Introductory Biochemistry (Chemistry 330)
Paper Chromatography

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This page contains some theory on chromatography, and procedures for doing cylindrical and circular paper chromatography and cellulose thin layer chromatography (TLC) of amino acids. The materials and equipment needed are listed at the end. You can also leave me a message, go to my home page, or go back to the lab schedule.

A number of modern analytical and preparative techniques are called chromatography ("color writing"). What they have in common is the separation of mixed samples using the components differing interactions with a stationary phase and a moving phase. These phases may be solid/liquid, liquid/liquid, solid/gas, etc. In paper chromatography, for example, amino acids can be separated because of their differing solubilities in the water of hydration around cellulose fibers and in solvents moving through these fibers. The relative solubilities can be changed by altering the polarity of the solvent or its pH, which will change the ionic state of samples. Under a suitable set of conditions, then, each molecule in a mixture will move at a different rate over the stationary phase and be at a unique distance from a common starting point, when solvent movement ceases. This fact can be conveniently expressed as a retardation factor (R_f), defined as follows:

Thus, if two samples of the same molecule (one known and the other unknown) are analyzed under identical conditions, both will have the same R_f. This ratio will also differ (hopefully) from the R_f for any other molecule present, supporting the identification of the unknown. A basic principle here is that difference is proof that the samples are different, but identical values only support identity, since two different structures may have the same R_f under one set of conditions. One practical example of how this principle is used is to understand the structure of proteins.

Proteins are polymers built from more than twenty different types of amino acid subunits joined by peptide (amide) bonds. These amino acids differ only in the chemistry of their side chains, which can range from ionic to polar to nonpolar, and acidic to neutral to basic (see below). Thus, the different properties and functions of proteins are the direct result of different amino acid sequences. Therefore, to understand a protein, we must first be able to determine which amino acids it contains and the order in which they occur. To do this we must be able to separate complex mixtures of amino acids and identify them. An early but still useful technique for this is paper chromatography.

PROCEDURES:

Prepare sample applicators by heating the center of a glass capillary in the edge of a small flame until it softens. Remove from the flame and pull quickly and firmly. Break the fine tube to produce two microtipped capillaries. Make enough applicators for your unknowns and standards, but share capillaries for the standards with your colleagues to cut waste.

Cylindrical Paper Chromatography

For cylindrical chromatography, you should use 2 (6 x 15 cm) sheets of filter paper, one for solvent A and one for B. Samples should be spotted on a line 4.5 cm from the top and 1 cm apart. Holes are punched on each side of the samples to improve resolution. The strips will be stapled into cylinders, and developed in 250 mL beakers with a watch glass or petri dish as a lid.

CAUTION: Don't touch the analytical area with your fingers (wear gloves!). They secrete amino acids, proteins, etc. which react with ninhydrin.

1. Add 20 mL of each solvent into a separate 250 mL beaker and cover with watch glasses. Label them "A" or "B" to match the solvent used.
2. Obtain 50-100 µL samples of any standard amino acids in a labeled spot plate, if desired. Share these knowns with you partners.
3. Label the side of each paper with your name and the solvent to be used (A or B).

Draw a light line 4.5 cm below the top and make a row of light dots 0.5 cm apart on this line with a pencil. Punch out every other dot with the hole's center slightly below the line and dots. Clamp the papers to the metal rack, if provided.

Apply 1-2 µL of each sample (2-4 applications) on (or above) the dots selected, drying (hair drier?) between applications. The final spots should be as small as possible for the best resolution. Apply twice as much of an unknown. Dry the sample spots with a hair drier or oven. After sample application, pull the paper over an edge to make it curl into a cylinder, and staple with the edges just touching, but not overlapped. (Gloves!) Briefly hold each paper over a steam bath to ensure hydration of the fibers. Develop as shown here and described below. To check procedures and colors, use a scrap strip of paper. Test the applicators and your technique by applying samples in widely separated and labeled positions. Then, while the analytical papers are developing, dry the test strip, spray it with ninhydrin, and heat it to develop the colors. Correct any problems before doing the developed papers.

1. Lower each paper into their beaker of solvent and cover with the watch glass. Note the time. The surface of the solvent must be below the sample spots!
2. Remove the cylinder (in the hood!) when the solvent reaches the top of the paper. (If you remove it early, mark the solvent front immediately so you can measure for the R_f calculations.) Note the time(s).
3. Dry completely (hood, then oven) until you can not detect the odor of HAc or ammonia.
4. Note the times and temperatures used.

1. Spray each paper until visibly damp, but not wet, with 0.2% ninhydrin.
2. Allow to dry in the hood, and develop the colors overnight at room temp., by heating with a hair drier or in an oven at 100°C for 5-10 minutes.

