Identification of Unknown Amino Acid
Elizabeth Amundson
Lab Partner: Mary Witucki

Introduction: The goal of this lab was to determine which two amino acids where contained within an unknown mixture by comparing the results of a primary amine test, an amide test, a benzene ring test, a thiol test, and paper chromatography to that of amino acids with known compositions. I hypothesize that alanine and lysine will test positive for primary amines because they are the only amino acids being tested in this reaction that contain a primary amine (-NH2). I hypothesize that glutamine will test positive for amides because it is the only amino acid being tested in this reaction that has an amide, or (R1-N-R2). I hypothesize that tryptophan will test positive for benzene rings because it is the only amino acid being tested that contains a benzene ring. I hypothesize that cysteine will test positive for thiols because it is the only amino acid being tested with a thiol, or (R-S-H). I also hypothesize that the hydrophilic amino acids will have smaller Rf values than the hydrophobic amino acids because the chromatography paper is more hydrophilic than the solvent being used, so they will have a higher affinity for the paper and will not travel very far with the solvent. It is important to be able to identify unknown amino acids so that it can be determined which amino acids are found in cells and proteins.

Methods:
Paper Chromatography 1. Pick either unknown A, B, or C. 2. Draw a pencil line along a long side 2cm from the edge and dot 5uL of the following samples on the line 2cm apart. a. Unknown b. Alanine c. Cysteine d. Glutamine e. Leucine f. Lysine g. Proline h. Tryptophan 3. Roll the paper into a cylinder and staple it so that it stays so that it can be placed in a TLC chamber containing a mixture of 1-butanol, acetic acid, and water (3:1:1) and allow it to develop until the solvent front is 1cm from the top of the paper. 4. Spray the paper with 2mg/mL ninhydrin in 95% ethanol and place in a 120C oven until the color develops.
Primary Amines: (test unknown C, alanine, lysine, and proline) 1. Add 500L of ninhydrin solution (4mg/mL in water) to tubes containing 100L of each amino acid. 2. Place each tube in a boiling water bath for 5 minutes and observe the color that develops.
Amides: (test unknown C and glutamine) 1. Add 500L of 20% NaOH to tubes containing 250L of each amino acid. 2. Dip a small piece of red litmus paper in water and place it into a Pasteur pipet mounted in a support. 3. Place the Pasteur pipet into each tube and place the tubes into a boiling water bath for 5 minutes. 4. Note the color of the litmus paper after 5 minutes.
Substituted Benzene Rings: (test unknown C and tryptophan) 1. Add 250L of each amino acid to a tube with 200L of concentrated nitric acid. 2. Heat in a boiling water bath for 3 minutes. 3. Add 500L of 20% NaOH and note the color change that takes place.
Thiol Groups: (test unknown C and cysteine) 1. Add 100L of amino acid to a tube of 500L of 1mg/mL DTNB. 2. Allow the mixture to stand for a few minutes and then note any color changes.
Results:
Primary Amine test:
*The presence of a purple color indicates that a primary amine is in solution. Amino Acid | Color | Presence of Primary Amine | Unknown C | Dark Blue/Purple | + | Alanine | Dark Blue/Purple | + | Lysine | Purple | + | Proline | Yellow | - |

Amide test:
*The presence of blue litmus paper is an indication that amides are present in solution Amino Acid | Color of Litmus | Presence of Amide | Unknown C | red | - | Glutamine | blue | + |

Benzene Ring test:
*The presence of a yellow or orange color indicates the presence of benzene rings Amino Acid | Color | Presence of benzene ring | Unknown C | Dark Yellow | + | Tryptophan | Dark Yellow | + |

Thiol test:
*The presence of a dark yellow color indicates that a thiol is present in solution Amino Acid | Color | Presence of Thiol | Unknown C | Light Yellow | - | Cysteine | Dark Yellow | + |

Unknown Alanine Cysteine Glutamine Leucine Lysine Proline Tryptophan
3.6 cm
4.0 cm
3.3 cm
2.9 cm
1.6 cm
5.6 cm
1.4 cm
3.3 cm
4.1 cm
7.7 cm Unknown Alanine Cysteine Glutamine Leucine Lysine Proline Tryptophan
3.6 cm
4.0 cm
3.3 cm
2.9 cm
1.6 cm
5.6 cm
1.4 cm
3.3 cm
4.1 cm
7.7 cm

*Rf is calculated by the equation: (Distance solute traveled)/(Distance solvent traveled)
Ex. For Alanine… (3.3cm)/(7.7cm) = .43 Amino Acid | Distance (cm) | Rf | Solvent Front | 7.7 cm | ------------- | Unknown C | 3.6 cm / 4.0 cm | .47 / .52 | Alanine | 3.3 cm | .43 | Cysteine | 2.9 cm | .38 | Glutamine | 1.6 cm | .21 | Leucine | 5.6 cm | .73 | Lysine | 1.4 cm | .18 | Proline | 3.3 cm | .43 | Tryptophan | 4.1 cm | .53 |

Discussion: 1. Rf values can be seen in the above table in the results sections. 2.

3. Since the paper is cellulose, it is hydrophilic. This means that amino acids with hydrophilic side chains, like cysteine, glutamine, and lysine have relatively low Rf values because they do not travel very far due to their affinity for the paper. However, the nonpolar amino acids like, alanine, leucine, proline, and tryptophan, travel farther up the paper because they are more attracted to the 1-butanol, acetic acid, and water (3:1:1) solvent mixture than is more hydrophobic than the paper. 4. Our cysteine did not produce 2 spots on the chromatography paper, but it should. This is because he thiol group on the cysteine reacted with the solvent to create a spot for the product of the thiol reaction and a spot for the whole cysteine. 5. The two amino acids in the unknown C mixture are alanine and tryptophan. It can be concluded that alanine is in the mixture because in the primary amine test the alanine and unknown c had the exact same color, and their Rf values are very close at .47 and .43. It can also be concluded that tryptophan is in the mixture because the unknown and tryptophan both tested positive for benzene rings and the unknown’s second spot on the chromatography paper has almost the same Rf as tryptophan at .52 and .53.