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Determining the Effects of Cooking on the Vitamin C Content of Green Pepper

Article · January 1998

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 A N A L

501
modular • laboratory • program • in • chemistry
publisher: H. A. Neidig editor: Henry D. Schreiber

Determining the Effects of Cooking on the

Vitamin C Content of Green Pepper
prepared by Todd P. Silverstein, Willamette University,
and Eric R. Johnson, Ball State University

Purpose of the Experiment

Use an oxidation–reduction titration to determine how four different cook-

ing methods affect the vitamin C content of green pepper.

Background Information tends to donate electrons in a chemical reaction. It

Many raw fruits and vegetables contain high levels of
vitamins and minerals, but cooking can remove much
of this nutritionally important content. Some of the vita-
min loss that occurs during cooking is caused by oxi-
dation, degradation, and vaporization. However, most
of the loss of water-soluble vitamins, including vitamin
C, is due to the fact that these vitamins simply dissolve
in the cooking water. We can quantify the vitamin C
loss caused by cooking by comparing the amount of
vitamin C in cooked green pepper to the amount of vi-
tamin C in raw green pepper.
In this experiment, you will cook green pepper using
four methods: baking, boiling, steaming, and microwaving.
You will then compare the different degrees of vitamin C
loss caused by the different cooking methods.
plays a critical role in the synthesis of several impor-
tant proteins, including adrenaline, a hormone, and
collagen, a structural protein. Because collagen is vital
for strong skin, tendons, and ligaments, a lack of vita-
min C in the human diet leads to scurvy, a skin
disease.
The two critical hydrogens in ascorbic acid are part
of –OH groups, shown underlined in Figure 1. One of
these hydrogens is responsible for the acidity of ascor-
bic acid (see Equation 1). The other is responsible for
the oxidation of the ascorbate anion (see Equation 2).
The importance of these two hydrogens is stressed in
the abbreviation for ascorbic acid as H2Asc.
As with all sugars, ascorbic acid is highly wa-
ter-soluble. Its acidity stems from the fact that it loses one
of its hydrogens fairly easily, as shown in Equation 1:

H2Asc → HAsc:– + H+ (Eq. 1)

Ascorbic Acid, an Important Biological
Reducing Agent
Ascorbic acid (H2Asc), more commonly known as vita-
min C, is an oxidized form of glucose, the common
6-carbon sugar (see Figure 1 on the next page).
Ascorbic acid is a good reducing agent, meaning that it
ascorbic acid ascorbate anion
Equation 1 also shows that when vitamin C dis-
solves in water, it exists mainly as the ascorbate anion
(HAsc:–) in solution. The ascorbate anion is itself eas-
ily oxidized, causing the loss of a pair of electrons and
producing an H+. In this process, the ascorbate anion

Copyright © 1998 by Chemical Education Resources, Inc., P.O. Box 357, 220 S. Railroad, Palmyra, Pennsylvania 17078
No part of this laboratory program may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photo-
copying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the United
States of America 02 01 00 99 98 — 12 11 10 9 8 7 6 5 4 3 2 1
2 ANAL 501/Effects of Cooking on Vitamin C Content

