ANALYTIC METHODS

II. PART

Jana Švarcová

Chromatography Electrophoresis Potentiometry Titration Spectrophotometry

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

Chromatography methods

chromatography

paper

gas (GC)

distributive

liquid (LC)

adsorption

thin layer

ion-exchange gel affinity Gas-liquid Gas-solid

Chromatography techniques by: physical state of mobile phase layout of stationary phase (column/planar) separation mechanism

Basic term

w – width of peakw1/2 – half-widthtR – retention time

abso

rban

ce

(AU

)

time (min)

substance

w

w1/2

tM

tR

h

tR

Typical chromatographic separation of substance

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/

HPLC

High Performance Liquid Chromatography

higher flow rate of mobile phase (high pressure ∼ 107 Pa)

the better separation

Gas chromatography

The mobile phase in gas chromatography is generally an inert gas

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

start

a b

http://web.natur.cuni.cz/~pcoufal/tlcpc.html

Retention factor RF - RF = a/b

a - distance the spot traveled

b – maximum distance the eluent traveled

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 (+)

Electrophoresis

Electroforeogram of serum proteins

proteins – fraction %

Potentiometry

Anlytical method – analytes are studied by measuring the potential (volts) in the electrochemical galvanic cell (the difference in electrode potentials)

Potentiometry – basic terms

Two electrodes (acording potential stability) Indicator electrode Reference electrode

The potential is related to the concentration of one or more analytes

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 formsEo –  the standard reduction potential

Potentiometry – analytical application

Potentiometric titration curve

Volume of titrant (ml)

P

ote

nti

al

(V)

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.

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

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)

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

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

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  Φo

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)

Absorption spectroscopy

 performed across the electromagnetic spectrum → choice of wavelength max absorption

sample

VIS – analytical applications

Blank Lambert-Beer low – unknown

concentrations Calibartion curve

Basic structure of spectrophotometers