T.

Kolusheva,
A. Marinova
Journal of the University of Chemical
Technology
and Metallurgy, 46, 1, 2011, 75-80

FAST COMPLEXOMETRIC METHOD

FOR ANALYSIS OF REDUCING SUGARS OBTAINED
DURING STARCH HYDROLYSIS
T. Kolusheva, A. Marinova

University of Chemical Technology and Metallurgy

8 Kl. Ohridski, 1756 Sofia, Bulgaria
E-mail: manahova@abv.bg

Received 22 October 2010

Accepted 20 January 2011

ABSTRACT
This paper establishes a complexometric method for serial fast analysis to determine the quantity of the reducing
sugars obtained during starch hydrolysis. The method is based on the interaction between the free carbonyl group of sugar
and the Fehling I and II solutions; the creation of an amount of Cu2O equivalent to the reducing sugar and the
complexometric titration of the surplus of Cu2+ by ethylendiamine tetraacetic acid in acetic acid media (pH 5-5,1) against
indicator pyridilazoresorcin. The possibility of using the empirical tables of Bertrand for measuring the quantity of the
reducing sugars determined by the developed method from the quantity of Cu in the sediment, obtained from Cu20, is
proved .The reproducibility and accuracy of the method are very good. The relative standard deviation, Sr is <1%, the
relative error, x is <1% for concentrations from 10 g l -1 to 200 g l -1 glucose. The absence of systematic errors in the
proposed procedure is proved.
Keywords: starch hydrolysis, reducing sugars, complexometric titration.

INTRODUCTION
In practice, it is very often required to determine
the quantity of the reducing sugars that exists in products of plant, animal and industrial origin, for different
purposes - for instance, when determining the utilization of sugars in fermentation processes, ensilaging of
fodders, determining the sugars in blood and urine, etc.
for a diagnostic purpose. Such a determination is necessary in the case of starch hydrolysis to low molecular
mass reducing sugars in the sugar, brewing, spirits, textile, and other industries, as well as for studying the
optimal conditions and the kinetics of the starch hydrolysis.
At present the sedimentary and titrimetric methods of Bertrand [1]; the method of Hagedorf-Jenssen
[1]; the iodometric method of Luff-Shoorl [1, 2] and
the method of Smgyi-Nelson [3, 4] are applied for the

analysis of reducing sugars (which contain a free carbonyl group).

The first two methods coincide to a great extent,
as they include the following basic procedures: extraction of the sugars from the given material; clarification
of the solutions in a proper way; filtering of the obtained sediment, hydrolysis of the soluble poly- and
oligosaccharides with HCl under heating and filtrating
of the obtained solution, containing monosaccharides.
Equal volumes of solutions of Fehling I and II are added
to a certain volume of the obtained filtrate. The red
sediment of the obtained Cu2O is filtrated. The sediment is rinsed many times with hot water and finally
in acetone. Under the sedimentary method [1] the filter
dries at 105oC for 30 min, after which it is weighed.
The quantity of the reducing sugars from the obtained
mass of Cu2O is estimated by the Bertrands tables. By
the titrimetric method the sediment of Cu2O is dissolved

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Journal of the University of Chemical Technology and Metallurgy, 46, 1, 2011

in Fl2(SO4)3. (NH4)2SO4.2H2O and the obtained filtrate

is titrated with KMnO4.
Although both of the cited methods are precise,
they require a lot of time and effort. The preparation of
the sample takes about 16 17 hours. Thus, these methods are not suitable for serial analyses.
The method of Hagedorf-Jenssen, which is a
modification of the method of I.Popov [1], is applied
primarily for the analysis of sugars in corn flour, fruit
and vegetables. The method utilizes the property of
K3[Fe(CN)6] to interact with the reducing sugars, thus
being reduced to K 4 [Fe(CN) 6 ]. The produced
K4[Fe(CN)6] sedimentates with ZnSO4 while the quantity of the unreduced K 3[Fe(CN) 6] is determined
iodometrically in acidic media. The quantity of the
reducing sugars (expressed in the form of maltose),
corresponding to the quantity of the reducing
K3[Fe(CN)6], is determined by a special empiric table.
This method, however, also requires long preliminary
preparation of the sample for analysis, as well as elimination of the protein substances in the sample, which
takes additional time. For these reasons this method
is also not effective for serial analyses. The next two
methods the iodometric method of Luff-Shoorl and
the method of Smgyi-Nelson have the same shortcomings. Both methods are applicable primarily for
determination of sugars in clinical chemistry. The
present paper establishes a method for the analysis of
the reducing sugars produced by enzyme or acidic starch
hydrolysis. The method is based on the interaction of
the reducing sugars with solutions of Fehling I and II,
the creation of an amount of Cu2O equivalent to the
quantity of sugars, the titrating of the surplus Cu2+ with
a standard solution of ethylendiamino tetraacetic acid
(EDTA) in acetic acid media under pH 5-5,1 and indicator of pyridilazoresorcin (PAR). The quantity of
the reducing sugars reacting with Cu2+ in the solutions
of Fehling is estimated by the table introduced by
Bertrand [1]. The proposed method is fast and suitable
for serial analyses.

