Analysis of Carbohydrate

Kimia Bahan Makanan (CHM342)

Nelson Gaspersz
 Source of
Carbohydrate
 Processed
Food of
Carbohydrate
Source
I. Qualitative Analysis
 Molisch's test (named after Austrian botanist Hans
Molisch) is a sensitive chemical test for the presence
of carbohydrates, based on the dehydration of the
carbohydrate by sulfuric acid or hydrochloric acid to
produce an aldehyde, which condenses with two
molecules of phenol (usually α-naphthol, though other
phenols (e.g. resorcinol, thymol) also give colored
products), resulting in a red- or purple-colored
compound.
All carbohydrates – monosaccharides, disaccharides,
and polysaccharides – should give a positive reaction, and nucleic
acids and glycoproteins also give a positive reaction, as all these
compounds are eventually hydrolyzed to monosaccharides by
strong mineral acids.
Pentoses are then dehydrated to furfural, while hexoses are
dehydrated to 5-hydroxymethylfurfural.
Either of these aldehydes, if present, will condense with two
molecules of naphthol to form a purple-colored product, as
illustrated below by the example of glucose
 Seliwanoff ’s test is a chemical test
which distinguishes
between aldose and ketose sugars.
Ketoses are distinguished from aldoses
via their ketone/aldehyde functionality.
 If the sugar contains a ketone group, it
is a ketose. If a sugar contains an
aldehyde group, it is an aldose. When
added to a solution containing ketoses,
a red color is formed rapidly
indicating a positive test. When added
to a solution containing aldoses, a
slower forming light pink is
observed instead.
The reagents consist of resorcinol and concentrated hydrochloric acid (or
H2SO4 & CH3COOH):
 The acid hydrolysis of polysaccharide and oligosaccharide ketoses yields
simpler sugars followed by furfural.
 The dehydrated ketose then reacts with two equivalents of resorcinol in a
series of condensation reactions to produce a molecule with a deep
cherry red color.
 Aldoses may react slightly to produce a faint pink color.
Fructose and sucrose are two common sugars which give a positive test.
Sucrose gives a positive test as it is a disaccharide consisting of fructose and
glucose.
 furfural
fructose
resorcinol
red-colored dye
 Anthrone’s test is a tricyclic sweet-smelling ketone. It is utilized
for a well-known cellulose measure and in the colorimetric
determination of carbohydrates. The anthrones are utilized as a
part of drug store as diuretic. They improve the movement of the
colon and are in charge of less water reabsorption.
 Starches are dried out with concentrated H2SO4 to frame
“Furfural”, which gathers with anthrone to shape a blue-green
shading complex which can be measured by utilizing
colorimetrically at 620 nm.
 Anthrone reacts with dextrins, monosaccharide, disaccharides,
polysaccharides, starch, gums and glycosides. If this happens, the
yield of shading is where is to frame sugar to starch.
 anthrone

