15.1: Paper Chromatography of Amino Acids Lab Procedure

Learning Objectives

• To use paper chromatography to identify amino acids.
• To identify components of unknown amino acid sample by calculating Rf values

 

Background

Chromatography is a collective term for a set of analytical techniques used to separate mixtures. Chroma means color and graph means to write or draw. Paper chromatography is an analytical technique used to separate mixtures of chemicals (sometimes colored pigments) using a partitioning method. The paper in this method is called the stationary phase because it does not move and serves as a substrate or surface for the separation. Analytes (substances being analyzed) are separated from each other based on a differential affinity to a solvent. The solvent dissolves and carries the analytes along the matrix of the stationary phase. Since the solvent moves through a wicking action, it is called the mobile phase.

Figure \(\PageIndex{1}\): Credit: Theresa Knott [CC BY-SA 3.0 or GFDL]

The figure above shows a chamber containing mobile phase and a prepared paper stationary phase. A line drawn at the bottom edge of the paper is the starting point/line. The starting line and sample dots must be above the level of the mobile phase when the paper is placed inside the chamber. If the starting line is below the liquid level, the samples will wash out into the mobile phase rather than elute up the stationary phase. Another line is drawn about 10 cm above the bottom edge of the paper. This is the finish line. Its location was chosen for this experiment because when the eluting solution reaches that line, any inks that are mixtures should be clearly separated.

Note

When the solvent front reaches the finish line, the paper should be removed immediately from contact with the mobile phase.

The figure below shows a typical paper chromatogram of the separation of pen inks. There are a few difficulties commonly encountered in the elution process. One problem is that spots tend the spread out as they elute, and can bleed into each other as they proceed up the paper. This can be confusing when interpreting the chromatogram. To avoid this problem, space the spots of sample far apart and make repeated, tiny applications of sample to prevent spreading. Another problem is an uneven solvent front. This can happen if the beaker is nudged – if the mobile phase sloshes inside, the elution trails may travel diagonally, which makes interpretation very difficult. This can also happen if the two edges of the chromatogram are allowed to touch when they are stapled or taped together to form a cylinder.

Figure \(\PageIndex{2}\):

A component with a given solubility travels along with the mobile phase at one rate, regardless of what other components are present in the sample. If the red part of purple ink travels at the same rate as pure red ink, and both stop in the same place, the two should be the same red ink. The two red spots should have the same Retention Factor, Rf. The Rf is the distance, D, traveled by the spot divided by the distance traveled by the eluting solution, or Solvent Front, F.

\[ R_f = frac{D}{F} \]

Comparing the Rf values allows the confirmation of a component in multiple samples because unique components have unique Rf values.

 

Experimental Procedure

Materials and Equipment

Materials: chromatography paper, amino acids (1% solutions): tyrosine, serine, glutamine, glycine, arginine, glutamic acid and unknown, eluting solution (isopropyl alcohol, 0.5 M NH4OH; 0.2% ninhydrin spray).

Equipment: 600-mL beaker, pencil, ruler, evaporating dish, toothpicks, hair dryer, stapler and paper towels.

 

Safety

Wear safety goggles at all times. Use eluting solution only in the hood. Do not breathe fumes from the eluting solution. Be sure to handle only the dry part or the chromatogram when removing it from the beaker. Wash hands thoroughly if the eluting solution touches your skin.

 

Part A: Preparation of Chromatography Paper

1. Wash your hands thoroughly to remove excess oils from your skin. Obtain a ruler and a piece of chromatography paper from the fume hood. Handle the paper only on the edges to avoid leaving fingerprints, as these may hinder the elution process.
2. Place the chromatography paper on a sheet of clean notebook paper or paper towel to avoid picking up dirt or contaminants from the bench top. Orient the paper into a “landscape” position and write your name on the top edge of the paper in one corner. Using a pencil and ruler to measure accurately, draw a straight line across the paper, about 1.5 cm above the bottom edge. This is the starting line. At the top of the paper draw a straight line across the paper about 2.0 cm from the top, this is finish line.
3. On the starting line, measure in from one side about 2.5 cm and lightly draw a small “X” centered on the starting line. Draw six more, 1.5 cm apart.
4. In the center of each X, make a small spot of amino acid sample in this order: tyrosine, serine, glutamine, glycine, arginine, glutamic acid and unknown.
5. When you have finished, you should have something that looks like \ref{chrom} below.
6. Go back over each amino acid spot a second time to ensure there is enough amino acid in the spot.
7. Gently curl the paper into a cylinder, with the spots on the outside. Staple the ends together near the top and bottom, taking care that the two edges of the paper do not touch. If they do touch, the eluent will creep on a diagonal, and the spots will run together, or not in straight lines.

Figure \( \PageIndex{3} \label{chrom} \): Prepared chromatography paper.

 

Figure \( \PageIndex{4} \): Chromatogram in solvent chamber

 

Part B: Acquisition of Chromatogram

1. Take a 600-mL beaker and pour 10-mL of 0.5 M NH4OH and 20 mL of isopropyl alcohol (eluting solution) into the beaker. Obtain your evaporating dish and use it to cover the beaker.
2. Gently place the paper cylinder into the beaker and cover the top with the plastic wrap. Remember that the spots must be above the liquid level for the experiment to work. Watch the eluent creep up the paper until it is about 2 cm from the top. It will take about 45-60 minutes for the solvent front to reach the finish line.
3. When the solvent front reaches the finish line, remove the paper from the beaker, being careful to touch only the top. Let excess eluent drip into the beaker. Gently remove the staple and lay the chromatogram on a piece of paper towel. Use a hair dryer to dry the chromatogram completely. Pour the eluting solution in the organic waste container under the fume hood.
4. Working in the fume hood, spray the chromatogram lightly with the ninhydrin solution. Dry the sprayed chromatogram with a hair dryer, distinct colored spots will appear as a result of the ninhydrin reacting with the amino acids.
5. CAUTION: Use the ninhydrin inside the fume hood. Do not breathe the fumes or get spray on your skin.

 

Part C: Interpretation of Chromatogram

1. Circle around each color spot.
2. Use a ruler and draw a plus sign in the center of each spot. Measure the distance from the starting line to each plus sign. Record this distance for each spot on your lab report. These are the DD values, in cm.
3. Measure the distance between the starting line and the finish line or, the farthest up that the solvent front reached. Record this distance. This is the FF value, in cm.
4. Calculate the retention factor (Rf) for each spot and record the values in your lab report.
5. You and your lab partner will hand in your lab reports at the same time, with the paper chromatogram stapled to one of the lab reports.