Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that are or can be colored, especially pigments. The way paper chromatography works is pretty simple. A small concentrated spot of a solution that contains the sample of the solute is applied to a strip of chromatography paper about two centimeters away from the base of the plate or paper, usually using a capillary tube for maximum precision of amount spotted. This sample is absorbed onto the paper and may form an interaction with it. The paper is then dipped into a suitable solvent, such as ethanol, H2O, or another aqueous solvent, taking care that spot is above the surface of the solvent. The spotted chromatogram is then placed in a sealed container. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper. Differential adsorption of the solute components into the solvent can also occur and explain the separations observed. As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent and due to differences in their attraction to the fibers in the paper. The more soluble the component the further it goes. Paper chromatography takes anywhere from several minutes to several hours.
In some cases, paper chromatography does not separate pigments completely; this occurs when two substances appear to have the same values in a particular solvent. In these cases, two-way chromatography is used to separate the multiple-pigment spots. There are two types of paper chromatography: ascending and descending chromatography. In ascending chromatography, the solvent is in pool at the bottom of the vessel in which the paper is supported. The ascending chromatogram is folded over the glass rod and the other half becomes the descending chromatogram. This technique gives as quick separation as that of the individual techniques. In descending chromatography, the solvent is kept in a trough at the top of the chamber and is allowed to flow down the paper. The liquid moves down by capillary action as well as by the gravitational force, thus this method is also known as the gravitational method. In this case, the flow is more rapid as compared to the ascending method, and the chromatography is completed more quickly. The apparatus needed for this case is more sophisticated. The developing solvent is placed in a trough at the top which is usually made up of an inert material. The paper is then suspended in the solvent. Substances that cannot be separated by ascending method can sometimes be separated by the descending method.
Application in Wine microbiology:
Malolactic fermentation (MLF, or "malo") is an important winemaking process conducted on most red grape wines and some white grape wines. It is also used with some fruit wines. Paper chromatography is used to measure MLF progress. To measure microbial populations or the concentration of substrates and products, we need paper chromatography. Before you ever add the sulfur, you’ll need to make sure that both your primary fermentation and ML conversion are complete. This technique separates out tartaric, citric, malic and lactic acids from a wine sample. In this test, drops of the tested wines are placed on the chromatographic filter paper and dried, with reference standards of tartaric, citric and malic acids. The spotted paper is then (usually) rolled up like a cylinder and left vertically to absorb chromatographic solvent via capillary action. The solvent travels vertically up the paper, separating out the organic compounds present. When the solvent has almost penetrated to the top of the paper (usually after 8 hours), the paper is removed and left to dry. The chart can then be viewed and the presence of each individual acid assessed. The background appears blue, with yellowish spots appearing up the paper. Each acid can only travel a certain distance up the paper. Tartaric, citric, malic, lactic, and succinic acids are represented in order at heights progressively up the paper.