General aspects of GC analysis of volatiles

Transcription

1 General aspects of GC analysis of volatiles Kaunas University of Technology Content Introduction: food volatiles, principles of GC separation Main parameters for the optimisation of volatile analysis columns carrier gas samples inlet systems temperature programming detectors Identification retention time, Kovats and retention indices spectral data (GC/MS) Multidimensional GC, GCxGC What are the volatile compounds? Physically it is rather imprecise term, because there is no precise boundary in the boiling temperature between volatile and non-volatile compounds For foods: the compounds which are present in detectable concentrations in headspace (both in instrumental and sensory analysis) may be considered as volatile compounds Depends on the matrix (food), compound concentration, physical and chemical properties Why to analyse volatile compounds? Responsible for food aroma (also off-odours - deffects) May indicate about desirable and undesirable changes (food spoilage) May indicate about reactions taking place in foods May provide scientific information for the control of food flavour during processing/storage May provide scientific information for designing food flavourings Therefore, we need about volatiles as much information as possible 1

2 Main steps in volatile analysis Sample preparation Isolation Preliminary fractionation Concentration Separation (GC) Identification (GC-MS other specroscopic techniques) Quantification GC main techniques for the analysis of volatiles Sample preparation GC analysis Extract of volatiles Large number of compounds Different chemical properties (functional groups, reactivity, etc.) Different physical properties (boiling temperature, flashpoint, polarity, etc.) Different concentrations Gas chromatography - definition Gas-liquid chromatography (GLC), or simply gas chromatography (GC), is a type of chromatography in which the mobile phase is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen, and the stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside glass or metal tubing, called a column. Martin and Synge for the developments in GC separation were avarded Nobel prize in 1952 (chemistry) 2

3 The principle of chromatographic separation When the components moving through the column an equilibrium is formed between components, which are adsorbed by the column stationary phase and the components which dissolve in column mobile phase: Component separation in GC The time, during which a component is retained in the column is called retention time (t R ) in analytical chromatography and retention factor R f in preparative chromatography High K the compound moves fast low t R (high R f ) Low K the compound moves slowly high t R (low R f ) Stationary Phase Collect/ Detect Components mobile phase mobile phase mobile phase Component Component mobile phase mobile phase Component mobile phase Inject/ Add Sample Stationary Phase Principal scheme of gas chromatography Blue molecules are better soluble in a liquid (or less volatile) than green molecules Column effectiveness Are the peaks separated efficiently? If no how to improve the separation Separation Does the peak always reach baseline (0)? Peak shape: is it Gaussian? 3

4 Separation Trennzahl TZ = number of baseline separated peaks between one pair of system homologues (Kaiser) Analysis of volatile compounds by GC Composition (quantitative information) identification of the components TZ = t w R ( B ) 0,5( A) t + w R ( A) 0,5( B ) 1 A and B two members of homologue, differ in one methylene group Content (quantitative) - measurement of component concentration The number of peaks which may be recorded between the two contiguous homologues members Injector Carrier gas Configuration of a GC Data acquisition and processing Sample Detector Essential parameters for the optimisation of GC analysis Carrier gas Sample inlet Systems GC oven Analytical column Columns Injection techniques Analysis temperature program Detection system 4

5 Column technology Separation of volatile compounds proceeds in column, therefore selection of a right column is a crucial step in the GC analysis of volatile Packedcolumnsvsopentubes Stationary phases Testing Packed Column Carrier gas flow Glass tube (2-4 mm i.d.) Lenght 2-4 m Non reactive patricles (different shapes and sizes possible) Stationary Phase Characterisation of the column by 1) Amount and weight of stationary phase 2) Type of carrier particles 3) Dimensions e.g. 1% OV-1 on Chromosorb W Capillary Columns Wall Coated Open Tubes WCOT Carrier gas Very pure fused silica tube material coated outside with polyimide to protect the column and to give flexibility Stationary phase is just on the inner wall of the tube, there is a free gas flow and no EDDY DIFFUSION, that means a drastically increase in length is possible Types of WCOT WCOT are classified by 1) Inner diameter: MICROBORE 250 µm inner diameter and less (down to 10 µm!!!) high speed, high resolution types with low capacity MEDIUMBORE 320 µm id a good compromise between capacity and performance MEGABORE 530 or 750 µm id working horses for high sample amount and capacity 2) Film Thickness: Normal film thickness: µm Thin Film : down to 1 nm (nanometer) Thick Film: up to 10 µm Length of column: standard size m Longest column (Guiness books of records) Chrompack: 2.2 km For high speed analysis: 1 or 2 m with small inner diameter and thin film (limited by data acquisition!) 5

