of static (liquid) phase, sq. em. C, = mass of solute per unit volume of mobile phase C, = mass of solute per unit volume of static (liquid) phase g = thermal gradient, O C. per em. GGC = gradient gas (-liquid) chromatography H = height equivalent to a theoretical plate, cm. gas (-liquid) IGC = isothermal chromatography Q = molar heat of vaporization of solute from solvent, tal./ mole R = gas constant, cal./deg.-mole T = temperature, O K. T, = characteristic temperature = cross-sectional area
A,
AT, = radial
temperature difference in column, Tperiphery -
TO,,,,,
t Vb V,
= time,sec. = band velocity, cm./sec. = furnace or heater velocity,
V,
= =
z
(T
cm./sec. linear gas velocity, cm./sec. lenuth of column traversed Fy band or furnace = sigma, standard deviation of normal error curve, em. ACKNOWLEDGMENT
This research was supported by a giant from the Petroleum Research Fund administered by the American Chemical Society. Grateiul acknowledgment is hereby made t o the donors of this fund.
LITERATURE CITED
(1) Bohenien, J., S)turnell, J . H., “Gas Chromatography, D. H. Desty, ed., p.
6, Academic Press, New York, 1958. J. C., A 4 ~CHEM. ~ ~ 34, . 122 (1962). (3) Janak, Jaroslav, “Chromatography,” E. Heftmann, ed., p. 654, Reinhold, Yew York, 1961. (4) Porter, P. E., Deal, C. H., Stross, F. H., J . Am. Chem. Soc. 78, 2999 (1956). (5) Tudge, A. P., Can. J . Phys. 40, 557 (1962). (6) ZhukhovitslriI, A. A., Zolotareva, 0. V,. Sokolov. V. A , . Turkel’taub. N. M.: Doklady’ Akad. k a u k S.S.8.R:
( 2 l Giddings,
77,453 (1951).
RECEIVEDfor review August 13, 1962. Accepted December 6, 1962.
Paper Chromatographic Analysis of Terpene Alcohols as Their o-Nitrophenyl- and p- Phenylazophe nyl uretha n Derivatives JOHN A. ATTAWAY, RICHARD W. WOLFORD, and GILBERT E. ALBERDING Florida Citrus Commission, lake Alfred, Fla.
GEORGE J. EDWARDS University of Florida Citrus Experiment Station, lake Alfred, Fla.
b A paper chromatographic procedure has been developed for the separation and identification of terpene and related alcohols as their o-nitrophenyland p-phenylazophenylurethans. This method was successfully applied to the identification of gas chromatographically separated alcohols too impure or too low in concentration for infrared confirmation. The procedure was also useful for the analysis of alcohols without prior separation by gas liquid chromatography.
T
of oxygenated terpenes found in volatile constituents of fruit juices, naval stores, perfumes, and many other sources presents a very challenging problem to the analytical chemist. The mixtures normally encountered contain alcohols, esters, aldehydes, ketones, and possibly organic acids. Many of the individual components are quite similar in their chemical and physical properties and, consequently, are very difficult to separate. Several of the terpene alcohols are closely related isomers. The use of gas liquid chromatography has greatly simplified the problem, but HE ANALYSIS
234
ANALYTICAL CHEMISTRY
even this powerful tool does not completely resolve natural mixtures of osygenated terpenes into individual components. A recent publication from this laboratory (3) has shown the presence of as many as 53 components in orange juice extracts, partially or completely resolved by gas chromatography. Gas chromatographic peaks from these mixtures frequently contained multiple components and represented more than one chemical class. Minor components, particularly those not completely resolved, frequently could not be identified through their infrared spectra because of impurities or lack of sufficient sample. Because it is unsafe to rely on retention times alone for identification of components in such highly complex mixtures, the objective of this study was to provide a method for the absolute identification of terpene alcohols in the presence of other similar compounds. A previous publication (1) had shown that o-nitrophenyl- and p-phenylazophenylurethans of Cg to Clz saturated aliphatic alcohols could be successfully separated and identified in quantities as small as 1 pgram using paper chromatographic techniques. Consequently, it was desired to adapt this technique to the more complex terpene alcohols to
