Determination of Prohibited Fragrances Using Headspace Solid-Phase Microextraction

Determination of prohibited fragrances using headspace solid-phase microextraction

B.Sc. Thesis

Paulina Rebecka Malmén Gunhild Thunold Helleve Espen Landro

Supervisor: Eke Zsuzsanna, PhD

 Eötvös Loránd University   Faculty of Science   Budapest, 2009  Acknowledgements

First and foremost we are very grateful to Eke Zsuzsanna, PhD, Head of the Joint Research and Training Laboratory on Separation Techniques (EKOL) at Eötvös Loránd University, Hungary – our Hungarian supervisor and gas chromatography guru. We warmly thank her for her guidance, encouragement and friendship.

We would like to thank Ove Jan Kvammen, Head of Institute for Aquaculture Technology, Chemical Engineering and Biomedical Laboratory Science at Bergen University College, Norway; his work to make connections with universities abroad and encouraging students to go out in the world and experience other countries has benefited us greatly. Our thanks also go to Kristin Kvamme, our Norwegian supervisor at Bergen University College, for her quick responses to our questions.

We warmly thank the other students at the EKOL laboratories for their friendship and willingness to help no matter how stupid the questions or problems have been. A special thanks to Szekeres Zoltan for guiding us through the mysteries of gas chromatography.

Gunhild Thunold Helleve Rebecka Malmén Espen Landro

2 Table of contents

Acknowledgements...... 2 Table of contents...... 3 1. Introduction...... 3 2. Tested compounds...... 3 3. Gas chromatography...... 3 3.1 Carrier gas...... 3 3.2 Sample injection...... 3 3.2.1 Split injection...... 3 3.2.2 Splitless injection...... 3 3.3 Columns...... 3 3.4 Flame ionization detector...... 3 4. Solid-phase microextraction...... 3 5. Experimental...... 3 5.1 Materials...... 3 5.2 Equipment...... 3 5.3 Establishment of GC parameters...... 3 5.3.1 Identification of elution order and retention times...... 3 5.3.2 Inlet temperature...... 3 5.3.3 The role of solvent trapping...... 3 5.4 Background check...... 3 5.4.1 Water...... 3 5.4.2 Solvent change...... 3 5.5 Fibres...... 3 5.6 Optimization of HS-SPME...... 3 5.7 Optimization of ultrasonic agitation...... 3 5.8 Final experimental setup...... 3 6. Detection limits and linearity...... 3 6.1 Linearity...... 3 6.2 Detection limits...... 3 Summary...... 3 Összefoglalás – summary in Hungarian...... 3 Sammendrag – summary in Norwegian...... 3 References...... 3 APPENDIX 1...... 3 APPENDIX 2...... 3 STATEMENT...... 3 STATEMENT...... 3 STATEMENT...... 3

3 1. Introduction

The European Union has formed several directives and legislations concerning cosmetic products, based on “Council Directive 76/768/EEC of 27 July 1976 on the approximation of the laws of the Member States relating to cosmetic products” [1]. Annex II in this directive contains a list of substances prohibited in cosmetic products, and this list is updated by the Commission of the European Communities whenever new scientific information about potentially health threatening compounds is given. As stated in an article in The Official Journal of the European Union [2], “the main aim of the directive (Directive 76/768/EEC) is to protect consumer health while harmonising legal provisions on cosmetic products within the common market”. The “Twenty-sixth Commission Directive 2002/34/EC of 15 April 2002” [3] lists substances which were incorporated in Annex II of the above-mentioned directive [1]. Many compounds on the list are allergens, which mean that they may cause an allergenic reaction. Even though they are prohibited it may occur that producers of cosmetic products add these fragrances to their products, on purpose or unintentionally, thus there is a need for developing a simple, applicable and efficient method for detecting these allergens. After having developed a method for the determination of 23 restricted fragrance compounds in household and personal care products (PCPs) [4] the Joint Research and Training Laboratory on Separation Techniques aims at finding an appropriate method for the determination of banned fragrance compounds specified in Directive 2002/34/EC [3]. The aim of this final bachelor project was to evaluate the applicability of headspace solid-phase microextraction (HS-SPME) for the detection of trace amounts of these compounds. Taking into consideration the relatively short period of time and the limited availability of gas chromatography with mass spectrometric detector, the task was to test this technique only using gas chromatography with flame ionization detector and reference materials.

4 2. Tested compounds

Number Name CAS No. Boiling point 1 4-Ethoxyphenol 622-62-8 121 °C / 9 mmHg 2 7-Methoxycoumarin 531-59-9 334-335°C 3 7-Methylcoumarin 2445-83-2 171-172 °C / 11 mmHg 4 Diphenylamine 122-39-4 302 °C 5 Ethyl acrylate 140-88-5 99 °C 6 Dimethyl citraconate 617-54-9 210-211°C 7 Benzyl cyanide 140-29-4 233-234 °C 8 trans-2- Hexenal diethyl acetal 67746-30-9 76°C/ 15 mmHg 9 Diethyl maleate 141-05-9 225 °C 10 trans-2-Heptenal 18829-55-5 90-91 °C/ 50 mmHg 11 Allylisothiocyanate 57-06-7 150 °C 12 Verbena oil 8024-12-2 13 Methyl crotonate 623-43-8 118-120 °C 14 Dihydrocoumarine 119-84-6 272 °C 15 trans-2-Hexenal dimethyl acetal 18318-83-7 16 4-tert-Butylphenol 98-54-4 226-238 °C

5 3. Gas chromatography

The science of gas chromatography can be described as a separation of compounds between two phases; a gas phase in motion which transports the sample and a stationary phase which is absorbing or adsorbing analytes and make them elute one by one with their own characteristic retention time [5]. The retention time depends on the distribution between the mobile phase and the stationary phase. Figure 1 shows an overview of the main components of a gas chromatograph (GC): An injection port, a column and a detector connected to a computer with adequate software which handles the information collected in the detector, and also controls GC parameters such as pressure and temperature.

Figure 1 Overview of the main components in a GC.

The sample is introduced to the carrier gas in the injection port. The carrier gas is a pressurized gas stream which transports the sample through the system, and the sample separation takes place in the column. The detector senses the compounds’ presence different from the carrier gas and converts the information into an electrical signal. The computer software reads the information and converts it into a chromatogram [6]. The GC requires three heated zones: Firstly, the injection port can be heated to vaporize the sample as it enters the GC. Secondly, there is an oven in the column in order to control the separation by controlling the temperature. The column temperature is one of the main parameters during the measurement. Thirdly, the detector is heated to keep it clean; this

6 temperature is usually kept higher than the column oven temperature to keep the components from condensing in the detector [6]. The main components of the GC will be more thoroughly described in chapters 3.1- 3.4.

3.1 Carrier gas

A stream of gas is continuously sweeping through the column, consisting of helium, nitrogen or hydrogen. The carrier gas must be dry and pure or with a minimum of contamination, and it needs to be controlled to a stabile pressure or mass flow [6]. What type of gas to use depends on the specific application and the type of detector. The van Deemter curve, shown in figure 2, could be a helpful tool in the decision. The van Deemter curve shows the height equivalent to a theoretical plate (HETP) against linear velocity for three common carrier gases.

Figure 2 van Deemter curve showing the height equivalent to a theoretical plate (HETP) against average linear velocity for hydrogen, nitrogen and helium [7].