Figure 1 Conversion of D-glucose to ascorbic acid

oxidizes to form dehydroascorbate (Asc), as shown in
Equation 2:
HAsc:– → Asc + H+ + 2 e– (Eq. 2)
ascorbate anion dehydroascorbate
This oxidation half-reaction is the key to vitamin
C’s biological activity as well as the key to the titration
we will use for analyzing vitamin C content.
Titrating Vitamin C
To analyze green pepper for vitamin C content, we will
use a standardized solution of N-bromosuccinimide
(NBS, also succN–Br). NBS is an excellent electron
acceptor (oxidizing agent), as shown in Equation 3:
succN–Br + H+ + 2 e– →
N-bromosuccinimide (NBS)
succN–H + Br–
succinimide
When added to a solution containing vitamin C,
NBS rapidly oxidizes all of the ascorbate anion, con-
verting it to dehydroascorbate. Simultaneously, the
NBS is reduced to succinimide (succN–H; see Equa-
tion 4).
succN–Br + HAsc:– → succN–H + Br– + Asc (Eq. 4)
color change indicates the end point of the reaction,
the point at which no more ascorbate anion is left in so-
lution. Equation 6 shows the overall NBS/I – reaction.
succN–Br + H+ + 3 I– + starch →
succN–H + starch • I3– + Br– (Eq. 6)
(dark blue)
When we systematically add NBS to solution con-
taining dissolved vitamin C and iodide–starch indicator
solution until we observe the end point of the reaction,
we are performing an oxidation–reduction titration.
At the end point of the titration, we can determine the
volume of NBS required to completely react with all of
the ascorbate anions in solution.
Calculating Vitamin C Content
Your laboratory instructor will tell you the concentration
(Eq. 3)
of the standardized NBS solution. This concentration
was determined by titrating a known amount of pure
ascorbic acid with the NBS solution. The concentration
is expressed in units of milligrams of vitamin C oxi-
dized by one milliliter of NBS solution (mg vitamin C
per mL NBS). Based on the NBS solution concentra-
tion and your titration data, you will calculate the vita-
min C content of your green pepper samples, using the
following four steps.

Equation 4 represents the redox reaction obtained by
adding the oxidation (Equation 2) and reduction
(Equation 3) half-reactions.
To help determine when the titration is complete,
1. Your titration results will be expressed in milliliters
of NBS (mL NBS) necessary to completely react with
all of the ascorbic acid in the sample. Hence, multiply-
ing the titration volume of NBS by the NBS concentra-
© 1998 Chemical Education Resources

we add an iodide–starch indicator solution to the reac-
tion mixture. When all of the ascorbate anions from the
dissolved vitamin C have been oxidized, any additional
NBS oxidizes iodide anions (I–) in the indicator
(Equation 5).
3 I– → I3– + 2 e– (Eq. 5)
The triiodide (I3–) product binds to the starch, produc-
ing a dark blue starch • triiodide complex. The resulting
tion will yield the total number of milligrams of vitamin C
present in the titrated sample, as shown in Equation 7a.
 mL   mg vitamin C  = total mg vitamin C (Eq. 7a)
  
