Title: Study of different chromatographic techniques
Introduction
History of Chromatography
Chromatography, literally "color writing", was first employed by Russian scientist Mikhail Tswett in 1903/1906. He continued to work with chromatography in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls. Since these components have different colors (green, orange, and yellow, respectively) they gave the technique its name
Definition:
It is a physical method of separation in which the components of a mixture are separated by differences in their distribution between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves through it in a definite direction. The substances must interact with the stationary phase to be retained and separated by it.
The stationary phase may be a solid, or a liquid supported on a solid or gel, the mobile phase may be either a gas or a liquid.
Terminologies used in chromatography
1. Chromatogram: It is the visual output of the chromatograph.
2. Chromatograph: It is equipment that enables a sophisticated Separation.
3. Stationary phase (bounded phase): It is a phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
4. Mobile phase: It is the phase which moves in a definite direction.
5. Analyte (Sample): It is the substance to be separated during chromatography.
6. Eluent: It is the solvent that will carry the analyte. Fluid entering a column.
7. Eluate: It is the mobile phase leaving the column. Fluid exiting the column
8. Retention time: It is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.
9. Elution: The process of passing the mobile phase through the column.
10. Retardation factor (Rf): Fraction of an analyte in the mobile phase of a chromatographic system.
Chromatography is used by scientists to:
- Analyze – Examine a mixture, its components, and their relations to one another
- Identify– Determine the identity of a mixture or components based on known components
- Purify – Separate components in order to isolate one of interest for further study
- Quantify – Determine the amount of the a mixture and/or the components present in the sample
Chromatography is usually introduced as a technique for separating and/or identifying the components in a mixture. The basic principle is that components in a mixture have different tendencies to adsorb onto a surface or dissolve in a solvent.
Basic principles
All chromatographic methods require one static part (the stationary phase) and one moving part (the mobile phase). The techniques rely on one of the following phenomena: adsorption; partition; ion exchange; or molecular exclusion.
1. Adsorption:
Adsorption chromatography was developed first. It has a solid stationary phase and a liquid or gaseous mobile phase.
Each solute has its own equilibrium between adsorption onto the surface of the solid and solubility in the solvent, the least soluble or best adsorbed ones travel more slowly. The result is a separation into bands containing different solutes. Liquid chromatography using a column containing silica gel or alumina is an example of adsorption chromatography.
2. Partition
In partition chromatography the stationary phase is a non-volatile liquid which is held as a thin layer (or film) on the surface of an inert solid. The mixture to be separated is carried by a gas or a liquid as the mobile phase. The solutes distribute themselves between the moving and the stationary phases, with the more soluble component in the mobile phase reaching the end of the chromatography column first e.g. Paper chromatography
3. Ion exchange
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Fig. 3. Ion exchange Ion exchange chromatography is similar to partition chromatography in that it has a coated solid as the stationary phase. The coating is referred to as a resin, and has ions (either cations or anions, depending on the resin) covalently bonded to it and ions of the opposite charge are electro statically bound to the surface. Domestic water softeners work on this principle. |
4. Molecular exclusion
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| Fig. 4. Molecular exclusion chromatography |
Molecular exclusion differs from other types of chromatography in that no equilibrium state is established between the solute and the stationary phase. Instead, the mixture passes as a gas or a liquid through a porous gel. The pore size is designed to allow the
large solute particles to pass through uninhibited. The small particles, however, permeate the gel and are slowed down so the smaller the particles, the longer it takes for them to get through the column. Thus separation is according to particle size.
Types of Chromatography
1. Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)
2. Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase)
3. Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)
4. Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)
Chromatographic techniques
A) Paper chromatography
This is probably the first, and the simplest, type of chromatography technique. In this method a drop of a solution of a mixture of dyes or inks or pigment is placed on a piece of chromatography paper and allowed to dry. The mixture separates as the solvent front travels beyond the mixture. Separation is most efficient if the atmosphere is saturated in the solvent vapour.
Paper chromatography works by the partition of solutes between water in the paper fibres (stationary phase) and the solvent (mobile phase).
Common solvents that are used include pentane, propanone and ethanol. Mixtures of solvents are also used, including aqueous solutions, and solvent systems with a range of polarities can be made.
As each solute distributes itself (equilibrates) between the stationary and the mobile phase, the distance a solute moves is always the same fraction of the distance moved by the solvent. This fraction is variously called the retardation factor or the retention ratio, and is given the symbol R or Rf:
It is possible that two solutes have the same Rf values using one solvent, but different values using another solvent (eg this occurs with some amino acids). This means that if a multi component system is not efficiently separated by one solvent the chromatogram can be dried, turned through 900, and run again using a second solvent
B) Thin layer chromatography (TLC)
Thin layer chromatography is similar to paper chromatography, but the stationary phase is a thin layer of a solid such as alumina or silica supported on an inert base such as glass, aluminum foil or insoluble plastic. The mixture is ‘spotted’ at the bottom of the TLC plate and allowed to dry. The plate is placed in a closed vessel containing solvent (the mobilephase) so that the liquid level is below the spot.
TLC has advantages over paper chromatography in that its results are more reproducible, and that separations are very efficient because of the much smaller particle size of the stationary phase.
The solvent ascends the plate by capillary action, the liquid filling the spaces between the solid particles. This technique is usually done in a closed vessel to ensure that the atmosphere is saturated with solvent vapour and that evaporation from the plate is minimized before the run is complete. The plate is removed when the solvent front approaches the top of the plate and the position of the solvent front recorded before it is dried to calculate Rf value of individual component.
Many spots are not visible without the plates being ‘developed’. This usually involves spraying with a solution that is reversibly adsorbed or reacts in some way with the solutes.
C) Gas chromatography (GC)
This technique uses a gas as the mobile phase, and the stationary phase can either be a solid or a non-volatile liquid. Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography.
Gas chromatography is always carried out in a column, which is typically "packed" or "capillary". Gas chromatography (GC) is based on a partition equilibrium of analyte between a solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium).
The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column).
Components of GC:
1. Carrier gas:
2. Flow regulators & Flow meters:
3. Injection devices
4. Columns
5. Temperature control devices
6. Detectors
7. Recorders
8. Integrators
Process of Separation
Volatile liquid or gas injected through septum into heated port
The injector oven is usually 50–100 °C hotter than the start of the column.
Sample rapidly evaporates and is pulled through the column with carrier gas
Column is heated to provide sufficient vapor pressure to elute analytes
Separated analytes flow through a heated detector for observation
D) Liquid chromatography (LC)
Liquid chromatography is similar to gas chromatography but uses a liquid instead of a gaseous mobile phase. The stationary phase is usually an inert solid such as silica gel (SiO2.xH2O), alumina (Al2O3.xH2O) or cellulose supported in a glass column.
The adsorbing properties of silica and alumina are reduced if they absorb water, but the reduction is reversed by heating to 200–400 °C. Silica is slightly acidic, and readily adsorbs basic solutes. On the other hand, alumina is slightly basic and strongly adsorbs acidic solutes.
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