Vitamin C Titration Protocol

Vitamin C Titration Protocol

Vitamin C titration protocolThe amount of vitamin C can be determined using a classic starch-iodine titration.  Iodine reacts with starch to make an intense blue-black color, but vitamin C reduces iodine to iodide, thus preventing it from creating the blue color.  When a known volume of solution containing vitamin C is combined with a small quantity of starch/dilute HCl, dropwise addition of iodine will not cause a color change as long as any vitamin C is present.  However, as soon as the vitamin C has been exhausted, the iodine will be free to react with the starch, and a blue color will appear.  The amount of vitamin C present will be proportional to the amount of iodine required to bring about the appearance of the blue color.  If the iodine has been standardized so that the amount required to titrate a known amount of vitamin C, then the amount of vitamin C present in the unknown can be determined.

If you need practice reading burets (i.e., interpolating between marking lines), here is a page of buret images to read.

buret supported in a
buret clamp and
ring stand
250 mL beaker
three 250 mL flasks
10 mL pipets

scrap white paper
standardized 0.01 N iodine solution
in 250 mL flasks with a funnel
Starch-HCl Reaction mix in repipet
soln. or suspension of sample



1. Place 250 mL beaker under spout to catch excess iodine solution.
close the stopcock

2. VERY IMPORTANT: Close stopcock. Otherwise, iodine will flow out all over your bench as you try to fill the buret.

3. Place a funnel in the buret, and fill with 0.01 N by adding iodine titrant. Add iodine slowly enough so that the funnel is nearly empty at all times. Do not fill up the funnel or the buret may over fill and spill iodine every where…

4. Stop adding iodine solution when the buret is filled to close to the top of the graduations. (Do not try to fill to the 0.00 mL.)

5. Shoot several streams of titrant (iodine solution) to drive bubbles out of the buret spout. (If bubbles reappear in the spout, grease the joint between the valve and the spout with stopcock grease.) Practice controlling the stopcock so that a single drop is added to the flask.


1. Place the desired quantity of reaction mix in 250 mL flask,
using a repipet (Use 10 mL for clear samples, 15 mL for others).
The upper image shows the operation of the repipet.
The lower image shows the delivery of 10 mL of reaction mix to a 250 mL flask.


2. Pipet in a precisely measured aliquot of sample specimen to be titrated into the 250 mL flask. (See separate protocol for specific volumes. The total vitamin C should not exceed 15 mg/flask).



1. Follow the format for recording your data as demonstrated : Record the starting volume in the buret to nearest hundredth of a mL (remember that the graduations go from top to bottom of buret, the numbers increases as you go down, and that you read at the bottom of the meniscus). Before you begin each titration, judge whether you have enough titrant in the buret to finish the flask. When in doubt, refill the buret, and record the new start reading.

2. Place prepared flask of sample under spout.


3. If you have not mastered it yet, practice again controlling the stopcock so that a single drop is added to the flask.

4. Add titrant while simultaneously swirling the flask. Be cautious on the first flask, as it may take much less titrant than you anticipate.

5. When the color change begins to show while swirling, reduce the rate of titrant addition. Continue to reduce the rate of iodine addition as you approach the endpoint (color will take longer to disappear).

6. Begin adding titrant drop-wise, swirling to remove color after each addition.
comparing endpoints


7. Stop when a trace of blue is stable (hopefully after a single drop has been added). The flask at the left displays a good endpoint. The flask in the middle has not been titrated, and for the flask at the right, the endpoint was over shot (too dark a blue).

8. Record the finish buret reading to nearest hundredth mL.

9. Repeat this process for the second and third flask, using the finish reading of the previous flask for the start of the current flask.

10. The second and third flasks can be titrated more quickly since you can estimate where the endpoint will be. Shoot in titrant at full speed until about three mL short of predicted endpoint. Reduce speed markedly and finish carefully as in steps 5 through 7.

1. Determine the mL titrant used for each flask, determine the mean and mean deviation for the three. (If the deviation is greater than 3%, consider repeating the titration.)

2. Calculate the mean mg of vitamin C in aliquot titrated by multiplying the mean mL titrant required times the conversion factor for the iodine titrant used.

3. Calculate the mg vitamin C/100 mL sample by multiplying the mg vitamin C/aliquot X 100 mL/aliquot volume. If sample is a slurry (of vegetable, etc), further multiply mg vitamin C/100 mL times the inverse of fraction of slurry which is the starting material (i.e., if 1 g per 5 mL total, multiply times 5) for the mg vitamin C per 100 g of starting material

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