Fluca AG; Phosphate buffer containing 1/15 mol l-1

solutions of KH2PO4 and Na2HPO4; Fehling I solution
prepared by dissolving 34.639 g CuSO4.5H2O, p.a. in
distilled water and marking up to a volume of 500 ml;
Fehling II solution prepared by dissolving 173 g
C4H4O6KNa.4H2O (potassium-sodium tartarate) and
51.6 g NaOH in distilled water and made up to a volume of 500 ml; Acetate buffer solution, pH 5-5.1, prepared by carefully adding 400 ml solution containing
80g NaOH to 600 ml CH3COOH, containing 140 ml
CH3COOH, p.a., 99 % Pyridilazoresorcin (PAR):KNO3
(1:100) prepared by mixing of 0.25 g PAR with 25 g
KNO3 ; Standard solution of ethylendiaminetetraacetic
acid (EDTA), C Na2 H 2Y = 0.05 mol l-1. This solution must
be titrated before using it against 0.05 mol l-1 Pb (NO3)2
solution in the presence of xylenol orange.

EXPERIMENTAL

EP
(CCu .VCu CEDTA .VEDTA
)ACu .103.100
mCu=
Vs

The following reagents are used

A purum-grade starch, produced by Fluca is
used as a substrate; D-glucose, p.a. grade, produced by

76

Analytical procedure
A sample of 1.00 ml hydrolysate is taken after
various intervals of time and is placed in a 100ml measurement flask. Immediately after sampling, 0.5 ml 2
mol l-1 HCl is added in order to stop further hydrolysis in the enzymatic case. Under acidic hydrolysis the
addition of HCl is not necessary. 25 ml distilled water, 25.00 ml Fehling I and 25.00 ml Fehling II solutions are added to the sample. The resulting solution
is homogenized. The flask is placed in a boiling water
bath for 5 minutes. Then the solution in the flask is
cooled to room temperature and filled up to the mark
with distilled water. The resulting sediment of Cu2O is
filtered through a paper filter suitable for the filtering
of fine crystalline residues.
A sample of filtrate with a volume of 25.00 ml is
transferred to a 300 ml Erlenmeyer flask. 8 ml acetate
buffer and 0.10 0.15g PAR are added. The surplus Cu2+
is titrated with a standard solution of EDTA until the color
of the solution changes from red to yellow-green.
The quantity of Cu, g, (contained in Cu2O)
equivalent to the oxidized sugar is calculated by the
formula

where: Ccu is the concentration of Cu2+; mol l-1 of the

titrated solution with volume 25.00 ml; VCu is the vol-

T. Kolusheva, A. Marinova

ume of the Cu2+ solution, 25.00 ml; CEDTA is the concentration of the EDTA solution, mol l-1; V EP
is the
EDTA
titrated volume of the EDTA solution, ml; ACu is the atomic
mass of Cu; Vs is the volume of the sample of filtrate,
25.00 ml.
The quantity of the reducing sugars (inverted sugar
or glucose) is calculated from the resulting quantity of
Cu, by the use of the tables of Bertrand [1].
The experiments described throughout the method
for analysis of the reducing sugars were done with hydrolysates obtained under enzyme starch hydrolysis with
a thermo-stable bacterial -Amylase, producer strain of
Bac. Subtilis XK-86.
The optimal conditions we established for hydrolysis are as follows: concentration of substrate 250 g l-1, pH
= 7, supported by 1/15 mol l-1 phosphate buffer, concentration of the enzyme 12 units/ml suspension; temperature of hydrolysis 90C [7, 8].

