stir until it dissolves. Add 6F NaOH dropwise until the brown precipitate which forms just redissclves; if the solution is too acid the complex will not fullv form. This gives a green solution 0.050P in the indicator; 2 to 3 drops in 50 to 75 ml. are sufficient for a titration. The use of this indicator is illustrated by the data given for the titration of ferric iron with CrClz A solution of ferric chloride in HC1 vim prepared and standardized against K2Cr207. A chromous chloride solution was prepared by electrolytic reduction of CrC13 and was standardized by coulometric titration with Brz. One-millilitcr aliquots of the iron solution were titrated as follows: 50 ml. of 1F H2S04and 3 drops of indicator were placed in a cell and oxygen was removed with K2. Chromous chloride n-as added until thl: first perceptible blue color, the iron solution was added, and the solution was iitrated again to the blue color. The color change is rapid, even a t room temperature, and iy easily seen, even though the solution is colored green by the chromic ions. The color change is obscured if more than 3 ml. of 0.1F FeCl, are titrated in 50 ml. Figure 1 shows that the indicator end point effectively coincides with the equivalence point in this titration. The
Table I.
i
I
-2ooc
I
-3001-
Results of Titrations
Fe taken,
Cr(I1) solution,
Fe found,
4.93
1.010 1.032
4.96 4.92 4.94
l0OC
--
1.036
av. 4.94
Pale Green
LITERATURE CITED
(1) Brandt, W. W., Dwyer, F. P., Gyarfas, E. C., Chem. Rev. 54, 959 (1954). (2) Kolthoff, I. ill., Stenger, V. A.,
“Volumetric Analysis,” 2nd ed., Vol.
630, Interscience, New York, -600LL _ * ’ (3)1957. Ibid., Vol. I, 109 111, p.
0
2
m1. c r i m B
IO
h
Titration of 0.896 ml. of 0.08906F FeCI3 in 50 ml. of 1F HnS04 with 0.08533F CrClz Figure 1.
results of some visual titrations are shown in Table I. The use of this complex, as well as substituted phenanthroline complexes of vanadium, as indicators for other oxidation-reduction titrations is being investigated and the results will be reported later.
p. ff. (4) Laitinen, H. A., “Chemical Analysis,” pp. 448-50, McGraw-Hill, New York,
1960.
WILLIAM P. SCHAEFER
Division of Chemistry and Chemical Engineering Ca!ifornia Institute of Technology Pasadena Calif. RECEIVEDfor i-evieiv July 10, 1963. Accepted July 26, 1963. Research supported by the National Science Foundation Contribution KO 3001 from the Gates and Crellin Laboratories of Chemistry.
Separatioin of the Methyl Esters of Resin Acids by Gas Liquid Chromatography SIR: At the Forest l’roducts Laboratory, work has been under way with the objective of developing a gas liquid chromatography method for the separation of the fatty and resin acids found in extractives from wood and wood products. At this stage in the study several results may be stated. First, the polar liquid Ihase, diethylene glycol succinate (DEGS), gives better separation of the methyl esters of the principal resin acids ( 7 ) than previously used polar solvents (6) and appears also to be capable of separation of some pairs of these esters to an extent not indicated by a recent publication ( 1 ) . Second, methyl levopimarate, though quite labile both chemically-e.g,, acid medium-and thermally, has been found to traverse the GC column unchanged both chemically and reproducibly in the chromatographic sense. Finally, when stored in capped vials as a solution in a low boiling solvent (Skellysolve B, n-hexane), methyl levopimarate is unstable and slowly converts to methyl dehydroabietate. The change is accompanied by four additional peaks having retention times shorter than that of the parent material; one of the four
peaks has a retention value corresponding to that of methyl pimarate. EXPERIMENTAL
Apparatus. A Research Specialties Company Model 600 gas chromatograph equipped with thermal conductivity detector is being used. Operation has been in the isothermal mode with 275O, 200°, and 215” C. as the respective temperatures of injection port vaporizer, column, and detector. The detector is operated with a bridge current of 200 ma. Sample size (0.25 to 1.0 pl. of solution) and solute concentration are adjusted so that the detector m y be operated a t full sensitivity (XI scale) into a recorder (Leeds and Northrup, Speedom x Type G, AZAR) with 1-mv. full scale sensitivity and 1/4-inch-per-minute chart speed. The helium carrier gas is adjusted to a nominal flow (soap-film flowmeter) of 150 ml. per minute, where p i / p , = 2.18 ( p o = atmospheric pressure). Microliter size hypodermic syringes (Hamilton Co.) were used for sample inj eetions. The apparatus described by Lohr and Kaier (11) was adapted to the instrument for the purposes of peak trapping from the effluent steam. A i-tube-
