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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

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.

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.

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 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 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.

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.

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.

A COLOUR reaction, known as Scherer's test, is extensively used as a specific, qualitative test for inositol. It is based on the formation of the coloured alkaline earth metal salt (usually calcium salt) of rhodizonic acid on the oxidation of inositol with concentrated nitric acid1,2. While studying phos-pholipids containing inositol we needed to identify accurately very small amounts of inositol by paper chromatography, or by spot tests on filter paper. P. Fleury et al. 3 reported a method in which they used mercuric oxide as a catalyst of the oxidation, and barium acetate as for the alkaline earth metal. This method, however, is tedious and sometimes gives uncertain results; the smallest quantity detectable is 5–10 µgm. This may be ascribed to the formation of yellow mercuric salt at the first stage of the reaction. A more sensitive, reliable and easier method was, therefore, devised by modifying that of Fleury. This modified method can, in addition, be applied to the detection of inositol diphosphate located on paper chromatograms. The procedure is as follows.

TWO of the sulphonphthalein dyes, bromophenol blue1 and bromocresol green2, have been used widely for the detection of protein after electrophoresis on filter paper. The uptake of bromocresol green by protein under these conditions has been studied recently by Franglen and Martin3. Extending this work, the purity of other sulphonphthalein dyes has been investigated with paper chromatography and filter paper electrophoresis; the methods used and the results obtained are described below.

ATTEMPTS at the separation of higher fatty acids by paper chromatography have not, in the main, been successful, although Isherwood and Hanes1 have studied the problem in relation to a number of straight-chain fatty acids, and Nunez and Spiten2 have evolved a technique, using paper impregnated with olive oil, for chromatographic separation of such compounds. Apart from other considerations, the low aqueous solubility and the low dissociation constants of these acids presented the problem of finding an indicator which would be sensitive enough to enable their presence to be detected on paper. It has been found that the methyl red/bromthymol blue indicator of Duncan and Porteous3 permits of the detection of small amounts (for example, about 80 µgm. of C16) of acids up to C18 on paper. Considerably more care is required, however, in the colour development with the higher acids than with the lower members of the series.

IN work concerned with the composition and estimation of neurohypophysial hormones in the posterior pituitary, the hypothalamus and body fluids, it would be desirable to have a chromatographic procedure which permits the identification in and recovery of small amounts of the active peptides from impure extracts. It would also be useful to know whether the substituted synthetic oxytocins which have recently been prepared1 can be distinguished by a simple chromatographic method. Taylor2 separated oxytocin, and arginine- and lysine- vasopressin by ion-exchange chromatography on resin columns. He used du Vigneaud's highly purified peptides, and the smallest amounts applied were equivalent to 0.6 mgm. of this material. Benfey3 purified Stehle and Fraser's4 preparations of ox posterior pituitary hormones (200 U./mgm. pressor activity and 250 U./mgm. oxytocic activity) by paper chromatography using ninhydrin as the colour reagent. The smallest amounts of the hormone preparations used were 1 mgm. However, since only terminal amino-groups react with ninhydrin, it is obviously disadvantageous to use this reagent when attempting to visualize very small quantities of the posterior pituitary peptides. Rydon and Smith5 have shown that micrograms of peptides of all kinds, including cyclopeptides, can be detected by chlorination followed by spraying with a starch–potassium iodide solution. Reindel and Hoppe6 improved this procedure in two ways. First, before exposure to chlorine, they moistened the paper with a mixture of watery ethanol and acetone, which prevents background staining. Secondly, they treated the N-chloropeptides with a mixture of potassium iodide and either benzidine or o-tolidine in acetic acid and thereby increased the sensitivity of the method.

PHENOL and collidine have remained the most widely used pair of solvents for two-dimensional paper chromatography of amino-acids since their introduction by Consden, Gordon and Martin1. The use of the latter solvent, however, is attended by certain disadvantages such as double spots and haloes2, an offensive smell and possible toxic effects3,4 and, in some countries, difficulty of supply. Alternative solvents have been suggested, for example, butanol – acetic acid5, acetone – water2 and pyridine – amyl alcohol6. Some defects of the latter system have recently been reported7.

