394
ANALYTICAL CHEMISTRY
boiling fraction may be obtained in the case of certain resins, or an impure material may be purified by the process. INSTRUMENTATION
For the benefit of those who have had limited experience with vacuum systems, the authors include a short description of the system currently used.
’
The most important feature is the filament-heating circuit which consists of a Type V-10 Variac whose secondary taps are connected to the primary of a 1-kv.-amp welding transformer (Eider Engineering, 760 South 13th St., Newark, N. J.). The secondary coil of this transformer has 10 turns of 6/8 X 3/g inch copper busbar with a connecting tap on every coil. With 110 volts on the primary, secondary voltages from 0.7 to 7 volts in 0.7-volt steps can be selected a t will. Storage battery cables connect the binding posts of the evaporator with the welding transformer. This circuit permits one to bring to white heat a tungsten or molybdenum strip 2 X 0.5 X 0.005 inch thick for the purpose of cleaning it. It also permits the use of a boatrtype filament with enough holding capacity for a lar e charge of ma8 inches tall) terial. The bell jar is small (5.5-inch opening and is evacuated by means of an oil diffusion pump of small mm. size, together with a fore pump. Vacuums of about of mercury can be obtained. This is a very small and simple system. It is, nevertheless, adequate for the job, inexpensive, and easy to clean.
6,
ACKNOWLEDGMENT
The authors would like to express their thanks to the microscopy department under John J. Kelsch, and especially to Ralph Bainbridge and Vincent Salines, who supplied the photographs for this paper, and to Frank JV, Dunne, who built the sniall evaporator described here. They are also grateful to Dan Hurley and Anthony Sonnessa, who did the x-ray work. LITERATURE CITED
Blout, E. R., and Fields, RI., Science, 107, 252 (1948). Brown, J. IT., Trans. Roy. Soc., Can., 111, 26, 173-5 (1932). Reinkober, O., 2. Physik, 5, 192-7 (1921). Scott, J. F., Sinsheimer, R. L., and Loofbourow, J. R., Science, 1 0 7 , 3 0 2 (1948).
(5) Sinsheimer, R. L., Scott, J. F., and Loofbourow, J. R., iYatur~, 164, 7 9 6 (1949). (6) Wagner, E. L., and Hornig, D. F., J . Chem. Phys., 18, 296-301 (1950). ( 7 ) Ihid., pp. 305-12.
RECEIVED for review April 15, 1952. Accepted Sovember 18, 1952. Prerented before the Pittsburgh Conference on Analytical Chemistry and Spectroscopy, March 1962.
Identification of Some Organic Acids by Paper Chromatography A. R . JONES, E. J. DOWLING, A N D W . J. SKRABA Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tenn.
In the course of investigations of the acidic products formed by the action of gamma-rays from cobalt-60 on aqueous acetic acid solution, the paper chromatographic R values for a number of common acids were determined in several solvent combinations. A pseudo-two-dimensional chromatograph was devised for presenting and using R value data for characterization. .4new acidic solvent combination with great resolving power for nonvolatile acids, and a new basic solvent combination w-hich increases the sensitivity of the paper chromatographic method as applied to volatile acids, were developed.
D
U R I S G preliminary investigations of the identity of substances by paper chromatography, it is advantageous to be able to compare the R values for the unknowns with R values reported in the literature for knoiin substances. In the course of research on the acidic products formed by the irradiation of dilute aqueous solutions of acetic acid with gamma-rais from cobalt-60, the paper chromatographic R values for a number of common acids nere determined in several solventq and are reported in Tables I and 11. Although R values in any one solvent combination m a r not characterize an unknown material because the solvent conibination lacks sufficient resolving polqer, the use of several solvent Combinations minimizes the possibility of error. This is particularly true vhen the unkrionn substance is chromatographed in different chemical forms in different solvent combinations-e.g., as a free acid and as the acid anion. MATERIALS AND REAGENTS
The chromatographic developments were carried out in cylindrical jars 18 inches high by 12 inches in diameter, having ground lips and covered a i t h desiccator lids. A stainless steel scaffold in each jar supported the paper sheets and, in the case of descending chromatograms, the solvent trays. Whatman No. 1 filter paper was used in all experiments. The test acids were commercial products purified before use. Paper chromatograms of the volatile acids were sprayed with
