Muller counters. The flow of argon saturated with propanol vapor provides an air-lock. Pulses from the Geiger tubes are fed to a precision, linear ratemeter and recorder with chart speed equal to that of the filter paper strip. The efficiency of counting tritium is low. The sensitivity limit lies aroung 3 m mu c/cm/sup 2/ of Whatman No. 1 filter paper. (auth)

A silicic acid paper chromatographic method for the separation of four major classes of cholesterol esters, namely saturated cholesterol esters, cholesterol oleate, cholesterol linoleate and cholesterol linolenate plus cholesterol arachidonate is described. This method uses a benzene-hexane solvent. The use of combined column and paper chromatography as well as gas chromatographic analysis is presented for definitive identification of the various spots. The predominance of cholesterol linoleate in human plasma, and cholesterol arachidonate in rat plasma is confirmed.

1.1. Filter paper electrophoretic analysis of the serum and urine proteins in thirty-five cases of multiple myeloma has been carried out. In all thirty-five cases characteristic protein abnormalities were observed; seventeen cases showing homogeneous peaks in both the serum and urine, eleven cases in serum only, seven cases in urine only. 2.2. All urine samples showing a positive Bence-Jones protein reaction also displayed a characteristic electrophoretic abnormality. Eight of the twenty-four electrophoretically positive urine samples were Bence-Jones protein negative by the usual tests. Because of the relative simplicity of urine electrophoresis by the filter paper technic, this test is considered to be a practical clinical procedure. 3.3. Two cases are presented in detail because certain atypical clinicopathologic features of their disease patterns possibly assist in an understanding of the relationship of the plasma cell dyscrasias to chronic lymphatic leukemia and the lymphosarcomas. A third case, which demonstrates the value of serial electrophoretic studies in myeloma, is also presented. 4.4. Consideration is given to some of the physicochemical properties of the myeloma serum and urine proteins, and to the possible nature of their interrelationship.

1. 1. Twenty-four substances were separated from a sample of cured Robinson's Medium Broadleaf Maryland tobacco by solvent extraction and paper chromatography. Even minute quantities of these substances give color with Amine-CNBr reagents. The evidence indicates that they are all pyridine alkaloids or derivatives thereof. 2. 2. By use of R f values, color tests with various indicator reagents, and ultraviolet absorption spectra, nicotine, nornicotine, anabasine, oxynicotine, myosmine, 3-acetylpyridine, 2,3′-dipyridyl, and nicotinic acid have been identified. The possible presence of nicotinamide and anatabine is also indicated. 3. 3. The quantity of each of the identified substances in each solvent extracted fraction and in the original tobacco is estimated from the ultraviolet absorption spectrum of the material extracted from the paper chromatogram. 4. 4. Two rapid chromatographic methods are suggested to be applied to large numbers of tobacco samples in order to obtain qualitative information as to their principal alkaloids.

Results comparable to conventional colorimetric and spectrophotometric methods have been obtained by paper chromatographic and electrophoretic methods. In several instances, where stability studies are involved or when the combination of drugs do not lend themselves to assay by the usual methods, it is concluded that the chromatographic and/or electrophoretic procedures are a practical applicable solution. Some of the modifications that are needed for applying these chromatographic and electrophoretic methods to pharmaceutical assays are discussed. In general, separations of the various compounds are accomplished by ascending or descending paper partition chromatography, by selection of appropriate solvent systems, or by paper electrophoresis with selected pH buffer systems. The developed papergrams are sprayed with an appropriate color reagent and assayed quantitatively by photoelectric densitometry. The compounds treated in this manner are: phenylephrine hydrochloride, chlorpheniramine maleate, dextromethorphan hydrobromide, N-acetyl-p-aminophenol, meperidine hydrochloride, atropine, ethinyl estradiol, procaine and tetracaine hydrochlorides.

Assumptions concerning the similarity of paper chromatography and paper electrophoresis as regards the nature of the interaction of the substances analysed with the immobile phase were verified. glueckauf's method for constructing distribution isotherms from chromatographic measurements was applied to electrophoretic measurements. It was proved that the isotherms obtained in this way are identical with the isotherms obtained by the direct method, when the dependence of the mobility in free solution on the concentration can be neglected. The values of RF, the electrophoretic mobility in free solution Uo and the velocity uo obtained both by direct measurement and by calculation were proved to be identical.