THE isolation of dihydrosphingosine from cerebrosides of beef brain and spinal cord by Carter et al. 1 raised questions regarding its distribution in different sphingolipids and its metabolic relationships to sphingosine. However, no method for the routine differentiation of sphingosine and dihydrosphingosine appeared until very recently with the gas chromatographic method of Sweeley and Moscatelli2. Wittenberg3 separated the N-succinyl derivatives of sphingosine and related compounds by reversed phase chromatography on diatomaceous earth treated with dichlorodimethylsilane, but he was unable to separate sphingosine and dihydrosphingosine. This communication describes a reversed-phase paper chromatographic method for the separation of the dinitrophenyl derivatives of sphingosine and dihydrosphingosine.

THE separation of amino-acids by partition chromatography on paper was first described by Consden, Gordon and Martin1. A large number of separations of organic compounds has been described since then. I have recently discovered that cations can be separated successfully if the solvent is adjusted suitably to prevent hydrolysis. The solvent selected for study Was butanol saturated with N aqueous hydrochloric acid.

AFTER capsulated B. anthracis had grown in a modified synthetic medium of Brewer et al.1, a mixture of growth products was isolated from the glass-filtered medium which gave a precipitate with copper sulphate. Crude polyglutamyl peptide was obtained from this precipitate2. An attempt was made to separate the components of the original mixture of growth products by paper electrophoresis using a tank as described by Flynn and Mayo3, and it was found that the peptide could not be detected using naphthalene black, bromo-phenol blue, ninhydrin or ultra-violet light4. We found it could be detected by taking advantage of the acidic nature of the peptide, using the following technique : (1) paper strips with the peptide applied are run in a suitable buffer, for example, veronal/veronal sodium at pH. 8.6 or acetic acid/sodium acetate at pH 4.5, and dried at 100°; (2) the buffer is washed out with two changes of 80 per cent ethanol; (3) the strips are treated with N/50 hydrochloric acid in 90 per cent ethanol; (4) the acid is thoroughly removed with ethanol.; (5) after drying, the strips are dipped into N/300 alcoholic sodium hydroxide containing 0.04 per cent bromo-cresol purple, which is repeated if necessary until colour differentiation is obtained, and the strips are blotted.

Neher and Wettstein1 have used a solution of antimony trichloride in organic solvent to identify certain steroids on paper chromatograms. Although Jaminet2 has used an acetic anhydride solution of antimony trichloride for the spectrophotometric identification of cardiac glycosides, he has not reported its use with paper chromatograms. Work in this laboratory has shown that antimony trichloride may be successfully applied to this field.

TRANSAMINASE reactions are known to occur in practically all animal tissues, especially in heart muscle, brain, kidney, liver and testes. Reactions of this type were first suggested by Needham1, who noted that when glutamic acid was added to minced pigeon-breast muscle it disappeared without a parallel formation of ammonia. Four or five different transaminase systems have so far been thoroughly investigated by various workers2; but the methods employed for the study of these reactions have required manometric equipment3 and intensive analytical procedures4. The present communication deals with the application of paper chromatography technique to the separation of 2 : 4 dinitrophenyl-hydrazones of the keto-acids in the reaction mixture, since it has proved in our hands a satisfactory tool for the study of transaminase reactions. The reaction studied was: L-Aspartate + α-Ketoglutarate = Oxalacetate + L-Glutamate.

FOR Chromatographic identification of iodide and iodinated protein derivatives, it is often necessary to locate inorganic and organic iodide tracers on the same chromatogram. In paper chromatography a number of methods have been used for the detection of iodide. These include spraying with silver nitrate1 ; potassium chlorate, acid and starch2 ; 1 per cent aqueous palladium chloride3 ; a mixture of ferric nitrate and hydrogen peroxide4 ; the ceric-arsenite reagent5; and a mixture of FeCl3.6H2O, K3Fe(CN)6 and NaAsO2 (FFCA reaction)6. Most of these tests for iodide interfere with those commonly used for the identification of iodinated amino-acids, namely, ninhydrin7, diazotized sulphanilic acid3, or its diethyl amide8. The ceric-arsenite and the FFCA reagents are the most sensitive (0.01 µm. I−) and can moreover be used for the detection of inorganic iodide, together with iodinated amino-acids on the same chromatogram6,9. The FFCA reagent, however, is not specific for iodine but also gives positive reactions with certain reducing substances6. The ceric-arsenite reaction has several disadvantages and is generally not used in the spraying technique9.

