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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.

PROF. K. P. LINSTEAD kindly sent me a manuscript copy of the communication by Burstall, Davies, Linstead and Wells1, and I should like to comment on this work in relation to my own.

The principal aim of the present report is to present a series of data which demonstrate the applicability of simple chromatographic methods to the problems of investigating biochemical changes which occur during development of Drosophila melanogaster and studying biochemical differences between mutants of this organism. Extensive analyses of the data and their significance in relation to the work of other investigators will be reserved, for the most part, for subsequent and more detailed presentations.

THE formation of more than one zone containing phosphate when the sodium orthophosphates are chromatographed on paper suggests that these spots contain different molecular species originating in proton transfer equilibria of the kind: where B−n is a base1,2. The speed of proton transfers, for example, the half-life for the reaction of hydronium and hydroxyl ions is 10−8 sec3, argues against this as such equilibria yield multiple spots only if the transition time is comparable with the time required for the formation of the chromatogram4. It is natural to question next if the zones are actually of different species since discontinuities in either the immobile or mobile phases can also produce the result4. Chromatograms of orthophosphoric acid and of the primary, secondary, and tertiary sodium salts were formed on 55 cm × 2.8 cm strips of Whatman No. 1 filter paper, acid washed, which had also been washed in 2 M acetic acid followed by three successive rinsings in distilled water5. The chromatograms were formed either with pyridine/ethyl acetate/water (the faster solvent) or butanol/water. Fig. 1 is a photograph of 10 µl. of 0.01 M trisodium phosphate (No. 321) and 10 µl. of 0.01 M sodium dihydrogen phosphate (No. 322) formed simultaneously with BuOH/15 per cent H2O. The origin was the vertical pencil line adjacent to the numbers. When revealed, the colour process was that employing ammonium molybdate and benzidine hydrochloride6. In certain select cases, the multiple spots were irradiated in the University's Triga reactor7,8. Both sodium and phosphorus undergo (n,γ)·reactions to form radioactive isotopes which decay as follows9,10

WITHIN the last twenty years, chromatography has become increasingly important as an analytical tool. The purpose of this article is to review the uses of partition chromatography on paper, which, since its inception four years ago1, has had a wide application and which promises to become as well established as the older forms of chromatography. It should be emphasized, however, that, as mixtures, especially from biological sources, may be very complex, preliminary separations2-5 may often be necessary before paper chromatography can be used to the best possible advantage. For recent developments in all types of chromatography, the reader is referred to a number of articles indicated in footnotes 6–9.

IN the procedure for the quantitative analysis of sugars, described by Flood, Hirst and Jones1, the paper strips containing the sugars are cut out and extracted by allowing condensed water to run down the paper. It has been found, however, that destruction of certain sugars occurs on boiling a solution under these conditions. To obviate this difficulty, we have devised a technique of extraction at room temperature similar to that used by Dent2 for amino-acids.

High-Voltage Paper Electrophoresis of Non-Volatile Organic Acids and their Mixtures with Amino-Acids

THE interesting suggestion that the oligosaccharides formed from sucrose may be the precursors of polysaccharides such as inulin and levan requires verification by the establishment of the identity and type of linkage of each of these compounds1. The recent discovery of their presence in plant extracts2 and human urine3 is a certain indication of their significance in metabolic processes.

High-Voltage Paper Electrophoresis of Inorganic Cations: Alkali, Alkaline-Earth and Other Metals

WE have shown1 that primycin, an agent of strong antituberculotic effect belonging to the group of guanidino antibiotics, is not identical with any of the known antibiotics. On the basis of its colour reactions and of its infra-red and ultra-violet spectra2, the molecule of primycin (C19H37O7N)n contains an N—H group and several O—H groups; it is inclined to molecular associations and it discloses adsorptive properties. On subjecting primycin to paper chromatography, the formation of a second spot was observed. This was due to the so-called ‘secondary adsorption’ of an accompanying substance present in minute amounts.