Where the HETP value is at its minimum the efficiency is the greatest. The minimum HETP for nitrogen occurs over a narrow range of the average linear velocity, and at a relatively low speed. The efficiency will decrease rapidly if the average linear velocity increases because of the steep slope of the curve, and nitrogen is therefore not commonly used as carrier gas for capillary columns. The minimum HETP for hydrogen, on the other hand, occurs over a broader range and at higher linear velocities. This makes the efficiency curve more “flat” in this area, and hydrogen as carrier gas is therefore a better choice than nitrogen

7 or helium [5]. Hydrogen is also less expensive to purchase compared to helium, and is the most common carrier gas used in European laboratories.

3.2 Sample injection

The purpose of the inlet system is to introduce the sample in a manner which is repeatable and does not change the chemical structure by decomposing the compounds. To yield good peak shapes and separated peaks the injection has to be done in a way which make the sample occupy the shortest possible length of the column. If this band of solute maintains short as it passes through the column until reaching the detector, sharper peaks are generated and a better separation is obtained [8]. Modern GC instruments are equipped with autosamplers that inject samples automatically and make it possible to do multiple liquid injections without human intervention. Several samples can be measured in sequence as this is programmed in advance. The autosampler is removable to make manual injections possible in case of measurements where this is required. In this project both automatic and manual injections were used depending on the state of the sample to be injected. Liquid injections were performed by the autosampler, while samples collected by extraction were injected manually by placing the fibre in the inlet. In addition the inlet mode had to be chosen, deciding how much of the sample which should be allowed to enter the column after injection. For the split/splitless injector there are two main inlet modes; split injection where the sample is divided between the column and a vent flow, and splitless injection where the entire sample enters the column [6].

3.2.1 Split injection

Split injection is preferred when the sample contains more than 0.1 % of the analytes of interest or if the sample is not able to be diluted; the reason is that only 0.2-2 % of the sample actually reaches the column with the split injection [9]. The capillary column has a limited capacity and cannot separate big and/or concentrated amounts of samples. Therefore, after the sample enters the inlet, the vapour is divided between the column and a vent flow only allowing small amounts to enter the column. The injector temperature is relatively high

8 to evaporate the sample before it reaches the split point. The ratio of column flow and split flow is a parameter controllable from the software, and it is calculated by dividing the split vent flow by the column flow [6, 9]. In this project the split ratio was set to 1:10 and it was not optimized further since most of the measurements were done in splitless mode.

3.2.2 Splitless injection

For trace analysis where the sample contains less than 0.01 % of analytes, it is preferred to use splitless injection. The splitless injection occurs slowly, different from the split mode were the sample rapidly enters the column. A cloud of vaporized sample follows the carrier gas to the column. The split valve is closed during injection, thus the sample either enters the column or goes out through the septum purge, which removes any vapour that escape from the injection liner. At a set time the split valve opens to empty any remaining vapour after injection. [6, 9] When using the splitless mode the inlet should have a temperature lower than for the split mode to prevent decomposition of the compounds as they spend more time in the injection port before entering the column. The initial column oven temperature should be set below the boiling point of the solvent to make it condensate as soon as it enters the column. The solutes will then be trapped in the condensed solvent at the beginning of the column, forming a narrow band. This is called solvent trapping. As previously mentioned a narrow band generates sharp chromatographic peaks [9]. Splitless mode was chosen during the experiments each time the sample concentration was 10 ppm or less, and whenever headspace solid-phase microextraction was used for collecting samples before measurement in the GC. Because desorption of the compounds, which is adsorbed on the fibre during extraction, takes time, the inlet temperature should be kept low to avoid decomposition. Low column oven temperature is also important in order to retain the compounds at the beginning of the column.

9 3.3 Columns

There are two main types of columns, packed columns and open tubular columns. The main differences between these two types are column length and inner diameter. Since capillary columns have much smaller diameters than packed columns they can only handle small amounts of sample. Nevertheless, open tubular or capillary columns are the most commonly used columns due to chromatographic benefits such as better separation of the peaks. Even though the capillary columns have less sample capacity this limitation can be solved using an appropriate injector. [6] In packed columns inert particles are coated with a given stationary phase, as shown in figure 3. The packed column requires relatively high pressure to maintain a suitable flow, compared to the capillary column which can maintain an appropriate flow in a much longer column at the same pressure. [6]

Figure 3 The in section of a packed column [6].

Open tubular columns are made of fused silica where the inside wall is coated with either a liquid stationary phase or a solid stationary phase. The in section of an open tubular column is shown in figure 4.

Figure 4 The in section of an open tubular column [6].

10 There are different types of capillary columns, such as the wall-coated open tubular column (WCOT) which has a uniform layer of liquid stationary phase on the inner wall, and the porous layer open tubular column (PLOT) which has a solid stationary phase on the inner wall. The porous layer is supporting the stationary phase, or, in some columns the porous layer is the stationary phase. The support-coated open tubular column (SCOT) is similar to the PLOT column. The inner wall is covered with a porous layer which functions as a sorbent, such as porous polymers, aluminium oxide or zeolites [8, 9]. Based on earlier work where similar compounds were analysed [4], a WCOT column was chosen since this type of column is suitable for volatile fragrances similar to the ones tested in this project. The temperature of the column oven, which as previously mentioned is an important parameter, must be carefully adjusted. If the temperature is too low analytes spend much time in the stationary phase and rarely enters the mobile phase, if the oven is too hot analytes will be spending more time in the mobile phase than in the stationary phase. In both cases this leads to bad peak shapes and/or poor separation [8]. The maximum temperature for each specific column must also be considered to make sure the column is not overheated. The temperature programme in the software makes it possible to control the temperature during the separation in the column. This is done by programming temperature rate per minute and temperature ramps with hold time in order to get better separation and sharper peaks.

3.4 Flame ionization detector

There is a wide range of detectors to choose from and they all have the same purpose; to recognize and respond to the compounds in the sample as they elute and convert this information to an electrical signal. The electrical signal is then converted by the software which results in a chromatogram. The chromatogram displays peaks with their peak area proportional to the amount detected as a function of retention time. The flame ionization detector (FID) has a wide dynamic range, which means that it can provide an accurate quantification for a wide concentration range of the samples. The FID is selective for materials which ionize in a flame of air and hydrogen. Even though it is not a universal detector it is considered to be one, since it has a broad selectivity and detects almost all compounds with a C-H bonding. The flame ionization detector responds to nearly all

11 organic compounds, but gives hardly any or little response to H2O, CO2, N2, O2, CS2, inert gases and heavily halogenated compounds. The eluate is burned in the air/hydrogen flame inside the detector. Ions are formed from the organic analyte and this provides a current which then is converted to voltage. After passing through a filter which removes noise, a digital signal is produced and shown in the software. The current formed from the analyte is proportional to the amount of sample in the flame. Nitrogen gives the best detector performance when FID is used, but as mentioned in chapter 3.1 nitrogen is not a very applicable carrier gas, and that is why it is added to the hydrogen as make up gas instead [6, 9, 10].

