3Lecture C1 – Part 1:Introduction and Theory of ChromatographyCU- Boulder CHEM 5181Mass Spectrometry & ChromatographyProf. Jose L. JimenezFall 2007Reading: Braithwaite & Smith Chapters 1 & 24Concept 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 & Smith5ChromatogramWhy is Chromatography So Successful?ThermogramSelectivityZiemann et al.UC-Riverside6Schematic 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 ComputerLaser7Schematic 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 Phase8Schematic of Column Chromatography III• If analyte has some affinity to the stationary phase, it will be retarded– Equilibrium– Kinetics• Molecular mass transfer: diffusion– Emerge at the detector after “retention time” tRSPMCCKs=9Retention and Mobile Phase Time• Simplest chromatogram w/ 2 components– Unretained peak (tM)– Analyte peak (tR)– Corrected retention time (t’R)10Chromatography Simulator• http://www.chem.uoa.gr/Applets/AppletChrom/Appl_Chrom2.htmlInvestigate- Effect of K- Effect of N- Effect of RR11Equilibrium: 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=12Using 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 phase13The 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.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 Absorption15Separation 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• Kinetics16Partitioning in Real Chromatography17Factors 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– Temperature18Clicker 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 know19Intermolecular 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 dipole20Intermolecular 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 phase21Intermolecular 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+=ααε22Separating Efficiency – Peak WidthAssume Gaussian Peaks FWHM = wh= 2.36σHWHM = 1.18σBase Width of Peak (wb)• Width at intersection of tangents at inflexion points (+/-σ) and the baseline• wb= 4 σ• 95.5% of molecules are within wb– Assuming that the Gaussian model holds!Figure 2.3, Braithwaite & SmithWhen in doubt, use NORMDIST in Excel23Resolution in Chromatography• Objective: accurate measurement of individual peak areas (spectra)FWHMtRs*2Δ=• What is the resolution if this is a mass spectrum? • If it is a chromatogram?00.511.522.546 51 56m/z OR t'RSignal Intensity (Arbitrary Units)24Clicker Question• Analysis B is more desirable than AA. In MS & Chrom.B. In MS but not Chrom.C. In Chrom but not MSD. In neither MS nor ChromE. I need a coffee00.050.10.150.20.25100 110 120 130 140 150 160 170 18000.10.20.30.40.50.60.70.80.9100 110 120 130 140 150 160 170 180AB25Resolution vs Peak Integration• http://www.vias.org/simulations/simusoft_peakoverlap.html26Can One Have Too Much Resolution?5.12.1 <<sR27Progress of a Chromatographic Analysis• If you have too much resolution, you can– Shorten the column– Increase temperature (GC), flow rate=> Shorten the analysis28Issues for Start of Class• For review lecture: clicker question: send 3 slides about instrumentation, interpretation, chromatography (CP)– Return clickers at end of review lecture• No additional HW– Will distribute past HWs• FCQ evaluations– This year they are online for the first time– Do separately for Joel and Jose• Zoomerang.com questionnaire– Will send invitation by email, done on web– Typically more detailed and