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Colorants in the sugar industry: Laboratory preparation and spectrometric

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 Colorants in the sugar industry:
Laboratory preparation and spectrometric analysis
Amel Mersad, Richard Lewandowski, Bertrand Heyd, Martine Decloux*

ENSIA (Ecole Nationale Supérieure des Industries Agricoles et Alimentaires) 1, Avenue des Olympiades 91744
Massy cedex, France Tel : +33 1 69 93 5092 Fax : +33 1 69 93 50 44 E-mail : decloux@ensia.inra.fr
*Corresponding author

Abstract

Colorants are a source of significant problem during white

sugar production. In the first part of the paper, nature and
origins of colorants during the various stages of sugar pro-
duction are reviewed. A reproducible method was devel-
oped to produce these colorants to gain understanding of
their kinetic formation. Amongst the colorants known to
form during sugar processing, it was observed that using
the same precursors, HADP and melanoidins formed more
rapidly than caramels, and at a lower temperature.
Absorbance in the visible range (at 420 nm using ICUMSA)
remains the officially approved method for measuring col-
orants, but increasingly the focus has been turned on
absorbance in the UV region. The Hλf vs. Lλf diagram
method proposed by Bento (1995) was used to distinguish
the different types of colorants. This method was improved
using principal component analysis (PCA) method which
makes a finer distinction between colorants than the
method proposed by Bento (1995), because it endows
absorbances with the same degree of importance at all
wavelengths of the UV spectrum. It was demonstrated that
this method allows to distinguish between different com-
pounds and to group similar compounds. It was also shown
that the permeate of raw cane sugar remelt syrup of 20
kg/mol and 50 kg/mol ultrafiltration membranes moved to
caramel, which confirms that these colorants have a lower
molar mass than melanoidins and HADP.

Introduction (1995) proposed a mathematical method using their indi-

Colorants constitute a major problem during sugar pro-
cessing. Crystallization is adversely affected because they
reduce the purity of white sugar. At present, no analytical
method has officially been approved for the separate iden-
tification of these colorants in mixtures. Indeed, coloration
in the sugar industry is only estimated using the ICUMSA
method, based on a global measurement of absorbance at
420 nm wavelength. It is therefore impossible to determine
which of these colorants is the most difficult to remove dur-
ing sugar refining, as a quantitative estimate of decolorisa-
tion process efficiency is problematic. Some authors have
nonetheless used spectrometric analyses (in the UV as well
as in the visible range) to distinguish sugar colorants. Bento
vidual UV spectra as a means of differentiation.
The first part of this paper reviews the various colorants
identified during sugar processing. The aim was to clarify
their nature, origins, the conditions and reactions involved
in their formation, and the different stages of their forma-
tion during sugar processing. The most harmful colorants
were prepared separately in the laboratory with a repro-
ducible technique mimicking the conditions prevailing dur-
ing sugar processing. The spectra of each colorant in the
UV and visible ranges were then measured at different pH
values. Taking the Hλf vs. Lλf diagram method, one step
further in terms of analysing the UV spectra, an improve-
ment was proposed using principal component analysis
(PCA) method.
Sugar colorants
Types of colorants
Colorants in the sugar industry can be of two types, natural
and those which form during sugar processing.
Natural colorants
Cane sugar contains more natural colorants than beet
sugar. These colorants are found in the juice after milling.
Table 1 lists the most common colorants encountered,
namely chlorophylls, carotenes, xanthophylls, and
flavonoids, as well as some of their properties. Among these
colorants, flavonoids pose a real problem in cane sugar pro-
cessing because they make a considerable contribution to
the coloration of raw sugar (up to 30 % at pH 7) according
to Smith and Paton, (1985).
Sugar processing colorants
These colorants form during both cane and beet sugar pro-
cessing. They are the products of a number of reactions
between the precursor molecules initially present in beet or
cane. They are referred to as "sugar processing colorants".
Four types can be distinguished, as a function of the
mechanisms through which they are formed: melanins,
melanoidins, caramels and hexose alkaline degradation
products (HADP).
Melanins result from the enzymatic oxidation (or enzymat-
ic browning) of phenolic compounds in quinones (always
red), to produce indole polymers (Chichester, 1972). This
enzymatic browning is catalyzed by polyphenoloxidases
(PPO), a generic term which refers to several enzymes.
Enzymatic oxidation of phenols can generate other com-
pounds as well as melanins. Indeed, red quinones can bind
to reticulated proteins to form coloured polymers (Cheftel
and Cheftel, 1980; Catherine, 1999). They also condense
among themselves to form colorants which are not neces-
sarily indole polymers.
Caramels have been the subject of considerable study in the
food industry. They are used as additives and their
composition varies considerably, depending on the condi-
tions of preparation and the catalysts used (Greenshields
and Mac Gillivray, 1972; Dufresnoy, 1978; Cottier et al.,
1989). Indeed, the products of Maillard's reaction are called
caramels, (ammoniacal caramel class III) (Thornton, 1990).
The sugar industry mainly targets caramel produced solely
by the thermal degradation of sucrose, without any cataly-
sis. This type of caramel covers most polymers of 5 hydrox-
ymethyl-furfural (HMF) (Greenshields and Mc Gillivray,
1972 ; Dufresnoy, 1978; Kelly and Brown, 1978). Indeed,
Cottier et al. (1989) found 70 to 90 % of HMF in the vapours
of caramels produced by heating sucrose or glucose at
180 °C. According to Kroh (1994), sucrose is hydrolyzed to
invert sugars (glucose and fructose), which then, on heat-
ing, undergo a series of dehydration reactions and cyclisa-

