Materials Included In Kit

Additional Materials Required

Prelab Preparation

Chromatography Solvent

1. To a 400-mL beaker, add 120 mL of toulene, 60 mL of glacial acetic acid and 60 mL of ethyl acetate.
2. Stir, then transfer to a glass or polypropylene storage bottle.

TLC Plates

1. To prepare TLC plates, cut the TLC sheet into plates 10 cm x 4 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 10 mL of the chromatography solvent to a 250-mL beaker.
2. Cover the beaker with a watch glass, or better yet, Parafilm.^®

Carboxylic Acid Solution

1. To a clean 100-mL beaker, add 3 g of citric acid.
2. Use a graduated cylinder to add 50 mL of distilled or deionized water to the 100-mL beaker.
3. Stir to thoroughly dissolve the citric acid.
4. Repeat steps 1–3 for succinic acid and for tartaric acid.

Safety Precautions

Toluene is a flammable organic solvent and dangerous fire risk. Keep away from flames and other sources of ignition. Toluene is moderately toxic by ingestion, inhalation and skin absorption. Acetic acid is corrosive to skin and tissue, is a moderate fire risk and is moderately toxic by ingestion and inhalation. Ethyl acetate is a volatile and highly flammable liquid, a skin and eye irritant and is mildly toxic by inhalation and skin absorption. 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. 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. The TLC sheets may be placed in the trash according to Flinn Suggested Disposal Method #26a. Save the chromatography solvent for future use.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. Fruit juice samples are not included in the kit. Both parts of this laboratory activity can reasonably be completed in one 50-minute class period. The prelaboratory assignment may be completed before coming to lab, and the data compilation an calculations may be completed the day after the lab.
• Various juice drinks can be selected based on their acid content. Apple juice is high in malic and citric acids, orange juice is high in citric and malic acids, grape juice is high in tartaric and citric acids, and cranberry juice is high in succinic and citric acids.
• 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 strip is critical. The strip 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.
• Solvent used for development can 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
• Demonstrate the spotting technique for the students. When spotting the TLC sheet, keep the spot as small as possible and make sure the spot will be above the solvent line in the developing jar. Briefly and gently touch the top of the micropipet tube to the TLC surface. Let the solvent evaporate before touching the capillary tube to the TLC plate again. Touch the capillary to the same spot again. Remove the capillary and gently blow on the spot to evaporate the solvent. The spot should not be more than 2 mm in diameter when completed.

Teacher Tips

• Other TLC kits include Introduction to Thin Layer Chromatography, AP4504; Thin Layer Chromatography, AP9095; and Identification of Unknown Substances II, FB1648.

Answers to Prelab Questions

TLC can be used to identify the components contained in unknown painkillers. Over-the-counter painkillers contain one or more active ingredients. The most common active ingredients are aspirin, acetaminophen and caffeine. The active ingredients contained in painkillers can be determined by comparing the R_f values obtained from unknown components with those of known solutions of aspirin, acetaminophen and caffeine.

The plate on the left contains the chromatographs of the three drugs caffeine, aspirin and acetaminophen. The plate on the right contains the chromatographs of the the sample drug and a duplicate sample.

1. Measure the R_f values for the 3 drugs and the two sample chromatographs.
2. Based on your R_f values, determine which of the 3 drugs are present in the sample.
   {12136_PreLabAnswers_Figure_5}
   Based on the data and the calculated R_f values, the over-the-counter drug appears to contain caffeine and acetaminophen.

Sample Data

Answers to Questions

1. Why are the acid spots yellow on a blue-green background?

   Once the plates are soaked in the bromcresol green solution, any spot on the plate containing a carboxylic acid will change the color of the indicator to yellow, its acid-form color.

2. Calculate the R_f values for each acid and the spots identified in the fruit juice chromatograph.

   See results table.

3. Based on these results, which, if any, of the three fruit acids are found in the fruit juice sample?

   Answers will vary with selected fruit juice sample.

Introduction

Thin-layer chromatography (TLC) is a valuable analytical technique for separating and analyzing organic compounds. In dye analysis, for example, TLC is used to identify dyes in fabrics or fibers (forensics) and to determine the composition of natural dyes in plants and animals. In this laboratory, thin layer chromatography will be used to separate and identify various carboxylic acids in fruit juices.

