Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. To prepare 500 mL of 0.5% sodium chloride solution, fill a 500-mL volumetric flask one-third to one-half full with distilled or deionized water. Add 12.5 mL of 20% NaCl solution using a serological pipet and dilute with distilled or deionized water to the mark. Mix thoroughly.
2. To prepare 100 mL of FD&C Yellow dye solution, add 0.5 g of the dye to a separate beaker with 100 mL of distilled or deionized water. Mix thoroughly.
3. To extract the colors from the candy coated chocolates, place each in a spot well plate and squirt about 0.5 mL (keep the dye concentrated) of deionized water. Use the toothpick to apply it to the chromatography paper.

Safety Precautions

The FD&C dyes are slightly hazardous by ingestion, inhalation and eye or skin contact. Yellow No. 5 may be a skin sensitizer. All dyes are irritating to skin and eyes. Avoid contact with eyes, skin and clothing. Wear chemical splash goggles, chemicalresistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal 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. Excess dye solutions and sodium chloride solution may be stored for future use or rinsed down the drain with excess water according to Flinn Suggested Disposal Method #26b.

Lab Hints

• This laboratory activity was specifically written, per teacher request, to be completed in one 50-minute class period. It is important to allow time between the Prelab Homework Assignment and the Lab Activity. To save time, have students measure sample markers and solvent form with a pencil on the chromatography paper before lab day.
• Enough chromatography paper strips are included for 12 groups of students to develop 16 chromatograms each. Extra strips are provided in case of mistakes.
• M&M™ candies were provided in this lab for testing. Students may also bring coated candies of their own, such as Skittles™.
• As an inquiry opportunity, challenge students to investigate other concentrations of sodium chloride eluent. They may try 0.1% aqueous sodium chloride.
• Students should avoid over-handling the chromatography strips. Oil from the skin can interfere with the capillary action that draws water through the paper.
• Good technique is important to achieve clean separations in paper chromatography. Common sources of student error include “overloading” the paper by placing too much dye on the initial spot and the band broadening that occurs because the initial spot is too large.
• It is critical to allow enough time for the development of the chromatography paper. The chromatography paper must be left in the chromatography chamber long enough for the solvent to be drawn up near the top of the strip. Do not stop the development until the solvent front nears the top of the strip. Underdevelopment will lead to incomplete separation. Do not allow the solvent front to move off the paper, however, because that prevents measurements of R_f factors.
• The developing solvent and dyes will continue to move even after the paper strip is removed from the solvent. It is necessary for students to mark the solvent front and positions of the dye bands or spots immediately after the strip is removed from the flask.

Further Extensions

Alignment to the Curriculum Framework for AP^® Chemistry—Big Idea 2

Enduring Understandings and Essential Knowledge
Matter can be described by its physical properties. The physical properties of a substance generally depend on the spacing between the particles (atoms, molecules, ions) that make up the substance and the forces of attraction among them. (2A)
2A3: Solutions are homogenous mixtures in which the physical properties are dependent on the concentration of the solute and the strengths of all interactions among the particles of the solutes and solvent.

Forces of attraction between particles (including the noble gases and also different parts of some large molecules) are important in determining many macroscopic properties of a substance, including how the observable physical state changes with temperature. (2B)
2B2: Dipole forces result from the attraction among the positive ends and negative ends of polar molecules. Hydrogen bonding is a strong type of dipole–dipole force.
2B3: Intermolecular forces play a key role in determining the properties of substances, including biological structures and interactions.

Learning Objectives
2.7 The student is able to explain how solutes can be separated by chromatography based on intermolecular attractions.
2.10 The student can design and/or interpret the results of a separation experiment (filtration, paper chromatography, column chromatography, or distillation) in terms of relative strength of interactions among and between the components.
2.13 The student is able to describe the relationships between the structural features of polar molecules and the forces of attraction between particles.

