Carbohydrates reactions
Reactions performed under acidic conditions
Heating of monosaccharides under acidic conditions (concentrated sulphuric, hydrochloric
acids) leads to their dehydration. From pentoses the furfural is generated, and in case of hexoses 5-hydroxymethylfurfural. Pentoses more easily undergo dehydration compering to hexoses and
ketoses undergoes dehydration more easliy than aldoses. Disaccharides react more slowly in
comparison to all monosaccharides. This phenomenon is due to the fact that oligo- and
polysaccharides have to be hydrolysed first and then undergo dehydration.
H
OH HO C
O
O
H
H
C
H
H
C
H
C
C
H
-H2O
C
O
H
C
H
H
C
H
C
C
H
OH OH
O
-2H2O
H
H
C
H
C
C
H
OH OH
CH
OH OH
O
H
C
C
H
CH
Furfural
Pentoza
pentose
HOH2C OH HO C
O
C
HC
O
HOH2C
-H2O
C
O
H
C
H
H
C
H
C
C
H
O
-2H2O
HOH2C
O
C
C
C
H
CH
CH
OH OH
5-Hydroksymetylenofurfural
5-hydroxymethylfurfural
Heksoza
hexose
fig. 1. Heating of saccharides with concentrated acids leads to the loss of one water molecule in the
first phase and then two water molecules. As a result furfural is formed - a heterocyclic aldehyde – or
its derivatives.
Furfural and 5-hydroxymethylfurfural are colourless substances. They form colour
products of the condensation with phenols: -naphthol, resorcin and orcin.
OH
OH
OH
OH CH3
-naphthol
resorcin
OH
orcin
1. Saccharides detection –Molisch’s test
Experiment procedure
Add 2 drops of alcoholic -naphthol solution (α-naphthol dissolved in ethanol) to the
carbohydrate solution. Mix the contents of the test tube and then cautiously add down the
sides of the test tube, using the pipette, approximately l cm3 of concentrated sulphuric (VI)
acid. A positive reaction is indicated by appearance of a purple ring at the interface between
the acid and test layers. Sometimes the green ring is also observed which indicates the
presence of impurities in α-naphthol solution. The negative test result eliminates the presence
of saccharides in the test solution.
2. Reaction of ketose and aldose differentiation through Seliwanoff’s test
Ketoses undergo dehydration when 12% solution of HCl is added and the mixture is
heated in the water bath ( heating time- 30 seconds). Product of this reaction condensates with
resorcin to produce a deep cherry red color. Aldoses may react slightly to produce a faint pink
color, however longer heating is required.
Experiment procedure
Add 2 drops of alcoholic resorcin solution and l cm3 of 12% HCl to l cm3 of fructose solution.
Put the mixture into water bath for approx. 30 seconds. Simultaneously, perform the same
procedure for the glucose solution.
3. Reaction of pentose and hexose differentiation- Bial’s test
When pentoses are heated with conc. HCl furfural is formed, further it condences with
orcin in the presence of ferric ions to give a blue-green colour. This test is used for qualitative
and quantitative determination of ribose in nucleic acids and pentoses in urine or in plasma
samples.
Experiment procedure
Add l cm3 of Bial’s reagent to l cm3 of pentose solution. Place the test tube in the
water bath for approx. 1 minute.
Reactions in basic conditions
The ring forms of saccharides are predominant in neutral or weak-acid medium. In the
presence of basic solutions the hemiacetal bond, that closes the ring, is less stable and this
phenomenon is the reason of increasing number of chain structures. Carbohydrates easily
undergo enolisation in basic medium (reactions of keto-enol tautomerism where enol forms
are generated). As a result of further isomerisation glucose can be transformed into mannose
and fructose ( fig. 3). All three carbohydrate forms are in equilibrium and are epimers. When
more concentrated basic solutions are used (0,5 mol/dm3) the enol bond can translocate to
different locations in carbon chain. Heating carbohydrates with concentrated basic solutions
leads to bond cleavage. It generates two-, three-carbons forms of strong reductive properties
(glycolic aldehyde, triose, tetrose, formaldehyde, glycolic acid) that condense with each other
producing resinous structures which give dark brown colour. The monosaccharides are
particularly sensitive to basic solution treatment because under these conditions the
enolisation process can be easily started at free carbonyl group. The carbonyls groups of
oligosaccharides and polysaccharides are blocked by glycosidic bond and for this reason these
compounds are resistant to basic treatment.
O
C
O
C
H
C
OH
C
H
OH
HO
C
H
H
C
OH
H
C
OH
C
OHH+
H
OH
HO
C
H
H
C
OH
H
C
OH
CH2OH
CH2OH
D-Glukoza
D-glucose
1,2-Endiol
OH-
HO
C
H
H
HO
C
H
H
C
OH
H
C
OH
H+
CH2OH
D-Mannoza
D-mannose
CH2OH
C
O
HO
C
H
H
C
OH
H
C
OH
OHH+
CH2OH
D-Fruktoza
D-fructose
fig.3 Epimerisation of glucose, mannose and fructose
1. Moore test – treatment of carbohydrates with concentrated basic solutions
Experiment procedure
Add l cm3 of 10% NaOH to l cm3 of glucose solution then heat the mixture (in the water
bath) until the dark brown colour is visible.