NBS  mL NBS  in sample
2. Because you will always titrate a 5.00-mL sample,
you can calculate the vitamin C concentration in the
green pepper extract (in mg/mL) by dividing the mass
of vitamin C from Equation 7a by 5.00 mL (Equation 7b).
 ANAL 501/Effects of Cooking on Vitamin C Content
mg vitamin C
= mg vitamin C / mL (Eq. 7b)
5.00 mL
3. You can then calculate the total amount of vitamin
C (in mg) in the extract by multiplying the vitamin C
concentration (in mg/mL) from Equation 7b by the total
volume of the extract (in mL), which you can measure
using a graduated cylinder, as shown in Equation 7c.
(total mL of extract)(mg vitamin C / mL) =
total mg of vitamin C (Eq. 7c)
You can combine steps 1 through 3, as shown in Equa-
tion 7d:
 mL   mg vitamin C   total mL of extract  =
    of the pepper. Carefully weigh each sample to the
NBS  mL NBS   5.00 mL of extract 
total mg of vitamin C (Eq. 7d)
4. Finally, you will express the vitamin C content of
your green pepper samples in units of mg vitamin C
per g green pepper. To do so, you will divide the total
mass of vitamin C (in mg) from Equation 7c by the
mass of the sample, as shown in Equation 7e.
total mg of vitamin C vitamin C
= (Eq. 7e)
g of green pepper content
By comparing the experimentally determined vita-
min C contents of variously cooked and raw green
pepper samples, you will be able to determine which
cooking method causes the greatest vitamin C loss.
Procedure
Preview
• Obtain two portions of green pepper, one for
each partner
• Partners prepare pepper samples: one cooked;
one raw
• Extract the vitamin C from the raw or cooked
green pepper
• Titrate the vitamin C in your green pepper extract
© 1998 Chemical Education Resources
Chemical Alert
N-bromosuccinimide (NBS)—strong irritant
1M H2SO4—corrosive, irritant
Caution: Wear departmentally approved safe-
ty goggles while doing this experiment.
3
Note: You need to do this experiment with a
partner. You and your partner will choose (or be
assigned) one of the four cooking methods. One
of you will prepare a cooked sample of green
pepper, as instructed below, while the other will
prepare a raw sample. Each of you will then ti-
trate the pepper sample you prepared.
I. Preparing and Cooking the Green Pepper
You and your partner should cut two portions of green
pepper, approximately 20 g each, from the edible part
nearest 0.01 g, and record the mass of your sample on
your Data Sheet. Keeping the samples separate, chop
them into small pieces. Keep one sample raw as a con-
trol, and cook the other one.
You and your partner will choose (or be assigned)
one of four cooking methods: baking, boiling, steam-
ing, or microwaving. Whichever of you is preparing the
cooked sample should then follow the appropriate
cooking procedure described below. Meanwhile, the
partner preparing the raw pepper should proceed di-
rectly to the extraction procedure (Part II).
A. Baking
Place a weighed, chopped pepper sample in a
250-mL beaker. Bake it in an oven at 180–200 °C for
about 15 min. Using tongs, remove the beaker from
the oven and allow it to cool to room temperature.
B. Boiling
Boil about 150 mL of distilled or deionized water in
a 400-mL beaker. Wrap a weighed, chopped pepper
sample in a double layer of cheesecloth. Secure the
mouth of the cheesecloth “bag” with string or a rubber
band. Immerse the bag in the boiling water for 20 min.
Then, using tongs, remove the bag from the water. Be
sure to save the cooking water!
Acidify the cooking water by adding 5.0 mL of 1M
H2SO4. Use gentle boiling to reduce the volume of the
solution to about 50 mL. Use a graduated cylinder to
carefully measure, to the nearest 0.5 mL, the final vol-
ume of the acidified cooking water. Record the volume
on your Data Sheet.
C. Steaming
Wrap a weighed, chopped pepper sample in a dou-
ble layer of cheesecloth. Secure the mouth of the
cheesecloth “bag” with string. Add about 100 mL of dis-
tilled or deionized water to a 400-mL or 600-mL beaker.
Cover the beaker with a watch glass, concave side up,
4 ANAL 501/Effects of Cooking on Vitamin C Content
as in Figure 2. Stretch a wide rubber band across the
diameter of the watch glass. Take the end of the string
tied to the cheesecloth bag, and secure it under the
rubber band on the underside of the watch glass. This
will suspend the bag over the water, as shown in Fig-
ure 2. Be sure that the bag does not touch the water.
Heat the water to boiling, then continue boiling
gently for about 10 min, thus generating steam. Turn
off the heat source. Using tongs, remove the cheese-
cloth bag from the beaker. Be sure to save the cook-
ing water!
Acidify the cooking water by adding 5.0 mL of 1M
H2SO4. Use gentle boiling to reduce the volume of the
solution to about 50 mL. Use a graduated cylinder to
carefully measure, to the nearest 0.5 mL, the final vol-
ume of the acidified cooking water. Record the volume
on your Data Sheet.
D. Microwaving
Place a weighed, chopped pepper sample in a
250-mL beaker. Cook the sample in the microwave
oven until it is tender (about 45 s in a 700W oven). Re-
move the sample from the oven.
II. Extracting Ascorbic Acid from Green
Pepper
To extract the vitamin C from your raw or cooked green
pepper, place the sample in a mortar, and add a few
drops of distilled or deionized water. Then use a pestle
to thoroughly grind the sample to a paste. Do not worry
