How many components were in the dye mixture that you used? Did the manufacturer of the pen use a single color ink or a mixture of different color inks?

Experiment 12.

Paper Chromatography

LEARNING OBJECTIVES

Students will be able to:

1. Learn the theoretical and practical concepts of paper chromatography.

2. Apply the concept of intermolecular forces to separation processes.

3. Use the process of paper chromatography to separate various mixtures of substances in a qualitative way.

BACKGROUND

A. USES

Paper chromatography is an analytical tool used in forensic science and organic chemistry laboratory. This technique was first introduced by Tyndall when he experimented with separating pigments from plants. Chemists use this technique for separating and identifying mixtures that are or can be colored, especially inks and food colorings. This method has been largely replaced by TLC, thin layer chromatography, where a glass plate is coated with thin layer of alumina, Al2O3. Meanwhile, paper chromatography is still a powerful teaching tool. Two-dimensional chromatography involves using two solvents and rotating the paper 90° in between solvent changes. This enhances the separation of the mixture. This technique can also be applied to colorless mixtures such as amino acids and analgesics. The spots can be developed under a UV lamp, where they are seen as violet spots. The spots can also be colored by combining them with other chemicals (derivatization) such as iodine vapor.

B. THEORY

A small sample of a mixture is placed on porous paper which is in contact with the solvent. The solvent moves through the paper due to capillary action and dissolves the mixture spot. The components of the sample start to move along the paper in the same direction as the solvent. If the solvent or solvent mixture is polar, the polar constituents of the mixture will move along with the solvent (mobile phase). The others will be retained longer and move at a slower rate.

Components of the mixture with a stronger attraction to the paper than to the solvent will move more slowly than the components with a strong attraction to the solvent. The differences in the rates with which the components travel along the paper leads to their separation. Particular mixtures will have chromatographic patterns that are consistent and reproducible as long as the paper, solvent, temperature, and time are constant. Different solvents will change the patterns of component separation.

Quantitative analysis of chromatographic data

Retention factors, Rf, are calculated to give a quantitative measure of a component’s properties in a mixture.

When chromatograms are made with the same solvent and developed for the same amount of time, Rf values are identical for the same components in the mixture.

Paper chromatography presents some advantages and disadvantages. Paper chromatography is cheap, requires very small amount of sample size, and quick to perform and easy to learn. The sample can also be recovered after the experiment. With a correctly chosen mobile phase (chromatographic solvent), an analyst can rapidly determine the number of constituents in the sample. It has its limitations. It is an analytical tool to identify the components of a mixture, not to separate compounds at large scales. A better preparative tool, such as ultra HPLC, is employed by pharmaceutical companies to purify the active compound present in a complex matrix.

MATERIALS

400-mL beakers, cut filter papers, pencil, toothpicks, ruler, Al-foil.

CHEMICALS

For Procedure A. A 5% salt solution and a 50:50 acetone:water mixture.

Water soluble food colors (red, yellow, green, and blue), pencil, toothpicks, ruler,

For Procedure B. 50:50 hexane:ethylacetate mixture, 0.1% solutions of acetyl salicylic acid (aspirin), ibuprofen, acetaminophen, caffeine, and Excedrin in isopropyl alcohol.

Optional Excedrin® tablets and isopropyl alcohol.

SAFETY

1. No eating and drinking in the laboratory. Obtain and wear eye goggles at all times.

2. Some of the solvents mixtures contain harmful solvents for your contact lenses. Perform the elutionprocess in the fume hoods.

3. After you finish the experiment, clean up and wash your hands.

PART A. PAPER CHROMATOGRAPHY OF FOOD DYES

PROCEDURE A-1.

In a fume hood, using a Pasteur pipet, place one drop of each of the mobile phases on the workbench and allow them to evaporate. Identify the faster and slower evaporating solvents and label them as “the more polar” and “the less polar”, respectively.

PROCEDURE A-2.

1. Obtain two 400-mL beakers. Add 15-20 mL of mobile phase A to one and 15-20 ml of mobile phase B to the other. Cover each beaker with aluminum foil. This will help saturate the headspaces with solvent vapors and run the chromatograms faster. The solvent choices are: A. 5% aqueous NaCl solution.

