Date: 17- Feb-2017

Name: Brittnee Xuxa Issarie

Lab Partner: Tarik, Akeila, Kareem

Demonstrator: Teshelle

Title: Estimation of ascorbic acid from 3 different canned juices and extraction of DNA from a
banana.

Aim: 1) To extract DNA from a banana

2) To estimate the amount of ascorbic acid present in 3 canned juices; apple, orange and
grapefruit.

Introduction:

Ascorbic acid, also known as Vitamin C, has the chemical formula C6H8O6. It serves as a
powerful reducing and anti-oxidizing agent and is naturally occurring in many fruits and
vegetables (Mayo clinic 2013). It is water soluble and can be used by the body to defend against
pathogens and also to synthesize collagen which is needed for healthy teeth, bones and skin.
Since it is soluble in water, the vitamin cannot be stored or made by the human body and must be
an eminent part of the diet as it is essential (Wax 2015). In the human body, ascorbic acid plays
many vital roles. Its necessary for the growth and repair of tissues, bones cartilage and teeth,
aids in the absorption of iron by the body, responsible for the healing of wounds and it also forms
part of a protein used to synthesize skin, tendons and blood vessels (Wax 2015). Additionally, it
acts as an antioxidant which protects against damage caused by free radicals. By doing this, it
slowers the aging process in humans. Ascorbic acid plays a very important role in the food
industry as it acts as a source of nutrient as well as it protects the color and smell of the product
thereby improving the product (Science in Hong Kong 2008). Due to its structure, ascorbic acid
is quite unstable and cannot withstand heat. During manufacturing and processing in the food
industry, levels on vitamin C present in food may be significantly reduced and hence must be
restored by the industry. In foods and beverages, it can play roles of acidifiers, buffering and
reducing agents or even solvents. Since it is an effective reducing agent, it can be important to
other industries such as that of the cosmetic by tightening the skin, keeping it firm, slowering the
aging process (Science in Hong Kong 2008).

Diagram 1 showing the chemical structure of ascorbic acid (vitamin C), (date accessed 27-Feb-
2017).

In this experiment, an indicator called dichlorophenol-indophenol was used to detect the

presence of ascorbic acid in the 3 canned juices. Initially, its of a blue hue due to oxidation and
changes to red when acids are present. However, there is a loss of color when the chemical
Vitamin C is present as reduction occurred and as a result it appears a yellow-tinge color
(Nuffield Foundation 2011).

DCPIP (blue) + H+ DCPIPH (pink)

DCPIPH (pink) + vitamin C DCPIPH2 (colorless)

In the titration performed, not all the ascorbic acid was used up and hence there were available
electrons to reduce DCPIPH to give the colorless end point (Iris 2017). The sample to be tested
was acidified using metaphosphoric acid to remove any precipitate proteins and ferric ions.

Method:

1) Estimation of ascorbic acid in 3 different canned juices.

Twenty mL of orange canned juice was poured into a measuring cylinder and the
transferred to a 500mL beaker. Two mL of glacial acetic acid was added to the beaker to
acidify the orange juice. The volume was made up to 50mL with distilled water. The
mixture was shaken thoroughly and carefully. Twenty-five mL of the mixture was
transferred using another measuring cylinder to a 50mL conical flask. There, 20mL of 5%
metaphosphoric acid was added. The volume was made up once more to 50mL by the
addition of 5mL distilled water. The solution was then transferred into a burette. Three
 5mL volumes of the dichlorophenol-indophenol solution were titrated. The ascorbic acid
content of the orange juice was calculated in mg/mL. The procedure was repeated using
grapefruit and apple canned juices.
2) Extraction of DNA from bananas.
One hundred mL of distilled water and approximately 50g of banana was placed into a
large zip lock bag and crushed until the banana was completely pulverized. The mixture
was then poured into a beaker. Two mL of soap was mixed with 0.5g of salt in a small zip
lock bag. Four mL of distilled water was added. The mixture was mixed gently to avoid
the formation of foam, until the soap and salt dissolved. Ten mL of the banana mixture
was added to the small zip lock bag which contained the soap solution. It was then mixed
for 10 minutes. A filter was inserted into a clean plastic cup where it was ensured that the
filter did not touch the bottom of the cup. The mixture was gently stirred into the filter
and left to sit for a minute. The filter was then removed and set aside. A test tube of cold
ethanol was fetched. A pipette was used to collect the filtrate which was then added to the
alcohol. The test tube containing the alcohol and filtrate was placed in a beaker and
allowed to sit undisturbed for 4 minutes. A glass rod was dipped into the tube and rotated
slowly to spool out the bananas DNA.
3) Titration with standard ascorbic acid.
The standard ascorbic acid solution was added to a burette. Twenty mL of dichlorphenol-
indophenol solution was pipetted into an Erlenmeyer flask. It was then titrated with the
ascorbic acid until the last trace of pink color disappeared. The titration was repeated
twice. The mean and standard deviations of ascorbic acid equivalence were calculated per
1mL of dye.

