The method is simple and requires relatively simple equipment. This method requires a n additional step in the procedure-making negative photocopies of the chromatograms-but this is the only disadvantage. On the other hand, by copying on a high contrast paper it is possible to increase the transmittance gradation of weak spots. Thus, the method can be made more sensitive and useful for smaller amounts or concentrations of the substance to be determined. The original chromatograms usually lose their color intensity in a short time, while the photocopies remain as a permanent record and allow measurements to be repeated or checked a t any time. ACKNOWLEDGMENT
The author expresses his thanks to Gotfred Haugaard for his helpful discussions and to Helen Brezicki, Chester Hargreaves, Leon Marker, and Eve
Marker for their advices in the preparation and correction of the manuscript. LITERATURE CITED
(1) Block, R. J., ANAL. CHEM.22, 1327 (1950). (2) Block, R. J., Science 108,608 (1948). (3) Bolling, D., Sober, H. A., Block, R. J., Federation Proc. 8, 185 (1949). (4) Brimley, R. C., Nature 163, 215 I 1 949 ). (5) Brown, J. A , , Marsh, M. M., ANAL. CHEM.25, 1865 (1953). (6) Bull, H. B., Hahn, J. W.,Baptist, V. H.. J . Am. Chem. SOC.71. 550 (1949 j. (7) Consden, R., Gordon, A. H., Martin, A. J. P., Biochem. J . 38, 224 (1944). \ - - - ~ I -
(8) Decker, P., Riffart, IT.,Wagner, H., Klin. Wochschr. 29, 418 (1951). (9) Fisher, R. B., Parsons, D. S., Holmes, R., Nature 164, 183
11949). (10) Fisher, R. B., Parsons, D. S., Morrison, G. A., Zbid., 161,764 (1948). (11) Fowden, L., Biochem. J . 48, 327 (1951).
(12) Gustafsson, C., Sundman, J., Lindh, T., Paper and Timber (Finland) 1, 1 (1951). (13) Klatzkin, C., Nature 169, 422 (1952). (14) Kowkabany, G., Cassidy, H. G., ANAL.CHEM.24, 643 (1952). (15) Landua, A. J., Awapara, J., Science 109,385 (1949). (16) McFarren, E. F., Brand, K., Rutkowski, H., ANAL.CHEM.23, 1146 (1951).
McFarren, E. F., Mills, J., Zbid., 24, 650 (1952).
Markham, R., Smith, J. D., Biochem. J . 45, 294 (1949). Martin, A. J. P., Mittelmann, R., Zbid., 43, 353 (1948).
Patton, A. R., Chism, P., ANAL. CHEII.23, 1683 (1951). Redfield, R. R., Barron, E. S. G., Arch. Biochem. and Biophys. 35, 443 (1952).
Rockland, L. B., Dunn, M. S..
J . Am. Chem. Soc. 71, 4121 (1949). (23) Vaeck, S. V., Nature 172, 213 (1953). (24) Woivod, A. J., Biochem. J . 45, 412 (1949).
RECEIVED for review June 21, 1956. Accepted April 22, 1957.
Chromatographic Separation of 2,4-Dini trop henylhydrazones E. L. PIPPEN, E. J. EYRING, and MASAHIDE NONAKA Western Utilization Research Branch, Agricultural Research Service,
b Chromatography
of 2,4-dinitrophenylhydrazones of aliphatic aldehydes and ketones on silicic acid-Celite columns was studied. Columns packed to a height of 75 cm. permitted separation of hydrazone mixtures of adjacent members of the homologous series of saturated normal aldehydes as high as C11. In addition, the feasibility of separating various combinations of the derivatives of 34 aliphatic aldehydes and ketones is described.
I
CONNECTION with flavor studies under way a t this laboratory it was necessary to separate a complex mixture of aliphatic 2,4-dinitrophenylhydrazones. Existing reports (1, 2, 6, 6) concerning chromatographic separation of aliphatic 2.4 - dinitrophenylhydrazones, while helpful, were limited primarily to studies of somewhat simple mixtures of carbonyl compounds having one to six carbon atoms. The possibility of separating known mixtures, particularly of some higher aldehydes and ketones, and certain other combinations of 2,4dinitrophenylhydrazones not previously reported was therefore studied. The results not only describe N