a solution of 50 mg. of bromophenol blue in 100 ml. of water, thr solution being made acidic with 200 mg. of citric acid. Indicator solutions (0.04%) of bromophenol blue, chlorophenol red, or bromocresol green in alcohol, the solutions being made definitely basic with sodium hydroxide solution, were used for spraying paper chromatograms of nonvolatile acids. Chromatograph solvents were prepared as follows: Solvent Combination A, 100 volumes of n-butyl alcohol. 15 volumes of water, and 1 volume of diethylamine. Solvent Combination B, 15 volumes of ether, 3 volumps of acetic acid, and 1 volume of water (4). Solvent Combination D, equal volumes of 1-pentanol and 5 .If aqueous formic acid (3). Solvent Combination E. 2 volumes of 2-ethyl-I-butanol and 3 aqueous formic acid. volumes of 5 Solvent Combination F, 8 volumes of 95% ethyl alcohol. 1 volume of water, and 1 volume of concentrated ammonium hydroxide (1). The dried papers were sprayed with indicator solution until clearly defined spots were obtained. Care was taken to avoid making the paper wet with the indicator solution. The paper? were resteamed and resprayed if solvent removal was not complete. GENERAL PROCEDURE
Ether or ether-acetone solutions (2 mg. per ml.) of the test acids were spotted by means of micropipets on paper sheets 18 em. wide and sufficiently long to provide a chromatograph path of 30 em. They were allowed to dry in air. Before the solutions of the volatile test acids were placed on the paper, the spot posi-
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Table I. R Values for Some Nonvolatile Organic -4cids on Paper Chromatograms Run in Several Solvent Combinations NO. in Fig. Bo 1 1 72-77 2 58-64 3 22-24 ? 39-43 J 97-99 6 88-93 7 85-88 8 49-52 9 85-88 65-67
Acid
70 Rf in Solvent Combination Ca 74-78 72-73 37-39 55-55 93-97 84-89 81-83 59-65 83,6-85.5 76-77.5
Db 67-69 66-68 18-23 36-43 87-88 77-79 69-71 43-44,5 70-72 58-61
E O
60-64 53.5-57 13-14.5 27-28.5 80.5-81.5 72,575 64.5-66 27-29 64-65 49/670 50.6/70 66-67 17-18.5 34.5-36 39-40 83.5-88 69.5-72.5 72-74 48-49 4.5-6.5 11.5-12.5 37-40
70 R h f d i c d in Solvent Combination F8 48/68-52/711 125-126 41.5-42.5 116-118 159-161 141-143 134.5-135.5 190-193 125-128
of the developing tank. As the paper stretched slightly development, during care was exercised to prevent the paper from touching t h e a q u e o u s phase. The lid was replaced, and development was allon-ed to proceed for ascending chromatograms until the solvent front had advanced 20 to 25 cm. For descending c h r o m a t o g r a m with solvent F, the development was allowed to proceed for 20 to 24 hours. The
grams was immediately traced with a marking pen. The papers were hung in a hood, and the solvents were removed by steam distillation (Q), by blowing saturated steam directly at the face of the paper. The paper was kept dry by irradiating it with a battery of infrared lamps. RESULTS AND DISCUSSION
Of the combinations investigated for nonvolatile acids, 2-ethyl-1-butanol-aqueous formic acid and FumariE ethyl alcohol-ammonium hydroxide gave the least Glutaric Glycolic variation in R values from one experiment to anItaconic other, and had the greatest resolving poner for Lactic mixtures of the acids. Lerulinic 10 87-88 87.5-89 65-70 214-219 The minimum and maximum R values obtained 100 Malic 11 32-37 49-55 27.5-32 Maleic 12 30-32 53-59 52-54 100-106 in several experiments for each known substance 3Ialonic 13 58-60 61-68 47-53 82-83.5 have been reported rather than the calculated Mesaconic 14 97-98 93-94 83-87 148-152 Methylsuccinic 15 90-92 88-92 74-75 137-138 average R values, not only to represent the epperiPyruvic 63-64 16 73-79 74-79 55-60 119-113 Succinic mental error, but because the data for a knonn Tartaric 17 12-16 29-34 13.5-15 s9-90.5 substance determined with any two solvent comTartronic 18 24-25 39-41 I 6-80 20-2 1 Tricarballylic 19 59-62 67.5-69.5 5 2 , 5 4 4 58-62 binations could be plotted against each other as a Ether-acetic acid-water. ordinates and abscissas (Figure 1) to give an area b 1-Pentanol-5 M aqueous formic acid. within or close to which it would be likely that 2-Ethyl-1-butanol-5 M aqueous formic acid. d R ~ d i o represents ratio of distance moved b y test acid t o t h a t moved b y pure malic any new ordinate-abscissa pair for that substance acid, a