if a small amount of vegetable skin and fiber remains
unground.
Figure 2 Apparatus for steaming
Add 4–5 mL of distilled water to the mortar, and
grind the sample briefly. Carefully decant the liquid into
a clean, dry 100-mL beaker.
Note: When decanting, carefully pour only the
supernatant liquid into the beaker, leaving the
pulp in the mortar. If necessary, use a glass rod
or the pestle to direct the stream of liquid into the
beaker.
Grind the pulp remaining in the mortar. Add an-
other 4–5 mL of distilled water to the mortar, grind
briefly, and decant the liquid into the same 100-mL
beaker. Repeat the grinding, water addition, and de-
canting sequence one more time. Acidify the solution
in the beaker by adding 5.0 mL of 1M H2SO4.
Transfer the pulp and skin from the mortar onto a
triple-layer square of cheesecloth (approximately 8 cm
× 8 cm). Form the cheesecloth square into a closed
bag. Squeeze the liquid remaining in the pulp into the
extract beaker. Add 1–2 mL of distilled water to the
empty mortar, re-wet the pulp in the cheesecloth with
the water, and squeeze the liquid into the beaker.
Finally, add 3–4 mL of distilled water to the mortar,
re-wet the cheesecloth, and squeeze the liquid into the
beaker. Discard the cheesecloth with the pepper rem-
nants into the container, labeled “Discarded Cheese-
cloth” and provided by your laboratory instructor.
Note: Your laboratory instructor will inform you
whether you are to use the Büchner funnel with a
bored rubber stopper or a FiltervacTM adapter.
If your aspirator does not have a trap attached
to it, as shown in Figure 3, your laboratory in-
structor will show you how to assemble such a
trap. Tape the flasks to prevent flying glass in
case of a flask imploding. The trap prevents wa-
ter from the aspirator being drawn back into the
filter flask, if the water pressure changes during
the filtration.
© 1998 Chemical Education Resources
Set up a vacuum-filtration assembly as shown in
Figure 3. Use a 125-mL filter flask and a 4.5-cm diam-
eter Büchner funnel fitted with filter paper. Wet the filter
paper with 1 mL of distilled water. Scoop small
amounts of celite onto the dampened filter paper until
you have a thin “pad” of celite about 0.5–1 cm thick.
Dampen the celite with 1–2 mL of distilled water, turn
on the aspirator, and make sure that the water is drawn
through the filter into the flask. Also make sure that the
 ANAL 501/Effects of Cooking on Vitamin C Content
celite forms an even pad covering the entire filter pa-
per. Turn off the aspirator, carefully remove the
Büchner funnel from the filter flask, and discard the
water in the flask into the sink.
Replace the Büchner funnel on the filter flask, and
turn on the aspirator. Slowly and carefully pour the
pepper extract from the 100-mL beaker onto the celite
pad in the Büchner funnel. Rinse the beaker several
times, using 1–2 mL of distilled water each time, pour-
ing the rinses onto the celite pad. The filtered extract in
the filter flask should be nearly clear, with a slight yel-
lowish or greenish tinge. Turn off the aspirator, and re-
move the Büchner funnel. Pour the filtered extract from
the flask into a 100-mL graduated cylinder. Rinse the
flask several times, using 1–2 mL of distilled water
each time, and add the rinses to the graduated cylin-
der. Read and record the final extract volume to the
nearest 0.5 mL on your Data Sheet. Discard the filter
paper and used celite in the container provided by your
laboratory instructor and labeled “Discarded Filter
Paper”.
III. Assaying the Ascorbic Acid in the
Extracts
Use the following procedure to titrate the vitamin C in
your cooked or raw green pepper extract from Part II. If
you boiled or steamed your sample, you must also use
this procedure to titrate the vitamin C dissolved in your
cooking water.
Use a 100-mL graduated cylinder to prepare
enough indicator solution for ten titrations, by mixing
10.0 mL of 0.25M KI, 10.0 mL of 0.7M acetic acid, and
40.0 mL of 1% starch in a 250-mL Erlenmeyer flask.
For each titration, use a 10-mL graduated cylinder to
Büchner funnel
© 1998 Chemical Education Resources
taped
filter
flask
Figure 3 A vacuum-filtration apparatus
5
add about 6 mL of your indicator solution to a clean
125-mL Erlenmeyer flask.
Note: Your laboratory instructor will demon-
strate correct use of a volumetric pipet.
Using a volumetric pipet, add 5.00 mL of your
acidified green pepper extract or cooking water to a
125-mL Erlenmeyer flask containing 6 mL of the indi-
cator solution.
Caution: NBS is a strong irritant. Use it care-
fully. If you spill any on your skin or clothing, rinse
thoroughly with water, and immediately inform
your laboratory instructor.
Pour about 70 mL of the standardized NBS solu-
tion into a clean, dry 100-mL beaker labeled
“NBS-Containing Solution”. Obtain and record the
concentration of the NBS solution on your Data Sheet.
Note: Make sure your buret is clean before be-
ginning your titrations.
Obtain a clean 50-mL buret. Close the stopcock.
Using a short-stem funnel, add 3–5 mL of standard-
ized NBS solution from your beaker into the buret. Tilt
and rotate the buret so that the solution rinses the en-
tire inside surface. Drain the rinse solution into the con-
tainer provided by your laboratory instructor and la-
beled “Discarded NBS-Containing Solutions”.
clamp
water
aspirator
taped
trap
6 ANAL 501/Effects of Cooking on Vitamin C Content

