_____University of Puget Sound
Department of Chemistry
Chem 230
EXP. 2–MICROSCALE DETERMINATION OF VITAMIN C
LABORATORY OBJECTIVES AND ASSESSMENTS
1. Know what a titration is and how to use a titration to determine the amount of an
analyte in a sample.
a. Determine the amount of Vitamin C present in supplement tablets and a sample
of fruit juice using a “Rapid-Screening” technique.
b. Describe the titration process using the concepts of excess and limiting reagents,
equivalence point and indicators.
2. Understand how to use statistics to describe the accuracy and precision of the
titration method for determining Vitamin C.
a. Use a spreadsheet to calculate relevant statistics for a data set.
b. Calculate the standard deviation of each titration experiment.
c. Use the Grubbs test to demonstrate and remove any outlying data points.
d. Calculate the confidence interval of the Vitamin C tablet and compare to a
“true” value using a Student’s t-test.
e. Identify sources of random error that contribute to the observed uncertainty
3. Understand how to standardize a solution.
a. Describe and demonstrate how to prepare a Vitamin C standard solution using
the proper analytical tools (analytical balance, weighing by difference,
volumetric flask, etc.)
b. Describe and demonstrate how to use the Vitamin C standard solution to
standardize the titrant (I3-) solution.
4. Understand how to use a standardized titrant solution for determination of an
unknown.
a. Describe and demonstrate how to use the standardized triiodide titrant (I3-)
solution to determine the amount of Vitamin C in known and unknown
samples.
INTRODUCTION
Vitamin C, ascorbic acid, plays an essential role in the enzyme-catalyzed reactions
that your body uses to produce strong connective tissue, including your skin. Humans
and guinea pigs cannot make their own ascorbic acid; they have to get it in their diet.
Ascorbic acid deficiency leads to the disease called scurvy. Among the symptoms of
scurvy are weakness, skin lesions, and slow wound healing. All these effects are a result
of weak connective tissue.
To maintain good health, the current recommended daily allowance (U.S. RDA) of
vitamin C is 75mg for adult women and 90 mg for adult men. Foods like fruits and
vegetables with a high water content often contain large amounts of vitamin C. It is
very soluble in water. If high doses of vitamin C are ingested, much of it is rapidly
excreted in urine.
Ascorbic acid (C6H8O6) is a reducing agent that is readily oxidized to
dehydroascorbic acid (C6H8O7). See Harris (pg 367) for structures.
C6H8O6
+
H2 O
+
I2
C6H8O7
+
2H+
+
2I-
(1)
Ascorbic acid reacts with a number of oxidants and several of these oxidation–
reduction reactions are the basis for methods to determine the ascorbic acid content of
samples. The oxidizing agent you will use is iodine (I2). In iodide containing solutions,
c230 Exp. 2-Microscale Determination of Vitamin C
1
iodine (I2) is in equilibrium with the triiodide ion (I3-). (We will use the terms iodine
and triiodide interchangeably.)
I2 + II3(2)
Since the oxidized form I3 is colored and the reduced form is colorless it is well suited for
a titration method for determining ascorbic acid. The color can be amplified by adding
starch to the solution because triiodide forms an intensely colored blue-black complex
with starch that is easy to see.
I3-
+
Starch
Starch – I3- complex
(3)
(Blue-black color)
The titrations in this lab will be rather different from future titrations. Instead of
dispensing your titrant (I3-) from a buret, you will be counting drops from a micropipet.
How accurate is this method? To answer, you'll have to use statistical ideas like
confidence intervals and the student’s t-tests.
The second difference in this titration lab has to do with how you standardize the I3titrant. Instead of finding its molarity, you'll measure the number of drops you need to
react with a known mass of ascorbic acid, frequently called a titer. The mass of ascorbic
acid titrated by a drop of I3- is called the “ascorbic acid titer”, C(mgVitC/drop I3-) about
which we will have more to say in the Calculations section below.
EXPERIMENTAL PROCEDURE
RECORD ALL DATA IN YOUR LABORATORY NOTEBOOK.
Please read Harris, pp 83-93 (4-1 and 4-3) on some basic statistics before coming to lab.
YOU WILL WORK WITH A PARTNER TO MAKE THE SOLUTIONS BUT WORK INDIVIDUALLY
ON TITRATIONS
1. Preparation of the ascorbic acid Standard Solution
(Work with a partner)
a. Dissolve ~100 mg (weigh by difference using the analytical balance) in a
clean 250-mL volumetric flask. Record mass in notebook.
