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Biochemistry Lab
(Experiment 1)
Qualitative Analysis of Carbohydrates
In Experiment 1, you will
- Examine the carbohydrates: monosiccharides, polysaccharides, and celluloses
- Identify pentose/hexose and aldose/ketose using following tests:
Molisch's test: presence of all carbohydrates.
Anthrone test: presence of all carbohydrates.
Fehling’s test: presence of reducing sugars.
Barfoed’s test: presence of reducing sugars.
Benedict’s test: presence of reducing sugars.
Bial test: presence of pentose.
Seliwanoff's test: presence of ketose.
Iodine test: presence of polysaccharides.
Carbohydrates
A carbohydrate (saccharide, meaning "sugar") is an organic compound that consists only of
carbon, hydrogen, and oxygen, usually with the empirical formula [Cm(H2O)n]. Polysaccharides serve
for the storage of energy (e.g., starch and glycogen), and as structural components (e.g., cellulose in
plants and chitin in arthropods). Monosaccharides are the simplest carbohydrates in that they
cannot be hydrolyzed to smaller carbohydrates with The general chemical formula of [(C•H2O)n]. They
are aldehydes or ketones with two or more hydroxyl groups.
The 1 and 2 anomers of glucose (C1 is anomeric carbon). Lactose is a disaccharide, which consists
of a molecule of D-galactose and a molecule of D-glucose bonded by beta-1-4 glycosidic linkage.
Reducing Sugars
A reducing sugar is any sugar that either has an aldehyde group or is capable of forming one
in solution through isomerism. The aldehyde functional group allows the sugar to act as a reducing
agent. The cyclic hemiacetal forms of aldoses can open to reveal an aldehyde and certain ketoses
can undergo tautomerization to become aldoses. However, acetals, including those found
polysaccharide linkages, cannot easily become a free aldehyde. The reducing sugar can reduce
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copper(II) ions to copper(I), which then forms a brick red copper(I) oxide precipitate.
Cu(II) reduction of reducing sugar.
[Cu-based Tests for Reducing Sugars]
Test Ligand for Cu2+ Target Medium
Benedict’s test Citric acid Reducing carbohydrates Alkaline
Fehling’s test Tartaric acid Reducing carbohydrates Alkaline
Barfoed’s test - Reducing monosaccharides Acidic
1. Benedict’s Test
Objective:
It is a qualitative and semi qualitative test for reducing carbohydrates. Benedict’s test is
more specific than Fehling’s test. More over in Benedict’s test uric acid and creatinine does not
interfere its results.
Principle:
Cupric ion (Cu2+) during the process of heating is converted to red cuprous oxide (Cu2O),
which precipitates immediately. The precipitation of cupric hydroxide is avoided by sodium citrate.
In Benedict’s test alkaline medium is provided by sodium carbonate. Note that ultimate quantity of
cuprous oxide produced at the end of the reaction depends on the amount of reducing sugars present
in the sample used. As benedict reagent has blue color, the final color is the mixture of blue color of
Benedict’s reagent and red color of Cu2O. Therefore, it may vary from green to brick red depending
upon the concentration of reducing sugar. If the sample contains the reducing sugar more than 2%
the final color of solution will remain red because the production of red cuprous oxide will not affect
the already present color of solution.
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Interpretation:
Appearance of green, yellow, orange or red precipitate indicates that carbohydrate is
reducing one. This is semiqualitative test. If the solution are taken in correct proportion and
procedure is followed strictly, the approximate concentration of carbohydrate can be judge from color
of precipitate.
2. Fehling’s test
Objective:
Fehling's test is commonly used to identify the reducing sugars.
Principle:
Fehling's solution is made initially as two separate solutions, known as Fehling's A and
Fehling's B. Fehling's A is a blue aqueous solution of copper(II) sulfate, while Fehling's B is a clear
solution of aqueous potassium sodium tartrate (also known as Rochelle salt) and a strong alkali
(commonly sodium hydroxide).
Cupric (Cu2+) hydroxide has a tendency to precipitate but sodium potassium tartrate prevents
the precipitation of cupric hydroxide by forming a soluble deep blue complex with cupric ions. This
complex dissociates to provide cupric ions for oxidation. The reduction occur best in alkaline
medium which is provided by potassium hydroxide. Cupric hydroxide on heating with reducing
carbohydrate reduces to cuprous oxide (Cu2O). The color of solution changes from blue to red.