THE filter paper chromatographic procedure developed by Consden, Gordon and Martin1 has proved to be very useful in the qualitative and semi-quantitative analysis of amino-acid mixtures, and appears to have possibilities for extension to a variety of other compounds2, 3.

THE author in his preface has rightly stated that the chemist interested in paper-making finds ample technical literature at his disposal, whereas the engineer is not provided with any books dealing with the peculiar mechanical problems of his industry. Since the publication of H of man's treatise on “The Manufacture of Paper” in 1873 no serious attempt hasbeen made to supply the paper-maker with an intelligent and comprehensive text-book devoted to a study of the economic and efficient control of the machinery peculiar to the manufacture of paper. Paper-making and its Machinery: Including Chapters on the Tub-sizing of Paper, the Coating and Finishing of Art Paper, and the Coating of Photographic Paper. By T. W. Chalmers. (The Engineer Series.) Pp. xi + 178 + vi plates. (London: Constable and Co., Ltd., 1920.) Price 26s. net.

IN order to obtain a good separation in filter paper chromatography, the size of the spot of liquid applied to the paper must be small, which often necessitates repeated, time-consuming applications of material. Methods have been evolved with the purpose of having spots or a. band laid down automatically by a burette1,2. There still seems to be a need, however, for a technique by which a larger volume of fluid can be deposited quickly and simply, yet permitting small-sized bands or spots for good separation.

BY the use of rectangular strips of filter paper, rather than filter paper disks, in combination with a horizontal solvent flow at an elevated temperature, amino-acids are rapidly separated as round compact spots rather than circular bands1. This is desirable since such spots in many cases lend themselves to quantitative study by the maximum colour-density technique. Recently, such a procedure has been applied to the analysis of tryptophan in an alkaline hydrolysate of α-casein2.

The use of filter paper in selective adsorption experiments, probably first described by Schonbein (1), has become a powerful tool in research, especially since the studies of Consden, Gordon, and Martin (2). Although in most modifications of paper chromatography only very small amounts of material can be resolved, Mitchell and his colleagues must be credited with preparative applications; recently these authors carried out successful enzyme separations in their chromatopile (3, 4).

IN a previous communication1 we reported that, using a butanol–ethanol–water–ammonia solvent, paper chromatography of diabetic urines and, in certain circumstances, of glucose samples, gave the normal glucose spot together with two extra spots of lower mobility. Subsequent studies of the inter-convertibility of the spots at various pH values have revealed that three spots, possessing the same RF values and chemical properties as those previously encountered, can be obtained when aqueous solutions containing glucose and ammonium acetate are chromatographed; the substances responsible for the two extra spots are formed when, following common practice, the solution under test is being dried on the paper in a current of warm air. The replacement of ammonium acetate by ammonium salts of other carboxylic acids and by ammonium carbonate led to similar results; in the case of the latter salt it was possible to observe that the compound responsible for the ‘middle-spot’ was formed prior to that responsible for the ‘back-spot’. From a larger-scale experiment in which glucose was heated with aqueous ammonium acetate, a product was isolated which was shown chromatographically to give mainly the ‘back-spot’. This product had a very small optical rotation, and gave the known crystalline α-diglucosylamine octa-acetate2 on acetylation; chromatography of the saponified acetate yielded one spot which coincided exactly with the ‘back-spot’. It seemed, therefore, most likely that this product was the equilibrium mixture of the α- and β-diglucosylamines prepared by Brigl and Keppler2, a view which was endorsed when these two diglucosylamines were used as reference compounds on chromatograms. We have shown further that glucosylamine, prepared by the method of Ling and Nanji3, is identical chromatographically with the ‘middle-spot’.