U. S. Department o f Agriculfure, Albany 7 0, Calif.
the relative chromatographic behavior of many new combinations of 2,4dinitrophenylhydrazones but also show that it is possible, by using longer columns, to separate the 2,4-dinitrophenylhydrazone derivatives of the homologous series of normal, saturated aldehydes from Cg through C1,. EXPERIMENTAL WORK
'Reparation of 2,4-Dinitrophenylhydrazones. Most of the hydrazones used in this study n-ere prepared from commercially available carbonyl compounds. The carbonyl compound was added to a solution containing a 10% excess of 2,4-dinitrophenylhydrazine (2 grams per liter in 2-l' hydrochloric acid). After the hydrazone precipitated it n a s filtered off and washed thoroughly with water, followed by one or more recrystallizations from alcohol. The 2,4-dinitrophenylhydrazones of glycolaldehyde, methyl cyclopropyl ketone, 2-methylbutanal, 2-methyl-2butenal (tiglaldehyde), n-pentanal, n-heptanal, and 2-hexenal were authentic samples available within the laboratory. 3-iVethylmercaptopropional was prepared as described by Patton (4). Melting points of all hydrazones were checked to ensure their authenticity. I n some instances chromatog-
raphy followed by recrystallization was necessary to remove impurities. Diacetyl mono-2,4-dinitrophenylhydrazone was obtained by adding the hydrazine solution slowly to an excess of diacetyl dissolved in water. Ordinarily when diacetyl-2,4-dinitrophenylhydrazone is prepared, both carbonyl groups in the diacetyl molecule react to form the bis-2,4-dinitrophenylhydrazone. If only one carbonyl group reacts, a hydrazone is obtained n hich is referred to here as the mono derivative. The yellow precipitate was filtered off, washed with mater, taken up in hot alcohol, and filtered to remove any insoluble his derivative. The mono derivative in the filtrate was crystallized several times from alcohol. It melted a t 178" C. Analvsis showed C. 45.1%: H I 3.79%; and f, 21.1%. Calculateb for C1,HIoO5S4:C, 45.117,; H, 3.79%; N. 21.05%. 'Chromatography of 2,4-Dinitrophenylhydrazones. Chromatography was carried out on glass columns having an outside diameter of 35 mm. The procedure, solvent mixtures, adsorbent (silicic acid-Celite, 2 to 1 weight ratio), etc., used were those of Gordon and coworkers ( 2 ) . Solvents were redistilled before use; the petroleum ether used boiled a t 40' to 50" C. TWOcolumn lengths, Fvhich permitted packing the adsorbent to maximum VOL. 2 9 , NO. 9 , SEPTEMBER 1957
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Table 1. Chromatographic Separation of 2,4-Dinitrophenylhydrazones of CdCIZ Normal, Saturated Aldehydes on Columns Packed to Height of 75 Cm.
(Ethyl ether in developing solution was 27, volume) System (as Mix- 2,4--l)initroture phenyl Band No. hydrazone) Separation No." 1 n-Butanal Complete 5 n-Pentanal 4 3 n-Hexanal n-Heptanal 2 n-Octanal 1 2 n-Octanal Complete 4 n-Konanal 3 n-Decanal 2 n-Undecanal 1 3 n-Undecanal l'itrt i d 2 1 n-Dodecanal Identity was confirmed by mixed melting point and paper chromatography. Fastest running band = I , next fastest 2, etc.
heights of 23 and 75 cm., were used. To prepare a mixture for chromatography, 10 to 15 mg. of each of the hydrazones was weighed into a common container. Chloroform was used to dissolve the mixture and transfer it to the column. Recovery and Identification of 2,4Dinitrophenyihydrazones. When separation of the components on the column was complete, the fractions rvere either collected by eluting them from the column or, if convenient, were dug out of the column t o save time and solvent. I n the latter case. the hydrazone was recovered from the adsorbent by extraction with chloroform. Hydrazones were recovered by evaporating the solvent with a stream of nitrogen on the steam bath. If separation was good, the residue obtained represented hydrazone pure enough to permit identification by melting point alone, provided the system consisted of derivatives of distinctly different melting points. Often hydrazone mixtures were run in which two or more components had melting points so nearly identical that this criterion alone did not serve as a conclusive means of identification. Occasionally a fraction isolated after chromatography exhibited a different crystalline form and melting point than the starting material. In these instances, its identity was confirmed by one or more additional steps. such as mixed melting point determinations. recrystallization with subsequent melting and mixed melting point dcterminations, and comparison with authentic samples by paper chromatography. Paper Chromatography of 2,4-Dinitrophenylhydrazones. Paper chro-
1306
ANALYTICAL CHEMISTRY
matography, as a n aid in identification of hydrazones, was carried out essentially as described by Lynn, Steele, and Staple (3). Whatman No. 1 filter paper was used. RESULTS A N D DISCUSSION
Methods in the literature have not demonstrated the feasibility of separating 2,4-dinitrophenylhydrazones of ad-
Table II.
jacent members of the homologous series of normal, saturated aldehydes having six or more carbon atoms. White (6) had difficulty separating the C4-Csmixtures. Using the method of Gordon and con-orkers ( 2 ) , which calls for a column packed to a height of about 22 cm., the authors found it possible to separate adjacent members of the series up to C?, but not the mixed C& derivatives.
