chloride, and the solute was identified by comparison of the infrared spectrum with spectra of authentic reference materials. RESULTS AND DISCUSSION
The ob$erred relative retention times of the three types of materials are listed in Table I. All terpenes were eluted from both columns in less than 8 minutes. The retention time of tetralin (taken as 1) was the reference standard. Under the experimental conditions described, the specific retention volume per gram of liquid phase was 264 ml. per gram for Dow Corning 710 and 164 ml. per gram for Hyprose S.P. 80. As shown in Figure 1, the complete separation of alcohols was achieved with Hyprose S.P. SO but not with Dow Corning 710 column. The aldehydes and esters examined, however, were completely separated in either column. Peak 7 in Figure 1B (geraniol) is skewed toward peak 6 (nerol, the cis isomer of geraniol). The infrared curve for peak 7 is qualitatively very similar to t h a t for pule geraniol. Both
isomers exhibit medium strength absorption bands at 1670 em.-‘ but only the trans form, geraniol, has a band a t about 890 em.-’ The ratio of &SO cm.-l to AI6i0 cm.-1 for pure geraniol was approximately 0.8 whereas this ratio for fraction 7 was lower, indicating the presence of some nerol in fraction 7 . I n general, the behavior of three types of compounds was quite different on the tm-o columns. For example, with the exception of the esters shown in Table I, all compounds had longer retention times on the Hyprose column. W t h the Don. Corning 710 column, all the terpenes within one mixture were eluted in the order of their boiling points, but with the Hyprose S.P. 80 column this was not the case. I n Figure 2 the logarithms of the relative retention times observed on the two columns are plotted against each other. The resulting points for the esters, aldehydes, and alcohols lie approximately in three different straight lines. Determination of the chemical nature of oxygen-containing terpenes can be facilitated in many
cases by comparing their behavior on a polar and a nonpolar column. LITERATURE CITED
(1) Hishta, C., Messerly, J. P., Reschke, R. F., ANAL.CHEX.32,1730 (1960). (2) James, A. T., Martin, A. J. P., Baochem. J . 50, 679 (1952). (3) LittleiTood, A. B., in “Gas Chromatography,” D. H. Desty, ed., p. 23,
Butterworth, London, 1958.
( 4 ) Pollard, F. H., Hardy, C. J;: in
“F‘apour Phase Chromatography, D. H. Desty, ed., p. 115, Academic Press, Sew York, 1957. (5) Ring, R. D., in “Gas Chromatogranhv.” V. J . Coates. H. J. Noebels. I: rST Fagerson, eds., p. 195, Academic Press, New York, 1958. HEIXOS r s ~ Eastern Utilization Research and Develoament Division U. S.Departhent of Agriculture Philadelphia 18,Pa. 1 Senior Research Fellow, American Spice Trade Association. Work was supported in part by funds from the American Spice Trade Association. Mention of a specific product does not constitute endorsement of that product over similar ones not mentioned.
Silicic Acid Chromatography of Methoxypiperonylic Acids SIR: 9 neiv lignan, possessing one unsubstituted and one methoxy-substituted methylenedioxyphenyl group, upon oxidation with potassium perrnanganate in acetone, gave piperonylic acid as expected, but only a minute amount of the niethoxypiperonylic acid. -4means of separating and identifying micro amounts of the methoxypiperonylic acids in the presence of piperonylic acid n-as needed. This can be done by thin-layer chrornatography, using a spray of chromotropic acid in sulfuric acid as the chroniogenic reagent. This reagent gives colors v i t h methylenedioxyphenyl compounds, which are found in many natural products. It will also respond to methylenedioxyl and other groupings that give formaldehyde on treatiiient with acid (g). The methosypiperonylic acid in niilligram amounts n-as isolated by silicic acid column chromatography, and its identity n-as confirmed as 6-methosypiperonylic acid by the ultraviolet and infrared spectral data reported below.
