obtaining (dCo/dt)op from Equations 2, 5, and 18 and combining terms, one finds
I,'
=
nPAk,Co*(an,F/RT)K'u
exp(-an,F/RT)
(EDp
- EO) (20)
where
Equation 20 is consistent with Equation 6, evaluated at E D p . Thus, by experimentally varying the scan rate and plotting ln(I,'/u) against (EDp - E " ) , a linear plot can be obtained with slope a function of an. and intercept a function of k, and an,. This last relationship may have one distinct advantage over a similar one proposed by Gokhshtein and Gokhsh-
tein and (3) and Nicholson and Shain (4) which relates the conventional peak current to ( E , - E"). T h a t is, because the scan rate must be varied over several orders of magnitude to obtain kinetic data, the interference of charging current may be a serious limitation with conventional voltammetry. The derivative measurement minimizes charging current interferences (6) and, therefore, may prove more useful experimentally. Further work is currently in progress in this laboratory to correlate experimentally irreversible behavior with the theoretical relationships described in this communication. I n addition, the application of derivative voltammetry for trace analysis with irreversible systems is being investigated. The results of these studies will be reported in the near future.
LITERATURE CITED
(1) Buck, R. P., ANAL.CHEM.36, 947
(1964). (2) Delahay, P., "New Instrumental Methods in Electrochemistry," Chap. 3., Interscience, Sew York, 1954. (3) Gokhshtein, A. Y., Gokhshtein, Y. P., Doklady Akad. ,Vauk SSSR 131, 601 i 1960). (4) Xicholson, R . S., Shain, I., Au.ir,. CHEM.36, 706 (1964). ( 5 ) Perone, S. P., Birk, J. R., Ibzd., 37, 9 (1965). (6) Perone, 6. P., Mueller, T. R., Ibid., p. 2. (7) Reinmuth, W. H., Ibid., 33, 1793 (1961). S.P. PEROXE C. V. EVIXS Department of Chemistry Purdue University West Lafavette. Ind. I N V E S T I G A T I ~ N supported in part by Public Health Service Research Grant T o . CA-07773-01 from the Sational Cancer Institute. .
I
,
Gas Liquid Chromatographic Separation of Resin Acid Methyl Esters with a Polyamide Liquid Phase SIR: I n recent years a number of gas liquid chromatographic column systems have been applied to the separation of resin acid methyl esters (1, 4, 6-8). I n spite of these many reports, a liquid phase which cleanly resolves all the seven major resin acids has yet to be found. I n the course of our continuing efforts to find a rapid and simple method of analyzing complex mixtures of the resin acids, we have found that Versamide 900 is a very useful substrate. EXPERIMENTAL
Gas liquid chromatographic separations were made using an F&M Model 500 gas chromatographic instrument equipped with a thermal conductivity detector and utilizing W2 tungsten filaments. Columns were packed by blowing the packing material from a homemade or commercial reservoir inch 0.d. copper into the precoiled refrigeration tubing under a nitrogen pressure of 50 to 55 p.s.i. A mechanical vibrator was employed to aid in fluidizing the packing as it was being loaded into the column. Coating of the Versamid 900 on the solid support was accomplished in the following way. Equal parts of the polyamide and 2-ethylhexanol were heated on a hot plate until homogenous. The resulting solution was then diluted with hot ethanol in a n amount sufficient to give a fairly fluid slurry with the solid support. After slurrying with solid support, the solvent was stripped off i n vacuo with a rotary evaporator. The coated support was reslurried with hot ether and again stripped of solvent. Before use in packing columns the packing material was screened to a 60/80 mesh particle size. Coating on Chromosorb W and Anakrom ABS
supports gave packings with essentially identical performance characteristics. Columns were conditioned for 12 to 16 hours a t 250" C. Peak assignments were verified b y use of samples of purified resin acids. The acids and acid mixtures (rosin) were esterified with freshly prepared diazomethane. Determination of Relative Response of Resin Acid Methyl Esters. Pure resin acid methyl esters were weighed (10 mg.) and dissolved in 2 ml. of a methanol solution containing 5 mg. per ml. of eicosane. Aliquots of each ester solution were passed through t h e gas chromatograph and the areas of t h e ester and eicosane peaks were compared. T h e relative response was calculated by dividing the area of the eicosane peak into the area of the ester peak. T h e relative responses of the methyl esters of the following resin acids were determined : levopimaric acid, 0.56; abietic acid, 0.80; isopimaric acid, 0.79; pimaric acid, 0.80; a n d dehydroabietic acid, 0.78. RESULTS A N D DISCUSSION
