Materials Included In Kit

Additional Materials Required

Prelab Preparation

Prepare the 1% amino acid solutions: Add 1 g of each amino acid to beaker containing 100 mL of distilled water. Label each beaker with the name of the amino acid. Weigh out 0.5 g of both alanine and methionine and add to a beaker containing 100 mL of distilled water to prepare the unknown sample. Place the beakers in a central location since all student groups will need access to them.

Prepare 2% ninhydrin solution: Add 4 g of ninhydrin to 200 mL acetone. Stir to dissolve. Store for up to two weeks in an appropriately labeled bottle. Transfer to spray bottles before laboratory.

Prepare chromatography solvent: Combine 210 mL of n-butyl alcohol, 70 mL of glacial acetic acid, and 70 mL of distilled water. Mix well and store in a labeled bottle.

Safety Precautions

The chromatography solvent contains n-butanol, glacial acetic acid and water. It is acidic and corrosive to skin and eyes. Avoid all contact with body tissues. The ninhydrin solution is flammable and a fire risk. Both solutions are toxic by ingestion and inhalation. Do not breath the fumes. This lab should be performed in a fume hood or well-ventilated laboratory. 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. Completed chromatograms may be thrown away in the regular trash. Amino acid solutions may be rinsed down the drain with an excess of water according to Flinn Suggested Disposal Method #26b. The ninhydrin solution may be stored in a properly labeled bottle for future used. Alternatively, the solution may be disposed of according to Flinn Suggested Disposal Method #18a. The chromatography solvent may be stored and reused or disposed of according to Flinn Suggested Disposal Method #18b.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. This laboratory activity can reasonably be completed in one full 50-minute class period and approximately half of the following lab period to develop the amino acids using the ninhydrin solution and to take measurements. The prelaboratory assignment may be completed before coming to lab, and the data compilation and calculations may be completed the day after the lab.
• The spray bottles included with the kit may be shared among several groups.
• Do not store the ninhydrin solution in the plastic spray bottles. Pour the solution back into the original glass bottle and save for future use if desired.
• It is important to begin the lab as soon as the class period starts since it usually takes about 50 minutes for the solvent to reach the desired point on the chromatography paper. If needed, remove the chromatography sheets after students leave and allow the paper to dry in a well-ventilated area such as a fume hood. Students should write their names or group number in pencil on the chromatogram.
• If a drying oven is being used to heat the chromatograms, 100 °C for a few minutes should be sufficient. Once the purple color is obvious, the papers may be removed.

Teacher Tips

• In forensic chemistry, ninhydrin is most often used to detect latent fingerprints left behind on porous surfaces (e.g., cloth, paper, cardboard). Prints on nonabsorbent surfaces (e.g., mirrors, tiles, glass), are detected using fingerprint powders, such as graphite or aluminum dust.
• Additional unknowns may be made by simply mixing together two or more amino acid solutions.
• Another general protein classification test is the biuret test, which can be used as more of a quantitative test than the ninhydrin test. With the biuret test, the intensity of the purple product depends on the protein being tested and how much protein is present in the test sample.
• Have students research the various amino acids used in this lab.

Answers to Prelab Questions

1. Based on information regarding chromatography in the Background section, why must the markings on the paper be made in pencil rather than pen or marker?

   Pen and marker ink are likely to contain pigments that are soluble in the solvent that will spread and separate thus interfering with the amino acid samples.

2. The solvent in this activity will flow upward, against gravity, via capillary action. Describe capillary action using your own words. Reference your textbook for additional information, if necessary.

   Capillary action refers to the spontaneous rise of the liquid in small tubes or fibers, such as the wetting of a solid like the chromatography paper used in this activity. Capillary action is responsible for the rise of sap in plant fibers and the flow of blood through the capillaries in the body. Attractive forces, such as adhesion, between the molecules of the liquid and the walls of the vessel are responsible for capillary action.

3. The R_f value for an amino acid sample is 0.60. If the solvent traveled 72 mm from the origin, how far did the sample travel from the origin?
   {12643_Answers_Equation_3}

Sample Data

Data Table 1

Data Table 2
Solvent distance traveled from the origin ___72___ mm

Answers to Questions

1. Based on your observations and data, identify the amino acids in the unknown sample.

   Comparing the distances measured in mm from the origin to the known samples, it appears that the unknown contains both alanine and methionine.

2. Many different organic solvents may be used for paper chromatography. If this experiment were repeated with a different solvent, would you expect the R_f values to change or remain the same? Explain.

   The solvent distance traveled from the origin in mm would likely be different for a different solvent. Since Rf is calculated by dividing the sample distance from the origin by the solvent distance from the origin in mm, the R_f values for each sample are likely to be different. However, the distance the sample travels may also change; therefore the ratio of distances may or may not differ.