Table 1. Natural colorants in cane sugar

Colorants Properties

Chlorophylls Type a : C55H72O5N4Mg (5) Green photosynthetic pigments (4)

Type b : C55H70O6N4Mg (5)

Carotene C40H56 (5) Yellow carotenoid colorants

Long unsaturated and unbranched carbon chains (3)

Xanthophylls C40H56O2 (5) Yellow carotenoid colorants (4)

Flavonoids General structure C6 structures are A and B aromatic nuclei, common to different flavonoids. C3
C6-C3-C6 (3) structure differ from one type to another type of flavonoids (2)
Most frequent in cane sugar: Generally linked to one or several sugar molecules (2)
* Flavones acidic colorants (1) are stable in acidic media and completely dissociate at
pH > 9 (1)

* Flavonols common in vegetables. derived from Flavones (2)

* Chalcones less common yellow colorants. become orangish red with ammonia (2).

* Catechines polymers of condensed tannin react with iron to form other colorants (2)
heated in acidic media, they become yellowish brown and insoluble (2)

* Anthocyanes cationic nature

decompose at pH • 8
red in acidic media
become blue and lose color with increasing pH (1).
form complexes with some metals and become stable at high pH (3)

(1):(Smith and Paton 1985); (2):(Ribereau-Gayon 1968); (3):(Chichester 1972); (4) :(Duval and Duval 1978)
tion to form 5 hydroxymethyl-furfural (HMF), hydroxy-
acetyl-furan (HAF) and hydroxydimethyl-furanone (HDF),
together with other aromatic compounds in smaller quanti-
ties such as carbocyclics and pyrones. According to Kroh
(1994), the presumed precursors of coloured polymers dur-
ing caramelisation are HMF and HAF. This agrees with the
views of Kelly and Brown (1978), who described HMF as a
precursor of coloration with a maximum UV absorbance at
285 nm. In an acid aqueous solution, HMF is unstable and
decomposes into organic acids.
Melanoidins are by definition the products of Maillard
reactions. These reactions start with the condensation of a
carbonyl compound (reducing sugars, vitamins, quinones,
etc.), with an amine (amino acids, proteins, ammonia, etc.).
A host of reactions then follow resulting in coloured poly-
mers (melanoidins) and various volatile or odorous com-
pounds (aldehydes, ketones, pyrazine) via intermediates
such as HMF, reductones, aldimines and others (Gillett,
1953; Hodge, 1953; Greenshields and Mc Gillivray, 1972;
Cheftel and Cheftel, 1980; Moll, 1993). The products
formed and intermediate products are also precursors of
melanoidins since they contain nitrogen (pyrazine) or car-
bonyl groups (HMF, aldehydes, melanoidins themselves,
etc.) in their structure.
Hexose alkaline degradation products (HADP) are brownish
yellow colorants produced by the decomposition of mono-
saccharides in alkaline media through a series of ionization,
enolization and isomerization reactions leading to the for-
mation of the intermediate enediol anion (de Bruijn et al.,
Figure 1. Formation of colorants in cane and beet sugar industries
1986). This anion, in turn, undergoes a sequence of complex
reactions, forming several carboxylic acids (predominantly
lactic acid, plus saccharinic acid, formic acid, acetic acid,
oxalic acid and others) (Pieck, 1963; Kelly and Brown, 1978).
Part of the carboxylic acids formed are colourless and of low
molar mass (with carbon chains not exceeding six atoms).
They are referred to as HADP • C6. The remainder poly-
merise with each other to form polycarboxylic chains with a
high molar mass, and are called HADP > C6. These unsatu-
rated polymers formed are the source of the brownish yellow
coloration (de Bruijn et al., 1986)
Formation of colorants during sugar processing
Figure 1 shows a schematic representation of the simulta-
neous formation of colorants during processing, in the
presence of natural colorants. These are mainly extracted
from cane during cutting and milling. They are present in
juice after milling but for the most part are eliminated dur-
ing clarification (Chen and Chou, 1993). However, because
flavonoids are phenol compounds, they are also involved in
the formation of melanins.
Melanins form at temperatures between -18 °C and 55 °C
and at pH values between 4.5 and 8 (Catherine, 1999).
Enzymatic browning reactions are therefore very active
during cutting and milling (cane) and slicing into cos-
settes (beet), especially as these stages cause contact
between the enzymes (PPO) and their substrates
(flavonoids and phenols in cane and organic acids and
amines in beet). During the processing stage following,
high temperatures denature enzymes. However, quinones
formed by the enzymatic browning of phenols may react
with amines to form melanoidins.
