development of an optimised hptlc method for analysis - camag

Universität Basel – Philosophisch-Naturwissenschaftliche Fakultät – Departement Pharmazeutische Wissenschaften – Institut für Pharmazeutische Biologie

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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

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May – September 2005

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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.

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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 Nevertheless the particular monographs have a touch of outdated and are no longer keeping with the times.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.

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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

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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

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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.

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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.

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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.

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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.

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2 Material and Methods

2.1 Material

2.1.1 Essential oils Table 1 Samples

Sample number Sample description Distributor Lot Nr.

S403 Anise Oil, Ph.Eur.3 Essencia 2002.10.0481 S156 Matricaria oil, blue, DAB2001 Essencia 602280 S158 Clary sage oil, PhE4 Essencia 600860 S170 Fennel seed oil, bitter Essencia - S181 Nutmeg oil, PhE4 Essencia 603580 S188 Turpentine oil Essencia 601740 S1941 Cassia oil Polarome 680086 S1941 Cassia oil Polarome 680086 S1942 Cinnamon bark oil, Ceylon Polarome 311709 S1943 Cinnamon leaf oil, BLCH Polarome 305904 S1944 Citronella oil Polarome 311912 S1945 Citronella oil, Java Polarome 311589 S1946 Citronella oil, Java Polarome 311277 S1947 Citronella oil, Ceylon Polarome 311801 S1948 Citronella oil, Ceylon Polarome 311912 S1949 Clove leaf oil, crude Polarome 311830 S1950 Clove leaf oil, crude Polarome 311899 S1951 Coriander seed oil Polarome 311942 S1952 Corn mint oil, crude Polarome 311637 S1953 Corn mint oil, redistilled Polarome 311931 S1954 Cumin seed oil Polarome 311764 S1955 Eucalyptus oil Polarome 312654 S1956 Eucalyptus oil Polarome 312653 S1957 Fennel oil, sweet Polarome 311913 S1958 Fir needle oil, Canada Polarome 311514 S1959 Grapefruit oil, Florida Polarome 312630 S1960 Grapefruit oil, South Africa Polarome 312631 S1961 Juniper berry oil Polarome 311606 S1962 Lavender oil, Russian Polarome 311729 S1963 Lavender oil, traditional Polarome 311856 S1964 Lemon oil Polarome 680161 S1965 Lemon oil Polarome 680157 S1966 Lemon oil, Argentina Polarome 312568 S1967 Lemon oil, Argentina Polarome 311435 S1969 Orange oil, bitter, Coast ivory Polarome 311091 S1970 Orange oil, California Polarome 680091 S1971 Orange oil, California Polarome 312490 S1972 Orange oil, Florida Polarome 680071 S1973 Orange oil, Navel Polarome 312680 S1974 Orange peel oil, Brazil Polarome 680106

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S1975 Orange peel oil Polarome 680109 S1976 Peppermint oil Polarome 311748 S1977 Pine needle oil Polarome 312673 S1978 Rosemary oil, Tunisian Polarome 305637 S1979 Spearmint oil Polarome 312706 S1980 Spearmint oil, 80% Polarome 311379 S1981 Star anis oil Polarome 311876 S1982 Star anise oil Polarome 311936 S1983 Star anise seed oil Polarome 312686 S1984 Tea tree oil Polarome 360145 S1985 Thyme red oil, Spain Polarome 305646 S2032 Pine needle oil, DAB 2004 Essencia 60-4180-0 S2033 Citronella oil, winterianus, Ph.Eur. 5.0 Essencia 60-0290-0 S2034 Matricaria oil, Roman Essencia 60-1180-0 S2035 Nutmeg oil, Ph.Eur. 5.0 Essencia 60-3580-0 S2036 Mint oil, Indian, Ph.Eur. 5.2 Essencia 60-3850-0 S2067 Lavender oil, Maillette, Ph.Eur. 5.0 Essencia 60-0550-0 S2068 Cinnamon leaf oil, Ph.Eur. 5.0 Essencia 60-2260-0 S2069 Anise oil, Ph.Eur. 5.0 Essencia 60-2420-0 S2072 Matricaria oil, CT Bisabolol, Ph.Eur. 5.1 Essencia 60-2280-0 S2078 Fennel oil, bitter Essencia 60-0350-0 S2080 Caraway oil Essencia 60-2000-0 S2081 Coriander oil Essencia 60-1080-0 S2082 Cassia oil, Ph.Eur. 5.0 Essencia 60-1060-0 S2083 Cinnamon bark oil, mind. 60% Essencia 60-3280-0 S2088 Star anise oil, Ph.Eur. 5.0 Essencia 60-0240-0 S2089 Grapefruit oil, Israel Essencia 60-1940-0 S2092 Lime oil, distilled Essencia 60-1780-0 S2095 Mandarin oil Essencia 60-0120-0 S2104 Clary sage oil, Ph.Eur. 5.0 Essencia 60-0860-0 S2105 Orange oil, bitter Essencia 60-0170-0 S2160 Fennel seed oil, sweet Essencia 60-0360-0 S2161 Fennel oil, bitter Hänseler 01-4500-0 S2162 Star anise oil Hänseler 01-3450-0 S2163 Caraway oil Hänseler 01-3850-0 S2164 Cinnamon oil Hänseler 01-4150-0 S2165 Citronella oil, nardus Essencia 60-0280-0 S2166 Citronella oil Hänseler 01-4300-0 S2167 Clove oil Hänseler 01-3900-0 S2168 Clove flower oil, Ph.Eur. 5.0 Essencia 60-0610-0 S2169 Clove flower oil Essencia 60-0600-0 S2170 Mountain pine oil, Tirol, Ph.Helv.9 Essencia 60-0450-0 S2171 Spruce needle oil, Siberian, DAB 2002 Essencia 60-0430-0 S2172 Swiss stone pine oil Essencia 60-1750-0 S2173 Silver fir needle oil Essencia 60-0370-0 S2174 Spruce needle oil, Mariana Essencia 60-4180-0 S2175 Pine needle oil Hänseler 01-5375-0 S2176 Mountain pine oil Hänseler 01-5350-0 S2177 Coriander oil Hänseler 01-4350-0