furfural
blue green-colored dye
 Benedict's reagent (often told as Benedict's Qualitative
Solution or Benedict's Solution) is a chemical reagent named
after an American chemist, Stanley Rossiter Benedict.
 It is a complex mixture of sodium carbonate, sodium
citrate and copper(II) sulfate pentahydrate. Benedict's reagent is
a chemical reagent commonly used to detect the presence
of reducing sugars, however other reducing substances also give
a positive reaction. This includes all monosaccharides and
many disaccharides, including lactose and maltose. Such tests
that use this reagent are called the Benedict's tests.
 Generally, Benedict's test detects the presence
of aldehydes, alpha-hydroxy-ketones, also by
hemiacetal, including those that occur in certain
ketoses. Thus, although the ketose fructose is not
strictly a reducing sugar, it is an alpha-hydroxy-ketone,
and gives a positive test because it is converted to the
aldoses glucose and mannose by the base in the reagent.
 A positive test with Benedict's reagent is shown by a
colour change from clear blue to a brick-red
precipitate.
The principle of Benedict's test is that
when reducing sugars are heated in the
presence of an alkali they get converted
to powerful reducing species known
as enediols.
RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+
Brick-red
precipitate
 The color of the obtained precipitate
gives an idea about the quantity of sugar
present in the solution, hence the test is
semi-quantitative. A greenish precipitate
indicates about 1 g% concentration;
yellow precipitate indicates 1.5 g%
concentration; orange indicates 2.5 g%
and red indicates 3,5 g% or higher
concentration.
 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.
 (Disaccharides may also react, but the reaction is much
slower.) The aldehyde group of the monosaccharide which
normally forms a cyclic hemiacetal is oxidized to
the carboxylate.
RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+
 In this test the presence of aldehydes but not ketones is
detected by reduction of the deep blue solution of copper(II)
to a red precipitate of insoluble copper oxide (Cu2O). The test
is commonly used for reducing sugars but is known to be NOT
specific for aldehydes. For example, fructose gives a positive
test with Fehling's solution as does acetoin.
Two solutions are required:
Fehling's "A" uses 7 g CuSO4.5H2O
dissolved in distilled water containing 2
drops of dilute sulfuric acid.
Fehling's "B" uses 35 g of potassium
tartrate and 12 g of NaOH in 100 ml of
distilled water.
 The famous German chemist Emil Fischer developed and used the
reaction to identify sugars whose stereochemistry differed by only
one chiral carbon. Glucosazone and fructosazone are identical.
Osazones formation test involves the reaction of a reducing
sugar (free carbonyl group) with excess of phenylhydrazine when
kept at boiling temperature.
 All reducing sugars form osazones. Therefore, sucrose, for example,
does not form osazone crystals because it is a non reducing sugar as
it has no free carbonyl group. The reaction involves formation of a
pair of phenylhydrazone functionalities, concomitant with
the oxidation of the hydroxymethylgroup in alpha carbon (carbon
atom adjacent to the carbonyl center).
 The reaction can be used to identify monosaccharides. It involves
two reactions. Firstly glucose with phenylhydrazine gives
glucosephenylhydrazone by elimination of a water molecule from
the functional group. The next step involves reaction of
one equivalent of glucosephenylhydrazone with two equivalents
of phenylhydrazine (excess).
 First phenylhydrazine is involved in oxidizing the alpha carbon to
a carbonyl group, and the second phenylhydrazine involves in
removal of one water molecule with the new-formed carbonyl
group of that oxidized carbon and forming the similar carbon
nitrogen bond.The alpha carbon is attacked here because its more
reactive than the others.
 phenylhydrazine

D-glucose glucose osazone

Osazones are highly coloured and crystalline compounds and can be easily
detected. Each sugar has a characteristic crystal form of osazones.
 Maltose forms petal-shaped/sun flower-shaped crystals
 Lactose forms powder puff-shaped crystals
 Galactose forms rhombic-plate shaped crystals
 Glucose, fructose and mannose form broomstick or needle-shaped
crystals.
 Tollens' reagent is a chemical reagent used to determine
the presence of an aldehyde, aromatic aldehyde and alpha-
hydroxy ketone functional groups.
 The reagent consists of a solution of silver nitrate and
ammonia. It was named after its discoverer, the German
chemist Bernhard Tollens.
 A positive test with Tollens' reagent is indicated by the
precipitation of elemental silver, often producing a
characteristic "silver mirror" on the inner surface of the
reaction vessel.
 This reagent is not commercially available due to its short shelf life, so it
must be freshly prepared in the laboratory. One common preparation
involves two steps. First a few drops of dilute sodium hydroxide are added
to some aqueous silver nitrate. The OH−ions convert the silver aquo
complex form into silver oxide, Ag2O, which precipitate from the solution
as a brown solid:
2 AgNO3 + 2 NaOH → Ag2O (s) + 2 NaNO3 + H2O
 In the next step, sufficient aqueous ammonia is added to dissolve the
brown silver(I) oxide. The resulting solution contains the
[Ag(NH3)2]+ complexes in the mixture, which is the main component of
Tollens' reagent. Sodium hydroxide is reformed:
Ag2O (s) + 4 NH3 + 2 NaNO3 + H2O → 2 [Ag(NH3)2]NO3 + 2 NaOH
diamminesilver(I) complex
 The iodine test is used to test for the
presence of starch. When treated with
KI solution-iodine dissolved in an
aqueous solution of potassium iodide-
the triiodide anion (I3−) complexes
with starch, producing an intense
blue/purple colour. To put it simply,
when the iodine solution comes into
contact with starch, it turns dark
blue/purple. Otherwise, it will remain
brown in color.
 Left to right : Iodine solution, starch
solution, starch solution with iodine

 Amylose, a linier chain component of starch, gives a deep blue color.

