VITAMIN C TITRATION PROTOCOL ©David B. Fankhauser, Ph.D., Professor of Biology and Chemistry University of Cincinnati Clermont College, Batavia OH 45103
detail of set up for Vitamin C titration	This page has been accessed  times since 11 May 2004. 30 January 1980, rvsd 29 March 1994, 24 March 1996, 2 April 98, 2 May 02, 26 Mar 03	endpoints: accurate versus overshot

	EQUIPMENT:	SUPPLIES:
set up:               detail of setup:	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

I.  FILL THE BURET:
 

	1. Place 250 mL beaker under spout to catch excess iodine solution.
	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. The lower image shows a spout filled with iodine

II.  PREPARE THE SAMPLE:
 

	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).

III.  TITRATE THE SAMPLE:
 

	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.
	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.