Title(s): Estimation of Ascorbic Acid and DNA Extraction from Bananas

Date: 09/03/18

Name: Oluchi Baird

ID Number: 816002641

Lab Partner: Arianne -

Demonstrator’s Name: Gervonne Barran

Aim(s):

- To determine the ascorbic acid content of 4 different fruit juices by titrimetric analysis
using dichlorophenol-indophenol
- To extract and observe DNA precipitation from bananas using simple, household
reagents/items

Introduction:

Compounds which exhibit the biological activity of L-ascorbic acid are generically
termed vitamin C. Vitamins are essential for the maintenance of a healthy human diet due to
their association with specific coenzymes that take part in metabolism and other activities.
Vitamin C, in particular, is a strong reducing agent and water-soluble vitamin that is
necessary for the normal metabolic functioning of the body (Jaffe, 1994). As a component of
enzymes involved in collagen synthesis, vitamin C also plays a major role. This vitamin
lowers cholesterol levels and blood pressure (Rath, 1993), and serves as the major antioxidant
in the body where it serves several other functions, such as iron absorption, wound healing,
and the activation or enhancement of the immune response (Deekshika et al, 2015). Vitamin
C, however, cannot be synthesized by humans due to mutation in the gene which codes for L-
gulonolactoneoxidase, the enzyme responsible for its biosynthesis via the glucuronic acid
pathway (Woodall and Ames, 1997). Hence, vitamin C intake occurs via diet and the
consuming of supplements or foodstuff which contain it, such as citrus fruits and fruit juices
(Bendich, 1997). A vitamin C deficiency can result in a condition known as scurvy, which
has symptoms such as muscle degeneration, joint pains, gums which bleed easily and
haemorrhaging (Anon., n.d.). L-ascorbic acid is a six-carbon compound that is related to
glucose, with molecular formula C6H8O6 and structural formula as seen below:
 Ahern. Ascorbic Acid. http://oregonstate.edu/instruct/bb350/ahernmaterials/a16/16p24.jpg

The analytical method used for the determination of ascorbic acid concentration of the
fruit juices in this lab was to perform a redox titration using dichlorophenol indophenol
(DCPIP). This method of titrimetric analysis relies on oxidation and reduction, which occur
simultaneously. Oxidation involves the gain of oxygen, or loss of hydrogen while reduction,
conversely, is the gain of hydrogen, or loss of oxygen. The vitamin C is oxidized by the
DCPIP as it loses two hydrogen atoms to become dehydroascorbic acid, while the DCPIP is
reduced by the vitamin C as it gains the two hydrogens which were lost by the vitamin to
produce a colour change of pink to colourless.

The DCPIP in this experiment functioned as an indicator, in conjunction with the

glacial acetic acid that was used. DCPIP is an organic dye with both acidic and basic redox
properties, appearing blue in basic solutions and red/pink in acidic solutions, depending on
the concentration of the solution. Determination of the end point can prove difficult using
DCPIP with coloured samples since the colour produced at the end point is sometimes
masked or distorted by the colour of the sample being analysed (Anon., n.d.). In this
experiment, metaphosphoric acid was used to bring about the precipitation of ferric ions and
proteins within the samples. It defends against atmospheric oxidation of the vitamin C within
the solutions while not significantly affecting the rate of reaction (Musulin and King, 1936).
The glacial acetic acid was used to acidify the juice sample so that the colour change to be
observed was from pink to colourless. This was done to facilitate the reduction of the DCPIP,
which occurs readily in its acidic form.
 Mills. DCPIP Reduction. https://upload.wikimedia.org/wikipedia/commons/a/aa/DCPIP-
reduction-2D-skeletal.png

