Introduction:

This lab was comprised of several different experiments which were carried out to
identify unknown compounds by employing different analytical techniques. Identification of
the unknown compound is achieved via the collection of qualitative data, which is then
compared to the expected chemical reactions and observations for the known compound. The
presence of carbohydrates, proteins and lipids were tested for in this lab by performing the
following tests, using arabinose, glucose, sucrose, fructose, maltose, gum arabic, starch,
dextrin, albumin, and urea.
To test for the presence of carbohydrates in a solution, the Molisch test can be used. It
is a general test for carbohydrates, so called as it identifies carbohydrates whether free or in
combination with another compound, such as in glycolipids (Arshad, 2016). This test
functions on the basis that the carbohydrate is dehydrated by the addition of concentrated
sulphuric acid, which results in the formation of furfural or hydroxymethylfurfural. A reaction
of the furfural or hydroxymethylfurfural with αnaphthol is then expected to give a red/violet
solution. In general, the Molisch test is used to determine whether carbohydrates are present
in various substances, such as elastin protein. (Stein and Miller, 1938).
The Benedict’s test is one which is used to test for the presence of reducing sugars in
a solution. A reducing sugar is one which acts as a reducing agent and includes all
monosaccharides such as glucose, which all have freely reactive carbonyl groups in their
structure. For example:

Anon., Oxidation of Monosaccharides. http://slideplayer.com/slide/8697101/

Maltose is an example of a disaccharide which tests positive for reducing sugars using
the Benedict’s test due to the exposed carbonyl groups contained in its structure. However,
maltose demonstrates less reactivity in comparison. This test functions on the basis that in an
alkaline solution, and upon heating, enolization of the sugars occurs which leads to the
reduction of cupric oxide to cuprous oxide, which is red in colour. However, due to variations
in sugar concentration, resulting colours can range from yellow to green as well (Anon.,
2011). In general, the Benedict’s test has been used to determine the presence of reducing
sugars such as glucose in solution, and in so doing, test for diabetes in persons. For example,
Saul Roseman once reflected upon how all army recruits during World War II had their urine
tested by this means (Simoni et al, 2002). However, due to limitations regarding sugar
specificity, enzymatic methods such as with Clinistix and Tes-Tape have displaced the
Benedict’s test as it provides higher accuracy (Ackerman et al, 1958). Yet finally, urine
glucose monitoring has been supplanted by blood glucose monitoring.
The detection of ketose requires the Seliwanoff’s test, a method of qualitative analysis
that is specific to ketose. This test functions on the basis that the ketose is dehydrated upon
the addition of concentrated hydrochloric acid (contained in Seliwanoff’s reagent) to give
derivatives of furfural. These derivatives, hydroxymethylfurfural, form more rapidly than if
the sugar was an aldose, and further reacts with resorcinol (also contained in Seliwanoff’s
reagent) to form a deep-red coloured complex in solution. This reaction occurs with aldoses
at a much slower rate, and so may result faint pink colour.

Anon., Seliwanoff’s Test. http://science-biochem.blogspot.com/2012/03/seliwanoff-

