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1 Preparazione del campione per l’analisi cromatografica.

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Presentation on theme: "1 Preparazione del campione per l’analisi cromatografica."— Presentation transcript:

1 1 Preparazione del campione per l’analisi cromatografica

2 Estrazione solido-liquido Soxhlet Ultrasuoni Estrazione accelerata con solvente (ASE) Estrazione assistita da microonde (MAE) Estrazione con fluidi supercritici (SFE) Estrazione in fase gas Spazio di testa statico Spazio di testa dinamico Microestrazione in fase solida (SPME)

3 What is Headspace Gas Chromatography? The 'headspace' is the gas space in a chromatography vial above the sample. Headspace analysis is therefore the analysis of the components present in that gas. Headspace GC is used for the analysis of volatiles and semi-volatile organics in solid, liquid and gas samples. It is most suited for the analysis of the very light volatiles in samples that can be efficiently partitioned into the headspace gas volume from the liquid or solid matrix sample. Higher boiling volatiles and semi-volatiles are not detectable with this technique due to their low partition in the gas headspace volume. Headspace analysis also lends itself to automation for quality control or sample screening. This is made possible by modern instrumentation being able to reproducibly prepare samples in an efficient manner. Complex sample matrices, which would otherwise require sample extraction or preparation, or be difficult to analyse directly, are ideal candidates for headspace since they can be placed directly in a vial with little or no preparation. This saves both time and money.

4 Basic Principles of Headspace Analysis Phases of the Headspace Vial G = the gas phase (headspace) The gas phase is commonly referred to as the headspace and lies above the condensed sample phase. S = the sample phase The sample phase contains the compound(s) of interest. It is usually in the form of a liquid or solid in combination with a dilution solvent or a matrix modifier. Once the sample phase is introduced into the vial and the vial is sealed, volatile components diffuse into the gas phase until the headspace has reached a state of equilibrium as depicted by the arrows. The sample is then taken from the headspace.

5 Headspace solid-phase micro extraction


7 Purge and trap system

8 METHOD 5035° CLOSED-SYSTEM PURGE-AND-TRAP AND EXTRACTION FOR VOLATILE ORGANICS IN SOIL AND WASTE SAMPLES Acetone Acetonitrile Acrolein Acrylonitrile Allyl alcohol Allyl chloride t-Amyl ethyl ether t-Amyl methyl ether Benzene Benzyl chloride Bis(2-chloroethyl)sulfide Bromoacetone Bromochloromethane Bromodichloromethane Bromoform Bromomethane n-Butanol 2-Butanone (MEK) t-Butyl alcohol Carbon disulfide Carbon tetrachloride Chloral hydrate Chlorobenzene Chlorodibromomethane Chloroethane 2-Chloroethanol 2-Chloroethyl vinyl ether Chloroform Chloromethane Chloroprene Crotonaldehyde 1,2-Dibromo-3-chloropropane 1,2-Dibromoethane Dibromomethane 1,2-Dichlorobenzene 1,3-Dichlorobenzene 1,4-Dichlorobenzene cis-1,4-Dichloro-2-butene trans-1,4-Dichloro-2-butene Dichlorodifluoromethane 1,1-Dichloroethane 1,2-Dichloroethane 1,1-Dichloroethene cis-1,2-Dichloroethene trans-1,2-Dichloroethene 1,2-Dichloropropane 1,3-Dichloro-2-propanol cis-1,3-Dichloropropene trans-1,3-Dichloropropene 1,2,3,4-Diepoxybutane Diethyl ether Diisopropyl ether (DIPE) 1,4-Dioxane Ethylbenzene Ethylene oxide Ethyl methacrylate Ethyl tert-butyl ether (ETBE)

9 Segue Hexachlorobutadiene 2-Hexanone Iodomethane Isobutyl alcohol Isopropylbenzene Malononitrile Methacrylonitrile Methylene chloride Methyl methacrylate 4-Methyl-2-pentanone (MIBK) Methyl tert-butyl ether (MTBE) Naphthalene Nitrobenzene 2-Nitropropane N-Nitroso-di-n-butylamine Paraldehyde 2-Pentanone 2-Picoline 1-Propanol 2-Propanol Propiolactone Propionitrile (ethyl cyanide) n-Propylamine Styrene 1,1,1,2-Tetrachloroethane 1,1,2,2-Tetrachloroethane Tetrachloroethene Toluene o-Toluidine 1,2,4-Trichlorobenzene 1,1,1-Trichloroethane 1,1,2-Trichloroethane Trichloroethene Trichlorofluoromethane 1,2,3-Trichloropropane Vinyl acetate Vinyl chloride o-Xylene m-Xylene p-Xylene

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15 15 Step 1. Standby During the standby mode, the purge gas flow is stopped, the trap is cooled, and the system is readied for the start of an analysis. The desorb gas bypasses the trap and is directed onto the column as the carrier gas flow. The gas flow rate through the column can be measured.

