Biol 211, Cell Biology, Fall 2008

Exercise #2: Production of an Amino Acid Hydrophobicity Scale Using Paper Chromatography

Investigator: Ramsey Hanna

Purpose of the Experiment

The scientific objective of this laboratory exercise is to create a hydrophobicity scale of 15 amino acids using simple paper chromatography.

Materials

• Stock solutions of 5 different amino acids
• A stock of 70% 1-propanol to serve as the mobile phase
• A solution of ninhydrin in acetone to stain the chromatogram
• A 12.5 cm X 23 cm piece of chromatography paper
• A large jar to serve as the chroamtography vessel
• A large petri dish to serve as the vessel lid
• A porcelain depression dish to hold you amino acide samples
• A specialized capillary pipettor to apply samples to the paper
• Pasteur pipettes with bulbs
• A drying oven

Procedure. Preparation of the Mobile Phase

1. Glove yourself. Keep the gloves on throughout the lab period.

2. Add 20 ml of the mobile phase into a clean glass chromatography jar. Place the jar on the lab bench surrounding the room so it will remain undisturbed. Cover the jar with a petri dish cover.

Procedure. Preparation of the Stationary Phase

1. Obtain one sheet of chromatography paper from the instructor bench. The paper measures 12.5 cm (h) x 23 cm (w). Write your name in the upper right hand corner of the paper. Using a plastic ruler, draw a light pencil line across the paper 12 mm from the bottom edge, then place 10 small pencil dots at 2 cm intervals on this line. To the side of each dot place a number 1 through 10

Procedure. Collect aliquots of each of the 5 amino acids.

1. Using a wax pencil, label depressions of a spot plate A through E.

2. Using the pasteur pipets that come with each amino acid, half-fill each of the labeled depressions with the amino acids assigned to your group according to the table to the right.

3. Half-fill a sixth depression with water.

Procedure. Add a sample of each amino acid to the chromatography paper.

1. With a capillary pipette calibrated for 2 µl, spot 2 µl of each amino acid on its appropriate pencil mark. Between samples, clean the pipette by pulling distilled water from depression F into the pipet several times. Expel the rinse water on a piece of filter paper.

2. Once all samples have been delivered to the paper, allow the samples to dry, then gently roll the paper into a cylinder (sample side out), then loosely staple the ends together.

Procedure. Run the Chromatogram.

1. With the spots towards the bottom and without moving the jar, gently and in one smooth motion place the paper cylinder into the middle of the chromatography jar containing the mobile phase. Cover immediately and do not disturb.

2. Allow the organic solvent to rise to within about 12 mm from the top of the paper. Since we only have about 60 - 70 minutes to allow the chromatogram to develop, there will only be enough time for the mobile phase to migrate to within about 8 cm of the top of the paper.

3. When the solvent front has migrated a sufficient distance, without moving the jar, carefully remove the paper from the jar with small forceps, immediately remove the staples and trace the solvent front lightly with pencil. Be sure to trace the solvent front accurately. Let the paper cylinders air-dry under the hood for approximately 5 minutes until dry.

Procedure. Stain the Chromatogram.

1. Grasp the dried chromatography paper by its edges using a pair of forceps and smoothly and evenly dip it into the staining solution, then remove it quickly.

2. Dry the chromatogram under the hood for approximately 5 minutes, then place it in a 105 degree oven. As the chromatogram heats in the oven, the amino acid spots will begin to appear. When fully developed, remove it from the oven.

Procedure. Collect and Record the Raw Data

1. Accurately trace the outer edge of each spot with a pencil line. Then, estimate the center of each spot and mark it with a pencil point.

2. For each amino acid sample applied to the chromatography paper, collect two pieces of raw data. The Dsample and the Dsolvent, both in mm.

From the raw data, calculate the relative mobility of eavh amino acid(retention factor, Rf)

Results