Separation of Terpene Hydrocarbons by Gas Liquid Chromatography Utilizing Capillary Columns and Flame Ionization Detection RICHARD A. BERNHARD Department of Food Science and Technology, Universify of California, Davis, Calif.
b Fourteen terpene hydrocarbons were examined b y gas liquid chromatography on LAC-2-R446, Tween-20, n-butylcyclohexyl phthalate, and Apiezon L stationary liquid phases employing capillary chromatographic columns. The apparatus employed and procedures for preparation of the chromatographic columns are described. Data for relative corrected retention volumes, peak sharpness, number of theoretical plates, relative peak separation, and resolution are presented in tabular form. The order of elution from these columns is discussed in light of the relative polarizabilities of these terpenes. High column efficiency alone (as expressed b y number of theoretical plates) does not ensure complete resolution of these compounds, but proper selection of the stationary liquid phase is still the most significant operational parameter.
EXPERIMENTAL
T
ubiquity of the terpenes in the plant kingdom has prompted liter. ally thousands of investigations in the past hundred years. The prime deterrent to a more accurate knowledge of the chemistry of the terpenes has been the chemists’ inability to separate and identify these compounds from the complex mixtures that occur in nature. Terpene compounds are best characterized by one word, isomers: geometric, positional, and optical isomers that have similar physical and chemical properties, IS’ith the advent of gas liquid chromatography (GLC) and its many attractive properties, the chemist was provided with a n analytical tool well suited to further his investigation of this series of compounds. I n the years 1955-56 Bernhard (3) attempted to separate and characterize some common terpene hydrocarbons isolated from the essential oil of the lemon. H e was able to achieve adequate separation of a number of compounds employing rather crude chromatographic equipment. Prior to this investigation, many researchers attempted to separate and characterize the terpenes using other chromatographic methods. Among the most HE
1576
notable of these investigations were those of Kirchner, Miller, and Keller (12) and Clements (4). I n 1958 and thereafter, numerous researchers applied GLC techniques to the separation of terpenoids. Recently von Rudloff (18), Zubyk and Conner (21), and Klouwen and ter Heide (13) have investigated the effect of various column parameters on the separation of terpenoids. It is obvious from examination of even such recent papers as those mentioned above (23, 18, 21) that a considerable increase in the resolution of the compounds in question is still highly desirable. With the advent of the flame ionization detector (15, 16) and its obvious advantages, and the development of capillary columns (8, 9), a new avenue of analysis emerges that promises some unique advantages in the esamination of the terpenes.
ANALYTICAL CHEMISTRY
Gds Chromatographic Apparatus. The chromatograph was designed and constructed for this laboratory in cooperation with Loenco, Inc., U t a dena, Calif. T h e detector was a single flame unit having a burner jet constructed of stainless steel tubing (0.010-inch i.d.), and insulated from tne burner chamber by a Vycor glass ring. T h e cylindrical brass burner chamber had a gas escape stack atop the enclosure. The burner feed line was insulated from the chromatographic column by interposing a 0.5-inch length of 3-mm. 0.d. Vycor glass tubing between the column connection and the burner jet base. The entire detector assembly was housed in a separate thermostated oven. The electrometer was a Loenco direct-coupled feedback type having a current amplification of lo9. Sensitivity for full scale deflection was 1 x 10-12 ampere. A 300-volt battery was used to provide a potential for ion collection and electrode polarity. Best results were obtained when the burner tip was made negative with respect to ground. The column oven heater was constructed in a cylindrical fashion. S i chrome mire was wound about a central mandrel, which was then covered by
an insulated brass sleeve. Two cartridge heaters were attached to the inner side of the mandrel to provide trim heat. Both column and detector ovens were designed for operation between 20’ and 225’ C. Temperature was controlled t o 10.2’ C. by application of a fixed voltage to the main heaters and by using electronic proportional controllers to supply power to the trim heaters. Chromatographic columns were stainless steel tubing 0.010-inch i.d. (Type 321 stainless steel tubing, R . J. Gallagher Co., Houston, Tex.) wound on aluminum spools. These spools mere of such inside diameter that they could be readily slipped over the mandrel-type heater and yet provide close thermal contact between the heater and spool. This arrangement provided a convenient way to contain the long capillary columns and permitted rapid column change. The hydrogen and air flow rates were measured with a soap-bubble flowmeter. Because of the very low carrier gas flow rates required for efficient capi!lary column operation, this flow rate was not measured. Instead, the average lin-ar carrier gas velocity, U , ( 1 1 ) , was determined (by timing the elution of methane, which does not partition appreciably a t the temperatures employed in this study). This can be converted into a flow rate if desired. A C , of approximately 10 cm. per second mas used, since this appears to give a favorable HETP for capillary columns ( 7 ) . Coating Columns. A 5% (w./w.) solution of t h e stationary liquid phase dissolved in acetone works well for the three polar substances reported here. Pentane or hevane is a satisfactory solvent for Apiezon L. Coating mas accomplished by attaching a small reservoir (ea. 1.5 ml.) t o t h e end of the capillary tubing, applying gas (nitrogen) pressure, and flushing the empty column with acetone several times. The solution containing the liquid phase was then passed through the clean column in much the same manner with but one exception; nitrogen pressure was regulated to limit the linear velocity to less than 10 em. per second (at higher velocities the coating appears to be less satisfactory). This process was repeated until a minimum of 3 nil. of solution had been passed through a 100-foot column. Xitrogen v a s allowed t o flow through the coated
column at room temperature for 12 to 16 hours t o evaporate the solvent. The inlet fittings were then removed and attached to the other end of the capillary column, and the nitrogen flow n-as reversed for a n additional 12 to 16 hours. This seems to smooth out the inhomogeneities in the coating and produces a more efficient capillary column. With nitrogen flowing through it, the coated column was then heated in the column oven a t 100' C. for 5 days (or until all appreciable bleeding had stopped). This aging appears to bt. essential for column stability. The four stationary liquid phases used in this study were LAC-2-R446 (14) (the adipate polyester of diethylene glycol partially cross-linked with pentaerythritol, Cambridge Industries Co., Inc., 101 Potter St., Cambridge 42, Mass.) ; Tn-een-20 (polyosyethylene sorbitan monolaurate, AitlasPowder Co., \\'ilmington. Del.) ; n-butylcyclohexyl phthalate (Barrett Chemicals, Kew York, N. Y.), and Apiezon L (James G. Biddle Co., Philadelphia, Pa.). Sample Injection. A stream splitter employing a variable split ratio was used t o adjust t h e amount of sample introduced into t h e injection system. T h e sample injection system was heated independently of t h e column oven. -411 pertinent operating parameterse.g., flow rate, temperature, chart speed, etc.-are presented in Table I. In each case these conditions were chosen after considerable trial to optimize the conditions of separation. RESULTS A N D DISCUSSION
Selection of the stationary liquid phases used in this study was based on rather careful screening made by the author (2). Polar phases tend to give better separation of terpenoids than nonpolar types such as the silicone oils and Apiezon greases. Data are presented in tabular form and provide the relative corrected retention volumes, V i / V i ( I ) , of the 14 terpene hydrocarbons examined. Retention volumes were measured relative to D-limonene, since this substance is reasonably stable, is present in numerous essential oils, and has a central position in the chromatogram (Figure 1, peak 11). To provide a quantitative basis for comparison among the various separations, data are also presented for relative peak sharpness, Q; the number of theoretical plates in the column, 16 Q* ( 6 ) ; the relative peak separation, Slz; and resolution, R (11). Jones and Kieselbach (11) point out that i t is not possible t o assign a value of R adequate to ensure good resolution in all cases, b u t an R value of about 2.5 represents complete resolution within the precision of the usual recording instrument. The values for the parameters discussed above are presented in Tables I1 to V for the terpene mixtures examined.
I
I
1
I
0
IO
I
I
I
I 20
1
I
30
40
-
TIME Iminulerl
Figure 1 . Chromatogram of synthetic terpene mixture on LAC-2-R446 capillary column See Table II for peak identification and Table I for operating Conditions
Of the four stationary liquid phases examined, LAC-2-R446 (14) appears t o be the most efficient for the separation of the terpene hydrocarbons. The separation of a-pinene from trans-pmenthane is incomplete (Figure 1) ( R = 0.75), as is that of A3-carene from myrcene ( R = 0.33). All of the remaining compounds have adequate R values (Figure 1 and Table 11) for good
Table 1.
separation. It does not appear necessary to have an R value of 2.5 to achieve apparent complete resolution using this system; a value of approximately 2 appears sufficient [note the separation between D-limonene (peak 11) and 0phellandrene (peak 12), R = 1.9 (Figure l)]. For packed chroinatographic columns, James (IO) has stated that relative
Operating Conditions for Capillary Columns
Stationary liquid phase Detector
LAC-2-R446 Tween-20 Hydrogen Hydrogen flame flame Column length, ft. 100 100 Column diameter, i.d., inch 0.010 0.010 Column temperature, ' C. 70 50 Injection block temperature,o' C. 150 150 Detector oven temperature, C. 95 55 Hydrogen flow rate, ml./min. 20 20 Air flow rate, ml./min. 1500 1500 Nitrogen velocity, cm./sec. 10 10 Injection split ratio 1:600 1:600 Sample size, pl. 0.2 0.1 Recorder range, mv. 10 10 Chart speed, in./hr. 15 15
Table 11.
