I n the development of the scheme a letter was assigned to each of the 18 paraffin isomers which can be obtained o n reduction of the Cu aldehyde isomers. The carbon-hydrogen groups were represented by the number of each group which was present in an isomer and the following group order was used: C H r , C H r , CH-, a-hydrogen, -(CHJs-, and -(CHZ).--. When described in this manner, each of the Ca aldehyde isomers has a number or “profile.” ks an example, if n-octane is represented by the letter A, the profile for n-octyl aldehyde is 160201A. Doing this for the 39 CS aldehydes reveals that there are 34 different profiles resulting in a duplication in only five isomers. I n no case are there more than two isomers with the same profile. Thus, the identification problem is simplified considerably. To test the reliability of the infrared and nuclear magnetic resonance procedures, the three available isomer CI aldehydes were inspected for the information described above (Table I). This analytical scheme waa applied to fractions cut from a mixture of isomeric Cs aldehydes (Figure 2) aa described above. The first fraction, peak A, was examined on the ‘/r-inch partifion column and found to be almost 100% pure aldehyde isomer as shown in Figure 3. A Wolff-Kishner reduction yielded a hydrocarbon which was 96%
2,4-dimethylhexane (hydrocarbon C). Peak A was also analyzed by infrared and nuclear magnetic resonance (Table
11). Out of the 34 different profiles, the onlv Dossible one is 3222000 or 3.5-dimethilhexanal, CHsCH(CHa)CH;CH(CH8)CHoCHO. Peak B (Figure 2) was also separated by this scheme and, on reduction, nine CI paraffins were obtained, the most abundant of which was only 40%, which showed the fraction was not a single isomer. About 20% of this hydrocarbon mixture was 2,4dimethylhexane, the same as the reduction product from peak A. Because peaks A and B had different chromatographic retention times, the major portion of peak B could not be 3,5-dimethylhexanal. Only three C8 aldehyde isomers can reduce to 2,4dimethylhexane: 3,5-dimethylhexanal, %ethyl-4-methylpentanal, and 2,4-dimethylhexanal. Pure 2 - e t h y l 4 methylpentanal had a retention time different from peak B; therefore, peak B contained 20% 2,4-dimethylhexanal. Although no attempt wm made to identify all the isomers in the aldehyde mixture, this schematic approach should make possible the identification of all the components, provided they can be separated in sufficient purity. The separated components should have a purity of 90% or better for infrared and nuclear magnetic resonance to be
applicable. The method requires a minimum of about 0.2 ml. When working with these small volumes, the cut should be analyzed first by nuclear magnetic resonance and then by infrared. This amount is sufficient for the reduction and identification of the hydrocarbon by gas-liquid partition chromatography after these examinations. ACKNOWLEDGMENT
The authors are indebted to L. R. Cousins and R. J. Martin for the infrared and nuclear magnetic resonance data. LITERATURE CITED
(1) Bens, E. M., McBride, W. R., ANAL. CWEM. 31, 1379 (1959). 2) Brown, F.,J. Biochem. 47, 590 (1950). 3) Desty, D. H.,Goldup, A,, Swanton, W. T.,Nature 183, 107 (1959). (4) Desty, D. H., Wh an, B. H. F., ANAL.CEEM.29,320c57). (5) Huang-Minlon, J . Am. Chem. SOC. 68,2487(1946). (6) Keulemys, A. I. M., “Gas Chromatography, pp. 34-53, Reinhold, New York, 1957. (7) OstJeux, R., Guillaume, J., Laturaze, J., J. Chromatog. 1,70 (1958). (8) Zlatkis, A,, Ling, S., Kaufman, H. R., ANAL.CHEM.31,945 (1959).
R E C ~ I V Efor D review October 21, 1959 Accepted January 13, 1960.
Chromatographic Separation of 2,4-Dinitrop henylhydrazine Derivatives of Highly Oxygenated Carbonyl Compounds M. L.