IT has been shown by paper electrophoresis that xanthine dehydrogenase activity is bound to the globulin fractions of rat serum1. Using differential centrifugation, it was demonstrated that xanthine oxidase and xanthine dehydrogenase activities are present in the supernatant fluid corresponding to the ‘cell sap’ containing the soluble proteins of the rat liver2.

ESCULETIN was found in an extract of plant tissue which contained caffeic acid. The esculetin was not a natural product but was formed from caffeic acid during the paper-chromatographic purification procedures. This conversion was confirmed by two-dimensional chromatography of authentic caffeic acid.

THE separation of several non-volatile carboxylic acids by paper chromatography has been described by Lugg and Overell1,2. These workers found that it was impossible to carry out the separation with neutral solvents because the ionization of the acids caused the spots to ‘tail’. They overcame the difficulty by swamping both the stationary and mobile phases with formic or acetic acid. Removal of the swamping acid was then a necessary and sometimes tedious operation before the non-volatile acids could be revealed by spraying with an acid-base indicator.

WE have studied a strain of Chaetomium globosum which was cultured on the basal medium of Buston and Basu1 (NaNO3, 2 gm.; KH2PO4, 1 gm.; KCl, 0.5 gm.; MgSO4.7H2O, 0.5 gm. ; FeSO4, 0.01 gm., in 1,000 ml. water) with the addition of 5 per cent sucrose and 2 per cent yeast extract. 50-ml. portions contained in 500-ml. flasks were inoculated with 1 ml. of a spore suspension and incubated at 25°. Samples were withdrawn aseptically from the culture flasks at intervals and spotted on a paper chromatogram2 and developed with n-butanol – water for 56 hr.

SAMPLES of human urine have been examined for indole and phenol acids as a matter of routine in this laboratory for several months; the methods used for extraction and two-dimensional paper chromatography were described earlier1,2. The chromatograms were satisfactory until certain days in April, when the normally pink and purple spots given by indoles with p-dimethylaminobenzaldehyde showed a diminished colour intensity and brown discoloration. The colours became so weak in May that indole spots could not be seen with certainty. Unlike the indoles, the phenolic acids gave colours which were normal immediately after spraying with diazotized sulphanilic acid, but which faded and were discoloured afterwards to an increasing extent. No improvement was obtained when the components of the chromatographic solvents were purified extensively.

THE separation of phospholipids by paper chromatography has generally been performed on modified papers. Marinetti and Stotz1 and Lea et al.2 report the use of silicic acid-impregnated paper, and Brown et al.3 have used silicic acid-impregnated glass-fibre ‘paper’. Rouser et al.4 investigated the possibility of using paper impregnated with mineral oil, rubber, silicone, and formamide, and paper oxidized by periodic acid, but found all these to be unsuitable. Recently, however, in this laboratory5 a method has been evolved for the separation and differential staining of phospholipids on untreated paper. During work on this method difficulty was encountered with the separation of lecithin from phosphatidyl ethanol-amine, and a procedure for separating them on paper impregnated with zinc salts is described here.

SEVERAL papers1,2 have been published on the separation of strychnine and brucine by paper chromatography, but none of these gives a satisfactory separation of these alkaloids, particularly in regard to the quantitative determination of strychnine after its elution from the chromatogram. The difference between the R F values of strychnine and brucine is only 0.15–0.18 even under the optimum conditions.

THE application of paper chromatography to the qualitative separation of phenols and phenolic derivatives has been reported by a number of workers1. The methods described employ the one-dimensional partition technique and they separate, with varying degrees of efficiency, phenolic derivatives having fairly pronounced structural differences.

INORGANIC chloride is commonly present in mixtures of biological origin and, if not removed, may interfere with the identification of the organic constituents. A silver nitrate reagent has been developed which will detect as little as 1 µgm. of chloride ion on a paper chromatogram.

THE technique of paper chromatography devised by Consden et al.1 for the qualitative analysis of ammo-acids has since found wider spheres of application2. Bate-Smith3 described its application to anthocyanins, flavones and other polyphenolic substances.