6 Comparison- packed vs. capillary column Polarity of stationary phase Packed column 3 m length, 2 mm id OV1 Apolar - + Polar Capillary column 35 m length, 0.28 mm id, OV1 Stationary phases 6

7 Cross linking of stationary phase Due to the cross linking a chemical bonding between the surface and the stationary phase is achieved. Due to this effect the temperature range, the stability and the life time of the column is increased. The cross linking is made by radiation with γ-rays Sample: the mixture consisting of 3 components is eluting through the column OH O bp 212 C bp 207 C bp 177 C OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH For which compound retenion time will be the lowest (moves fastly)? For which compound retenion time will be the highest (moves slowly)? The answer OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH Temperature range of stationary phases O OH Will move most fast, the lowest Rt, because it is pure hydrocarbon and does not form strong intermolecular bonds with stationary phase Will move more slowly, medium RT; may form partial hydrogen bonds (donor of electron pair) with stationary phase some intermolecular interactions Will move most slowly, the highest RT; may form effective hydrogen bonds (donor of electron pair and hydrogen) with stationary phase strong intermolecular interactions Lower Temperature: is given by the melting point of the phase (-60 C to +40 C depending on the polarity) Upper Temperature: is given by the vapour pressure of the stationary, liquid (!) phase. The vapour pressure increases in an exponential manner with the temperature!!!!! Maximum upper Working Temperature: is defined as the temperature where the column can be used for 6 month and loosing 50% of the stationary phase 7

8 pa FID1 A, (G:\DATA\FSME\FSMESTA.D) min Column bleeding Influence of film thickness Abundance Ion ( to ): SB_REKL.D Temp. program: 15 m/0.3 mm, SE C (1 ) at 10 C/min to 310 C (5 ) 20 o C programmed 2 o C/min to 80 C Bleeding Time--> Abundance Influence of polarity-fame TIC: FSSTD.D HP-5 MS order of elution: unsatuated BEFORE saturated Capacity of capillary columns Time--> HP-WAX unsatuated AFTER saturated C 14:0 C 14:1 C 16:0 C 16:1 C 18:0 C 18:1 C 18:2 C 18:3 C 20:0 C 20:1 C 20:2 C 20:3 C 20:4 C 22:0 C 22:1 C 24:0 C 22:6 C 24:1 8

9 Comparison of columns Special columns for the separation of enantiomers: carvone Special columns for the separation of enantiomers: limonene Cyclodextrin columns Fresh citrus, orange like Harsh, turpentine-like, lemon note 9

10 Enantioselective separation of linalool Composition of enatiomers in natural and synthetic oils (authenticity testing) natural synthetic Carrier gas Mobile GC phase Should be inert do not react with the majority of organic compounds Usually pressure range psi Carrier gases Mainly used: Nitrogen A: inexpensive D: slow separation see van-deemter Hydrogen A: good separation D: explosive mixtures with air/oxygen Helium A: similar separation behaviour like H 2, Excellent for GC/MS D: price, extra gas cylinder Purity: Main impurities: 4.6 means % less than % impurities 5.0 means % Water Oxygen Mineral Oil Always try to use metal tubes instead of plastic materials (reduces background signal and avoids contamination 10

11 Van Deemter equation Describes the relation between the linear gas velocity and the HETP (Height Equivalent of a Theoretical Plate) A theoretical plate is the length of the column where a balance between the mobile and the stationary phase is reached Comparison of different carrier gases HETP N 2 He H 2 Constant flow Constant pressure systems! Viscosity of gases increase with the temperature due to higher movements of gas molecules Linear velocity [cm/s] Effect of carrier gas on separation Injector (injection systems) The sample is injected with microsyringe (μl) The amount of injected sample depends on the column and desirable separation 11

12 Inlet systems Compounds for GC must be volatile and MUST not decompose during vaporisation!! Samples for standard injection devices must be either liquid by itself (e.g. essential oil) or should be dissolved in an appropriate solvent SPLIT-SPLITLESS INJECTOR COOL ON-COLUMN INJECTOR PTV DIRECT INJECTION INLET FOR SOLID SAMPLES Split-splitless injector Compounds were injected (~ 1 µl = 1 mg, if density is 1 g/ml) and vaporized at temperatures between C 2 different modes of injection for different samples and concentration ranges Split mode: Concentration of compounds of interest relatively high Split ration 1:5 1:1000 Splitless mode: for trace analysis All sample compounds are transferred to the column Switching the Split Valve in the Splitless Mode Improper purge valve setting 12

13 On column injector Advantage: suitable for thermolabile compounds Disadvantage: Samples have to be clean Avoid any contamination with non volatiles (Sugar, minerals ) ONLY for trace compounds Comparison OC - SSL HANDLING OF SYRINGE IS VERY IMPORTANT! Temperature control Effect of temperature Isothermal Gradient a) isothermal 45 o C b) isothermal 145 o C Temp (deg C) Time (min) c) Programmmed from 30 o C to 180 o C 13

14 Temperature program Solvent vapours during injection syringe Carrier gas septum Septum purge GC column Split vent Split Different types of liners Single taper Double taper Jennings Deactivated glass wool 14

15 Choosing the proper liner There are three liner characteristics that must be considered for each application: Linervolume Liner treatments or deactivation Any liner design feature that might affect carrier gas flow through the inlet or sample vaporization Vapor volumes of solvents Flow calculator Temp. Injector: 250 C; Column Head pressure: 138 kpa01 If the vapour volume is too great for the liner, reproducibility and sensitivity can be compromised, due to backflash or loss of the sample into the septum purge or split lines. How to test the system? Test mixture for activity To maintain a proper GC system it is necessary to check it on a regular base WHAT is regular? Depends on the type of analysis (polar, non polar, dirty samples, high number of injections, different methods for different boiling ranges and different analytes...) Check it with a test mixture Record the performance Keep a log book with the history of ever column (date of purchase, date of installation, approximate number of injections, types of samples...) Even test every new column you buy! n-alkanes from C 9 -C 16 α-pinene β-pinene p-cymene 1,8-Cineole Menthol Dodecanoic acid methyl ester Acenaphtene Dodecanole Inject 1 µl splitless of this test mixture diluted in diethyl ether or n-pentane at a concentration of 10 mg/l (injected amount of each compound is 10 nanogram) 15

16 Abundance Test mixture for activity Liner aktiv active GC-Parameters: HP5-MS 30 m, 0,25mm, 0,25 µm df Dirty liner with glass wool Dirty liner without glass wool Abundance ? [min] Liner desaktiviert deactivated? Temp. program: 30 C (0 min) with 5,3 C/min to 250 C Injection 1µL splitless Injector: 250 C CHP: 0,81 bar constant pressure deactivation process ensures high performance! [min] Cleaning of liners Rinsing a column Remove liner from injector Remove glass wool plug Immerse liner in a mixture of 30 % H 2 O 2 & H 2 SO 4conc. ATTENTION VERY CORROSIVE!!!!!!! Wash carefully with distilled water followed by acetone Dry in an oven at 300 C Vacuum hose to pump Pasteurpipette GC column Immerse the liner in pure Hexamethyldisilazane (HDMS) Put it back into the hot oven for a couple of minutes Remove and install still warm into the injector 30 ml Headspace Vial with septum at the screw cap Side of Detector!!! 4 ml Vial 16

17 Rinsing a column Remove GC column from GC Immerse the detector end in the vial with the rinsing solvent Puncture the septum of the 30 ml vial and put the other column end into it Suck each of the following solvents through the column (in this order) 1) Methanol 2) Ethyl acetate 3) Hexane After sucking the solvent install the column (with the detector side!!!) in the injector Apply carrier gas at normal flow rates for a couple of minutes at room temperature Ramp the oven at 2 C/min to the maximum oven temperature required Hold it for 20 min then cool down Test the column ATTENTION: Only chemically bonded column can be rinsed! Recipe for column deactivation Rinse column as described previously Suck a 10 % solution of Diphenyltetramethyldisilazane (DPTMDS) until 5-10 % of the column is filled (start at the detector end!!!) Install the column in the injector (with the detector end!) and apply carrier gas for a couple of minutes at room temperature. Reduce carrier gas pressure to 0.1 bar and close the open end with a septum Increase oven temperature to 200 C (15`) and then to 300 C for 30` Afterwards cool down to room temperature Rinse once with a few ml of Dichloromethane and condition the column as described K. Grob, Journal of HRC & CC 5, (1982) 13, Syringes and Injection Techniques Syringes and injection techniques Needle Type A Standard 5 or 10 µl Plunger Sample Type B small volumes 1 µl Minimum 50 nanoliter Glass body Type A: Hot needle technique Rinse the syringe several times with a solvent (should be the same as used for sample) Push the plunger to the end, solvent remains in the needle, move plunger back until you see the air plug Fill a little bit more than desired volume of sample (watch for bubbles!) Adjust exactly the desired volume, wipe off solvent from needle and push plunger back until you can see the air in the glass body Sample Tungsten wire Sample Air Solvent 17

