Search

Under conditions in which insulin molecule has a maximal positive charge, comparison of paper chromatographic and paper electrophoretic behaviors was performed between fish and beef insulins. In 33 per cent acetic acid fish insulin migrates faster than beef insulin, whereas it shows lower Rf when is chromatographed using acetic acid-butanol-water (1:3:4) solvent.

A new solvent system was given for the chromatography of food preservatives; benzoic acid, salicylic acid, p-hydroxybenzoic acid and it's esters, p-chlorobenzoic acid, sorbic acid, resorcinol and β-naphtol with the ascending method on Toyo Roshi No. 50 paper at 20-25°. Cyclohexanol-ammonium carbonate buffer gave results in 30 hours. Spots were detected by absorption when the paper was examined in ultraviolet light and with color reagcnts of the methyl red or diazo spray reagent. Rf values for this solvent were listed for these substances. Each substance was separated except in the case of mixture of benzoic acid and sorbic acid. However in this case, these two substances were identified by measuring spectrophotometrically the alcohol solution extracted from the part containing the substances on paper chromatogram.Horizontal paper electrophoresis on Toyo Roshi No. 51 paper for 1.5-2 hours at 20V per cm with buffer consisted of dimethylformamide, pyridine, acetic acid and water (pH 5.6) or of butanol, ammonia, acetic acid and water (pH 5.0) was useful. MNC (the ratio of the migration of the substances of New Coccine) and colors given with the methyl red, FeCl3 and diazo spray reagents were tabulated adding dehydroacetic acid to above substances. Data was also given for citric acid and tartaric acid.Sake (Japanese wine), soy etc., was acidified with HCl, extracted with ether, purified as ammonium-salts and submitted to paper chromatography and paper electrophoresis with similar technique.

For the quantitative analysis of fatty acid composition of fatty oils, the application of paper-chromatography using hydrogenation on papers was examined, in addition to the usual reversed phase paperchromatography consisted of hydrocarbon as the stationary phase and acetonitril acetic acid as the mobil solvent. Complete hydrogenation on papers was achieved after 2 hr at 30, using palladium black as catalyst. By applying this analytical method, the composition of fatty acids of wheat germ oil and of skipper oil was determined as follows. Wheat germ oil : C16F0 : 12.3%, C18F0 : 3.7%, C18F1 : 20.6%, C18F2 : 45.6%, C18F3 : 12.3%, C20F0 : 5.4%. Skipper oil : C14F0 : 8.6%, C16F0 : 14.6%, C16F1 : 3.8%, C18F0 : 1.6%, C18F1 : 8.7%, C18F2, : 6.8%. C18F3 : 1.2%, C20F1 : 15.7%, C20F4, 5 : 7.1%, C22F1 : 17.2% C22F5, 6 : 14.7%

The RF values of a series of eleven phenols have been determined using paper-chromatographic techniques on filter paper loaded with ion-exchange resin. The data have been used to predict the separation of various mixtures of these phenols by development with aqueous solutions of acetic acid.

A method using ascending paper chromatography has been developed for determining radiochemical impurities, such as51Cr3+ and51CrO42−, in51Cr-DTPA and51Cr-EDTA (51CrY) complexes. The chromate concentration of the sample to be checked is increased to about 5·10−2M by adding inactive chromate as a carrier, then the sample is applied onto a chromatographic paper strip (Whatman No. 1), and the impurities are precipitated at the start line with sodium hydroxide and lead acetate solution. The chromatogram is developed for 3–4 hours using a solvent mixture consisting of 25% ethanol and 75% bidistilled water, adjusted topH=8 with ammonia; the impurities remain fixed in the start line and thus separated from the chromium complexes, which migrate with anR1-value of 0.8–0.9.

The paper chromatographic behaviour of a variety of amines both alone and in the presence of acids in acidic, neutral and basic running solvents is reported. Using a n-butanol: acetic acid: water solvent, multiple spot formation by several pure sympathomimetic amines was observed in the presence of the stronger organic acids. Many weaker acids affected the amines in a neutral solvent. The results emphasise the need for correct controls and the cautious interpretation of chromatograms of biological extracts since the number of spots produced is not necessarily indicative of the number of bases present.

