Dynamic headspace (DHS) technique: set-up and parameter control for GC/MS analysis of odorant formulations

Transcription

1 Dynamic headspace (DHS) technique: set-up and parameter control for GC/MS analysis of odorant formulations Ellen Vercruyssen Supervisors: Prof. dr. Jo Schaubroeck, dr. Jan Van Biesen Master's dissertation submitted in order to obtain the academic degree of Master of Science in de industriële wetenschappen: chemie Department of Industrial Technology and Construction Chairman: Prof. dr. Marc Vanhaelst Faculty of Engineering and Architecture Academic year

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3 Acknowledgements I am very grateful for the opportunity that I have received to accomplish this task in Scentarom. The combination of the subject and the entourage at Scentarom resulted in the feeling that this was the best internship I could ever imagine. I would like to express my appreciation for Dr. J. Van Biesen because he was always available when I wanted to discuss the content of my thesis. He was full of involvement, patience and enthusiasm during my process. Also my special thanks to Dr. J. Schaubroeck and Dr. G. Diricks for the time they put into reading my thesis and for the adaptations they ve suggested. Last but not least I would like to thank my colleague A. Vermoesen for the support during my internship and the adjustments she made to my thesis. I

4 Abstract The DHS-technique has been explored as possible tool to obtain additional analytical information on fragrances and flavours present in a complex matrix. The focus of present work is on mastering the governing (odour) contamination present as ambient background in a fragrance company, while performing an in-depth analysis. Furthermore all experimental parameters, important for the DHS technique itself, have studied and evaluated in the context of specific problems encountered in the loaded environment of a fragrance production house. II

5 Table of Contents Acknowledgements... I Abstract... II Table of Contents... III List of Figures... V List of Tables... VI List of abbreviations... VII Introduction... 1 I Literature/method description... 2 I.1 GC/MS... 2 I.1.1 Gas chromatograph... 2 I Carrier gas... 2 I Sample introduction... 3 I Types of sample introduction in GC... 3 I PTV combined with TDU... 3 I Column... 7 I Detector... 7 I.1.2 The GC/MS Interface... 7 I.1.3 MS... 7 I Sample inlet... 7 I Ionization system... 8 I Mass analyzer... 8 I Ion detector... 9 I.1.4 Computer... 9 I.2 Sample preparation I.2.1 Static headspace I.2.2 Dynamic headspace I Description I Trapping of components I.3 Flow charts II Experimental part III

6 II.1 Short experimental method descriptions II.1.1 Blank TDU II.1.2 Blank DHS II.1.3 DHS analysis of sample II.2 Contamination II.2.1 Introduction II.2.2 Sources where contamination can occur II.2.3 Measurement of contamination II Preservation conditions of adsorption material (tenax in tenax tube and tenax in tenax liner) II TDU-tube in TDU-tray II PTV-liner in PTV II TDU-tube in TDU II Unlocking of TDU II Transporting TDU-tube to DHS carriage II Septum of the sample vial II DHS station and DHS carriage II O-rings on TDU-head II Carrier II.2.4 Conclusion II.3 Parameters II.3.1 Introduction II.3.2 Test sample II.3.3 Split ratio R in PTV II.3.4 Sample temperature (T sample i and T sample tr ) during incubation step and trapping step II.3.5 Total incubation time Δt i II.3.6 Trapping gas flow F (V=constant) II.3.7 Trapping gas volume V (F=constant) II.3.8 Reproducibility II.4 Comparing DHS analysis and SHS analysis II.5 Shampoo Conclusion Bibliography Attachments IV

7 List of Figures Figure 1-Schematic of GC/MS... 2 Figure 2-Sample introduction for DHS: thermal desorption (TDU-PTV-Column)... 4 Figure 3-Structure Tenax TA... 4 Figure 4-Pneumatic diagram of the flow of the GC, TDU and PTV during desorption step and injection step... 6 Figure 5-Schematic figuration of electron ionization... 8 Figure 6-Schematic figuration of a quadrupole analyzer... 9 Figure 7-Scheme of an electron multiplier... 9 Figure 8-DHS device Figure 9-Schematic overview when trapping all components Figure 10-GC/MS with configuration for DHS analysis Figure 11-Schematic overview of GC/MS and sample preparation Figure 12-Schematic overview of trapping and releasing Figure 13-Schematic overview of parameters of DHS and GC/MS Figure 14-Figuration of PTV-liner, TDU-tube and sample vial Figure 15-Chromatogram of contamination of TDU-tube (containing tenax) in tray Figure 16-Results of PTV-liner in PTV Figure 17-Results of contamination of TDU-tube in TDU Figure 18-Chromatogram of contamination when the TDU is open for 45 seconds Figure 19-Chromatogram of test of transporting TDU-tube to DHS carriage Figure 20-Figuration of septum of sample vial Figure 21-Result for contamination of septum of the sample vial Figure 22-Results for contamination of blank DHS Figure 23-Contamination of O-rings on TDU head Figure 24-Results of contamination of carbohydrate and silica Figure 25-Chromatograms of blank TDU Figure 26-Total area of test sample and total area of contamination Figure 27-Chromatogram of blank analysis of split ratio 1/10, 1/25, 1/50 and 1/ Figure 28-Influence of incubation temperature on the relative area and the total area of the components trapped Figure 29-Influence of incubation time on the relative area and the total area of the components trapped Figure 30-Influence of trapping gas flow on the relative area and the total area of the components trapped Figure 31-Influence of trapping gas volume on the relative area and the total area of the components trapped Figure 32-Reproducibility of DHS on the absolute area of each component and the total area of all components trapped Figure 33-Comparing DHS with SHS on the absolute, relative and total area of the components trapped Figure 34-Chromatograms of shampoo with concentration of 100 % shampoo, 50 % shampoo 50 % H2O, 50 % shampoo 50 % silica and 25 % shampoo 25 % H2O 50 % silica V

8 List of Tables Table 1-Overview sample introduction when using TDU/PTV combination... 5 Table 2-Overview of the split ratio and the start split ratio Table 3-Overview of the parameters of steps of the DHS and the start parameters Table 4-Overview of components in test sample Table 5-Overview of the different split ratios and corresponding concentrations that have been tested Table 6-Overview of the different incubation temperatures that have been tested Table 7-Overview of the different incubation times that have been tested Table 8-Overview of the different trapping gas flows that have been tested Table 9-Overview of the different trapping gas volumes that have been tested Table 10-Total area of the individual analyses VI

9 List of abbreviations GC/MS: Gas Chromatography/Mass Spectrometry GC: Gas Chromatograph MS: Mass Spectrometer COC: Cold On Column PTV: Programmed Temperature Vaporizer TDU: Thermal Desorption Unit PDMS: polydimethylsiloxane FID: Flame Ionisation Detector TCD: Thermal Conductivity detector m/z: mass-to-charge ratio EI: Electron Ionisation SHS: Static Headspace DHS: Dynamic Headspace DC: direct current RF: radio frequency VOC: volatile organic component VII

10 Introduction Scentarom is a company which produces flavours and fragrances. These flavours and fragrances are mixtures of different components and these components have a typical odour. The amount of different components is dependent on the nature of the flavour or fragrance. Because of the complex nature of the matrix to which these flavours and fragrances are added, there are no straightforward methods for the analysis of the different components in it. GC/MS is an analytical instrument with a high sensitivity, but fragrances and flavours cannot be injected directly in the GC/MS (because of the matrix) and therefore a sample preparation is necessary. In the open environment within Scentarom there is a strong background odour present and this odour can cause contamination when using adsorption material during measurements. This contamination will be examined in this work. Present work is preceded by Nele Piens thesis Static headspace characterisation of odorants in complex matrices by hyphenated GC-MS techniques. The SHS-type performed by Nele Pien is a SHS with the use of trapping material. The trapping material is placed in a glass jar containing the sample and then sealed with aluminium foil for a certain period of time at a certain temperature. During this period of time the volatile components of the sample will be adsorped on the trapping material. After this period of time the trapping material is analysed through GC/MS. In this work attention is focussed on dynamic headspace (DHS), as more sensitivity could be expected. DHS is a dynamic technique in which more parameters have to be explored when compared to the SHS-type analysis described in the previous paragraph. 1

