Multiple-Paper Chromatogram WILLIAM L. POR Exstern Regional Research Laborotoi
A method was required by which mixtures of organic acids or alkaloids in semimicroquantities wuld be resolved into their pure components to yield sufficient amounts for the preparation of derivatives for identifieation. A new apparatus, the “Chmmatopack,” consists of a pack of Whatman No. 1 filter paper strips wmpressed between two stainless steel plates. The zones obtained from mixtures of pure compounds were separated and yielded up to 10 mg. of each constituent. The method lends itself to easy detection of zone positions with resolution equal to that obtained with single sheets of paper but producing semimicro quantities of pure materials. This extends the usefulness of paper ehmmatography from a submicroqualitativetool to a semimicropreparativetool.
T
water, is carefully added to the tray and the chromatogram is developed until the solvent front has moved to just below the top of the pack. A t this time, the pack is removed, the plates are loosened, and the filter uauer uack is withdrawn. A sheet from’e&h &de and from the center of the pack is removed, dried, and sprayed far detection of the hands. The pack is cut and the different eones are isolated. These fractions are eluted and subieeted to qualitative analysis. No quantitative analyses have been carried out to date, but several investigations are in progress.
HE usefulness of the papergram technique in the resolution of mxtures of many kinds of materials has been estahlished. The original technique of Consden et al. ( 1 ) has been modified in order to handle smaller and smaller amounts of materials (6). However, for preparative work and for identification experiments, where known materials are not availahle, these procedures are not satisfactory and modifications such aa the “Chromatopile” (S,4 ) have been adapted. In this laboratory the Chromat,opile has proved somewhat inconvenient bemuse of the formation of zones shaped like “inverted cones,” which made it difficult to separate the fractions, and the operation of detecting the mnes hy removing m n y sheets from the pile was relatively tedious. As a remilt. s. new ____,
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EXPERIMENTAL
simplest apparatus and lends itself to easy detection of the zones and to their ultimate isolation and elution. The setup has been termed a “Chromtttopack.” A photograph of the Chromatopack is shown in Figure 1.
The technique was tested using dyes, nonvolatile organic acids, and tobacco alkaloids. Chromato scka consisting of 200 strips (18 X 2 incges), each containing the solute from 0.05 ml. of sample solution, were assembled, placed in the glass cylinder, and developed until the solvent front had moved in excess of 28 cm. At thie time the pack waa disaasemhled and the test sheets were removed, dried, and sprayed for detection of the zones.
It consists of a sck of long Strips of Whatman No. 1paper (18 X 2 inches or wiser) clamped between two stainless steel plates. The strips are cut from the usual 18.25 X 22.5 inch sheets by means of a paper cutter. For use, the sample is placed on a line 2.0 em. from one end of each of 100 or more strips, using about 0.01 ml. of solution per centimeter of width, A No. 26 hypodermic needle and aO.5-ml. syringe with the plunger removed are used to distribute the mme solution. Ten blank strips are placed on each side of the pack of sample strips, The entire pack is carefully aligned and placed between the stainless steel plates, so that the end on which the sample was deposited is ahout 5 mm. from the end of the plates. After tighteuing the nuts to compress the sheets, the entire assembly is placed upright in a 12 by 24 inch (30 X 60 cm.) glass cylinder with the sample end resting in a stainless steel tray. The solvent, either the organic phase sf the equilibrated two-phase solvent or a one-phase solvent containing
Figure 1. FilterPaperChromat opaok after Removal from Solvent Chamber
The individual components of the mixtures, their concentration, range of spot movement, and their Ra values, a8 well as the solvent and spray materials, are summarized in Table 1. DISCUSSION
The procedure of placing the sample an the separate sheets is nomewhatt&ous,hutit can heeased hy placing the sample on the I& .; BheeL of paper (18.25 X Table I. Summary of Experimental Results Obtained Using Chromatopack 22.5 inches) prior to cutting Type of Camwonents Range of Solvent Solvent. spray the strips. If precut strips Mixture of Mirturea Concentration Spots Front Cornpostwn Mixture are available, a microburet Pork 07 rnl. M d d . em. Cm. RF having a mechanical delivery 0.3-1.8 28 0.04 l-Butanol 40 None needed Dyes Fuchsin G ? 10.6-12.4 0.41 Abs. ethyi aloahol. 10 attachment can be used. The Methylene blue Cry.thl violet 5 22.9-28.0 0.91 water, 50 relative inefficiency of this step Organia Tartaric 5 11.0-14.5 33.1 0 . 4 0 5 Mformioaeid, 50 Bromaphci8 more than overoome by the acids no1 blue Malic 15 16.9-18.7 0.53 1-Pentenol.50 ease of assembly, detection of S”CCiIliC 15 20.9-24.3 0.68 Addnl. aba. ethyl aloohoi t o form one aone position, and isolation of phase the pure components which Tobacco Nornicotine 5 0.3-4.0 34.0 0.06 1-Butanol.85 Iodine have been resolved from the alkaNicotine 5 7.5-19.2 0.36 Benzene 5 loids Niootyrine 5 21.5-31.1 0.80 ButTera,’30 mixture. In the preliminary experiMixture of 9.5 ml. of 0.2 M acetic acid, and 90.5 mi. of 0.2 Msodium acetkte: pH. 5.6 (6) ments with the organic acids, 0
412
V O L U M E 23, NO. 3, M A R C H 1 9 5 1 the organic phme of the twc-phase system of Lugg and Overell (2)contained insufficient formic acid to suppress the ionization of the organic acids and resulted in tailing and poor resolution. the formation of a Addition of enough ethyl to aingle-phase system produced the results shown in Table I. ACKNOWLEDGMENT
The author expresses his appreciation for the help given by Margaret L. Buch in the cvperimental development of the Chromatopack technique.
