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AP Chemistry Laboratory
Publication No. 10535A
Catalog No. AP9093
In this experiment, liquid chromatography is used to separate the substances that are present in grape-flavored Kool-Aid®. First, the dyes responsible for the purple color, FD&C Blue #1 and Red #40 are separated. Then, in a second experiment, the other components of Kool-Aid®, the flavorings and citric acid, are separated as well.
• Liquid chromatography
Chromatography is an important analytical tool used to separate the components of a mixture. These components become separated or partitioned between a stationary phase and a moving phase of the chromatography system. The moving phase is either a gas or a liquid and the stationary phase is usually a solid. The mixture to be separated is combined with the mobile phase. As the mobile phase "solution" flows over the stationary phase, the components of the mixture continuously equilibrate between the phases, based on their particular affinity for each phase. A higher attraction for the mobile phase leads to a higher concentration of a component in the mobile phase and a faster transport through the entire system. Components more strongly attracted to the stationary phase take longer to migrate through the system. This results in the components becoming separated into bands that flow through the system at different rates. If the separation, or resolution, is sufficient, the bands will exit the system as distinct fractions (Figure 1). Components
Y _ polar
Stationary Phase (nonpolar)
(j 00 Yy y
Figure 2. Basic chromatography system
Usually, the solid phase is relatively polar and the solvent nonpolar in liquid chromatography. This experiment utilizes a form of chromatography called reverse phase liquid chromatography (RPC). In RPC, the stationary phase is a nonpolar solid and a polar solvent is used as the mobile phase. When a mixture is injected into the RPC column and washed through it, several processes occur (see Figure 3). The more polar components of the mixture are attracted more strongly to the polar solvent, so they will move more quickly through the column with the solvent. The less polar components will move more slowly, as they spend more time adsorbed onto the nonpolar column medium. Ideally, the components should emerge at different times. A measure of the degree of separation that is achieved is called the resolution of the system. As the band of each component moves down the column, the band widens due to diffusion. As bands widen they can overlap each other and may prevent clean separation or resolution of the components .
B. Elution begins.
Note that bands widen as they move down the column.
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