Melanoidins form at ambient temperatures, but are pro-
moted by its rise. They develop slowly in an acidic medi-
um and more intensely in a basic medium (Adrian, 1993).
Consequently, melanoidins may already start to form at
slicing into cossettes (beet) or milling (cane), when the pH
is slightly acidic and the temperature low. The reactions
intensify during diffusion (beet) and extraction (cane),
when temperatures rise (70 to 75 °C). During beet sugar
processing, Maillard's reactions are further intensified
during calco-carbonic purification where the conditions
(pH > 11 and T = 85 °C) are favourable to their formation.
Melanoidins are less of a problem during cane sugar pro-
cessing, where clarification is achieved under much less
alkaline conditions. However, in some cases during cane
sugar refining, pH may rise to 10.5, thus favouring the for-
mation of melanoidins. In both cases (cane and beet),
melanoidin formation peaks during juice evaporation
when the temperature reaches 120 °C and the polymerisa-
tion continues during crystallisation. Because beet juice is
naturally richer in nitrogenous compounds and amino
acids than cane, melanoidins form more easily and in larg-
er quantities.
HADP always arise in an alkaline medium, the optimum
value being about pH = 11. Hexose alkaline degradation
occurs at room temperature, but intensifies as the temper-
ature rises. In the knowledge that sucrose also hydrolyses
in an alkaline medium, particularly at high pH values
(Mauch, 1971), the main stage of HADP formation during
Figure 2. Experimental reactor
reflux electric laboratory
150 rd/min stirrer
condenser
electrical
135°C
regulator
thermometer
cold water
Temperature
cylindrical colorants
regulation
reactor precursors
heating mantle
beet sugar processing is therefore the calco-carbonic
purification (pH > 11 and T = 85 °C). During cane sugar
processing (clarification) and refining (purification), less
HADP form than with beet sugar, although levels are slight-
ly higher during refining.
With respect to caramels, Ahari and Genotelle (1961)
noted the need to heat sucrose beyond its melting point
(185°C) for these colorants to form. Intense heating does
indeed supply the energy necessary for the successive
dehydration reactions observed during caramelisation.
According to Pieck (1963) and Kelly and Brown (1978),
caramels tend to form in an acidic medium. Consequently,
during cane and beet sugar processing, the only stage
when the temperature is sufficiently high to form caramels
is evaporation, although the conditions are not acidic but
neutral to slightly alkaline.
Characteristics of sugar processing colorants
Particular attention was paid to the characterisation of pro-
cessing colorants. Indeed, they form throughout the pro-
duction of white sugar and their action continues during
crystallization, because the inclusion of some of them in
crystals diminishes their purity.
Measurement of indicator values (I.V.)
Godshall (1997) distinguished colorants in terms of their
indicator value (I.V.), being the ratio between absorbance at
λ2
∫Abs•dλ
Lλf or Hλf = C (1) where λ1 (2)
C=
Abs0 (λ2 − λ1)
420 nm and pH 9 and absorbance at 420 nm and pH 4. In
this case, spectrometry was coupled with the pH parameter
which, according to these authors, has a significant influ-
ence on the absorbance of these colorants. The indicator
values for processing colorants are summarised in Table 2.
Hλf vs. Lλf diagram method
In 1995, Bento proposed a mathematical method applicable
to the distinction of sugar colorants, which involved defin-
ing two fractions, one at high wavelengths (Hλf) and the
other at low wavelengths (Lλf), as follows:
λ1 and λ2 are equal to 200 nm and 273 nm respectively
for Lλf
λ1 and λ2 are equal to 273 nm and 340 nm respectively
for Hλf
Abs represents the absorbance obtained at different wave-
lengths
Abs0 represents the absorbance obtained at λ = 273 nm
The ratio "C" represents the average of different
absorbencies between the two corresponding wavelengths.
To a certain extent, the Llf and Hlf fractions illustrate the
comparison between the average "C" and absorbance at
273 nm. The 273 nm wavelength was chosen because it
enabled the clearest distinction between the colorants
under study. The next step consists in constructing an Hlf
vs. Llf diagram and placing the colorants at their respec-
tive co-ordinates. It is thus possible to differentiate between
caramels, melanoidins and HAPD, depending on their dis-
tinct positions on the diagram.
Materials and methods
The aim of this research work was first of all to trace the
