Materials Included In Kit

Additional Materials Required

Prelab Preparation

Spotter
An important piece of equipment used when doing TLC is the spotter. The spotter is used to apply (or “spot”) the sample onto the TLC plate. To prepare the spotter:

1. While holding both ends of the capillary tube with gloved hands, place the center of the capillary tube into a Bunsen burner flame. Hold the tube there until the middle begins to soften (Figure 5a).
   {12605_Pre-LabPreparation_Figure_5}
2. Quickly remove the tube from the flame and pull both ends outward, forming a very small tube opening in the middle of the capillary tube (Figure 5b).
3. Gently and carefully break the tube in the center to make two spotters (Figure 5c).
4. Prepare at least six spotters for each pair of students.

TLC Plates
To prepare TLC plates, cut the TLC sheet into plates 10 cmX2 cm. (Remember to use a pencil when marking the TLC sheets or plates.) When cutting, be careful not to scrape any of the silica gel from the plates; this will adversely affect results. (Some silica gel will chip from the edges of each plate—this should not present a problem.)

Chromatography Chamber

1. Add 6 mL of the acetone chromatography solvent (a 50/50 acetone/water solution) to each 250-mL beaker.
2. Cover the beaker with a watch glass, or better yet, Parafilm.^®

Safety Precautions

The chromatography solvent is flammable and a dangerous fire risk; toxic by ingestion and inhalation. This lab should be performed only in an operating chemical fume hood or well-ventilated area. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory.

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. Acetone chromatography solvent may be saved for future used or disposed of according to Flinn Suggested Disposal Method #18a. Dye solutions may be disposed of according to Flinn Suggested Disposal Method #26b and the TLC plates may be disposed of according to Flinn Suggested disposal Method #26a.

Lab Hints

Good technique is required for the students to see well-separated compounds on their strips. Some of the common causes for poor separations or results are:|wrong solvent mixture| too much starting material placed on the initial spot|initial spot too large| initial spot is below the solvent level in the chromatography chamber In this activity, it is extremely important that the initial sample spots are as small as possible. If the spots are too large or if there is too much material (dye) on the initial spot, the students may only see a streak of color up the entire chromatogram.|Allowing enough time for the development of the plate is critical. The plate must be left in the chromatography chamber long enough for the solvent to be drawn up near the top of the plate. Do not stop the development until the solvent front is near the top. Underdevelopment will lead to incomplete separation.|Solvent used for development may be recycled. Do not discard leftover chromatography solvent. Save it for use by another class or other chromatography development. Do not leave the chromatography solvent uncovered for long periods of time: the acetone may evaporate faster than the water, changing the polarity of the solvent.|Unknown #1 contains both safranin and eosin Y, which are both pink, fluorescent dyes. However, there are two ways for students to know that there are actually two pink dyes in the mixture: the difference in the Rf values, and the fact that the dyes fluoresce different colors—eosin Y fluoresces pink/orange, while safranin O fluoresces pink/red.|If two chromatography chambers are prepared per group, more chromatography solvent will be required. To prepare 100 mL of chromatography solvent, add 50 mL of acetone to 50 mL of distilled water and stir to mix thoroughly.

Further Extensions

Encourage students to try other dyes and/or solvent mixtures to achieve different or better separations.

Sample Data

Answers to Questions

1. Since the samples are dyes, they are relatively easy to see. Draw representations of each of the TLC plates, including the dye colors and locations, as well as the starting spot and final solvent front locations in the space below. To compare and identify compounds separated by TLC, calculate the Rf (rate of flow) values for each dye, using Equation 1.
   {12605_Answers_Equation_1}

   Record Rf values for each of the dyes in the data table above.

2. Which dyes appear to be in your unknown?

   The unknown dye mixtures contain the following dyes:
   Unknown #1 Eosin Y, Methylene Blue, Safranin O
   Unknown #2 Fast Green FCF, Fluorescein
   Unknown #3 Eosin Y, Fluorescein

   There may be some slight interactions between the dye molecules in each mixture, resulting in wider spots and slightly different Rf values of the various dyes from the individual dyes. However, the Rf values are different enough that these small variations will not significantly affect interpretation of the results.

3. Knowing that the solvent is quite polar, what can you infer about the relative polarities of the various dyes?

   The dyes that moved up the plates the fastest, fast green FCF and eosin Y, have a polarity similar to the solvent.

4. Use Figures 1 and 3 from the Background section to identify chromophores, auxochromes, and solubilizing groups for each of the dyes used in the activity. How might each be responsible for the position of the dye on the chromatogram?
   {12605_Answers_Figure_6 TLC Results}

   Since the solvent is highly polar, the molecules with the most polar effects from the auxochromes and solubilizing groups would travel farthest. This appears consistent with the greater number and types of these groups on the fast green FCF, eosin Y, and fluorescein dye molecules compared to the other two.

References

Epp, D. N. The Chemistry of Vat Dyes; Terrific Science: Middletown, OH, 1995; p 7.
Russo, T.; Meszaros, M. W. Vial Organic; Flinn Scientific: Batavia, IL, 1996; pp 25–33.
Shriner, R. L., et. al. The Systematic Identification of Organic Compounds; John Wiley & Sons: New York, 1964; pp 33–37.
Zubrick, J. W. The Organic Chem Lab Survival Manual, 3rd ed.; John Wiley & Sons: New York, 1992; pp 245–246.

Introduction

Chromatography is one of the most useful method of separating organic compounds for identification or purification. Measure the migration distance of five common dyes and calculate their R_f values to identify three unknown dyes.