(2)

OOC

OOC

4-

H
C

C
H

Cu

H
C

COO

C
H

COO-

COOH
CHOH + Cu2O +2
4

CH2OH

CHO

+ CHOH

CH2OH

HO

H
C

COO-

HO

C
H

COO-

The resulting residue of Cu2O equivalent to the

oxidized sugar is filtered. The surplus Cu2+ in the filtrate that has not reacted with the sugar is titrated
complexometrically by EDTA in acetic acid media (pH
5 5.1), against the indicator of PAR, according to the
reaction:
Cu2+ + H2Y2- Cu Y2- + 2 H+

(3)

RESULTS AND DISCUSSIONS

Principle of the Method
Under this method we prepared solutions of
Fehling I and II, taking certain quantities of CuSO4 and
potassium-sodium tartarate.
The complex Cu2+ salt of potassium-sodium
tartarate with deep blue color is obtained as a result of
the mixing of the two solutions

2+

(1) Cu

+2

HO

H
C

COO-

HO

C
H

COO-

4-

OOC

OOC

H
C

C
H

Cu

H
C

COO

C
H

COO-

blue complex

An oxidation-reduction process follows by which

Cu is reduced to Cu+ from the free carbonyl group of
sugar.
Thus, red Cu2O is obtained and sugar is oxidized
to an acid under the following reaction:
2+

The quantity of Cu in the residue of Cu2O is calculated and with it the quantity of the reducing sugars
in starch hydrolysate, is calculated using the Bertrand
tables [1] (as invert sugar or glucose).
Conditions for back titration of Cu2+
The conditions for the complexometric titration of Cu2+ that are equivalent to the oxidized reducing
sugars in the starch hydrolysate were determined experimentally by varying the different parameters: the
necessity of stopping the hydrolysis immediately after
obtaining the analysis sample; the manner and the duration of the heating of the sample for carrying out reaction (2); the proper acidity of the media for reaction
(3), the volume of the solutions of Fehling I and II for
increasing the preciseness of the analysis. Due to the
high rate of the enzyme hydrolysis it is necessary to
stop the hydrolysis reaction immediately after sampling.
0.5 ml 2mol l1 HCl with 1 ml sample volume are sufficient to stop the hydrolysis.
Under acidic hydrolysis this requirement is not
applicable since the hydrolysis stops simultaneously with
the cessation of heating.
Another factor which had to be taken into consideration was the manner and the duration of heating

77

Journal of the University of Chemical Technology and Metallurgy, 46, 1, 2011

Table 1. Statistical data for the precision and reproducibility of the method for analysis of
reducing sugars.

Used
amount
of glucose
g l-1

Obtained
Amount
of glucose
g l-1

10,2
30,1
50,3
70,2
90,1

x x
10,3 0,1
29,9 0,2
50,1 0,2
70,5 0,2
90,4 0,2

Number
of
analysis

Standart
deviation
S

5
5
5
5
5

0,10
0,15
0,16
0,16
0,20

Relative Relative
standart
error
deviation x,%
Sr, %
0,97
0,50
0,32
0,22
0,22

+0,98
-0,66
-0,40
+0,42
+0,33

Table 2. Analytical results for concentration of reducing sugars / invert sugar / in enzymatic
starch hydrolysate determined by two methods

Duration of
Proposed
enzymatic complexometric
hydrolysis
method
min
g l-1
10
53,5
20
60,5
30
63,5
40
65,5
50
66,5
60
67,4

Comparative
iodometric
method
g l-1
53,2
60,6
63,3
65,3
66,7
67,6

Relative
error
%
+0,56
-0,16
+0,31
+0,31
-0,30
-0,29

Method Relative
of
error
Bertrand
%
53,4
60,3
63,7
65,6
66,4
67,6

+0,18
+0,33
+0,31
-0,15
+0,15
-0,30

Glucose, gl-1
#




#



#




"

Volume of the sample, ml

Fig. 1. Function CGl = f(Vsample) systematic error when studying glucose after enzymatic hydrolysis of starch. Substrate
concentration 250 g l-1; pH 7; enzyme concentration 12 units/ml suspension; -amylase from Bac. subtilis; 90oC. Curve 1-10
min after the beginning of hydrolysis; Curve 2-30 min after the beginning of hydrolysis.