Science Practices
1.4 The student can use representations and models to analyze situations or solve problems qualitatively and quantitatively.
4.2 The student can design a plan for collecting data to answer a particular scientific question.
4.3 The student can collect data to answer a particular scientific question.
5.1 The student can analyze data to identify patterns or relationships.
5.2 The student can refine observations and measurements based on data analysis.
5.3 The student can evaluate the evidence provided by data sets in relation to a particular scientific question.
6.2 The student can construct explanations of phenomena based on evidence produced through scientific practices.
6.4 The student can make claims and predictions about natural phenomena based on scientific theories and models.

Answers to Prelab Questions

1. Observe a student’s chromatography experimental setup for FD&C dye mixture separation in Figure 2. Answer Questions a–d. For free demonstration videos on food dye chromatography, visit flinnsci.com.

   Chromatography strip was folded at the top, hanging over the flask lip. Starting at the top of the strip, the student measured 20 mm and folded the paper across the width of the strip.

   {12348_PreLabAnswers_Figure_2}
   1. Label the Figure 2 experimental set-up from the available word bank: Erlenmeyer flask (250-mL), eluent, chromatography paper (152 mm tall and 19 mm wide), watch glass, dye sample. Define the terms in this word bank.

      Erlenmeyer flask—common glassware used in a laboratory with a larger, flat bottom and a narrow, open mouth at the top.
      Eluent—known as the mobile phase (solvent). The eluent carries the analyte up the chromatography paper and separates it depending on the intermolecular forces between it and the sample.
      Chromatography paper—this is the adsorbent; a solid material that will attract and adsorb the materials to be separated.
      Watch glass—common glassware used in the laboratory for various purposes.
      Dye sample—the analyte in this experiment.

   2. Prior to applying the dye sample, the student made sure to place the sample marker above the eluent, approximately 15 mm from the bottom, marked with a pencil. Why is the eluent level below the sample marker?

      If the eluent level is too high, the samples will dilute into the solvent and render the adsorbent and sample marker unusable.

   3. Using a toothpick, the student spotted the chromatography strip with a given FD&C dye mixture on the sample marker three times, placed the chromatography strip into the Erlenmeyer flask containing an eluent, and covered the flask with a watch glass. Why was the flask covered with a watch glass?

      The flask was covered with a watch glass to prevent eluent evaporation since some eluents have boiling points near room temperature. In some chromatography experiments, a mixture of two solvents are used. As an example, ethyl ether and acetone is a typical chromatography solvent mixture. Leaving the chromatography experimental set-up open to the air may result in evaporation of ether in this mixture since it has a lower boiling point, therefore changing the eluent concentration.

   4. After development of the chromatogram, about 20–30 minutes, the student removed the strip from the setup and with a pencil lightly drew a line to mark the distance the solvent traveled—called the solvent front. Why is this step important?

      Marking the line where the solvent traveled is important because it is measured in cm and used to calculate the R_f value of the chromatogram according to the following equation:
      R_f = distance traveled by sample/distance traveled by solvent

2. An eluent solution of aqueous 2% sodium chloride was used in the experimental setup Question 1. Answer Questions a–c.
   1. Chromatography paper is very hydrophilic. It is made from a long chain of glucose molecules, a polymer called cellulose. In the molecular followng diagram, draw the intermolecular forces between three water molecules and the unit of cellulose.
      {12348_PreLabAnswers_Figure_4}
   2. Identify the intermolecular force between sodium chloride and water molecules in the 2% eluent and draw a molecular diagram of this representation. Repeat this exercise with an eluent of 2% isopropyl alcohol.
      {12348_PreLabAnswers_Figure_5}
   3. Observe the FD&C Red 40 molecule in the Background section of this lab. Predict the intermolecular attractions with the two solvents from Question 2a.

      In the sodium chloride solution, FD&C Red 40 dye molecule would experience ion–dipole interactions due to the charged sulfonate groups, although the overall strength of these interactions will vary due to the number of groups. Hydrogen bonding will occur between water and the functional groups. Red No. 40 would experience weaker ion–dipole and hydrogen bonding interactions with the sodium chloride solution because the molecule has fewer charged side groups. Red No. 40 only has two charged sulfonate groups.
      
