Instrumental Review PDF

Instrumental Review Ion sources: gas-phase source has sample vaporized then ionized, desorption source has sample converted to gaseous ions electron impact: hard ionization (more fragmentation) - sample is heated to vapor then ionized by bombarding with electrons which are emitted from a heate...

Instrumental Review Ion sources: gas-phase source has sample vaporized then ionized, desorption source has sample converted to gaseous ions electron impact: hard ionization (more fragmentation) - sample is heated to vapor then ionized by bombarding with electrons which are emitted from a heated tungsten filament and accelerated by applied voltage - path of electrons and molecules are at right angles - fragmentation happens because molecules are excited when hit by electrons, and fragment when they relax chemical ionization: soft ionization - gaseous atoms are ionized by collision with ions produced by electron bombardment of a reagent gas, too low energy excitation to fragment a lot electrospray ionization: also used for proteins, oligonucleotides, polymers, etc - sample pumped through a capillary needle with an applied voltage, resulting in a charged spray of droplets that then pass through a desolating capillary so solvent is evaporated off but charge remains on the molecule - often just results in multiply charged ions, no fragmentation matrix-assisted desorption ionization: good for proteins, oligonucleotides, biopolymers - analyte is in solution under vacuum, and a laser is focused at it so the matrix absorbs the radiation, then both analyte and matrix are desorbed and ionized, then often TOF - spectra show no fragmentation, just multiple ionization and sometimes dimerization/trimerization secondary ion mass spectrometry also a method Instrument: inlet system - ion source - mass analyzer - detector - signal processor/readout (under vacuum so ionization is not lost from collisions) Resolution R = m/delta m m= Magnetic sector analyzer: uses a magnet to cause the beam from the ion source to travel in a circular path so ions are accelerated through a slit into the metal analyzer tube, and ions are scanned for mass at the exit slit by hitting a collector electrode Double focusing mass spectrometer: ions travel through an electrostatic analyzer with an applied voltage to select for only ions with a certain KE, then through the magnetic analyzer TOF mass spectrometers: ions aer produced periodically by pulses of electron bombardement, and resulting ions are accelerated into a tube by electric field applied, then separation by m/z Ion trap: gaseous ions are confined by electric and magnetic fields, then sequentially ejected by mass by increasing the voltage Orbitrap: ions in the trap travel around an electrode in circular motion proportional to m/z, and current is generated and detected Tandem mass spectrometry: ion source through mass analyzer 1 then interaction cell to further fragment it and through MA 2 to detector (e multiplier) Triple quadrupole mass spectrometer: 1 for mass separation first stage, 2 for collision focusing, 3 for second-stage mass separation (used in tandem MS) QTOF is same as QQQ but last Q replaced by TOF QQQ lowest resolution, TOF much higher, TOFTOF and QQTOF best High specificity and sensitivity - lots of applications Isotopic abundance and M+1 calculation Need R=20000 to get unit resolution Surface Spectroscopy Surface spectroscopy: something shot at a sample, causes surface damage and something else is released (ions, electrons, neutral molecules, or photons) Ions most damaging, then electrons, then photons Spot analysis: focus on a spot on the surface, observe the secondary beam 2D imaging analysis: map the surface by moving primary beam across in a pattern, observe changes in secondary beam 3D depth profiling: beam makes a hole in the surface by sputtering, then primary beam shot into the hole to get data on depth X-ray photoelectron spectroscopy: X-ray or UV beam displaces inner electron which is emitted and analyzed, determines the KE of emitted electrons (each element has characteristic binding E, some have multiple) to produce spectrum of electron beam power vs wavelength KE of electron = hv - binding E 2-5 nm surface layer penetration Instrument: solid sample in high vac chamber to avoid attenuation of the electron beam or contamination, x-ray source travels to crystal disperser in crystal monochromator, which is focused at the sample, then emitted electrons travel through a lens and through an electrostatic field to the transducer and analyzer Applications: determine element composition/oxidative states/structure Secondary Ion MS: ion beam (ie Ar+ ions) bombards surface, causes sputtering (mostly neutral atoms but some ions) and