Transcript CHROMATOGRAPHY

ANALYTICAL METHODS

Decision Making – Matrix

Sample Preparation & Extraction

Non-Instrumental Analysis

Instrumental Analysis

Data Analysis
ANALYTICAL METHODS
MATRIX
Non-biological - Everything imaginable
- chemicals/pure drugs (tablets, powders,
liquids)
- delivery or storage containers (bubble
wrap, paper, cellophane, bottles)
- surfaces (car interior surface, floor mats,
prison blankets, mattresses, cups,
syringes, ashtrays)
- food – turkey, eclairs, spagetti
ANALYTICAL METHODS
MATRIX
Non-biological
1) Pure Drug
- visual inspection (photograph)
Solid
Liquid


Dissolve



UV

MS
ANALYTICAL METHODS
MATRIX
Non-biological
2) Drug Residue on a Surface
Rinse with EtOH

UV

GC/MS
ANALYTICAL METHODS
MATRIX
Non-biological
3) Food
Three-way Extraction
Blend/Mix

Extraction


Acid + Total base & Neutral

GC/MS & LC
ANALYTICAL METHODS
MATRIX
Biological
1) Stomach Contents
- visual inspection
- colour tests
- 3-way Extraction – GC/MS & LC
ANALYTICAL METHODS
MATRIX
Biological
2) Urine
- 3- way extraction
- alcohol – GC
- IA (drugs of abuse)
- Confirmation of specific drugs –
GC, LC, GC/MS
ANALYTICAL METHODS
MATRIX
Biological
3) Blood
- extraction (basic) – screen – GC & GC/MS
- alcohol – GC
- IA (drugs of abuse, acetaminophen,
salicylate)
- Confirmation of specific drugs – GC,
LC, GC/MS
ANALYTICAL METHODS
EXTRACTION
Samples may require preparation prior to
extraction
- Grind or blend in water to obtain liquid
extract; then filter
ANALYTICAL METHODS
EXTRACTION
Extraction
 To pull or draw out
 Based on chemistry
 Required for analysis of drugs/poisons from
biological or coloured samples
 Unnecessary for
pure pharmaceuticals or drugs/poisons dried
onto surfaces (glasses, syringes)
presumptive tests (colour tests, Reinsch test
for heavy metals)
ANALYTICAL METHODS
EXTRACTION
Ionic
R-COOH
R-NH3
 R-COO- at high pH
 R-NH4+ at low pH
 Polar vs Non-Polar
 Liquid/Liquid - Aqueous vs Solvent
 Solid Phase Extraction
ANALYTICAL METHODS
EXTRACTION
+ base
or acid
+ solvent
ANALYTICAL METHODS
EXTRACTION
Acid Extracts Acidic drugs
R-COO- + R-NH3 + H+  R-COOH + R-NH4+


solvent

analysis
aqueous
ANALYTICAL METHODS
EXTRACTION
Base Extracts Basic drugs
R-COO- + R-NH4+ + OH-  R-COO- + R-NH3


aqueous solvent

analysis
ANALYTICAL METHODS
SAMPLE PREPARATION
Theoretically can separate drugs into 5
categories
– Strongly acidic (low pH)
– Weakly acidic (pH 5 to approaching 7)
– Neutrals (pH around 7)
– Weakly basic (pH 8-9)
– Strongly basic (pH > 9)
ANALYTICAL METHODS
EXTRACTION
In Reality:
1) Blood & Urine
- single basic or acidic extraction
2) Urine (sex.assaults), Stomach Contents, Food
- 3-way extraction
- acid, weakly basic & neutral, basic
FIGURE 5.1.2
THREE WAY SEPARATION FOR LIQUIDS AND POWDERS
Sample (A)
1)
2)
3)
4)
Aqueous (A)
1)
2)
3)
4)
Toluene/Butanol (B)
add 6N NaOH
add toluene/butanol
vortex
centrifuge
Aqueous (A)
1)
2)
3)
4)
add sat. NaHCO3
add toluene/butanol (9:1)
vortex
centrifuge
1) wash
AMPHOTERIC BASE &NEUTRAL
Toluene/Butanol (C)
add conc. HCl
add toluene/butanol
vortex
centrifuge
BASE
wash
Aqueous (A)
Toluene/Butanol (D)
Discard
1) wash
MS
HPLC
ACID
1) add diazomethane
2) dry
3) add toluene
MS
MS
HPLC
TBN
1) add acetic anhyd.
2) dry
3) add toluene
MS
ANALYTICAL METHODS
EXTRACTION
Basic Extraction – GC & GC/MS Screen
Blood + Base (NH4OH) + Toluene

