UNIVERSIDAD DE ZAMBOANGA

School of Allied Medicine (SAM)

Pharmacy Department

PHARM BIO SCI 3 LABORATORY

PHARMACEUTICAL BIOCHEMISTRY
Activity No. 3 - CARBOHYDRATES
GROUP WORK OUTPUT

BS Pharmacy 2-A
MWF 9-11 am

Submitted to:

Sabtula, Ben-Frazier U.
Pharm-Biochemistry Instructor

Submitted by:

Acabal, Jasher Dave C (Performer, Encoder and Researcher)

Tuknol, Apasra (Performer and Observer)
Latosa, Ariel (Errand and Performer)
Ramos, Ronald (Performer)
Sarahadil, Ayman (Performer and observer)
AmilHamja, Alnaliza (Performer)

PROTEIN MAZIMIZERS
Group name
 UNIVERSIDAD DE ZAMBOANGA
School of Allied Medicine (SAM)
Pharmacy Department
PHARM BIO SCI 3 LABORATORY
PHARMACEUTICAL BIOCHEMISTRY

ACTIVITY No. 5 CARBOHYDRATES

A. MONOSACCHARIDES and DISACCHARIDES

1. MOORES TEST

We prepared and mixed 1.O of 5% glucose solution with 1.O ml concentrated NaOH in the test
tube and boiled at least 5 minutes. We observed when reducing sugar is heated with an alkali it turns
yellow to orange, finally dark brown liberating odor of caramel, which becomes more marked upon
acidification due to the liberation of aldehyde which subsequently polymerizes to form resinous
substance, caramel.

1.0 mof 5% Glucose After heating

solution
+ Conc. NaOH 5 minutes

2. MOLISCHS TEST

We placed 1.0 ml of 5% glucose solution in a test tube and added 1 drop of molischs reagent and
we mixed it thoroughly. After a certain seconds we inclined the tube and allowed 1.0 ml of
concentrated sulfuric acid to flow on the side of the tube and now by not shaking the mixed reagent
and solution we immediately placed it the test tube rack and noted the color or appearance of reddish
violet or purple color ring at the junction of the two liquids.

Then therefore carbohydrates (glucose) are dehydrated by concentrated sulfuric acid to form
hydroxymethylfurfural and reacted with alpha-napthol ( Molischs reagent) to yields the positive result
of purple condensation product- not specific for carbohydrates. This reaction has the same
explanation with the moores test.
 5% Glucose + After adding 1.0 ml
Molischs reagent conc. Sulfuric acid

B.REDUCTION TESTS:

3. FEHLINGS TEST

We placed 5.O ml of Fehlings A,in which is a blue aqueous solution of copper (II) sulfate and
B is a clear solution of aqueous potassium sodium tartrate (also known as Rochelle salt, which made
by dissolving of potassium sodium tartrate of sodium hydroxide in distilled water ) and a strong alkali
(commonly sodium hydroxide) in the test tube and we diluted it at 4.0 cc of water, produced the blue
color solution, then we boiled at 1.0 ml of the mixture in a water bath and yet there was no
discoloration happened afterwards Equal volumes of two mixtures are mixed together to get the final
Fehlings solution, which is deep blue color, then if the color is unchanged after boiling (brick-red
color), then therefore we instantly got a 1.0 ml product produced in put it in a test tube again and
added 5.0% glucose solution drop by drop,at the 1st drop, the color was indigo, the 2nd was ruby
color, 3rd drop was red , 4th was red precipitate at the bottom, orange at the top and 5th was red
precipitate at the bottom and dark orange at top, until it became 20 drops the color was red
precipitate at the bottom, orange at the top. In which the formation of red precipitate indicates the
presence of glucose.
Therefore, Fehlings test can be used as a generic test for monosaccharides. It will give a
positive result for aldose monosaccharides (due to the oxidized aldehyde group) but also for ketose
monosaccharides, as they are converted to aldoses by the base in the reagent, and then give a
positive result. For this reason, Fehlings reagent is sometimes referred to as general test for
monosaccharide in other words Fehlings test is considered positive when the solution turns from blue
to orange or rep precipitate.
Mixed Fehlings A and B 1st Drop (Indigo)

3rd drops (Pale

2 drops ruby color
redcolor)

4th drops (orange red)

5th drops (copper color)
 20 drops (red precipitate
bottom, and golden yellow
solution

Explain the Principles involved. Why the solution are kept separate?