      In the isopropyl alcohol solution, FD&C Red 40 dye molecule would experience ion–dipole interactions due to the charged sulfonate groups and polar alcohol group in isopropyl alcohol. In addition to its charged side chains, it is a large organic molecule with significant nonpolar rings and groups. These nonpolar regions interact with relatively nonpolar isopropyl alcohol molecules and thus have a greater affinity for this solvent than for either the NaCl solution or the hydrophilic paper.

3. See the student’s developed chromatogram in Figure 3. She traced the shape of the dye band, which is the streaked mark the dye leaves at it travels. Answer Questions a–d.
   1. Label the stationary phase, mobile phase, solvent front in Figure 3.
   2. Calculate the R_f value. R_f = distance traveled by sample/distance traveled by solvent.

      R_f = 4.8/5.6 = 0.86

   3. In a separate experiment, the student measured the dye band traveling distance as 2.8 cm. In contrast to the results in Figure 3, what can you conclude about the intermolecular attractions to the eluent and paper in this second experiment?

      First, calculate the R_f value: R_f = 2.8/5.6 = 0.50
      
      Based on the smaller R_f value, the dye analyzed in this experiment has a stronger attraction for the paper than the eluent, thus it traveled a shorter distance.

   4. If the student were to witness two dye spots/bands on the resulting chromatogram, what does it tell us about the composition of the sample.

      If she witnessed two dye bands on the chromatogram, this means that this dye sample is a mixture of at least 2 dyes.

      {12348_PreLabAnswers_Figure_3}
4. Predict what would happen in the following scenarios:
   1. The student used a black marker instead of pencil as a sample marker on the chromatography paper.

      If a black marker was used instead of pencil as a sample marker, the eluent would start separating the black marker into its many dyes. Black markers are composed of many different color inks (dyes). The student would not be able to analyze her sample on this chromatogram due to the contamination.

   2. The student used filter paper instead of the given chromatography paper for dye separation.

      If filter paper was used instead of chromatography paper, the experiment would still work due to the hydrophilic nature of its composition.

5. The student analyzed a final FD&C dye mixture sample and recorded her results in the following data table. Select the best eluent (i.e., that provided the best dye mixture separation based on R_f values).
   {12348_PreLabAnswers_Table_1}

   The 0.5% sodium chloride solution was the more optimal solvent, compared to the 2% isopropyl alcohol and the 8% sodium chloride solutions, because the dyes traveled farther on the paper and there were greater distances separating the dye bands. These observations are evident in the reported R_f values of the 0.5% sodium chloride solution; the best separationout of the three solvents tested. The reported R_f values for 2% isopropyl alcohol prove that dye separation was poor, especially for the Blue 1 and Yellow 5, where the reported R_f values were 0.94 and 0.92, respectively.

Sample Data

Using the experiment setup provided in the homework set, students should successfully set up the chromatography experiment. An eluent of 0.5% aqueous sodium chloride and developing time of 25 to 30 minutes provide chromatograms of each color, plus the control. Tartrazine is FD&C food dye Yellow 5. This information is available in the SDS on the Flinn Scientific website (flinnsci.com). Tartrazine is listed as one of the synonyms for FD&C Yellow 5 in section 3. The data table reports R_f values of all M&M™ candy coated chocolate colors and the Rf value for pure FD&C Yellow 5. The cooperative class activity leads to a few conclusions and remaining questions.

The yellow, green and brown candies contain FD&C Yellow 5 dye (tartrazine), while orange and red do not. People with allergies or sensitivities to tartrazine should avoid consuming yellow, green and brown candies. When the yellow, green and brown yellow band R_f values are compared to pure Yellow 5 control, each had lower R_f values, likely owing to a “dragging effect” attributed to the sugars and other binding additives in the candies. This “dragging effect” is observed to have a similar empirical effect on the R_f values measured for yellow, green and brown candies. That is, the R_f value for each of these candies are close in value. Alternatively, when Yellow 5 in yellow, green and brown candies were compared, they were fairly close. In contrast, the blue candy showed a high affinity for the chromatography paper. A small blue spot remained at the bottom of the paper, close to the solvent line and the rest of the sample proceeded with the solvent along the paper.

