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CHROMATOGRAPHY ©
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CHROMATOGRAPHY it is technique for separating the components of the mixture based on the relative amount of each substance divided between the moving fluid stream, known as the mobile phase and the adjacent stationary phase.
http://www.waters.com/waters/en_PL/HPLC---High-Performance-Liquid-Chromatography-Explained/nav.htm?cid=10048919&locale=en_PL
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CHROMATOGRAPHY
Method used for separation of mixture of the compoundsbetween two phases:
❖ Stationary phase ( solid or liquid located on neutral
medium)
❖ Mobile phase ( liquid or gas)
Choice of the method depends on:
❖ the amount of analysed mixture
❖ kind of compounds
❖ complexity of separation process
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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 ofthe 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 intime and space.
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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
Components
Affinity to Stationary Phase
Affinity 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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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
• used solvent
• 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 an 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
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Preparation of chromatographic plates
• Glass plates - 1-2 mm thick covered with silica gel
•Investigated solution are placed on the plate
using 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 (for example: mixture of concentrated sulphuric and
acetic acid, anisic aldehyde and ethanol).
©
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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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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Chromatography column in 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 concentration gradients; repeatability of retention times;
quantitative determination of the compounds (area under the peak)
➢ Detection: spectroscopy (UV-vis), electrochemical, spectrofluorometric
➢ Drawback: separation only of small quantities
LIQUID CHROMATOGRAPHY LC
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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36http://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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