Transcript الشريحة 1
Analytical Toxicology
Instrumentation and Methodologies
Sampling from liquid phases
Liquid extractions:
1. Batch liquid-liquid extraction
Solid phase extractions:
2. SPE – Solid Phase Extraction (transfer to another solvent)
3. SPME – Solid Phase Micro-Extraction (solventless)
Headspace sampling:
•
Static headspace (high concentrations)
•
Dynamic headspace (low-moderate concentrations)
•
Purge-and Trap (low concentrations)
Solvent Extraction
1
Add immisible
extracting solvent
2
Shake or mix
thoroughly
3
Separate the 2
phases
Repeat these steps 1 to 3 times
Combine the extracts and
evaporate to reduce the volume
Solvent Extraction
Distribution coefficient:
Fraction remaining
after n extractions:
C2
D
C1
q
n
C1, aftern extractions
C1,initial
1
1 DV2 / V1
n
Miscibility of solvents
Miscible if the two solvents can be mixed in all
proportions without forming two phases
Solid Phase Extraction - SPE
Conditioning The sorbent is wetted and
rinsed by the eluting solvent
The sorbent is conditioned by
a pure solvent like the matrix
Acetonitrile
Retention
A liquid sample passed
through a short column of
solid sorbent, where the
desired compounds are sorbed
Water
samples
Rinse
Unwanted compounds are
rinsed by elution with a
suitable solvent
Methanol/
water
Elution
The analytes are eluted by a
suitable eluent
Acetonitrile
Water
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
Ion exchange sorbents:
• Strong Anion eXchanger
• Strong Cation eXchanger
• Weak anion- and cation exchangers
Polar sorbents (normal phase):
• Cyanopropyl
• Bare Silica
• Diol
• Aminoalkyl
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
Protecting
metal tube
Silica fiber
1 cm
Solid sorbent
coating
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 = ebc
P
Po
Measure at lmax
A
l
Single beam spectrophotometer
Tungsten
Halogen
Lamp
D2 Lamp
Grating
Cell
Detector
Double beam Spectrophotometer
Reference Cell
D2 Lamp
Detector
Chopper
Beam Splitter
Sample Cell
Which drugs can be determined
using UV-Vis?
Energy
Energy
s
s
p
p
p*
n
p*
n
s-s*
s-s*
p-p*
p-p*
n-s*
n-s*
n-p*
n-p*
s*
s*
Luminescence Spectroscopy
VR
IC
T1
S2
ISC
S1
Absorption
S0
FL
IC and EC
Ph
F = KP0ebc
Absorption
Fluorescence
I
Phosphorescence
Wavelength
Fluorometers
Sample
cell
Source
Fex
Fem
Black
Surface
Detector
Spectrofluorometers
Source
Sample cell
Black
Surface
lex
lem
Detector
Luminometers
000000000
Atomic Absorption Spectrometers
6s
5s
3d
4s
3p
E
3s
Solution of Analyte
Nebulization
Spray
Desolvation
Solid/Gas Aerosol
Volatilization
Gaseous molecules
Atoms
Atomic Ions
Excited Molecules
hn molecular
Excited Atoms
hn Atomic
Excited ions
hn Ionic
Sample introduction
High Pressure
Gas Flow
Solution sample
High Pressure
Gas Flow
Solution sample
AAS Spectrometers
Detector
Pr
P
Half-Silvered
Mirror
Flame or Graphite Furnace Atomizer
Atomic Emission Spectroscopy
DCP
ICP Spectrometer
Detector
Mutichannel ICP Spectrometer
CCD or CID Detector
Grating
ICP-MS
Injector
Syringe
Septum
Carrier
Gas
Vaporizatio
n Chamber
To
Column
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 (<100-300
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 260-320
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:
Stationary phases
Typical Separation
TPGC versus Isothermal
Head Space GC