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17 During the wet purge, the purge gas flow passes through the purge vessel, removes volatile analytes from the sample, and sweeps the analytes through the heated valve onto the adsorbent trap. The analytes are collected on the trap and the purge gas exits through the purge vent. The purge gas flow typically is set at 30-50mL/min. and can be measured at the purge vent. Samples usually are purged for 10-15 minutes. During the purge mode, the desorb (carrier) gas is directed onto the column. During the wet purge, a large amount of water is removed from the sample and collects on the trap. The dry purge removes the excess water that accumulated. During the dry purge, the purge gas bypasses the purge vessel and is directed to the trap. The dry purge gas removes water and carries it out the exit vent. The desorb (carrier) gas is directed onto the column. Only traps that incorporate hydrophobic adsorbents can be dry purged. Once the analytes have been trapped and excess water removed, the purge gas flow is stopped. During this static period, the trap is rapidly heated to ~5°C below the desorb temperature of the adsorbent materials used. The desorb preheat step uniformly volatilizes the sample to create a narrow sample band and a more efficient sample transfer onto the GC column. Without a desorb preheat step the peaks would tail, resulting in poor chromatography.

18 While the sample transfer occurs, the trap is heated to its final desorb temperature. Desorb temperatures range from 180°C-250°C, determined by the adsorbent materials and the model of concentrator. The desorb flow rate is extremely important; it must be high enough to ensure that the sample remains in a narrow band during the transfer to the GC column. The optimum desorb flow rate for a purge and trap system is >20mL/min.; however, this flow rate is too high to use with capillary columns and must be reduced to retain column efficiency. The optimum flow rate for 0.53mm ID columns is 8-10mL/min. For narrow bore capillary columns (0.18-0.32mm ID), the desorb flow rate usually is 1- 2mL/min. when direct interface is used. This low flow rate requires a longer desorb time due to the slow transfer of the sample from the trap, which, in turn, creates a wide sample bandwidth resulting in broad peak shapes for all early eluting compounds. Cryofocusing (i.e., cold trapping) can be used to reduce band broadening, by installing a secondary cold trap or by cooling the GC column to subambient temperatures.

19 Water and methanol can cause the biggest problems in purge and trap concentration. Current designs of purge and trap systems have added features to eliminate water prior to delivering the sample to the chromatographic system. Moisture control systems (MCS) remove water by condensation, prior to the desorb step. Such systems typically are composed of a piece of metal tubing that is heated during purge and then cooled to 30°C. The sample, desorbed from the heated trap, travels through the MCS, where a large portion of the water is condensed from the saturated carrier gas. These systems are very effective for GC methods that do not have polar/active compounds, such as ketones, in the analyte list. Fritless spargers are used for samples that have high particulate content, or for industrial wastewater samples that may foam. They create fewer bubbles, which decreases purging efficiency but eliminates plugged frits and reduces foaming problems. Needle spargers are used when purging soil, sludge or solid samples. A narrow gauge needle is inserted into the sample and used to release a small stream of purge gas. If you are running an unattended autosampler, you can insert a plug of deactivated fused silica or glass wool into the top of the purge vessel to prevent foam from entering the purge and trap lines. Anti-foaming agents such as polydimethylsiloxane and silicon dioxide methylcellulose are designed to reduce foaming of surfactants in a liquid matrix. These are effective at preventing a sample from foaming, but they generally produce artifact peaks that can interfere with the target analytes. An anti-foam blank must be run prior to samples to determine the contribution of artifact peaks from the anti-foaming agent.

