solid phase extraction and application
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MODH SUDIP C.
PA/2010/11
NIPER HYDERABAD
SOLID PHASE EXTRACTION AND APPLICATION
seminar on
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Introduction
Principle
Types Solid phases
Experimental steps
Trace enrichment
Solid phase micro extraction (SPME)
Comparison with HPLC
Application
Contents
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What is the solid phase extraction?
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DEFINATION OF SOLID PHASE EXTRACTION (SPE):
“A solid phase extraction consists of bringing a liquid or gaseous test sample in contact with a solid phase, whereby the analyte is selectively adsorbed on the surface of the solid phase”
Other solvents (liquids or gases)added to remove possible adsorbed matrix components
Eluting solvent added to desorb analyte selectively
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Liquid-solid extraction
Column extraction
Digital chromatography
Bonded phase extraction
Selective adsorption techniques
SYNONYMS
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Active substance can be:
Unretained- while matrix interference are adsorbed
Retained-while matrix interference are washed through
Strategies for solid phase extraction
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Retained-while matrix interference are washed through
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Principle of Solid Phase Extraction:
Partitioning of compounds between two phases of solid and liquid
Must having greater affinity for the solid phase than for the sample matrix Compounds retained on the solid phase can be removed by eluting solvent with a greater affinity for the analytes
pH changes can be useful8
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In modern SPE the adsorbent is packed between two flitted disks in polypropylene cartridge and liquid phases are passed through the cartridge either by suction or by positive pressure
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Cartridge
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OVERVIEW OF SPE
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Activated charcoal
Alumina
Silica gel
Magnesium silicate (Florisil)
Chemically bonded silica phases and polymers
E.g. styrene divinylbenzene
Solid phases
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According to chemical nature of The functional group bonded to the silica or the copolymer
The resulting phases are classified as Non-polar Polar Ion exchangers
It gives different mode of chromatography
Other solid supports Polymeric resins, cellulose and zirconia
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Zirconia coated silica as a stationary phase
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Reaction of phenobarbitone with pentafluorobenzyl bromide onto the adsorbent
Amphetamine by Chiral derivatization of solid Phases
Doxorubicin by the metal-loaded phases in which metal cation is loaded onto a reagent-labelled phase
Molecularly imprinted polymers synthetic polymeric materials with specific cavitiesdesigned for a template molecule
Examples of selective stationary phases
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Technical Data - Solid Phase Extraction (SPE) Media ProductSorbent Abbreviations Description ODS Octadecyl silica 5% carbon loadODS-4 Octadecyl silica 14% carbon load, end capped*ODS-5 Octadecyl silica 18% carbon load, end cappedC-8 Octyl silica 8.5% carbon load, end cappedFLO Florisil™ Magnesium silicate NH2 Weak anion exchanger Primary amineSAX Strong anion exchanger Quaternary amine (-NR3
+)SCX Strong cation exchanger Aromatic benzene sulfonic acidSIL Normal phase silica
* End capping masks residual silanol groups, reducing ionic affinity for amines. 17
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1. Activation of sorbent by appropriate solvent that conditions the surface of the solid
2. Removal of solvent by liquid similar to the sample matrix
3. Application of sample, the analytes retained by the sorbent
4. Removal of interfering compounds retained in step 3 with a solvent, but shouldn’t remove the analytes (washing step)
5. Elution of the analytes with an appropriate solvent (desorption or elution step) and collecting for analysis
Experimental procedure of five steps:
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Other technique can be used
Supercritical fluid
Thermal desorption for analytes of high volatility and thermal stability
Thermal desorption with GC for occupational hygiene analysis
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Analyte eluted with an organic, relatively volatile solvent is evaporated to dryness
Then residue dissolved in appropriate solvent
Due to evaporation step, speed with SPE is lost
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IMMUNOAFFINITY PHASES:
Highly selective packings of Immunoaffinity phases of specific antibody immobilised on solid support such as agarose or silica
Useful for selective extraction of biological importanceSubstance
Diagnosis of cancer
ELISA TEST
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IMMUNOAFFINITY PHASES
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TRACE ENRICHMENT WITH SPE
Sensitivity depends on
Physicochemical properties of the Analyte
Detection system
Selective clean-up
Isolation and concentration step
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SPME is the technique in which by using special instrument, sampling is possible in a vapour state
Solid phase microextraction:
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Design of SPME
Syringe like instrument
Fused silica fiber of a small size and cylindrical shape
connected to stainless-steel tube for additional mechanical strength and repeated sampling
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Fused silica fibre coated with thin film of several polymeric stationary phases
Reusable and replaceable
Small size and cylindrical geometry of fiberPlacement into sample or headspace is easy
