Chromatostrips for identifying Constituents of Essential Oils JOHN 31. M I L L E R
AND
J. G. KIRCHNER
Fruit a n d Vegetable C h e m i s t r y Laboratory, Bureau of Agricultural arid Zndicstrial Chertzistry, V. S . D e p a r t m e n t of Agriculture, Pasadena, Calif. The coniponents of essential oils cannot be positively identified by means of R / values on chromatostrips, although many compounds can he eliniinated hy comparison of R / values. Many reactions can be perfarmed directly on the chromatostrip and the products chromatographed or the reaction can be carried out on a micro scale in a test tube and the reaction mixticre chromatographed. These reactions include oxidation, reduction, dehydration, hydrolysis, and formation of derivatives. A list of R / values, in a variety of sohents, of m a n y terpenes and oxygenated terpenes found in essential oils is included.
T
HE work reported was done in coiiiiection n i t h the tievelopment of new analytical methods for the identification of volatile c-onstituents of canned grapefruit juice. The problem of identifying individual compounds isolated from essential oils is often complicated by similarities in molecular formula and physical properties. It is sometimes difficult to apply diagnostic tests for functional groups, because of the great reactivity of the terpenes and oxygenated terpenes found in these oils. Kirchner, Miller, and Keller (Z), Kirchner and llillcr ( I ) , and Miller and Kirchner ( 3 ) have described chromatographic methods t h a t are useful in the isolation of pure ronipounds from essential oils. I t u as found that the use of chromatostrips (an adsorbent-coated glass strip) can be extended to assist in the identification of co:npounds not only by direct comparison to known Rj values, but also by carrying out reactions directly on the chromatogram or t u chromatographing the results of micro scale reactions in test tubes. Because of the large number of terpenes, the identity of an unknown compound cannot be established with certainty by comparison with the R/ values of known compounds. Hoxvever, tentative identity may be accomplished in this fashion and a large number of compounds eliminated by comparison of their I21 values in a number of solvents. Tables I and I1 contain the Rj values of a number of pure terpenes and oxygenated terpenes on silicic acid chromatostrips in solvents that have been found useful for characterizing these types of materials. In every instance when compounds on chromatostrips are compared, the unknown compound should be compared with the known on the same batch of strips a t the same time, and a m h e d chromatogram run on the known and unknown. The reproducibility of R , values on a given batch of chromatostrips is about ~ t 0 . 0 5 . ___.
R j X 100 of Some Terpenes in Yarious Solrcnts on Silicic -4cid Chromatostrips I< P Q R
Liiiioncne Terpinolene a-Pinene Camphene p-C ymene Cedrene or-Caryophyllene 8-Caryophyllene 7-Caryophyllene @-Pinene
41 64 83 84 38
82 50
55 67 85 76 41
80 35 60
54 60 84 79 62 83 47
52 82 87 80 75 80 K , hexane P, 2,2-dimethylbutane Q ,cyclohexane R, methylcyclohexane S. isopentane 62 80
Table 11. K, X 100 Yalues of Some Oxygenated Terpcnes and Other Essential Oil Constituents in Various Solvents" on Silicic -tcid Chromatostrips
Citronellol Geraniol Linalool m-Terpineol SOD01
U S E OF R, VALUES
Table I .
