Introduction

A biomolecule is a molecule, found in living organisms, which has unique properties and shape that
determine how they perform in certain functions and reactions within the organisms body.
Examples of biomolecules are proteins, carbohydrates (sugars, starch, fibre), lipids, and nucleic
acids.
Proteins are complex molecules and are also large. They are vital for maintaining proper function
and structure of bodily tissues and organs and can act as biological catalysts (enzymes) for metabolic
reactions. Proteins are comprised of smaller units, amino acids, that are linked to each other via
peptide bonds and form a long chain. Each amino acid has the following structure:

"Khan Academy." Khan Academy. Accessed February 11, 2017. https://www.khanacademy.org/test-

prep/mcat/biomolecules/amino-acids-and-proteins1/a/chemistry-of-amino-acids-and-protein-structure.
Diagram Showing General Structure of an Amino Acid

The peptide bonds are covalent and are formed by nucleophilic addition-elimination reactions
between the carboxylic group of one amino acid, and the amino group of the other, with water
being released as the by-product. In the bond formation, the amino group of the second amino acid
acts as the nucleophile and it attacks the carboxylic group (electrophile) of the first amino acid.
Afterwards, the carbonyl bond reforms and a hydroxide ion is eliminated. The hydroxide ion attracts
a proton (H+) and forms water (by-product) this causes the Nitrogen to become neutralized, resulting
in bond formation between both amino acids.

"Khan Academy." Khan Academy. Accessed February 10, 2017. https://www.khanacademy.org/test-

prep/mcat/biomolecules/amino-acids-and-proteins1/a/chemistry-of-amino-acids-and-protein-structure.
Mechanism of Peptide Bond Formation

Certain chemical tests can be executed to determine the presence of or to differentiate between
proteins. The Biuret Reaction tests for the presence of urea and/or peptide bonds in solution. The
urea decomposes in heat to form a compound, Biuret, which reacts with ions in an alkaline solution
to give a characteristic deep purple colour. Protein precipitation involves reactions by which the end
product causes any proteins in solution to precipitate out, this can be done by several different
compounds. Protein precipitation can be useful in purifying drinking water, processing biological
products and in concentrating DNA or RNA samples. The last experiment concerning proteins in this
laboratory exercise is the Ninhydrin reaction. It involves the products of amino acids reacting with
ninhydrin, further reacting with excess ninhydrin to form a purple compound. This test is useful for
recognition and estimation of amino acids in chromatographic procedures.

Carbohydrates are biological carbon compounds that contain a significant number of hydroxyl
groups. They are the most abundant of the biomolecules, and can be classified into
monosaccharides, disaccharides and polysaccharides. Monosaccharides are the simplest form of
sugar (one sugar unit) and are relatively small. Some examples are glucose, fructose, galactose, each
has a very specific structure. They are used as fuel for cellular metabolism and in biosynthetic
reactions. Two monosaccharides can also bond together to form one disaccharide. Disaccharides can
provide energy to muscles when broken down, they also fuel the nervous system, metabolize fat and
prevents tissues from breaking down proteins. Examples of disaccharides are sucrose (made from
glucose and fructose) and lactose (made from glucose and galactose). Several monosaccharides can
also join together to form polysaccharides which are used for structural components of the body
such as cellulose in plants and chitin in animals. Examples of polysaccharides are amylose (starch),
amylopectin and glycogen (animal storage). Polysaccharides can also act as energy storages and can
be broken down into monosaccharides. A monosaccharide can have a carbonyl group that is either a
ketone or an aldehyde. Those that have an aldehyde as the carbonyl group are aldose
monosaccharides, those that have a ketone are ketose monosaccharides.

Monosaccharides can either be straight chained or ringed in formation, where the ringed formation
is the more abundant of the two. The ring structure is formed due a bond formation between
Carbons 1 and 5 of the straight chain.