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S2178 Eucalyptus oil, radiata Essencia 60-4040-0 S2179 Eucalyptus oil, globulus, Ph.Eur. 5.0 Essencia 60-2740-0 S2180 Eucalyptus oil, citriodora Essencia 60-3990-0 S2181 Eucalyptus oil Hänseler 01-4450-0 S2182 Juniper berry oil, Ph.Eur. 5.0 Essencia 60-3220-0 S2183 Juniper oil Hänseler 01-4775-0 S2184 Lavender oil, Bulgarian Essencia 60-1310-0 S2185 Lavender oil, France, Ph.Eur. 5.0 Essencia 60-0540-0 S2186 Lavender oil Hänseler 01-4850-0 S2187 Lemon oil, Messina extra Essencia 60-0040-0 S2188 Lemon oil Hänseler 01-3960-0 S2189 Lime oil, squeezed cold Essencia 60-1250-0 S2190 Lime oil, squeezed cold, stabilized Essencia 60-1430-0 S2191 Matricaria oil Hänseler 01-4925-0 S2192 Mint oil, rectified, China Essencia 60-0750-0 S2193 Mint oil, Nagaoka, Ph.Eur. 5.2 Essencia 60-0770-0 S2194 Nutmeg flower oil Essencia 60-3370-0 S2195 Orange oil, blood, Messina Essencia 60-1840-0 S2196 Orange oil, bitter, South America Essencia 60-0840-0 S2197 Orange oil, bitter, Guinea Essencia 60-0150-0 S2198 Orange oil, sweet, Florida Essencia 60-0130-0 S2199 Orange oil, sweet, Messina, Ph.Eur. 5.0 Essencia 60-0140-0 S2200 Orange peel oil, sweet Hänseler 01-3550-0 S2201 Peppermint oil, USA, Ph.Eur.5.0 Essencia 60-0690-0 S2202 Peppermint oil, French, Ph.Eur. 5.0 Essencia 60-1900-0 S2203 Peppermint oil Hänseler 01-5100-0 S2204 Rosemary oil, CT Campher, Ph.Eur. 5.0 Essencia 60-4210-0 S2205 Rosemary oil, CT Cineol Essencia 60-0790-0 S2206 Rosemary oil, Tunisian Hänseler 01-5600-0 S2207 Sage oil, Spanish Essencia 60-0830-0 S2208 Sage oil, officinalis, Ph.Helv. 9 Essencia 60-0845-0 S2209 Sage oil Hänseler 01-5750-0 S2210 Spearmint oil, USA Essencia 60-0501-0 S2211 Spearmint oil, Chinese Essencia 60-0520-0 S2212 Mandarin oil, squeezed cold Essencia 60-0090-0 S2213 Tea tree oil, Ph.Eur. 5.0 Essencia 60-1640-0 S2214 Tea tree oil Hänseler 01-4940-0 S2215 Thyme oil, vulgaris, Switzerland Essencia 60-1040-0 S2216 Thyme oil, zygis, Ph.Eur. 4.7 Essencia 60-0951-0 S2217 Thyme oil Hänseler 01-6550-0 S2218 Turpentine oil, Ph.Eur.5.0 Essencia 60-3300-0 S2219 Turpentine oil Hänseler 01-6350-0 S2220 Clary sage oil, Ph.Eur. 4.1 Essencia 600860 S2265 Eucalyptus oil, globulus, Ph.Eur. 4 Essencia 602740 S2266 Eucalyptus oil (Mix of S2178, S2179,

S1955, S1956, S2181 and S2265) Various -

S2267 Spearmint oil (Mix of S1979, S1980, S2211 and S2210)

Various -

S2268 Tea tree oil (Mix of S1984, S2214 and S2213)

Various -

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S2269 Thyme oil (Mix of S2217, S2215, S2216 and S1985)

Various -

S2270 Clove oil (Mix of S2169, S2168, S1949, S1950 and S2167)

Various -

S2271 Coriander oil (Mix of S2081, S2177 and S1951)

Various -

S2272 Caraway oil (Mix of S2080 and S2163) Various - S2273 Cinnamon leaf oil (Mix of S1943 and

S2068) Various -

S2274 Cinnamon bark oil (Mix of S1942, S2083 and S2164)

Various -

S2275 Citronella oil, nardus/Ceylon (Mix of S1947, S1948 and S2165)

Various -

S2276 Citronella oil, winterianus/Java (Mix of S2166, S1944, S1945, S1946 and S2033)

Various -

S2277 Matricaria oil (Mix of S2191 and 2072) Various - S2278 Lavender oil (Mix of S2186, S2184, S2185,

S2067, S1962 and S1963) Various -

S2279 Clary sage oil (Mix of S2104 and S2220) Various - S2280 Juniper oil (Mix of S2182, S2183 and

S1961) Various -

S2281 Rosemary oil (Mix of S2204, S2205, S1978 and S2206)

Various -

S2282 Turpentine oil (Mix of S2219 and S2218) Various - S2283 Peppermint oil (Mix of S1976, S2203,

S2202 and S2201) Various -

S2284 Mint oil (Mix of S1952, S2036, S2193, S2192 and S1953)

Various -

S2285 Orange oil, bitter (Mix of S1969, S2105, S2197 and S2196)

Various -

S2286 Orange oil, sweet (Mix of S1970, S1971, S1972, S1973, S2198, S2199, S1975, S1974 and S2200)

Various -

S2287 Grapefruit oil (Mix of S1959, S1960 and 2089)

Various -

S2288 Mandarin oil (Mix of S2095 and S2212) Various - S2289 Lime oil, squeezed (Mix of S2189 and

S2190) Various -

S2290 Lemon oil (Mix of S1964, s1966, S2187 and S2188)

Various -

S2291 Anise Oil, Ph.Eur.3 Essencia 2002.10.0481 S2292 Anise oil (Mix of S2069 and 2291) Various - S2293 Star anise oil (Mix of S1981, S1982, S1983,

2088 and 2162) Various -

S2294 Fennel oil, bitter (Mix of S2078 and S2161) Various - S2295 Fennel oil, sweet (Mix of S1957 and 2160) Various - S2296 Nutmeg oil (Mix of S2035 and S2194) Various - S2297 Cassia oil (Mix of S1941 and S2082) Various - S2310 Fennel seed oil, bitter Essencia -

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2.1.2 Standards Table 2 Standards

Standard number Standard description Distributor Lot Nr.

R1986 Guaiazulene Merck K29155433 117 R1987 Anethole Essencia 702700 R1988 Fenchone Chemika 62329/1 15001 R1989 Linalol Essencia 701460 R1990 Geranyl acetate Essencia 70-0080-0 R1991 Bisabolol Essencia 703030 R1992 Bornyl acetate Essencia 703410 R1993 Terpinen-4-ol Essencia 703800 R1994 Linalyl acetate Essencia 700100 R1995 Carvone Merck K27960616 R1996 β-Caryophyllene Essencia 2002.08.0209 R1997 Thymol Fluka 420973/1 40802 R1998 Menthyl acetate Aldrich KO 02122KO R1999 Carvacrol Essencia 70-2300-0 R2000 Menthol Fluka 360728/1 11502 R2001 β-Pinene Essencia 2002.10.0348 R2002 Eugenol Merck S2170029 114 R2003 Coumarin Merck S31222 119 R2004 Methyl anthranilate Essencia 70-0600-0 R2005 Bergaptene Fluka 425788/9 34301 R2006 trans-cinnamic aldehyde Merck 8.02505.0250 R2007 Borneol Aldrich 12408PI R2008 Cineole Aldrich 10107HG R2009 Citronellal Aldrich 05322AF R2010 Anisaldehyde Essencia 700620 R2076 Citronellol Aldrich 00829AQ R2077 Myristicine ChromDex 13935-703 R2079 Carveol (+) 97% Uni Wien - R2090 Citral nat. Essencia 70-3750-0 R2091 Lemarome N Essencia 70-1410-0 R2093 Terpineol perfume Essencia 70-2280-0 R2094 Methyl N-Methyl anthranilate Fluka 399299/1 24201 R2221 Anethol (trans-) 99% Aldrich S05492-081 R2222 Fenchone (+)- Fluka 62329/1 15001 R2223 Fenchone (1R)-(-)- Aldrich 02508HV R2224 Caryophyllene oxide Essencia 70-4100-0

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2.1.3 Thin layer chromatography plates Table 3 Thin layer chromatography plates

Plate Manufacturer Lot Nr. HPTLC-Fertigplatten, Nano-DURASL-20 UV254 Macherey-

Nagel 309252

TLC glass 10x20, Si 60 F254 Merck 840316352 HPTLC glass 10X10, Si 60 F254 Merck OB464935 HPTLC glass 10X10, Si 60 F254 Merck OB515602 HPTLC glass 20X10, Si 60 F254 Merck OB526793 HPTLC glass 20X10, Si 60 F254 Merck OB545060

2.1.4 Chemicals used Table 4 Chemicals used

Name, purity or quality Manufacturer Lot K27583063 Acetic acid Merck K29535963 130

Acetone per analysi Acros A019663101 Fluka 422315/1 53601 Anisaldehyde Roth 02569422

Cyclohexane per analysi Merck K10066566 815 Diethyl ether per analysi, stabilized with BHT Acros 0557085 Diisopropyl ether per analysi Merck K25739667 901 Ethanol 96% Merck K33957583 441 Ethyl acetate per analysi Merck K33137923415 Heptane Merck K25693579 841 Isopropyl alcohol per analysi Merck K32940634407