Concepts

• Chromatography
• Separation techniques
• Polar vs. nonpolar solvent

Background

Dye molecules are generally large molecules which vary greatly in structure and composition. A typical dye molecule contains at least three functional chemical groups, each responsible for a particular property of the dye. These three groups include the chromophore, which is the color-producing portion of the dye, the auxochrome, which influences the intensity of the dye and provides a site for bonding (as to fabric), and the solubilizing group, which allows a dye to be water-soluble. Typical examples of each of these groups can be found in Figure 1.

It is the variation in number and arrangement of these chemical groups that determines the polarity of the dye molecule. In general, the chromophores tend to be non-polar, while the auxochromes and solubilizing groups tend to increase the polarity of the dye molecule, although, again, this is subject to the location and arrangement of the groups. The structures of the dye molecules used in this activity can be seen in Figure 2. Many different types of chromatography are used but most work on the concept of adsorbance. The two important components of chromatography are the adsorbent and the eluent. A good adsorbent is usually a solid material that will attract and bind the components in a mixture. Paper, silica gel, or alumina are all very good adsorbents. The eluent is the solvent that 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 solubilities in 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 change the solubility of the molecules but will greatly change the degree to which they are held by the adsorbent. This affinity for the eluent versus the adsorbent is what separates the molecules.

To separate complex organic molecules, thin-layer chromatography (TLC) is frequently used. In TLC, the adsorbent is usually silica gel (SiO₂) or alumina (Al₂O₃) coated on a glass plate or plastic sheet and the eluent is an organic solvent. The polarity of the eluent is very important in TLC since a small change in polarity can dramatically increase or decrease the solubility of some organic molecules. Many times, a mixture of a nonpolar solvent (petroleum ether) and a polar solvent (acetone) is used to achieve an optimum polarity. When placed in a chromatography chamber as shown in Figure 3, the eluent (chromatography solvent, which is petroleum ether and acetone) moves up the plate, being drawn by both capillary action and by the silica gel itself. The molecules, which were “spotted” onto the TLC plate, separate as they are carried with the eluent up the plate at different rates. Those molecules that have a polarity closest to the polarity of the eluent will be the most soluble, and will move up the plate the fastest. The choice of the eluent or solvent is the most difficult task. Choosing the right polarity is critical because this determines the level of separation that will be achieved. Common solvents used in TLC, in order of increasing polarity, are petroleum ether or hexanes, cyclohexane, toluene, chloroform, ethyl ether, acetone, ethanol and methanol. Sometimes mixtures of solvents are used to achieve the desired degree of polarity. A general rule of thumb is if the substances to be separated are polar, the developing solvent should be slightly less polar. Likewise, nonpolar substances would require slightly polar solvents.

Materials

Safety Precautions

The chromatography solvent is flammable and a dangerous fire risk; toxic by ingestion and inhalation. This lab should be performed only in an operating chemical fume hood or well-ventilated area. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

1. Obtain two TLC plates. Using a pencil, draw a horizontal line 1 cm from the bottom edge of each TLC plate.
2. Again using a pencil, label the top of the first plate with the numbers “1,” “2” and “3.” Label the top of the second plate with the numbers “4” and “5” and the letter “M” (for “mixture”) (see Figure 4).
   {12605_Procedure_Figure_4_TLC plates with labels}
   The numbers on the plates correspond to the dye samples according to the following key:

   1 = Methylene Blue
   2 = Safranin
   3 = Eosin Y
   4 = Fluorescein
   5 = Fast Green FCF

3. Place one drop of Sample 1 (methylene blue) on a watch glass or glass plate.
4. You are now ready to “spot” the TLC plates. The sample spot will be placed on the pencil line under its corresponding number. Touch the narrow end of a spotter to the drop of sample. The sample will be drawn up the tube due to capillary action. Gently and very briefly touch the tip of the spotter to the line on the TLC plate (under the corresponding number), keeping your index finger over the end of the tube, so that only a small amount of solution is transferred. It is extremely important to keep the spot as small as possible, as the dyes are very concentrated. It is also important not to disrupt the silica gel, so a gentle touch is required.
5. Repeat steps 3 and 4 for the other dye samples (safranin, eosin Y, fluorescein and fast green FCF) according to the key provided in step 2.
6. Repeat steps 3 and 4 for one of the unknown dye mixtures (the spot will be placed on the pencil line under “M”).
7. Remove the watch glass or Parafilm cover of the chromatography chamber. Carefully place the first TLC plate in the chromatography chamber with the sample end down (as shown in Figure 2). Important: (1) Do not get any solvent on the upper portion TLC plate, and (2) the sample spots must remain above the level of the solvent. If the solvent level is too high, the sample will dilute into the solvent! Carefully place the second TLC plate in the chamber, making sure the two plates do not touch. Replace the watch glass or Parafilm cover.
8. The solvent will be drawn up the TLC plates. As it is drawn up, it will carry the dyes in each sample up the plates at different rates depending on the characteristics of the individual compounds.
9. When the solvent front is within 1–2 cm of the top of the TLC plate, the run is stopped by removing the plate from the chamber.
10. Mark the location of each of the separated bands on the TLC plates and the final solvent front, again using a pencil. This is done because some of the color and brightness of each of the spots may be lost over time.
11. Measure the distance from the pencil line where the dyes were spotted to the solvent front near the top of the TLC plate. Record in the data table on the Thin-Layer Chromatography Worksheet.
12. Repeat step 11 for each of the dyes, measuring from the pencil line to the center of each colored band.
13. (Optional) If an ultraviolet light is available, shine it on each of the TLC plates in a darkened room. Note any differences in color. Can you make any additional inferences about your unknown sample, or are your conclusions further confirmed?
14. The TLC plates may be placed in the trash. The chromatography solution should be returned to the instructor.

Student Worksheet PDF

12605_Student1.pdf