I n one veiy important instance the li, vulue serves as a mcsiiis of distinguishing two classes of m n t ri iai-hydrocarbons from 11011hydrocarbons. Hydrocarbons hare an appi eciable R f value 13 rth hexane as solvent, whereas n o n h ~tlrocarbons have an R/ of 0 Kirchner and lliller ( 1 ) have used this property to sepalxte hydrocarbons from oxygenated coinpouritis for the preparatioii of terpeneless oils. Chromatograplung a large iiuinber of these tn-o
59 65 90
80 69
85 33 65 90
85
26 27 20 36 29 27 34 31
farveol V e t h y l heptenol 3-Hexen-1-01 20 .ildehydes Citrala 45 Lauric aldehyde b .58 31 Cinnainaldehyde b Furfuralb 21 lietones 45 CarvoneC Methyl heptenone 48 Pulegone 08 Camphord 56 lsters 51 Geranyl acetate 55 Keryl acetate 56 Citronellyl acetate Octyl acetate 72 58 Terpin11 acetate Methyl anthranilate6 42 E t h y l anthranilate8 41 Y-Methyl methyl i8 anthranilate8 66 Carvgl acetate Oxides 48 1,8-Cineoli 21 Linalool monoxide Compounds detected by
S 74 89 80 79 57 78 27 75
36
3 $4 40 4i 34 46 44 20
32
42 41 43 4.5 36 52 49 44 32
30 33 34
i2
62 ti2 i A 53
70
6'3
70
56 75 52 37
51 83 45 17
75
62 57 72 47
65 0 :f7 60 79 50
0 0 0
71 69 75 92 73 49
73 73 84 87 86 46 53
0
3.; 0 50 0 42 0 26 0 25
79 84
0 56 0 64
70
41
i4
69 84 78 88
67 56 63 60
i6 69 79
41
7;.
27 36 25 48 25 41 29 34 26
62 76 70 41
70
70
J .I
30 48 35 43 45 37 30
62 $11 A8 44
47
A1 6.5
53
ii?
58
71 -13 il'
50 37
??
r).)
53 46 71 67
40 42 29
3 8 36 41 41 31 45 20 60 51 39 34
5fi
d9
ti6
66 55 66 96
J3
51 38 28 32
3i 6i
R!) 6(+ 66 98 66 52 56
54
36 29
:; ;2 --
64 72 70 44
74 65 :39
28
::j
;!I
83
75 91 76
56
59
81 86
72 69 81 85 7.5 6.5 67
86 89 92 86 72 84
78 'J6 84
90
G9
91
77
91 !10
00 62 70 '11
90
58
14
19 12 15
s
12 1'
14 13
0
15
4 0 0
50
0
0
a3
1107
13
35 60 28 27
:w
66 6.5 7:1 €2 83 70 83 0 12 8 42 13 18 16 0 3 20 I O fliiorestc.in-liroininF test unless otherni.a
66 16
A . 15% ethyl acetate in hexane (1.. ' Y . ) B 10% ethyl acetate in chloroform (alcohol-free) ( v . / v . ) C: 15% ethyl acetate in benzene (v. k.) D. 5% ethyl acetate in chloroform (alcohol-free) ( \ . , l v , ) E , 50% I-nitropropane in hexane ( v . 'v,) F, 30% ethyl acetate in hexane ( v . / v . ) G , 15% ethyl carbonate in chloroforiii (alcohol-free) ( V J Y . ) H, 30Y0 isopropyl formate in hexane (v./v.) J. 50% isopropyl ether in hexane (v. 'Y.) K hexane L,' chloroform (alcohol-free)
75
!I
1108
ANALYTICAL CHEMISTRY
classes of compounds has not revealed any exceptions to this property. Carbon tetrachloride, benzene-free petroleum ethers, and other low boiling aliphatic hydrocarbons will separate hydrocarbons from nonhydrocarhons on silicic acid columns. REACTIONS ON CHROMATOSTRIPS
The chromatographic method offers a simple means of separating the products of a reaction. ,Inumber of reactions have been developed whereby the pure compound is adsorbed on a chromatostrip, covered with the reagent, and chromatographed in an appropriate solvent. I n cases where this technique is not suitable, t,he reagent and compound are mixed on a micro scale in a small test tube. The crude mixture is then applied directly to the chromatostrip. The high resolving power and the rapidity with which individual chromatograms can be run make the chromatostrip ideally suited for this type of work. I n addition, the inert character of the chromatostrip makes it ideally suited for use with strong or corrosive reagents, so that the entire reaction mixture can be chromatographed without danger of destroying the chromatogram. I n combination with the RJ values of the pure compound the chromatographed results of a reaction often positively identify the compound. I n other instances the results of a reaction afford valuable clues to the identity of the compound. Citral can be cited as an example. I n addition to the R, values in numerous solvents, citral can be oxidized to geranic acid (n-ith 30% hydrogen peroxide and exposed to ultraviolet light) and reduc.ed to geraniol. The R, values of the two reaction products establish with a fair degree of certainty the identity of the original compound.
