Analytical ToxicologyAnalytical Toxicology
Instrumentation and MethodologiesInstrumentation and Methodologies
Sampling from liquid phases
Liquid extractions:1. Batch liquid-liquid extraction
Headspace sampling:• Static headspace (high concentrations)• Dynamic headspace (low-moderate concentrations)• Purge-and Trap (low concentrations)
Solid phase extractions:2. SPE – Solid Phase Extraction (transfer to another solvent)3. SPME – Solid Phase Micro-Extraction (solventless)
Solvent Extraction
1 Add immisibleextracting solvent
Repeat these steps 1 to 3 times
Combine the extracts and evaporate to reduce the volume
2 Shake or mixthoroughly
3 Separate the 2phases
Solvent Extraction
Distribution coefficient:
Fraction remaining after n extractions:
1
2
C
CD
n
sn
VDVC
Cq
12initial,1
extractionnafter,1
/1
1
Miscibility of solvents
Miscible if the two solvents can be mixed in all proportions without forming two phases
Solid Phase Extraction - SPE
Retention A liquid sample passed through a short column of solid sorbent, where the desired compounds are sorbed
Conditioning The sorbent is wetted and rinsed by the eluting solvent
Rinse Unwanted compounds are rinsed by elution with a suitable solvent
Elution The analytes are eluted by a suitable eluent
Acetonitrile
Water
Water samples
Methanol/water
Acetonitrile
The sorbent is conditioned by a pure solvent like the matrix
Solid Phase Extraction - SPE
Solid phases for SPE
Most materials are bonded phases attached covalently to big porous silica particles(o.d. 50 m surface 50 m2g-1)
Non-polar sorbents (reversed phase):• C18 – C8 – C6 – C4 – C2• Cyclohexyl• Phenyl• Cyanoprolyl
Polar sorbents (normal phase):• Cyanopropyl• Bare Silica• Diol• Aminoalkyl
Ion exchange sorbents:• Strong Anion eXchanger• Strong Cation eXchanger• Weak anion- and cation exchangers
Endcapped sorbents:• accessible –SiOH are reacted
with trimethyl silane
SPE – Modes and eluents
Change of sample polarity:• dilute with appropriate solvent• exchange solvent by SPE
Solid Phase Micro-Extraction - SPME
Metal rod
Silica fiber
Solid sorbentcoating
Protecting metal tube
1 cm
Sorption of analytes:• In situ extraction from headspace
or liquid samples• 1-60 min• NO SOLVENTS USED !
Thermal desorption:• Splitless injection in GC• (Interface for HPLC are known)
Solid Phase Micro-Extraction - SPME
UV-Vis Absorption SpectroscopyUV-Vis Absorption Spectroscopy
A = A = bcbc
Po
P
Measure at Measure at maxmax
A
DetectorCellGrating
D2 Lamp
Tungsten Halogen
Lamp
Single beam spectrophotometerSingle beam spectrophotometer
Double beam SpectrophotometerDouble beam Spectrophotometer
Detector
D2 Lamp
Reference Cell
Chopper
Sample Cell
Beam Splitter
Which drugs can be determined Which drugs can be determined using UV-Visusing UV-Vis??
n
n
n
Energy
n
n
n
Energy
Luminescence SpectroscopyLuminescence SpectroscopyVR
IC
ISC
IC and ECAbsorption
FL
Ph
T1
S1
S0
S2
Absorption
Fluorescence
Phosphorescence
I
Wavelength
F = KP0bc
FluorometersFluorometers
Sample cell
Detector
Fem
Fex
Source
Black Surface
SpectrofluorometersSpectrofluorometers
Source
Sample cell
Detector
em
ex
Black Surface
LuminometersLuminometers
000000000000000000
Atomic Absorption SpectrometersAtomic Absorption Spectrometers
3s3s
4s4s
5s5s
6s6s
3p3p
3d3d
EE
h molecular
Solution of Analyte
Spray
Nebulization
Atoms
Solid/Gas Aerosol
Gaseous molecules
Desolvation
Volatilization
Excited Molecules
h Atomic
Atomic Ions
Excited Atoms
h IonicExcited ions
Sample introductionSample introduction
High Pressure Gas Flow
High Pressure Gas Flow
Solution sampleSolution sample
High Pressure Gas Flow
High Pressure Gas Flow
Solution sampleSolution sample
AAS SpectrometersAAS Spectrometers
Flame or Graphite Furnace Atomizer
DetectorDetector
Pr
P
Half-Silvered Mirror
Atomic Emission SpectroscopyAtomic Emission Spectroscopy
DCPDCP
ICP SpectrometerICP Spectrometer
Detector
Mutichannel ICP SpectrometerMutichannel ICP Spectrometer
Grating
CCD or CID Detector
ICP-MSICP-MS
InjectorInjector
Carrier Gas
Syringe
Vaporization Chamber
To Column
Septum
Thermal Conductivity detectorThermal Conductivity detector
Flame Ionization DetectorFlame Ionization Detector
Packed ColumnsPacked Columns
These columns are fabricated from glass, stainless steel, These columns are fabricated from glass, stainless steel, copper, or other suitable tubes. Stainless steel is the most copper, or other suitable tubes. Stainless steel is the most common tubing used with internal diameters from 1-4 mm. common tubing used with internal diameters from 1-4 mm. The column is packed with finely divided particles (<100-300 The column is packed with finely divided particles (<100-300 mm diameter) which is coated with stationary phase. mm diameter) which is coated with stationary phase. However, glass tubes are also used for large scale separations. However, glass tubes are also used for large scale separations. Several types of tubing were used ranging from copper, Several types of tubing were used ranging from copper, stainless steel, aluminum and glass. Stainless steel is the most stainless steel, aluminum and glass. Stainless steel is the most widely used because it is most inert and easy to work with. widely used because it is most inert and easy to work with. The column diameters currently in use are ordinarily 1/16" to The column diameters currently in use are ordinarily 1/16" to 1/4" 0.D. 1/4" 0.D.
Capillary/Open TubularCapillary/Open Tubular
Open tubular or capillary columns are finding broad Open tubular or capillary columns are finding broad applications. These are used for fast and efficient applications. These are used for fast and efficient separations but are good only for small samples. The separations but are good only for small samples. The most frequently used capillary column, nowadays, is most frequently used capillary column, nowadays, is the fused silica open tubular column (FSOT) which is the fused silica open tubular column (FSOT) which is a WCOT column. The external surface of the fused a WCOT column. The external surface of the fused silica columns is coated with a polyimide film to silica columns is coated with a polyimide film to increase their strength. The most frequently used increase their strength. The most frequently used internal diameters occur in the range from 260-320 internal diameters occur in the range from 260-320 micrometer. micrometer.
Liquid Stationary PhasesLiquid Stationary Phases
In general, the polarity of the stationary phase should In general, the polarity of the stationary phase should match that of the sample constituents ("like" dissolves match that of the sample constituents ("like" dissolves "like"). Most stationary phases are based on "like"). Most stationary phases are based on polydimethylsiloxane or polyethylene glycol (PEG) polydimethylsiloxane or polyethylene glycol (PEG) backbones:backbones:
Stationary phasesStationary phases
Typical SeparationTypical Separation
TPGC versus IsothermalTPGC versus Isothermal
Head Space GCHead Space GC
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