11 I Literature/method description I.1 GC/MS Gas chromatography/mass spectrometry (GC/MS) (figure 1) is a synergetic combination of two powerful analytical techniques. There is a GC which separates components of a mixture in function of time, depending on the boiling point and the polarity of these components. After separation, the MS provides information that helps to identify (structural identification) the components in the mixture. [1] Figure 1-Schematic of GC/MS [2] I.1.1 Gas chromatograph Gas chromatograph is a separation technique used to analyse volatile substances in the gas phase. Components are dissolved in a solvent and vaporized when heated in the inlet port (injector), so the components are in the gas phase. The gas chromatograph uses a carrier gas to sweep the components to the column, that contains a coating of a stationary phase. Separation of components is determined by the distribution of each component between the carrier gas and the stationary phase. A component that has a low interaction with the stationary phase, will elute quickly and reaches the detector first. A polar component interacts with a polar stationary phase. A non polar component will interact with a non polar stationary phase. Also the boiling point has an influence on the elution time of the component. Only components that can be vaporized without decomposition will be suitable for GC analysis. [1, 2] I Carrier gas Helium is the most commonly used carrier gas. Also nitrogen, argon and hydrogen can be used as carrier gas. The choice of carrier gas depends upon the desired performance and the detector being used. The carrier gas must be inert, so it won t react with the components in the sample. It also may not be retarded by the stationary phase of the column, so the carrier gas will reach the detector first. The linear velocity of the carrier gas is an important parameter. It can be determined by injecting the 2

12 carrier gas and measuring the time from injection to detection (=retention time). The linear velocity is the retention time divided by the column length. [1, 2] I Sample introduction I Types of sample introduction in GC There are several types of sample introduction. The most common and oldest technique is the split/splitless injection. Here the liquid sample is introduced into a heated room by a syringe. The temperature of this room is normally minimum 50 C higher than the temperature of the column. The sample will evaporate immediately and will be swept to the column by the carrier gas. The injection of the sample needs to be done quickly to avoid peak broading. When using a splitless injection every component of the sample will reach the column. To avoid overloading and contamination the injection can be done by a split injection. Only a small part of the components will reach the column when using a split injection. Another type of sample introduction is the Cold On Column (COC) injection. Here the liquid sample is introduced into a precolumn by a syringe at a low temperature. The precolumn is an uncoated column with a wider diameter than the analytical column. This is used to avoid contamination of the column and to refocus the liquid sample at the entrance of the analytical column. The temperature of the COC is kept at a constant, near ambient temperature during the injection. After the injection the GC will heat up. Because the sample is introduced directly into the column there is no discrimination of volatile components. The most advanced injection technique of sample introduction is a PTV-injector. PTV stands for Programmed Temperature Vaporizor injection. The different between a PTV and a COC injection is that the PTV contains a temperature program that can be activated in the software. PTV can heat up very quickly. It can also be used as a COC injection. PTV can be used in split or splitless mode. [3] I PTV combined with TDU Use of a PTV can be combined with a thermal desorption unit (TDU). TDU is a highly efficient thermal desorption system. It allows the controlled transfer of thermal desorbed material to an injection system (like PTV). When TDU is combined with PTV sample injection (figure 2), the sample introduction device contains 2 units, a PTV and a TDU. Both PTV and TDU have a liner which can contain an adsorption or an absorption material. There are several kinds of liners, for example: empty glass liners, empty glass mulitbaffled liners, liners packed with glass beads or Tenax TA or carbotrap or PDMS (polydimetylsiloxane) foam. The liner in TDU can also contain a part of the sample itself when the sample is a solid. In this work tenax TA is used in TDU and PTV. Tenax TA is a porous material with a specific surface area of 35m 2 /g. It has a low affinity for water and methanol and adsorbs components in the C 5 -C 20 range. Tenax TA is based on 2,6-diphenylphenylene oxide polymer (figure 3). It is used for it stability when heated (upper limit of 350 C). The tube of PTV contains less tenax than the tube of TDU. The tube of PTV is also smaller than the tube of TDU. [3, 4] 3

13 TDU PTV Column Figure 2-Sample introduction for DHS: thermal desorption (TDU-PTV-Column) [5] Figure 3-Structure Tenax TA [6] There are 2 steps in the sample introduction when using the PTV in combination with the TDU: Step 1. Thermal desorption (TDU) The thermal desorption unit contains a liner that can contain an adsorption material used in the sample preparation (see chapter I.2 Sample preparation, page 10). During the desorption step, TDU is heated. While heating, the components will be desorbed from the adsorption material in TDU, carried by helium gas and trapped on the adsorption material in the cold PTV. Step 2. Sample injection (PTV) PTV will be heated. While heating, the trapped components will be desorbed from the tenax in PTV, carried by helium gas to the column. [3] TDU and/or PTV can be used in split or splitless mode. There are different possible modes of operation. The use of which methods depends on the type and concentration of the components present in the sample. The 2 most important ones are mentioned in table 1. [3] 4

14 Table 1-Overview sample introduction when using TDU/PTV combination Mode A B Step 1 Desorption Splitless Splitless Step 2 Injection Splitless Split Software TDU Splitless Splitless PTV Solvent Venting Solvent Venting Mode A (splitless desorption, splitless injection): Mode A is used for a quantitative transfer from TDU to the column. This is used when there has to be a maximum sensitivity. Step1: Nearly every component, desorbed from the tenax, will be carried by the helium gas and trapped on the tenax in the cold PTV. Step 2: Every component, trapped on the tenax of PTV of Step 1, goes quantitatively to the column. [3] For an efficient thermal desorption the flow in the TDU needs to be at least 20 ml/min. This flow is too high for the column (should be 1 ml/min). The TDU and/or PTV therefore need to be in split during the desorption step. This is done by choosing solvent venting in the software program, allowing the PTV to be used in split or splitless mode. In solvent venting the valve, regulating the split, closes during a small defined period of time. [3] In figure 4 a pneumatic diagram of the GC, TDU and PTV during desorption step and injection step is shown. 23 ml/min 20 ml/min 3 ml/min PTV 16 ml/min Pneumatic diagram during desorption step 1 ml/min 5

15 7 ml/min 4 ml/min 3 ml/min PTV Pneumatic diagram during injection step 1 ml/min PV 1: Proportional valve PV 2: Proportional valve FS: Flow sensor PS: Pressure sensor SPR: Purge flow regulator for the septum purge TPR: Purge flow regulator for the TDU SV 3: Valve split/splitless of the GC pneumatic control SV 4: Valve to switch between PTV split and TDU split Figure 4-Pneumatic diagram of the flow of the GC, TDU and PTV during desorption step and injection step [3] During thermal desorption step, the TDU is in splitless mode and SV4 valve is closed in TDU. The SV3 valve needs to be open, so there is no overflow in the column. During sample injection, the PTV is in splitless mode, SV3 valve is closed in PTV. After the splitless mode time the SV3 valve is open again. [3] Mode B (splitless desorption, split injection): Mode B is used whenever circumstances necessitate that not all the adsorbed material on the TDU needs to reach the column. Step 1: Every component, desorbed from the tenax in the TDU, will be carried by helium gas and trapped on the tenax in the cold PTV. Step 2: The components present on the tenax in the PTV, are splitted. This means that only a small part reaches the column. The other part is vented through the split vent. [3] During the thermal desorption step, the TDU is in splitless mode and the SV 4 valve is closed in TDU. The SV3 valve needs to be open, so there is no overflow in the column (see figure 4, desorption step) During the sample injection, the PTV is in split mode, the SV 3 valve is open in PTV. This SV 3 valve controls the split ratio. The split ratio is the ratio of the flow in the column to the flow through the PTV. [3] 6

