Development of an optimised HPTLC method for analysis

Transcription

Universität Basel – Philosophisch-Naturwissenschaftliche Fakultät – Departement
Pharmazeutische Wissenschaften – Institut für Pharmazeutische Biologie
___________________________________________________________________________
Development of an optimised HPTLC
method for analysis of essential oils
Diploma thesis of Katherine Gessler
Supervision:
Prof. Dr. Matthias Hamburger, Universität Basel
Dr. Eike Reich, CAMAG Muttenz
___________________________________________________________________________
May – September 2005
Expression of thanks
I would like to thank …
… Dr. Matthias Hamburger for leading and realising the project of this diploma thesis.
… Dr. Eike Reich, CAMAG for his specialized knowledge, his endurance and his dragging
along enthusiasm.
… the employees of CAMAG Laboratory Services for their interest, patience and their help.
… the staffers of CAMAG for their warmly welcome.
… Reto Della Casa, Essencia for the large amount of oil samples, the interesting background
information and handy relationships
…. the employees of Essencia for their friendly receipt and the music.
… Dr. Bernd Wissmann, Polarome for his spontaneous invitation to collecting my first
samples.
… Rita Tengler, Hänseler for the unstinting arrangement of oil samples.
… Seraina Caprez for sharing ups and downs during this summer.
… all those who supported me to facilitate this diploma thesis.
2
Abstract
In the European Pharmacopoeia 5.2 the general regulation of thin layer chromatography
described in chapter 2.2.27 was revised. Nevertheless the particular monographs have a touch
of outdated and are no longer keeping with the times. Gas chromatography is the standard
method to analyse essential oils; even it is an expensive and time-consuming method. Modern
thin layer chromatography with all his advantages may be seen as a complementary
technique; but a standard method suitable for all essential oils is still missing.
Aim of this diploma thesis was to optimize the established thin layer chromatography analysis
methods of essential oils such as there is a standard method suitable for all essential oils. With
this standard method every essential oil should be doubtless referable.
The mobile phase, the derivatization and the derivatization reagent were optimised and
parameters influencing the reproducibility proved. Six different oil types were classified and
all essential oils were divided after their types. By the introduction of four standard
substances, such as menthol, caryophyllene oxide, menthyl acetate and β-caryophyllene, the
important zones of all essential oils could be described.
3
Table of Contents
1
Introduction ........................................................................................................................ 6
1.1
Essential oils............................................................................................................... 6
1.2
Methods for analysis .................................................................................................. 7
1.3
Thin layer chromatography ........................................................................................ 7
1.3.1
Stationary phase ................................................................................................. 7
1.3.2
Method development.......................................................................................... 8
1.3.3
Automatic Developing Chamber ADC 2 ........................................................... 9
1.4
Aim of this diploma thesis ......................................................................................... 9
2 Material and Methods....................................................................................................... 10
2.1
Material .................................................................................................................... 10
2.1.1
Essential oils..................................................................................................... 10
2.1.2
Standards .......................................................................................................... 14
2.1.3 Thin layer chromatography plates........................................................................... 15
2.1.4 Chemicals used........................................................................................................ 15
2.1.5 Equipment and accessories...................................................................................... 16
2.2
Methods.................................................................................................................... 17
2.2.1 Sample preparation and standard preparation ......................................................... 17
2.2.3 Derivatization reagents and application .................................................................. 17
3 Results .............................................................................................................................. 18
3.1
Current situation: Pharmacopoeia ............................................................................ 18
3.2
Specifying of unmodified parameters ...................................................................... 20
3.3
Optimization of the derivatization............................................................................ 22
3.3.1
Different derivatization reagents...................................................................... 22
3.3.2
Variations of anisaldehyde reagent R............................................................... 23
3.3.3
Influence of temperature and age of anisaldehyde reagent R........................... 24
3.3.4
Different heating times..................................................................................... 25
3.3.5
Subsequent treatment ....................................................................................... 26
3.4
Optimization of the mobile phase ............................................................................ 27
3.4.1
Mobile phases found in literature..................................................................... 27
3.4.2
Method development........................................................................................ 28
3.4.3
Variations of the toluene-ethyl acetate ratio .................................................... 29
3.5
Reproducibility......................................................................................................... 31
3.5.1
Influence of humidity ....................................................................................... 31
3.5.2
Mobile phase .................................................................................................... 34
3.5.3
Drying time ...................................................................................................... 35
3.5.4
Dipping versus spraying................................................................................... 36
3.5.5
Reproducibility of derivatization with anisaldehyde reagent R ....................... 37
3.5.6
Merck versus Macherey –Nagel....................................................................... 38
3.6
Comparison of different samples of the same oil..................................................... 39
4
3.7
Standard method suitable for all essential oils ......................................................... 40
3.7.1
Standards assignment ....................................................................................... 40
3.7.2
Standard method............................................................................................... 40
3.7.3
Classification of oil types................................................................................. 41
3.7.3.1 Type 1: Oils visible at 366 nm before derivatization ................................... 42
3.7.3.2 Type 2: Oils with one main zone ................................................................. 43
3.7.3.3 Type 3: Two main zones .............................................................................. 46
3.7.3.4 Type 4: Caryophyllene oxide and β-caryophyllene zone............................. 47
3.7.3.5 Type 5: Caryophyllene oxide, β-caryophyllene and bornyl acetate zone .... 48
3.7.3.6 Type 6: Menthol and a menthyl acetate zone............................................... 49
4 Discussion ........................................................................................................................ 50
5 References ........................................................................................................................ 51
6 Annex ............................................................................................................................... 52
6.1
Preparation of samples and standards ...................................................................... 52
6.2
List of tables ............................................................................................................. 59
6.3
List of Figures .......................................................................................................... 60
5
1 Introduction
1.1 Essential oils
In the European Pharmacopoeia 5.2 essential oil is defined as follows:
An essential oil is an odorous product, usually a complex composition, obtained from a
botanically defined herbal raw material by steam distillation, dry distillation or by a suitable
mechanical process without heating. Essential oils are usually separated from the aqueous
phase by a physical process that does not significantly affect their composition.
Essential oils may be subjected to a suitable subsequent treatment. [1]
Essential oils evaporate completely without leaving behind a residue; at room temperature
they are usually liquid. The relative density is typically less than 1; they have a high refractive
index and a high optical activity. During long storage without protection against light and
oxygen essential oils oxidise; colour, consistence and odour may change.
Because of the commonly high price of essential oils, they are often falsified with substances
which have similar chemical and physical properties. [2]
Corresponding to their multifarious composition the range of medical use of essential oils is
wide. Externally they are used because of their spasmolytic, anti-inflammatory, anodyne,
antibacterial, antiviral and antimycotic qualities. Internally against flue-like respiratory
ailment, disorders of blood flow, tonsillitis, colon irritable, stomach trouble and stomach
diseases as well as appetizer, digestive or laxative. For internal use they are commonly
handled as capsule (e.g. Colpermin®, Tillotts Pharma, containing peppermint oil); but also as
tablet, sugar-coated tablet, spirituous extract, suppository, unguent or even pure oils
themselves. [3] [4]
Essential oils are not only used medically; but also in the perfume industry, for aromatherapy
or for esoteric purposes. [5] [6]
Side effects appear normally only after overdoses of medicaments containing essential oils.
Described side effects are contact dermatitis, photosensitization, stomach trouble, diarrhoea,
paralysis and spasms. Essential oils may have hepatotoxic, nephrotoxic and carcinogenic
effects. [3] [4]
Essential oils mostly are compounded out of terpenes, such as monoterpenes, sesquiterpenes
and rarely diterpenes, which are only few vapour-volatile. Also oxides, epoxides and
peroxides are contained, as well as esters, lactones, aldehydes, ketones and its derivatives. [7]
On the World Wide Web essential oils are praised and sold against every discomfort without
any side effects; often without warning of incorrect use or use without medical supervision.
Also the quality of such essential oils should be given a critical look.
6
1.2 Methods for analysis
Different methods to analyse essential oils are used, of which gas chromatography is the
usually-used because of the volatile components of essential oils. This type of analysis gives
information about the individual components of an essential oil and its relative amounts.
With thin layer chromatography an easy and fast identification of essential oils is enabled;
adulteration and falsification often are detected. More polar and less volatile components of
essential oils may be shown as with gas chromatography.
Gas chromatography is an expensive and time-consuming method; per run which take 45-60
minutes only a single sample can be analysed. Thin layer chromatography separates up to 17
samples on a sole plate; is a rapid, reliable, cost effective, and extremely flexible method of
analysis. Results are fast and clearly visible. With the introduction of automated equipment
chromatograms can be well documented and methods of thin layer chromatography fulfil
GMP guidelines.
Nevertheless gas chromatography is the standard method to analyse essential oils; thin layer
chromatography may be seen as a complementary technique.
Olfactive detection is used for pre-screening of an essential oil sample; but it is subjective and
not accurate enough. Gas chromatography analysis provides more detailed information while
thin layer chromatography is preferred for a preliminary analysis of many samples.
1.3 Thin layer chromatography
1.3.1 Stationary phase
Separation mechanisms in chromatography are adsorption and partitions equilibriums, as well
as ion-exchange and complex processes. The theory of these mechanisms is not elaborated on
in this diploma thesis.
For around 90% of all separations silica gel is used as stationary phase. The SiOH-groups on
the surface of the stationary phase build hydrogen bonds among each other and interact with
polar substances. Furthermore silica gel acts as a weak sieve because of its pores.
Due to it compounds for the separation of essential oils silica gel is used as stationary phase.
Silica gel offers ideal suppositions for the typical separation problems of essential oils, such
as separation of isomers or separation of fractions with different degrees of saturation.
Even though in the methods described in the European Pharmacopoeia thin layer
chromatography (TLC) plates are used, most of the tests done in line of this diploma thesis
were done with High Performance Thin Layer Chromatography (HPTLC) plates.
Due to its lesser particle size HPTLC plates provide a better performance of separation than
TLC plates; more samples may be applied on the same plate. The plates are smaller and the
separation distance is shorter; therefore the solvent consumption and the running time are also
smaller.
7
1.3.2 Method development
The development of a new or optimised chromatographic method of analysis contains several
steps; choice of the plate material, the mobile phase, the chamber type, saturation of the
chamber, application of the samples, development, derivatization and detection. But also the
humidity and the temperature of the laboratory where the thin layer chromatography is done
may influence the obtained results. To create reproducible and comparable chromatograms
the parameters of all those steps have to be determined and abided exactly.
It is important to change only one parameter at a time to get comparable results. In the present
diploma thesis always twin trough chambers saturated for 20 minutes were used.
First step is to test the methods found in literature search. Were no suitable methods found,
the CAMAG-optimization scheme, shown in figure 1, assists to develop a new method.
Figure 1 The CAMAG-optimization scheme
On different plates the same sample is developed with different neat mobile phases from all
eight selectivity groups defined by Snyder, 1978 [8]. The solvent strength can now be reduced
by addition of hexane or increased with water or methanol. Mixtures of solvents from
different selectivity groups can upgrade the separation. The sharpness of the zones may be
influenced by the addition of modifiers, such as acids or bases.
The derivatization also has to be optimized so that an optimal detection in daylight, under 366
nm or 254 nm is possible; the time to dry the plate after development, the derivatization
reagent, the application of the derivatization reagent and the conditions until the
documentation of the detected result have to be specified.
In the end the developed method must be validated before the method can be used routinely.
The result should be reproducible and the mobile phase stable. With a two dimensional
development the stability of the test solution in the chromatographic system needs to be
tested. The test solution has not been modified during the chromatographic process if all
zones are located on the line between the application point and the break-even point of the
two solvent fronts.
8
1.3.3 Automatic Developing Chamber ADC 2
In the CAMAG Automatic Developing Chamber ADC 2 the developing
chamber is part of a closed circuit in which a stream of air with defined
humidity is generated by means of a saturated salt solution or a molecular
sieve. So it is possible to develop chromatograms under exact humidity
conditions and to eliminate on this way the influence of the environment
humidity.
A picture of the ADC 2 is shown in figure 2.
Figure 2 CAMAG ADC 2
1.4 Aim of this diploma thesis
In the European Pharmacopoeia the analysis of essential oils by thin layer chromatography is
described separately in the corresponding monographs separately. Also in the Pharmacopoea
Helvetica, in the “Deutscher Arzneimittel Codex”, in the “Deutsches Arzneibuch” and in the
Pharmeuropa thin layer chromatography methods are suitable only for one corresponding
essential oil.
In the European Pharmacopoeia 5.2 the general methodology of thin layer chromatography
described in chapter 2.2.27 was revised. Now the use of HPTLC plates is permitted, which
may result saving of time and developing solvent. Nevertheless the particular monographs are
mostly outdated and are no longer keeping up with the state of the art in TLC.
They are expensive because of the large amount of standards which is used and the saturation
of the chamber which takes a lot of time. The applied volume is mostly not adapted, resulting
in an overload of the plates. The derivatization often leads to deviations from the described
colours.
An adaptation on recent methods offers the chance to standardise existing methods. With a
standardised method different essential oil may be investigated at the same time; to test every
species of essential oil with a separate method is any longer necessarily.
Even the optimised method has not to be inevitable a displacement of the existing methods,
but a supplement to already existing methods. The standardised method may be useful in
control of charges or to do stability tests for example.