The carbohydrate is simultaneously oxidized to corresponding aldonic acid (glucose to gluconic acid).
Interpretation:
Appearance of yellow or brick red precipitates indicates the presence of reducing
carbohydrate.
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3. Barfoed’s test
Objective:
To determine whether the reducing sugars is a monosaccharide or disaccharide. This test is
basically mean to detect monosaccharide in acidic medium. It can also be used to distinguish
between monosaccharide and disaccharide by controlling the time of heating.
Principle:
Barfoed's Test is a chemical test used for detecting the presence of monosaccharides. It is
based on the reduction of copper(II) acetate to copper(I) oxide (Cu2O), which forms a brick-red
precipitate (positive test for reducing monosaccharides). Reducing disaccharides may also react,
but the reaction is much slower. This test differs from the Fehling’s and Benedict’s tests in aspect
that the reduction of cupric ions is carried out in a mildly acidic medium. Monosaccharides can
reduce cupric ions even in acidic conditions. Since acidic medium is unfavorable for reduction, only
the strongly reducing carbohydrates, react very fast and give a positive test within three minutes.
Disaccharides can also give this test positive provided they are boiled for sufficient time, enough to
hydrolyze them in the presence of acidic medium.
Barfoed’s reagent, cupric acetate in acetic acid, is slightly acidic and is balanced so that it can
only be reduced by monosaccharides but not by less powerful reducing sugars. The aldehyde group
of the monosaccharide, which normally forms a cyclic hemiacetal is oxidized to the carboxylate.
Interpretation:
•
If the red precipitates appear at the bottom of test tube
in five minutes. It indicates that the carbohydrate is a
monosaccharide.
•
If the reed precipitates appear after 15 minutes of
heating, it indicates that the carbohydrate is a
disaccharide.
[Phenol-Sulfuric Acid Method]
The PhenolBSulfuric Acid method is an example of a colorimetric method that is widely used to
determine the total concentration of carbohydrates present in foods. A clear aqueous solution of the
carbohydrates to be analyzed is placed in a test-tube, then phenol and sulfuric acid are added. The
solution turns a yellow-orange color as a result of the interaction between the carbohydrates and the
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phenol. The absorbance at 420 nm is proportional to the carbohydrate concentration initially in the
sample. The sulfuric acid causes all non-reducing sugars to be converted to reducing sugars, so
that this method determines the total sugars present. This method is non-stoichemetric and so it is
necessary to prepare a calibration curve using a series of standards of known carbohydrate
concentration.
Hydroxymethylfurfural (HMF)
HMF (5-(Hydroxymethyl)furfural) is an
organic compound derived from dehydration of
certain sugars. The molecule consists of a
furan ring, containing both aldehyde and alcohol
functional groups.
4. Molisch's test
Objective:
It is a qualitative test for detection of carbohydrate in the given solution is positive for all
carbohydrate whether free or bound to other substances such as protein (glycoprotein, myoprotein) or
lipid (glycolipid).
Principle:
Molisch's Test is the reaction of 1-naphthol with carbohydrates in the presence of sulfuric acid.
Polysaccharide and disaccharides are hydrolyzed by conc. sulfuric acid to monosaccharides and are
dehydrated to furfural derivatives, which will condense with the 1-naphthol to give a purple-colored
dye.
Interpretation:
It shows positive test for all carbohydrates. Monosaccharides give a rapid positive test while
disaccharides and polysaccharides react slower. A positive test is indicated by the formation of a
purple product at the interface of the two layers (left: negative result and right: positive result).
Appearance of reddish violet colored ring at the junctions of two liquid indicates the presence of
carbohydrate in the test tube. No such ring will appear in test tube. In the presence of excessive 1-
napthol a green ring may also be seen, this should be ignored.
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5. Anthrone test
Objective:
Anthrone test is a general reaction test for carbohydrates.