formation of sugar colorants from their precursors. To
achieve this, a controlled and reproducible method of
preparation for each type of colorant was developed. These
methods took account of the techniques in line with sugar
processing conditions and ensuring conditions favourable
to the formation of each colorant. Spectrometric analyses
of the three model colorants under visible and UV spectra
were performed, in order to characterise them (absorbance
peaks and sensitivity to pH). Finally, the UV spectra
obtained was exploited by mathematical calculations in
order to distinguish between the colorants. The Hλf vs. Lλf
diagram method proposed by Bento (1995) was used, and
the application of a statistical method, principal component
analysis (PCA), was tested to enable a distinction between
the colorants.
Synthesis of sugar colorants
Sugar processing colorants are a centre of interest of vari-
ous studies and many researchers have formed them in their
laboratory, as Ahari and Genotelle (1961), Smith (1966),
Shore et al. (1984), Bento (1995), Keremat and Nursten
(1994), Hirano et al., (1996) and Bento and Sá (1998).
We do not always come accross the same preparatory
methods and for each colorant, techniques vary with
authors. The changes we made for this work are described.
Reactor
Instead of directly incubating colorant precursors in an
oven, a cylindrical reactor (Pyrex glass) with a capacity of
1 L (Fig.2) was used. The reactions were carried out under
atmospheric pressure. A reflux condenser was used to
control evaporation and thus ensure a reproducible
method. The lid of the reactor had holes in it, thus allowing
the introduction of measurement probes to monitor the dif-
ferent stages of the reaction. In order to ensure that it was
airtight, the reactor was sealed with a gasket and screw lid.
It was heated with a heating mantle (Prolabo) equipped
with an electrically regulated thermometer. The tempera-
ture probe was placed inside the reactor to regulate the
temperature of the solution (± 1 °C). The reactor was
equipped with an electrical laboratory stirrer (Heidolph
RZR 2021) to mix the solution and prevent the accumula-
tion of mass at the bottom of the reactor, especially in the
Figure 3. Temperature and colour evolution during carameli-
sation reaction at different temperature set-points
150 brown-yellow colour
140 faint yellow colour variable dark brown colour
130 faint yellow colour brown colour
120 faint yellow colour
110
Temperature (°C)
100 white colour
90
80
70
60
temperature set-points
50
140°C
40
white colour 130°C
30 120°C
20 100°C
10
0 20 40 60 80 100 120 140 160 180 200 220 240
Time (min)
case of caramels.
Choice of experimental conditions
A reaction time of 3 h and a stirrer speed of 175 min-1 were
chosen for the preparation of the three sugar processing
colorants. Temperature, pH and precursors used were cho-
sen individually for each type of colorant.
In the case of caramels, it was unsure whether it was
necessary to heat the precursors to a very high temperature.
Several different temperatures (100, 120, 130 and 140 °C)
were then tested. Despite knowing that caramel formation
is favoured by acidic conditions, HCl was not added
because, during sugar processing, acidic conditions are
avoided to prevent sucrose hydrolysis. 320 g pure sucrose
was placed in the reactor and then moistened using 30 mL
ultra pure water. The mixture was reflux heated for 3 h at
100, 120, 130 and 140 °C. At the end of the reaction, 200 g
of the caramel obtained were weighed and diluted with 200
g ultra pure water and then cooled. In order to prevent the
pursuit of polymerisation, the pH of the solution was adjust-
ed to 7 before refrigeration.
For melanoidins, 500 mL of a solution of glucose (1
mol/L) and glycine (1 mol/L) using ultra pure water was
then placed in the reactor. The pH of the solution was
adjusted to 8, which is the optimum pH to promote
Maillard’s reactions (Cheftel and Cheftel, 1980). According
to Adrian (1993), melanoidins form slowly at room temper-
ature, whereas at 100 °C all the amino acids present in the
solution react. This temperature was therefore chosen,
because it also had the advantage of being comparable to
the conditions prevailing during sugar processing. The glu-
cose-glycine solution was reflux heated for 3 h at 100 °C. At
the end of the reaction, 200 g of the solution were weighed
and cooled. To prevent further polymerisation, the pH was
adjusted to 7 and the solution was refrigerated.
To obtain HADP, 500 mL of a glucose solution (10 g/L)
 Figure 4. Temperature and colour evolution during caramels, melanoidins and HADP formation

