1 - a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. 2 CHROMATOGRAPHY

 Stationary phase ( solid or liquid located on neutral medium)  Mobile phase ( liquid or gas)


© Chromatographic separation involves placing the test mixture on the liquid sample or solid stationary phase and then passing the liquid or gaseous mobile phase through it or above it, that is detector Stationary Mobile of the mixture from a phase phase stationary phase.

The mixture of components with different ratios of division are eluted (migrate) at different speed. These differences in the speed of migration lead to the separation of components

in time and space. 4 4 Investigated compound is located in mobile phase() and migrates with it across stationary phase which is porous medium: adsorbent, exchanger or .

© 5 Illustration of Chromatography

Stationary Phase


Mobile Phase Mixture Components Affinity to Stationary Affinity to Mobile Components Phase Phase

Blue          

white         Red        © Yellow           6 Mobile phase or carrier - solvent moving through the column Stationary phase or adsorbents - substance that stays fixed inside the column Eluent - fluid entering the column Eluate - fluid exiting the column (that is collected in flasks) Elution - the process of washing out a compound through a column using a suitable solvent Analyte - mixture which individual components have to be separated and analyzed

7 Classification of chromtographic methods

1. According to physico – chemical forces: . chromatography . chromatography . Partition chromatography . (size exclusion) chromatography . . Capillary chromatography

2. According to parameters of the proces:

. High Pressure/Performance Liquid Chromatography (HPLC) . Fast liquid chromatography (FPLC)

© 8 Classification of chromtographic methods

3. According to applied techniques:   Planar chromatography (thin layer, paper)

4. According to mobile phase type:  , GC  Liquid Chromatography, LC  Supercritical Fluid Chromatography, SFC

5. According to mobile and stationary phases type:  Gas – liquid chromatography (GLC)  Liquid – liquid chromatography (LLC)  Gas – solid chromatography (GSC)  Liquid - solid chromatography (LSC) © 9 Choice of the chromatographic method depends on:

 the amount of analysed mixture  kind of compounds  complexity of

10 Types of chromatography

Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)

Gas Chromatography – separates vaporized samples with an inert carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase)

© 11 Types of chromatography

Adsorption Chromatography uses solid material as stationary phase (adsorbents). The separation of the mixture is caused by a different affinity of individual components (adsorbates) of a mixture to the surface of stationary phase.

Planar chromatography: • – separates liquid samples with a liquid solvent (mobile phase) on a paper strip (stationary phase) • Thin-Layer Chromatography – separates liquid samples with a liquid solvent (mobile phase). Glass plate is covered with a thin layer of oxide or (stationary phase)

12 Adsorption chromatography

If the stationary phase is solid, chromatography is called adsorption chromatography.

Separation of the mixture is caused by different affinities of mixture components (adsorbates) to the surface of the stationary phase known as adsorbent.

Adsorption of analysed sample particles, can be induced by:

- physical forces (physical adsorption) - chemical influence ()

13 The effectiveness of adsorption depends on:

• kind of adsorbent and its properties • solvent type • properties of adsorbed

14 Classification of adsorbents according to:

1. Adsorption activity: . Weak ( starch, saccharose, talc) . Middle ( calcium carbonate, sodium carbonate) . Strong ( , activated silica acid)

2. Chemical properties:

. acidic ( SiO2) . basic ( CaO) . neutral ( activated )

. amphoteric ( Al2O3)

15 Classification of adsorbents

3. Chemical nature: . Organic ( carbon, starch, saccharose, polyamides )

. Inorganic ( Al2O3, MgO, natural and synthetic silicates )

. Mixed (talc + saccharose, CaCO3 + talc ) . Specific (silica gel with specific pores )

4. Polarity : . Highly polar ( aluminium oxide, silica oxide) . Weak polar (calcium carbonate, MgO) . Non-polar ( , talc, )

© 16 used in chromatogrphy are arranged in an eluotropic series, depending on the ability of adsorption with respect to the substances dissolved in them and the ability of elution. They are used to develop the chromatogram and to elute the separate components.


2. Carbon tetrachloride (CCl4) 3. Benzene 4. Diethyl ether 5. Acetone 6. Chloroform 7. Ethyl acetate 8. 9. Methanol 10. Water

© 17 Properties of adsorbed molecules

Adsorption ability of adsorbed molecules depends on::  Size and spatial orientation  Number and location of double bonds  Number of polar and nonpolar substituents  Number and kind of functional groups

The degree of adsorption of the adsorbate increases with:  Number of double bonds  Number of functional groups  Number of substituents of the same type

© 18 Thin Layer Chromatography – TLC (partition chromatography) Separation of the mixture depends on the difference in of the mixture’s components between two non miscible phases, from which one is a liquid coated on a medium (stationary phase), and a second one is a mobile phase (liquid,gas).

Partition Coefficient is equal to the ratio of the of the same substance in the stationary phase and the mobile phase. Nernst law of partition:

K = Cs / Cm K – partition coefficient at equilibrium state, depends only on the temperature and properties substance-forming and does not depend on the amount of solute.