Principle:
The total saccharides moiety in a sample can be estimated by the anthrone method, which is
a simple colorimetric method with relative insensitivity to interferences from the other cellular
components. The sample is mixed with sulfuric acid and the anthrone reagent and then boiled until
the reaction is completed. The solution is then allowed to cool and its absorbance is measured at
620 nm. There is a linear relationship between the absorbance and the amount of sugar that was
present in the original sample. This method determines both reducing and non-reducing sugars
because of the presence of the strongly oxidizing sulfuric acid. Like the other methods it is nonstoichemetric
and therefore it is necessary to prepare a calibration curve using a series of standards
of known carbohydrate concentration. The pentose and hexose sugars are converted to furfural and
hydroxymethylfurfural, respectively. When anthrone (an aromatic compound) is added, it reacts with
these digestion products to give colored compound. The amount of total carbohydrates in the
sample is then estimated via reading the absorbance of the resulting solution against a glucose
standard curve.
Interpretation:
Carbohydrates react with anthrone reagent to give green color complex.
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6. Bial test
Objective:
Bial's test is a chemical test for the presence of pentoses.
Principle:
The components include orcinol, hydrochloric acid, and ferric chloride. A pentose, if present,
will be dehydrated to form furfural, which then reacts with the orcinol to generate a colored substance.
Pentoses furfural orcinol Blue-green
Interpretation:
The solution will turn bluish and a precipitate may form. A positive test is indicated by the
formation of a bluish product. All other colors indicate a negative result for pentoses. Note that
hexoses generally react to form green, red, or brown products.
[hexoses](green brown) [pentoses](bluish green)
7. Seliwanoff's test
Objective: Seliwanoff’s test is a chemical test, which identifies the ketoses and aldoses.
Principle:
Monosaccharides are resistant to the action of dilute hot mineral acids. Strong acids remove
the water and dehydrate hexoses to form Furfurals. When furfurals condense with phenols, they form
a colored product. This test is based on the fact that, when heated, ketoses are more rapidly
dehydrated than aldoses.
A positive test is indicated by the formation of a red product.
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Ketoses furfural Red color
Interpretation: Appearance of a cherry red color or pink color with in 30 sec indicates the
presence of ketohexoses.
Procedure
A. Molisch Test
1. Place 5 mL of each solution to be tested in a test tube.
2. Add 3 drops of the Molisch reagent. Swirl to mix.
3. Tilt the test tube at a 45 degree angle and carefully add 40 drops of concentrated sulfuric acid by
slowly dripping it down the side of the test tube. Do NOT mix the solutions. The sulfuric acid should
form a layer beneath the carbohydrate layer.
4. Set the test tubes carefully in the rack and observe for evidence of a reaction over the next 15-20
minutes. Record your results.
5. Dispose of the contents of the test tubes in the waste bottle labeled "E26 Carbohydrate Waste".
B. Seliwano Test
1. Place 1 mL of each solution to be tested in a test tube.
2. Add 4 mL of the Seliwano reagent. Swirl to mix.
3. Place the test tubes in a boiling water bath for 2-3 minutes. Do not overheat.
4. Record your results.
5. Dispose of the contents of the test tubes in the waste bottle labeled "E26 Carbohydrate Waste".
C. Benedict Test
1. Place 1 mL of each solution to be tested in a test tube.
2. Add 5 mL of the Benedicts reagent. Swirl to mix.
3. Place the test tubes in a boiling water bath for 5 minutes. Do not overheat.
4. Record your results.
5. Dispose of the contents of the test tubes in the waste bottle labeled "E26 Carbohydrate Waste".
D. Barfoed Test
1. Place 1 mL of each solution to be tested in a test tube.
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2. Add 5 mL of the Barfoed reagent. Swirl to mix.
3. Place the test tubes in a boiling water bath for 5 minutes. Do not overheat.
4. Record your results.
5. Dispose of the contents of the test tubes in the waste bottle labeled "E26 Carbohydrate Waste".
E. Bial Test
1. Place 2 mL of each solution to be tested in a test tube.
2. Carefully add 3 mL of the Bials reagent. (Bials reagent is dissolved in concentrated HCl. Swirl to
mix.