150
caramels caramels
140 faint yellow colour brown-yellow colour
caramels
130 brown colour
120
Melanoidins black colour
Temperature (°C)

110 HADP black colour

Melanoidins black colour
100 HADP black colour
90 Melanoidins light brown colour
80 HADP black colour

70 Melanoidins faint yellow colour

HADP yellow colour
60
50 Caramels white colour
Melanoidins faint yellow colour Caramels 130°C
40 HADP colourless Melanoidins 100°C
30 HADP 100°C
20
0 20 40 60 80 100 120 140 160 180 200 220 240

was prepared using ultra pure water and placed in the reac-
tor. The pH was adjusted to 11 because at this value, the
alkaline degradation of hexose is maximum (Kelly and
Brown, 1978). Because HADP form under alkaline condi-
tions and produce acids, it was necessary to maintain an
alkaline pH throughout the reaction. A pH probe (Metler-
Toledo InPro 3100 325 with Pt100 temperature probe)
resistant to 130 °C, was then placed in the reactor to moni-
tor the pH, and a round flask with a tap, containing NaOH,
was used to adjust the pH. After several tests, we found that,
to maintain an alkaline pH, it was necessary to add 2 mL
NaOH 10 M every five minutes during the first 30 min of the
reaction. The glucose solution (pH = 11), was reflux heated
for 3 h at 100 °C. As in the case of melanoidins, a tempera-
ture of 100 °C is enough to form HADP, and is similar to the
conditions prevailing during sugar processing. Indeed,
according to de Bruijn et al. (1986), HADP are formed at
room temperature. At the end of the reaction, 200 g of the
coloured solution obtained were weighed and cooled. As in
the other tests, to prevent further polymerisation, the pH
was stabilised to 7 before refrigeration.
Spectrometric analysis
A spectrometer (Jasco V550 SSE-343 – precision of 0.02
absorbance unit) was used to measure the visible and UV
spectra. The precise limit between the UV and visible
regions is not clearly defined, different authors proposing a
range from 340 nm to 400 nm. The limit was fixed at 390
nm because this value is the most frequently cited in the lit-
erature. The spectra was measured on diluted colorants so
that their absorbency was between 0 and 1 in both domains:
UV (from 220 to 390 nm) and visible (from 390 to 900 nm)
separately. For UV spectra, caramel and HADP solutions
were diluted 500-fold, while the melanoidin solution was
diluted 1000-fold. For visible spectra, the caramel solution
was diluted 5-fold, the HADP solution 50-fold and the
melanoidin solution 100-fold. In order to assess the influ-
ence of pH on the absorbance of colorants, the pH was
adjusted to 7, 4 and 9 for each type, and the UV and visible
spectra were then measured. The pH meter used was a com-
bined electrode pH/temperature (WTW pH330/SET – preci-
sion 0.05 pH unit). The necessary precautions were taken to
dilute the samples in exactly the same manner at adjust-
ment of the pH. Each analysis was repeated at least three
times so as to be sure of the result. The Bento (1995) method
cited above was then applied to situate the colorants on the
Hλf vs. Lλf diagram.
Results and discussion
Colorant formation
Caramels
There was no formation of colour at 100 °C for caramels. At
120 °C, faintly coloured, yellowish caramel was observed. At
 Figure 5. UV-Visible spectra of sugar processing colorants tions are in fact successive dehydration reactions. The
quantity of water released will be markedly larger more
than that required for the hydrolysis of sucrose (3 molecules
0.5
released for 1 molecule required for hydrolysis, according
to Kelly and Brown, 1978). To confirm this explanation, it
0.4
was observed that the sucrose had dissolved 10 min after
0.3 the reaction. According to Charles’s equation (Chen and
Caramels - pH7
0.2 Chou, 1993), it is necessary to attain a temperature of 155
0.1 °C to dissolve sucrose at a concentration of 91.4 g per 100
0.0 g of solution. It is therefore clear that the concentration of
the solution decreases with the release of water molecules.
-0.1
220 320 420 520 620 720 820 Once the temperature was stabilised, it rose gradually to
0.5 reach 130 or 140 °C and then the boiling was halted. It was
0.4
from that moment onwards that we noticed the appearance
of a brown colour indicating that HMF, HAF and probably
0.3
Melanoidins - pH7 other monomers were polymerising to form colorants. The
Abs