test.html
In general, the Seliwanoff’s test can be used for the detection of ketoses such as
fructose in substances. For example, this test has been used for the detection of fructose in
blood and urine by Kronenberger and Radt in 1927 (Roe, 1934).
The modified Barfoed’s test performed in this lab is a qualitative carbohydrate test
that is used for determining the presence of monosaccharides in a solution. This test functions
on the basis that upon heating the combined mixture, the weakly acidic Barfoed’s reagent,
containing ethanoic acid and copper (II) acetate, will be reduced by the monosaccharide in
the solution. The formation of a red copper oxide precipitate results, which confirms the
presence of the monosaccharide/reducing sugar. The specificity of the test results from the
inability of disaccharides to cause a reduction in the Barfoed’s reagent since it is a weaker
reducing agent. The addition of the phosphomolybdate colour reagent in this experiment
served to oxidize the copper oxide produced back to copper (II) oxide, while in the process,
was reduced to give the positive testing blue colour (Anon, n.d.). In general, this modified
Barfoed’s test is applied in determining the presence of monosaccharides while disaccharides
are also present, i.e., distinguishing between monosaccharides and disaccharides in a solution.
For example, the modified Barfoed’s test was used in the determination of blood sugar levels
and can be used when studying saccharases and glucosides (Tauber and Kleiner, 1932).
Another test performed during the experiments was the Bial’s test, which is
qualitative and used for the identification of a pentose in solution. This is a test which
functions on the basis that the pentose is dehydrated by the Bial’s reagent, which contains
orcinol, hydrochloric acid and ferric chloride. This dehydration results in the formation of
furfural, which further undergoes a condensation reaction with orcinol in the presence of
ferric chloride to produce a blue-green coloured solution. Additionally, this test can be used to
distinguish between a pentose and a hexose as when a hexose is present, a muddy brown
precipitate is formed. In general, the Bial’s test is used to for the detection of pentose in
solution. For example, Bial’s reagent was used in the chemical analyses of three types of
viruses, for which the conclusion was that all three contained “…similar complexes of
protein, lipid, and carbohydrate, containing small amounts of nucleic acid of the deoxyribose
type” (Taylor, 1944).
Sixth is the iodine test, which is used to confirm the presence or absence of starch.
Starch contains amylopectin and α-amylose, which are both polymers of glucose. The α-
amylose in starch is what reacts with the iodine, forming a starch-iodine complex which has a
blue-black colour (Ophardt, 2017).

Anon., Starch/Iodine Complexation.

http://www.smallscalechemistry.colostate.edu/labtop/soft_machines/sugar_starch/si.html
Glycogen is also branched and contains glucose monomers linked together by α-1,4-
glycosidic bonds with α-1,6-glycosidic branching (Berg et al, 2002).
 Berg, JM et al. Glycogen Structure. https://www.ncbi.nlm.nih.gov/books/NBK21190/
Dextrin is a less complex, smaller derivative of starch, yielded upon the hydrolysis of
starch (BeMiller, 2003).
In general, the iodine test is applied in the detection of starch, and has been used in
determining the presence of amylase in urine and blood serum, where the blue-black colour
complex formed can be photometrically measured before and after the incubation of starch in
solution, along with the specified enzyme (Smith and Roe, 1948).
Glucose monomer units make up starch, glycogen and cellulose, however, these three
compounds differ in the types of bonds which link their monomers together. Another
difference is that while starch is the major energy-storing/carbohydrate source molecule in
plants, cellulose comprises the major structural components of plant cell walls, while in fungi
and animals, glycogen serves as the major energy store/carbohydrate source. The bonding in
starch consists of alpha-1,4 glycosidic bonds in amylose, and alpha-1,4 glycosidic bonds with
alpha-1,6 glycosidic bonds creating branching in the polysaccharide. Cellulose is a fibrous
polymer of glucose, containing strictly alpha-1,4 glycosidic bonds. Hence, there is no
branching in cellulose. Glycogen, however, displays branching much like amylopectin, but
more frequent. Though coiling of the saccharide chains is seen in starch and somewhat in
glycogen, cellulose displays no coiling in its structure (Lakna, 2017).
The acid hydrolysis of sucrose followed by the Benedict’s and Seliwanoff’s test
provide qualitative results that can ultimately determine the presence of reducing sugars in
solution. This test functions on the basis that the acid hydrolysis of sucrose, a non-reducing
sugar will cause the molecule to split into its monomers, glucose and fructose, which are
strong reducing sugars. If the Benedict’s test or Seliwanoff’s test were carried out on sucrose,
a negative result would be yielded for both as no reducing sugars and particularly, no
fructose, would be present. However, upon hydrolysis of the sucrose and the yielding of its
constituent sugars, a positive test can be achieved (Anon., n.d.).
 Ophardt, Charles. Hydrolysis of Sucrose.
https://chem.libretexts.org/Core/Biological_Chemistry/Carbohydrates/Disaccharides/Sucrose
In general, the acid hydrolysis of sucrose can be used to create a reducing sugar
solution from a non-reducing sugar solution. This test can also be carried out in investigating
experiments, such as the investigation into sugar transformation in Canna indica leaves
(Putman and Hassid, 1953).
To test for the presence of lipids in solution, the Sudan III test can be carried out. This
test functions on the basis that the lipid-solubility of the Sudan III reagent, which is red in
colour, will allow the Sudan III to stain the lipids red. Due to this ability of the Sudan III
reagent, the amount of location of the lipids may also be identified (SEP staff, 2013). In
general, the Sudan III test can be used in the determination of the presence of lipids and to
assess lipid content, for example, in tissues such as in that of broad bean (Anon., 2009).
Another qualitative lipid test is the emulsion test, which functions on the basis that
lipids dissolve in ethanol but not in water. Hence, if lipids are present, they will disperse and
form tiny emulsion droplets in the water after being dissolved in the ethanol and shaken
vigorously in attempts to mix the solution with the water. In general, the emulsion test is used
to determine the presence of lipids in a solution (Anon., n.d.).
To test for protein in solution, the Biuret’s test is applied. The limitation of this test is
that it only takes effect when there are 2 or more peptides linked together since it is
performed for the detection of a peptide bond. The Biuret’s test functions on the basis that in
an alkaline solution, proteins present will react with copper (II) oxide ions to produce a
violet-coloured complex, biuret (Anon., 2013).
 Anon.
Biuret Test. http://amrita.olabs.edu.in/?sub=73&brch=8&sim=140&cnt=1
In general, the Biuret test can be used to detect the presence of peptide bonds and
therefore, proteins in a solution. It has been applied in detection urine that contains protein,
however, the standard was found to be unsatisfactory due to impurities and as such, Biuret
was abandoned. However, it has also been performed in the determination of serum total
protein, albumin, and globulin (Kingsley, 1939).
The Ninhydrin test is one that tests for the presence of amino acids in a solution, or
proteins which contain a free amine group in their structure. In slightly acidic to slightly
alkaline solution solutions, amino acids react with ninhydrin to produce a purple coloured
complex, diketohydrin, that is known as Rhuemann’s purple. A reaction also occurs between
ninhydrin with proline and hydroxyproline, which are imino acids. However, the complex
produced has a yellow colour instead (Anon., 2011).