3. Carefully heat (with agitation) each tube over a bunsen burner until the mixture just begins to boil.
4. Record your results.
5. Dispose of the contents of the test tubes in the waste bottle labeled "E26 Carbohydrate Waste".
G. Hydrolysis of Disaccharides
1. Perform the Benedict and Seliwano test prior to doing this section.
2. Place 10 mL of disaccharide in a test tube.
3. Add 5 drops of concentrated HCl to the test tube.
4. Place the test tubes in boiling water for 10 minutes.
5. Cool the solutions.
6. Neutralize the acid with 10% NaOH solution (15-20 drops), using red litmus paper as an indicator.
7. Using 2 mL portions of the resulting solutions run the Benedict and Seliwano
tests.
8. Record your results.
9. Dispose of the contents of the test tubes in the waste bottle labeled "E26 Carbohydrate Waste".
H. Hydrolysis of a Polysaccharide
1. Perform the Iodine and Benedict test prior to doing this section.
2. In a 250 mL beaker place 2.5 mL of a 1% starch solution, 200 mL of water, and 10 drops of
concentrated hydrochloric acid.
3. Remove a 2-3 mL portion of the sample. Perform the Iodine test on the removed sample. Record
your results.
4. Cover the beaker with a watch glass, and gently boil the starch solution for 15-30 minutes.
Consider time zero to be when the solution rst boils.
5. After the solution starts to boil, remove 2-3 mL of the solution and perform the Iodine test. Record
your results.
6. Perform the iodine test every 5 minutes until a clear results is achieved. Record your results.
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7. If your solution is running dry, add 50 mL of water to the beaker.
8. After the heating is completed, take a 2-3 mL portion and neutralize it with 10% NaOH solution,
using red litmus paper as an indicator.
9. Perform the Benedict test on the sample.
10. Record your results.
11. Dispose of the contents of the test tubes in the waste bottle labeled "E26 Carbohydrate Waste".
I. Fruit Juices
1. Perform the Benedict and Seliwanoff’s test prior to doing this section.
2. Using the fruit juice samples provided run the Benedict and Seliwano tests using 1 mL samples of
each juice.
3. Dispose of the contents of the test tubes in the waste bottle labeled "E26 Carbohydrate Waste".
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Materials
Monosaccharides
Disaccharides
Polysaccharides
Dextrins are a group of low-molecular-weight
carbohydrates produced by the hydrolysis of starch or glycogen.
Dextrins are mixtures of polymers of D-glucose units linked by
1-(1N4) or 1-(1N6) glycosidic bonds.
Cellulose is a polysaccharide consisting of a linear
chain of several hundred to over ten thousand 2(1N4) linked Dglucose
units. Cellulose is an important structural component
of the primary cell wall of green plants. Cellulose is mainly
used to produce paperboard and paper. Cellulose is a straight
chain polymer: and the molecule adopts an extended
conformation, aided by the equatorial conformation of the
glucose residues. The multiple hydroxyl groups on the glucose
from one chain form hydrogen bonds with oxygen atoms on the
same or on a neighbor chain, holding the chains firmly together
side-by-side and forming microfibrils with high tensile strength.
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Laboratory exercise
Using the provided solutions of mono-, di-, and poly-saccharides, perform the tests in
the table below and write down your observations.
1. Molisch's Test
Sugars Color Change Sugars Color Change
Fructose Maltose
Glucose Sucrose
Arabinose Starch
Cellulose
2. Anthrone Test
Sugars Color Change Sugars Color Change
Fructose Maltose
Glucose Sucrose
Arabinose Starch
Cellulose
3. Fehling’s Test
Sugars Color Change Sugars Color Change
Fructose Maltose
Glucose Sucrose
Arabinose Starch
Dextrin Cellulose
4. Barfoed’s Test
Sugars Color Change Sugars Color Change
Fructose Maltose
Glucose Sucrose
Arabinose Starch
Dextrin Cellulose
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5. Benedict’s Test
Sugars Color Change Sugars Color Change
Fructose Maltose
Glucose Sucrose
Arabinose Starch
Dextrin Cellulose
6. Iodine Test
Sugars Color Change Sugars Color Change
Starch Starch
Dextrin Cellulose
Filter paper
7. Bial Test
Sugars Color Change Sugars Color Change
Arabinose Starch
Glucose Cellulose
Filter paper
8. Seliwanoff's Test
Sugars Color Change Sugars Color Change
Glucose Fructose