ASPARTAME The Study of a Peptide Bond INTRODUCTION Aspartame, commonly known as NutraSweet or Equal, is the most popular artificial, low-calorie sweetener available to consumers today. Chemically, aspartame is much different than sucrose, the natural sugar. Its chemical name is L-aspartyl-L-phenyl-alanine methyl ester; it is categorized as a modified dipeptide consisting of two amino acids, aspartic acid and phenylalanine. The phenylalanine possesses a methyl ester group on its carboxyl end. Weight for weight, aspartame is almost 200 times sweeter than sucrose. Aspartame is used in almost phenomenal amounts, where an estimated 100 million Americans consume the artificial sweetener every year. Furthermore, approximately 5 million pounds of aspartame are sold each year at a price of $20 per pound. Since the patent for NutraSweet expired in December of 1992, prices are expected to decline in the future. The use of aspartame does register some drawbacks. First of all, aspartame seems to have a relatively short shelf life since it can be converted to a tasteless cyclic form in solution in as little as 3 months. 1 Second, aspartame is not stable in the presence of heat or strongly acidic/basic media. Aspartame's use as a calorie control substance is somewhat deceiving. Research shows that the intake of this artificial sweetener can produce a craving for carbohydrates. Furthermore, many research experiments have led to the agreement that aspartame does not aid in weight loss or weight control. In essence, the term "diet" in reference to the use of this low-calorie sweetener is misleading. The most serious drawback concerning aspartame is the question of its safety, a topic under great debate. The proposed health risks range from headaches and seizures in humans to brain tumors in rats. 2 By July of 1988, 4 years after its approval, the Food and Drug Administration received over 6000 complaints concerning side effects (mainly headaches) from the consumption of the sweetener. Only 251 reports of seizures were brought to the attention of the Adverse Reaction Monitoring System, a subgroup of the FDA. 3 The conclusion reached after the evaluation of these cases was that the occurrences of seizures were not decisively linked to the intake of aspartame. In the end, if the hydrolysis of aspartame is studied, doubts about its safety can be better understood.
Aspartame breaks down into its respective amino acids and methanol when it is metabolized in the body. Methanol is normally toxic to the body, but research has shown that the amount formed is so small that idoes not cause any harmful effects. Other artificial sweeteners available now to consumers include saccharin and sorbitol. Sorbitol is similar in structure to glucose, a monosaccharide. Instead of possessing an aldehyde group in its structure, sorbitol is a polyalcohol. The use of saccharin is hampered by its controversial cancer link. Sorbitol also has its limitations due to the symptoms that can appear with heavy consumption. These include abdominal cramps, intestinal bloating, and diarrhea. Furthermore, unlike its competition, it is NOT noncaloric. The newest artificial sweeteners that are clost to being approved or found some limited approval by the FDA include Sunnette (noncaloric, 200X sweeter than sucrose); Sucrolose (chlorinated sucrose derivative, 600X sweeter than sucrose); and Alitame (amino acid derivative, 2000X sweeter than sucrose.) An aspartame solution and two hydrolyzed aspartame solutions will be test for the presence of an alcohol. Remember that the methanol is a product in the hydrolysis of aspartame. The ceric nitrate reagent turns from yellow to reddish orange in the presence of alcohol. The potassium permanganate reagent acts as an oxidizing agent. A positive test form methanol is indicated by a change in color from purple to brown.
The following laboratory experiment will involve the technique of thin layer chromatography (TLC). Chromatography is a technique sued widely to purify, to separated or to identify substance. While several types of chromatography are available, the theory behind each type is the same. Chromatography involves two phases; the stationary phase that works to adsorb the substance to some extent, and the moving phase which works to move the substances along the stationary phase. The technique works on the concept that compounds of similar structure or polarity attract and hold together. For example, if the stationary phase is made up of a very polar compound/s, a substance being tested that possesses polar groups will tend to be held to this stationary phase. Polar substances, in this will not be carried will by the moving phase. Non-polar substances, on the other hand, would move easily along a polar stationary. They would not be readily adsorbed by the polar stationary phase. TLC can utilize commercial chromatography plates that have some type of chemical coating that functions as the stationary phase. The plates for this experiment possess acetylated cellulose coating. The substances to be tested will be spotted on this plate. Then the plate will be set in a covered jar that contains a small amount of solvent. This is called the developing chamber. The solvent makes up the moving phase, as it functions to carry the substances up the TLC plate. When the solvent nears the top of the plate, the plate is removed from the jar and solvent front (the place where the solvent stops) is marked. The spots represent how far the substances traveled and should be marked or circles lightly with a pencil. By knowing the distance the solvent moves and the distance that each substance moves, Rf values can be calculated. Rf values have no units and are solvent dependent. Rf = Distance substance traveled Distance solvent traveled PROCEDURE 1. Prepare a chromatography chamber, 400 ml beaker and enough solvent (n-butanol, glacial acetic acid, and water in a 6:2:2 ratio) so that the level is 0.4-0.6 cm high. The developing chamber should be kept under the hood. Cover the beaker with parafilm immediately after adding the solvent. Let it sit for several minutes to become vapor saturated. 2. With gloves on, obtain of chromatography paper. HANDLE THEM ONLY AT THE EDGES TO AVOID CONTAMINATION!!! 3. Draw a light pencil line 1 cm up from the bottom of the plates which is 5 cm across. This is your line of origin. Mark 9hash marks lightly on the line, equally spaced on the paper. 4. Obtain each of the solutions in Table 1 on a spot plate.