9
2 Material and Methods
2.1 Material
2.1.1 Essential oils
Table 1 Samples
Sample
number
S403
S156
S158
S170
S181
S188
S1941
S1941
S1942
S1943
S1944
S1945
S1946
S1947
S1948
S1949
S1950
S1951
S1952
S1953
S1954
S1955
S1956
S1957
S1958
S1959
S1960
S1961
S1962
S1963
S1964
S1965
S1966
S1967
S1969
S1970
S1971
S1972
S1973
S1974
Sample description
Distributor
Lot Nr.
Anise Oil, Ph.Eur.3
Matricaria oil, blue, DAB2001
Clary sage oil, PhE4
Fennel seed oil, bitter
Nutmeg oil, PhE4
Turpentine oil
Cassia oil
Cassia oil
Cinnamon bark oil, Ceylon
Cinnamon leaf oil, BLCH
Citronella oil
Citronella oil, Java
Citronella oil, Ceylon
Clove leaf oil, crude
Coriander seed oil
Corn mint oil, crude
Corn mint oil, redistilled
Cumin seed oil
Eucalyptus oil
Eucalyptus oil
Fennel oil, sweet
Fir needle oil, Canada
Grapefruit oil, Florida
Grapefruit oil, South Africa
Juniper berry oil
Lavender oil, Russian
Lavender oil, traditional
Lemon oil
Lemon oil
Lemon oil, Argentina
Lemon oil, Argentina
Orange oil, bitter, Coast ivory
Orange oil, California
Orange oil, Florida
Orange oil, Navel
Orange peel oil, Brazil
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
2002.10.0481
602280
600860
603580
601740
680086
680086
311709
305904
311912
311589
311277
311801
311912
311830
311899
311942
311637
311931
311764
312654
312653
311913
311514
312630
312631
311606
311729
311856
680161
680157
312568
311435
311091
680091
312490
680071
312680
680106
10
S1975
S1976
S1977
S1978
S1979
S1980
S1981
S1982
S1983
S1984
S1985
S2032
S2033
S2034
S2035
S2036
S2067
S2068
S2069
S2072
S2078
S2080
S2081
S2082
S2083
S2088
S2089
S2092
S2095
S2104
S2105
S2160
S2161
S2162
S2163
S2164
S2165
S2166
S2167
S2168
S2169
S2170
S2171
S2172
S2173
S2174
S2175
S2176
S2177
Orange peel oil
Peppermint oil
Pine needle oil
Rosemary oil, Tunisian
Spearmint oil
Spearmint oil, 80%
Star anis oil
Star anise oil
Star anise seed oil
Tea tree oil
Thyme red oil, Spain
Pine needle oil, DAB 2004
Citronella oil, winterianus, Ph.Eur. 5.0
Matricaria oil, Roman
Nutmeg oil, Ph.Eur. 5.0
Mint oil, Indian, Ph.Eur. 5.2
Lavender oil, Maillette, Ph.Eur. 5.0
Cinnamon leaf oil, Ph.Eur. 5.0
Anise oil, Ph.Eur. 5.0
Matricaria oil, CT Bisabolol, Ph.Eur. 5.1
Fennel oil, bitter
Caraway oil
Coriander oil
Cassia oil, Ph.Eur. 5.0
Cinnamon bark oil, mind. 60%
Star anise oil, Ph.Eur. 5.0
Grapefruit oil, Israel
Lime oil, distilled
Mandarin oil
Clary sage oil, Ph.Eur. 5.0
Orange oil, bitter
Fennel seed oil, sweet
Fennel oil, bitter
Star anise oil
Caraway oil
Cinnamon oil
Citronella oil, nardus
Citronella oil
Clove oil
Clove flower oil, Ph.Eur. 5.0
Clove flower oil
Mountain pine oil, Tirol, Ph.Helv.9
Spruce needle oil, Siberian, DAB 2002
Swiss stone pine oil
Silver fir needle oil
Spruce needle oil, Mariana
Pine needle oil
Mountain pine oil
Coriander oil
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Polarome
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Hänseler
Hänseler
Hänseler
Hänseler
Essencia
Hänseler
Hänseler
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Hänseler
Hänseler
Hänseler
680109
311748
312673
305637
312706
311379
311876
311936
312686
360145
305646
60-4180-0
60-0290-0
60-1180-0
60-3580-0
60-3850-0
60-0550-0
60-2260-0
60-2420-0
60-2280-0
60-0350-0
60-2000-0
60-1080-0
60-1060-0
60-3280-0
60-0240-0
60-1940-0
60-1780-0
60-0120-0
60-0860-0
60-0170-0
60-0360-0
01-4500-0
01-3450-0
01-3850-0
01-4150-0
60-0280-0
01-4300-0
01-3900-0
60-0610-0
60-0600-0
60-0450-0
60-0430-0
60-1750-0
60-0370-0
60-4180-0
01-5375-0
01-5350-0
01-4350-0
11
S2178
S2179
S2180
S2181
S2182
S2183
S2184
S2185
S2186
S2187
S2188
S2189
S2190
S2191
S2192
S2193
S2194
S2195
S2196
S2197
S2198
S2199
S2200
S2201
S2202
S2203
S2204
S2205
S2206
S2207
S2208
S2209
S2210
S2211
S2212
S2213
S2214
S2215
S2216
S2217
S2218
S2219
S2220
S2265
S2266
S2267
S2268
Eucalyptus oil, radiata
Eucalyptus oil, globulus, Ph.Eur. 5.0
Eucalyptus oil, citriodora
Eucalyptus oil
Juniper berry oil, Ph.Eur. 5.0
Juniper oil
Lavender oil, Bulgarian
Lavender oil, France, Ph.Eur. 5.0
Lavender oil
Lemon oil, Messina extra
Lemon oil
Lime oil, squeezed cold
Lime oil, squeezed cold, stabilized
Matricaria oil
Mint oil, rectified, China
Mint oil, Nagaoka, Ph.Eur. 5.2
Nutmeg flower oil
Orange oil, blood, Messina
Orange oil, bitter, South America
Orange oil, bitter, Guinea
Orange oil, sweet, Florida
Orange oil, sweet, Messina, Ph.Eur. 5.0
Orange peel oil, sweet
Peppermint oil, USA, Ph.Eur.5.0
Peppermint oil, French, Ph.Eur. 5.0
Peppermint oil
Rosemary oil, CT Campher, Ph.Eur. 5.0
Rosemary oil, CT Cineol
Sage oil, Spanish
Sage oil, officinalis, Ph.Helv. 9
Sage oil
Spearmint oil, USA
Spearmint oil, Chinese
Mandarin oil, squeezed cold
Tea tree oil, Ph.Eur. 5.0
Tea tree oil
Thyme oil, vulgaris, Switzerland
Thyme oil, zygis, Ph.Eur. 4.7
Thyme oil
Turpentine oil, Ph.Eur.5.0
Turpentine oil
Eucalyptus oil, globulus, Ph.Eur. 4
Eucalyptus oil (Mix of S2178, S2179,
S1955, S1956, S2181 and S2265)
Spearmint oil (Mix of S1979, S1980, S2211
and S2210)
Tea tree oil (Mix of S1984, S2214 and
S2213)
Essencia
Essencia
Essencia
Hänseler
Essencia
Hänseler
Essencia
Essencia
Hänseler
Essencia
Hänseler
Essencia
Essencia
Hänseler
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Hänseler
Essencia
Essencia
Hänseler
Essencia
Essencia
Hänseler
Essencia
Essencia
Hänseler
Essencia
Essencia
Essencia
Essencia
Hänseler
Essencia
Essencia
Hänseler
Essencia
Hänseler
Essencia
Essencia
Various
60-4040-0
60-2740-0
60-3990-0
01-4450-0
60-3220-0
01-4775-0
60-1310-0
60-0540-0
01-4850-0
60-0040-0
01-3960-0
60-1250-0
60-1430-0
01-4925-0
60-0750-0
60-0770-0
60-3370-0
60-1840-0
60-0840-0
60-0150-0
60-0130-0
60-0140-0
01-3550-0
60-0690-0
60-1900-0
01-5100-0
60-4210-0
60-0790-0
01-5600-0
60-0830-0
60-0845-0
01-5750-0
60-0501-0
60-0520-0
60-0090-0
60-1640-0
01-4940-0
60-1040-0
60-0951-0
01-6550-0
60-3300-0
01-6350-0
600860
602740
-
Various
-
Various
12
S2269
S2270
S2271
S2272
S2273
S2274
S2275
S2276
S2277
S2278
S2279
S2280
S2281
S2282
S2283
S2284
S2285
S2286
S2287
S2288
S2289
S2290
S2291
S2292
S2293
S2294
S2295
S2296
S2297
S2310
Thyme oil (Mix of S2217, S2215, S2216
and S1985)
Clove oil (Mix of S2169, S2168, S1949,
S1950 and S2167)
Coriander oil (Mix of S2081, S2177 and
S1951)
Caraway oil (Mix of S2080 and S2163)
Cinnamon leaf oil (Mix of S1943 and
S2068)
Cinnamon bark oil (Mix of S1942, S2083
and S2164)
Citronella oil, nardus/Ceylon (Mix of
S1947, S1948 and S2165)
Citronella oil, winterianus/Java (Mix of
S2166, S1944, S1945, S1946 and S2033)
Matricaria oil (Mix of S2191 and 2072)
Lavender oil (Mix of S2186, S2184, S2185,
S2067, S1962 and S1963)
Clary sage oil (Mix of S2104 and S2220)
Juniper oil (Mix of S2182, S2183 and
S1961)
Rosemary oil (Mix of S2204, S2205, S1978
and S2206)
Turpentine oil (Mix of S2219 and S2218)
Peppermint oil (Mix of S1976, S2203,
S2202 and S2201)
Mint oil (Mix of S1952, S2036, S2193,
S2192 and S1953)
Orange oil, bitter (Mix of S1969, S2105,
S2197 and S2196)
Orange oil, sweet (Mix of S1970, S1971,
S1972, S1973, S2198, S2199, S1975, S1974
and S2200)
Grapefruit oil (Mix of S1959, S1960 and
2089)
Mandarin oil (Mix of S2095 and S2212)
Lime oil, squeezed (Mix of S2189 and
S2190)
Lemon oil (Mix of S1964, s1966, S2187
and S2188)
Anise Oil, Ph.Eur.3
Anise oil (Mix of S2069 and 2291)
Star anise oil (Mix of S1981, S1982, S1983,
2088 and 2162)
Fennel oil, bitter (Mix of S2078 and S2161)
Fennel oil, sweet (Mix of S1957 and 2160)
Nutmeg oil (Mix of S2035 and S2194)
Cassia oil (Mix of S1941 and S2082)
Various
-
Various
-
Various
-
Various
Various
-
Various
-
Various
-
Various
-
Various
Various
-
Various
Various
-
Various
-
Various
Various
-
Various
-
Various
-
Various
-
Various
-
Various
Various
-
Various
-
Essencia
Various
Various
2002.10.0481
-
Various
Various
Various
Various
Essencia
-
13
2.1.2 Standards
Table 2 Standards
Standard
number
R1986
R1987
R1988
R1989
R1990
R1991
R1992
R1993
R1994
R1995
R1996
R1997
R1998
R1999
R2000
R2001
R2002
R2003
R2004
R2005
R2006
R2007
R2008
R2009
R2010
R2076
R2077
R2079
R2090
R2091
R2093
R2094
R2221
R2222
R2223
R2224
Standard description
Distributor
Lot Nr.
Guaiazulene
Anethole
Fenchone
Linalol
Geranyl acetate
Bisabolol
Bornyl acetate
Terpinen-4-ol
Linalyl acetate
Carvone
β-Caryophyllene
Thymol
Menthyl acetate
Carvacrol
Menthol
β-Pinene
Eugenol
Coumarin
Methyl anthranilate
Bergaptene
trans-cinnamic aldehyde
Borneol
Cineole
Citronellal
Anisaldehyde
Citronellol
Myristicine
Carveol (+) 97%
Citral nat.
Lemarome N
Terpineol perfume
Methyl N-Methyl anthranilate
Anethol (trans-) 99%
Fenchone (+)Fenchone (1R)-(-)Caryophyllene oxide
Merck
Essencia
Chemika
Essencia
Essencia
Essencia
Essencia
Essencia
Essencia
Merck
Essencia
Fluka
Aldrich
Essencia
Fluka
Essencia
Merck
Merck
Essencia
Fluka
Merck
Aldrich
Aldrich
Aldrich
Essencia
Aldrich
ChromDex
Uni Wien
Essencia
Essencia
Essencia
Fluka
Aldrich
Fluka
Aldrich
Essencia
K29155433 117
702700
62329/1 15001
701460
70-0080-0
703030
703410
703800
700100
K27960616
2002.08.0209
420973/1 40802
KO 02122KO
70-2300-0
360728/1 11502
2002.10.0348
S2170029 114
S31222 119
70-0600-0
425788/9 34301
8.02505.0250
12408PI
10107HG
05322AF
700620
00829AQ
13935-703
70-3750-0
70-1410-0
70-2280-0
399299/1 24201
S05492-081
62329/1 15001
02508HV
70-4100-0
14
2.1.3 Thin layer chromatography plates
Table 3 Thin layer chromatography plates
Plate
HPTLC-Fertigplatten, Nano-DURASL-20 UV254
TLC glass 10x20, Si 60 F254
HPTLC glass 10X10, Si 60 F254
Manufacturer
MachereyNagel
Merck
Merck
Merck
Merck
Merck
Lot Nr.
