Mia Srimiati, SGz, MSi
Qualitative Analysis of
Carbohydrates
CARBOHYDRATE
Carbohydrates are important in food as the
main source of energy, to provide important
textural properties, and as dietary fiber that
plays a role in health.
CARBOHYDRATE
Other roles of carbohydrates:
Bulk (large size)
Viscosity (viscosity)
emulsion and foam stability (pectin, gum)
water holding capacity
freezing and thawing stability
browning (including taste and aroma)
any desired texture (from crunchy to smooth)
reduce water activity to inhibit microbial growth
CARBOHYDRATE
Classification of carbohydrates based on chemical structure:
• Monosaccharides and disaccharides (low molecular weight) simple
sugar
Monosaccharides D-glucose, D-Fructose, D- Galactose, D-Manose
Disaccharides Sucrose (glucose + fructose), Maltose
(glucose + glucose), Lactose (glucose + galactose)
• Oligosaccharides (medium molecular weight) the number of
molecules 2- 10 monosaccharides
- Fructo Oligosaccharide (FOS), Galacto Oligosaccharide (GOS)
• Polysaccharides (high molecular weight) complex carbohydrates
- Starch, Glycogen, Inulin, Cellulose, Chitin, Mucopolysaccharide
CARBOHYDRATE
Classification of carbohydrates :
Digestable carbohydrate / available
carbohydrate
Easily converted into monosaccharides, which are
absorbed and provide metabolic energy and a
feeling of fullness.
Examples: glucose, fructose, lactose, dektrin, starch
Carbohydrates that can not be digested (non-
digestable carbohydrate / unavailable carbohydrate)
Cannot be used directly, it needs to be
broken down / fermented with the help of
intestinal microorganisms
all polysaccharides apart from starch which
comprise the majority of dietary fiber
CARBOHYDRATE
CARBOHYDRATE ANALYSIS
The importance of carbohydrate analysis :
Monitor food label claims
Analyze sweeteners, bulking agents, fat
substitutes
Determine the authenticity of a food product
Monitor the fermentation process in the
production of alcoholic beverages
CARBOHYDRATE ANALYSIS
CARBOHYDRATE ANALYSIS
CARBOHYDRATE ANALYSIS
Qualitative and quantitative analyzes are used to
determine the composition of food, drink and
ingredients.
Qualitative analysis ensures that ingredient labels
present accurate composition information.
Qualitative analysis ensures that ingredient labels
present accurate composition information.
CARBOHYDRATE ANALYSIS
IMPORTANT REACTION
REDUCTION :
Carbohydrates with free aldehyde or ketone
groups can reduce metal hydroxides
Fehling's reagent: Alkaline metal is dissolved with
Na-Tartarat
Benedict's reagent: Alkaline metals are dissolved
with Na-Citrate
Barfoed reagent:
Cu-Acetate in dilute acid
Cu can be reduced by monosaccharides
CARBOHYDRATE QUALITATIVE ANALYSIS
Molisch test Colorimetric Method
General Carbohydrate Test
The Molisch test is named after its discoverer Hans
Molisch (botanist from Australia)
This test is based on the reaction of carbohydrate
dehydration by sulfuric acid to form a purple furfural ring.
A positive reaction is indicated by the appearance
of a purple ring on the surface between the acid
layer and the sample layer
The samples tested were mixed with Molisch's reagent,
namely α-naphthol dissolved in ethanol.
After mixing or homogenization, concentrated H2SO4 is
slowly poured through the walls of the test tube so that
it does not mix with the solution or only forms a layer.
CARBOHYDRATE QUALITATIVE ANALYSIS
Molisch Test
Procedure
1. Enter into the test tube 1 ml sample
2. Add 2 drops of Molish reagent and shake.
3. Add 1 ml H2SO4
4. Observe the results
CARBOHYDRATE QUALITATIVE ANALYSIS
Benedict Test
Chemical test to determine the content of reducing
sugars (carbohydrates). Reducing sugars include all
types of monosaccharides and some disaccharides
such as lactose and maltose
In Benedict's test, this reagent will react with
aldehyde groups, except for aldehydes in the
aromatic group, and alpha hydroxy ketones.
CARBOHYDRATE QUALITATIVE ANALYSIS
Benedict Test
Although fructose is not a reducing sugar, because it
has an alpha hydroxy ketone group, fructose will turn
into glucose and mannose in an alkaline environment
and give positive results with benedict's reagent.
