Liquid Chromatography Lab

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Liquid Chromatography – Laboratory #18

Introduction: We are using liquid chromatography to separate the colored substances in grape-flavored drinks. We separate the component dyes, and then we separate the flavorings and citric acids.

Background: Chromatography is a process that is used to separate a substance into its component parts. The separation occurs between the stationary and moving phase of the lab. The moving phase consists of a fluid and the stationary phase consists of a solid. The mixture we are trying to split up is integrated into the mobile phase. When the mobile phase interacts with the solid phase, the components of the mixture are attracted to the solid phase in varying degrees. Components with higher levels of attraction for the mobile phase result in a faster speed of transport throughout the solid phase. Components with higher levels of attraction for the solid phase result in a slower speed of transport throughout the solid phase. These differing levels of attraction end up in separation of the mixture into component bands, which exit the system as distinct liquids.


Liquid chromatography labs are composed of six main parts:
a. A separation column filled with a fine-grain solid.
b. A solvent (mobile phase) that moves through the separation column. c.An injection system to transport the solvent to the separation column. d. A pump to force the solvent through the separation column. e. A detector to determine when the components exit the separation column. f. A recorder

Although in most chromatography labs the solid phase is polar and the mobile phase is nonpolar, we are using Reverse Phase Liquid Chromatography, where the mobile phase is polar and the solid phase is nonpolar.

When the mobile phase interacts with the solid phase, the polar parts of the mobile phase are attracted to each other, so they wash through the column quickly. The less polar components of the mobile phase are attracted to the nonpolar solid in the column, so they wash through the column slowly. This results in a separation of the components, whose degree is measured as the resolution. [pic] Pre-lab Questions

1. What is the process of chromatography used for?
2. In the chromatography, components of a mixture distribute themselves between the stationary phase and the mobile phase. Explain how the components can be separate with these two phases. 3. In the liquid chromatography column used in this experiment, the solid has a C18 hydrocarbon bonded to it. Would a C18 hydrocarbon be polar or nonpolar? Explain. 4. Below are the typical data for this experiment. 1 mL of a Kool-Aid solution was loaded on a Sep-Pak C18 Column. The red and blue dyes were eluted from the column with a constant flow of 18% isopropyl alcohol. The eluted solution was collected in a 10 mL graduated cylinder. The volumes of eluant were recoreded at the beginning and end of each color band.

The first step in calculating the selectivity and resolution of the system is determining the volumes of eluant corresponding to the band widths and band centers for each eluted dye. a. Bandwidth W is the volume in mL of eluant containing each dye as it emerges from the column. Calculate the bandwidth W for each dye for each of the three runs and then determine the average bandwidth W average for each dye. b. Center of band, called Average Retention Volume V Rave corresponds to the center of each band. The average retention volume is calculated by taking the average starting volume for each band and adding one half the corresponding average band width. V Rave = V start + (½) W ave

Calculate the average retention volume volume V Rave for the red and blue dyes. c. For each dye, a capacity factor k' can be calculated. This term is a relative measure of the attraction of the dye for the stationary phase as compared to its attraction for the mobile phase. The equation fo capacity...
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