12 4. Solid-phase microextraction

Solvent free preparation methods can be classified in three main groups according to the phases in which the extraction takes place; gas phase extraction, membrane extraction and sorbent extraction [11]. Sample preparation involves making the compounds of interest suitable for measurement in different analytical instruments by separating components from a complex matrix. A good sample preparation method should have excellent analytical performance, and offer high efficiency, selectivity and applicability to the compounds of interest. Solid-phase microextraction (SPME) is a solvent free, simple and fast sample preparation technique with many preferable properties particularly suitable for GC measurements [11]. Sorbent extraction involves that a sorbent with high affinity towards organic compounds extracts compounds from their matrices. Different adsorbents are suitable for different organic groups. The adsorbent in SPME is a coated fibre which is placed in a holder device to ease the extraction and the injection. The extraction technique for SPME can be done in two ways; exposing the fibre directly into the sample, or in the headspace of the sample. The headspace solid-phase microextraction (HS-SPME) method is suitable for extracting volatile compounds, and it is also better for the fibre since the quality will be maintained for longer. A coated fibre contains 1 cm of fused silica, coated with a polymer and bonded to a stainless steel plunger, and this is installed in a holder device. The plunger is able to move the fibre in and out of a hollow needle to keep the fibre protected [12]. Polymer coatings have different chemical properties, recognizable from different colour codes, and acts as a sorbent. The sorbent extracts compounds to the fibre. The maximum compound concentration on the fibre occurs when equilibrium is reached between the sample matrix and the fibre coating. This is also the maximum extraction time required. When equilibrium is reached the concentration is constant and independent of further extraction [11]. The next step in this technique is desorbing concentrated extracts from the coated fibre into an analytical instrument, in this case the GC. As previously mentioned a standard splitless injection port was used with a liner which has a diameter that allows the needle to penetrate. As soon as the coated fibre is exposed to the heat in the injection port the extracted components start desorbing and follow the carrier gas to the column. The injection

13 temperature has to be considered as one of many important parameters, as mentioned in chapter 3.2.2. Furthermore the HS-SPME technique has to be optimized regarding the parameters that affect the equilibrium between the matrix and the headspace and/or the coated fibre and the headspace. As shown in figure 5 two distribution coefficients, K1 and K2, are established when the fibre punctures the septum of the vial which contains the sample. Some parameters directly affect one or both of the distribution coefficients, others only establish the equilibrium faster.

Figure 5 Overview of the process which occurs during a HS-SPME in a vial with the sample.

Based on the law of mass conservation the number of moles in the system is considered to be constant during the extraction and can be described with the following overall equation:

C0 Vs  C f V f  Cs Vs  Ch Vh

C0 is the initial concentration of the analyte in the sample, and Vf, Vs, Vh are the volumes in the fibre, the sample and the headspace, respectively. Cf, Cs and Ch are the equilibrium concentrations of analytes in the coated fibre, the matrix and the headspace. The distribution constants (K1 and K2) can be defined as:

14 C f Ch K1  and K 2  Ch Cs

The amount of analytes (n) extracted by the coating can be described with the following equation:

C V V  K  K n  0 f s 1 2 K1  K 2 V f  K 2 Vh Vs

Meaning that the amount extracted on the fibre is proportional to the initial concentration of the analyte in the sample, C0. Parameters that affect the distribution constants are temperature, salt content, pH and organic solvent content in the liquid phase. Increasing the temperature affects both K1 and K2. Depending on the compounds the effect can be either positive or negative. The salt content and pH affect only the second distribution coefficient. In general the effect of salting increases the amount of extracted compounds with the increase of compound polarity. Decreasing of pH increases the amount of acids which takes part in the partitioning on the coating resulting in a greater sensibility. Extraction while the vial is held in an ultrasonic cleaner has turned out to be very effective. Ultrasound is a sound wave with a frequency so high that the human ear is not able to hear its pitch. The frequency used for industrial cleaning lies between 20-50 kHz. An ultrasonic cleaner is an apparatus with a small tank containing water. When water is exposed to the ultrasonic wave, cavitation occurs. Cavitation bubbles appear because of the decreasing density from the negative pressure in the water. As the wave passes by, the bubbles increase to an unstable state and will eventually collapse. The collapse causes shock waves with extremely high temperature and pressure [13]. The resulting stirring effect helps the faster establishment of the equilibrium. This can ameliorate the extraction method significantly, even though neither of the distribution constants is affected.

15 5. Experimental

5.1 Materials

Tested compounds (distributor; cat.no.; purity): 4-Ethoxyphenol (Aldrich; 25.859-8; 99%); 7-Methoxycoumarin (Aldrich; WS 1.580-9; 98%); 7-Methylcoumarin (Aldrich; 220329-10G; 98%); Diphenylamine (Aldrich;11.276-3; 99%); Ethyl acrylate (Aldrich; E 970-6; 99%); Dimethyl citraconate (AccuStandard; ALR-038N; 97%); Benzyl cyanide (Aldrich;18.572-8; 99%); trans-2-Hexenal diethyl acetal (AccuStandard; ALR-045N; 97%); Diethyl maleate (Aldrich; D9.770-3; 97%); trans-2- Heptenal (Aldrich; 324140-5G; 97%); Allylisothiocyanate (Aldrich; 37.743-0; 95%); Verbena oil (Fluka; 94877; -); Methyl crotonate (Aldrich; 13.945-9; 98%); Dihydrocoumarin (Aldrich; D10.480-9; 99%); trans-2-Hexenal dimethyl acetal (AccuStandard; ALR-046N; 97%); 4-tert- Butylphenol (Aldrich; B9.990-1; 99%).

Solvents and other materials (distributor; cat.no.; purity): Acetone (Merck; 1.0012.3500; Suprasolv®); methanol (Merck; 1.06011.2500; Suprasolv®); distilled water; tap water; still mineral water (Szentkirályi).

16 5.2 Equipment

 Gas chromatograph: Agilent Technologies/ 7890A GC System  Gas chromatograph-mass spectrometer: Agilent Technologies/ 5975C inert XL MSD with triple axis detector/ Agilent Technologies/ 7890A GC System  Autosampler: Agilent Technologies/ 7683 Series  Injector: Split/splitless  Column: Restek/ Rtx®-VMS, 20 meter, 0.18 mmID, 1 um df, max. prog. temp. 260 ºC, min. bleed at 240 ºC  Column: Agilent Technologies J&W Scientific/ HP-5MS, 30 meter, 0.25 mmID, 0.25 µm, temp. limits: -60 ºC to 325 ºC (350 ºC)  Software: Agilent ChemStation for GC Systems Rev. B. 04.01 [481]  Ultrasonic bath: Realsonic cleaner  Fibre: Supelco Co./ 65 µm polydimethylsiloxane/divinylbenzene (PDMS/DVB)  Fibre: Supelco Co./ 7 μm polydimethylsiloxane (PDMS)  Fibre: Supelco Co./ 30 μm polydimethylsiloxane (PDMS)  Fibre: Supelco Co./ 100 μm polydimethylsiloxane (PDMS)  Fibre: Supelco Co./ 85 μm polyacrylate (PA)  Headspace vials: 20 ml Symmetron; cat.no. 70254  Septum used with the vials: La-pha-pack; cat.no. 20 03 0975

17 5.3 Establishment of GC parameters

16 of the 29 prohibited substances amended in Annex II [3] were available at the university laboratory. Since there was no literature available concerning detection of the 16 fragrances, the first method, method A, was created from an already established method for other fragrances. Method A is described in Appendix 1. The parameters were changed/optimized as the experiments proceeded.

