Materials Included In Kit

Additional Materials Required

Prelab Preparation

Prepare the grape Kool-Aid as directed on the package, but omit the sugar. To prepare less than a whole package, use 0.5 g/250 mL distilled or deionized water.

Safety Precautions

Isopropyl alcohol is a flammable liquid and a fire hazard. Do not use near flames or other ignition sources. It is slightly toxic by ingestion and inhalation. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please consult current Safety Data Sheets for additional safety information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The 70% isopropyl alcohol may be diluted and then disposed of according to Flinn Suggested Disposal Method #26b. The 28%, 18% and 5% isopropyl alcohol solutions and all the eluant solutions may be disposed of according to Flinn Suggested Disposal Method #26b. The cartridges may be saved and reused many times.

Lab Hints

• The 6 syringes and cartridges can be shared among the 12 student groups. As calculations are made, other students can use the equipment to generate their individual data.
• The cartridges will not perform properly if the required pretreatment and between-injection washings are not done.
• The cartridges can be reused many times. Simply pretreat the cartridges, allow them to dry and store.
• Students may perform the pretreatment procedure several times in order to master the technique of eluting the solution at a fairly constant rate. Count the rate of delivery of drops into a 10-mL graduated cylinder. A rate of 5–10 mL per minute is suggested.
• If students observe the eluting solution against a white background, they will see the color due to the dyes more easily.
• Try separating the dyes of other beverages. Load the Sep-Pak C₁₈ column with 1 mL of the new beverage. Have the students try various concentrations of isopropyl alcohol solutions to separate the dyes. Start with a 5% isopropyl alcohol eluant and increase this by 5% increments of concentration until the separation is good.
• The dyes can also be separated by thin-layer chromatography (TLC). Have the students compare and contrast the methods and separation results.

Answers to Prelab Questions

1. What is the process of chromatography used for?

   Chromatography is used to separate the components of a mixture.

2. In chromatography, components of a mixture distribute themselves between the stationary phase and the mobile phase. Explain how the components can be separated with these two phases.

   In the chromatography columns used in this experiment, the column material is nonpolar. The nonpolar components of the mixture are attracted more strongly to it. The solvent is relatively polar, and attracts the more polar molecules. As the solvent washes down the column, the solutes spend time both adhering to the column packing and being dissolved in the liquid. The more polar components spend more time dissolved in the liquid, so they emerge more quickly from the column.

3. In the liquid chromatography column used in this experiment, the solid has a C₁₈ hydrocarbon bonded to it. Would a C₁₈ hydrocarbon be a polar or a nonpolar substance? Explain.

   The difference in electronegativity between carbon and hydrogen is very slight, so hydrocarbons are nonpolar substances.

Following are typical data for this experiment. 1 mL of a Kool-Aid solution was loaded on a Sep-Pak C

₁₈

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 recorded at the beginning and end of each color band.

This process is represented graphically below in Figure 4. The

x

-axis represents the milliliters of eluate that emerge from the column and the

y

-axis represents the concentration of each dye as it emerges with the eluant.

The first step in calculating the selectivity and resolution of the system is determining the volumes of eluant corresponding to the bandwidths and band centers for each eluted dye.
1. Bandwidth, W. This 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_ave, for each dye.

   W_{Red 1} = (1.8 – 1.0) mL = 0.8 mL W_{Red 2} = (1.8 – 1.1) mL = 0.7 mLW_{Red 3} = (1.8 – 1.0) mL = 0.8 mL W_ave = (0.8 + 0.7 + 0.8) mL / 3 = 0.8 mLW_{Blue 1} = (3.8 – 2.0) mL = 1.8 mL W_{Blue 2} = (4.2 – 2.2) mL = 2.0 mLW_{Blue 3} = (4.3 – 2.4) mL = 1.9 mL W_ave = (1.8 + 2.0 + 1.9) mL / 3 = 1.9 mL

2. Center of band is called Average Retention Volume, V_Rave. This volume 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 bandwidth.

   V_Rave = V_start + ½W_Rave

   Calculate the average retention volume, V_Rave, for the red and blue dyes. Red Dye: V_Rave = 1.0 mL + 0.5(0.8) mL = 1.4 mL Blue Dye: V_Rave = 2.2 mL + 0.5(1.9) mL = 3.2 mL

3. 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 for capacity factor is
   {12652_Answers_Equation_1}
   where V_Rave is the average retention volume for each dye and V_M is mobile phase or eluant volume in the cartridge. V_M can be estimated to be one-half the cartridge volume, with the stationary phase occupying the other half. For the Sep-Pak cartridges, this V_M value is 0.49 mL. Calculate k′ for each dye.
   {12652_Answers_Equation_2}
4. A selectivity or separation factor, α, can now be calculated. This is the ratio of the k′ values for each dye, with the larger value in the numerator. For good separation, a mobile phase is usually chosen that gives a value between 2 and 10. Calculate α for this separation.

