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CHROMATOGRAPHY ©

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Chromatography - a physical method of mixture 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.

https://www.chemicool.com/definition/chromatography.html

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CHROMATOGRAPHY

Stationary phase ( solid or liquid located on neutral

medium)

Mobile phase ( liquid or gas)

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Chromatographic separation involves

placing the test mixture on the liquid

or solid stationary phase and then

passing the liquid or gaseous mobile

phase through it or above it, that is

elution of the mixture from a

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

sample

Stationaryphase

Mobile phase

detector

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Investigated compound

is located in mobile

phase(solvent) and

migrates with it across

stationary phase which

is porous medium:

adsorbent, ion

exchanger or molecular

sieve.

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Illustration of Chromatography

ComponentsAffinity to Stationary

PhaseAffinity to Mobile

Phase

Blue

white

Red

Yellow

Mixture Components

Separation

Stationary

Phase

Mobile

Phase

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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

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1.According to physico – chemical forces:

Adsorption chromatography

Ion exchange chromatography

Partition chromatography

Gel (size exclusion) chromatography

Affinity chromatography

Capillary chromatography

2. According to parameters of the proces:

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

Classification of chromtographic methods

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

4. According to mobile phase type: Gas Chromatography, 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)

Classification of chromtographic methods

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Choice of the chromatographic method depends on:

the amount of analysed mixture

kind of compounds

complexity of separation process

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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)

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Types of chromatography

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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:

• Paper 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 aluminium oxide or silica gel (stationary phase)

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 (chemisorption)

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The effectiveness of adsorption depends on:

• kind of adsorbent and its properties

• solvent type

• properties of adsorbed molecules

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Classification of adsorbentsaccording to:

1. Adsorption activity:

Weak ( starch, saccharose, talc) Middle ( calcium carbonate, sodium carbonate) Strong ( aluminium oxide, activated silica acid)

2. Chemical properties:

acidic ( SiO2) basic ( CaO) neutral ( activated carbon) amphoteric ( Al2O3)

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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 ( activated carbon, talc, graphite)

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Solvents 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.

1. Hexane2. Carbon tetrachloride (CCl4)3. Benzene4. Diethyl ether5. Acetone6. Chloroform7. Ethyl acetate8. Ethanol9. Methanol10. Water

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Properties of adsorbed molecules

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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

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Thin Layer Chromatography – TLC (partition

chromatography)

Separation of the mixture depends on the difference in partition

coefficient 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 concentration 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 solutions 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 ©

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, peptides,

saccharides, nucleotides

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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

A

B

START

END

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Invastigatedsample

Height reached bysolvent

1>Rf<0

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Preparation of chromatographic plates

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

•Investigated solution are placed on the plateusing micropipette

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

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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

After removing plate from the chamber and drying –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).

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http://machan11.tripod.com/History,%20Definitions%20and%20Types.htm

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GAS CHROMATOGRAPHY

Fast and effective method of separation for volatile compounds mixtures.

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.)

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Scheme of gas chromatograph

Oven

Chromatogram =

result

Sample’s injection

Carrier gas as

mobile phase

Column Column with stationary phase

(liquid on inert base or solid -

adsorbent).

,

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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.

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Chromatography column in gas chromatograph

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Obtaining chromatogram in gas chromatography

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Carrier gas must be chemically inert ( hydrogen, helium, nitrogen,

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.

GAS CHROMATOGRAPHY cont.

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Quantitative measurement of mixture components can be done byusing 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.

Detectors in gas chromatography

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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.

Czas

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HPLC Agilent Company

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GAS CHROMATOGRAPHY

• 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 chemistry (time retention and area or hight of the peak).

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Gas chromatograph

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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.

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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; easyappliacation of concentrated gradients;

quantitative determination of the compounds

Detector: spectroscopy (UV-vis), electrochemical, spectrofluorometric

Drawback: separation only of small quantities

HPLC columns

http://www.analytical-sales.com/HPLC-Columns.html

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Column chromatography – some time ago ;-)

Chromatographic

columns

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38http://web.nmsu.edu/~kburke/Instrumentation/Waters_HPLC_MS_TitlePg.html

It is used for the determination of:

- Biologically active compounds such as amino acids, proteins, polysaccharides, vitamins, steroids and nucleic acids- Pharmaceutical Preparations- Plant protection products - pesticides- Polycyclic hydrocarbons -benzopyrene- Complexes- Rare earth elements

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THE END

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