SEVERAL spray reagents are available for the specific detection of ketoses and ketose-containing sugars on paper chromatograms. Probably the most widely used are resorcinol and naphthoresorcinol, originally prepared as sprays in 2 N hydrochloric acid by Forsyth1. Afterwards, Partridge2 replaced the hydrochloric acid with trichloracetic acid in order to minimize attack on the filter paper. Heyrovsky3 has recently reported that β-indolyl acetic acid in trichloracetic acid is also a very sensitive ketose-specific reagent. In work in this laboratory on two fructose-containing disaccharides it has been found that all three of the above reagents, when prepared in trichloracetic acid, failed completely to detect these sugars on paper chromatograms. When the trichloracetic acid was replaced with 2 N hydrochloric acid strong positive tests were obtained. The results are listed in Table 1.

IN recent studies we were concerned with the detection of traces of nor-adrenaline in preparations of adrenaline derived from natural sources. Colorimetric methods1,2 proved to be of limited value with low concentrations of nor-adrenaline; but excellent results were obtained with paper chromatography, using modifications of the methods described by James and others3,4. The adrenaline samples are dissolved in methanol containing a minimum quantity of hydrochloric acid, and then transferred to paper strips for descending chromatography, or to circular disks of filter paper for chromatography by Rutter's technique5. Water-saturated phenol is used as the developing solvent in an atmosphere of hydrochloric acid4. The paper chromatograms are then dried and sprayed with an appropriate colour reagent.

WE wish to describe briefly a convenient paper chromatographic separation and identification of pyridine derivatives in use in this laboratory for some time which employs a modification of the wellknown Konig colour-reaction1. Leifer et al.2. have recently described the paper chromatography of nicotinic acid and nicotinamide, but the more involved microbiological method was used for defining the spots. In this laboratory the solvent used for the development of the compounds listed in the accompanying table is n-butyl alcohol saturated with 1.5 N ammonia. After evaporation of the solvent at room temperature, the dry paper is placed for fifteen minutes in a closed glass cylinder containing a few crystals of cyanogen bromide. The vapour pressure of this substance is high enough to provide a sufficient concentration for the first phase of the Konig reaction. The paper is then sprayed with a 0.25 per cent solution of benzidine in 1 : 1 ethanolwater. Amounts down to 5γ can be detected easily. It should be pointed out that although nicotinic acid and iso-nicotinic acid cannot be distinguished on the basis of RF values, they can be distinguished by distinctive differences in colour. This technique should be generally applicable to all non-volatile pyridine derivatives giving a positive Konig colour-reaction.

FILTER paper is frequently used as a means of supporting radioactive substances, such as proteins or the effluent from a column, for counting in liquid scintillation counters. While this is satisfactory for most isotopes in common use, the efficiency of counting tritium is undesirably low, being a fraction of the efficiency obtainable with tritiated toluene. Glass fibre paper, however, has been found to allow a much higher counting efficiency, approaching the theoretical maximum.

P work from this laboratoryl-S has demonstrated the heterogeneity of crystalline trypsin by paper and starch column electrophoresis. This article presents an electrophoretic method of greater resolution. Using this technique it was possible to demonstrate in a sample of crystalline trypsin the occurrence of fractions showing different ratios of proteolytic to amidasic activity. In the experiments recorded here a buffer of pyridine acetate of pH 4·9 (0,48 : 0·56 : 98·96 v/v of acetic acid: pyridine: water) was used for electrophoresis in a Durrumtype apparatus. To a strip of Whatman 3 filter paper of 12 x 50 cm, 0·12 ml. of an 8 per cent trypsin solution in 0·001 N hydrochloric acid were applied. An electrical tension of 450 V was used and the experiment was allowed to run for 5 h in the cold room. In these conditions at the end of the experiment the temperature of the buffer was 9° C. The strip, while still wet, was cut in 1-cm wide transversal segments and transferred individually to test-tubes containing 3ml. of a 0·25 M pyridine formate buffer of pH 2·7. This extraction was allowed to continue for about 20 h in the cold room. The extract of each tube was then analysed for protein content, proteolytic activity against azo casein and amidasic activity towards cebenzoyl-L-arginine amide (BAA). The protein content was determined by the FolinLowry4 method as applied to the 'auto analyseI" system·. Proteolytic activity against azo casein was measured by the method of Charney and Tomarelli". The hydrolysis of BAA was followed colorimetrically in the auto analyser'. Chromatography of trypsin in carboxy methyl cellulose was carried out according to the method of Liener8• The trypsin samples used in this work were purchased from the 'Worthington Biochemical Corporation ('Cryst. Trypsin, lyophilized, lot TL 747-58'), Freehold, New Jersey, and from Novo Industri ('Cryst. Trypsin Novo, batch 114-3'), Copenhagen, Denmark. The results obtained by the fractionation of the Worthn~on trypsin are shown in Fig. 1. The presence of three

