The experimental details given here are given in good faith and are believed to be safe and workable methods. However, the authors cannot take responsibility for the consequences of performing these experiments.
The experiments are written for experienced science teaching staff to use as instructions for a supervised class of students. The experiments are not designed for students or inexperienced members of the public to perform without supervision. If you wish to attempt the experiments, ensure that you have completed a legally adequate risk assessment beforehand and that you work within the constraints of the risk assessment.
Uses of chromatographyEdit
Chromatography separates a mixture of solutes in a solution. The solvent may be water, but it is often a buffer solution, a mixture of organic solvents, or even a gas.
Separation can be used to identify the components of the mixture, or it can be used to isolate pure chemicals.
Sample preparation varies widely from one type of chromatography to the next, but all types of chromatography require the sample to have a small volume and therefore a high concentration. This is because the separation is done by allowing the solutes to move apart. If they are in a small volume to begin with, it does not take much movement to separate the mixture.
In paper and thin-layer chromatography, the sample is often applied many times to the same spot, and the solvent is allowed to evaporate between applications. This concentrates the sample at that point.
In gas and column chromatography it is usually necessary to concentrate the sample as much as possible before application. Some chromatography columns will adsorb the solutes strongly at the top of the column if the sample solvent is chosen carefully. This process concentrates the sample at the top of the column before a second solvent is used to move the solutes through the column to achieve separation.
Standard procedures and equipmentEdit
Paper chromatography of mine water sampleEdit
The mine water sample consists of a number of metal ions in the form of their sulphate salts. To separate them, we can use paper chromatography.
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Diethyl ether: Methanol: Water: conc HCl (50: 30: 15: 4 by volume). To minimise esterification, mix the reagents in the order water, acid, ether, alcohol. Measure water and acid using burettes or pipettes.
Place 50 cm³ solvent in the bottom of the tank.
The solvent is very volatile and will saturate the air in the tank (‘equilibration’). Improved separation is obtained if the solvent is allowed to equilibrate with the tank atmosphere for one hour.
Spot the solutions along the starting line of the chromatography paper. Each solution should be spotted twice so that the chromatogram can be cut into two halves afterwards.
Spot two samples for the minewater, one with a single addition of solution and one with multiple additions. For the standards, use multiple additions to create 5 mm spots.
Form the paper into a cylinder. Place the cylinder, spotted end down, into the tank. Make sure the paper does not touch the sides of the tank.
Close the lid of the tank.
Prepare the locating reagents.
After three hours, remove the chromatogram from the tank. Open out the paper. Mark the solvent front with a pencil. Leave to dry.
Cut the chromatogram into two and treat the two halves as follows. Spray both sides of the paper. Take great care to avoid excessive spraying. An assistant will use the spray in a fume cupboard:
1) Spray with potassium thiocyanate in acetone. (50 cm³ saturated KCNS in water [200 g in 100 cm³ water], 50 cm³ propanone).
2) Spray with oxine (8-hydroxyquinoline – 0.5 g in 80:20 ethanol:water). Hold over '880' ammonia fumes. Place under UV light to observe fluorescence.
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Observations and measurementsEdit
Results, calculation and evaluationEdit
- The Rf value of each solute can be calculated from the following formula:
Distance from origin to solute
Distance from origin to solvent front
- Which solutes can you identify?
- How do your Rf values compare to the published ones?
- How do your results compare with those of other students?
The underlying scientific principles behind chromatographyEdit
Chromatography involves the physical separation of a mixture of compounds. Chromatography can be used as a purification method but also sees wide use for the identification of compounds based on their chromatographic behavior.
There are many variations of chromatography, but all involve the dissolution of an analyte into a fluid known as the mobile phase and the passage of this fluid solution across a stationary phase, often a solid or liquid-coated solid.
As the mobile phase comes into contact with the stationary phase, some of the analyte molecules dissolve or adsorb onto the mobile phase. The more the molecules of that substance are retained, the slower their progress through the chromatographic apparatus. Different substances will then move through at different rates, ideally resulting in distinctly identifiable retention times for each substance.
Alternatively, if the solutes are retained on the chromatogram instead of eluted from its far end, one can calculate the ratio of the distance traveled by the solute compared to the distance traveled by the solvent. The is the Rf value.
Commonly used chromatographic techniques are identified through the nature of the stationary and mobile phases used, the method for passing the mobile phase through the apparatus, and how separated components are identified.
As the chromatogram develops (l-r) the two components of the mixture separate.
In paper chromatography the stationary phase is paper and the mobile phase is generally some liquid that will dissolve the analyte and wet the paper, often water or an aqueous solution.
Thin layer chromatographyEdit
In thin layer chromatography (TLC) a plastic or glass plate is coated with the stationary phase, often alumina, silica, or alkylated silica. The analyte is dissolved in a quick-drying solvent and spotted near the bottom of the plate. The edge of the plate beneath the spot or spots is then dipped and left in a solution of the mobile phase, either an organic solvent or aqueous solution (depending on the nature of the analyte and stationary phase). Capillary action is then allowed to draw the solvent front through the spotted analyte, carrying with it and in the process separating out the analyte's components.
In gas chromatography (GC) the analyte and mobile phase must both be gases or be readily introduced into the gas phase by heating. The mobile phase gas must be inert and not reacting with the sample to be analysed. Examples of inert gases are helium and nitrogen gas.
The gases are passed through a long, narrow (and most often, coiled) tube either packed with a porous stationary phase or whose inner walls are coated with a stationary phase, and the analyte components are detected as they emerge from the far end of the tube. The tube is commonly known as GC column.
Often a time-varying temperature gradient, from lower temperature to higher temperature, is applied to the tube. This first allows the analyte components to partition into the stationary phase and then, as the temperature rises, to differentially force them back into the mobile phase.
Common detectors for gas chromatography are flame ionization detector (FID), electron capture detector (ECD) and mass spectrometry (MS). Different types of sample analysis would require the use of a different type of detectors.
Column chromatography, like gas chromatography, uses a tube packed with a stationary phase, but the mobile phase is a liquid instead of a gas (It is sometimes known as 'liquid chromatography or LC). Instead of temperature gradients, a gradient in the composition of the liquid phase can be used to separate components.
Column chromatography can be performed on larger molecules which may not be readily introduced into the gas phase. On the other hand, because of the increased viscosity of liquids compared with gases, liquid chromatography can be a more ponderous process. HPLC (variously high-pressure liquid chromatography or high-performance liquid chromatography) speeds the process and improves its selectivity and sensitivity to a significant degree by forcing the mobile phase through the chromatographic column with high-pressure pumps.
The first root of the word chromatography, chroma, indicates that the separated components in some forms of the technique can be identified by their colour alone. But chromatography has now long been performed on colourless compounds that can be identified in other ways.
Analyte components on thin-layer chromatography plates are often identified under ultraviolet light, or by chemical staining in, for example, an iodine chamber or potassium permanganate. Gas chromatographic analytes are detected by changes in the ionisation levels of a flame at the output end of the column or by changes in the electrical conductivity of the gas mixture at the end of the column. Liquid chromatography fractions are often analysed through spectrophotometric techniques, notably UV-visible spectroscopy. When separation with GC or LC is performed in tandem with mass spectrometry (the "hypenated" techniques of GC-MS and LC-MS), masses of individual fractions is rapidly determined. These methods are frequently employed in analytical and forensic science.
- Smith, I & Feinberg, JG (1965) Paper and thin layer chromatography and electrophoresis. 2nd edition. p104-109, p222. Shandon Scientific, London.