Chromatography of Two-Component Systems of 2,4-Dinitrophenylhydrazones on Columns Packed to Height of 23 Cm.
Ether in
Faster Moving Component
Slower Moving Component A
Deve2ers
Completely Separated
Formaldehyde Acetaldehyde Acetaldehyde 2-Propenal (acrolein) Acetone Diacetyl (mono derivative) n-Butanal (butyraldehyde) n-Butannl Methyl cyclopropyl ketone Methyl ryclopropyl ketone 2-Methyl-2-hutenal 2-Methyl-2-butenal Methyl isopropyl ketone Methyl isopropyl ketone Methyl n-propyl ketone (2-pentanone) Diethyl ketone (3-pentanone) Diethyl ketone (3-pentanone) 2-Methyl butanal n-Hexanal Methyl n. butyl ketone (2-hexanone) Methyl n-butyl ketone (2-hex anon^) n-Heptanal Methyl n-amyl ketone (2-heptanone) Methyl n-amyl ketone (2-heptanone) Methyl n-amyl ketone (2-heptanon~) Methyl n-hexyl ketone (2-octanone) Methyl n-hexyl ketone (2-octanone 1 Methyl n-hexyl ketone (2-octanone) Methyl n-heptyl ketone (2-nonanone)
Diacetyl (mono derivative) Glycolaldehyde
3-Methylmercaptopropional
Acetone Pyruvic acid Glycolaldehyde 2-Butenal (crotonaldehyde) Methyl cyclopropyl ketone 2-Butenal Acetone 2-Butenal n-Butanal Methyl cyclopropyl ketone Methyl-n-propyl ketone Pentanal (valeraldehyde) n-Pentanal Methyl n-propyl ketone 3-Methyl butanal n-Heptanal Methyl n-propyl ketone n-Hexanal 2-Methyl-butanal n-Heptanal Methyl n-butyl ketone n-Octanal (caprylaldehyde'i n-Octanal Methyl n-amyl ketone n-Nonanal Methyl n-hexyl ketone
10 4
6
4
20 10 4
2 2 4
2
2 2 2
2
2 2
2 2
2
2 2
2 2 2 2 2
2 2
R. Partially Separated"
Propand n-Hexanal Methyl isepropyl ketone Methyl n-heptvl ketone (2-nonanonc~)
2-Propenal 2-Hexenal Diethyl ketone n-Dodecanal
2 2 2
2
C. 30Apparent Separation 3-Methylmerraptopropional
n-Pentanal 3-Methyl butanal 3-Methyl butanal n-Hexanal 2-Methyl but anal n-Heptanal n-Heptanal Methyl n-butyl ketone (2-hexanone)
Diacetyl (mono derivative) 2-Methyl 2-butenal n-Pentanal 2-Methyl propanal Methyl n-propyl ketone n-Hexanal Methyl isopropyl ketone Methyl n-butyl ketone Methyl isobutyl ketone
ti
2
2 2 2 2 2 2 2
0 By taking small fractions and recrystallizing them it was possible to obtain a portion of each component in the pure state. Longer columns might result in better or complete separation.
