Lecture Chromo-7:Electric Field-Driven SeparationsIon Mobility Spectrometry, Gel Electrophoresis and Capillary ElectrophoresisCU- Boulder CHEM 5181Mass Spectrometry & ChromatographyProf. Jose-Luis JimenezFall 2003R. Weinberger: Practical Capillary Electrophoresis, Academic Press, 1993Objective of Today’s Lecture• Learn about separations driven by electric fields – Not under vacuum! (unlike MS)– In a liquid: “Electrophoresis”– In a gas: “Ion Mobility Spectrometry”• We teach them under chromatography, but chromatographic principles (partitioning between phases) do not applyBooks on ElectroseparationsAuthor Publisher Title $ on AmazonComments Rubinson & Rubinson Prentice-Hall Contemporary Instrumental Analysis, 2000 (QD79.J5 R83 2000 - In Library) $127 Good Basic Chapter of CE and IMS Weston & Brown Academic HPLC & CE, 1997 (QD79.C454 H63) $82 Good reference with lots of pointers to the research literature Cunico, Gooding, & Wehr Bay Bioanalytical Labs Basic HPLC and CE of Biomolecules, 1998 (QP519.9.H53 C86) $40 Good, practical reference, read it if you use these techniques a lot for biomolecules Weinberger Academic Press Practical Capillary Electrophoresis, 2nd Ed. 2000 (QP519.9.C36 W45 2000) $105 General reference on CE Part 1: Ion Mobility Spectrometry“Electrophoresis in the gas-phase”Ion Mobility Spectrometry IVdistance++1. Electric force pulls2. “Drag” of gas retards3. Ratio is Ion mobility+++++++-------Questions• How many collisions does an ion experience every ms?• Do the ions continuously acceleterate?Ion Mobilityv = KEd = KtE•Where:• d is the distance traveled (cm)• K is the ion mobility (cm2V-1s-1)– Increases with number of charges– Decreases with cross sectional area (“bigger target”)• t is the time (s)• E is the electric field strength (V cm-1)• If d is fixed, and K1> K2?Ion Mobility Spectrometry II(IMS)From RubinsonIdea: “race” of ions; most mobile get to detector firstDoes this remind you of something?Example of Ion Mobility SpectrumFrom RubinsonPros and Cons of IMS• Advantages–Very fast– Continuous– Low detection limits (ppb)– Relatively cheap• Disadvantages– Low resolution– Potential interferences• Chemical ionization reactions! => “Matrix Effects”• Clustering of ions: A+(H2O)2, A+(H2O)3 ,A+(H2O)4A Common Application of IMSA Common Application of IMSPart 2: Gel ElectrophoresisElectrophoresis IVdistance+1. Electric force pulls2. “Drag” of gas retards3. Ratio is Ion mobility+++++++-------+Concept of Electrophoresis• Used (mostly) to separate charged molecules• Based on differences on:– molecular movement through a fluid (“carrier electrolyte” or “buffer”)– under an electric field• No partitioning between mobile and stationary phases– Not a chromatographic technique– Result called an “electropherogram”Physical Basis of Electrophoresis• Solutions can be electrically conductive– Due to the migration of individual ions• Different ions migrate at different rates– Electrophoresis means “ion migration”– Analytical EP uses the differential ion migration rates as a means of separationElectrophoresis vs. ChromatographyFrom WestonDefinition of Ionic Mobility in SolutionIon+Fdragvv: relative velocity between the ion and the fluidFelecFdrag=6πηrvFelec= qErqEvπηµ6==Ionic MobilityUnits: cm2/V.sMobilities for Group 1A MetalsFrom WeinbergerMobility vs. MW for biomoleculesFrom WeinbergerClassical Slab-Gel Electrophoresis• Multiple lanes• One or more lanes used for standard mixtures (calibration)• Often used to classify according to molecular size, due to the distribution of gel pores (like size exclusion C)• Gel plate should also dissipate the Joule (V=RI) heat generated From Kealey – Instant Notes on Analytical ChemistryBios Scientific Publishers, Oxford, UK, 2002Slab Gel Electrophoresis IIFrom RubinsonExample of Gel Electrophoresis• Separation of PCR products• Sizing standards on outer lanes• Two replicate lanes of each sampleFrom WeinbergerCombined TLC- Gel ElectrophoresisFrom BraithwaitePart 3: Capillary Electrophoresis“High Performace Electrophoresis” or“Ion Mobility Spectrometry in a Liquid”Conceptual Schematic of HPCEFrom KealeyCapillary for HPCEFrom WeinbergerWhy small capillaries in HPCE?Temperature in HPCEFrom RubinsonHistory of HPCEFrom WeinbergerGrowth of HPCENumber of mentionsof CE in Chemical Abstracts(Research Phase, use continues to increaseafter abstracts taper off)From CunicoEfficiency: ChromatographyuCuCuBAHMS+++=Efficiency: CENLHtNR== and σH??Comparison of LC and ElectrophoresisFrom WeinbergerPositive vs. negative ions in CEVdistance+Which direction will eachType of ion move in?+++++++--------Electroosmotic Flow (EOF) IFrom WestonElectro-Osmotic Flow IIFrom KealeyFlow ProfilesFrom WestonOrder of Elution with EOF• What would be the order of elution between?– Small cations– Small anions– Large cations– Large anions– Neutral moleculesFrom SettleTypes of HPCE• Capillary Zone Electrophoresis (CZE)• Capillary Isotachophoresis (CITP)• Capillary Isoelectric Focusing (CIEF)• Micellar Electrokinetic Capillary Chromatography (MEKC)• Capillary Gel Electrophoresis (CGE)• Capillary Electroosmotic Chromatography (CEC)ClassificationFrom WestonSelecting a HPCE ModeFrom WeinbergerCapillaryZoneElectrophoresisFrom RubinsonCapillaryZoneElectrophoresisFrom KealeyResponse vs. Time and LengthFrom WeinbergerSpecies Ionic Mobility vs pHFrom WeinbergerEffect of pH on CZE SeparationFrom WeinbergerTypical Buffers and Additives for CEFrom KealeyIsoelectric Points for ProteinsFrom KealeyIsoelectric Focusing ITrick: modify the pH on-line => change the mobility on-lineFrom WeinbergerIsoelectric Focusing IIFrom RubinsonIsoelectic Focusing IIIFrom WeinbergerMicellar Electrokinetic Capillary Chromatography (MEKC)MEKC: separation due to partitioningbetween two mobile phases moving at different velocitiesFrom KealeyPartition in Micellar LCFrom WeinbergerMicellar Electrokinetic CEFrom SettleOther Types of CE• Capillary Gel Electrophoresis (CGE)– Like size exclusion chromatography• Capillary Electroosmotic Chromatography (CEC)– Chromatography (partition)– Electroosmotic “pump”– Allows miniaturization and improved resolutionCE Detectors IFrom KealeyCE Detectors: Detection LimitsFrom SettleAccuracy and Precision of HPCEFrom WeinbergerAppendix:Tuning and Troubleshooting CETuning of HPCEFrom WeinbergerCE