DEPARTMENT OF PURE AND APPLIED CHEMISTRY

Visayas State University, Baybay, Leyte

CHEM31a Biochemistry
Laboratory Report

Name : Mark Ryan R. Tripole Date Performed : 05/06/2015

Course/Yr : BS Chemistry II Date Submitted : 05/11/2015
Group No : 6 Score

Experiment No. 3
Carbohydrates

OBJECTIVES
 The use of chemical tests to identify some physical and chemical characteristics of typical
carbohydrates
 Differentiate between monosaccharides, disaccharides and polysaccharides using the tests
included above
 Identify an unknown carbohydrate

I. Results
PART A: Structural Formulas for Carbohydrates

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II. Discussion

INTRODUCTION

Considered as one of the main biomolecules, carbohydrates are also considered as the most
abundant class of compounds in the biological world, coincidentally making up more than 50% of the
dry weight of the Earth’s biomass. The term “carbohydrate” is derived from the French “hydrate de
carbon” and is a term that is also a synonym of “saccharide” (inclusive of sugars, starch and
cellulose). Both of these terms are actually used interchangeably when referring to them by modern
scientific standards. These biomolecules are basically important constituents of all living organisms,
and a have a wide variety of different functions. As an example, consider the most abundant
carbohydrate in nature, D-glucose. At the microscopic level, D-glucose is oxidized by cells in living
organisms as the first step in a whole plethora of steps that provide the cells with energy. The
application of this is actually different for different kinds of cells, considering that the eukaryotic
spectra of cells can be subdivided roughly into animal and plant cells. Animal cells basically convert
any of the extra D-glucose that it takes in into glycogen, which is a polymer form of glucose units
stuck together. Whenever the need for any extra energy arises, this glycogen is broken back down
into the individual glucose units that can be used for energy synthesis. Plants essentially do the same
thing, converting any of the extra accumulated D-glucose into starch, but another function that they
can have is the production of the polymer cellulose, which makes up the structure of the plant.
Moreover, the arthropod species also use a carbohydrate makeup that is similar in structure to
cellulose, which is called chitin. This chitin is what makes up the exoskeletons of insects,
crustaceans and other members of the arthropod species.
Given that carbohydrates are one of the most important biomolecules to be studied, over the
years it has been given three classifications. Despite these classifications, it is to be kept in mind
that the basic backbone for any carbohydrate is C n(H2O)n. The first classification denotes molecules

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that fall under the monosaccharides or essentially the single sugars. These molecules can either be
polyhydroxy aldehydes (“aldoses”) or polyhydroxy ketones (“ketoses”), and follow the standard
counting for the number of carbons up to 6 (3 carbons being trioses, 4 carbons being tetroses, 5
carbons being pentoses and 6 carbons being hexoses). These monosaccharides can join up with each
other to form bigger carbohydrates through glycosidic bonding and it is these molecules the form the
next classification of carbohydrates. The disaccharides are carbohydrates that are formed from the
fusion of two monosaccharides. A whole plethora of important molecules fall under this category like
maltose (formed from 2 glucose units), lactose (formed from galactose and glucose) and sucrose
(formed from glucose and fructose). In close relation with this are the oligosaccharides or those
containing more than two monosaccharide units. Generally speaking, the two terminologies are
interchangeable but the disaccharide classification is given more preference because of the
importance of the molecules that fall under them. The last classification denotes molecules that can
be considered as polysaccharides. These are complex units of monomers all joined together to form
a complex structure that could possible be from hundreds to thousands of units long. Falling under
this category are starch molecules (mixture of amylose and amylopectin), cellulose and glycogen.
Below is a simple illustration differentiating the three classifications of carbohydrates:

The experimental procedure as performed was geared towards the familiarization of the
rudimentary techniques that could be performed in the laboratory to detect for carbohydrates. The
diverse nature of carbohydrates by virtue of their structure and the way the molecules within them
bond to each other dictate the many characteristics that each carbohydrate has. The tests that will
be discussed in this report have all been geared towards specific aspects of these carbohydrates
(such as the samples being reducing sugars or not, or whether they have immensely big structures or
not (polysaccharides)). Each of these different characteristics will be discussed along with each
classification tests based on what they are each testing for respectively.

