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Thirty-seven paper and thin-layer chromatographic systems in general use for the analysis of basic drugs have been examined. Their discriminating powers were measured, both individually and when used in combination. The better systems were found to be thin-layer systems of silica gel sprayed with 0·1n NaOH, dried and run using one of the following solvents: (1) chloroform-methanol (90:10), (2) cyclohexane-toluene-diethylamine (75:15:10) and (3) acetone. A thin-layer cellulose system using n-butanol-water-citric acid (87:13: 0–48) was suitable if speed was not a requirement and a reversed phase paper system run with an aqueous buffer solution (pH 4·58) at 95† was the fastest system examined. Any of these five systems could be used in combination since their correlation coefficients were never higher than 0·61.

The nature of the artifacts formed in the paper chromatography of solutions of catechol amines, histamine, and various tryptamine derivatives in trichloroacetic acid (TCA) has been considered. With TCA solutions of histamine the artifact is not the corresponding N-acyl derivative. It is suggested the production of these artifacts is assisted by the formation of molecular complexes between the above compounds, acting as electron donors, and the TCA acting as electron acceptor. A solvent is described which eliminates multiple spot formation when TCA solutions of the above type are subjected to paper chromatography

A reliable method for the determination of the amylose and amylopectin fractions of starch has been developed using paper chromatography. Starch is separated into amylose and amylopectin fractions on a filter paper strip by descending paper chromatography in iodine vapor, using 40% HClO4 solution as the solvent. These fractions are collected separately by irrigating the strip with 40% HClO4 solution. After adding iodine and potassium iodide to the eluate, the iodine complexes of these components are collected by centrifugation and are treated to remove the iodine. The concentration in these iodine-free fractions is determined colorimetrically by the anthrone-sulfuric acid method. In this way, the anlylose and amylopectin contents of starch are determined without reference to authentic amylose and amylopectin preparations separated from the same starch specimen by the Schoch method. The values by this method represent more true starch contents than those obtained by the other current methods.

This paper reports minimum quantities, in micrograms, of 22 organic chlorinated pesticides that can be detected by the silver nitrate/2-phenoxyethanol chromogenic agent, both before and after chromatographing, and after the papers have been washed with water to remove excess silver nitrate.

A simple quantitative chromatographic method is described for the separation of epitetracycline and tetracycline in pharmaceutical preparations. The method is applicable qualitatively to human serum and quantitatively to the biological determination of tetracycline in urine. Whatman's modified cellulose phosphate cation-exchange paper is developed by the descending technique using 0·1 per cent w/v ammonium chloride solution. The spots are located as yellow fluorescent spots under ultra-violet light and ammonia vapour, eluted in 10 per cent w/v ammonium chloride, and the extinction determined at 356 mμ. The method is not suitable for the separation of chlortetracycline or oxytetracycline from tetracycline.

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.

A simple and highly sensitive method for the identification and quantitative estimation of 5-hydroxyindoleacetic acid (5-HIAA) concentration in urine is described. 5-HIAA was concentrated following extraction into ethyl acetate, and separated from other indolic acids by paper chromatography. A color reagent, p-dimethylaminocinnamaldehyde, was used to detect 5-HIAA on paper, in concentrations as low as 0.1 µg./cm.2 Urinary excretion levels could be measured below the normal range as well as in the normal range and above.

A method is presented for the concentration of native gastric juice by dialysis against a 30% solution of Carbowax 6000 in distilled water, prior to paper electrophoresis. Detailed descriptions of the dialysis procedure and subsequent vertical paper electrophoresis are given. The data show that the dialysis procedure is comparable to the lyophilization technic and has the advantage of faster processing, which makes the method applicable to routine clinical use in many diagnostic applications.

Some of the paper chromatographic techniques and detection methods used for pesticide residue analysis are reviewed. Procedures utilized for quantitative estimation pesticide residues separated by paper chromatography, such as measurement of spot size, combusion prior to analysis, and elution before chemical analysis, are also discussed. The advantages and disadvantages of paper chromatographic techniques and the problems inherent in the paper chromatographic separation and detection of minute amounts of pesticide residues are outlined. Additional applications and modifications of paper chromatography to extend its usefulness in pesticide residue analysis are suggested.

The Panel on the Laboratory in Biology was formed by the Commission on Undergraduate Education in the Biological Sciences (CUEBS) with the charge of clarifying the function of the laboratory in the changing biology curriculum. This paper, the first product of our deliberations, seeks to make a single point which we feel is of such great importance to the future of undergraduate biology that all other considerations in laboratory instruction must be deemed inconsequential beside it. Stated simply, the point is that the best use of the laboratory in undergraduate instruction is to engage the student in the process of active investigation. This paper will be devoted, therefore, to the development and support of the concept of such a laboratory program, which we will refer to as an "investigative laboratory." Additional papers, describing specific investigative laboratory programs in which three of the authors of this paper have participated, will appear separately.