Table I lists the retention times for the seven most common resin acids ) relative t o methyl pimarate ( T ~ ~and to the next earlier emerging ester (rI2). Data for a diethylene glycol succinate (DEGS) column of similar efficiency is given for comparison with the Versamide 900 column. DEGS columns have recently been recommended for analysis of oleoresins (6). The r12values obtained with the DEGS column are in good agreement with those reported by Nestler and Zinkel (6). A Golay resolution of 4 (S), corresponding to 98% separation, of the most difficultly separable pair would require about 5500
plates using DEGS as the substrate but only 3500 plates using Versamide 900. As shown in Figure 1, good separation of the resin acids present in slash pine gum was achieved in 35 minutes with a 15-foot Versamide 900 column. Only the methyl levopimarate-methyl palustrate pair failed to separate. The separations of this pair which are reported in the literature (4, 8 ) apparently resulted from the use of old solutions of methyl levopimarate (6). I n all these cases the same relative retention is reported for levopimarate and for dehydroabietate. Hudy (4) observed typical acid catalyzed isomerization of levopimarate during GLC with the appearance of peaks of abietate and neoabietate, while Nestler (6) reported on the basis of
Table I. Relative Retention Data for Resin Acid Methyl Esters on DEGS and Versamide 900 Column
T'ersamide 900a
DEGSb
rlpc
rl,
718
712
Pimarate 1.00 . . . 1.00 , . . Levopimarate/ palustrate 1 . 2 1 1 . 2 1 1.31 1.31 Isopimarate 1.32 1.09 1.41 1 08 Dehydroabietate 1 . 5 1 1.14 2.10 1.07 Abietate 1.74 1 . 1 5 1.96 1.39 Neoabietate 1.96 1.13 2.27 1 . 0 8 " 10 ft. X ' / 4 inch 57, Versamide 900 on Chromosorb W at 250" C., 3000 plates for abietate. b 10 ft. X '/4 inch 20y0 DEGS on Chromosorb W at 220" C., 2700 plates for abietate. Retention relative t o pimarate. Retention relative to preceding peak.
VOL. 37, NO. 8, JULY 1965
1063
W
Figure 1 .
Chromatogram of methyl esters of pine gum
Instrument, F 8 M M o d e l 500 Column, 1 5 ft. X 1 /4 inch 5 % Versamide 900 on 6 0 / 8 0 Chrom W. Column temperature 2 5 0 ’ C.
ultraviolet spectra that levopimarate was not isomerized on a DEGS column. With the Versamide 900 column a sample of pure methyl levopimarate gave only a single peak. The material collected from the peak also gave a single peak when it was rechromatographed, but the ultraviolet spectrum of the collected material had a somewhat broader absorption band than pure levopimarate. This suggested that some isomerization to palustrate had occurred. Estimation of the composition of mixtures of levopimarate and palustrate from ultraviolet spectra is very difficult because the maxima for the pure compounds are broad and only 7 or 8 mp. apart. Optical rotation is much better for this purpose. Methyl levopimarate has an [Cy], of -269’ and methyl palustrate has an [Cy], of +68’. The material collected from the levopimarate peak had an [Cy], of -134.5’ which corresponds to a 60:40 mixture of levopimarate and palustrate. Similar results were obtained with Chromosorb W and Anachrom ABS supports. This isomerization was also observed with Craig polyester substrate. Collection with the detector filaments turned off had no effect. Although little is known about thermal isomerization of methyl levopimarate, the apparent selective isomerization to palustrate was unexpected in view of the known behavior of levopimaric acid (2, 6). Previous work (9) had shown that the seven major resin acids all gave the same specific response with the thermal conductivity detector. However, using 1064
ANALYTICAL CHEMISTRY
Det block temperature 285’ C. injecture temperature 2 7 5 ’ C. He flow rate, 1 2 0 ml./min. Attenuation, 2 Fil. current, 200 ma.