creation of secondary ions that are analyzed Low E electron flood gun is used for charge neutralization of the surface, but can cause surface damage Photon spectroscopy: primary beam and detected are photons, up to 1000 nm surface penetration, low surface damage so no vac needed Surface Plasmon Resonance (SPR): photons pass through a prism and activate surface plasmon waves Surface plasmon waves: solid surface is coated with conducting material, and free electrons on the metal film interact with photons, creating surface electromagnetic waves that propagate in the xy plane of the metal film Useful as biosensors Sum Frequency Generation (SFG): two photons interact at the surface and produce a single photon whose frequency is the sum of incident Ellipsometry: polarized light (laser) probes dielectric properties of samples Scanning electron microscopy: excited by finely focused beam of electrons, detect emitted electrons Electron microscopy has a higher resolution than optical microscopy, better picture Chromatographic Separation Mobile is forced through stationary phase in column Elution: solutes washed through the column Partition coefficient: the ratio of concentration in one solvent to another when in equilibrium Detector responds to concentration To improve column performance: improve separation or reduce band width/ broadening Dead time: time for unretained species to reach detector (flow rate) RT independent of loading amount Fronting and tailing can be due to distribution constant being concentration dependent (ie sample overload for fronting) Column bleeding: small amount of stationary phase is carried out of the column during elution, treated with cross-linking stationary phases Theoretical plates: column made of numerous layers called theoretical plates Plate height H, plate count N, length of column packing L N = L/H Higher efficiency: more plate count, less plate height N = equation *example question Optimal flow rate for best column efficiency GC has faster separation, higher efficiency because of longer column length Smaller particle size - better column efficiency but higher pressure Optimization: reduce peak broadening, improve peak separation, reduce total time, reduce max pressure, reduce max length Resolution equation Isocratic elution: constant mobile phase composition Gradient elution: varying composition HPLC is gradient elution, GC is temp programming, SFC pressure programming Qualitative analysis (compare RT), quantitative (peak area, peak height) *example question GC Instrument: carrier gas tank - flow regulators - sample injection chamber - column - detector Chemically inert carrier gas, pressure controlled, molecular sieve to filter impurities Injection needs high efficiency and reproducibility Needle into hot chamber, mix with carrier gas and into column Open tubular/capillary column: longer, slower flow rate, skinnier, walls coated with stationary phase Packed column: packed with material coated with stationary phase Many detection systems (can be tandem with other techniques) Flame ionization detector: column effluent goes into flame, collector captures the charged ions/electrons produced from the flame and monitors current change to determine sample amount, high sensitivity but destructive Electron capture detector: radioactive emitter produces electrons and ionizes carrier gas, analytes capture electrons and decrease the current, high sensitivity/ selectivity Mass spectrometer detector: challenges is thermal stability of analyte molecules Polydimethyl siloxane common stationary phase Surface-modified columns - longer lifetime Qualitative (identify), quantitative (peak area/peak height) Fast analysis, mostly for small volatile and thermostable molecule analysis Tandem GC with polar and non polar columns LC High performance LC (or just LC) - smaller packing particles, lower plate height, better column performance, higher pressure Efficiency of separation depends on difference in elution time and broadness of the peaks Longer time passing through column - broader band Resolution - how far apart two bands are relative to their widths, measures ability of the column to separate the solutes Broadening outside of the column can occur - due to injection/detector (equation) or connecting tubing (equation) Column and flow rate affect plate height (equation depends on multiple paths, longitudinal diffusion, and equilibration time) Longitudinal diffusion: solute molecules diffuse away from the concentrated center of the band in both directions due to the concentration gradient (inversely prop to flow rate) Greater for gas than liquid so optimal flow rate is higher for gas (equation) There is finite equilibration time between phases - solute must diffuse from the mobile