Materials:

Burette, pippete, conical flask, measuring cylinder, beaker, apple juice, grapefruit juice, orange
juice, glacial acetic acid, metephosphoric acid, dichlorophenol-indophenol, distilled water, a
large banana, clear liquid soap containing EDTA, table salt, isopropyl, large zip lock, small zip
lock, filter paper, glass rod, test tube.

Results:
Table 1 illustrating the titration results obtained from performing a titration with standard
ascorbic acid.

Trial 1 Trial 2 Trial 3

Initial burette reading/ 30.3 17.0 27.5
mL
Final burette 40.2 27.5 37.2
reading/mL
Volume used/mL 09.9 10.5 09.7

Table 2 illustrating the titration results obtained from performing a titration with canned
grapefruit juice.

Trial 1 Trial 2 Trial 3

Initial burette reading/ 05.6 20.0 34.3
mL
Final burette 20.0 34.3 48.6
reading/mL
Volume used/mL 14.4 14.3 14.3

Table 3 illustrating the titration results obtained from performing a titration with canned orange
juice.

Trial 1 Trial 2 Trial 3

Initial burette reading/ 16.0 24.5 33.1
mL
Final burette 24.5 33.1 41.7
reading/mL
Volume used/mL 8.5 8.6 8.6

Table 4 illustrating the titration results obtained from performing a titration with canned apple
juice.

Trial 1 Trial 2 Trial 3

Initial burette reading/ 06.0 24.5 25.2
mL
Final burette 24.5 42.0 42.9
reading/mL
Volume used/mL 18.5 17.5 17.0

After performing the DNA extraction with the large banana, a white clump composed of string-
like strands appeared.

Calculations:

Mass of ascorbic acid on juice packages:

Grapefruit: 37mg in 250mL

Orange: 124mg in 250mL

Apple: 46.5mg in 250mL

Standardization:

To find how many mg of ascorbic acid reacts with 1ml of DCPIP

DCPIP concentration= 3mg in 20mL

Standard ascorbic acid solution= 3/20=0.1mg/mL

Average titre values= (9.9+10.5+9.7)/3= 10.033mL

Calculation of standard deviation:

Using formula, SD=

SD per mL of DCPIP=0.4163334/20 =0.0208 mg

If 10.033mL of ascorbic acid reacts with 20ml of DCPIP

Given 1.0ml of ascorbic acid contains 0.1mg

1.0033mg will be found in 20ml DCPIP

Therefore, for 1.0mL DCPIP 1.0033/20 = 0.050165mg

Therefore mean SD = 0.050165 0.0208 mg ascorbic acid

Average titre value for grapefruit juice dilutions = (14.4+14.3+14.3) / 3 = 14.33ml

We can say 14.33ml of juice dilution reacts with 5 ml DCPIP

We know that 0.050165 0.0208mg of ascorbic acid reacts with 1ml of DCPIP

Therefore, ascorbic acid in 14.33ml juice dilution = 0.050165 x 5 = 0.250825mg

If 14.33ml of juice dilution contains 0.250825

Then 50ml of this dilution will contain (0.250825 x 50)/14.33 = 0.87517mg ascorbic acid

This 50ml juice dilution was obtained from 25ml of a previous dilution

This 25ml came from another 50ml

Therefore, if 25ml contains 0.87517mg ascorbic acid, 50ml will contain

50/25 x 0.87517= 1.7503mg

For original 20ml of juice, since 20ml made up to 50ml, it will the same ascorbic acid

Therefore, ascorbic acid in 20ml juice =1.7503mg

Therefore ascorbic acid in 1ml = 1.7503/20 = 0.087517mg/ml

RDA for 75mg = 75/0.087517= 857ml of juice

Average titre value for orange juice dilutions = (8.5+8.6+8.6)/3 = 8.566ml

We can say 8.566 ml of juice dilution reacts with 5ml DCPIP

We know that 0.0501650.0208mg of ascorbic acid reacts with 1ml of DCPIP

Therefore, ascorbic acid in 8.566ml juice dilution = 0.050165 x 5 = 0.250825mg

If 8.566ml of juice dilution contains 0.250825

Then 50ml of this dilution will contain (0.250825 x 50)/8.566 = 1.4640mg ascorbic acid

This 50ml juice dilution was obtained from 25ml of a previous dilution

Therefore 25ml came from another 50ml

Therefore if 25ml contains 1.4640mg of ascorbic acid, 50ml will contain

50/25 x 1.4640 = 2.928mg

For original 20ml of juice, since 20ml made up to 50ml, it will contain the same ascorbic acid

Therefore ascorbic acid in 20ml juice = 2.928mg

Therefore, ascorbic acid in 1ml = 2.928/20 = 0.1464mg/ml

RDA for 75mg = 75/0.1464= 512ml of juice

Average titre value for apple juice dilutions = (18.5+17.5+17.7)/3 = 17.9ml

We can say 17.9ml of juice dilution reacts with 5ml DCPIP

We know that 0.0501650.0208mg ascorbic acid reacts with 1ml of DCPIP

Therefore ascorbic acid in 17.9ml juice dilution = 0.050165 x 5 = 0.250825mg

If 17.9ml of juice dilution contains 0.250825

Then 50ml of this dilution will contain (0.250825 x 50)/17.9 = 0.7005mg ascorbic acid