Concepts

• Solubility
• Polarity
• R_f value
• Chromatography
• TLC

Background

There are many different types of chromatography (chromato = color + graphy = to write), but most work on the principle of adsorption. A good adsorbent is usually a solid material that will attract and bind the components to be separated in a mixture. Paper, silica gel or alumina are all very good adsorbents. The other key element in chromatography is the eluent. The eluent is the solvent that carries the materials to be separated through the adsorbent.

In chromatography, the compounds to be separated are slightly soluble in the eluent and will spend some of the time in the eluent and some of the time in the adsorbent. When the compounds to be separated spend different amounts of the time being adsorbed, they are then separated from one another. The polarity of the molecules to be separated and the polarity of the eluent are very important in the separation process. Remember the saying “like dissolves like”; the affinity of a molecule for the eluent versus the adsorbent is what separates the molecules.

Thin-layer chromatography (TLC) is a form of chromatography that uses a thin layer of adsorbent material on an inert support. The TLC sheets used in this experiment contain a thin layer of silica gel on a plastic sheet. The eluent for the TLC is an organic solvent that travels up the TLC plate as it is adsorbed by the silica gel. TLC is frequently used as an analytical method to verify the presence of certain molecules. The polarity of the eluent is very important in TLC and a small change in polarity can dramatically change the ability to separate compounds. Many times, a mixture of a nonpolar solvent (hexane) and a polar solvent (acetone) are used to achieve an optimum polarity. The use of “known” substances as a baseline for comparison is critical for making a strong case for the composition of unknowns. In order to compare substances collected over different time periods, a relative value called the R_f value is calculated for a given substance for specific chromatographic conditions. Typically, an unknown substance is spotted on a TLC plate alongside of known substances. Then the solvent is allowed to travel up the TLC plate carrying the substance components up the plate to different levels. The distance a component moves compared to the distance the solvent moves is recorded as a ratio and is called the R_f value.

In this laboratory, you will determine the various carboxylic acids in fruit juices. The main carboxylic acids found in edible fruits are citric acid, malic acid and tartaric acid, with trace amounts of benzoic, oxalic and succinic acids, among others.

Excluding benzoic, all these carboxylic acids are similar in structure and composition, being short-chained and either di- or tri-protic acids (see Figure 1). With many single and double carbon to oxygen bonds in each of these molecules, all of these molecules are highly polar. The solvent of choice for the separation of these polar organic molecules is a 2:1:1 mixture of toluene, concentrated acetic acid and ethyl acetate (see Figure 2). This solvent mixture of low polarity toluene, moderately polar ethyl acetate, and highly polar acetic acid is found to give clear separation of these molecules having similar polarity. As with all TLC separations, this solvent mixture was arrived at after extensive trials of various combinations of pure solvents.

After the compounds of a mixture have been separated by TLC, they have to be observed. Some organic compounds are highly colored and readily visible, but most, like our fruit acids, are colorless and cannot be observed directly with the naked eye. Several techniques are used to observe the colorless organic compounds.

1. Iodine. Add a few crystals of iodine (I₂) to a covered jar or beaker. Place the completed TLC sheet in the jar along with the iodine. Iodine forms a complex with most organic compounds and its vapors will be adsorbed onto most spots containing organic compounds, indicating their position on the TLC sheet. Keep the TLC sheet in the iodine developing chamber for only a few minutes or until dark spots appear. These dark spots will be the various organic compounds that were separated.
2. Long-wave UV light. The Flinn TLC sheets contain a fluorescent powder that glows bright green when placed under a long-wave UV lamp. The spots that contain separated compounds will not glow green and will frequently be dark.
3. Heat. Some organic compounds will quickly char and turn black when heated. Placing the TLC sheet on a warm hot plate will decompose some organic compounds, turning them black.
4. Chemical reaction. Sometimes the location of individual compounds can be visualized by chemical reaction if a specific reagent is used as a spray. For instance, ninhydrin dissolved in butanol sprayed on a TLC sheet containing several amino acids will stain the amino acids violet.

