# Determination of Vitamin C Excretion Rate

See  VITAMIN C TITRATION PROTOCOL for detailed directions on how to titrate for the quantity of vitamin C in a sample.  The same titration technique is use to measure vitamin C in aliquots of urine.  The principle is to completely void your bladder and drink a full 12 ounces of water at time zero.  At exactly one hour later, collect 100% of the urine produced during the preceeding hour, measure and record the volume, and titrate 10 mL aliqouts  to determine the amount of vitamin C in 10 mL.  When you multiply the vitamin C in 10 mL times the number of 10 mL aliquots in your one hour sample, you get the hourly excretion rate of vitamin C.  Compare your excretion rate with those of your class mates.  Look for correlations between diet, smoking habits, health, etc for possible explanations of high or low excretion rates.

Here is a Vitamin C Excretion Rate data sheet into which you should enter your data.  It will guide you through the steps and calculations.

EQUIPMENT:
buret
buret clamp
ring stand
250 mL beaker
urine hydrometer and cylinder
10 mL pipets

SUPPLIES:
standardized 0.01 N iodine
funnel
Starch-HCl Rxn mix in repipet
freshly collected urine
white scrap paper

Here is a pdf summarizing the interpretation of urinalysis and the list of activities which you are to perform to measure Vitamin C Excretion Rate. Here is a sample data page from a recent quiz.

1. Completely void the bladder at the beginning of the Lab, note the exact time to the minute.

2. Consume 12 full ounces of water (soft drink is OK, but it should not be caffeinated). (less and you may

3. Exactly one hour later, collect and measure all urine produced by completely voiding into a 400 mL beaker (more for some…).

4. Record the total volume produced.

5. Measure the specific gravity of your urine using the hydrometer. Note that it should be between 1.000 and 1.040. (Make an illustration.)
DO NOT DROP THE HYDROMETER INTO A DRY CYLINDER, THEY BREAK!
Here are a variety images of hydrometers:
Hydrometer out of its cylinder
Hydrometer reading 1.015 in sugar solution
After recording the specific gravity, dip a urinalysis “Multistix 10 SG” strip into the urine, allow to sit for the prescribed time, and score and record the results based on this color chart.

6. Titrate 10 mL aliquots of the urine in triplicate, carefully recording start and finish volumes for each, as given in the titration protocol.

7. Determine the average mL iodine required per 10 mL of urine, multiply times the conversion factor to yield the amount of vitamin C in the 10 mL aliquot.

8. Determine the number of aliquots produced per hour by dividing the total volume of urine produced in an hour by the aliquot size (10 mL).

9. Determine the total vitamin C excreted per hour by multiplying the number of aliquots in the hour’s sample by the Vitamin C per aliquot.

I.  FILL THE BURET

II.  PREPARE THE SAMPLE:
1. Place 10 mL of reaction mix in 250 mL flask, using a repipet.
2. Pipet in a 10 mL aliquot of urine into each of three flasks.

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. Add titrant while simultaneously swirling the flask.  Be cautious on the first flask, as it may take much less titrant than you anticipate.
4. When the color change begins to show while swirling, reduce the rate of titrant addition.  Continue to reduce the rate as you approach the endpoint (color will take longer to disappear).
5. Begin adding titrant drop-wise, swirling to remove color after each addition.
6. Stop when a trace of blue is stable (hopefully after a single drop has been added).  The blue color may fade after 10-15 seconds.  Not to worry.
7. Record the finish buret reading to nearest hundredth mL.
8. Repeat this process for the second and third flask, using the finish reading of the previous flask for the start of the current flask.

IV.  CALCULATE THE RESULTS:
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 10 mL urine by multiplying the mean mL titrant used times the conversion factor for the iodine titrant used.
3. Calculate the mg vitamin C/hour multiplying the mg vitamin C/10 mL X (total mL urine produced in an hour)/10 mL aliquot volume.