16 I Column After the sample introduction the components reaches the column. This column contains a thin layer of a non-volatile chemical, called the stationary phase and can be polar or non polar. The non-volatile chemical can be coated onto the walls of the column or can be coated onto an inert solid that is added to column. Components in the gas phase are carried through the column by the carrier gas. They will selectively interact with the stationary phase of the column. The strength of interaction depends on the polarity of the stationary phase and the polarity of the component. If the component is a polar component, it will interact strongly with a polar stationary phase. The column is heated with a temperature program. Because of its stability and the possibility of decomposition of its components, the temperature of the column has a limit. The column used in Scentarom is CP-Sil 8 CB. CP-Sil 8 CB is a non polar column. CP-Sil 8 CB has a stationary phase of dimethyl polysiloxane with 5 % of the dimethyl replaced with diphenyl. Dimethyl polysiloxane is a non polar component, but by replacing 5% of dimethyl in diphenyl the column will be slightly less non polar, but still very non polar. Scentarom also uses carbowax as column. Carbowax is a more polar column than CP-Sil 8 CB. In this work only CP-Sil 8 CB will be used. [1, 2] I Detector There are several detectors used in gas chromatography. The most common used detectors are flame ionisation detector (FID) and thermal conductivity detector (TCD). When the GC is combined with a mass spectrometer, the GC separates the components and the mass spectrometer serves as a detector. I.1.2 The GC/MS Interface When the GC is combined with a MS, an interface is required. The interface transports the effluent from the gas chromatograph to the mass spectrometer. It is a small tube on a high temperature about 150 C. The temperature of the interface must be programmed at a certain temperature, so the components neither condense in the interface nor decompose before entering the MS. [1, 7] I.1.3 MS The mass spectrometer measures the mass-to-charge ratio (m/z) of gas phase ions and provides a measure of the abundance of each ionic species. Components that are separated by the GC go in the MS. In the MS, the components will be ionized through electron impact (bombardment). The charged ions will be placed in a modulated electromagnetic field and sorted by their mass-to-charge ratio in high vacuum. [7] I Sample inlet In stand alone MS, the entire process from ionisation to detection takes place in gas phase and in vacuum. In sample inlet, components go to the gas phase. Liquids and solids will go in a gas chamber to proceed in gas phase. Volatile components will evaporate immediately by vacuum. This is called cold inlet system. Non volatile components will go in gas phase by heating. This is called heated inlet system. When using the hyphenated technique GC/MS, components leaving the column are already in gas phase. Only a small quantity will reach the ion source. Carrier gas will be eliminated. [7] 7

17 I Ionization system The most common used ionization technique is the electron ionization (EI) (figure 5). Gaseous molecules are brought in vacuum in the ionization chamber by column flow. In this chamber a beam of electrons are produced by a heated filament and molecules will be ionized and defragmented by the electrons. Resulting ions are then accelerated by the repeller and brought to the mass analyzer. The repeller is positively charged compared to this ionization chamber. [7, 8] Figure 5-Schematic figuration of electron ionization [9] The beam of electrons are perpendicular to the beam of molecules, so electrons clash with molecules. Electrons that will not clash with the molecules will be collected on the trap. When clashing a molecule, an electron is removed from the molecule and produces a positively charged ion corresponding to the relative molecular mass of the sample being analysed. Also the molecule will disintegrates in different pieces. Every piece is typical for the molecule, thus the mass spectrum gives a fingerprint of the molecule. [8] I Mass analyzer As mass analyzer a quadrupole analyzer (figure 6) is the most common used. A quadrupole analyzer consists of four parallel rods that have fixed DC and alternating RF potentials applied to them. Ions produced from the EI are focussed and passed along the middle of the 4 rods of the quadrupole analyzer. The motion of the ions depends on the electric fields. So only ions with a particular m/z will have a stable trajectory and will reach the detector, corresponding to a specific RF potential. Other ions will disappear among the rods of the quadrupole analyzer. To bring ions of different m/z into focus on the detector the RF is varied and in this way a mass spectrum is built up. The quadrupole analyzer is used in hyphenated techniques, because of it high scan speed: it can measure up to 500 mass units in 0,005s. In Scentarom the speed of the mass analyzer is up to 4,45 scans per second. 1 scan consists of a mass spectrum of an m/z of 35 to 350. [7, 10] 8

18 Figure 6-Schematic figuration of a quadrupole analyzer [11] I Ion detector The most common used ion detector is the electron multiplier (figure 7). It will generate an increased signal through which the ions can be detected. The electron multiplier consists of a series of dynodes and is operating in a vacuum. When the ions reach the first dynode, the dynode will emits secondary electrons. These generated electrons are multiplied in a cascade by the following dynodes. At the end of the dynodes a cluster of secondary electrons will reach the anode that measures the signal. The amplified signal will lead to the computer coupled at the MS and GC. [12] Figure 7-Scheme of an electron multiplier [13] I.1.4 Computer The computer is connected to the MS by a converter which converts the signal of the MS in a signal that can be registered by the computer. The signal is expressed into values of mass versus abundance. The computer also registers the chromatogram of the GC. Furthermore, the computer contains a data base of all possible mass spectra of many different components. It compares the spectrum of the MS with the spectra in the data base. When there is a match, the computer can tell which component it is. In the computer there is also a software for creating several methods and adjusting parameters for obtaining required results. 9

19 I.2 Sample preparation Sample preparation is needed whenever a sample contains analytes present in a complex matrix. In this case the sample cannot be used as such and preliminary steps must be taken to prepare the sample so it is fitted to the instrumental analyses to which it is to be subjected. During sample preparation there is an enhanced risk of contamination and especially for SHS and DHS, as sensibility increased considerably. Contamination can often become a major problem. A few sample preparation techniques will here be described briefly. I.2.1 Static headspace The word headspace here refers to the gas phase located above a liquid or solid phase present in a sealed vial. The partition of volatile organic components (VOC s) into the gas phase depends on several factors a.o. the solubility in water or solvent (hydrophilic or hydrophobic), polarity, ionic nature of analyte and solvent, molecular weight and temperature. A liquid and/or solid sample is placed in a sealed vial. The sealed vial has an inert septum, mostly as a result of teflon coating, and thus should be inert for adsorption of volatile components. In static headspace an equilibrium is reached between the sample and gas phase. The gas phase can be partially removed with a syringe and injected in the GC. Another method for removing the gas phase is, placing an adsorbent material in a sealed vial so volatile components will adsorb on the adsorbent material. The adsorbent material with the trapped volatile components will be placed in a TDU. [14, 15] I.2.2 Dynamic headspace I Description Dynamic headspace (DHS) is a dynamic extraction technique. In DHS, VOC s are continuously swept away from the headspace onto a trap by a flow of inert carrier gas. An equilibrium is never reached between the gas phase and the sample. The inert carrier gas can be nitrogen or helium. Sometimes the inert carrier gas can be air, but there is a risk for oxidation. To release the components from the trap, rapid heating is the most efficient method when using an adsorption material. Releasing the components from the trap can be done in the TDU (see chapter I Sample introduction, page 3). The trap used in experimental work is tenax TA. [14, 16] In practice, the DHS device contains 2 units (figure 8): a DHS station and a DHS carriage. The sample is placed in a vial in the DHS station while the DHS carriage holds the tube containing the tenax TA. This tube shall later be placed in the TDU unit for desorption. The DHS carriage can move forward, so it can be placed above the sample in the DHS station. DHS can be used for analysis of flavours and fragrances. 10

20 DHS carriage DHS station Figure 8-DHS device [17] I Trapping of components Figure 9-Schematic overview when trapping all components [18] Figure 9 gives an overview of the several steps of DHS when trapping all components. This includes also all components which are already in the headspace above the sample before the DHS run starts. Step 1: Before the analysis The glass vial with the sample is sealed with a septum. Some analytes are already in the headspace above the sample (gas phase). Step 2: Incubation The glass vial is placed into the DHS station. During incubation step the sample is heated up. The incubation step includes also an agitation step, so the sample can be agitated if desired. Step 3: Trapping A TDU tube with an adsorption material is placed into DHS carriage above the glass vial. A double needle is pushed through the septum into the glass vial, 1 needle for the inlet of the carrier gas in the glass vial and 1 needle for the outlet of the carrier gas with the analytes. The carrier gas flows over the sample and transfers the components from the headspace onto the adsorption material in the tube above it. The carrier gas used is mostly the same as the carrier gas that will be used in the subsequent GC analysis. During trapping the components the volume and the flow of the carrier gas can be selected in the software. 11

21 During trapping 3 parameters are linked to each other: Trapping gas volume (V) Trapping gas flow (F) Trapping time Only 2 of the 3 parameters can be adjusted. Here the trapping gas volume (V) and the trapping gas flow (F) can be adjusted. By adjusting the trapping gas volume at a constant gas flow, the trapping time will change. Step 4: Drying (optional) For removing water or solvent, drying is used. Drying is done in a empty glass vial (the drying vial ). A defined gas flow is transferred through the drying vial and over the adsorption material in the tube. This step is optional. Step 5: Desorption and GC analysis Adsorption material with the analytes, is transferred to the TDU and here the desorption and injection step will start (see chapter I PTV combined with TDU, page 3). [18] In this work a DHS analysis is performed in 2 steps: incubation step (incl. agitation step) and trapping step. Every step has a specific time and temperature. Incubation step Trapping step Agitation step Δt i1 Δt i2 Time Δt i Δt tr Temperature T sample i T sample tr T tr Every step has several parameters that can be adjusted. These parameters will be described in the experimental part of this work. 12