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PART B – Benedict’s Test

This is functional test that can be used in the laboratory to test for carbohydrates that can be
termed as reducing sugars. Before moving further into the results, the definition of a “reducing
sugar” must first be understood. To be classified as a reducing sugar, two things have to be
considered when looking at a sugar molecule. One thing to look for is if the sugar has an aldehyde
group, and if it doesn’t, the second thing to look out for is whether the sugar can somehow form an
aldehyde group through isomerism (which would be the case for the ketoses, or from the opening of
the hemiacetal from the cyclic structure which will be discussed later). The main reason why the
aldehyde group is the star of this concept is because it is the aldehyde group that gives the sugar
molecule its reducing powers. And it is this reducing power that makes it a reducing sugar, giving rise
to a positive result in the Benedict’s test, where the sugar essentially reduces the cupric ion (Cu 2+) to
the cuprous ion (Cu+) while the aldehyde group is oxidized. The visual representation of this positive
result would be the observation of a brick red precipitate of cuprous oxide (Cu 2O).
The Benedict’s reagent is a solution that is usually made from anhydrous sodium carbonate,
sodium citrate and copper (II) sulphate pentahydrate. Looking at the components of the reagent, one
can easily tell that overall the solution formed by the reagent has an alkaline pH level and the
importance for this will be discussed shortly. The importance of this will be discussed in turn as the
samples are analyzed. By following through with the definition of reducing sugars as stated in the
previous paragraph, reducing sugars include all monosaccharides and quite a number of the
disaccharides as well. Included among these are the alpha-hydroxy-ketones as well (or the ketoses)
which is why fructose is considered to be a reducing sugar.
Based on the results for this portion, the samples for glucose, fructose, maltose and lactose
gave positive results for the Benedict’s test. It is to be kept in mind that rarely will one find the
chain form of a particular sugar in solution, because the sugar will almost always be in its cyclic
form, being a form where it is most stable. The anomeric position where the hemiacetal lies is the
most important factor here, because if there is a free hydroxyl group at this position, the ring can
open and the sugar can momentarily exist in its open chain state with an aldehyde group. This is
possible with the samples that gave the positive results by virtue of their structure as illustrated
below:

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 When these positions open up the aldehyde group is oxidized and the molecule is converted
into a carboxylic acid, or in this case something called an aldonic acid. The cupric ions are reduced
cuprous ions in this process. To avoid redundancies, only glucose will be shown as a representative
example:

The same equilibrium exists with the other molecules as well and so they basically undergo
the same reaction flow with the oxidation of the aldehyde group and the reduction of the cupric
ions. Fructose has to go through a few more steps though before it can become a reducing sugar. It is
considered as such because it undergoes two base-catalyzed keto-enol tautomerization reactions
that result in the conversion to an aldohexose (which hasn’t been yet defined because of the nature
of the stereochemistry of C2 in the aldohexose form is unknown as will be indicated using the wavy

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line in the next diagram). This keto-enol tautomerism, aided by the base in the solution, is shown
below:

Once the fructose has been converted to the aldohexose form, then it can easily be oxidized
and can act as a reducing sugar and thus giving the red positive result. Concerning those samples
that gave a negative result, this can be seen based on their structures and these are illustrated in
the diagram on the following page.

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 Looking at the structure of sucrose, it can be seen that there are no free hydroxyl groups on
any of the anomeric positions because it is at these positions where one can find the glycosidic bond.
This α,β-1,2 connection between the glucose and the fructose prevents the ring opening equilibrium
and thus there won’t be any aldehyde group to oxidize in the case of sucrose. For starch and
glycogen, one has to keep in mind that these two are polysaccharides that are up to thousands of
units long, and considering that there’s only one existing anomeric position in the whole chain, this
will be overwhelmed by the size of the chain and so as a whole they won’t act as reducing sugars.
PART B – Barfoed’s Test