BENZIDINE has been found to be a useful reagent for sugar detection on paper chromatograms1,3 and for quantitative determination in test-tubes4. Horrocks3 first found that benzidine–acetic acid detected reducing aldoses on paper chromatograms, but ketoses were detected only at high concentration. Bacon and Edelman1 added trichloracetic acid to the benzidine–acetic acid reagent to increase its acid strength and claimed that it detected less than one microgram of glucose in free or combined form. Its sensitivity to fructose was considerably less. An acid is required for the formation of furfuraldehyde from sugars. Furfuraldehyde, or a degradation product of it, then reacts with an aromatic amine or phenol (benzidine in this case) to form a coloured product. It is important that the acid should he strong enough to cause the reaction to proceed, but, in the case of paper chromatography, it should not cause partial destruction of the paper or produce a dark background colour. For this purpose, organic acids (acetic, phthalic, or trichloracetic acid) have been used. However, when mineral acids at low concentrations are used, an improved reagent is obtained without paper destruction or serious background interference.

VARIOUS methods have been described for the chromatographic separation of pyrethrin-type compounds. Alumina, silica and kieselguhr have been used as adsorbents for separation of ‘pyrethrin I’ from ‘pyrethrin II’1–3. A separation of cinerin I from ‘pyrethrin I’, and ‘cinerin II’ from ‘pyrethrin II’, has almost certainly been achieved by Ward2, using displacement chromatography on alumina. The ‘pyrethrins’ have also been separated by paper chromatography using alumina-impregnated paper1 and ‘Vaseline’-impregnated paper4. Hitherto, untreated paper has only been used with success for the separation of the ‘pyrethrin’ 2,4-dinitrophenylhydrazones, using light petroleum (b.p. 80°–100° C.) as solvent1. The present communication records a simple chromatographic system for separating pyrethrin-type compounds on untreated paper.

THE application of the principle of electrophoretic separation on paper to steroids is limited by their relatively low polarity, so that they do not readily migrate under the influence of an electric current, and also by their sparing solubility in aqueous media. By converting the steroids into derivatives with polar groupings, some separation has been achieved (Voigt and Beckmann1, 1953, using the hemisuccinates; McKinley2, 1955, using the mono- and di-phenylhydrazone derivatives). Our early experiments, begun before the publication of these authors' results, were along similar lines, and with somewhat similar results. It did not seem likely, however, that electro-phoretic separation would be so convenient a method as chromatography, which has been adapted by Lembart and Schneider3 for the separation on paper of mixtures of pure steroid conjugates. The natural occurrence of more polar steroid derivatives, the sulphates and glucuronides in urine, suggested to us that these might undergo electrophoretic separation on paper. Although the individual steroids could not be resolved, the two groups of conjugates were sharply separated.

IN a recent communication by Proom and Woiwod commenting on circular paper chromatography, they state: "although the method is admirable for the separation of two or three substances, it is not a satisfactory substitute for large–scale paper chromatography in the separation of more complex mixtures such as protein hydrolysates".

SINCE the work of Lederer1 and Arden et al.2, the separation of inorganic cations by paper chromatography has been developed rapidly. The alkali salts, however, do not appear to have been studied, because of the difficulties of detection of alkali cations on the filter paper. We have recently discovered a simple electrolytic method of detecting the alkali and alkaline earth cations, and have studied the paper chromatography of the alkali salts.