spot of which was applied t o each sheet of paper irrigated in descending: fashion with ethyl alcohol-ammonium hydroxide solvent. would be found. Such a plot was useful 4 hen an e E t h y l alcohol-ammonium hydroxide. unknown compound had to be compared a i t h subf Two spota, probably cis a n d trans isomers. Two spots, monomer a n d dimer (lactyllactic acid). stances for which R values had already been deh KO spot could be obtained. termined. The authors generally used those X values along one axis obtained for the anion of an Table 11. Rf Values for Diethylamine Salts of Some acid and along the other axis the R values obYolatile Organic Paper Chromatographed with Solventa .i tained for the free acid. as is shown in the fimre. " Acid % RP Two-dimensional chromatography requires roughly twice as Acetic 27-29 ~. -. much time as is necessary to produce two one-dimensional paper Propionic 40-42 Butyric 50-53 chromatograms simultaneously. Valeric 59-52 The method also requires the manipulation of large sheets of n-Butyl alcohol-diethylamine-water. b Extreme limits for 5 determinations of each acid. n e t paper and the use of cumbersome scaffolding in bulky cabinets in which it is difficult to ensure a solvent saturated atmosphere. Khile, in general, the greatest resolution of a mixture M ill be accomplished by t n o-dimensional chromatography, it tions mere dampened with diethylamine. The spot's diameters has been reported in an excellent paper by Stark, Goodban, and were approximately 1 cm. Owens ( 7 ) that separate one-dimensional chromatograms reSeven samples were placed on each sheet approsimately 5 cm. from the paper-solvent contact. From this point until the solved certain mixtures more satisfactorily. papers were removrd from the developing solvent, the work n-as The volatile acids were applied to positions previously dampcarried out in an air-conditioned room a t 25' C. The solvent was ened with diethylamine and the chromatograms were developed prepared and placed in a developing jar about an hour before use. in a solvent containing free diethylamine, so that the acids esisted The walls of the jar were thoroughly wetted. The desiccator lid was removed and the spotted papers were quickly inserted so as in the completely ionized salt form. Chromatography of the to come in contact with the solvent. Care was taken that the anions of volatile acids has been proposed previously ( 2 ) 5, 6); paper dipped into only the organic phase of two-phase solvent however, the method herein described offered several advantages. systems. The aqueous and organic layers of such systems were Use of the diethylamine salts increased the sensitivity of the not separated, because more consistent R values were thus obtained. Instead, the whole mixture was placed in the bottom method several times as compared v i t h the ammonium salts. C
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ANALYTICAL CHEMISTRY
396 Solvent removal by steam distillation ( 4 ) minimized “ghost” spots and thus made pretreatment of the paper XTith oxalic acid unnecessary (6). LITERATURE CITED
Brown, F., .l‘uture, 167, 411 (1951). (2) Brown, F., and Hall, L. P., Ibid., 166, 66 (1950).
(1)
(3) Buch, &I. L., Montgomery, R., and Porter, IT. L . , A s a ~CHEM., . 2 4 , 4 8 9 (1952).
Denison, F. IT., J r . , and Phares, E. F., Ihid., 24, 1628 (1952). (5) Hiscox, E. R., and Berridge. K.J., .Vatwe, 166, 522 (1950). (6) Kennedy, E. P., and Barker. H. .I., - 4 x . i ~CHEX, . 23, 1033 (1951). (7) Stark, J. B., Goodban, .1.E., and Owens. H. S.,I b i d . , 2 3 , 413 (4)
(1951).
RECEIVED for reriem September 19, I%?.
.iccepted December 1.5, 1932.
Two-Dimensional Chromatography of Amino Acids on Buffered Papers A. L. LEVY AND DkVID CHUNG Zforrnone Research Laboratory, D e p a r t m e n t of Biochemistry, Z-nirersity of California, Berkelel., Calif.
Previous systems for two-dimensional chromatography of amino acids-e.g., collidine-phenol-0.3% ammonia-were not entirely satisfactory owing inter alia, to irregularly shaped spots, lack of reproducibility, incomplete separation, discoloration of the paper, and unpleasant odor. The solvent system described (4:1:5 butanol-acetic acid-water-1 :1 rn-cresol-phenol, pH 9.3 borate buffer, Figure 3), overcomes these difficulties, allowing completion of the chromatogram in 40 hours. As analysis of peptide and protein hydrolyzates is established routine in many laboratories, iniprovements in technique are to be welcomed.
