NAME: COLENE HOYTE I.D.

#: 810003702

LAB PARTNER: CHERELLE DIDIER, KAILA JONES DEMONSTRATOR’S NAME: AVION

TITLE: CARBOHYDRATES, LIPIDS, PROTEINS AND AMINO ACIDS

AIM: To investigate the presence and or absence of carbohydrates, lipids, proteins and
amino acids.

THEORY: This experiment is designed to determine the presence or lack thereof

carbohydrates, lipids, proteins and amino acids through specific tests. These
tests are designed to show whether or not these substances are present. The
experiment sets out to determine the presence of carbohydrates by performing
Molisch, Benedict’s, Seliwanoff’s, Modified Barfoed’s, Bial’s and Iodine Tests.
However, to detect the existence of lipids the experiment does so by way of a
Sudan III and an Emulsion Test. To establish whether or not there was a presence
of proteins a Biuret Reaction as well as a Protein Precipitation was set up and
observed, and to examine whether or not amino acids are present a Ninhydrin
Reaction process is conducted.

PROCEDURE: TEST 1 – MOLISCH TEST

All test tubes were carefully washed before use. Ten (10) drops of sucrose was
then placed into a clean test tube. One millilitre (1ml) of Molisch reagent was
then added to the test tube and the contents were mixed. The test tube was
then inclined and about one millilitre (1.0 ml) of concentrated H 2SO4 was
carefully poured down the side of the test tube. Observations were then made
and accurately recorded. The test was then repeated using glucose, maltose,
arabinose and a starch solution. These results were also carefully observed and
accurately recorded.

TEST 2 – BENEDICT’S TEST

All test tubes were carefully washed before use and a water bath was accurately
set up. One millilitre (1.0ml) of Benedict’s solution was placed in clean test tube.
Eight drops of the solution to be tested was then added and mixed well. With the
use of a test tube holder the test tube was then placed in a boiling water bath
for five (5) minutes. The test tube was then allowed to slowly cool and
observations of the content’s colour and whether or not a precipitate was
formed was also noted. This process was carried out on 0.1M, 0.05M, 0.02M,
0.01M AND 0.001M dilutions of glucose solution. Results obtained were
accurately recorded.

TEST 3 – SELIWANOFF’S TEST

All test tubes were carefully washed and labelled before use and a water bath
was accurately set up. A few drops of glucose was placed in a test tube labelled
A and a few drops of fructose was dropped in a test tube labelled B. One
millilitre (1.0ml) of Seliwanoff’s reagent was then added to test tube A and to
test tube B. Both test tubes were then heated in a boiling water bath for one
minute. Accurate observations of colour change were then recorded.

TEST 4 – MODIFIED BARFOED’S TEST

All test tubes were carefully washed before use and a water bath was accurately
set up. One millilitre (1.0 ml) of the solution to be tested was placed in a clean
test tube and 0.5 ml of Barfoed’s reagent was added to the tube. With the use of
a test tube holder the tube was placed in a boiling water bath and heated for
three minutes. When the time elapsed it was then placed in a beaker of cold
water for two minutes. One millilitre (1.0ml) of phosphomolybdate colour
reagent was then added and mixed thoroughly. The test was then repeated
using sucrose, fructose and maltose solutions. A control was also performed by
carrying out the test on one millilitre (1.0ml) of water.

TEST 5 – BIAL’S TEST

All test tubes were carefully washed before use and a Bunsen burner was set up.
One millilitre (1.0ml) of the reagent was placed in a clean test tube and one
millilitre (1.0ml) of the solution to be tested was then added to the tube. The
test tube was then gently heated until boiling and then was allowed to cool. This
process was performed using glucose, arabinose and gum arabic solutions.
Results were then accurately recorded.

