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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 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 • 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
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)
injector detector cleaner PC column thermostate Pressure controller Flow controller GASCHROMATOGRAPH (GC) Gas tank
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
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
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
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
solvent Quick injection Slow injection Eluent gas moves the sample to the column.
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
200:1 5:1
Splitless injector Purge Off Purge On
On-column PTV (Programmed Temperature Vaporizer) Injection directly to the column
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
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
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)
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
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
Polyethyleneglycols (PEG) Special separation • Disadvantage: • Lower thermal stability • „oxygen-sensitivity” Carbowax
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
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
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
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
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
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
HPLC Gas removal pump automated injector detector (thermostate)
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 !!!
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
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
Reciprocating pump V time Pulsation: double pistons (phase-deviation) Volume: 10-100 l Change of flow rate: easy
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
Sample loading • Quick • Sample should be mixable with eluent Sample volume: 10-50 l Micro syringe: „Six-port” valve
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
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
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
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
Dioda Array Detector (DAD) polychromator lence Light source cuvette Diode array Advantage: Spectra and chromatogram at the same time
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