   α = k′_Blue / k′_Red = 5.4 / 1.8 = 3.0

5. The resolution, R, a measure of how well the two dyes are separated by the column and eluant, is given by the equation
   {12652_Answers_Equation_4}
   where the numerator is the volume between the band centers and the denominator represents the average bandwidth. The greater the selectivity, the larger the numerator and therefore the greater resolution. The resolution can also increase as the efficiency of the column increases, since this results in a lower average bandwidth. Calculate R for this separation.
   {12652_Answers_Equation_5}

Sample Data

Part 1. Isocratic Separation

Calculated Values
α ___3.9___
R ___1.3___

Part 2. Step Gradient Separation Calculations
Determine the following values and show calculations. Refer to question six in the Prelab Assignment. Enter the results in the Part 1 data table.

1. Bandwidth, W, for each dye.

   W_{Red 1} = (1.8 – 1.0) mL = 0.8 mL
   W_{Red 2} = (1.8 – 1.1) mL = 0.7 mL
   W_{Red 3} = (1.8 – 1.0) mL = 0.8 mL
   W_ave = (0.8 + 0.7 + 0.8) mL / 3 = 0.8 mL
   
   W_{Blue 1} = (3.8 – 2.0) mL = 1.8 mL
   W_{Blue 2} = (4.2 – 2.2) mL = 2.0 mL
   W_{Blue 3} = (4.3 – 2.4) mL = 1.9 mL
   W_ave = (1.8 + 2.0 + 1.9) mL / 3 = 1.9 mL

2. Average Retention Volume, V_Rave, for each dye.

   Red Dye: V_Rave = 0.9 mL + 0.5(0.9) mL = 1.4 mL
   Blue Dye: V_Rave = 2.5 mL + 0.5(2.1) mL = 3.5 mL

3. Capacity Factor, k′, for each dye.
   {12652_Data_Equation_6}
4. Selectivity, α, for the two dyes with this isocratic separation.

   α = k′_Blue / k′_Red = 7.0 / 1.8 = 3.9

5. Resolution, R, for the two dyes with this isocratic separation.
   {12652_Data_Equation_8}

Answers to Questions

1. What is meant by polarity of molecules? What causes differences in polarity?

   A molecule that is polar has a separation of charge. This is caused by having atoms of differing electronegativity in the molecule. The more electronegative atoms pull the electrons away from the less electronegative atoms causing one part of the molecule to be slightly positive, and another part to be slightly negative.

2. In discussing solubility, the rule “like dissolves like” is frequently used. What does this mean?

   The rule that like dissolves like refers to polarity of molecules. Polar molecules dissolve well in polar solvents like water. Nonpolar molecules like hydrocarbons dissolve in nonpolar solvents.

3. Draw the structural formula of isopropyl alcohol. Explain how it differs in polarity from water.
   {12652_Answers_Figure_5}
   Isopropyl alcohol is much less polar than water because of the hydrocarbon part of the molecule. The highly electronegative oxygen will pull electrons toward itself and develop a slight negative charge.
4. For good separation of the dyes, the resolution should be greater than one. What was the value you calculated? Did the two dyes overlap as they emerged from the column, or was the separation a good one?

   Student answers will vary.

5. In the step gradient separation, four separate fractions were collected. How were these related to the polarities of the column and of the eluting solvent?

   The column is nonpolar and the polarity of the eluting solvent begins high with H₂O and is steadily decreased by using more isopropyl alcohol. As the mobile phase polarity decreases the eluted fractions are increasingly nonpolar.

References

Bidlingmeyer, B. A.; Warren Jr., F. V. “An Inexpensive Experiment for the Introduction of High Performance Liquid Chromatography” J. Chem. Educ. 1984, 61, 716–720.

Institute for Chemical Education, Fun With Chemistry; Vol. 1, Sarquis, Mickey and Sarquis, Gerry, Ed.; University of Wisconsin—Madison, 1991, 77–82.

Vonderbrink, Sally Ann.Laboratory Experiments for Advanced Placement Chemistry, Flinn Scientific, Batavia, IL, 1995, 149–156; 309–312.

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

• Liquid chromatography
• Resolution
• Selectivity

Background

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 (see Figure 1).