 Amylopectin, a branched chain component of starch, gives a purple color.
 Glycogen, gives a reddish brown color.
 Dextrins, Amylo, Eryhthro and Achrodextrins, form as intermediates
during hydrolysis of starch gives violet, red, and no color with iodine
II. Quantitative Analysis
Determination of carbohydrates from
polysaccharides and oligosaccharides groups,
need pretreatment, i.e. hydrolysis to obtain
monosaccharides.
Hidrolysis
Oligo/polysaccharides  monosaccharides
(Starch) acid/enzyme (glucose)

Monosaccharides Determination:
- Chemical Method
- Physic/Optic Method
- Enzymatic Method
- Chromatography Method
 Cupri oxide is reduced by reducing sugar

Reagent :
• Luff Reagent (mixed CuSO4, Na2CO3, and
citric acid)
• Soxhlet Reagent (mixed CuSO4with
K – Na –tartrate). K-Na-tartrate prevent
CuSO4 precipitated in reagents
 Cupri oxide : - oxidator
- reduced by reducing sugar
to formed cupro oxide
(Brick-red precipitate)
Detemination of cupro oxide formed:
 Weigh after dried
 Dissolve again, then titrated
 Determined of cupri oxide difference before
and after reacting with reducing sugar
Determination of reducing sugar in solution:
 Luff Schoorl Method
 Munson Walker Method
 Lane-Eynon Method
For Lufff Schoorl method CuO was determined
before and after reacting with reducing sugar
= (mL blanko titration – mL sample titration)

Reaction :
R – COH + CuO  Cu2O + R – COOH
H2SO4 + CuO  CuSO4 + H2O
CuSO4 + 2KI  CuI2 + K2SO4
2CuI2  Cu2I2 + I2
I2 + Na2S2O3  Na2S4O6 + NaI
I2 + starch : blue
Reduction of ferricyanide  ferrocyianide by reducing sugar.
2K3Fe(CN)6 + 2KI  2K4Fe(CN)6 + I2
2K4Fe(CN)6 + 3 ZnSO4  K2Zn2[Fe(CN)6]2 + 3 K2SO4
Reducing can determined :
 Based on  I2
 Based on  Na2S2O3 for titration

Indicator : starch (blue colour disappear)

K4Fe(CN)6 formed is calculated from difference between
K3Fe(CN)6 before and after reduction reaction.

Standardization experiments are required

Excess I2 is titrated with Na2S2O3
Specific for aldose, but ketose slightly oxidized
Must be removed substances that can react with
Iodine: ethanol, aceton, mannitol, glycerin, Na
lactate, Na formate and Urea
Sample Titration :
R–COH + I2 + 3NaOH  R–COONa + 3NaI + 2H2O
I2(sisa) + 2Na2S2O3  Na2S4O6 + 2NaI
I2 + starch  I2-starch (blue)

Blanko Titration :
I2(total) + 2Na2S2O3  Na2S4O6 + 2NaI
 Somogyi-Nelson method is based on the reduction
of Cu2+ ions to Cu+ ions in the presence of reducing
sugars. Cu+ ions further reduced the
arsenomolibdate complex.
 Reduction of arsenomolibdate complex produced a
stable blue-colored dye and can be measured
spectrophotometrically at 500 nm.
 The arsenomolibdate complex is prepared by
reacting ammonium molybdate and sodium arsenate
in sulfuric acid
O O
RCH+ 2
Cu
SO4 RCON
a+C
uO+
2 3
HO
2
 The determination of total reducing sugar by using the DNS
method (3.5-dinitrosalicyl Acid) (Miller, 1959). This method
can be used to determined the sugar content of reducing
glucose, fructose, maltose.
 The DNS will be reduced by sugar to 3-amino-5-
nitrosalicyllic acid, which gives red brick or brown red
colour with maximum absorption wavelength of 540 nm.
 To determined the total amount of reducing sugar, a series
of standard solutions such as glucose or maltose are
required.
 In the Morgan-Elson method, the amino sugars or
N-acetyl sugars are heated in alkaline solution to
formed chromogen, which is produced red/purple
compound when reacted with
N,N-dimethyl-p-aminobenzaldehyde in acid solution.
 The sugars contained in the sample were
determined by comparing their absorbance with
absorbance of the standard sugar solution (D-
glucosamine, D-galactosamine, or N-acetyl-D-
glucosamine) through the calibration curve using a
spectrophotometer at 530 nm (amino sugar) and 544
or 585 nm (N-acetyl sugar).
 Especially for sugar determination in mixture
 because enzyme is specific
Example: glucose and fructose determination

