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GORDON, MARTIN AND SYNGE1 and Consden, Gordon and Martin2 have shown that good separations of amino-acids can be obtained on filter paper by allowing a suitable solvent, which has previously been saturated with water, to flow over the paper in a closed container, the air in which is saturated with the vapours of water and the solvent. Filter paper contains 20–25 per cent water under these conditions, and separation depends upon the differences in partition coefficient of the amino-acids between the stationary water phase and the moving solvent. It has further been shown2 that for any individual amino-acid the value

By so-called ‘one-dimension partition chromatography’ on a paper support (first used successfully by Consden, Gordon and Martin1 for the separation of amino-acids, and recently adapted by Partridge2 for the separation of reducing sugars), we have succeeded in separating the individual members of a mixture of carboxylic acids of the types commonly occurring in plant tissues.

The quantum spin properties of nitrogen-vacancy defects in diamond enable diverse applications in quantum computing and communications1. However, fluorescent nanodiamonds also have attractive properties for in vitro biosensing, including brightness2, low cost3 and selective manipulation of their emission4. Nanoparticle-based biosensors are essential for the early detection of disease, but they often lack the required sensitivity. Here we investigate fluorescent nanodiamonds as an ultrasensitive label for in vitro diagnostics, using a microwave field to modulate emission intensity5 and frequency-domain analysis6 to separate the signal from background autofluorescence7, which typically limits sensitivity. Focusing on the widely used, low-cost lateral flow format as an exemplar, we achieve a detection limit of 8.2 × 10−19 molar for a biotin–avidin model, 105 times more sensitive than that obtained using gold nanoparticles. Single-copy detection of HIV-1 RNA can be achieved with the addition of a 10-minute isothermal amplification step, and is further demonstrated using a clinical plasma sample with an extraction step. This ultrasensitive quantum diagnostics platform is applicable to numerous diagnostic test formats and diseases, and has the potential to transform early diagnosis of disease for the benefit of patients and populations. Lateral-flow in vitro diagnostic assays based on fluorescent nanodiamonds, in which microwave-based spin manipulation is used to increase sensitivity, are demonstrated using the biotin–avidin model and by the single-copy detection of HIV-1 RNA.

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.

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.

THE photolysis of riboflavine has always been considered1 to be a complex process, but as a rule only the final products, lumichrome (6 : 7 dimethyl alloxazine) and lumiflavine (6 : 7 : 9 trimethyl iso-alloxazine), have been isolated.

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.

PAPER-PARTITION chromatography of tissues of genetically known strains of Drosophila melanogaster as well as those of certain plants has shown that each strain gives a constant and distinctive pattern. The chromatographic patterns of heterozygotes carrying recessive genes can be easily distinguished from those of either parental homozygous strain, even though the phenotype of the heterozygote is indistinguishable from that of the homozygous dominant. This is the case for: (a) the fluorescent patterns observed under ultra-violet light in the chromatograms of adult Drosophila melanogaster heterozygous for the following mutants: bent, eyeless2, short-vein, spineless-aristapedia, vestigial; (b) the ultra-violet-fluorescent and the ninhydrin-positive patterns of root-tip smears of heterozygotes for the mutant male-sterile2 in tomato1; (c) the ultra-violet-fluorescent and the ninhydrin-positive patterns of root-tip smears and leaf extracts of heterozygotes for the yellow-green mutant of musk melon2 (see also chromatogram).

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

SEVERAL types of paper columns have been described for separating closely related substances on a preparative scale; but with these methods it has proved difficult to get the same degree of separation as is achieved by simple paper-strip experiments1. This disadvantage is overcome by the present method, the principles of which have been reported in a previous communication2. The technique has now been improved and simplified.

THE use of 'Gammexane' has been recommended for controlling insect pests in libraries and record offices, where insect infestation is a serious problem especially in a tropical climate. 'Gammexane' smoke generators appeared to be particularly suitable for this purpose owing to the ease and safety with which they can be handled. But no information about its effect on paper1 was available, and the use of a new insecticide for the disinfestation of documents of a unique and irreplaceable nature had therefore to be scrutinized from the preservation point of view.

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.

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.

GRANT et al.1–3 have reported that latent fingerprints on paper may be detected by autoradiography of the paper after it has been treated with 35SO2. Work carried out at the Health Physics and Medical Division, AERE, Harwell, has shown that a modified version of the technique of Grant et al. can be used to detect latent fingerprints on a wide range of papers. I have also found that the modified technique can be used to visualize latent fingerprints on finely woven fabrics.

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.

TEMPERATURE is a variable which has received relatively little attention in chromatographic studies. This is particularly true of paper chromatography in which more attention is given to controlling temperature as an aid to reproducibility than to using temperature to effect improved resolution.

A Two-Dimensional Paper Chromatographic Method Combining Ion-Exchange and Partition Techniques

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.

THE identification of sulphonamides in biological fluids is usually attempted by microscopical examination of crystals and by the use of various, rather non-specific, colour reactions1. Since it is common for only small quantities of fluid to be available, it was decided to investigate the possibilities of paper partition chromatography.

THE possibility of combining the advantages of circular paper chromatography with those obtained by using an automatic fraction collector for separation and isolation of substances from a mixture has been investigated. The essential features of the technique consist in the application of the method of preparative circular paper chromatography1 for the separation of the substances on a paper pack, followed by the elution of the components by means of a device for facilitating the flow of the solvent from the edge of the paper pack. Fig. 1 is a diagrammatic sketch of the apparatus devised.

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.

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).