78

T. Kolusheva, A. Marinova

of the sample for the complete oxidation-reducing process between the Cu-complex and the reducing sugars
(see reaction 2). According to the existing literature,
the duration of this reaction is 3 min under direct heating of the sample. Such heating, however, contains a
certain risk of loss of part of the analysis sample due to
sprinkling of the solution during the boiling.
Therefore, we heated the sample in a water bath
and determined that the results after 5min heating in
the water bath are identical to those obtained with 3
min direct heating of the sample.
The high alkalinity of the sample after adding
the solutions of Fehling (pH 10-11) made necessary the
utilization of a more concentrated acetic acid buffer
2 mol l-1. This creates suitable conditions for the completion of the complexometric reaction between Cu 2+ and
EDTA (reaction 3). By means of experiments, we proved
that 8 ml 2 mol l -1 acetate buffer are sufficient for this
purpose.
The volume of the solutions of Fehling I and
II through which Cu 2+ is added to the sample was
also determined. 25 ml volume of both solutions is
sufficient in order for part of Cu 2+ to be able to react
with the reducing sugars and at the same time enough
surplus of Cu 2+ to remain in the solution. This surplus is titrated by approximately 20 30 ml solution
of EDTA.
After determing the optimal conditions of the
analysis we investigated the ratio Creducing sugars= f (VS),
where Creducing sugars is the concentration of the reducing
sugars in the sample, VS is the volume of the sample.
The graphical representation of the function is given in
Fig. 1 The strictly linear relationship Creducing sugars= f (VS),
passing through the origin of the coordinate system
proves the quantitative realization of reactions (1), (2)
and (3) under the specified conditions.

viation, Sr is less than 1 %, which shows very good reproducibility of the results obtained by back complexometric
titration of Cu 2+, corresponding to the reducing sugars.
The relative error, x <1% is indicative that the new method
we propose is characterized by good precision for the concentration interval from 10gl -1 to 200 gl -1 glucose.
As a robustness check, we determined the concentration of the reducing sugars in an enzymatic
starch hydrolysate (Table 2) by three methods. The
iodimetric method of Luff-Shoorl [1, 2] and the sedimentary method of Bertrand [1] were used as comparative methods. The calculated relative error between the results obtained by the three methods is
also less than 1 %.

Analytical characteristics of the method

REFERENCES

In order to make an exact estimate of the reproducibility and the precision of the method, we prepared 5
model solutions of the reducing sugars, containing different quantity of glucose, dissolved in phosphate buffer
1 15 mol l -1, pH 7. We investigated the samples of the
solutions by our new procedure.
The obtained results and their analytical characteristics are presented in Table 1. The relative standard de-

1. S.V.Iliev, Rukovodstvo po biohimia, Nauka I Izkustvo,

Sofia, 1991, (in Bulgarian).
2. L.Iotova, I.Dobrev, I.Ivanov, Praktikum po biohimia,
Sofia, 2000, (in Bulgarian).
3. M.J. Smgyi, Biol. Chem., 195, 1952,19.
4. D. Yankov, E. Dobreva, V. Beschkov, E. Emanuilova,
Enzyme Microb. Technol., 8, 1986, 665.
5. M.Y.Fedorov, Rukovodstvo po mikrobiologia,

CONCLUSIONS
We developed a new method for analysis of
reducing sugars (such as inverted sugar or glucose) in
enzymatic or acidic starch hydrolysates;
We proved the lack of systematic errors. The
reproducibility and precision of the results are very good,
Sr < 1 % glucose; x <1% for the concentration interval from 10 g l -1 to 200 g l -1 glucose;
The possibility of using of the empirical tables
of Bertrand for measuring the quantity of the reducing;
The method is very fast, it does not require
special reagents and equipment, and is thus very suitable for serial analyses of different kinds of technologies, in which starch hydrolysis to low molecular mass
reducing products is widely applied. Our method can
be successfully used for scientific studies of the optimal
conditions and the kinetics of starch hydrolysis when
using hydrolytic enzymes, produced by different microorganisms.

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Journal of the University of Chemical Technology and Metallurgy, 46, 1, 2011

Moskva, Gos. Izd., 1991, p.260 (in Russian).

6. E. Dobreva, E. Emanuilova, P. Kosturkova,
M.Beschkov, Acta Biotechnol., 5, 1985, 187-190.
7. T.Kolusheva, A.Marinova, Study of the Optimim

80

Conditions of Starch Hydrolysis by means of Thermostable -amylase, J. Univ. Chem. Technol. Met.
(Sofia), 42, 1, 2007, 93-96.
8. H.-S. Kim, D.D. Miller, J. Nutr., 135, 2005, 434.