Mixtures of ketones and acetic acid, namely 2-pentanone-acetic acid 30:2, 3-methyl-2-butanone-acetic acid 30:3, 4-methyl-2-pentanone-acetic acid 30:2, and 2, 6-dimethyl-4-heptanone-acetic acid 30:7 were found to give excellent separations of individual phospholipides on paper. It was possible to separate lysolecithin, sphingomyelin, phosphatidyl ethanolamine, lecithin, and phosphatidic acid in a unidimensional chromatogram. The individual phospholipides had to be present at concentrations from 10 to 25 /μg./20 μl. in order to achieve good separations.

A simple and rapid method for the separation and identification of fatty acids and glycerides has been developed by using circular paper chromatography. Petroleum hydrocarbon, dodecylbenzene, and tetralin were used as the stationary solvents. As the developing solvents, 90% acetic acid and methanol-acetic acid, both saturated with a stationary solvent, were suitable for the separations of fatty acids and of glycerides respectively. The fatty acids were detected on paper by successive treatment with 0.5% lead acetate, water, and ammonium sulfide solution. The selective detection of unsaturated acids by spraying with a 2% solution of iodine in ethanol was also applied. The separation of various “critical partners” of fatty acids was achieved by the method of double development, involving the first low-temperature development with petroleum ether under dry ice and the second reversed-phase development with the usual solvent systems at room temperature. The unsaturated glycerides were chromatographed as their mercuric acetate addition compounds, and were detected by spraying with a 0.2% solution of diphenylcarbazone in ethanol or by soaking in a Sudan black solution. The clear-cut separations of fatty acids and glycerides into their homologs can be attained by these circular chromatographic techniques, since the separation of unsaturated triglycerides mainly depends on their unsaturation and that of mono-, di-, and triglycerides mainly depends on the number of their free hydroxyl groups. This method was successfully applied to the analysis of various fats, fatty acids, and their products, and was confirmed to be of great value as a simple, rapid, and practical method for their analysis.

By the combination of the method for isolation and concentration by using NH4SH and the calculation RMo at the paper electrophoresis, following injurious nine metals were separated and identified : Hg, Pb, As, Sb, Cu, Zu, Ag, Cd and Bi. Three test solutions for the paper electrophoresis were prepared by the NH4SH method. Cu, Zn, Ag, Cd and Pb, belong to the 1st section, were separated and identified according to RMo by using 1N citric acid, 5N acetic acid and 5N formic acid as the electrolyte for the paper electrophoresis. Hg and Bi, the 2nd section, were separated and identified as stated above by using 0.1M lactic acid or 5N formic acid as the electrolyte. As and Sb, the 3rd section, were separated and identified as stated above by using 0.1M lactic acid as the electrolyte.

The chromatography of piperidine, cyclohexylamine, benzylamine, and morpholine hydrochlorides on unwashed Whatman No. 1 and No. 17 papers in t-butanol: acetic acid: water (2:1:1) leads in each case to partial conversion to the acetate salt, the mobility of which differs from that of the hydrochloride. The interconversion is reduced but is not prevented by the use of acid-washed papers and is increased again when the acid-washed papers are treated with sodium, potassium, calcium, or magnesium acetate prior to chromatography. The salt interconversion is continuous during development of the chromatograms, extended development leading to complete disappearance of the hydrochloride. Quantitative chromatography of piperidine and morpholine hydrochlorides revealed that the extent of salt interconversion is ca. 12% on unwashed and calcium ion washed Whatman No. 17 papers, ca. 6% on sodium ion washed papers, and ca. 1% on acid-washed papers. Piperidinium ions become bound to unwashed, acid-washed, and salt-washed Whatman No. 17 papers during chromatography in aqueous solution just as they do to the hydrogen, sodium, and calcium forms of the carboxylate ion exchanger Amberlite IRC-50 under similar conditions, indicating that a cation exchange mechanism involving carboxyl groups in the paper is responsible for the observed salt interconversion.

Ascending paper chromatographic methods for the separation of 2,4-dinitrophenylsulfide derivatives of mercaptans and mercapto-acids are presented. A solvent system composed of methanol as the stationary phase and heptane as the mobile phase gave satisfactory separations of the normal C 1 to C 7 mercaptan derivatives on What-man No. 1 paper. Two procedures were devised to separate the mercapto-acids derivatives. One method employed a solvent system of n -butanol:ethanol:water (20:5:25) with the water-rich layer as the stationary phase and the butanol-rich layer as the mobile phase. The solvent system for the second procedure was composed of equal parts of benzene and 1% acetic acid with the benzene-rich layer as the mobile phase in this case.