BACK EXTRACTION (clean up)
Toluene + Acid (H2SO4)

Aqueous fraction + Base (NaOH) + Toluene

GC & GC/MS
ANALYTICAL METHODS
NON-INSTRUMENTAL ANALYSIS
Colour Tests
 Some drugs have ‘side groups’ which will
react with a chemical indicator to produce
colour
 Were used to quantitate drugs in the past
based on degree of colour developed
 Used now as an initial screen
 e.g. Chlorinated drugs/chemicals
ANALYTICAL METHODS
NON-INSTRUMENTAL ANALYSIS
Advantages
Rapid
Disadvantages
+ Indicator
Not
screening test
quantitative
Useful
Not
when
tablets in
stomach
dissolved
necessarily
specific

Not sensitive
ANALYTICAL METHODS
NON-INSTRUMENTAL ANALYSIS
Reinsch Test
 Uses Metal-Metal interactions to screen for the presence of
significant quantities of metals in urine/stomach contents.
 Uses Conc HCl and SnCl2 as a catayst to precipitate metals
onto a copper wire.
 Indications of positive tests are:
»
Bluish to purple - Antimony
»
Dull black- Arsenic (Sensitivity - 200 g As2O3)
»
Shiny black - Bismuth
»
Silvery - Mercury
 Presence of metals on wire can be identified by Electron
Microscopy
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - SPEC
Spectrophotometry (UV ,VIS & IR)
 Instrument used to measure the absorbance of
light by compounds within a liquid
 Based on the BEER-LAMBERT LAW –
absorbance is proportional to concentration
 Note: not linear at high concentrations
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - SPEC
Spectrophotometry (UV ,VIS & IR)

Electromagnetic radiation extends from
Cosmic rays to radiowaves

Most useful spectra for Toxicological
analyses are UV spectra, Visible spectra &
Infrared spectra
Electromagnetic Radiation
Spectrum
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - SPEC

Characteristic UV absorbance spectra – drug
identification (200-360 nm)

Carbon monoxide saturation can be measured
using absorbance of visible light (380-700nm)

Alcohol in breath can be measured using IR
absorbance (700-14,000nm 1-10 m for
EtOH)
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - SPEC

Measure the absorbance of light over a range
of wavelengths

UV spectrum - Plot Absorbance vs
wavelength

Many drugs have characteristic UV
absorbance spectra which can be used to
identify a suspected drug or confirm a
submitted drug sample
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - SPEC
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - SPEC
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - SPEC
IR Wavelengths Used by the
Intoxilyzer 5000C
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - SPEC
Spectrophotometry
Advantages
Disadvantages

Fast
 Non specific

Easy
 Not applicable to all
compounds

Non-Destructive
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - IA
Immunoassay

A technique that uses antibodies to isolate a
compound of interest

Combined with radioactivity or enzyme-based
colour changes (Enzyme-linked immunosorbant
assay- ELISA) to measure amount of
compound in the sample

Primary use in forensic science is as a
screening tool
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - IA
Direct Method
(many radioimmuno assays)
Sample (Drug) + Ab*
Ab*-Drug + Ab*
Isolate and Measure Ab*-Drug
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - IA
Indirect Method (Competitive)
Enzyme-linked-immunosorbant Assay (ELISA)
Step 1
Add Enzyme-Drug + drug to Ab (bound to well)
Step 2
Wash
Enzyme-Drug-Ab + Drug-Ab
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - IA
Indirect Method (Competitive)
Step 3
Add Enzyme dependant flourescence
***Enzyme-Drug-Ab + Drug-Ab
Step 4
Measure Fluorescence
 Drug -  flourescence
 Drug -  flourescence
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - IA
Advantages
 Requires small amount of sample