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21 The low soil method utilizes a hermetically-sealed sample vial, the seal of which is never broken from the time of sampling to the time of analysis. Since the sample is never exposed to the atmosphere after sampling, the losses of VOCs during sample transport, handling, and analysis are minimized. The applicable concentration generally fall in the 0.5 to 200 μg/kg range. This method can be used for most volatile organic compounds that have boiling points below 200EC and that are insoluble or slightly soluble in water. Volatile, water-soluble compounds can be included in this analytical technique. However, quantitation limits (by GC or GC/MS) are significantly higher because of poor purging efficiency. The purging efficiency can be improved for water soluble analytes, e.g. ketones and alcohols, when purging at an elevated temperature of 80°C as compared to 20° or 40°C. As with any preparative method for volatiles, samples should be screened to avoid contamination of the purge-and-trap system by samples that contain very high concentrations of purgeable material above the calibration range of the low concentration method. In addition, because the sealed sample container cannot be opened to remove a sample aliquot without compromising the integrity of the sample, multiple sample aliquots should be collected to allow for screening and reanalysis. To ensure minimal loss of volatile constituents prior to analysis the entire sample vial is placed, unopened, into the instrument auto sampler device. Immediately before analysis, organic-free reagent water, surrogates, and internal standards (if applicable) are automatically added without opening the sample vial. The vial containing the sample is heated to 40EC and the volatiles purged into an appropriate trap using an inert gas combined with agitation of the sample. Purged components travel via a transfer line to a trap. When purging is complete, the trap is heated and backflushed with helium to desorb the trapped sample components into a gas chromatograph for analysis by an appropriate determinative method.

22 Soxhlet extractor


24 Ultrasound effects Disgregazione delle particelle solide Degasaggio Effetti termici Aumento del coefficiente di diffusione

25 METHOD 3550C ULTRASONIC EXTRACTION This method describes a procedure for extracting nonvolatile and semivolatile organic compounds from solids such as soils, sludges, and wastes. The ultrasonic process ensures intimate contact of the sample matrix with the extraction solvent. Because of the limited contact time between the solvent and the sample, ultrasonic extraction may not be as rigorous as other extraction methods for soils/solids. Therefore, it is critical that the method be followed explicitly, in order to achieve the maximum extraction efficiency. The choice of extraction solvent will depend on the analytes of interest and no single solvent is universally applicable to all analyte groups. As a result of concerns about the efficiency of ultrasonic extraction, particularly at concentrations near or below about 10 μg/kg, it is imperative that the analyst demonstrate the performance of the specific solvent system and operating conditions for the analytes of interest and the concentrations of interest. This demonstration applies to any solvent system that is employed, including those specifically listed in this method. At a minimum, such a demonstration will encompass the initial demonstration of proficiency described in Method 3500, using a clean reference matrix. It is highly recommended that the extracts be subject to some form of cleanup prior to analysis.

26 Microwave-Assisted Extraction

27 Applications of MAE Microwave-assisted extraction (MAE) is a relatively new extraction technique, which utilizes microwave energy to heat the solvent and the sample to increase the mass transfer rate of the solutes from the sample matrix into the solvent. Microwave extraction has been used for many years to extract compounds from plastics, biological samples, foods, animal feeds, paper, wastewater and many other types of samples. In early 2008, the U.S. Environmental Protection Agency (USEPA) approved Method 3546 for microwave extraction of organic compounds from soils, sludges and sediments. It is a proven technique that is fast, uses significantly less solvent than traditional techniques and is cost-effective. Method 3546 is only applicable to solid samples with small particle sizes. If practical, soil/sediment samples may be air-dried and ground to a fine powder prior to extraction. Alternatively, if worker safety or the loss of analytes during drying is a concern, soil/sediment samples may be mixed with anhydrous sodium sulfate or pelletized diatomaceous earth. (Drying and grinding samples containing PCDDs/PCDFs is not recommended, due to safety concerns.) The total mass of material to be prepared depends on the specifications of the determinative method and the sensitivity needed for the analysis, but an amount of 2 - 20 g of material is usually necessary and can be accommodated by this extraction procedure.