309252
840316352
OB464935
OB515602
OB526793
OB545060
2.1.4 Chemicals used
Table 4 Chemicals used
Name, purity or quality
Acetic acid
Manufacturer
Merck
Acetone per analysi
Anisaldehyde
Acros
Fluka
Roth
Merck
Acros
Merck
Merck
Merck
Merck
Merck
Acros
Cyclohexane per analysi
Diethyl ether per analysi, stabilized with BHT
Diisopropyl ether per analysi
Ethanol 96%
Ethyl acetate per analysi
Heptane
Isopropyl alcohol per analysi
Methanol per analysi
n-Heptane per analysi
Pentane per analysi
Phosphomolybdic acid
Sulphuric acid
Toluene per analysi
Merck
Merck
Riedel-de-Haen
Merck
Merck
Acros
Lot
K27583063
K29535963 130
A019663101
422315/1 53601
02569422
K10066566 815
0557085
K25739667 901
K33957583 441
K33137923415
K25693579 841
K32940634407
A019863901
A0205287001
K25693579 841
K11687677 919
11280
K28679231 101
0445584
A0206963001
15
2.1.5 Equipment and accessories
Table 5 Equipment and accessories
ADC light µP controlled
Analytical balance AG245
Automatic Development Chamber ADC 2
Automatic Development Chamber ADC2
Automatic TLC Sampler ATS 4
Automatic TLC Sampler ATS 4
Digital camera G5
Hair dryer Starline 301
Oven for plate drying, Thermocenter
Reprostar 3
TLC Scanner 3
Twin Trough Chamber 10x10 cm
winCATS Software
Chromatogram Immersion Device III
Manufacturer
CAMAG
Mettler Toledo
CAMAG
CAMAG
CAMAG
CAMAG
Canon
Solis
Salvis
CAMAG
CAMAG
CAMAG
CAMAG
CAMAG
CAMAG
CAMAG
Serial Number
Preproduction model
1114402254
120424
120425
061104
090119
070705
041118
Version 1.3.3
-
16
2.2 Methods
2.2.1 Sample preparation and standard preparation
In a first step the samples and standards were prepared as described in the European
Pharmacopoeia to test the already existing analysis methods. Then the applied volumes were
adapted to use for HPTLC and the dissolutions of the samples and standards were adapted
accordingly. The complete listing of the preparation of all samples and standards is shown in
table 22 and table 23 in the annex.
2.2.3 Derivatization reagents and application
Table 6 Derivatization reagents
Derivatization
reagent
Anisaldehyde
reagent R
[1]
Preparation
0.5 ml anisaldehyde R,
10 ml acetic acid 99% R,
85 ml methanol R and 10
ml sulphuric acid R are
mixed in chronological
order.
Methyl 40.25 methyl 4acetylbenzoate
acetylbenzoate reagent R
reagent R
are solved in a mixture of
[1]
5 ml sulphuric acid R and
85 ml cooled methanol R.
Freshly prepared 200g/l
Phosphomolybdic
solution of
acid R
Phosphomolybdic acid R
[1]
in ethanol R96%
Vanillin reagent R
2 ml sulphuric acid R are
[1]
added slowly and
carefully to 100 ml of a
solution of Vanillin R
(10g/l) in ethanol R 96%
17 ml cold water, 2 ml
Sulphuric acid
sulphuric acid (95-97%),
reagent for dipping
5 ml methanol
[7]
17 ml acetic anhydride, 1
Sulphuric acid
reagent for spraying ml sulphuric acid (9597%)
[7]
Mangan (II)-chloride 100 mg mangan (II)chloride, 15 ml water, 15
sulphuric acid
ml methanol, 1 ml
reagent
[9]
sulphuric acid
Sulphuric acid in
10 ml sulphuric acid, 90
methanol, 10%
ml methanol
Application
Spraying;
heating 5-10 minutes at 100-105°C
Spraying;
heating 10 minutes at 100-105°C
A Spraying;
heating 15 minutes at 150°C
B Spraying;
heating 10 minutes at 100°C
Spraying;
Heating 10 minutes at 100-105°C
Spraying;
Heating 5 minutes at 100°C
Spraying;
Spraying;
Dipping;
Anisaldehyde reagent R was not only sprayed; but also used as dipping solution. Dipping was
done with the CAMAG Chromatogram Immersion Device III, speed 5, time 0.
17
3 Results
The chromatograms were derivatized with anisaldehyde reagent R and detected after
derivatization in white light and with 254 nm UV light if nothing different is indicated. The
shown figures are cut-outs of the chromatograms from the start position to the solvent front.
3.1 Current situation: Pharmacopoeia
To obtain an impression of the already existing analytical methods for essential oils, those
listed in the European Pharmacopoeia were performed exactly as described in chapter 2.2.27
of the European Pharmacopoeia 5.2 and in the corresponding monographs. The received
results were compared to the schemes or the descriptions written in the European
Pharmacopoeia.
a)
b)
c)
Figure 3 Methods of the European Pharmacopoeia 5.0
a) White light after derivatization; Track 1: Eugenol R2002-01, Trans-cinnamic aldehyde R2006-01 (in
order of increasing RF-value); Track 2: Cassia oil S1941-01; Track 3: Cinnamon leaf oil S1943-01; Track
4: Cinnamon bark oil S1942-01; Track 5: Linalol R1989-04, Eugenol R2002-02; β-Caryophyllene R199601; (in order of increasing RF-value)
b) 366 nm, left before derivatization, right after derivatization; Track 1: Bergaptene R2005-01, Linalol
R1989-04, Methyl anthranilate R2004-01, Linalyl acetate R1994-03 (in order of increasing RF-value);
Track 2: Orange oil, bitter S1969-01; Track 3: Orange oil sweet S1970-01; Track 4: Bergaptene R2005-02,
Linalol R1989-04, Linalyl acetate R1994-03 (in order of increasing RF-value)
c) White light after derivatization, left single development, right double development; Track 1: Linalol
R1989-01, Linalyl acetate R1994-01 (in order of increasing RF-value); Track 2: Lavender oil S1963-01
As displayed in figure 3 a) track 1, the eugenol zone was not violet like described in the
European Pharmacopoeia, but more brown. There is no corresponding eugenol zone on track
2 where the cassia oil was applied; but other weak zones are visible as described in the
European Pharmacopoeia. The trans-cinnamic aldehyde zone on track 2 is too large and
therefore blurred.
Also on track 3 and 4, cinnamon leaf oil and cinnamon bark oil, the applied volume was too
large. The linalol zone is not visible on track 3; on track 4 it is more violet than blue as
described in the European Pharmacopoeia.
18
In figure 3 b) is exposed on the left side the chromatogram obtained doing the method
prescribed in the European Pharmacopoeia at 366 nm: The methyl anthranilate zone on track
1 has no corresponding zone on track 2 where the bitter orange oil was applied. Because the
chromatogram obtained with the test solution shows also a band corresponding to that due to
bergaptene, it is probably not a flower but bitter-orange peel oil. The sweet orange oil on track
3 has no corresponding zone to the intense blue fluorescent bergaptene zone on the
chromatogram obtained with the test solution on track 4.
On the right side is shown the plate after derivatization at 366 nm: The linalol and the linalyl
acetate zone of the chromatograms obtained with the test solutions are both more pink than
brownish-orange as described in the European Pharmacopoeia. The zones on track 2 and 3
corresponding to the linalyl acetate zone are redder. The separation of both oil is satisfiable;
bitter orange oil and sweet orange oil are unequivocal distinguishable.
As shown in figure 3 c) the linalyl acetate zone of the reference solution (track 1, upper zone)
and the linalyl acetate zone of the test solution (track 2) were on the same position, not as
schematically described in the European Pharmacopoeia. There a double development is
prescribed. In the mentioned figure is displayed on the left side a single developed plate and
on the right side a double developed plate. Even the better separation on the double developed
plate is well visibly, the double development needed a lot of time; but it is not necessary for
the separation of the two main components, linalol and linalyl acetate.
The methods of the European Pharmacopoeia 5.0 are all well suitable to analyse the
correspondent essential oils. But certain problems may arise: overload, non-conformance of
colours or description. If the amount of essential oil or reference solution leads to an overload
of the plate, the zones are blurred and the separation is diminished. The obtained results were
not on every case congruent with the descriptions of the European Pharmacopoeia. Deviations
from colour and sometimes even positions were found. Also the description of a
chromatogram in form of a text is not functional; a scheme, as written in newer monographs
is more useful.
19
3.2 Specifying of unmodified parameters
To get comparable results only one parameter at a time was changed. All other parameters
were maintained as described in table 7, apart of the tests where those parameters were
investigated single.
Table 7 Unmodified parameters
Stationary phase
Chamber
Amount of mobile phase
Saturation
Fist application position X
Application position Y
Band length
Solvent front position
Plate drying after development
Derivatization
Derivatization reagent
Detection
HPTLC Silica gel F254
Twin trough chamber (TTC)
TTC 10x10: 5 ml per trough
TTC 20x10: 10 ml per trough
20 minutes with filter paper
15 mm
8 mm
8 mm
70 mm
5 minutes, cold
Dipping:
speed 5, time 0
Heating 5 minutes at
100°C
Spraying:
1-3 minutes
Anisaldehyde reagent R
White light, 366 nm
The unmodified parameters listed in the table above were taken from the HPTLC SOP of
CAMAG, which bases on long-standing experiences. The plate drying after development and
the advantages of dipping instead of spraying were investigated in line of this diploma thesis,
see chapter 3.3.2 and 3.3.3.
The dilution of samples and standards was chosen in such a way that the application of 2 µl
gave an optimal pronounced chromatogram. For some oils this was tightrope walk between
too large and blurred main zones and no longer visible weak zones. For example mint oil on
the first track in figure XY a): The blurred blue menthol zone in the lower third of the
chromatogram arose due to a test solution which was too concentrated. But reducing the
concentration of the solution would made weak zones above the menthol zone no longer
visible.
As test samples for the optimization of the derivatization were selected five different essential
oils with characteristic zones and a typical chromatogram. Mint oil and nutmeg oil both have
a strongly pronounced zone, menthol and myristicine. Citronella oil has two well visible
zones, the citronellol and the citronellal. All four zones are unequivocal identifiable.
Matricaria oil and pine needle oil have weaker zones, which acted as a kind of control; a
suitable method had to make all those zones visible.
For the optimization of the mobile phase the range of test samples was widened and some
samples were substituted. Star anise oil, sweet orange oil, tea tree oil and clove oil were
added; the Roman Matricaria oil was substituted with blue Matricaria oil, which is used more
often concerning its anti-inflammatory qualities. Star anise oil was added to observe the
anethole zone which is very volatile; sweet orange oil to have a sample of citrus oil in the
selection; tea tree oil due to its only weak visible cineole zone and clove oil to see the
difference to cinnamon oil if exists. Additionally were two standard substances applied,
menthol and guaiazulene. Menthol was selected due to its quality to develop a well visibly
zone, which is depending on the mobile phase more or less blurred. Guaiazulene was chosen
20
because it is the only standard visible in daylight without derivatization and its quality to run
direct under the solvent front. It made a first measurement of the suitability of a mobile phase
possible direct after the development.
21
3.3 Optimization of the derivatization
3.3.1 Different derivatization reagents
Different derivatization reagents found in the literature were tried.
a)
b)
c)
d)
Figure 4 Different derivatization reagents
a) Anisaldehyde reagent R; b) Sulphuric acid in methanol, 10%; c) Phosphomolybdic acid R; d) Vanillin
reagent R
a), b): Dipped; c), d): Sprayed 2 minutes
Track 1: Mint oil S2036-01; Track 2: Nutmeg oil S2035-01; Track 3: Matricaria oil S2034-01; Track 4:
Citronella oil S2033-01; Track 5: Pine needle oil S2032-01
In figure 4 are displayed some of the results received from testing different derivatization
reagents found in literature.
It was tried to get a better result with sulphuric acid in methanol (10%) by heating longer and
at higher temperature; but no significant better result as shown in figure 4 b) was obtained.
Heating longer and at higher temperature after derivatization with phosphomolybdic acid R
makes the background more yellow and the zones more brown than grey; the result is not
better than those in figure 4 c).
Derivatization with vanillin reagent R made several zones visible (figure XY c)); but they are
blurred and not as colourful as after derivatization with anisaldehyde reagent R.
As exposed in figure 4 a), derivatization with anisaldehyde reagent R gave the sharpest and
most colourful zones.
Derivatization with methyl 4-acetylbenzoate reagent R, mangan (II)-chloride sulphuric acid
reagent, sulphuric acid reagent for dipping and sulphuric acid reagent for spraying gave no
satisfactory results (without figures).