SILICAGEL G. Brinkmann Instruments, Inc., Great Neck, N. Y . , or Microchemical Specialties Co., Berkeley 3, Calif. SILICICACIDwas a specially prepared grade for chromatographing lipides (Bio-Rad Laboratories, Richmond, Calif.), used without drying. It lost 17.8y0 water on drying overnight a t 110’ c. SOLVENTSwere reagent grade and distilled before use. 3,4-llethylenediouyphenyl Acids. Melting points were: piperonylic, 228-0’ C.; 2-methoxypiperonylicJ 1567’ C.; 5rnethouypiperonylic, 2067’ C.; 6-methouypiperonylic, 150-1’ C. Solutions in acetone, 1% (w./v.), were used for thin-layer chromatography. CHROMOTROPIC-SULFURIC ACIDSPRAY RE.~GEST.One volume of freshly made 10% (w./v.) aqueous solution of sodium 1,8 - dihydroxynaphthalene - 3,6 - disulfonate (Eastman Kodak P230) was
Table I.
PROCEDURES AND RESULTS
Thin-layer Chromatography. A silica gel layer, 250 microns thick, applied on a glass plate 200 mni. square as described by Stahl ( 3 ) , was activated for 45 minutes at 105’ C., spotted 2.5 em. from one side with 1 to 3 pl. (10 to 30 kg.) of the acids, and allowed to develop at room temperature in a closed chamber (with a solventsaturated paper lining) containing a 1-em. depth of ethyl acetate-hexaneacetic acid (50:50:0.5) until the solvent front moved 11 to 14 em. past the starting line. The solvent front was marked, and the plate was removed. The sol-
Chromatographic Data on Piperonylic and Methoxy-Substituted Piperonylic Acids
EXPERIMENTAL
Apparatus and Reagents. THIKLAYER C H R O M A T O G R B P H T . The apparatus of Stahl(5) was used. COLVMP;CHROMATOGRAPHY. A smaller version of the all-glass apparatus of Gordon and Beroza (4) was used. (Tube was 1 cm. i.d. b y 30 cm. long; a plug of glass wool replaced the sintered glass disk.)
added to 5 volumes of sulfuric acid, prepared by adding carefully 5 volumes of concentrated sulfuric acid to 3 volumes of water. Renen-ed reekly.
Acid Piperonylic 5-Methoxypiperonylic 2-Methoxypiperon ylic
6-Methoxypiperonylic
Thin-laver Rj val;e 0.64 0.54 0.47 0.37
Column ______--_ Eluent Peak elution 280/300 mp (yoethyl volume absorbance acetate in ratio iso-octane) (ml.1 c0.6 10 13 3.1 10 110 1.1 20 35 0.3 20 140
VOL. 34, NO. 8, JULY 1962
1029
vent was allowed to eraporate (5 minutes), and the plate was sprayed with the chromotropic-sulfuric acid reagent. Heating in an oven a t 105’ C. for 30 minutes revealed the spots. The Rf values are given in Table I. The compounds formed round compact spots that readily separated whether chromatographed together or separately. One-hundred microgram amounts of the niethouypiperonylic acids and 50 kg. of piperonylic acid were chromatographed without appreciable tailing. The methylenedioxyphenyl group is very stable, and i t was necessary to use the corrosive chromotropic-sulfuric acid spray to make the compounds 1 isible. The strong acid in the reagent hydrolyzes the niethylenediouyl group to give formaldehyde, 11-hich reacts with the chromotropic acid to give a colored spot (5’). The spray nil1 detect compounds containing meth: lenediouyl groups but will also respond to other compounds R hich qplit off formaldehyde on treatment with acid-e.g., anisyl alcohol, vanillyl alcohol, and s-trithiane ( 2 ) . As little ar I pg. or an acid was detectable. At the high concentrations the spots were an intense blue, the 6-niethoxy-substituted acid ha\ ing an orange . the acids halo. At the 1- to 5 - , ~ glevel appeared as purple spots eucept for the 6-niethouy compound which was orange. Because R, values by thin-layer chromatography are not as reproducible as on paper, it is important to compare unknown with known acids in a parallel run. Column Chromatography. freefloning slurry of 5 grams of silicic acid in isooctane was poured into the column. After the gel had settled, the column was prewashed ivith 20 i d . of 70y0ethyl acetate in iso-octane and n i t h 20 ml. of iso-octane. Samplea, 0.5 mg., of the four acids in A \
Table II.
