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by: Humberto Bosco DVM


Marketplace > Texas A&M University > Geology > GEOL 689 > SPTP RADIOGENIC ISOTOPE GEOCH
Humberto Bosco DVM
Texas A&M
GPA 3.99


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Class Notes
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This 27 page Class Notes was uploaded by Humberto Bosco DVM on Wednesday October 21, 2015. The Class Notes belongs to GEOL 689 at Texas A&M University taught by Staff in Fall. Since its upload, it has received 15 views. For similar materials see /class/225979/geol-689-texas-a-m-university in Geology at Texas A&M University.




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Date Created: 10/21/15
Capmary E ectrophoresws Tasks mmws mums gtTh2urv m o W m 1 work Maw mums gtAdvama es and msamamaues gtW2WM useCE m mm a me mamuns mcmmng cw an N03 N01 so and FOR Smme amuma ed Hm Capillary Electrophoresis Ta sks pWEDNESDAY 5 Prepare sample vials for analysis 5 Prepare column and lamp 5 Prepare the method you will use to analyze your samples gtStandards have been made for you gtSET UP THE RUN olN LAB Go through the details ofthe method used Manually integrate the standards and your samples to determine concentration Data processing may be more dif cult than you think Capillary Electrophoresis Overview fused silica capilla Detector anode cathode Separation occurs within buffer based on size and charge Positive negative and neutral molecules can be separated Capillary Electrophoresis Overview Anode Cathode Separation occurs within buffer based on size and charge Positive negative and neutral molecules can be separated Capillary Electrophoresis Types of Analytes Proven I Emerging Prot lns Pharmaceuticals Peptldes Pesticides and Aman aCIdS daughter products Nucleic acids hormones Inorganic ions chemica and organic bases biological defense Organic acids Whole cells Caffeine Capillary Electrophoresis Advantages High separation ef ciency Small sample size required 110 nl Fast separation 1 to 45 min Automation Quantitation large linear range Complex sample matrix Couple to mass spectrophotometer Fundamental data Capillary Electrophoresis Disadvantages immature and undeveloped sensitive buffer systems pH nonstandard retention times Capillary Electrophoresis Overview Migration of ions in solution underthe influence of an electric field pressure injection Pressurized or elevated sample vial for loading capillary open capillary Polylmide coming Fused 15 um thick silica 330 um diameter Opening of 25775 um Fused Silica capillary diameter 50 cm length Cross section quotquotquotquotquotquotquotquotquotquotquotquotquot 39 of capillary Direction of flow Thermostated compartment Background electrolyte home ui39l Background electrolyte home 30kV variable power supply 1981JW Jorgenson first describes electrophoresis in glass capillaries Anal Chem Harris Figure 26 14 Capillary Electrophoresis Open Capillary Highperformance IquId Capillary 39 Unprecedented Chromatography electrophoresis resolution 1 4 100 plates 92 000 plates 3 No need for solute to equilibrate between g the mobile and stationary phase 3 RecallNumber of platesthe number of 39 5quot239 I 5639 39 60 I 64l I I58 I 397392 interactions ie transitions between Time min mobile and stationary phases a solute has during it s residence time in the column measure of ef ciency Harris Figure 26 1 6 Capillary Electrophoresis Open Capillary i a 7 single intact cell 5 iu m cellular debris Harris Figure 26 15 Capillary Tube Conditions Positive negative and neutral molecules can be separated Separation can take place in a fused silica capillary tube coated with polyamide to give strength Capillary 30100 cm long with an internal diameter of 25100um various types of capillaries The hydroxyl group