22
3.3.2 Variations of anisaldehyde reagent R
With different variations of anisaldehyde reagent R it was tried to improve the derivatization
with anisaldehyde reagent R.
a)
b)
c)
Figure 5 Variations of anisaldehyde reagent R
a) Anisaldehyde reagent R diluted with methanol, 1:1; b) Anisaldehyde reagent R, methanol, water, 1:1:2;
c) Anisaldehyde reagent R without acetic acid
a), b): Sprayed 2 minutes; c): Dipped
Derivatization with anisaldehyde reagent R diluted with methanol made visualised not as
many zones in daylight as derivatization with anisaldehyde reagent R undiluted; but at 366 nm
all zone were visible sharper and more colourful than after derivatization with anisaldehyde
reagent R undiluted. Derivatization with anisaldehyde reagent R diluted with methanol 1:2
and 1:5 (without figures) gave a still weaker result than those shown in figure 5 a).
Derivatization with anisaldehyde reagent R diluted with water 1:1 visualised only a few zones
visible; with anisaldehyde reagent R diluted with water 1:5 not a single zone was visible
(results without figures).
A better result was achieved with a mixture of one volume of anisaldehyde reagent R, two
volumes of methanol and one volume of water. The mixture of one volume of anisaldehyde
reagent R, one volume of methanol and two volumes of water also was tried; but the obtained
result (without figure) was not significantly better than those exposed in figure 5 b).
In figure 5 c) is displayed the result obtained by derivatization with anisaldehyde reagent R
prepared without acetic acid. In daylight were only a few zones visible; also at 366 nm were
not as many zones visible as after derivatization with anisaldehyde reagent R. Remarkable is
that the colours of some zones have changed totally; for example the citronellal zone on track
4 is now blue instead of red.
Derivatization with anisaldehyde reagent R still gave the best result, the zones are brighter
and more colourful than on the plates derivatized with the variations of anisaldehyde reagent
R described above.
23
3.3.3 Influence of temperature and age of anisaldehyde reagent R
There were done some tests to test the influence of temperature of anisaldehyde reagent R
while dipping and to look into the influence of age of the derivatization reagent.
a)
b)
Figure 6 Influence of temperature of anisaldehyde reagent R, freshly prepared
a) Anisaldehyde reagent R, cold; b) Anisaldehyde reagent R, room temperature
As displayed in figure 6 a) and b), the colours are brighter and the zones are more clearly
defined on the plate derivatized with cold anisaldehyde reagent R than on the plate derivatized
with anisaldehyde reagent R at room temperature.
a)
b)
Figure 7 Influence of the age of anisaldehyde reagent R, cold
a) Anisaldehyde reagent R three days old; b) Anisaldehyde reagent R seven days old
As exposed in figure 7 a), the colours are brighter and more intense on the plate derivatized
with freshly prepared cold anisaldehyde reagent R than on the plates derivatized with older
anisaldehyde reagent R, shown in figure 7 a) and b). Not definite visible was the influence of
the age of anisaldehyde reagent R at 366 nm.
So it is best to use always cold anisaldehyde reagent R directly out of the fridge and to store
the derivatization reagent in the cold and not too long, depending on the frequency of use.
Anisaldehyde reagent R is a clear and transparent liquid; it should no longer be used, after
getting a pinkish colour.
24
3.3.4 Different heating times
To look at the influence of duration of heating a plate after dipping in anisaldehyde reagent R
the same five oils were applied in three sets on a plate. After development the plate was cut
into three equal pieces, each piece was derivatized with dipping in anisaldehyde reagent R and
then heated over different times.
a)
b)
c)
Figure 8 Different heating times after dipping in anisaldehyde reagent R
a) Heated 5 minutes at 100°C; b) heated 10 minutes at 100°C; c) heated 15 minutes at 100°C
As displayed in figure 8 heating longer made some more zones visible; but also the
background became darker. Because all important zones are already visible after heating 5
minutes, it is not necessary to heat longer. Heating at higher temperature made the
background darker but not more zones visible (results without figure).
25
3.3.5 Subsequent treatment
Derivatization with anisaldehyde reagent R gave a pinkish-grey background that diminished
the brightness and the colourfulness of the zones. The colour of the background decreased
while storing the plates wrapped with aluminium foil. So it was tried whether chromatograms
become more pronounced with different ways of storing.
The same five oils were applied in three sets on a plate. After derivatization the plate was cut
into three equal pieces and each piece was stored differently.
a)
b)
c)
d)
Figure 9 Subsequent treatment
a) Anisaldehyde reagent R, detected direct after derivatization; b) stored 120 minutes wrapped in
aluminium foil; c) stored 120 minutes covered with a glass plate and wrapped in aluminium foil; d) stored
120 minutes covered with a glass plate
Track 1: Nutmeg oil S2035-02; Track 2: Matricaria oil S2072-03; Track 3: Orange oil, sweet S1970-02;
Track 4: Mint oil S2036-03; Track 5: Pine needle oil S2032-02; Track 6: Nutmeg oil S2035-02; Track 7:
Matricaria oil S2072-03; Track 8: Orange oil, sweet S1970-02; Track 9: Mint oil S2036-03; Track 10: Pine
needle oil S2032-02; Track 11: Nutmeg oil S2035-02; Track 12: Matricaria oil S2072-03; Track 13:
Orange oil, sweet S1970-02; Track 14: Mint oil S2036-03; Track 15: Pine needle oil S2032-02
The pinkish-grey background obtained by derivatization with anisaldehyde reagent R,
exposed in figure 9 a), was clearer and less pinkish after the three different after treatments, as
shown in figures 9 b)-d). But also the colours have faded, they have lost brightness and
previously weak zones were no longer visible.
It was decided to capture the obtained results immediately after derivatization and not to
perform any subsequent treatment.
26
3.4 Optimization of the mobile phase
To get comparable results only one parameter at a time was changed, here the mobile phase.
All other parameters were abided as described in table 7. All plates were derivatized with
dipping in anisaldehyde reagent R.
A mixture of ethyl acetate and toluene is used in 19 of 24 monographs described in the
European Pharmacopoeia 5.2 and it is suitable for all tested essential oils. Nevertheless it was
evaluated, whether the result can be improved when using other mobile phases.
3.4.1 Mobile phases found in literature
In literature different mobile phases, pure or mixtures were found. All mobile phases
containing benzene or chlorinated components were not tested because they are not suitable as
mobile phase of a standard method concerning their cancerogenic effects and the problematic
disposal of chlorinated solvents.
The results of testing the mobile phases found in literature are shown in figure 10 a)-d).
a)
b)
c)
d)
Figure 10 Mobile phases found in literature a) Ethyl acetate [11]; b) Cyclohexane-ethyl acetate, 90:10 [10];
c) Diisopropyl ether-acetone, 75:25 [7]; d) Cyclohexane [7]
Track 1: Matricaria oil S2072-03; Track 2: Star anise oil S2088-03; Track 3: Citronella oil S2033-02;
Track 4: Sweet orange oil S1970-02; Track 5: Tea tree oil S1984-02; Track 6: Cinnamon leaf oil S2068-02;
Track 7: Clove oil S1949-01; Track 8: Menthol R2000-02, guaiazulene R1986-02
The best result was obtained with cyclohexane-ethyl acetate 90:10 as mobile phase, displayed
in figure 10 b).
27
3.4.2 Method development
Additionally a method development following the CAMAG-optimization scheme exposed in
figure 1 and described in chapter 1.3.2 was performed. The results are shown in figure 11.
Cyclohexane
Heptane
Diisopropyl ether
Cyclohexaneethyl acetate
90:10
Heptane-isopropyl
alcohol 95:5
Heptane-ethyl
acetate 95:5
Toluenediisopropyl etherethyl acetateformic acid
80:10:10 :10
Isopropyl alcohol
Acetone
Ethyl acetate
Toluene
Toluenediisopropyl ether
95:5
Toluene-isopropyl
alcohol 95:5
Toluene-acetone
95:5
Toluene-ethyl
acetate 95:5
Toluenediisopropyl etherethyl acetate
80:10:10
Figure 11 Method development
Track 1: Matricaria oil S2072-03; Track 2: Star anise oil S2088-03; Track 3: Citronella oil S2033-02;
Track 4: Sweet orange oil S1970-02; Track 5: Tea tree oil S1984-02; Track 6: Cinnamon leaf oil S2068-02;
Track 7: Clove oil S1949-01; Track 8: Menthol R2000-02, Guaiazulene R1986-02
With toluene-diisopropyl ether 95:5 as mobile phase a better separation of cinnamon leaf oil
and clove oil was possible than with toluene-ethyl acetate 95:5 as mobile phase.
With a mixture of toluene-diisopropyl ether-ethyl acetate 80:10:10 as mobile phase it was
tried to unite the good characteristics of both mobile phases to upgrade the separation. The
result was a better separation of clove oil as with toluene-ethyl acetate 95:5; but the zones
were not as sharp as before. So with the addition of formic acid it was attempted to influence
the sharpness of the zones. Because the consequence of this addition was a separation getting
worse, it was decided to keep a mixture of toluene and ethyl acetate as mobile phase.
28
3.4.3 Variations of the toluene-ethyl acetate ratio
The European Pharmacopoeia uses toluene-ethyl acetate in four different mixing proportions.
With each of those four mixing proportions was developed an identically applied plate. The
obtained RF-values of the applied standards are compared in table 8.
As came out from these results the amount of ethyl acetate has an influence on the RF-values
of the applied standards. The more ethyl acetate in the mobile phase, the higher the RF-values
are.
Table 8 RF-values after development with toluene-ethyl acetate in different mixing proportions
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Anisaldehyde
Methyl anthranilate
Bergaptene
Cineole
Carvone
Menthyl acetate
Carvone
Cineole
Menthol
Citronellal
Citronellol
Guaiazulene
Bornyl acetate
Bisabolol
Anethole
Linalol
Linalyl acetate
EtOAc-Tol
5:95
0.34
0.36
0.18
0.32
0.34
0.53
0.34
0.32
0.20
0.51
0.14
0.77
0.46
0.27
0.68
0.25
0.48
EtOAc-Tol
7:93
0.37
0.41
0.24
0.36
0.38
0.55
0.38
0.36
0.22
0.55
0.18
0.75
0.51
0.34
0.68
0.28
0.53
EtOAc-Tol
10:90
0.50
0.47
0.30
0.45
0.49
0.65
0.49
0.43
0.28
0.62
0.22
0.77
0.58
0.41
0.71
0.35
0.60
EtOAc-Tol
15:85
0.50
0.54
0.38
0.51
0.55
0.69
0.55
0.51
0.37
0.67
0.28
0.79
0.65
0.52
0.73
0.41
0.68
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Toluene-ethyl acetate 95:5
Figure 12 Variations of toluene-ethyl acetate; x-coordinate: RF-value; y-coordinate: standards
29
In figure 12 the results were pictured to obtain a visual impression of the differences. Because
all standards got a higher RF-value with a higher amount of ethyl acetate in the mobile phase,
the separation was not unequivocal better with toluene-ethyl acetate 85:15 than with tolueneethyl acetate 95:5.
The RF-value of guaiazulene had differed only 0.04 units because guaiazulene runs in the
solvent front.
In 12 of 24 monographs described in the European Pharmacopoeia 5.2 toluene-ethyl acetate
95:5 is used as mobile phase, the other mixing proportions were used just twice or three times.
It was decided to use toluene-ethyl acetate 95:5 as mobile phase.
30
3.5 Reproducibility
To get comparable results only one parameter at a time was changed. The other parameters
were abided as described in table 7. All plates were derivatized with dipping in anisaldehyde
reagent R.
The reproducibility of a method is fulfilled, if the difference of the RF-values reached in this
test amounts not more than 0.02 units.
3.5.1 Influence of humidity
The humidity of the environment influences the activity of the silica gel layer. Higher
humidity means higher RF-values. This tendency was confirmed in the following tests.
The reproducibility of development at specific humidity conditions was tested. To do this test
the CAMAG Automatic Developing Chamber 2 was used. Three different salt solutions and a
molecular sieve were exerted; so the test was done at 1% humidity with the molecular sieve,
at 34% humidity using a magnesium chloride solution, at 46% humidity using a potassium
thiocyanat solution and at 68% using a sodium chloride solution. Three identical applied
plates were developed under each of those four humidity conditions. The results are shown in
table 9 and schematically in figure 13.
31
Table 9 Reproducibility of development at specific humidity conditions
RF-value
34% humidity
46% humidity
1% humidity
1
2
3
4
5
6
7
8
9
Menthol
Linalol
Bisabolol
Eugenol
Linalyl acetate
Menthyl acetate
Myristicine
Anethole
β-Caryophyllene
Test 2
Test 3
Test 1
Test 2
Test 3
Test 1
Test 2
Test 3
Test 1
Test 2
Test 3
0.18
0.22
0.26
0.35
0.47
0.50
0.56
0.67
0.79
0.18
0.22
0.26
0.34
0.46
0.50
0.55
0.67
0.80
0.18
0.22
0.26
0.35
0.47
0.50
0.56
0.67
0.79
0.19
0.23
0.28
0.36
0.48
0.52
0.57
0.67
0.78
0.19
0.23
0.28
0.36
0.48
0.52
0.57
0.68
0.78
0.19
0.23
0.28
0.36
0.48
0.52
0.57
0.68
0.78
0.20
0.24
0.30
0.36
0.50
0.54
0.59
0.68
0.78
0.20
0.24
0.30
0.36
0.49
0.53
0.58
0.67
0.77
0.20
0.23
0.30
0.35
0.49
0.53
0.58
0.67
0.78
0.23
0.27
0.34
0.40
0.54
0.59
0.62
0.69
0.77
0.23
0.27
0.34
0.39
0.53
0.57
0.61
0.70
0.78
0.23
0.27
0.34
0.40
0.54
0.58
0.61
0.70
0.78
0.80
0.80
0.70
0.70
0.60
0.60
0.50
0.50
0.40
0.40
0.30
0.30
0.20
0.20
0.10
0.10
0.00
0.00
1
2
3
4
5
Test 1
Test 2
6
7
8
9
Test 3
0.80
0.70
0.60
0.60
0.50
0.50
0.40
0.40
0.30
0.30
0.20
0.20
0.10
0.10
0.00
2
3
4
Test 1
5
Test 2
6
Test 3
2
7
8
9
c)
3
4
Test 1
0.70
1
1
a)
0.80
0.00
68% humidity
Test 1
1
2
3
Test 1
5
Test 2
4
5
Testl 2
6
7
8
9
Test 3
6
b)
7
8
9
Test 3
d)
Figure 13 Reproducibility of development at specific humidity conditions; x-coordinate: RF-value; ycoordinate: standards; a) 1%; b) 34%; c) 46%; d) 68% humidity
The requirement of reproducibility - the obtained RF-values differ not more than 0.02 units was fulfilled with all four humidity conditions.