5.3.1 Identification of elution order and retention times

To be able to identify the peaks of the 16 fragrances, liquid injections were performed in order to establish the elution order of the substances and their retention times. Firstly 16 stock solutions were made using acetone as solvent. Approximately 0.1000 g of each compound was transferred to a 50 ml volumetric flask and diluted with acetone. The concentration of each stock solution was approximately 2000 ppm. Each was diluted to 50 ppm and transferred to a vial before measurement. To establish the retention time each compound was measured separately; to find the elution order a 50 ppm mixture was measured and to identify the chromatographic background a sample containing pure acetone was run. Method A was used for all these measurements, see Appendix 1 for details. 12 of the 16 fragrances were identified as can be seen in figure 6. The molecular structures of these 12 compounds are presented in Appendix 2.

Figu re 6 Chromatogram of a mixture containing 16 compounds where 12 compounds are identified.

18 Verbena oil was chosen to be excluded because it contained many different substances which led to too many peaks. It also appeared that three of the fragrances, Dimethyl citraconate, trans-2-Hexenal diethyl acetal and trans-2-Hexenal dimethyl acetal, had similar retention times close to the first peak in the chromatogram in figure 6. They could either be hidden behind an acetone peak or not be separated well enough. Since the rest of the compounds were identified the elution order was established: Ethylacrylate, Methyl crotonate, Allylisothiocyanate, trans-2-Heptenal, Diethyl malete, Benzyl cyanide, 4-tert-Butylphenol, 4- Ethoxyphenol, Dihydrocoumarin, Diphenylamine, 7-Methylcoumarin, 7-Methoxycoumarin. Because verbena oil was excluded a new stock solution of the remaining 15 substances was made. The concentration of the stock solution was 140 ppm for each compound. To find out whether verbena oil could have interfered with the three missing peaks a sample of 50 ppm of the mixture was prepared and run in the gas chromatograph with the same method, method A, but the compounds could still not be seen. A 10 ppm mixture with only the three problematic compounds was prepared and measured three times based on method A, but with a new parameter changed for every measurement. The first measurement had a change in initial hold time, from 1 minute to 3 minutes, and split mode was changed to splitless mode. For the second run, a different column (HP-5MS) was tried out with the same method described above. The third method included the initial column and the splitless mode, but the initial hold time was changed from 3 to 5 minutes. The chromatograms from these measurements did not give any trace of the compounds of interest (Dimethyl citraconate, trans-2-Hexenal diethyl acetal and trans-2- Hexenal dimethyl acetal). To find out if the compounds were present in the mixture a run using a gas chromatograph-mass spectrometer (GC-MS) was done. For the measurement method A was used considering GC parameters. The MS was operated in SCAN mode, acquiring masses from 50-500 atomic mass units (amu). GC-MS provides qualitative information as well as quantitative [9], but not even the GC-MS could find any trace of the missing compounds. After testing a more concentrated mixture (50 ppm) of the of three compounds with even longer hold time (6 minutes), and another mixture from new stock solutions which were diluted to 100 ppm and run with method A, the compounds could still not be found. The conclusion was made that the reference materials of Dimethyl citraconate, trans-2-Hexenal diethyl acetal and trans-2-Hexenal dimethyl acetal most probably were too old, or in any case

19 could not be seen in any chromatograms alongside with the other twelve compounds. Hence it was decided to eliminate them from further testing. A new stock solution with a mixture of the 12 identifiable fragrances was therefore made. Several methods were created in order to find the circumstances which provided best separation of the peaks and large peak areas. The parameters optimized were the initial oven temperature and the ramp temperatures.

5.3.2 Inlet temperature

There was an issue concerning the GC’s inlet temperature when using HS-SPME. For all methods mentioned earlier the inlet temperature was set at 220 °C, and as the experiments proceeded it became clear that it was the least volatile (!) compounds which appeared in the chromatograms. This indicated that the inlet temperature of the GC could have been too high at 220 ºC, thus causing decomposition of more volatile compounds. Three experiments with different inlet temperatures were carried out for comparison. The methods were based on method A but with the following changes; initial oven temperature: 40 ºC with 2 minutes hold time; ramp one: 20 ºC/minute to 70 ºC with 1 minute hold time; ramp two: 15 ºC /minute to 240 ºC with 3 minutes hold time. The inlet mode was changed to splitless mode and inlet temperatures were 220, 200 and 180 ºC, respectively. It was concluded that an inlet temperature at 200 ºC gave the highest areas for most of the compounds. Thus this method, method B, was chosen to be the most optimal method, applicable for 12 of the tested compounds. Method B is fully described in Appendix 1.

5.3.3 The role of solvent trapping

The retention times, which were established in chapter 5.3.1, were set using liquid injections. During liquid injections the compounds are being retained at the beginning of the column due to solvent trapping, described in chapter 3.2.2. When using HS-SPME less solvent is present and the effect of solvent trapping therefore decreases causing shorter retention times for the compounds. This affects only the most volatile compounds since they

20 are the first to enter the column. The peak shapes from these compounds are slightly distorted and less sharp than the peaks obtained by liquid injection. During the HS-SPME experiments Ethyl acrylate and Methyl crotonate seemed to be absent in most of the chromatograms, but unidentifiable peaks did appear at the beginning of the chromatograms. It was suspected that there could be a shift in the retention times for these two compounds due to the above-mentioned reasons. Ethyl acrylate and Methyl crotonate were extracted by HS-SPME and compared to a sample containing water and methanol to identify impurities in the mixture. The compounds gave distorted peaks with different retention times compared to the liquid injection chromatogram shown in figure 7. The chromatogram where the new retention times were identified using HS-SPME is displayed in figure 8.

Figure 7 The identification of retention times for 12 compounds for liquid injection, method B.

21 Figure 8 Retention times for Ethyl acrylate and Methyl crotonate for HS-SPME, method B.

The retention times for liquid injections were around 5.482 for Ethyl acrylate and 6.548 for Methyl crotonate, whereas the retention times for HS-SPME were closer to 5.119 and 6.383, respectively. It was therefore necessary to go back and consider the HS-SPME chromatograms over again, and this time the Ethyl acrylate and Methyl crotonate peaks were identified. Since two of the compounds had been mistakenly ignored, a wrong decision concerning the GC’s inlet temperature was made during optimization of HS-SPME with ultrasonic agitation (described in chapter 5.7). At that time, the results from three different runs based on method B but regulating the inlet temperatures (180, 200 and 220 °C), showed that 200 °C gave the most peaks. All of the following experiments were therefore run with an inlet temperature at 200 °C. However, with this new discovery it turned out that two more compounds were visible in the 220 °C measurement than in the 200 °C measurement. Due to this new information all the chromatograms were processed over again and can therefore be presented in the data.

22 5.4 Background check

When establishing the elution order and the retention times of the tested compounds, it was important to decide which of the peaks who represented the compounds, and which of the peaks who belonged to the solvent used in the stock solutions and the water used during HS- SPME – causing background noise.