Ultraviolet Molar Absorptivities of Piperonylic and MethoxySubstituted Piperonylic Acids in Absolute Ethanol Solution
Acid Piperonylic
5-Methoxypiperonylic 2- RIethoxypiperonylic 6-?vIethoxypiperonylic
Wavelength, llaximum Minimum Maximum Minimum Maximum Minimum Plateau Maximum Minimum Maximum Minimum Maximum Minimum
chloroform were placed on the column, followed by 1-ml. portions of chloroform. The chloroform was eluted by washing with 20 ml. of iso-octane. The column was developed at a flow rate of about 1 ml. per minute with 175 ml. of 10% ethyl acetate in iso-octane and then with 2001, ethyl acetate in iso-octane. The acids were detected in the effluent by their 280/300 mp absorbance ratios ( I ) . The elution volumes and absorbance ratios are given in Table I. ULTRAVIOLET SPECTRA. Molar absorptivities were determined from solutions containing 0.02 mg./ml. of the acid in absolute ethanol. Maxima and minima are listed in Table 11. INFRARED SPECTRA. Disks containing 1 mg. of the acid in 300 mg. of potassium bromide gave major peaks a t the following cm.-’ values: Piperonylic acid: 1670, 1450, 1300, 1260, 1035. 5-Methoxypiperonplic acid: 1680, 1640, 1460, 1430, 1370, 1330, 1275, 1226, 1204, 1118, 1041.
Molar Absorptivity 5260 2570 5890
mp
294,s 275.5 258,O 234.0 270.5 239.0 291-284 260.5 242.0 311 5 275 0 255.5 239.0
1580
7270 1470 2430 6760 4040 6760 960 5680 3210
2-Rlethoxypiperonylic acid: 1700, 1480, 1285, 1081, 1035. 6-Methoxypiperonylic acid: 1720, 1488, 1468, 1432, 1352, 1268, 1239, 1197, 1167, 1030. ACKNOWLEDGMENT
Rafael Sarmiento of this Division assisted in the thin-layer chromatography work. Henry Fales of the Sational Institutes of Health kindly supplied some of the compounds. LITERATURE CITED
(1) Beroza, M., ANAL. CHEM.2 2 , 1507
(1950). (2) Beroza, Ll., Ibid., 26,1970 (1954). (3) Eegriwe, E., 2. Anal. Chem. 110, 22 (1937). (4) Gordon, S . , Beroza, Ll., ANAL. CHEM.24, 1968 (1952). ( 5 ) Stahl. E., Chemzlzei Ztg. 82,323 (1958). MORTON BEROZA
WILLIAM A. JOXES Entomology Research Division U. S.Department of Agriculture Beltsville, RId.
Identification of Alcohols by Color Reactions SIR: Many alcohols n hen treated with certain test reagents produce colored products. Often neither the mechanism of the reaction nor the identity of the products are known, but the color which is generated and the intensity serve as an index for qualitative or quantitative analysis of the alcohol. Reagents which have been used with the simple alcohols to produce colored solutions include, ceric ammonium nitrate ( I ) , salicylaldehyde ( 2 ) , catechol (S), 1-naphthol (4, S-halosuccinimides (6), and resorcinol (6).
This work describes the color reactions of many simple alcohols with aromatic reagents, particularly phenols, in the presence of concentrated sulfuric
1030
ANALYTICAL CHEMISTRY
acid. The variety of color developed and the variation of intensity indicate that these reactions can be used as an aid in the identification of alcohols and particularly in differentiating among simple alcohols.
minutes the color was recorded, and the intensity was evaluated empirically in terms of numbers from 1 (low intensity) to 10 (high intensity). The results are registered in Table I.
EXPERIMENTAL
As seen in Table I, a series of alcohols may produce characteristically colored solutions with a variety of reagents and sulfuric acid, whereby any unknown alcohol of this group can be identified tentatively. For example, 2-naphthol u-ill produce an olive solution with methanol, no color with ethanol, a red-green solution with 1-propanol, a yellow solution with 1-butanol, a rust solution with 1-pentanol, and an orange solution
Twenty alcohols and also water were treated in turn with 25 color inducing agents in the presence of sulfuric acid. The color reagent (approximately 5 mg. for a solid and 5 p1. for a liquid), in most cases a phenol, was added to the alcohol (3.0 ml.) and shaken vigorously. Concentrated sulfuric acid (5.0 ml.) was then poured slowly down the inclined test tube and carefully mixed with the other reagents. After 10
DISCUSSION
