Lab 5 - Amino Acids - Student Instructions ed

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Apr 3, 2024

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Biology 2112/2912 - Lab 5 - Amino Acid Chromatography Student Instructions Learning goals of this week’s lab: ● Identify structural features of various amino acids ● Use thin-layer chromatography to isolate and identify unknown amino acids ● Develop protocol-writing skills in preparation for enzyme lab Materials required for this week’s lab: 1. Micropipettes (2-20 µL) 2. Glass slides coated in silica gel 3. Amino acids - standards 4. Amino acids - unknowns Overview of Lab & Background: Building on last week’s lab exploring the structure of biological macromolecules, this week you will explore how the structure of amino acids can be used to distinguish them via thin-layer chromatography . Amino acids are the building blocks of proteins. Amino acids have a common structure, and generally differ from each other by the atomic composition of the “R” group (or residue). Here are two different forms of a typical amino acid, the de-ionized form and the ionized form: In aqueous solution at pH 7, amino acids typically exist in their ionized form. (They are known as Zwitterions, because they have both a negatively and a positively charged group on the same molecule.) There are 20 amino acids, which differ only in their R groups. “R” can be a single H atom (as in glycine), a short hydrocarbon chain (as 1

in alanine), or a ring structure (as in tryptophan). The chemistry of amino acids is pri- marily determined by the properties of the R groups. Amino acids can be placed into three major groups: 1. Non-polar amino acids have a non-polar R group and are less soluble in water than the other amino acids 2. Polar amino acids have a polar R group and are very soluble in water 3. Electrically-charged amino acids have R groups that are ionized at intra- cellular pH levels and are also very soluble in water. These characteristics of the R groups also determine the solubility of individual amino acids in different solvents. For this lab, you should be familiar with the R group structure for the amino acids Leucine, Proline , Glutamic acid , & Lysine . Additionally, you should be able to use the terms above (non-polar, polar, and charged) to describe the different groups of amino acids. Chromatography is a method of separating a mixture of molecules by their solu- bility properties. A chromatograph has two phases: the stationary phase (usually polar), and the mobile phase (relatively nonpolar). In thin-layer chromatography (the method we will use in this lab to separate a mixture of amino acids), the stationary phase is sil- ica gel on glass (essentially Si-O-H) and the mobile phase is a mixture of n-butyl alco- hol, acetic acid, and water in the ratio 2:1:1, v/v/v. Each amino acid is both adsorbed onto the stationary phase and is soluble in the mobile phase . For each amino acid, the R group determines how strongly it adsorbs to the silica gel and how soluble it is in the solvent. Therefore, the amino acids will migrate differ- ent distances on the plate and this will allow us to identify them . A key metric to identify your amino acids is the Rf value - the ratio of the distance a molecule travels to the distance the solvent travels. The molecule (e.g. the amino acid) cannot move faster than the solvent front; therefore, the Rf value for the molecule will always be between zero and one. Lab Activities: 1) Activity 1 - Making Predictions about the Behavior of Amino Acids For this first activity, use your web browser to look up the structures of the amino acids below. In Table 1 below, enter a description of the R group for each of the amino acids (feel free to draw it if you like) and also enter a prediction about how fast or how slow this amino acid will move on the chromatogram relative to the 2

solvent. Your lab instructor can help you complete this table. For this activity, work in pairs . Table 1: Amino Acid Structures and Predictions Amino Acid Description (or drawing) of R group Prediction Regarding Movement Leucine Proline Glutamic acid Lysine 2) Activity 2 - Chromatography of Amino Acids The second activity for today’s lab involves setting up thin-layer chromatography for standard (e.g. known) and unknown amino acids. For this activity, you should work as a bench . Each bench will use four slides, with four known standards and four unknowns to identify. Your instructor will demonstrate how to spot the standards and unknowns Protocol 1. Label four glass slides (precoated with silica gel) #1 to # 4. Use a pencil to write lightly on the gel. 2. On each slide, mark the ORIGIN by drawing two tick marks on each side of the gel 2 cm from the bottom edge of the slide. This is where you will spot the samples. 3. On slide #1, spot 2 µL of two standards: Leucine (Leu) & Proline (Pro) 3

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