CARBOHYDRATE QUALITATIVE ANALYSIS
Benedict Test
Procedure
1. Put 2 drops of the sample to the test tube
2. Add 1 ml Benedict.
3. Heat in a water bath.
4. Observe the results
CARBOHYDRATE QUALITATIVE ANALYSIS
Barfoed Test
test to distinguish monosaccharides and
disaccharides by controlling pH conditions and
heating time
The principle is based on the reduction of Cu2
+ to Cu +
Monosaccharide samples have a faster time to
form a brick red color in the barfoed test
CARBOHYDRATE QUALITATIVE ANALYSIS
Barfoed Test
Procedure
1. enter 5 drops of the sample solution into the test
tube.
2. add 1 ml of Barfoed reagent
3. heat in a water bath, count the time until a brick
red discoloration forms.
4. observe the results.
CARBOHYDRATE QUALITATIVE ANALYSIS
Iodine Test
Starch in an acidic atmosphere when heated
can be hydrolyzed into simpler compounds,
the results of the breakdown of starch when
tested with iodine will give blue, brown,
yellow to colorless
CARBOHYDRATE QUALITATIVE ANALYSIS
Iodine Test
Procedure
1. 1 drop of sample on a Druppel plate.
2. add 1 drop of iodine solution.
3. observe the color that occurs.
CARBOHYDRATE QUALITATIVE ANALYSIS
Fehling Test
The Fehling test aims to determine the presence of an
aldehyde group. The reagents used in this test were Fehling
A (CuSO4) and Fehling B (NaOH and KNa tartaric).
The reaction that occurs in the Fehling test is :
The heating in this reaction aims to break the bonds of the
aldehyde groups in the sample and can react with OH ions
to form carboxylic acids. Cu2O (brick red precipitate) that is
formed is a byproduct of the reaction to form carboxylic
acids.
CARBOHYDRATE QUALITATIVE ANALYSIS
Fehling Test
Procedure
1. In the test tube, add 2 ml of the
test carbohydrate solution
2. add 2 ml Fehling A & Fehling B
keep in boiling water bath for a
few minutes.
3. When the solution in the test tube
comes to a boil, stir it.
4. observe for changes in color
formation or precipitates.
Production of a yellow or
brownish-red precipitate indicates
the presence of reducing sugars in
the given sample.
CARBOHYDRATE QUALITATIVE ANALYSIS
Tollens Test
to distinguish which is an aldehyde or ketone compound.
Tollens reagent is also called ammonia silver a mixture of
AgNO3 and ammonia
The active group in Tollens' reagent is Ag2O, which if reduced
will produce a silver precipitate that sticks to the test tube wall
(silver cemin)
The reaction with Tollens' reagent is able to convert the C-H
bonds in the aldehyde to the C-O bonds
CARBOHYDRATE QUALITATIVE ANALYSIS
Tollens Test
Procedure
1. 20 drops of NaOH solution were added to
20 drops of the 10% w / v solution
2. AgNO3 then NH4OH is added dropwise
until the precipitate is gone
3. 1 drop of liquid sample or 1 spatula of
solid sample dissolved in a small amount
of water or 95% ethanol.
4. The sample was added dropwise to the
Tollen reagent while shaking it, observing
the formation of Ag deposits
5. The test tube is heated in boiling water,
then observed
CARBOHYDRATE QUALITATIVE ANALYSIS
Ozazon Test
Aldose and Ketose react with
Phenyl hydrazine to form
phenylhydrazone.
Excess phenyl ghidrazine will
form oxy-phenyl hydrazine
Oxy-phenyl hydrazine with
phenyl hydrazine formed
ozazone (crystals)
CARBOHYDRATE QUALITATIVE ANALYSIS
Ozazon Test
Procedure
1. Put the CHO solution into a test
tube.
2. Add Phenyl hydrazine-HCL and Na-
Acetate and two drops of acetic
acid. Warm it until it dissolves.
3. Strain and heat over a bath of
boiling water for 30 minutes.
4. Cool look under a microscope
CARBOHYDRATE QUALITATIVE ANALYSIS
Trommer Test
The nature of Cu (OH) 2 is soluble in poly alcohols
The reducing property of poly alcohols is not for glycerol.
Procedure:
1. Put sample into 2 test tubes
2. Each tubes was added 2 ml of glycerol and 2 ml of 2%
glucose.
3. Add 2 drops of 5% CuSO4 and 10% NaOH each. Seen in
blue
4. Preheat, red / yellow glucose tube.
5. The glycerol tube is colorless.
CARBOHYDRATE QUALITATIVE ANALYSIS
Seliwanof Test
Specific for ketoses. 4-hydroxy methyl is red. furfural and
resorcinol form complex compounds
Procedure:
1. Put 1 ml of Barfoed solution into a test tube,
add the sample solution.