Fill the flask about half full with DI water. Shake and swirl the flask until
the solid is dissolved.
b. Fill to the line with DI water. After thorough mixing, transfer to a labeled
flask and set aside while you go on to the next procedure.
2. Preparation of the Vitamin C Tablet Solution (Record label information.)
(Work with a partner)
a. Weigh a vitamin C tablet and record the weight to at least the nearest
milligram in notebook. Record the amount of ascorbic acid on the
manufacturer’s label and the lot number of the Vit. C bottle in notebook.
b. Place the tablet in a clean 500-mL volumetric flask. Fill the flask about
half full with DI water, shake and swirl until the tablet is broken down.
The tablet may leave a cloudy solution due to fillers and binders used in
the manufacture of the tablet.
c. Fill to the line with DI water and mix thoroughly.
d. Transfer the solution to a clean, dry flask, label and set it aside while you
go on to the next procedure.
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2
3.
Preparation of Juice Sample (Record label information.)
(Work with a partner)
Record the serving size and stated amount of Vit. C per serving from the
label in notebook. If filtering is needed use filter paper and a funnel to
obtain ~5-10 ml of filtrate.
FOR THE REMAINING TITRATIONS-WORK INDIVIDUALLY BUT USE THE
SOLUTIONS PREPARED WITH YOUR PARTNER.
CALIBRATION OF “MEASURING-PIPET”
4.
Calibration of “Measuring-pipet” (the graduated-stem plastic pipet)
Note: It is essential that you always hold your pipets in a reproducible VERTICAL
position at all times.
a. Fill a 250-mL beaker with DI water to stabilize to room temperature.
Before calibrating your 1-mL Measuring-pipet, practice drawing up 1.00
mL and without any loss, deliver the mL to 50-mL flask.
b. Record the temperature of DI water that has stabilized to a constant
temperature.
c. Measure and record the mass (±0.001g) of the 50-mL flask with a stopper.
d. At your workstation, draw up and transfer 1.00 mL of DI water into the
flask. Stopper, measure and record the mass in the table in your
laboratory notebook.
e. Repeat the procedure to obtain five trials.
f. Enter the data into your Calibration section of your spreadsheet. (See
Figure 1.)
TITRATION OF VITAMIN C STANDARD
5.
Standardization of the Titrant Solution I3- (e.g., Determination of Ascorbic Acid
Titer)
a. Obtain a few milliliters of the triiodide titrant in a clean, dry 13 x 100-mm
test tube. Don’t store the triiodide titrant in plastic or keep it in a plastic
pipet for longer than required for a titration. The iodine is adsorbed by
plastic and this will change its concentration.
WARNING: Solutions containing iodine will stain your skin and clothing.
The stains are harmless, except to your pocketbook, if you ruin some
article of clothing. Handle with care. If applied in time, a rinse with
sodium thiosulfate solution can sometimes prevent staining.
b. Use your calibrated “Measuring-pipet” to put 1.00 mL of the standard
ascorbic acid solution in a clean well of your 12-well plate.
c. From a plain plastic pipet, add one drop of starch solution.
d. Using a “Titrating-pipet”, titrate the ascorbic acid by adding dropwise the
triiodide titrant solution, stirring constantly with a toothpick. Record the
number of drops required to turn the colorless solution blue.
e. Repeat the standardization titration until you have ten (10)
determinations.
f. Try one additional titration without the starch indicator to the first
permanent color. How do the titrations compare?
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3
TITRATION OF VITAMIN C IN TABLET
6.
Determination of Ascorbic Acid in Vitamin C Tablet
a. Use your calibrated Measuring-pipet to put 1.00 mL of the solution made
with the Vitamin C tablet into a well of your 12-well plate.
b. Add one drop of starch solution.
c. Using your Titrating-pipet, titrate the ascorbic acid by adding the triiodide
titrant solution dropwise, stirring with a toothpick as before. Record the
number of drops required to turn the solution blue.
d. Repeat until you have ten titrations.
TITRATION OF VITAMIN C IN SERVING OF JUICE
6.
Determination of Ascorbic Acid in Juice
a. For these sample determinations, use your calibrated Measuring-pipet to
put 1.00 mL of your clarified juice in a clean well of your 12-well plate.
b. Add one drop of starch solution.
c. Titrate the juice sample by adding dropwise the triiodide titrant solution
(stirring with a toothpick) and record the number of drops required to
turn the solution blue. NOTE: The number of drops of I3- solution should
be between 35 and 45. If it is not, you may adjust the volume of your juice
sample to get the titration in this range.
d. Repeat until you have ten titrations.