 Principle :
glucose and fructose phosphorylated form
glucose–6-phosphate (G6P) and fructose-6-
phosphate (F6P) with hexokinase enzyme aid
and Adenosine–5-triphosphate (ATP)
Glucose + ATP  G-6-P + ADP
Fructose + ATP  F-6-P + ADP
G-6-P-DH
G-6-P + NADP  gluconate-6-P + NADPH + H+

G-6-P oxidazed by NADP formed gluconate-6-phosphate

by aid of glucose-6-phosphate dehidrogenase (G-6-P-DH).
NADPH which formed equivalent with glucose that react 
measured by spectrophotometer ( = 334, 340, 365 nm).
F-6-P need to converted into G-6-P by aid of
phosphoglucose isomerase (PGI).
PGI
F-6-P  G-6-P
( = 334, 340, 365 nm)
G-6P-DH
G-6-P + NADP  gluconate-6-P + NADPH + H+
Principle :
Lactose can hydrolized formed glucose and -galactose by
-galactosidase and water. Then, -galactose oxidazed by NAD
formed galatonate acid and NADH with aid of galatose
dehydrogenase (GAL-DH). NADH which formed equivalent with
lactose that react  measured by spectrophotometer ( = 334,
340, 365 nm).

-galactosidase
Lactose + H2O  Glucose + -galactose + H2O
GAL- DH
-galactose + NAD  galatonate acid + NADH + H+

(= 334, 340, 365 nm)
Paper Chromatography/Thin Layer Chromatography:
measured Rf value for each carbohydrate component
𝐦𝐢𝐠𝐫𝐚𝐭𝐢𝐨𝐧 𝐝𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐨𝐟 𝐬𝐮𝐛𝐬𝐭𝐚𝐧𝐜𝐞
𝐑𝐟 =
𝐦𝐢𝐠𝐫𝐚𝐭𝐢𝐨𝐧 𝐝𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐨𝐟 𝐬𝐨𝐥𝐯𝐞𝐧𝐭 𝐟𝐫𝐨𝐧𝐭

Rf value for each sugar types for same treatment

affected by:
o Various solvent
o Chamber size
o Temperature
o Type of stationary phase
o The properties of the analyzed substance
 Paper chromatography is an analytical method used to separate
colored chemicals or substances. It is primarily used as a teaching
tool, having been replaced by other chromatography methods, such
as thin-layer chromatography.
 The paper chromatography variant, two-dimensional
chromatography involves using two solvents and rotating the paper
90º in between. This is useful for separating complex mixtures of
compounds having similar polarity, for example, amino acids.
 The setup has three components. The mobile phase is a solution that
travels up the stationary phase, due to capillary action. The mobile
phase is generally an alcohol solvent mixture, while the stationary
phase is a strip of chromatography paper, also called a chromatogram.
 The chromatographic method is called adsorption chromatography if
the stationary phase is solid.
 Stationary phase: paper
composed of pure cellulose
(e.g. whatman paper no.1
medium velocity).
 Drops sample (as small as
possible), drops 3-4 x (big
droplets  tailings/
separation are not perfect)
 Insert the paper into a
chamber containing the
solvent  the solvent
migrated on the paper until
the mark (Solvent front)
 Identification :

