REACTIONS OF CARBOHYDRATES

Anda, Zhyra Alexis P.

Barrinuevo, Clarisse R.
Caribala, Camille N.
Dazo, Angelica Arlou N.
De La Salle University-Dasmarias
Dasmarias City, Cavite, Philippines

ABSTRACT
The experiment about reactions of carbohydrates is to detect the presence of carbonyl in
carbohydrates using different tests. In doing this experiment, keen observation is needed the
most. The experiment was divided into two parts. For the first part, identification of unknown
carbohydrate samples, a 1.00 ml of the known carbohydrate sample and two unknown samples
was transferred on a separate labelled test tubes. A 0.50 ml Molisch reagent and 0.50 ml of
concentrated H2SO4 was added to each sample. Six tests were used using the fresh batch of
sample, namely Molisch test, Iodine test, Benedicts test, Barfoeds test, Seliwanoffs test, and
2,4-DNP test. For molisch test, deep puprle color occured for a positive result. For the negative
result, the color is transparent or colorless and a bubbly appearance will occur. For Iodine test,
blue black complex occured for the positive result. For Benedicts test, a brick red change in color
occured while for the negative result, a blue change in color will occur. For Barfoeds test, brick
red change in color occured for positive result while for a negative result, a blue change in color
will occur. For Seliwanoffs test, a cherry res solution change in color occured for the positive
result while for the negative result, a transparent or colorless color will occur. On the other hand,
nine carbohydrate samples were test using the same test to identify the two unknown
carbohydrates samples. The known samples that were used are glucose, galactose, ribose,
fructose, lactose, sucrose, starch, unknown 1, and unknown 2. For molischs test, all the samples
had a positive reaction because molisch test shows positive result for all carbohydrates. For
iodines test, all samples did not show any reaction or no reaction happened except for starch.
For benedicts test, only sucrose and starch did not show positive reaction because benedicts
test shows positive reaction for all reducing sugars. For Barfoeds test, only lactose, sucrose, and
starch did not show positive reaction; because barfoeds test shows positive result for reducing
monosaccharides. For seliwanoffs test, only fructose, sucrose, and unknown 1 showed positive
reaction because seliwanoffs test shows positive reaction for ketoses. For 2,4-DNP test, only
starch did not show a positive reaction. The two unknown samples were identified as Fructose
and Ribose. For the second part of the experiment, hydrolysis of starch, 25.0 ml of 5% starch
solution was transferred in a 100-ml beaker and was then added with 5 ml of concentrated HCl.
The solution was heated covered with aluminum foil using water bath. A 0.50 ml was then
transferred in a separate test tubes and were test using Benedict reagent and Iodine reagent with
2 minutes interval of heating the solution until getting the positive result. The experimenters got
the positive result after eighteen minutes which is a brick red color. Overall, the experiment is a
success one because the data that were gathered are correct and by so doing the said
experiment, proper handling of reagent and proper decorum was observed.

