THIN LAYER CHROMATOGRAPHY- TLC

THIN LAYER CHROMATOGRAPHY- TLC GREEN LAB Students work in teams of four CCO SATISFIED Chromatography IMPORTANT TECHNIQUES Analysis- quantitative; TLC HAZARDS None EXPECTED RESULTS WASTE The student will use TLC to determine the retention factor of common food dyes, and rank their relative polarities. TLC will be used to separate and identify the color components of common powdered drink mixes. Glass disposal or trash can. MATERIALS TLC plates: four per student pair Powdered drink mixes Food dyes Elution solvent (2-propanol: water; 90:10) Medium jars Pencils Rulers Paper towels Scissors Forceps or tweezers

INTRODUCTION Thin-layer chromatography (TLC) is used separate mixtures of 2 or more compounds by distribution between two phases, one of which is moving (the solvent) and the other that is stationary (the solid media). Prepared plates are made of a porous adsorbent solid media (silica gel SiO 2 xh2o) which is adhered to a thin piece of glass or plastic. The chromatography process works by differences in polarity of the compounds present. Polar compounds include carboxylic acids, amines, alcohols, esters aldehydes and ketones. Less polar compounds include aromatic compounds, halocarbons, ethers, olefins and hydrocarbons. Organic molecules will bind to fine particles of the silica gel by intermolecular forces. Non polar compounds such as aromatic compounds bind to the silica gel via weak van der Waals forces. Polar compounds will bind to the silica gel more strongly via dipole-dipole interactions, hydrogen bonding or salt formation. TLC can be used to determine if a substance is pure (one spot appears on the plate after developing) or a mixture (two or more spots on plate). When separating a mixture, one chooses a solvent that will cause the compounds, which hopefully all have different polarities to move across the TLC plate at different rates.

When examining TLC plates in this laboratory exercise, the following general rules will apply: The role of the solvent is to move the compound through the solid media. A non-polar solvent will dissolve non-polar compounds. The non-polar compound/s will move most rapidly through the solid media with the solvent. A polar compound is more tightly bound by the silica gel via stronger intermolecular forces. A polar compound is therefore found closer to the baseline of the TLC plate. A good solvent allows mixtures to be resolved to distinct spots on the TLC plate, by separating them based on their relative polarities. Solvent front Less polar compounds More polar compounds Base line A silica gel TLC plate PURPOSE TLC can be used to determine if a substance is pure (one spot on plate) or a mixture (two or more spots on plate). 1. Working together, as a group of four, determine the Retention Factors (Rf) for four food dyes: Brilliant Blue, Erythrosine, Allura Red AC and Tartrazine. Each student in the group will choose one of these dyes and run at least one TLC plate for that dye. The group will share this data with each other and with the class. 2. The class will examine all the data and discuss the results. (Students may rerun plates if needed). The class data will be used to find average retention factors for each dye. 3. Each group will choose one unknown mixture to separate using TLC. Each student in the group will run one TLC plate of this unknown and determine the retention factors for the component dyes in this unknown.

4. Each student will compare the retention factors of the pure dyes, with the retention factors of the dyes in the unknown mixture and identify the dyes used in the unknown. 5. Students will obtain data from other groups to complete the study of all mixtures and identify the dyes present. The four food dyes, Brilliant Blue, Erythrosine, Allura Red AC and Tartrazine are synthetic organic compounds used to impart uniform color to food, beverages, cosmetics and pharmaceuticals. Their production and use are regulated by the United States Food and Drug Administration under the Federal Food, Drug, and Cosmetic Act. For this reason they are better known and listed on packaging by their FD&C number designations (Blue #1, Red #3, Red #40 and Yellow #5). Blue #1, Red #3, Red #40 and Yellow #5 are primary colors. Commercial items often use a combination of these dyes to make secondary colors such as green and purple. The unknown mixtures for this lab will have at least two of these dyes present. HYPOTHESIS Examine the structures of the food dyes. Count the number of polar functional groups and nonpolar functional groups and aromatic rings in each dye. Subtract the number nonpolar groups and aromatic rings from the number of polar groups count = polar groups (nonpolar groups + aromatic rings) Use this count to rank the dyes from least to most polar. Assume that the lowest count is the least polar dye. (Don t worry if this answer is right or wrong, you are just using this information to make a best estimate of the polarities of these dyes for now; your instructor may provide more insight into this during lab if there is time). State your hypothesis in your notebook as follows: dye is predicted to be the least polar and will be closest to the top of the developed TLC plate. dye is predicted to be the most polar and will be closest to the base line of the developed TLC plate.