WOLFROM and G.
P. ARSENAULT
Department of Chemistry, The Ohio State University, Columbus
V The chromatographic separation of 2,4-dinitrophenylhydrazine derivatives of highly oxygenated two- and threecarbon carbonyl compounds .effected on silicic acid-Celite.
1
N CONNECTION with
was
the ignition decomposition of cellulose nitrate (I@, i t was necessary to separate a complex mixture of 2,4-dinitrophenylhydrazine derivatives of short carbon chain (two and three carbon atoms) sugars and oxidation products thereof, without carbon fragmentation. The chromatographic separation of 2,4-dinitrophenylhydrazine derivatives of carbonyl compounds has been studied extensively
IO, Ohio
and detailed reviews have appeared (1, 4). The compounds of interest (Table I) are somewhat rare 2,4-dinitrophenylhydrazine derivatives. A thorough study of their chromatographic separation has not been reported, although the chromatographic properties of methylglyoxal bis(2,4--dinitrophenylhydrazone) (6, 8), glyoxal his(2,4-dinitrophenylhydrazone) @), hydroxypyruvaldehyde bis(2,4-dinitrophenylhydrazone) (8), and glycolaldehyde 2,kiinitrophenylhydrazone (10) have been recorded. Silicic acid has often been used to separate 2,4-dinitrophenylhydrazine derivatives. I n the work herein reported, silicic acid was used and was
deactivated by the addition of water. This adsorbent, silicic acid-Celite (5 to 1; 8% water), was highly satisfactory for the separation of 2,4dinitrophenylhydrazine derivatives of highly oxygenated carbonyl compounds. A chromatographic adsorption series of the 2,4-&nitrophenylhydrazine derivatives is shown in Table 11, listed in descending order of adsorptive strength. The adsorptive strength of a compound was determined by the position of its adsorption zone after development with the given developer. Substances written in a vertical sequence within a group are separable under the conditions specified, whereas those written in a horizontal sequence are not, VOL. 32, NO. 6, MAY 1960
693
Table I.
Melting Points of 2,4-Dinitrophenylhydrazine Derivatives Used in Chromatographic Study hlelting - Point, ' C. 2,4-Dinitrophenylhydrazine
Derivative A . Hydrazone of ._ 'Glycoialdehyde Acetol nL-Glyceroseb DihydroxyacetoneC B. Bis(hydrazone) of Hydroxypyruvaldehyde Mesoxaldehyde (42-bis) hlethylglyoxal Glyoxal C. Tris(hydrazone) of Mesoxaldehyde
Determinedo
159-61 137.5-8,s 165-8 159-67d 267-8 (decompd. ) 262-9 (decompd. ) 304 5-5.5 (decompd.) 330-8 (decompd.)
-
Reported
155-6 ( 3 )
134.5-30.5 (8) l i 0 (8) 168-9 (8)
265 (decompd.) (6) 299-300 (9) 326-8 (9)
306-8 (decompd.je
Kofler stage melting point; corrected. Anal. Calcd. for CQHIJi,Oe: C , 40.00; H, 3.73; S , 20.74. Found: C, 39.91; H, 3.76; B,20.64. c Anal. Calcd. for CBHI&,OS: C, 40.00; H, 3.73; N, 20.74. Found: C, 40.18; H, a
b
3_ 97:.
s.20.60.
h,felfing point unchanged by three recrystallizations from ethanol and two recrystallizations from ethyl acetate. d e
S e w compound.