22 Figure 10 gives an overview of the GC/MS (Agilent) used in Scentarom. The MPS (Gerstel) is the robot that prepares and injects the samples. Dynamic headspace is a fully automated technique through the use of the MPS robot Gas chromatograph 2. Mass spectrometer 3. Dynamic headspace (DHS) 4. Thermal desorption unit (TDU) 5. MPS robot 6. Tray TDU-tube Figure 10-GC/MS with configuration for DHS analysis [19] 13

23 I.3 Flow charts I.1.1 Gas chromatography (GC) Injection technique Injector types Columns Split/splitless (Cold) Oncolumn PTV I.1.2 Mass spectrometry (MS) Filtration Soxhlet Off-line Extraction Distillation Liquid-liquid SPE... Purge & trap I.2. Sample preparation Dynamic headspace (DHS) "Sweep" Headspace (HS) Thermal extraction (TE) On-line SPME Pyrolysis Static headspace (SHS) "Classical" Using traps... Figure 11-Schematic overview of GC/MS and sample preparation [15] 14

24 Methods Adsorption Absorption Trapping Zeolit Materials Tenax Carbo(Trap) PDMS Releasing Desorption Thermal (TDU) Solvent Figure 12-Schematic overview of trapping and releasing [15] 15

25 II Experimental part The following subjects will be discussed: Short overview of the several experimental methods used Study and description of contamination caused during DHS analysis Parameters description and adjustment for optimal analytical results Short comparison between DHS and previous SHS techniques used in Scentarom Analysis of odorants in a shampoo using DHS technique II.1 Short experimental method descriptions The following methods are used in this experimental part and will be explained: Blank TDU Blank DHS DHS analysis of sample Figure 13 gives an overview of the several methods. There are 10 time laps described in the figure. 1. Incubation step DHS During the incubation step the sample vial will be heated up and agitated if desired. 2. Trapping step DHS During trapping step the components, present in the test sample, will be trapped on the tenax in the DHS carriage. 3. Transport of TDU-tube from DHS to TDU 4. Pressurize The pressurize time lap is the time the GC/MS needs to set all the parameters at start position. 5. TDU heating During TDU heating the TDU will heat up at a constant rate. 6. TDU hold time The TDU hold time is the time the TDU stays on it highest temperature. Time lap 4 and 5 are the desorption step (see chapter I PTV combined with TDU, page 3). 7. TDU cooling During TDU cooling the TDU cools down. 8. PTV heating + start GC runtime During the PTV heating, the PTV will heat up with a constant speed. The GC runtime starts with 1 minute solvent delay. This means that the detector starts to collect the data after 1 minute. During solvent delay the solvent will reach the detector. 9. PTV hold time PTV hold time is the time the PTV stays on it highest temperature. Time lap 8 and 9 are the injection step (see chapter I PTV combined with TDU, page 3). 10. Running 16

26 II.1.1 Blank TDU The method blank TDU consists of 7 time laps (figure 13): During blank TDU time lap 1,2 and 3 are not performed. The GC starts directly with pressurize. 4. Pressurize The duration of this time lap is 1 min. The temperature of the TDU will be 50 C, the temperature of the PTV will be 20 C, the GC-oven will have a temperature of 50 C and the flow will be 24 ml/min. 5. TDU heating The duration of the TDU heating is 2 min. The TDU will heat up with a velocity of 100 C/min and the flow will be 54 ml/min. 6. TDU hold time This is the time lap the TDU stays on a temperature of 250 C and a flow over the TDU of 54 ml/min. The duration of this time lap is 5 min. 7. TDU cooling The TDU will cool down to 90 C in 15 s. The flow is 54 ml/min. 8. PTV heating + start GC runtime Here the PTV will heat up with a speed of 10 C/s for 25 s. The flow over the PTV will be 14 ml/min. The runtime of the GC starts and after 1 minute solvent delay the detector will collect the data of the analysis. 9. PTV hold time This is the time lap the PTV stays on a temperature of 250 C. The duration of this time lap is 10 min. The flow goes from 14 ml/min to 24 ml/min. 10. Running The GC will run for 23 min 40 s with a velocity of 10 C/min until a temperature of 240 C is reached. This is the green line of GC-oven in figure 13. Data are collected as a control for the blank-level of the blank TDU. II.1.2 Blank DHS The method blank DHS consists of 10 time laps (figure 13): 1. Incubation step DHS During the incubation step the transfer heater will be 150 C, the incubation temperature 30 C, the agitator speed 500 rpm. The duration of the incubation step is 1 min. 2. Trapping step DHS During the trapping step the transfer heater will be 150 C, the incubation temperature 30 C and the trapping flow 50 ml/min. The duration of the incubation step is 2 min. 3. Transport of TDU-tube from DHS to TDU During the transport the DHS flow will be 1 ml/min. 4. Pressurize The duration of this time lap is 1 min. The temperature of the TDU will be 50 C, the temperature of the PTV will be 20 C, the GC-oven will have a temperature of 50 C and the flow will be 24 ml/min. 5. TDU heating The duration of the TDU heating is 2 min. The TDU will heat up with a velocity of 100 C/min and the flow will be 54 ml/min. 17

27 6. TDU hold time This is the time lap the TDU stays on a temperature of 250 C and a flow over the TDU of 54 ml/min. The duration of this time lap is 5 min. 7. TDU cooling The TDU will cool down to 90 C in 15 s. The flow is 54 ml/min. 8. PTV heating + start GC runtime Here the PTV will heat up with a speed of 10 C/s for 25 s. The flow over the PTV will be 14 ml/min. 9. PTV hold time This is the time lap the PTV stays on a temperature of 250 C. The duration of this time lap is 10 min. The flow goes from 14 ml/min to 24 ml/min. 10. Running The GC will run for 23 min 40 s with a velocity of 10 C/min until a temperature of 240 C is reached. This is the green line of GC-oven in figure 13. Data are collected as a control for the blank-level of the blank DHS. II.1.3 DHS analysis of sample The method DHS analysis of sample consists of 9 time laps (figure 13): 1. Incubation step DHS During the incubation step the transfer heater will be 150 C, the incubation temperature 30 C, the agitator speed 500 rpm. The duration of the incubation step is 1 min. 2. Trapping step DHS During the trapping step the transfer heater will be 150 C, the incubation temperature 30 C and the trapping flow 50 ml/min. The duration of the incubation step is 2 min. 3. Transport of TDU-tube from TDU to DHS During the transport the DHS flow will be 1 ml/min. 4. Pressurize The duration of this time lap is 1 min. The temperature of the TDU will be 50 C, the temperature of the PTV will be 20 C, the GC-oven will have a temperature of 50 C and the flow will be 24 ml/min. 5. TDU heating The duration of the TDU heating is 2 min. The TDU will heat up with a velocity of 100 C/min and the flow will be 54 ml/min. 6. TDU hold time This is the time lap the TDU stays on a temperature of 250 C and a flow over the TDU of 54 ml/min. The duration of this time lap is 5 min. 7. TDU cooling The TDU will cool down to 90 C in 15 s. The flow is 54 ml/min. 8. PTV heating + start GC runtime Here the PTV will heat up with a speed of 10 C/s for 25 s. The flow over the PTV will be 14 ml/min. 9. PTV hold time This is the time lap the PTV stays on a temperature of 250 C. The duration of this time lap is 10 min. The flow goes from 14 ml/min to 24 ml/min. 18

28 10. Running The GC will run for 74 min 20 s with a velocity of 3 C/min until a temperature of 240 C is reached. This is the red line of GC-oven in figure

29 DHS Trap 25 C Transfer heater 150 C DHS Incubation temperature (T sample i ) Agitator speed Trapping flow 30 C 500 rpm 5 ml/min 50 ml/min 1 ml/min 5 ml/min Incubation temperature (T sample tr ) 30 C TDU transfer 300 C GC/MS TDU temperature PTV temperature GC temperature 50 C 20 C 50 C 100 C/min 250 C 90 C 10 C/s 123 C 250 C 10 C/min 250 C 3 C/min 100 C 250 C Data Flow 24 ml/min 50 ml/min Solvent delay SS 0 min 1 min 3 min 4 min 5 min 7 min 12 min 12 min 15 s 10 ml/min 24 ml/min SS SS SS SS 0 s 24 s 1 min 10 min 25 s 23 min 40 s 65 min 20 s 74 min 20 s Figure 13-Schematic overview of parameters of DHS and GC/MS Sample preparation GC/MS analysis