All liquid chromatography systems consist of six basic components (see Figure 2): (1) a separation column, consisting normally of a fine, granular solid packed in a column; (2) a solvent, the mobile phase that washes along the column; (3) an injection system, needed to place the sample mixture on the column; (4) a pump, or solvent delivery system, that forces the solvent through the column; (5) a detector, used to indicate when the components emerge from the column; and (6) a recorder. 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.

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 C₁₈ columns are used for the separation. The Sep-Pak column is packed with a silica solid that has a C₁₈ 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 solvent, or eluant. Measurements are made during the separation that allow for the calculations of the selectivity and the resolution of the separation process. In Part 2, four eluants of different polarities are used to separate the polar components citric acid and salt, the slightly polar dyes and the nonpolar flavoring oils.

Materials

Prelab Questions

See Student PDF.

Safety Precautions

Isopropyl alcohol is a flammable liquid and a fire hazard. Do not use near flames or other ignition sources. It is slightly toxic by ingestion and inhalation. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part 1. Isocratic Separation
(Flow rate and solvent concentration are held constant.)

Pretreat the Sep-Pak C₁₈ cartridge.

1. To help eliminate remixing of closely eluting bands in the cartridge, cut off the exit tube of the cartridge (the shorter end) at the point where it meets the body of the cartridge.
2. Fill the syringe with 10 mL of the 70% isopropyl alcohol.
3. Attach the tip of the syringe cartridge to the long end of the Sep-Pak cartridge, and pump the isopropyl alcohol through the syringe cartridge at a rate of 5–10 mL per minute.
4. Collect the eluted alcohol in a 10-mL graduated cylinder to monitor the flow rate.
5. Repeat steps 2–4 using distilled or deionized water.

Inject the sample.

1. Use the 10-mL syringe to slowly inject 1 mL of the Kool-Aid sample onto the column. (Optional) Use a clean 3 mL syringe to inject the Kool-Aid sample.
2. Discard the column effluent (the portion that washed out as the sample was injected).
3. Remove the cartridge from the syringe.
4. If the 10-mL syringe was used in step 1, rinse the syringe with 10 mL of distilled water three times to remove any traces of Kool-Aid sample.

Elute the sample.

1. Use the 10-mL syringe to slowly elute the dyes. Fill the syringe with the 18% isopropyl alcohol eluant and attach the syringe to the Sep-Pak cartridge.
2. Pump the 18% isopropyl alcohol through the cartridge at a steady rate of 5–10 mL per minute.
3. Collect the column effluent in a 10-mL graduated cylinder.
4. Record, in the Part 1 data table, the volume of effluent collected as the first and last of the colored drops of each of the dyes emerge. If there is not a perfect separation between the blue- and red-colored bands, record data for the beginning and end of the intermediate purple band. The center of the purple band will serve as the end of the first band and beginning of the last.

Regenerate the column and repeat the measurements.

1. Repeat the measurements two more times.
2. Between injections, wash the column with 10 mL of distilled water at the same flow rate of 5–10 mL per minute. If colored material builds up on the column, repeat the pretreatment procedure (step 2).

Part 2. Step Gradient Separation
In this procedure, the composition of the eluting liquid is changed. Since the column is nonpolar, first a very polar solvent, water, is used. Then its composition is changed to less polar by adding more isopropyl alcohol. This procedure allows the separation of the citric acid and flavoring oils as well as the dyes.

Pretreat the cartridge.
Follow the same pretreatment as in Part 1.

Inject the sample and elute the components.

1. Slowly inject 1 mL of the Kool-Aid sample onto the column.
2. Elute the polar components of the mixture (citric acid and any sugar present) by passing 5 mL of water through the column.
3. Collect the effluent in a small beaker.
4. Elute the red dye by passing 10 mL of 5% isopropyl alcohol through the column. Note that large amounts of the 5% isopropyl alcohol can be used without eluting the blue dye. Collect this effluent in a second beaker.
5. Use 10 mL of the 28% isopropyl alcohol to elute the blue dye. Collect it in a third beaker.
6. Use 10 mL of 70% isopropyl alcohol to elute the nonpolar flavor oils and other nonpolar additives. Collect this fraction in a fourth beaker.
7. Record the color of each eluted fraction in the Part 2 data table.

Evaporate the solvents and examine the components.

1. Allow the solutions to evaporate by leaving them in the fume hood until the next laboratory period. Be sure to label those solutions containing isopropyl alcohol as the solvent.
2. Observe and describe the contents of each of the beakers. Look for color, odor and appearance. Enter these observations in the Part 2 data table.

Student Worksheet PDF

12652_Student1.pdf