INTRODUCTION
Carbohydrates are carbon compounds that contain great amounts of hydroxyl groups. The
simplest carbohydrates also comprise either an aldehyde moiety ( polyhydroxyaldehydes ) or a ketone
moiety (polyhydroxyketones). All carbohydrates can be categorized as monosacchrides, oligosaccharides
or polysaccharides. Somewhere from two to ten monosaccharide units, linked by glycosidic bonds, make
up an oligosaccharide. Polysaccharides are much bigger, having hundreds of monosaccharide units. The
existence of the hydroxyl groups permits carbohydrates to interrelate with the aqueous nature and to
participate in hydrogen bonding, both inside and between chains. Derivatives of carbohydrates can
contain nitrogen, phosphate and sulfur compounds. Carbohydrates also can unite with lipids to form
glycolipids or with proteins to form glycoproteins.
Molisch's test is a sensitive chemical test for the presence of carbohydrates, based on the
dehydration of the carbohydrate by sulfuric acid or hydrochloric acid to produce an aldehyde, which
condenses with two molecules of phenol (usually -naphthol, though other phenols
(e.g. resorcinol, thymol) also give colored products), resulting in a red- or purple-colored compound. The
test solution is combined with a small amount of Molisch's reagent (-naphthol dissolved in ethanol) in
a test tube. After mixing, a small amount of concentrated sulfuric acid is slowly added down the sides of
the sloping test-tube, without mixing, to form a layer. A positive reaction is indicated by appearance of a
purple ring at the interface between the acid and test layers. Benedict's reagent is a chemical reagent
commonly used to detect presence of reducing sugar(s), however other reducing substances also give a
positive reaction. This includes all monosaccharide 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 color change from clear blue to a brick-red precipitate. Barfoed's test is a chemical test used for
detecting the presence of monosaccharide. It is based on the reduction of copper (II) acetate to copper (I)
oxide (Cu2O), which forms a brick-red precipitate.
RCHO + 2Cu2+ + 2H2O RCOOH + Cu2O + 4H+
(Disaccharides may also react, but the reaction is much slower.) The aldehyde group of the
monosaccharide which normally forms a cyclic hemiacetal is oxidized to the carboxylate. A number of
other substances, including sodium chloride may interfere. Seliwanoffs test is a chemical test which
distinguishes between aldose and ketose sugars. Ketoses are distinguished from aldoses via
their ketone/aldehyde functionality. If the sugar contains a ketone group, it is a ketose and if it contains an
aldehyde group, it is an aldose. This test is based on the fact that, when heated, ketoses are more rapidly
dehydrated than aldoses. The Iodine test is used to test for the presence of starch. Iodine solution
iodine dissolved in an aqueous solution of potassium iodide reacts with the starch, producing a purpleblack color. The color can be detected visually with concentrations of iodine as low as 0.00002 M at
20 C. However, the intensity of the colour decreases with increasing temperature and with the presence
of water-miscible organic solvents, such as ethanol. Also, the test cannot be done at very low pH values
due to the hydrolysis of the starch under these conditions. 2,4-Dinitrophenylhydrazine (DNPH, Brady's
reagent) is the chemical compound C6H3(NO2)2NHNH2. Dinitrophenylhydrazine is relatively sensitive
to shock and friction; it is a shock explosive so care must be taken with its use. It is a red to orange solid,
usually supplied wet to reduce its explosive hazard. It is a substituted hydrazine, and is often used to
qualitatively test for carbonyl groups associated with aldehydes and ketones.

MATERIALS AND METHODS

For the Identification of Unknown Carbohydrate Samples, about 1.00ml of the known
carbohydrate samples and two unknown samples were transferred on a separate labelled test tube.
0.50ml of Molisch reagent then 0.50ml of concentrated H 2SO4 was added to each sample. The remaining
tests were conducted using fresh batch of samples each time:
Iodine test- 0.50ml of iodine reagent was added to each sample.
Benedicts, Barfoeds and Seliwanoffs test- 0.50ml of the respected reagents were added to each
sample then heated using water bath.
2,4- DNP test- 0.50ml of 2,4-DNP was added then the spot plate was placed on top of the water
bath for 3-5 minutes. After that, it was observed.
The identity of the unknown samples was determined by comparing it to the known carbohydrate
samples.
For the Hydrolysis of starch, 25.0ml 0f 5% starch solution was transferred in a 100-ml beaker
then 5.00ml of concentrated hydrochloric acid was added. The beaker was covered with an aluminum foil
and heated using a water bath. Two 0.50ml volume of the sample was transferred in separate test tubes
after two minutes. 0.50ml of Benedicts reagent was added to one tube and iodine reagent to the other.
Heating was continued while doing the same test as in the previous step every two minute interval until
positive reaction was seen for both reactions.
RESULTS AND DISCUSSIONS

A. Identification of Unknown Carbohydrate Samples

Table 1. Positive Reactions for Carbohydrates Test (6pts)
Test
Molisch

Positive Color Change

Deep Purple

Iodine

Blue Black

Benedict

Brick Red

Barfoed

Brick Red

Seliwanoff

Cherry Red

2.4-DNP

Yellow Black

Molisch's Test is a sensitive chemical test for all carbohydrates, and some compounds comprising
carbohydrates in a shared form, based on the dehydration of carbohydrate by sulfuric to generate an
aldehyde (furfural or a derivative) that is then compresses with the phenolic structure resulting in a deeppurple colored compound.
Seliwanoff's test is used to differentiate aldohexoses from ketohexoses. A ketohexose just like
fructose will create a deep red color with Seliwanoff's reagent while an aldohexose will exhibit a light pink
color and takes a longer time to produce the color. This test is based on the fact that, when settled on fire,
ketoses are more precipitously dehydrated than aldoses.