30 II.2 Contamination II.2.1 Introduction Scentarom produces flavours and fragrances for various applications. As a consequence a strong background odour is present in the laboratories and production facilities. Thus, whenever the adsorbent material (tenax) is in contact with the open environment within Scentarom, contamination inevitably takes place. Detailed study of this contamination is absolutely necessary in order to master (and if possible to eliminate) its influence on later analytical results. II.2.2 Sources where contamination can occur To understand the sources of contamination next chapters has to be read first: chapter I PTV combined with TDU, page 3 chapter I.2.2 Dynamic headspace, page 10 Sealing (graphite) TDU head (inox) O-rings (rubber) Septum (rubber + teflon) Tenax Tenax Vial (glass) Tenax liner (glass) Tenax tube (glass) PTV-liner TDU-tube sample vial Figure 14-Figuration of PTV-liner, TDU-tube and sample vial Figure 14 gives a figuration of PTV-tube, TDU-tube and sample vial used in GC/MS. For photos of PTVtube, TDU-tube and sample vial: see attachment 2, page 55. The following steps can cause contamination: Preservation conditions of adsorption material (tenax in tenax tube and tenax in tenax liner) TDU-tube in TDU-tray PTV-liner in PTV TDU-tube in TDU Unlocking of TDU Transporting TDU-tube to DHS carriage Septum of sample vial DHS carriage DHS station 21

31 O-rings on TDU head Carriers II.2.3 Measurement of contamination II Preservation conditions of adsorption material (tenax in tenax tube and tenax in tenax liner) There is a strong background odour present within Scentarom. This background odour causes a lot of contamination when the tenax in tenax tube and tenax liner are in contact with this background odour. To reduce the contamination of the tenax in tenax tube and tenax liner a serie of blank TDU has be performed before starting a DHS analysis. Former test shows that the contamination can be reduced by keeping the tenax in a glass jar sealed with aluminium foil. There will be less contamination, so less blank TDU will have to be performed to reduce the contamination. (see thesis Pien and chapter II.2.4 Conclusion, page 30) II TDU-tube in TDU-tray The TDU-tube needs to be placed in the TDU-tray when starting a DHS analysis. When performing a DHS analysis the TDU-tube goes from TDU-tray to the DHS station. The time the TDU-tube is in TDUtray is 5 min. The contamination of the TDU-tube in TDU-tray is given in figure 15. Figure 15-Chromatogram of contamination of TDU-tube (containing tenax) in tray Conclusion: TDU-tube in TDU-tray causes contamination. This contamination can be eliminated by performing analyses in a sequence. When performing a sequence of analyses, the movement of the TDU is done by the MPS robot. This robot moves the tenax tube in the TDU directly to the DHS station without staying in the tray. The several steps of the MPS robot when performing a sequence of measurements are given in attachment 1, page 54. II PTV-liner in PTV When there is no analysis performed the PTV-liner stays in the PTV for technical and practical reasons. The PTV is not airtight, so during standby the PTV-liner is exposed to the open environment 22

32 Area (absolute) within Scentarom. This contamination is tested when the PTV-liner was respectively 1 day, 3 days and 1 week present in the PTV. The results are given in figure 16. 1,00E+09 Total area of contamination of PTV-liner in PTV 9,00E+08 8,00E+08 7,00E+08 6,00E+08 5,00E+08 4,00E+08 1 day 3 days 1 week 3,00E+08 2,00E+08 1,00E+08 0,00E+00 Figure 16-Results of PTV-liner in PTV The chromatograms of these tests are given in attachment (see attachment 3, page 55-56). Conclusion: The PTV-liner causes contamination. When no analyses are performed, the tenax in the tenax liner adsorbs odour molecules present in the open environment within Scentarom. The tenax liner needs to be free of contamination before starting a DHS analysis. To reduce the contamination of the PTVliner in the PTV a sequence of blank analyses (i.e. TDU blank) needs to be done. After a few analyses the tenax in the PTV-liner will be blank. In all further tests the PTV-liner will remain in the PTV during each analysis. II TDU-tube in TDU The TDU-tube is kept outside Scentarom to reduce the contamination of the tenax in the tenax tube. The contamination is tested when the TDU-tube was 3 days, 1 week and 2 months exposed in an open environment outside Scentarom. The results are given in figure

33 Area (absolute) 4,50E+08 Total area of contamination of TDU-tube in TDU 4,00E+08 3,50E+08 3,00E+08 2,50E+08 2,00E+08 3 days 1 week 2 months 1,50E+08 1,00E+08 5,00E+07 0,00E+00 Figure 17-Results of contamination of TDU-tube in TDU The chromatograms of these analysis are given in attachment (see attachment 4, page 56-57). Conclusion: The tenax in the tenax tube adsorbs odour molecules even when the tenax is not exposed to the open environment within Scentarom. To eliminate this contamination the tenax in the tenax tube needs to be free of contamination before starting a DHS analysis. To reduce the contamination of the TDU-tube in TDU, a sequence of blank TDU needs to be done. After a few analyses the tenax in the TDU-tube will be blank. To reduce the total time of measurements, the TDU-tube and PTV-liner will be made free of contamination during the same sample analysis sequence. II Unlocking of TDU When the TDU-tube goes to the DHS carriage, the TDU will be unlocked and open for 45 seconds. The contamination of this step is tested and the result is given in figure

34 Figure 18-Chromatogram of contamination when the TDU is open for 45 seconds Conclusion: When the TDU is unlocked and open for 45 seconds, it causes negligible contamination. II Transporting TDU-tube to DHS carriage When the TDU-tube is transported to DHS carriage, the TDU-tube will be exposed to the open environment within Scentarom for 1 minute. The contamination of this step is tested and the result is given in figure 19. Figure 19-Chromatogram of test of transporting TDU-tube to DHS carriage Conclusion: Transporting the TDU-tube to the DHS carriage will not cause much contamination, so this step has also negligible effect on the presence of contamination. 25

35 Area (absolute) II Septum of the sample vial Needles Movement of needles in DHS carriage rubber teflon Figure 20-Figuration of septum of sample vial The sample vial septum contains rubber and teflon (figure 20). Rubber is known for its adsorption of odour molecules. In this test an empty sample vial with the same septum is measured for several consecutive times. The result is given in figure 21. 8,00E+07 Total area of contamination of different blank DHS analyses 7,00E+07 6,00E+07 5,00E+07 4,00E+07 3,00E+07 2,00E+07 1,00E+07 0,00E Figure 21-Result for contamination of septum of the sample vial Conclusion: The contamination increases slightly. This can be explained by the fact that when the rubber and teflon are pierced, the contamination of the rubber (odorous material adsorbed on the rubber) can 26

36 Area (absolute) go into the sample vial through the pierced teflon. The described contamination can be avoided through the use of new septa for each analysis. Changing the septa for every analysis is however not expected to be essential. II DHS station and DHS carriage The DHS station and DHS carriage are not airtight. There are o-rings made of rubber present in the DHS carriage, these rubber o-rings can also cause contamination. The o-rings cannot be removed for technical reasons, so the contamination caused by the o-rings in the DHS carriage cannot be reduced. The contamination of the DHS station and DHS carriage is tested and the results are given in figure 22. 3,50E+07 Total area 3,00E+07 2,50E+07 2,00E+07 1,50E+07 Transporting TDU-tube to DHS carriage Blank DHS 1,00E+07 5,00E+06 0,00E+00 Figure 22-Results for contamination of blank DHS The chromatogram of blank DHS is given in attachment (see attachment 5, page 57). Conclusion: The DHS station and carriage causes a lot of contamination. The amount of contamination is shown by the difference in total area in figure 22. This significant contamination cannot be reduced. However the influence of the contamination on the analytical results can be minimized by increasing the split ratio. (see chapter II.3.3 Split ratio R in PTV, page 33). II O-rings on TDU-head The O-rings of the TDU-head are made of rubber. Rubber is known for its adsorption of odour molecules. The contamination is tested when the TDU head is exposed in the open environment within Scentarom for respectively 1 min and 1 day. It is also tested when the TDU head is wrapped in 27

37 Area (absolute) aluminium foil and exposed in the open environment within Scentarom for respectively 1 day and 1 week. The results are given in figure Total area of O-rings on TDU head min 1 day 1 day in aluminium foil 1 week in aluminium foil Figure 23-Contamination of O-rings on TDU head The chromatograms of these tests are in attachment (see attachment 6, pages 57-58). Conclusion: The O-rings on the TDU head causes contamination. To avoid this contamination the TDU heads are kept in an open environment outside Scentarom. II Carrier When preparing a test sample for DHS analysis, the sample needs to be diluted. Silica and a carbohydrate are used as carrier. Silica and carbohydrate are solids that can adsorb odour molecules, so can cause contamination. The contamination of carbohydrate and silica is tested when exposed in open environment within Scentarom and when exposed in an open environment outside Scentarom. The results are given in figure