The compound to be reduced is spotted at the origin of the strip and covered by a drop of a 10% solution of lithium aluminum hydride in ether and the strip is developed in a suitable solvent. Care must be taken to avoid too great an excess of reagent, as it reacts vigorously with the moisture in the solvents and in some cases with the solvent. The excess can be drawn off the strip by means of a medicine dropper. I n the case of esters it was necessary to carry out the reaction in a test tube. Dehydration. A small amount of the compound is spotted a t the origin of the strip and covered with a drop of concentrated sulfuric acid. The strip is then developed with hexane. Hydrolysis. -4drop of the compound and a drop of potassium hydroxide in ethylene glycol ( 4 ) (6 grams in 100 ml.) are heated in a small test tube until a ring of liquid condenses on the cool end of the tube. A small amount of the condensate or the centrifuged mixture is then chromatographed. Preparation of 3,s-Dinitrobenzoates. A drop of the compound, 5 drops of pyridine, and a few crystals of 3,5-dinitrobenzoyl chloride are mixed and heated. The mixture is ,hen chromatographed. Preparation of Phenyl Carbamates. A drop of the compound is added to 5 drops of hexane and 1 drop of phenyl isocyanate in R small test tube. After heating, the mixture is chromatographed Preparation of Phenylhydrazones. The compound t,o be tested is spotted on a strip and covered with a drop of phenylhydrazine. It is then chromatographed. Preparation of Semicarbazones. A 10% solution of semicarbazide hydrochloride in water is neutralized with sodium hydroxide and spotted directly on the strip with the compound. The strip is then chromatographed with ethyl acetate.
Oxidation. The material to be oxidized is adsorbed a t the origin of a strip and covered with a drop of a saturated solution of chromic anhydride in glacial acetic acid. The strip is then developed with a suitable solvent such as 15% ethyl acetate in hexane or l o yoethyl acetate in chloroform. Some hydrocarbons have been oxidized wifh 30% hydrogen peroxide and the strip exposed to ultraviolet llght for 10 minutes to facilitate oxidation. Reduction. A drop of a solution of 5 grams of aluminum isopropoxide in 50 nil. of benzene is added to a drop of the compound in a small test tube. The mixture is heated until a distillate appears on the cool part of the test tube. The distillate is centrifuged back to the bottom of the tube and the mixture is chromatographed.