Rapid test; results can be obtained in
hours

ELISA can provide results for a battery of
drugs in one assay

Can provide reliable quantitation for some
drugs, especially in serum
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - IA
Disadvantages
Cross reactivity can lead to false positives
Cross reactivity can result in elevated
quantitation
Some assays provide unreliable results from
postmortem samples (e.g. Ethanol in PM blood)
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS
CHROMATOGRAPHY
chromatography is a separation
process that is achieved by
distribution of the substances
between a mobile phase and a
stationary phase
Amobile  A stationary
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS
Amobile  A stationary
Mobile
Stationary
Liquid
Solid
Gas
Liquid
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS
COMPONENTS
I NJECTI O N
DETECTI O N
SEPARATI O N
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - TLC
Thin Layer Chromatography
Injection: Dotting of solution
Separation:
Silica bound to glass plate
Stationary Phase: Silica (solid)
Mobile Phase: Solvents (liquid)
Detection: Colour, fluorescence, UV, dyes
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS – TLC
Mix
Codeine
Butalbital
Salicylate
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - GC
Gas Chromatography
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - GC
Injection:
 flash evapourates sample (eg. 240oC)
 introduces sample onto column with
carrier gas (mobile phase)
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - GC
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - GC
Separation:
Column (packed or capillary)
Stationary Phase – Liquid
Mobile Phase - Gas
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - GC
Separation:
 based on vapour pressure of
components to be separated
& affinity for mobile phase
 time spent in liquid phase vs.
gas phase
 dependant on temperature
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - GC
Separation:
 Isothermal
 useful for separation of known
component from mixture
Temperature program
 useful for separation of a variety of
components of different size
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - GC
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - GC
Mobile Phase (Carrier Gas) – Gas
 does not interact with components
 dry (no water)
 free of oxygen
He, Ar, N2, H2
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - GC
Detector
monitors the carrier gas as it emerges from
the column and generates a signal in
response to variations in its composition due
to eluted components
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - GC
Detectors
 fast speed of response
 high sensitivity
 linear response
 good stability
 ease of operation
 uniform response to a wide variety of
chemical species
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - GC
Common Types of Detectors




Flame Ionization Detector (FID)
Electron Capture Detector (ECD)
Nitrogen Phosphorous Detector (NPD)
Thermal Conductivity Detector (TCD)
 Mass Spectrometry (MS)
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - HPLC
High Performance Liquid Chromatography
 Preparative
 separation, purification, large amounts
 **Analytical**
 separation, quantitation, identification
Useful for non volatile, large molecules
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - HPLC
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - HPLC
Injection
Sample (Syringe)

Injection Valve

PUMP
(Mobile phase)
Sample Loop
(10-500L)