Matta, Michael S., Antony C. Wilbraham, and Dennis D. Staley. Introduction to general, organic, and biological chemistry.
Lexington, MA: D.C. Heath, 1996.
Diagram Showing Formation of Ringed (cyclic) Monosaccharide from Straight Chained Monosaccharide

There are several experimental tests which can be carried out involving Carbohydrates. The Molisch
Test identifies the presence of carbohydrates in solution by the reaction causing a brown-red change
in colour upon the formation of hydroxymethyl furfural due to hydrolysis of glycosidic bonds (of the
carbohydrates). The Benedicts Test is well known for differentiating between reducing and non-
reducing sugars (carbohydrates) in solution. Reducing sugars will have the ability to reduce Cu2+ ions
in solution, causing a colour change from blue to red-brown. Seliwanoffs test is Ketose specific and
gives a positive result as a colour change of light yellow to orange upon reaction of a ketose with the
Seliwanoffs reagent. The Modified Barfoeds test can distinguish between monosaccharides and
disaccharides. The test has a colour change of light blue to dark blue which indicated the presence of
monosaccharides because they can more readily reduce the Cupric ions in solution than
disaccharides.

Bials Test is a test for identifying pentoses, the positive green colour change is brought about by the
condensation of furfural (pentose converted by HCl) with orcinol in the presence of ferric ions. The
Iodine Test determines the presence of starch in solution and gives a positive colour change to dark
blue or black. Acid Hydrolysis aims to separate sucrose into its monosaccharides glucose and
fructose, as the heat and acid hydrolyze the bond.

Lipids are biomolecules that are insoluble in aqueous solution but soluble in organic solvents. These
include fats, phospholipids, triglycerides and sterols. Lipids are energy reserves when in the form of
triglycerides (or TAGs; triacylglycerol). Lipids also serve as components of biological membranes and
aid in emulsification, digestion and absorption.

Lorenwuster Follow. "Structure of organic compounds." Share and Discover Knowledge on LinkedIn SlideShare. September
29, 2012. Accessed February 10, 2017. http://www.slideshare.net/lorenwuster/structure-of-organic-compounds.
Diagram of General Structure of Lipids

The Sudan III Test uses a red fat soluble dye to indicate presence of lipids. The Sudan III dye reacts
with lipids and stains them red. The Emulsion test also determines the presence of lipids. It suspends
the solution in alcohol which causes the lipids to dissolve because they are soluble in alcohol. The
solution formed when the lipids dissolve in the alcohol then emulsify within the water from the
original mixture. This emulsion is visible and appears white and opalescent.
Discussion
The tests conducted in this experiment that dealt with Carbohydrates are; Molisch Test, Benedicts
Test, Seliwanoffs Test, Modified Barfoeds Test, Bials Test, Iodine Test and Acid Hydrolysis of
Sucrose.
The Molisch Test is done to determine Carbohydrate presence in solution. The biochemical basis of
this test operates on hydrolysis of the glycosidic bonds of carbohydrates. Carbohydrates lose water
and dehydration occurs in the presence of concentrated H2SO4(aq), and the pentoses and hexoses
(simple sugars with five and six carbon atoms respectively) form five member rings that contain
oxygen. The dehydrated pentoses form the aldehyde compound furfural, C5H4O2, and the
dehydrated hexoses form 5-hydroxymethylfurfural, C6H6O3. The Molisch reagent used is a solution of
-naphthol dissolved in ethanol. It is with two molecules of the -naphthol that the aldehydes
formed condense with to form a usually red-purple product, but was seen as red-brown in this
experiment, this maybe because the initial carbohydrate solutions were not clear, or other
substances in the solution could have caused a slightly different colour change. Subsequently, it is
important to note that colour change is subjective to the experimenter.

Nicolenk. "Lab review 1." Biochemistry is a good thing. February 21, 2013. Accessed February 10, 2017.
https://biochemistryisagoodthing.wordpress.com/2013/02/17/lab-review-1/.
Structural Chemical Equations of Reactions in Molisch Test

A positive reaction would show the appearance of a red-purple solution or a purple ring (recorded as
being seen as red-brown in this lab exercise). Monosaccharides give a more rapid test than
disaccharides and polysaccharides because they are already in the simplest form and are available to
react more readily. In this experiment, the Arabinose, Glucose, Maltose, Starch and Sucrose
solutions all gave positive results for this test, indicating that they all have carbohydrates in solution.
Distilled water did not change in colour because it is not a solution of carbohydrates.