provide a procedure for identification of traces of gas chromatographically separate alcohols, as well as a means of qualitatively analyzing terpene alcohols without the use of gas chromatographic instrumentation. REAGENTS
The alcohols were obtained from several sources including complimentary samples from The Glidden Co., Jacksonville, Fla.; Fritzsche Brothers, New York, N. Y.; and Hoffman-La Roche, Nutley, N. J. The isocyanates used in the preparation of the urethans were obtained from Distillation Products Industries, Rochester, N. Y. PROCEDURE
The urethans were synthesized as follows: 2 grams of isocyanate, 4 ml. of pyridine, and 1.5 ml. of the alcohol were mixed and allowed to stand a t room temperature for 3 days. Primary alcchols reacted quickly, but some tertiary alcohols reacted slowly. One milliliter of water was then added to the mixture, which was allowed to stand for several hours to decompose excess isocyanate. The mixture was washed with a slight excess of saturated aqueous citric acid to remove the pyridine, and then extracted with hot Skellysolve C. ‘The
extract was passed through a 6-inch by 3/4-inch column of activated alumina t o remove impurities, including unreacted alcohol, and to facilitate crystallization. The p-phenylazophenylurethans were crystzlline solids. Their melting points are shoan in Table 1. The o-nitrophenylurethms n-ere difficult t o crystallize, and relatively few gave acceptable melting points. These are shown in Table 11. Since many o-nitrophenylurethans could not be characterized by melting point, their infrared spectra were determined and checked against those of known o-nitrophenylurethans (2). The paper chromatograms were developed and the spots detected according t o Attaway et al. ( I ) using Whatman S o . 3MlI paper impregnated with vaseline and 90-11, methanol-water, as developing solvent. The development time was increased to 5 hours t o give better separation. The adaptability to gas liquid chromatography mas studied using a n F & M Model 502 Linear Temperature Programmed Dual Column Gas Chromatograph. The column mas 12 ft. by 1/4 inch packed with Carbowax 20M, 20% on 40- 60-mesh Chromasorb P. Fifty microliter aliquots of various mixtures containing 4 t o 10 known terpene and aliphatic alcohols plus nonalcoholic contaminants were separated gas chromatographically using this instrument. Individual alcohols, or alcohol pairs where separation was incomplete, were collected in 5-mm. 0.d. glass U-tubes, cooled in dry ice and acetone where necessary. The stainless steel adaptor was removed from the exit port so that the collecting tube could be held flush against the collecting block t o avoid prior condensation. Also, the tube was fitted with a rubber sleeve t o secure s snug fit. The alcohols condensed as a thin film or a collection of minute drodets. They n-ere converted into urethak as follows: 2 to 3 drops of dry pyridine were carefully passed through the Utube t o wash the alcohol into a I-ml. vial. A few crystals of the isocyanate were added t o the solution. The vial was capped and placed on the shelf for 3 days. A few drops of a saturated aqueous solution of citric acid were added and allowed t o stand overnight. It was then extracted with about ml. of Skellysolve C. The ex.tract was concentrated and paper chromatographed.
~~~~~
Table I. Melting Points of p-Phenylazophenylurethans
Parent alcohol Phenethanol Cinnamyl alcohol Thymol
a
Melting point, C. (uncorrected) 121-23 134-38 161-63
3-Hexen-1-01 2-Hexanol Methvl hep”teno1 Linalool
107-11 78-79 68-70
Carveol Isopulegol Borneol Fenchvl almhol Terpinen4-01
107-09 81-83 151-52 134
-4one dimensional, descending paper chromatographic study was made of the a-nitrophenyl- and p-phenylazophenyluretlian derivatives of 23 alcohols. Included were primary, secondary, and tertiary terpene and nonterpene aliphatic alcohols, primary and tertiary sesquiterpene alcohols, secondary and tertiary monocyclic terpene alcohols, bicyclic terpene alcohols, aromatic alcohols, and phenols. The o-nitrophenylurethans separated more completely than the p-pbenylazophenylurethans
Melting point, C. (uncorrected) 112-14 114-16
130-31
128-30 74 160 152-54
Tetrahydrolinalool Xerolidol Farnesol Santalol Cedrol
121-22 oil4 59-60 106-08
139-40
146-48
Infrared spectrum indicates impurities.