413 LITERATURE CITED
(1) Consden, R., Gordon, A. H., and Martin. A. J. P., B i o c h . J . , 38, 224 (1944). (2) LUgg, J. w. H., and Overell, €3. T., Australian J . SCi. Research, Series A,1, 98 (1948). (3) Mitchell, H. K.,and Haskins, F. A , , Science, 110,278 (1949). (4) Hitohell, H. K.,Gordon, H.. and Haskins, F. A., J . BioZ. Chem. 180, 1071 (1949). ( 5 ) Porter, W. L., Naghski, J . , and Eisner, Abner, Arch. Biochem., 24, 461 (1949). (6) Rockland, L. B., and Dunn, JI. S., Science, 109,539 (1949). RECEIVED July 19. 19jO.
Paper Chromatography of Organic Acids .1. B. STARK, A . E. GOODBAN, AND H. S. OWENS Western Regional Research Laboratory, .41bany, Calif. The study of the paper chromatography of organic acids was undertaken to provide a simple method for the rapid identification of organic acids in sugarbeet processing liquors. The use of several developing solvent mixtures has been investigated. The RF values for 18 organic acids were measured. The effect on RF values of hydration of paper, water content of solvents, temperature of development, and presence of inorganic acids was studied. In many cases an inversion of RF values was obtained by altering the composition of the developing solvent. The method is suitable for detection of many organic acids o r their salts in plant extracts or other biological media. The presence of acid impurities or their production as reaction products may he detected.
A
INVESTIGATION under way in this laboratory on changes in composition of sugar-beet processing liquors required a technique suitable for rapid and accurate identification of organic acids. The detection and identification of amino acids and other compounds by paper chromatography (3-6, 10, 11) suggested its application. During this work, methods were published for paper chromatography of acids in the form of derivatives (2, 6) or labeled with radioisotopes ( I ) . The authors have studied the behavior of several nonvolatile acids in various solvent mixtures containing formic or acetic acid (9). The method discussed here is similar to that of Lugg and Overell (8, 9) but offers some advantages in identification. It hss been used to identify some of the acids in sugar-beet processing liquors and to determint= the efficacy of fractionation proredures. ,"i
METHOD
The paper sheets, 40 X 57 cm., Whatman No. 1, were developed by an ascending technique similar to that described by Williams and Kirby (18). Glass jars, lo1/* X 18 inches, with glaas covers were used to contain the solvent and paper. The sheets were clipped to a hexagonal rack made from 5-mm. glass rod. Only one solvent phase was present in the developing tank. The paper sheets were not humidified, nor was the temperature of the developing bath controlFd; hfwever, the laboratory temperature averaged about 25 * . 3 C. The papers were spotted with 0.003 to 0.01 ml. of solution 3 cm. from the bottom and 2 cm. apart. The standard acid solutions were 0.075 N . Development time was usually 16 hours, but periods as short as 2 hours were also satisfactory. Following development, the papers were removed from the tank and either dried overnight in a hood or for 45 minutes at 100" C . in a circulating air oven. Better definition of spots was usually obtained on papers dried a t room temperature; some acids are volatile or subject to decomposition at higher temperatures. Occasionally it was found advisable to hydrate the paper by exposure to water vapor before drying was finished, to remove traces of some acidic substances that might be present or formed on heating the paper and solvent. The position of the acids on the chromatogram was shown by spraying the dried paper with
either bromocresol green or bromophenol blue, 400 mg. per liter in 95% alcohol. The indicator solutions were made slightly alkaline with sodium hydroxide. The acid spots were yellow against a blue or purple background. They were circled and the centers marked. RF values were calculated for the individual spots. Many of the solvents contained alcohols, and some esterification with formic or acetic acid took place. The small amount of esters present did not appreciably affect the RF values, but the reduction in acid concentration caused considerable tailing. This was not serious during a period of a week or two, and if the solvent was not replaced in this time, a portion wm titrated and make-up acid added. Every month the solvent should be replaced by fresh solution. The solutions were made up in stock bottles and the formic or acetic acid was added when the solvent was placed in the jar. Solvents should be checked for the prep ence of nonvolatile acids and purified, if necessary, before use. EFFECT OF CONDITIONS OF EXPERIMENT
Effect of Hydration of Paper, Temperature of Development, and Water Concentration. A preliminary study wm made of the effects of hydration of the paper, of changes in water concentration in the phenol-water-formic acid system, and of temperature during development in solvents F and G. These results are shown in Table I. In general, humidification of the paper has an influence similar to increasing the water concentration of the developer-that is, the RF values of acids such as citric and aconitic are increased, while those that are high generally remain practically unchanged. In most cases temperature changes have only a slight influence. Becauee of the slight effect of temperature change and humidification, the chromatographic papers were not humidified and development was a t room temperature. Acid Concentration. Several acids were developed with 75% phenol and 25y0 water containing 0.2, 1, and 2% acetic acid.