Fehlings solution consists of Fehlings A (copper(II) sulphate solution) and Fehlings B (sodium
tartarate solution), equal amounts of which are added to the test solution. After boiling, a positive
result is indicated by the formation of a brick-red precipitate of copper(I) oxide. Methanal, being a
strong reducing agent, also produces copper metal; ketones do not react. The test is now rarely used,
having been replaced by Benedicts test.
The reaction requires strongly basic conditions, which is provided by sodium hydroxide in
Fehling's Solution B. However, Cu(II) will precipitate in basic solution as the hydroxide complex. To
prevent this, tartrate is added to Solution B. Tartrate forms a soluble complex with Cu(II), so that when
the Solution A (copper sulfate) is added to the basic Solution B (tartrate and hydroxide) the Cu(II)
remains in solution. However, the tartrate complex is only kinetically favoured: the precipitating
hydroxide is actually more stable. So in a mixture of Fehling's Solution A and Fehling's Solution B that
is left to stand the Cu(II) hydroxides will eventually precipitate.

The two separate solutions are indefinitely stable and can be left on a shelf until it's time to use it.

4. BENEDICTS TEST

We mixed 1.0 ml Benedicts reagent with 2 drops of glucose solution, and we permitted to cool
at least 2 minutes and we perceived the positive result of brown or cherry red color. The precipitate is
moulded due to the reduction of the Cu2+ ions to Cu+ ions in the process of hydrolysis. The color
formed depends upon the amount of reducing sugar present in the mixture. Reducing sugars reduce
soluble blue copper sulfate, containing copper(II) ions to insoluble red-brown copper oxide containing
copper(I).

Therefore, when the mixture is heated carbohydrates which reacts with Benedicts reagent to
reduce the blue copper (II) ions to form a brick-red precipitate of copper (I) oxide are classified as
reducing sugars
 1.0 ml of Benedicts reagent +
2 drops Glucose solution

What are the differences between Benedicts and Fehlings Tests? Which one is more
sensitive?

Chemically, Benedict's solution and Fehling's solution are very similar, with copper sulfate as a source
of copper (II) ions, sodium carbonate and sodium hydroxide respectively as alkali, and sodium citrate
and sodium potassium tartrate respectively as chelators. They are also both used to test aldehyde
groups which can reduce the copper (II) ions into copper (I) ions, giving a precipitate of Cu2O. Hence,
they are often used in testing for presence of aldehydes, and by extension, reducing sugars. Other
alternative test to benedicts test for reducing sugar is the Fehlings test for an non-sugar is an
alternative. To answer which was more sensitive, is that Benedictss test, because Fehlings test need
more or strong alkaline in order for a reaction to take place, unlike Benedicts test where even in a
weak alkaline environment, a reaction still occur, and addition it also undergoes a series a change in
color.

5. NYLANDERS TEST

We mixed 1.0 ml of 5% glucose with 0.1 ml of Nylanders reagent in the test tube and created
dispersed color in nature purple and white dispersion region of the Nylanders reagent and finally
heated at least 5 minutes in the time given, we distinguished the black precipitate of metallic bismuth
is formed, in view of that Nylander reagent consist of bismuth nitrate , potassium sodium tartrate
( Rochelle Salt) and 19 % potassium hydroxide gives the alkaline environment in order for the
reaction to occur. The potassium tartrate serves as the solvent for the bismuth subnitrate.

The conclusion of the test is that when sugar is present, there is a brown/purple to black
coloration of the fluid where the metallic bismuth settles down. If the dark coloration occurs as the
fluid is cooling down, it does not prove the presence of sugar.

1.0 ml Glucose + 0.1 ml After heating 5 minutes

Nylanders reagent
Write the reaction involved in the test.
Bi(OH)2NO3 + KOH -------> Bi(OH)3 + KNO3

Reducing sugar
2 Bi(OH)3-----------> 2 Bi + 3 O + 3 H2O
Heat (black precipitate)

6. BARFOEDS TEST
We mixed 1.0 ml of Barfoeds reagent with 0.1 ml of 5% glucose solution as what we observed
the bluish solution transpired. We progressed to the heating process at least 30 seconds and stood
up for 15 minutes. As we observed the solution gave the positive result of a scanty brick-red
precipitate. This indicates the presence of reducing sugars.
Therefore, Barfoeds Test is similar to the Benedicts test, but differs in the specific reagents
used (copper (II) acetate and acetic acid) which is less reactive than the Benedicts reagent. It is used
to differentiate between monosaccharides and disaccharides. When boiled in water,
monosaccharides will react in under 10-15 minutes, while disaccharides will take longer. If heated
long enough (15-20 minutes) disaccharides will give a positive result due to hydrolysis to form
monosaccharides. As in the Benedicts test above, the primary reaction is the reduction of Cu +2 ions
to Cu2O which forms a brick red precipitate. The test is like the response of Fehlings answer for
aldehydes.
 1.0 ml Barfoeds reagent + After Heating
0.1 ml of 5% Glucose
solution

7. PICRIC ACID TEST

We set 1.0 ml glucose solution in the test tube and supplemented a 0.5 ml of Saturated Picric
acid solution when progressively mixed the yellow color take placed. In order to react the solution of
picric acid, the solution must be alkaline this was the purpose of adding the 10% of sodium carbonate
(NaCo3) in our experiment. After heating the solution it turned into a picramic acid which gave the
positive result of mahogany red precipitate.
Therefore, when Picric acid reacts with the aldehyde or keto group of the reducing sugar, it
turns into a picramic acid which give the positive color of mahogany red. And heating was done
because in higher temperatures, reaction between substances takes place faster.