A019863901 Methanol per analysi Acros A0205287001

n-Heptane per analysi Merck K25693579 841 Pentane per analysi Merck K11687677 919 Phosphomolybdic acid Riedel-de-Haen 11280 Sulphuric acid Merck K28679231 101

Merck 0445584 Toluene per analysi Acros A0206963001

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2.1.5 Equipment and accessories Table 5 Equipment and accessories

Manufacturer Serial Number ADC light µP controlled CAMAG Preproduction model Analytical balance AG245 Mettler Toledo 1114402254 Automatic Development Chamber ADC 2 CAMAG 120424 Automatic Development Chamber ADC2 CAMAG 120425 Automatic TLC Sampler ATS 4 CAMAG 061104 Automatic TLC Sampler ATS 4 CAMAG 090119 Digital camera G5 Canon - Hair dryer Starline 301 Solis - Oven for plate drying, Thermocenter Salvis - Reprostar 3 CAMAG 070705 TLC Scanner 3 CAMAG 041118 Twin Trough Chamber 10x10 cm CAMAG - Twin Trough Chamber 20x10 cm CAMAG - Twin Trough Chamber 20x20 cm CAMAG - winCATS Software CAMAG Version 1.3.3 Chromatogram Immersion Device III CAMAG -

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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 Preparation Application

Anisaldehyde reagent R [1]

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.

Spraying; heating 5-10 minutes at 100-105°C

Methyl 4-acetylbenzoate reagent R [1]

0.25 methyl 4-acetylbenzoate reagent R are solved in a mixture of 5 ml sulphuric acid R and 85 ml cooled methanol R.

Spraying; heating 10 minutes at 100-105°C

A Spraying; heating 15 minutes at 150°C

Phosphomolybdic acid R [1]

Freshly prepared 200g/l solution of Phosphomolybdic acid R in ethanol R96%

B Spraying; heating 10 minutes at 100°C

Vanillin reagent R [1]

2 ml sulphuric acid R are added slowly and carefully to 100 ml of a solution of Vanillin R (10g/l) in ethanol R 96%

Spraying; Heating 10 minutes at 100-105°C

Sulphuric acid reagent for dipping [7]

17 ml cold water, 2 ml sulphuric acid (95-97%), 5 ml methanol

Spraying; Heating 5 minutes at 100°C

Sulphuric acid reagent for spraying [7]

17 ml acetic anhydride, 1 ml sulphuric acid (95-97%)


Mangan (II)-chloride sulphuric acid reagent [9]

100 mg mangan (II)-chloride, 15 ml water, 15 ml methanol, 1 ml sulphuric acid


Sulphuric acid in methanol, 10%

10 ml sulphuric acid, 90 ml methanol

Dipping; Heating 5 minutes at 100°C

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.

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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 R1996-01; (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.

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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.

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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 HPTLC Silica gel F254 Chamber Twin trough chamber (TTC)

Amount of mobile phase TTC 10x10: 5 ml per trough TTC 20x10: 10 ml per trough

Saturation 20 minutes with filter paper Fist application position X 15 mm Application position Y 8 mm Band length 8 mm Solvent front position 70 mm Plate drying after development 5 minutes, cold

Dipping: speed 5, time 0 Derivatization Spraying: 1-3 minutes

Heating 5 minutes at 100°C

Derivatization reagent Anisaldehyde reagent R Detection 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

21

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.

22

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).

23

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.

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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.

25

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).

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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.

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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).

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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

Isopropyl alcohol

Acetone

Ethyl acetate

Toluene

Cyclohexane-ethyl acetate

90:10

Heptane-isopropyl

alcohol 95:5

Heptane-ethyl acetate 95:5

Toluene-diisopropyl ether

95:5

Toluene-isopropyl alcohol 95:5

Toluene-acetone 95:5

Toluene-ethyl acetate 95:5

Toluene-diisopropyl ether-

ethyl acetate-formic acid

80:10:10 :10

Toluene-diisopropyl ether-

ethyl 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.

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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

EtOAc-Tol 5:95

EtOAc-Tol 7:93

EtOAc-Tol 10:90

EtOAc-Tol 15:85

1 Anisaldehyde 0.34 0.37 0.50 0.50 2 Methyl anthranilate 0.36 0.41 0.47 0.54 3 Bergaptene 0.18 0.24 0.30 0.38 4 Cineole 0.32 0.36 0.45 0.51 5 Carvone 0.34 0.38 0.49 0.55 6 Menthyl acetate 0.53 0.55 0.65 0.69 7 Carvone 0.34 0.38 0.49 0.55 8 Cineole 0.32 0.36 0.43 0.51 9 Menthol 0.20 0.22 0.28 0.37 10 Citronellal 0.51 0.55 0.62 0.67 11 Citronellol 0.14 0.18 0.22 0.28 12 Guaiazulene 0.77 0.75 0.77 0.79 13 Bornyl acetate 0.46 0.51 0.58 0.65 14 Bisabolol 0.27 0.34 0.41 0.52 15 Anethole 0.68 0.68 0.71 0.73 16 Linalol 0.25 0.28 0.35 0.41 17 Linalyl acetate 0.48 0.53 0.60 0.68

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0.70

0.80

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Toluene-ethyl acetate 95:5 Toluene-ethyl acetate 93:7Toluene-ethyl acetate 90:10 Toluene-ethyl acetate 85:15

Figure 12 Variations of toluene-ethyl acetate; x-coordinate: RF-value; y-coordinate: standards

30

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 toluene-ethyl 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.

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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.

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Table 9 Reproducibility of development at specific humidity conditions

RF-value 1% humidity 34% humidity 46% humidity 68% humidity

Test 1 Test 2 Test 3 Test 1 Test 2 Test 3 Test 1 Test 2 Test 3 Test 1 Test 2 Test 31 Menthol 0.18 0.18 0.18 0.19 0.19 0.19 0.20 0.20 0.20 0.23 0.23 0.23 2 Linalol 0.22 0.22 0.22 0.23 0.23 0.23 0.24 0.24 0.23 0.27 0.27 0.27 3 Bisabolol 0.26 0.26 0.26 0.28 0.28 0.28 0.30 0.30 0.30 0.34 0.34 0.34 4 Eugenol 0.35 0.34 0.35 0.36 0.36 0.36 0.36 0.36 0.35 0.40 0.39 0.40 5 Linalyl acetate 0.47 0.46 0.47 0.48 0.48 0.48 0.50 0.49 0.49 0.54 0.53 0.54 6 Menthyl acetate 0.50 0.50 0.50 0.52 0.52 0.52 0.54 0.53 0.53 0.59 0.57 0.58 7 Myristicine 0.56 0.55 0.56 0.57 0.57 0.57 0.59 0.58 0.58 0.62 0.61 0.61 8 Anethole 0.67 0.67 0.67 0.67 0.68 0.68 0.68 0.67 0.67 0.69 0.70 0.70 9 β-Caryophyllene 0.79 0.80 0.79 0.78 0.78 0.78 0.78 0.77 0.78 0.77 0.78 0.78

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1 2 3 4 5 6 7 8 9

Test 1 Test 2 Test 3 a)

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1 2 3 4 5 6 7 8 9

Test 1 Test 2 Test 3 b)

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1 2 3 4 5 6 7 8 9

Test 1 Test 2 Test 3 c)

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1 2 3 4 5 6 7 8 9

Test 1 Testl 2 Test 3 d) Figure 13 Reproducibility of development at specific humidity conditions; x-coordinate: RF-value; y-

coordinate: 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.