of the fused silica dissociates leaving the tube with a net negative charge Capillary Tube Fused silica capillary tube with exposed hydroxyl groups Dissociation of hydroxyl group leaving negative charge on the inside of tube Capillary Electrophoresis Endosomatic Flow Hydrogen ion cation bilayer will ow toward the cathode causing endosomatic flow or bulk flow Turn off voltage and you stop the endoosmotic ow Capillary Electrophoresis Endosomatic Flow When the voltage is applied the anion H and cation move in a different directions ln CE the endoosmotic gt electrophoretic migration and both cations and anions migrate toward the cathode This is why non standard retention times Capillary Electrophoresis Eectroosmotic Flow Cation bilayer Q J 9 results in uniform ow Anode I lCathode a Electroosmotic velocity profile Result I I increased resolution for CE High gt Low pressure pressure l Hydrodynamic velocity profile b laminar flow Harris Figure 26 18 Capillary Electrophoresis Types of Capillaries r 50llm Side View 1 Straight cell Any length 2 Extended light path 1 bubble D Light path Fixed length 501quot l 2 Greater sensitivity 150 m ltD 3 A longer capillary will allow greater separation a O between peaks Harris Figure 26 25 Capillary Electrophoresis Types of Capillaries 3 High Sensitivity cell Zcell Light path Balance signalnoise ratio I b Capillary Electrophoresis Sample Injection Types 1 Hydrodynamicpressure difference Gravity Pressurevacuum to one end of capillary 2 Electrokineticcapillary is dipped in sample and voltage applied Differing mobility fused silica capillar Injected sample will not have the same composition as the original sample Gel Electrophoresis anode cathode Detector Capillary Electrophoresis Direct Detection 1 Ultraviolet detectors Background electrolytelow absorption reference wavelength Solutesstrong absorption sample wavelength 2 Fluorescent Naturally fluorescent analytes Capillary Electrophoresis Direct Detection 3 Amperometric Analytes that can be oxidized or reduced 3 Ag AgCl reference Pt auxiliary Nectrode electrode Electrophoresis cathode grounded Capillary Butter compartment Cu working electrode Micropositioner Harris Figure 26 26 Capillary Electrophoresis Direct Detection 3 Conductivity 4 Mass Spectrometry Low detection limits 110 nM Molecular weight and structural information Tandem UV and MS detectionmethod development and optimization Expensive 5 11 mi 1 i 77 IH 11 Capillary Electrophoresis Indirect Detection 1 UV Fluorescent Conductivity etc Background electrolytestrong steady signal reference wavelengthhigh Solutesblock signal sample wavelengthlow 00 O 0 00000 000 o 0000 O O 000 O 0 GOO GOO GOO Bright solvent Dark analyte Bright solvent zone zone zone Harris Figure 26 27 Capillary Electrophoresis Indirect Detection Background electrolyte strong steady signal reference wavelength high response Solutes block signal sample wavelengthlow response Absorbance at 254 nm gt Switch ref low and sample Time mm high to invert peaks Harris Figure 26 28 12 Capillary Electrophoresis Method Development Select Buffer SystemKEY sample matrix order ofanalytes sensitivity signal to noise ratio can you use replenishment Optimization temperature voltage injection type and duration wavelength column type and length Every environmental sample is different Capillary Electrophoresis Troubleshooting View stored data ls instrument cleansometimes daily Degraded or blocked column Degraded buffer oQuality ofwater Lamp life Quality of power sourceyour data will only be as clean as your current Capillary Electrophoresis THE MOVIE Cameca 5X50 Electron Microprobe Laboratory Capabllltle using wavelength dispersive X ray spectrometry WDS ie quotTraditionalquot electron probe microam ysis electron microscopy SEM dispersive X ray spectrometry