"The Molisch Test." The Molisch Test. Accessed February 10, 2017. http://www.harpercollege.edu/tm-
ps/chm/100/dgodambe/thedisk/food/molisch/molisch.htm.
Negative Molisch Test Results (left) and Positive Molisch Test Results (right)
The Benedicts test distinguishes carbohydrates into reducing sugars and non-reducing sugars. A
reducing sugar has a free aldehyde or ketone group attached to it and can donate electrons to
another molecule, it can act as a reducing agent. All monosaccharides and few disaccharides (e.g.
lactose, maltose) are reducing sugars. Non-reducing sugars do not have free aldehyde or ketone
groups attached to them and cannot donate electrons. Benedicts reagent contains copper (II)
sulphate, CuSO4(aq) which gives the reagent its signature blue colour as well as anhydrous sodium
carbonate, Na2CO3, and sodium citrate C6H5Na3O7. Upon heating Benedicts reagent with reducing
sugars, the CuSO4(aq) reacts with the electrons from the free ketone or aldehyde group on the
reducing sugar to form cuprous oxide, Cu2O which is a red-brown precipitate. This is formed as the
free carbonyl group reduces the Cu2+ ions from the CuSO4(aq) to Cu+ ions, in this process, the
carbohydrate becomes oxidized hence a redox reaction has occurred. The Na2CO3 provides alkaline
conditions which are required for the redox reaction and the C6H5Na3O7 forms a complex with the
Cu2+ ions so that they do not deteriorate to Cu+ ions prior to being added to the carbohydrate
solution.

"Benedict's Test: Principle, Reagent Preparation, Procedure and Interpretation." LaboratoryInfo.com. June 09, 2016.
Accessed February 10, 2017. http://laboratoryinfo.com/benedicts-test-principle-reagent-preparation-procedure-
interpretation/.
General Equation for Main Reaction Occurring in Benedict's Test

Colour change due to the precipitate formed is dependent on the volume or concentration of
reducing sugars in the solution. No reducing sugar present will not produce a colour change and the
solution will remain blue (negative test). Small amounts of reducing sugars will result in a blue to
green colour change as the brick-red precipitate mixes with the remaining unreacted blue Benedicts
reagent. An intermediate amount of reducing sugars will give a colour change to orange, as there is
more brick-red precipitate present than unreacted blue Benedicts solution, the blue has a lesser
effect on the overall colour of the final solution. A great amount or a concentration solution of
reducing sugars will have a completely brick-red colour change as all or almost all the Benedicts
reagent would have reacted, causing there to be no or negligible amounts of blue Benedicts reagent
present. The results of this experiment showed that the blank exhibited no colour change due to an
absence of reducing sugars. The 0.001M solution had a negligible amount of reducing sugar, there
was barely a colour change. The 0.01M solution turned from light blue to a light brownish-blue,
indicating a higher amount of reducing sugar. The 0.02M solution turned from light blue to pale
orange and had a fair amount of reducing sugar. The 0.05M solution showed to change from light
blue to orange, indicating a moderate concentration of reducing sugar. Lastly, the 0.1M solution
exhibited a colour change of light blue to dark orange, signifying a relatively high concentration of
reducing sugars.

Aryal, Sagar. "Benedict's Test- Principle, Composition, Preparation, Procedure and Result Interpretation." Online
Microbiology Notes. November 27, 2016. Accessed February 10, 2017. http://www.microbiologyinfo.com/benedicts-test-
principle-composition-preparation-procedure-and-result-interpretation/.
Image Showing Varying Results of Benedict's Test
Seliwanoffs test distinguishes ketose sugars from aldose sugars based on reaction rate. A ketose
sugar is a monosaccharide that contains one ketone group and an aldose is a monosaccharide that
contains an aldehyde group. The test is based on the principle that, when heated, ketose sugars
become dehydrated faster than aldose sugars. Seliwanoffs reagent contains hydrochloric acid, HCl,
which is a non-oxidizing acid, and resorcinol, C6H6O2. The HCl reacts with the ketone group on the
ketose sugar due to the dehydration reaction, to form a derivative of furfural, 4-hydroxy-methyl-
furfural. The furfural then reacts with the resorcinol to form a red complex via condensation
reaction. This reaction occurs at a much slower rate for the aldose sugar since the initial dehydration
reaction is not as fast in aldoses. While the aldose does have the ability to form the same red
complex, the time taken for this to occur is much longer, hence, if left undisturbed for a significant
time, the aldose solution would eventually form the red complex.

