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 paper gas (GC) distributive liquid (LC) adsorption thin layer ion-exchangegelaffinityGas-liquid Gas-solid Chromatography techniques by:  physical state of mobile phase  layout of stationary phase (column/planar)  separation mechanism

5. Basic term w – width of peak w 1/2 – half-width t R – retention time absorbance (AU) time (min) substance w w 1/2 tMtM tRtR h tRtR Typical chromatographic separation of substance

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

7. HPLC  High Performance Liquid Chromatography  higher flow rate of mobile phase (high pressure ∼ 10 7 Pa)  the better separation

8. Gas chromatography  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 start ab http://web.natur.cuni.cz/~pcoufal/tlcpc.html Retention factor R F - R F = a/b a - distance the spot traveled b – maximum distance the eluent traveled

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 Electroforeogram of serum proteins 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  Nernst equation – calculation of the electrode potential E R – the universal gas constant (8,314 J.K -1.mol -1 ) T – absolute temperature 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/red forms E o – the standard reduction potential

15. Potentiometry – analytical application  Potentiometric titration curve Volume of titrant (ml) Potential (V)

16. Electrodes  Referent electrodes:  calomel  Ag/AgCl  Indicator electrodes:  Ion-selective Potential in turn is described by the Nernst equation and 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 – organic chemical compound which causes the colour of the solution to change depending on the pH (sensitivity to different concentration of H + ions)

19.  Calculate the mass of sulfuric acid in the sample solution when the consumption of standard titrant solution NaOH was 24.22 ml at a concentration of 0.1022 mol/l. Acid-base titration

20. Spectrophotometry - VIS  Analytical applications - measure concentrations of absorbing (coloured) materials based on developed calibration curves  To obtained the unknown concentration of sample – calibration curve (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 beam of monochrom. radiation Φ o beam of radiation leaving the sample Φ l  Lambert-Beer low: A = ε × c × l ε – Molar absorptivity c - sample concentration (mol/L) l – length of light path through the sample (cm)

22. Absorption spectroscopy  performed across the electromagnetic spectrum → choice of wavelength max absorption sample

23. VIS – analytical applications  Blank  Lambert-Beer low – unknown concentrations  Calibartion curve

24. Basic structure of spectrophotometers