Modification of Gas Chromatographic Substrates for the Separation of Aliphatic Diamines EDGAR D. SMITH' and RICHARD D. RADFORD Chemstrand Research Ceder, Inc., Durham, N. C.
b The modification of gas chromatographic substrates to reduce undesirable adsorption effects in gasliquid partition chromatography is not new. The profound influence of alkali pretreatment on the separation of strongly adsorbed amines has not been adequately stressed, however. In the present work, the relative adsorption properties of the four most commonly used gas chromatographic substrates for aliphatic diamines are demonstrated. Alkali pretreatment of these substrates plays amajor role in the development of satisfactory gas chromatographic packings for the separation of these diamines. The exact role of the alkali in the final partition column is not understood, but is apparently much more complex than heretofore thought. The preparation and properties of three highly efficient columns for the separation of diamines are briefly discussed.
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(10) has recently reviewed the literature on various techniques for modifying the activity of gas chromatographic substrates. Most of this work has been concerned with the elimination or reduction of "tailing" to facilitate quantitative peak calculations. With specific reference to the separation of amines, several workers (1-9, 11) have pretreated their substrates with alkali in the manner first suggested by James (9). Decora and Dinneen (3) coated extracted Tide with 10% by weight of potassium hydroxide (KOH) before overapplication of the liquid partition phase, and obtained marked improvement in the separation of pyridines over previous substrates. They state, however, that asymmetric peaks were obtained for more basic amines, which proved true for our diamines. Ring and Riley (11) used Flexol 8N8 on KOH-treated C-22 firebrick for amine separation, but this column bled too badly to be tested on our higher boiling diamines. In none of the work cited above was TTENSTEIN
Present address, Graduate School of Technoloy University of Azkansas, Little Roc , Ark. 1 160
ANALYTICAL CHEMISTRY
it suggested that excess alkali on the gas chromatographic substrate was of paramount importance in achieving the desired separations, probably because these earlier workers were concerned with the separation of less strongly adsorbed amines. The present authors, however, have found that this alkali treatment is a primary requisite in the gas chromatographic separation of aliphatic diamines. For example, many partition liquids will retain hexamethylenediamine indefinitely a t 150" to 200" C. when coated on Chromosorb W, but will give symmetric peaks at 10- to 30-minute retention times with the same packing on substrates modified with KOH. The quantity of excess alkali is apparently not critical, but is far greater than the stoichiometric amount necessary for neutralization of the "acid sites" of the substrate. This is shown by the fact that simple washing of the substrate with 5% alcoholic KOH, followed by water rinsing and drying, does not confer the same beneficial effects as actual coating of the substrate with excess alkali. In fact, the data obtained suggest the possibility that the liquid substrates themselves may be interacting with the alkali,
FIR E ERIC K
CHROMOSORB
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in some as yet unexplained manner, to produce superior partition liquids. The objective of this study was to develop column packings for the quantitative separation of hexamethylenediamine (HMD) from tetramethylenediamine, pentamethylenediamine, and cis- and trans-1,Zdianiinocyclohexane (DCH). Since DCH boils closer to HMD than the other compounds mentioned above, most of the exploratory work was carried out on mixtures of these two aliphatic diamines. A wide variety of partition liquids was tested on firebrick, Chromosorb, Chromosorb W, and Celite with almost complete lack of success. Where distinct elution peaks could be obtained, they tailed badly and the recorder response was low, indicating that incomplete elution of the diamines was being obtained. This finding led to the testing of the four usually recommended chromatographic substrates as bare columns to determine their relative adsorption properties for DCH and HMD. Onemeter columns of firebrick, Chromosorb, Chromosorb W, and Celite were packed and conditioned a t 200" C. for 1 to 2 hours in a hydrogen atmosphere. The temperature was then reduced to 80" C. and a standard 50:50 mixture of DCH and HMD was injected. Figure 1 shows the gas chromatograms obtained. It is apparent that all of the substrates tested have an appreciable adsorption affinity for the diamines and the relative adsorption strengths are in the order of firebrick, Chromosorb (red), Chromosorb W, Celite. In addition, Chromosorb and Celite were found to have some selectivity in their adsorption. This adsorption separation has been pointed out by Bens (1) and might be used to advantage if tailing could be controlled. The diaminocyclohexane emerged first, as would be expected from its 12' C. lower boiling point. To determine the effect of KOH treatment alone, Celite and Chromosorb W were retested under the same conditions as the bare columns after coating with 1, 5, 10, and 20% KOH. Surprisingly, these treatments appeared to have little effect on the retention times or tailing of the DCH and H M D peaks, but sensitivity (peak height)