Additionally, since vitamin C is essentially an acid, its stability is greater in acidic solutions
as opposed to basic or neutral solutions where it is readily oxidized by dissolved oxygen. The
acetic acid functions to lower the pH, thereby limiting the action of the polyphenol oxidase
enzyme and thus, limiting the unwanted oxidation of the ascorbic acid (Anon, n.d.). The
reaction between DCPIP and vitamin C is 1:1 and occurs as follows:

ascorbic acid + HDCPIP (pink) → DCPIPH2 (colourless) + dehydroascorbic acid

The second portion of this lab execution involved the extraction of DNA from
bananas. DNA is the material found within cells that facilitates the transmission of genetic
information from one generation to the next. It consists of genes, which are segments of the
deoxyribonucleic acid that determine the traits possessed by organisms. These genes, which
code for proteins and contain the information which directs growth, functioning and the
characteristics of an organism, sometimes exist in more than one form. The variants of a
particular gene are known as alleles, and these variants are responsible for the differences
observed in specific characteristics among different organisms, such as hair colour and eye
colour, for example (Sayres, n.d.). The cells of bananas also contain DNA, and though it is
normally not visible with the naked eye, due to the relatively large amount that is extracted
from the many cells which make up the banana, we are able to view the DNA without the aid
of a microscope.

The banana was crushed well in order to break apart the cells so that the DNA might
be freed, and the reagents used played specific roles in the precipitation and extraction of the
DNA. For instance, the use of soap in the experiment aided in the breakdown of the cell’s
lipid membranes. The ability of soap to breakdown lipids facilitates this release of DNA as
the cell’s outer membrane and nuclear membrane are made of these lipid molecules.
Particularly, a compound referred to as ethylenediamine tetra-acetic acid (EDTA) which is
found in soap, plays an important role in the successful precipitation of the DNA. Due to its
ability to donate electrons and form coordinate bonds with metals, EDTA can be classified as
a chelating ligand. This complex structure can readily donate electrons from any of its four O
atoms or two N atoms. Nucleases are also present in the solution after the cell membranes are
broken down, and poses a threat of DNA disruption. However, the EDTA readily binds with
essential nuclease cofactors such as Mg2+ ions and in so doing, allows for greater DNA
precipitation (Anon., n.d.). Cold alcohol was also used, but instead, to aid in the precipitation
or clumping together of the DNA. This occurs since DNA is not soluble in alcohol. Lastly,
the salt was added as it aids in the separation of DNA from other materials in the cell. This
was done in preparation of the addition of the soap so that upon the release of the DNA from
the cells, the salt causes the DNA strands to stick together more readily, allowing it to
become more visible (Josephs, 2011; Sayres, n.d.).

Materials:

Estimation of Ascorbic Acid

- 5% metaphosphoric acid - 4 different fruit juices

- Dichlorophenol-indophenol - standard ascorbic acid solution (1 mg/ml)

Extraction of DNA from Bananas

- A large banana
- 3/4 cup of distilled water
- 1 teaspoon of clear, colourless shampoo or liquid soap that contains EDTA
- 1/4 teaspoon of table salt
- 15 ml of 90% isopropyl, chilled and in chilled test tube
- A large Ziplock bag
- 1 #4 cone paper coffee filter
- 250 ml beaker
- 1 plastic pipette, or medicine dropper
- A thin, glass rod

Method:

Estimation of Ascorbic Acid:

Standardization

1. The standard ascorbic acid solution was added to a clean burette.

2. 20 ml of dichlorophenol-indophenol solution was pipetted into an Erlenmeyer flask.
3. Titration with the ascorbic acid was carried out until the disappearance of the last
trace of pink colour from the solution.
4. The titration was performed at least 3 times.
5. The mean ± S.D. ascorbic acid equivalence per 1 mL of dye was calculated.

Estimation of Ascorbic Acid in Fruit Juices

1. 2 ml of one type of fruit juice was poured into a 50 ml beaker. The fruit juice was
acidified using 0.2 ml glacial acetic acid.
2. Using distilled water, the volume was made up to 50 ml.
3. The solution was mixed thoroughly. A measuring cylinder was used to measure and
transfer 25 ml of the preparation into another 50 ml conical flask.
4. 20 ml of 5% metaphosphoric acid was added.
5. The volume was made up to 50 ml by the addition of 5 ml of distilled water.
6. The solution was transferred into a burette.
7. At least 3 titrations were carried out using 5 ml volumes of the dichlorophenol-
indophenol solution.
8. Steps 1-7 were repeated for the other 3 fruit juices.
9. The ascorbic acid content in mg/ml was calculated for the fruit juices used.