Any pair of the group of 11 phosphate esters studied may be separated on a paper chromatogram with one of three solvent mixtures: ethyl acetate – acetic acid – water, methyl Cellosolve – methyl ethyl ketone – ammonia water, ethyl acetate – formamide – pyridine. Separation of the components of more complex mixtures usually requires the use of two-dimensional paper chromatography. Position constants for the individual esters with each of the three solvents are listed, and diagrams showing the type of separation pattern obtained by two-dimensional chromatography of mixtures are presented.

This paper describes a paper chromatographic method of identification of morning glory (Ipomoea purpurea) seeds used by individuals seeking hallucinatory experiences. The procedure is applicable to a single seed, seed fragments and products of “tea”. A 50 percent aqueous alcoholic extract is chromatographed in a butanol : acetic acid : water system and the pattern visualized by ultraviolet fluorescence, p-dimethylaminobenzaldehyde and ninhydrin spray reagents. The chromatographic patterns of various morning glory seeds and other seeds of criminalistic interest are shown. Milligram quantities of Ipomoea purpurea (two varieties) are readily differentiated from all other seeds examined.

The RF values of 86 phenols, phenolic acids, coumarins, phenolic aldehydes and acetophenones have been determined experimentally in the benzene—acetic acid—water (125:72:3, by vol.) solvent system, and the resulting values have been studied as a means of estimating free hydroxyl groups in these compounds. The results indicate that his solvent system is applicable for the estimation of the number of free phenolic hydroxyl groups present in many different types of phenolic compounds. The color reactions and fluoroscence of many of these compounds have also been recorded.

The authors studied the use of chloranilic acid as a reagent in the paper chromatography of some of the heavier metals. Sensitivity was found to vary from 0.01 μg for ferric iron, to 5.0 μg for mercury (II) and zinc. It was found that iron gave tailing spots with most of the solvent systems, which interfered with the separation of the other cations. This interference was avoided by using acetone—acetic acid, which displaces iron to the solvent front. Efficient two-dimensional separations were obtained by carrying out a second run with ethanol—acetic acid.

Phenol and butanol-acetic acid-water are suitable solvents for bidimensional paper chromatography of amino compounds in the routine clinical laboratory. A chart illustrating the positions of 56 amino compounds on bidimensional chromatograms with these solvents is presented. The behaviour of the compounds studied can be correlated with their chemical structure. Members of homologous series behave in a predictable manner. The effect of inorganic salts is discussed and the importance of maintaining the room temperature at a suitable level stressed. The former affects separation in phenol, the latter in butanol-acetic acid. Desalting of urine and packed red and white blood cells is unnecessary when runs are made at the proper temperatures. Illustrative chromatograms of normal and pathological untreated urines are presented.

An acidic and a basic solvent containing the same proportions of propanol and water have been used for the study of about 100 organic acids by paper chromatography. The acid and the base were solutions fo the gases sulphur dioxide or ammonia in water and therefore were readily removed after chromatography. Sulphurous acid has the advantage of being a stronger acid than those normally used, e.g. formic and acetic acid. The R M values of the organic acids in the two solvents can be arranged into clear-cut groups dependent on the number of carboxyl and other substituted groups present, in such a way that the number of carboxyl groups in an unknown acid can be confidently predicted. An indication can also be obtained of other groups such as alkyl, aryl, amino, hydroxy, bromo etc. which may be present, and the manner in which they are arranged. The results include nine homologous series whose members differ from one another only in the addition of -CH 2 - to the molecule.

Solubilized vat dyes wre separated by paper chromatography, using a methanolic solution of ammonia as solvent system. A system containing pyridine, isoamyl alcohol and ammonia, as well as a mixture of methanol and acetic acid were also used. Both descending and centrifugal chromatography were employed. The relation between dye constitution and chromatographic behaviour has been examined.

The chromatography of a series of primary aromatic amines is studied on papers composed of pure cellulose and carboxy-methylcellulose with water, acetic acid and an acetate buffer as solvents. Migration data and possible separations are reported, and results are compared with those obtained on carboxymethylcellulose thin layers.