Table II. Chromatographic Adsorption Series of Some 2,4-Dinitrophenylhydrazine Derivatives"
(.4rranged in decreasing order of adsorptive strength) Adsorbent. 5.0 grams of 5: 1 mixture of silicic acid-Celite containing 870 xater. Column. 8-mm. diameter, 20 cm. of adsorbent. Solute. 0.5, 0.1, or 0.95 mg., respectively, of mono-, bis-, or tris(2,4dinitrophenylhydrazone) dissolved in 2Oy0bof nitrobenzene in benzene. Developer, noted below following group heading. Group I(35 ml. of 20% of ether in benzene) nL-Glycerose Dihydroxyacetone Group I1 (20 ml. of 2% of ether in benzene followed by 25 ml. of 5% of ether in benzene) DL-Glycerose, dihydroxyacetone Glycolaldehyde Acetol Hydroxypyruvaldehyde, mesoxaldehyde (tris) Group I11 (14ml. of 20'3, of nitrobenzene in benzene)" Hydroxy yruvaldehyde Mesoxalghyde (tris) Group IV (65 ml. of benzene) DL-Glycerose, dihydroxyacetone, glycolaldehyde, acetol, hydroxypyruvaldehyde, mesoxaldehyde (tris) hlesoxaldehyde (lJ2-bis) Glyoxal Methylglyoxal 2,CDinitrophenylhydrazine derivatives, listed as parent carbonyl compounds, are shown in Table I. * Percentage by volume composition before mixing. c Developer is followed by 12 ml. of benzene to wash column free of nitrobenzene. 0
694
0
ANALYTICAL CHEMISTRY
in this study are listed in Table I, with the melting points reported in t h e literature. Each of these nine derivatives, with the exception of DL-glycerose(glyceraldehyde) 2,4dinitrophenylhydrazone, showed a single zone when chromatographed by the method described herein. Two zones were obtained when the chronatogram of DLglycerose 2,Pdinitrophenylhydrazone was developed with 20% of ether in benzene. The material in each zone, upon rechromatography on separate columns, afforded in each case two zones. DL-Glycerose 2,4-dinitrophenylhydrazone therefore exhibits the phenomenon of double zoning. This may be due to ring-chain isomerism in this substance, but if so, it is not reflected in the melting point, ivhich was t h e same for the material in both zones. Equilibration under melting point conditions is not excluded, however.
Preparation of Adsorbent. The silicic acid used (100-mesh, analytical reagent, Mallinckrodt Chemical Works, St. Louis, hlo.) was dried although they can be separated under overnight a t 200' C. t o remove i t s other conditions of development. Two "free" water (11). To the dried silicic compounds were considered separable acid were then added one fifth of its when their respective, highly colored weight of Celite (No. 535, Johns-Manadsorption zones after development ville Co., New York, -U. Y.) and enough water to bring the water content to 8% were separated by at least 1 em. of the over-all h-eight of the mixture. of clean, white adsorbent. Any mixThe adsorbent was then mixed thorture of the 2,4-dinitrophenylhydrazine oughly and stored in a hermetically derivatives could be separated, since closed container. This adsorbent may it was established that the admixture of be characterized in the following several components does not interfere manner: The chromatogram of glyoxal with their respective adsorptive bis(2,4-dinitrophenylhydrazone), after strengths. Thus, reference to the addevelopment with 65 ml. of benzene, yielded a zone located 8 to 11 cm. from sorption series indicates the method to the column top. A shipment of silicic use in separating a mixture of derivaacid received in the latter part of the tives. An evample is given to illustrate work required the preparation of a the complete separation, by four sucsilicic acid-Celite (5 t o 1) adsorbent cessive column chromatograms, of a containing 9.3% water in order t o have synthetic mixture of the nine derivathe characteristics just stated. tives. Chromatographic Procedure. T h e EXPERIMENTAL nitrobenzene and benzene T\ ere of good commercial grades and were not Preparation of 