For this determination, a staining solution of bromcresol green will be used. This stain is excellent for functional groups whose pK_a is approximately 5.0 and lower. Thus, this stain provides an excellent means of selectively visualizing carboxylic acids. These appear as bright yellow spots on either a dark or light blue background and typically, it is not necessary to heat the TLC plate following immersion. This TLC visualization method has a long lifetime (usually weeks) thus, it is not often necessary to circle such spots following activation by staining.

Experiment Overview

In this laboratory, thin layer chromatography will be used to separate and identify the various carboxylic acids in fruit juices. Standards will be run in order to establish R_f values for each of the carboxylic acids. These R_f values will be used to identify the acid components of various fruit juices.

Materials

Prelab Questions

TLC can be used to identify the components contained in unknown painkillers. Over-the-counter painkillers contain one or more active ingredients. The most common active ingredients are aspirin, acetaminophen and caffeine. The active ingredients contained in painkillers can be determined by comparing the R_f values obtained from unknown components with those of known solutions of aspirin, acetaminophen and caffeine.

The plate on the left contains the chromatographs of the three drugs caffeine, aspirin and acetaminophen. The plate on the right contains the chromatographs of the the sample drug and a duplicate sample.

1. Measure the R_f values for the three drugs and the two sample chromatographs.
2. Based on your R_f values, determine which of the three drugs are present in the sample.
   {12136_PreLab_Figure_3}

Safety Precautions

The chromatography solvent is a dangerous fire risk, flammable and 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 a TLC plate. The plates are fairly fragile and, if cracked or soiled, will yield incorrect results. Handle the plates by the edges only. Do not touch the powdery side of the plate. Place the plate powdery-side up on a piece of clean paper.
2. Use a pencil and ruler to gently draw a faint line 1 cm from the bottom of the plate and make other light markings as shown in Figure 3. Do not dig into the white powder on the plate. Note: Do not use a pen!
   {12136_Procedure_Figure_3}
3. Again using a pencil, label the top of the first plate with the numbers “1,” “2,” “3” and “F.” The numbers on the plates correspond to the weak acid samples and friut juice sample according to the following key:

   1 = Citric acid
   2 = Succinic acid
   3 = Tartaric acid
   F = Fruit juice

4. Using separate Beral-type pipets, place two drops of sample 1 (citric acid) in the first well of a well plate or reaction strip. Add two drops of sample 2 (succinic acid) to the second well, two drops of sample 3 (tartaric acid) to the third well, and two drops of the sample fruit juice to the fourth well.
5. Add 1 drop of bromcresol green to each of the four sample wells. Mix the solutions in each well with a separate tooth pick.
6. 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 end of a spotter to the drops of sample 1. 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. It is also important not to disrupt the silica gel, so a gentle touch is required.
7. Repeat step 6 for the other carboxylic acid samples (succinic acid, and tartaric acid) according to the key provided in step 3.
8. Repeat step 5 for the fruit juice sample (the spot will be placed on the pencil line under “F”). For this sample, let the drop dry, then repeat step 6 three more times with a new spot on top of the previous fruit juice sample spot.
9. Remove the watch glass or Parafilm cover of the chromatography chamber, then add 10 mL of the chromatography solvent. Carefully place the TLC plate in the chromatography chamber with the sample end down (as shown in Figure 4). 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! Replace the watch glass or Parafilm cover.
   {12136_Procedure_Figure_4_Chromatography chamber}
10. The solvent will be drawn up the TLC plate. As it is drawn up, it will carry the carboxylic acids in each sample up the strips at different rates depending on the characteristics of each acid.
11. When the solvent front is within 1–2 cm of the top of the chromatography strip, the run is stopped by removing the strip from the flask. Allow the strip to air dry.
12. While the plate is drying, place about 15 mL of the 0.12% bromcresol green solution in a large plastic weighing dish.
13. Once the TLC plate is dry, use tweezers to pick up the plate by its top edge.
14. Submerge the plate in the bromcresol green solution, then remove. Hold the plate over the weighing dish to allow the excess solution to drain from the TLC plate.
15. Set the plate aside to air dry.
16. Using a pencil and ruler, mark the solvent line and each of the yellow acid spots. Place the mark in the middle of each acid spot.
17. Use the ruler to measure the distance, in mm, from the bottom line to the solvent line and record this value in the data table.
18. Measure the distances from the bottom line to the pencil mark for each of the yellow acid spots. Record these values in the data table.
19. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

12136_Student1.pdf