5.4.1 Water

The sample preparation method HS-SPME involves exposing a fibre to the gaseous headspace above a liquid, in this case water, containing the sample. To be able to interpret the chromatograms which were created after desorbing the analyte from the fibre in the GC, pure water samples were extracted as well and run for background check. When using HS-SPME water itself does not affect gas chromatographic measurements, but if it is contaminated with organic compounds this could interfere with the results. Thus different types of water were tested to decide which gave the least disturbance in the chromatograms. Originally distilled water was used in this project, but since it was taken from a laboratory where volatile organic compounds are kept, it appeared to be quite contaminated and therefore affecting the chromatograms. Freshly distilled water was kept out of the above- mentioned laboratory and used for extraction; boiled distilled water and mineral water were tested as well. It was concluded that mineral water gave least disturbance in the chromatograms. Consequently this was used for extraction for the rest of the experiments. Mineral water’s natural salt content (it contains small concentrations of salt) is also preferable since it increases the ionization strength.

23 5.4.2 Solvent change

The elution order and retention times for 12 of the fragrances had been established in chapter 5.3.1. However, when using HS-SPME it appeared that acetone, which had been used as solvent in the stock solutions, created too much noise in the chromatograms. Another major problem is that acetone extracts easily to the fibre preventing extraction of other compounds. Water was tested as a solvent, but it was not applicable since the 12 fragrances of nonpolar nature were not soluble in water. Based on a similar project [4] with HS-SPME measurements of fragrances where methanol was used as solvent, methanol was decided to be the next solvent tested. New stock solutions were made in the same way as described in chapter 5.3.1. To test methanol’s influence on the chromatograms two extractions (at 90 °C for 40 minutes) were therefore performed, one with 5000 μl pure tap water and one with 5000 μl tap water and 50 μl methanol. The results from the two chromatograms were compared and it was concluded that methanol did not influence the chromatogram. The stock solutions with methanol as solvent were used throughout the rest of the project.

5.5 Fibres

A set of different colour-coded fibres were tested – three kinds of polydimethylsiloxane (PDMS) fibres with different diameters, 7, 30 and 100 μm, one polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre with diameter 65 μm and one polyacrylate (PA) fibre with diameter 85 μm. The fibre which proved to be the most suitable for extracting the 12 fragrances by headspace sampling was the 65 μm PDMS/DVB fibre. This is a fibre suitable for nonpolar compounds [14], such as the ones being tested in this project. Moreover, tests with the different fibres using the same parameters were compared and the PDMS/DVB came out as the best one. The large diameter also benefits the extraction because of a larger adsorption volume [11]. Before every measurement the fibre was purified as recommended by the producer, which was at least 30 minutes at 200-270°C. A blank was always run first to check that the fibre had been properly purified.

24 To increase efficiency during extraction, several PDMS/DVB fibres were run parallel. These fibres had also been used before and were, during the testing period, available to other students at the laboratory. The fibres are very fragile, and are easily reduced in efficiency, which means that they were of an unknown quality during these experiments. When two measurements with similar parameters were run, very different results could appear. It was decided to investigate whether using different fibres of the same type influenced the ability to reproduce the same result. Two fibres were marked as A and B and separated from the rest. They were measured three times each with ultrasonic agitation for 45 minutes (method described in chapter 5.6), with a 50 ppm concentration. The results are presented in four diagrams, figures 9-12, showing large variation within the three parallels run with the same fibre. In order to make the data easier to read each fibre’s results are divided and presented in two diagrams due to the large variation of the area. Notice the difference in the area scale for the large-area diagrams and the small-area diagrams of the two fibres.

Figure 9 HS-SPME, 45 min ultrasonic agitation with fibre A for three parallel measurements of the 12 compounds. This diagram shows the compounds with area from 0-200.

25 Figure 10 HS-SPME, 45 min ultrasonic agitation with fibre A for three parallel measurements of the 12 compounds. This diagram shows the compounds with area from 0-1800.

4-Ethoxyphenol and Dihydrocoumarine could not be seen at all in the chromatograms for fibre A (figures 9 and 10), but gave some small peaks after extraction with fibre B, as shown in figure 11. 7-Methoxycoumarin did not appear in any of the HS-SPME measurements.

Figure 11 HS-SPME, 45 min ultrasonic agitation with fibre B for three parallel measurements of the 12 compounds. This diagram shows the compounds with area 0-250.

26 Figure 12 HS-SPME, 45 min US-agitation with fibre B for three parallel measurements of the 12 compounds. This shows the compounds with area from 0-3000.

The difference in the area scale for the large area diagrams and the small area diagrams of the two fibres shows that there is a big difference within the fibres as well as between the fibres. As can be seen in figures 11 and 12 less variation occurs between the parallels for fibre B. This and the tall area peaks imply that fibre B is in a slightly better condition than fibre A. The standard deviations for the compounds were calculated in order to measure the variation. Each compound’s standard deviation, based on the three parallels, measured with two fibres, was calculated. The standard deviation is calculated from the equation below, based on the empirical variance and standard deviation.

n 2  xi  x S  i1 n 1

The residual standard deviation was also calculated by dividing the standard deviation by the average value for all of the compounds. The results are presented in table 1.

27 Standard Standard Avarage deviation, Average deviation, Name of compounds value, fibre A fibre A RSD, % value, fibre B fibre B RSD, % Ethyl acrylate 53 41 77 86 36 41 Methyl crotonate 59 39 66 91 36 40 Allylisothiocyanat 286 198 69 437 196 45 trans-2-Heptenal 1302 452 35 3094 1116 36 Diethyl maleate 48 19 39 87 29 34 Benzyl cyanide 255 78 31 394 132 34 4-tert-Butylphenol 125 35 28 236 82 35 4-Ethoxyphenol 0 0 9 9 100 Dihydrocoumarine 0 0 5 5 100 Diphenylamine 141 43 31 229 91 40 7-Methylcoumarin 4 1 29 12 8 68 7-Methoxycoumarin 0 0 0 0 Table 1 Average value and standard deviation for 12 compounds. The compounds were measured three times each using HS-SPME with 45 minutes ultrasonic agitation.

The calculations describe a large variation within the measurements. Fibre B, as suspected, has less variation. The conclusion drawn from this is that reproducible measurements are hard to yield when using different fibres. Using only one fibre could give more accurate results, but measurements showed that this also gives a certain variation. When choosing a fibre the quality should be tested in advance. Furthermore the quality of the fibre seems to decrease the more it is being used.

5.6 Optimization of HS-SPME

Firstly an extraction method which consisted of exposing the fibre in the headspace of a sample for 25 minutes at approximately 90 °C, was chosen. These particular parameters were based on an article concerning solid-phase microextraction for musk compounds [15]. The extraction samples consisted mainly of 5000 µl mineral water and 50 µl of the mixture of 12 compounds. This amount was chosen based on the volume of the vial used. By using 5000 µl water there will still be room for the sample and the fibre will have enough room to extract in the headspace. This also means that all extractions performed with different types of fibres and the same extraction methods are comparable. After choosing the PDMS/DVB fibre different combinations of extraction parameters were run, all of them with method B (described in Appendix 1). Extractions with room

28 temperature, 60 °C and 90 °C were run for 30, 60 and 90 minutes, in all 9 measurements. Extraction in room temperature resulted in biggest peaks after 90 minutes; extraction while holding the temperature constant at 60 °C resulted also in biggest peaks after 90 minutes. Holding the temperature constant at 90 °C resulted in best peaks after 60 minutes extraction. The explanation why 60 minutes appeared to be better than 90 minutes for 90 °C extraction might be the decomposition of compounds spending too much time in the heated headspace, or it could simply be the large variation within the fibres as explained in chapter 5.5. Three extractions with ultrasonic agitation (US) were performed in an ultrasonic bath for 15, 30 and 45 minutes, respectively; resulting in largest peak areas after 45 minutes. The best methods for all of the above-mentioned extractions are presented in two diagrams, figures 13 and 14, where the compounds are divided into two groups to be able to compare them within the same scale because of the large area variation.