2,4-Dinitrophenylredistilled. Dry ether was used. T h e hydrazine Derivatives. Most of the chromatographic columns (8-mm. derivatives used in this study mere diameter, 20 cm. of adsorbent) were prepared from commercially available constructed from glass tubing, packed, carbonyl compounds. T h e 2,4and developed at 200-mm. pressure. dinitrophenylhydrazones were preT h e column of adsorbent was prewashed with sufficient benzene t o pared in refluxing ethanol following wet the column completely. A mono-, the procedure of Brady and Elsmie bis-, or tris(2,Pdinitrophenylhydra(2) and were recrystallized from ethanol zone), 0.5, 0.1, or 0.05 mg., respectively, to constant melting point. The bis(2,4 was dissolved in 0.2 ml. of warm nitrodinitrophenylhydrazones) of glyoxal and benzene and 0.8 ml. of benzene was methylglyoxal were prepared in 30y0 added t o this solution. The nitroperchloric acid following the procedure benzene-benzene solution was placed of Keuberg, Grauer, and Pisha (7) and immediately on the adsorbent column were recrystallized from nitrobenzene and the chromatogram was developed. The rate of flow of developer through to constant melting point. The prepthe column was ca. 30 ml. per hour. aration of hydroxypyruvaldehyde bisAfter the chromatogram was developed, (2,4-dinitrophenylhydrazone), mesoxaldehyde 1,2-bis(2,4-dinitrophenylhydra- the glass column was cut where the colored zone appeared and the zone zone), and mesoxaldehyde tris(2,4-diwas separated mechanically from the nitrophenylhydrazone) was complicated glass cylinder thus obtained. Elution and will be reported elsewhere (IS). was performed with tetrahydrofuran. The melting points of the nine 2,4A procedure identical with that just dinitrophenylhydrazine derivatives used described was followed for the chroma-
tography of larger quantities of 2,4dinitrophenylhydrazine derivatives on columns up to 54 mm. in diameter, except that the larger columns were extruded. Chromatographic Separation of 2,4Dinitrophenylhydrazine Derivatives.
The chromatographic adsorption series (Table 11) was obtained by chromatographing each derivative individually. The present study, however, was far more extensive, and a large number of synthetic mixtures containing two or more of the derivatives were chromatographed and separated. The identity of the coniponent present in each of the zones obtained by separating a synthetic mixture of derivatives was deduced by reference to the adsorption series and then demonstrated by comparative chromatograms. Thus it was ascertained that any mixture of these derivatives could be separated and that the influence of the presence of one or more derivatives on the chromatographic propertjes of a given derivative was negligible. The following example illustrates the method used in separating a complex, synthetic mixture of the 2,4-dinitrophenylhydrazine derivatives. The mix-
ture, made up of all nine derivatives, 0.5 mg. each of the mono derivatives, 0.1 mg. each of the bis derivatives, and 0.05 mg. of mesoxaldehyde tris(2,4dinitrophenylhydrazone), was dissolved in 1 ml. of 20% nitrobenzene in benzene and chromatographed as per Group I V (Table 11). Four zones thus resulted, the uppermost of which was located 0.0 to 2.5 cm. from the top of the column and contained a mixture of six derivatives. This six-component mixture was then chromatographed as per Group 11, affording a separation into four zones. The material in the uppermost zone, located 0.0 to 0.6 cm. from the top of the column, was then chromatographed as per Group I, whereas the material in the lowest zone, located 13.4 to 18.6 cm. from the top of the column, was then chromatographed as per Group 111. The resolution of the synthetic mixture of nine derivatives into its components was thus completed, a total of four successive chromatograms having been used for this purpose.
