a l o s project workshop iv s olid- p hase e xtraction
TRANSCRIPT
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A L O S Project Workshop IVA L O S Project Workshop IV
SSOLID-OLID-PPHASE HASE
EEXTRACTIONXTRACTION
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Principles of solid phase extractionPrinciples of solid phase extraction Features and benefitsFeatures and benefits Silica products and polymers Silica products and polymers PracticePractice ApplicationsApplications
Outline
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Principles
Why do sample preparation?
Remove interferences from sample More accurate results Concentrating analytes to improve
detection Protecting equipment to reduced costs
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SPESPE is an extraction process is an extraction process
whereby an aqueous sample is whereby an aqueous sample is filteredfiltered through a thin bed through a thin bed
ofof
sorbent particles, sorbent particles,
the analytes of interest are the analytes of interest are removedremoved from the liquid matrix, from the liquid matrix,
and and concentratedconcentrated onto the sorbent. onto the sorbent.
Once concentrated, the analytes are Once concentrated, the analytes are removedremoved by an eluting by an eluting
solvent.solvent.
Principles of SPE
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Comparison of LLE vs SPE Disk LLELLE
Uses 200 - 500 ml Uses 200 - 500 ml solventsolvent
Shaking / continuous Shaking / continuous processprocess
Forms emulsions Forms emulsions
Little selectivity Little selectivity
Takes 1 - 2 hours / Takes 1 - 2 hours / samplesample
Uses 2 - 20 ml solventUses 2 - 20 ml solvent
Filtration processFiltration process No emulsions formedNo emulsions formed
Wide selectivity Wide selectivity
(adsorbent) (adsorbent) Takes 10 - 20 min. / Takes 10 - 20 min. / samplesample
SPE diskSPE disk
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Problems with the LLE Procedure
Tedious and time-consumingTedious and time-consuming Shaking and separation timeShaking and separation time Evaporation timeEvaporation time
Expensive-labor and materialsExpensive-labor and materials Time factorTime factor Solvent cost and exposureSolvent cost and exposure Solvent disposalSolvent disposal
Poor resultsPoor results Forming of emulsionsForming of emulsions Irreproducible extractionsIrreproducible extractions Low recoveriesLow recoveries
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What are the Benefits of SPE?
SPE uses less solvent than LLESPE uses less solvent than LLE SPE is faster (at least 5 times)SPE is faster (at least 5 times) High capacity High capacity Total SPE costs are considerably less than LLETotal SPE costs are considerably less than LLE High selectivity: broad choice of bonded phases and High selectivity: broad choice of bonded phases and
solventssolvents Automation much easierAutomation much easier
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SPE Column
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SPE Column accessories
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Eluent
Säule
Kanüle
Saugflaschebzw.Reaglas mitAnsaugstutzen
Abb.2:Aufbau einer Festphasenextraktionsapparatur
Abb.3:Arbeitsablauf bei Anwendung der Festphasenextraktion
Vakuum; zur Sicherheitsflasche undWasserstrahlpumpe
Konditionieren Probenaufgabe Waschen Elution
Filterelement
Sorbens
Filterelement
SPE- Experimental Set-up
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Silicagel-Phases
Reversed Phase C18
Adsorption Si-OH
Normal Phase NH2
CN C-OH(OH)
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Anion Exchange
N+
NH2
Cation Exchange C6H5-SO3H COOH SO3H
Biochromatography WP PEI (NH2)
WP Butyl (C4) WP CBX (COO) Sephadex G-25
Phases
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Interactions
Non polar: van der Waals ~20 KJ/mole
Polar: Dipole / Dipole ~ 40 KJ/moleHydrogen bond ~40 KJ/mole
Electrostatic: Ionic ~600 KJ/mole!
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Reversed Phase Principle
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Mixed Mode Principle
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Polymer- Phase Principle
(-CH-CH2)n-
N-CH3
C=O
CH3
R
R
R
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Polymer- Phase Principle
(-CH-CH2)n-
N-CH3
C=O
CH3
R
R
SO3H or CH2N+R3
SO3H or CH2N+R
3
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Practice
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4 Steps in SPE
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Conditioning of silica-based sorbents
Add an organic solvent to rinse and activate
the alkyl-chains (C18, C8 etc.)!
Don´t let the column run dry during conditioning!
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Experiment 1:Rapid Extraction of natural dyes in beverages
Sample Preparation:1 mL blackberry-juice is dissolved in 2 mL of distilled water.Column Conditioning:A 3 mL C18 (Baker: 7020-03) SPE cartridge is conditioned with 1 mL methanol followed by one column volume of distilled water.Sample Addition/Wash:The prepared sample is aspirated through the column. A 5 mL distilled water wash is used to remove sugars, sugar colouring and organic acids.Elution:The dye(s) [anthocyanines, flavonoids, tannins and/or alkaloids)] is (are) eluted with a column volume of methanol. Sometimes propanol-2 will be more successful.Analysis:For detailed analysis- an absorption spectrum can be taken from the eluate.
- TLC-experiments can be done.
