Carbohydrate Testing

After performing Benedict's test on the extracts from bananas and

their peels we found that our precipitate was an orange-red color
thus indicating that the carbohydrate contains a free aldehyde
group. For both the peels and the actual fruit the amount of
precipitate visually increased from unripe to ripe to overripe. The
unripe peels and fruit showed less precipitate than the ripe peels
and fruit, which in turn showed less than the overripe peels and
fruit. Since our extract did show a positive result it agrees with our
prediction that one of the sugars present in bananas is structurally
similar to fructose. We cannot tell if sucrose is present from this test
because sucrose did not react under the conditions provided (Table

1).Our results from Barfoed's test showed that the unripe fruit was
the only one that did not change color and therefore contained poly-
and disaccharide sugars. The rest of the fruit solutions and all the
peel solutions showed a change in color, indicating that they
contained monosaccharide sugars (Table 1). These results do not
confirm our prediction that there would be more complex sugars
present in the later stages as compared to the unripe stage of
development. As the fruit and the peels ripened the sugars became
less complex, thus the reason for the absence of poly- and
disaccharide sugars in the later stages.
After performing Selivanoff's test we verified that the sugars in the
unripe, ripe, and overripe banana's fruit were monosaccharide
ketoses. The ripe and overripe peels also tested positive for
monosaccharide ketoses, while the unripe peel tested positive for
disaccharide ketoses (Table 1). Since Selivanoff's test and Barfoed's
test provided results that slightly contradicted each other in the
unripe peel and fruit, we are not sure about the accuracy of our
tests. Barfoed's test gave us results that signified that poly- and/or
disaccharides were present, while Selivanoff's test signified
monosaccharides were present. We had performed two different
trials for each test and obtained almost the same results each time.
Therefore we did obtain consistency, yet the two tests still
contradicted.

After completing Bial's test we were able to determine which type of

furanose rings were present during the different stages of the
ripening process. The peel of the unripe and ripe bananas turned
the solution an olive color, thus indicating that the sugars were in
the shape of a pentose-furanose ring. The peel of the overripe
banana and the fruit at all three stages turned the solution brown,
indicating that the sugars were in the shape of a hexose-furanose
ring. These results show that the sugar's structures change slightly
in the peel throughout the ripening process but remain the same in
the actual fruit (Table 1).
Our results from the Iodine test showed that starch is not present in
all three stages of the ripening process for both the banana's peel
and fruit (Table 1). The solutions for the fruit at the unripe, ripe,
and overripe stages darkened slightly and took on the yellowish cast
of the iodine, but none of them turned the bluish-black color that
would indicate starch.

Although it was not possible to figure out exactly which sugar(s)

were present at each stage of development from looking at the
results of just one test, the combined results gave us a clearer
picture of the carbohydrates present. The results for the unripe peel
and unripe fruit were the only ones that gave contradicting results
between tests. For both cases, the contradiction came between the
results of Benedict's and Selivanoff's tests. Although a sugar cannot
be a monosaccharide and be a disaccharide, these extracts gave
results that claimed both were true in those cases. However, for the
other extracts, the structure of the carbohydrate was determinable
from the results of the tests. For the ripe peel, the results described
a reducing sugar that was a mono-ketose, not starch, a
monosaccharide, and a hexose-furanose. These criteria exactly
match the characteristics of fructose, the structure of which we
found in the CEM 351 textbook (Jones, 1174). For all the remaining
tests, we consistently found that the extracts contained a
carbohydrate that was a monosaccharide ketose in pyranose, which
was not one of the known sugars tested in the first carbohydrate
lab.

Photosynthesis Testing
After completing our pigment identification we were not able to
identify the different pigments in the fruit and peels of bananas
during the stages of the ripening process. We performed two tests
for each of the six solutions, one for the peel and one for the fruit at
each stage, and each test showed no pigments in our extracts.
Since both times we performed the test we obtained the same
results, we had consistency but our consistency did not tell us what
pigments were present. Our paper chromatography test only tested
for the pigments chlorophyll A and B, xanthophyll, and carotene.
Since our tests gave no results this could have happened due to the
fact that bananas do not have these pigments in large enough
quantities to produce a result or that they do not contain these at
all. In this case, to obtain more accurate results we should have
tested for all pigments. Our lack of pigments could also be because
we were unable to successfully make a suitable extract to test for
banana pigments, as we used a method that works for spinach but
is unproven to work for bananas. Since our test did not produce us
any results we were not able to calculate the Rf values.

We graphed the wavelength of light versus the corresponding

absorbance levels (Figure 9). Our graph shows that for each
solution the absorbency was highest in the lower wavelengths and
steadily decreased as the wavelengths increased. This is concurrent
with our predictions because we assumed that the bananas would
have greater amounts of chlorophyll in the unripe stage and
decrease as the banana ripened. We assume that more absorbance
means that there more photosynthesis taken place because light is
a key component to photosynthesis.
Although our paper chromatography testing did not produce the
results we would have liked, our absorbance spectrum test gave
ample evidence to support our hypothesis.