Peak 1
2 3 4 5 6 7
8
9 10
11 12
13 14 a
n-Butylcvclohexyl phthalate Hydrogen flame 1.50 0,010 96 150
Apiezon L Hydrogen flame 100
95' 20
1500
9
0.010 71
157 90 30 1500
1: 600
11 I : 600
10 15
10 30
0 .1
0.2
Chromatography of Terpene Mixture on LAC-2-R446 at 70' C.Q
Compound trans-p-Menthane a-Pinene cia-p-Menthane Camphene @-Pinene Sabinene A3-Carene Myrcene a-Phellandrene a-Terpinene n-Limonene 8-Phellandrene r-Terpinene p-Cymene
?.P., C. 170
156
171-2 l59.G 164 164
171 167 171.5 173
177.7 178 183 176.7
T'i/Vi 0.479 0.491 0.538 0.575 0.674 0.715 0.794 0.802
0.850 0.904 1.00 1.05 1.24 1.43
Q 29 32 35 30 34 36
36 34 46
41 41
39 41 47 iiverage
16 Q' 13,000
16,000
19,500 14,000
18,000 21,000 21,000 18,000
33,000 27,500 27,000 24,000
27,000 35,000 22,000
S12
: x 103) ...
23.4 90.2 T4.3 171.8 61.0
111.3 9.69 60.0 64.2 105.7 49.7 185.8 146.4
R
...
0.75 3.1 2.2 5.8
2.2 4.0
0.33
2.7 2.7 4.3 1.9 7.7 6.9
See Table I for operating conditions.
VOL. 34, NO. 12, NOVEMBER 1962
1577
retention volumes must differ by roughly 20% for complete separation of zones, differences of 10 to l5yOwill show two united peaks, and differences of less than 7% will show only a single united peak. Sample calculations made with capillary columns reveal that a difference of approximately 5% in relative retention volumes mill give complete separation of peaks (Table I1 and Figure 1, peaks 11 and 12); as little as 2.4% difference will show a distinct shoulder (peaks 1 and 2) ; and only when differences are less than 1% will they coincide conipletely (the difference between peaks 7 and 8 is approximately 1%). The average theoretical plate value for the LAC-2-R446 capillary column mas 22,000. This is about half of reported values for capillary columns coated with nonpolar stationary liquid phases ( 7 ) . An informal poll by the author of participants a t a recent conference on gas chromatography (17) indicated that most researchers found lower theoretical plate values for capillary columns coated with polar phases than with nonpolar phases. When Tween-20 was used as the
Table 111.
Peak 1 2
3 4 5
.
6
9 10 11 12
Peak 1
7 8 9 10
See Table 111 for peak identification and Table I for operating conditions
stationary liquid phase, resolution of a-pinene-trans-p-menthane and myrcene-a-phellandrene was, within the limits of experimental error, nil (Table I11 and Figure 2). The relative cor-
567 600 630 io4
0.737
0.818 0 863 0 916 1 00 1 03 1 22 1.29
25 23 24 23 24 21 21 23 24 21 23 21
10,000 8.300 9,200 8.200 9 .000 fi:900 6,800 8.100 9,200 6 , 700 8.600 7.200
Average
8 !200
579
498 118 0 46.6 109.0 54i 623 912 26 9 189 0 59 2
1 3 1 2
2 7
1.1
2.3
11
1 4 2 2 0 55 4 4 1.3
Chromatography of Terpene Mixture on n-Butylcyclohexyl Phthalate a t 96" C:
Compound ol-Pinene t runs-p-Menthane ; camphene cis-p-Ment hane &Pinene Sabinene Myrcene
A3-Carene a-Phellandrene a-Terpinene n-Limonene 11 p-Phellandrene 12 e;-Terpinene; p-cymene a
0 0 0 0
T;:/V;
0.626
0.685
0.727 0 , 778
0.791 0.836 0.874 0,907 0.955 1.00 1.06 1.21
See Table I for operating conditions.