The Barfoed’s test is basically similar to that of the Benedict’s test, except that the only main
difference is that the Barfoed’s version tests for monosaccharides, which is bought about by the
reagent used. The cupric component still exists in the reagent, the only difference is that it is made
with a portion of acetic acid, and this makes the pH of the Barfoed’s reagent much lower (rated
generally at 4.5 for usual formulations) than the Benedict’s reagent (rated at 10.6). The whole
concept is basically the same as the Benedict’s test; there will be the oxidation of the aldehyde
group to the carboxylic acid group producing an aldonic acid and reduction of the cupric ion to its
cuprous form to form the red precipitate of cuprous oxide. The lower pH and the shorter incubation
time means that only the monosaccharides can react in this solution when heated. In reality, it
doesn’t really prevent disaccharides from reacting; it’s just that the milder oxidations make the
reaction with disaccharides all too slow to be that obvious. This is the reason why only the glucose
and the fructose gave a positive result for this test.
PART C – Seliwanoff’s Test
This test is one of the quantitative tests for carbohydrates that more or less on the different
rates of dehydration between the aldoses and the ketoses. But before going much deeper into the
specifics, a few things are to be noted about Seliwanoff’s reagent. It is a reagent made of the
compound resorcinol in somewhat highly concentrated hydrochloric acid. The acidic conditions
provided by the reagent and heating basically dehydrate the aldose or the ketose into what is called
a hydroxymethylfurfural (an unsaturated cyclic molecule that contains both the aldehyde functional
group and the alcohol functional group). The furfural formed from the dehydration then goes on to
form a colored complex with the resorcinol in the solution. It is to be noted that though this happens
to both the aldo-sugars and the keto-sugars, it happens much faster with the ketoses and this is why
only fructose and sucrose gave positive results for this test. The reaction pathway for fructose is
shown below:

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 In the case of sucrose, the acidic conditions and the heat basically break it down and split
apart the monosaccharide components, which are glucose and fructose. The fructose then goes
through the same process as before being converted into a furfural and then forming a red colored
complex with the resorcinol that is in the reagent.
PART D – Iodine Test

This particular test is a qualitative test that is geared towards testing for the presence of
polysaccharide chains in the samples and so the test does not work with monosaccharides and most
oligosaccharides. The basis of this test comes from the action of the arrangement of the
polysaccharides on the iodide anions upon interaction. Of all the samples that were used in the
experiment, only the starch and the glycogen provided positive results. Before moving further, the
basic structure of both starch and glycogen has to be taken into account. Starch is basically a
mixture of two carbohydrates, amylose and amylopectin in a 4:1 ratio. As has been shown in the
earlier pages, the structure of amylose is basically the recurrence of glucose units in a straight chain
connected in an α-1,4 configuration all throughout. Because of the way that the molecules are
arranged spatially, amylose chains tend to take up a helical shape. How the iodine test actually
works is that the iodide ions (usually triiodide but in some cases pentaiodide as well) get stuck within

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this helical structure of starch. This forces the iodide atoms into a linear arrangement in the central
groove of the amylose coil. What happens then is basically the transfer of charge between the iodine
and the starch. This results in the changes in electron confinement and the spacing of the energy
levels that exist between the molecules. The complex that exists between starch and the iodine has
high level spacing that absorbs visible light and this is responsible for the characteristic blue-black
color that can be observed. An illustration of this concept is shown below:

Shown here is a partial helice of amylose with the

triiodide ions forced into a linear pattern along the
inner groove. The same thing applies to an
amylopectin chain as well.

The same thing happens with the glycogen, which is essentially similar to amylose save for
the branching coming from the α-1,6 connections that are formed between the glucose molecules.
The concept is pretty much the same and shall not be explained again. Now obviously this test is only
geared towards detecting the presence of polysaccharides, so it’s no surprise to see everything else
on the list come up with a negative result.

PART E – Hydrolysis of di- and polysaccharides

Before moving forward, it is imperative to be completely knowledgeable about the meaning

of the term hydrolysis. Basically speaking, hydrolysis is a reaction involving the breaking of a bond in
a molecule using water. Looking at the bonds that exist between disaccharides and the individual
units in polysaccharides, it can be seen that they are held together by what are known as glycosidic
bonds. Under normal conditions these are pretty stable and quite hard to break, but given the right
conditions, they can be broken through hydrolysis. It is to be noted here that not all hydrolyze at the
same rate. There are a few factors to be kept in mind when looking at the carbohydrates from a
hydrolysis point of view. Like for example, the type of the glycosidic bond present. β-configurations
are essentially more stable compared to the α-configurations. The second factor would be the
location of the glycosidic bond between the molecules, which includes other aspects of the polymer
such as the length, etc. And the last factor to be looked at is the inductive effect that holds certain
part of the polysaccharide together. Combined together, these are all of the factors that basically
influence the rate and the extent to which a particular carbohydrate hydrolyzes in solution.