32
In table 10 and figure 14 the results of the previous tests are summarized. The RF-values
obtained for a standard with all four humidity conditions were averaged and the mean values
were opposed.
Table 10 RF-values after development with different humidity
1% humidity
1
2
3
4
5
6
7
8
9
Menthol
Linalol
Bisabolol
Eugenol
Linalyl acetate
Menthyl acetate
Myristicine
Anethole
β-Caryophyllene
RF-value
34% humidity 46% humidity
0.18
0.22
0.26
0.35
0.47
0.50
0.56
0.67
0.79
0.19
0.23
0.28
0.36
0.48
0.52
0.57
0.67
0.78
68% humidity
0.20
0.24
0.30
0.36
0.49
0.54
0.59
0.67
0.77
0.23
0.27
0.34
0.40
0.54
0.58
0.61
0.70
0.78
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
1
1% humidity
2
3
4
34% humidity
5
6
7
46% humidity
8
9
68% humidity
Figure 14 Influence of humidity on the RF-value; x-coordinate: RF-value; y-coordinate: standards
The described tendency was confirmed; the higher the humidity is the higher are the RFvalues.
The RF-value of β-Caryophyllene differed only 0.02 units over the four tests because it runs in
the solvent front.
The following tests to investigate the reproducibility were performed with the CAMAG
Automatic Developing Chamber 2; the humidity was adjusted either with magnesium chloride
solution or with potassium thiocyanate solution.
33
3.5.2 Mobile phase
To test the reproducibility of the mixture of the mobile phase, three identical plates were
developed with the CAMAG Automatic Developing Chamber 2 under constant humidity
conditions. The adjustment of the humidity was done with magnesium chloride solution
during 10 minutes; that results in a humidity of 34%.
The mobile phase, toluene-ethyl acetate 95:5, was freshly prepared for each development. In
table 11 the obtained RF-values were opposed, in figure 15 the results of this test are shown
schematically.
Table 11 Reproducibility with a mobile phase each time freshly prepared
1
2
3
4
5
6
7
8
9
RF-value
Test 2
0.20
0.25
0.31
0.39
0.51
0.54
0.60
0.69
0.78
Test 1
0.22
0.25
0.31
0.39
0.51
0.54
0.60
0.69
0.77
Menthol
Linalol
Bisabolol
Eugenol
Linalyl acetate
Menthyl acetate
Myristicine
Anethole
β-Caryophyllene
Test 3
0.20
0.24
0.29
0.37
0.49
0.53
0.58
0.68
0.79
Difference
0.02
0.01
0.02
0.02
0.02
0.01
0.02
0.01
0.02
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
1
2
3
Test 1
4
5
Test 2
6
7
8
9
Test 3
Figure 15 Reproducibility with a mobile phase each time freshly prepared; x-coordinate: RF-value; ycoordinate: standards
The requirement of reproducibility - the obtained RF-values differ not more than 0.02 units was fulfilled. Anyhow the differences were higher than in the test where the reproducibility
with different humidity conditions was tested, compare table 10.
Therefore it is suggested to prepare a volume of developing solvent that is sufficient for one
working day or one series of tests to minimize volume errors.
34
3.5.3 Drying time
Some of the samples and standards resulted in blurred zones. In the following test the
influence of the duration of drying time was examined. The same set of samples was applied
four times on four plates. The development was done with the CAMAG Automatic
Developing Chamber 2 to have the same humidity conditions for the four plates. The
adjustment of the humidity was done with potassium thiocyanat solution during 10 minutes;
that results in a humidity of 46%.
The drying time was varied as follows: one minute, two minutes, three minutes and five
minutes as usual.
a)
b)
c)
d)
Figure 16 Different drying times
a) 1 minute; b) 2 minutes; c) 3 minutes; d) 5 minutes
Track 1 Borneol R2007-02, Bornyl acetate R1992-03; Track 2 Pine needle oil S2032-02; Track 3:
Citronellal R2009-02; Track 4: Citronella oil S2033-01; Track 5: Bisabolol R1991-01, Bornyl acetate
R1992-03, Guaiazulene R1986-02; Track 6: Matricaria oil S2072-02; Track 7: Myristicine R2077-01;
Track 8: Nutmeg oil S2035-01; Track 9: Menthol R2000-02, Cineole R2008-03, Carvone R1995-02,
Menthyl acetate R1998-01; Track 10: Mint oil S2036-02; Track 11: Linalol R1989-01, Linalyl acetate
R1994-01; Track 12: Lavender oil S2067-02; Track 13: Linalol R1989-01, Eugenol R2002-05, βCaryophyllene R1996-03; Track 14: Cinnamon leaf oil S2068-01; Track 15: Linalol R1989-01, Anethole
R1987-03; Track 16: Anise oil; (all standards are listed in order of increasing RF-value)
As shown in figure 16 the variation of drying time gave no visible difference. Those zones
having a disposition to be blurred after five minutes drying time, for example the menthol
zone, are also blurred on the plate dried only one minute.
35
3.5.4 Dipping versus spraying
To test the reproducibility of spraying the same five oils were applied in three sets on a plate.
After development with toluene-ethyl acetate 95:5 the plate was cut in three equal pieces and
each piece was sprayed for 1.5 minutes with anisaldehyde reagent R and heated 5 minutes at
100°C. For detection the three pieces were rearranged as exposed in figure 17.
Figure 17 Reproducibility of spraying with anisaldehyde reagent R
Track 1: Mint oil S2036-01; Track 2: Nutmeg oil S2035-01; Track 3: Matricaria oil S2034-01; Track 4
Spraying with anisaldehyde reagent R as derivatization gave not a reproducible result. The
background has not on every plate the same colour and the brightness and the colours of the
zones differ.
It was very difficult to spray every time the same amount equable of anisaldehyde reagent R
on the plate; so the results were not reproducible.
Therefore in a next test derivatization by spraying the anisaldehyde reagent R was compared
to do the derivatization by dipping using the CAMAG Chromatogram Immersion Device III.
Dipping
Spraying
Figure 18 Dipping versus spraying
Track 1: Mint oil S2036-01; Track 2: Nutmeg oil S2035-01; Track 3: Matricaria oil S2034-01; Track 4
As shown in figure 18, dipping makes more zones visible than spraying; also the zones are
more colourful and bright than on the sprayed plate. With equipment like the CAMAG
Chromatogram Immersion Device III, which was used in line of this diploma thesis, it is
possible to do an exact and reproducible dipping. Speed and time of dipping are adjustable.
So it was decided to use dipping (speed 5, time 0) instead of spraying to do the derivatization
with anisaldehyde reagent R.
36
3.5.5 Reproducibility of derivatization with anisaldehyde reagent R
To test the reproducibility of derivatization with anisaldehyde reagent R three different
persons did the derivatization; dipping speed 5, time 0, heating 5 minutes at 100°C.
Person 1
Person 2
Person 3
Figure 19 Reproducibility of derivatization with anisaldehyde reagent R
Track 1: Lemarome N; Track 2: Lemon oil; Track 3: Linalol, linalyl acetate; Track 4: Orange oil bitter;
Track 5: Orange oil sweet; Track 6: Bornyl acetate, borneol; Track 7: Pine needle oil; Track 8:
Citronellol, citronellal; Track 9: Citronella oil; Track 10: Linalol, linalyl acetate; Track 11: Lavender oil;
Track 12: Linalol, anethole; Track 13: Anise oil; Track 14: Star anise oil; Track 15: Bisabolol, Bornyl
acetate, Guaiazulene; Track 16: Matricaria oil; Track 17: Terpineole, Guaiazulene; Track 18: Mandarin
oil; All standards are listed in order of increasing RF-value.
As seen in figure 19 the colours and the sharpness of some zones (for example guaiazulene,
the orange zone on the top of track 15 and 17) differ. Also the pinkish-grey background has
not the same intensity on all three plates. To do this test not all parameters important for
reproducibility were abided; such as the temperature and the age of the used anisaldehyde
reagent R. This is visible on the third plate, which was not dipped as deep as the first and the
second plate, respectively the tank of the immersion device was not filled enough.
This test shows the importance of abiding all specified parameters exactly. Likewise must be
considered that the sensation of colours is from everyone apprehended differently.
Even though the colours of the zones and the background differ; all important zones to
identify an essential oil are visible and unequivocal referable.
37
3.5.6 Merck versus Macherey –Nagel
All of the tests performed as part of this diploma thesis utilized Merck HPTLC plates. To
assure the robustness of the proposed method also plates of another manufacturer were tested.
Merck
Macherey-Nagel
Figure 20 Merck versus Macherey-Nagel
Track 1: Myristicine R2077-01; Track 2: Nutmeg oil S2035-01; Track 3: Menthol R2000-02, Cineole
R2008-03, Carvone R1995-02, Menthyl acetate R1998-01; Track 4: Mint oil S2036-02; Track 5: Linalol
R1989-01, Linalyl acetate R1994-01; Track 6: Lavender oil S2067-02; Track 7: Linalol R1989-01, Eugenol
R2002-05, β-Caryophyllene R1996-03; Track 8: Cinnamon leaf oil S2068-01; Track 9: Linalol R1989-01,
Anethole R1987-03; Track 10: Anise oil; (all standards are listed in order of increasing RF-value)
In figure 20 are exposed two equal applied plates from different manufacturers, Merck and
Macherey-Nagel. At daylight the colours are the same, but not at 366 nm after derivatization.
To do an expressive statement pursued tests would be necessary.
38
3.6 Comparison of different samples of the same oil
All different samples of the same kind of oil were applied side by side for comparison. In
figure 21 are shown anise oil and star anise oil as two examples of oils of which all samples
have given a similar chromatogram.
a)
b)
Figure 21 Anise oil and star anise oil
a) Track 1: Anise oil S2291-01; Track 2: Anise oil S2069-03
b) Track 1: Star anise oil S1981-02; Track 2: Star anise oil S1982-01; Track 3: Star anise oil S1983-01;
Track 4: Star anise oil S2088-03; Track 5: Star anise oil S2162-01
a)
b)
Figure 22 Spearmint oil
a) Track 1: Spearmint oil S1979-01; Track 2: Spearmint oil 80% S1980-01; Track 3: Spearmint oil USA
S2210-01; Track 4: Spearmint oil Chinese S2211-01
b) Track 1: Juniper berry oil S1961-02; Track 2: Juniper berry oil Ph.Eur. 5.0 S2182-01; Track 3:
Juniper oil S2183-01
In figure 22 are shown examples of oils with divergent chromatograms:
a) The menthol and the menthyl acetate zone of the spearmint oil on track 3 are lesser visible
than those on track 1, 2 and 4. Reasons for this may be the origin of the oil or that the oil was
rectified in order to diminish the amount of the containing menthol.
b) The juniper oil on track 3 has two well visible zones between the caryophyllene oxide and
the β-caryophyllene zone, which are not or only weak visibly the other two tracks. There is a
pink zone in the first two tracks, which is not visible on the last track.
39
3.7 Standard method suitable for all essential oils
3.7.1 Standards assignment
Due to position and frequency in monographs of essential oils written in the European
Pharmacopoeia menthol, caryophyllene oxide, menthyl acetate and β-caryophyllene were
designated as standards for the standard method suitable for all essential oils. With those four
standards, shown in figure 23, the position of the important zones in the chromatograms of all
oils investigated in line of this diploma thesis can be described.
Figure 23 Standards for the standard method
1 β-Caryophyllene, 2 Menthyl acetate, 3 Caryophyllene oxide, 4 Menthol
3.7.2 Standard method
This method was suitable for all essential oils investigated in line of this diploma thesis.
The essential oils distilled from the different conifer woods, such as fir needle oil, mountain
pine oil, pine needle oil, silver fir needle oil, spruce needle oil or Swiss stone pine oil can be
discerned with this method as an essential oil of a conifer wood; but an unequivocal
graduation of the origin of the different conifer wood oils is not possible.
As plate material HPTLC plates silica gel 60 F 254 in the format of 10x10 or 20x10 are used.
Prewashing the plates is not necessary unless chromatography produces impurity fronts due to
contamination of the plate.