Nicolenk. "Lab review 1." Biochemistry is a good thing. February 21, 2013. Accessed February 10, 2017.
https://biochemistryisagoodthing.wordpress.com/2013/02/17/lab-review-1/.
Reaction of Ketose and Seliwanoff's Reagent in the Seliwanoff's Test

In this experiment, the results showed that the glucose solution remained light yellow but began to
turn orange after a long period of time, indicating that glucose is not a ketose sugar and there were
no ketoses in the solution. Whereas, the fructose solution produced a colour change from light
yellow to orange, hence fructose is a ketose sugar.

Nicolenk. "Lab review 1." Biochemistry is a good thing. February 21, 2013. Accessed February 10, 2017.
https://biochemistryisagoodthing.wordpress.com/2013/02/17/lab-review-1/.
Image of Positive Colour Change (left to right) for Seliwanoff's Test

The Modified Barfoeds test is used to differentiate disaccharides from monosaccharides. For the
test to be effective, the carbohydrate must be a reducing sugar since it utilizes a reduction reaction
to identify the carbohydrate. On this basis, monosaccharides will give a positive test result, which is
a faster reaction rate, than disaccharides, because monosaccharides are the simplest form of
carbohydrate sugars and are also reducing sugars. Additionally, some disaccharides are not reducing
sugars and will give no reaction. Barfoeds reagent is made up of cupric acetate Cu(CH3COO)2, and
acetic acid, CH3COOH, in solution. Monosaccharides are oxidized readily in weakly acidic solutions
and will react readily with the cupric acetate, causing the Cu2+ ions to be reduced to Cu+ resulting in
formation of Cu2O. The resulting product causes a red-brown colouration. (This reaction can occur
with reducing disaccharides, but occurs at a much slower rate.) The Cu+ ions subsequently reduce
the phosphomolybdic acid in the phosphomolybdate colour reagent to form phosphomolybdenum
blue. This results in the formation of a deep blue colouration in the monosaccharide solution.
Reducing disaccharides can also cause this reaction but, as iterated, the reaction rate will be much
slower or may not occur at all.
"Carbohydrates - Barfoed's Test." Carbohydrates - Barfoed's Test. Accessed February 10, 2017.
http://www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/carbo/barf/barfoed.htm.
General Reaction of Monosaccharide with Copper (II) Ions in Modified Barfoed's Test

"Find and share online flashcards and notes from StudyBlue. Any subject, anywhere, anytime." STUDYBLUE. Accessed
February 10, 2017. https://www.studyblue.com/#flashcard/view/9726316.
Image Showing Modified Barfoed's Test Colour Change of Positive Result (left to right)

In the laboratory exercise conducted, the blank showed no colour change as it contained no
monosaccharides. The fructose solution and glucose solution both showed a positive colour change
as both are monosaccharides. The maltose and sucrose solutions both gave negative results because
they are disaccharides. Even though maltose is a reducing sugar, its reaction rate is very slow in this
test and would have turned dark blue if it were left for a long time period.

Bials test is used to distinguish pentose sugars via hydrolysis reactions. The Bials reagent consist of
hydrochloric acid; HCl, orcinol; C7H8O2, and ferric chloride; FeCl3. The HCl hydrolyses pentose sugars
to form a derivative of furfural, this compound then reacts with the C7H8O2 in solution. In the
presence of ferric ions, the reaction subsequently forms a yellow-green complex via condensation.
Any polysaccharides made up of pentose units are hydrolysed in order to break glycosidic bonds that
join the pentose units together. The separated units then undergo the same reaction to form the
yellow-green complex. This test distinguishes pentose sugars because they help form the furfural
derivative necessary for reaction completion. Although, hexose sugars may also form a colour
change, the final colour is a different colour, it is red-brown as opposed to the yellow-green colour
change produced by the pentose solution. This is because the hexose sugars react to form a different
compound.

