Liquid Chromatography Lab

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Liquid Chromatography
AP Chemistry Laboratory

# 18
Publication No. 10535A

Catalog No. AP9093

Introduction
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.

Concepts
• Resolution

• Liquid chromatography

• Selectivity

Background
Chromatography is an important analytical tool
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
X-
D.Second component emerges.

Note that bands widen as they move down the column.
Figure 3. Components of Mixture Moving Through Liquid Chromatography Column

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Liquid Chromatography

Page 3

Experiment Overview
The purpose of this experiment is to use liquid chromatography as a tool to separate the components of unsweetened, grape-flavored Kool-Aid® or any grape flavored drink. Miniature liquid chromatography columns called Sep-Pak CI8 columns are used for the separation. The Sep-Pak column is packed with a silica solid which has a CI8 hydrocarbon bonded to it, so it is very nonpolar. In Part 1, the two dyes in the drink are separated using dilute isopropyl alcohol as the