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

Analytical methods

Titration

Spectrometry

Chromatography

Electrophoresis

Potentiometry

Basic dividing of analytical methods

Qualitative analysis

+/-

Quantitative analysis

Amount

Titration

Volumetric analysis

Quantitative method

Titrant – titration reagent

Known concetration and volume

The equivalente point

burette

Titrationreagent

Titrationflask

Analyte

Types of titration

By reaction

Acid – base titrations

Redox titrations

Precipitation titrations

Complex titrations

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

Titration curve

Titration curve

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

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

Exitation by electromagnetic radiation

E

FL - fluorescencePh - phosphorescence

Non-radiative processes:

VR - vibrate relaxation IC - internal conversion ISC – intersystem crossing

Transmittance and Absorbance

Transmittance - relative proportion of the transmitted

radiation

Absorbance – Optical density – Absorption of

monochromatic wave

Lambert-Beer law

Lamber law

Beer law

Lamber-Beer law

Calibrate curve – standard curve

Wave length and arrange

Choosing of wave length

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

Chromatography – categorization I

Nature of the mobile phase

Gas chromatography

Liquid chromatography

Chromatography – categorization II

Form of the chromatographic bed of stationary phase Column chromatography Surface chromatography

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

ChromatographyRetention characteristics

Retention factor Rf

k

Starting point

Solvent front

TLC

Before separation After separation

ChromatographyRetention characteristics

Qualitative analysis Retention time

Retention factor R

Retention volume

Quantitave analysis Peak area

Calibration curve

Electrophoresis

Instrumental electromigration separation method

Separation of proteins and nucleic acids

Different mobility under influence of direct electric

field

Charge

Size

Electrophoresis - classification

Zone electrophoresis Electrophoresis on a supportConstant pH

Isoelectric focusation - IEF Isoelectric point (pI)- without chargepH gradient

Electrophoresis - classification

According to gel running setupVerticalHorizontal

Gel typeAgarose elektrophoresisPAGENativeSDS

2D –Electrophoresis

1 . Isoelektric focusation2. SDS PAGE 3. MS

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

Potenciometry

Ion select electrode (ISE) Ionts Selectively permeable membranes Membrane potencial

Glass electrode (ISE) pH

Direct potenciometry Potenciometric titration

Thanks for your attention