A procedure for the detection and identification of α-glycerolphosphonolipids in the presence of their phosphoric acid analogues, viz. α-lecithins and α-cephalins, is described. It combines the saponification of these compounds by a modified Dawson procedure with the separation and identification of the saponification products by descending paper chromatography with n-butanol, glacial acetic acid, and water (5:3:1, v/v/v), and staining of the chromatograms with ninhydrin and molybdate sprays.The relative mobilities of the saponification products, viz, α-glycerol-(2-trimethylammoniumethyl)phosphonate, α-glyceryl-(2-aminoethyl)phosphonate, α-glycerolphosphorylcholine, and α-glycerylphosphorylethanolamine with respect to each other and to α-glycerophosphoric acid have been determined.

The ion exchange capacities of Whatman No. 1 and Whatman CM81 chromatographic papers, with and without pre-washing, were determined chromatographically using lead nitrate and lead chloride. Mean values with standard deviations are reported. Statistical analysis indicates that pre-washing with acetic acid, perchloric acid, and disodium dihydrogen ethylene, diaminetetraacetate affects the capacity by the same amount at the 95% confidence limit.

The R F values of six cobalt (III) complexes in Iutanol/HCl, butanol/NH 3 , butanol/acetic acid, acetone-HCl and water were measured. In organic solvents the least iomyed complex travels fastest unless decomposed. In water the highly ionized complexes travel faster than the monovalent or neutral complexes.

The variation with absorbance of the zone mobilities and obstructive factors for a number of inorganic ions has been investigated in acetic acid and in ammonium hydroxide at several concentrations. The results agree with the form of the semiempirical equation of Crawford and Edward. For a given ion the apparent obstructive factor defined as[Formula: see text]remains fairly constant as the concentration of the background electrolyte is changed, except in the very dilute region. However, the apparent obstructive factor varies with the ion and the background electrolyte indicating that specific effects, which are not considered in the equation, are important.

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 identification of 19 protein composing amino acids by paper electrophoretic method was investigated.By using 5N acetic acid as the electrolyte, 19 amino acids were grouped into four groups, that is A (Lys, Arg, His, Gly, Ala), B (Val, Leu, Ileu, Ser, Thr), C (Ser, Thr, Met, Glu, Pro, Phe), D (Tyr, Cys, Asp, Try, Hypro), and the individual amino acid in these groups was identified by the further electrophoresis with 3% aqueous ammonia as the electrolyte.Amino acids of E group (Thr, Met, Phe) and F group (Tyr, Hypro), could not be separated in this case, were tried with 1% borax as the electrolyte.By the above mentioned method, the amino acids, in this study, could be separated and identified mutually, except the discrimination between Leu and Ileu, Met and Phe.

As the developing solvent in the paper chromatography of phenols, we found that the mix-ture of n-butanol, benzene, acetic acid, and water (1:5:1:0.5 by vol. %) is better than other solvents so for reported, (Table 1) and p-benzoquinone and p-nitrosodimethylaniline are preferable as the spraying reagent (Table 2).

We studied on the identification of melamine and urea resin in wet strength paper for utilization of waste paper. A suitable test is based upon the use of an acid dye sold under the name of Alizarine saphirol B. The identification of melamine and urea resins in wet strength papers can also be made by preparation of specific crystalline derivatives.Melamine can be identified by first hydrolyzing the resin treated paper with acetic acid to produce melamine, which can be identified as melamine crystals by sublimating the residue in the presence of aluminium crystals. Melamine can also be identified as melamine picrate by treating the above solution with picric acid. The xanthydrol test is specific for urea resins, since xanthydrol condensers with urea to form crystalline derivatives.

A tentative method for paper chromatographic study of tea catecbins was established after several meetings of tea chemical engineers.The procedure is as follows :1. Preparation of sample solution.Add 10 c.c. of boiling water to 1g. of sample, keep in a boiling water bath for 5min. Drop 0.02 c.c. of this infusion on the original point of filter paper.2. Two dimensional development.Filter paper : Toyo No.3, 20×20cm.Developing temperature : 20°C (Unless 20°C. adopted, describe the temperature used.)Original point of development : Near a corner of the paper 2cm from either edge.The-first run :Developing solvent : Phenol containing 25% (vol.) water.Developing distance : Reach the solvent front 2cm, below the upper edge of the paper.Developing direction : Along the fibre arrangement, of the paper.After the first run is over, dry the paper in an electric heater, cut off the contaminated part of the paper by phenol front. The second run :After standing one night, perform the second run.Developing solvent : n-Butanol : acetic acid : water (4:1:2 vol.).Developing distance : Same length as the first run.Coloring reagent :A 1% solution of MOHR'S salt plus same volume of methanol.