1578
ANALYTICAL CHEMISTRY
20
Figure 2. Chromatogram of synthetic terpene mixture on Tween-20 capillary column
Chromatography of Terpene Mixture on Tween-20 a t 50" C:
Compound a-Pinene; tl-uns-p-menthane Camphene cis-p-Menthane P-Pinene Sabinene Al-Carene Rlyrcene; a-phellandrene a-Terpinene D-Imionene 0-Phellandrene y-Terpinene p-Cymene
Table IV.
5 6
IO TIME (minutes1
See Table I for operating conditions.
(I
2 3 4
0
Q 32 38 40 42 43
16~2 16,000 23,000 26,000 28,000 29,000 38 23,000 35 20,000 22,000 37 23,000 38 39 25,000 35 20,000 30 15,000 Average 22,500
.SI*
( x 103)
R
... 95.1 63.6
...
67.7
16.8 56.9 45.1 38.2 52.8 47.1 63.9 135.0
3.6 2.6 2.8 0.i2 2.2 1.6 1.4 2.0 1.8
2.3 4.1
rected retention volumes for the pair, a-pinene and trans-p-menthane, were essentially identical under the conditions employed in these experiments. The same is true for the pair, myrcene and a-phellandrene (Table 111). I n general, values for R mere lovrer for the Tween-20 phase than for the LhC-2-R466 phase, despite the fact that the operating temperature was significantly lower for this separation (at a temperature of 70" C., comparable to that used with the LAC-2-R446 column, separation was poor). Values for Q were appreciably lower also. Admittedly, the efficiency of this particular column, as expressed by 16 Qz, was not good by capillary column performance standards, but still it was some 4 to 10 times better than a typical packed column (3, 7 ) . Separation of the terpene mixture on a n-butylcyclohexyl phthalate column revealed that trans-p-menthane and camphene had essentially identical relative corrected retention volumes, as did the pair, 7-terpinene and p-cymene (Table IV). There was a distinct doublet-type peak in the separation of sabinene from p-pinene ( R = 0.72) (Table IV). The greater column efficiency (16 Q 2 ) for this column is undoubtedly due in part to the increase in column length employed in these analyses (Table I). Values of Q are noticeably higher for this column than for the Tween-20 column, but not so good as those for the LXC-2-R446 column. Some experiments were made using a column coated with the nonpolar phase Apiezon L analyzing a simpler eightterpene mixture (Table V). Resolution of the two p-menthanes and p-pinene was incomplete, and resolution of pphellandrene from D-limonene was also poor (comparable to the Tween-20 column in this regard).
It is interesting to examine the various orders of elution of the terpene hydrocarbons from the four stationary liquid phases employed in this study. On polar columns. the two p-menthanes elute rapidly, despite their higher boiling points (Tables I1 to 117 (21). possible explanation for this may be that neither of these compounds is very polarizable, and therefore, there is very little bonding interaction between them and the stationary liquid phases. If this is the case, the trans, having a lower boiling point than the cis, should elute’first, as it does. The trans has the diequatorial Conformation, while the cis exists predominantly in the axial methyl, equatorial isopropyl conformation [the energy difference between the latter more stable conformation and the equatorial methyl and axial isopropyl conformation is about 1 kcal. (20)l. If significant bonding interaction did take place, one would expect the diequatorial form to participate more readily than others (j), and thus elute last. Klouwen and ter Heide ( I S ) have reported a series of terpene polarizabilities. Fmploying polar stationary liquid phases, they conclude that cyclohexadiene derivatives with conjugated double bonds inside the six-membered ring-e.g., a-terpinene and a-phela lower polarity than landrene-show the isomer with isolated ethylenic linkages in the ring-i.ci., y-terpinene. They found a-phellandrene less polarizable than a-terpinene, and state that most of the bicyclic members of the terpenes do not differ appreciably in polar character (except for sabinene, which behaves like a monocyclic terpene). These authors conclude that terpenes with exocyclic ethylenic double bonds are more easily polarized than those with endocyclic double bondse.g., 01- and /?-pinene. Sabinene was rclported to have a stronger polar character than /?-pinene. A\ll of the above conclusions are in agreement with the orders of elution described in the present study. Caniphrne elutes after cis-p-menthane on LA\C-2-R446 and before it on the other two polar columns. Also the order d3-carene-myrcene is reversed on n-butylcyclohe.;yl phthalate columns (’Table IV). p-Cymene, a nonterpenoid compound n-ith a carbon skeleton similar to u-limonene, appears to be more strongly polarizable than Dlimonene, 8-phellnndrene, and y-terpinene
Table V.
Peak 1 2 3 4 5
A \
6
ka
Chromatography of Terpene Mixture on Apiezon L a t 71 (’ C..
