CHROMATOGRAPHY
advertisement
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 (10-500L) 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)