shaped trap mas used to collect solvent bleeding from the packing. Column. The column used was 6foot stainless steel tubing, nominal ’/(-inch o d. The effective dimensions of t h e packing were 0.54 cm. ( i d . of tubing) b 182.7 em. Iong; 14.6 grams of D8GS packing were loaded into this column volume. The packing was prepared from materials available commercially: DEGS, “pretested polyester,” (Applied Science Laboratories, Catalog S o . M306) ; KSG, nitrile silicone gum, General Electric Company, XE60 (Analytical Engineering Laboratories, Inc., Catalog No. GS91.4) ; solid support, Anakrom ABS, 70- to 80-mesh (Analytical Engineering Laboratories, Inc.). The support was coated in each case by slurrying it and the appropriate liquid phase in acetone and removing the acetone in a rotary evaporator with heating. While the solvent loading was initially a nominal 20% by weight for each packing, subsequent bleeding reduced this figure. By trapping the column effluent almost continuously during operation, it is estimated that the loading was reduced to approximately 16% (maximum) for the DEGS data of Table I and 225’ C. data of Table 11; the loading for the rernaindc t VOL. 35, NO. 11, OCTOBER 1963
1747
RESULTS AND DISCUSSION
Table 1.
Relative Retention Data for Methyl Esters of Resin Acids"
DEGS Ester 18:Ob
Table 11.
rLp
rla
1.00
Pimarate 4.08 Levopimarate 5.47 Palustrate 5.50 Isopimarate 5.98 Abietate 8.64 Dehydroabietate 9.52 Neoabietate 10.2 0 Column temperature, 200° C. 1.72 for methyl stearate.
NSG
...
1.00 1.01
1.34 1.01 1.09 1.45
0.75 1.09
a12
rLP
a19
0.183
...
...
...
1.00
... ... ...
...
*..
... ...
1.58 1.40 ... 1.74 1.10 1.24 1.13 ... 1.86 1.07 Stearic acid, tn' = 4.3 minutes, rlb(lG:O = 1) =
Temperature Function of Relative Retention for Test Pairs of Fatty Acid and Resin Acid Esters on DEGS
Temperature, C. 200 216 Resin acid esters: methyl dehydroabietate-methyl abietate cy19 1.11 1.09 1.07b 9.94 9.42 8.80 riac nc (plates) 2060 1880 1800 1.13 0.94 R 0.66 Fatty acid esters: methyl oleate-mctliyl stearate t ~-~ ' d(min.) 6.02 3.34 1.96 . , 18'3
225" 1.07
8.67
2080
0.72
2.19 1.19 n d (plates) 1690 1630 1560 1480 R 1.50 1.42 1.31 1.4 a From a previous set of measurements (1.2). Calculated from initialretention times, ~ R I '(6). c Methyl dehydroabietate. Methyl stearate. 0112
=
rla
1.17
of the data in Table I1 and for that in Table I11 is something less t,han the above figure. For the NSG data (Table I) the loading was not larger than lo%, excessive bleeding having occurred. Each packing was conditioned and operated a t a number of temperatures. With the DEGS column, the maximum temperature of exposure was 236' C.; for the NSG, this was 265' C. Normal use of each was at 200' C. Methyl Ester Standards. The fatty acid methyl esters were of 99+% purity (Applied Science Laboratories, State College, Pa.) Levopimaric (9), neoabietic (IO), and isopimaric (2) acids were isolated from oleoresin; dehydroabietic acid was prepared from dehydroabietonitrile (4), and abietic acid was
1.17
1.18
prepared by isomerization of levopimaric acid (8). Palustric and pimaric acids were obtained from R. V. Lawrence, Naval Stores Laboratory, U. S. Department of Agriculture, Olustee, Fla. Methylation of the resin acids was accomplished using diazomethane in a method similar to that of Schlenk and Gellerman (IS). Freshly distilled diazornethane in ethyl ether was added dropwise to a solution containing 10 mg. of the resin acid per ml. (9:l) ethyl ether-methanol a t 0' C. until the solution had a yellow tinge against a white background. The slight excess of diazomethane and solvent were removed with a stream of high-puSity nitrogen. Methyl levopimarate, isopimarate, and dehydroabietate were each recrystallized before use.