Column (stationary phase)
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - HPLC
Pump
 pushes mobile phase thru column
under high pressure
 flow rates: 0.01 – 10 mL/min
 pressure: 1 – 5,000 psi
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - HPLC
Mobile Phase
 Liquid
 can vary composition to optimize
separation
 alter polarity & pH
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - HPLC
Mobile Phase
Common Properties
 pure
 compatible with the detector
 dissolves sample
 low viscosity
 chemically inert
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - HPLC
Stationary Phase - Column
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - HPLC
Stationary Phase
 surface chemistry on the packed particles
determines the type of interactions to
take place
 normal phase = polar
 reverse phase = non-polar
 cation exchangers
 anion exchangers
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - HPLC
Separation
 based on interaction between column packing and
mobile phase
 normal phase HPLC
Stationary phase = polar, Mobile phase =
nonpolar
 reverse phase HPLC
Stationary phase = non-polar, Mobile phase =
polar
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - HPLC
Separation
Isocratic
 same pH
 useful for separation and quantitation of a
known analyte
Gradient
changing of pH
 useful for separation of a large number of
analytes – screening
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - HPLC
Common Detectors
 Refractive Index
 UV-Vis
Fixed wavelength
 Variable wavelength
 Diode array
 Fluorescence
 MS
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - MS
Mass Spectrometry
 Powerful detector
 can be teamed up with GC or HPLC
GC/MS
LC/MS
Requires very low pressure (10-5 Torr)
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - MS
Uses the difference in mass-to-charge ratio
(m/z) of ionized atoms or molecules to
separate them from each other
 Useful for quantitation
 Useful for obtaining chemical and
structural information
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - MS
Components
 Instrument interface
 Ionization source
 Mass separator
(quadrupole, ion trap)
 Ion detector
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - MS
Instrument interface
 Goal – introduce only the analyte
into the Mass Spectrometer
 Several types
Molecular separator
Permeation interface
Open Split
Capillary Direct Interface
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - MS
Ionization
Chemical Ionization
 Methane is introduced into an electron ion
impact source to create positive ions
 The methane ions (reagent ion) then interact
with the compound of interest to form
ions of the compound
 Softer ionization
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - MS
Ionization
Electron Impact Ionization
 uses an electron beam to ionize gas phase
atoms or molecules
 breaks compound into smaller positively
charged components
 Destructive
 Finger print generation
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - MS
Ionization
Electrospray Ionization
 very fine needle and series of skimmers
 sample is sprayed into the source chamber
to form droplets that carry a charge
The solvent evapourates leaving highly
charged analyte molecules
Useful for large molecules (LC/MS)
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - MS
Mass Separator – Ion Trap
 Uses 3 electrodes to trap ions in a small
volume
 Compact size
 Can trap and accumulate ions of interest to
increase signal to noise ratio, ejects
unwanted ions
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - MS
Mass Separator – Quadrupole
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - MS
Ion Detectors
Variety of Detectors
Electron multiplier tube, Farraday cup, Channeltron,
Daly detector, microchannel plate
Purpose of all
detect ions
enhance signal
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - MS
Two Operational Modes
1)
Full Scan
collect mass data over known range
maximum qualitative info
fingerprint
ANALYTICAL METHODS
INSTRUMENTAL ANALYSIS - MS
Two Operational Modes
2)
Selective Ion Monitoring (SIM)
sample at predetermined mass
values
maximum quantitative info
ANALYTICAL METHODS
DATA ANALYSIS
QUALITATIVE INFORMATION
1) Retention Data
 characteristic of analyte
 dependent on interaction between mobile
and stationary phase
2) Detector Info
 GC/MS or LC/MS Full scan, LC-UV/VIS
spectrum, UV sectrum
should be compared to a known standard
ANALYTICAL METHODS
DATA ANALYSIS
RETENTION TIME (tR )
The time between sample injection and an
analyte reaching a detector at the end of the
columnn
Different analyte = different retention time
ANALYTICAL METHODS
DATA ANALYSIS
RETENTION TIME (tR )
ANALYTICAL METHODS
DATA ANALYSIS
Rt A = Rt B on one column or one mobile phase
Unlikely Rt A = Rt B on second column or
second mobile phase
Should be compared to a known standard
ANALYTICAL METHODS
DATA ANALYSIS
QUANTITATIVE INFORMATION
From peak information – detector response
Assumes response correlates to amount
Peak height – simple, rapid, variable
Peak area – requires data handling, manual or
automated
ANALYTICAL METHODS
DATA ANALYSIS
CHROMATOGRAM
the signal produced from the eluting
component present in the mobile phase,
plotted against time
ANALYTICAL METHODS
DATA ANALYSIS
CHROMATOGRAM
SIGNAL
QUANTITATIVE
Peak
Base line
TIME
Rt
QUALITATIVE
ANALYTICAL METHODS
DATA ANALYSIS
Quantitation
 Standards = compound of interest in sample (X)
 Known concentrations
 Concentrations similar to X
 Extracted under same conditions
 Run under same conditions at same time
ANALYTICAL METHODS
DATA ANALYSIS
Methods of Quantitation
Internal Normalization
% area peak unknown X 100 = % Concentration
% area standard
ANALYTICAL METHODS
DATA ANALYSIS
Methods of Quantitation
External Standard Method
 Series of standards of known concentration
 Plot Response Standard vs Conc. Standard
 Extrapolate Unknown Concentration from graph
using unknown response
ANALYTICAL METHODS
DATA ANALYSIS
External Standard Method
R
e
s
p
o
n
s
e
Standard Curve
unknown
response
x
x
x
x
Concentration
ANALYTICAL METHODS
DATA ANALYSIS
Methods of Quantitation
Internal Standard Method
 Series of standards of known concentration
 Add a uniform known amount of internal
standard to each standard and unknown
 Plot Response Ratio (Int. Std/Std) vs. Conc.
Ratio (Int. Std/Std)
 Extrapolate Unknown Concentration from graph
using unknown response ratio (Int.
Std/Unknown)
ANALYTICAL METHODS
DATA ANALYSIS
Internal Standard Method
Response
Ratio
Standard Curve
unknown
response
ratio
x
x
x
x
Concentration Ratio
ANALYTICAL METHODS
DATA ANALYSIS
Internal Standard Method
Conc. Unknown =
Conc. Ratio (from Std. Curve) X Conc. Internal Standard
ANALYTICAL METHODS
DATA ANALYSIS
Internal Standard
 Stable
 Measurable
 Does not interfere in terms of Rt or chemically
 Similar chemically to compound of interest
 Similar concentration to unknown
IDEAL = Deuterated internal standard
(MS only)