Extraction of DNA from Bananas:

1. 100 ml of distilled water, along with approximately 50g of banana was placed into a
large Ziplock bag and crushed until the banana is completely pulverized. The mixture
was poured into a beaker.
2. 2 ml of soap and 0.5g of salt were mixed in a small Ziplock bag, followed by the
addition of 4 ml of distilled water.
3. To avoid the creation of foam, mixing was done gently. This was continued for a few
minutes until the soap and salt were well-dissolved.
4. 10 ml of the banana mixture was added to the small Ziplock bag and this was mixed
for at least 5 minutes.
5. A filter was inserted into a clean, plastic cup, ensuring that it did not touch the bottom
of the cup.
 6. The final mixture created (step 4) was poured into the filter. After 5 minutes had
elapsed, the filtrate was observed to have been collecting at the bottom of the cup.
The mixture in the filter was stirred and allowed to sit for an additional minute.
7. The filter was removed and set aside.
8. A test tube of cold alcohol was retrieved. Using a pipette or eyedropper, the filtrate
was collected and added to the alcohol.
9. The test tube containing the alcohol and filtrate was placed in a beaker or test tube
holder where it was allowed to sit, undisturbed, for roughly 4 minutes.
10. A glass rod was dipped into the test tube and used to spool out the banana’s
precipitated DNA through the slow rotation of the rod.

Results:

Estimation of Ascorbic Acid

Table 1. Standardization of DCPIP Solution

Trial Number Initial Reading (ml) Final Reading (ml) Volume Used (ml)
Trial 1 6.00 39.50 33.50
Trial 2 0.00 34.50 34.50
Trial 3 7.50 39.70 32.20
Average 37.90

Table 2. Titration Using Apple Juice Sample

Trial Number Initial Reading (ml) Final Reading (ml) Volume Used (ml)
Trial 1 8.00 23.70 15.70
Trial 2 23.70 39.50 15.80
Trial 3 2.20 18.00 15.80
Average 15.77
Table 3. Titration Using Grapefruit Juice Sample

Trial Number Initial Reading (ml) Final Reading (ml) Volume Used (ml)
Trial 1 3.60 25.70 22.40
Trial 2 0.00 22.80 22.80
Trial 3 22.80 44.20 21.40
Average 22.20

Table 4. Titration Using Pineapple Juice Sample

Trial Number Initial Reading (ml) Final Reading (ml) Volume Used (ml)
Trial 1 4.30 12.40 8.10
Trial 2 12.40 20.20 7.80
Trial 3 20.20 27.80 7.60
Average 7.83

Table 5. Titration Using Orange Juice Sample

Trial Number Initial Reading (ml) Final Reading (ml) Volume Used (ml)
Trial 1 7.50 15.00 6.50
Trial 2 15.00 24.20 9.20
Trial 3 24.20 30.80 6.60
Average 7.43
Extraction of DNA from Bananas

Observation: a white precipitate was formed; air bubbles were produced.

Calculations:

Estimation of Ascorbic Acid in Fruit Juices

Mass of Ascorbic Acid on Juice Packages:

Apple – 24 mg in 250 ml

Grapefruit – 29.5 mg in 250 ml

Pineapple – 40 mg in 250 ml

Orange – 25 mg in 250 ml

Standardization:

DCPIP concentration = 3 mg in 20 ml

Standard ascorbic solution = 0.1 mg/ml

Mean = sum of titre values / number of titrations = 34.50 + 32.20 + 33.50 / 3 = 33.40 ml
Applying the formula gives

S.D. = √ (33.50-33.40)2 + (34.50-33.40)2 + (32.20-33.40)2 = √ ( 2.66 / 2) = 1.1533 mg

3-1

S.D. per ml of DCPIP = 1.1533 / 20 = 0.0577 mg

1 ml of ascorbic acid contains 0.1 mg, hence 33.40 ml of ascorbic contains 0.1 x 33.40 = 3.34
mg

Ascorbic acid and DCPIP react in a 1:1 ratio, thus, 3.34 mg will be found in 20 ml DCPIP.