Figure 13 Comparison of four different extraction methods showing the compounds with small peak area.

29 As seen in figure 13, 7-Methoxycoumarin did not appear in any of the extraction methods, but was left in the mixture to see if it would be possible to extract it with an optimized method. Dihydrocoumarine was, in small amounts, extracted in all methods except for 90 °C for 60 minutes.

Figure 14 Comparison of four different extraction methods showing the compounds with large peak area.

Even though ultrasonic agitation did not yield best areas for all compounds it gave the best areas for most of them. 4-tert-Butylphenol and Diphenylamine still gave good peaks with ultrasonic agitation, even if they obtained larger area with other extractions methods. To establish equilibrium within a reasonable time agitation was needed, thus the 45 minute US agitation was chosen to be further optimized.

30 5.7 Optimization of ultrasonic agitation

After the ultrasonic agitation was established as the most efficient way for extraction, further optimizing tests were performed. The next parameter to check was the optimal extraction time. Several extractions were done in addition to the three described in chapter 5.6, with increasing extraction time; 60, 75 and 90 minutes. Table 2 shows peak areas for the compounds during the six extraction methods.

Name of compound: Area from 15 Area from 30 Area from 45 Area from 60 Area from 75 Area from 90 min US min US min US min US min US min US Ethyl acrylate 92 66 26 22 105 20 Methyl crotonate 100 94 213 41 121 30 Allylisothiocyanat 48 726 1287 341 590 211 trans-2-Heptenal 1501,822 1998 5864 1923,4 1532,3 1073,4 Diethyl maleate 29,661 71 214 161 223 241,3 Benzyl cyanide 168,615 416 1222 709,4 904,9 765,3 4-tert-Butylphenol 93,25 157 641 572 871,6 1528,5 4-Ethoxyphenol 0 0 19 32,6 13,1 16,6 Dihydrocoumarine 19,578 0 33 47,8 23,9 10,8 Diphenylamine 140,317 230 874 791 711,9 3277,4 7-Methylcoumarin 9,403 17 36 55,9 26,3 59,2 7-Methoxycoumarin 0 0 0 0 0 0

Table 2 Areas for the 12 tested compounds with different extraction times during HS-SPME ultrasonic agitation, method B.

Figures 15 and 16 are based on the data in table 2. To give a more suitable presentation, two diagrams were made with different area scales, large area scale (figure 15) and small area scale (figure 16). When comparing the different methods to each other the 45 minutes and the 90 minutes US agitation come out equal. Since the least time-consuming method is preferable, 45 minutes US agitation was chosen to be the preferred extraction method in further measurements.

31 Figure 15 Comparison of different extraction times showing the compounds with large peak area.

Figure 15 shows that trans-2-Heptanal, Allylisothiocyanat and Bencylcyanide, all have their best peak areas at 45 minutes extraction time, while the two remaining compounds give better peak areas after 90 minutes extraction.

32 Figure 16 Comparison of different extraction times showing the compounds with small peak area.

Within the lower peak areas there are bigger variation when considering the best extraction time, only Methyl crotonate has the best extraction time for 45 minutes. Diethyl maleate and 7-Methylcoumarin have their best peak area after 90 minutes. The extraction curve for Diethylmaleate states that 15 and 30 minutes is too short time for the equilibrium to be established. Dihydrocoumarine and 4-Etoxyphenol have their best peak area at 60 minutes. Ethyl acrylate is the only one with its best peak area after 75 minutes. 7-Methoxycoumarin could still not be seen in any of the measurements, which means that it could not be extracted using these parameters. As described in this chapter 45 minutes US agitation was concluded to be the optimal extraction method. At that time the variation between the fibres and within the fibres (as described in chapter 5.5) had not been tested, meaning the standard deviation for these were not known. Therefore the conclusion made seemed to be right. Since the standard deviation now is known, it cannot be stated that 45 minutes US agitation is the best method because of the large variation within the fibres. Furthermore, the heater in the laboratory’s ultrasonic cleaner, used for agitation, did not work properly and the temperature could not be controlled. Temperature variation during

33 agitation was ignored in all of the measurements and was later discovered to be a more influential factor that first assumed. As described in chapter 4, high temperature is created when the cavitation bubbles in the ultrasonic cleaner explode. The water is heated, which affects the extraction. The temperature in the ultrasonic cleaner used in this experiment increased with a little less than a degree per minute. This means that the stable maximum temperature of 45 °C was reached in about 25 to 30 minutes. If two extractions were done in sequence within short time, the water temperature could vary between room temperature and 45 °C from the first to the second extraction. Experience from earlier extraction parameters showed that higher temperature increased the amount of extract on the fibre. This was taken into further consideration after the tests of fibre A and B were run (described in chapter 5.5) and may, most likely, explain some of the huge variance discovered in this experiment.

5.8 Final experimental setup

The final, optimized method, which is applicable for 11 of the fragrances, (since 7- Methoxycoumarine was not extractable during these parameters), is presented as follows:

1) Model sample: 5000 µl mineral water + 50 µl mixture of 12 compounds with methanol. 2) Sample preparation: Polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre with diameter 65 μm was exposed in the headspace for 45 minutes using ultrasonic agitation and no temperature control. 3) Method: Method B, see Appendix 1.

34 6. Detection limits and linearity

To establish the detection limits and linearity for the compounds, the stock solution was diluted to concentrations of 100, 75, 50, 25, 10, 5, 1 and 0,1 ppm and run accordingly to the parameters stated in chapter 5.8, Final experimental setup.

6.1 Linearity

The chromatograms were evaluated and each compound were put in a graph according to the size of the areas, as can been seen in figures 17 and 18.

Figure 17 The areas yielded at different concentrations for compounds with large peak areas.

35 Figure 18 The areas yielded at different concentrations for compounds with small peak areas.

As seen in figure 17 and 18 some trace of linearity exist for some of the compounds in some of the concentration intervals.

6.2 Detection limits

The detection limits were investigated by keeping the amount of mixture of 12 compounds with methanol at 50 µl for all experiments. The concentration of the methanolic solution added varied from 5 to 5000 µg/kg. The detection limits in table 3 are the lowest concentrations detectable with a signal to noise ratio at least 3 to 1.

36 Concentration Concentration limits for 1 g limits for 1 g Concentration PCP sample, Concentration PCP sample, Compounds limits, ppm μg/ kg Compounds limits, ppm μg/ kg Ethyl acrylate 10 500 4-tert-Butylphenol 5 250 Methyl crotonate 5 250 4-Ethoxyphenol 100 5000 Allylisothiocyanat 0.1 5 Dihydrocoumarin 100 5000 trans-2-Heptenal 0.1 5 Diphenylamin 5 250 Diethyl maleate 5 250 7-Methylcoumarin 100 5000 Benzyl cyanide 1 50 7-Methoxycoumarin - -

Table 3 Detection limits for the different compounds.

