- list and describe methods of separating mixtures.
- explain the principles involved in chromatographic separation.
- identify the mobile and stationary phases in a chromatography set up.
- calculate Rf values from appropriate data.
- column chromatography
- fractional distillation
- gas chromatography
- paper chromatography
- retention factor
- thin-layer chromatography
Mixtures occur very commonly in chemistry. When a new substance is synthesized, for example, the new substance usually must be separated from various side-products, catalysts, and any excess reagent still present. When a substance must be isolated from a natural biological source, the substance of interest is generally found in a very complex mixture with many other substances, all of which must be removed. Chemists have developed a series of standard methods for the separation of mixtures. In fact, the separation of mixtures into their constituent substances defines an entire sub-field of chemistry referred to as separation science.
Mixtures of solids may often be separated on the basis of differences in their solubilities. If one component of the mixture is soluble in water while the other components are insoluble in water, the water-soluble component can be removed from the mixture by dissolving the mixture in water and filtering the mixture through filter paper. The component dissolved in water will pass through the filter while the undissolved solids will be caught in the filter. Since the solubility of substances is greatly influenced by temperature, it may also be possible to separate the components by controlling the temperature at which the solution occurs or at which the filtration is performed. Often times, a sample is added to water and heated to boiling. The hot sample is then filtered to remove completely insoluble substances. The sample is then cooled to room temperature or below, which causes crystallization of those substances whose solubilities are very temperature dependent. These crystals can then be separated by another filtration, and the filtrate (the material that went through the filter) will then contain only those substances whose solubilities are not as temperature dependent.
Homogeneous solutions are most commonly separated by distillation. In general, distillation involves heating a liquid to its boiling point, then collecting, cooling, and condensing the vapor produced into a separate container. A common distillation setup is illustrated below.
In solutions of non-volatile (resistant to vaporization) solid solutes in liquid solvent, when the solution is boiled, only the solvent boils off and all of the solid remains in the solution. As the solvent vaporizes and all of the solute remains behind, the same amount of solute is now dissolved in less solvent. Since the concentration increases, the boiling point of the solution is also increasing. As the solution boils, increased temperature is necessary to keep the solution boiling because its boiling point has increased. This is a quick method of determining if a liquid is a pure substance or a solution: start boiling the solution, and if it continues to boil at the same temperature, it is a pure substance, whereas if its boiling point increases, it is a solution.
For a mixture of liquids in which several components of the mixture are likely to be volatile (easily vaporized), the separation is not as easy. If the components of the mixture differ reasonably in their boiling points, it may be possible to separate the mixture simply by monitoring the temperature of the vapor produced as the mixture is heated. Liquid components of a mixture will each boil in turn as the temperature is gradually increased, with a sharp rise in the temperature of the vapor being distilled indicating when a new component of the mixture has begun to boil. By changing the receiving flask at the correct moment, a separation can be accomplished. This process is known as fractional distillation.
Chromatography is another method for separating mixtures. The word chromatography means color-writing. The name was chosen around 1900 when the method was first used to separate colored components from plant leaves. Chromatography in its various forms is perhaps the most important known method for the chemical analysis of mixtures. Paper and thin-layer chromatography are simple techniques that can be used to separate mixtures into the individual components. The methods are very similar in operation and principle. They differ primarily in the medium used.
Paper chromatography uses ordinary filter paper as the medium upon which the mixture to be separated is applied. Thin-layer chromatography (TLC) uses a thin coating of aluminum oxide or silica gel on a glass microscope slide or plastic sheet to which the mixture is applied. A single drop of the unknown mixture to be separated is applied about half an inch from the end of a strip of filter paper or TLC slide. The filter paper or TLC slide is then placed in a shallow layer of solvent in a jar or beaker. Since the filter paper and the TLC slide coating are permeable to liquids, the solvent begins rising up the paper by capillary action.
As the solvent rises to the level of the mixture spot, various effects can occur, depending on the constituents of the spot. Those components of the spot that are completely soluble in the solvent will be swept along with the solvent front as it continues to rise. Those components that are not at all soluble will be left behind at the original location of the spot. Most components of the mixture will move up the paper or slide at an intermediate speed somewhat less than the solvent front speed. In this way, the original mixture spot is spread out into a series of spots or bands, with each spot representing one single component of the mixture, as seen in the illustration of a paper chromatography strip below.
The separation of a mixture by chromatography is not only a function of the solubility in the solvent used. The filter paper or TLC coating consists of molecules that may interact with the molecules of mixture as they are carried up the medium. The primary interaction between the mixture components and the medium is due to the polarity of the components and that of the medium. Each component of the mixture is likely to interact with the medium to a different extent, thus slowing the components of the mixture differentially depending on the level of interaction.
In chromatography analysis, there is a mathematical function called the retention factor. The retention factor, Rf, is defined as
Rf=distance traveled by spotdistance traveled by solvent front
Rf is the ratio of the distance a substance moves up the stationary phase to the distance the solvent have moved. The retention factor depends on what solvent is used and on the specific composition of the filter paper or slide coating used. The Rf value is characteristic of a substance when the same solvent and the same type of stationary phase is used. Therefore, a set of known substances can be analyzed at the same time under the same conditions.
In the case shown below, the Rf for the green spot is
and for the yellow spot
Rf = 1.8 cm5.7 cm=0.32
Paper chromatography and TLC are only two examples of many different chromatographic methods. Mixtures of gases are commonly separated by gas chromatography. In this method, a mixture of liquids are vaporized and passed through a long tube of solid absorbent material. A carrier gas, usually helium, is used to carry the mixture of gases through the tube. As with paper chromatography, the components of the mixture will have different solubilities and different attractions for the solid absorbent. Separation of the components occurs as the mixture moves through the tube. The individual components exit the tube one by one and can be collected.
Another form of chromatography is column chromatography. In this form, a vertical column is filled with solid absorbent, the mixture is poured in at the top, and a carrier solvent is added. As the mixture flows down the column, the components are separated, again, by differing solubilities in the carrier solvent and different absorbencies to the solid packing. As the liquid drips out the bottom of the column, components of the solution will exit at different times and can be collected.
This video presents thin layer chromatography with fluorescent materials, and column chromatography with UV active materials. There is no narration on the video so it would be advantageous to watch with a chemistry teacher (6f): http://www.youtube.com/watch?v=gzp2S0e9o8s (4:27).
- Mixtures of solids may be separated by differing solubilities of the solids.
- Components of a solution composed of a non-volatile solid solute and a liquid solvent can be separated by distillation.
- Mixtures of liquids with reasonably different boiling points can also be separated by distillation.
- Solutions with several components can be separated by paper or thin-layer chromatography.
- Gas chromatography and column chromatography are also used to separate the components of a solution.
This video describes methods used to separate the components of a mixture (1a I&E): http://www.youtube.com/watch?v=jWdu_RVy5_A (2:30).
Further Reading / Supplemental Links
An interactive video on separating mixtures is available at the link below.
- In a paper chromatography experiment to separate the various pigments in chlorophyll, a mixture of water and ethanol was used as the solvent. What is the stationary phase in this separation?
- Do you think that paper chromatography or TLC would be useful for separating a very large quantity of a mixture? Explain why or why not.
- If the mobile phase in a chromatographic experiment moved 15.0 cm and one of the compounds in the mixture moved 12.7 cm, what is the Rf value for this compound?
- If the stationary phase in a paper chromatography experiment was very polar and the solvent was moderately polar, would the polar components in the mixture be closer to the bottom of the paper or toward the top of the paper?