Name: _______________________ Period: _______ Date: _______________ Ink Chromatography One of the main jobs of biochemists is to unravel the complexities of chemical compounds and reduce them to their individual components. The term chromatography comes from two Greek words, "chromat" meaning color and the word "graphon" meaning to write. Separation of the components of chemical compounds can be done by using several methods. Liquids can be separate by High Performance liquid Chromatography (HPLC), while the components of gases are separated by Gas Chromatography. Chromatography is a method for analyzing complex mixtures (such as ink) by separating them into the chemicals from which they are made. Chromatography is used to separate and identify all sorts of substances in police work. Drugs from narcotics to aspirin can be identified in urine and blood samples, often with the aid of chromatography. Chromatography was first used to separate pigments (colors) in leaves, berries, and natural dyes. In paper chromatography, the solid surface is the cellulose fibers in the chromatography paper. A solvent or developer (water, alcohol, or acetone) is placed in the bottom of the chromatography chamber. The paper acts as a wick to pull the solvent up the paper. The solvent front will "wick" up the chromatography paper by capillary action. A minute drop of the ink or chemical mixture to be separated is placed near the bottom of the strip of chromatography paper, but slightly above the level of the solvent in the chamber. As the solvent passes over the drop of ink, the components of the ink dissolve in the solvent. Because the components of the ink do not all dissolve at the same rate, as the components of the mixture move upward, they show up as colored streaks. The separated substances on the chromatography paper form a color pattern called a chromatogram. To determine the rate of migration for each pigment or component of the ink, the Rf value for each pigment must be calculated. The Rf value represents the ratio of the distance a pigment moved on the chromatogram relative to the distance the solvent front moved. Each pigment or compound will have a unique Rf value that scientists can use to identify the substance. The Rf value is calculated using the following formula: Rf = distance traveled by the compound / distance traveled by the solvent Paper Chromatography Paper chromatography is a method chemists use to separate compounds from one another, but not change them. In this section we shall explore how this separation is made--using different inks as mixtures and dyes as different compounds. Molecules with similar arrangements of their atoms or molecular structures are attracted to each other. o Water molecules have the structure shown below in which the two hydrogen atoms form a 104 angle with the oxygen at the vertex. Because of this structure the oxygen end of the molecule has a small negative electrical charge and the hydrogen end has a small positive charge. Liquid water is held together by the attraction between the charges on different molecules. This is shown below for a small cluster of water molecules. A molecule with these charged regions is called a polar molecule. Methanol (CH3OH) has a similar structure (see below), and the methanol molecules are very soluble in water because of the mutual attraction between the two polar molecules. A more complex, yet still similar molecule is cellulose, a molecule which is the basic component of paper. It is a very long molecule (a polymer) in which thousands of rings of six atoms each are linked together like beads. A portion of a cellulose molecule is shown below. The polar -OH regions of these molecules are attracted to OH groups on adjacent cellulose chains helping to hold the fibers together in paper. Not surprisingly, water molecules, being polar, are also attracted to these regions and when paper is wet it loses strength because the water molecules get between the cellulose chains and weaken the attraction between them. When the end of a piece of paper is dipped into water the water molecules keep finding new places (polar regions) to stick to and so the water molecules climb up the paper being replaced by new water molecules below. Other molecules which might be dissolved in the water will also be carried along up the paper. This is applied to the separation of dyes in a technique known as paper chromatography. A spot of dye is placed on the paper above the level of the water. As the water moves up, the dye molecules will move with it if they are more strongly attracted to the water molecules than to the paper molecules. If the dye molecules are more strongly attracted to the paper than to the water, they will move more slowly than the water or even not at all. What if the dye is a mixture? If two or more dyes have been mixed to form an ink, then they may move at different rates as the water moves up the paper. If this happens, they will separate and we can identify them.