Chromatography
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Transcript of Chromatography
Adsorption Chromatography Separation based on their adsorption onto the surface
of solid (stationary phase). A solid which is insoluble in the solvent chosen may
be used as the fixed phase. The mode of interaction between the components of
the mixture and the fixed phase is adsorption, Hence the method is called adsorption chromatography.
Ex; Column chromatography, TLC etc.,
Parti t ion chromatography
Partition chromatography, is a chromatographic technique in which the solute is separated based on their partition between a liquid mobile phase and a liquid stationary phase coated on a solid support. The support material used in partition chromatography is usually silica
Fall, 2008 4
Thin-Layer ChromatographyHere the mobile phase is a liquid
Flowing past a thin layer of powder on a solid support.Substances that are less attracted to the solid or are more soluble in the liquid move faster Stationary phase
• glass or plastic plates coated with thin layer of adsorbent
• Silica gel, alumina, celluloseMobile phase
• Solvent or mixture of solvents
Thin layer chromatography
• It involves the separation of substances of a mixture over a thin layer of an adsorbent.
• A thin layer(about 0.2mm thick) of an adsorbent (silica gel or alumina) is spread over a glass plate of suitable size. The plate is known as thin layer chromatography plate.
• The solution of the mixture to be separated is applied as a small spot about 2 cm above one end of the TLC plate.
• The glass plate is then placed in closed jar containing the solvent (Below 2cm height).
• As the solvent in the jar moves up the plate, the components of mixture move up the plate to different distances, depending on this degree of adsorption separation takes place.
The relative adsorption of each component of the mixture is expressed in terms of its retention factor i.e., Rf value
Rf valueRf values are used in identification of each of the component.
• The retention factor, or Rf, is defined as the distance traveled by the compound divided by the distance traveled by the solvent
• For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, the Rf is 0.75:
Paper Chromatography
• In this technique, the stationary phase is considered to be the cellulose network of the paper.
• The mobile phase known as the developing solvent consists of either one solvent or a mixture of different solvents.
Paper Chromatography
• In this the mixture of compounds is applied on the paper as a spot little above the lower end and then this end is dipped in the solvent. When the solvent has risen more than two third length of the paper, then it is removed from the solvent. The paper is dried and is known as chromatogram.
• Now, the spots of different compounds can be visualised using some suitable chemicals.
Rf value
The ratio of the distance travelled by the compound in a particular solvent to that the distance travelled by the solvent is a constant and is known as retention factor (Rf).
For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, the Rf is 0.75:
Ion Exchange Chromatography
Ion Exchange Chromatography
• Ion exchange chromatography is a special name given to column chromatography when the stationary phase is an ion exchange resin.
• Synthetic ion exchange resins are high molecular weight polymeric materials containing large number of ionic functional groups per molecule. Cation exchangers contain sulphonic acid groups (RSO3
– H+) or carboxylic acid groups (RCOO– H+). Anion exchange resin contains amines attached to the polymer molecule RN(CH2)3
+OH–).
Ion Exchange Chromatography
• In ion exchange chromatography a column is packed with an acid resin and treated first with hydrochloric acid to make sure that all exchange points were occupied by hydrogen ions.
• A mixture of rare earths as their chlorides is sent down the column. This resulted in the displacement of hydrogen ions by rare earth cations.
• The rare earth ions could then be eluted one after another. Since elution with water was very slow, a solution of citric acid was used as eluting solvent.
• The cations moved at different rates depending on the stability of the corresponding complex with citric acid.
Gas Chromatography
Gas Chromatography (GC)Gas Chromatography (GC)GC is currently one of the most popular methods for separating and analyzing compounds.
This is due to its high resolution, low limits of detection, speed, accuracy and reproducibility.
GC can be applied to the separation of any compound that is either naturally volatile (i.e., readily goes into the gas phase) or can be converted to a volatile derivative.
A simple GC system consists of:
. G 1. Gas source2. Injector or sample application system 3. Chromatographic column 4. Detector & computer or recorder
GC GC structurestructure
Gas sourceGas source
• It provides all the necessary gas supplies.• The most widely used gases are H, He, N2 and air.
InjectorInjector
• We use a syringesyringe to inject sample onto the column. • It is situated inside a thermostaticallythermostatically controlled
enclosure.
ColumnColumn• It contains the column and an oven.• The column is the essential device to
achieve the necessary separation.• The oven is used to control the column
temperature.• The column has two kinds: Packed columnPacked column Capillary columnCapillary column
DetectorDetector• There are wide range of detectors available
each having unique operating parameters and its own performing characteristics.
• The output of detector is electronically modified.
Types of detectorsTypes of detectors
• The two types of detectors are TCD:TCD: Thermal Conductivity Detector Thermal Conductivity Detector ECD:ECD: Electron Capture Detector Electron Capture Detector
TCD Detector
A TCD detector consists of an electrically-heated wire. The temperature of the sensing element depends on the thermal conductivity of the gas flowing around it.
Changes in thermal conductivity, such as when organic molecules displace some of the carrier gas, cause a temperature rise in the element which is sensed as a change in resistance.
The TCD is not as sensitive as other detectors but it is non-specific and non-destructive.
ECD DetectorUses a radiactive Beta emitter (electrons) to ionize some of the carrier gas and produces a current between a biased pair of electrodes.
When an organic molecule that contains electronegative functional groups, such as halogens, phosphorous and nitro groups, pass by the detector, they capture some of the electrons and reduce the current.