28 Accelerated solvent extraction (ASE)


30 Extractions that normally take hours can be done in minutes using Accelerated Solvent Extraction System (ASE ® ). Compared to techniques like Soxhlet and sonication, ASE generates results in a fraction of the time. In addition to speed, ASE offers a lower cost per sample than other techniques by reducing solvent consumption by up to 90%. Extractions in minutes Dramatic solvent reduction Wide range of applications Approved for use by the U.S. EPA and CLP Program Automated Extraction of up to 24 samples Sample Cell sizes: 1, 5, 11, 22, and 33 mL Collection bottle 40 or 60 mL Operating pressure 500–3000 PSI (35–200 Bar)

31 METHOD 3545A PRESSURIZED FLUID EXTRACTION This method is a procedure for extracting water insoluble or slightly water soluble organic compounds from soils, clays, sediments, sludges, and waste solids. This method uses elevated temperature (100-180 EC) and pressure (1500-2000 psi) to achieve analyte recoveries equivalent to those from Soxhlet extraction, using less solvent and taking significantly less time than the Soxhlet procedure. This procedure was developed and validated on a commerciallyavailable, automated extraction system. This method is applicable to the extraction of semivolatile organic compounds, organophosphorus pesticides, organochlorine pesticides, chlorinated herbicides, polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDDs/PCDFs), and diesel range organics (DRO), which may then be analyzed by a variety of chromatographic procedures. The quantitative analysis of DRO is operationally defined on the basis of the retention times of characteristic components. This definition can be found in Method 8015. This method may also be applicable for the extraction of additional target analytes, provided that the analyst demonstrates adequate performance for the intended application

32 Supercritical fluid extraction (SFE)

33 1. CO2 2. Modifier or washing solution 3. CO2 Large volume solvent delivery pump 4. Modifier delivery pump 5. Stop valve 6. Safety valve 7. Pre-heating coil 8. Extraction vessel (1000 mL) 9. Temperature control jacket 10. Temperature meter 11. Back pressure valve 12. 6-way switching valve 13. Pressure meter 14. Fraction vessel 15. Back pressure valve 16. Temperature controller

34 Advantages * Dissolving power of the SCF is controlled by pressure and/or temperature * SCF is easily recoverable from the extract due to its volatility * Non-toxic solvents leave no harmful residue * High boiling components are extracted at relatively low temperatures * Separations not possible by more traditional processes can sometimes be effected * Thermally labile compounds can be extracted with minimal damage as low temperatures can be employed by the extraction Disadvantages * Elevated pressure required * Compression of solvent requires elaborate recycling measures to reduce energy costs * High capital investment for equipment Solvents of supercritical fluid extraction The choice of the SFE solvent is similar to the regular extraction. Principle considerations are the followings. * Good solving property * Inert to the product * Easy separation from the product * Cheap * Low PC because of economic reasons

35 FluidCritical Temperature (K)Critical Pressure (bar) >Carbon dioxide>304.1> 73.8 >Ethane>305.4>48.8 >Ethylene>282.4>50.4 >Propane>369.8>42.5 >Propylene>364.9>46.0 >Trifluoromethane (Fluoroform)>299.3>48.6 >Chlorotrifluoromethane>302.0>38.7 >Trichlorofluoromethane>471.2>44.1 >Ammonia>405.5>113.5 >Water>647.3>221.2 >Cyclohexane>553.5>40.7 >n-Pentane>469.7>33.7 >Toluene>591.8>41.0

36 Liquid-liquid extraction

37 Kuderna-Danish apparatus

38 QuEChERS: an acronym for Quick, Easy, Cheap, Effective, Rugged and Safe, covers a variety of sample preparation and clean-up techniques Add 15 mLs of 1% HOAc in ACN to15 ml homogenized/ hydrated sample in a 50 mL centrifuge tube. Add ISTD. Shake. Add 6 g MgSO4 & 1.5 g NaOAc. Shake vigorously for 1 minute. Centrifuge at >1500 rcf for 1 minute. Transfer 1 mL aliquot of supernatant to a dispersive clean-up tube containing MgSO4, PSA (C18, GCB or ChloroFiltr can be added for additional clean-up). Shake for 30 seconds. Centrifuge at >1500 rcf for 1 minute

39 Solid Phase Extraction (SPE)


41 Small volume samples

42 Large volume samples


44 Empore disks

45 Disk: extraction apparatus




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51 51 SPME - HPLC

52 Solid Phase Matrix Dispersion (SPME)

53 L I M Q I U C I R D O - L E I X Q T U R I A D C T I O N LLME

54 Molecular imprinted polymers



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