3. In the Procedure section it cautions that fingerprints on the chromatography paper will react with ninhydrin and appear. Explain.

   Oils found on the skin and in fingerprints contain amino acids. These oils will easy affix to porous surfaces such as chromatography paper. The amino acids in the oil will react with the ninhydrin and appear purple in color when heated just like the known amino acid samples in this activity.

4. The chromatography solvent used in this lab is very polar as it contains an alcohol, an acid and water. Based on this information, which amino acid probably is the least polar of those tested?

   In a polar solvent, most non-polar samples will travel the furthest away from the origin. Methionine traveled the furthest distance so it appears to be the least polar amino acid of those tested.

5. Considering your answer to Question 4, would you expect the R_f value for this amino acid to be large or small in comparison to the other samples used in this experiment?
   {12643_Answers_Equation_4}
   So the larger the distance traveled from the origin by the amino acid, the larger the R_f value.

References

Flinn ChemTopic™ Labs, Volume 20, Biochemistry—the Molecules of Life; Cesa, I., Ed.; Flinn Scientific: Batavia, IL, 2002.

Flinn ChemTopic™ Labs, Volume 2, Elements, Compounds and Mixtures; Cesa, I., Ed.; Flinn Scientific: Batavia, IL, 2005.

Introduction

Amino acid samples travel at different rates in an organic solvent as they migrate up chromatography paper. In this experiment, several known amino acid samples will be run on a chromatogram, compared and used to identify an unknown mixture of amino acids.

Concepts

• Chromatography
• Amino acids
• Adsorption

Background

Proteins represent the most diverse class of biological compounds within cells. It is estimated that a single bacteria cell contains more than 3,000 different types of proteins. The word protein is derived from the Greek word “proteios,” meaning first or primary. Proteins are of primary importance in terms of both their occurrence within cells and their function in cell activities. The functions of proteins are at the very center of life itself—proteins catalyze all of our metabolic reactions, carry oxygen to our body tissues, protect the body from infection and maintain cell and tissue structure.

Proteins are composed of amino acid molecules joined together in a chain-like fashion via peptide linkages. Amino acids are thus often referred to as the “building blocks” of protein structure. The size of the amino acid chain in a single protein molecule can vary from 50 amino acid residues in insulin to more than 500 in hemoglobin and more than 5,000 in some viruses. When fewer than 50 amino acids are joined together the resulting molecules are called polypeptides.

All amino acids have two structural features in common—they contain a carboxylic acid group (–COOH) on one end and an amine group (–NH₂) on the other end. Peptide linkages are created when the carboxyl group of one amino acid reacts with the amine group of the next amino acid in the sequence. As each amino acid is added to the growing polypeptide chain, a molecule of water is formed as a byproduct, as shown in Figure 1.

All proteins are derived from about 20 different, naturally occurring amino acids, which can be arranged in an almost infinite number of ways, giving rise to the thousands of unique proteins found in nature. The primary structure of a protein is determined by the number and identity of amino acids within the protein and the order in which they are joined together via peptide linkages. Higher levels of protein structure (called secondary, tertiary, and quaternary structure) result as the polypeptide chains form ribbons, sheets and coils that ultimately fold in on themselves to form more compact and more stable three-dimensional arrangements.

Differences in the molecular structure of various amino acids contribute to their differing polarities. These variations in polarity make it possible to separate amino acids via chromatography. The word chromatography is derived from two Greek words meaning color (chroma) and writing (graphein)—“color writing.” The term was coined by the Russian chemist Michael Tswett in 1903 to describe a new technique he had invented to separate the pigments in green plant leaves. Tswett found that in addition to chlorophyll, the green main pigment, plant leaves also contained red and yellow secondary pigments. The results were literally “written in color” when a plant extract was passed through a column containing a clay-like absorbent solid.

Paper chromatography is an example of a more general type of chromatography called adsorption chromatography. The paper acts as an adsorbent (not to be confused with absorption), a solid which is capable of attracting and binding the components in a mixture (see Figure 2). The sample is “spotted” onto the surface of a special type of chromatography paper and a solvent is then allowed to seep or flow through the paper. 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 chromatography, 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, non-polar substances would require slightly polar solvents.

The solvent will always travel farther than the samples. The relationship between the difference in distance traveled by the solvent versus the sample is referred to as the R_f value (rate of flow). How far each amino acid sample travels depends on several factors, including how high the solvent is allowed to rise on the paper, the type of absorbent solid, the type and concentration of the solvent, temperature and the distance of the origin from the solvent.

Several tests may be used to detect the presence of proteins or amino acids. One type is a ninhydrin test which will be used in this activity to make the amino acid spots visible.