1 ml DCPIP will contain 3.34 / 20 = 0.1670 mg

Hence, mean ± S.D. ascorbic acid equivalence per 1 ml of DCPIP = 33.40 ± 0.1670 mg

Estimation of Ascorbic Acid in Apple Juice:

Mean for apple juice = 15.70 + 15.80 + 15.80 / 3 = 15.77 ml

Hence, 15.77 ml of apple juice reacts with 5 ml of DCPIP. Since 33.40 ± 0.1670 mg of
ascorbic acid reacts with 1 ml of DCPIP, ascorbic acid in 15.77 ml of apple juice = 33.40 x 5
= 167 mg

Since 15.77 ml of apple juice contains 167 mg ascorbic acid, then 50 ml of the apple juice
dilution contains (167 x 50) / 15.77 = 529.49 mg ascorbic acid

This 50 ml apple juice dilution was obtained from 25 ml of a previous dilution, which itself
came from another 50 ml dilution.

Therefore if 25 mL contains 529.49 mg ascorbic acid, 50 ml will contain (50 / 25) x 529.49 =
1058.98 mg

The original 2 ml of apple juice was made up to 50 ml and so, contains the same ascorbic
acid. Hence, ascorbic acid in 2 ml of apple juice = 1058.98 mg. Ascorbic acid in 1 ml of
apple juice = 1058.98/2 = 530 mg/ml

RDA for 75 mg = 75 / 530 = 0.14 ml of apple juice

Estimation of Ascorbic Acid in Grapefruit Juice:

Mean for grapefruit juice = 22.40 + 22.80 + 21.40 / 3 = 22.20 ml

Hence, 22.20 ml of grapefruit juice reacts with 5 ml of DCPIP. Since 33.40 ± 0.1670 mg of
ascorbic acid reacts with 1 ml of DCPIP, ascorbic acid in 22.20 ml of grapefruit juice = 33.40
x 5 = 167 mg

Since 22.20 ml of grapefruit juice contains 167 mg ascorbic acid, then 50 ml of the grapefruit
juice dilution contains (167 x 50) / 22.20 = 376.13 mg ascorbic acid

This 50 ml grapefruit juice dilution was obtained from 25 ml of a previous dilution, which
itself came from another 50 ml dilution.

Therefore if 25 mL contains 376.13 mg ascorbic acid, 50 ml will contain (50 / 25) x 376.13 =
752.26 mg

The original 2 ml of apple juice was made up to 50 ml and so, contains the same ascorbic
acid. Hence, ascorbic acid in 2 ml of grapefruit juice = 752.26 mg. Ascorbic acid in 1 ml of
grapefruit juice = 752.26 /2 = 376 mg/ml

RDA for 75 mg = 75 / 376 = 0.20 ml of grapefruit juice

Estimation of Ascorbic Acid in Pineapple Juice:

Mean for pineapple juice = 8.10 + 7.80 + 7.60 / 3 = 7.83 ml

Hence, 7.83 ml of pineapple juice reacts with 5 ml of DCPIP. Since 33.40 ± 0.1670 mg of
ascorbic acid reacts with 1 ml of DCPIP, ascorbic acid in 7.83 ml of pineapple juice = 33.40
x 5 = 167 mg

Since 7.83 ml of pineapple juice contains 167 mg ascorbic acid, then 50 ml of the pineapple
juice dilution contains (167 x 50) / 7.83 = 1066.41 mg ascorbic acid

This 50 ml pineapple juice dilution was obtained from 25 ml of a previous dilution, which
itself came from another 50 ml dilution.