Paper-electrophoretic separation of intrinsic factor-related “primary” and “secondary” B12 binders in pooled normal human gastric juice was achieved by using Teorell-Stenhagen buffers of pH 4.0–5.0, at which pH the “primary” binder moves to the cathode, while the “secondary” one travels to the anode. The isoelectric point of the “primary” binder is about pH 5.5, while that of the “secondary” one is pH 2.5–3.0. The electrophoretic mobility of none of the major carbohydrate-, proteinor peptide-nondialyzable components of gastric juice correlates with that of the “primary” or “secondary” B12 binders when run on electrophoresis in the pH range of from 2.5 to 10.0. This supports previous conclusions that none of the major components of gastric juice resolved by paper electrophoresis represents the intrinsic factor-related B12 binders.

The possible degradation of tryptophan and seven of its metabolites during and after paper chromatography with n-butanol-acetic acid-water has been investigated. It has been found that extensive degradation results from developing and drying the chromatograms at temperatures over 27° and storing them in room light for extended periods before the second development. Lesser amounts of degradation were found after limited exposure of compounds to ultraviolet light between developments and occasionally with the use of unwashed paper. Procedures to eliminate degradation have been described.

The results obtained in an investigation of rhodium(III) oxalates indicate that heating of pure rhodium(III) hydroxide with oxalic acid solution produces RhOx 3 −3 to a great extent, but that the reaction does not go to completion, other aquo-coordinated oxalato complexes of rhodium being formed in smaller quantities. Evidence is presented that in order to obtain complete conversion of rhodium(III) into potassium rhodiotrioxalate the chloride must be heated under reflux with four to five times its weight of potassium oxalate for at least 6 hours. The absorption spectrum, the stability to heating and aging at room temperature in solution, as well as the electrophoretic, paper- and ion-exchange chromatographic properties of the rhodiotrioxalate ion were studied. It was shown that the dextro - and laevo -forms of RhOx 3 −3 have identical electrophoretic mobilities.

A study has been made of some of the fundamental factors involved in obtaining good separations and reproducible chromatograms, with the technique of contrifugally accelerated paper chromatography. A study was also made of variables which might influence R F values. Mixtures of bromphenol blue, methyl orange and methyl red were fractionated as well as solutions of lencine, methionine and glycine. For the separation of the dyes, Whatman No. 1 filter paper was used with a veronal buffer solution of ionic strength 0.05 and having a pH of 8.7. For this system, it was found that the R F values of the dyes were essentially unaffected by changes in rotational speed of the rotor from 300 to 925 r.p.m. It was found that the rate of addition of solvent was important in developing satisfactory chromatograms. A flow rate of 1.15 ml/min was found to be satisfactory at all rotational speeds above 250 r.p.m. The shape of the developed chromatogram was elliptical rather than circular, for all four types of paper which were studied. For Whatman No.1, the major axis of the ellipse was parallel to the machine direction of the paper. The R F values for the dye components were essentially the same for the following papers: Whatman No. 1, Eaton-Dikeman No. 613, and Cremer-Tiselius-Munktells. The amino-acid mixture was fractionated using Whatman-3MM paper and a solvent system of butanol-glacial acetic acid and water, in the volume ratio 40:10:10, respectively. The disk was rotated at 975 r.p.m. With a solvent flow rate of 1.2 to 2.1 ml/min, sharply defined zones were obtained. The time of development was 15 min for a flow rate of 1.2 ml/min and 7 min for a flow rate of 2.1 ml/min. The R F values obtained were 0.85 for lencine, 0.56 for methionine and 0.20 for glycine.

On account of differences in lipophilic character it is possible to separate p -nitrophenyl methylphosphonates and their sulfur analogs by reversed phase paper chromatography. Whatman No. 1 filter paper was impregnated with silicone oil 550; the upper layer of a chloroform-ethanol-water (5:5:3, v/v) system served as mobile phase. The relation R M = log (1/ R F —1) holds for homologous series of O-alkyl p -nitrophenyl methylphosphonates (I) and S-alkyl p -nitrophenyl methylphosphonothiolates (II) up to the n -pentyl homologues. Using a combination of Gibbs' and Dragendorff reagents it was possible to distinguish the phosphonates, phosphonothiolates and phosphonothionates.

Filter paper impregnated with salts has been used for paper chromatography of inorganic cations. The RF values of a number of cations on such paper differ considerably from those on untreated paper and in some cases better separations are possible. The RF value of cations varies with the cation of the salt used for impregnation in the same manner as has been observed for “salting out” in solvent-solvent extraction.

After conversion to 2,4-dinitrophenylhydrazones, keto acids in biological fluids were extracted into isopropyl ether followed by NaHCO 3 and finally into ethyl acetate. The derivatives were chromatographed two-dimensionally on Whatman No. SG 81 paper. Limitations of the extraction and chromatographic procedures are discussed but it is considered that the method is superior to orthodox one-dimensional paper chromatography.