phase to surface of stationary for equilibration to occur, and this depends on distance traveled and inversely on diffusion rate (equation) Open tubular columns - higher resolution, shorter time, higher sensitivity (if small radius), but lower sample capacity Reduce extra column broadening (any additional column traveling) Instrument: bubble helium through to degas (called sparring), also filter (lots of complex stuff before injector, column, and detector) Gradient elution - vary ionic strength or pH or polarity Analytical column - straight, 10,000ish plates Analytical micro LC - smaller diameter, higher N Guard column - short, larger particle size, used to prevent impurities from reaching the analytical column Micro LC vs capillary vs nano (decreasing flow rates from above 10 microliters per min to below) Lots of techniques that can be detectors (UV-Vis, IR absorption, fluorescence, ESI-MS) Functionalized silica as stationary phase Longer binding chain - stronger retention Normal phase is polar stationary non polar mobile, reverse is reverse Polarity of solvents ranges from hexane to water Partition chromatography: liquid stationary phase, partition coefficient Absorption chromatography: solid stationary phase, can be very strong retention and even separate isomers Ion exchange: for cations or anions, interact with charged resin Ion exclusion: retain and separate neutral species, ions flow through Size exclusion: larger molecules travel faster Affinity chromatography: covalent bonding between analyte and affinity ligands (ie antibody), solid stationary, specific *example calculation Electroanalytical chemistry Voltammetry: method in which current is measured when potential is applied to an electrode, causing oxidation or reduction Voltammogram: current vs potential graph Current increases when number of electrons increases, concentration can be measured (sweep voltammetry?) Cyclic voltammetry: triangle waveform (linear sweep but stop and reverse) is applied, cathodic and anodic processes occur in succession Red/ox currents peak, then decrease if diffusion is too slow to replenish analyte at the electrode surface Reversible reaction: fast enough to maintain equilibrium concentrations of reactant and product at electrode surface Epa-Epc=0.059/n Randles-Sevcik equation Irreversible reactions: increased separation and broadening of cathodic and anodic peaks, not fast enough to maintain equilibrium concentrations at the electrode Can make them surface confined? Square wave voltammetry: increased sensitivity, derivative peak shape obtained by applying a square wave superimposed on a staircase voltage ramp With each cathodic pulse, a rush of analyte to be reduced comes to the surface  During anodic pulse, reduced analyte is reoxidized Voltammogram shows difference between cathode/anode currents Stripping voltammetry: even more sensitive, analyte is concentrated by reduction at a fixed voltage for a fixed time, then the potential made more positive and current measured as analyte is reoxidized and current is recorded as a function of time - the first step makes it so sensitive Negative voltage reduces all ions present Concentration is measured by the current peaking during reoxidation Pulse voltammetry: rapid scan switching gives higher current response, a small pulse is applied and current is measured before and after, and the difference is recorded as a function of the linearly increasing excitation voltage - produces a curve with height proportional to concentration More sensitive because the higher current Electrochemistry Current shows number of electrons transferred and voltage free energy change Power = J / S Galvanic cell: spontaneous reaction generates electricity *example? Potentiometry: using electrodes to measure voltages that provide chemical information Indicator electrode responds to analyte activity SHE difficult, Ag-AgCl and calomel are better For silver silver chloride, concentration of Cl- is fixed (saturated) so potential changes when concentrations of analyte change only (E fixed also) Same for calomel with Cl- Electrolysis: process by which a chemical reaction is forced to occur at an electrode by a voltage Moles of reactant equation *example calc For electrolysis Ecell = Ec - Ea - IR - over potentials Overpotential: voltage required to overcome the activation energy of an electrode reaction Ohmic potential (IR): voltage needed to overcome internal resistance of the cell Concentration polarization: concentration of electroactive species near an electrode is not the same as its concentration in bulk solution This also opposes electrolysis Controlled-potential electrolysis: potential of an electrode is measured with respect to a reference electrode to which negative current