ACKNOWLEDGMENT
One of the authors (G. P. A.) acknowledges the support of the Standard Oil Foundation, Inc. (Indiana), fellowship held during 1957-58. LITERATURE CITED
(1) Arsenault, G. P., .Ph .D. dissertation, The Oh10 State Univexesity, Columbus, Ohin. -, 1- R . W-. (2) Brady, 0. L., Elsmie, G. V., Analyst 1I
. 51, 77 (1926). (3) collatz, H., Neuberg, I. S., Biochem. 2. 255,27 (1932). (4) Lederer, E., Lederer, M., “Chromatography. Renew of Principles and Applications,” 2nd ed., p. 169, Elsevier, New York, 1957. (.-, 5 ) Malmbere. E. W.. J . Am. Chem. SOC. 76,980 (19B4). (6) Neuberg, C., Collatz, H., Biochem. 2. 223, 494 (1930). (7) Neuberg, C., Grauer, A., Pisha, B. V., Anal. Chim. Acta 7,238 (1952). (8) Reich, H., Samuels, B. K., J . Org. Chem. 21, 68 (1956). (9) Strain, H. H., J. Am. Chem. SOC.57, 758 (1935). (10) Sundt, E., Winter, M., ANAL. CHEM.30,1620 (1958). A mixture of methylglyoxal bis(2,4(11) Trueblood, K. N., Malmberg, E. W., Ibid., 21,1055 (1949). dinitrophenylhydrazone) and 2,4(12) Wolfrom, M. L:, Arsenault, G. P., dinitroaniline could not be separated J . Am. Chem. SOC.,in press. when benzene was used as developer. (13) Wolfrom, M. L., Arsenault, G. P., J. Org. Chem., in press. This was also true of a mixture of glyoxal bi~(2~4-dinitrophenylhydrazone) RECEIVED for review September 3, 1959. Accepted January 25, 1960. and 2,4-dinitrophenylhydrazine.
Separation of the Alkali Metal Cations by Electrochromatography in Paper MURRAY M. TUCKERMAN’ and HAROLD H. STRAIN Division o f Chemistry, Argonne National laboratory, Lemont, 111.
b The mobilities of sodium, potassium, rubidium, and cesium cations were measured in various organic solvents with paper as the stabilizing medium. A useful separation of these alkali metal ions was obtained in nitromethane, 0.2M in ammonium formate and 0.4M in trichloroacetic acid, in 3 hours with a potential gradient of 15.0 volts per cm.
A
t o separate the alkali metal ions by ionophoresis with paper as the stabilizing medium have been made by Harasawa and Sakamoto (8) with ammonium hydroxide solution; by Schier (6) with ammonium carbonate solution; by Evans and Strain ( I ) with aqueous hydrochloric acid and various lactate, citrate, tarTTEMPTS
Present address, Temple University School of Pharmacy, Philadelphia, Pa.
trate, or EDTA buffers; and by Seiler, Artz, and Erlenmeyer (7) with a combination of ascending chromatography and ionophoresis in a n ethanol-acetic acid-aqueous ammonium acetate system. Although lithium and sodium were well separated from each other and from other members of the group, potassium, rubidium, and cesium were not separated from one another. The order of decreasing mobility was determined as cesium, rubidium, potassium, sodium, lithium. Because the electrochromatographic separations in aqueous media were not successful, the migration in organic solvents was investigated. For practical reasons, the solvents were restricted to compounds that were readily available in high purity and contained various functional groups (Table I). Lithium was not included in this study for lack of a radioactive isotope suitable for its detection. hioreover,
it is readily isolated by electrical migration in aqueous media (1). MATERIALS, APPARATUS, AND PROCEDURES
The solutions of the cations to be compared with respect t o relative mobility were prepared from radioactive salts. Sodium and potassium were used as 0.01M solutions of the activated nitrates in dimethylformamide. Rubidium was used in early experiments as a 0.01M aqueous solution of the activated chloride; in later experiments, as a 0.01M solution of the activated nitrate in dimethylformamide. The chloride was converted to the nitrate by treatment of the aqueous chloride solution with a calculated 1% excess of silver nitrate, removal of the precipitate by centrifugation, treatment of the filtrate with hydrogen sulfide, removal of the precipitate by centrifugation and filtration, and evaporation of the filtrate t o obtain the crystalline nitrate. Cesium was used in the early experiments as “carrier-free” cesium-I37 nitrate tracer VOL. 32, NO. 6, MAY 1960
695