Table II.
Melting Points of o-Nitrophenylurethans
Parent alcohol Phenethanol Cinnamyl alcohol Borneol Menthol Cedrol Isopulegol Fenchyl alcohol Carveol
Table 111.
Alcohol Phenethanol Cinnamyl alcohol Thvmol 3-Hexen-1-01 2-Hexanol Methyl heptanol Linalool Carveol Isopulegol Borneol Fenchyl alcohol Terpinen-4-01 Nerol Geraniol 7,8-Dihydrolinalool Citronellol Terpineol Menthol Tetrahydrolinalool Nerolidol Farnesol Santalol Cedrol a
O
7,8-Dihydrolinalool Citronellol Terpineol Menthol
v
RESULTS AND DISCUSSION
Parent alcohol Nerol Geraniol
Melting point, C. (uncorrected) 53-54 98 80-82 134-44 98-99 108-09 86-88 54-59
The Rf values of the former (Table 111) ranged from a maximum of 0.80 for phenethanol to a minimum of 0.14 for cedrol, while the values for the latter ranged from 0.80 for phenethanol to 0.31 for cedrol. All R, values were based on a t least 12 individual determinations, with compounds of similar R, values run on adjacent spots t o ensure accurate comparison. Many of the alcohol derivatives differed sufficiently in Rf value to effect separation (Figure 1). Some observations regarding the relationship of R, value of the derivative
R, Values of Some Terpene Alcohol Urethans“ R I Values 0p-PhenylNitrophenyl azophenyl urethan urethan Type Aromatic 0.80 0.80 Aromatic 0.75 0.79 Phenolic 0.72 0.78 0.67 0.74 Unsatd. aliphatic 0.61 0.74 Satd. aliphatic Unsatd. aliphatic 0.60 0.73 Di-unsatd. aliphatic 0.52 0.70 Di-unsatd. alicyclic 0.51 0.70 Unsatd. alicyclic 0.50 0.68 0.47 0.65 Satd. bicyclic Satd. bicyclic 0.45 0.64 Unsatd. alicyclic 0.44 0.63 Di-unsatd. aliphatic 0.44 0.63 Di-unsatd. aliphatic 0.43 0.63 Unsatd. aliphatic 0.43 0.79 Unsatd. aliphatic Unsatd. alicyclic Satd. alicyclic Satd. aliphatic
0.38 0.37 0.35 0.32
0.61 0.58 0.56 0.54
Tri-unsatd. aliphatic Tri-unsatd. aliphatic Unsatd. tricyclic Satd. tricyclic
0.19? 0.17 0.15 0.14
0.39 0.40 0.34 0.31
Papers dipped in 770 vaseline solution developed with methanol-water (90-11 v.v.)
VOL. 35, NO. 2 FEBRUARY 1963
235
to the structure of the parent alcohol were made, although the small number of individuals in any class other than saturated aliphatic alcohols (1) made any generalization untenable. Within the same homologous series, separations were based on chain length; however, unsaturated alcohol derivatives had higher RJ values than saturated alcohol derivatives of the same chain length. For example, 3-hexen-1-yl o-nittophenylurethan could not he separated from n-pentauyl o-nitrophenylurethan, but was completely separated from the n-hexyl derivative (Figure 1). Phenol and aromatic alcohol derivatives had consistently higher RJ values (Table 111) than derivatives of other classes. The C,O terpene alcohol derivatives were difficult to separate, although some separations were made. The separation of linalyl, dihydroliualyl, and tetrahydrolimdyl o-nitrophenyliiretham was apparently caused hy the differences in unsaturation. The separation of linalyl and terpinyl derivatives may have involved both the degree of unsaturation and a difference between an aliphatic and an alicyclic molecular structure. The higher molecular weight sesquiterpene alcohol derivatives were lowest in R, value with apparent,ly some slight difference between the aliphatics, farnesol and nerolidol, as compared to the tricyclics, santalol and cedrol.