CALCULATIONS
1.
Transfer the data for the experimental results from the tables in your notebook to
the Excel spreadsheet(s) you created. See Figure 1 for example of the layout and
formatting of Sheet 1 of the spreadsheet, the Calibration of the Measuring-pipet
and the Titration of the Vitamin C Standard. See Figure 2 for example of the
layout and formatting of Sheet 2 of the spreadsheet for the Determination of
Vitamin C in Tablet and Determination of Vitamin C in a Serving of Juice.
2.
Calibration of Measuring-pipet: From your spreadsheet calculations determine
the mean value of the volume delivered and the standard deviation for the 1.00mL “Measuring-pipet”. Use this value in all of your calculations.
3.
Standardization: For this calculation and the following ones, we employ the
following symbols.
M(mg) = mass of ascorbic acid in a well plate (mg)
N = number of drops of I3- used in a titration
C(mg/drop) = “ascorbic acid titer”, mass of ascorbic acid titrated by each drop of I3-.
For example, for the standardization step (5), the mass of ascorbic acid in the
1.00-mL samples from the Measuring-pipet you used for standardization will be
called Mstd(mg). Using the True volume from the calibration of the ”Measuringpipet“ we can calculate Mstd(mg) of ascorbic acid, in mg,
Mstd(mg) = Mean true V x (mg Asc. Acid/mL of Std Asc. Acid solution)
c230 Exp. 2-Microscale Determination of Vitamin C
(4)
4
For each titration, divide this by the number of drops of I3-, triiodide titrant, Nstd
giving the ascorbic acid titer, caa (mg/drop) for each titration,
C(mg/drop) = Mstd(mg)/Nstd
(5)
Enter formula in your spreadsheet (Figure 1) to calculate “C(mg/drop)” for Ascorbic
acid samples.
Figure 1. Example of partial spreadsheet for the Calibration of the 1.00-mL “Measuring-pipet”
and the spreadsheet for the Standardization of your ascorbic acid solution for the
Vitamin C experiment. These will be on Sheet 1 of your Excel document.
4.
Grubbs-test: Look carefully at the data from each of the titrations to see whether
any titration result looks like an outlier. Run a Grubbs test for all trials with
respect to C(mg/drop) with the spreadsheet you created following the steps below.
Show one Grubbs test sample calculation in your lab notebook.
• Compare your results with the values in Table 4-4 in Harris (pp 92) to decide
if you should keep or reject the data.
• If a data point was rejected drag the contents of that cell in the “G” column
over to the “H” column. This removes the outlier from the calculation but
does not delete it all together in case you need to consider it later.
Q.1. STANDARDIZATION-Determination of C(mg/drop): Calculate the mean C(mg/drop),
standard deviation, and 95% confidence interval of the mean using Excel
functions and cell formula calculations. Report the confidence interval in two
ways: as mean ± number with matching decimal places, and as an interval
(number to number).
5.
DETERMINATION of mg Vitamin C in Tablet: The number of drops used in
titrating the tablet solution, Ntab, will allow you to calculate the mass of ascorbic
acid in each well plate using the ascorbic acid titer, C(mg/drop).
mtab(mg)= ntab x C(mg/drop)
c230 Exp. 2-Microscale Determination of Vitamin C
(6)
5
Again C(mg/drop) is the mean value of ascorbic acid titer you obtained previously.
The number of drops for these titrations, Ntab, multiplied by C(mg/drop) equals the
mass of Asc. Acid in each mL titrated, mtab(mg). The values of mtab(mg) can be
used to determine the mass of ascorbic acid in the entire tablet. To find this value,
multiply mtab(mg) by the ratio of the volume of the volumetric flask to the well
plate volume (eq. 7). Enter the appropriate formula in the “Mentire tab“ column to
calculate this value.
Mentire tab(mg/tablet) = mtab(mg) x (500.0mL/Volume “Measuring-pipet”)
(7)
Determine the mass of ascorbic acid in the Vitamin C tablet for each trial and
calculate the mean value, etc. (Q.2.)
Figure 2.
Partial layout and formatting for Sheet 2, Determining Vitamin C in a Tablet and
Determination of Vitamin C in a Serving of Juice.
Q.2. Vitamin C Tablet: Calculate the mean ascorbic acid (mg) in your vitamin C tablet,
standard deviation, and 95% confidence interval of the mean. Report the
confidence interval two ways as before. Compare, statistically using the student ttest, with the amount listed on the label. Is there a statistically significant
difference between your results and the amount listed on the bottle? Based on
your results, what percent by mass of the tablet is actually vitamin C?
