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CU-Boulder CHEM 5181 - Introduction and Theory of Chromatography

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4Lecture C1 – Part 1:Introduction and Theory of ChromatographyCU- Boulder CHEM 5181Mass Spectrometry & ChromatographyProf. Jose L. JimenezFall 2006Reading: Braithwaite & Smith Chapters 1 & 25Concept of ChromatographyIUPAC Definition:Chromatography is:a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase, SP) while the other moves(mobile phase, MP or ‘eluant’) in a definite directionBraithwaithe & Smith6Separation MechanismIn MS we used ‘handles’ provided byElectric and magnetic forces on ions. In Chrom. the ‘handles’are provided bycomponent affinity forthe SP and MP (exceptions…) Need to consider:• Equilibrium• Kinetics7ChromatogramWhy is Chromatography So Successful?ThermogramSelectivityZiemann et al.UC-Riverside8Growth of ChromatographyNumber of mentionsof HPLC or CEin Chemical Abstracts(Research Phase, use continues to increaseafter abstracts taper off)9Equilibrium: the Distribution Ratio• Stationary phase, mobile phase, & analyte form a ternary system• Each analyte is distributed between the two phases (in equilibrium):– Distribution ratio–CS: concentration of analyte on the stationary phase–CM: concentration of analyte on the mobile phaseMCCKs=10Using the Distribution Ratio: Single Step (“Plate”)Mobile phaseStationary PhaseAnalyte AAnalyte B• Q: If each symbol represents a µmol, given 10 cm2 of surface to which analytes adsorb, and 100 cm3of liquid in which analytesare dissolved, what are KA& KB?• Large K has more affinity for stationary phase• Small K has more affinitiy for the mobile phase11The first step in a separationX = 0 , t = 0X = 1 , t = 1X = 0 , t = 1PumpQ: does molecular-level kinetics matter?Key concepts:• Enriched in component which prefers mobile phase• Not very good separation in 1-step (like thermogram). It is repetition that makes it great.12Partitioning in Real Chromatography13The Distribution Ratio & SeparationsC: Large number of sequential separation steps=> magnify small differences in retention=> Measure Retention TimeClicker:(a)KA< KB< KC(b)KA> KB> KC(c)KA= KB= KCMCCKs=14Mechanisms of Partitioning to the Stationary PhaseStick to the surfaceDissolve into the bulkQ: (A) Left is Absorption, Right is Adsorption(B) Left is Adsorption, Right is Absorption15Factors Influencing Retention…• are those that influence distribution (equil.)– Stationary phase: type & properties– Mobile phase: composition & properties– Intermolecular forces between• Analyte & mobile phase• Analyte & stationary phase– Temperature16Intermolecular Forces I• Based on electrostatic forces– “Like-attracts like” or “oil and water” (similar electrostatic properties)• Polar/polar & non-polar/non-polar– Molecules with dissimilar properties are not attracted• Polar retention forces– Ionic interactions (IC)– Hydrogen bonding (permanent dipoles)– Dipole-Induced dipole17Intermolecular Forces II (Dipole)• Polar forces (cont.):– Energy of dipole-dipole interaction– Factor of 10 variation on permanent dipole moment• Factor of 104variation on interaction energies–As r6=> mainly at the surfaces⎟⎟⎠⎞⎜⎜⎝⎛−=kTrSAD6222µµεAµµ:dipole moment, A: analyte, S: stationary phase18Intermolecular Forces III (London)• London’s Dispersion Forces– Most universal interaction between molecules• Only one for non-polar species– Due to induced dipoles• Relatively weak– Energy of interaction:–αis the polarizability, I: ionization potential, A: analyte, S: stationary phase)(236SASAPALIIrII+=ααε19Clicker QuestionA. The chemical nature of the mobile phase affects distribution in gas-chromatography (GC) & liquid chromatography (LC)B. The mobile phase affects distribution in GC but not LCC. The mobile phase affects distribution in LC but not GCD. The mobile phase affects doesn’t affect distribution in either GC or LCE. I don’t know20Mass Transfer Kinetics: Fick’s LawStat. Phase From Bird, Stewart & Lightfoot, Transport Phenomena, 2ndEd. 2002YDSWAABAy00−=ωρdydDjAABAyωρ−=• Initially no substance A• Pulse at the surface• Transient concentration• Steady state profile at long times– WAy: mass flux of A–ωA: mass fraction of A– DAB: diffusivity of A in B– S: surface area; ρ : density– jAy: molecular mass flux of A21Mass Transfer Kinetics: Fick’s LawStat. Phase From Bird, Stewart & Lightfoot, Transport Phenomena, 2ndEd. 2002YDSWAABAy00−=ωρdydDjAABAyωρ−=• Initially no substance A• Pulse at the surface• Transient concentration• Steady state profile at long times– WAy: mass flux of A–ωA: mass fraction of A– DAB: diffusivity of A in B– S: surface area; ρ : density– jAy: molecular mass flux of A22Situation in Column ChromatographyStat. Phase t = 1t = 2Mass transfer takes time => separation + limit on resolutiont = 3t = 4t = 5t = 6 23Lecture C1 – Part 2:Introduction and Theory of ChromatographyCU- Boulder CHEM 5181Mass Spectrometry & ChromatographyProf. Jose L. JimenezFall 2006Reading: Braithwaite & Smith Chapters 1 & 224Issues for Start of Class• Last HW after next lecture (Tue Dec 5)• Dec 7 presentations– 10 min per person, ~8 slides• Rehearse timing• Focus on topics related to class– Why is it important? What they did? How? Results? Implications / future work?• FCQs on Dec 7 after presentations– Please bring a #2 pencil25Reminders on Preparing Prez26Review Clicker Question• In chromatographic separations…A. Equilibrium is always reached at every position along the columnB. The solubility of the analyte in the stationary phase is the most important parameterC. The kinetics of mass transfer play an important roleD. A and CE. I don’t know27Schematic of Column Chromatography• Sequence of events– At t=0 we will open the gate and let the analyte into the column– Analyte will be carried by mobile phase– Analyte may partition to stationary phase– Analyte will be detected by its absorption of light at the detectorStationary PhaseStationary PhaseMobilePhaseDetectorData to ComputerLaser28Schematic of Column Chromatography II• For simplicity, we will assume that the mobile phase moves in steps rather than continuously• If analyte had no affinity to the stationary phase, it would just follow the mobile phase– Emerge at the detector after tM(mobile phase time)Stationary PhaseStationary Phase29Retention and Mobile


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