Barfoed's reagent is composed of copper acetate in acetic acid and will not be reactive as
Benedict's reagent. It is also stable so that it can be only reduced by monosaccharides but not less
authoritative reducing sugars. Dissacharides may also react with this reagent, but the response is not as
fast when compared to monosaccharides.
2,4-DNP test can react with both ketone and aldehyde. Therefore it's also a general test for all
carbohydrates except starch. A positive reaction results to a yellow-black color. Thus, absence of
yellow-black color shows negative result.

Table 2. Reaction of Carbohydrate Samples

Samples
Molisch
Glucose
+
Galactose
+
Ribose
+
Fructose
+
Lactose
+
Sucrose
+
Starch
+
Unknown 1
+
Unknown 2
+
Identity of Unknown 1
Identity of Unknown 2

Iodine
+
-

Benedict
+
+
+
+
+
+
+
Fructose
Ribose

Barfoed
+
+
+
+
+
+

Seliwanoff
+
+
-

2,4-DNP
+
+
+
+
+
+

Legend: + positive, slow reaction / - no reaction

The Unknown 1 and Unknown 2 was identified through the same reactions brought by the tests
which are Molisch, Iodine, Benedict's, Barfoed's, Seliwanoff, and 2,4-DNP. All of the Carbohydrates are
positive to Molisch test. The only carbohydrate that was positive with iodine test is the starch. The only
ones who had a negative reaction with Benedict test was Sucrose and Starch. In Barfoed's test, the ones
that showed a negative reaction are lactose, sucrose and starch. Next is Seliwanoff which is used to
identify aldehexoses to ketohexoses. The only carbohydrate that showed a positive result is the fructose.
Lastly, the 2,4 DNP test which is used for aldehydes and ketones only had a negtive result with lactose,
sucrose and starch.
When it comes to Diabetes test, urine taxing is a necessary part of complete physical test routine.
Urine test helps in diagnosing the state of the body and give important diagnosis.
Benedict's test is used to sense the occurrence or absence of sugar in the urine is tremendously
easy and ought to be leant by every diabetic. Even when sugar has been found to be in the urine, the
analysis of diabetes should be completed by blood-sugar evaluation.

Table 3. Hydrolysis of Starch

Time (min)

Color w/ Benedicts test Time (min)

Color w/ Iodine test

2 mins

Light Blue

2 mins

Orange

4 mins

Light Blue

4 mins

Yellow Orange

6 mins

Light Blue

6 mins

Orange

8 mins

Blue Green

8 mins

Red Orange

10 mins

Dark Blue Green

10 mins

Reddish Brown

12 mins

Blue

12 mins

Reddish Brown

14 mins

Bluish Brown

14 mins

Reddish Brown

16 mins

Reddish Brown

16 mins

Reddish Brown

18 mins

Brick Red

18 mins

Brick Red

The transformation in iodine color with the accumulation of starch and enzyme solution will signify
starch hydrolysis. If the iodine turns blue-black, it indicates the presence of starch, therefore the
denatured enzyme. If the iodine remains orange-yellow color, it shows the absence of starch, thus the
proper function of amylase. In Benedict's test, as the temperature increases, formation of brick red color
begins; this is due to recognition of glucose.
All monosaccharides and numerous disaccharides diminish feeble oxidizing agents like Cu2+ ion.
These carbohydrates are called reducing sugars Benedict's reagent changes color form blue to brick red
in the presence of reducing sugars. Reducing sugar is a sugar that, in a mixture, has an aldehyde or a
ketone group. The conversion of a keto to an enol form of sugars under alkaline environments is a
significant contemplation in reduction tests. The capability of a sugar to trim down alkaline test reagents
depends on the promptness of an aldehyde or keto group for reduction reactions.
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