0.2 temperature was maintained constant throughout the reac-

0.1 tion and the coloration became even more intense. A logical
0.0 explanation for the rise in the boiling point after the stabil-
-0.1 isation phase has not been found. To achieve this, the con-
220 320 420 520 620 720 820 centration of the solution must be increased, whereas the
0.5 reaction took place under total vapour recycling. Moreover,
0.4 according to Cloizeaux and Jannink (1987), polymerisation
does not involve the uptake of water molecules. Indeed, it
0.3
occurs through chain reactions of monomers with double
0.2
HADP - pH7
bonds, which open during the binding of monomers. In
0.1 some cases, the polymerisation even gives rise to the release
0.0 of water. One possibility is that the production of acids and
other monomers was quite considerable and that the molar
-0.1
concentration of the solution increased significantly. At 100
220 320 420 520 620 720 820
and 120 °C, no stabilisation of temperature was observed
UV-visible wavelengths (nm) because the chosen temperatures were lower than the boil-
ing point of the solution (124 °C). These temperatures were
130 °C, reproducible caramels were obtained. Finally, at attained as from 20 min of the reaction and maintained
140 °C, brown caramels were obtained but were too differ- until the end of the reaction.
ent to be reproducible. Indeed, the six caramels prepared at
this temperature did not give the same intensity of col- Melanoidins
oration. Thus, for the analyses, the three caramels, A, B and
C, prepared at 130 °C were selected. These caramels were Figure 4 shows the temperature and coloration kinetics
highly viscous and translucent. It was necessary to dilute during the formation of melanoidins. Initially, the equimo-
them with water to 50 % immediately to prevent solidifica- lar solution of glucose and glycine at pH 8 displayed a very
tion. Before being adjusted to 7, their pH was 3.72, thus pale yellow colour. A temperature of 100 °C was attained in
confirming that the formation of caramels is accompanied 20 min and maintained until the end of the reaction with-
by the production of acids. out the solution boiling, because of the rise in the boiling
Figure 3 represents the evolution of temperatures and of point. The solution started to colour 15 min after the begin-
coloration during the formation of caramels at the different ning of heating and then darkened very rapidly, turning
temperatures tested (100, 120, 130 and 140 °C). The same black in five minutes. The coloured solution obtained was
kinetics were observed at 130 and 140 °C. Indeed, a tem- black, opaque, liquid and acid (pH = 4.77) before the pH
perature of 124 °C was reached in 20 min, during which the was adjusted to 7 for storage. The kinetics were perfectly
sucrose was dissolved. This temperature was maintained reproducible for the three melanoidins prepared, A, B, and C.
for 70 min while the solution was allowed to boil. However,
the boiling point of a sucrose solution at 91.4 g per 100 g of HADP
solution is 130 °C according to the Adamson equation
(McGinnis, 1982). Furthermore, the hydrolysis of sucrose The temperature and coloration kinetics observed during
into glucose and fructose doubles the molar concentration the formation of HADP are shown in Figure 4. The glucose
and can therefore be expected to increase the boiling point solution (10 g/L) at pH 11 was initially transparent. As soon
of the solution. A plausible explanation for the observed rise as heating was started, the pH fell, thus marking the onset
in the boiling point (24 °C) was that caramelization reac- of acid formation. After 10 minutes, the solution acquired a
 Table 2. Characteristics of sugar processing colorants

Colorants Chemical composition Absorbances peaks Indicator value (IV)

Melanoidins polymers of HMF, reductone and others 300 nm(2) (5)

200 nm (5) 1 to 1,2(4)
Caramels polymers of HMF and HAF 285 nm(1) (5)
220 nm and 200 nm(5) 1 to 1,5(4)
HADP Carboxylic acids polymers 265 nm at pH 7(3)
260 nm(5) 1,5 to 3(4)

(1):(Kelly et Brown, 1978); (2):(Cheftel et Cheftel, 1980); (3):(de Bruijn et al., 1986); (4):(Godshall, 1997); (5):(Cosmeur, 1999)

yellow coloration, which intensified and turned dark

brown and then black in 5 minutes. This indicated poly- Figure 6. Influence of the pH on the visible spectra of
colorants formed in the laboratory
merisation of the acids formed, giving rise to chains of
polyunsaturated hydrocarbons, which generate colour.
After 30 min, the set-point temperature (100 °C) was
0.9
reached and the pH was 10.3. From that moment
onwards, the pH of the solution did not fall, so did not 0.7 pH4
require the addition of any NaOH, thus indicating the
Caramels pH7
total consumption of acid precursors. Contrary to the 0.5 pH9
findings of de Bruijn et al., (1986), the coloured solution
obtained was not brownish yellow but dark, opaque and 0.3
liquid, like the melanoidin solution. After cooling, the pH
of the HADP solution was 12.6, which shows clearly that 0.1
the pH is inversely proportional to the temperature. The
pH was then adjusted to 7 for storage. The kinetics were -0.1
perfectly reproducible for the three HADP prepared, A, B, 390 440 490 540 590 640 690 740 790 840 890
and C.
To conclude, by heating moistened sucrose (91.4 g per
100 g of solution) for 3 h, it was possible to produce 0.9
caramels at 130 °C without necessarily acidifying the
medium. The presence of this colorant is therefore not 0.7 pH 4
impossible during sugar processing. However, HADP and Melanoidins pH 7
melanoidins also form during sugar processing. Because 0.5 pH 9
Abs

