Introduction to Peptide Sequencing
Amino Acid Analysis Using Paper Chromatography



SAFETY PRECAUTION: Ninhydrin spray should only be used in the hood and should only be handled while wearing gloves. 

Day 1:  Hydrolysis of an unknown dipeptide and Equal sweetener.

1. Obtain a sample of the unknown dipeptide. The sample will contain approximately 2 mg of the dipeptide. The sample will be in a plastic microcentrifuge tube.  Be sure to record the unknown sample number in your notebook.

2. To the unknown sample in the microcentrifuge tube, add 2 ml of 6N HCl and mix gently to dissolve the sample. Note that 6N HCl is the same as 6M HCl - this is true for any monoprotic acid.  Using a Pastuer pipette, transfer the sample to a  4-ml hydrolysis vial and seal with a Teflon-lined screw cap.

3,  Place approximately 10 mg of the Equal sweetener in a microcentrifuge tube, add 2 ml of 6N HCl and mix gently to dissolve the sweetener.  Using a Pastuer pipette, transfer the sample to another 4-ml hydrolysis vial and seal with another Teflon-lined screw cap.

4. Label the top of both vials (NOT THE SIDES) with your names (or initials) and either "Unk" or "Eq" and place in a 110° C heating block overnight.

5.  Note, I will turn off the heat in the morning.  Either you or your partner MUST come to the lab before noon to pick up your vials and place them in your drawer.


Day 2: Amino acid paper chromatography
 

Paper Chromatography of Amino Acids:

1. Recover the 4-ml vials containing your acid hydrolyzed dipeptide and acid hydrolyzed Equal sweetener (should be colorless samples).

2. Obtain a 8.5in by 8.5in piece of 3 MM Whatman filter paper and draw a straight line across the bottom using a pencil. The line should be ~3.5 cm from the bottom of the paper and will serve as the origin for spotting your samples. Be sure to handle the paper with gloves. Why?

3. On both edges of the paper perpendicular to the origin, draw two straight lines from top to bottom that intersect the line at the origin ~2.5 cm from either edge of the paper. All of the samples should be spotted on the origin between these two lines.

4. Spot three 1drop spots of each of the amino acid standards along the origin  (i.e., 3 x 1 drop spots). Spotting can be done with a capillary tube or even your Pasteur pipette. However, each spot should be no more than approximately 1 mm in diameter and each spot must dry before spotting the next drop!  Using your pencil, indicate the identity of each spot below the origin. Also be sure that your names are on the paper.

5. Next to the standards, spot a 3 x 1 drop spots of your unknown hydrolyzed dipeptide and 3 x 1 drop spots of the hydrolyzed sweetener.  Note: we may even spot diet Coke!  Why?

6. When all of the spots have dried, you will need to roll your paper into a cylinder (see instructor for procedure), staple the ends and place your paper in one of the 2L beakers in the hood.

7. In the hood, Place the paper (origin side down) in the beaker containing 2 cm of the mobile phase solvent (butanol:acetic acid:water (60:15:25)).  You will need to figure out what volume of solvent you will need and you will need to prepare the solvent mixture.

8.  Cap the beaker with aluminum foil and allow the mobile phase to migrate up through the paper until it reaches ~2.5 cm from the top (~3-4 hrs). Remove the paper from the beaker, mark the position of the solvent front with a pencil and allow the paper to dry in the hood.

9. At the beginning of the next laboratory period, while wearing gloves, spray the paper chromatogram lightly with ninhydrin reagent, dry in the hood and heat in a 100 degrees C oven for 2-3 minutes.

10. Circle the position of all of the spots with a pencil and calculate the Rf values.  What is the amino acid composition of your unknown dipeptide? Of Equal? 
What dipeptide do you think is in Equal?  Discuss any error/uncertainty in your identification. How might the experiment be modified to reduce this error/uncertainty?  Describe a method to determine the sequence of your unknown dipeptide.  ANSWER ALL OF THESE QUESTIONS AS PART OF YOUR RESULTS AND DISCUSSION.