Thin layer chromatography post lab questions

BIOL 1406 Austin Community College Lab 7 Thin Layer Chromatography Report
Subject
Science

Course
Biol 1406

School
Austin Community College

Department
BIOL

Question Description
I need you to do the boxes that says (Your Turn). and Post lab I will attach the lab here and also my teacher have us a powerpoint for this lab.Lab 7: Thin Layer Chromatography Objectives After completing this exercise, you should be able to: • • • • Explain how chromatography is used to separate a mixture of molecules into its components, and describe the role of the mobile and stationary phases in this process. Explain how thin layer chromatography works. Explain how absorption spectra can be used to identify molecules. Interpret an absorption spectrum, identifying the major absorption maxima in a spectrum. Prelab A. Thin Layer Chromatography Chromatography is a technique used to separate molecules that are in a mixture. Chromatography means “color writing” and was originally used to separate pigments from plant extracts. There are many different types of chromatography. In this lab, you will use thin layer chromatography (TLC) to separate photosynthetic pigment molecules from an extract of spinach leaves. In the next lab, you will use size exclusion column chromatography to isolate a protein from a protein mixture. In today’s TLC experiment, the pigments will separate from each other according to their charges (or lack of charges). Your thin layer chromatography setup consists of two different parts. One part of the setup is called the stationary phase and the other is called the mobile phase. A diagram of the TLC setup is pictured on the next page. • The stationary phase of your thin layer chromatography is a TLC plate, which is a rectangular strip of plastic with a thin coating of silica on one side. Silica is a charged molecule. You will apply a horizontal line of spinach leaf extract on the silica coating near one end of the plate. The location on the plate where the line of extract is applied is called the origin. • The mobile phase of your thin layer chromatography is a mixture of three nonpolar organic solvents: petroleum ether, acetone, and chloroform. The solvent mixture is housed in a sealed glass jar called a developing chamber. • The end of the TLC plate closest to the line of spinach extract will be immersed in the solvent. Notice that the level of the solvent is below the origin. The solvent mixture will slowly travel up the plate by capillary action, which is similar to the way that water would travel up a dry paper towel. • As the mobile phase travels up the TLC plate, it pulls the pigments in the spinach extract up the plate as well. Recall that this TLC will separate the pigments by charge. As they travel up the plate, the pigment molecules that are charged will be more attracted to the silica coating, slowing their migration. Pigment molecules that are less charged or completely uncharged will be more attracted to the mobile phase, making their migration faster. Thus, charged pigments will appear as horizontal bands closer to the origin, while less charged or uncharged pigments will appear as bands farther away from the origin. BIOL 1406 Lab Manual Hays Campus Edition Fall 2018 Lab 7 Page 1 The TLC is stopped when the leading edge of the solvent, called the solvent front, is near the opposite end of the TLC plate. From the array of pigment bands that appear, you will identify the bands corresponding to carotene, chlorophyll a, and chlorophyll b and three other pigments. Then, you will extract the three pigments from the silica and plot absorption spectra for them using a spectrophotometer. This procedure is similar to early experiments that isolated plant pigments involved in photosynthesis. At beginning of chromatography At end of chromatography Developing chamber Solvent front TLC plate Origin: Starting point for pigment mixture Solvent at bottom of chamber Separated pigment bands Extra solvent B. Potential Problems in Thin Layer Chromatography Here are some potential problems that can occur in TLC and what you can do to prevent them from happening: • At start of TLC, pigment mixture dissolves into solvent: There is too much solvent in the developing chamber. Make sure that the pigment mixture is applied so that it is above the level of the solvent. • During TLC, pigment bands appear uneven: o o o o Wear gloves. Oils from bare hands can interfere with the even migration of molecules along the silica. The silica coating on the plate was damaged while applying the pigment mixture onto the plate. Use a light touch to avoid scraping off the silica coating. Apply the pigment mixture in as straight of a horizontal line as possible, leaving empty space on both sides of the pigment line so that it does not touch the edges of the plate. After inserting the TLC plate into the developing chamber,