Chemistry 108
Name_______________________________
Lab #5 Prelab: EXTRACTION AND SEPARATION OF PLANT PIGMENTS Purpose of the lab: The purpose of this lab activity is for the student to learn about extraction and chemical separation technology. Specifically, the student will learn how to do a liquid phase-extraction and Thin Layer Chromatography in order to separate a mixture of molecules. Introduction Chromatography is a useful analytical technique that allows various components of a mixture to be separated based on their polarity and/or size. There many different types of chromatographic separation apparatuses and methods. We will use a technique called thin-layer chromatography (TLC). The thinlayer chromatographic surfaces that we will use in this experiment are plastic slides (TLC slides) with a coating of very small porous silica (SiO2) particles. The particles are so small that when they are not adhered to a surface, they behave like dust. A small drop of the mixture to be separated is applied (spotted) near one end of the TLC slide. The spotted end of the TLC slide is then dipped into a developing solvent (see figure 1a), called the mobile phase¸ which flows up the TLC surface by capillary action. As the developing solvent flows up the TLC surface, it can carry along the components of the mixture. Each component of the spotted mixture will move upward at a different rate.
The fact that different molecules move along the chromatographic surface/solvent interface at different rates is the key feature of chromatographic separation. Think of the component molecules as constantly moving back-and-forth from being adsorbed to the TLC surface to being carried upward with the mobile phase solvent. In the case of TLC, the more soluble the component is in the solvent, the faster it travels up the surface. If it is not very soluble, it will remain adsorbed on the TLC surface (stationary phase) longer and therefore travel at a slower rate. In addition to the component’s affinity for the solvent, the component’s affinity for the stationary phase also plays a part in the rate at which the component
Chemistry 108
Plant Pigments
moves. If there is a strong affinity between a component molecule and the stationary phase surface, the molecule will move upward more slowly. The affinity to the surface is determined by: (1) non covalent attractive interactions, and (2) in cases of porous stationary phases, the size of the molecule. If a molecule has a size such that it is easily “stuck” in a pore (hole), it will move upward more slowly since it takes more time to become “un-stuck”. The rates of flow are measure in terms of Rf (retardation factor) values. An Rf is the relative distance that a sample component has moved relative to the distance moved by the mobile phase solvent. The following illustration will demonstrate how the Rf is calculated. Rf is measured by dividing the distance the component traveled by the distance the solvent traveled. Therefore, an Rf value can never be greater than 1. The Rf value for a particular component is characteristic for that component in that particular solvent. Therefore, it will always be the same (considering that the mobile and stationary phases are the same) and can be identified in other mixtures
Rf = Distance component traveled Distance solvent traveled Example: Calculation of TLC Rf values for a sample containing 4 components: Solute
Distance Traveled (cm)
Rf value
A
2.0 cm
2.0 cm = 0.33 6.0 cm
B
3.0 cm
3.0 cm = 0.50 6.0 cm
3.5cm
3.5 cm = 0.58 6.0 cm
5.5cm
5.5 cm = 0.92 6.0 cm
C
D
Chemistry 108
Plant Pigments
Prelab Questions 1) Consider the following TLC chromatograph. Calculate the Rf values for each of the spots in the illustration below.
Spot
Distance Traveled (cm)
A
3.10 cm
B
2.58 cm
C
4.20 cm
D
5.08 cm
Rf value
2) In the first step of this lab, you will crush the spinach while it is in methanol. Crushing breaks (opens) the plant cells and the methanol removes much of the water from the cells. Make a drawing showing how methanol molecules (CH3OH, common name methyl alcohol) are attracted to water molecules (and vice versa). Name the non covalent attractions that are present between water and methanol. Which of these attractive forces are the strongest?
Chemistry 108
Plant Pigment Lab
In the second step of the lab, we will extract the pigment molecules in a technique called liquid-phase extraction. In this step you will separate the hydrophobic plant pigment molecules from other hydrophilic component molecules and solids. This is done by placing the pulverized spinach in a flask that contains an extremely hydrophobic liquid called “petroleum ether” and a hydrophilic liquid. These two liquids will not mix and form two layers because one is hydrophobic and the other hydrophilic. When the flask is shaken vigorously, then left to settle so that the two liquids separate into layers again, the spinach pigment molecules will be extracted into the hydrophobic petroleum ether layer. These various pigment molecules that are extracted into the petroleum ether layer will be separated from each other in a final TLC Chromatography step of the lab described earlier. 3) Petroleum ether contains molecules like CH3CH2CH2CH2CH2CH3 and CH3C(CH3)2CH2CH(CH3)CH2CH3. (Note: Although the name implies that petroleum ether is in the ether family, it is not; it is a mixture of non-aromatic hydrocarbons. The name is historical from when “ether” was the same as “spirits” and had to do with the fact that the hydrocarbon mixture had a high vapor pressure.) Look at the structure of β-carotene (one of our plant pigments) below and thoroughly explain why β-carotene is much more soluble in petroleum ether than in water. Do not simply say that “like dissolves like” (although it is true), name and explain the nature of the intermolecular force that is involved here.
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