A FEW drops of the alcohol in a small test-tube are treated with 0.5 ml. of pure carbon disulphide and 0.1 gm. of pure powdered potassium hydroxide and the mixture shaken for some minutes. The liquid phase is decanted to another test-tube, evaporated and the residual xanthogenate dissolved in a drop of water. The resulting solution is developed by paper partition chromatography (ascending method) using alkaline butanol (2 per cent potassium hydroxide) as solvent. About twenty hours is required. The location of the xanthogenates on the paper is detected easily by their dark brown luminescence under ultraviolet light or by the yellow or blue colours produced by spraying with Grote's reagent1. In the case of solid alcohols, the xanthogenate formed is dissolved in formamide and developed in the same way. The RF of formamide solutions of potassium xanthogenates, however, indicates, assuming the following reaction, the values of the corresponding ammonium xanthogenates

Franklin and Quastel1 have recently applied the technique of paper chromatography to proteins. Protein–haemin complexes were prepared, and the position of each protein on the chromatogram could thus be revealed by the benzidine reaction. The method has limited application since many proteins do not react with haemin. Jones and Michael2 have used acid dyes to stain proteins on the chromatogram.

PHOSPHATE esters separated on paper chromatograms are usually detected by degrading them on the paper with acid to give ortho-phosphoric acid and detecting this by the reaction with molybdate and a reducing reagent. The procedure of Hanes and Isherwood1 is based upon this and is widely used. This technique suffers from two disadvantages. In the first place, the prolonged initial ‘digestion’ necessary to break down the more resistant esters frequently leaves the paper in a very fragile state; and secondly, further analyses cannot be carried out on the spot after treatment. In order to avoid these disadvantages the following technique was developed. It depends upon the fixation of ferric ions by the esters and the reaction of the free ferric ion with salicylsulphonic acid. If the paper is not strongly buffered it is sprayed with 0.1 per cent FeCl3.6H2O in 80 per cent alcohol, dried in air at room temperature and then sprayed with 1 per cent salicylsulphonic acid in 80 per cent alcohol. Upon drying, the phosphates appear as white spots on a pale mauve background, ortho-phosphate having a band of deeper mauve surrounding it. The colour formation occurs only when the pH of the residual moisture in the paper is about 1.5–2.5. This can be checked conveniently with thymol blue, since the colour formation occurs most satisfactorily when the paper just turns this indicator red. Further indications of unsatisfactory pH are that below this range no colour is formed, whereas above the range the colour is orange-yellow and definition of the spots is very poor.

ON electropapergrams, the alpha-2-globulin protein peak has been shown to coincide exactly with the locus of maximum non-specific alkaline phosphatase activity1. This enzyme may be detected during the run by an adaptation of the Huggins and Talalay sodium phenolphthalein phosphate method, which determines alkaline phosphatase by ascertaining the colour due to free phenolphthalein produced at pH. 9.2 by incubating a serum sample with the colourless substrate (a blank correction is made for non-enzymatic substrate hydrolysis). Because of its broad optimum pH range, alkaline phosphatase has considerable activity at pH. 8.6, which is most often used for paper electrophoresis, and a significant proportion of free phenolphthalein is coloured at this pH. To detect alkaline phosphatase during paper electrophoresis, 0.1–1.0 per cent of sodium phenolphthalein phosphate is included in the buffer; the region of enzymatic activity is seen as a well-localized red band. At pH 9.2 the band is considerably more impressive in colour.

LEIFER et al.1 have reported the paper chromatography of nicotinic acid with the use of microbiological assay. While our work was in progress, Huebner2 described a spot test for detection of some pyridine derivatives. The present work describes sensitive qualitative techniques developed in this Laboratory for the detection of tertiary and quaternary pyridinium compounds. The laborious procedure involved in the microbiological method of Leifer et al.1 is obviated by rapid colour or fluorescent tests. For the colour test, advantage was taken of Konig's3 reaction, namely, the formation of coloured compounds when tertiary pyridine derivatives are treated with cyanogen bromide and a primary aromatic amine. The fluorescence test allows the detection of quaternary nicotinamide derivatives. Use is made of the formation of bluish-white fluorescent substances when quaternary pyridine compounds with a side chain CO—NH—R in the β position are treated with methyl ethyl ketone and ammonia4.