flows (three electrode cell) - controls cathode potential to prevent unwanted side reactions Electrogravimetric analysis: analyte is deposited on an electrode, and increase in mass is measured Coulometry: number of moles of electrons that participate in a chemical reaction are measured to obtain original concentration, precise, sensitive Coulometric titration: time needed for complete reaction measures the number of electrons consumed Controlled-potential coulometry - more selective but slower, electrons consumed are measured by integrating current vs time Amperometry: current at the working electrode is proportional to analyte concentration Voltammetry: collection methods in which the dependence of current on the applied potential of the electrode is observed Polarography: voltammetry with a dropping mercury electrode Stripping voltammetry: sensitive, analyte is concentrated into a single drop or thin film of mercury or other electrodes by reduction at fixed voltage for fixed time - potential then made more positive, and current is measured as analyte is reoxidized Peak current during oxidation is proportional to analyte concentration Microelectrodes: fit in small places SC Chromatography SCF: substance at temp and pressure above critical point, distinct liquid and gas phases don’t exist Higher diffusion and lower viscosity than liquids, good for HPLC (temp and pressure are within the range) Higher density than gas so it can dissolve large molecules SFC is hybrid between GC and LC, SF is mobile Oven to regulate temp, restrictor to regulate pressure (higher pressure than normal), decreases it to convert to gas before detector Pressure programming - higher P means higher density and stronger mobile phase so faster elution Flame Ionization Detector, or UV or IR, also MS SFC has lower plate height and faster linear velocity - faster diffusion rate and sharper peak than HPLC Applicable to larger molecules Works really well for extraction - fast, easy to change pressure to change solvent strength, recovery of SCF, can be cheap Can extract coffee eg, also good for dry cleaning Capillary Electrophoresis Electrophoresis: differential rate of migration of charged species in an electric field Larger charge to size ratio, faster migration High resolution, accurate quantification Fused silica capillary extends between two buffer reservoirs with Pt electrodes in them, sample is introduced at one end and detection at the other, voltage is applied to the electrodes to get sample to move Applied voltage makes an electric field that causes ions to move Electrophoretic flow equation CE only has one phase, separate based on charge to size ratio Electroosmotic flow: silica of the capillary wall binds OH, causing aggregation of cations as a compact inner layer and so cations in the diffusion layer are solvated so the net flow is toward the cathode which is negatively charged Electrical double layer near charged surface - compact inner layer and diffusion layer, linear decrease in potential with distance vs exponential Since electrophoretic flow causes cations to be attracted to the cathode and anions to the anode, cathodes have an additive total migration rate, while the velocity of neutrons is only due to electroosmotic flow, and the total velocity of anions is the smallest since EP flow cancels out a little of the EO flow since they are in opposite directions No extracolumn broadening, sharper peaks Anions vs cations vs neutral Polyimide coating over fused silica capillary tube to protect it, tube connects anode reservoir and cathode reservoir Instrument - total volume 4-5 microliter capillary made of fused silica Electroosmotic flow needs to be faster than electrophoretic to detect anions and neutral molecules 5-50 nL injected Can be electrokinetic injection or pressure injection Lots of different detectors again, spectrometric or electrochemical Capillary zone electrophoresis: buffer composition is constant, applied field causes each of the different ionic components of the mixture to migrate according to mobility and separate into zones that may be completely resolved or not For separating anions, flow can be reversed by treating the walls of the capillary with alkyl ammonium salt to create a negatively charged mobile solution layer attracted to the anode Isotachophoresis - sample is injected between a leading high mobility buffer and trailing low-mobility buffer Capillary isoelectric focusing - continuous pH gradient exists along the length of the capillary, and analyte ions migrate to the pH that corresponds to their isoelectric points Capillary gel electrophoresis - in a porous gel polymer matrix with a buffer mixture to fill the pores, which slows the migration of ions by their charge/size ratio