Picric acid (Yellow) Picramic acid (Mahogany red)

8. SELIWANOFFS TEST
 We prepared at least 6 test tubes and placed 1.0 ml of seliwanoffs reagent in each 6 test
tubes. They have the same initial color (Golden yellow) as when they mixed with 2% sugars
(Fructose, Glucose, Galactose, Sucrose, Maltose, Lactose) as that order. After mixing sugar solution
and seliwanoffs reagent in the different test tubes we heated it individually in a prepared water bath
with boiled water, as we put the different test tube in the water bath we detected the changed in color
from golden-yellow turned to deep-red color in the 1 st test tube which was fructose with the time
elapsed of 03.46 minutes, so it is a ketohexose, 2 nd test tube which was glucose a deep-red
precipitate nor cherry red precipitate is not observed, the time elapsed 06.13 minutes, same with 3 rd
test tube which was galactose the only time elapsed is 04.58 minutes, and 5 th test tube which was
maltose do not gave the positive result of cherry red, even deep red precipitate the only time elapsed
is 07.31 minutes, and same with the 6th test tube was lactose do not gave the positive result, but we
noticed when we checked the 4th test tube which was sucrose it produced the deep- red precipitate
which indicated as a positive result and considered as a ketohexose containing disaccharides. As we
are going to analysed one hindrance maybe of not achieving the pink, deep-red, or cherry color of the
other sugars like galactose, glucose, maltose and lactose is due to the percentage of the solution, so
then we could say the color formed depends upon the amount of reducing sugar present in the
mixture, unlike fructose and sucrose gave the positive result because accordingly they have high
content of sugar, theoretically epeaking fructose is the most sweetest sugar with 173.3% of
sweetness and sucrose 100% sweetness present.

Therefore, aldoses get converted to ketoses and gave the positive result reaction with
seliwanoffs reagent; this test reagent is caused the dehydration of ketohexose to form 5-
hydroxymethylfurfural and reacted with resorcinol present in the test reagent to produce a deep-red
product. In other words it is a timed color reaction specific for ketoses. In our experiment we gained a
positive result for fructose and sucrose (fast reacting sugar) which are the fact ketoses in nature.
They turned deep-red and cherry color as they heated passing seliwanoffs test.

Fructose Sucrose

Fructose and Sucrose (fast reacting sugars)

Maltose Glucose
 Lactose Galactose

Galactose, lactose, maltose, and glucose are slowest reacting sugars

Observation based on the filtered and dissolved precipitate in Ethyl Alcohol. Does it impart a red color
to the solution? We could not answer yes in the mere fact there was no required amount of ethyl
alcohol to be poured out through filter paper, yet there was a red precipitate present in the filter paper
most especially in the sucrose and fructose but then again the amount of ethyl we poured out is not
been measured thats why we chemically analysed the fact that, the more ethyl alcohol been poured
out to the precipitate in the filter paper will give the golden yellow solution instead red solution. This
are the illustration we have done on filtration and dissolving the precipitate through ethyl alcohol. And
to answer the question if red color occurs or impart in the solution:

Filtered Precipitate Imparted solution

Galactose
Glucose
Lactose ppt.
ppt.
ppt. Galactose
Glucose
Lactose
sol.sol.
sol.
Fructose ppt. Fructose sol.
 Sucrose ppt. Sucrose sol.

Maltose ppt. Maltose sol

C. POLYSACCHARIDES:

1. IODINE TEST on Starch Powder

 We placed a small pinch of starch powder on a glass over a piece of white paper, and
followed by dropping a lugols solution known as lugols iodine on it. Fortunately we observed some
changes happened on the starch, as we all know the powder being used is high in present of starch it
gave the positive result of black in color. Black/purple in color foamy is we called it the unknown
reaction. Before performing the test, we had apre-conclude answer that the starch will give a positive
black color while the glycogen will not base on the theoretical research we conducted. Upon
performing the procedure, we had gained a starch which is black.

Therefore, to put it simply, when the iodine solution comes into contact with starch, it
turns black. Otherwise, it will remain brown in color. And precisely this is considered as
polysaccharides which had the property of adsorption for iodine. So, they adsorb iodine and give
coloration. As we go deeper there are some effects that must be considered in this test. Based on our
research, specifically in https://allmedicalstuff.com/iodine-test-for-starch: Noted below
Effects:
Effect of pH on iodine test- At low pH, like when acid (HCl or any other) is added to the solution, the
color will turn black. While in case of base such as NaOH complex will break and colour will
disappear.

