Biology Lab: Paper Chromatography Mr. Spinelli Background: In order to study the function of individual biomolecules (and therefore learn how they interact to create life functions) it is important that scientists be able to separate and isolate them. Scientists have developed techniques that allow them to exploit some of the characteristics of these molecules for the process of separation. The most common characteristics used to separate one molecule from another are size and charge. Chromatography is one technique by which molecules can be separated from each other based on size and charge, by forcing molecules to travel through a polar (charged) matrix. Q1: If a large molecule and a small molecule were forced to travel through a matrix for the same period of time, which one would move farther? Q2. Why is this? Q3: If a polar molecule and a non-polar molecule (both the same size) were forced to travel through a polar matrix for the same period of time, which one would move farther? Q4. Why is this? *General procedure: (groups of two students per team) – 1. Plant pigments (many different molecules of different size and charge) will be extracted from spinach leaves and swiss chard leaves by crushing them with a mortar and pestle with ethanol.* 2. The extracted pigment will then be added to chromatography paper with a Q-tip.* 2. A solvent will then be absorbed up into chromatography paper (the polar matrix), picking up the plant pigments as it travels past them. As the pigments and solvent travel through the paper, some molecules will be held back more than others.* 3. Observe the final position of the different plant pigments.* There are many different types of plant pigments as you can see from looking at the different colors of plants and plant parts. Many of these pigments are present at the same time in the same location, but you can’t see them all because they are masked by the dominant pigment in plants; chlorophyll. The purpose of this lab will be to separate the mix of plant pigments into four individual molecules (chlorophyll a, chlorophyll b, beta carotene and xanthophylls) and infer there relative charge differences based on their final positions. Data to record in your lab notebook: 1. Your chromatography paper Test tube (covered 2. A sketch of your paper along side the actual with parafilm after paper with pigments outlined and identified. setup) 3. A description of what you saw during the lab and what your final paper looks like (describe Chromatography the movement and colors that you saw and see. paper cut to a point on 4. Calculate the Rf values for each pigment the lower ½ inch (procedure on back) Pigment origin (mark this with a pencil line Chromatography solvent (1/2 pipet full, about ½ ml) * Listen for special procedural steps from your teacher. Solvent front (f) Pigment x Distance the solvent traveled (df) Pigment y Distance pigment y traveled (dy) Origin (O) To calculate the Rf value for pigment y: Divide the pigment distance (dy) by the solvent distance (df) dy/df = Rf value for pigment y Questions Make sure you read your textbook to answer some of these questions. 1. What pigments were present in the smallest amounts in the leaf? 2. What pigments were present in the largest amounts in the leaf? 3. What is the role of chlorophyll a in photosynthesis? 4. What is the role of the other pigments? 5. Many leaves change color in the fall. How is it possible for this color change to occur? 6. Why do some pigments move fast on the paper whereas some move slowly? 7. Suppose you were to analyze the pigments from different species. Why are the R f values calculated useful in addition to the color of the pigments?
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