This 50ml of juice dilution was obtained from 25ml of a previous dilution

This 25ml came from another 50ml

If 25ml contains 0.7005mg ascorbic acid, 50ml will contain

50/25 x 0.7005=1.401mg

For original 20ml of juice, since 20ml made up to 50ml, it will contain the same ascorbic acid

Therefore ascorbic acid in 20ml juice = 1.401mg

Therefore ascorbic acid in 1ml = 1.401/20 = 0.07005mg/ml

RDA for 75mg = 75/0.07005= 1070ml of juice

Discussion:

According to this juice packages values, there were 46.5mg of ascorbic acid present in 250ml of
apple juice. However, when titration was performed on this juice in the laboratory, we found that
75mg contained 1070ml of ascorbic acid.

Apple: 46.5mg in 250ml (published data)

75mg in 1070ml (experimental data)

 75/1070 = 1ml
250ml = 75/1070 x 250 = 17.52mg

Based on the above calculation, only 17.52mg of ascorbic acid was present in the 250ml apple
juice. The experimental data was significantly less than that of the published data on the package,
that is, 46.5>17.52.

Additionally, the orange juice published data present on the package remarked 124mg of
ascorbic acid present in 250ml of juice. However, when the experiment was done, the following
calculation was observed.

Orange: 124mg in 250ml (published data)

75mg in 512ml (experimental data)

75/512 = 1ml
75/512 x 250= 36.62mg

Based on the calculation performed above, the experimental data was significantly less than that
of the published, that is, 36.62 < 124.

Furthermore, the grapefruit juice package presented that 37mg of ascorbic acid was present in
the 250ml of juice but on performing the titration, it value was not attained. The calculation is
presented below:

Grapefruit: 37mg in 250ml (published data)

75mg in 857ml (experimental data)

75/857 = 1ml
250ml = 75/857 x 250 = 21.88mg

From the above calculation, the published data was greater than the value calculated when the
experiment was carried out, that is, 37>21.88.

In the extraction of DNA from the large banana, the banana was squashed to allow the cells and
cell walls of the banana to break apart.
Cold alcohol was added when this experiment was being carried out as DNA cannot be dissolved
in alcohol. However, DNA clumps are soluble in some liquids and this was avoided since the
addition of alcohol aided the DNA in clumping together (Sayres).

The use of soap was necessary to break down or hydrolyze the lipids. This proved useful in this
particular experiment as the membranes which surround the nucleus is made up of lipids and the
aim was to extract the DNA from the nucleus of the banana (Sayres). Hence, the DNA was
released upon the addition of soap as the membranes and nucleus were broken.

The use of salt simply aided the experimenter to view the clumps of DNA once extracted as it
allowed the strands of DNA to adhere making them clearly visible to the naked eye (Sayres).

The experiments performed in this laboratory may have had errors due to impurities being
present in solutions as well as the dichlorophenol-indophenol being exposed to some degree of
light. DCPIP is sensitive to light and the beaker which it was poured into was wrapped in foil to
avoid it being deactivated by light but some light may have penetrated the solution when
carrying out the experiment. Other precautions observed included reading all measurements at
eye level, wearing protective clothing and removing the funnel after pouring solution in burette
before beginning the titration.

REFERENCES

"Vitamin c (ascorbic acid)." Mayo clinic. November 01, 2013. Accessed February 27,
2017. http://www.mayoclinic.org/drugs-supplements/vitamin-c/safety/hrb-20060322.
Wax, Emily. "Vitamin c ." Medline plus. February 2, 2015. Accessed February 27, 2017.
https://medlineplus.gov/ency/article/002404.htm.
 "The application of Vitamin C--- From food and beverages to cosmetic products."
Science in Hong Kong. 2008. Accessed February 27, 2017.
http://www2.caes.hku.hk/sciencehk/files/2010/08/2006169614.pdf.
"Measuring the vitamin C content of food and juices." Society of biology. November 24,
2011. Accessed February 27, 2017. http://www.nuffieldfoundation.org/practical-
biology/measuring-vitamin-c-content-foods-and-fruit-juices.
Iris, Ivy. "ANALYSIS OF CONCENTRATION OF VITAMIN C IN COMMERCIAL
AND FRESH FRUIT JUICES BY TITRATION METHOD (DCPIP TITRATION)."
Academia. 2017. Accessed February 27, 2017.
http://www.academia.edu/7664372/ANALYSIS_OF_CONCENTRATION_OF_VITAMI
N_C_IN_COMMERCIAL_AND_FRESH_FRUIT_JUICES_BY_TITRATION_METHO
D_DCPIP_TITRATION_.
Sayres, Melissa Wilson. "Extracting DNA from a banana and other fruits." ASU School
of life sciences. Accessed March 4, 2017. https://askabiologist.asu.edu/activities/banana-
dna.