THE DETERMINATION OF ASCORBIC ACID (VITAMIN C) IN FOOD PRODUCTS
OBJECTIVE
Determine the contents of vitamin C (ascorbic acid) in various food products using small scale redox titration.
INTRODUCTION
It was as long ago as in mid eighteenth century that the beneficial effects of vitamin C were discovered. This is when citrus fruits rich in vitamin C were used to prevent scurvy in the sea sailors. Since then, vitamin C has been claimed a miracle agent in curing and preventing common colds and known as one of the anti-cancer antioxidants.
Pure ascorbic acid is a white, crystalline solid with a molecular formula C
6
H
8
O
6.
It is well soluble in water. Its main chemical property is that it oxidizes extremely easily in solution, thus reducing anything that can accept electrons. The oxidation process is impeded in acidic environment and at low temperatures. The process is accelerated by addition of light, heat, in basic solutions, and in the presence of metal ions such as Cu 2+ or Fe 3+ .
Chemically, ascorbic acid is similar to sugar molecules. In the body, it is oxidized to dehydroascorbic acid, the fully functional molecule affecting biochemical reactions.
To read more about vitamin C and its physiological role as well biochemical processes involving this nutrient click HERE .
Vitamin C is not produced or stored in the human body; therefore there is a need for a constant and regular supply of this vitamin in our everyday diet. Vitamin C is present in many fruits and vegetables, the best sources being broccoli, sweet peppers, parsley, Brussels sprouts, strawberries, papayas, kiwi, oranges, lemons, and grapefruits. Amazingly, potatoes are a reasonable source of vitamin C, particularly when cooked with skin on. Because vitamin C is so easily destroyed by the food processing techniques (due to its water solubility and reducing properties), you may find or ascorbic acid listed as an ingredient on a nutritional label of grocery items, as foods are frequently artificially enriched in this nutrient.
Analysis of vitamin C
Redox titration is used here to determine the vitamin C content in a sample. The oxidizing agent, iodine, reacts in an acidic environment with the reducing agent, ascorbic acid, to produce iodide ion and dehydroascorbic acid.
ascorbic acid dehydroascorbic acid
Rather than using tedious large -scale burette titrations, you will perform the redox titration using a small scale burette.
This technique is fast and easy and allows for comparison of the vitamin C content in many

different food items.
The procedure consists of two steps:
1) the standardization of I
2
solution
2) the analysis of a sample
After the analysis is completed some simple calculations are required.
In this experiment, you will determine the vitamin C content in a serving (typically 250mL) of various juices and /or concentrates. You may also choose to bring your own items, including some solids (breakfast cereals, vegetables, fruits etc.).
If you need to develop a technique for analyzing solids (for extra credit), remember that they need to be processed (crushed, chopped, mashed, etc.) and sometimes vitamin C must be waterextracted from them. You will also need the to know the exact mass of the food sample and/or the volume of extract prepared as well as the volume of an aliquot tested, in order to calculate the vitamin C content in a serving (e.g. 30 g is a serving for Total cereal, but will you test 30 g or less
?).
The amount of food sampled for testing will depend on the expected vitamin C content. The richer the food in vitamin C, the smaller sample can be taken to analyze using our method. The less vitamin C, the more sample must be used in order to obtain good results in titration. It may also be possible to keep smaller sample size of a food item lower in vitamin C, but use a less concentrated
I
2
solution.
PROCEDURE
A. Standardization
To standardize the iodine solution here means to determine how many drops of iodine solution are required to react with 1 mg of vitamin C. In every 1.0 ml of standard ascorbic acid solution used here there is 1.0 mg of vitamin C.
Therefore, the number of drops of iodine solution used to completely react with 1.0 ml of the standard solution is equivalent to 1.0 mg of vitamin C.
1. Prepare (and clean and dry if necessary) a plastic 24-well tray and a short stem beryl pipette
(=small scale burette).
2. From a provided burette, dispense 1.0 ml portions of the standard ascorbic acid solution into three wells of your well plate
3. Add 1 drop of 1 M sulfuric acid and two drops of starch solution to each well with standard solution.
4. Fill the short stem beryl pipette with provided iodine solution: Step 1 Step 2
5.
To titrate the first solution in the well plate: gently squeeze the small scale burette (tip down, above the solution in the well) to dispense one
drop of iodine solution at a time, stirring with a toothpick or small plastic stirrer after every addition, until the solution turns jet-black and the color persists for at least 10 seconds (this means that the iodine is no longer reacting with vitamin C and is forming a colored complex with starch = iodine indicator). Make sure to count all drops and record the number of drops of I
2 on your data sheet.
6. Repeat titration for the remaining two samples of solution.
B. Vitamin C in food
1. Use at least two different types of food or juices for testing (e.g. orange and lemon juice, or fresh orange juice vs. reconstituted from concentrate orange juice, or juice vs. green pepper, etc.). See
Table 1 for sample size and prep.