It is difficult to relate extractable auxin directly to the growth of plant tissues. The diffusible auxin, on the other hand, can be correlated with the growth in younger stem tissues (2, 6). The chemical nature of this important substance is poorly characterized. Using paper chromatography Raadts and Soding (5) identified part of the diffusible auxin from the Avena coleoptile tip as IAA and reported the presence of another diffusible auxin (Wanderung auxin). By paper chromatography Shibaoka and Yamaki (9) could not find the Wanderung auxin. They found only IAA. The diffusible auxin from sunflower leaf (8) and Coleus stem tip (7) has been reported to be IAA also by paper chromatography. The present study was undertaken to examine the nature of diffusible auxin in etiolated and green plants, and to determine the effect of light on the diffusible auxin.

A paper chromatographic method for the analysis of barbiturate in blood is presented. The Rf values and ratios of 8 standard barbiturates are given and compared with the values obtained from patients suspected of having taken barbiturate overdoses.

A method for paper electrophoresis of hemoglobins in a discontinuous buffer system is presented. Tris-EDTA- borate buffer 0.12M., pH 9.1, is applied to the paper. Barbital buffer 0.06 M., pH 8.6, is used in the buffer vessels. The discontinuous buffer system enables the detection of hemoglobin A2 in small samples of hemolysate. Resolution of abnormal hemoglobins is superior to that in barbital or Tris-EDTA-borate buffer alone. It offers special advantage for the examination of aged samples of hemoglobin. Reasons for the behavior of the hemoglobins in this buffer system are discussed.

A simple, rapid, and efficient method has been developed for determining biphenyl in citrus fruit and in biphenyl-impregnated kraft papers. Biphenyl is separated from citrus tissue by steam distillation and from biphenyl-impregnated papers by solution in iso-octane. Plastic sheets coated with silica gel, to which a polyvinyl alcohol binder and a fluorescent indicator have been added, are used for cleanup by thin layer chromatography (TLC). The TLC spots are extracted and measured spectrophotometrically at 248 m/µ. Recoveries of added biphenyl ranged from 92 to 98%. The new method is simple and brief; it is more accurate than cleanup by oxidative degradation since the variable background is practically eliminated. The new method is easier and faster than the silicic acid chromatostrip technique, and is just as accurate.

The results obtained here consistently failed to show deviations from Beer's law for optical densities less than 1.4, and the use of scanning as a convenient method for differential protein estimations would appear to be justified. The methods described may enable other workers to make similar tests of the method with a minimum of preliminary development, and some may succeed in obtaining significant failure of Beer's law in their apparatus. The use of apparatus of this type would enable the variation of the deviations with arrangement of the optical system, etc., to be worked out, but the following experimental conditions are already known to lead to significant deviations: 1. Use of dry paper instead of lightly oiled paper. 2. An inadequate light ifiter for the photocell. 3. An illuminated slit too long to be completely covered by the protein pattern. 4. Variations in protein density over the slit, either because the slit is too wide or because the protein pattern has been applied unevenly. 5. Use of low quality ifiter paper containing "pin holes." 6. A dye uptake which is not proportional to protein content of the paper [Martin and Franglen (14)]. The failure of Crook, Harris, and Warren (4) to substantiate Beer's law does not indicate the most general situation for the application of the scanning method. The application of Beer's logarithmic law to the scanning of dyed protein patterns has been investigated by methods described in detail. No deviations could be found for optical densities less than 1.4 for amidoschwarz 1OB or less than 1.1 for azocarmine B staining. The scanning method can be used for evaluation of protein fractions if care is taken.

Rm values have been determined for four homologous series of androgen esters, using a Bush paper chromatography system, to assess the value of the ratio of the solubilities in the individual solvents (solubility ratio), as an estimate of distribution coefficient. The distribution coefficients were proportional to Rm values in all four series, and for three of these the proportionality constant was the same. Limitations in the use of RM values to predict biological effects have been pointed out.

Filter paper has been tested as a medium for the collection of blood samples for the subsequent elution and analysis of phenylalanine, tyrosine, glucose, and urea nitrogen. The use of paper did not measurably alter the precision of the above methods. The paper punch as a means of quantification has been compared with the more commonly used technic of volumetric measurement. The precision obtained with punched spots was found to be similar to that found with measured spots for the above four procedures and for weighed samples. The hematocrit value was found to have an influence on the volume of blood contained in a punched spot. Differences in spreading through filter paper between 100 individual blood samples tested did not seem to be responsible for a variance in excess of that introduced by the analytic procedures themselves.

