Amino acids are the building blocks of proteins. They are used to build tissues, enzymes, skin, hair and many other functions in our bodies. The structure of every amino acid is similar in that they contain an amino group (-NH2) and a carboxylic acid group (-COOH). Amino acids have different side groups attached which are called R groups. The R group determines whether the amino acid is polar, non-polar, acidic, basic, or neutral.
Chromatography is a technique used to separate and identify components in a mixture. In today’s experiment, you will be separating the amino acids present in the artificial sweetener aspartame (Nutrasweet). In chromatography, a solvent is allowed to flow up a paper chromatogram like a wick. The solvent is the mobile phase because it moves, and the paper is the stationary phase because it does not move. When the amino acids are "spotted" on the chromatogram, they will travel up the chromatogram with the solvent at different speeds depending on their polarity/solubility. The amino acids that are more soluble in the solvent will move higher on the paper. The amino acids that are less soluble in the solvent will be more attracted to the paper and will remain closer to the original starting point. As a result, a mixture of amino acids can be separated using this technique.
Rf Values: The Rf value is calculated from the chromatogram and can be used to help identify unknowns. The Rf value is the relationship of the distance traveled by he sample compared to the distance traveled by the solvent.
Rf = distance traveled by the sample
distanced traveled by solvent
To determine the distance traveled by the sample, measure the distance in centimeters from the starting line to the center dot of each spot. To determine the distance traveled by the solvent, measure the distance (in cm) from the starting line to the solvent front line. Note: You will have two Rf values for the NutrasweetÒ sample since it contains two amino acids.
In the example above, the solvent front traveled 8 cm, the one component in A traveled 4 cm, and the one component in sample B traveled 5 cm. By comparing the Rf values for each sample, it can be seen that component C1 is identical to sample A and that component C2 is identical to sample B. Assuming that the identity of sample A and B are known, then the identity of the two components in sample C can be determined.
A. Chromatography of Amino Acids
1. Preparation of Developing Chamber:
Place 15 mL of developing solvent into a 400-mL beaker.
Cover the beaker with plastic wrap and secure it with a rubber band. This is your developing
chamber. Label the beaker with your name and place it in the hood.
2. Preparation of Chromatogram:
a. Using plastic gloves, obtain a piece of
Whatman No. 1 chromatography paper. (Be sure not to
touch it with your bare hands because amino acids from your skin can be transferred to the
paper.) Use a pencil to draw a line across the paper that is about 2 cm from the long edge of
the paper (see diagram below). Make a small "x" at eight points along that line equal distance apart
and label them as shown in the figure below. Put your initials in the upper right-hand corner of the paper.
b. Sample Application: Using the capillary tubes
provided, apply a small amount of each of the
following amino acids to the appropriate "x" (Your instructor will demonstrate the proper
technique for applying the samples to the chromatogram.):
an unknown (Ukn) (Be sure to record your unknown number!)
aspartic acid (Asp)
glutamic acid (Glu)
3. Developing the Chromatogram: Once the sample spots
dry, roll the paper into a cylinder and
staple the edges. Do not let the edges overlap. Stand the cylinder next to the beaker and check
to see that the sample spots are higher than the level of the solvent in the beaker. If the solvent
level is higher than the level of the spots, use a pipette to remove some of the solvent from the
beaker until the level is lower than that of the spots. Remove the plastic wrap "lid" and carefully
lower the paper cylinder into the developing chamber (beaker) with the spotted edge down. Do
not allow the paper to touch the sides of the beaker. Cover the beaker with the plastic wrap and
leave undisturbed. Allow the solvent front to rise until it is approximately 2-3 cm from the top
edge of the paper. Do not let the solvent run over the top of the paper.
4. Visualization of Amino Acids: When the solvent level is
2-3 cm of the top edge, remove the
paper from the developing chamber. Remove the staples and spread the chromatogram out on a
paper towel. Immediately, mark the solvent front with a pencil. Allow the chromatogram to dry
completely. In the hood, spray the paper lightly with ninhydrin solution. Allow the solution to dry
completely. You will be able to see distinct colored spots where the ninhydrin reacted with the
amino acids. (The NutrasweetÒ should show two spots.)
5. Recording your data: Outline each spot with a pencil and
a dot at the center of each spot.
Record the color of each spot. From the chromatogram, calculate the Rf values for the known
amino acid samples, the unknown, and the NutrasweetÒ spots.
6. Identifying your Unknown Amino Acid: Compare the
and Rf values of your unknown
amino acid to those of the known amino acids. Identical amino acids will travel the same
distance, have similar Rf values, and form the same color with ninhydrin. From these
comparisons, identify your unknown amino acid. In the same manner, identify the two amino
acids present in the NutrasweetÒ .
B. Using your text book as a reference, draw the structures of
the amino acids you used in this
experiment. Label them as polar, non-polar, acidic, basic, or neutral.
Issues to be addressed in your conclusion...
What order did the known amino acids travel up the chromatogram.
Do you see a trend between the order that the amino acids traveled and their polarities?
Explain how you determined the identity of your unknown.
What amino acids are present in the NutrasweetÒ