Ninhydrin is a pale yellow solid. It reacts with amine groups in amino acids and proteins to produce a purple product (Equation 2). The reaction is very slow at room temperature so heat must be used to speed up the reaction.

Experiment Overview

Various amino acid samples, including an unknown mixture, will be spotted on chromatography paper and run with chromatography solvent. The following lab period, the samples will be treated with ninhydrin solution and heated to make the amino acid spots visible. At this point, the distances traveled from the origin by the samples may be measured in millimeters. Using these measurements, R_f values for each sample will be calculated and the unknown sample can be identified.

Materials

Prelab Questions

1. Based on information regarding chromatography in the Background section, why must the markings on the paper be made in pencil rather than pen or marker?
2. The solvent in this activity will flow upward, against gravity, via capillary action. Describe capillary action using your own words. Reference your textbook for additional information, if necessary.
3. The R_f value for an amino acid sample is 0.60. If the solvent traveled 72 mm from the origin, how far did the sample travel from the origin?

Safety Precautions

The chromatography solvent is acidic and corrosive to skin and eyes. Avoid all contact with body tissues. The ninhydrin solution is flammable and a dangerous fire risk. Both solutions are toxic by ingestion and inhalation. Do not breath the fumes. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Follow all laboratory safety guidelines.

Procedure

1. On a 20 cm wide by 10 cm high piece of chromatography paper, use a pencil to draw a straight line about one centimeter from the bottom of the paper extending from the left side to the right side (see Figure 3). Note: Do not touch the paper with your fingertips.
   {12643_Procedure_Figure_3}
2. Place nine pencil dots on the line spaced approximately 2 cm apart.
3. Have one partner write with a pencil the name of a different amino acid sample under each of the nine dots as shown on the data table while the other partner adds 20 mL of chromatography solvent to a 600-mL beaker. Note: If needed, names may be abbreviated to save space (e.g., Al, Ar, Asp).
4. Using a microtip pipet, obtain a small amount of the first amino acid listed in the paper.
5. Gently touch the tip of the pipet to the chromatography paper directly above the pencil dot—spot each sample only once. The sample spot should not be more than 0.5 cm in diameter. It may be helpful to practice properly administering the sample on a sheet of paper before adding it to the chromatography sheet.
6. Repeat steps 4 and 5 using the remaining eight amino acid solutions. Try to keep the sample spots uniform in size. Note: Remember to only touch the chromatography paper on the edges because fingerprints will show up when the paper is treated with ninhydrin solution.
7. With the pencil line/sample side facing outward, roll the chromatography paper into a loose cylinder and carefully staple the top and bottom edges together avoiding the end samples (see Figure 4).
   {12643_Procedure_Figure_4}
8. Place the paper cylinder, pencil line side down, into the beaker containing the chromatography solvent. Do not allow the paper to touch the sides of the beaker (see Figure 5).
   {12643_Procedure_Figure_5}
9. Cover the beaker with a watch glass or aluminum foil.
10. Allow the samples to run on the chromatography paper until the solvent level is about a centimeter from the top of the paper. If the solvent has not reached this point by the end of class, notify your teacher.
11. Remove the chromatography paper from the beaker and mark the solvent height with a pencil line (before it evaporates). Then carefully remove the staples.
12. Place the chromatography paper in a well-ventilated area to dry as directed by your teacher.

Complete the next steps during the following lab period.

13. Spray the chromatography paper using a spray bottle containing 10 mL of a 2% ninhydrin solution.
14. Allow the chromatography paper to dry in a well-ventilated area for 10–20 minutes or until the paper is completely dry.
15. Heat the chromatography paper for a few minutes until color develops. This may be done in a drying oven or by holding the paper about 10 cm above a hot plate set to a low setting. Follow your teacher’s instructions and be careful not to scorch the paper.
16. Place a pencil dot at the centermost point of each amino acid spot. Note: The unknown sample may have more than one center point.
17. Sketch your completed chromatogram in Data Table 1.
18. Measure the approximate distance in millimeters the solvent traveled from the pencil line to where it stopped on the paper and record on the worksheet.
19. Measure the distance in millimeters from the origin to where the first amino acid sample traveled.
20. Record the distance measured for each amino acid sample in Data Table 2.
21. Calculate the R_f value for each of the nine amino acids and record in Data Table 2.
22. Attach the chromatogram to the Amino Acid Chromatography Worksheet once it is completed.
23. Keep your chromatogram and turn it in with your Amino Acid Chromatography Worksheet. Follow your teacher’s instructions regarding disposal of the chromatography solution and ninhydrin solution in the spray bottles.

Student Worksheet PDF

12643_Student1.pdf