Nicolenk. "Lab review 1." Biochemistry is a good thing. February 21, 2013. Accessed February 10, 2017.
https://biochemistryisagoodthing.wordpress.com/2013/02/17/lab-review-1/.
Reactions Involved with Pentoses and Hexoses in Bial's Test
Nicolenk. "Lab review 1." Biochemistry is a good thing. February 21, 2013. Accessed February 10, 2017.
https://biochemistryisagoodthing.wordpress.com/2013/02/17/lab-review-1/.
Image Showing Colour Changes Observed in Bial's Test

In the experiment conducted, the blank gave no colour change as it contains no carbohydrates; no
pentoses or hexoses, to produce a reaction. Arabinose solution produced a blue-green colour
change which indicates that the solution contained a small amount of pentose sugars, Arabinose is a
pentose sugar. Gum Arabic solution provided a green colour change, Gum Arabic is a polymer of
arabinose, rhamnose and galactose. As such, when hydrolysed, it will give a positive result. Lastly,
glucose solution gave a brown colour change, since glucose is a hexose, it can react in this test as
iterated before.

The Iodine Test indicates the presence of starch in solution and is starch specific. Starch is
constituted of straight chained amylose. It is the amylose that is responsible for the positive
reaction. Amylose is composed of -glucose monomers which are connected by -1,4 glycosidic
bonds. Amylose exits as helically coiled structures in solution, hence, when iodine is added to the
solution, it becomes trapped within the helical structure. This results in the formation of a complex
which is blue-black in colour because the iodine is blue-black in colour. However, the extent of
colour change depends on the compound in solution.

"Reversible guest exchange mechanisms in supramolecular hostguest assemblies." Reversible guest exchange
mechanisms in supramolecular hostguest assemblies - Chemical Society Reviews (RSC Publishing). Accessed February 10,
2017. http://pubs.rsc.org/en/content/articlelanding/2007/cs/b603168b#!divAbstract.
Illustration of Iodine Trapped in Amylose Helix

"Iodine/Potassium Iodide Test." Iodine/Potassium Iodide Test. Accessed February 10, 2017.
http://www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/food/iki/iki.htm.
Image of Results of Iodine Test; (left to right) Test on Glycogen, Test on Starch, Test on Dextrin

In the laboratory exercise, starch stained blue-black in colour. However, the glycogen and dextrin
both exhibited colour changes as well. Glycogen showed a colour change to a rust colour, because
the compound is composed of branched chains of glucose monomers and therefore does not form a
helix as in amylose. Hence, glycogen dos not trap iodine but can still form a complex with the iodine
which gives it the rust colour change. Dextrin gives a violet colour when mixed with Iodine. This is
because, while dextrin is similar to starch in structure, it is smaller and less complex than starch and
is formed during hydrolysis of starch. The dextrin forms a complex with iodine that results in the
violet colour.

Acid hydrolysis of sucrose is performed in disaccharides and polysaccharides. They breakdown to

form their monomer monosaccharide units, the test is capable of enabling breakdown of sucrose
into fructose and glucose. Hydrochloric acid breaks down the 1-2 glycosidic bond that links the
glucose monomer and fructose monomer together. This results in the formation and release of two
monosaccharides. Sodium hydroxide is added to neutralize the pH of the solution. This is done
because the acid in the solution can disturb the chemistry of the Seliwanoffs and Benedicts reagent
that were subsequently added. Since two monosaccharide sugars were formed from the sucrose,
when the Benedicts test is conducted, the solution will be able to produce a brick-red precipitate
due to the presence of the reducing sugar, glucose. This allows the Cu2+ ions to be reduced to Cu+
which form the brick-red precipitate. Likewise, on completion of the Seliwanoffs test, the fructose
present will give a positive result and will show a colour change to orange.