The resolving ability of a column having adsorbent packed to a 75-cm. height was then tested. The first mixture tried consisted of the Cc to Cs aldeIipdes. Results shown in Table I tkmonstrate that this mixture was sucwssfully resolved into its several components. Additional data presented in this table show that successful resolution was obtained through Clr. but beyond this point difficulty was mcountered. Presumably, increasing the height of column packing beyond 75 cm. would extend its resolving ability beyond this point. However, longer cxolumns result in more diffuse bands than short ones. This promotes a tendency for overlapping of fractions. thus partly counteracting the increased resolving ability of the longer column. [inless the amount of diffusion can be reduced, it may be the limiting factor i n determining the maximum practical ~ d s o r b e nheight t which can be used. Gordon and coworkers ( 2 ) reported that pure methyl ethyl ketone-2,4clinitrophenylhydrazone split into two hands when chromatographed. Neither Roberts and Green ( 6 ) nor White (6) q,(>cificallymentioned that methyl ethyl
ketone-2,4-dinitrophenylhydrazone separated into two bands on chromatography. I n the authors’ hands these derivatives did exhibit anomalous behavior, in that they formed a band with an unusually long tail which, however, uever completely separated from the main part of the band. In addition to thc systems of 2,4 - dinitrophenylhydrazone. reported above, chromatographic properties of 34 other 2,4-dinitrophenylhydrazone derivatives of aliphatic aldehydes and ketones are shown in Table 11. The chromatographic properties of most of these have not been previously reported. Hon-ever. a few hydrazones n hose chromatographic properties are well known. were deliberately included to serve as reference compounds. The relative nature of the data makes it possible to conclude how any compound rill run ivith respect to the others, hencr permits prediction of the results when any mixture of the compounds listed is chromatographed. For example, it is apparent from these data that we can expect to resolve completely any mixture of 2,4-dinitrophenylhydrazones of methyl n-alkyl ketones from
methyl n-propyl to methyl n-heptyl. These data have been useful to the authors not only as an aid to identification of substances listed in Table 11, which were resolved from a mixture of unknowns, but also t o predict the identity of other compounds on the basis of their chromatographic properties. ACKNOWLEDGMENT
The authors thank Geraldine E Secor for r1rment:il analyses. LITERATURE CITED
(1) Elvidge, J . .I.> Wholley, Margaret, Chem. B I n d . 1955, 589. ( 2 ) Gordon, B. E., Wopat, Fred, Jr., Burmhani, H. D., Jones, L. C., J r . , ANAL.CHEX.23, 1754 (1951). (3) Lynn, JT S., Jr., Steele, L. A., Staple, Ezra. Zbid..28. 132 (1956’1. (4) Patton, Stuart, Food ’Technol. 10, 60 (1956). (5) Roberts, J. D., Green, C., IND.E ~ Q . CHEM.,ANAL.ED. 18, 335 (1946). (6) White, J. W.) Jr., . ~ N A L . CHEM.20, 726 (19481. RECEIVED for review December 10, 1956. Accepted hIarch 28, 1957.
Partially Deactivated Silica Gel Columns in Chromatography Chromatographic Behavior of Benzo[a]pyrene H. J. CAHNMANN National lnstitufes o f Health, Bethesda, Md.
b Various activity grades of silica gel were prepared by the controlled deactivation of activated silica gel with known amounts of water. Moderately deactivated silica gel gave better defined chromatographic zones than a nondeactivated gel in the fractionation of many polycyclic aromatic hydrocarbons. The shapes of the elution curves of benzo [alpyrene, which was chosen as a typical example, show that by the gradual addition of water to activated silica gel an optimal degree of deactivation is reached, beyond which further deactivation yields poorer adsorbents. RF values of benzo[a]pyrene for various activity grades of silica gel are presented and the advantages offered by the use of a partially deactivated silica gel are discussed.
complex mixtures such as crude tars or oils had to be analyzed qualitatively and semiquantitatively for individual polycyclic aromatic hydrocarbons in this laboratory, preliminary experiments were carried out to determine the chromatographic behavior of a number of such hydrocarbons, either alone or in mixtures, on various adsorbents and under various operating conditions. I n these experiments silica gel, partially deactivated by the addition of appropriate amounts of water, gave better defined chromatographic zones in most instances than other adsorbents. The superiority of partially deactivated silica gel was confirmed later when well over 100 chromatographic fractionations were carried out with more complex mixtures of known or unknown composition. HEN
The use of partially deactivated siiicic acid as a chromatographic adsorbent was first reported by Trappe (29). Shortly afterwards Brockmann and Schodder (4) described grades of partially deactivated aluminum oxide that frequently offer advantages over the fully activated oxide 13, 25), and therefore have found widespread use as chromatographic adsorbents. (The term “fully actiT-ated” is used for an adsorbent mhoqe adsorptive strength has attained a iliaximum and cannot be increased by further heating to any temperature.) In view of this fact it is surprising that partially deactivated silicic acid (of which silica gel is a special form) has only rarely found application in adsorption chromatography. It is the more 3urprising as the dependence of the adsorptive strengths of VOL. 2 9 , NO. 9 , SEPTEMBER 1957
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