Effect of temperature on iodine test- At low temperate solution gives blue color when treated with
iodine solution.While in case of high temperature, complex breaks and color disappears.

Pinch of starch + Lugols

iodine

2. IODINE TEST on Starch Paste

We placed 1.0 ml of 1% starch paste in a test tube, and added some drop of lugols
solution also known as Lugols iodine. We heated and noted the color of blue-black precipitate
that was the given positive result, thus upon adding Iodine solution to a solution or directly onto
 materials like starch paste, a BLUE-BLACK COLOR is a positive result: starch is present. As we
cooled the heated solution with starch paste we observed a sticky or becoming viscous of a
solution appeared. Hence At low temperate solution gives blue color when treated with iodine
solution. While in case of high temperature, complex breaks and color disappears.

Hereafter for boiled starch --> H bonds broken --> exposed amylose and amylopectin
structure --> allow amylase to hydrolyse starch. In contrast, for unboiled starch --> amylase unable to
hydrolyse starch as the starch granules are intact.

The different changes now explained some polysaccharides have the property of
adsorption for iodine. So, they adsorb iodine and give coloration. The use of Lugol's iodine reagent
(IKI) is useful to distinguish starch and glycogen from other polysaccharides. Lugol's iodine yields a
blue-black color in the presence of starch. Glycogen reacts with Lugol's reagent to give a brown-blue
color. Other polysaccharides and monosaccharides yield no color change; the test solution remains
the characteristic brown-yellow of the reagent. It is thought that starch and glycogen form helical coils.
Iodine atoms can then fit into the helices to form a starch-iodine or glycogen-iodine complex. Starch
in the form of amylose and amylopectin has fewer branches than glycogen. This means that the
helices of starch are longer than glycogen, therefore binding more iodine atoms. The result is that the
color produced by a starch-iodine complex is more intense than that obtained with a glycogen-iodine
complex. If you want to distinguish between monosaccharide, disaccharides and polysaccharide, you
should perform iodine test. This test would be positive for polysaccharide and negative for mono and
disaccharides.
When iodine dissolved in potassium iodide solution reacts with starch or glycogen, it reacts with it and
the color of solution is changes, indicating the presence of these polysaccharides.

Before heating After heating starch paste +

Lugols solution
Starch paste + lugols
solution

3. HYDROLYSIS OF STARCH

We placed 20.0 ml of starch solution in a beaker and added 2 drops of concentrated

HCL. We boiled at intervals of 3 minutes; test a drop with Lugols and a small portion with
Benedicts Test, until Iodine Test becomes negative and the Benedicts Tests becomes positive.
When starch solution is added with conc. HCL then boiled, then added with Lugols solution, it
becomes dark-violet precipitate. But when we examined it on the light, we dont see the blue color
which is supposed the positive results.

Based on our observation on the changes happened this experiment showed that
starch can be rehydrated with HCL and heat. This breaks down the molecules up into simple
glucose. Amylose molecules are made up of single strands of glucose molecules shaped like
springs. When iodine is added to a starch, it adheres to the beta amylose molecules because of
their solubility. The starch pushes the iodine into a line in the middle of the amylose coils and
creates a transfer of charge between the iodine and starch. This causes a change in the
arrangement of electrons and energy level spacings. The new spacings absorb visible light
differently and create the deep blue color. While in the Benedicts solution,our results, it forms a
light blue precipitate because this is due to the reaction not taking place its since starch is a
polysaccharide, it is unsurprising that the starch solution tested negative for simple sugars.
because benedicts test only reacted to the glucose which is the simple sugar and starch doesnt
react with benedict solution because starch are non-reducing sugar, and take note that Benedicts
test only reacted to reducing sugar. And the time which was being asked specifically its minutes
when does completely hydrolysed it was 5 minutes and 45 seconds as we noted it.

Therefore, we conclude starch molecules break down by reacting with water

molecules. If any molecule reacts with water molecules to break apart, then this is called a
hydrolysis reaction, starch can also be broken down by heating it with acid. This is called acid
hydrolysis.

20.0 ml Starch sol. + 2 drops After heating and Adding

of conc. HCL lugols sol.
 before hydrolysis After Hydrolysis
05.45 minutes

CONCLUSION:

Therefore, we conclude that there are many qualitative tests for carbohydrates. This
test is design to identify the specific carbohydrates, whether it is a monosaccharide,
disaccharides, polysaccharides, reducing or non-reducing sugar. Moores test, Molischs test,
Fehlings test, Benedicts test, Nylanders test and Picric acid test are test for reducing sugars.
Barfoeds test is also for reducing sugar but only specific for monosaccharide. Seliwanoffs test in
specific for ketoses and Iodine tests for starch.