TEST 6 – IODINE TEST

All test tubes were carefully washed and labelled before use. A few drops of
iodine solution were added to the following polysaccharides: starch, glycogen
and dextrin. Observations were then carefully made and recorded.
TEST 7 – SUDAN III TEST

All test tubes were carefully washed before use. Two millilitres (2.0ml) of water
was placed in a clean test tube. Two (2) drops of oil were then added to the test
tube and a few drops of Sudan III were also added and the test tube shaken to
mix the contents of the tube. Observations were then recorded and results
accurately discussed.

TEST 8 – EMULSION TEST

All test tubes were carefully washed before use. Two millilitres (2.0ml) of
absolute ethanol were added to the clean test tube. Two (2) drops of oil was
then also added to the tube. Vigorous shaking of the tube then occurred and an
equal volume of cold water was then added. Observations were then made and
results recorded accurately.

TEST 9 – THE BIURET REACTION

All test tubes were carefully washed and labelled before use and a water bath
was accurately set up. To one millilitre (1.0ml) of albumin solution four millilitres
(4.0ml) of biuret reagent was added. In a separate test tube, a control was
prepared using distilled water instead of albumin. The test tubes were then left
at room temperature for ten (10) minutes. Once the time elapsed the tubes
were then heated in a water bath at thirty seven degrees Celsius (37 0 C) for ten
(10) minutes. In another new and clean test tube one level spatula of urea was
added to the tube. The test tube was then heated over a low flame until the urea
melted or showed signs of effervescence. The test tubes were then left for ten
(10) minutes at room temperature and then heated in a water bath for ten (10)
minutes at thirty seven degrees Celsius (37 0C) using one millilitre (1.0ml) of urea
solution together with four millilitres (4ml) of Biuret reagent. Observations were
then accurately recorded.

TEST 10 – PROTEIN PRECIPITATION

All test tubes were washed carefully before use and a water bath was accurately
set up. Seven (7) test tubes were respectively labelled from one (1) to seven (7)
and three millilitres (3ml) of albumin solution was then pipette into each test
tube. The following were then added to the respective test tubes based on their
given numerical markings.

1. Two millilitres (2ml) of 10% (v/v) TCA

 2. Two millilitres (2ml) of 1M HCL
3. Two millilitres (2ml) of saturated ammonium sulphate solution
4. Two millilitres of 1M NaOH
5. Two (2) drops of 5% (w/v) lead acetate
6. One (1) drop of 2% (w/v) CuSO4
7. Ten millilitres (10ml) of cold ethanol (EtOH)

Test tubes one to six were then placed in a boiling water bath for five (5)
minutes. Observations were then made and recorded with any specific details
pertaining to possible uses of protein precipitation.

TEST 11 – NINHYDRIN REACTION

All test tubes were carefully washed before use and a Bunsen burner was set up.
Four (4) drops of each of the three (3) test solutions X, Y and Z were placed on
one filter paper and were allowed to be dried. One (1) drop of ninhydrin solution
was then added to each of the spots and gently warmed over a Bunsen burner.
The colours produced were then accurately recorded.

RESULTS: TEST 1

MOLISCH REAGENT POSITIVE OR COLOUR

NEGATIVE OBSERVATIONS
Sucrose Positive Colourless to reddish
violet in colour
Glucose Positive Colourless to reddish
violet in colour
Maltose Negative Colourless
Arabinose Negative Colourless
Starch Negative Colourless
Table
1 showing detailed results from TEST 1 The Molisch Test.

A reddish violet colour at the junction of the two liquids indicates a positive test.
 TEST 2

CONCENTRATION OF COLOUR CHANGE OBSERVATIONS

GLUCOSE DILUTION
(M)
0.1 Reddish brown Reddish brown
precipitate formed
0.05 Blue with reddish Reddish brown
brown colouring precipitate formed
0.02 Blue solution Reddish brown
precipitate ring
formed at the top and
at the bottom
0.01 Blue solution Reddish brown
precipitate ring
formed at the top
0.001 Remained blue No precipitate was
formed.