Anon. Ninhydrin Test. http://vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1

In general, the Ninhydrin test is used to detect the presence of proteins. It has also
been used in forensic experiments such as to reveal latent fingerprints and has become the
most commonly used method since its development in 1954 (Anon., 2009).
Several protein reactions were performed using albumin and various reagents,
namely: TCA, ammonium sulphate, HCl, NaOH, copper sulphate, lead acetate and ice-cold
ethanol. These reactions can be grouped into the following:
 - Protein precipitation by ‘salting out’
- Protein precipitation by the addition of strong acids,
- And protein precipitation by the addition of heavy metals
Precipitation by ‘salting out’ functions on the basis that specific salt concentrations
correspond with the precipitation of proteins. The type of salt and molecular weight of the
protein are considered as those with high molecular weights show the first signs of
precipitation. A low salt concentration is required by proteins with high molecular weights,
whereas a high salt concentration is required by those with low molecular weights. The
relationship between these two can then be described as inversely proportional.
The ions of the salt are attracted to the functional groups contained in the protein, and
so a low concentration of the salt facilitates increased solubility of the protein. The opposite
occurs with a high salt concentration as protein dehydration occurs and the protein is
precipitated out. Ammonium sulphate is used with this method.
Precipitation can also occur by the addition of a strong acid, which would include
TCA and HCL for this experiment. This method functions based on the denaturation of the
protein which results in changes in its solubility. When the protein becomes denatured, it can
experience loss of solubility as one of the effects, which is demonstrated by the precipitation
of the protein. Denaturation is a process whereby only the primary structure of the protein is
retained and can also result from the addition of an organic solvent, such as cold ethanol, or a
strong base, such as NaOH.
Precipitation occurs through the addition of heavy metals as well, where the ionic nature of
the heavy salts causes disruption of the salt bridges in proteins. The result of this reaction is
typically an insoluble metal protein salt. The precipitation of the protein by this method in the
lab is achieved by the use of copper sulphate and lead acetate (Anon, n.d.).