OWING to the frequency with which caffeine is used for the illicit medication of racehorses, the positive identification of µg quantities of this compound is a matter of some importance. Although a caffeine spot on a paper chromatogram exhibits a deep blue fluorescence under ultra-violet light, and may be eluted for subsequent identification by spectrophotometry1 or by crystal tests2, in common with most other xanthine derivatives caffeine does not react with the usual spray reagents, and there is need of a colour reaction which can be carried out directly on the paper.

WHEN ethyl pyruvate and 4,5-diaminopyrimidine were refluxed in water, between pH 1 and 3, a solid of molecular weight about 320 was formed which was seen as two spots1 after dissolution in 0.1 N sodium hydroxide and chromatography on a sheet of Whatman No. 1 paper in water at pH 5.5 or 8.5. The spots (Fig. 1A) had RF values of 0.55 and 0.65 respectively and appeared identical when viewed in ultra-violet light. When eluted, and rechromatographed, each spot generated only one spot, which corresponded in RF to the spot from which it was eluted (Fig. 1B and C).

WHILE investigating the chromatographic separation of sugars upon filter paper impregnated with sodium borate buffer (0.05 N borax, pH 9.18) it was found that the carbohydrates showed up prominently on spraying with 80 per cent aqueous ethanol containing either 1 mgm./ml. of phenolphthalein or phenol red just made alkaline with sodium hydroxide. Phenolphthalein gave rise to white spots on a purple background and was rather more effective than phenol red, which gave yellow spots on a purple background.

THE separation of mixtures of amino-acids into discrete spots on paper chromatograms has hitherto almost invariably been carried out using solvents only partially miscible with water. However, in work concerned with the separation of certain peptides we found that the usual solvents (namely, butanol, benzyl alcohol, phenol, collidine) were inadequate, and in consequence sought alternative solvents.

THE standard method of rendering visible radioactive compounds on paper chromatograms is to place the paper against a fast X-ray film for the requisite exposure period. The energies of β-particles emitted by tritium are so low (E max = 18 keV.) that the paper itself and the inevitable air gap between paper and film absorb most, if not all, of the radiation, reducing the sensitivity of the technique below useful limits. Wilson1 has described the preparation of scintillation autographs, in which the chromatographie strip, in close contact with a fast film, is immersed in a tank of scintillation fluid; but his method cannot be applied to the study of compounds, such as steroid hormones, which are soluble in toluene, the primary solvent of most scintillating fluids.

A WIDE variety of phosphate esters have great importance as intermediates in biological reactions. Development of adequate procedures for the chromatography and detection of these phosphate esters has, in many respects, lagged behind. With the exception of the method of Runeckles and Krotkov1, which was developed as a modification of the procedure of Wade and Morgan2, the detection of phosphate esters on paper chromatograms has been very laborious and relatively insensitive. Most of the molybdate procedures, in addition to requiring many manipulations, have the disadvantage of employing perchloric acid, which weakens the paper and causes some charring during heating.

DETERMINATION of individual amino-acids in a chromatographed mixture by measuring nitrogen content was discouraged by Martin and Mittelmann1, who reported interference from the solvents used. The method has now been re-investigated. Chromatograms were prepared using Whatman No. 1 paper and the apparatus described by Datta, Dent and Harris2; the solvents were (a) n-butanol-acetic acid3, and (b) a mixture of phenol and water (4 : 1) in the presence of acetic acid. The papers were dried overnight at room temperature, then sprayed with Moore and Stein4 reagent diluted with methyl alcohol to contain 0.1 gm. ninhydrin/100 ml. The colours obtained with this reagent were found to be more stable than those given by a solution of ninhydrin in n-butanol, and there was less vertical streaking of the spots on the paper. The papers were heated, the spots outlined with pencil and identified by comparison with a series of similarly prepared ‘maps’. Demarcation of the spots is helped by the fact that adjacent spots often give slightly different colours with ninhydrin. The identified spot and a piece of paper of the same area were cut from the same sheet, the latter for a blank determination.