Clamp the rinsed buret to a ring stand. Close the Calculations

stopcock. Using the short-stem funnel, add 45–50 mL
of the standardized NBS solution to the buret. Drain Do the following calculations, and record your results
some of the NBS solution into the “NBS-Containing as instructed below.
Solution” beaker in order to fill the buret tip. Be sure
there are no air bubbles in the tip. Measure the volume, 1. For each of your consistent pepper extract and
to the nearest 0.02 mL, of NBS solution in the buret. cooking water titrations, calculate the vitamin C con-
Record this as the initial buret volume on your Data centration in units of mg vitamin C / g green pepper.
Sheet. Record the results on your Data Sheet.
2. Using the results of Calculation 1, calculate the
Note: When you think the following titration has average vitamin C concentration in the pepper, and re-
reached its end point, set aside the titration flask cord it on your Data Sheet. Do the same for your cook-
for a minute in order to make sure that the blue ing water titrations, if applicable.
color is permanent. If the color disappears, add 3. On the Class Data Report Sheet posted in the lab-
another drop of NBS solution from the buret, and oratory, record your average vitamin C concentration
check again. Repeat this procedure until the for either the raw or cooked pepper, whichever you ti-
color persists for at least several minutes. trated. Your partner will report the other value. If you
boiled or steamed your pepper, record your average
Titrate your pepper extract with NBS solution. Dur- vitamin C concentration for your cooking water as well.
ing the titration, frequently swirl the flask. Periodically
4. When all student pairs have recorded their data on
check the color of the titration solution, by setting down
the Class Data Report Sheet, copy the data onto your
the flask on a piece of white paper. When the color in
own Class Data Table.
the solution begins to linger for longer periods of time,
the reaction is near its end point. From this point on, 5. For each mode of preparation (including raw), av-
add NBS solution dropwise until the reaction is com- erage all the reported average values to obtain an av-
plete. Remember to frequently swirl the flask. erage value for the whole class. Record these values
The titration end point is indicated by the perma- on your Class Data Table.
nent appearance of a dark blue color in the titration so-
6. Define the class-wide average vitamin C content
lution. This color is due to the starch • triiodide complex.
of raw green pepper as 100%. For each of the four
Measure the volume, to the nearest 0.02 mL, of NBS
cooking methods, use the class-wide average vitamin
solution left in the buret. Record this reading as the fi-
C content for that method to calculate the percent vita-
nal buret volume on your Data Sheet.
min C retention resulting from that cooking method. Do
Using fresh 5.00-mL samples of your pepper ex-
the same for the cooking water from boiling and
tract, repeat the above titration until you obtain three
steaming. Record these values on your Class Data
net NBS titration volumes that are consistent to within
Table.
3% of each other. Be sure to record all volume data for
these titrations on your Data Sheet.
If you boiled or steamed your sample, be sure to
use the above procedure to titrate the vitamin C in your
cooking water as well as in your cooked pepper ex-
tract. Record volume data on your Data Sheet.
When your titrations are complete, transfer all so-
lutions to the “Discarded NBS-Containing Solutions”
© 1998 Chemical Education Resources

container. Clean all glassware used in the analyses.

Caution: Wash your hands thoroughly with

soap or detergent before leaving the laboratory.
 ANAL 501/Effects of Cooking on Vitamin C Content 7

Post-Laboratory Questions
(Use the spaces provided for the answers and additional paper if necessary)

1. Using the class-wide averages for percent vitamin 4. Explain the difference between the amounts of
C retention listed in the far right-hand column of your vitamin C in the boiling and steaming water.
Class Data Table, compare the four cooking methods.
Briefly explain.