Compound a-Pinene Camphene trans-p-Menthane @-Pinene czs-p-Menthane D-T,imonene @-Phellandrene yTerpineiie
?.P., C. 156 159 6 170 164 171-2 177 7 178 183
Vi/Vg 0 476 0 559 0 641 0 667 0 687 1.00 1 03
Q 16Q* 14 3,100 15 3.600 27 11,700 19 5,800 20 6,400 23 8.500 19 5.800 1 24 23 8,500 Average 6,700
Si? ( X lo3)
R
174 0 147 0 40 5
30 0 456 0 30 0 204 0
2 G 4 0 0 77 0 60
10 4 0 57 4 7
Pee Table I for operating conditions.
The order of elution on Xpiezon L appears to be that of the boiling points of these hydrocarbons, with the euception of trans-p-menthane (Table V). This would indicate that even on a socalled nonpolar column, this separation is not completely free of some bonding effects (19). The affinity of the terpenes for the LAC-2-R446 column over the other columns used in this study is graphically demonstrated b y reference to Figures 1 and 2. The analysis time on the LXC-2-R446 is roughly twice that of the Tween-20 column despite the fact that they are both the same length and the Tween-20 column rvas operated a t a significantly lower temperature (Table I), From this study one may conclude that the separation of the terpene hydrocarbons is greatly enhanced by employing capillary columns; that column efficiency as expressed by 16 &2 is not the sole factor determining degree of separation; and that selection of the proper stationary liquid phase is still by far the most significant factor in obtaining maximum resolution
(9) Golay, 11.J. E.. Ihzd., p. 36. (10) James, A . T., A \ . . ~ L CHEM. . 28, 1564 (1R.Sfi’l ,--__,. (11) Jones, W. L., Iiieselbach, It., Ibid.,
30, 1590 (1958). 112) Kirchner. J. G.. Miller. J. AI.. Keller, G. J., Ibzd., 23, 420 (1951). (13) Klouwen, 11. H., ter Heide, R., J . Chromatog. 7,297 (1962). (14) Lipsky, S. R., Landowne, R. A,, Biochem. Bzophys. Acta 27, 666 (1958). (15) McWilliam, I. G., Deaar, R. A., “Gas Chromatography 1958,” D. H. Desty, ed., p. 142, Academic Press, New York, 1958. (16) McWilliam, I. G., Dewar, R. A,, S a t w e 181, 760 (1958). (17) Second Research Conference in Gas Chromatography Cniv. of California, Los Angeles, Jan. 29-30, 1962. (18) von Rudloff, E , Can. J . Chetn. 38, 631 (1960). (19) Wehrli, A., Kovats, E., Helv. Chim. ilcta 42. 2728 11959). (20) Win&ein, S:,Holness, X. J.. J . Ani. Chem. SOC. 77, 5562 (1955). (21) Zubvk, IT. J., Conner, A. Z., SAL. CHEJI.32,912 (1960). RECEIVED for review July 5 , 1962. Accepted August 31, 1962. Kork supported by a grant from the Research and Development Department of the Sunkist Growers, Inc., Ontario, Calif.
LITERATURE CITED
(1) Ambrose, D., Keulemans, -4.I. AI., Purnell, 1., H., ANAL. CHEM.30. 1582
(1958).
(2) Bernhard, R. A., Food Res. 25, 531
11960).
Correction
(3j Bernhard, R. A , , J . Assoc. O$ic. A g r .
Chemists 40,915 (195ii. ,
(4) Clements, R. L., bczence 128, 899
(1958). ( 5 ) Dauben, W.G., Bozak, R. E., J . Org. Chem. 24. 1596 (19591. (6) Desty, D. H.,‘Xntu;e 179, 242 (1957). ( 7 ) Desty, D. H., Gold;p, A., “Gas Chromatography 1960, R. P. W. Scott, ed., pp. 162-83, Butterworth. Washington,-D. C., 1960. ( 8 ) Dijkstra, G., de $Fey, J., “Gas Chromatography 1958, D. H. Desty, ed., p. 56, Academic Press, Sew York, 1958.
Conductivity Method Determination of Urea
for
In this article by Kei-Tsung Chin and . 33, 1757 K y b e Kroontje [ k i a ~CHEx (1961)], on page 1758, column 3 , paragraph 5 , the sentence should read: “The experiments reported n ere conducted at 27“ C. Kith a conductivity cell having a cell constant of 0.30723 cm.+’
VOL. 34, NO. 12, NOVEMBER 1962
1579