Procedures have been described for testing for phenylpyruvic acid, sugars, protein, and chondroitin sulfuric acid in urine dried on filter paper. Additional paper chromatographic methods for identification of phenylalanine, -hydroxyphenylacetic acid, glucose, and galactose have been described.

By the mid-197o's, an estimated 63 million Americans will be enrolled in the nation's schools and colleges. Total attendance in higher education alone will exceed i61,2 million. With almost one million earned baccalaureates, over a quarter of a million master's degrees, and nearly 50,000 doctorates awarded each year, servicing the academic world will be lucrative for many educational vendors, including equipment manufacturers, textbook publishers, as well as a new breed of supplier-commercial firms which provide ghost-written term papers for students.' For years, educators have known about fraternity files and the exchange of papers among students. The assistance rendered by relatives and friends in the preparing of assignments has sometimes been met with academic oversight and even acquiescence. Only the most flagrant cases of plagiarism from known and traceable published sources have resulted in disciplinary action (warning, reprimand, grade reduction, failure, revocation of privileges, probation or, in rare cases, suspension or

A procedure is described for the determination of polychlorinated biphenyls (PCBs) in paper and paperboard. PCBs are extracted from the paper sample by reflux with alcoholic KOH. Column chromatography on Florisil is used for cleanup, if needed. Compounds are determined by GLC with electron capture detection, using conditions previously devised for organochlorine pesticides. An exhaustive extraction utilizing sulfuric acid was developed in order to obtain comparative data on extraction effectiveness. Recoveries of Aroclors 1242 and 1254 from fortified paper samples averaged 95.5 and 96.8% with the alcoholic KOH reflux and sulfuric acid digestion extractions, respectively. Aroclor 1242 determined after alcoholic KOH extraction of paperboard was 96% of the amount found after sulfuric acid digestion of comparable samples. Aroclor 1242 had been incorporated into the paperboard during manufacture.

A reproducible technic is presented for the staining of lipoproteins on paper. This method gives a greater total uptake of dye than previously described procedures, especially by the α-lipoproteins. The staining is accomplished with a solution of Sudan Black B in a mixture of methyl and isopropyl alcohols and the excess dye is removed by wash solution of 30% isopropanol.

We describe a quantitative paper-chromatographic method for assaying 3-methoxy-4-hydroxymandelic acid (VMA) in urine. VMA is extracted from acidified urine into ethyl acetate, separated from interfering amine, acid, and alcoholic metabolites by paper chromatography, and reacted with diazotized p-nitroaniline. The colored derivative is eluted and spectrophotometrically analyzed. Recoveries of replicates of standard and sample solutions on the same chromatogram exceeded 95%.

Previous methods for estimating vanillin and ethyl vanillin in mixtures were long and complicated. However, this paper presents a simple chromatographic separation and quantitative determination of these aromatics, using Mitchell equipment for paper chromatography and 8 × 8” papers. Development for 2 hours separates the compounds adequately; they are then extracted and measured by absorbances at 348 mμ in alkaline solution. Recoveries of added vanillin and ethyl vanillin to vanilla extract are excellent. Aside from normal manipulative errors, the only significant inaccuracy is caused by the small amount of natural p-hy-droxybenzaldehyde in vanilla (usually equivalent to about 5% of the vanillin content). The method does not separate p-hydroxybenzaldehyde and vanillin. Total errors in the method do not exceed 0.01%. Comparison of the method with the two AOAC methods for vanillin shows that the paper chromatographic method gives results close to those by the ultraviolet absorption method, in most cases, and significantly lower results than the photometric method, which is known to give erroneously high results.

The alkaloids selected were atropine, cocaine, codeine, morphine, dionine, dilaudid (dihydromorphinone), heroin, strychnine, ineperidine and papaverine. After the alkaloids (100 pig. in a 2.5% solution of appropriat.e solvent) had been applied at the bottom of rectangular sheets of filter paper, they were chromatographed in a container made from a conventional aquarium covered with a heavy glass plate. Since variations � � �

The identification and determination of vanillic, homovanillic, p-hydroxybenzoic, p-hydroxyphenylacetic, m-hydroxybenzoic and o-hydroxyphenylacetic acids by the use of two-dimensional paper chromatography has been investigated

1. A modification of the usual paper electrophoretic procedure has been made by including in the buffer medium 0.05 per cent of certain alkylphenyl ethers of polyethylene glycol. 2. This modified procedure yields electrophoretic patterns of blood serum proteins that may be subdivided into 12 fractions— albumin, α1-globulin, three α2-globulins, three β-globulins, and four γ-globulins. 3. The method was applied to blood serum from 70 patients apparently free of significant disease and to sera from a number of patients with varied disease. More specific location of the altered protein levels was possible in a number of disease states. 4. Glycoprotein distribution was not seriously disturbed by the presence of the nonionic surfactants, but lipoproteins migrated at a decreased rate.