Nicolenk. "Lab review 1." Biochemistry is a good thing. February 21, 2013. Accessed February 10, 2017.
https://biochemistryisagoodthing.wordpress.com/2013/02/17/lab-review-1/.
Hydrolysis of Sucrose into Glucose and Fructose

In this experiment, the Hydrolysate (sucrose solution post acid hydrolysis) showed positive test
results for both the Benedicts test and Seliwanoffs test, because the hydrolysis broke the sucrose
down to glucose and fructose. The sucrose solution gave no reaction with the Benedicts test,
because sucrose is not the reducing sugar and the glucose unit in the sucrose was unavailable for
reduction reaction. The sucrose solution initially did not produce a colour change with Seliwanoffs
test, because the fructose monomer was occupied within the sucrose. However, a colour change
was observed after a long time period because Seliwanoffs reagent has hydrochloric acid, which
overtime, can hydrolyse some of the glycosidic bonds present.

The Sudan III Test utilizes the Sudan III dye. This dye is soluble in fat and can therefore identify the
presence of lipids, lipoproteins and triglycerides in solution. The Sudan III dye reacts with any fatty
compounds present in solution and becomes dissolved in the fat globules causing the lipids to be
stained red, making them visible in solution. In this experiment, the oil stained red with the Sudan III
dye because the oil is a lipid and contains triglycerides. Since oil is less dense than water and is
insoluble in water, the red stained oil forms a layer of globules above the water and may even
appear as a red layer above the water in the test tube.
Nicolenk. Biochemistry is a good thing. Accessed February 10, 2017.
https://biochemistryisagoodthing.wordpress.com/?s=sudan%2Btest.
Image of Positive Results for Sudan III Test
In this experiment, the Sudan III dye, when introduced to the oil mixture, stained the lipids orange-
red and the lipids floated to the top of the water.

The Emulsion test indicates the presence of lipids in a solution. The lipids and triglycerides in
solution are alcohol soluble and hence dissolve in ethanol to form a solution. When in water, oil
forms small globules or droplets on the waters surface because the triglycerides rearrange to allow
their hydrophobic tails to point inwards. The globules formed have the ability to scatter light and
causes the mixture to appear cloudy.

Nicolenk. Biochemistry is a good thing. Accessed February 10, 2017.

https://biochemistryisagoodthing.wordpress.com/?s=sudan%2Btest.
Image of Positive Emulsion Test

"Lipids." Home. Accessed February 10, 2017. https://dlc.dcccd.edu/biology1-3/lipids.

Arrangement of Lipid/Triglyceride in Water

In the experiment done, the oil beomes dissolved in the ethanol to form a solution and because oil is
less dense that water, it forms dispersed, small droplets which alter the light that passes through the
overall mixture, causing the white, cloudy appearance.
The Biuret test can identify peptide bonds which are present in proteins and is specific for peptide
bonds in proteins and not in amino acids. Biuret is a compound that is formed upon heating urea
and reacts with Cu2+ ions which results in a purple complex. In the reaction, four biuret molecules
form a complex with one cupric ion. The proteins in turn mimic the formation of the complex and
hence also give off a purple complex upon reaction with the Cu2+ ions. The Biuret reagent is
composed of potassium hydroxide; KOH, hydrated copper (II) sulphate; CuSO4.5H2O and potassium
sodium tartrate; KNaC4H4O6.4H2O. The potassium sodium tartrate is used to stabilize the cupric ions.
The dipeptide bonds in the proteins can react with the Cu2+ ions to form the complex and the lone
pairs (present on the nitrogen of the peptide bond) forms a linkage with the cupric ions in the Biuret
reagent and gives the purple colour. The deepness of the colour of the complex is proportional to
the concentration of dipeptide bonds present in solution.

Nicolenk. Biochemistry is a good thing. Accessed February 10, 2017.

https://biochemistryisagoodthing.wordpress.com/?s=biuret%2Breaction.
Diagram Showing Arrangement of Complex Formed

Tok, Panji. "Biuret Test." Biuret Test. January 01, 1970. Accessed February 10, 2017.
http://birdingpark.blogspot.com/2013/11/biuret-test.html.
Image Showing Negative (left) and Positive (right) Test Results for Biuret Test

In this experiment, the blank gave no colour change because it contains no proteins and hence no
dipeptide bonds. The albumin produced a purple colour change because it reacts with the Biuret
reagent and forms the purple complex due to the presence of dipeptide bonds and cupric ions. The
urea also gives a colour change to purple because the heated urea forms the Biuret compound
which reacts with the cupric ions in the reagent to form the purple complex.