Reducing action of sugars in basic solution
Aldoses, as well as the aldehydes, due to the presence of carbonyl group can be oxidise
to carboxylic acids even when a weak oxidation agent is used. Ketoses can undergo this
reaction only in basic conditions ( Fehling’s test, Benedict’s test). The positive test result for
ketoses is possible due to the keto-enol tautomerism of ketoses that leads to enols formation
and then from enols to aldoses ( the epimerization). If a disaccharide or oligosaccharide
contains free anomeric carbon ( as e.g. maltose, lactose) it can also show reducing properties
under these conditions. Saccharose is the example of carbohydrate that does not contain free
anomeric carbon, so it cannot be oxidise and is a non-reducing sugar.
Treatment of monosaccharides with weak oxidative agents (Cu2+, Ag+, Bi3+) leads to the
formation of aldonic acids. Strong oxidative agents (HNO3 at elevated temperatures) lead to
the formation of aldaric acids. Under basic conditions (particulary concentrated one)
monosaccharides can be cleaved into shorter forms that may also demonstrate reducing
properties. It can be observed that: the more acidic medium the weaker reducing properties of
saccharides. This dependance is used to differentiate reducing monosaccharides (aldoses)
from disaccharides. In acidic medium aldoses (after short time heating) can undergo oxidation
while disaccharides need longer heating to be hydrolysed, and then their hydrolysis products
can show reducing action. Ketoses, as it was mentioned before, undergo oxidation only in
basic medium.
1. Fehling’s test
In the presence of a sugar, the copper(II) ions are reduced to copper(I) ions. Red
copper(I) oxide precipitates out of the reaction mixture. Fehling's reagent is always prepared
fresh in the laboratory. It 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 and colourless solution of aqueous potassium sodium tartrate and a strong base
(commonly sodium hydroxide). The tartrate ions by complexing copper prevent the formation
of the blue precipitate of Cu(OH)2 or black precipitate of CuO.
OH
Cu
2+
Complex
Kompleks Cu2+
+
C
C
H
Glukoza
glucose
OH
O
Cu2O
+
C
O
C
OH
Kwas glukonowy
Gluconic acid
Fig.4 Oxidation of aldoses to the aldonic acids in basic medium
Experiment procedure
Add l cm3 of Fehling’s solution to l cm3 of glucose solution. Heat the mixture in the water
bath. The presence of red/orange precipitate of Cu2O is the positive test result.
2. Benedict’s test
The main reaction of Benedict’s test follows the same scheme as in Fehling’s test. Benedict's
reagent is a mild, alkaline oxidizing agent consisting of a solution of sodium citrate and
sodium carbonate mixed with a solution of copper sulfate. To obtain less alkaline reaction
medium and prevent fragmentation of sugars chain sodium carbonate is used as a component
of Benedict’s reagent.
Although Benedict’s test is based on the same principle as Fehling's test, this first one is
more sensitive. Benedict’s test can be used to test for the presence of reducing compounds
(including sugars) in urine samples.
Experiment procedure
Add a few drops of glucose solution to 3 cm3 of Benedict’s reagent. Agitate to mix the
contents and place the test tube in a boiling-water bath for 5 minutes. A colour change from
blue to orange, red, or dark brown indicates that reaction has taken place and reducing sugares
were present in the test solution.
3. Barfoed’s test
Barfoed's reagent serves to distinguish between reducing monosaccharides and
disaccharides. The test is similar to the reaction of Fehling's or Beneditc’s solution, but it is a
weaker oxidizing agent than these two reagents. Barfoed's reagent is a slightly acidic solution
(diluted lactic acid solution) containing Cu2+ ions. Under these conditions the reaction is
much slower and reaction rate is different for monosachcarides and disaccharides since
hemiacetal group of monosaccharides is more active than hemiacetal group in disaccharides.
Barfoed's reagent will oxidise monosaccharides and produce the red-coloured
precipitate of Cu2O. Disaccharides may also react, but the reaction is much slower.
Experiment procedure
Take 2 test tubes and add l cm3of Barfoed’s reagent to each of them. Then add to one of the
test tubes 0,5 cm3 of glucose and 0,5 cm3 of lactose to the second one. Place all the tubes in a
boiling-water bath at the same time for approx. 3 minutes. Compare the results after 3 minutes
of heating and then place the tubes in water bath again for 15 minutes. Note the appearance of
any red precipitate.
4. Detection of polysaccharides– Iodine test (Lugol's iodine)
As a result of incorporating of iodine ions into some polysaccharides structures the complexes
of a characteristic colour are formed. The colour depends on the chain length. Amylose gives
blue colour, almylodextrin – purple, glycogen – red. Lugol's iodine yields a blue-black colour
in the presence of starch. Heating causes the shift of absorption maximum to the ultraviolet
and the colour turns clear. After cooling the colour turns blue-black again.
Experiment procedure
Add 2-3 drops of Lugol's iodine solution to l cm3 of starch solution. A positive test
for starch gives dark blue colour that turns clear when the mixture is heated.