B. 50:50 (volume by volume) acetone: water mixture.

2. Cut two 10 cm long by 5 cm wide pieces of filter paper to use as stationary phases.

3. With a pencil, draw two lines: one 2 cm from the bottom (solvent front) and the other 2 cm from thetop (solvent end) of the filter paper.

Figure 1. Schematic for paper chromatography.

R: red food color Y: yellow food color G: green food color B: blue food color S: chosen sample

4. Using toothpicks or Pasteur pipets, make small spots of food dyes on the bottom line. Label them as R (red), Y (yellow), G (green), and B (blue). Also make a spot using your pen (sample) and label it as S. Let them dry.

Figure 2. Spotting the samples on paper.

5. Make a second spot of the appropriate food dye or ink on top of the original spots. This will double the concentration of test material without increasing the size of the spots.

6. Insert a filter paper into each beaker, secure it by hanging it from wooden splints, and wait until the solvent reaches the top line.

7. Remove the filter papers from the beaker, place them in the fume hoods and allow some time forthem to dry.

8. Using a centimeter ruler measure the distances (from the spotting line to the centers of the spots) traveled by the dyes.

Note: A group of students can use 5% NaCl as a mobile phase and another group can use the (50:50) acetone:water mixture as the mobile phase. Afterwards, the students can share their data.

PROCEDURE A-3. Interpreting the Data.

The Rf value for each spot should be calculated. Known Rf values can be compared to those of unknown substances to aid in their identifications.

Figure 3. Elution profile of food coloring using 5% NaCl solution as a mobile phase.

PART B. PAPER CHROMATOGRAPHY OF ANALGESICS

PROCEDURE B-1. OPTIONAL or Thin Layer Chromatography. https://www.youtube.com/watch?v=qdmKGskCyh8

THE EXPERIMENT SHOULD BE PERFORMED IN A FUME HOOD.

1. Into a 400-mL beaker, add about 15-20 mL of a 50:50 (volume / volume) hexane:ethyl acetate mixture and cover it with aluminum foil.

2. Cut a 10 cm long by 5 cm wide piece of filter paper to use a stationary phase.

3. With a pencil, draw two lines: one 2 cm from the bottom (solvent front) and the other 2 cm from thetop (solvent end) of the filter paper.

4. Using toothpicks, make small spots of analgesics on the bottom line. Label them, with a pencil, asA (acetyl salicylic acid, aspirin), I (ibuprofen), Ac (acetaminophen), C (caffeine) and E (Excedrin®). Let them dry.

5. Insert the filter paper in the beaker, secure it by hanging it from wooden splints, and wait until thesolvent reaches the top line.

6. Remove the filter paper from the beaker, place it in the fume hood, and allow some time for it to dry.

7. Under the UV lamp, observe the spots. Outline them in pencil. Using a cm ruler measure the distances traveled by the compounds.

8. Identify the ingredients present in Excedrin®.

9. Circle the ingredients below that are present in the Excedrin sample: RESULT:

Acetyl salicylic acid Ibuprofen Acetaminophen Caffeine

DATE:

Paper Chromatography Part A

NAME

Sample	Color	Distance the Sample Traveled	Distance the Mobile Phase Traveled	Rf

Paper Chromatography Part B

Sample	Color	Distance the Sample Traveled	Distance the Mobile Phase Traveled	Rf

POSTLAB QUESTIONS NAME:

1. What is the purpose of double-spotting the chromatography paper at the beginning of the experiment?

2. Define the mobile and stationary phases in chromatography.

3. Which of the solvents or solvent mixtures is the less polar? The more polar? What makes a substance polar or nonpolar?

4. There are two substances, Q and T, in a mixture. Substance Q is more polar than substance T. Which one will have a larger Rf when pure water is used as an elution phase? Why?

5. You identified the most and the least traveled dyes, and the most and least polar solvents. What causes the components to separate? Based on these observations and using the concept of “LIKE DISSOLVES LIKE” identify: