Introduction to Chromatography
General Chemistry (CHMA1001)February 25, 2019
Chromatography
Ø Known as color writing. A lab technique that is based on PHYSICAL ELUILIBRIA.
(Chemical equilibria – Keq and Ka )
Ø Involves the separation of mixtures into individual components by passing a fluid (gas or liquid) along a stationary phase.
Ø Samples may be gaseous, liquid or solid in nature and can range in complexity from a simple blend of two entantiomers to a multi component mixture containing widely differing chemical species
http://slideplayer.com/slide/9274099/28/images/2/Separation+of+plant+pigments+by+thin-layer+chromatography.jpg
Chromatography allows for:
1. Qualitative analysis – What is
present?
Rf; tR; type of color (hue)
2. Quantitative analysis – How much is
present?
Size of spot and intensity of the color
TLC), size of peak (GC; HPLC); expressed
in terms of amounts (ppm; mg, intensity of
color)
Physical Equilibria
• The distribution of analytes between phases can often be described as an analyte in equilibrium between the two phases;
Amobile Astationary
Physical Equilibria• Components physically separate
themselves by distributing themselves between two phases 1) Stationary phase - which can be a
solid or a liquid supported on a solid; and
2) Mobile phase – which can be a gas or a liquid that continuously flows through the stationary phase.
Mechanism of Physical Equilibra used in the separation process are
• Adsorption – “stick to”
• Absorption – “dissolve into”
Adsorption Chromatography• Each component interacts
with its environment differently under the same conditions.
• Components move at a different rate, depending upon their interaction with the stationary phase.
• Adsorption is dependent upon the nature of the component, nature of the adsorbent and the temperature.
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/be_types.htm
Absorption Chromatography• The solute molecules
distribute themselves between two immiscible liquid phases, the stationary phase and the mobile phase according to their solubilities.
• The two phases must have different polarities (only for LC).
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/be_types.htm
Adsorption vs Absorption
• ADsorption– Sticks to – Example
of Kleenex sticks to a sweater. When a lint roller is used the lint has a stronger attraction (affinity for) the lint roller
– Only moves when in the mobile phase
• ABsorption– Dissolves into -
Example of eating a cookie and how it dissolves into.
– Like has the greatest affinity for like (polar to polar or non polar to non polar)
– Only moves when in the mobile phase
ElutionThe process of moving the solutes through and out of the chromatographic system is called elution. Solutes having left the chromatographic system are said to have been eluted. The mobile or moving phase is sometimes referred to as the eluting phase.
In a multi-component sample separation is based on their attraction for (ability to adsorb or absorb) the stationary phase
http://biotech.matcmadison.edu/resources/proteins/labManual/chapter_4/section4_4.htm
DiffusionSolutes when contained in a fluid naturally diffuse and spread driven by their concentration gradient.In a chromatographic column a discrete solute band will diffuse in the gas or liquid mobile phase.
http://www.chromatography-online.org/Principles/Peak-Dispersion/Longitudinal-Diffusion.html
Main Types of Chromatography Used in Labs
1) Column Chromatography (CC)2) Thin Layer Chromatography (TLC)3) Gas Chromatography (GC)4) High Pressure (Performance) Liquid
Chromatography (HPLC)
Column ChromatographyThe stationary phaseis a powdered adsorbent which is held in a vertically positioned glass column.
The separation mechanism is usually ADSORPTION.
The mixture to be analyzed is loaded on top of this column.
http://www.m2c3.com/chemistry/VLI/M4_Topic2/M4_Topic2_print.html
Column ChromatographyThe mobile phase is a solvent poured on top of the loaded column; it flows down under the force of gravity.The sample components set up multiple, repeating physical equilibria between the stationary and mobile phases. If analytical conditions are correct, some components elute in the early fractions; other components elute from the column only after more mobile phase has flowed.
http://orgchem.colorado.edu/hndbksupport/colchrom/colchrom.html
Polarity• Polarity results from the
uneven partial charge distribution between various atoms in a compound.
• Atoms, such as nitrogen, oxygen, and halogens, that are more electronegative have a tendency to have partial negative charges.
• Atoms, such as carbon and hydrogen, have a tendency to be more neutral or have partial positive charges.
Column ChromatographyØThe polarity of the solvent which is passed
through the column affects the relative rates at which compounds move through the column.
ØOften a series of increasingly polar solvent systems are used to elute a column.
Ø If a solvent is too polar, movement becomes too rapid, and little or no separation of the components of a mixture will result. If a solvent is not polar enough, no compounds will elute from the column.