PROCEDURE: While wearing gloves, use a pencil to draw a baseline approximately 1 cm from the end of two silica TLC plates. On plate 1, use a glass spotter to apply the pure dye that you have chosen to study. One or two light touches of the tip of the spotter to the TLC plate are sufficient to apply the dye. Allow the spot to dry. Repeat this using a clean glass spotter to apply the mixture your group will study to plate 2. Spot of one dye on the baseline Spot of mixture on the baseline 1 cm 1 cm Plate 1: pure dye Plate 2: unknown mixture

Trisodium (4E)-5-oxo-1-(4-sulfonatophenyl)-4-[(4- sulfonatophenyl)hydrazono]-3-pyrazolecarboxylate

DEVELOPING A PLATE 1 Each student will obtain a small jar to use as the developing chamber. A piece of paper towel or filter paper is inside of each chamber. The paper will absorb the eluting solvent and help keep the chamber saturated with solvent vapor. The eluting solvent is poured into the developing chamber to a depth slightly less than 1 centimeter (black line). Check your chamber, it may already have enough solvent. Use forceps or tweezers to place plate 1 in the chamber and tightly cover the top of the chamber with the jar cap Allow the solvent rise to near the top of the plate (about 1.5 cm from the top). Transfer the plate to a paper towel to dry, with tongs and mark the solvent front with a pencil. Repeat this process for the second plate. Allow the plates to dry. Circle the spots with a pencil Describe the appearance of each of the plates, noting if distinct spots, color bands or just a smear was obtain as the dye components traveled with the solvent up the TLC plate. If a spot or spots are seen, indicate the color of each and the order in which it appears from the bottom of the plate in your notebook. It is best to draw a sketch on the TLC plate in your notebook and label the base line, spot/s and solvent front. 1 Williamson, K., Minard, R., Masters, K. (2007). Macroscale and Microscale Organic Experiments 5 th edition. Boston: Houghton Mifflin.

DETERMINING THE R F VALUES For each spot Measure the distance from the baseline, (drawn on the plate) to the solvent line. This is the distance the solvent moved. Measure the distance from the baseline (drawn on the plate) to the where spot stopped. This is the distance the spot moved. Distance solvent line moved Distance spot or band moved (measured to center of the spot or band) Baseline (Spot started ) Divide the distance the solvent moved into the distance the spot moved. This ratio is the R f value. R f value = Distance spot moved Distance solvent line moved Calculate the R f values for each spot and record the data in the table. Tape the TLC plates in your notebook/s. You may photograph them for your report.

DATA R f values for food dyes Color GROUP 1 GROUP 2 GROUP 3 GROUP 4 GROUP 5 GROUP 6 AVERAGE R f BLUE #1 Brilliant Blue RED #3 Erythrosine RED #40 Allura Red YELLOW #5 Tartrazine R f values for dyes in Grape Kool Aid AVERAGE R f Dye is most likely R f values for dyes in Lemon-Lime Kool Aid AVERAGE R f Dye is most likely R f values for dyes in : AVERAGE R f Dye is most likely

REPORT Rank each dyes from lowest to highest polarity and give the corresponding R f value to support your ranking. Identify the components of each mixture. CRITICAL THINKING QUESTIONS: (please answer in complete sentences) 1. Which food dye is the most polar? What functional groups are present in this dye? 2. Which food dye is the least polar? What functional groups are present in this dye? 3. Can you make any generalizations about the polarities of certain colors? (for examples, are red dyes generally more polar than blue dyes?) 4. To what class of compounds does the solvent 2-propanol belong? What functional group is present in the solvent? 5. How did the results of the experiment compare to your hypothesis? Instructors may add supplemental questions. CLEAN UP Leave the eluting solvent in the jar and return it to the lab bench. It can usually be reused by the next lab section. Return the dyes to the boxes and return these to the bench as well. Used spotters may be placed in the glass disposal box. END