Separating Color Pigments by Chromatography

1. Paper & Ion Exchange Chromatography Separating Color Pigments by Chromatography

2. Uses in Science • Separate and purify biological molecules in biotechnology research. • Several types of chromatography including: • Paper Chromatography • Ion Exchange Chromatography

3. Big Picture • Chromatography involves the dissolution of an analyte into a fluid known as the mobile phase and the passage of this fluid solution across a stationary phase, often a solid or liquid-coated solid. • What is an analyte? • An analyte, or component, is a substance or chemical constituent that is of interest in an analytical procedure. • As the mobile phase comes into contact with the stationary phase, some of the analyte molecules dissolve or adsorb onto the mobile phase. Components of the sample will separate readily according to how strongly they adsorb on the stationary phase versus how readily they dissolve in the mobile phase.

4. Mobile v. Stationary Phase

5. Paper Chromatography • Stationary phase is a is a highly absorbent paper. • The mobile phase will consist of 2 solutions: • Acidic Solution • Basic Solution Recall, that acidic solutions have more dissolved H⁺ ions, whereas, basic solutions have more dissolved OH⁻ ions. How might this affect solubility of a solute?

6. Rule of Thumb • Generally, in an acidic solvent, an alkaline solute will be more soluble, & vice versa. • The more soluble a solute, the higher it travels up the paper.

7. Limitations of Paper Chromatography • Only water soluble solutes can be used. • Rfvalues are not always accurate. • This makes paper chromatography most useful to distinguish the differences b/t 2 residues NOT similarities.

8. What is the Rf value? • Ratio of distance traveled by the solute to the distance traveled by the solvent front. • Rf = distance traveled by solute distance traveled by solvent If the Rf of an unknown does not match that of a known material, they are not the same compound.

9. Calculating Rf Molecules that have small Rf values did not travel very far on the stationary phase.

10. Applications of Paper Chromatography • Determine what chemicals may be polluting a stream. • Determine types of drugs in a urine sample. So, scientists take a known sample and compare it with the results of an unknown sample.

11. Ion Exchange Chromatography • A process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger (in this case – charged beads) • In this lab, + and – dyes are mixed with the beads. • The ion beads are negatively charged.

12. Applications of Ion Exchange Chromatography • Water Softeners • Remove calcium, magnesium, & other cations (+) in hard water. • Separate & Purify Metals • PUREX process (plutonium-uranium extraction process), used to separate plutonium & uranium from the spent fuel products from a nuclear reactor, and to be able to dispose of the waste products. Then, the plutonium and uranium are available for making nuclear-energy materials, such as new reactor fuel and nuclear weapons.