A L O S Project Workshop IV

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A L O S Project Workshop IV
SOLID-PHASE EXTRACTION
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ALOS ROSKILDE-MEETING November 2005
Outline

• Principles of solid phase extraction
• Features and benefits
• Silica products and polymers
• Practice
• Applications

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ALOS ROSKILDE-MEETING November 2005

• 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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ALOS ROSKILDE-MEETING November 2005
Principles of SPE

• SPE is an extraction process
• whereby an aqueous sample is filtered through a
  thin bed of
• sorbent particles,
• the analytes of interest are removed from the
  liquid matrix,
• and concentrated onto the sorbent.
• Once concentrated, the analytes are removed by an
  eluting
• solvent.

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ALOS ROSKILDE-MEETING November 2005
Comparison of LLE vs SPE Disk

• LLE
• Uses 200 - 500 ml solvent
• Shaking / continuous process
• Forms emulsions
• Little selectivity
• Takes 1 - 2 hours / sample

SPE disk

• Uses 2 - 20 ml solvent
• Filtration process
• No emulsions formed
• Wide selectivity
• (adsorbent)
• Takes 10 - 20 min. / sample

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ALOS ROSKILDE-MEETING November 2005
Problems with the LLE Procedure

• Tedious and time-consuming
• Shaking and separation time
• Evaporation time
• Expensive-labor and materials
• Time factor
• Solvent cost and exposure
• Solvent disposal
• Poor results
• Forming of emulsions
• Irreproducible extractions
• Low recoveries

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ALOS ROSKILDE-MEETING November 2005
What are the Benefits of SPE?

• SPE uses less solvent than LLE
• SPE is faster (at least 5 times)
• High capacity
• Total SPE costs are considerably less than LLE
• High selectivity broad choice of bonded phases
  and solvents
• Automation much easier

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ALOS ROSKILDE-MEETING November 2005
SPE Column
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ALOS ROSKILDE-MEETING November 2005
SPE Column accessories
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ALOS ROSKILDE-MEETING November 2005
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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ALOS ROSKILDE-MEETING November 2005
Phases

• Anion Exchange
• N
• NH2
• Cation Exchange
• C6H5-SO3H
• COOH
• SO3H
• Biochromatography
• WP PEI (NH2)
• WP Butyl (C4)
• WP CBX (COO)
• Sephadex G-25

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ALOS ROSKILDE-MEETING November 2005

• Interactions
• Non polar van der Waals 20 KJ/mole
• Polar Dipole / Dipole 40 KJ/mole
• Hydrogen bond 40 KJ/mole
• Electrostatic Ionic 600
  KJ/mole!

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ALOS ROSKILDE-MEETING November 2005
Reversed Phase Principle
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Mixed Mode Principle
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ALOS ROSKILDE-MEETING November 2005
Polymer- Phase Principle
R
(-CH-CH2)n- N-CH3 CO CH3
R
R
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ALOS ROSKILDE-MEETING November 2005
Polymer- Phase Principle
R
(-CH-CH2)n- N-CH3 CO CH3
R
SO3H or CH2NR3
SO3H or CH2NR3
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• Practice

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4 Steps in SPE
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ALOS ROSKILDE-MEETING November 2005
Conditioning of silica-based sorbents

• Add an organic solvent to rinse and activate
• the alkyl-chains (C18, C8 etc.)!
• Dont let the column run dry during
  conditioning!

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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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ALOS ROSKILDE-MEETING November 2005
Experiment 1 Rapid Extraction of natural dyes in
beverages washing step
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ALOS ROSKILDE-MEETING November 2005
Experiment 1 Rapid Extraction of natural dyes in
beverages elution step
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ALOS ROSKILDE-MEETING November 2005
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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ALOS ROSKILDE-MEETING November 2005
Experiment 1 Rapid Extraction of natural
synthetic dyes in beverages elution step
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ALOS ROSKILDE-MEETING November 2005
Experiment 1 Rapid Extraction of natural
synthetic dyes in beverages
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ALOS ROSKILDE-MEETING November 2005
Application Database

• Environmental
• Water, Soil
• Pharmaceutical/clinical
• Serum, plasma, urine, blood
• Food/feed
• Juice, grain, milk
• Biological/biotech
• Water, plasma, urine

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ALOS ROSKILDE-MEETING November 2005

• Environmental
• - PAH s from water and soil
• - PCBs from oil-
• - Pesticides from water/soil-
• - etc.
• Food/Feed/Beverages
• - Aflatoxine from corn meal
• - Caffeine from di-caffeinated diet cola
• - Vitamin E from juice
• - etc.
• Pharmaceutical/Clinical/Biological
• - Benzodiazipines from serum
• - Anabolic Steroids/Urine
• - Aflatoxine from liver
• - 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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ALOS ROSKILDE-MEETING November 2005
Experiment 1 Rapid Extraction of natural dyes in
beverages washing step
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ALOS ROSKILDE-MEETING November 2005
Experiment 1 Rapid Extraction of natural dyes in
beverages elution step
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ALOS ROSKILDE-MEETING November 2005
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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ALOS ROSKILDE-MEETING November 2005
Experiment 1 Rapid Extraction of natural
synthetic dyes in beverages elution step
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ALOS ROSKILDE-MEETING November 2005
Experiment 1 Rapid Extraction of natural
synthetic dyes in beverages
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ALOS ROSKILDE-MEETING November 2005
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 (c0,1
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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ALOS ROSKILDE-MEETING November 2005
Experiment 2 Rapid Extraction of iron-ions from
water samples or waste water
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ALOS ROSKILDE-MEETING November 2005
Experiment 2 Rapid Extraction of iron-ions from
water samples or waste water
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ALOS ROSKILDE-MEETING November 2005
Experiment 2 Rapid Extraction of iron-ions from
water samples or waste water
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ALOS ROSKILDE-MEETING November 2005
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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ALOS ROSKILDE-MEETING November 2005
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 (82,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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ALOS ROSKILDE-MEETING November 2005
Experiment 4 Rapid Extraction of ß-carotine
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ALOS ROSKILDE-MEETING November 2005
Experiment 4 Rapid Extraction of ß-carotine
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ALOS ROSKILDE-MEETING November 2005
Experiment 4 Rapid Extraction of ß-carotine
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ALOS ROSKILDE-MEETING November 2005
Students-Experiment Rapid Extraction of Haribos
wine gum dyes
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ALOS ROSKILDE-MEETING November 2005
Students-Experiment Rapid Extraction of Haribos
wine gum dyes
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ALOS ROSKILDE-MEETING November 2005
Students-Experiment Rapid Extraction of Haribos
wine gum dyes