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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

RF-value 1% humidity 34% humidity 46% humidity 68% humidity 1 Menthol 0.18 0.19 0.20 0.23 2 Linalol 0.22 0.23 0.24 0.27 3 Bisabolol 0.26 0.28 0.30 0.34 4 Eugenol 0.35 0.36 0.36 0.40 5 Linalyl acetate 0.47 0.48 0.49 0.54 6 Menthyl acetate 0.50 0.52 0.54 0.58 7 Myristicine 0.56 0.57 0.59 0.61 8 Anethole 0.67 0.67 0.67 0.70 9 β-Caryophyllene 0.79 0.78 0.77 0.78

0.000.10

0.200.30

0.400.50

0.600.70

0.80

1 2 3 4 5 6 7 8 9

1% humidity 34% humidity 46% humidity 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 RF-values. 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.

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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

RF-value Test 1 Test 2 Test 3 Difference1 Menthol 0.22 0.20 0.20 0.02 2 Linalol 0.25 0.25 0.24 0.01 3 Bisabolol 0.31 0.31 0.29 0.02 4 Eugenol 0.39 0.39 0.37 0.02 5 Linalyl acetate 0.51 0.51 0.49 0.02 6 Menthyl acetate 0.54 0.54 0.53 0.01 7 Myristicine 0.60 0.60 0.58 0.02 8 Anethole 0.69 0.69 0.68 0.01 9 β-Caryophyllene 0.77 0.78 0.79 0.02

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0.30

0.40

0.50

0.60

0.70

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1 2 3 4 5 6 7 8 9

Test 1 Test 2 Test 3

Figure 15 Reproducibility with a mobile phase each time freshly prepared; x-coordinate: RF-value; y-coordinate: 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.

35

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, β-Caryophyllene 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.

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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 Citronella oil S2033-01; Track 5: Pine needle oil S2032-01

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 Citronella oil S2033-01; Track 5: Pine needle oil S2032-01

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.

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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.

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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.

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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.

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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.

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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)

Dilution Dilution Essential oil solved in 1 ml toluene Essential oil solved in 1 ml toluene Anise oil 50 µl Lime oil 100 µl Caraway oil 30 µl Mandarin oil 500 µl Cassia oil 75 µl Matricaria oil 50 µl Cinnamon oil 40 µl Mint oil 20 µl Citronella oil 5 µl Nutmeg oil 50 µl Clary sage oil 100 µl Orange oil 200 Clove oil 10 µl Peppermint oil 20 µl Coriander oil 10 µl Rosemary oil 50 µl Eucalyptus oil 20 µl Sage oil 100 µl Fennel oil 20 µl Spearmint oil 20 µl Grapefruit oil 10 µl Star anise oil 50 µl Juniper oil 50 µl Tea tree oil 20 µl Lavender oil 25 µl Thyme oil 20 µl Lemon oil 500 µl Turpentine oil 100 µl Table 13 Standard method: Preparation of the standard solutions

Menthol 2.5 mg of R2000 dissolved in 1 ml toluene Caryophyllene oxide 2 mg of R2224 dissolved in 1 ml toluene Menthyl acetate 5 µl of R1998 dissolved in 3 ml of toluene β-Caryophyllene 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.

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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, β-Caryophyllene 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

two blue zones blue fluorescent zone blue fluorescent zone (Start position)

blue fluorescent zone (Start position)

intense blue fluorescent zone blue fluorescent zone (Start position)

weak blue zone two intense blue fluorescent zones several weak blue zones blue fluorescent zone (Start position)

weak blue zone two intense blue fluorescent zones weak blue zone blue fluorescent zone (Start position)

blue zone blue zone 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.

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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 S2269-01; 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 Tea tree oil Thyme oil Clove oil Coriander oil pink zone (β-caryophyllene) blue zone (menthyl acetate) pink zone (caryophyllene oxide) blue zone (menthol) Start position

weak pink zone (β-caryophyllene) Brownish-grey zone (terpinen-4-ol) weak brown zone (terpineole) Start position

weak pink zone (β-caryophyllene) orange zone (thymol) weak pink zone weak blue zone weak brown zone Start position

weak pink zone (β-caryophyllene) dark grey zone (eugenol) Start position

weak grey zone (geranyl acetate) grey zone (linalol) two weak grey zones 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 β-caryophyllene 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.

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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 Nutmeg oil Cassia oil pink zone (β-caryophyllene) blue zone (menthyl acetate) pink zone (caryophyllene oxide) blue zone (menthol) Start position

grey zone (cineole) weak grey zone Start position

brown zone (carvone) Start position

weak pink zone brown zone (myristicine) weak zone weak zone brown zone (terpinen-4-ol) weak zone Start position

weak pink zone violet zone grey zone green zone some weak zones 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.

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a) b) c) d)



a) Track 1: Standards; Track 2; Anise oil S2292-01; b) Track 1: Standards; Track 2: Star anise oil S2293-01; 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 Star anise oil Fennel oil bitter Fennel oil sweet pink zone (β-caryophyllene) blue zone (menthyl acetate) pink zone (caryophyllene oxide) blue zone (menthol) Start position

violet zone red zone (anethole) weak zone weak zone weak zone Start position

reddish zone weak violet zone red zone (anethole) weak zone weak zone Start position

red zone (fenchone) Start position

red zone (fenchone) weak zone 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.

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3.7.3.3 Type 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 Cinnamon bark oil Cinnamon leaf oil Citronella oil nardus Citronella oil winterianus

pink zone (β-caryophyllene) blue zone (menthyl acetate) pink zone (caryophyllene oxide) blue zone (menthol) Start position

pink zone (β-caryophyllene) weak grey zone (cinnamic aldehyde) dark grey zone (eugenol) Start position

pink zone (β-caryophyllene) dark grey zone with reddish core (eugenol) weak violet zone other weak zones Start position

weak pink zone (β-caryophyllene) two weak zones weak brown zone weak brown zone blue zone (citronellol) Start position

weak pink zone (β-caryophyllene) blue zone (citronellal) weak blue zone weak violet zone blue zone (citronellol) 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.

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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 Juniper oil pink zone (β-caryophyllene) blue zone (menthyl acetate) pink zone (caryophyllene oxide) blue zone (menthol) Start position

pink zone (β-caryophyllene) pink zone (caryophyllene oxide) several weak brown zones Start position

pink zone (β-caryophyllene) grey zone (linalyl acetate) pink zone (caryophyllene oxide) grey zone (linalol) several weak brown zones Start position

violet zone blurry grey zone (linalyl acetate) pink zone (caryophyllene oxide) grey zone (linalol) brownish-grey zone Start position

violet zone brown zone (bornyl acetate) pink zone (caryophyllene oxide) several weak grey zones 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.

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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 Rosemary oil Conifer wood oil pink zone (β-caryophyllene) blue zone (menthyl acetate) pink zone (caryophyllene oxide) blue zone (menthol) Start position

pink zone (β-caryophyllene) brown zone (bornyl acetate) pink zone (caryophyllene oxide) grey zone weak grey zone brownish-grey zone Start position

pink zone (β-caryophyllene) brown zone (bornyl acetate) pink zone (caryophyllene oxide) 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.

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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 pink zone (β-caryophyllene) blue zone (menthyl acetate) pink zone (caryophyllene oxide) blue zone (menthol) Start position

pink zone (β-caryophyllene) blue zone (menthyl acetate) weak blue zone pink zone (caryophyllene oxide) weak blue zone blue zone (menthol) Start position

pink zone (β-caryophyllene) blue zone (menthyl acetate) weak blue zone pink zone (caryophyllene oxide) blue zone (menthol) Start position

pink zone (β-caryophyllene) blue zone (menthyl acetate) brown zone (carvone) blue zone blue zone (menthol) 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.

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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.

