Preparazione del campione per
l’analisi cromatografica
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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)
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.
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.
Headspace
solid-phase
micro
extraction
Purge and trap system
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)
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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11
12
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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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16
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.
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 12mL/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.
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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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.
Soxhlet extractor
Ultrasound effects
•
•
•
•
Disgregazione delle particelle solide
Degasaggio
Effetti termici
Aumento del coefficiente di diffusione
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.
Microwave-Assisted Extraction
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.
Accelerated solvent extraction (ASE)
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)
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
Supercritical fluid extraction (SFE)
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
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
Fluid
Critical 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
Liquid-liquid
extraction
Kuderna-Danish apparatus
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
Solid Phase Extraction (SPE)
Small
volume
samples
Large volume
samples
Empore disks
Disk: extraction
apparatus
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SPME - HPLC
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Solid Phase Matrix Dispersion (SPME)
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LLME
Molecular imprinted polymers