Table 2 showing detailed observations from TEST 2 The Benedict’s Test.

Colour changes went from colourless blue reddish brown. Basically the
more concentrated the glucose solution the more precipitate formed.

The lowest concentration of glucose detectable by Benedict’s test was 0.01 M

TEST 3

 The test tube containing a few drops of glucose and Seliwanoff’s reagent
showed no colour change as no precipitate was formed. This was due to
the fact that glucose is not a ketose and therefore the formation of 4-
hydoxy-methyl-furfural and its subsequent reaction with resorcinol to
produce an observable colour change did not occur.
 The test tube containing a few drops of fructose and Seliwanoff’s
reagent displayed a colour change from yellow to a reddish orange
coloured precipitate. This occurred because fructose is a ketose and a 4-
hydoxy-methyl-furfural and its subsequent reaction with resorcinol was
produced.
 TEST 4

SUGAR COLOUR COLOUR COLOUR COLOUR AFTER

BEFORE AFTER AFTER ADDITION OF
HEATING HEATING COOLING PHOSPHOMOLYBDATE

Sucrose Blue No visible No visible Pale blue colouration

reaction reaction
Glucose Blue No visible Brick red Colour changes from
reaction precipitatedark blue on standing;
formed the glucose turned dark
blue.
Fructose Blue Brick red Brick red Turned dark blue
precipitate precipitate
formed at formed
the top
Maltose Blue No visible No visible Pale blue colouration
reaction reaction
Water Blue No visible No visible Pale blue colouration
reaction reaction

Table 3 showing detailed findings from TEST 4 The Modified Barfoed’s

Test.

TEST 5

SOLUTIONS COLOUR
CHANGES/OBSERVATIONS
Glucose Dark yellow coloration
Arabinose Pale yellow coloration
Gum arabic Medium yellow
Table 4 showing results fromcolouration
TEST 5 The Bial’s Test

Table 4 showing the results from TEST 5 The Bial’s Test.

TEST 6

 When the iodine was added to the starch the result was a blue
black colouration.
 When iodine was added to glycogen a brick red colouration
resulted.
  When iodine was added to dextrin a light purple colour resulted
and upon standing approached colourless.

TEST 7

 When two drops of oil were added to two millilitres of water, the oil floated
to the top.
 When Sudan III was added to the test tube and lightly shaken, a deep burnt
orange colour was the result with numerous amounts of bubbles to the
surface of the contents in the test tube.
 The Sudan III stained the globules of fat red making them more visible.

TEST 8

 When two drops of fat were added to two millilitres of absolute ethanol the
numerous globules of fat gathered at the bottom of the test tube.
 When an equal volume of cold water was added to tube a cloudy (murky)
appearance developed along with one large globule which floated to the
surface. However upon shaking the one large globule was broken up into
several smaller globules which also stayed afloat.

TEST 9

 The albumin solution changed to purple on the addition of the biuret.

However, the solution remained purple after heating.
 The water changed from transparent (colourless) to light blue upon the
addition of biuret and there was no change after heating.
 The urea changed to a purple colour when cooled and having biuret added.
This purple colouration lasted even after heating. However, when urea was
heated a pungent odour was detected, indicating the presence of ammonia.
 TEST 10

TEST REAGENT ADDED TO OBSERVATIONS

TUBE 3ML OF ALBUMIN
SOLUTION
1 2ml of 1M HCl in water When heated there was no
bath for five minutes observable change in colour; a
colourless precipitate remained
2 2ml of 10% (v/v) TCA in a When heated no observable colour
water bath for five change occurred; a colourless
minutes precipitate resulted
3 2ml of ammonium When heated there was no
sulphate solution in a observable colour change; a
water bath for five colourless precipitate remained
minutes
4 10ml of cold ethanol When heated no observable colour
change occurred; a colourless
precipitate remained
5 1 drop of 2% (v/v) CuSO4 A cloudy, gelatinous white precipitate
in a water bath for five with globules suspended in the
Possibleminutes
uses for protein precipitation are: making it opaque was seen.
solution,
6 2 drops of 5% (v/v) lead A cloudy, white gelatinous precipitate
acetate in a water bath was suspended in the solution
for five minutes making its appearance opaque

TablTable 5 showing detailed observations of TEST 10 The Protein

Precipitation.

