Chromatographic Study of Aspartame Degradation

Jordan F. StaceyWest Liberty University

Economically- Commonly known as Nutrasweet or Equal- Most popularly used artificial sweetener- Estimated 100 million Americans consume artificial sweeteners

daily- About 5 million pounds of aspartame are sold annually at $20

per lb

Aspartame

Chemically- Much different than sucrose (natural sugar)- Modified dipeptide consisting of two amino acids

- Aspartic Acid- Phenylalanine

- Weight for weight Aspartame is almost 200x sweeter than sucrose

- Relatively short shelf life (3 months)- Non stable in the presence of heat or strong acid/base

concentrations

Aspartame

Health- FDA approved in 1984- Originally used as a calorie control substance- Further research shows intake of aspartame can

cause cravings, specifically for carbohydrates- Some proposed health risks associated with

consumption

Aspartame

In Class

- Analyzing the break down of an aspartame solution, as well as two hydrolyzed aspartame solutions by testing for the presence of an alcohol (Methanol)

- Thin Layer Chromatography- Two sets of tests using the same prepared solutions- Ceric Nitrate Test

- Yellow Reddish orange in the presence of alcohol- Potassium Permanganate Test

- Acts as an oxidizing agent - Purple Brown in the presence of alcohol

Thin Layer Chromatography

- TLC is a chromatographic technique used to separate the components of a mixture

- Uses a thin stationary phase that is supported by an inert backing

- It can be done a monitoring scale- Monitoring the progress of a reaction

- Can also be used on a preparative scale- Purifies small amounts of a compound

- TLC functions on the same principle as all chromatography- A compound will have different affinities for the

mobile and stationary phases and this affects the speed at which it migrates

Thin Layer Chromatography

- TLC uses commercial chromatography plates- The substances that are being tested will be spotted

on this plate- Plates have a chemical coating that functions as the

stationary phase- Acetylated Cellulose Coating

- Plate is then set in a covered jar with a specific solvent

- The solvent is used as the mobile phase - Carries the substances up the TLC plate- TLC Plate + Jar = Developing Chamber

Thin Layer Chromatography

- Once solvent nears the top of the plate, the plate is removed

- Solvent front is marked- Spots are marked to show how far the substances

travelled- Retention Factor (Rf) values are calculated - Rf = Distance substance traveled/Distance

solvent traveled- Retention factors have no units and are dependent on

the solvent at hand

Thin Layer Chromatography

PROS• Simple• Inexpensive• Little materials needed• Can be applied to other

techniques once best solvent is found

• Solvents for plate can be changed easily

• Easy to check purity under UV light

• Rf values allow for simple identification

CONS• TLC plates do not have

long stationary phases• Length of separation is

limited• Detection level is

higher• TLC operates as an

open system and is subject to outside influences (humidity, temperature, etc)

Common Errors in TLC

- Streaking- If sample is too concentrated, the substance will

travel up the plate as a streak versus a single separated spot

- Over-large Spots- Spotting sizes of the sample should be about 1-2

mm in diameter- Uneven Advance of Solvent Front

- No flat bottom- Enough Solvent- Evenly cut plate

- Spotting- Sample should be ABOVE the solvent level, if

solvent is above the sample, samples will be washed off into the solvent

Experiment

1. Prepare Chromatography Chamber using TLC jar/400mL beaker

2. Add solvent (6:2:2 ratio of n-butanol, glacial acetic acid, and water) to a line 0.4-0.6 cm high from the base of the jar

3. Cover jar and place in a hood to allow for the jar to be vapor saturated

4. Obtain 2 previously cut TLC plates (10 x 5 cm) DO NO TOUCH THE PLATE FRONTS

5. Pencil line 1 cm from bottom of plate with 4 equally marked spots on plate one, and 5 on the second

Experiment

6. Obtain previously prepared solutions 1. Aspartame 2. Phenylalanine3. Aspartic Acid4. Base Hydrolyzed Aspartame5. Diet Soft Drink (Bev. A)

1. 5 mL + 10 drops ethanol6. 10 mL Bev. A + 10 drops of 6M NaOH 20 minutes

divide add 10 drops ethanol to ½ for TLC (Bev. B)

7. 20 mL Bev. A + 10 drops of 6M NaOH boil 15-20 divide add 10 drops ethanol to ½ for TLC (Bev. C)

8. Aged Aspartame-Sweetened Beverage (Bev. D) 1. 5 mL + 10 drops ethanol

7. Spot Plates (Plate #1 1-4)(Plate #2 5-8, 1)8. Add TLC plates to vaporized chamber

Experiment

9. Remove plated once solvent reaches 1-2 cm from the top10. Mark Solvent front and let dry under the hood11. Spray plates with ninhydrin solution (or stain)12. Place plate in the oven at 100 C for a few minutes13. Measure the distance of both the solvent and the substances 14. Conduct the KMnO4 test15. Conduct the Ceric Nitrate Test

KMnO4 Test

1. Prepare fresh aspartame solution 8mg Aspartame in 10 mL H2O

2. Add 6 drops of solution to one depression in the spot plate

3. Add 6 drops of solution D to another depression4. Add 6 drops of Bev. B to third depression5. Add 6 drops 24% methanol solution to fourth

depression6. In fifth depression, add 6 drops distilled water (blank)7. To each of the 5 filled depressions, add 8 drops of

acetone followed by 2 drops of KMnO4 reagent stir8. Over 15 minutes, note any change in color to the

solutions

Ceric Nitrate Test

1. Add 5 drops of the Ceric Nitrate reagent (previously prepared) into each of four depressions

2. To the first 3 depressions, add 5 drops of distilled water3. To the second depression, add 5 drops of the freshly

prepared Aspartame4. Add 5 drops of solution D to the 3rd depression5. Lastly, add 5 drops of 24% methanol to the fourth

depression6. Record color changes

Works Cited

1. Chromatographic Study Of Aspartame Degradation: The Study of a Peptide Bond, In Class Handout, West Liberty University, November 2013

2. UC Davis Chemwiki, Thin Layer Chromatography, University of California, November 2013