FLAVOUR AND FRAGRANCE JOURNAL, VOL.

2, 17-19 (1987)

Determination of the Enantiomeric Composition of

( t)-Terpinen-4-01from Sweet Marjoram Origunum Mujorana L.
using a Chiral Lanthanide Shift Reagent
Uzi Ravid, Michal Bassat and Eli Putievsky
Department of Medicinal, Spice and Aromatic Plants, Agricultural Research Organization, Newe Ya'ar, Post Haifa 31 -999, Israel

Raphael Ikan and Vera Weinstein

Department of Organic Chemistry, Natural Products Laboratory, Hebrew University of Jerusalem, Jerusalem 91 -904, Israel

'H-NMR examination of (+)-terpinen-4-01, performed in the presence of various molar ratios of a chiral
lanthanide shift reagent Eu(hfc)3, showed that the enantiomeric composition of (+)-terpinen-4-01isolated from
the essential oil of sweet marjoram is 73%(+): 27%(-). Peak assignment of the enantiomers was achieved by
spiking the natural (+)-enantiomer with the commercial (-)-alcohol. The method will readily reveal adulteration
of natural oils with racemic synthetics.

KEY WORDS (+)-Terpinen-4-01 Sweet marjoram Origanum rnujorunu L. 'H-NMR Chiral Eu(hfc)3
Enantiomeric composition

INTRODUCTION EXPERIMENTAL

Terpinen-4-01 [4-methyl- 1-(1-methylethyl)-cyclo- Sweet marjoram (Origanum majorana L.) was

hex-3-ene-1-01] occurs in the essential oils of
marjoram, thyme, cypress, juniper berry, Ceylon
cardamom, nutmeg and others.' The (+)-
enantiomer was found in the oil of Melaleuca
finariifolia Smith (37% of the total The
(-)-enantiomer was isolated from the oil of
Eucalyptus dives (3-4%),4 and the racemate was
found in oil of Liquidambar formosana Hance
(8%).5
Terpinen-4-01 was found also in several bark
beetle species' and is the main component in the
aggregation pheromone of Polygraphus polig-
raphus. P. poligraphus responded well to traps
baited with (-)-terpinen-4-01, while the addition
of the (+)-enantiomer inhibited beetle response.'
(-)-Linalyl acetate isolated from clary sage,
Salvia dominica, lavender and lavandin oils,' and
(-)-linalol isolated from sweet basil oil9 show
high optical purity by 'H-NMR spectroscopy in
the presence of E ~ ( h f c ) ~(+)-Linalol
. isolated
from corinader oil9 is a mixture of enantiomers.
grown in an experimental field at the Newe Ya'ar
Agricultural Experiment Station. Seeds of a
commercial variety of sweet marjoram received
from Desert Seeds Co., California, were sown in
the autumn of 1977. The fresh foliage was
steam-distilled for one hour in a 130-1 direct
steam pilot plant apparatus. The isolated oil
(yield, 0.28%),was dried over anhydrous sodium
sulphate and stored at 4-6°C. Terpinen-4-01
was isolated from the oil by flash chromatography
under pressure (2 atm.) on silica gel Davisil60 A,
200-425 mesh (Aldrich Chemical Co.), eluant: 1
to 20% solution of ethyl acetate in hexanes. The
chemical purities of natural and commercial sam-
ples of terpinen-4-01 were detected by a Varian
3700 gas chromatograph equipped with FID, and
a Hewlett-Packard 3390A integrator. Samples of
0.1 p1 of the natural fractions and of commercial
terpinen-4-01 were injected on a packed glass
column (3 m x 4 mm i.d.) with 5 % Carbowax
20M on acid-washed, silanized Chromosorb W

0882-5734/87/010017-03SO5.00 Received 16 December I986

0 1987 by John Wiley & Sons, Ltd. Accepted 3 March I987
18 U. RAVID E T A L .

(80-100 mesh) and on a fused silica capillary

column (25 m) coated with Carbowax 20M. Oper-
ating conditions were: temperature programme,
70-190°C (SUCmin-I); carrier gas, nitrogen, 30
and 5 ml min-I, respectively. Commercial sam-
ples of (+)-terpinen-4-01 were obtained from CA
Aromatics [[aID2'+ 20.2" (C = 2.10, EtOH)]
and from Aldrich Chemical Co. [[aID2' + 18.6"
(C = 0.93, EtOH)] and (-)-terpinen-4-01 was
obtained from Bedoukian Co. [[alD'' - 24.05",
(C = 2.05, EtOH)]. + +
Tris [3-(heptafluoropropylhydroxymethylene-
(+)-camphorato], europium(II1) [Eu(hfc),], was
obtained from Aldrich Chemical Co. Optical
rotations were measured on a Perkin-Elmer
Model 531 polarimeter. The specific rotation of
terpinen-4-01 isolated from sweet marjoram
was +12.2" ( C = 1.36, EtOH). 'H-NMR
spectra were obtained at 200 MHz with a
Bruker Model WP-2OOSY Fourier transform
spectrometer. Terpinen-4-01(10 pl) was dissolved
in a solution of 2% TMS in deuterochloroform
(350 pl) in the NMR tube. Ten-milligram aliquots
of Eu(hfc), were added and dissolved in the
solution. Molar ratios of the lanthanide shift
reagent to substrate ([ L]/[S]) were 0.45, 0.60,
0.75 and 0.90.