Discussion:

Test number one was the Molisch’s test, which was used as a general test to identify
the presence of carbohydrates in solution. A positive result is indicated by a reddish-violet
colour where the two liquids meet, which was expected for all carbohydrates tested, namely:
sucrose, glucose, maltose, arabinose, and starch. Glucose, maltose, arabinose, starch and
sucrose gave positive results to varying degrees, while the blank, as expected, gave a negative
result.
 Glucose is a hexose monosaccharide with molecular formula, C6H12O6. From the
formula, it is seen to contain carbon, hydrogen, and oxygen as all carbohydrates do (Miller,
2017).

Anon. Glucose. https://burningscience.wordpress.com/biological-molecules-

monosaccharides/
The glucose solution experienced a colour change to a two-layered solution, with a
dark brown colour at the bottom layer and a light brown colour at the top. The sulphuric acid
used is denser when compared to glucose, and so a purple-coloured ring layer would have
been formed at the junction of the acid and glucose layers. This colour change would have
resulted from the occurrence of a condensation reaction between furfural derivates and the α-
naphthol contained in the Molisch’s reagent (Khan, 2013).
Maltose is a disaccharide with molecular formula, C12H22O11.

Stewart. Maltose. https://sites.google.com/site/mrstewartjfr/biochemistry-notes

The two glucose monomers which form maltose are linked together by a α-1,4 glycosidic
bond, formed through hydrolysis (Crow et al, 2012). The maltose experienced a colour
change to a purple solution, again, due to the condensation reaction occurring between
furfural derivates and the αnaphthol contained in the Molisch’s reagent.
Similarly, arabinose would have tested positive due to the same reasons. Arabinose,
however, is a pentose monosaccharide with molecular formula, C5H10O5.
 Anon. Arabinose.
https://web.squ.edu.om/med-lib/med_cd/e_cds/Electronic%20Study%20Guide%20of
%20Biochemistry/ch26/arabinos.htm
A colour change to a brown solution was observed. It is assumed that the purple colour was
indeed formed, however, obscured due to charring (Foulger, 1931). Hence, this colour change
was taken as a positive result.
A starch solution was also tested using Molisch’s reagent, which resulted in a colour
change to a grey solution being formed. Starch is a polysaccharide containing the polymers
amylose and amylopectin. These are both derivatives of glucose, with amylose being
unbranched and amylopectin displaying branching throughout its chain. In amylose, the
glucose monomers are linked together by α-1,4 glycosidic bonds whereas in amylopectin, the
glucose monomers are linked together by α-1,4 glycosidic bonds with α-1,6 glycosidic bonds
creating branches (Anon., 2013).

Anon. Amylose and Amylopectin. https://biochem1362.wordpress.com/tag/starch/

The starch solution experienced a colour change to a grey-ish solution.
Polysaccharides, such as starch, must undergo hydrolysis to be broken down to its constituent
monosaccharide so that it can then be dehydrated to produce a positive test result (Chhabra,
2014). Hence, this colour change was assumed to be in the process of slowly unfolding since
it is not expected to rapidly produce a positive result as with monosaccharides. The
possibility also exists that the purple colour was extremely pale, and so, mistaken for being
grey.
 Lastly was sucrose, a which is a disaccharide consisting of glucose and fructose
monomers. Its molecular formula is
C12H22O11, and in its structure, it contains a
α-1 –> β-2 glycosidic bond which links the
two constituent monomers (Anon.,
2008).

Tortland, Paul. Chemical Structure of Table Sugar, Sucrose.