The samples and standards are applied as bands of 8 mm length by spray-on technique on 8
mm distance from the lower edge of the plate. The first and the last band are applied with a
minimum of 15 mm distance from left and right edge of the plate. A minimum of 10 mm
distance between two bands has to be abided because of the tendency of some oil components
to give blurred zones. The desired developing distance (7 cm from the lower edge of the
plate) is marked with a pencil.
The developing solvent - toluene-ethyl acetate 95:5 - is prepared in a volume that is sufficient
for one working day or one series of tests to minimize volume errors. 5 mL per trough are
sufficient for a 10x10 cm Twin Trough Chamber, 10 mL per trough for a 20x10 cm Twin
Trough Chamber.
In the rear trough a properly sized filter paper is placed, which is thoroughly wetted with the
developing solvent. The solvent volume in both troughs is equalized by tilting the chamber to
the side. The lid is closed and after a saturation time of 20 minutes, the plate is placed in the
front trough. During the development the lid has to remain closed. After development the
plate is dried 5 minutes in a stream of cold air.
40
The developed and dried plate is documented under white light, under UV 366 nm and under
254 nm.
To derivatize the plate the tank of an immersion device is charged with enough anisaldehyde
reagent R to ensure complete immersion of chromatogram. The plate is dipped with an even
movement without stopping. The back of the plate is wiped off and the excessive
derivatization reagent is evaporated in the fume hood. Then the plate is placed for 5 minutes
on a plate heater which was heated up before on 100°C.
The derivatized plate is documented under white light and under UV 366 nm.
In table 12 the dilutions of the essential oils are listed and in table 13 the preparation of the
standard solutions is exposed.
Table 12 Standard method: Dilutions of the essential oils (all oils were diluted in 1 ml toluene)
Essential oil
Anise oil
Caraway oil
Cassia oil
Cinnamon oil
Citronella oil
Clary sage oil
Clove oil
Coriander oil
Eucalyptus oil
Fennel oil
Grapefruit oil
Juniper oil
Lavender oil
Lemon oil
Dilution
solved in 1 ml toluene
50 µl
30 µl
75 µl
40 µl
5 µl
100 µl
10 µl
10 µl
20 µl
20 µl
10 µl
50 µl
25 µl
500 µl
Essential oil
Lime oil
Mandarin oil
Matricaria oil
Mint oil
Nutmeg oil
Orange oil
Peppermint oil
Rosemary oil
Sage oil
Spearmint oil
Star anise oil
Tea tree oil
Thyme oil
Turpentine oil
Dilution
solved in 1 ml toluene
100 µl
500 µl
50 µl
20 µl
50 µl
200
20 µl
50 µl
100 µl
20 µl
50 µl
20 µl
20 µl
100 µl
Table 13 Standard method: Preparation of the standard solutions
Menthol
Caryophyllene oxide
Menthyl acetate
β-Caryophyllene
2.5 mg of R2000 dissolved in 1 ml toluene
2 mg of R2224 dissolved in 1 ml toluene
5 µl of R1998 dissolved in 3 ml of toluene
20 µl of R1996 dissolved in 5 ml toluene
3.7.3 Classification of oil types
To do the classification of oil types and to get chromatograms of oils representative of their
species, all oils of the same species were mixed in equal proportions. In this way standard oils
of every variety were received. The chromatograms exposed in figure 24-31 were obtained of
this standard oils.
41
3.7.3.1 Type 1: Oils visible at 366 nm before derivatization
a)
b)
c)
d)
e)
f)
Figure 24 Standard method: Oils visible at 366 nm before derivatization
Left: 366 nm before derivatization; right: white light after derivatization
Standards: Menthol R2000-03, Caryophyllene oxide R2224-02, Menthyl acetate R1998-04, βCaryophyllene R1996-06; in order of increasing RF-value
a) Track 1: Standards; Track 2: Bitter orange oil S2285-01; b) Track 1: Standards; Track 2: Sweet
orange oil S2286-01; c) Track 1: Standards; Track 2: Mandarin oil S2288-01; d) Track 1: Standards;
Track 2: Lime oil S2289-01; e) Track 1: Standards; Track 2: Lemon oil S2290-01; f) Track 1: Standards;
Track 2: Grapefruit oil S2287-01
Table 14 Description of the chromatograms of citrus fruits under 366 nm before derivatization
Bitter orange
oil
Sweet orange
oil
Mandarin oil
Lime oil
Lemon oil
Grapefruit oil
weak blue zone
weak blue zone
two intense blue
fluorescent
zones
two intense blue
fluorescent
zones
blue zone
two blue zones
blue fluorescent
zone
blue fluorescent
zone (Start
position)
several weak
blue zones
blue fluorescent
zone (Start
position)
weak blue zone
blue zone
blue fluorescent
zone (Start
position)
blue fluorescent
zone (Start
position)
intense blue
fluorescent zone
blue fluorescent
zone (Start
position)
blue fluorescent
zone (Start
position)
As exposed in figure 24 a) and b) and table 14 sweet and bitter orange oil is well
distinguishable; sweet orange oil has only a blue fluorescent zone on the start position while
bitter orange oil has a blue fluorescent zone on the start position, one direct over the start
position and two more in the lower third of the plate.
Mandarin oil, displayed in figure 24 c) and schematically in table 14, has beside the blue
fluorescent zone on the start position another intense blue fluorescent zone in the middle of
the length of run.
At 366 nm lemon oil has a single weak zone after the start position, while lime oil, while lime
oil has several weak zones there. This difference is shown in figure 24 d) and e) and in table
14.
The zones of grapefruit oil are only at 366 nm visible; hardly in white light.
42
3.7.3.2 Type 2: Oils with one main zone
a)
b)
c)
d)
Figure 25 Standard method: Oils with one main zone (1)
Standards in order of increasing RF-value: Menthol R2000-03, Caryophyllene oxide R2224-02, Menthyl
acetate R1998-04, β-Caryophyllene R1996-06
a) Track 1: Standards; Track 2: Tea tree oil S2268-01; b) Track 1: Standards; Track 2: Thyme oil S226901; c) Track 1: Standards; Track 2: Clove oil S2270-01; d) Track 1: Standards; Track 2: Coriander oil
S2271-01
Table 15 Description of the chromatograms with one main zone (1)
Standards
pink zone (βcaryophyllene)
Tea tree oil
weak pink zone (βcaryophyllene)
Thyme oil
Clove oil
blue zone
(menthyl acetate)
orange zone
(thymol)
weak pink zone
pink zone
(caryophyllene
oxide)
blue zone
(menthol)
Start position
Coriander oil
weak grey zone
(geranyl acetate)
dark grey zone
(eugenol)
Brownish-grey
zone (terpinen-4ol)
weak brown zone
(terpineole)
weak blue zone
grey zone (linalol)
weak brown zone
two weak grey zones
Start position
Start position
Start position
Start position
Tea tree oil has beside the weak β-caryophyllene zone on the top and the greyish zone on the
start position the brownish-grey main zone of terpinen-4-ol above the menthol zone on the
track with the corresponding standards. As shown in figure 25 a) and schematically in table
15 under the main zone also a weak brown zone may be visible.
The thymol zone above the caryophyllene oxide zone is the main zone of thyme oil, exposed
in figure 25 b) and schematically in table 15. At the position of β-caryophyllene and
caryophyllene oxide are also two pink zones visible, as well as a weak blue and a weak brown
zone over and under the menthol zone.
Clove oil has a dark grey zone on beside the caryophyllene oxide zone on the first track with
the standards. As displayed in figure 25 c) and schematically in table 15 also a weak βcaryophyllene zone is visible on the solvent front.
Coriander oil presents beside the linalol zone above the menthol zone three weak grey zones,
one over the linalol and two under the linalol zone. Those results are shown in figure 25 d)
and in table 15 as schematic description.
43
a)
b)
c)
d)
a) Track 1: Standards; Track 2: Eucalyptus oil S2266-02; b) Track 1: Standards; Track 2: Caraway oil
S2272-01; c) Track 1: Standards; Track 2: Nutmeg oil S2296-01; d) Track 1: Standards; Track 2: Cassia
oil S2297-01
Standards
Eucalyptus oil
Caraway oil
Cassia oil
weak pink zone
brown zone
(myristicine)
weak zone
blue zone
(menthyl acetate)
pink zone
(caryophyllene
oxide)
Nutmeg oil
weak pink zone
grey zone (cineole)
blue zone
(menthol)
weak grey zone
Start position
Start position
brown zone
(carvone)
Start position
weak zone
violet zone
grey zone
green zone
brown zone
(terpinen-4-ol)
weak zone
some weak zones
Start position
Start position
Figure 26 a) shows the chromatogram of eucalyptus oil; well visibly are the cineole zone
direct under the pink caryophyllene oxide zone and the weak grey zone under the menthol
zone.
As displayed in figure 26 b) and table 16 caraway oil presents only one zone, the carvone
zone beside the pink caryophyllene oxide zone.
Main zone of nutmeg oil is the myristicine zone above the menthyl acetate zone. Some other
weak zone may be visible as exposed in figure 26 c) and table 16.
Cassia oil has a typical chromatogram with three nested zones beside the pink caryophyllene
oxide zone as shown in figure 26 d) and table 16. Other weak zones may be visible under the
nested zone.
44
a)
b)
c)
d)
a) Track 1: Standards; Track 2; Anise oil S2292-01; b) Track 1: Standards; Track 2: Star anise oil S229301; c) Track 1: Standards; Track 2: Fennel oil bitter S2294-01; d) Track 1: Standards; Track 2: Fennel oil
sweet S2295-01
Standards
Anise oil
violet zone
red zone (anethole)
Star anise oil
reddish zone
weak violet zone
red zone (anethole)
Fennel oil bitter
Fennel oil sweet
red zone (fenchone)
red zone (fenchone)
blue zone
(menthyl acetate)
pink zone
(caryophyllene
oxide)
weak zone
weak zone
weak zone
weak zone
blue zone
(menthol)
weak zone
weak zone
Start position
Start position
Start position
Start position
Start position
In figure 27 a) and b) as well as in table 17 were shown the chromatograms of anise oil
respectively star anise oil. The differences were only weak; the chromatogram of star anise oil
displayed additionally a weak violet zone above the red anethole zone, while the
chromatogram of anise oil showed a weak brown zone beside the caryophyllene oxide zone.
Likewise the chromatograms of bitter and sweet fennel, displayed in figure 27 c) and d) and
schematically in table 17, oil exposed narrowly no difference; after derivatization at 366 nm
was a faint zone visible under the caryophyllene oxide zone which was at 366 nm orange.
45
3.7.3.3Type 3: Two main zones
a)
b)
c)
d)
Figure 28 Standard method: Oils with two main zones
a) Track 1: Standards; Track 2: Cinnamon bark oil S2274-01; b) Track 1: Standards; Track 2: Cinnamon
leaf oil S2273-01; c) Track 1: Standards; Track 2: Citronella oil nardus S2275-01; d) Track 1: Standards;
Track 2: Citronella oil winterianus S2276-01
Table 18 Description of the chromatograms with two main zones
Standards
blue zone (menthyl
acetate)
pink zone
(caryophyllene
oxide)
Cinnamon bark oil
weak grey zone
(cinnamic aldehyde)
dark grey zone
(eugenol)
Cinnamon leaf oil
Citronella oil nardus
two weak zones
dark grey zone with
reddish core
(eugenol)
weak violet zone
Citronella oil
winterianus
blue zone (citronellal)
weak blue zone
weak brown zone
blue zone (menthol)
weak brown zone
blue zone (citronellol)
weak violet zone
blue zone (citronellol)
Start position
Start position
other weak zones
Start position
Start position
Start position
The difference between cinnamon bark and cinnamon leaf oil is unequivocal; the eugenol
zone of cinnamon leaf oil has a reddish core, as shown in figure 28 b) and table 18; while the
eugenol zone of cinnamon bark oil is continuous dark grey coloured, shown in figure 28 a).
Citronella oil is classified in trade into two types: Ceylon citronella oil, obtained from
Cymbopogon nardus Rendle, is the inferior type, while Java Type citronella oil obtained from
Cymbopogon winterianus Jowitt, is considered superior. [12]
The citronella oil described in the European Pharmacopoeia is obtained by steam distillation
from the aerial parts of Cymbopogon winterianus Jowitt. In figure 28 c), figure 28 d) and in
table 18 the differences of the two citronella types are shown.
46
3.7.3.4 Type 4: Caryophyllene oxide and β-caryophyllene zone
a)
b)
c)
d)
Figure 29 Standard method: Oils with caryophyllene oxide and β-caryophyllene zone
a) Track 1: Standards; Track 2: Turpentine oil S2282-01; b) Track 1: Standards; Track 2: Lavender oil
S2278-01; c) Track 1: Standards; Track 2: Clary sage oil S2279-01; d) Track 1: Standards; Track 2:
Juniper oil S2280-01
Table 19 Description of the chromatograms with caryophyllene oxide and β-caryophyllene zone
Standards
Turpentine oil
Lavender oil
Clary sage oil
violet zone
Juniper oil
violet zone
pink zone
(caryophyllene
oxide)
pink zone
(caryophyllene oxide)
grey zone (linalyl
acetate)
pink zone
blurry grey zone
(linalyl acetate)
pink zone
brown zone (bornyl
acetate)
pink zone
grey zone (linalol)
grey zone (linalol)
blue zone (menthol)
several weak brown
zones
several weak brown
zones
brownish-grey zone
several weak grey
zones
Start position
Start position
Start position
blue zone (menthyl
acetate)
Start position
Start position
As shown in figure 29 a) and table 19 turpentine oil has apart from the caryophyllene oxide
and the β-caryophyllene zone some weak brown zones in the lower part of the chromatogram.
