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Analytic methods II. part

Analytic methods II. part. Jana Švarcová. Chromatography Electrophoresis Potentiometry Titration Spectrophotometry. Chromatography methods. B asic theory – separation of mixtures  distributed between two phases stationary phase (SF)  mobile phase (MF) – carries the mixtures

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Analytic methods II. part

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  1. Analytic methods II. part Jana Švarcová

  2. Chromatography • Electrophoresis • Potentiometry • Titration • Spectrophotometry

  3. Chromatography methods • Basic theory – separation of mixtures  distributed between two phases • stationary phase (SF)  • mobile phase (MF) – carries the mixtures • The separation is based on differential partitioning between the mobile and stationary phases • Differential rates of migration as the mixture moves over adsorptive materials provide separation • Various components of mixtures have different affinities for the stationary phase

  4. Chromatography methods chromatography • Chromatography techniques by: • physical state of mobile phase • layout of stationary phase (column/planar) • separation mechanism liquid (LC) gas (GC) Gas-solid distributive adsorption ion-exchange gel affinity Gas-liquid paper thin layer

  5. Basic term Typical chromatographic separation of substance tR w – width of peak h substance w1/2 – half-width tR – retention time tM w1/2 absorbance (AU) tR w time (min)

  6. Liquid chromatography • MF - a liquid of low viscosity which flows through the stationary phase bed column Computer data - results Reservoir of mobile phase sample detector pump waste http://www.pharmacelsus.de/hplc/

  7. HPLC • High Performance Liquid Chromatography • higher flow rate of mobile phase (high pressure ∼ 107 Pa) • the better separation

  8. Gaschromatography • The mobile phase in gas chromatography is generally an inert gas

  9. TLC - thin layer chromatography • Plane layout of SF - layer of solid particles spread on a support • Compounds in the sample mixture travel different distances according to how strongly they interact with the stationary phase a - distance the spot traveled a b b – maximum distance the eluent traveled start start http://web.natur.cuni.cz/~pcoufal/tlcpc.html Retention factor RF - RF = a/b

  10. Electrophoresis • A class of separation techniques - analytes are separated by their ability to move in gel in response to an applied electric field • Separation – size of charge, shape and size of molecule • Migration – cations migrate towards the cathode (-), anions towards the anods (+)

  11. Electrophoresis Electroforeogramofserumproteins proteins – fraction%

  12. Potentiometry • Anlytical method – analytes are studied by measuring the potential (volts) in the electrochemical galvanic cell (the difference in electrode potentials)

  13. Potentiometry – basic terms • Two electrodes (acording potential stability) • Indicator electrode • Reference electrode • The potential is related to the concentration of one or more analytes

  14. Potentiometry • Nernstequation – calculation of the electrode potential E R – theuniversalgasconstant (8,314 J.K-1.mol-1) T – absolutetemperature F – the Faraday constant, the number of coulombs per mole of electrons (96 500 C.mol-1) a – the chemical activity for the relevant species; ox/redforms Eo –  the standard reduction potential

  15. Potentiometry – analyticalapplication • Potentiometrictitrationcurve Potential (V) Volume oftitrant (ml)

  16. Electrodes • Referent electrodes: • calomel • Ag/AgCl • Indicatorelectrodes: • Ion-selective Potential in turn is described by theNernstequationand is directly proportional to the pH difference between solutions on both sides of the glass.

  17. Titration • laboratory method of quantitative chemical analysis - is used to determine the unknown concentration of an identified analyte • the titrant – reagent (is prepared as a standard solution) • A known concentration and volume of titrant reacts with a solution of analyte to determine concentration • Titre – the volume of titrant reacted • Detection of the equivalence point • appropriate pH indicator is added (reflecting the pH range of the equivalence point) • Different methods to determine the endpoint include • Spectroscopy • Potentiometer • Conductivity Burette

  18. Acid–base titration • Indicators – organicchemicalcompoundwhich causes the colour of the solution to change depending on the pH (sensitivity to differentconcentrationof H+ions)

  19. Acid-base titration • Calculate the mass of sulfuric acid in the sample solution when theconsumptionofstandard titrantsolutionNaOH was 24.22 ml at a concentration of 0.1022 mol/l.

  20. Spectrophotometry - VIS • Analyticalapplications - measure concentrations of absorbing (coloured) materials based on developed calibration curves • To obtainedthe unknown concentrationof sample – calibrationcurve (graph of the transmittance or absorbance versus the wavelength) • Absorption of VIS light by a sample • 390 – 750 nm

  21. Spectrophotometry - measurement • the absorbance of a sample will be proportional to the number of absorbing molecules in the spectrometer light beam  • transmittance T • absorbance A l beamof monochrom. radiationΦo beam of radiation leaving the sample Φ • Lambert-Beerlow: A = ε × c × l ε – Molar absorptivity c - sample concentration (mol/L) l – length of light path through the sample (cm)

  22. Absorptionspectroscopy •  performed across the electromagnetic spectrum→ choiceofwavelengthmaxabsorption sample

  23. VIS – analytical applications • Blank • Lambert-Beerlow – unknownconcentrations • Calibartioncurve

  24. Basic structureofspectrophotometers

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