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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.

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.

IN previous experiments1 the 0.02 M phosphate buffer of pH 6.8 was used for electrophoresis of bile pigments on paper. Under these conditions (in the absence of proteins) the conjugated bilirubin migrating anodically separates from the unconjugated bilirubin which remains on the starting line. The presence of bile salts clearly influences this separation. Purified preparations2 of conjugated bilirubin which are devoid of bile salts migrate in the form of a tail beginning just from the starting line. If bile salts are present a sharply denned zone of conjugated bilirubin separates, migrating in front of the bile salts. The velocity of migration of this zone in the presence of bile salts is considerably higher than the rate of migration of pure preparations of the pigment.

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).

POLYCHLOROBIPHENYLS (PCB) cause chloracne and liver damage in man and induce oestradiol degradation1. They are widely distributed in the ecosystem in birds, fish and shellfish1–4. The miserable episode of “Yusho”, a mass food poisoning involving more than 1,000 people which was caused by chronic ingestion of rice oil contaminated with ‘Kanechlor-400’, a commercial brand of PCB, occurred in western Japan in 19685,6.

WE have already described a fortuitous combination of events which led to the discovery that American newspapers and other paper products contain a potent analogue of the juvenile hormone of the European bug, Pyrrhocoris apterus L. (Heteroptera ; Pyrrhocoridae)1. When reared in contact with ‘active paper’ or when exposed to surfaces impregnated with extracts of the ‘paper factor’ (PF), fifth-stage Pyrrhocoris larvae undergo one or more supernumerary larval moults and finally die without completing metamorphosis or attaining sexual maturity. This same result is observed when 1 µg of partially purified PF is topically applied to young fifth instar larvae1,2.

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.

IN the first report1,2, on ‘crossing paper electrophoresis’, a direct demonstration of enzyme-substrate complex has been described. The enzymes tested included crystallized pure trypsin, chymotrypsin and ribonuclease.

Dewar and Gunew1 have described the use of a serrated glass strip as a collector for column chromatography. In this laboratory a method has been developed in which a moving strip of paper is used for the same purpose.

PAPER electrophoresis of proteins has on the whole proved to be a more satisfying means of separation than paper chromatography, owing to the physico-chemical properties of the proteins that make them rather unfit for the latter method1,2. Nevertheless there do exist methods of paper Chromatographic separation of, for example, enzymes, blood and serum proteins, mainly based on a kind of adsorption analysis, in addition to the more usual procedures of partition-, ion exchange- and adsorption-chromatography on columns.

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 acid phosphatase test has proved its usefulness in seminal stain examinations and is now in widespread use1–4. The desirability of preparing a stable form of the reagent was obvious, more especially for ‘scene-of-crime’ searching, post-mortem examinations and for scientists and clinicians without laboratory facilities.

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.

THE paper chromatography of steroids, for example, cortical steroids, has already been investigated by Zaffaroni et al.1 and by Bush2. Although the separation on paper of the natural œstrogens free or in compounds has already been investigated by Bush3, Heftmann4, Nyc et al.5 and Boscott6, a satisfactory separation of the three natural steroids on non-treated paper has, to our knowledge, not yet been published. The method described below gives excellent results. It has been especially devised for separating and purifying the small amounts of œstrogens normally present in biological fluids.

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.

METHODS for the analysis of small amounts of nucleic acids and their components by paper partition chromatography have been described by Vischer and Chargaff1 and Chargaff et al.2. Photographic methods for their identification on paper have also been used3,4, based on the specific absorption in the ultra-violet range of the spectrum of the nucleic acid components. The present communication describes a simple and sensitive method for rapid analysis in routine work.

THE analysis of the protein composition of blood serum or plasma by means of zone electrophoresis on filter paper has stimulated interest in clinical laboratories throughout the world. In addition to numerous publications dealing with the applications of the technique in various pathological conditions, critical assessments of its validity and accuracy have appeared1–4, in which attention is properly directed to the limitations of the method from the point of view of the numerical evaluation of the separated protein fractions. It has seemed desirable to undertake a study of the accuracy and reproducibility of this method of protein analysis, using two of the most commonly employed protein-dyeing techniques.

PAPER partition chromatography is not successful with many organic compounds for two reasons: (1) the compounds have low solubility in water, and hence move on the solvent front in normal partition systems; (2) low reactivity of the compounds makes detection of the position of spots difficult.

FOR the purpose of studying the metabolism in insects of a radioactive bromine analogue of DDT, (Br*C6H4)2 CH.CCl3 (I), a method was required for the separation and estimation of (I) and the possible metabolites, (Br*C6H4)2 C : CCl2 (II), and (Br*C6H4)2 CH.COOH (III), on the micro-mole scale. It was important that the method itself should not bring about any decomposition of unchanged (I).

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.