Protect the papers from contact with your hands before, during, and after analysis. Cover the important areas with magic tape to save them. Protect the developed papers from light. The colors will fade with time and exposure.

Never circle a spot; put a dot in the center of it for measuring.

Analysis:

1. Measure the distance from the application point to the center of the final spot, and the distance from the application point to the solvent front for each spot detected.
2. Calculate the R_fs of all spots on both papers.
3. To identify which amino acids are present in any unknown, use R_fs and colors.
Remember: The key is to exclude those that can not be present.
4. Present your data in a professional manner! Label everything! Clearly explain your reasoning for conclusions you draw!

1. Add 20 mL of each solvent, each into separate, glass petri dish lids and cover with watch glasses. Label them "A" or "B".
2. Obtain 50-100 µL samples of the standard amino acids in a labeled spot plate, if desired. Share these with your partners.

Find the center of two 11 cm diameter Whatman #l papers. Scribe a sample line with a 5 mm radius, and a solvent line with a 5 cm radius. Push the pin through the center to enlarge the hole for a wick, and label the side of each paper with your name and solvent letter. Clamp the filters to the metal rack, if provided.

Apply 1-2 µL of each sample (2-4 applications) on (or above) the dots selected, drying (hair drier?) between applications (The final spots should be as small as possible for the best resolution). Apply twice as much of an unknown.

Insert a wick of 6 cm of rug yarn, doubled over, with a flattened hair pin, and slip it off, leaving the loop on one side of the circle and the two ends on the other. (Wear gloves!) Dry the sample spots with a hair drier or oven, and briefly rehydrate over a steam bath. It is a good idea to use a scrap of paper to test your applicators, technique and reagents. Apply samples in widely separated and labeled positions. While the other papers are developing, dry it, spray with ninhydrin, and heat it to check procedures and colors.

Place the paper over the solvent with the wick down, and cover with a watchglass or petri dish lid. Note the time. Remove the circle (in the hood!) when the solvent reaches the outer line. (If you remove it early, mark the solvent front immediately so you can measure for the Rf calculations.) Note the time(s). Remove the wick by pulling the tails with a tissue (wear gloves). Dry completely (hood, then oven) until you detect no odor of HAc or ammonia. Record the times and temperatures.

Detection: See above.

Alternative: Circular Thin Layer Chromatography

Cellulose TLC sheets are cut into 10 x 10 cm squares. The center is found by laying a ruler from one vertex to the opposite one, first one way and then the other. The pin of a compass is pushed through the backing at the center and the other pin or pencil tip is used to gouge out the cellulose layer in a ring with a 5 cm radius. This results in a self limiting solvent barrier.

Other procedures are much the same as for the paper media above. A 5 mm radius sample line is then lightly scribed with the compass. The pin is then used to enlarge the center hole for the wick. First, apply the samples about every 5 mm. Then, a wick of yarn, doubled over, is inserted. The lids of glass petri dishes serve as both solvent chambers and lids. The loop on the plastic backing side of the TLC plate is cut off to remove the yarn wick. Development, detection and analysis are the same as for the paper media above.

Materials:
Equipment Needed:

• Glass capillary tubes, spotting racks and clips
• 6 x 15 cm sheets of Whatman #1 paper
• 11 cm Whatman #1 circular filter papers
• Cellulose TLC sheets (Eastman or ?) cut to 10 x 10 cm squares
• Station of chicken paper supplied with hole punch, 15 cm ruler, drawing compass, white rug yarn, flattened round wire hairpins and Cub® stapler
• Spraying station in hood with hair drier; an oven at 100°C
• 250 mL beakers, watch glasses, 10 cm diameter, glass petri dishes

Reagents Needed: (Go back to cylindrical chromatography procedures.)

1. Solvent A = 250 mL 1-butanol (n-Bu): 250 mL tertiary butanol (t-Bu): 100 mL conc. acetic acid (HAc): 200 mL distilled water (dH₂O)(5:5:2:4) and
2. Solvent B = 250 mL n-Bu: 250 mL t-Bu: 150 mL concentrated ammonia (NH₃): 150 mL distilled H₂O (5:5:3:3). Warm to room temp. and mix thoroughly before use.

0.2% ninhydrin - 2.0 g ninhydrin in 1 L 95% ethanol - store @ 5°C in dark bottle. 0.01% collidine may be added to improve stability.

Amino acid standards and samples: dissolve 0.010 g of each amino acid in 10 mL 0.10 M HCl (or 1.0 mM Na₂EDTA) = 1 mg/mL Lysine, Proline, Glycine, Aspartate, Tyrosine and Phenylalanine.

¹Helser, T.L. (1990) "Amino Acid Chromatography: The 'Best' Technique for Student Labs." J. Chem. Educ., 67, #11, 964-966.

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  Author of this page: Terry Helser - helsertl@oneonta.edu
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  Last Modified on 8/26/97