.Ifter the products of the reaction have been chromatographed, the position of the various spots is determined by spraying with a suitable reagent or viewing the strips in ultraviolet light'. The results of these reactions with a number of typical compounds are shown in Table 111. DISCUS S I 0 3
I n most cases the reactions do not go to completion, so that there is present a mixture of the original compound. the products, the reagent, and any necessary solvents. Fortunatelj-, all the inorganic compounds such as water, bases, salts, and acids are not moved in the solvents used in this work and thus remain a t the origin. 1Iost of the simple organic compounds such as acetic acid, ethylene glycol, etc., are strongly adsorbed on the silicic acid and hence have low R f values Jvhich do not interfere in t,he detection of the desired products. I n the case of the formation of derivatives such as 3,j-diriitroberizoates and phcnylhydra-
Table 111. Results of Reactions for Chromatostrip Identification of Terpenes and Other Essential Oil ConstituentsQ on Silicic Acid Chromatostrips
Compound Carveol Linalool Geraniol a-Terpineol Nopol Methyl heptenol Octyl alcohol Nerol Pulegone Methyl heptenone Carrone Citral Lauric aldehyde Cinnamaldebyde
Redtiction Lithium Aluminum aluminum isopropoxidec hydride
Oxidationh. CrOa Carvone Citral Citral Xi0 reaction No reaction Methyl heptenone
S o reaction
Citral ?io reaction S o reaction
KOreaction Pulegolb
K O reaction KOreaction
Citronellol Furfural Linalool monoxide Terpinyl acetate
Methyl heptenol Carreol Geraniol
Carveolb Geraniol b
Cinnamyl alcohol
Cinnamyl alcoholb
S o reaction
Reductionb Terpineol5
Linalyl acetate Carvyl acetate Geranyl acetate Neryl acetate
SerolC
Derivatives Dehydrationb, Hydrolysisc, HzSO4 KOH Semicarbazoneb
3,5-DinitroPhenylbenzoatec hydrazoneb
Phenyl isocyanate0
Hydrocarbon Hydrocarbon Hydrocarbon Hydrocarbon
N o reaction Benzoate S o reaction
No reaction N o reaction Hydrocafbon N o reaction No reaction
Carbamate Carbamate Carbamate Soreaction Carbamate Carbamate
Benzoate
No No So S o
Benzoate S o reaction Semicarbazone
reaction reaction reaction r.iacrion
No reaction 2 Semicarbazonee Semicarbazone Semicarbazone
S o reaction
Trace of hydrocarbon Trace of hydrocarbon XOreaction
Semicarbazone
Hydrazone Hydrazone
Carbamate
X o reaction Hydrazone Hydrazone Hydrazone
Hydrazone
Terpineol Linalool Carx-eo1 Geraniol Nerol
reaction" indicates t h a t no reaction products were observed. I n oxidation of citral t o geranic acid, results of oxidation axe not visible because of interference from acetic acid; therefore it is marked no reaction. I n dehydration reaction, it means no hydrocarbons are formed. b Reaction directly on chromatostri 0 Reaction on micro scale in test tug,, then mixture chromatographed. a "So
.
V O L U M E 25, N O
1109
7, J U L Y 1 9 5 3
zones, the reagents are strongly adsorbed and remain a t the origin. I n the chromatographic systems studied the R f value of a compound appears to be related t o the size of the molecule and the number arid kind of functional groups which the molecule contains. h n i n c r e w in molecular weight tends to increase the I?, value, as does a decrease in the number of functional groups. Changing the nature of the functional group by means of some reaction. such as reducing a ketone to an alcohol, always results in a change of R, value, so that the separation by chromatography is easily effected. I n some cases where the product of a reaction is a highly oxygenated compound, such as the oxidation of ritral to geranic acid by chromic acid, the I?, value of the product is lonand is obscured by the acetic acid used in the reagent. Such reactions are listed in Table I11 as “no reaction.” The authors have found these reactions and their subsequent aiialysis on chromatostrips to be useful in a number of mays: t 1 j I n inany cases the compound may be positively identified h\- means of the reactions. (2) A wealth of information can be obtaine(l on a compound with the expenditure of a small amount of (*ompound. (3) Considerable time can be saved. Thus if a derivative is desired from a particular compound, its formation
may be readily checked on a chromatostrip without the necessity of extensive purification, recrystallization, etc. -4s an example, a-terpineol does not form a 3,5-dinitroberizoate, and this was quickly checked by means of the chromatostrip reaction. (4) Il-here it is desired t o perform macro reactions, the conditions of the reaction and its progress may sometimes be checked rapidly. The list of reactions presented in this paper is intended to illustrate what can be accomplished in the way of identifying constituents of essential oils by the use of chromatostrips. 0.A lation with hydrogen peroxide. nitric acid, and potassium permanganate has been tried with success, and many other reactions of a specific or general nature may be employed as the occasion arises. LITERATURE CITED
(1) Kirchner, J. G., and 3Iillei, .J. 11 , I d Eng. Chem., 44, 318 (1952). (2) Kirchner, J. C.. AIiller, J. AI., and Keller, C. J.. ANAL CHEX.,23,.