Table 1 Solution Preparation_ Solutions Description 1 Aspartame-prepared 2 Phenylalanine prepared 3 Aspartic acid prepared Base-hydrolyzed aspartame: prepared 4 Dissolve 12 mg of aspartame in 20 ml_ of water and 10 ml of acetone; add 10 drops of 6 M NaOH, stir and heat for 60 minutes; add H 2 O, if necessary, during the heating. Beverage "A" (diet soft drink): prepared 5 Measure 5 ml of diet soft drink and add 10 drops of ethanol. Beverage "B": prepared 6 Measure 10 ml of diet soft drink into a 50 ml beaker, add 10 drops of 6 M NaOH and stir for 20 minutes; divide in half; add 10 drops ethanol to one half this is used for TLC only. Beverage "C": prepared 7 Measure 20 ml of diet soft drink into a 50 ml beaker; add 10 drops of 6 M NaOH and heat to a boil for 1 5-20 minutes; let cool anddivide into 2 portions. Add 10 drops of ethanol to the portion that will be used for TLC. Beverage "D" ("aged" aspartame-sweetened beverage): prepared 8 Measure 5 ml and add 10 drops of ethanol. 5. Spot the TLC plates according to Table 2. Make sure that the spots dry before applying more solution. Remember that the beverage and aspartame solutions should contain ethanol to facilitate the spotting procedure. The ethanol-spiked solutions should not be used for the ceric nitrate and potassium permanganate tests. Table 2 Spotting descriptions Spot No. of spotting times Solution Paper #1 #1 6 Aspartame (solution #1) #2 6 Phenylalanine (solution #2) #3 6 Aspartic acid (.solution #3) #4 12 Hvdrolyzed aspartame (solution #4) #5 12 Beverage "A" (solution #5) #6 12 Beverage "B" (solution #6) #7 12 Beverage "C" (solution #7) #8 6 Aspartame (solution #1) #9 12 Beverage "D" (solution #8)
6. Align the bottom edges so that they are flushed. Staple the edges without overlapping Place paper cylinder in the developing chamber Make sure that the solvent is below the line of origin. Be careful that the solvent does not cover the 1 cm line. Cover the chamber immediately after the placement and try not to disturb the chamber. 7. When the solvent reaches approximately 1-2 cm from the top, remove the plate, MARK THE SOLVENT FRONT, and let the chromatography paper dry under the hood. 8. Carefully spray the plate with a ninhydrin solution. Then place the plate in an oven at 100 C for a few minutes. 9. Measure the distance the solvent moved as well as the distances moved by each substance (to the middle of the spots). If more than one spot is seen, measure the distance for each spot. Calculate Rf values. Turn in your paper chromatograph with your data sheets. 10. KMnO 4 Test. While the chromatogram is being developed, the oxidation tests can be performed. Using a dropper, add 6 drops of fresh aspartame solution to one depression in a spot plate, 6 drops of your cooled, hydrolyzed aspartame solution (solution #4) to another depression, and 6 drops of the base-hydrolyzed beverage solution (solution #6) to a third depression. To a fourth depression, add 6 drops of a 50% methanol solution. Add 6 drops of distilled water to a depression to use as a blank. Add 6 drops of an "old" aspartame-sweetened beverage (solution #8) to another part of the spot plate. 11. To each of the five substances, 2 drops of KMnO 4 reagent and stir. Notice any change in color that takes place over a 15-minute time period. 12. Ceric Nitrate Test. Place 5 drops of the ceric nitrate reagent into each of four depressions on a spot plate. To the first of the three, add 5 drops of distilled water. Add 5 drops of the freshly prepared aspartame solution to the second depression and add 5 drops of the hydrolyzed aspartame (solution #4) to the third area of the spot plate. Finally, to the fourth depression add 5 drops of 50% methanol. Record the observed colors on your data sheet