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THE high resolving powers of columns of cross-linked sulphonated polystyrene resins when applied to the separation of mixtures of amino-acids has been demonstrated by the classic work of Moore and Stein1 and afterwards by other workers2,3.

IN 1955, Scott1,2 described a method for the preparative separation of polysaccharides containing different amounts or types of anionic residues, based on the formation of insoluble complexes with cetyl pyridinium chloride (CPC) or similar long-chain cations. The resulting complexes can be selectively dissociated by varying concentrations of electrolytes (sodium sulphate, sodium chloride, magnesium chloride)3,4.

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.

METHODS have been devised for the separation of bile acids and their conjugates with glycine or taurine using partition chromatography on columns1,2 or paper3,4. These methods, however, may confuse certain glycine conjugated acids with the unconjugated bile acids containing a greater number of hydroxyl groups4. Thus ambiguous separations may result if unconjugated bile acids are also present in the mixture.

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

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.

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.

THE blue colour of bromocresol green-dyed protein developed by exposure to ammonia vapour is not stable, and elution of the dyed protein has been used to obtain an electrophoretic curve from paper electro-phoretograms of serum dyed with bromocresol green. One disadvantage of this is that the paper strip is destroyed when it would have been of value as a record for reference.

SEVERAL paper-chromatographic procedures for the separation of the lower fatty acids as their ammonium or ethylamine salts have been described1. They suffer, however, from two disadvantages: formic acid cannot be separated from acetic acid and the time required is about 20 hr. By running the chromatograms at 50–60° C. the time required for the separation of the acids C2 to C6 could be reduced to 1–2 hr., according to a recent report2.

THE separation of vanillin and syringaldehyde produced by the oxidation of lignins with nitrobenzene in alkaline medium is of importance in lignin chemistry. Spruce lignin gives only vanillin1, whereas hardwood lignins give both aldehydes in relative quantities depending on the species2. Only limited success has attended efforts to separate them by chemical means2,3. Separation by vacuum sublimation2 employs 0·4 gm. of the mixed aldehydes and requires specialized equipment. The present communication shows that separation can be effected by paper partition chromatography using petroleum ether saturated with water as the mobile phase.

RECENTLY, it has been obvious that in most biological materials N-acetylsialic acid (N-acetyl-neuraminic acid) and N-glycolloylsialic acid occur together. By the introduction of a micromethod for the determination of glycollic acid, Klenk and Uhlenbruck1 have made possible the evaluation of the amount of N-glycolloylsialic acid in isolated sialic acids. In search of a more direct method, we tried partition chromatography, employing the solvent systems previously used for the separation of sialic acids but with unfavourable results2,3. As the pK a of sialic acids are low, about pH 2.7, it is necessary to use rather a strong acid in the solvents to prevent the dissociation of the carboxylic group. Of the different acids tried, only trichloracetic acid and hydrochloric acid were suitable. On crystallization of the sialic acids we had observed that N-glycolloylsialic acid was much more insoluble in alcohols than N-acetylsialic acid. Therefore, we tried mixtures of alcohols with the acids mentioned above. The best results were obtained with n-butanol/n-propanol/ 0.1 N hydrochloric acid (1:2:1, v/v). In this solvent the R F values were for N-acetylsialic acid 0.45, and for N-glycolloylsialic acid 0.34, on Whatman paper No. 1. The solvent system applied cannot be used for the separation of diacetylsialic acids and is unsuitable for the separation of free acids and the methyl esters of the sialic acids.

VERTEBRATE neurohypophysial hormones have a common structural pattern characterized by a chain of nine amino-acid residues with a disulphide bridge connecting the amino-acids in positions 1 and 6. There are usually two hormones in each species, the active principles rarely varying from one species to another within a given class. Thus oxytocin and arginine vasopressin have been chemically identified in five species of mammals belonging to the orders Primates, Artiodactyla, Perissodactyla and Cetacea. Isotocin (Ser4-Ile8-oxytocin) and vasotocin (Arg8-oxytocin) have up to now been isolated from six species of bony fish belonging to the families Gadidae, Scombridae and Cyprinidae. The structural variations which occur between one vertebrate class and another are confined to one or two amino-acid substitutions in positions 3, 4 or 8 (for a review of this subject see ref. 1).

ONE of the advantages of the ion-exchange cellulose papers as chromatographic media is their ability to exhibit both ion-exchange and cellulosic properties. Thus, it has been shown that ion-exchange separations can be combined with those of a partition type as a two-dimensional chromatogram to form a rapid and efficient means of resolving mixtures of inorganic ions1.

DURING the past few years, a large number of papers have appeared dealing with the quantitative determination of amino-acids in paper chromatograms in situ or after elution; but no simple satisfactory general method has yet been developed. Chemical reactions of the amino-acids on paper cannot easily be controlled, and results obtained from such reactions must be considered semi-quantitative. Elution of amino-acids after location of the spots in ultra-violet light seems to be the most promising method for quantitative estimation hitherto described. This method is based on the interaction between cellulose and amino-acids, which produces fluorescent substances1. The amino-acids take part only in the formation of intermediate compounds, from which they can be recovered quantitatively or almost quantitatively. The reactions are complicated and not fully understood2.

THE term ‘development’ is used here to describe the process of chromatographic separation. Anion exchangers based on a cellulose matrix have been found to be fundamentally more useful for the separation of amino-acids than anion exchangers based on a resin matrix of the polystyrene type1,2.

DURING an investigation of the possibility of cross-linking nitrocellulose molecules in solution with silicon esters1,2, some experiments were also made with cellulose itself in the form of filter paper or unsized kraft paper.

THE recent finding of relatively large concentrations of inosine triphosphate (ITP) (44–51 µg of phosphorus/g of haemoglobin) in erythrocytes obtained from two members of a family1 led to a closer investigation of the nucleotide composition of normal erythrocytes. A detailed analysis of several samples of fresh human erythrocytes revealed the presence of small amounts of ITP in an average concentration of 1.8 µg of phosphorus/g of haemoglobin2. From these studies it was concluded that ITP is a normal constituent of human erythrocytes.

IN the course of investigations into the application of paper chromatography to the identification of reducing substances in urine it was found that, when ammoniacal silver nitrate was used as a developing reagent, urates, normally present in urine, produced a dark brown spot with an RF value of approximately 0·20. This spot, which generally ‘tails', tends to mask the spots produced by other reducing substances with RF values in the region of 0·20, namely, those of glucose, sorbose, arabinose and fructose.