L CELITE
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TIME IN MINUTES Figure 1. Comparative gas chromatograms of DCHIHMD mixtures on various substrates 1-meter columns at 80' C. and 43 c c per minute hydrogen
was much improved, indicating a marked decrease in adsorption. Figure 2 shows the results of similar screening after each of the four substrates was coated with 1.6% KOH and 3% Carbowax 20M. A low percentage of partition phase and alkali was used in these tests to accentuate any residual adsorption effects. The separations and tailing were progressively better from firebrick to Celite, as was expected from the bare column data. Chromosorb W was lower priced and gave a lower pressure drop, so was used for subsequent work. A series of packings containing 2.5, 5, and 10% KOH and 20% Carbowax 20M was next prepared. Excellent separation of DCH and HMD was obtained a t 120' C. on all of these columns, with no significant differences observed. However, a fourth Chromosorb W packing thoroughly washed with 5% KOH solution, rinsed with one volume of distilled water to remove readily extractable KOH, and then coated with 20% Carbowax 20M and dried failed to yield any detectable DCH or HMD peaks in 30 minutes at 120" C. This behavior was the same as had been observed for Carbowax 20M on Chromosorb W with no KOH. It is apparent that simple washing with KOH does not overcome the adsorption properties of the substrate, and hence it seems very unlikely that the beneficial effects of KOH treatment are due to simple
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Regardless of the mechanism ipvolved, this work has shown that coating Chromosorb W with 5% by weight of KOH prior to appl:cati&n of the liquid partition phase gives gas chromatographic packings of very puperior properties for the separation of aliphatic diamines. Figure; 3 shows the gas chromatogram for'Cc to Clo diamines on a 10% Carbowax ZOM-Chromosarb' W 5% KOH column. Peak symmetry and separations were good and retention time was reasonable. Similar columns of Apiezon L and Dow Corning 710 fluid under the same operating conditions gave similar chromatograms. Less basic potassium carbonate was tried with Carbowax 20M on a 1-meter column and gave similar nontailing peaks. The work was not extended to less basic coatings. A secondary, though very important, objective of this work was to develop a packing capable of separating the cis- and trans-isomers of DCH. These isomers boil so closely together that they cannot be separated by ordinary fractional distillation and, of course, they are very similar in chemical properties. It was found that the three packings described above ditrered widely in their selectivity for these isomers, in marked contrast to their approximately equivalent bahavior for the homologous series of aliphatic diamines. No separation of the DCH isomers was observed on Carbowax 20M, whereas separation was obtained on the Apiezon L and Dow Corning 710 columns. The former column gave considerable overlapping of the cis-DCH (higher
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Figure 2. Comparative gas chromatograms of DCH/HMD mixtures on 3% Carbowax 20M 1.6% KOH packings
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100' C., EO cc per mlnute of hydrogen, 2-meter column
neutralization of "acidic sites" as has been suggested by Hardy and Pollard (6). The limiting amount of free or extractable KOH below 1.6% that would give symmetric peaks was not determined.
EMERGENCE TIME
Figure 3. Gas chromatogram of synthetic C&o aliphatic 5% KOH on diamine mixture on 10% Carbowax 20M Chromosorb W 1 6 0 ' C., 1 1 0 cc par minute of hydrogen, 2-meter column
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EMERGENCE TIME
Figure 4. Gas chromatogram of cis- and frunsDCH and HMD mixture 1 2 0 ' C., 100 c c per minute hydrogen, E-meter column
VOL 33, NO. 9, AUGUST 1 9 6 1
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boiling) peak with that of HMD, while complete separation of all three diamines was finally obtained on the DC 710 column. Figure 4 shows the separation obtained using an 8-meter column of 10% DC 710 on Chromosorb W 5% KOH. This separation was found to be entirely satisfactory for quantitative analysis, even though base line separation was not achieved a t the retention times chosen.
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EXPERIMENTAL
The sources for the various packing substrates and partition solvents used in this work are listed below. Packing Substrates. Chromosorb. W. H. Curtin Co., Houston, Tex., Cetalog No. WHC-8786G7B, 30/60-
mesh size. Chromosorb W. W. H. Curtin Co., Houston. Tex.. Cataloe No. WHC8786G7D, 30/6b-mesh &e. Firebrick. Coast Engineering Laboratory, Hermosa Beach, Calif., GC-22 Super Support, 42/60-mesh size. Celite. Perkin-Elmer Corp., Norwalk, Conn., Catalog KO.154-0048. Partition Liquids. Carbowax 20M. Union Carbide Chemicals Co., New York 17, N. Y. Dow Corning 710 Fluid. Dow Corning Corp., Midland, Mich. Apiezon L Grease. James G. Biddle Co., Philadelphia 7, Pa.