The chromatographic behaviour of alkali and alkaline earth metals on paper treated with di(2-ethylhexyl) phosphoric acid (HDEHP) has been investigated, using acetic acid as the eluent in the range of concentrations from 2 M to 0.01 M . The quantity (1/ RinF — 1) has been related to the hydrogen ion concentration and to the effective HDEHP concentration on the paper. A behaviour similar to that show in liquid—liquid extraction with HDEHP solutions was found, provided that the acetic acid concentration was kept low. The peculiar behaviour shown with concentrated acetic acid has been discussed. No direct proportionality has been obtained between the HDEHP molarity in the solution used to treat the paper and the effective concentration of HDEHP on the paper which acts during elution. The instability of HDEHP on paper with very concentrated acetic acid has been also investigated. Some chromatographic separations of alkali and alkaline earth metals have been carried out, and others have been suggested.

Reversed-phase chromatography of alkaline earth metals on paper treated with di-(2-ethylhexyl) orthophosphoric acid (HDEHP) has been investigated, using hydrochloric acid as eluent in a range of concentrations from 10 M to 1 · 10 −4 M . The quantity (I R F —1) has been related to the hydrogen ion concentration in the mobile phase, and to the effective HDEHP concentration on the paper. The chromatographic behavious has been correlated with that of liquid—liquid extraction systems. Chromatographic separations of alkaline earths from each other, and from other cations, were performed. Two of them were obtained with acetic acid elutions.

When a measurement of serum protein fractions by the paperelectrophoretic method is made, it is assumed that the amount of dyestuff adsorbed onto serum protein of the paper is proportional to the content of the latter. This assumption, however, is not necessarily correct, since the amount of dye per unit of protein does not keep constant, if the concentration of adsorbed protein increases more than a definite one. This was shown by the present author's experiment. Accordingly, in order to get the true values, extinction readings must be corrected in regard to protein concentration by the calibration curve which was obtained here.When the dyestuff of the paper was washed out by 0.5% acetic acid aqueous solution containing free chlorine in it, the adsorbed dyestuff of the protein as well as of the paper is simultaneously decolored to such an extent as to interfere with the extinction readings. Thus the percentage of each serum protein fraction will not indicate the true one. In order to prevent this effect by decoloration of the dyestuff, a drop of 5% sodium thiosulfate solution was added to 2 liters of acetic acid solution, the water of which was from city water supply. By this method the author obtained the satisfactory pattern of the serum protein fractions which indicated minimum standard errors.

A rapid separation of Cs/sup 137/, Sr/sup 90/, Ce/sup 144/, and Zr/sup 95/ by paper electrophoresis was studied. The filter strips were placed in a background electrolyte. After removal from the solution, they were blotted between sheets of dry paper. The strips were sandwiched by plastics plate and cooling bath, and screwed tightly. They were allowed to equilibrate for 30 minutes. The sample solution (0.005 ml) containing Cs/sup 137/, Sr /sup 90/ , Ce/ sup 144/, and Zr/sup 95/ was spotted at a point 10 cm apart from one end of the filter strip through a hole of plastics plate, and then the hole was covered with cellophane tape. The most suitable conditions for the separation were investigated in detail, using tantaric acid, citric acid, acetic acid and formic acid. Separation of trace Cs/sup 137/, Sr/sup 90/, Ce/sup 144/ and Zr/sup 95/ from each other was carried out in only 25 minutes using 0.05M ammonium citrate, pH 3.5, as the electrolyte at 1080 v, and the mixture of those with 0.05M carrier of each was separated in only 20 minutes at 1440 v. (auth)

