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Chromatographs

Chromatographs . column. eluent tank. injector. pump. detector. PC. CHROMATOGRAM. Qualitative & Quantitative information. GasChromatography (GC) 1952: A.T. James & A.J.P. Martin. High performancy Qualitative & Quantitative information Complicated samples Separation.

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Chromatographs

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  1. Chromatographs column eluent tank injector pump detector PC CHROMATOGRAM • Qualitative • & • Quantitative information

  2. GasChromatography (GC) 1952: A.T. James & A.J.P. Martin • High performancy • Qualitative & Quantitative information • Complicated samples • Separation • 1956: van Deemter: kinetic theory • M. Golay: capillary columns MOBILE PHASE: GAS STATIONARY PHASE: solid or liquid on solid support (GSC, GLC) COLUMN ELUTION TECHNIQUE • Base of separation: • Boiling point (vaporization) • Structure GASCHROMATOGRAPHY: analysis in vapor phase ~12 billion organic compounds ~ 50 000: evaporative without destruction • Evaporization depends on: • Molar mass • polarity Thermal stability

  3. GASCHROMATOGRAPHY (GC) Sample introduction to the mobile phase: gas/vapor • Sample can be: • gas • liquid: vaporization • solid: dissolution in liquid Pressure and flow regulators Gas tank Gas cleaner Gaschromatograph (GC)

  4. injector detector cleaner PC column thermostate Pressure controller Flow controller GASCHROMATOGRAPH (GC) Gas tank

  5. Eluent gas • Depending on the type of detector: • H2 • Ar • N2 • He Reductor valve: Type depends on the quality and pressure of the gas Flow-rates Inside apparatus: Pressure and flow controllers

  6. Sample introduction • Injection in a very short time • Vapor/gas phase • Mixible with eluent gas volume 0,1 l-1 ml Liquid vaporization: 100-10000 X volume increase Syringe For gas & liquid sample „six-port” valve

  7. Septum (rubber) Eluent gas inlet Heating block (25 – 300 oC) liner (glass) column FLASH INJECTOR • Samle introduction • Vaporization • Inlet to column Packed columns: greater diameter: greater sample volume Capillary columns: small sample volume

  8. Flash injector vaporization Injection • Sample vaporization • Liquids: 100 – 1000 X volume increase • Mixing with eluent • Stick needle into the septum • Push the syringe piston • Remove syringe

  9. solvent Quick injection Slow injection Eluent gas moves the sample to the column.

  10. SPLIT • SPLITLESS • ON-COLUMN • PTV Type of injectors Split-injector Carrier gas Septum wash split-gas Split/splitless ratio: determines amount of sample moving to the column

  11. 200:1 5:1

  12. Splitless injector Purge Off Purge On

  13. On-column PTV (Programmed Temperature Vaporizer) Injection directly to the column

  14. Columns Capillary polyimid, 350 oC quarz d Stationary phase microbore: d < 150 m standard capillary: 150 m < d < 500 m widebore: d > 500 m Adsorption mechanism: PLOT (Porous Layer Open Tubular) Distribution mechanism: WCOT:Wall Coated OT SCOT: Support Coated OT

  15. SiOH SiOH SiOH SiOH SiOH SiOH SiOH Si-O-Si(CH3)3 Si-O-Si(CH3)3 Si-O-Si(CH3)3 Interaction: between stationary phase and sample Active side: silanol groups • „tailing” • Non-symmetric peaks desactivation: sylil reagents Quartz surface

  16. Stationary phases I. • Thermal stability • No „bleeding” • Known chemical structure • Chemical inertnees • Low price Adsorbents (GSC) porous, with large special suface modified adszorbents: Based on carbon or silicagel • inorganic adsorbents: • silicagel • aluminium-oxide • zeolits (molekulasziták) • Organic adsorbents: • active carbon • polymers Analytes: Hydrocarbons with small molar mass, He, Ne, Ar, Kr, Xe (PLOT)

  17. Stationary phases II. (GLC) (absorption: dissolution of gas and liquids in liquids) Polymers: WCOT: polymers on the surface of capillary) Relative small number: 12-15 substituted polysiloxans (silicons): long lifetime R: substituents on polysiloxans Thermal stability: up 250-300 C Methyl: -CH3 Phenyl: • Substituents:: • Methyl • phenyl • Cianopropyl • Trifluoropropyl Cianopropyl: -CH2CH2CH2CN Trifluoropropyl: -CH2CH2CF3

  18. O CH Si O 3 CH 3 Si O CH Si CH 3 O 3 CH Si 3 CH 3 • methyl-phenyl • cianopropyl-phenyl • etc. substitution: how much % of Si atoms 100 % metil 5 % fenil & 95 % metil

  19. Polyethyleneglycols (PEG) Special separation • Disadvantage: • Lower thermal stability • „oxygen-sensitivity” Carbowax

  20. Polarity of stationary phase: • Structure of stationary phase • Quality of functional groups • Number of functional groups • Apolar stationary phases: • 100 % methyl • 5 % phenyl • Polar phases: • cyanopropyl • PEG • Midium polar phases: • 35 % phenyl • 50 % phenyl Selectivity depends on: the interaction between stationary phase and analyte • Interactions depend on: • Quality of analytes • Structure of stationary phase

  21. Thermostate column • Type of working: • Izotherm • Programmable heating T (oC) thermostate • Large temperature range -50 – 400 C • Programmable heating: 0- 40 oC/min • „cooling” t (min) Decrease of analysis time Good peak shape