The solid substrates were tested as received for inherent adsorption properties by packing into ‘/,-inch by 1meter aluminum U-tube columns. All gas chromatograms were run on a Perkin-Elmer Model 154-B Vapor Fractometer after the columns were conditioned for at least 1 to 2 hours at about 200’ C. The solid substrates were coated with KOH by slurrying a weighed quantity with a methanolic solution of the KOH. The excess methanol was first removed by evaporation in a water bath with continuous stirring. The moist packing material was then dried in a circulating air oven for 1 to 2 hours a t 100’ C. The calculated quantity of partitioning phase was applied to the pretreated packing in a similar manner. Water was used as the solvent for Carbowax 20M, benzene for Apiezon L, and methylene chloride for the Dow Corning 710 fluid. After evaporation of the excess solvent, the packings were finally dried in a circulating air oven a t 120’ C. No particular precautions were used to protect the packings from COZ during their preparation or later use, so some of the KOH may have been present as the carbonate. ACKNOWLEDGMENT
The authors express their appreciation to M. B. Powell for her careful work in preparing the many gas chromatographic packings studied in this
work, and for running and recording the actual chromatograms. LITERATURE CITED
(1) . . Bens, E. M.. AN^. CHEM.33. 178 (1961) (2) Brooks, V. T., Collins, G. A,, Chent. and Znd. (London) 38, 1021 (1956). (3) Decors, A. W., Dinneen, G. V.,
:
“Solid Support for Gas-Liquid Chromatography of Strongly Basic Nitrogen Cbmuounds.” 2nd Bisnnual Internatj6nal G& Chromatography Symr m , Michigan State University, ast Lansing, Mich., June 1959. (4) Glueckauf, E., Trans. Faraday Soe. (5)51,Hardy, 34 (1955). C. J., Pollard, F. H., J .
Chromalog. 2, 1-43 (1959). (6) Honegger, V. T., Honegger, R., Nature 184, 551-2 (1959). (7) James, A. T., ANAL. CHEM.28, 1564 (1956). (8) James, A. T., Biochem. J . 52, 242 (1952). (9) James A. T., Martin, A. J. P., Smith, 6. H., Zbid., 52, 238 (1952). (10) Ottenstein, D. M., “Influence of the
Chromatographic Support on the Separation of Polar Compounds,” Seventh Detroit Anachem Conference, Detroit, Mich., October 1959. (11). Ring, R. D., Riley, F. W., “GasLiquid Chromatographic Analysis of Amine Mixtures,” Sixth Detroit Anachem Conference, Detroit, Mich., October 1958. RECEIVED for review July 25, 1960. Accepted May 23, 1961. Contribution 70 from Chemstrand Research Center. Inc.
Chromatog ra phic Separation and Identification of Normal Aliphatic Alcohols as Esters of p-Nitrophenylazobenzoic Acid by infrared and X-Ray Diffraction ROLF BOSVIK, KNUT V. KNUTSEN, and C. F. ERIK von SYDOW Swedish Institute for Food Preservation Research (SIK), Gothenburg, Sweden
b Alcohols in fruit flavors can be separated and identified as esters of p-nitrophenylazobenzoic acid by combined paper partition chromatography, infrared analysis, and x-ray diffraction. Both infrared spectra and x-ray diffraction patterns show marked differences among the esters.
A
are generally present in fruit flavors, although mostly in very small amounts. Paper partition chromatography is very useful for separation and identification of the alcohols, and several methods have been described. A general trend is to chromatograph compounds having colors of high intensity and distinctive melting points for a wide melting point LCOHOLS
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ANALYTICAL CHEMISTRY
range. Esters of p-nitrophenylazobenzoic acid (p-NPAZB acid) are easily prepared and seem to be suitable derivatives. Their use for characterization of alcohols using melting point data was first suggested by Hecker (I), and later Winter et al. (8) proposed chromatographic separation of the esters. The possible use of paper partition chromatography, which seemed to be the most effective and practical procedure, was investigated. Identification of compounds using R, values alone, is often insufficient due to the fact that the conditions during chromatography are hard to control, and that several compounds may have nearly the same Rf value. These difficulties can be reduced by using other supplementary methods. In the present
work infrared spectrophotometry and x-ray diffraction proved effective supplementary techniques. EXPERIMENTAL
Reagents. The p-nitrophenylazobenzoyl chloride (p-NPAZB chloride) used for the preparation of the esters was synthesized according to Hecker ( I ) , starting with the ethyl ester of p-nitrobenzoic acid. This ester was transformed into the nitroso compound, which was condensed with p-nitroaniline to give the ethyl ester of p-NPAZB acid. After saponification, the acid chloride was obtained after reaction with thionyl chloride. The alcohols used as references were commercial samples, purified, and checked in this laboratory by gas-liquid chromatography.