6.
Juice: The number of drops used in titrating each juice solution, Njuice, will allow
you to calculate the mass of ascorbic acid in each well plate using
mjuice(mg)= Njuice x C(mg/drop)
c230 Exp. 2-Microscale Determination of Vitamin C
(8)
6
Find mjuice for each titration of the juice. Then for each juice trial, use the volume
of the sample, volume of a serving and mjuice to calculate the amount(mg) of
ascorbic acid in a serving of your juice. Calculate the mean value, etc. (Q.3.)
M(mg/juice serving) = mtab(mg) x (serving size/Volume juice titrated)
(9)
Similar to the manner used for the tablet determination set up your spreadsheet
to record your data and calculate the results for a serving of your juice.
Q.3. Juice: Calculate the mean concentration (mg/mL) of ascorbic acid in your juice
sample, standard deviation and 95% confidence interval of the mean. Report the
confidence interval two ways as before. Based on your results, how much of your
juice do you need to consume to have the RDA of Vitamin C? Based on the label,
how much of your juice do you need to consume to have the RDA of Vitamin C?
Compare these amounts (first get them in matching units!). Did the juice have the
stated amount of Vit. C per serving?
ADDITIONAL QUESTIONS
Q.4. Calculate the mean amount of I2 per drop of triiodide reagent, in mmol and mg .
Q.5. List the three major sources of random error in this experiment?
Q.6. An error of one drop in fifty drops results in what percent relative uncertainty?
What was your relative uncertainty ( Std.Dev. × 100% ) from the Standardization of
Mean
the Ascorbic Acid?
€
c230 Exp. 2-Microscale Determination of Vitamin C
7
WHAT TO DO
To Be Turned in From Exp. 1-Statistical Analysis of Volumetric Tools of the Laboratory:
Please hand in the following items in a packet STAPLED and in the following order:
a) Printout of Plot for Calibration of Buret,
b) Page(s) of Questions (Copy pages from notebook OK),
c) Copy page(s) from laboratory notebook of the completed table of volume and
mass data.
d) Stapled at the end-any remaining copy pages.
Be Sure To Put Your Name on Every Page.
For Experiment 2
Before Lab:
Read this experiment and Harris and prepare your notebook as
follows
You must also include the following information:
• Completed title bar.
• Reagent Table containing ascorbic acid and iodine. See Laboratory Guideline
#IV. GUIDELINES FOR MAKING TABLES INCLUDING A TABLE OF
REAGENTS.
BE SURE TO NOTE THE DISPOSAL PROCEDURES IN YOUR TABLE.
• Write a summary of the procedure in your notebook. Include a balanced
equation for the overall titration reaction.
• Prepare a table in your notebook to record data in the laboratory divided
into four sections.
One section with the raw data for the calibration of the 1.00-mL
“Measuring-pipet”. (masses, volumes, etc. for each trial.)
One section with the raw data for the ascorbic acid standardization
(masses, volumes and number of drops for each trial.)
One section with the raw data for the tablet.
One section with the raw data for the juice.
LEAVE EXTRA SPACE IN YOUR NOTEBOOK TABLES WHEN PREPARING!
• Following the example given in Figures 1 and 2 generate an Excel
spreadsheet file. Submit a printed copy of this spreadsheet as prelab. You
should bring a copy on a flash drive or email yourself an electronic copy to
use during lab.
• Bring your copy of Harris to lab and your computer. Do not bring your
calculator.
• With your lab partner bring a labeled juice sample with the nutritional
information label.
The TA will check the copies of these pages from your notebook at the beginning of
the lab period.
During the Lab:
Carry out the procedure as written. Record all data as you
collect in your notebook. Modify your procedure if you find it necessary, but be sure to
record the modifications. Record all other pertinent observations and measurements as
you do the experiment. Show sample calculations in your laboratory notebook. If time
permits complete the spreadsheet with your data in lab.
c230 Exp. 2-Microscale Determination of Vitamin C
8
To Be Turned In: NOTE: Specific pages and order of assignment to be turned in
will be state on the Moodle page for the next experiment.
Complete your calculations in your spreadsheet. Your report will consist of:
• Answers to the Questions.
• Summary table in notebook showing results (including comparison with
label info for 3 and 4) from 1) Calibration, 2) Asc. Acid Standardization, 3)
Vit. C in tablet and 4) Vit. C in juice sample.
• Completed spreadsheet with your results sent to your TA as an email
attachment.
• Completed spreadsheet printout.
• Copies of the relevant tables and pages from your notebook.
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