Looking back at the experiment, the purpose of the concentrated hydrochloric acid was
actually to facilitate the hydrolysis by acting as a catalyst in the reaction. The hydrolysis of sucrose
would lead to the separation of the disaccharide into its monomer components, namely glucose and
fructose. A simple diagram showing the hydrolysis of sucrose is shown below:

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 Comparing the two solutions (one with the sucrose as is and the other with the hydrolyzed
sucrose) using the Benedict’s test, the sample with the hydrolyzed sucrose gave a positive result.
Basing on an earlier part of the procedure, it can be seen that sucrose gave a negative test with the
Benedict’s reagent. As has already been mentioned before, this is due to the fact that there is no
anomeric carbon with a free hydroxyl attached to it on the sucrose, so essentially the molecule
would not be able to go through the ring opening isomerism and prep itself for oxidation. Performing
hydrolysis first on the sucrose using a strong acid means the conversion of a di- or polysaccharide into
the smaller monosaccharide components, which the experiment has already proven to give positive
results with the Benedict’s reagent. Same application with starch, the amylopectin and amylose units
are broken down into their individual glucose units which will of course give a positive result in the
Benedict’s test. Aside from the Benedict’s test, there’s also the iodine test that was performed on
the hydrolyzed samples. The difference between the hydrolyzed samples and the non hydrolyzed
samples is the fact that as the complex amylose/amylopectin breaks down, the surface area of the
molecule on the whole decreases. As these complex molecules are broken down further and further,
they start to lose the ability to “contain” the iodide ions and so the iodine will turn up negative.

PART F - Fermentation

Fermentation is basically a metabolic process that functions in the conversion of sugars to

alcohol and carbon dioxide. This can be found predominately through the action of yeast on certain
sugars, but it also happens in bacteria and in the muscles (in the case of lactic acid fermentation).
For this portion of the experimental procedure, the group was tasked with observing for the
fermentation of different kinds of sugars using yeast. The products of fermentation are mostly the
same, producing ethyl alcohol and carbon dioxide, with a formation of bubbles being an indication of
a positive result. A basic equation for what goes on with glucose is shown below:
yeast
C6H12O6 2C2H5OH + 2CO2

Fermentation basically works on the premise that the yeast will be able to act effectively as a
fermenting agent and be able to react with the different samples as presented in the laboratory.
Depending on the yeast used, there will be different proportions of different enzymes with each,
meaning that one type of yeast might actually be able to ferment one type of sugar while another
type won’t be able to do the same. Looking at the results based on the experiment, only lactose was
the one that provided a negative result for the fermentation test and this will be explained shortly.
Yeasts are basically eukaryotic microorganisms, and being living cells, they aren’t exempt
from breaking down complex carbohydrates into simpler sugars, just like most living things. Most

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yeasts (the common species being saccharomyces cerevisiae) are commonly able to break down the
simpler sugars like glucose and fructose naturally, and so when handling larger carbohydrates, the
yeasts use enzymes in an attempt to break them down and get them to their glucose or fructose
components. In the case of sucrose for example, yeasts use an enzyme called invertase to be able to
break down the bonds between the monosaccharide components in sucrose. Once the sucrose
components are split apart, the glucose component is then fermented by an enzyme in the yeast
called zymase. The thing with sucrose is that it’s readily fermented in the presence of yeast because
even a slightly elevated pH levels sucrose will generally break into its two single sugars
automatically. For the rest of the molecules save for lactose, the principle is pretty much similar
save for the enzyme that comes into play for each type of sugar (like maltase for maltose for
example, etc. An explanation that will shed light on why the yeast failed to ferment lactose comes
basically from an understanding of the structure and the components of lactose. Lactose is basically
a disaccharide sugar made of glucose and galactose connected in a β-1,4 arrangement. Yeasts
commonly do not have the necessary enzyme that will break lactose into its constituent sugars, and
so while yeasts can process galactose and glucose as themselves directly, they won’t be able to
interact with lactose directly as is. To circumvent this, researchers have actually found a way to
genetically engineer yeast in such a way that they are able to use lactose, by having the yeast
secrete the enzyme lactase. Regardless, the absence of the necessary enzymes is the main reason
why yeast doesn’t ferment lactose directly. All the other samples used in the experiment fermented
with yeast by virtue of their structures and the fact that the polysaccharides were broken down into
their glucose monomers which undergoes fermentation quite readily with yeast.