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Experiment 1:Rapid Extraction of natural dyes in beverages: sample load
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Experiment 1:Rapid Extraction of natural dyes in beverages: sample load
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Experiment 1:Rapid Extraction of natural dyes in beverages: washing step
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Experiment 1:Rapid Extraction of natural dyes in beverages: elution step
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Experiment 1:Rapid Extraction of natural + synthetic dyes in beverages:
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Experiment 1:Rapid Extraction of natural + synthetic dyes in beverages: sample load
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Experiment 1:Rapid Extraction of natural + synthetic dyes in beverages: elution step
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Experiment 1:Rapid Extraction of natural + synthetic dyes in beverages
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EnvironmentalEnvironmental Water, SoilWater, Soil
Pharmaceutical/clinicalPharmaceutical/clinical Serum, plasma, urine, bloodSerum, plasma, urine, blood
Food/feedFood/feed Juice, grain, milkJuice, grain, milk
Biological/biotechBiological/biotech Water, plasma, urineWater, plasma, urine
Application Database
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EnvironmentalEnvironmental- PAH ‘s from water and soil- PAH ‘s from water and soil
- PCB‘s from oil-- PCB‘s from oil- - Pesticides from water/soil-- Pesticides from water/soil- - etc.- etc.
Food/Feed/BeveragesFood/Feed/Beverages- - Aflatoxine from corn mealAflatoxine from corn meal
- Caffeine from di-caffeinated diet cola- Caffeine from di-caffeinated diet cola - Vitamin E from juice- Vitamin E from juice - etc.- etc.
Pharmaceutical/Clinical/BiologicalPharmaceutical/Clinical/Biological- - Benzodiazipines from serumBenzodiazipines from serum
- Anabolic Steroids/Urine- Anabolic Steroids/Urine - Aflatoxine from liver- Aflatoxine from liver
- etc.- etc.
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Thank you for your attention
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ALOS: ROSKILDE-MEETING November 2005
Experiment 1:Rapid Extraction of natural dyes in beverages
Sample Preparation:1 mL blackberry-juice is dissolved in 2 mL of distilled water.Column Conditioning:A 3 mL C18 (Baker: 7020-03) SPE cartridge is conditioned with 1 mL methanol followed by one column volume of distilled water.Sample Addition/Wash:The prepared sample is aspirated through the column. A 5 mL distilled water wash is used to remove sugars, sugar colouring and organic acids.Elution:The dye(s) [anthocyanines, flavonoids, tannins and/or alkaloids)] is (are) eluted with a column volume of methanol. Sometimes propanol-2 will be more successful.Analysis:For detailed analysis- an absorption spectrum can be taken from the eluate.
- TLC-experiments can be done.
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ALOS: ROSKILDE-MEETING November 2005
Experiment 1:Rapid Extraction of natural dyes in beverages: sample load
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ALOS: ROSKILDE-MEETING November 2005
Experiment 1:Rapid Extraction of natural dyes in beverages: sample load
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Experiment 1:Rapid Extraction of natural dyes in beverages: washing step
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Experiment 1:Rapid Extraction of natural dyes in beverages: elution step
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Experiment 1:Rapid Extraction of natural + synthetic dyes in beverages:
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ALOS: ROSKILDE-MEETING November 2005
Experiment 1:Rapid Extraction of natural + synthetic dyes in beverages: sample load
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Experiment 1:Rapid Extraction of natural + synthetic dyes in beverages: elution step
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Experiment 1:Rapid Extraction of natural + synthetic dyes in beverages
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Experiment 2:Rapid Extraction of iron-ions from water samples or waste water
Sample Preparation:Dissolve 100 mg Fe(NO3)3 or FeCl3 in 10 mL of distilled water or use an amount of
approximately 100mL waste water, which contains iron (III) ions.Column Conditioning:A 3 mL sulfonic acid (Baker: 7090-03) SPE column is conditioned with 2 mL methanol followed by one column volume of distilled water.Sample Addition/Wash:2 mL of the sample is aspirated through the column.The column is washed with 2 mL of distilled water.Elution:Fe3+ -ions are eluted in 1-2 mL hydrochlorid acid (c=0,1 M).The eluate is coloured yellow.Analysis:For detailed analysis - add NH4SCN to the eluate. The colour changes to deep red.
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Experiment 2:Rapid Extraction of iron-ions from water samples or waste water
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Experiment 2:Rapid Extraction of iron-ions from water samples or waste water
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Experiment 2:Rapid Extraction of iron-ions from water samples or waste water
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Experiment 2:Rapid Extraction of iron-ions from water samples or waste water
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Experiment 3:Rapid Extraction of sugars (glucose)
Sample Preparation:Dissolve 100 mg glucose in 10 mL water.Column Conditioning:A 3 mL Amino (Baker: 7088-03) SPE column is conditioned with 2 mL ethanol.Sample Addition/Wash:2 mL of the prepared sample is aspirated through the column.Elution:Sugar is eluted with 2 mL water.Analysis:For detailed analysis - use few drops of Fehling-solutions.For detailed analysis - an absorption spectrum can be taken from the eluate. - TLC-experiments can be done.
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Experiment 4:Rapid Extraction of ß-carotine from Multivitamine-juice or carot-juice
Sample Preparation:5 mL of Multivitamine-juice or carot-juice is filtered or centrifuged.Column Conditioning:A 3 mL C18 (Baker: 7020-03) SPE cartridge is conditioned with methanol followed by one column volume of distilled water.Sample Addition/Wash:The prepared sample is aspirated through the column. A 2 mL distilled water wash is used to remove all the other compounds.Elution:ß-carotine is eluted with about 2 mL heptane-acetone mixture (8:2,v,v). the eluate is coloured yellow.Analysis:For detailed analysis - an absorption spectrum can be taken from the eluate.
- TLC-experiments can be performed.
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Experiment 4:Rapid Extraction of ß-carotine
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Experiment 4:Rapid Extraction of ß-carotine
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Experiment 4:Rapid Extraction of ß-carotine
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Students-Experiment:Rapid Extraction of Haribo’s wine gum dyes
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Students-Experiment:Rapid Extraction of Haribo’s wine gum dyes
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Students-Experiment:Rapid Extraction of Haribo’s wine gum dyes