these three colorants have common precursors, competi-

tion necessarily exists. Unlike caramels, HADP and 0.3
melanoidins form much more rapidly and easily, without
requiring any significant heating. Therefore the presence 0.1
of caramels in sugar processing is probable, but they are
present in much smaller quantities than melanoidins and -0.1
HADP, particularly since the temperature rarely exceeds 390 440 490 540 590 640 690 740 790 840 890
120 °C.
0.9
Results of spectrometric analyses
0.7
The UV (220 to 390 nm) spectra of colorants, at pH 7, pH4
HADP pH7
were extended into the visible region up to 900 nm and 0.5
are shown in Figure 5. For the three types of colorants pH9
analysed, the absorbance in the visible region (and 0.3
notably at 420 nm) was negligible, at the same dilution,
compared with UV absorbance. The spectra of the three 0.1
caramels prepared at 130°C were superimposed, as were
those of the three melanoidins and three HADP at 100°C, -0.1
thus confirming that the method of preparation developed 390 440 490 540 590 640 690 740 790 840 890
was entirely reproducible for these three colorants. Visible wavelengths (nm)
 Figure 7. Comparison of the visible spectra obtained with the melanoidins. This may be due to the linearity of HADP mol-
sugar processing colorants prepared in the laboratory ecules (which are polymers of carboxylic acid), whereas
caramels are polymers of HMF and HAF, which are cyclic
components already containing conjugations. Melanoidins
are also polymers of HMF alongside amines, and also con-
0.9 tain triacetone polymers and cyclic aromatic compounds
Caramels - pH7 (pyrazin).
0.7 Melanoidins - pH7 By observing the superimposition of the UV spectra of the
three colorants at pH 7 (Fig.9), it can be noted that the spec-
0.5 HADP - pH7 tra intersect and their forms differ. The peaks are also locat-
Abs

ed at different wavelengths. These differences led us to make

0.3 a more detailed examination of the Hλf vs. Lλf
diagram proposed by Bento (1995) and cited above.
0.1
Figure 8. Influence of the pH on the UV spectra of colorants
formed in the laboratory
-0.1
390 440 490 540 590 640 690 740 790 840 890
Visible wavelengths (nm)
0.9
pH 4
Visible spectrometric analyses 0.7
pH 7
Caramels
0.5 pH 9
In the visible region (Fig.6), a rise in the pH value induced a
significant increase in the absorbance intensity of caramels
0.3
and HADP between 390 and 500 nm. This effect was much
less marked with respect to melanoidins. Furthermore, the 0.1
indicator values for HADP A, B and C were the highest
(between 2.13 and 2.26), followed closely by those of caramels -0.1
(between 1.78 and 2.00), while those of melanoidins were 220 240 260 280 300 320 340 360 380
much lower (between 1.08 and 1.09). These values are in
accordance with those found by Godshall (1997) (Table 2),
except for caramels where the authors obtained slightly high-
0.9
er indicator values than Godshall’s (1 to 1.5). By observing the
pH 4
superimposition of the visible spectra for the three colorants 0.7 Melanoidins pH 7
Abs

at pH 7 (Fig.7), it can be seen that the forms were virtually

0.5 pH 9
identical. Thus, except for the undoubted influence of the pH
on absorbance intensity, it is very difficult to distinguish the
colorants by simply comparing the spectra. Moreover, even if 0.3
this pH effect exists, the indicator values did not differ great-
0.1
ly from one colorant to another. Indicator value measurement
is mainly used to distinguishing sugar processing colorants -0.1
from natural colorants; the latter are highly sensitive to pH 220 240 260 280 300 320 340 360 380
when compared with the former (I.V.= 5 to 14 for flavonoids
versus I.V.= 1 to 3 for sugar processing colorants).

UV spectrometric analyses 0.9

0.7 pH 4
In the UV region, the effect of the pH on the absorbance HADP
pH 7
intensity of the three colorants analysed was negligible 0.5
(Fig.8). These colorants exhibited practically the same pH 9
absorbance peaks as those given in the literature (Table 2). 0.3
The two peaks for caramels were situated at around 285 nm
and 228 nm. The absorbance peak was located at 265 nm for 0.1
HADP and 300 nm for melanoidins. The precise composition
-0.1
of the colorants formed is not known, but based on the prin-
220 240 260 280 300 320 340 360 380
ciple of absorbance under UV (Hamon et al., 1990), it could
be supposed that HADP contain fewer conjugate double UV wavelengths (nm)
bonds than caramels which, in turn, contain fewer than
 Figure 9. Comparison of the UV spectra obtained with the Figure 10 shows the positioning of the colorants prepared
sugar processing colorants prepared in the laboratory above and those prepared by Bento (1995) in the Hλf vs. Lλf
diagram. The raw sugar analysed by Bento (1995), as well
as the raw sugar (from La Reunion) were also positioned on
the diagram. On the Hlf vs. Llf diagram, the position of the
0.9 caramels prepared was distinct but not far from that of the
Caramels - pH7 caramels analysed by Bento (1995). The same applied for
0.7 HADP and melanoidins. This suggests that depending on
Melanoidins - pH7 the method of preparation of colorants, non-identical but
0.5 not entirely different products arise. Similarly, for raw
HADP - pH7
Abs