PART G – Testing Food for Carbohydrates

Given the importance of carbohydrates as biomolecules both internally and externally, it

would be pretty obvious that the very food that we eat contains these substances. In the
experimental procedure, three different food samples were tested separately with each of the tests
as already mentioned above and the results pain quite an informative picture as to the possible
contents of the particular food.
Brown sugar gave positive results for most of the tests, save for the iodine test which is to be
expected because brown sugar doesn’t really have any polysaccharides in the mixture. Based on
research, brown sugar is mostly sucrose with some glucose and fructose in small quantities as a result
of what is known as the invert sugar. These components combined actually account for the positive
results as shown in the table in the results section.
As for the syrup, the sample tested positive for all the tests indicating the presence of a wide
array of possible carbohydrates. Depending on the brand, some of the most common ingredients of
the syrup used in pancakes are high fructose corn syrup (which accounts for the Seliwanoff’s,
Benedict’s, Barfoed’s as well as the fermentation tests) and cellulose gum (accounting for the iodine
test) which is commonly used as a thickener and stabilizer in food products. There could be other
more complex carbohydrates present in the sample of syrup but then this varies greatly with the
brand and the type of the syrup used.
Taking a look at the oatmeal sample, it gave negative results for all the other tests except for
the fermentation and the iodine tests, indicating the presence of a predominantly polysaccharide
composition. Oats are basically considered to be soluble fibers, and fibers are basically plant derived
cellulose that is made of recurring units of glucose molecules.

IV. Conclusion
Carbohydrates are important biomolecules, in the sense that they are pretty abundant on the
surface of the planet. Having a wide variety of industrial applications as well as already naturally

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occurring applications, the actual importance of carbohydrates is already quite obvious. Ranging
from the glycogen that exists as the energy storage within animals, to the cellulose that forms the
integral structure of plants, carbohydrates can definitely be seen as something that is akin to the
basic make up of life as we know it. This experimental procedure has aided in being able to
understand the structures of these carbohydrates and how these physical properties dictate how the
sugar will behave in different situations and the like. Like how sugars are considered to be reducing
sugars if they have an aldehyde group that can be oxidized or if they are able to create one through
isomerism (tautomeric conversion of fructose to an aldose). Moreoever, the knowledge of the
fundamental classification test for these compounds has been invaluable, given that the knowledge
and familiarity with these tests also reinforce the knowledge of the properties of the carbohydrate
samples tested.

VI. References
 "Reactions of Carbohydrates"
http://www.namrata.co/category/practical-biochemistry/reactions-of-carbohydrate
 "Laboratory Tests for Cabohydrates"
http://www.slideshare.net/katealyssacaton/seliwanoff-benedicts-test
 "Testing for Cabohydrates"
https://biokamikazi.files.wordpress.com/2013/10/merged_document.pdf
 "Benedict's Test"
www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/carbo/bened/benedict.htm
 "Drawing Sugar Structures"
www.chtf.stuba.sk/~szolcsanyi/education/files/Chemia%20heterocyklickych%20zlucenin/Pred naska
%206/Odporucane%20studijne%20materialy/Drawing%20sugar%20structures.pdf
 "Carbohydrates"
chemed.chem.purdue.edu/genchem/topicreview/bp/1biochem/carbo5.html
 "Amylose"
en.m.wikipedia.org/wiki/Amylose
 "Benedict's Test"
www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/carbo/bened/benedict.htm
 "Benedict's Reagent"
www2.sunysuffolk.edu/sabatil/by14/by14lab3/benedict.htm
 "Reducing Sugars"
en.m.wikipedia.org/wiki/Reducing_sugar
 "Reducing Sugar"
www.ausetute.com.au/redsugar.html
 "Barfoed's Test"
www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/carbo/barf/barfoed.htm
 "Functional Tests for Cabohydrates"
https://www.msu.edu/course/lbs/145/luckie/Lab1.html
 "Seliwanoff's Test"
en.m.wikipedia.org/wiki/Seliwanoff's_test
 "Fermentation"
https://answers.yahoo.com/question/index?qid=20130424141552AAyv5js
 "Iodine Test for Starch and Glycogen"
generalchemistrylab.blogspot.in/2011/12/iodine-test-for-starch-and-glycogen.html?m=1
 "Fermentation of Lactose"
https://answers.yahoo.com/question/index?qid=20080529121326AA9hSgF

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