https://naturalnutmeg.com/good-carbs-bad-carbsits-anything-but-simple/
A colour change to a light purple solution occurred. The intensity of the purple colour,
or lack thereof, can be accredited to the time it takes for hydrolysis to produce the constituent
monomers for dehydration as well as the oxidizing and reducing strengths of the various
reagents involved.
Furthermore, the test performed on the blank gave a negative result as no purple
colour change was produced. This was expected as water does not contain carbohydrates.
One limitation associated with the Molisch’s test is that it is not specific to
carbohydrates, and so only a test to prove the absence of carbohydrates can be considered
conclusive. Another limitation is that if carbohydrates are present, the Molisch’s test lacks the
ability to distinguish between what types may be present and so, further testing would be
required (Khan, 2013). Some sources of error may include not allowing enough time for the
reaction to occur and so, false results would be obtained.
The Benedict’s test was the second test performed, which is used in the determination
of the presence of reducing sugars. A positive test result was expected for all glucose
concentrations which fall within the detectable range for the Benedict’s test, and is indicated
by the formation of a red, yellow, or green precipitate. The concentrations of glucose tested
were 0M, 0.001M, 0.01M, 0.02M, 0.05M, and a 1M solution. As expected, the blank gave a
negative result, as well as the 0.001M glucose solution with all other concentrations tested
positive for reducing sugars.
The blank gave a negative test result as the water used contained no glucose or any
other reducing sugar. On the other hand, the 0.001M glucose solution which also remained
blue upon the addition of Benedict’s reagent may have tested negative due to containing
glucose at too low a concentration, i.e., the glucose in the solution is undetectable using
Benedict’s reagent.
With respect to the remaining glucose solutions of varying concentrations which
tested positive, this is so since the copper (II) oxide contained in the reagent oxidizes the free
carbonyl groups in glucose to carboxylic acids. By the principle of redox reactions, the
copper (II) oxide is reduced (Paik, 2014). The Benedict’s test is semi-quantitative, though, as
a rough estimate of the concentration of reducing sugars contained in a sample can be made
based off precipitate colour, where:
- Green colour indicates up to 0.5% glucose
- Green precipitate indicates between 0.5-1.0% glucose
- Yellow precipitate indicates 1.0-1.5% glucose
- Orange precipitate indicates 1.5-2.0% glucose, and
- Brick red precipitate indicates 2.0% and above glucose (Chhabra, 2014).
The results are consistent with what is expected as the intensity of the precipitate
increased with the increasing concentration of the glucose, going from no precipitate (as with
0M and 0.001M) to an orange-red precipitate, a reddish precipitate, a reddish-brown
precipitate and finally, a red-brown/brick red precipitate respectively. The more diluted the
glucose solution, i.e. the lower the concentration, the less free carbonyl groups there are
available for reaction with the Benedict’s reagent.
Limitations of this experiment include the general nature of the test in identifying
reducing sugars and the lack of specificity in determining the carbohydrate present. However,
further testing can be done to identify contained carbohydrates. Sources of error may include
using varying volumes for the different concentrations, the volume of reducing sugar present
directly influences the colour of the precipitate formed (Khan, 2013).
The third test performed was Seliwanoff’s test for the detection of ketoses in solution.
A positive test result for this experiment is indicated by the formation of a cherry-red
coloured complex when the hydroxymethylfurfural reacts with the resorcinol contained in
Seliwanoff’s reagent. As expected, a positive result was obtained for fructose, which
underwent a colour change to cherry-red while a negative result was obtained for glucose,
which remained colourless.
Fructose is a ketose sugar, and so it is expected that a positive test result would be
yielded. Glucose gave a negative test result as unlike fructose, it is an aldose sugar. However,
upon further heating, glucose can also experience a colour change to pink or red as
conversion of the aldose to a ketose occurs (Chhabra, 2014).
 Limitations of this experiment include a lack of specificity since after a period, an
aldose cans till form the red complex. Additionally, the Seliwanoff’s test is a general test for
the presence of ketose since it does not indicate which ketose is present (Khan, 2013).
Sources of error may include allowing the time for the reaction to occur to exceed one minute
since a positive test with glucose can result.
The modified Barfoed’s test performed was carried out for the determination of the
presence of monosaccharides/reducing sugars in solution. The carbohydrate contained must
be a reducing sugar, since a redox reaction is required, where the reagent is reduced as it
oxidizes the monosaccharide. Cu2+ ions are converted to Cu+ ions, forming Cu2O. This