Investigation of various steps involved in separation of serum proteins by paper electrophoresis and their quantitation by dyeing with bromphenol blue has revealed that the results obtained are quite dependent on a number of variables. It is essential, therefore, that rigid control of these variables be exercised and that normal values be obtained by the specific technic adopted. Normal values have been given for the technic employed and data on stability of sera at room and refrigerator temperatures have been presented.

When solutions of adrenaline, noradrenaline, histamine, 5-hydroxy-tryptamine and related amines in trichloroacetic, trifluoroacetic or picric acid are chromatographed in various organic solvent systems, the active material divides into two areas. If a basic amino acid is included in the solution before chromatography, the separation becomes more complete. It is suggested that the second area consists of a loose complex between the amine and the corresponding acid. When solutions of the amines in hydrochloric, acetic or oxalic acid are chromatographed, the active material resides only in one area.

An easy procedure for the estimation of 3,4-dihydroxyphenylalanine (dopa), 3,4-dihy-droxyphenethylamine (dopamine), and homovanillic acid (HVA), besides 3-methoxy-4-hydroxymandelic acid (VMA), by means of the "two solutions paper electrophoresis" (1) is described. Increased amounts of these phenolic compounds were found by this method in the urine of a patient with neuroblastoma, and the case report is presented.

In an attempt to screen children for amino-aciduria related to various inborn errors of metabolism, a two-dimensional paper chromatography method was developed. This method requires no expensive equipment, resolves a standard mixture of 17 α-amino acids, is consistently reproducible, and requires less than 7 hr from start to visualization of the chromatogram.

A one-dimensional paper chromatographic method is presented for determination of urinary homovanillic acid. The method is faster and gives higher values than does two-dimensional chromatography. Urinary homovanillic acid values are given for patients with metastatic malignant melanoma and for presumed normals. Homovanillic acid level was elevated in the case of amelanotic melanoma. Determinations of urinary homovanillic acid may be a valuable adjunct to the therapy and study of metastatic malignant melanoma.

Studies of serum glycoproteins by paper electrophoresis in rheumatic fever showed the following: Total glycoproteins were generally increased in concentration, the alpha-2 glycoprotein fraction showed moderate to marked increases in value, and alpha-1 and beta glycoprotein components generally showed moderately decreased concentrations.

Analytical difficulties in the analysis of D&C Red No. 21 by column chromatography are overcome by using an ascending paper chromatographic technique, based on that of Graichen, followed by elution of the zones and spectrophotometric determination. Results compare well with those by column chromatography.

A modification of McDonald's method for the determination of serum lipoproteins is described in which serum is prestained with Sudan Black B dye before paper electrophoretic separation. Normal values were determined in 24 adults. Average value for α lipoproteins was 39.2 per cent and for β lipoproteins 60.8 per cent. In 31 patients with proved coronary artery disease, 22.7 per cent α lipoprotein and 77.3 per cent β lipoprotein were found. These results are in general agreement with those of other investigators using different methods.

The migration distance of several scores of dyes in 30% and 4% acetic acid solutions in a horizontal-type apparatus permitting evaporation was measured. In these occasions, an extremely relationship was found to exist between the degree of influence of osmotic flow into filterpaper due to evaporation from the paper surface and the displacement ratios obtained from adsorption analysis with the same filterpaper.

A procedure is presented for the determination of 3-methoxy-4-hydroxymandelic acid (or vanillylmandelic acid) in urine. For separation from other phenolic acids, paper electrophoresis at low constant current is employed. Data are presented to show that this method is useful as a confirmatory test for pheochromocytoma, in which elevated urinary levels of vanillylmandelic acid are found.

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A new technic has been described for the chromatographic separation of estrogens. It includes the use of a dual-purpose system where the impregnating material of the stationary phase (p-toluene sulfonic acid) is used as a color reagent after the separation is complete. This novel approach precludes the need for a spraying or dipping step to achieve the desired color reaction. Quantitation by densitometry and fluorescence is also detailed.

Nine substituted urea herbicides and/or their TCA salts and TCA can be separated from one another by the use of two solvent systems, one aqueous and the other nonaqueous, with the exception of monuron and 3-(2-chlorophenyI)-l-methylurea; the latter, however, is not a commercial pesticide. The compounds are detected in the chromatogram by screening under ultraviolet light, except TCA, or by spraying with the silver nitrate-2-phenoxyethanol chromogenic agent followed by exposure to germicidal ultraviolet light, except fenuron and the fenuron moiety in fenuron TCA.