Student Procedure

1. The materials needed for this lab are listed:

   Tartrazine sample, 1 mL
   Candy coated chocolate samples, 2
   Chromatography paper, 3
   Erlenmeyer flasks, 3
   Spot plate, 12-well
   Watch glasses, 3

2. Position the chromatography paper strip so it is 152 mm tall and 19 mm wide. Note: Handle the paper by the edges so the analysis area is not accidentally compacted or contaminated.
3. Using a ruler and a pencil, draw a faint line 15 mm from the bottom of the paper across the width of the strip. Measure 9.5 mm from the edge and place a dot on the line. This is the starting point for the sample.
4. Using the same ruler, measure 20 mm from the top of the strip and fold across the width of the strip. This will allow the strip to hang on the lip of the flask.
5. Repeat steps 2 and 3 for a two more paper strips.
6. Obtain two candy coated chocolates and tartrazine sample.
7. Add 1–2 drops of DI water to two wells on the well plate. Add each color candy to the separate wells. Allow enough soaking for the color to dissolve in the water droplets. Using a clean toothpick, spot the chromatography strip by placing a toothpick into the candy wells and then touching the tip of the toothpick gently onto the designated pencil dot. Allow the sample to dry. Perform the same technique with the tartrazine solution. Repeat the procedure two to three more times. Note: This step is necessary to increase the concentration of the sample but do not allow the size of the spot to increase.
8. While the samples are drying, obtain three 250-mL Erlenmeyer flasks and watch glasses to cover the tops of the flasks.
9. Pour 20 mL of the assigned 0.5% chromatography solvent into each flask. Cover the flasks with the watch glasses.
10. Once the chromatography papers are dry, remove the watch glasses from the tops of the flasks. Carefully hang the chromatography strips into the flasks with the sample end down.
11. Carefully place the watch glasses back on the top of the flasks. Allow the chromatograms to develop. Record observations of the samples as the solvent travels up the papers and the chromatograms develop.
12. When the chromatography solvent is within 1–2 cm of the fold in the chromatography strips, stop the runs by removing the strips from the flasks.
13. With a pencil, lightly draw a line to mark the distance the solvent traveled. This is called the solvent front.
14. Measure the distance from the pencil line at the bottom of the strip to the solvent front.
15. With a pencil, trace the shape of each dye band or spot to mark its location on the chromatography strip. This should be done immediately because the color and brightness of some spots may fade over time.
16. Measure and record the distance in millimeters that each dye band or spot traveled. Measure from the line at the bottom of the paper to the center of each band or spot.

References

Kandel, M. Chromatography of M&M Candies. 1992, 69 (12), 988–989.

AP^® Chemistry Guided-Inquiry Experiments: Applying the Science Practices; The College Board: New York, NY, 2013.

Introduction

Delve into essential chromatography concepts covered on the AP^® Chemistry exam with this investigation! A small percentage of the population experiences allergies or sensitivities to tartrazine—one of the seven approved FD&C food dyes. Because many sweets contain FD&C food dyes, this demographic avoids sweets or carefully monitors available food ingredient labels. In order to obtain “pretty” colors, manufacturers often create various mixtures from the approved dyes. As a result, this poses a dilemma for those with tartrazine allergies. Start making connections between the commercial uses of food dyes and their structures and properties with this lab. Select various colored M&Ms™ and use chromatography to determine which contain the FD&C dye, tartrazine. Is tartrazine the sole ingredient in your selected candy samples? Or is tartrazine in a mixture with other FD&C dyes?