To make it more informative these figures can be transformed into concentration in a PCP sample, supposing 1 g is used for the measurement. Benzyl cyanide is used as an example. In this case the detection limit can be estimated with the following equation:

Concentration: 1 ppm = 1 μg/ml

Volume: 50 μl = 0.05 ml

1l 0.05ml ml 1000 g  50g 1g kg kg

This means that if a PCP sample was measured according to these limits it would be 50µg/kg benzyl cyanide, at minimum, present in the 1 g sample. The detection limits presented in table 3 have an insecurity due to the large standard deviation, and the method cannot be used for quantification.

37 Summary

To protect the health of consumers when using cosmetic products the European Union adopted Council Directive 76/768/EEC in 1976. Annex II of this directive lists substances which must not form part of the compounds of these products. Based on new scientific data the directive is regularly updated. In 2002 several fragrances were added to the list by Commission Directive 2002/34/EC. Even though they are prohibited, these fragrances may appear in cosmetic products both on purpose and unintentionally. For checking the compliance of the products with the above-mentioned directives adequate analytical methods are essential. The aim of this final bachelor project was to evaluate the applicability of headspace solid-phase microextraction (HS-SPME) for this task. One of the biggest advantages of this solvent free sample preparation technique is the enrichment that can be achieved by using a fibre, a small volume sorbent. Since fragrances tend to be in the headspace there is no need to expose the fibre directly to the sample. This can increase its lifetime significantly. As described in this thesis, after having established the appropriate gas chromatographic parameters 5 different types of fibres were tested. Also extraction circumstances, such as extraction time, temperature and ultrasonic agitation were investigated. With the final method developed, 11 of the 12 available compounds can be detected from water, though their quantification is impossible because of a really poor reproducibility. The estimated detection limits vary between 5 and 5000 µg/kg. The reproducibility is not satisfying even when the same fibre, applying the same extraction conditions, is used for all parallel measurements. This is even worse if there is big variance in the quality of the fibre, or if the temperature is not controlled properly during extraction time. The prohibition of the compounds listed in the updated Annex II of Directive 76/768/EEC is independent of concentration. Thus it can be concluded that as far as it can be tested without real samples, HS-SPME proved to be applicable to test the presence of banned fragrances. However, being aware of the extremely poor reproducibility, which prevents quantitative analysis, is of utmost importance.

38 Összefoglalás – summary in Hungarian

Az Európai Közösségek Tanácsa 1976-ban fogadta el a 76/768/EGK számú irányelvet a kozmetikai termékeket használó fogyasztók egészségének védelme érdekében. E rendelet II. melléklete azon anyagok listája, melyeket kozmetikai termékek nem tartalmazhatnak. Az irányelvet a legújabb tudományos kutatások alapján rendszeresen módosítják. A 2002/34/EK irányelvvel a II. mellékletet számos illatanyaggal bővítették. Annak ellenére, hogy ezen anyagok alkalmazása tiltott, mégis akár szándékosan, akár véletlenül is előfordulhatnak a kozmetikai termékekben. A termékek fenti irányelveknek való megfelelőségének ellenőrzéséhez elengedhetetlenül szükségesek a megfelelő analitikai módszerek. Szakdolgozati munkánk célja az volt, hogy megvizsgáljuk alkalmas lehet-e erre a célra a gőztérből végzett szilárd fázisú mikroextrakció (HS-SPME). Ennek az oldószermentes mintaelőkészítési technikának az egyik legfőbb előnye, hogy a kis térfogatú szorbenst, fibert alkalmazva jelentős dúsítás érhető el. Mivel az illatanyagok jellemzően kikerülnek a minta gőzterébe, ezért a fibert nem szükséges a mintával közvetlen érintkezésbe hozni. Ezzel annak élettartama jelentősen megnő. Miként azt dolgozatunkban ismertetjük, a gázkromatográfiás paraméterek meghatározását követően 5 különböző fajta szorbenst teszteltünk. Vizsgáltuk továbbá az extrakció körülményeit, úgymint az extrakció hőmérsékletét, időtartamát és az ultrahangos kevertetést. Javasolt módszerünkkel a rendelkezésre álló 12 komponensből 11-et lehet meghatározni vízből. Ugyanakkor a módszer mennyiségi meghatározásra nem alkalmas a rossz reprodukálhatósága miatt. A becsült detektálási határok egyes komponensekre 5 és 5000 µg/kg közöttiek. A reprodukálhatóság ugyanazt a fibert azonos körülmények között alkalmazva sem kielégítő. Mindez tovább romlik, ha nagy az eltérés az alkalmazott fiberek állapotában, vagy ha az extrakció hőmérséklete nem megfelelően szabályozott. A 76/768/EKG irányelv II. mellékletének tiltása ugyanakkor koncentráció független. A csak referencia anyagokkal végzett mérések alapján vállalható mértékben azt a következtetést vonhatjuk le, hogy a HS-SPME alkalmas tiltott illatanyagok kimutatására. Ugyanakkor elengedhetetlenül fontos tisztában lenni azzal, hogy a módszer rendkívül nagy szórása a mennyiségi meghatározást nem teszi lehetetővé.

39 Sammendrag – summary in Norwegian

For å beskytte forbrukere av kosmetikk og hudpleieprodukter ble direktiv 76/768/EEC utarbeidet av Den europeiske union i 1976. Annex II omhandler duftkomponenter som ikke skal tilsettes slike produkter. Direktivet blir regelmessig oppdatert basert på nytt vitenskaplig materiale. I 2002 ble flere duftstoff lagt til denne listen i direktiv 2002/34/EC. Selv om disse stoffene er forbudt å bruke, kan de likevel forekomme i kosmetikk og hudpleieprodukter, enten utilsiktet eller med vilje. Det er derfor et behov for å utvikle en egnet analytisk metode for å kunne påvise eventuelle spor av duftstoffene fra det sistnevnte direktivet i slike produkter. Formålet med denne bacheloroppgaven var å evaluere anvendeligheten av ”headspace solid-phase microextraction” (HS-SPME) til dette formålet. En av fordelene med en slik løsningsmiddelfri prøvepreparering er at man benytter et lite fiber som fungerer som sorpsjonsmiddel. Siden slike duftstoffer er flyktige og lett fordamper kan de ekstraheres direkte, og fibret trenger ikke å komme i direkte kontakt med prøvene i det hele tatt. Dette øker levetiden til et slikt fiber betraktelig. Som beskrevet i denne avhandlingen ble fem forskjellige fiber testet etter at det først ble etablert passende parameter for gasskromatografisk analyse. Ekstraksjonsforhold som ekstraksjonstid, temperatur og ultrasonisk røring ble også undersøkt. Med den endelige metoden kan 11 av 12 av de tilgjengelige stoffene bli detektert fra vann, selv om kvantifisering ikke er mulig på grunn av dårlig reproduserbarhet. De estimerte deteksjonsgrensene varierer mellom 5 og 5000 μg/kg for de forskjellige stoffene. Evnen til å reprodusere resultatene var ikke tilfredsstillende selv ved parallelle kjøringer med bruk av samme fiber og samme ekstraksjonsmetode. Dette ble enda tydeligere i tilfeller der det var stor ulikhet i fiberkvalitet eller der temperaturen ikke ble kontrollert under ekstraksjon. Siden det er et totalforbud mot duftstoffene som i 2002 ble tilføyd Annex II i direktiv 76/768/EEC, var det en metode for å påvise duftstoffenes eventuelle tilstedeværelse i produktene og ikke konsentrasjonsnivå som ble utviklet. Det kan derfor konkluderes med at så lenge stoffene kan bli testet ved bruk av referanseprøver er HS-SPME en tilstrekkelig metode for deteksjon av de ulovlige stoffene. På grunn av ekstremt dårlig reproduserbarhet kan metoden som ble utviklet overhodet ikke brukes i kvantitativ analyse.