The chromatograms of lavender oil and clary sage oil look similar as displayed in figure 29
b), figure 29 c) and table 19. Single difference is the linalyl acetate zone which is more blurry
and shows a tailing on the chromatogram of clary sage oil.
In figure 29 d) and table 19 is displayed that juniper oil has beside the both pink
caryophyllene oxide and β-caryophyllene zones also a weak brown zone under the menthyl
acetate zone and several weak grey or brown zones around the menthol zone.
47
3.7.3.5 Type 5: Caryophyllene oxide, β-caryophyllene and bornyl acetate
zone
a)
b)
Figure 30 Standard method: Oils with caryophyllene oxide, β-caryophyllene and bornyl acetate zone
a) Track 1: Standards; Track 2: Rosemary oil S2281-01; b) Track 1: Pine needle oil S2175-01; Track 2:
Mountain pine oil S2176-01; Track 3: Spruce needle oil S2171-01; Track 4: Fir needle oil S1958-01; Track
5: Silver fir needle oil S2173-01; Track 6: Swiss stone pine oil S2172-01
Table 20 Description of the chromatograms with caryophyllene oxide, β-caryophyllene and bornyl acetate
zone
Standards
pink zone (β-caryophyllene)
Rosemary oil
Conifer wood oil
brown zone (bornyl acetate)
pink zone (caryophyllene oxide)
grey zone
weak grey zone
brown zone (bornyl acetate)
blue zone (menthyl acetate)
blue zone (menthol)
brownish-grey zone
Start position
Start position
Start position
As shown in figure 30 a) and table 20 has rosemary oil under the pink caryophyllene oxide
zone two grey zones, the lower zone is weaker than the upper zone. Direct under the menthol
zone is a brownish-grey zone visible.
In figure 30 b) are some examples of conifer wood oils exposed. Due to the many kinds of
different conifer wood oils, it was not elaborated on in line of this diploma thesis. As already
displayed, the conifer wood oil can be identified with the method here submitted; but it is not
possible to separate the different conifer wood oils.
48
3.7.3.6 Type 6: Menthol and a menthyl acetate zone
a)
b)
c)
Figure 31 Standard method: Oils with menthol and menthyl acetate zone
a) Track 1: Standards; Track 2: Mint oil S2284-01; b) Track 1: Standards; Track 2: Peppermint oil
S2283-01; c) Track 1: Standards; Track 2: Spearmint oil S2267-01
Table 21 Description of the chromatograms with menthol and menthyl acetate zone
Standards
Mint oil
Peppermint oil
Spearmint oil
blue zone (menthyl
acetate)
blue zone (menthyl
acetate)
weak blue zone
blue zone (menthyl
acetate)
brown zone (carvone)
pink zone (caryophyllene
oxide)
oxide)
weak blue zone
blue zone (menthol)
blue zone (menthyl
acetate)
weak blue zone
oxide)
blue zone (menthol)
blue zone
blue zone (menthol)
blue zone (menthol)
Start position
Start position
Start position
Start position
As displayed in figure 31 a) and table 21 mint oil has two weak blue zones under and above
the pink caryophyllene oxide zone.
Peppermint oil is shown in figure 31 b) and also in table 21 schematically; its difference to
mint oil is the absence of the second additional zone between caryophyllene oxide and
menthol.
In figure 31 c) and table 21 is exposed spearmint oil. The menthol and the menthyl acetate
zone both have not exact the same highness as on the first track where the standard were
applied. Conspicuous is the brown carvone zone in the middle of the chromatogram at the
position of caryophyllene oxide.
49
4 Discussion
Aim of this diploma thesis was to optimize the established analysis methods of essential oils
such as there is a standard method suitable for all essential oils. With this standard method
every essential oil should be doubtlessly referable.
In a first step the already existing methods were tested and the obtained results were opposed
to the schemes respectively to the descriptions of essential oil written in the corresponding
monographs. The following problems became visible during these tests: Due to overload,
zones became blurred and the separation was diminished. Deviations of colours or even
positions were found while opposing the results to the descriptions written in the European
Pharmacopoeia.
To get comparable results only one parameter at a time was changed during the tests to
optimize the existing methods. Some parameters such as the chamber type, the amount of
mobile phase, the saturation time, the application position, the band length, the solvent front
position and the detection were left unchanged.
The other parameters such as the mobile phase, the way of derivatization and the
derivatization reagent were optimized.
Different derivatization reagents were tested; on the one hand those written in the European
Pharmacopoeia in diverse monographs of essential oils, on the other hand such found in
literature. Derivatization with anisaldehyde reagent R out of the European Pharmacopoeia
gave the best result. Thereupon variations of anisaldehyde reagent R were probed: The
composition was modified, different temperatures and ages of anisaldehyde reagent were
tested. Also on the way of derivatization was looked at; different heating times and
temperatures were tried. With a subsequent treatment it was attempted to attenuate the
background and make thereby the obtained zones sharper and more colourful; but no better
result was reached.
To optimize the mobile phase different developing solvents found in literature were probed;
additionally a method development was done. Because a mixture of toluene and ethyl acetate
gave the best separation, different toluene-ethyl acetate ratios were tested.
The best result was obtained with toluene-ethyl 95:5 as mobile phase, freshly prepared cold
anisaldehyde reagent R and heating 5 minutes at 100°C.
In a further step the optimized method was submitted several reproducibility tests. To
eliminate the influence of humidity it should be worked at certain humidity conditions. The
developing solvent should be prepared in a volume that is sufficient for one working day or
one series of tests to minimize volume errors. Although the drying time did not seem to have
influence, it is suggestive to abide a specified drying time. So confounders not shown up in
the test may be avoided. Due to the irregularities of derivatization with spraying it is
suggested to use an immersion device and therefore to do the derivatization with dipping.
Pursued tests are necessary to prove the reproducibility of derivatization with anisaldehyde
reagent R and to display the reproducibility of the compiled standard method with thin layer
chromatography plates of other manufacturers.
The essential oils were divided in six types of essential oils. With the present standard method
based on four standard substances, such as menthol, caryophyllene oxide, menthyl acetate and
β-caryophyllene the important zones of the chromatogram of every essential are describable
and every oil is unequivocal identifiable.
50
5 References
[1]
European Pharmacopoeia 5.2
[2]
Burger, A., Wachter, H., 1998. Hunnius-Pharmazeutisches Wörterbuch. 8. Auflage.
Walter de Gruyter, Berlin, New York, pp. 993-994.
[3]
Arzneimittel-Kompendium der Schweiz
[4]
http://www.who.int/medicines/library/trm/medicinalplants/vol2/097to105.pdf
[5]
http://www.naturalhealthcourses.com/Reading_Room/contraindications.htm
[6]
http://www.nutrasanus.com/caraway-seed.html
[7]
Stahl, E., York, H., 1967. Terpenderivate, ätherische Öle, Balsame und Harze. In:
Stahl, E. (Editor), Dünnschicht-Chromatographie – Ein Laboratoriumshandbuch.
Springer-Verlag Berlin, Heidelberg, New York, pp 203-253.
[8]
Snyder, L.R., 1978. Classification of the solvent properties of common liquids. Journal
of Chromatographic Science 16, 223-234.
[9]
Jork, Funk, Fischer, Wimmer.1989. Dünnschichtchromatographie, Band 11a. VCH
Verlagsgesellschaft mbH, Weinheim, pp331-334
[10]
Pachaly P., 1999. DC-Atlas Dünnschicht-Chromatograpie in der Apotheke.
Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, Kümmelöl
[11]
Pachaly P., 1999. DC-Atlas Dünnschicht-Chromatograpie in der Apotheke.
Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, Menthol
[12]
http://en.wikipedia.org/wiki/Citronella_oil
51
6 Annex
6.1 Preparation of samples and standards
Table 22 Preparation of the samples
Sample
Number
S156-01
S158-01
S170-01
S170-02
S181-01
S188-01
S403-01
S403-02
S1941-01
S1941-02
S1942-01
S1942-02
S1943-01
S1943-02
S1945-01
S1946-01
S1947-01
S1948-01
S1948-02
S1949-01
S1950-01
S1951-01
S1951-01
S1952-01
S1953-01
S1953-02
S1954-01
S1955-01
S1955-02
S1956-01
S1957-01
S1958-01
S1959-01
S1960-01
S1961-01
S1961-01
S1961-02
S1962-01
S1963-01
S1963-02
S1964-01
Sample Description
Sample Preparation
Matricaria oil, blue, DAB2001
Clary sage oil, Ph.Eur. 4
Nutmeg oil, Ph.Eur. 4
Turpentine oil
Anise Oil, Ph.Eur. 3
Cassia oil
Cassia oil
Coriander seed oil
Coriander seed oil
Corn mint oil, crude
Cumin seed oil
Eucalyptus oil
Eucalyptus oil
Eucalyptus oil
Fennel oil sweet
Fir needle oil Canada
Grapefruit oil Florida
Grapefruit oil South Africa
Juniper berry oil
Juniper berry oil
Juniper berry oil
Lavender oil Russian
Lemon oil
20 µl of S156 dissolved in 1 ml toluene
100 µl of S158 diluted to 1 ml with toluene
50 µl of S170 dissolved in 2.5 ml toluene
100 mg of S403 diluted to 1 ml with toluene
50 µl of S1941 diluted to 1 ml with acetone
10 mg of S1948 dissolved in 1 ml ethanol
100 µl of S1961 dissolved in 2.5 ml heptane
100 µl of S1961 dissolved in 2.5 ml heptane
52
S1965-01
S1965-02
S1966-01
S1967-01
S1969-01
S1969-02
S1970-01
S1970-02
S1971-01
S1972-01
S1973-01
S1974-01
S1975-01
S1976-01
S1976-02
S1977-01
S1978-01
S1979-01
S1980-01
S1981-01
S1981-02
S1982-01
S1983-01
S1984-01
S1984-02
S1985-01
S1985-02
S2032-01
S2032-02
S2033-01
S2033-02
S2034-01
S2034-02
S2035-01
S2035-02
S2036-01
S2036-02
S2036-03
S2067-01
S2067-02
S2068-01
S2068-02
S2069-01
S2069-02
S2069-03
S2072-01
Lemon oil
Lemon oil
Lemon oil Argentina
Lemon oil Argentina
Orange oil California
Orange oil, Florida
Orange oil, Navel
Orange peel oil, Brazil
Orange peel oil
Peppermint oil
Peppermint oil
Pine needle oil
Spearmint oil
Spearmint oil 80%
Star anise oil
Star anise oil
Star anise oil
Star anise seed oil
Tea tree oil
Tea tree oil
Citronella oil, winterianus,
Ph.Eur. 5.0
Citronella oil, winterianus,
Ph.Eur. 5.0
Lavender oil, Maillette, Ph.Eur.
5.0
Lavender oil, Maillette, Ph.Eur.
5.0
Matricaria oil, CT Bisabolol,
1 ml of S1965 dissolved in 1 ml toluene
20 mg of S1969 diluted to 1 ml with ethanol
200 µl of S1970 dissolved with 1 ml ethanol
200 µl of S1970 dissolved with 1 ml toluene
50 µl of 1978 diluted to 1 ml with toluene
20 µl of 1980 dissolved in 1 ml toluene
20 µl of S1984 dissolved in 1 ml heptane
20 mg of S1985 diluted to 1 ml with pentane
53
S2072-02
S2072-03
S2078-01
S2080-01
S2080-02
S2080-03
S2081-01
S2082-01
S2082-01
S2082-02
S2083-01
S2083-02
S2088-01
S2088-02
S2088-03
S2089-01
S2092-01
S2092-02
S2095-01
S2095-02
S2104-01
S2105-01
S2105-02
S2160-01
S2161-01
S2162-01
S2163-01
S2164-01
S2165-01
S2166-01
S2167-01
S2168-01
S2169-01
S2170-01
S2171-01
S2172-01
S2173-01
S2174-01
S2175-01
S2176-01
S2177-01
S2178-01
S2179-01
S2180-01
Ph.Eur. 5.1
Ph.Eur. 5.1
Ph.Eur. 5.1
Caraway oil
Caraway oil
Caraway oil
Coriander oil
Cassiaöl Ph.Eur. 5.0
Cassia oil Ph.Eur. 5.0
Cassia oil, Ph.Eur. 5.0
Grapefruit oil, Israel
Lime oil, distilled
Lime oil, distilled
Mandarin oil
Mandarin oil
Orange oil, bitter
Orange oil, bitter
Fennel seed oil, sweet
Fennel oil, bitter
Star anise oil
Caraway oil
Cinnamon oil
Citronella oil, nardus
Citronella oil
Clove oil
Clove flower oil, Ph.Eur. 5.0
Clove flower oil
Mountain pine oil, Tirol,
Ph.Helv.9
Spruce needle oil, Siberian, DAB
2002
Swiss stone pine oil
Silver fir needle oil
Spruce needle oil Mariana
Pine needle oil
Mountain pine oil
Coriander oil
Eucalyptus oil, radiata
Eucalyptus oil, globulus, Ph.Eur.