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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

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6 Annex

6.1 Preparation of samples and standards Table 22 Preparation of the samples

Sample Number Sample Description Sample Preparation

S156-01 Matricaria oil, blue, DAB2001 20 µl of S156 dissolved in 1 ml toluene S158-01 Clary sage oil, Ph.Eur. 4 100 µl of S158 diluted to 1 ml with toluene S170-01 Fennel seed oil, bitter 50 µl of S170 dissolved in 2.5 ml toluene S170-02 Fennel seed oil, bitter 30 µl of S170 dissolved in 1.5 ml toluene S181-01 Nutmeg oil, Ph.Eur. 4 100 µl of S181 diluted to 1 ml with toluene S188-01 Turpentine oil 100 µl of S188 diluted to 1 ml with toluene S403-01 Anise Oil, Ph.Eur. 3 100 mg of S403 diluted to 1 ml with toluene S403-02 Anise Oil, Ph.Eur. 3 50 µl of S403 dissolved in 1 ml toluene S1941-01 Cassia oil 50 µl of S1941 diluted to 1 ml with acetone S1941-02 Cassia oil 75 µl of S1941 dissolved in 1 ml toluene S1942-01 Cinnamon bark oil, Ceylon 100 µl of S1942 diluted to 1 ml with acetone S1942-02 Cinnamon bark oil, Ceylon 40 µl of R1942 dissolved in 1 ml toluene S1943-01 Cinnamon leaf oil, BLCH 100 µl of S1943 diluted to 1 ml with acetone S1943-02 Cinnamon leaf oil, BLCH 40 µl of R1943 dissolved in 1 ml toluene S1945-01 Citronella oil, Java 5 µl of S1945 dissolved in 1 ml toluene S1946-01 Citronella oil, Java 5 µl of S1946 dissolved in 1 ml toluene S1947-01 Citronella oil, Ceylon 5 µl of S1947 dissolved in 1 ml toluene S1948-01 Citronella oil, Ceylon 10 mg of S1948 dissolved in 1 ml ethanol S1948-02 Citronella oil, Ceylon 5 µl of S1948 dissolved in 1 ml toluene S1949-01 Clove leaf oil, crude 10 µl of S1949 dissolved in 1 ml toluene S1950-01 Clove leaf oil, crude 10 µl of S1950 dissolved in 1 ml toluene S1951-01 Coriander seed oil 10 µl of S1951 dissolved in 1 ml toluene S1951-01 Coriander seed oil 10 µl of S1951 dissolved in 1 ml toluene S1952-01 Corn mint oil, crude 20 µl of S1952 dissolved in 1 ml toluene S1953-01 Corn mint oil, redistilled 100 µl of S1953 dissolved in 1 ml toluene S1953-02 Corn mint oil, redistilled 20 µl of S1953 dissolved in 1 ml toluene S1954-01 Cumin seed oil 60 µl of S1954 dissolved in 1.5 ml toluene S1955-01 Eucalyptus oil 10 mg of S1955 diluted to 1 ml with toluene S1955-02 Eucalyptus oil 10 µl of S1955 dissolved in 1 ml toluene S1956-01 Eucalyptus oil 10 µl of S1956 dissolved in 1 ml toluene S1957-01 Fennel oil sweet 30 µl of S1957 dissolved in 1.5 ml toluene S1958-01 Fir needle oil Canada 135 µl of S1958 dissolved in 1.5 ml toluene S1959-01 Grapefruit oil Florida 15 µl of S1959 dissolved in 1.5 ml toluene S1960-01 Grapefruit oil South Africa 15 µl of S1960 dissolved in 1.5 ml toluene S1961-01 Juniper berry oil 100 µl of S1961 dissolved in 2.5 ml heptane S1961-01 Juniper berry oil 100 µl of S1961 dissolved in 2.5 ml heptane S1961-02 Juniper berry oil 50 µl of S1961 dissolved in 1 ml toluene S1962-01 Lavender oil Russian 40 µl of S1962 dissolved in 1.5 ml toluene S1963-01 Lavender oil, traditional 20 µl of S1963 dissolved in 1 ml toluene S1963-02 Lavender oil, traditional 40 µl of S1963 dissolved in 1.5 ml toluene S1964-01 Lemon oil 500 µl of S1964 dissolved in 1 ml toluene

53

S1965-01 Lemon oil 1 ml of S1965 dissolved in 1 ml toluene S1965-02 Lemon oil 500 µl of S1965 dissolved in 1 ml toluene S1966-01 Lemon oil Argentina 500 µl of S1966 dissolved in 1 ml toluene S1967-01 Lemon oil Argentina 500 µl of S1967 dissolved in 1 ml toluene S1969-01 Orange oil, bitter, Coast ivory 20 mg of S1969 diluted to 1 ml with ethanol S1969-02 Orange oil, bitter, Coast ivory 200 µl of S1971 dissolved in 1 ml toluene S1970-01 Orange oil, California 200 µl of S1970 dissolved with 1 ml ethanol S1970-02 Orange oil, California 200 µl of S1970 dissolved with 1 ml toluene S1971-01 Orange oil California 200 µl of S1973 dissolved in 1 ml toluene S1972-01 Orange oil, Florida 200 µl of S2198 dissolved in 1 ml toluene S1973-01 Orange oil, Navel 200 µl of S2199 dissolved in 1 ml toluene S1974-01 Orange peel oil, Brazil 200 µl of S1974 dissolved with 1 ml toluene S1975-01 Orange peel oil 200 µl of S1975 dissolved with 1 ml toluene S1976-01 Peppermint oil 5 mg of S1976 diluted to 1 ml with toluene S1976-02 Peppermint oil 20 µl of S1976 dissolved in 1 ml toluene S1977-01 Pine needle oil 135 µl of S1977 dissolved in 1.5 ml toluene S1978-01 Rosemary oil, Tunisian 50 µl of 1978 diluted to 1 ml with toluene S1979-01 Spearmint oil 20 µl of S1979 dissolved in 1 ml toluene S1980-01 Spearmint oil 80% 20 µl of 1980 dissolved in 1 ml toluene S1981-01 Star anise oil 100 mg of S1981 diluted to 1 ml with toluene S1981-02 Star anise oil 50 µl of S1982 dissolved in 1 ml toluene S1982-01 Star anise oil 50 µl of S2162 dissolved in 1 ml toluene S1983-01 Star anise seed oil 50 µl of S1983 dissolved in 1 ml toluene S1984-01 Tea tree oil 20 µl of S1984 dissolved in 1 ml heptane S1984-02 Tea tree oil 20 µl of S1984 dissolved in 1 ml toluene S1985-01 Thyme red oil, Spain 20 mg of S1985 diluted to 1 ml with pentane S1985-02 Thyme red oil, Spain 20 µl of S1985 dissolved in 1 ml toluene S2032-01 Pine needle oil, DAB 2004 45 µl of S2032 dissolved in 1.5 ml toluene S2032-02 Pine needle oil, DAB 2004 135 µl of S2032 dissolved in 1.5 ml toluene

S2033-01 Citronella oil, winterianus, Ph.Eur. 5.0 15 µl of S2033 dissolved in 1.5 ml toluene

S2033-02 Citronella oil, winterianus, Ph.Eur. 5.0 5 µl of S2033 dissolved in 1 ml toluene

S2034-01 Matricaria oil, Roman 150 µl of S2034 dissolved in 1.5 ml toluene S2034-02 Matricaria oil, Roman 50 µl of S2034 dissolved in 1 ml toluene S2035-01 Nutmeg oil, Ph.Eur. 5.0 150 µl of S2035 dissolved in 1.5 ml toluene S2035-02 Nutmeg oil, Ph.Eur. 5.0 50 µl of S2035 dissolved in 1 ml toluene S2036-01 Mint oil, Indian, Ph.Eur. 5.2 47 µl of S2036 dissolved in 1.5 ml toluene S2036-02 Mint oil, Indian, Ph.Eur. 5.2 50 µl of S2036 dissolved in 1.5 ml toluene S2036-03 Mint oil, Indian, Ph.Eur. 5.3 20 µl of S2036 dissolved in 1 ml toluene