In paper strip electrophoretograms on Whatman 3 MM paper, clam poison migrated toward the cathode when formic acid (1 N and 0.1 N), acetic acid (1 N), sodium acetate–acetic acid (0.1 M), monosodium dihydrogen phosphate–disodium monohydrogen phosphate (0.1 M), and sodium tetraborate (0.1 M) were used as electrolytes. Formic acid (1 N) proved the most suitable of these for examination of the quantitative electrophoretic behavior of clam poison. A method of pretreatment for the paper was developed to minimize introduction of extractable impurities into material recovered after electrophoresis by aqueous elution. Pure clam poison, on electrophoresis for 1.75 hours at 900 v on an 8 × 18-in. sheet of pretreated paper, migrated as a single, positively charged, Weber-positive band with apparent mobility 18.1 cm, which exhibited no fluorescence. Recovery was quantitative on both a weight and toxicity basis, and the toxicity, specific rotation, and infrared spectrum of the recovered toxin were identical with those of the starting material, thus providing an additional check on the homogeneity of the sample. Quantitative electrophoresis of toxin from clam poison tailings which had been purified by paper chromatography removed impurities which the latter method was incapable of eliminating, with resultant improvement in both the specific rotation and toxicity. Two such treatments provided toxin possessing a toxicity and specific rotation within the accepted limits for pure clam poison.

The even-numbered C2–C22saturated and the C18-unsaturated fatty acid esters of beta- and gamma-sitosterol, stiginasterol, ergosterol, stigmastanol, and gamma-sitostanol were used separately and in model mixtures for the evaluation of existing and the development of new paper chromatographic methods for the separation and identification of these compounds. Satisfactory separations of the esters were obtained by the ascending reversed phase chromatography on cellulose or glass fiber paper using one of the following systems depending upon the number of carbon atoms in the fatty acid moiety of the ester: C2–C12, aqueous acetic acid/chloroform/paraffin oil; C8–C18, aqueous propionic acid/paraffin oil; C8–C22, acidified aqueous methanol/chloroform/paraffin oil or aqueous butyric acid/paraffin oil. The rate of migration of the esters was inversely proportional to the molecular weight and was increased by the introduction of unsaturation.The increased movement due to unsaturation has been correlated with the number of ethylenic hydrogens, and the possibility that polarity of these atoms might account for this phenomenon is discussed. The reversed phase paper partition chromatography of sterols as their esters is suggested as a potential tool for evaluation of polarity of the various attributes of the sterol nucleus in terms of the chain length of the ester fatty acid. The rule of independent contributions of the constituents of a molecule to its distribution isotherm was observed not to hold in this fatty ester series.

Amino acids have been chromatographed in n-butanol, n-butanol + acetic acid, and methanol + hydrochloric acid systems on titanium arsenate and plain papers using the ascending technique. The effect of solvent composition on the migration of spots has been studied systematically. A number of important binary and ternary separations have been achieved.

Although prostaglandin (PG) mixtures have previously been resolved by chromatography on silica-impregnated paper, drawbacks inherent in each technique have kept them from becoming generally accepted for routine analytical separations. Singh and co-workers (1,2) obtained excellent separation of prostaglandin mixtures on silica-impregnated glass fiber paper. However, this paper was not commercially available and its preparation is tedious. On the other hand, Stamford and Unger (3) separated PGE and PGF on commercially available paper using benzene/chloroform/acetone/methanol/acetic acid as developing solvent. Nevertheless, this solvent does not resolve less polar prostaglandins and fatty acids. More generally acceptable solvent systems cannot be used quantitatively with Stamford and Unger's technique due to irreversible binding of prostaglandins at the origin. Tobias and Paulsrud (11) have separated prostaglandins on commercial silicic acid-impregnated glass fiber sheets, but these are extremely brittle and difficult to accommodate to standard paper radiochromatogram scanners. This communication describes the quantitative chromatographic separation of PGF 1α , PGE 2 , PGA 1 , and arachidonic acid on commercially available Whatman SG-81 silica-impregnated paper using a wide variety of developing solvents. Irreversible binding of prostaglandins at the origin, previously a serious drawback, has been eliminated by applying the sample onto premoistened paper. This method is quantitative, sensitive, reproducible, and applicable to a variety of solvent systems. In addition, it is simple and inexpensive. Although loading capacity is somewhat limited, this is no problem with prostaglandins since they can be readily concentrated in organic solvents.

SUMMARY: Washed cells of micro-organisms were extracted with 10% acetic acid and the extracts subjected to two-dimensional paper partition chromatography. With closely related strains within species already defined serologically and physiologically, the patterns of amino acids and peptides revealed by ninhydrin were relatively constant; but with different species variations were noted, even between species hitherto not easy to differentiate.

A simple and rapid method is described for the separation of mixtures of dichlorophene and hexachlorophene. The compounds are resolved by ascending paper and plate chromatography, using n -heptane saturated with glacial acetic acid as the developer, and detecting the spots with a solution of ferric chloride and potassium ferricyanide. After elution from the plates, the isolated compounds are assayed by ultraviolet spectrophotometry.