2. Heat on a water handler for 3 minutes.
3. Cool, add 1 ml of phosphomolybdate solution.
4. The blue color is glucose.
CARBOHYDRATE QUALITATIVE ANALYSIS
Tauber Test
Specific for pentose
Procedure:
1. Put 2 ml of 2% Benzidine solution into a test tube, add the
sample solution.
2. Heat over a water handler to a volume of 1/2.
3. Cool and the volume is set to normal.
4. The red color is the presence of pentose
CARBOHYDRATE QUALITATIVE ANALYSIS
Bial Test
Specific for pentose to pentose, pentosan, glucoronate.
The reaction consists of orsinol, HCl, FeCl3
Procedure:
1. Enter into a test tube 0.5 ml of Orsinol solution,
2. Add 5 ml of sample solution.
3. Add 3 ml of concentrated HCl.
4. Heat over a closed water handler.
5. Simmer for 10 minutes.
6. The red color is the presence of pentose.
CARBOHYDRATE QUALITATIVE ANALYSIS
Mustat Test
Only galactose and lactose, which are insoluble in the nitric
acid solution of all carbohydrates
Procedure:
1. Enter into a test tube 25 ml of sample solution.
2. Add 5 ml concentrated nitric acid.
3. Heat over a water handler, bring to a boil to a volume of 5
ml.
4. Cool slowly.
5. Observe under a microscope.
6. Lactose and galactose crystals will be visible
CARBOHYDRATE QUALITATIVE ANALYSIS
Fermentation Test
Carbohydrate fermentation produces Alcohol, acids and H20
and C02
Procedure:
1. Grind 2 g of yeast (yeast) bread with 20 ml of carbohydrates
2%.
2. Put it in the fermentation tube.
3. Observe the formation of CO2 gas.
CARBOHYDRATE QUALITATIVE ANALYSIS
Sucrose Hydrolysis Test
Hydrolysis of sucrose will produce glucose and fructose
Procedure:
1. 25 ml of 0.1 M sucrose solution add to 100 ml
erlenmeyer.
2. Add 1 ml of concentrated HCl, heat on a bath for
45 minutes.
3. Cool and neutralize and diluted to a volume of 50
ml.
4. Check the solution with the Benedict, Barfoed and
Seliwanof tests
CARBOHYDRATE QUALITATIVE ANALYSIS
EXAMINED SOLUTIONS
MOLISCH Test
Negative Positive
Not carbohydrate Carbohydrate
Test IODIUM
Blue Colorless Red
STARCH MONO & DISAKARIDA GLICOGEN
CARBOHYDRATE QUALITATIVE ANALYSIS
MONO & DISAKARIDA
BARFOED Test
Positive
Negative Positive 5-7 7-12 Minutes
menit
SUKROSA MONOSAKARIDA DISAKARIDA
BIAL test
Positiv Negative
e
PENTOSE HEXOSE
CARBOHYDRATE QUALITATIVE ANALYSIS
HEXSOSE
SELIWANOFF Test
Negative Positive
GLUCOSE & GALACTOSE FRUCTOSE
Fermentation Test
Positive Negatif
CO2 GLUCOSE GALACTOSE CO2
CARBOHYDRATE QUALITATIVE ANALYSIS
DISCARIDES
Fermentation Test
Positiv Negative
e CO2
CO2
MALTOSE LACTOSE
CARBOHYDRATE QUALITATIVE ANALYSIS
SUCROSE
- BENEDICT (-)
- FERMENTATION (+)
- SELIWANOFF (+)
REFERENCES
Herrero M, Cifuentes A, Ibanez E, del Castillo DM.
2012. Advanced Analysis of Carbohydrate in Foods.
Dalam: Methods of Analysis of food Component
and Additives, 2nd Edition. Editor: Semih Otles.
Boca Raton: CRC Press
Peris-Tortajada M. 2015. Carbohydrates and Starch.
Dalam: Handbook of Food Analysis, 3rd Edition-Vol.
I. Editor: Nollet LML, Toldra F. London: CRC Press
BeMiller JM. 2017. Carbohydrate Analysis. Dalam:
Food Analysis, 5th Edition. Editor: S.
Suzanne Nielsen. New York: Springer
VIDEO LINK
Molisch TEST
https://www.youtube.com/watch?v=9kD9sRAf2TM
https://www.youtube.com/watch?v=7r98ml0Ff
NY
Benedict Test
https://www.youtube.com/watch?v=PAKCgrnKeBA
https://www.youtube.com/watch?v=bVvy2UTfcl4
https://www.youtube.com/watch?v=-OFCo_FItPg