38 Area (absolute) Total area of carbohydrate and silica 2,50E+08 2,00E+08 Carbohydrate exposed in open environment at Scentarom 1,50E+08 1,00E+08 Carbohydrate 2 days exposed in open environment outside Scentarom Silica exposed in open environment at Scentarom 5,00E+07 Silica 2 days exposed in open environment outside Scentarom 0,00E+00 Figure 24-Results of contamination of carbohydrate and silica The chromatograms of these test are in attachment (see attachment 7, page 59-60). Conclusion: Carbohydrate causes a lot of contamination. Also silica adsorbs components present in the open environment within Scentarom. To reduce this contamination carbohydrate and silica need to be kept in a glass jar in an open environment outside Scentarom. After each use, the carriers need to be exposed to open air outside Scentarom during several hours before closing the jar. 29

39 II.2.4 Conclusion To reduce the contamination of the TDU-tube in TDU and the PTV-liner in PTV a sequence of blank analyses needs to be done. The results of these blank analyses are given in figure 25. before after Figure 25-Chromatograms of blank TDU The first chromatogram in figure 25 is the contamination of the tenax before performing a blank TDU and the second chromatogram is the contamination the tenax after performing a blank TDU. After 2 measurements the tenax in the tenax tube and tenax liner are reduced to almost blank. These analyses must be done before performing a DHS analysis. Also the septum of the sample vial needs to replaced after every analysis. After using carbohydrate and silica these adsorbens needs to be exposed in an open environment outside Scentarom. 30

40 Thus: When performing a DHS analysis, the following sequence is performed. 1. Blank TDU Blank TDU is performed to eliminate the contamination on the tenax. This analysis will be repeated till the tenax is blank. When the tenax is blank (see figure 25), blank DHS will start. 2. Blank DHS Blank DHS is a DHS analysis with an empty sample vial. This is performed to evaluate the contamination of a DHS analysis. 3. DHS analysis of sample This is a DHS analysis of a sample in the sample vial. 31

41 II.3 Parameters II.3.1 Introduction The DHS technique can be described in 3 steps: incubation step, trapping step and drying step (see chapter I Trapping of components, page 11). Every step of the DHS device has several parameters that can be adjusted. The next parameters will be tested: Split ratio R in PTV (see chapter I PTV combined with TDU, page 3) Sample temperature (T sample i and T sample tr ) Incubation time Δt i (Δt i = Δt i1 + Δ i2 ) Trapping gas flow F (V=constant) Trapping gas volume V (F=constant) The trap temperature (T tr ) is also a parameter that can be tested. This parameter is not tested in this work. Indeed, the temperature in the laboratories of Scentarom is 25 C, so a lower temperature is not possible. A higher temperature is has no advantage, because the components with a low boiling point will not adsorb on the tenax in the tenax tube when trapping the components. Table 2-Overview of the split ratio and the start split ratio Range Start Split ratio (R) 1/1-1/1250 1/10 Table 3-Overview of the parameters of steps of the DHS and the start parameters Range Start Transfer Heater C 150 C Incubation step Incubation C 30 C (incl. agitation step) Temperature (T sample i ) Total incubation Time min 1 min (Δt i ) Agitator On Time (Δt i2 ) s 60 s Agitator Off Time s 1 s Agitator Speed rpm 500 rpm Trapping step Volume (V) ml 100,0 ml Flow (F) ml/min 50,0 ml/min Trap Temperature (T tr ) C 25 C Incubation C 30 C Temperature (T sample tr ) Time (Δt tr ) / 2 min Drying step Volume ml 0 ml (optional) Flow ml/min / Trap Temperature C / (T dr ) Incubation C / Temperature (T sample dr ) Time / / Table 2 gives the split range and start value. 32

42 Table 3 gives an overview of the several parameters used in the DHS. It also gives the range of the parameters. All these parameters will be tested, and the best result will be selected. The last column in table 3 shows the start values of the parameters. These values will be adjusted to the most efficient value. II.3.2 Test sample The test sample used for parameter control consists of 12 components (table 4). Table 4-Overview of components in test sample Number in chromatogram (see attachments) Component (structures see attachment 8, page 60) Boiling Point ( C) Molecular weight (g/mol) Retention time (min) 1 diacetyl ,2 2 ethyl acetate ,5 3 cis-3-hexenol ,7 4 benzaldehyde ,7 5 ethyl hexanoate ,0 6 phenylethyl alcohol ,1 7.I & 7.II citronellal ,8 + 26,3 8 eugenol ,3 9 b-ionone ,8 10 g-undecalactone ,5 11 thibetolide ,4 12 benzyl salicylate ,7 Boiling point and molecular weight were found in data sheets of the products. The elution time of the components depends a.o. on boiling point, polarity and molecular weight of each components and column characteristics. These components show a good dispersion in the chromatogram. These components are representative for the most common chemical classes used when producing fragrance and flavours. II.3.3 Split ratio R in PTV See chapter I PTV combined with TDU, page 3. Split ratio is an important parameter. The concentration of the samples has been adapted so as to keep the amount of all injected components on the column independent from the split ratio used. After each analysis a blank DHS was performed to evaluate the contamination (see figure 26) The parameters of DHS for performing a DHS analysis are in attachment (see attachment 9, page 61). The following split ratios have been tested (table 5): Table 5-Overview of the different split ratios and corresponding concentrations that have been tested Test 1 Test 2 Test 3 Test 4 Split ratio (R) 1/10 1/25 1/50 1/100 Concentration (ppm)

43 area (absolute) Results: total area 4,50E+08 4,00E+08 3,50E+08 3,00E+08 2,50E+08 2,00E+08 1,50E+08 Total area of test sample Total area of blank DHS 1,00E+08 5,00E+07 0,00E+00 Splitratio 1/10 Splitratio 1/25 Splitratio 1/50 Splitratio 1/100 split ratio Figure 26-Total area of test sample and total area of contamination Split ratio 1/10 Split ratio 1/25 34

44 Split ratio 1/50 Split ratio 1/100 Figure 27-Chromatogram of blank analysis of split ratio 1/10, 1/25, 1/50 and 1/100 Figure 27 shows the chromatograms of a DHS analysis of an empty glass vial, so the peaks on the chromatogram are contamination present in the glass vial or contamination present during performing a DHS analysis. Conclusion: By increasing the split ratio, less contamination will reach the column and will be detected. This means that the contamination is present in the DHS unit and not in the test sample. Increasing the split ratio from 1/50 to 1/100, has minor advantage. The chosen split ratio for further testing will be 1/50 and concentration of the samples will be 500 ppm. II.3.4 Sample temperature (Tsample i and Tsample tr) during incubation step and trapping step See chapter I Trapping of components, page 11. The sample temperature is the temperature of the sample in the glass vial during incubation step (T sample i ) and trapping step (T sample tr ) and are kept equal. The concentration of the samples will be 500 ppm, the split ratio 1/50. (see attachment 10, page 61). The following incubation temperatures will be tested (table 6): Table 6-Overview of the different incubation temperatures that have been tested Test 1 Test 2 Test 3 Test 4 Test 5 Sample temperature T sample i and T sample tr ( C)

45 area (absolute) area (relative in %) Results: 30 Relative area C 50 C 65 C 75 C 85 C 0 Components Total area 4,50E+09 4,00E+09 3,50E+09 3,00E+09 2,50E+09 2,00E+09 1,50E+09 1,00E C 50 C 65 C 75 C 85 C 5,00E+08 0,00E+00 Figure 28-Influence of incubation temperature on the relative area and the total area of the components trapped The chromatograms of these tests are in attachment (see attachment 11, page 62-63) 36

46 Conclusion: The sample temperature has a significant influence on the signal. When using a low incubation temperature the components with a smaller elution time will have a higher area. When using 30 C, benzyl salicylate is not detectable. When using 85 C diacetyl and ethylacetate are almost not detectable. An incubation temperature of 65 C and 75 C shows the best results. An incubation temperature of 65 C will be selected, as a higher temperature could decompose the components. When analysing a sample with components that have a lower retention time, an incubation temperature of 30 C will be preferred. II.3.5 Total incubation time Δti See chapter I Trapping of components, page 11. The total time Δt i consists of two time intervals: incubation time and agitation time. The total incubation time Δt i and agitation time Δt i2 can be selected. By adjusting those 2 parameters the incubation time Δt i1 will change automatically. The concentration of the sample is 500 ppm, the split ratio is 1/50 and the incubation temperature is 65 C. (see attachment 12, page 64). The following incubation times will be tested (table 7): Table 7-Overview of the different incubation times that have been tested Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Test 7 Total incubation time Δt i 0 min 15 s 1 min 2 min 5 min 10 min 15 min Agitation time Δt i s 1 min 2 min 5 min 10 min 10 min Incubation time Δt i1 (min) min 37