i
Table I lists the relative retention values obtained for the seven principal resin acid methyl esters. With the exception of methyl pimarate, all data have been computed from chromatograms in which one or both of the standards, methyl stearate (ria) and/or methyl levopimarate ( r L p ), were also present. The use of the two reference compounds has been found convenient and is felt to be necessary also because of the great differences between the properties of the two types of acids, chemically and structurally. Since reported data (1, 6) have been given with respect to methyl palmitate, conversion of the data in the tables to this basis may be made from T I E = 1.72 X r18. [Relative retention values for practically all members of the fatty acid esters in the series 12:O through 24:0, plus 18:1and 18:2, have been measured; these data are omitted here since they agree essentially Mith that summarized elsewhere (3) 1, The degree of separation realized between adjacent pairs of resin acid esters is more effectively seen from the respective separation factors, a12 (column 4, Table I). Each value of a! is computed relative to the previous compound as the standard-Le., as component 2. In the published data (1, 6), GC separation of the ester pair abietatedehydroabietate has not been achieved. Recent data (12) obtained in the course of this work reported all = 1.07 for this pair a t 225' C. Extension of the data to several lower column temperatures has been made with the results shown in Table 11. For comparison, similar data are included for the fatty acid pair methyl oleate-methyl stearate which is used as a test mixture in following the history of the packing. The improvement in both separation factor and resolution is very evident as column temperature is decreased; an
75
?
Figure 1.
Chromatogram of methyl levopimarate, in solution
97 days
1748
0
P
ANALYTICAL CHEMISTRY
Figure 2. Chromatogram of methyl oleoresin, filtered through alumina
effective separation of this pair is thus possible. Presumably, further improvement in separation would result by decreasing column temperature further, but this at the expeme of an increase in retention time which could be offset only by decreasing the liquid phase loading. iidditional work is planned in this respect. The retention values for methyl levopimarate as reported (6) appear to be attributed to a peak derived from isomerization or disproportionation. In the course of our work ( l a ) ,injection of a sample of pure, (crystalline methyl levopimarate dissolved in low boiling solvent (C, range) resulted in a single peak on the chromatogram. The solute band corresponding to the chromatogram peak identified as methyl levopimarate was collected and its identity verified by means of UV spectrometry. Various samples of methyl levopimarate, both pure and impurc:, were used in this phase of the study, all with identical results. We believe, therefore, that this resin acid ester ISstable to the gas chromatography conditions applied herein and traverseii the column unchanged chemically and with a reproducible GC retention value. Special attention has been given to minimizing the possibility of acid-catalyzed isomerization of the resin acid esters by use of catalyst-free prepurified DEGS on siliconized diatomaceous earth support (Anakrom ABS). I n an unknown mixture the accurate identification of methyl levopimarate from GC retention data alone is limited, however, by the fact that other resin acid esters have (methyl palustrate, Table I), or may have, relative retention values essentially equal to that of levopimarate. Verification of the peak due to the latter would be subject then to independent analysis or to gas chromatography with a liquid phase other than DEGS that affords the needed separation. The latter aspect is also under investig a t’ion. Sufficient evidence has been accumulated to show that solutions of a crystalline, pure methyl levopimarate in low boiling solvents such as purified Shellysolve B and n-hexane, though stored under nitrogen in capped glass vials, are not stable. The resin acid ester is converted to methyl dehydroabietate (predominmtly) plus four other compounds wilh retention times shorter than that of methyl levopimarate (Figure 1) One of these four (No. 3) has a relatiJe retention value corresponding to thai; of methyl pimarate. Peak 1’ is observed distinctly in a “vounger” solution where peak 1 is smaller than here. The identity of both methyl levopimarate and methyl clehydroabietate peaks was verified from the characteristic ultraviolet spectrum of each for samples trapped from
Table 111. Relative Retention Data for Methylated Oleoresin and Rosin
Peak no. 18:O1 2 3 4
5 6 7
Tis
TLP
Compound
1.00 3.19 3.54 4.08 4.57 5.55
0.180 Stearate 0.57 0.64 .. .
6.03 6.95 8.75 9.46
1 .09 1 25 1.58 1.71
...