Loading in desorption chamber of GC orInterphase of the HPLC without any modification of Plunger
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Working with SPME
Fiber is first drawn into the syringe needle
Lowered into the vial by pressing the plunger
Fiber cleaned before analysis to remove contaminants
Cleaning can be performed in the desorption chamber of HPLC by running solvent
Cleaned fiber coating is exposed to a sample matrix for a predetermined, fixed period
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Limits of detection at the µg/L level with using a flame Ionization detector
Limits of detection as low as ng/ L can be reached with an ion-trap mass spectrometer
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Extraction can be performed in two ways
1) Headspace SPME or HS-SPMEFiber is exposed in the vapour phase above a gaseous, liquid, or solid sample
2) Direct immersion or DI-SPMEFiber is directly immersed in liquid samples
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Available SPME Fibres, by Film Type
•Absorption Fibres-Polydimethylsiloxane (PDMS) 7, 30, and 100μm Unpolar-Polyacrylate (PA) Polar-Polyethylene glycol (PEG) Polar •Adsorption fibres (with particles) -Carboxen-polydimethylsiloxane (CAR-PDMS) Adsorption
-Polydimethylsiloxane- divinylbenzene (PDMS-DVB) Adsorption-Divinylbenzene/ Carboxen-Polydimethylsiloxane (DVB-CAR-PDMS) Adsorption
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SPME applied to liquid, gaseous or heavily contaminated samples
chemicals like
Substituted benzene compounds
Polyaromatic hydrocarbons
Nitro- and chlorophenols
Naphthols
volatile organochlorine compounds
polychlorinated biphenyl congeners
caffeine
Metallic ions38
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The SPE process can be performed in a two ways:
On-line Off-line
In offline SPE eluate from the cartridge is introduced into the chromatograph by means of an injection valve
In on-line SPE the extraction cartridge is inserted as part of chromatographic equipment, as loops or high pressure stream of the mobile phase
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Types of online SPE:
SPE-GC(SPME-GC)
SPE-HPLC
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ONLINE SPE-GC
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ONLINE SPE-HPLC
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10ppb Nitrosamines in Water: SPME-GC/MS
Chromatogram courtesy of J. Clark, Liggett Group, Inc.
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ONLINE SPE-HPLC
THT= Tetra hydro thiophene 44
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SPE-HPLC SPME-GC
Universality Compounds +++ + Detection Sensitivity ++ +++ Selectivity +++ ++ Identification + +++ Detection limit (µ/ L) 0.05-0.8 0.2-5 Reproducibility (%) 1-15 4-14Analysis time 90 20Sample volume (mL) 200 2Automation +++ +++Simplicity + +++
Comparison of SPE-HPLC and SPME-GC
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Theoretical basis as HPLC
Retention and selectivity remain unaffected by particle sizeEfficiency dependent on:
Particle sizeColumn geometry
Typical number of plates
HPLC ~ 10,000SPE < 50
Minimum Selectivity(alpha)for Rs=1.2
HPLC 1.06SPE 3.95
Comparison of SPE and HPLC
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Advantages of SPE offers over LLE are
Higher selectivity
Cleaner extracts
More reproducibility
The avoidance of emulsion formation
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Application of SPE in various fields
Impurity profiling of pharmaceuticals
Environmental applications
Applications in food chemistry
Analysis of wines and other alcoholic beverages
Application to biological fluids
Hair analysis49
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Impurity profiling of pharmaceuticals
Residual solvent analysis by USP 467Involves head space solid phase micro extraction with GC and FID detector
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Application to biological fluids:
Simultaneous qualitative and quantitative determination ofDrugs of abuse opiates, cocaine, or amphetamines Prescribed drugs tricycle antidepressants,phenotiazines, benzodiazepines in biological fluids was developed
Eg. A Weak Cation-Exchange Monolithic SPE Column for Extraction and Analysis ofCaffeine and Theophylline in Human Urine
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Urinary Excretion Pattern of Benzophenone-3 and its Metabolite 2,4-Dihydroxybenzophenone in Human Urine
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Hair analysis It is used for the long-term monitoring of drug and alcohol
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References
Anal Bioanal Chem (2007) 388:1643–1651DOI 10.1007/s00216-007-1301-4K. Dettmer & D. Hanna
Chromatographia Vol. 41, No. 7/8, October 1995Comparison of On-Line SPE-HPLC and SPME-GC for theAnalysis of Microcontaminants in WaterC. Rivasseau / M. CaudeLaboratoire de Chimie Analytique (associ6 au CNRS, URA 437) de l'Ecole Sup6rieure de Physique et de ChimieIndustrielles, 10 rue Vauquelin, 75005 Paris, France
ChromatographiaTao Zhu, Kyung Ho Row&Department of Chemical Engineering, Inha University, 253 Yonghyun-Dong, Nam-Ku, Incheon 402-751, Korea; E-Mail: [email protected]: 10 October 2008 / Revised: 21 January 2009 / Accepted: 13 February 2009
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INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRYANALYTICAL CHEMISTRY DIVISIONCOMMISSION ON GENERAL ASPECTS OF ANALYTICAL CHEMISTRYM. MOORS1, D. L. MASSART' and R. D. McDOWALL''Vrije Universiteit Brussel, Pharmaceutical Institute, Laarbeeklaan 103, B-1090 Brussels, Belgium'Department of Chemistry, University of Surrey, Guildford, Surrey, GU2 SHX, UK
SIGMA ALDRICH Chemie GmbH SIGMAEschenstraße 5, 82024 Taufkirchen GermanyAnalytical Chemistry Insights 2008:3 1–7
http://www.whatman.com/SPEColumnsandCartridges.aspx
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THANK YOU
All of you
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