Cs – concentration of particular compound (solute) dissolved in stationary phase

Cm -concentration of solute dissolved in mobile phase © 19 Thin-Layer Chromatography (TLC)

This is a fast method for the initial orientation in the amount and relative contributions of the components in the mixture of organic compounds. Advantages of TLC:  a short time to develop the chromatogram  simple methods of process control  minimum consumption of materials  ease to perform and low costs Application of TLC: • separation of amino acids, , saccharides, nucleotides Chromatography application for qualititive analysis – Rf coefficient.

Substance location on chromatogram is characterized by Rf values. (ratio of fronts) = retention factor

distance from START line to the middle of substance spot - A

Rf = distance from START line to the End line of solvent – B

Rf - has characteristic value for particular substance in specific process conditions 1>Rf<0

END Height reached by solvent B Invastigated A sample START

© 21 Preparation of chromatographic plates

• Glass plates - 1-2 mm thick covered with silica gel

•Investigated are placed on the plate using micropipette

Chromatography chamber must be : • leak proof • saturated by solvent vapors

© 22 Development of chromatogram

Development occurs during migration of solvent from start to the end. Solvent migrates because of:  capillary forces ( up technique)  gravity forces ( down technique)

Development of chromatogram,%20Definitions%20and%20Types.htm After removing plate from the chamber and –special reagent (detector) reacts with separated compounds of the mixture and give coloured spots (detector example: mixture of concentrated sulphuric and acetic acid, anisic aldehyde and ethanol).


Fast and effective method of separation for volatile compounds . Separated compound is carried out by gas (inert in relation to the stationary phase) through the column filled with stationary phase Depending on the stationary phase gas chromatography can be divided into: . Partition gas chromatography ( stationary phase – liquid on stationary carrier) . Adsorption gas chromatography ( stationary phase – adsorbent) . Capillary gas chromatography ( stationary phase – liquid is directly on the column walls.)

© 24 Scheme of gas chromatograph

Sample’s injection Chromatogram = result

Column Column with stationary phase Oven (liquid on inert base or solid - Carrier gas as adsorbent). mobile phase Typical stationary phases are large molecular weight polysiloxane, polyethylene glycol, or polyester polymers of 0.1 to 2.5 micrometer film thickness. Columns are, available in many stationary phases sizes. A typical capillary column is 15 to 60 meters in length and 0.25 to 0.32 mm inner © diameter. 25 Chromatography column in gas chromatograph © 26 Obtaining chromatogram in gas chromatography GAS CHROMATOGRAPHY cont.

Carrier gas must be chemically inert ( hydrogen, helium, , argon, carbon dioxide) and is introduced to the column. Investigated sample or mixture is injected to the column with gas carrier. Stream of gas is carrying samples to be separated. Compounds are divided according to their retention factor between gas and stationary phase. Separated substances are measured and registered at the outlet of the column using detector system. Operating temperature is 0oC – 400oC.

Stationary phase – micro-layer of organic liquids on a inert solid.

© 28 Detectors in gas chromatography

Quantitative measurement of mixture components can be done by using different chemical and physical properties of the compounds. Therefore, there are the following types of detectors:

1. Thermal conductivity detector – katharometer (TCD). 2. Flame-ionization detector - (FID). 3. Discharge ionization detector – (DID). 4. Electron capture detector (ECD) .

Detector signals are registered by sensitive writing system. As a result we are obtaining CHROMATOGRAM.

© 29 Chromatogram – is a visible record showing the result of separation of the components of a mixture by chromatography


Peak area is proportional to the amount of single analyzed sample.

© 30

HPLC Agilent Company


• Separation ability of column is increasing with length of the column, type and amount of stationary phase, column temperature, speed and pressure of carrier gas.

• Gas chromatography is applied in qualitative and quantitative (time retention and area or hight of the peak).

© 33 Gas chromatograph

© 34 Gel chromatography • Used for separation of big molecules such as protein, polymers, nucleic acids, sugars, glycols, silicones etc. • Column is filled with gel particles: polyacrylamide, polyethylene oxide, dextrin. As a mobile phase is used liquid. • Separation is based on sieve and capillary mechanisms*. • Large particles are moving fast and small particles are moving slowly. • Method also used for separation of DNA molecules.

© 35 HPLC – High Pressure/Performance Liquid Chromatography

Mobile phase – pressurized liquid solvent (20MPa-40MPa) Stationary phase – solid adsorbent material

. Analytical version – narrow columns (4-8 mm); length 5 – 25 cm . Preparation version - wide columns (20 – 500 mm); length 25 –100 cm  Benefits: fast, automated and precise compound separation; easy appliacation of concentrated gradients; quantitative determination of the compounds  Detector: (UV-vis), electrochemical, spectrofluorometric  Drawback: separation only of small quantities

HPLC columns

36 Column chromatography – some time ago ;-)

Chromatographic columns

© 37 It is used for the determination of: - Biologically active compounds such as amino acids, , , vitamins, and nucleic acids - Pharmaceutical Preparations - Plant protection products - - Polycyclic -benzopyrene - Complexes - Rare earth elements

38 VWD1 A, Wavelength=254 nm (US\16PUR051.D) mAU NAD 10 GDP GTP




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