18 Syringes and injection techniques Type B: Filled, cold needle technique Clean the syringe several time with a solvent Fill and discard with sample Pump several time with sample and adjust desired volume Push the plunger a little back Put the syringe in the injector and depress immediately the plunger to prevent peak splitting The samples are in the bottles with silicone septa Atosamplers Air Sample Tungsten wire Solvent effect Improper solvent effect 18

19 Wrong solvent The solvent (MeOH) is not wetting the surface of the non polar stationary phase and forms droplets where the analytes are dissolved TIC Extracted Ion 119 (p-cymene) Detector Two main types: 1. Simple (TCD/FID) indicate when and how ) 2. Spectroscopic provide information about compound structure TCD FID EC or ECD FPD FTIR MS UV, NMR Sensitivity Flame ionisation detector 19

20 FID response (signal) ECD Sensitive towards halides, peroxides, and nitro groups. Highly sensitive, but not always linear NPD 20

21 Identification of volatiles Chromatogram RT or related data (RI, KI) RT x = RT r at the same GC parameters (very unreliable) RT x = RT r at 2-3 columns (polar and apolar) better, but still not sufficient for positive identification Spiking additional prove Retention time, min GC parameters Identification Many compounds have the same RT Addition of reference compound ( spiking ) The peak increases supports assumption Assuming that the peak is isopentane? Adding reference compound - purepentane The peak changes surely not isopentane 21

22 Compilations of retention indices Isothermal analysis Programmed analysis Linear RI Coupling of GC and MS The effluents from the GC column are moved via interface to the mass spectrometer (mass detector) TIC m/z fragments GC-MS of pepper extract ? [min] X compound MS libraries Willey NIST NBS EPA 7 CH (R ) - (+) H 3 C CH 2 Reference in library 22

23 Abundance m/z--> #25411:.BETA.-PINENE Compilations of RI (KI) and MS Comparisson of 2 mass spectra β-pinene Abundance 9500 #25416:.GAMMA.-TERPINENE γ-terpinene m/z--> Identification of volatiles RT or related data (RI, KI) Spectroscopic data (mass spectra) MS pattern X = MS pattern reference = not sufficient RI x =RI r ; MS x =MS r most reliable data for a positive identification Types of Mass Spectrometers Quadrupole Ion Trap Time of Flight Fourier Transform MS Magnetic Sector 23

24 Diffusion pump Components of MS Vacuum system (rough pump + high vacuum pump) Ion source Analyser, separation according to mass and charge to rough pump to MS need a rough vacuum 5*10-3 torr Detector Data acquisition and handling Diffusion pump oil (mineral oil with very high boiling point) is heated at the bottom, pumped to the top and injected over the circular openings. Vacuum is generated by diffusion from areas of higher gas partial pressure to areas with lower gas partial pressure. Turbomolecular pump Based on the principle that single gas particles collide with the very fast moving areas of the rotor. By this collision they get an impulse to in the direction of the rough pump Turning speed of the rotor: U/min 1 Stator 2 Rotor 3 Motor 4 Connection to MS Electron Impact Ionisation (EI) Electrons are accelerated with 70 V (Energy = 70eV) These electrons can remove another electron from a neutral, non charged molecule and form a single charged radical e - + M -> M e - This radical can undergo an decay under the formation of more fragments. Under well defined conditions this fragmentation pattern is characteristic for a molecule 24

25 Abundance m/z--> Abundance m/z--> #83538: Tetradecane (CAS) $$ n-tetradecane $$ Isotetradecane #87359: Pyrene (CAS) $$.beta.-pyrene $$ Benzo[def]phenanth Scheme of an ion source Comparison of two different MS Tetradecane Pyrene Quadrupole-MS Ion trap Quadrupole rods (permanent magnet) are overlayed with a radio frequency to filter the desired masses. Only at certain times a particle at a defined mass-tocharge ratio (m/z) can reach the detector 25