( a non-polar solvent is first used to elute a less-polar compound. A more-polar solvent is added to the column to elute the more-polar compound.)
Column ChromatographyØQualitative information:
Based on color and order of elution - the progress of the separation can simply be monitored visually.
ØQuantitaitve information: (semi-quantitative at best – approximate concentration)Based on intensity of color/hue
ØFractions of the eluent are often collected sequentially and the composition of each fraction is analyzed by thin layer chromatography or some other method.
Column Chromatography• Slurry Method Preparation:
– A mixture of the adsorbent (e.g.alumina or silica) with the solvent is prepared and this slurry is poured into the column containing glass wool or cotton.
– Always ensure that there is a continuous flow of mobile phase. The column must not go dry.
– The advantage of slurry methods is that they eliminate air bubbles from forming in the column as it packs.
– Air bubbles may lead to channels
Column Chromatography
• Dry – Packed Method– Easier to prepare than slurry method– A dry column containing glass wool or
cotton is filled with solvent. Dry alumina is added to the column containing the solvent and allowed to settle.
– Disadvantage of dry – packed method is that there is more chance for the formation of bubbles and channels in the column.
Thin Layer Chromatography (TLC)• A TLC plate is a sheet of glass, metal, or plastic
which is coated with a thin layer of a solid adsorbent (usually silica or alumina) – The stationary phase.
• A liquid, or eluent, is the mobile phase (usually an organic solvent).
• A small amount of the mixture to be analyzed is spotted near the bottom of this plate.
• The mobile phase slowly rises up the TLC plate by capillary action.
• The separation mechanism is usually ADSORPTION.
Thin Layer Chromatography (TLC)
• The TLC plate is placed in a reservoir of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid.
• The components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others.
http://www.coleparmer.co.uk/products/chromatography/Chromatography.asp
Thin Layer Chromatography
http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html
Thin Layer Chromatography
• The Rf (retention factor) can provide corroborative evidence as to the identity of a compound.- (Qualitative information – “what is present”)
• If two substances have the same Rfvalue, they are likely (but not necessarily) the same compound.
Thin Layer Chromatography
• Rf is dependent on– The solvent system– Adsorbent– Thickness of the adsorbent– Amount of material spotted– Temperature
Factors are difficult to keep constant from experiment to experiment – considered relative (compared to standards run on same plate)
Thin Layer Chromatography• Some samples are colored.
Simply mark by circling the spots before fading.
• Others colorless samples may require UV to visualize samples or to spray with an appropriate reagent.
Thin Layer Chromatography• If a set of standards that cover the concentration
range of interest are separated on the same plate as the sample and the spots simultaneously developed with an appropriate coloring reagent, an approximate estimation of the concentration of the unknown can be made by comparing its intensity with those of the standards.(semi-quantitative analysis)
• For more accurate work, other methods of spot evaluation must be used and this will involve the use of scanning instrumentation (spot scanning techniques).
Gas Chromatography
ØThe stationary phase is usually a high-boiling point liquid. The stationary phase is held permanently in a metal or glass column that is coiled to conserve space.
ØThe mobile phase is an inert gas (such as Helium, nitrogen or argon) which continuously flows through the column.
Schematic Diagram of GC
http://pharmaresearchdevelopment.blogspot.com/2010/12/gas-chromatography-general-introduction.html
Gas Chromatography
• Involves a sample being vaporised and injected onto the head of the chromatographic column.
• The sample is transported through the column by the flow of inert, gaseous mobile phase.
• The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.
Gas Chromatography
ØThe components of the mixture distribute between the liquid stationary phase and the mobile gas (vapor) phase. The separation mechanism is ABSORPTION.
ØThe components of the mixture distribute between the solid stationary phase and the mobile gas(vapor) phase. The separation mechanism is ADSORPTION.
Gas Chromatography
ØThe gaseous mixture flows through a detector (for example -TCD – thermal conductivity detector, FID – flame ionization detector, ECD electron capture detector, etc.) at the end of the column and individual components show as different peaks on a recorder.
Chromatogram
http://www.wcaslab.com/gif/perm.gif
Qualitative Analysis
• Retention time (tR) is used for identification in comparison with the retention time of a standard.
http://www.clu-in.org/characterization/technologies/gc.cfm
Quantitative Analysis
• The size (area; height) of the peak is used for quantitative information.
• Comparison to standards (standard curve).
http://online.cit.edu.au/toolboxes/labtech/Laboratory/StudyNotes/snGCRetentTimePeaks.htm
Common Uses for GC
ØPesticidesØFatty acidsØAntioxidantsØEnvironmental contaminants
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