2. ENVIRONMENTAL ChEMISTRy & TEChNOLOGy 2.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Environmental Chemistry & Technology
P53 VOLATILE ORGANIC SubSTANCES PRESENT
IN SPICES AND SPRuCE NEEDLES
LUDMILA MRAVCOVá, MILADA VáVROVá, JOSEF
ČáSLAVSKý, MICHAELA STOUPALOVá, ILOnA
HLAVáČKOVá and HAnA VíTEČKOVá
Brno University of Technology, Faculty of Chemistry, Purkyňova
118, 612 00 Brno,
mravcova@fch.vutbr.cz
Introduction
Essential oils are volatile lipophilic substances, usually
colorless. Most often, essential oils consist of terpenes,
namely monoterpenic hydrocarbons, aldehydes, alcohols,
ketones, acids, esters. Their content substances are usually
classified as isoprenoids and phenylpropanoids groups 1 . Their
characteristic scent is conditioned by terpenic compounds, in
general 2 .
The TLC (thin layer chromatography) method can be
used for the identification of essential oil present in spice 1 .
This method is simple, without need of sofisticated and
expensive instrumentation. Defined amount of the analysed
mixture is applied on the starting line of a plate covered by
a thin layer of sorbent (stationary phase). Chromatographic
plate is then placed into the developing chamber with mobile
phase, which rises slowly and evenly through the thin layer,
transporting the individual components of the analyzed mixture
by various speed. Dried thin layer with perceptible stains
of particular compounds of the mixture situated in different
distances from the start – represents TLC chromatogram. The
identification of compounds is performed either via comparison
of their migration distances with standards, or by
comparison of their R F values with those obtained from the
literature 3 .
Another option to identify essential oils is application
of SPME (Solid phase microextraction) in connection with
GC/MS. Solid phase microextraction is simple and efficient
sorptive – desorptive technique used for solventless isolation/
preconcentration of target analytes from the sample matrix 4 .
In the field, this procedure could be also used as passive sampling
method. During this procedure, analytes are sorbed by
thin layer of stationary phase placed on the SPME fiber. The
SPME process continues until the equilibrium in the system
is reached. In physical-chemical terms, the SPME technique
state of equilibrium depends on the analyte properties and on
the type and thickness of polymer covering the silica fiber 4 .
Experimental
For the identification of essential oils present in spice
(caraway, cardamom, pepper, sweet pepper, calamint, cinna-
mon and muscat), two methods were used 5 :
•
•
TLC
SPME, GC/MS
s437
T L C
Spice essential oils isolation proceeded in the following
manner. Spice samples were extracted by ethanol for
10 minutes. After that, the extract was filtered and the spice
was reextracted twice for 20 minutes by petroleum ether.
Extracts were concentrated on the vacuum rotary vaporizer
to the defined volume.
By means of micropippete, concentrated extracts were
applied on the chromatographic plate (Alugram Sil G). The
distance of applied stains was between 0.5–1 cm, the volume
of applied sample was always 10 μl and 20 μl. Ethanol and
petroleum ether extracts I and II were applied on plates.
Plates were developed in a closed chromatographic
chamber, which was filled with a developing agent – mobile
phase formed by the mixture of toluene and ethyl acetate
(ratio 93 : 7). Developing was ascensive and was let in progress
until the mobile phase reached the distance of 1 cm
from the top of the plate. Plates were let to dry and then they
were sprayed by developer for the purpose of visualization of
stains created by separated substances. The used developer
consisted of ethanol and sulphuric acid (ratio 95 : 5), which
was mixed in 1 : 1 ratio with one-percent solution of vanillin
in ethanol. After the chemical detection, plates were dried
again in the drier at the temperature of 105 °C for 5 minutes.
Identification of vizualized stains was performed via comparison
of experimental R F values with those published in the
literature 6 .
S P M E – G C / M S
Weighted amounts of individual spices (1 g) were put
into vials. Substances from spice were sorbed from the headspace
by SPME fiber at the temperature of 40 °C. The compounds
were then directly injected into the gas chromatograph.
The SPME fiber used was 65 μm polydimethylsiloxan/
divinylbenzene (PDMS/DVB) from Supelco. Gas chromatograph
with mass spectrometric detector was Agilent 6890n
GC/5973 MSD. The HP-5MS column (Agilent Technologies,
USA), 30 m × 0.25 mm × 0.25 μm was used, the injector temperature
was 270 °C, oven program was: 45 °C, 2 min, 5 °C
to 200 °C, hold 2 min. He at a flow of 1 ml min –1 (constant
flow mode) was used as a carrier gas, transferline temperature
was 250 °C, ion source temperature was 230 °C, quadrupole
temperature was set to 150 °C. Direct interface connection
was applied, electron ionization at 70 eV was employed.
Results
TLC chromatograms evaluation was performed in
accordance with requirements of the Pharmaceutical Codex 6
, which is valid for phytopharmaca and recomends TLC as
an optimum screening method. R F values were calculated
for each of detected stains. This factor was also used for the
identification. Besides the retention factor, also the colours of
the stains were compared. For example, the comparison of all
sweet pepper extracts shows Table I. In column “Identified
Compound”, unambiguously identified content substance