Supplementary data are given for the paper-chromatographic investigation of mono-, di- and trihydric phenol derivatives and various compounds of biochemical interest, such as biogenic amines, aromatic and heterocyclic amino acids, hippuric acids, indole derivatives. Some further types of compounds such as alkaloids, hydroxylated quinoline and pyridine derivatives and antibiotics, not dealt with in previous investigations reported earlier in these Reviews, have now been included. The R F values of these compounds in six different solvent systems together with the corresponding colour reactions produced by ten standard reagents have been determined and are presented in 15 tables. Altogether approx. 270 compounds have been investigated and 20 hippuric acid derivatives synthetized. Application of these procedures to the clinical-chemical analysis of urine samples in order to detect metabolic excretion or detoxification products is indicated. The hippuric acid reagent was modified so that it contained p -dimethylamino-cinnamaldehyde in acetic acid anhydride. The Ehrlich reagent was prepared on the basis of p -dimethylaminocinnamaldehyde in hydrochloric acid; the colours produced with this reagent made it possible to differentiate the indole derivatives more distinctly. The relation between the chemical structure of the compounds studied and the R F values and colours produced with the detection reagents, is discussed.

A study is described of the chromatography on paper of some alkylhydrazines and some monoand diacylhydrazines by both the circular and ascending methods. For the acylhydrazines the solvent system preferred was the upper layer from isoamyl alcohol, acetic acid, and water in the volume proportions of 10:1.5 :10 respectively. The upper layer from a mixture of n -butyl alcohol, acetic acid, and water in the volume proportions of 4 : 1 : 5 respectively, was preferred for the alkylhydrazines. For detection of the hydrazine derivatives on the chromatogram a spray of p -dimethylaminobenzaldehyde hydrochloride in ethanol (Ehrlich's reagent) was most satisfactory, although ammoniacal silver nitrate or ninhydrin could be used in some cases.

The electrical migration of tetra-valent metal ions, Ge[IV], Ti[IV], Zr[IV], Th[IV] and U[IV] was observed by means of paper electrophoresis, using tartaric acid and tartrate or acetic acid and acetate buffers as background electrolytic solutions. These metals formed with the tartrate negatively charged complex ions which migrated toward the anode, but not with the acetate except the U[IV] ion, which was gradually oxidized to UO2+2 ion and then formed complexes with the acetate. The ratio of ionic radii of tartrate complexes was approximately estimated from the ratio of their migration distances under the same conditions.

A simple method for the iodination of proteins of the aqueous humour with radioactive iodine 131I is described. After iodination of the proteins with radioactive iodine the aqueous humour is analysed by electrophoresis on paper strips saturated with calf-serum in veronal buffer of pH 8.5 with ionic strength of 0.07 μ at a potential drop of 3 V cm−1. The time of electrophoresis is 18 hr. The electrophoreograms are dried and washed three times with a solution of acetic acid in dilute ethylalcohol, dried again and evaluated by direct measurement of the radioactivity of the 131I ohemically bound to proteins of the aqueous humour or by photometric measurement of the autoradiograms obtained by contact exposure of the eleotrophoreograms on X-ray film. The method is very sensitive; it is therefore sufficient to use 10 to 20 μl of aqueous humour for the iodination. Ten to 20 μ of the iodinated aqueous humour, i.e. 4 to 8 μl of the original aqueous humour with a protein content of 2 to 5 μg are applied to the paper. The results of the eleotrophoretic analysis of human, rabbit and bovine aqueous humour are in good agreement with the results of analogous analyses carried out by other methods.

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.

A paper partition chromatographic procedure is described for the separation and identification of aryldiamines, aminophenols and polyhydric phenols used as colouring agents of oxidation-type hair dyes. Twenty-nine compounds were chromatographed on Whatman No. 3 MM paper using, as developing solvent, the top layer of a mixture of 500 ml n-butanol, 100 ml ethanol, 400 ml water, 100 ml acetic acid and 10 g sodium sulphite. Good separations of most combination of compounds were achieved. Where resolutions could not be accomplished, positive identifications could be made through the use of several detecting reagents. The application of this procedure to the analysis of colouring agents of hair dye preparations is described.

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

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.

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.

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.