reaction may be possible with disaccharides which contain reducing sugar monomers,
however, at a slower rate. The reduction of phosphomolybdic acid which is contained in the
phosphomolybdate occurs to produce phosphomolybdenum blue. Again, this may be possible
with disaccharides, but if so, at a slower rate (Khan, 2013). A positive result is therefore
indicated by a deeply coloured blue solution. As expected, a negative result was obtained
when the blank was tested while glucose, fructose, maltose upon heating and sucrose upon
heating, gave positive results.
Glucose and fructose are both reducing sugar monosaccharides, and so, a positive
result was expected. Since maltose consists of two glucose residues, a positive result was
expected upon heating as the glycosidic bonds would have been cleaved, allowing the
reducing sugar glucose residues to react with the reagent. Sucrose, like maltose has two
reducing sugars as its constituents. Hence, upon cleavage of the glycosidic bond when heated,
can produce a positive result. Though sucrose and maltose may produce positive results when
heating is prolonged, the reaction occurs at a slower rate due to their disaccharide nature
(Chhabra, 2014).
Limitations for this experiment include lack of specificity regarding which reducing
sugar is present. If over-heated, there would also be lack of specificity regarding whether the
sugar present is a monosaccharide or disaccharide since some disaccharides are able to
slowly produce positive results (Khan, 2013). Sources of error may include over-heating.
Next was the Bial’s test, which was performed to determine whether pentoses were
present in solution. A positive test is indicated by the formation of a blue-green/greenish
yellow colour or precipitate which results from the reaction between the formed furfural and
the orcinol present in the reagent. As expected, a positive result was obtained for arabinose,
while negative results were obtained for the blank, glucose, and gum arabic when tested.
 Arabinose is a pentose sugar, and so, it is expected that positive results would be
yielded with a colour change to dark yellow-green. However, upon hydrolysis, gum arabic
should have given a positive result since this is an arabinose polymer. A colour change to
yellow was observed, possibly due to not being heated long enough to fully react and give the
yellow-green colour. Additionally, glucose is a hexose sugar rather than pentose and so, a
negative result was expected by there being no change and the colour of the solution
remaining yellow. Alternatively, a red-brown precipitate could have been formed in the case
of glucose as hexoses generally are considered to produce.
Limitations include lack of specificity, as further tests will be required to determine
which pentose is present (Khan, 2013). Sources of error in this experiment include under-
heating, as the gum arabic may not be afforded adequate time to react.
The iodine test was performed to determine the presence of starch in solution. The
positive result is dependent on the presence of amylose within starch, and is indicated by the
production of a blue-black coloured complex where the colour obtained is dependent upon
the length of the unbranched chains available for the formation of the complex. A positive
result was obtained for each of the solutions tested, i.e., for starch, glycogen, and dextrin.
Amylose contained in starch is helically coiled when in solution, and so the trapping
of iodine molecules within the structure of the coiled amylose results in the starch taking on
its blue-black colour. Upon performing the iodine test, a blue-black colour was obtained as
expected, which confirmed the presence of starch in the solution.
Glycogen, consisting of branched chains, is more similar to amylopectin and does not
exist helically coiled in solution like starch. Hence, it does not possess the ability to trap the
iodine and adopt the blue-black colour. However, a colour change to a rusty brown solution
was observed due to slight staining, as expected.
Again, as expected, a colour change to a bluish-violet colour was observed upon
testing the dextrin solution. The structures of starch and dextrin are similar, both possessing
the same general formula and as such, very light staining occurred (Khan, 2013).
With respect to the acid hydrolysis of sucrose, this test is performed to achieve
cleavage of the glycosidic linkages within sucrose, so that tests for reducing sugars may then
be carried out. The sucrose is boiled with acid in order that hydrolysis should occur.
Hydrolysis involves the breaking of a bond through the use of water. Upon cleavage of the
bonds and release of monomers, the pH was neutralized using sodium hydroxide.
Neutralization must occur as the alkalinity of Seliwanoff’s and Benedict’s reagent can be
disrupted by the acidity of the HCl used (Khan, 2013).
 Sucrose is a non-reducing sugar which will not normally have a free carbonyl group
available for reduction to occur. As expected, a negative result was obtained for both
Seliwanoff’s and Benedict’s test for the unhydrolyzed sucrose. This was observed since the