  22. Detectors Quantitative analysis: signal of detector is proportional with concentration of analytes in detector universal: signal for every compounds selective: signal for a groups of compounds specific: signal for special compounds destruktiv non destruktiv Dinamic range: change of concentration results a change in signal linearity: T= mc (deviation < 5 %) sensitivity: m (ratio of signal/concentration) Limit of detection (LOD):signal to noise ratio: 3 Limit of quantitation (LOQ):signal to noise ratio: 10

  23. Detectors Thermal Conductivity Detector (katharometer) Change of impedance Wheatstone-bridge W-filaments: 100-200 mA heating current Carrier gas: H2, He N2 dinamic range: 105 LOD: 5-50 ng non destructív universal

  24. Flame-ionization detector (FID) hydrogen/air microburner with a pair of electrodes Carrier gas: non ionizable gas: N2, Ar, He, H2 Organic compounds leaving the column are burning in burner jet, ions are forming Ions result a small current Carbon-detector: it is good for organics, except formic acid destructív Dinamic range: 105-106 LOD: 0,05-0,5 ng

  25. column thermostate High Performance Liquid Chromatography (HPLC ) Mobile phase: liquid Stationary phase: adsorbent (LSC) or liquid on a support(LLC) Column Elution technique Sample: liquid Gas removal pump eluent tank injector PC detector

  26. HPLC Gas removal pump automated injector detector (thermostate)

  27. Eluent • Should (have) be: • Low viscosity • inert: no reaction with analytes • Chemical stability • No corrosion • No toxycity • Higher boiling point • Low price • Good quality and purity • Compatible with detector • UV-absortion: low purity: HPLC grade Water and buffers too !!!

  28. Eluent P O L A R I T Y Analytes distributed between stationary and mobile phase: interaction of analytes with both phases Polarity of molecule & mobile phase & stationary phase • Change of polarity: • Change of quality of mobile phase • Mixing of solvents hexane chloroform tetrahidrofuran acetonitrile isopropanol ethanol methanol water Mixed solvents: should be mixcible Eluent strength: determined on silicagel on the bease of heat of adsorption of solvents izoeluotrope mixture: eluent strength is the same: k’, Rs: may change !!! Izocratic elution: fixed mobile phase composition Gradient elution: eluent strength is increasing in time Use of buffers: adjusting of pH in the case of analysis of ionisable components

  29. Pumps To carry of eluent • Should be: • pressure (400 bar) • Stable flow-rate • Compatible with different solvents:no corrosion • Small hold-up volume • No pulsation Flow-rates in classical analytical HPLC: 0,1-1,5 ml/min (0-5 ml/min) Syringe-type pump

  30. Reciprocating pump V time Pulsation: double pistons (phase-deviation) Volume: 10-100 l Change of flow rate: easy

  31. Gas removals Liquids: contain dissolved gases • Effect of gas bubbles: • In pump: • Pressure pulsation • Different flow-rates • Mechanical instability • In detector: • Increased noise (retention time changes) Remove of gas from the solvent: • Ultrasound: • cheap • Non effective • Vacuum: • Higher price • effective • He-purge: • Higher price • effective

  32. Sample loading • Quick • Sample should be mixable with eluent Sample volume: 10-50 l Micro syringe: „Six-port” valve

  33. Columns Function: separation Liquid chromatography: NP LC: Normal Phase RP LC: Reversed Phase NPLC: polarity of stationary phase > polarity of mobile phase RPLC: polarity of stationary phase < polarity of mobile phase • Material of column: • Stainless steel • Glass • PEEK (poly(ether-ether-ketone) Packing: regular spherical • Size of column: • diameter: 2-5 mm • length: 5-25 cm

  34. Modified silica gel OH OH OH OH OH SiO2 Modifying groups: C18: octadecyl: C18H37 C8: octyl: C8H17 C4: buthyl: C4H9 Amino: CH2CH2CH2NH2 Ciano: CH2CH2CH2CN Phenyl: C6H5 RPLC: C18 stationary phase & methanol/water mobile phase NPLC: silicagel stationary phase& hexane/alcohol mobile phase Guard column: avoid contamination of analytical column

  35. Detectors Quantitative analysis: signal of detector is proportional with concentration of analytes in detector universal: signal for every compounds selective: signal for a groups of compounds specific: signal for special compounds destruktiv non destruktiv Dinamic range: change of concentration results a change in signal linearity: T= mc (deviation < 5 %) sensitivity: m (ratio of signal/concentration) Limit of detection (LOD):signal to noise ratio: 3 Limit of quantitation (LOQ):signal to noise ratio: 10

  36. UV-Vis spectrophotometer Application: UV-Vis range Measuring side splitter cuvette I0 D E T E C T O R I rés I0 I0 fényforrás Reference side monocromator Lambeert-Beer: A = ε c l A = lg I0/I Cuvette: quartz l=5-10 mm Light source: UV: deuterium lamp Vis: wolfram lamp Detector: fotodiode Most usable HPLC detector 190 nm <  < 800 nm

  37. Dioda Array Detector (DAD) polychromator lence Light source cuvette Diode array Advantage: Spectra and chromatogram at the same time

  38. Paper and thin-layer chromatography Planar arrangement Stationary phase: paper silica gel or aluminium-oxide on a glass plate • Evaluation of chromatogram: • Dropping liquid sample on the one edge of the plate with capillary • Evaporation (drying) the solvent • Place the plate to the closed container saturated with vapors of developing solvent • Running of analytes: based on capillary activity • After development of chromatogram, remove plate from container and dry it • Locating analytes on the plate: spraying with chemical reagents, like iodine, sulfuric acid or UV-light Selection of mobile phase:like in Normal Phase HPLC Qualitative data: retardation factor (Rf) Quantitative data: intensity of spots • Advantages: • simple • cheap

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