S2067-01 Lavender oil, Maillette, Ph.Eur. 5.0 20 µl of S2067 dissolved in 1.5 ml toluene

S2067-02 Lavender oil, Maillette, Ph.Eur. 5.0 40 µl of S2067 dissolved in 1.5 ml toluene

S2068-01 Cinnamon leaf oil, Ph.Eur. 5.0 30 µl of S2068 dissolved in 1.5 ml toluene S2068-02 Cinnamon leaf oil, Ph.Eur. 5.0 40 µl of R2068 dissolved in 1 ml toluene S2069-01 Anise oil, Ph.Eur. 5.0 30 µl of S2069 dissolved in 1.5 ml toluene S2069-02 Anise oil, Ph.Eur. 5.0 150 µl of S2069 dissolved in 1.5 ml toluene S2069-03 Anise oil, Ph.Eur. 5.0 50 µl of S2069 dissolved in 1 ml toluene S2072-01 Matricaria oil, CT Bisabolol, 200 µl of S2072 dissolved in 1 ml toluene

54

Ph.Eur. 5.1

S2072-02 Matricaria oil, CT Bisabolol, Ph.Eur. 5.1 100 µl of S2072 dissolved in 1 ml toluene

S2072-03 Matricaria oil, CT Bisabolol, Ph.Eur. 5.1 50 µl of S2072 dissolved in 1 ml toluene

S2078-01 Fennel seed oil, bitter 30 µl of S2078 dissolved in 1.5 ml toluene S2080-01 Caraway oil 60 µl of S2080 dissolved in 1.5 ml toluene S2080-02 Caraway oil 120 µl of S2080 dissolved in 1 ml toluene S2080-03 Caraway oil 30 µl of S2080 dissolved in 1 ml toluene S2081-01 Coriander oil 15 µl of S2081 dissolved in 1.5 ml toluene S2082-01 Cassiaöl Ph.Eur. 5.0 50 µl of S2082 dissolved in 1.5 ml toluene S2082-01 Cassia oil Ph.Eur. 5.0 50 µl of S2082 dissolved in 1.5 ml toluene S2082-02 Cassia oil, Ph.Eur. 5.0 75 µl of S2082 dissolved in 1 ml toluene S2083-01 Cinnamon bark oil, mind. 60% 30 µl of S2083 dissolved in 1.5 ml toluene S2083-02 Cinnamon bark oil, mind. 60% 40 µl of R2083 dissolved in 1 ml toluene S2088-01 Star anise oil, Ph.Eur. 5.0 30 µl of S2088 dissolved in 1.5 ml toluene S2088-02 Star anise oil, Ph.Eur. 5.0 150 µl of S2088 dissolved in 1.5 ml toluene S2088-03 Star anise oil, Ph.Eur. 5.0 50 µl of S2088 dissolved in 1 ml toluene S2089-01 Grapefruit oil, Israel 15 µl of S2089 dissolved in 1.5 ml toluene S2092-01 Lime oil, distilled 15 µl of S2092 dissolved in 1.5 ml toluene S2092-02 Lime oil, distilled 100 µl of S2092 dissolved in 1 ml toluene S2095-01 Mandarin oil 100 µl of S2095 dissolved in 1 ml toluene S2095-02 Mandarin oil 500 µl of S2095 dissolved in 1 ml toluene S2104-01 Clary sage oil, Ph.Eur. 5.0 100 µl of S2104 dissolved in 1 ml toluene S2105-01 Orange oil, bitter 100 µl of S2105 dissolved in 1 ml toluene S2105-02 Orange oil, bitter 200 µl of S2105 dissolved in 1 ml toluene S2160-01 Fennel seed oil, sweet 30 µl of S2160 dissolved in 1.5 ml toluene S2161-01 Fennel oil, bitter 30 µl of S2161 dissolved in 1.5 ml toluene S2162-01 Star anise oil 30 µl of S2088 dissolved in 1.5 ml toluene S2163-01 Caraway oil 60 µl of S2163 dissolved in 1.5 ml toluene S2164-01 Cinnamon oil 40 µl of R2164 dissolved in 1 ml toluene S2165-01 Citronella oil, nardus 5 µl of S2165 dissolved in 1 ml toluene S2166-01 Citronella oil 5 µl of S2166 dissolved in 1 ml toluene S2167-01 Clove oil 10 µl of S2167 dissolved in 1 ml toluene S2168-01 Clove flower oil, Ph.Eur. 5.0 10 µl of S2168 dissolved in 1 ml toluene S2169-01 Clove flower oil 10 µl of S2169 dissolved in 1 ml toluene

S2170-01 Mountain pine oil, Tirol, Ph.Helv.9 135 µl of S2170 dissolved in 1.5 ml toluene

S2171-01 Spruce needle oil, Siberian, DAB 2002 135 µl of S2171 dissolved in 1.5 ml toluene

S2172-01 Swiss stone pine oil 135 µl of S2172 dissolved in 1.5 ml toluene S2173-01 Silver fir needle oil 135 µl of S2173 dissolved in 1.5 ml toluene S2174-01 Spruce needle oil Mariana 135 µl of S2174 dissolved in 1.5 ml toluene S2175-01 Pine needle oil 135 µl of S2175 dissolved in 1.5 ml toluene S2176-01 Mountain pine oil 135 µl of S2176 dissolved in 1.5 ml toluene S2177-01 Coriander oil 10 µl of S2177 dissolved in 1 ml toluene S2178-01 Eucalyptus oil, radiata 10 µl of S2178 dissolved in 1 ml toluene

S2179-01 Eucalyptus oil, globulus, Ph.Eur. 5.0 10 µl of S2179 dissolved in 1 ml toluene

S2180-01 Eucalyptus oil, citriodora 10 µl of S2180 dissolved in 1 ml toluene

55

S2181-01 Eucalyptus oil 10 µl of S2181 dissolved in 1 ml toluene S2182-01 Juniper berry oil, Ph.Eur. 5.0 50 µl of S2182 dissolved in 1 ml toluene S2183-01 Juniper oil 50 µl of S2183 dissolved in 1 ml toluene S2184-01 Lavender oil, Bulgarian 40 µl of S2184 dissolved in 1.5 ml toluene S2185-01 Lavender oil, France, Ph.Eur. 5.0 40 µl of S2185 dissolved in 1.5 ml toluene S2186-01 Lavender oil 40 µl of S2186 dissolved in 1.5 ml toluene S2187-01 Lemon oil, Messina extra 500 µl of S2187 dissolved in 1 ml toluene S2188-01 Lemon oil 500 µl of S2188 dissolved in 1 ml toluene S2189-01 Lime oil, squeezed cold 100 µl of S2189 dissolved in 1 ml toluene S2190-01 Lime oil, squeezed cold, stabilised 100 µl of S2190 dissolved in 1 ml toluene S2191-01 Matricaria oil 50 µl of S2191 dissolved in 1 ml toluene S2192-01 Mint oil, rectified, China 20 µl of S2192 dissolved in 1 ml toluene S2193-01 Mint oil, Nagaoka, Ph.Eur. 5.2 20 µl of S2193 dissolved in 1 ml toluene S2194-01 Nutmeg flower oil 50 µl of S2194 dissolved in 1 ml toluene S2195-01 Orange oil, blood, Messina 200 µl of S1972 dissolved in 1 ml toluene S2196-01 Orange oil, bitter, South America 200 µl of S2196 dissolved in 1 ml toluene S2197-01 Orange oil, bitter, Guinea 200 µl of S2197 dissolved in 1 ml toluene S2198-01 Orange oil, sweet, Florida 20 mg of S1969 diluted to 1 ml with ethanol

S2199-01 Orange oil, sweet, Messina, Ph.Eur. 5.0 200 µl of S1969 dissolved in 1 ml toluene