2. Perform titrations on two separate portions of the same sample (juice, etc.) and determine the average # of drops of iodine solution used.
3.
Record the manufacturer claim on vitamin C content per serving (check product label).
IMPORTANT: Since various foods contain different amounts of vitamin C, the amount of juice or solid taken for titration or the required concentration of the I
2
solution depends on how rich the food item is in vitamin C. If only 1 or 2 drops of the standard iodine solution are used for titrating a portion of food, you need to: a. ask the instructor to show you how to properly dilute the I
2
solution in a 3:1 ratio. However, I
2 solution will have to be standardized again, to find the proper number of drops per 1 mg of vitamin
C, OR b. use at least triple the sample size and repeat the titration with the original I
2
solution.
Table 1.
Sample Sample size/ preparation
fresh or reconstituted juice (lemon, orange and grapefruit)
2.0 g (or 2. mL juice)* +
1 drop of dilute sulfuric acid + 2 drops of starch
concentrate of juice, frozen, undiluted
1.0 g (or 1. mL) ** +
1 drop of dilute sulfuric acid+ 2 drops of starch
*Transfer juice with a plastic pipette directly to a 24-well tray tared on the balance to weigh it, or use a calibrated plastic pipette to deliver proper volume of juice to the well. Record the actual volume or mass of juice used.
** Use a spatula to transfer concentrate to a tared 24-well tray on a balance or, if thawed, use a pipette.
Vitamin C content of some solid food items and juices Vitamin C in fruit of the world
Clean up
Dispose of the solutions from the tray in an appropriate waste container. Wash all equipment used, using soapy water, rinse well and replace on the instructor's cart.
Calculations and report
Part A: Standardization
1) Calculate the average number of drops of iodine solution per 1 mg vit.. C:
Avg # drops per 1 mg vit C = (# drops well1 + # drops well2 + # drops well3) / 3 part B
2) Calculate the average # of drops of iodine solution used for titration of your samples:
Avg # drops B = (# drops for sample 1 + # drops for sample 2) / 2
3) Calculate the number of mg of vitamin C in a sample of food used:
mg vit. C in a sample tested = Avg # drops B / Avg # drops per 1 mg vit. C

4) Calculate the content of vitamin C in a serving (for juice = 250 ml):
vit. C in 250 ml juice = mg vit. C in a sample tested x (250 ml / volume of sample in mL)
Example: 0.5 mg vit C x (250 mL /2. ml) = 0.5 mg x 125 = 62.5 mg vit C/serving
4a)
For the concentrate:
find the serving size on the product label and convert it to grams = grams of concentrate
/serving.
Hint: if a serving is in fl. oz, convert the fl. oz to mL, then use density of 1.0 g/mL to calculate the mass, m= dxV
Then calculate the content of vitamin C in a serving:
vit. C in one serving of concentrate = mg vit. C in a sample tested x (grams of concentrate
/serving)
Example: (0.6 mg vit. C /1.0 g sample) x (180 g concentrate/serving) = 108 mg vit C/serving
In your REPORT show all calculations and results, and include answers to the questions on your data sheet.
Extra credit :
If you chose additional solid food items brought from home, for which the procedure had to be developed, please give an outline of the steps followed (include the sample preparation method
(chopped, raw, cooked...), amount taken for testing, etc.
DATA SHEET
Prelab
1. Using the links provided in this document, find the current RDA (Recommended Daily
Allowance) for vitamin C for adults of either sex.
Adult Males 90 mg/day
Adult Females 75 mg/day http://www.iom.edu/Object.File/Master/7/296/0.pdf
2. Using the links: http://www.msdsonline.com
or http://msds.pdc.cornell.edu/msdssrch.asp find the health hazards associated with ascorbic acid and iodine.
WE ARE NOT AWARE OF ANY HAZARDS FOR THE ABOVE PRODUCT.
THE ABOVE INFORMATION IS BELIEVED TO BE CORRECT BUT DOES NOT PURPORT TO
BE ALL INCLUSIVE AND SHALL BE USED ONLY AS A GUIDE. SIGMA, ALDRICH,
FLUKA SHALL NOT BE HELD LIABLE FOR ANY DAMAGE RESULTING FROM HANDLING
OR FROM CONTACT WITH THE ABOVE PRODUCT. SEE REVERSE SIDE OF INVOICE OR
PACKING SLIP FOR ADDITIONAL TERMS AND CONDITIONS OF SALE.
COPYRIGHT 2001 SIGMA-ALDRICH CO.
LICENSE GRANTED TO MAKE UNLIMITED PAPER COPIES FOR INTERNAL USE ONLY
HIGHLY TOXIC (USA)
TOXIC (EU)
MAY CAUSE HARM TO THE UNBORN CHILD.
TOXIC IN CONTACT WITH SKIN AND IF SWALLOWED.

CAUSES BURNS.
MAY CAUSE SENSITIZATION BY INHALATION AND SKIN CONTACT.
SEVERE LACHRYMATOR.
3. What precautions must be taken when working with aqueous iodine solution?
EXPOSURE CONTROLS/PERSONAL PROTECTION- - - - - -
SAFETY SHOWER AND EYE BATH.
USE ONLY IN A CHEMICAL FUME HOOD.
WASH CONTAMINATED CLOTHING BEFORE REUSE.
DISCARD CONTAMINATED SHOES.
WASH THOROUGHLY AFTER HANDLING.
DO NOT BREATHE DUST.
DO NOT GET IN EYES, ON SKIN, ON CLOTHING.
AVOID PROLONGED OR REPEATED EXPOSURE.
NIOSH/MSHA-APPROVED RESPIRATOR.
COMPATIBLE CHEMICAL-RESISTANT GLOVES.
CHEMICAL SAFETY GOGGLES.
KEEP TIGHTLY CLOSED.
STORE IN A COOL DRY PLACE.
HANDLE AND STORE UNDER ARGON.
Additional Prelab for Extra credit (4 pts):
If you choose to bring your own solid food items, you need to develop a preliminary procedure for testing (how to prepare the sample, amount of the food used for titration, etc.- use a step-wise, organized approach here)
If you want to test cooked food, please cook it at home. If you need to chop the food, please bring your own kitchen knife or chop it at home and refrigerate until ready for lab (but no for too long, why?).
Some ideas you may want to test:
- fresh food vs. stored in the refrigerator for a long time, or
- food item cooked in a microwave vs. the same item cooked using a traditional method (in a pot) -
- cooked using various methods (e.g. traditional, pressure cooker, etc.) vs. raw