1. In the biotechnology industry protein precipitation is used to eliminate

contaminants commonly contained in blood.

TEST 11

 X produced a dark purple/ dark brown colouration which was due to the nature
of X. It can be said that X contains an ammonium ion and thus when tested by
ninhydrin produces a dramatic purple colour.
 Y produced a light yellow / purple colouration which is due to the nature of Y.
When amino acid residues are attached with their N-terminus protected, so if
the next residue has been successfully coupled onto the chain, the test gives a
yellow result which is what occurred. The yellow colour could also be due to a
 reaction of ninhydrin with secondary amines which gives an iminium salt that
generally causes a yellow – orange colouration.
 Z produced a brownish purple colouration which is due to the nature of Z itself.
It can be said that either Z contained ammonium carbonate and acetate upon
hydrindantin, or by the interaction of alanine and triketohydrindene
hydrate which causes the colouring to be the same and shows the same
characteristics in dilute solution.

DISCUSSION: In this experiment the goal was to determine whether or not there was a
presence of carbohydrates, lipids, proteins and amino acids in specific solutions.
This was done through various tests with each test performing a particular task.
Test 1 the Molisch test was one that provided information on whether or not
there was a positive or negative result for carbohydrates. When furfural is
formed by hydrolysis of glycosidic bonds and dehydration on the resulting
monosaccharide by concentrated sulphuric acid, condensation occurs with the
Molisch reagent (1-naphthol) to give a coloured product. In this case only
sucrose and glucose produced positive reactions in the form of a colour change
from colourless to reddish violet.
Test 2 the Benedict’s test is one used to test for reducing sugars as the alkaline
solution of copper reduces the sugar by having the free ketone group turn to a
reddish brown cuprous oxide. Also the more concentrated a sugar solution is,
the more precipitate is formed. This was a conclusion made from the results
obtained. It took literally two (2) minutes for this change in colour to occur. The
sucrose indirectly produced a positive result with Benedict's reagent when
heated. The conditions of the heat broke the glycosidic bond in sucrose through
hydrolysis.  Test 3 was the Seliwanoff’s test which was performed specifically to
identify ketoses. When the formation of 4-hydoxy-methyl-furfural and its
subsequent reaction with resorcinol was formed a red coloured compound was
the result. This occurred in the glucose after cooling and in the fructose solution
after heating.

Test 4 the Modified Barfoed’s test was used to distinguish between

monosaccharides and disaccharides. Cupric ions are reduced to Cu 2O in an acid
solution and monosaccharides reduce more readily in weak acid solutions than
do disaccharides. A positive result is usually noted by a deep blue colour. This
result was obtained when the test was performed on glucose and fructose. Test
5 the Bial’s test is a colour reaction that was used specifically to identify
pentoses, which are converted to furfural by HCl. This compound condenses with
orcinol in the presence of ferric ions to give a green coloured product. The Bial’s
reagent which is strongly acidic hydrolyzes polysaccharides made up of pentose
units and hence such compounds give a positive test result. The results obtained
for this test were more yellow. However, glucose produced a dark yellow
colouration followed by gum arabic and then arabinose with the light yellow
colouration.