420 (1981).
( 3 ) Miller, J. XI., and Kirchner, J. G.. Ibid., 24, 1450 (1952). (4) Redemann, C. E., and Lucas, H. J., ISD. ENG.CHEY.,- ~ A L
E D ,9, 521 (1937). R E C E I V Efor D reriew January 26, 1453.
Accepted April 13, 1953.
Microanalysis of Organic Salts by the Use of Cation Exchange Resins CECIL T i . YAN ETTEY
AVD
. \ I i R Y B. WIELE, S o r t h e r n Regional Research Luboratory, Peoria, I l l .
The ability of unifunctioiial, high capacity synthetic resins to exchange hydrogen ions quantitatively for metallic or organic cations suggested their use for the determination of organic salts on a micro scale. Successful analyses of 46 salts of organic acids or bases are reported. The accuracj and precision of the method are indicated by an aterage recovery of 99.87q~,with a standard deviation of 0.21%, on 20 determinations of a sample of pure sodium oxalate. Results are expressed as “exchange equh alent.” The method offers the advantages of speed, simplicit?,and accuracy o+erconientional micromethods for determining organic salts.
T
HE most common micromethod for the determination of salts of organic acids is to weigh the ash from the compound as the sulfate, oxide. or free metal (4). The halogen or sulfate salt of a n organic base is commonly determined gravimetrically by precipitating the halide as the silver salt or the sulfate as barium sulfate. -in alternate method. n.hich is here described. consists of passing a solution of the organic salt through a cation exchange resin in the acid form and titrat’ing the effluent whirh contains acid equivalent to the salt content of the initial solution Wiesenherger (8) has applied this microanal>-tical techniqur to the determination of a number of inorganic salts. In a numl~er of papers. H:muelson has described similar techniques on a macro scale (5). I n addition to its simplicity and speed, the method has the follon-ing ndvant’ages: Some salts that cannot he determined bjthe ashing method without special treatment-such as salts of nickel (d)-can he determined by this method. The method is readily a1)plicable to the determination of the number of equivalents of cations in solutions without the need for evaporation. Ammonium salts of organic acids and salts of organic bases can be determined. If desired, the sodium salt of the acid can be recovered after titration of the effluent. The titration can be checked by rerunning the titrated effluent through the ion exchange column. I n most cases, the cations can be quantitatively displaced and specifically determined if desired. For example,
the ammonium ion could be eluted from the ion exchangc resin with an acid and determined by the Kjeldahl procedure. Although the method has not been compared directly with rwently reported methods for the determination of salts ( I , &7), i t apprars t’ohave advantages over them in certain applications. MATERIALS
Micro ion exchange column, as shown in Figure 1. This column was so constructed that the delivery tip was about 25 mm. below the top of the ion exchange bed. Consequent,lv, the length of the capillary side arm depends on the amount a6d mesh size of the resin used; 0 . i gram of 20- to 40-mesh ion exchange resin occupied a column length of 4 to 5 cm., whereas a like amount of 50- to 100-mesh resin required only 2.5 to 3 cni. Dones 50 ion exchange resin of the above mesh size, either 9 or 16% cross linked. Only a limited number of other ion exchange resins were tested and these were found to give low recovery (95 to 98%) of titratable acid in the efijuent. Standardized 0.01 1V sodium hydroxide. Mixed indicator, one part 0.0470 aqueous cresol red and three parts 0.04% aqueous thymol blue (color changes at pH 8.2 to 8.7) ( 2 ) . PROCEDURE
Preparation of Ion Exchange Resin. h stock supply of the ion exchange resin was prepared by allowing 8 to 10 grams of the resin t o stand for at least 30 minut,es in each of three 50-ml. portions of 3 N hydrochloric acid. It was then washed free of acid