solution appeared blue when the Benedict’s test was performed and appeared orange when
the Seliwanoff’s test was performed.
However, a positive test result was obtained for the Benedict’s test when the
hydrolysate was used as an orange-brown precipitate was produced. Likewise, a positive test
result was obtained for the Seliwanoff’s test when done using the hydrolysate as a colour
change to a faint cherry-red colour was observed. The positive results are indicative that the
acid hydrolysis was successful in splitting sucrose into its constituents, glucose and fructose,
which are both reducing sugars and test positive with one or both tests (Khan, 2013).
The Sudan III test identifies the presence and location of lipids. A positive test result
for this experiment is indicated by a red stain, producing a red colour in the lipids present. As
expected, the olive oil that was used turned an orange-red colour and a layer of micelles
formed at the top. Since olive oil contains triglycerides, has a lower density compared to
water and is insoluble in water, the lipids float at the top of the mixture and are stained red
due to the colour of the Sudan III dye.
When the emulsion test was performed to again, test for the presence of lipids, an
emulsion was formed. Lipid droplets were observed at the top of the otherwise colourless
solution. Regarding solubility, lipids and triglycerides are soluble in alcohol and so, formed a
solution with the ethanol used. However, neither are soluble in water. Hence, micelles were
dispersed and formed at the top of the solution due to the lower density of the lipid compared
to water. These micelles are formed when the hydrophobic tails of the triglyceride take on an
arrangement where they point inward. A positive test was indicated by the formation of th e
micelles at the surface of the solution, along with the cloudy, white appearance resulting from
the scattering of light as it shines through the water by the micelles (Khan, 2013).
 Asmitha. Micelles. https://learnwithease.weebly.com/carbon-and-its-compounds.html
To test for the presence of peptide bonds and therefore proteins in solution, the Biuret
test was performed. This test is semi-quantitative and specific to the peptide bonds in proteins
and so, cannot be used to test amino acids. A positive test is indicated by the formation of the
Biuret compound, which is violet/purple in colour. The dipeptide bonds that link together
protein are involved in the reaction with copper (II) ions from the reagent.
As expected, negative results were obtained for the blank solution tested while
positive results were obtained for both albumin and urea, with colour changes to deep purple,
and a violet-pink solution upon heating respectively.
Albumin has the typical structures of a protein – primary, secondary, and tertiary and
is linked together by peptide bonds. A positive test result was obtained as a colour change to a
purple solution occurred. This purple colour was due to the complex formation between
dipeptide bonds and cupric ions present.
Urea is a nitrogen-containing compound which has the molecular formula CH4N2O
and is typically odourless. Upon heating, urea undergoes decomposition as follows:
2H2NCO-NH2  H2NCONHCONH2 + NH3
Upon decomposition, urea gives a positive test result for the presence of
proteins/peptide bonds as it forms the Biuret compound and reacts with cupric ions to
produce the complex. A positive test result was obtained as expected, however, a pungent
odour was also observed upon heating. This results from the formation of ammonia from urea
when heating is performed. The greater the amount of peptide bonds present, the darker the
coloured complex would appear. Hence, it can be deduced that albumin contains more
peptide bonds than the heated urea.
Limitations in this experiment include the requirement of relatively large volumes of
protein for testing (Khan, 2013) while sources of error may include under-heating of the urea.
The Ninhydrin test was performed to determine the presence of amino acids in
solution. A positive test result for this experiment can be indicated either by:
- The formation of a purple coloured product in the case of primary amines, or
- The formation of a yellow coloured product in the case of imino acids,
hydroxyproline and proline.
The oxidizing agent ninhydrin causes the deamination of amino acids to produce
aldehydes and reduced aldehyde forms of ninhydrin. The release of ammonia and carbon
dioxide take place, where the ammonia then reacts with ninhydrin and the reduced products
to produce diketohydrin, which is purple. In the case of imino acids such as proline, the
reaction with ninhydrin results in a bright yellow coloured product, while for hydroxyproline,
similar results are achieved (Khan, 2013).
Positive test results were obtained for amino acids X, Y and Z where:
X – experienced a colour change to purple, indicating the presence of alpha amino acids
Y – experienced a colour change to a reddish-orange, indicating the presence of imino acids
which are most likely proline, and
Z – experienced a colour change to yellow, indicating the presence of imino acids which are
most likely hydroxyproline.