0.73
0.82 1 .OO
Pimarate
Levopimarate/ Dalustrate Isopimarate
Abietatk’ Dehydroabietate 10 10.2 1.85 Keoabietate Methyl stearate, t ~ =’ 2.94 minutes. 8
9
0
the chromatograph effluent. Experiments aimed at observing the effect of the sample container on the stability of the ester showed that conversion could not be prevented even if glass was rinsed with aqueous ammonia solution, and that the rate of conversion was affected by the nature of the sample container and was slower for samples stored in “test tubes” of Teflon and polyethylene. Work is in progress to determine the source of this problem and how to avoid it so that the integrity of wood extracts will not be impaired. As a demonstration of the practical value of the technique and data described above, samples of gum rosin and of the resin acids obtained from an oleoresin sample by amino-cellulose ion exchange (1-4) were methylated and chromatographed. A portion of each ester mixture was filtered through an alumina column to remove oxygenated compounds. Four samples were run; each wm “spiked” with a small amount of methyl stearate. The resulting data are summarized in Table 111 and illustrated by the chromatogram for the filtered methyl oleoresin sample (Figure 2). With one exception (the unfiltered methyl rosin sample), the four chromatograms were alike qualitatively, each exhibiting the same nine peaks listed in the table; peak 7 disappeared when the methyl rosin sample was filtered. Since the adjusted retention times for the four samples were within *l% of each other, they were averaged and the relative retention values computed. The slightly higher value of for the levopimarate-palustrate peak apparently reflects the fact that the rosin sample contained no levopimarate, and in the oleoresin, this peak contained both esters in the ratio of approximately 3 to 1. These supplementary data were obtained by a thin layer chromatography technique wherein the levopimarate-palustrate pair was separated (15).
Quantitative application of the data is not possible a t this stage in the study. However, as an indication of the
quantity of material applied to the column, solutions of known concentration of methyl stearate and methyl levopimarate were chromatographed and interpreted in terms of peak height and peak area. I n the sample size range of 10 to 400 pg. represented by these data, the response was closely linear with respect to both peak height and peak area. For methyl levopimarate, the slope was 0.44 pg. per sq. mm. and 0.57 pg. per division for methyl stearate. Thus, the respective ester peaks in Figure 1 represent 50 pg of methyl levopimarate and 34 p g . of methyl stearate. Preliminary work (12) with the nitrile silicone gum XE60 (NSG) a t 200” C. provided the data, in Table I for three resin acid esters. While the relative retention with respect to methyl levopimarate is less than that for DEGS, the separation factor for methyl abiet ate-me t iiyldehydroabietato is not, but the order of elution has been reversed. LITERATURE CITED
(1) Abranis, Ellis, Yuppi 46 (2), 136A (1963). ( 2 ) Baldwin, D. E., Loeblich, V. M., Lawrence, R. V , J . Oig. Cliem. 23, 25 (195s). ( 3 ) I+rchfie!d, H. ,P., Storm, E. E., “Hiochemic:il Applicxtions of Gas Chromatography,” p. 549, Academic Press, S e w York, 1962.
Hercules Powder Co., unpublished procedure. (5) Hudy, J. A., ANAL CIIEM.31, 1754
(4)
(1959). (6) Keulemans, A. I. hf., “Gas Chromatography,” 2nd ed., p. 16, Reinhold, S e w York, 1960. ( 7 ) Lawrence, Ray V.,Tuppi 45 ( 8 ) , 654 (1962). (8) Lawrence, R. V., unpublished proce-
dure.
(9) Loeblich, V. bI., Baldwin, D. E
,
O’Connor, R. T., Lawrence, It. V., J . Am. Chcm. SOC.77, 6311 (1955). (10) Loeblich, V. h l , Lawrence, R. V., J . Org. C h e w 21,610 (1956). (11) Lohr, L. J., Kaier, R. J., F & h1 Scientific Corp., Avondale, Pa., “Facts and Methods for Scientific Research,” Vol. 2, No 2, Fall 1961. (12) Xestler, F. H. &tax, Forest Products Laboratory, U.9 D A , , Madison, Wis., Qzarterly Progress Repl No. 2, Division of Wood Chemistry Research, January
1, 1963. (13) Schlenk, H., Gellerman, J. I,., ANAL. CHEK32, 1412 (1960). (14, Zinkel, I). F., Rowe, J. W., unpub-
liehed data
(15) . . Zinkel, D. F., RoN-e, J. W., J . Chromalob., (in press).
F. H. MAXNESTLER DUANE F. ZINKEL Forest Products Laboratory Forest Service U.S. Department of Agriculture Madison 5, Wis. RECEIVEDfor review May 29, 1963. Accepted July 20, 1063. ’Cl’ork supported in part by Pulp Cheniicnls Association, New York, K.Y. Mention of proprietary products does not constitute endoreement of the product by the U. S. Department of Agriculture. YOL
35,
NO. 11, OCTOBER 1963
1749