S
I N C E the classical paper by Consden, Gordon, and Martin ( 2 ) in 1944 on the analysis of protein hydrolyxates by tn-odimensional paper chromatography using the system collidinephenol-O.3% ammonia (coal gas), a variety of other solvent combinations have been employed for this purpose ( I ) . I n this communication, the authors wish to record a new two-dimensional system for the qualitative analysis of amino acid mixtures, which, in this laboratory, has proved more satisfactory than any others so far tried. The authors’ experience with the sepnrate solvents in one-dimensional runs, and with procedures for protein hydrolysis, is also briefly described. Butanol-Acetic Acid. The butanol-acetic acid-water (4:1:5) mixture of Partridge (9) has proved the most generally satisfactory solvent for one-dimensional papers, affording a reproduciblr pattern of compact spots, allowing detection of minimal quantiticls of amino acids, and being not unpleasant t o handle. The reproducibility of this system is doubtless related t o the buffering action of the acetic acid, and to its relative insensitivity to temperature change. For control purposes i t was found convenient to divide the natural amino acids into two individually resolvable groups of nine (mixtures A and B, Figure l), and to run them on either side of the unknovn. From the chromatogram it will be seen that the pairs threonine-glutamic acid, methionine-valine, isoleucine-phenylalanine, and to a lesser extent glycine-serine, are inseparable in butanol-acetic acid. Nixture .4 was prepared by dissolving 200 micromoles each of lysine, aspartic acid, glJ-rine, thre?nine, proline, valine, tryptophan, phenylalanine, and leucine in 2 ml. of 1 S hydrochloric acid, and making up to 10 nil. with 0.1 S hydrochloric acid: mixture B , by dissolving 200 micromoles each of cystine, histidine, arginine, serine, glutamic acid, alanine, tyrosine, methionine, and isoleucine in 2.4 ml. of 1 S hydrochloric acid and niaking up to 10 ml. with 0.1 A‘ hydrochloric arid. By making the amino acid solutions thus 0.02 Ai in 0.1 S hydrochloric acid, complete dissolution of the relatively insoluble amino acids such as cystine and tyrosine was effected, and bacterial contamination was inhibited. JVhen a neutral solution was required (as for application t o a single dimensional buffered paper) a portion was neutralized to bromothymol blue with 1 iV sodium hydroxide. and applied t o the paper before the cystine and tyrosine had time to crystallize.
I n the case of butanol-acetic acid chromatograms, the Rf’s were identical whether the amino acids were applied a t p H 1.0 or p H G to 7, except for those of alanine and glutamic acid which xere a little lower Tvhen neutralized. The chromatograms were run by the descending method on Whatman S o . 1 paper for 16 to 24 hours in a Chromatocab Model B 250 cabinet 27 x 18.5 X 25 inches (University -4pparatus Co., 2229 NcGee hve., Berkeley 3, Calif.). One-microliter aliquots (equal to 0.02 micromole of each amino acid) of solutions A and B were applied 2 inches from the solvent level, Tvith the aid of a Carisberg (Lang-Levy) constriction pipet ( 5 ) . The microliter pipets meritivned in this paper were all of this type, and n.ere obtaine 1 from EIerr Pedersen, the Carlsberg Laboratory, GI. Carlshergvej 10, Copenhagen, Valby, Denmark. The chromatogram \vas not improved b y equilibration (2 hours) in the cabinet prior to introduction of the butanol-acetic acid, as has sometimes been recommended. The papers were sprayed with 0.1% ninhydrin in ethyl alcohol (10) containing 5 % collidine ( I $ ) , and the color was developed by brief heating (1 t o 3 minutes) over a hot plate. Under these conditions the amino acid spots she\\- up in a variety of different colors, which vary with the temperature and length of time of heating, v i t h the paper used, and with the purity of the collidine. For this reason no attempt is made t o list the colors. HoiTever, no two neighboring spots in mixtures d and E have quite the same color, and this is particularly helpful in identifying such closely adjacent pairs as aspartic acid and glycine and phenylalanine and leucine. Finally, the papers were sprayed with 1% copper nitrate in ethyl alcohol ( 4 ) ,causing the spots to appear salmon pink on a pale blue-green background. This ( a ) prevented fading on storage (Q), ( b ) gave better contrast when photographed or contact-printed, and (c) prevented subsequent soiling of the chromatogram by fingerprints and other extraneous ninhydrin reacting materials (since chelated amino groups do not react with ninhydrin). Phenol and m-Cresol. I n order to separate the four pairs of amino acids unresolved by butanol-acetic acid, phenol saturated with water appeared to be the most desirable solvent. However, as the experience of many other workers has shown, this system gives the best results only xhen buffered by some means, and for thip purpose ammonia has generally been used. Recently, i\Ic>Farren( 7 ) employed aqueous buffers for this purpose, showing that both the phenol and the paper must be separately buffered. In the authors’ euperience, McFarren’s buffered papers