• Physic : irradiate the paper with UV light at

 = 254 – 370 nm
• Chemical : spray with chemical solution e.g.
- reducing sugar : Anilinphthalate, AgNO3
- non reducing sugar: Naphthoresorcinol in
Phosphoric acid
- Chloroglusinol and HCl can be used for:
`  Aldose pentose (violet)
 Ketose pentose (dark green)
 Ketoheksose (brown yellow)
 Methyl pentose (green)
- Iodine Steam
 Spray on the paper in the acid room
 After dry, the colored stains will be appear
 Calculate Rf sample, compare with Rf
standard

Solvent:
 Solvent: pure substance or mixture
 For simple sugar determination:
Mixed butanol : acetic : water or
acetic acid: pyridine : water (4:1:5)
Determination of refractive index with refractometer
 each type of sugar has a specific refractive

Benefit:
• large interval scale refractive index 1.30-1.75
• very few samples (few drops)
• precision :  0,0002

 denoted by 20
n D
= measured at t = 20ºC with sodium ray as a source of
monochromatic rays.
Determination of carbohydrates with polarimeter

Principle:
Carbohydrates are active optical (capable to rotated the
polarized light field), because it has C asymmetric.

Benefit:
 The sample is not damaged
 Can be done quickly

In order for the results accurated, hence:

 The solution should be clear and colorless
 The solution does not contain an active optical
others material
 The optimum sample concentration: not too
concentrated or aqueous
 Determination of carbohydrates with polarimeter

Biot Law : the rotational capacity of each individual

sugar is proportional to the
concentration of the solution and the
length of the liquid in the tube
100
  D
t 
lC
[] : specific rotation
t : temperature measurement (ºC)
D : Na ray (589 nm)
 : rotate angle observed
C : concentration (g sample/100 mL solvent)
I : tube length (dm)
 The effect of concentration on () is
very small  ignored

 Temperature effect  need correction:

 
D
t     1  0,000184t  20 
D
t
Determination of Crude Fiber

Crude Fiber :
Compounds that can not be digested in human or
animal digestive organs

In the analysis:
calculated the amount of substance which is not
soluble in acid/alkaline under certain conditions.
Steps for Crude Fiber Determination
1. Defatting : remove fat in the sample with fat
solvent
2. Digestion :
- dissolved with acid
- dissolved with alkaline

 in a closed state at a controlled temperature

(boiling)
 immediate filtering to prevent further damage
 Protein complicates filtration  needs pre-
digestion with proteolytic enzymes
 Residue = crude fiber containing 97% cellulose
and lignin + unidentified compounds
 25 mL milk + reagent (hydrolizing)  filtrate
 5 mL filtrate + reagent  titrated with Na2S2O3

100
A = (Tb – Ts) x N x 0,171 x 
5
A = Lactose content (g/100 mL)
Tb = mL blanko titration
Ts = mL sample titration

• Milk with protein content = 3,2%, fat = 3,5% from

100 mL milk  48,4 mL filtrate
48,4 100
lactose/100 mL milk = A x  x 
100 25
 Directly with Polarimeter/refractometer
 Chemical : hydrolisis (detemined of total reducing
sugar)
C6H12O11 + H2O  C6H12O6 + C6H12O6
Sucrose fructose glucose
(342) (180) (180)

 Sucrose = 0,95 x  reducing sugar

MW Sucrose 342
CF =  = 
2. MW reducing sugar 360
Starch hydrolised by acid/enzyme 
reducing sugar  calculated

m[C6H10O25] + mH2O  mC6H12O6

pati glukosa
MW = m.162 MW = m.180

 Starch = 0,9 x  reducing sugar

MW starch
CF = 
MW reducing sugar
m x 162
=  = 0,9
m x 180
Monosaccharide
Reductive group

Alpha D-glucose
Beta- D-fructose

Opened Glucose Pyran Ring Furan Ring

 Disaccharide
Reductive group

1-4 linkage
 Disaccharide
Glucose+ Fructose= Sucrose
Glucose+ Galactose=Lactose

Reductive group
close each other
(non-reductive)
 Starch/Amylum Reductive group

amylopectin

amylose

Glykogen in
animal body
glucose polymer with  (alpha-) bond