5. Based on your class’s experimental data, which

two cooking methods most efficiently preserve the vi-
tamin C content of green peppers?

2. (a) Foods are usually cooked at high tempera-

tures in the presence of oxygen. How might these two
factors account for some of the loss of vitamin C in
cooked food?

(b) Some of the vitamin C content lost during

cooking can be easily regenerated once the cooked
food is inside the human body. What general type of re-
agent might the body use to regenerate vitamin C in
this way?

3. In this experiment, experimental error generally

leads to an underestimate of vitamin C content. What
parts of the Procedure are most likely to involve this
type of experimental error?
© 1998 Chemical Education Resources

name section date

 name section date

Data Sheet
mass of green pepper, g _______________________

cooking method _______________________

extract volumes, mL
green pepper extract _______________________

cooking water* _______________________

standardized NBS solution concentration,

mg vitamin C per mL NBS _______________________

determination
1 2 3 4

volume NBS used, mL, with green pepper:

initial _______________ _______________ _______________ ______________

final _______________ _______________ _______________ ______________

net _______________ _______________ _______________ ______________

volume NBS used, mL, with cooking water*:

initial _______________ _______________ _______________ ______________

final _______________ _______________ _______________ ______________

net _______________ _______________ _______________ ______________

vitamin C concentrations, mg vitamin C/g green pepper

green pepper _______________ _______________ _______________ ______________

cooking water* _______________ _______________ _______________ ______________

© 1998 Chemical Education Resources

average vitamin C concentration, mg vitamin C/g green pepper

green pepper _______________

cooking water* _______________

*Measurements of cooking water are made only for steamed and boiled green pepper.

ANAL 501/Effects of Cooking on Vitamin C Content 9

10 ANAL 501/Effects of Cooking on Vitamin C Content

Class Data Table

vitamin C content % of vitamin C
(mg vitamin C/g green pepper) content of raw
preparation pair’s average pair’s average class average green pepper
method values values values control
pair 1: pair 9:
pair 2: pair 10:
pair 3: pair 11:
pair 4: pair 12:
raw

pair 5: pair 13:

pair 6: pair 14: average vitamin C average percent, %,
pair 7: pair 15: content, mg/g: defined as 100.0:
pair 8: pair 16:

pair 1:
baking

pair 5:
pair 9: percent vitamin C
pair 13: average: retention, %:

pair 2:
microwaving

pair 6:
pair 10: percent vitamin C
pair 14: average: retention, %:

pair 3:
pair 7:
boiling

pair 11: percent vitamin C

pair 15: average: retention, %:

pair 3:
pair 7:
boiling
water

pair 11: percent vitamin C

pair 15: average: retention, %:

pair 4:
steaming

pair 8:
pair 12: percent vitamin C
© 1998 Chemical Education Resources

pair 16: average: retention, %:

pair 4:
steaming

pair 8:
water

pair 12: percent vitamin C

pair 16: average: retention, %:
 name section date

Pre-Laboratory Assignment
1. What precautions should you take when handling 5. Why is the green pepper pulp wrapped in cheese-
each of the following? cloth, and then rinsed and squeezed three times?
(a) NBS

6. A student performing this experiment obtained the

(b) dilute (1M ) sulfuric acid following data:

mass of green pepper: 19.85 g

pepper extract volume: 48.3 mL
NBS titration volumes: 7.55 mL, 7.39 mL, and 7.28 mL
2. Write a balanced chemical equation describing
Her standardized NBS solution concentration was
the oxidation–reduction reaction that is the basis of the
0.210 mg vitamin C / mL NBS. Calculate the average
titration you will perform in this experiment. Use
vitamin C concentration, in mg vitamin C per g green
HAsc:– to represent the ascorbate anion and
pepper, for her pepper.
succN–Br to represent the NBS molecule.

3. Why are iodide anion (I–) and starch used in the vi-
tamin C titration?

4. Why is the green pepper ground with water, and

the resulting solution decanted three times?
© 1998 Chemical Education Resources

ANAL 501/Effects of Cooking on Vitamin C Content 11

 ISBN 0-87540-501-0

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