IN a recent publication1 from this laboratory, a technique for the study of the chromatographic behaviour of enzymes on paper was described. The ease with which enzymes can be readily identified on the paper chromatogram by the agar-plate method enabled us to study the chromatographic behaviour of some of the important enzymes. In the course of preliminary experiments with amylases, phos-phorylases and phosphatases from various sources, we found that some enzymes did not move at all while others moved considerable distances on the paper. This observation prompted us to attempt the separation of enzymes by paper chromatography.

THE fluorescence of amino-acids and peptides on dry filter paper1 is of great value for locating these substances after paper chromatography, and has been found useful in quantitative work2 since it avoids destructive treatment with reagents such as ninhydrin. It also enables substances to be eluted for further investigations from sheets of chromatograms where gross irregularity of solvent flow renders the use of marker strips3 inaccurate as guides for cutting. Unfortunately, marked variations in fluorescence are often observed when identical chromatograms are run on sheets of No. 4 Whatman paper from different packets of papers or even on adjacent sheets from the same packet4, and batches of papers are occasionally encountered on which no fluorescence at all can be observed at normal nitrogen-levels. As Phillips1, quoting De Ment5, considered fluorescence of amino-acids on paper to be due to excitation of the acids themselves by ultra-violet light, it seemed possible that some impurity present in ‘bad’ fluorescent papers might therefore be quenching fluorescence. Sheets from a particularly bad batch of No. 4 Whatman paper could not be improved, however, by washing with water, dilute sodium hydroxide, hydrochloric acid or with solutions of 8-hydroxyquinoline or dithizone.

THE applications and limitations of one- and two-dimensional paper chromatographic techniques have been well documented1. There remain, however, problems in the separation of certain compounds which cannot bo resolved satisfactorily by either of these conventional approaches. Weiss and Rossi2 recently described a chromatographic method which enables separation of several catecholamines on a single paper strip.

THE surface of filter paper, in contact with water, has a slight negative charge1, and this may account for the non-adsorption of certain colloidal electrolytes such as dyes of the ‘acid’ type, when developed with water, as these also carry a net negative charge in aqueous solution. By the application of principles similar to those described by Weiss2, a ‘primary adsorbate’ may be applied to paper, in this case being so arranged that the paper now has a net positive charge in contact with water. A suitable compound for this purpose is cetyl trimethyl ammonium bromide, the paper being soaked in 0.03 per cent aqueous solution and dried before use.

ELECTROPHORESIS has been of value in determining the physico-chemical constitution of cell-free extracts of micro-organisms1,2. These studies have been confined mainly to the bacteria and no such investigation of trypanosomal extracts has been made. Moving-boundary electrophoresis has been the method most frequently employed to analyse microbial extracts although the simplicity of paper electrophoresis would be of obvious advantage. The purpose of this communication is to describe the technique for paper electrophoresis and the resultant electro-phoretic patterns of trypanosomal extracts.

AGAR diffusion1 has been used for detecting antigens after paper electrophoresis. I have found that a precipitin reaction can be carried out directly on the paper.

THE detection and identification of pterins in small quantities was carried out by Schopf and Becker1 in the course of their classical work on this class of pigments. They employed a chromatographic technique using 0·004 N aqueous hydrochloric acid or 0·01 N methyl alcoholic hydrochloric acid solutions on micro-absorption columns of alumina or frankonite. The technique of paper chromatography has now been applied successfully to certain of the common pterins, and as a result a method has been elaborated for the rapid identification of very small quantities of these compounds such as may be obtained by extraction of individual butterfly wings. Indications that this method might be applied in the pterin series had been given by Crammer2, who mentioned that xanthopterin and leucopterin could be concentrated by a paper chromatographic technique.