40 References

[1] Council Directive of 27 July 1976 on the approximation of the laws of the Member States relating to cosmetic products; 76/768/EEC.

[2] Official Journal of the European Community. Opinion of the European Economic and Social Committee on the ‘Proposal for a Regulation of the European Parliament and of the Council on cosmetic products (recast)’. Brussels: the European Economic and Social Committee; 2009. Available from: http://eur- lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:C:2009:027:0034:0038:EN:PDF

[3] Twenty-sixth Commission Directive 2002/34/EC of 15 April 2002 adapting to technical progress Annexes II, III and VII to Council Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products.

[4] Benesóczki Dóra: Allergének meghatározása kozmetikumokból HS-GC-MS módszerrel. (Szakdolgozat) Budapest, ELTE, 2007.

[5] Hyver KJ, Sandra P: High Resolution Gas Chromatography. 3rd edition. USA: Hewlett-Packard Co; 1989.

[6] User Information Agilent 6890N Series Gas Chromatograph [Compact Disc; identification no. G1530-90210]. USA: Agilent Technologies, Inc.; 2001 May.

[7] Kremmer Tibor, Torkos Kornél, Szókán Gyula: Elválasztástechnikai módszerek elmélete és gyakorlata. Budapest; Eötvös kiadó, 2005.

[8] Jennings W, Mittlefehldt E, Stremple P: Analytical Gas Chromatography. 2nd edition. San Diego (California): Academic Press; 1997.

[9] Harris DC: Quantitative Chemical Analysis. 7th edition. USA: W.H. Freeman and Company; 2007.

41 [10] Buffington R, Wilson MK: Detectors for Gas Chromatography: A Practical Primer. Avondale: Hewlett-Packard Co.; 1991.

[11] Pawliszyn J: Solid Phase Microextraction: Theory and Practice. Waterloo (Ontario, Canada): Wiley-VCH, Inc.; 1997.

[12] Supelco. Bulletin 923. Solid Phase Microextraction: Theory and Optimization of Conditions [identification no. T198923]. Sigma-Aldrich Co.; 1998.

[13] Fuchs FJ: Ultrasonic Cleaning: Fundamental Theory and Application [Technical paper]. New York: Blackstone-Ney Ultrasonics; 2002 May.

[14] Supelco. Solid Phase Microextraction Fiber Assemblies [Data sheet, identification no. T794123G]. Sigma-Aldrich Co.; 1998.

[15] García-Jares C, Llompart M, Polo M, Salgado C, Macías S, Cela R: Optimisation of a Solid-Phase Microextraction Method for Synthetic Musk Compounds in Water. Journal of Chromatography A. 2002; Vol. 963, p.277-285.

42 APPENDIX 1

Method A: Liquid injection, split mode

ALS: Acetone washes: 1 preinjection, 3 postinjections Sample washes: 1 preinjection Sample pumps: 2 preinjections

Inlet: Mode: Split Heater: 220 ºC Split ratio: 10:1 Injection volume: 1 µl

Column: Rtx®-VMS: 20 meter, 0.18 mm ID, 1 um df, max. 260 ºC. Average velocity: 40 cm/second

Oven: Initial temperature: 40 ºC Initial hold time: 1 minute Ramp rate: 20 ºC/minute Final temperature: 240 ºC Final hold time: 4 minutes

Detector: Heater: 250 ºC

H2 flow: 30 ml/min Air flow: 400 ml/min Makeup flow: 30 ml/min

Signals: 20 Hz/ 0.1 min

43 Method B: Liquid injection, HS-SPME, splitless mode

ALS (applies only for liquid injections): Acetone washes: 1 preinjection, 3 postinjections Sample washes: 1 preinjection Sample pumps: 2 preinjections

Inlet: Mode: Splitless Heater: 200 ºC Injection volume: 1 µl

Column: Rtx®-VMS: 20 meter, 0.18 mmID, 1 um df, max. 260 ºC. Average velocity: 40 cm/second

Oven: Initial temperature: 40 ºC; Hold time: 2 minutes. Ramp 1: Rate: 15 ºC /minute; Temperature: 70 ºC; Hold time: 2 minutes Ramp 2: Rate: 12 ºC/minute; Temperature: 240 ºC; Hold time: 3 minutes

Detector: Heater: 250 ºC

H2 flow: 30 ml/min Air flow: 400 ml/min Makeup flow: 30 ml/min

Signals: 20 Hz/ 0.1 min

44 APPENDIX 2

4-Ethoxyphenol Molecule weight: 138.1638 g/mol Boiling point: 121 °C/ 9 mmHg Melting point: 64-67 °C

7-Methoxycoumarin Molecule weight: 176.1687 g/mol Melting point: 117-121 °C

7-Methylcoumarin Molecule weight: 160.1693 g/mol Boiling point: 171 - 172 °C / 11 mmHg Melting point: 128-130 °C

Diphenylamine Molecule weight: 169.2224 g/mol Boiling point: 302 °C Melting point: 50-53 °C

45 Ethyl acrylate Molecule weight: 100.1158 g/mol Boiling point: 99 °C Melting point: -71 °C Density: 0.918 g/ml at 25 °C

Benzyl cyanide Molecule weight: 117.1479 g/mol Boiling point: 233-234 °C Melting point: -24 °C Density: 1.015 g/ml at 25 °C

Diethyl maleate Molecule weight: 172.1785 g/mol Boiling point: 225 °C Melting point: -10 °C Density: 1.064 g/ml at 25 °C

trans-2-Heptenal Molecule weight: 112.1696 g/mol Boiling point: 90-91 °C/ 50 mmHg Density: 0.857 g/ml at 25 °C

46 Allylisothiocyanate Density: 1.013 g/ml at 25 °C Molecule weight: 99.1542 g/mol Boiling point: 150 °C Melting point: -80 °C

Methyl crotonate Molecule weight: 100.1158 g/mol Boiling point: 118-120 °C Density: 0.944 g/ml at 25 °C

Dihydrocoumarine Molecule weight: 148.1586 g/mol Boiling point: 272 °C Melting point: 24-25 °C Density: 1.169 g/ml at 25 °C

4-tert-Butylphenol Molecule weight: 150.22 g/mol Boiling point: 226-238 °C Melting point: 96-101 °C Density: 0.908 g/ml at 25 °C

47 STATEMENT

Name: ELTE Faculty of Science, Field of studies: B.Sc. Chemistry

Title of thesis: Determination of prohibited fragrances using headspace solid-phase microextraction

I hereby declare, as the author of this thesis, that it is a product of my own and that it contains my own ideas. I used the standard rules for references and quotations consistently. I never used other people’s ideas without proper reference.

…………………….., Budapest

______signature STATEMENT

Name: ELTE Faculty of Science, Field of studies: B.Sc. Chemistry

Title of thesis: Determination of prohibited fragrances using headspace solid-phase microextraction

…………………….., Budapest

______signature

49 STATEMENT

Name: ELTE Faculty of Science, Field of studies: B.Sc. Chemistry

Title of thesis: Determination of prohibited fragrances using headspace solid-phase microextraction

…………………….., Budapest

______signature