Lastly were the various protein reactions which were carried out using TCA,
ammonium sulphate, HCl, NaOH, copper sulphate, lead acetate, and ice-cold ethanol. These
reagents were used to perform several tests on albumin, as follows:
Hydrogen bonds are formed between proteins in solution with water molecules that
are present via their exposed ionic and polar groups. Upon the addition of ammonium
sulphate which contains highly-charged atoms in high concentrations, competition develops
for binding to the water molecules. The protein, therefore, loses water molecules and so, the
solubility of the protein decreases. Precipitate results as a positive indication that proteins are
present (Anon., n.d.).
In this experiment, a white precipitate in suspension was observed. As expected,
albumin tested positive for protein.
When the pH value of the albumin solution is altered by the addition of a strong acid
such as TCA, the optimum pH is reduced to isoelectric point where the positive and negative
charges within the protein are equalized. Weak bonds are affected, and precipitation results
(Anon. n.d.).
In this experiment, a colourless solution, containing a white precipitate in suspension
was observed. As expected, albumin produced a positive result for the presence of protein.
HCl is another strong acid, which means that a similar result was observed where a
slightly white, cloudy solution was observed, containing a suspended white precipitate,
appearing almost gel-like. This again confirms the presence of protein in albumin.
Copper sulphate and lead acetate both contain heavy metals, and so, can be used in
this experiment of precipitation by heavy metals. The Cu2+ and Pb2+ contained in these
compounds have high molecular masses/weights, as well as positive charges. The cations can
cancel the negative charges in the albumin to produce a precipitate (Panibe, n.d.).
Upon the addition of copper sulphate, a white precipitate with a bluish tint was
formed and suspended throughout the solution. Upon the addition of lead acetate, a slightly
white, cloudy solution was obtained with a white precipitate in suspension. As expected,
these indicate the presence of protein in albumin.
The addition of NaOH, through alkaline hydrolysis, results in saponification where
the sodium salts of constituent fatty acids are formed from the hydrolysed fats. According to
solubility rules, all sodium salts are soluble. This explains the clear, colourless solution
observed. The lipids identified as present are likely due to the presence of cholesterol or fatty
acids in the albumin. Conversely, the sodium ions contained in the base, due to their
attraction to the protein’s polar groups, could have increased the solubility of the protein.
Finally, cold ethanol is used to prevent the denaturing of proteins contained in the
albumin solution to be tested. However, precipitation still occurs which results in an evenly-
distributed white precipitate being formed.
Generally, protein precipitation is used in the coagulation of proteins to precipitate
toxins from the blood, and in the fractionation of membrane proteins.
For the determination of the class of carbohydrate to which an unknown belongs, the
following procedure can be carried out:
Step 1
Create a solution of the unknown carbohydrate and perform Molisch’s test. Expected
observation: purple ring formation at the junction of the 2 solutions. Inference: carbohydrate
present.
Step 2
On a sample of the unknown solution, carry out the iodine test. Expected observation:
no colour change/no visible reaction. Inference: monosaccharide or disaccharide
carbohydrate present. Unexpected observation: colour change to blue, brown, or red.
Inference: starch, glycogen, or dextrin present, respectively.
Step 3
On a sample of the unknown solution, perform Benedict’s test. Expected observation:
formation of green, yellow, orange, or red precipitate. Inference: reducing sugar present, i.e.
glucose, fructose, galactose, lactose, or maltose present. Unexpected observation: no reaction
occurs; blue solution due to reagent. Inference: sucrose is present.
Step 4
On a sample of the unknown solution, Barfoed’s test is carried out. Expected
observation: slight formation of red precipitate at the bottom of test tube. Inference: a
monosaccharide is present, i.e., glucose, fructose, mannose, or galactose. Unexpected
 observation: no precipitate formed; no visible reaction/negative reaction. Inference:
disaccharide is present, i.e., lactose, sucrose, or maltose present.

Step 5
Seliwanoff’s test is to be performed on a sample of the unknown solution. Expected
observation: cherry-red colour produced. Inference: fructose is present. Sucrose possibly
present. Unexpected observation: no cherry-red colour produced; no visible reaction/
negative result. Inference: glucose, mannose, or galactose present.
Step 6
Acid hydrolysis is carried out on a sample of the unknown solution given Benedict’s
test and Barfoed’s test are negative and Seliwanoff’s test is positive, the carbohydrate is likely
sucrose. Expected observation: positive reaction occurs between hydrolytic products and
Benedict’s and Barfoed’s reagents. Inference: presence of sucrose confirmed.
(Chhabra, 2014