47 area (absolute) area (relative in %) Results: 25 Relative area min 15 s 1 min 2 min 5 min 10 min 15 min Components Total area 3,50E+09 3,00E+09 2,50E+09 2,00E+09 1,50E+09 1,00E+09 5,00E+08 0 min 15 s 1 min 2 min 5 min 10 min 15 min 0,00E+00 Figure 29-Influence of incubation time on the relative area and the total area of the components trapped The chromatograms of these tests are in attachment (see attachment 13, page 64-66). 38

48 When there is no incubation step, the total area of the components is low. An incubation step of 15 s does not give much improvement. One minute incubation gives a significant increase of the total area, but further increase gives limited ameliorations (see figure 29). Conclusion: The incubation step is necessary to trap the components with a higher elution time. It has a minimum duration required to trap the components with a higher elution time. To reduce the total time of a DHS analyse an incubation time of 5 minutes will be selected. II.3.6 Trapping gas flow F (V=constant) See chapter I Trapping of components, page 11. When adjusting the trapping gas flow at a constant trapping gas volume, the trapping time (Δt tr ) will change. The concentration of the sample is 500 ppm, the splitratio is 1/50, the incubation temperature is 65 C and the incubation time is 5 minutes. (see attachment 14, page 67). The following trapping flows will be tested (table 8): Table 8-Overview of the different trapping gas flows that have been tested Test 1 Test 2 Test 3 Test 4 Test 5 Trapping gas flow F (ml/min) Trapping gas volume V (ml) Trapping time Δt tr (min)

49 area (absolute) area (relative in %) Results: 30 Relative area ml/min 10 ml/min 25 ml/min 50 ml/min 100 ml/min 0 Components Total area 3,00E+09 2,50E+09 2,00E+09 1,50E+09 1,00E+09 5 ml/min 10 ml/min 25 ml/min 50 ml/min 100 ml/min 5,00E+08 0,00E+00 Figure 30-Influence of trapping gas flow on the relative area and the total area of the components trapped The chromatograms of these test are in attachment (see attachment 15, page 67-69) 40

50 Conclusion: The trapping gas flow has a minor influence on the amount of the components trapped. However when using a lower trapping gas flow the trapping time will increase. To reduce the total time of an DHS analysis to a reasonable time, a flow of 50 ml/min will be selected. II.3.7 Trapping gas volume V (F=constant) See chapter I Trapping of components, page 11. When adjusting the trapping gas volume at a constant trapping gas flow, the trapping time (Δt tr ) will change. The concentration of the sample is 500 ppm, the split ratio is 1/50, the incubation temperature is 65 C, the incubation time is 5 minutes and the trapping flow is 50 ml/min. (see attachment 16, page 69). The following trapping flows will be tested (table 9): Table 9-Overview of the different trapping gas volumes that have been tested Test 1 Test 2 Test 3 Test 4 Test 5 Trapping gas volume V (ml) Trapping gas flow F (ml/min) Trapping time Δt tr 12 s 1 min 2 min 3 min 4 min 41

51 area (absolute) area (relative in %) Results: 30 Relative area ml 50 ml 100 ml 150 ml 200 ml 0 Components Total area 3,00E+09 2,50E+09 2,00E+09 1,50E+09 1,00E ml 50 ml 100 ml 150 ml 200 ml 5,00E+08 0,00E+00 Figure 31-Influence of trapping gas volume on the relative area and the total area of the components trapped 42

52 The chromatograms of these tests are in attachment (see attachment 17, page 70-71) Conclusion: The trapping gas flow has a considerable influence. When increasing the trapping gas flow the components with a higher elution time will have a higher area. The total area of components trapped changes dramatically with the volume V increased. This change seems to be asymptotic. The difference between 150 ml and 200 ml becomes minor, so 150 ml as trapping gas volume can be selected. II.3.8 Reproducibility The concentration of the sample is 500 ppm, the split ratio is 1/50, the incubation temperature is 65 C, the incubation time is 5 minutes, the trapping flow is 50 ml/min and the trapping volume is 150 ml. (see attachment 18, page 72). The DHS analysis is repeated seven times. Each time a new test sample was prepared. The results are given in figure

53 area (absolute) area (absolute) Results: 8,00E+08 Absolute area 7,00E+08 6,00E+08 5,00E+08 4,00E+08 3,00E+08 2,00E+08 1,00E+08 0,00E+00 Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Test 7 Components Total area 4,00E+09 3,50E+09 3,00E+09 2,50E+09 2,00E+09 1,50E+09 1,00E+09 Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Test 7 5,00E+08 0,00E+00 Figure 32-Reproducibility of DHS on the absolute area of each component and the total area of all components trapped 44

54 area (relative in %) Table 10-Total area of the individual analyses Total area Test Test Test Test Test Test Test Table 10 gives the total areas of the individual analyses. Test 2 has a higher total area than the other tests, so the Dixon-Q test and the 4s standard has been performed to evaluate the result of test 2. Conclusion: After performing the Dixon-Q test and the 4s standard there can be concluded that test 2 is no outliner. The reproducibility of these results are sufficient enough to use this method in further analyses, as Scentarom will be in the future more interested in relative ratio among constituent of compositions (see attachment 19, page 72). II.4 Comparing DHS analysis and SHS analysis A SHS-type analysis has been performed and described by Pien earlier [15]. The concentration of the test sample when performing a SHS is 100 ppm with a split ratio 1/10. The concentration of the test sample when performing a DHS is 500 ppm with a split ratio 1/50. Results are given in Figure 33: 30 Relative area DHS SHS 5 0 Components 45

55 area (absolute) area (absolute) 7,00E+08 Absolute area 6,00E+08 5,00E+08 4,00E+08 3,00E+08 2,00E+08 DHS SHS 1,00E+08 0,00E+00 Components Total area 3,00E+09 2,50E+09 2,00E+09 1,50E+09 1,00E+09 DHS SHS 5,00E+08 0,00E+00 Figure 33-Comparing DHS with SHS on the absolute, relative and total area of the components trapped The chromatograms of these tests are in attachment (see attachment 20, page 73). 46

56 Conclusion: The sensitivity of DHS and SHS are in the same magnitude for this test sample (fragrance compounds on carrier). DHS has a higher sensitivity for the central components like citronellal and phenylethyl alcohol. DHS however is a fully automated technique through the use of the robot, which gives an advantage over SHS when several samples must be treated. II.5 Shampoo When analysing the shampoo four different methods of diluting the shampoo were performed. This is done to see which method gives the best results. The split ratio is adapted accordingly to keep the responses within dynamic range. Four different methods: % shampoo % shampoo + 50 % H 2 O % shampoo + 50 % silica % shampoo + 25 % H 2 O + 50 % silica These 4 test shampoos were analysed with a different split ratio and the results are given in Fout! Verwijzingsbron niet gevonden.: 1. Analysed with split ratio 1/ Analysed with split ratio 1/ Analysed with split ratio 1/ Analysed with split ratio 1/ % shampoo 47

57 50 % shampoo + 50 % H 2 O 50 % shampoo + 50 % silica 48

58 25 % shampoo + 25 % H 2 O + 50 % silica Figure 34-Chromatograms of shampoo with concentration of 100 % shampoo, 50 % shampoo 50 % H2O, 50 % shampoo 50 % silica and 25 % shampoo 25 % H2O 50 % silica Conclusion: When analysing the shampoo diluted in silica, all peaks are separated and have symmetrical shape. When analysing the shampoo with a concentration of 100 % or diluted in 50 % H 2 O, not all peaks are separated or have a symmetrical shape, see figure 34 chromatogram one and two. The reason why these peaks are not separated is not known. Further tests has to be done. 49

