2. 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
3. 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
4. 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
5. 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
6. 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
7. 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
8. 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
9. 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)
10. 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
11. 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
12. Experiment
6. Obtain previously prepared solutions
1. Aspartame
2. Phenylalanine
3. Aspartic Acid
4. Base Hydrolyzed Aspartame
5. Diet Soft Drink (Bev. A)
1. 5 mL + 10 drops ethanol
6. 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
13. Experiment
9. Remove plated once solvent reaches 1-2 cm from the top
10. Mark Solvent front and let dry under the hood
11. Spray plates with ninhydrin solution (or stain)
12. Place plate in the oven at 100 C for a few minutes
13. Measure the distance of both the solvent and the substances
14. Conduct the KMnO4 test
15. Conduct the Ceric Nitrate Test
14.
15. 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 depression
4. Add 6 drops of Bev. B to third depression
5. Add 6 drops 24% methanol solution to fourth depression
6. 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 stir
8. Over 15 minutes, note any change in color to the solutions
16. 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 water
3. To the second depression, add 5 drops of the freshly prepared
Aspartame
4. Add 5 drops of solution D to the 3rd depression
5. Lastly, add 5 drops of 24% methanol to the fourth depression
6. Record color changes
17. 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
