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Amino Acids

SEPARATION OF AMINO ACIDS BY THIN LAYER CHROMATOGRAPHY


PURPOSE: To understand the concepts of chromatography and to identify unknown amino acids. CAUTION: 1) Refrain from prolonged exposure to the mobile phase solvent (1-butanol, glacial acetic acid and water). Keep the cover on the developing tank when not adding or removing a plate. The 1-butanol will evaporate. 2) Ninhydrin will stain your skin. Wear gloves to protect yourself from contamination and from contaminating your silica gel plate. BACKGROUND The discovery of chromatography in 1944 revolutionized the separation and detection of amino acids and dipeptides. The separation is based on the liquid-liquid partition of the compounds between two immiscible phases. Initially the separations were primarily conducted on filter paper and were called paper chromatography.

M A T E R I A L S NE E D E D
silica gel plate mobile phase: 1-butanol, glacial acetic acid and water (4:1:1) known solutions of amino acids unknown solutions of amino acids micropipette developing tank 2% ninhydrin solution heat gun pencil gloves

In paper chromatography the hydrated cellulose fibers of the paper act as the stationary phase. A polar solvent ascends in the vertically held paper by capillary action and is the mobile phase. In thin layer chromatography (TLC) a thin uniform layer of silica gel acts as the stationary phase. TLC is replacing paper chromatography because the plates are easier to use than the paper, they give a sharper separation and the amino acids or dipeptides can easily be collected from the plate. Many microscopic distributions of the compounds occur between the mobile and the stationary phases. In time equilibrium is established between the two phases and the more soluble compounds move farther along the plate; different compounds move

Amino Acids

different distances from the origin. The plate is dried, sprayed with a ninhydrin solution and heated in order to locate the amino acids. The ninhydrin reacts with the amino acids to form colored products. The ratio of the distance moved by the amino acids to the distance moved by the solvent front from the original spot on the paper is defined as the Rf value and is characteristic of the compound. R f compound = distance traveled by compound distance traveled by solvent Rf values depend on several factors: type of silica gel plus binder used, water content of the thin layer, concentration of solute, temperature, manner of development and distance of the starting point from the solvent. Known compounds are usually run on the same plate as the unknowns to assist in identification of the unknowns rather than relying solely on published Rf values. PROCEDURE 1. Put gloves on. If your developing tank isn't prepared, add enough solvent to a depth of approximately 1 cm or less. Get your silica gel plate. Carefully hold the plate by the sides to prevent disturbing the silica gel layer. Draw a pencil line about 1.5 cm from the bottom plate. 3. Mark one point on the line for each one of your known and unknown solutions. (If you have four known solutions and 2 unknowns, mark six points.) Leave margins of at least 1.5 cm on both sides. Number each point. 4. At point number 1 apply a very small drop of one of your known solutions. Do not wet

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Amino Acids

the silica beyond a diameter of 2-3 mm. Locating the center of large spots will be difficult later when the spot has moved along the paper. 5. After the liquid has evaporated (only a few seconds), add a second drop to the same spot. Record the name of the amino acid and the number of the spot. Repeat this procedure for the remaining solution. Remember to record the name of the amino acid or unknown number and the number of the spot. Allow all the spots to dry completely. Place your TLC plate in the developing tank with the mobile phase with the spots toward the bottom. Allow the solvent to ascend the silica gel to at least 3/4 of its height, which will to require 1 hour or less. (The farther the solvent ascends, the greater the separation. Immediately remove the plate, if the solvent reaches the top.)

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10. Remove the plate and quickly mark the farthest advance of solvent front with a pencil, unless it reached the top of the paper. 11. Dry the plate with a heat gun. Be careful to move the heat gun around and not heat one point continuously. Do this procedure in the hood. 12. Spray the plate with the 2% ninhydrin solution in the hood. 13. Do not allow the ninhydrin solution to stream down the plate, because this may move some of the compounds. 14. Dry the plate again with the heat gun. Do not over heat the plate. Long heating times may cause browning of the plate over the entire surface. 15. Circle each colored spot with a pencil. The ninhydrin spots fade gradually, so circle at once. 16. Measure the distance from the origin to the center of each colored spot and calculate the Rf values for all spots.

Amino Acids

17. Record the Rf values and the color of each ninhydrin spot. 18. Identify the unknown amino acids. QUESTIONS 1. Why does touching the silica gel with your hands potentially contaminate your plate? Why can an Rf value never be greater than 1? What would happen if so much solvent was used (mobile phase) that the original spots were covered with solvent? What would happen if you made the line and points with an ink pen rather than a pencil?

C a l c u l a t i n g Rf V a l u e s solvent front

ninhydrin spot

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You dropped and mixed up your samples. You know that one contains only valine, one contains valine and glycylvaline, one contains valine and alanine, one contains only glycine and valine, and one contains glycine and glycylleucine. How would you determine what your samples are using TLC and the data below? Can you figure out what they all are?

original line

R f Values for Amino Acids and Dipeptides Compound glycine alanine glycylvaline valine glycylleucine Rf 0.26-0.29 0.39-0.42 0.62-0.66 0.62-0.64 0.76-0.80 Color purple purple gray purple light brown

Rf values taken two days after solvents were mixed and with solvent advance 100 mm in 43 minutes at a temperature of 31 C.

denominator

numerator

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