either Versamide 900 or Craig polyester columns, methyl levopimarate gave only 70% response relative to the other resinates-Le., only 70% of the injected levopimarate emerged in the levopimarate-palustrate peak. Careful examination of the chromatograms revealed that the curves did not return t o the base line following the levopimaratepalustrate peak. When the column effluent was collected from the end of this peak to the point a t which all the neoabietate should have emerged, and rechromatographed, peaks of methyl abietate and neoabietate were found. Hence, the isomerization of methyl levopimarate occurs throughout the column yielding methyl palustrate which is not resolved from the levopimarate and methyl abietate and methyl neoabietate which are distributed over the length of the column and have no opportunity to separate. Combining the relative response data with the optical rotation data about 42y0 of the levopimarate emerged unchanged, 28% was converted to palustrate and 30% to a mixture of abietate and neoabietate. After one hour at 155’ C. 1eTopimaric acid was reported (8) to have isomerized to a mixture of 43% levopimaric acid, 24% palustric acid, and 33% abietic and neoabietic acids. Hence, the observed isomerization of levopimarate on the GLC columns appears to be due to acid catalysis by the substrates. Attempts to deactivate the Versamide with amines were unsuccessful. Obviously this on column isomerization limits the usefulness of Versamide for quantitative analysis of pine gum. However, the excellent
separation of abietate, dehydroabietate, and neoabietate makes it particularly suitable for use with rosins and modified rosins, which contain little or no levopimaric acid. Column life was only moderate, being limited by the loss of resolution of isopimarate from palustrate. Typically, a 5yo column had a useful life of 50 hours or more at 240’ C. LITERATURE CITED
( 1 ) Abrams, E., TAPPI 46, 136A (1963). ( 2 ) Baldwin, D. E., Loeblich, V. &I., Lawrence, R. V., J . Am. Chem. SOC. 78, 2015 (1956). ( 3 ) Dal Nogare, S., Juvet, R . S.,“Gas-
liquid Chromatogra hy Theory and Practice,” p. 26, fnterscience, New York, 1962. ( 4 ) Hudy, J. A., ANAL. CHEM.31, 1754
(1959). ( 5 ) peblich, V. M., Baldwin, D. E., 0 Connor. R. T.. Lawrence. R. V.,’ J . Am. Chem. S O C . ’ 6311 ~ ~ , (1955). ( 6 ) Nestler, F. H. M., Zinkel, D. F., ANAL.CHEM.35, 1747 (1963). ( 7 ) Norin, T., Westfelt, L., Acta Chem. Scand. 17, 1828 (1963). (8) von Rudloff, E., Sato, A., Can. J . Chem. 41, 2165 (1963). (9) Summers, H . B., Southern Utilization
and Development Division, Olustee, Fla., unpublished data, 1965. THOMAS W. BROOKS S. FISHER GORDON N. MASONJOYE,JR. Naval Stores Laboratory U. S. Department of Agriculture, Southern Utilization Research and Development Division Olustee, Fla. SUPPORTin the form of a fellowship sti end to T. W. B. from the Pulp Chemicag Association is acknowledged. Mention of proprietary products does not constitute endorsement of the product by the U. S.Department of Agriculture.