RESULTS AND DISCUSSION
Terpinen-4-01 is the major constituent in the
essential oil of the fresh leaves and flowers from
sweet marjoram (26.6%). a-Pinene ( l . l % ) ,
p-pinene (0.3%), myrcene (2.1%), a-phellandrene
(0.5%), a-terpinene (9.2%), limonene (2.0%),
y-terpinene (17%) p-cymene (2.9%), terpinolene
(3.8%), linalol (9.4%), linalyl acetate (2.5%) and
P-caryophyllene (5.3Yo) were also identified.
Sweet marjoram oil from various sources was
found to contain terpinen-4-01 as its major com-
ponent.I0-l4
A chemotype with low phenol content and a
moderate concentration of monoterpene alcohols
(mainly linalol and terpinen-4-01) was detected in
a few populations of Origanum vulgare'5*'6and
0. syriacum . I b The flavour of this chemotype
resembles that of a mixture of sweet marjoram and
a phenol (thymol or carvacro1)-containing herb.
The essential oil of 0. majoruna from Turkey
contains 65% carvacrol and 4% thymol." The
'H-NMR spectra of (+)-terpinen-4-01, isolated
from the essential oil of sweet marjoram, in the
PPM 4 3
Lil2
Fig. 1 . ' H - N M R spectra of (+)-terpinen-4-01 isolated
froin sweet marjoram in the presence of Eu(hfc)3 at
[L]/[S] = 0.75
presence of Eu(hfc)3 at increasing lanthanide-to-
substrate molar ratios ([L]/[S] = 0.45 to 0.90)
show that the (+)-alcohol is not optically pure,
and the enantiomeric composition is 73:27
(+)/(-) (Figure 1). The absorption of the allylic
methyl protons is split into two unequal sets of
doublets at [L]/[S] = 0.60 to 0.90, and the
absorption of the isopropyl protons is split into
two unequal signals. The magnitudes of the enan-
tiomeric shift differences (AAd) are greater for
the isopropyl protons (AAd = 0.23) than for the
allylic protons (AAd = 0.06) at [L]/[S] = 0.90
(Table 1). At smaller ratios of lanthanide-to-
substrate the enantiomeric isopropyl and ally1
proton signals give lower AA6 values. The mag-
nitudes of the chemical shifts (6) and of the
induced shifts (Ad) of the isopropyl protons in the
presence of Eu(hfc)3 at the same [L]/[S] molar
 ENANTIOMERS OF TERPINEN-4-OL 19

Table 1 . Chemical shifts (6) and enantiomeric shift differences (AA6) for the allyl and isopropyl protons of natural
terpinen-4-01 isolated from sweet marjoram in the presence of Eu(hfc)3 at increasing [L]/[S] molar ratios

6 (PPm) AA.3 (PPm)

[LlI[Sl CH,-C=C (CH3)z-CH - CH3-GC (CH3)2-CH-
0.45 1.99 2.32 - 0.09
0.60 2.14 3.22 0.03 0.15
0,75 2.23 3.78 0.05 0.19
0.90 2.34 4.43 0.06 0.23

ratios are also greater than those of the allyl REFERENCES

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To assign peaks to the enantiomers, the
natural (+)-terpinen-4-01 was spiked with a com-
mercial (-)-enantiomer in the presence of
) ~[L]/[S] = 0.60 and 0.75. It was found
E ~ ( h f c at
that the (+)-enantiomer is represented by the
downfield signals of the four signals arising from
the enantiomeric isopropyl protons and by the
upfield signal of the two signals from the allyl
protons. 'H-NMR spectra of commercial samples
of (-)-and (+)-terpinen-4-01 in the presence of
) ~ molar ratios of 0.45-0.90 show that
E ~ ( h f c at
the samples contain both enantiomers but in vari-
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Our results indicate that the determination of
the enantiomeric composition of natural
terpinen-4-01, linalo19 and linalyl acetate' by
'H-NMR spectroscopy using a chiral lanthanide
shift reagent is a fast and nondestructive method.
Adulteration of essential oils with synthetic
racemic alcohols and acetates may be detected
quantitatively by the above-mentioned method.
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