ELECTROPHORESIS and ionophoresis on paper are being widely used, and a number of different forms of apparatus for carrying out this technique have already been described. The purpose of this communication is to give further details of an apparatus already briefly described1, which has been used for a considerable time with satisfactory results. While we do not claim that it is superior to other types of apparatus, it has the advantage of simplicity, results are reproducible, and the movement of a given substance varies linearly with time. The cross-section is shown in the accompanying diagram. The apparatus consists of a flat rectangular glass tray in which stands a closely fitting glass frame which carries the paper strip and rectangular ‘Perspex’ electrolyte boxes. The dimensions of the apparatus are not critical, but glass drip-trays from refrigerators are very convenient, the outside measurements being about 35 cm. × 20 cm. × 5 cm. The frame is constructed of two rectangles of glass rod, mounted one over the other, and separated from each other by about 1.5 cm. by means of sealed-on pieces of glass, or by pieces of cork at the corners. The frame stands on short legs to keep the paper away from the floor of the tray. Each electrode box has a hole drilled through the end about 1 cm. above the bottom. This enables electrolyte-levels in both boxes to be balanced prior to electrophoresis, and also permits old electrolyte to be run out and replaced by fresh, should this be desired.

MANNOSIDOSTREPTOMYCIN in some streptomycin preparations is conveniently detected by the paper chromatographic procedure of Winston and Eigen1. In the presence of high concentrations of salts such as in ion-exchange column eluates, or in fermentation broths, this procedure is not applicable: attempts to desalt such solutions, by mixed-bed ion-exchange, electrolytic de-ionization or solvent extraction, have proved unsuccessful. We have found it possible to separate streptomycin, mannosidostreptomycin and allied substances by paper electrophoresis, having developed (independently) a technique similar to that used for new antibiotic broths by Hosoya et al. 2, and King and Doery3.

AN automatic scanning technique employing a Geiger–Muller tube for the location and estimation of radioactive substances separated on unidimensional paper chromatograms has been described1. The application of this technique to two-dimensional chromatograms is tedious because a large sheet of paper has first to be cut up into the equivalent number of unidimensional strips and each strip scanned separately. To obviate this difficulty, an alternative technique to the conventional form of two-dimensional paper chromatography has been successfully developed for the application of two or more solvent systems to the same labelled mixture.

THE identification of non-volatile organic acids on paper chromatograms is usually dependent on the use of a suitable pH indicator which is applied to the developed and dried paper by spraying1. This procedure is inconvenient and an even distribution of indicator on the paper is difficult to obtain.

IN the amino-acid analysis of biological fluids using paper chromatograms1, the ninhydrin reaction indicates by means of a purple coloration not only α-amino-acids but also many other types of amino-acids and amines2. Spots that do not correspond in their positions on the paper to known substances are often found in these fluids. The circumstances sometimes preclude the possibility of isolating and analysing such substances, so that we may be limited to provisional identifications based entirely on paper chromatographic procedures.

CATIONIC detergents, such as cetyltrimethylammonium bromide (‘Cetavlon’) and cetylpyridinium chloride, have been used extensively in recent years for the fractionation of polysaccharide mixtures1–4. The quaternary ammonium cations present in these detergents form salts with acidic polysaccharides which often have solubility properties quite different from those of the usual metallic salts; for example1, cetyltrimethylammonium chondroitin sulphate is soluble in aliphatic alcohols up to n-pentanol and is insoluble in water. Many purification procedures have utilized the water-insolubility of these quaternary ammonium polysaccharide salts, but surprisingly, no serious attempt seems to have been made to exploit their solubility in organic solvents. This communication describes some preliminary experiments directed towards developing convenient methods for the separation of acidic carbohydrates, making use of the solubility of their cetylpyridinium salts in organic solvents. Current interest in these laboratories in the purification and identification of sugar sulphates, suggested that it would be useful if this property could be utilized in a method for the paper chromatography of these substances. It has been found that when a small amount of detergent is incorporated into the mobile (organic) phase of certain solvent systems for paper chromatography, the R F values for sulphated derivatives are increased by a factor of about ten, and some useful separations can be achieved. Two such systems are

IN recent studies on the paper electrophoresis of carbohydrate compounds, we have found that replacing the cellulose filter paper by paper made from glass fibres1 greatly facilitates the identification of sugars and their derivatives, especially those which are difficult or impossible to detect on cellulose paper.