Download - Analytické metody En final II
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H A N A L U K Š A N O V Á
H AN A. L U K S A N O VA @ L F M O TO L . C U N I . C Z
I N S TI TU TE O F M E D I C AL C H E M I S TRY AN D C L I N I C ALB I O C H E M I S TRY,
2 T H M E D I C AL FAC U LT Y, C H AR L E S U N I V E R Z I TY
Analytical methods
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Analytical methods
Titration
Spectrometry
Chromatography
Electrophoresis
Potentiometry
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Basic dividing of analytical methods
Qualitative analysis
+/-
Quantitative analysis
Amount
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Titration
Volumetric analysis
Quantitative method
Titrant – titration reagent
Known concetration and volume
The equivalente point
burette
Titrationreagent
Titrationflask
Analyte
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Types of titration
By reaction
Acid – base titrations
Redox titrations
Precipitation titrations
Complex titrations
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Indication of equivalence point
Indicator Visualization of equivalence point
Color change
Weak organic acid and its conjugated base changes color
pH =pKHA ±1
Precipitation or turbidity
Instrumental methods pH
Conductivity
Redox potential
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Titration curve
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Titration curve
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Optical analytical techniques
Instrumental optical analysis
Interaction of electromagnetic radiation with a mass
Non – spectral optical analytical techniques
change of specific radiation characteristics after passing though a
sample
Spectral optical analytical techniques
Energy exchange between radiation and matter
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Optical analytical techniques
Spectral optical analytical techniques
Energy exchange between radiation and matter
Absorption techniques
absorbing of electromagnetic radiation
Emission techniques
light emission by sample
fluorescence and phosphorescence
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Exitation by electromagnetic radiation
E
FL - fluorescencePh - phosphorescence
Non-radiative processes:
VR - vibrate relaxation IC - internal conversion ISC – intersystem crossing
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Transmittance and Absorbance
Transmittance - relative proportion of the transmitted
radiation
Absorbance – Optical density – Absorption of
monochromatic wave
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Lambert-Beer law
Lamber law
Beer law
Lamber-Beer law
Calibrate curve – standard curve
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Wave length and arrange
Choosing of wave length
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Chromatography
Separate technique based to different equilibrate distribution individual components to stationary a mobile phase
Michail Semjonovič Cvět Separated plant pigments in CS2 by a column
CaCO3
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Chromatography – categorization I
Nature of the mobile phase
Gas chromatography
Liquid chromatography
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Chromatography – categorization II
Form of the chromatographic bed of stationary phase Column chromatography Surface chromatography
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Chromatography – categorization III
Type of separation process
Partition chromatography
different solubility in stationary and mobile phase
Adsorption chromatography
different ability to bind the stationary phase
Ion-exchange chromatography - IONEX
different ion interaction with stationary phase
Gel permeation chromatography
different compound size – molecular sieve
Affinity chromatography
selective interaction
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ChromatographyRetention characteristics
Retention factor Rf
k
Starting point
Solvent front
TLC
Before separation After separation
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ChromatographyRetention characteristics
Qualitative analysis Retention time
Retention factor R
Retention volume
Quantitave analysis Peak area
Calibration curve
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Electrophoresis
Instrumental electromigration separation method
Separation of proteins and nucleic acids
Different mobility under influence of direct electric
field
Charge
Size
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Electrophoresis - classification
Zone electrophoresis Electrophoresis on a supportConstant pH
Isoelectric focusation - IEF Isoelectric point (pI)- without chargepH gradient
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Electrophoresis - classification
According to gel running setupVerticalHorizontal
Gel typeAgarose elektrophoresisPAGENativeSDS
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2D –Electrophoresis
1 . Isoelektric focusation2. SDS PAGE 3. MS
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Potenciometry
Electroanalytical method
Equilibrium potential of an electrochemical cell
Electron conductor (conductor of first class)
Ionic conductor (conductor of second class)
Nernst equation
Dynamic equilibrium of an electrochemical
reaction
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Potenciometry
Ion select electrode (ISE) Ionts Selectively permeable membranes Membrane potencial
Glass electrode (ISE) pH
Direct potenciometry Potenciometric titration
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Thanks for your attention