Contents
Introduction
Principle
Types Solid phases
Experimental steps
Trace enrichment
Solid phase micro extraction (SPME)
Comparison with HPLC
Application
2
What is the solid
phase
extraction?
3
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
4
Liquid-solid extraction
Column extraction
Digital chromatography
Bonded phase extraction
Selective adsorption techniques 5
Strategies for solid phase extraction
Active substance can be:
Unretained- while matrix interference are adsorbed
Retained-while matrix interference are washed through
6
Retained-while matrix interference are washed through
7
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 useful
8
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
9
Cartridge
10
11
Experimental procedure of five steps:
[Link] 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
[Link] of interfering compounds retained in step 3 with
a solvent, but shouldn’t remove the analytes (washing step)
[Link] of the analytes with an appropriate solvent
(desorption or elution step) and collecting for analysis
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Types of SPE
•Reverse Phase
•Normal Phase
•Ion Exchange
OVERVIEW OF SPE
19
Instrumentation
INSTRUMENTATION
•Cartridges –Capacity and size
•Filteration apparatus
• SPE with vaccum technique
• Vaccum technique with large samples
• Pressure technique with syringe
Desirable
Desirable properties
Properties of SPE
of SPE Particles
partiu
•Porous, large surface area : Surface area > 100 m sq. Greater the
surface area ,more adsorption
•Reversible Adsorption
•Pure, Low impurities
•Good Chemical stability
•Good Surface contact with sample solution –Pretreatment for
increasing hydrophilicity
•High Percentage recovery
Solid
phases
Bonded silica phases
Organic Polymers
Modified Polymeric Sorbents – Introduction of specific
functional groups
Carbon
Porous Graphitized Carbons
Ion Exchange Sorbents
Resin Loaded Membranes 22
Properties of Bonded Phase Silica
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 23
Polymeric resins, cellulose and zirconia
Zirconia coated silica as a stationary phase
24
Examples of selective stationary phases
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 cavities
designed for a template molecule
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Technical Data - Solid Phase Extraction (SPE) Media Product
Sorbent Abbreviations Description
ODS Octadecyl silica 5% carbon load
ODS-4 Octadecyl silica 14% carbon load, end capped*
ODS-5 Octadecyl silica 18% carbon load, end capped
C-8 Octyl silica 8.5% carbon load, end capped
FLO Florisil™ Magnesium silicate
NH2 Weak anion exchanger Primary amine
SAX Strong anion exchanger Quaternary amine (-NR3+)
SCX Strong cation exchanger Aromatic benzene sulfonic acid
SIL Normal phase silica
* End capping masks residual silanol groups, reducing ionic affinity for
amines.
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SS
Sorbent analyte Interaction
•Solvation
•Covalent
•Non polar
•Polar
•Mixed mode
•Ion exchange
•Miscelleneous
Sample Pretreatment of Different biological Matrices
•Liquid Samples
•Protein Samples
•Solid Samples
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
29
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
30
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 importance
Substance
Diagnosis of cancer
ELISA TEST
31
IMMUNOAFFINITY
PHASES
32
TRACE ENRICHMENT WITH SPE
Sensitivity depends on
Physicochemical properties of the
Analyte Detection system
Selective clean-up
Isolation and concentration step
33
Solid phase microextraction:
SPME is the technique in which by using special instrument,
sampling is possible in a vapour state
34
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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36
Fused silica fibre coated with thin film of several polymeric
stationary phases
Reusable and replaceable
Small size and cylindrical geometry of fiber
Placement into sample or headspace is
easy
Loading in desorption chamber of GC or
Interphase of the HPLC without any
modification of Plunger
37
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
38
39
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
40
Extraction can be performed in
two ways
1) Headspace SPME or HS-SPME
Fiber is exposed in the vapour phase above a gaseous, liquid,
or solid sample
2) Direct immersion or DI-SPME
Fiber is directly immersed in liquid samples
41
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
42
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
38
Metallic ions
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
chromatogra
44
ph by means
of an
Types of online SPE:
SPE-GC(SPME-
GC) SPE-HPLC
45
ONLINE SPE-GC
46
ONLINE SPE-HPLC
47
10ppb Nitrosamines in Water: SPME-GC/MS
Chromatogram courtesy of J. Clark, Liggett Group, Inc.
48
ONLINE SPE-HPLC
THT= Tetra hydro thiophene 44
Comparison of SPE-HPLC and SPME-GC
SPE-HPLC SPME-GC
Universality Compounds +++ +
Detection Sensitivity ++ +++
Selectivity +++ ++
Identification + +++
Detection limit (µ/ 0.05-0.8 0.2-5
L)
Reproducibility (%) 1-15 4-14
Analysis time 90 20
Sample volume (mL) 200 2
Automation +++ +++
Simplicity + +++
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Comparison of SPE and HPLC
Theoretical basis as HPLC
Retention and selectivity remain unaffected by particle
size Efficiency dependent on:
Particle size
Column geometry
Typical number of plates
HPLC ~ 10,000
SPE < 50
Minimum
Selectivity(alpha)for
46
Rs=1.2 HPLC 1.06
SPE 3.95
52
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 analysis
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Impurity profiling of pharmaceuticals
Residual solvent analysis by USP 467
Involves head space solid phase micro extraction
with GC and FID detector
55
Application to biological fluids:
Simultaneous qualitative and quantitative determination
of Drugs 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 of
Caffeine and Theophylline in Human Urine
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Urinary Excretion Pattern of Benzophenone-3 and its
Metabolite 2,4-Dihydroxybenzophenone in Human
Urine
57
Hair analysis
It is used for the long-term monitoring of drug
and alcohol
58
References
Anal Bioanal Chem (2007) 388:1643–1651
DOI 10.1007/s00216-007-1301-4
K. Dettmer & D. Hanna
Chromatographia Vol. 41, No. 7/8, October 1995
Comparison of On-Line SPE-HPLC and SPME-GC for the
Analysis of Microcontaminants in Water
C. Rivasseau / M. Caude
Laboratoire de Chimie Analytique (associ6 au CNRS,
URA 437) de l'Ecole Sup6rieure de
Physique et de Chimie
Industrielles, 10 rue Vauquelin, 75005 Paris, France
Chromatographia
Tao Zhu, Kyung Ho Row&
Department of Chemical Engineering, Inha University, 253 Yonghyun-Dong, Nam-Ku,
Incheon 402-751, Korea; E-Mail: rowkho@[Link]
Received: 10 October 2008 / Revised: 21 January 2009 / Accepted: 13 February 2009 59
INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRY
ANALYTICAL CHEMISTRY DIVISION
COMMISSION ON GENERAL ASPECTS OF ANALYTICAL CHEMISTRY
M. 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 SIGMA
Eschenstraße 5, 82024 Taufkirchen
Germany
Analytical Chemistry Insights
2008:3 1–7
[Link]
[Link]
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