26 Abundance m/z--> Scan 3911 ( min): KOLBEN2S.D Abundance m/z--> Scan 3921 ( min): KOLBEN2S.D Resolution of mass spectrometer R = m/δm High resolution MS (e.g. magnetic sector MS) up for the exact determination of molecular weight Low resolution MS (Quadrupole): Constant resolution over the total mass range, it is possible to resolve one mass from another (e.g ) Scan or SIM? Scan: For the identification of unknown samples. Gives a spectrum which can be interpreted or identified via a mass spec library ATTENTION: Time of acquisition MUST with the chromatographic resolution, that mean at least 10 points over the peak for quantitative information SIM: (selected ion monitoring): The substance is already known, reduces the information, but increases the sensitivity drastically Not resolved substances Abundance TIC: KOLBEN2S.D Quantification GC peak area is proportional to the amount of the compound Pak area may be easily measured by the integrator Time-->

27 Quantification Peak areas of separated components are being summed up and the content of each components is expressed in percentage Quantitative analysis Absolute calibration GC analysis of the samples containing different concentration of the relevant reference compound, determination of peak areas and preparation of calibration curves Internal standard Measured amount of reference compound is added to the analysed mixture and according to the obtained chromatogram calibration curve is preparaed Internal normalisation Based on assumption that the sum of the areas and/or heights of all peaks is 100 %, while individual peak area and/or heights constitutes a definite part in the sum peak area Absolute calibration X Y Z Component content X, Y and Z the curves of absolute calibration of each component ω = i k A, i Ai 100 a A i peak area of the component i k A,i correction coefficient of the component i which depends on detector sensitivity a- sample amount in mg or ml Lavender essential oil Lavender essential oil with IS methyldecanoate Use of internal standard (IS) RF mia =m x A i /m i A RF= analyte response in relation to IS A i ir A = IS and analyte peak areas m i and m = weight of IS and analyte 27

28 Analysis of peppermint oil on two GC columns; C10-C18 alkane mixture is added Apolar OV-1 Multidimensional GC Polar Carbowax 20M J.V.Hinshaw, 2004 Threedimensional GC J.V.Hinshaw,

29 Transfer of a fraction to another column Heartcut of peppermint essential oil Polar column Apolar column` Comprehensive 2D gas chromatography (GCXGC) Cryogenic modulation The M are heated and cooled out of phase with each other. Peaks are trapped at the M1 when it is cooled down. The M2 is also cooled down and then the M1 is heated. The The trapped peaks move to the cooled M2 zone along with any material that leaks through while the M1 is hot. When the M1 is cooled off again, the two trapping zones are effectively isolated from each other. The M2 is heated, and the peaks trapped within are released into the C2 J.V.Hinshaw,

30 GCxGC GCxGC GCxGC in the analysis of cheese volatiles Gogus F.et al. Analysis of the volatile components of cheddar cheese by direct thermal desorption-gc GC-TOF/MS) J. Sep. Sci., 2006, 29, Isolated from Cheddar cheese using direct thermal desorption (DTD) and analysed using comprehensive 2-D GC (GC x GC) coupled with TOF MS (TOF/MS). In total 12 aldehydes, 13 acids, 13 ketones, 5 alcohols, 3 hydrocarbons and 9 miscellaneous compounds were identified at desorption temperatures of 100, 150, 200 and 250 C using mature Cheddar cheese. A temperature of 150 C was found to be optimum for the DTD of volatiles from mature Cheddar cheese. The major components were acetic acid, butanoic acid, 3-hydroxy-2-butanone and 2,3-butanediol. A DTD temperature of 150 C was used to observe the effect of maturation (mild, medium or mature) on the volatiles of Cheddar cheese. The major components of the volatiles of mild, medium and mature Cheddar cheese were almost the same. However, their percentage compositions were found to change with the stage of maturity. DTD is simple, fast and requires only a small amount of sample (approximately 10 mg) and works well with comprehensive GC x GC-TOF/MS. Comprehensive GC also separated a number of components which remained overlapped on the single column, such as octane and hexanal. Multidimensional gas chromatography (MD-GCO) 30