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