identified on paper chromatograms, even when the sample of alcohol was too small or too impure for the determination by infrared analysis. CONCLUSIONS
Both o-nitrophenyl- and p-phenylazophenylurethans give paper chromatographic separation of alcohols of various classes, with the o-nitrophenylurethans giving the best results. I n addition, o-nitrophenylisocyanate reacts well with microquantities of alcohols to give enough derivative for paper chromatographic analysis, making this technique an effective supplement to gas liquid chromatography, LITERATURE CITED
Figure 1. Typical separations of onitrophenylurethons (illuminated by ultraviolet light, 3600 A.) A. 8. C.
D.
Borneol, menthol, eedrol Carved, terpineol, demnol 3-Hexen-l-ol, linalool, terpineol. fornerol 3-Hexen-1 -01, hexonol, linolool, nanonol
tives of C, 'to C,, Saturated Aliphatic Alcohols." Submitted t o the Cohlentz
Society.
(3) Wolfprd, R. W., Attaway, J. A., Alberdmg, G. E., Atkins, C. D., presented at the 22nd Annual Meeting of the Institute of Food Technologists, Miami, Fla., June 10-14, 1962. Submitted for publication in J. Food Sei.
RECEIVEDfor review September 13,
Condensation of microliter quantities of alcohols and subsequent reaction with isocyanates gave quantities of urethans which could he detected and
Analysis of Polyester Resins
by Gas
1962. Accepted December 18, 1962. Cooperative research by the Florida Citrus Commission and Florida Citrua Experiment Station. Florida Agricult u r d Experiment Station Journal Series, No. 1502.
Chromatography
D. F. PERCIVAL California Research Corp., Richmond, Calif.
b The complexity of polyester resins has increosed in recent years because a greater number of acids and glycols have attained commercial significance. A method for the identification and semiquantitative determination of various constituents in these resins consists of isolation of resin from monomer solution by precipitation, then methanolysis and analysis of the resulting dimethyl esters and free glycols by gas chromatography without prior separation. Analytical control can thus b e exercised on various formulations.
R
work in our laboratories required a general, semiquantitative analysis of unsaturated polyesters. Therefore, we developed a method involving methanolysis of the polyester followed by gas liquid chromatography that involves little contact time (but 236
,CENT
ANALYTICAL CHEMISTRY
long reaction t i e ) , is general for most unsaturated polyesters, and gives semiquantitative results for both dibasic acids and polyols. Methanolysis converts the polyester to a mixture of glycols and methyl esters of the acids, which without further manipulation can be easily separated and identified by gas chromatography. Temperature programming is useful in this separation but not essential. From the peak areas one can roughly calculate the molar concentration of each constituent in the polyester by using a simple conversion factor. Our method is similar to one recently published by Esposito and Swann (2)for the qualitative identificsr tion of carboxylic acids in alkyds and polyesters. The principal difference is in reaction time for transesterifteation. Our method does not require any separation prior to gas chromatography,
APPARATUS AND MATERIALS
A Willdns Aerograph A350 temperature-programmed gas chromatograph was used in this study. The recorder was a Leeds and Northrnp Speedomax Type G. .
OPERATINO CONDITIONS Detection cell, C. D.c. current, mah Injection temp.,o C. Column temp., C. (8" C./min for programmed runs)
Helium flow rate, ml./min.
200 200
200 110-1x0 50
COLUMNPREPARATION.Twelve feet by 1/4 inch 0.d. copper tubing packed with 10 grams of GE silicone SF-96 on 50 grams of Fluoropak 80. EXPERIMENTAL
Any solvent or monomer in the commercial resin is removed by precipitation, A 10-gram sample of the resin is weinhed into a tared beaker and 100
