Introductory Biochemistry (Chemistry 330)

This page contains some theory on vitamins, particularly ascorbic acid (vitamin C), questions to guide experiments and a procedure and the materials for determining its concentration in fluid extracts. An appendix is available with help on pH, logs and the pH meter. You can also leave me a message, go to my home page, or go back to the lab schedule. 

Vitamins are essential carbon compounds which heterotrophs must obtain from their diets, though only in minute amounts because they are cofactors for enzymes. Occasionally an animal can synthesize part of its requirement (Vitamin D in man), and often symbiotic organisms provide part or all of the requirement (the B vitamins by gut microbes).

There is no such thing as a general vitamin, but only ones for specific species. Ascorbic acid (vitamin C) is required for only a few mammals like guinea pigs, fruit bats and man. Deficiency causes scurvy, a disease where the structural protein collagen can't be finished, and sufferers literally fall apart. In addition, large doses of 0.25 g or more a day have been shown to alleviate the symptoms of the "common" cold. Whether such doses help prevent cancer and other diseases is still debated,¹ and there is a risk of kidney and bladder problems because vitamin C is acidic and most of it is excreted.

Assays often use the fact that ascorbic acid is an "anti-oxidant," (that is, a reducing agent) which may cause its reported health benefits. It is readily oxidized by the dye 2,6-dichloroindophenol (DCP). The reaction is:
 

With this reaction, one can assay various foods, serum or urine for vitamin C content, or determine the best methods of food preparation to preserve it. For example, if you want to test for excretion of excess vitamin C, you may have to arrange to take urine samples and store them in a stable form both before and after taking supplements. Whatever you choose to investigate, you need to prepare a standard curve and use it to determine vitamin C (actually, all similar reducing agents) concentrations in your samples.

1. Is the vitamin content the same in fresh, frozen and canned juices?
2. Does vitamin C content vary in different parts of a fruit or vegetable?
3. Do you excrete most of the vitamin C you ingest?
4. Which sources are high (low) in vitamin C? Is the name "Hi-C" false advertising?
• How much of each food you tested must you consume to prevent scurvy? (10 mg/day)
• How much to meet your minimum daily requirement? (Women = 55 mg/day; Men = 60 mg/day)
5. Does cooking destroy the vitamin C? Does the metal in the pan affect this?
6. Do raw, boiled, steamed , fried or baked vegetables retain more vitamin C?
7. What methods of preparation and storage best preserve vitamin C content?

Procedure: (Microscale Titration - Standard Curve)

1. Obtain about 20 mL of dye in a small, very clean beaker.
2. Obtain about 2 mL of each ascorbic acid standard (usually 1.0, 0.5, 0.25 and 0.125 mg/mL solutions in 1% oxalic acid) in small test tubes that are very clean and dry.
3. With a Beral pipet, carefully transfer 0.5 mL of the standard with the lowest concentration into each of 3 wells out of the 6 on one side of the plate.
4. With another Beral pipet filled with DCP, add drops of dye to a well of sample while carefully counting drops and stirring with a toothpick. The end point is when the dye stops becoming colorless and stays a light pink for at least 30 seconds.
5. Repeat the titration for each of the other wells, and for the other standards available, in order of increasing vitamin C concentration. See the figure below.

 

 



6. Plot the average drops of DCP used (ordinate = Y-axis) against the concentration of vitamin C in mg/mL.

Each team will need 2 or 3 Beral pipets, flat toothpicks and a 24 well plate (Costar® or Falcon®). To prepare their own standards from vitamin C tablets, teams will need appropriate volumetric flasks to prepare dilutions. Each team may also need a mortar and pestle to extract juices.³
 

Reagents

1. 1% oxalic acid: Dissolve 5 g (Sigma #O-0505, oxalic acid dihydrate, ACS Reagent) in 500 mL of distilled or deionized water.
2. DCP: Dissolve 0.200 g 2,6-dichloroindophenol (DCP; Eastman #3463) in 0.5 L of distilled or deionized water. This is enough for two lab sections. Store refrigerated, if possible.⁴
3. 1 mg/mL ascorbic acid: Dissolve 0.100 g L-(+)-ascorbic acid (Eastman #4640) in 1% oxalic acid and dilute to 100 mL in a volumetric flask.⁴
• Or, dissolve a 100 mg tablet in water, filter to remove debris and add 1% oxalic acid.
4. Dilute 1:1 with 1% oxalic acid to obtain 0.5, 0.25 and 0.125 mg/mL solutions. 50 or 100 mL of each dilution is enough for a lab section.

These solutions can be used for at least a week if stored in a refrigerator.³
5. Clean Up: Swish the plate and pipets in hot, soapy water; then rinse thoroughly in tap water and deionized water (3 times each). Return materials where you found them.