59 Conclusion Study of the DHS technique for fragrant compositions in Scentarom reveals following features: A. Contamination Contamination is caused by following steps (+ appropriate reduction): Preservation conditions of adsorption material (tenax in tenax tube and tenax in tenax liner) These materials are now kept in a glass jar sealed with aluminium outside Scentarom. TDU-tube in TDU-tray This contamination is eliminated by performing analyses in sequence, so the TDU-tube doesn t have to stay in the TDU-tray. PTV-liner in PTV and TDU-tube in TDU The PTV-liner and TDU-tube cause contamination when no analyses are performed. This contamination is eliminated by performing a sequence of blank TDU. DHS station and DHS carriage This contamination cannot be reduced. However the influence of the contamination on the analytical results can be minimized by increasing the split ratio. O-rings on TDU-head This contamination will be avoided by keeping the O-rings in an open environment outside Scentarom. Carrier To reduce this contamination the carriers are kept in a glass jar in an open environment outside Scentarom. Steps that give negligible contamination: Unlocking of TDU Transporting TDU-tube to DHS carriage Septum of the sample vial B. The following experimental parameters were tested: Split ratio R in PTV Sample temperature (T sample i and T sample tr ) during incubation step and trapping step By increasing the sample temperature the components with a higher elution time will have a higher area. Total incubation time Δt i The incubation time is necessary to trap the components with a higher elution time. It has a minimum duration required to trap the components with a higher elution time. Trapping gas flow F The trapping gas flow has a minor influence on the amount of the components trapped. Trapping gas volume V The trapping gas flow has a considerable influence. When increasing the trapping flow the components with a higher elution time will have a higher area. 50

60 As a result, optimal experimental parameter settings are: Spit ratio: 1/50 Sample temperature: 65 C Total incubation time: 5 min Trapping gas flow: 50 ml/min Trapping gas volume: 150 ml C. Comparing DHS analysis to SHS-type analysis: The sensitivity of DHS and SHS-type are similar for this test sample and test conditions. However, DHS has a higher sensitivity for the medium volatile components. Furthermore, DHS is a fully automated technique through the use of the robot, which makes it the method of preference. 51

61 Bibliography 1. Fulton G. Kitson, B.S.L., Charles N. McEwen, gas chromatography and mass spectrometry, a practical guide. 1996, California: Academic Press, Inc. 2. Kyaw Thet, N.W. gas chromatography [cited february]; Available from: Gas_Chromatography. 3. Gerstel, Thermal Desorption Unit TDU with C506-Operation Manual. 2009: Gerstel. 4. Gerstel. Thermal Desorption Unit TDU-certified supplies [cited march]; Available from: 5. R.I.C., SHS-DHS-TD-TE-SBSE-HSSE Lagard, D., G.A. Reiner, and G.W. Christensen. Analysis of the headspace proximate a substrate surface containing fragrance-containing microcapsules [cited may]; Available from: 7. Verberckmoes, A., Spectroscopische technieken. 2012, Gent. 8. Electron impact. [cited march]; Available from: 9. Mass Spectrometry Tutorial. [cited march]; Available from: Gates, P. Quadruple & Triple Quadrupole (QQQ) Mass Analysis [cited march]; Available from: GC/MS-TOF Description. [cited march]; Available from: Hamamatsu, Electron multiplier tubes and ion detectors, in Photomultiplier Tubes: Basics and Applications p How electron multipliers work [cited march]; Available from: Kevin Goodner, R.R., Practical analysis of flavor and fragrance materials. 2011: Whiley. 15. Pien, N., Static headspace characterisation of odorants in complex matrices by hyphenated GC-MS techniques, in Hogeschool Gent. 2013: Gent. 16. Hübschmann, H.-J., Handbook of GC/MS: Fundamentals and Applications. 2009, Weinheim: Whiley-VCH. 17. Gerstel. gerstel dynamic headspace DHS. [cited march]; Available from: Gerstel, Dynamic Headspace DHS with TDU and MPS-operation manual EVISA. Gerstel GmbH & Co.KG - MulitPurpose Sampler MPS for GC and GC/MS [cited february]; Available from: CoKG/MultiPurpose-Sampler-MPS-for-GC-and-GCMS-. 52

62 Attachments Attachment 1-The several steps of the MPS for performing a DHS analysis Attachment 2-Figuration of PTV-tube, TDU-tube and sample vial Attachment 3-Chromatograms of contamination of PTV-liner in PTV Attachment 4-Chromatograms of contamination of TDU-tube in TDU Attachment 5-Chromatogram of blank DHS Attachment 6-Chromatograms of contamination of O-ring Attachment 7-Chromatograms of contamination of carrier Attachment 8-Overview of structures of different components Attachment 9-Parameters of DHS for adjusting split ratio Attachment 10-Parameters of DHS for adjusting incubation temperature Attachment 11-Chromatograms of analyses for adjusting incubation temperature Attachment 12-Parameters of DHS for adjusting incubation time Attachment 13-Chromatograms of analyses for adjusting incubation time Attachment 14-Parameters of DHS for adjusting trapping gas flow Attachment 15-Chromatograms of analyses for adjusting trapping gas flow Attachment 16-Parameters of DHS for adjusting trapping gas volume Attachment 17-Chromatograms of analyses for adjusting trapping gas volume Attachment 18-Parameters of DHS when performing a DHS Attachment 19- Reproducibility of DHS on the relative area of each component trapped Attachment 20-Chromatograms of DHS and SHS comparison

63 Attachment 1-The several steps of the MPS for performing a DHS analysis The dynamic headspace is a fully automated technique. It is automated by a robot above the GC/MS, called MPS (Figure 10). When analysing a sample, the following measurements has to be performed when trapping all components. First the tenax of the TDU and PTV has to be blank, to eliminate components present in the air and on the tenax. When the tenax is blank, the DHS method is started on a blank sample. A blank sample is an empty glass vial. And the next step is the DHS method for a sample. These methods will be performed in a sequence. It starts with placing an empty TDU tube in the TDU and placing an TDU tube with tenax in the tray on the MPS. The method for making the tenax of the TDU and PTV blank starts (blank TDU). The following steps will be done by the MPS: 1. Tenax tube will be removed from the TDU and placed in hold 2. Tenax tube (containing contamination) will be removed from the tray and placed in the TDU Now the desorption step and the injection step starts. After the injection step, the GC/MS starts to measure. The next step is performing a DHS analysis of a empty sample (blank DHS). It starts with placing an empty glass vial in the tray on the MPS. The following steps will be done by the MPS: 3. Empty sample vial is removed from the tray and placed in the DHS Incubation step starts. 4. A plastic TDU tube is removed from the DHS carriage and placed in hold in the DHS 5. DHS carriage is placed above the empty glass vial 6. TDU tube with tenax is removed from the TDU and placed in the DHS carriage Trapping step starts. 7. While the trapping step starts, the empty TDU tube is removed from hold and placed in the TDU. This is necessary because of the pressure over column. The GC needs to be closed at any time 8. After the trapping step, the empty TDU tube is removed from the TDU and placed in hold 9. TDU tube with tenax is removed from the DHS carriage and placed in the TDU The desorption step and injection step starts. 10. While the desorption step starts, the DHS carriage is moved backward and the plastic TDU tube is placed in the DHS carriage. Also the empty glass vial in placed back in the tray on the MPS After the injection step, the GC/MS starts to measure. The following step is performing a DHS analysis of a sample (DHS analysis of sample). It starts with filling up the empty glass vial, used in the previous method, with the sample and placing it in the tray on the MPS. Steps 3 to 10 will be repeated by the MPS and in this method a glass vial with sample will be used. After the measurement of the DHS the following steps will be done by the MPS: 11. After the measurement the TDU tube with tenax is removed from the TDU and placed in the tray 12. The empty TDU tube is removed from hold and placed in the TDU 54

64 Attachment 2-Figuration of PTV-tube, TDU-tube and sample vial PTV TDU sample vial Attachment 3-Chromatograms of contamination of PTV-liner in PTV 1 day 3 days 55

65 1 week Attachment 4-Chromatograms of contamination of TDU-tube in TDU 3 days 1 week 56

66 2 months Attachment 5-Chromatogram of blank DHS Attachment 6-Chromatograms of contamination of O-ring 1 min 57

67 1 day 1 day in aluminium foil 1 week in aluminium foil 58

68 Attachment 7-Chromatograms of contamination of carrier Carbohydrate exposed in open environment within Scentarom Carbohydrate exposed in open environment outside Scentarom Silica exposed in open environment within Scentarom 59

69 Silica exposed in open environment outside Scentarom Attachment 8-Overview of structures of different components Component Structure Component Structure diacetyl citronellal ethyl acetate eugenol cis-3-hexenol b-ionone benzaldehyde g-undecalactone ethyl hexanoate thibetolide phenylethyl alcohol benzyl salicylate 60