Amino Acids

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Separation of Amino Acids by Paper Chromatography
Chromatography is a common technique for separating chemical substances. The prefix “chroma,” which suggests “color,” comes from the fact that some of the earliest applications of chromatography were to separate components of the green pigment, chlorophyll. You may have already used this method to separate the colored components in ink. In this experiment you will use chromatography to separate and identify amino acids, the building blocks of proteins. The proteins of all living things are composed of 20 different amino acids, some of which are described below. Chromatography is partially characterized by the medium on which the separation occurs. This medium is commonly identified as the “stationary phase”. Stationary phases that are typically used include paper (as in this experiment), thin plates coated with silica gel or alumina, or columns packed with the same substances. The “mobile phase” is the medium that accompanies the analyzed substance as it moves through the stationary phase. Both liquids and gases can be used as mobile phases depending on the type of separation desired. To refer to gas or liquid chromatography, chemists often use the abbreviations GC or LC, respectively. These abbreviations explicitly identify the phase of matter of the mobile phase. The term “paper chromatography” used in this experiment’s title identifies the composition of the stationary phase. The compositions of the stationary and mobile phases define a specific chromatographic method. Indeed, many different combinations are possible. However, all of the methods are based on the rate at which the analyzed substances migrate while in simultaneous contact with the stationary and mobile phases. The relative affinity of a substance for each phase depends on properties such as molecular weight, structure and shape of the molecule, and the polarity of the molecule. The relationship between molecular shape and polarity will be discussed later in Chemistry 11 (Chapter 10 of your text). In this experiment, very small volumes of solutions containing amino acids will be applied (this process is sometimes called “spotting”) at the bottom of a rectangular piece of filter paper. For ready comparison of each trial, it is vital that each solution be applied on the same starting line. After the solutions have been applied, the paper will be rolled into a cylinder and placed in a beaker that contains a few milliliters of the liquid mobile phase. For this separation, a solution containing n-propanol, water and ammonia is the optimum mobile phase. As soon as the paper is placed in the mobile phase, the solution (sometimes called the eluting solvent) will begin to rise up the paper. This phenomenon is called capillary action, a concept that is described in Chapter 12 of your text. As the mobile phase rises on the paper it will eventually encounter the “spots” of amino acids. The fate of each amino acid in the mixture now depends on the affinity of each substance for the mobile and stationary phases. If an amino acid has a higher affinity for the mobile phase than the stationary phase, it will tend to travel with the solvent front and be relatively unimpeded by the filter paper. In contrast, if the amino acid has a higher affinity for the paper than the solvent, it will tend to “stick” to the paper and travel more slowly than the solvent front. It is these differences in the amino acid affinities that lead

to their separation on the paper. The affinities of these amino acids for the mobile phase can be correlated to the solubility of the different amino acids in the solvent (i.e., an amino acid that is highly soluble in the eluting solvent will have a higher affinity for the mobile phase than an amino acid that is less soluble in the solvent.). When the solvent front comes near the top of the filter paper, the paper is removed from the beaker and allowed to dry. At this point, the various amino acids are...