Test 6 the Iodine test was used to test for carbohydrates, polysaccharides to be
exact. Starch produced a strong blue black colouration whereas glycogen
produced a brick red colouration, not typical for carbohydrates. Dextrin
produced a light violet colour which eventually turned colourless over time.
Initially all the polysaccharides started out clear in appearance and then the
described colour changes above occurred. Test 7 the Sudan III test was used to
test for lipids. The Sudan III dye which was a red dye basically stained the fat
globules making them more visible. Test 8 the Emulsion test was also used to
investigate the presence of lipids. The cloudy appearance of the liquid and the
floating fat globules were sufficient evidence to note that lipids were present.
Test 9 the Biuret reaction was used to identify the presence of proteins. When
urea was heated, it decomposed and produced the compound “biuret”. This
compound reacted with cupric ions in alkaline solution to produce a deep purple
colour. Compounds containing two or more peptide bonds also give that
reaction. The reaction with proteins is specific for the peptide bond.

Test 10 the protein precipitation which was also used to determine the presence
of proteins follows a different process. Since a variety of compounds can
precipitate proteins from solutions such precipitation often results in the
irreversible denaturation of protein. The removal of trichloroacetic acid (TCA) by
ether extraction is particularly useful since it usually occurs after the
precipitation of protein. This precipitation test only obtained result when: A.)
one drop of 2% (v/v) CuSO4 in a test tube was placed in a water bath after 3ml
of albumin solution was added to it. This process created a cloudy white
gelatinous precipitate with globules suspended in the solution presenting an
opaque appearance. B.) produced the same precipitate with the exact
appearance. However, what caused this reaction was a combination of two
drops of 5% (v/v) lead acetate combined with 3ml of albumin solution mixed in a
test tube and placed in a water bath for five minutes.

Test 11 was used to detect the presence of amino acids from three test
solutions. These solutions were all unknowns and therefore were described by
alphabetical letter representations of X, Y and Z. It was found that X produced a
dark purple colouration which was believed to be due from the nature of X.
Results showed that X could possibly contain ammonium ions and thus when
tested by ninhydrin produced a dramatic purple colour. Y however produced a
yellowish colouration which derived also from the nature of the solution. It was
noted that perhaps Y developed such a colouration because of the reaction with
ninhydrin and secondary amines which gives an iminium salt that generally
causes a yellow – orange colouration. Z which generated a brownish purple
coloration by way of its nature, it can also be said that perhaps Z contained
ammonium carbonate and acetate. Upon contact with ninhydrin the colouration
resulted.
ADDITIONAL

DISCUSSION: Question 1

You are provided with an aqueous extract from an unknown plant organ labelled
X. Give a clear and detailed outline of the various tests you are required to carry
out in order to determine as precisely as possible the nature of the biomolecules
extracted from the organ. Give a possible structure and function of one such
molecule.

Answer 1
Question 2

You are provided with a solution of unknown carbohydrate. Construct a flow

chart of the tests you will perform in order to determine as precisely as possible
the class of carbohydrates to which the unknown belongs.

Answer 2

There are four (4) classes of carbohydrates.

1. Monosaccharides (simple sugars)

2. Disaccharides (two (2) monosaccharides linked together)
3. Oligosaccharides (a few simple sugars linked together)
4. Polysaccharides (millions of simple sugars linked together)

In order to identify the unknown a series of tests need to be done. These tests
are done to check for the presence of specific details that the unknown may or
may not contain.

Modified Barfoed’s Test

to distinguish between monosaccharides and disaccharides

Benedict’s Test

to test to see whether or not it is a reducing sugar or not

Seliwanoff’s Test

to test for the presence of ketoses

Iodine Test

to identify the presence of polysaccharides

References

Benedict, S. R. (1 December 1908). "A Reagent For the Detection of Reducing Sugars". J. Biol.
Chem. 5 (6): 485–487.

Mary Grace Saba, Miguel M. Sabillena, Regine San Jose, David Santos, Virli-Anne Sebastian
Isolation and Characterization of Carbohydrates (Enzyme Hydrolysis of Glycogen) Group #7

Nachweisreaktion von Zuckern nach Molisch (Uni Regensburg) (in German, including a photo of
the reaction tube, English version also available)