AIDS FOR THE ANALYST Multiple-Color Development Method for Use in Paper Chromatography. Charles C. Woodward and Glenn s. Rabideau, The Plant Research Institute, The University of TexaR, and Clayton Foundation for Research, Austin, Tex.
Many atteiiipts were made to improve the degree of separation of the.se compounds on the diromatogram. Present results indicate, hoxever, that where spots of bubstances of different types of compounds-eg., sugars, organic acids, or amino acids-overlap, they do not interfere with the determination of compounds in the other groups because. as each of these groups is determined sequentially, the previori4y indirated spots fade.
chromatography Kith filter paper is being employed P widely for the tentative identification of organic compounds of plant or animal origin. In order to identify several different types ARTITION
of metabolic products using a single chromatogram, a method is here reported by which amino acids, organic acids, and sugars can be detected using, sequentially, three color-developing indicators. Further treatment affords differentiation between I educing and nonreducing sugars.
Polyethylene-Coated Glass Flasks. George .4. Simmons, Jr., Department of C'hemiPtr>, Hi~iiiiiigham-Souther~~ rnllege, Birmingham 4,Ala. author was recently coufror~ted with the problem of vacuum-evaporating a strongly caustic solution without introducing silica. Obviously, a glass flask could not be used for the till pot. Polyethylene plastic bottles, although chemically inert and reasonable in cost, are so flexible that they collapse under pressure. A polyethylene-coated glass flask would combine the strength of glass and the inertness of the plaatic. The common techniques of coating by painting, spraying, or dipping could not be used, because polyethylene is resistant to practically all solvents and does not melt to a free-flowing liquid. The plaatic would adhere when brought in contact with hot glass, but coats formed by this simple process tended to separate from the glass when the temperature varied and could not be replaced. The following procedure gives a uniform and complete coat which does not loosen with temperature fluctuations. HE
A two-dimensional chromatogram was made using a sample of the alcohol extract of plant or animal material. The following solvents were used: (1) 3 grams of phenol, 1 ml. of water, and a volume of 90% formic acid equivalent to 1 volume yoof the he nol-water mixture; and (2) 1 ml. of isopropyl alcohol, 1 my 0; fed-butyl alcohol, 3 ml. of benzyl alcohol, 1 ml. of water, and i: volume of 90% formic acid equivalent to 1 volume % of the wateralcohol mixture. The chromatograms were dried after solvent development, spraved with a 0.2% ninhydrin [Dent, C. E., Biochem. J.,43, 169 11948)] solution in water-saturated I-butanol and dried a t 70' C. in an oven. S ts denoting amino acid iocations werr then marked with a sok?lead pencil, their identities established, and concentrations evaluated by visual comparison with chromatograms prepared by using standard samples of pure amino acids. Orgmic acid identifications were made by spraying the same chromatogram with 0.1 weight-volume % solution of methyl red in alcohol. This solution was adjusted with sodium hydroxide to a pH where the solution just turned orange on the paper. Methyl orange was also used as an organic acid indicator, but has the disadvantage of giving less intense spots than does methyl red. The chromatogram was allowed to dry in air and spots denoting organic acids were marked and evaluated for identity and concentration by comparison with spots produced by standard saniplee of pure organic acids similarly treated. Following acid detection, 3,5-dinitrosalicylate reagent was sprayed on the paper for the identification of sugars [Jeanes, hllene, Wise, C. S., and Dimler, R. S., ANAL.CHEW,23, 415 (1951)]. The chromatogram was air-dried and placed in an oven a t 105' until the dark brown spots, denoting reducing sugars, reached maximum intensitv. The chromatogram was then stored in darkness at room temperature for 2 weeks, during which time the nonreducing sugars appeared as gray-green colored spots. The original reducing sugar spots remained brown. Both a t the beginning and a t the end of this storage period the kinds and concentrations of sugars present were interpreted by comparison with spots produced by standard samples of pure sugars similarly treated.
The plastic is obtained as a granular molding powder (Du Pont's Alathon 1-P 1000 uncompounded polyethylene resin). These granules are placed, with about 1 part of ice water to 2 parts of plastic by volume, in a Waring-type blender in sufficient quantity to cover the mixing blades. The plastic is shredded to a fine powder, which, with the proper proportion of water to plastic, will creep up the glass walls of the blender. If too much water is present, the powdered plastic does not separate well from the granules; and if too little is present, the plastic forms a gummy mass. This finely powdered plaatic is periodically scraped from the walls of the blender and air-dried. The glassiffask is h a t e d to about 150' C, and then rotated while the powdered plastic (about 100 ml. for a 1-liter flask) is poured into it so that the plastic comes in contact with the area to be covered. A layer of plastic adheres at once, and the excess is poured out. The flask is heated in an oven to about 250' C. until the molten plastic begins to smoke and discolor. Slight air pressure is applied to flatten the globules of molten plastic into a smooth coat. A rubber stopper larger than the mouth of the flask can be used for applying the pressure. A glass tube through the stop er is connected to a compressed air line and pressure is ap lie$ with the hand by holding the stopper against the mouth o t t h e flask, which is wrapped with a cloth. The flask is heated to about 150' C. to remelt the cooled plastic and more powdered plastic is added as before, forming a second layer. This layer is melted at 150' C. and again smoothed with pressure. This results in a coat of plastic which is 1 to 2 mm. thick. If a thicker coating is desired, more layers of plastic may be added in the same manner.
Relative sensitivity of the color-developing spray reagents afforded accurate identification with the following minimum concentrations: anlino acids, 1 to 5y, organic acids, about 257, reducing sugars about 157, and nonreducing sugars 60y, per chromatogram. Although these relative sensitivities are somewhat below figures quoted elsewhere, some sensitivity was sacrificed in favor of the advantage of the multiple detection of substances on a single paper chromatogram. A better interpretation of organic acids present in low concentrations could be made, if, in addition to the regular two-dimensional chromatogram, a duplicate sample in one corner of the chromatographic paper was used for a one-dimensional chromatogram. When the &st development was complete and the paper airdried, the one-dimensional chromatogram strip was cut off and sprayed for organic acids with 0.1 weight-volume Cr, solution of bromocresol green or methyl red in alcohol. This afforded a better evaluation of the organic acids present in low concentration because of less diffusion of the spots.
The success of this procedure is due to the heating of the f i s t plastic layer to 250" C., creating a very strong bond between the glass and plastic. It is not advisable to heat subsequent layers to this temperature, especially the final layer, as the slight decomposition which occurs may destroy some of the chemical inertness of the plastic. A Miter round-bottomed flask coated by this method to a thickness of 1 to 2 mm. has been used repeatedly at temperatures from -20" to 50" C. with no signs of chemical attack or separation of the plastic. Such coated flasks are also shatterproof and show a definitely inrreased resistance to breakage. 248