S2200-01 Orange peel oil, sweet 200 µl of S2200 dissolved with 1 ml toluene S2201-01 Peppermint oil, USA, Ph.Eur.5.0 20 µl of S2201 dissolved in 1 ml toluene

S2202-01 Peppermint oil, France, Ph.Eur. 5.0 20 µl of S2202 dissolved in 1 ml toluene

S2203-01 Peppermint oil 20 µl of S2203 dissolved in 1 ml toluene

S2204-01 Rosemary oil, CT Campher, Ph.Eur. 5.0 50 µl of S2204 dissolved in 1 ml toluene

S2205-01 Rosemary oil, CT Cineol 50 µl of S2205 dissolved in 1 ml toluene S2206-01 Rosemary oil 50 µl of S2206 dissolved in 1 ml toluene S2207-01 Sage oil, Spanish 100 µl of S2207 dissolved in 1 ml toluene S2208-01 Sage oil, officinalis, Ph.Helv. 9 100 µl of S2208 dissolved in 1 ml toluene S2209-01 Sage oil 100 µl of S2209 dissolved in 1 ml toluene S2210-01 Spearmint oil, USA 20 µl of S2210 dissolved in 1 ml toluene S2211-01 Spearmint oil, Chinese 20 µl of S2211 dissolved in 1 ml toluene

S2212-01 Mandarin oil, redistilled, squeezed cold 500 µl of S2095 dissolved in 1 ml toluene

S2213-01 Tea tree oil, Ph.Eur. 5.0 20 µl of S2213 dissolved in 1 ml toluene S2214-01 Tea tree oil 20 µl of S2214 dissolved in 1 ml toluene S2215-01 Thyme oil, vulgaris, Switzerland 20 µl of S2215 dissolved in 1 ml toluene S2216-01 Thyme oil, zygis, Ph.Eur. 4.7 20 µl of S2216 dissolved in 1 ml toluene S2217-01 Thyme oil 20 µl of S2217 dissolved in 1 ml toluene S2218-01 Turpentine oil, Ph.Eur.5.0 100 µl of S2218 dissolved in 1 ml toluene S2219-01 Turpentine oil 100 µl of S2219 dissolved in 1 ml toluene S2220-01 Clary sage oil, Ph.Eur. 4.1 100 µl of S2220 diluted to 1 ml with toluene

S2265-01 Eucalyptus oil, globulus, Ph.Eur. 4 10 µl of S2265 dissolved in 1 ml toluene

S2266-01 Eucalyptus oil, Standard 10 µl of S2266 dissolved in 1 ml toluene S2266-02 Eucalyptus oil, Standard 20 µl of S2266 dissolved in 1 ml toluene S2267-01 Spearmint oil, Standard 20 µl of S2267 dissolved in 1 ml toluene S2268-01 Tea tree oil, Standard 20 µl of S2268 dissolved in 1 ml toluene

56

S2269-01 Thyme oil, Standard 20 µl of S2269 dissolved in 1 ml toluene S2270-01 Clove oil, Standard 10 µl of S2270 dissolved in 1 ml toluene S2271-01 Coriander oil, Standard 10 µl of S2271 dissolved in 1 ml toluene S2272-01 Caraway oil, Standard 30 µl of S2272 dissolved in 1 ml toluene S2273-01 Cinnamon leaf oil, Standard 40 µl of R2273 dissolved in 1 ml toluene S2274-01 Cinnamon bark oil, Standard 40 µl of R2274 dissolved in 1 ml toluene

S2275-01 Citronella oil, nardus/Ceylon, Standard 5 µl of S2275 dissolved in 1 ml toluene

S2276-01 Citronella oil, winterianus/Java, Standard 5 µl of S2276 dissolved in 1 ml toluene

S2277-01 Matricaria oil, Standard 50 µl of S2277 dissolved in 1 ml toluene S2278-01 Lavender oil, Standard 40 µl of S2278 dissolved in 1.5 ml toluene S2279-01 Clary sage oil, Standard 100 µl of S2279 dissolved in 1 ml toluene S2280-01 Juniper oil, Standard 50 µl of S2280 dissolved in 1 ml toluene S2281-01 Rosemary oil, Standard 50 µl of S2281 dissolved in 1 ml toluene S2282-01 Turpentine oil, Standard 100 µl of S2282 dissolved in 1 ml toluene S2283-01 Peppermint oil, Standard 20 µl of S2283 dissolved in 1 ml toluene S2284-01 Mint oil, Standard 20 µl of S2284 dissolved in 1 ml toluene S2285-01 Orange oil, bitter, Standard 200 µl of S2285 dissolved in 1 ml toluene S2286-01 Orange oil, sweet, Standard 200 µl of S2286 dissolved with 1 ml toluene S2287-01 Grapefruit oil, Standard 15 µl of S2287 dissolved in 1.5 ml toluene S2288-01 Mandarin oil, Standard 500 µl of S2095 dissolved in 1 ml toluene S2289-01 Lime oil, squeezed, Standard 100 µl of S2289 dissolved in 1 ml toluene S2290-01 Lemon oil, Standard 500 µl of S2290 dissolved in 1 ml toluene S2291-01 Anise Oil, Ph.Eur. 3 50 µl of S2291 dissolved in 1 ml toluene S2292-01 Anise oil, Standard 50 µl of S2292 dissolved in 1 ml toluene S2293-01 Star anise oil, Standard 50 µl of S2293 dissolved in 1 ml toluene S2294-01 Fennel oil, bitter, Standard 30 µl of S2294 dissolved in 1.5 ml toluene S2295-01 Fennel oil, sweet, Standard 30 µl of S2295 dissolved in 1.5 ml toluene S2296-01 Nutmeg oil, Standard 50 µl of S2296 dissolved in 1 ml toluene S2297-01 Cassia oil, Standard 75 µl of S2297 dissolved in 1 ml toluene S2310-01 Fennel seed oil, bitter 30 µl of S2310 dissolved in 1.5 ml toluene Table 23 Preparation of the standard solutions

Standard Number Standard Description Standard Preparation

R1986-01 Guaiazulene 1.34 mg of R1986 dissolved in 1 ml toluene R1986-02 Guaiazulene 3 mg of R1986 dissolved in 1.5 ml toluene R1986-03 Guaiazulene 1 mg of R1986 dissolved in 1 ml toluene R1987-01 Anethole 16 mg of R1987 dissolved 1 ml toluene R1987-02 Anethole 200 µl of R1987 diluted to 15 ml with

toluene; 1 ml of this solution diluted to 5 ml with toluene

R1987-03 Anethole 10 µl of R1987 dissolved 1.5 ml toluene R1988-01 Fenchone 5 µl of R1988 dissolved in 2.5 ml toluene R1988-02 Fenchone 5 µl of R1988 dissolved in 1 ml toluene R1989-01 Linalol 10 µl of R1989 dissolved in 1 ml toluene R1989-02 Linalol 6 µl of R1989 diluted to 1 ml with toluene R1989-03 Linalol 4 µl of R1989 diluted to 1 ml with pentane

57

R1989-04 Linalol 5 µl of R1989 diluted to 5 ml ethanol R1989-05 Linalol 5 µl of R1989 diluted to 5 ml toluene R1989-06 Linalol 10 µl of R1989 diluted to 15 ml with toluene;