Protein precipitation can be achieved with TCA, Trichloroacetic Acid because TCA has the ability to
expose the protein hydrophobic structure, causing them to precipitate out of solution. TCA varies
the pH of the overall solution by lowering it. At low pH values, proteins have a net positive charge
because the amide attached gains an extra proton. This leads to reduced solubility because the
protein is unable to interact with the medium it is in, and will hence fall out of or precipitate out of
solution. Similarly, the hydrochloric acid, when added to the protein solution, causes the overall pH
to become more acidic, causing the hydrophobic structures of the proteins to be exposed and as
such causing protein precipitation. Therefore, a white, cloudy coagulant was seen upon treating the
protein solution with TCA as well as HCl in this experiment.
Ammonium sulphate can also be used to achieve protein precipitation. This processes is known as
salting out and involves interaction of protein with the ammonium sulphate instead of with the
water in solution. This leads to decreased interaction between the water and the protein, resulting
in more hydrophobic areas of the protein being exposed and hence leading to precipitation. This is
the same basis for the use of sodium hydroxide and copper sulphate in protein precipitation. These
salts allow the protein to decrease its interactions with the water and to no longer be dissolved in
water. The precipitation was seen in this experiment as a white, cloudy coagulant.
When ethanol is added to the protein solution, it also causes the proteins to precipitate out of
solution. Ethanol is an organic solvent and tends to displace water from the protein surface upon
binding around the ethanol molecules. This decreases protein-water interactions and causes the
protein to precipitate out of solution. In the lab exercise, this precipitation was seen as white, cloudy
coagulants.
Lead acetate is a heavy metal salt. Heavy metal salts have the ability to denature and precipitate
proteins as the ions react with the negatively charged functional groups on the protein to form a
covalent bond which denatures the protein. Because the protein is denatured, it can no longer
interact with the water molecules to stay in solution. As such, the proteins fall out of the solution.
This was seen as a white, cloudy coagulant in the laboratory exercise.

"BCH 302 [PRACTICAL] Proteins I." Ppt download. Accessed February 10, 2017. http://slideplayer.com/slide/8086066/.
Image Showing Appearance of Protein Precipitation

The Ninhydrin test is used for identification of amines and - amino acids. Ninhydrin reagent is an
excellent oxidizing agent and is responsible for the oxidative deamination of the amino acids. The
amino group of the free amino acids form a purple-blue colour when reacted with ninhydrin.
Ninhydrin deaminate the amino acids to form aldehydes and reduced aldehyde forms of ninhydrin.
Ammonia and carbon dioxide are also released as products of the reaction. The ammonia formed
reacts with the ninhydrin and its reduced products to give diketohydrin which is a blue-purple
coloured substance. However, some amino acids react with ninhydrin differently and may form a
bright yellow colour change.

Nicolenk. Biochemistry is a good thing. Accessed February 10, 2017.

https://biochemistryisagoodthing.wordpress.com/?s=biuret%2Breaction.
Illustration of Reactions and Colour Changes in Ninhydrin Test

In this experiment, amino acid X reacted to give a purple colour change and hence can be labeled as
an amino acid. Amino acids Y and Z gave yellow colour changes and are amino acids but most likely
not amino acids.
Lab Questions
1. Starch and glycogen are both polymers of glucose. However, starch itself is composed of amylose
and amylopectin. In amylose, the glucose monomers are linked by 1,4 glycosidic bonds which
produce an unbranched chain of glucose which coils into a helix. In amylopectin, there are both 1,4
and 1,6 glycosidic bonds. Subsequently, some glucose molecules have a glycosidic link from carbon 1
to carbon 6 resulting in branching. Conclusively, starch can exist as a coiled helix. This contrasts to
glycogen which is similar to amylopectin but has more branches and does not form a helix.
Cellulose, on the other hand, is an unbranched polymer of beta glucose molecules which are linked
by 1,4 glycosidic bonds with Hydrogen bonds between adjacent cellulose molecules. The large
number of Hydrogen bonds allow cellulose to form strong fibres which are more structurally sound
than starch and glycogen.

2.Sucrose was boiled in acid during Acid Hydrolysis of Sucrose because the acid provided H+ ions
necessary for the reaction.