Therefore if 25 mL contains 472.55 mg ascorbic acid, 50 ml will contain (50 / 25) x 1066.41
= 2132.82 mg

The original 2 ml of pineapple juice was made up to 50 ml and so, contains the same ascorbic
acid. Hence, ascorbic acid in 2 ml of pineapple juice = 2132.82 mg. Ascorbic acid in 1 ml of
pineapple juice = 2132.82 /2 = 1066 mg/ml
RDA for 75 mg = 75 / 1066 = 0.07 ml of pineapple juice

Estimation of Ascorbic Acid in Orange Juice:

Mean for orange juice = 6.50 + 9.20 + 6.60 / 3 = 7.43 ml

Hence, 7.43 ml of orange juice reacts with 5 ml of DCPIP. Since 33.40 ± 0.1670 mg of
ascorbic acid reacts with 1 ml of DCPIP, ascorbic acid in 7.43 ml of orange juice = 33.40 x 5
= 167 mg

Since 7.43 ml of orange juice contains 167 mg ascorbic acid, then 50 ml of the orange juice
dilution contains (167 x 50) / 7.43 = 1123.82 mg ascorbic acid

This 50 ml orange juice dilution was obtained from 25 ml of a previous dilution, which itself
came from another 50 ml dilution.

Therefore if 25 mL contains 1123.82 mg ascorbic acid, 50 ml will contain (50 / 25) x 1123.82
= 2247.64 mg

The original 2 ml of orange juice was made up to 50 ml and so, contains the same ascorbic
acid. Hence, ascorbic acid in 2 ml of orange juice = 2247.64 mg. Ascorbic acid in 1 ml of
orange juice = 2247.64 /2 = 1124 mg/ml

RDA for 75 mg = 75 / 1124 = 0.07 ml of orange juice

Discussion:

Four different samples of fruit juices were analyzed in this lab by means of titration to
determine the ascorbic acid content of the fruit juices. The results can be summarized as
follows:

- The ascorbic acid equivalence per 1 ml of DCPIP was found to be 33.40 ± 0.1670 mg.
- The ascorbic acid content of the apple juice sample was found to be 530 mg/ml.
- The ascorbic acid content of the grapefruit juice sample was found to be 376 mg/ml.
- The ascorbic acid content of the pineapple juice sample was found to be 1066 mg/ml.
- The ascorbic acid content of the orange juice sample was found to be 1124 mg/ml.

Based on these results, it can be stated that the orange juice sample had the highest ascorbic
acid content at 1124 mg/ml, followed by the pineapple juice sample at 1066 mg/ml, the apple
juice sample at 530 mg/ml and lastly, having the lowest ascorbic acid content was the
grapefruit juice at 376 mg/ml. The orange juice and pineapple juice share relatively similar
ascorbic acid contents with the variation being only 58 mg/ml. Besides these, the ascorbic
acid contents of the fruit juices differ considerably, with the greatest disparity occurring
between the orange and grapefruit juice samples at 748 mg/ml.

Additionally, the RDAs for the apple, grapefruit, pineapple, and orange fruit juice
samples were found to be 0.14 ml, 0.20 ml, 0.07 ml and 0.07 ml respectively. These values
are logical and expected since the fruit juices having a higher ascorbic acid content, like
orange juice, would be required in a lesser amount than those having a relatively low ascorbic
acid content, such as grapefruit. It was estimated that the orange and pineapple juices would
be required in the same amount and this is supported by their similar ascorbic acid contents.
However, these values do not rank the juices as expected, since based on given ascorbic acid
content values, pineapple, grapefruit, orange than apple is the expected ranking of highest
ascorbic acid content to lowest. Any differences may be accredited to the oxidation of
ascorbic acid as it would have been exposed for quite a period of time during the completion
of the lab. The experiment can thus be improved by limiting the exposure of the ascorbic acid
to oxygen, heat and light even further as it is easily oxidized.

