Analytical Toxicology Instrumentation 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 immisible extracting solvent Repeat these steps 1 to 3 times Combine the extracts and evaporate to reduce the volume 2 Shake or mix thoroughly 3 Separate the 2 phases

Solvent Extraction Distribution coefficient: Fraction remaining after n extractions:

Miscibility of solvents Miscible if the two solvents can be mixed in all proportions without forming two phases

Solid Phase Extraction - SPE RetentionA liquid sample passed through a short column of solid sorbent, where the desired compounds are sorbed ConditioningThe sorbent is wetted and rinsed by the eluting solvent RinseUnwanted compounds are rinsed by elution with a suitable solvent ElutionThe 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 m 2 g -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 sorbent coating 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 Spectroscopy A =  bc A =  bc PoPo P

Measure at max A

DetectorCell Grating D 2 Lamp Tungsten Halogen Lamp Single beam spectrophotometer

Double beam Spectrophotometer Detector D 2 Lamp Reference Cell Chopper Sample Cell Beam Splitter

Which drugs can be determined using UV-Vis?   n  n   n  Energy   n  n       n     Energy

Luminescence Spectroscopy VR IC ISC IC and EC Absorption FL Ph T1T1 S1S1 S0S0 S2S2

Absorption Fluorescence Phosphorescence I Wavelength F = KP 0  bc

Fluorometers Sample cell Detector F em F ex Source Black Surface

Spectrofluorometers Source Sample cell Detector em ex Black Surface

Luminometers

Atomic Absorption Spectrometers 3s 4s 5s 6s 3p 3d E

h molecular Solution of Analyte Spray Nebulization Atoms Solid/Gas Aerosol Gaseous molecules Desolvation Volatilization Excited Molecules h Atomic Atomic Ions Excited Atoms h Ionic Excited ions

Sample introduction High Pressure Gas Flow Solution sample High Pressure Gas Flow Solution sample

AAS Spectrometers Flame or Graphite Furnace Atomizer Detector PrPr P Half-Silvered Mirror

Atomic Emission Spectroscopy

DCP

ICP Spectrometer Detector

Mutichannel ICP Spectrometer Grating CCD or CID Detector

ICP-MS

Injector Carrier Gas Syringe Vaporizatio n Chamber To Column Septum

Thermal Conductivity detector

Flame Ionization Detector

Packed Columns These columns are fabricated from glass, stainless steel, copper, or other suitable tubes. Stainless steel is the most common tubing used with internal diameters from 1-4 mm. The column is packed with finely divided particles (< mm diameter) which is coated with stationary phase. However, glass tubes are also used for large scale separations. Several types of tubing were used ranging from copper, stainless steel, aluminum and glass. Stainless steel is the most widely used because it is most inert and easy to work with. The column diameters currently in use are ordinarily 1/16" to 1/4" 0.D. These columns are fabricated from glass, stainless steel, copper, or other suitable tubes. Stainless steel is the most common tubing used with internal diameters from 1-4 mm. The column is packed with finely divided particles (< mm diameter) which is coated with stationary phase. However, glass tubes are also used for large scale separations. Several types of tubing were used ranging from copper, stainless steel, aluminum and glass. Stainless steel is the most widely used because it is most inert and easy to work with. The column diameters currently in use are ordinarily 1/16" to 1/4" 0.D.

Capillary/Open Tubular Open tubular or capillary columns are finding broad applications. These are used for fast and efficient separations but are good only for small samples. The most frequently used capillary column, nowadays, is the fused silica open tubular column (FSOT) which is a WCOT column. The external surface of the fused silica columns is coated with a polyimide film to increase their strength. The most frequently used internal diameters occur in the range from micrometer.

Liquid Stationary Phases In general, the polarity of the stationary phase should match that of the sample constituents ("like" dissolves "like"). Most stationary phases are based on polydimethylsiloxane or polyethylene glycol (PEG) backbones: In general, the polarity of the stationary phase should match that of the sample constituents ("like" dissolves "like"). Most stationary phases are based on polydimethylsiloxane or polyethylene glycol (PEG) backbones:

Stationary phases

Typical Separation

TPGC versus Isothermal

Head Space GC