A L O S Project Workshop IV

A L O S Project Workshop IV SOLID-PHASE EXTRACTION

ALOS: ROSKILDE-MEETING November 2005 Outline • Principles of solid phase extraction • Features and benefits • Silica products and polymers • Practice • Applications

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

ALOS: ROSKILDE-MEETING November 2005 Principles of SPE SPE is an extraction process whereby an aqueous sample is filteredthrough 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.

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

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

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

ALOS: ROSKILDE-MEETING November 2005 SPE Column

ALOS: ROSKILDE-MEETING November 2005 SPE Column accessories

ALOS: ROSKILDE-MEETING November 2005 SPE- Experimental Set-up

ALOS: ROSKILDE-MEETING November 2005 Silicagel-Phases • Reversed Phase • C18 • Adsorption • Si-OH • Normal Phase • NH2 • CN • C-OH(OH)

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

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!

ALOS: ROSKILDE-MEETING November 2005 Reversed Phase Principle

ALOS: ROSKILDE-MEETING November 2005 Mixed Mode Principle

ALOS: ROSKILDE-MEETING November 2005 Polymer- Phase Principle R (-CH-CH2)n- N-CH3 C=O CH3 R R

ALOS: ROSKILDE-MEETING November 2005 Polymer- Phase Principle R (-CH-CH2)n- N-CH3 C=O CH3 R SO3H or CH2N+R3 SO3H or CH2N+R3

ALOS: ROSKILDE-MEETING November 2005 Practice

ALOS: ROSKILDE-MEETING November 2005 4 Steps in SPE

ALOS: ROSKILDE-MEETING November 2005 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!

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.

ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural dyes in beverages: sample load

ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural dyes in beverages: washing step

ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural dyes in beverages: elution step

ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural + synthetic dyes in beverages:

ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural + synthetic dyes in beverages: sample load

ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural + synthetic dyes in beverages: elution step

ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural + synthetic dyes in beverages

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

ALOS: ROSKILDE-MEETING November 2005 • Environmental • - PAH ‘s from water and soil • - PCB‘s 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.

ALOS: ROSKILDE-MEETING November 2005 Thank you for your attention