EDS e r m r r I I39J I39 materials Fibuler sourcc Re ected light Snuxcc CCD Camera Helical Gear Beam Regulate a Sample exchange Airlock WDS Detccmi39 EDS Deluum Sample Holder Objecme Lem amp Scanning Coils WDS Specimneier and AutoZ Focus FILAIENT nus uzuum assnsroa cumoen 39 HIGH VOLTAGE sunu EQUIPOTENTIALS no PLATE Fig 3 Configuration of self biased electron gun from Cold stein 1975 reproduced with permission from Academi c Press OPEIA ING POINT l A W h I z I U l 3 I quot I I lt I I l 0 FILAMENT CURRENT I Fig LL Emlulun characteristic of the selfbiased electron gun Schema vnruuon of beam current lb Huh filament current If after Goldsteln 1975 SX 100 Beam regulation principle ng C ondens er 2 Limiting aperture w Probe Forming Lens aperture J Condenser 1 power supply u Beam stabilizer electronic feedback Ch Stable beam current over a wide range of beam intensity 7 Probe Forming Lens lllumlnallon Illumination Sourca Source Condenser Condenser Lens Lens Specimen Flnal Obiactive Lens Lens Projection Lensles Specimen Signal 39 Deleclor 4 1 Eye Fluorescent A Cmnpalhon my llmgrmm Mme pmh ul39phnlnmlhmugh 1 lighl mn39 wupu vcnm lhcpallu ol L IL EIYOHN Iluuugh a lrnsmiwun clcclrcn micmicupc lloquot v rhunr similarmcs a diagram ul 1 stunning clccrmn nncroxmpc l39 Included for Cnmpm nun Video amp in V Spacing Fig Slb Scanning electron microscope A high energy electron beam 5 50 kV hitting a sample target results in the generation of several products Electrons Secondary Backscattered Absorbed Auger X rays Characteristic Continuous Fluorescent Visible light Cathodoluminescence Heat a Incident b electron beam Monte Carlo Model of 100 639 paths 1 Emilled Electrons Lew Auger 1OA Secondary SE 500A Backscattered B SE Emilled X Ray Characteristic Continuous Fluorescent Other products Cathodoluminescence ApprOXImate scale Heat l I Absorbed Electrons Fig 34 Monte Carlo electron trajectory simulations of the interaction volume in Fe as a function of beam energy Probe forming lens Multiguide electron detector W PhotoCathode Dynodes Light Guide 9 SE e S 4 Photon Photo e39 Secondary e39 39S Scintillator Crystal TIdoped Nal Electron Beam Back Scattered Electrons 5 Xray ports t0 Spectrometers s Ax w p l Diodes Sample l0 a 1 i 5 S x LO U I U 3 g TYPICAL MEASURED CURVES FILIMENT TEME270039K FINAL AFERTURE OPTIMIIED run a o It I 2 OJ O E U W J U DI l l l 1 0l 0 m 10 IO INCIDENT SPECIMEN CURRENT mom ms I FIGURE 4 Mcasuml Ineldenl specimen currcnl a a function or beam dlamcm a Incident b electron beam Emilled Electrons gm 1 Auger 1OA Secondary SE 500A Backscattered B SE Monte Carlo Model of 100 e39 paths Emilled X Ray Characteristic Continuous Fluorescent Other products Cathodoluminescence ApprOXImate scale Heat Absorbed Electrons rm Scattered Primary Electron I d t 39Ejected Orbital quot039 en Electron Electron Electron Transition L to K XRay Photon Emitted Auger Electron Emitted O O Figure 65 Inner shell electron ionization in an atom and subsequent deexcitation by electron transitions The incident electron is elastically scattered The unscattered direction of the incident electron is shown by the dotted line The difference in energy from an electron transition is expressed either as the ejection of an energetic electron with characteristic energy Auger process or by the emission of a characteristic x ray photon V II IVI IV II IV ll IV ll II V quotI I V III quotI II I L Mu ab 39 M ab Figure 69 Electron transitions in the atom that give rise to M seIies Lseries and K series xrays By reference to a table of atomic energy levels the xray energy of each photon shown can be calculated as the difference between the associated energy levels Woldseth 1973 ATOMIC NUMBER Ka K ATOMIC NUMBER 39 6C 7N 80 9F IONe ll 12


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