Determination of Volatile Substances Proof of Food Adulteration
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1 ANALYSIS OF FOOD AND NATURAL PRODUCTS LABORATORY EXERCISE Determination of Volatile Substances Proof of Food Adulteration (method: gas chromatography with mass spectrometric detection) Exercise guarantor: Assoc.Prof. Ing. Jan Poustka, Ph.D.
2 CONTENT Basic knowledge required for entry test... 3 Other recommended questions... 3 The exercise tasks (alternatives)... 4 The time frame of laboratory exercise... 5 A. Analytical procedure determination of volatile substances using solid phase microextraction (SPME)... 6 A1. Method applicability... 6 A2. Method principle... 6 A3. Instruments, devices, materials... 6 A4. Chemicals... 6 A5. Working procedure... 6 B. Analytical procedure determination of volatile substances using direct analysis by split injection... 8 B1. Method applicability... 8 B2. Method principle... 8 B3. Instruments, devices, materials... 8 B4. Chemicals... 8 B5. Working procedure... 9
3 Basic knowledge required for entry test principle of sample preparation for volatile substances determination (isolation and extraction methods) principle of gas chromatography (stationary phases, mobile phases), principle of mass spectrometry electron ionization, mass spectrum Other recommended questions a) Which groups of volatile compounds in food you know? What are volatile substances in foods typical? How important is knowledge of the composition of volatile substances in food technology? What methods are used to isolate volatiles? Which methods are used for the determination of volatile substances? What the difference is between targeted and profile analysis of volatile substances? b) What is the principle of solid phase microextraction - SPME? What types of technical implementation of SPME you know? What are the main advantages and disadvantages of SPME application?
4 The exercise tasks (alternatives) A. Head space (alter. direct) solid phase microextraction (HS-SPME alter. DI- SPME) of aromatic compounds from e.g. flavoured water or fruit juices; and determination of their profiles. B. Determination of the profile of volatile compounds in natural and artificial flavouring essence - their comparison, alter. in flavoured samples, fruit juices, etc.
5 The time frame of laboratory exercise 30 min Introduction + examination of required knowledge 20 min Discussion: methods of determination, results evaluation 60 min Instructions: practical realization sample preparation, sampling 60 min GC-MS direct analysis GC-MS analysis - SPME Evaluation and discussion of findings: 60 min Software functions for data processing Displaying of MS spectra, chromatograms for selected ions Deconvolution principle recognition of coeluting compounds Automatic spectrum search in library Interpretation of spectral information and identification suggestion 10 min Exercise finishing (glassware and place cleaning)
6 A. Analytical procedure determination of volatile substances using solid phase microextraction (SPME) A1. Method applicability The method is applicable to all analytes that exhibit sufficient affinity to the sorption phase of sampling fibre and are sufficiently volatile and thermostable under the conditions of measurement. A2. Method principle The sample is extracted after stabilization of equilibrium from headspace by sorption fibre (or directly from the sample). After the sorption substances are released by thermal desorption in the GC injector; separated and detected. A3. Instruments, devices, materials - gas chromatograph coupled to mass spectrometric detector - fused silica capillary column HP-INNOWAX: 30m 0,25mm 0,25μm with stationary phase polyethylene glycol - helium (99,9990 %) - digital thermometer with stainless steel sensor - manual SPME folder and SPME fibres (65 µm PDMS/DVB) - vials with septum screw cap (volume 4 ml) - pipettes 1 a 2 ml - heated magnetic plate equipped with thermostat and stirrer - laboratory stand A4. Chemicals - A5. Working procedure For the measurement, the following samples are prepared: 1. Control tempering bottle for sorption temperature setting - to bottle pipette 2 ml of tap water or the test sample and place magnetic stirrer (When applying DI-SPME pipette 3.5 ml).
7 2. The bottle with the sample - pipette 2 ml of sample (e.g. flavoured mineral water), which is free of dissolved gases (using ultrasound) and add before closing stirrer (When applying DI-SPME pipette 3.5 ml). HS-SPME realization: (instructional video is available on the website - ) 1. Placed on the heating plate the control bottle, cover it by thermostabilization cylinder and after previous septum perforation by pin; introduce the temperature sensor in. Start with mixing (set appropriate rotation) and temperature (40 C). 2. The sample bottle is placed on the heating plate, cover it thermostabilization cylinder and let equilibrate for 5 minutes. 3. Into the vial with the sample after previous septum perforation by pin placed fibre inserted in the manual SPME holder which is fasten to the rack on the stand. Then sorption fibre slide out of the protective SPME needle holder. 4. Sorption is performed for 5 minutes, then the fibre is inserted into the protective needle, pulled out the SPME holder, which is transferred immediately and introduced into the GC injection port and possibly fasten. Then fibre is slide out and at the same time the GC-MS analysis is started. Desorption time: for splitless period - 5 min; further the fibre is left in injection port throughout analysis - cleaning (split mode). GC-MS analysis: Separation is carried out on a gas chromatograph Hewlett-Packard HP 6890 using fused silica capillary column HP-INNOWAX (30 m 0.25 mm 0.25 mm). Subsequent detection (measurement of mass spectra) is performed using HP5973 mass selective detector. Conditions of separation and detection: Injection: splitless 5 min; temperature 250 C Temperature program: 45 C (1min), 20 C/min to 250 C (3,75min) overall analysis time 15 min Carrier gas: He, constant flow rate 1 ml/min Temperature of tranferline: 230 C MS mode: scan amu
8 B. Analytical procedure determination of volatile substances using direct analysis by split injection B1. Method applicability The method is applicable to all analytes that exhibit sufficient volatility and thermostability under the conditions of measurement. B2. Method principle Option A - solid and semi-solid samples. The sample is extracted with a solvent (mixture), which corresponds to the expected physic-chemical properties of investigated substances. Then follows ultrafiltration, which is necessary for the removal of solids parts of sample. Option B - liquid samples. The sample is diluted with a suitable solvent, possibly filtered. The obtained extract (after appropriate dilution) is then analyzed by gas chromatography coupled to mass spectrometry - using split injection. B3. Instruments, devices, materials - gas chromatograph coupled to mass spectrometric detector equipped by autosampler - fused silica capillary column HP-INNOWAX: 30m 0,25mm 0,25μm with stationary phase polyethylene glycol - helium (99,9990 %) - microsyringes - pipettes 1, 2 a 5 ml - Pasteur pipettes with balloon - extraction cuvette - filter syringes - syringe filters - sampling needle - 2 ml vials for autosampler B4. Chemicals - ethanol (superfine alcohol), methanol
9 B5. Working procedure Option A - solid and semi-solid samples. Into extraction plastic tube with a screw cap 0.5 g of sample is weighted, then added 2 ml of methanol and, after the capping the tube is vigorously shaken for 2 minutes. Then, using a syringe with attached sampling needle part of the extract is sampled, replace the needle for the syringe filter and pass through ca 0.5 ml of extract into the bottle. This extract using microsyringe is diluted x with methanol to another vial, which is capped. Injection volume to gas chromatograph is 1 µl. Option B - liquid samples. Sample is directly diluted x with methanol using a microsyringe into the vial, which is then capped.. Injection volume to gas chromatograph is 1 µl. Conditions of separation and detection: Injection: split (1:50); temperature 250 C Temperature program: 45 C (1min), 20 C/min to 250 C (3,75min) overall analysis time 15 min Carrier gas: He, constant flow rate 1 ml/min Temperature of tranferline: 230 C MS mode: scan amu
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