Low Temperature Liquid Chromatographic Separation of Oxygenated
Terpenes from Terpene Hydrocarbons
T. Yamasaki* / M. Sakai / T. Kanamori / M. Sogo
Department of Bioresources Science, Faculty of Agriculture, Kagawa University, Miki-Tyo, Kagawa-Ken 761-07, Japan
Key Words
Column liquid chromatography
Silica gel columns at low temperatures
Terpene hydrocarbons
Oxygenated terpenes
Summary
The separation of/3-pinene and menthone by HPLC on a
silica gel column can be significantly improved with
decreasing the column temperature. On a perparative
scale complete separation of oxygenated terpenes from
terpene hydrocarbons could be carried out under such
conditions.
I ntroduction
Terpeneless essential oils are prepared by fractional distil-
lation, by extraction with dilute alcohol or other solvents,
by column adsorption chromatography, by the adsorption
of terpene vapor, or by a combination of these methods.
However, with a single step oxygenated terpenes, cannot be
quantitatively separated from natural essential oils, because
of several disadvantages, such as the similarity of boiling
points, minor difference in solubilities, and incompleteness
of resolution or adsorption.
Kubeczka [1] demonstrated the excellent fractionation of
essential oils into three classes, oxygenated terpenes, mono-
terpene hydrocarbons, and sesquiterpene hydrocarbons,
using reversed-phase HPLC. However, according to his
procedure, the complete separation on a preparative scale
and the elimination of high-boiling polar substances may be
difficult.
In 1958 Clements separated several monoterpene hydro-
carbons by silica gel chromatography, at -78.5~ [2]. In
1980 Jinno et al. reported that the resolution of benzene
and pseudocumene by HPLC on a silica gel column signi-
ficantly improved with decreasing the temperature [3].
The present paper deals with the possibility of separating
oxygenated terpenes from terpene hydrocarbons by low-
temperature HPLC.
478 Chromatographia Vol. 21, No. 8, August 1986
0009-5893/86/8 0478-02 ~; 03.00/0 9 1986 Friedr. Vieweg & Sohn VerlagsgesellschaftmbH
Experimental
Apparatus
A low-temperature HPLC system was assembled in our
laboratory consisting of a JASCO Tri Rotar pump, a JASCO
VL-611 injector with a 5ml loop (6.37m long x 1.0ram
i.d.), a pre-cooling coil (3.63m Iong X 1.0ram i.d.), a
column, a heating coil (5.00m long x 0.5mm i.d.),a JASCO
UVlDEC-100-VI spectrophotometer, and a JASCO RC-
250 desktop recorder. The pre-cooling coil and the column
were cooled in an ethanol bath using NESLAB CC-100
Cyocool immersion cooler. The eluate was conducted to
the spectrophotometer via the heating coil immersed in a
water bath kept at 20~
Column
Fully porous silica gel (YMC, pore size 120A, particle size
88 to 63/1m) was washed with water and dried at 120~
for 48hr. The silica gel was mixed with chloroform (4.6g/
150ml for a 100ram IongX 7.5mm i.d. column, and
20.1g/500ml for a 50ram long x 20ram i.d. column), and
packed into the columns using methanol at a pressure of
120kg/cm 2, in a YMC Model-I column packing system.
The silica gel columns prepared in this way were washed
with chloroform, n-hexane, and n-pentane. The solvents
and the slurry were deaired by an ultrasonic cleaner before
use.
Elution conditions
The flow rate of the mobile phase was 1 ml/min. The UV
detector was used at 22Onto.
Gas Chromatographic Analysis
Fractions of the HPLC run were collected and analysed by
a Hitachi Model 163 s chromatograph equipped with a
FID in order to confirm the elution order. A 2m long X
3ram i.d. glass column was packed with 2% polyethylene
glycol 6000 P on Uniport HP support. The column tempera-
ture was programmed at 4~ from 50 to 150~
Short Communication
 ~9 , (A) P~ (B) el
(A),(a) _ _ .~1~

I I I
:
i
III IV(a i ~
i

i
I

o lOO 200 30O

(A) ,(b) Elution volume (ml)
Fig. 2
I I
Preparative scale separation of oxygenated terpenes from terpene
I
hydrocarbons.
Column: 50mm long X 20mm i.d. Mobile phase: (A) n-pentane
(A),(c) ,l(B)(a)l (B) diethyl ether. Column temperature: (a) 20~ (c) -80~
A 2ml n-pentane solution of 250rag each of ~-pinene, ~-pinene,
"),-terpinene, and d-limonene, 100rag each of myrcene, p-cymene,
and Iongifolene, and 90rag each of 1,8-cineol, menthone, citronel-
0 3b
Elution volume (ml)
6b 9b Iol, citronellal, geraniol, dl-perillaldehyde, borneol, d-camphor, and
s was injected.
Fig. 1 I: large amount of ~-pinene,#-pinene and Iongifolene, and a small
The effect of column temperature on the resolution of ~-pinene(I) amount of the other terpene hydrocarbons;
and menthone (11). I1: small amount of a-pinene, /~-pinene and Iongifolene, and a large
Column: lOOmm long X 7.Smm i.d. Mobile phase: (A) n-pentane amount of the other terpene hydrocarbons;
(B) diethyl ether. Column temperature: (a) 20~ (b) -30~ (c) II1: small amount ofd-limonene, p-cymeneandmyreene;
-80~ IV: mobile phase only;
Sample:0.2mln-pentanesolutioncontaining10mgof eachterpene. V: oxygenated terpenes.

Results and Discussion Acknowledgement

Fig. 1 shows the chromatograms obtained w i t h the HPLC A part of this w o r k was supported by the Grant-in-Aid for
system at d i f f e r e n t column temperatures. It is evident f r o m Scientific Research f r o m the Japanese Ministry of Educa-
these results that the resolution of ~-pinene and menthone tion.
improves w i t h decreasing column temperature, indicating
that the migration o f the compounds, e~pecially of the
oxygenated c o m p o u n d , menthone, was slower at lower References
temperatures. Menthone did not elute at all at - 8 0 ~ using
[1] K.-H. Kubeczka, in "Flavour' 81"', P. Schreier, ed., Walter
n-pentane as the mobile phase. de Gruyter, Berlin--New York, 1981; p. 345.
Complete separation of the class of oxygenated terpenes [2] R . L . Clements, Science 128, 899 (1958).
[3] K. Jinno, H. Nomura, Y. Hirata, J. High Resolut. Chrom-
from the class o f terpene hydrocarbons could also be car- atogr./Chromatogr. Commun. 3,305 (1960).
ried o u t on preparative scale w i t h the present HPLC system,
as shown in Fig. 2. Received: May 2, 1986
Accepted: May 18, 1986
In our w o r k l o w temperature, an inexpensive silica gel, and A
low-boiling eluants were utilized. Our results indicate that
low-temperature HPLC may have a favorable application
in the preparation o f terpeneless oil f r o m natural flavors o r
essential oils. We have already applied the technique t o the
solution of practical problems and plan t o report separately
on our results.

Chromatographia Vol. 21, No. 8, August 1986 Short Communication 479