5.0
Eucalyptus oil, citriodora
54
S2181-01
S2182-01
S2183-01
S2184-01
S2185-01
S2186-01
S2187-01
S2188-01
S2189-01
S2190-01
S2191-01
S2192-01
S2193-01
S2194-01
S2195-01
S2196-01
S2197-01
S2198-01
S2199-01
S2200-01
S2201-01
S2202-01
S2203-01
S2204-01
S2205-01
S2206-01
S2207-01
S2208-01
S2209-01
S2210-01
S2211-01
S2212-01
S2213-01
S2214-01
S2215-01
S2216-01
S2217-01
S2218-01
S2219-01
S2220-01
S2265-01
S2266-01
S2266-02
S2267-01
S2268-01
Eucalyptus oil
Juniper berry oil, Ph.Eur. 5.0
Juniper oil
Lavender oil, Bulgarian
Lavender oil, France, Ph.Eur. 5.0
Lavender oil
Lemon oil, Messina extra
Lemon oil
Lime oil, squeezed cold
Lime oil, squeezed cold, stabilised
Matricaria oil
Mint oil, rectified, China
Mint oil, Nagaoka, Ph.Eur. 5.2
Nutmeg flower oil
Orange oil, blood, Messina
Orange oil, bitter, South America
Orange oil, bitter, Guinea
Orange oil, sweet, Florida
Orange oil, sweet, Messina,
Ph.Eur. 5.0
Orange peel oil, sweet
Peppermint oil, USA, Ph.Eur.5.0
Peppermint oil, France, Ph.Eur.
5.0
Peppermint oil
Rosemary oil, CT Campher,
Ph.Eur. 5.0
Rosemary oil, CT Cineol
Rosemary oil
Sage oil, Spanish
Sage oil, officinalis, Ph.Helv. 9
Sage oil
Spearmint oil, USA
Spearmint oil, Chinese
Mandarin oil, redistilled, squeezed
cold
Tea tree oil, Ph.Eur. 5.0
Tea tree oil
Thyme oil, vulgaris, Switzerland
Thyme oil, zygis, Ph.Eur. 4.7
Thyme oil
Turpentine oil, Ph.Eur.5.0
Turpentine oil
Eucalyptus oil, globulus, Ph.Eur.
4
Eucalyptus oil, Standard
Eucalyptus oil, Standard
Spearmint oil, Standard
Tea tree oil, Standard
20 mg of S1969 diluted to 1 ml with ethanol
55
S2269-01
S2270-01
S2271-01
S2272-01
S2273-01
S2274-01
S2275-01
S2276-01
S2277-01
S2278-01
S2279-01
S2280-01
S2281-01
S2282-01
S2283-01
S2284-01
S2285-01
S2286-01
S2287-01
S2288-01
S2289-01
S2290-01
S2291-01
S2292-01
S2293-01
S2294-01
S2295-01
S2296-01
S2297-01
S2310-01
Thyme oil, Standard
Clove oil, Standard
Coriander oil, Standard
Caraway oil, Standard
Cinnamon leaf oil, Standard
Cinnamon bark oil, Standard
Citronella oil, nardus/Ceylon,
Standard
Citronella oil, winterianus/Java,
Standard
Matricaria oil, Standard
Lavender oil, Standard
Clary sage oil, Standard
Juniper oil, Standard
Rosemary oil, Standard
Turpentine oil, Standard
Peppermint oil, Standard
Mint oil, Standard
Orange oil, bitter, Standard
Orange oil, sweet, Standard
Grapefruit oil, Standard
Mandarin oil, Standard
Lime oil, squeezed, Standard
Lemon oil, Standard
Anise oil, Standard
Star anise oil, Standard
Fennel oil, bitter, Standard
Fennel oil, sweet, Standard
Nutmeg oil, Standard
Cassia oil, Standard
Table 23 Preparation of the standard solutions
Standard
Number
R1986-01
R1986-02
R1986-03
R1987-01
R1987-02
Standard Description
Standard Preparation
Guaiazulene
Guaiazulene
Guaiazulene
Anethole
Anethole
R1987-03
R1988-01
R1988-02
R1989-01
R1989-02
R1989-03
Anethole
Fenchone
Fenchone
Linalol
Linalol
Linalol
3 mg of R1986 dissolved in 1.5 ml toluene
16 mg of R1987 dissolved 1 ml toluene
200 µl of R1987 diluted to 15 ml with
toluene; 1 ml of this solution diluted to 5 ml
with toluene
10 µl of R1987 dissolved 1.5 ml toluene
5 µl of R1988 dissolved in 2.5 ml toluene
6 µl of R1989 diluted to 1 ml with toluene
4 µl of R1989 diluted to 1 ml with pentane
56
R1989-04
R1989-05
R1989-06
Linalol
Linalol
Linalol
R1989-07
R1990-01
R1991-01
R1992-01
R1992-02
R1992-03
R1992-04
R1993-01
R1994-01
R1994-02
R1994-03
R1994-04
R1995-01
R1995-02
R1995-03
R1996-01
R1996-02
R1996-03
R1996-04
R1996-05
R1996-06
R1996-07
R1997-01
R1997-02
R1997-03
R1998-01
R1998-02
R1998-03
R1998-04
R1998-05
R1999-01
R2000-01
R2000-02
R2000-03
R2000-04
R2001-01
R2001-02
R2002-01
R2002-02
R2002-03
R2002-04
R2002-05
R2002-06
Linalol
Geranyl acetate
Bisabolol
Bornyl acetate
Bornyl acetate
Bornyl acetate
Bornyl acetate
Terpinen-4-ol
Linalyl acetate
Linalyl acetate
Linalyl acetate
Linalyl acetate
Carvone
Carvone
Carvone
β-Caryophyllene
β-Caryophyllene
β-Caryophyllene
β-Caryophyllene
β-Caryophyllene
β-Caryophyllene
β-Caryophyllene
Thymol
Thymol
Thymol
Menthyl acetate
Menthyl acetate
Menthyl acetate
Menthyl acetate
Menthyl acetate
Carvacrol
Menthol
Menthol
Menthol
Menthol
β-Pinene
β-Pinene
Eugenol
Eugenol
Eugenol
Eugenol
Eugenol
Eugenol
5 µl of R1989 diluted to 5 ml ethanol
5 µl of R1989 diluted to 5 ml toluene
10 µl of R1989 diluted to 15 ml with toluene;
1 ml of this solution diluted to 5 ml with
toluene
1.5 µl of R1991 dissolved in 1.5 ml toluene
5 mg of R1992 diluted to 1 ml with toluene
4 µl of R1993 dissolved in 5 ml heptane
5 µl of R1996 diluted to 5 ml with ethanol
5 µl of R1999 diluted to 5 ml with pentane
7.5 mg of R2000 dissolved in 1.5 ml toluene
5 µl of R2002 diluted to 5 ml with acetone
57
R2003-01
R2003-02
R2003-03
R2003-04
R2004-01
R2004-02
R2005-01
R2005-02
R2005-03
R2005-04
R2006-01
R2006-02
R2006-03
R2006-04
R2006-05
R2007-01
R2007-02
R2007-03
R2008-01
R2008-02
R2008-03
R2008-04
R2008-05
R2009-01
R2009-02
R2010-01
Coumarin
Coumarin
Coumarin
Coumarin
Methyl anthranilate
Methyl anthranilate
Bergaptene
Bergaptene
Bergaptene
Bergaptene
Cinnamic aldehyde
Cinnamic aldehyde
Cinnamic aldehyde
Cinnamic aldehyde
Cinnamic aldehyde
Borneol
Borneol
Borneol
Cineole
Cineole
Cineole
Cineole
Cineole
Citronellal
Citronellal
Anisaldehyde
R2010-02
R2010-03
R2076-01
R2077-01
R2079-01
R2090-01
R2091-01
R2093-01
R2093-02
R2094-01
R2221-01
R2222-01
R2223-01
R2224-01
R2224-02
R2224-03
Anisaldehyde
Anisaldehyde
Citronellol
Myristicine
Carveol (+) 97%
Citral nat.
Lemarome N
Terpineol perfume
Terpineol perfume
Methyl N-Methyl anthranilate
Anethol (trans-) 99%
Fenchone (+)Fenchone (1R)-(-)Caryophyllene oxide
Caryophyllene oxide
Caryophyllene oxide
5 mg of R2003 diluted to 1 ml with acetone
5 µl of R2004 diluted to 10 ml ethanol
1 mg of R2005 diluted to 1 ml with ethanol
1 mg of R2005 diluted to 5 ml with ethanol
1 mg of R2005 dissolved in 1 ml ethanol
5 µl of R2006 diluted to 1 ml with acetone
15 µl of R2008 dissolved in 5 ml heptane
5 µl of R2009 dissolved in 2.5 ml ethanol
30 µl of R2010 diluted to 15 ml with toluene;
1 ml of this solution diluted to 5 ml with
toluene
10 µl of R2221 dissolved 1.5 ml toluene
58
6.2 List of tables
Table 1 Samples ....................................................................................................................... 10
Table 2 Standards ..................................................................................................................... 14
Table 3 Thin layer chromatography plates............................................................................... 15
Table 4 Chemicals used............................................................................................................ 15
Table 5 Equipment and accessories ......................................................................................... 16
Table 6 Derivatization reagents................................................................................................ 17
Table 7 Unmodified parameters............................................................................................... 20
Table 8 RF-values after development with toluene-ethyl acetate in different mixing
proportions ....................................................................................................................... 29
Table 9 Reproducibility of development at specific humidity conditions ............................... 32
Table 10 RF-values after development with different humidity ............................................... 33
Table 11 Reproducibility with a mobile phase each time freshly prepared ............................. 34
Table 12 Standard method: Dilutions of the essential oils (all oils were diluted in 1 ml
toluene)............................................................................................................................. 41
Table 13 Standard method: Preparation of the standard solutions........................................... 41
Table 14 Description of the chromatograms of citrus fruits under 366 nm before derivatization
.......................................................................................................................................... 42
Table 15 Description of the chromatograms with one main zone (1) ...................................... 43
Table 18 Description of the chromatograms with two main zones.......................................... 46
Table 19 Description of the chromatograms with caryophyllene oxide and β-caryophyllene
zone .................................................................................................................................. 47
Table 20 Description of the chromatograms with caryophyllene oxide, β-caryophyllene and
bornyl acetate zone........................................................................................................... 48
Table 21 Description of the chromatograms with menthol and menthyl acetate zone ............ 49
Table 22 Preparation of the samples ........................................................................................ 52
Table 23 Preparation of the standard solutions ........................................................................ 56
59
6.3 List of Figures
Figure 1 The CAMAG-optimization scheme............................................................................. 8
Figure 2 CAMAG ADC 2 .......................................................................................................... 9
Figure 3 Methods of the European Pharmacopoeia 5.0 ........................................................... 18
Figure 4 Different derivatization reagents ............................................................................... 22
Figure 5 Variations of anisaldehyde reagent R ........................................................................ 23
Figure 6 Influence of temperature of anisaldehyde reagent R, freshly prepared ..................... 24
Figure 7 Influence of the age of anisaldehyde reagent R, cold ................................................ 24
Figure 8 Different heating times after dipping in anisaldehyde reagent R............................... 25
Figure 9 Subsequent treatment................................................................................................. 26
Figure 10 Mobile phases found in literature a) Ethyl acetate [11]; b) Cyclohexane-ethyl
acetate, 90:10 [10]; c) Diisopropyl ether-acetone, 75:25 [7]; d) Cyclohexane [7] .......... 27
Figure 11 Method development ............................................................................................... 28
Figure 12 Variations of toluene-ethyl acetate; x-coordinate: RF-value; y-coordinate: standards
.......................................................................................................................................... 29
Figure 13 Reproducibility of development at specific humidity conditions; x-coordinate: RFvalue; y-coordinate: standards; a) 1%; b) 34%; c) 46%; d) 68% humidity...................... 32
Figure 14 Influence of humidity on the RF-value; x-coordinate: RF-value; y-coordinate:
standards........................................................................................................................... 33
Figure 15 Reproducibility with a mobile phase each time freshly prepared; x-coordinate: RFvalue; y-coordinate: standards.......................................................................................... 34
Figure 16 Different drying times.............................................................................................. 35
Figure 17 Reproducibility of spraying with anisaldehyde reagent R ....................................... 36
Figure 18 Dipping versus spraying .......................................................................................... 36
Figure 19 Reproducibility of derivatization with anisaldehyde reagent R............................... 37
Figure 20 Merck versus Macherey-Nagel................................................................................ 38
Figure 21 Anise oil and star anise oil....................................................................................... 39
Figure 22 Spearmint oil............................................................................................................ 39
Figure 23 Standards for the standard method........................................................................... 40
Figure 24 Standard method: Oils visible at 366 nm before derivatization............................... 42
Figure 25 Standard method: Oils with one main zone (1) ....................................................... 43
Figure 28 Standard method: Oils with two main zones ........................................................... 46
Figure 29 Standard method: Oils with caryophyllene oxide and β-caryophyllene zone ......... 47
Figure 30 Standard method: Oils with caryophyllene oxide, β-caryophyllene and bornyl
acetate zone ...................................................................................................................... 48
Figure 31 Standard method: Oils with menthol and menthyl acetate zone.............................. 49
60

Development of an optimised HPTLC method for analysis

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