Enzyme Testing
Each banana peel and its respective fruit turned the litmus paper a
shade of orange. This informs us that the pH levels were about 4.
Bananas and peels in all stages of the ripening process reacted to
catechol, so all of them have polyphenoloxidase (PPO).
Upon completing the tests for the effects of heat and inhibitors,
both the heated solutions and the inhibited solutions were lighter
than the control, but the heated solutions were darker than the
inhibited solutions. This data shows that enzymes in heated
solutions catalyze faster than enzymes in inhibited solutions (Table
3).After testing the effects that different pH's had on the
absorbances of banana extracts, we concluded that enzymes in
bananas catalyze more the closer they got to neutral conditions,
around pH 7 (Table 4). The banana extracts tended to absorb less
the further away from neutral conditions, in both directions. This
confirms that the rate of the reaction was catalyzed most effectively
under neutral conditions.

Weaknesses that were present in our experiment were mainly due

to the time constraint. There were two weeks in between our
carbohydrate testing and our photosynthesis testing and then
another two weeks in between the photosynthesis testing and
enzyme testing. The lengthy amount of time in between the
different tests would have caused our samples to spoil and so we
had to attain new materials for each test. We could not have used
the spoiled samples because obviously then we would not have had
bananas at all three stages of the ripening process. Instead we
would have just been testing the overripe stage over and over
again. We assumed since we received the bananas from the same
location every time that essentially they were the same.
One problem that arose during our experiment was the fact that we
were not the only group to use the lab equipment and perform an
experiment. Numerous groups were constantly moving in and out of
the lab and possibly exposing unpredicted chemicals into our
specimen. Many other groups were working with fruits too and as
explained in our introduction, this may have caused more ripening
to occur then was planned. Also in the adjacent laboratory there
was another biology class performing breeding experiments on the
Drosophila melanogaster or the common fruit fly. The lab groups
even sometimes came into our lab and used our fume hood. Some
of the flies could have managed to infiltrate our specimen and thus
cause some contamination. Regardless of these complications, our
hypothesis was overall confirmed from the tests conducted and the
data collected.
Benedict's reagent (often sold as Benedict's Qualitative Solution or
Benedict's Solution) is a chemical reagent named after an American
chemist, Stanley Rossiter Benedict.[1]
Benedict's reagent is a chemical reagent commonly used to detect
presence of reducing sugars, however other reducing substances also give
a positive reaction.[2] This includes all monosaccharides and
many disaccharides, including lactose and maltose.
Generally, Benedict's test will detect the presence of aldehydes, and alpha-
hydroxy-ketones, including those that occur in certain ketoses. Thus,
although the ketose fructose is not strictly a reducing sugar, it is an alpha-
hydroxy-ketone, and gives a positive test because it is converted to the
aldoses glucose and mannose by the base in the reagent.
A positive test with Benedict's reagent is shown by a colour change from
clear blue to a brick-red precipitate.
One litre of Benedict's reagent can be prepared from 100 g of
anhydrous sodium carbonate, 173 g of sodium citrate and 17.3 g
of copper(II) sulfate pentahydrate.[3] It is often used in place of Fehling's
solution.
The principle of Benedict's test is that when reducing sugars are heated in
the presence of an alkali, they get converted to powerful reducing
compounds known as enediols. Enediols reduce the cupric ions (Cu2+)
present in the Benedict's reagent to cuprous ions (Cu+) which
get precipitated as insoluble red copper(I) oxide(Cu2O).
The color of the obtained precipitate gives an idea about the quantity of
sugar present in the solution, hence the test is semi-quantitative. A
greenish precipitate indicates about 0.5 g% concentration; yellow
precipitate indicates 1 g% concentration; orange indicates 1.5 g% and red
indicates 2 g% or higher concentration.

Benedict's solution[edit]
Benedict's solution is a deep-blue alkaline solution used to test for the
presence of the aldehyde functional group, - CHO.[2] The substance to be
tested is heated up to 95 C (for example, in a water bath) with Benedict's
solution; formation of a brick-red precipitate indicates presence of the
aldehyde group in relatively high concentrations.[2] Since simple sugars
(e.g., glucose) give a positive test, the solution is used to test for the
presence of glucose in urine, a symptom of diabetes. One liter of
Benedict's solution contains 173 grams sodium citrate, 100 grams sodium
carbonate, and 17.3 grams cupric sulfate pentahydrate. It reacts chemically
like Fehling's solution; the cupric ion (complexed with citrate ions) is
reduced to cuprous ion by the aldehyde group (which is oxidized), and
precipitates as cuprous oxide, Cu2O.

Experiment Observation Inference

Substance in water + 3ml Benedict's solution, then boil Red ppt or green ppt or yellow Reducing sugar e.g.
for few minutes and allow to cool. ppt obtained Glucose is present

Substance in water + 3ml Benedict's solution, then boil Solution remains clear or is a Reducing sugar is not
for few minutes and allow to cool. little blue present

Quantitative reagent[edit]
Benedict's quantitative reagent contains potassium thiocyanate and is
used to determine how much reducing sugar is present.[2] This solution
forms a copper thiocyanateprecipitate which is white and can be used in
a titration. The titration should be repeated with 1% glucose solution
instead of the sample for calibration.