Concepts

• Chromatography
• Intermolecular forces
• R_f values
• Commercial chemistry
• Polar vs. non-polar

Background

Food dye chromatography is an incredibly useful method to explore separation of a dye mixture in this lab class. The concepts covered will strengthen understanding of intermolecular forces and solubility and will introduce the world-wide application of chromatography while bridging chemistry and consumer goods. Candy manufacturers add approved food dyes, known as FD&C dyes, to candy coatings, which are rendered safe for human consumption. Did you know that only seven food dyes are approved by the USDA as safe for human consumption in the U.S.? Just seven. Where, astonishingly, hundreds of color additives were utilized to make food more visually appealing just a century ago. Later, however, food safety regulators prohibited the use of many of these colorants due to their harmful toxicity levels to humans.

All FD&C-approved food dyes are charged, water-soluble organic compounds that bind to natural ionic and polar sites in large food molecules, including proteins and carbohydrates. Notice the organic structure of FD&C Red 40 in Figure 1.

There are extensive series of alternating single and double bonds (also known as conjugation) with a large molar mass when compared to smaller, organic molecules like benzene (78.11 g/mole). The resultant beautiful, intense colors of the FD&C dyes are due to the molecule’s conjugation. Notice the bonding sites on the FD&C Red 40 molecule? These bonding sites play a key role in paper chromatography.

Chromatography is one of the most useful methods of separating and purifying organic compounds. There are many different types of chromatography but most depend on the principle of adsorption. The two important components of chromatography are the adsorbent and the eluent. Absorbents are usually solid materials that will attract and adsorb the materials to be separated. The eluent is the solvent, which carries the materials to be separated through the adsorbent. Chromatography works on the concept that the compounds to be separated are slightly soluble in the eluent and will spend some of the time in the eluent (or solvent) and some of the time on the adsorbent. When the components of a mixture have varying affinities for the eluent, they can then be separated from one another. The polarity of the molecules to be separated and the polarity of the eluent are very important. Changing the polarity of the eluent will only slightly affect the solubility of the molecules but may greatly change the relative attraction for the adsorbent. Affinity of a substance for the eluent versus the adsorbent allows molecules to be separated by chromatography.

The choice of the eluent is the most difficult task in chromatography. Choosing the right polarity is critical because this determines the level of separation that will be achieved. Different samples will spend varying amounts of time interacting with the paper and the solvent. Through these different interactions, the samples will move different distances along the chromatography paper. The distance a sample moves along the chromatography paper is compared to the overall distance the solvent travels—this ratio is called the R_f value or retardation factor.

Experiment Overview

Complete the following homework set before lab day. The dry portion of this experiment—the homework set—solidifies the chromatography separation technique and knowledge needed for lab day (wet portion). On lab day, determine if those with allergies or sensitivities to the FD&C dye tartrazine are able to consume various colors of candy coated chocolates by determining its presence in the candies. Each student group will analyze two candy samples and compare their results to a control—tartrazine sample. Results will be shared with the class to arrive at a conclusion—which candies are safe to consume? Turn in any graphs or figures you were asked to create and use a separate sheet of paper, if necessary.

Prelab Questions

1. Observe a student’s chromatography experimental set-up for FD&C dye mixture separation in Figure 2. Answer Questions a–d. For free demonstration videos on food dye chromatography, visit flinnsci.com.

   Chromatography strip was folded at the top, hanging over the flask lip. Starting at the top of the strip, the student measured 20 mm and folded the paper across the width of the strip.