1 ml of this solution diluted to 5 ml with toluene

R1989-07 Linalol 5 µl of R1989 dissolved in 1 ml toluene R1990-01 Geranyl acetate 2 µl of R1990 dissolved in 1 ml toluene R1991-01 Bisabolol 1.5 µl of R1991 dissolved in 1.5 ml toluene R1992-01 Bornyl acetate 2 mg of R1992 dissolved in 1 ml toluene R1992-02 Bornyl acetate 5 mg of R1992 diluted to 1 ml with toluene R1992-03 Bornyl acetate 5 µl of R1992 dissolved in 1.5 ml toluene R1992-04 Bornyl acetate 5 µl of R1992 dissolved in 3 ml toluene R1993-01 Terpinen-4-ol 4 µl of R1993 dissolved in 5 ml heptane R1994-01 Linalyl acetate 15 µl of R1994 dissolved in 1.5 ml toluene R1994-02 Linalyl acetate 20 µl of R1994 diluted to 1 ml with toluene R1994-03 Linalyl acetate 5 µl of R1994 dissolved in 1 ml toluene R1994-04 Linalyl acetate 6 µl of R1994 dissolved in 2 ml toluene R1995-01 Carvone 4 µl of R1995 dissolved in 5 ml toluene R1995-02 Carvone 5 µl of R1995 dissolved in 1.5 ml toluene R1995-03 Carvone 5 µl of R1995 dissolved in 3 ml toluene R1996-01 β-Caryophyllene 5 µl of R1996 diluted to 5 ml with ethanol R1996-02 β-Caryophyllene 5 µl of R1996 dissolved in 1.5 ml toluene R1996-03 β-Caryophyllene 80 µl of R1996 dissolved in 1.5 ml toluene R1996-04 β-Caryophyllene 25 µl of R1996 dissolved in 1.5 ml toluene R1996-05 β-Caryophyllene 40 µl of R1996 dissolved in 5 ml toluene R1996-06 β-Caryophyllene 20 µl of R1996 dissolved in 5 ml toluene R1996-07 β-Caryophyllene 10 µl of R1996 dissolved in 5 ml toluene R1997-01 Thymol 1 mg of R1997 diluted to 1 ml with toluene R1997-02 Thymol 15 mg of R1997 diluted to 1 ml with toluene R1997-03 Thymol 15 mg of R1997 dissolved in 1 ml toluene R1998-01 Menthyl acetate 5 µl of R1998 dissolved in 2.5 ml toluene R1998-02 Menthyl acetate 5 µl of R1998 diluted to 5 ml with toluene R1998-03 Menthyl acetate 5 µl of R1998 dissolved in 1.5 ml toluene R1998-04 Menthyl acetate 5 µl of R1998 dissolved in 3 ml toluene R1998-05 Menthyl acetate 5 µl of R1998 dissolved in 6 ml toluene R1999-01 Carvacrol 5 µl of R1999 diluted to 5 ml with pentane R2000-01 Menthol 10 mg of R2000 dissolved in 1 ml toluene R2000-02 Menthol 7.5 mg of R2000 dissolved in 1.5 ml toluene R2000-03 Menthol 2.5 mg of R2000 dissolved in 1 ml toluene R2000-04 Menthol 5 mg of R2000 dissolved in 4 ml toluene R2001-01 β-Pinene 5 µl of R2001 diluted to 5 ml with toluene R2001-02 β-Pinene 5 µl of R2001 dissolved in 1.5 ml toluene R2002-01 Eugenol 5 µl of R2002 diluted to 5 ml with acetone R2002-02 Eugenol 5 µl of R2002 diluted to 5 ml with ethanol R2002-03 Eugenol 15 µl of R2002 dissolved in 2 ml toluene R2002-04 Eugenol 1.5 µl of R2002 dissolved in 1.5 ml toluene R2002-05 Eugenol 10 µl of R2002 dissolved in 1 ml toluene R2002-06 Eugenol 5 µl of R2002 dissolved in 1 ml toluene

58

R2003-01 Coumarin 5 mg of R2003 diluted to 1 ml with acetone R2003-02 Coumarin 6 mg of R2003 dissolved in 1 ml toluene R2003-03 Coumarin 60 mg of R2003 dissolved in 1 ml toluene R2003-04 Coumarin 30 mg of R2003 dissolved in 1 ml toluene R2004-01 Methyl anthranilate 5 µl of R2004 diluted to 10 ml ethanol R2004-02 Methyl anthranilate 1 µl of R2004 dissolved in 1 ml toluene R2005-01 Bergaptene 1 mg of R2005 diluted to 1 ml with ethanol R2005-02 Bergaptene 1 mg of R2005 diluted to 5 ml with ethanol R2005-03 Bergaptene 1 mg of R2005 dissolved in 1 ml toluene R2005-04 Bergaptene 1 mg of R2005 dissolved in 1 ml ethanol R2006-01 Cinnamic aldehyde 5 µl of R2006 diluted to 1 ml with acetone R2006-02 Cinnamic aldehyde 5 µl of R2006 diluted to 1 ml with ethanol R2006-03 Cinnamic aldehyde 5 µl of R2006 dissolved in 1 ml toluene R2006-04 Cinnamic aldehyde 50 µl of R2006 dissolved in 1 ml toluene R2006-05 Cinnamic aldehyde 25 µl of R2006 dissolved in 1 ml toluene R2007-01 Borneol 5 mg of R2007 diluted to 1 ml with toluene R2007-02 Borneol 4 mg of R2007 dissolved in 1.5 ml toluene R2007-03 Borneol 2 mg of R2007 dissolved in 1.5 ml toluene R2008-01 Cineole 5 µl of R2008 dissolved in 1 ml toluene R2008-02 Cineole 10 µl of R2008 diluted to 5 ml with toluene R2008-03 Cineole 15 µl of R2008 dissolved in 1.5 ml toluene R2008-04 Cineole 15 µl of R2008 dissolved in 5 ml heptane R2008-05 Cineole 5 µl of R2008 dissolved in 2 ml toluene R2009-01 Citronellal 5 µl of R2009 dissolved in 2.5 ml ethanol R2009-02 Citronellal 5 µl of R2009 dissolved in 1.5 ml toluene R2010-01 Anisaldehyde 30 µl of R2010 diluted to 15 ml with toluene;

1 ml of this solution diluted to 5 ml with toluene

R2010-02 Anisaldehyde 1.5 µl of R2010 dissolved in 1.5 ml toluene R2010-03 Anisaldehyde 10 µl of R2010 dissolved in 1 ml toluene R2076-01 Citronellol 5 µl of R2009 dissolved in 1.5 ml toluene R2077-01 Myristicine 10 mg of R2077 diluted to 10 ml with toluene R2079-01 Carveol (+) 97% 15 µl of R2079 dissolved in 1 ml toluene R2090-01 Citral nat. 5 µl of R2090 dissolved in 1 ml toluene R2091-01 Lemarome N 5 µl of R2091 dissolved in 1 ml toluene R2093-01 Terpineol perfume 5 µl of R2093 dissolved in 1 ml toluene R2093-02 Terpineol perfume 5 µl of R2093 dissolved in 2 ml toluene R2094-01 Methyl N-Methyl anthranilate 2 µl of R2094 dissolved in 1 ml toluene R2221-01 Anethol (trans-) 99% 10 µl of R2221 dissolved 1.5 ml toluene R2222-01 Fenchone (+)- 5 µl of R2222 dissolved in 1 ml toluene R2223-01 Fenchone (1R)-(-)- 5 µl of R2223 dissolved in 1 ml toluene R2224-01 Caryophyllene oxide 25 mg of R2224 dissolved in 1.5 ml toluene R2224-02 Caryophyllene oxide 2 mg of R2224 dissolved in 1 ml toluene R2224-03 Caryophyllene oxide 1 mg of R2224 dissolved in 1 ml toluene

59

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 16 Description of the chromatograms with one main zone (2) ...................................... 44 Table 17 Description of the chromatograms with one main zone (2) ...................................... 45 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

60

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: RF-

value; 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: RF-

value; 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 26 Standard method: Oils with one main zone (2) ....................................................... 44 Figure 27 Standard method: Oils with one main zone (3) ....................................................... 45 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

development of an optimised hptlc method for analysis - camag

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