Question

Describe how the solvents worked as the mobile phase of the liquid chromatography experiment. Why was it necessary to use different concentrations of aqueous isopropanol in the step-gradient separation?

Answer 1

Liquid chromatography is a technique used to separate a sample into its individual parts. This separation occurs based on the interactions of the sample with the mobile and stationary phases. Because there are many stationary/mobile phase combinations that can be employed when separating a mixture, there are several different types of chromatography that are classified based on the physical states of those phases. Liquid-solid column chromatography, the most popular chromatography technique and the one discussed here, features a liquid mobile phase which slowly filters down through the solid stationary phase, bringing the separated components with it.

Components within a mixture are separated in a column based on each component's affinity for the mobile phase. So, if the components are of different polarities and a mobile phase of a distinct polarity is passed through the column, one component will migrate through the column faster than the other. Because molecules of the same compound will generally move in groups, the compounds are separated into distinct bands within the column. If the components being separated are colored, their corresponding bands can be seen. Otherwise as in high performance liquid chromatography (HPLC), the presence of the bands are detected using other instrumental analysis techniques such as UV-VIS spectroscopy

In the first step, the mixture of components sits atop the wet column. As the mobile phase passes through the column, the two components begin to separate into bands. In this example, the red component has a stronger affinity for the mobile phase while the blue component remains relatively fixed in the stationary phase. As each component is eluted from the column, each can be collected separately and analyzed by whatever method is favored. The relative polarities of these two compounds are determined based on the polarities of the stationary and mobile phases. If this experiment were done as normal phase chromatography, the red component would be less polar than the blue component. On the other hand, this result yielded from reverse phase chromatography would show that the red component is more polar than the blue component.

Chromatography is a separation method that exploits the differences in behavior of substances between a mobile phase (solvent) and a stationary phase (substrate) to separate the components in a mixture. The stationary phase may interact with the substances in the mixture based on charge, relative solubility or adsorption. The mobile phase carries each substance along at a rate that depends on how much the substance is attracted to the stationary phase. The components of the mixture will be separated if their interactions with the substrate and solvent are significantly different

a single solvent is used as the mobile phase to separate the dyes from the other ingredients in the mixture. This is known as an isocratic elution. In part 2 a gradient elution is used in which the composition of the mobile phase is changed during the separation process so make

1) Make a 5% isopropanol solution by mixing 3.5 mL of 70% isopropanol and 46.5 mL of distilled water in a 100 mL Beaker.

(2) Make a 28% isopropanol solution by mixing 20 mL of 70% isopropanol with 30 mL of distilled water in a 100 mL beaker

1)        Obtain a dropper bottle of distilled water for use as the first solvent for the gradient elution.

2)        Using a 10 mL syringe, dropper bottle or wash bottle as a solvent pump, flush the C18 Sep-Pack cartridge with undiluted 70% isopropanol at rate of 5-10 mL per minute. Collect the eluate into a 10 mL graduated cylinder so you can monitor the rate of flow of the isopropanol.

3)        Wash the Sep-Pack cartridge with 10 mL of distilled water.

4)        Use a 1 mL syringe to draw up 1 mL of the grape Kool-Aid sample. Slowly inject the 1 mL of Kool-Aid sample into the Sep-Pack cartridge. Collect and discard the effluent that washes out of the column as you inject the sample.

5)        Label four 50 mL beakers 1-4.

6)        Set up beaker 1 to collect the first eluate. Pass 5 mL of distilled water through the column to elute the polar components of the mixture.

7)        Set up beaker 2 to collect the second eluate from the column. Pass 5 to 10 mL of 5% isopropanol through the column to elute the red dye.

8)        Set up beaker 3 to collect the third eluate. Pass 5 to 10 mL of 28% isopropanol to elute the blue dye.

9)        Set up beaker 4 to collect the fourth eluate. Pass 8 mL of 70% isopropanol through the column to elute the flavor and other nonpolar ingredients.

10)    Place the 4 beakers of eluate in a hood or well-ventilated area away from open flame, and allow the solvents to evaporate. When the beakers are dry, record your observations of the contents.

11)    Dispose of the solutions as directed.