With respect to the extraction of DNA from bananas, a white precipitate was
produced as expected. The white precipitate results from the clumping together of the DNA
as they are released out of the banana’s cells and into the alcohol medium, in which they are
insoluble. The air bubbles which were observed can be accredited to oxygen that becomes
trapped due to spooling.

Sources of error in this lab include:

- Contamination of apparatus or reagents used, for example, in the case of improper

cleaning of the burette before use.
- Parallax errors when recording volumes, as in the case of not taking readings at eye
level and below the meniscus.
- Human reaction time error with respect to identification of the end point.
Overshooting (or undershooting) may occur.
- Dilution errors.

Some precautions include:

- Using soap which is not cloudy, as this would cause difficulty in observing the DNA
precipitation.
 - Pre-rinsing the burette with the solutions to be placed in it so as to prevent dilution or
contamination.
- Ensure that readings and measurements are done at eye-level to avoid parallax errors.
- Titrate drop-wise as the reaction approaches the end-point to avoid overshooting.

Limitations to these experiments include:

- Incomplete transfer of reagents due to retention by apparatus beyond “the standing

drop”.
- Oxidative degradation of ascorbic acid.
- A possible lack of complete decolourization of DCPIP due to the method’s low
sensitivity.

In conclusion, vitamin C or ascorbic acid is essential to good human health as it

supports the biosynthesis of collagen, which has several important roles in the body, and
enhances the immune system, among other things. The ascorbic acid content can be said to
vary significantly among various fruits, vegetables and foodstuff in which it is found.
Additionally, all living organisms contain genetic material, mainly in the form of DNA,
which can be extracted or precipitated out of cells as demonstrated in this experiment.

References:

Anon. (n.d.). Ascorbic Acid Content of Fruit Juice.

http://www.markedbyteachers.com/gcse/science/ascorbic-acid-content-of-fruit-juice.html
(March 11, 2018)

Anon. (n.d.). Ascorbic Acid Titration.

http://slb.faculty.arizona.edu/sites/slb.faculty.arizona.edu/files/152/AscorbicAcid.pdf (March
12, 2018)

Anon. (n.d.) Vitamin C. http://chemistry.oregonstate.edu/courses/ch130/old/VITCTEXT.htm

(March 11, 2018)

Anon. (n.d.) What is the Function of EDTA in DNA Extraction?

https://study.com/academy/answer/what-is-the-function-of-edta-in-dna-extraction.html
(March 15, 2018)

Bendich A. (1997): Vitamin C Health and Disease. New York, pp.367-379.

Deekshika et al. (2015). Estimation of Ascorbic Acid Content in Fruits & Vegetables from
Hyderabad, India – A Theoretical Assessment of Vitamin C Activity.
https://www.ijcmas.com/vol-4-1/Deekshika%20B,%20et%20al.pdf (March 12, 2018)

Jaffe G.M. (1984): Vitamin C, In: Machalinal ed. Handbook of vitamins. New York:
Mercell Dekker Inc., pp.199 – 244.

Josephs, Molly. (2011). Find the DNA in a Banana.

https://www.scientificamerican.com/article/find-the-dna-in-a-banana-bring-science-home/
(March 14, 2018)

Musulin, R. and King, C. (1936). Metaphosphoric Acid in the Extraction and Titration of
Vitamin C. http://www.jbc.org/content/116/1/409.full.pdf (March 13, 2018)

Rath, M. (1993): Eradicating Heart Disease. Gealth Now, San Francisco, C.A.

Sayres, Melissa. (n.d.). Extracting DNA from a Banana and other Fruits.
https://askabiologist.asu.edu/activities/banana-dna (March 14, 2018)

Woodall A.A. and B.N. Ames. (1997): Diet and oxidative damage to DNA. The importance
of ascorbate as an antioxidant. In: Packer L. and J.Fuchs, Eds. Vitamin C in health and
disease. New York: Marcel Dekker INC, pp. 193 – 203.