   {12348_PreLab_Figure_2}
   1. Label the Figure 2 experimental set-up from the available word bank: Erlenmeyer flask (250-mL), eluent, chromatography paper (152 mm tall and 19 mm wide), watch glass, dye sample. Define the terms in this word bank.
   2. Prior to applying the dye sample, the student made sure to place the sample marker above the eluent, approximately 15 mm from the bottom, marked with a pencil. Why is the eluent level below the sample marker?
   3. Using a toothpick, the student spotted the chromatography strip with a given FD&C dye mixture on the sample marker three times, placed the chromatography strip into the Erlenmeyer flask containing an eluent, and covered the flask with a watch glass. Why was the flask covered with a watch glass?
   4. After development of the chromatogram, about 20–30 minutes, the student removed the strip from the set-up and with a pencil lightly drew a line to mark the distance the solvent traveled—called the solvent front. Why is this step important?
2. An eluent solution of aqueous 2% sodium chloride was used in the experimental setup Question 1. Answer Questions a–c.
   1. Chromatography paper is very hydrophilic. It is made from a long chain of glucose molecules, a polymer called cellulose. In the following molecular diagram, draw the intermolecular forces between four water molecules and the unit of cellulose.
      {12348_PreLab_Figure_5}
   2. Identify the intermolecular force between sodium chloride and water molecules in the 2% eluent and draw a particulate diagram of this representation. Repeat this exercise with an eluent of 2% isopropyl alcohol.
   3. Observe the FD&C Red 40 molecule in the Background section of this lab. Predict the intermolecular attractions with the two solvents from Question 2a.
3. See the student’s developed chromatogram in Figure 3. She traced the shape of the dye band, which is the streaked mark the dye leaves at it travels. Answer Questions a–d.
   1. Label the stationary phase, mobile phase, solvent front in Figure 3.
   2. Calculate the R_f value. R_f = distance traveled by sample/distance traveled by solvent.
   3. In a separate experiment, the student measured the dye band traveling distance as 2.8 cm. In contrast to the results in Figure 3, what can you conclude about the intermolecular attractions to the eluent and paper in this second experiment?
   4. If the student were to witness two dye spots/bands on the resulting chromatogram, what does it tell us about the composition of the sample.
      {12348_PreLab_Figure_3}
4. Predict what would happen in the following scenarios:
   1. The student used a black marker instead of pencil as a sample marker on the chromatography paper.
   2. The student used filter paper instead of the given chromatography paper for dye separation.
5. The student analyzed a final FD&C dye mixture sample and recorded her results in the following data table. Select the best eluent (i.e., that provided the best dye mixture separation based on R_f values).
   {12348_PreLab_Table_1}

   Total time for chromatograms to develop ranged from 25–30 minutes. Based on the data collected for the 8% and 0.50% sodium chloride solutions, the more dilute solvent separated the mixture of dyes better. The three dyes were more distinguishable from one another in the 0.50% solution than in the 8% solution. This was evident with the overlapping of the Yellow 5 and Red 40 dyes with the 8% solvent.
   
   Based on the data collected for the 2% isopropyl alcohol solution, two separate dye spots were visible: blue and yellow on top, and red on the bottom. The Blue 1 and Yellow 5 dyes overlapped resulting in a blue front followed by a green line then a yellow tail end. The blue-yellow color band traveled straight for a portion of the time, and then the edges began trailing while the middle continued at a “faster” rate.

6. Select two candy coated chocolate samples and use paper chromatography to determine which ones contain tartrazine. As a control, obtain a sample of tartrazine from your instructor and analyze it by paper chromatography. Helpful tips:
   1. Think safety first. Make sure you have the proper PPE available to perform this lab (i.e., goggles, apron and gloves).
   2. Make a list of the equipment and glassware needed for this lab.
   3. Based on your answer to Question 5, select the eluent and eluent concentration to be used in candy chromatography.
   4. Tartrazine is an FD&C dye, and is also a synonym. Which FD&C dye is tartrazine? Flinn Scientific has a full library of SDS documents on flinnsci.com.
   5. What information do you need from the control and how will this data be compared to the results?
   6. To extract the colors from the candy coated chocolates, place each in a spot well plate and squirt about 0.5 mL (keep the dye concentrated) of deionized water. Use the toothpick to apply it to the chromatography paper.
   7. The developed chromatogram will look very similar to Figure 4. This is an example of how to measure a chromatogram that does not provide even, circular sample spots.
      {12348_PreLab_Figure_4}
7. Consult your instructor for appropriate disposal procedures.

Safety Precautions

The FD&C dyes are slightly hazardous by ingestion, inhalation and eye or skin contact. Yellow No. 5 may be a skin sensitizer. All dyes are irritating to skin and eyes. Avoid contact with eyes, skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.