sugar, the co-ordinates were comparable to those of Bento

0.3 (1995) but did not coincide.
Bento (1995) analysed clarified sugarcane syrup with its
0.1 discoloration products using different decolouring agents,
and marked them on the Hλf vs. Lλf diagram (Figure 11).
-0.1 With the exception of lime and acrylic resin, discoloration
220 240 260 280 300 320 340 360 380 always shifted clarified syrup towards a weaker Hλf and a
stronger Lλf. The difference in behaviour observed with
UV wavelengths (nm) lime and acrylic resin can be explained as follows: the pres-
ence of lime ensures an alkaline medium and induces the
formation of HADP, so that treated syrup moves towards the
Exploitation of UV colorant spectra using mathmatical latter (i.e. weaker Lλf and weaker Lλf). As for acrylic resin,
calculations according to Bento (1995), it does not retain certain col-
orants with a strong Hlf, thus explaining the movement
Hλf vs. Lλf diagram method towards a strong Hλf and strong Lλf rather than a weak Hλf
and strong Lλf.

Figure 10. Positions of colorants and raw sugar with its permeates on Hlf vs. Llf diagram

H λf
2.0

1.6

1.2 Mel S
Mel B
RS B RS La Réunion
0.8
HADP B P50
Car B Car S P20
0.4 HADP S
discoloration
L λf
0.0
0.0 0.4 0.8 1.2 1.6 2.0

Car B : Caramels prepared by (Bento, 1995)

Car S : Caramels prepared in the laboratory
Mel B : Melanidins prepared by (Bento, 1995)
Mel S : Melanidins prepared in the laboratory
HADP B : HADP prepared by (Bento, 1995)
HADP S : HADP prepared in the laboratory
RS B : Raw sugar analysed by (Bento, 1995)
RS La Réunion : Raw sugar from La Réunion
P20 : Raw sugar permeate with 20 kg/mol membrane
F50 : Raw sugar permeate with 50 kg/mol membrane
 Figure 11. Positions of cane syrup and its discoloration products by different discoloration agents in the Hlf vs.Llf diagram
(Bento, 1995)
H λf
0.76
Cane syrup
0.72
0.68
0.64
Ca(OH) 2
Activated carbon
0.60
1.15 1.20 1.25
Discoloration tests were also carried out on raw sugar
using crossflow filtration membranes (20 kg/mol and 50
kg/mol). Permeates obtained were analysed and located on
the Hλf vs. Lλf diagram (Figure 10). The discoloration sense
(towards a weak Hλf and strong Lλf) was precisely the same
as that found by Bento (1995).
The problem with this method is that the average "C",
calculated in Equation 2, may be identical for two com-
pounds which have not the same spectra. This implies that
two compounds with different spectra may have the same
co-ordinates on the Hλf vs. Lλf diagram. As a result, com-
pounds which should be distinguished from one another,
are not. Furthermore, the choice of the 273 nm wavelength
to define the boundary between Hλf and Lλf, is entirely
arbitrary. It was therefore decided to apply a more general
statistical method that could compare individuals while tak-
ing account of all wavelengths in the UV spectrum under
consideration, with the same degree of importance. This
method is Principal Components Analysis, or PCA.
Principal components analysis
The aim of this statistical method is to analyse data in the
form of a matrix in order to make a distinction between
compounds (Saporta, 1990; Bouroche and Saporta, 1992).
In this case, the distinction between compounds was made
on the basis of their UV spectra. The matrix contained the
absorbance of the compounds to distinguish at each
nanometre of the UV region (between 220 and 390 nm).
This method was applied to the 15 compounds as follows:
- CA, CB and CC (the three prepared caramel solutions)
Acrylic resin
H 2O 2
Styrenic resin
Granular carbon L λf
1.30 1 .35 1.40 1.45
- MA, MB, MC, (the three prepared melanoidin solutions)
- HA, HB, HC,(the three prepared HADP solutions)
- Mix, a mixture of colorants CA, MA and HA
- S1, S2, S3, three successive analyses of raw sugar
(from La Réunion)
- P20 and P50, permeates of this raw sugar obtained
using crossflow filtration membranes, at 20 kg/mol and
50 kg/mol, respectively.
The calculations were performed using the OCTAVE
program. Each compound analysed was projected on the
three principal axes of the PCA method. These three axes
are able of providing 97% of the total initial information (52
% on the 1st principal axis, 31 % on the 2nd principal axis
and 14 % on the third principal axis). Figure 12 shows the
projections of colorants, raw sugar and permeates in plane
1 (defined by axis 1 and axis 2) and plane 2 (defined by axis
1 and axis 3). Plane 3 (defined by axes 2 and 3) was not used
as it did not provide any additional, significant information.
It is interesting to note that on these two planes, five
individual groups were clearly distinct:
- a homogeneous group representing the caramels,
- a homogeneous group representing the melanoidins,
- a homogeneous group representing the HADP,
- one group representing the mixture,
- a homogeneous group representing the analysis of raw
sugar with its permeates (P20 and P50).
The presence of these homogeneous groups confirms
that the laboratory method applied to prepare the colorants
was reproducible. Furthermore, these results proved the
ability of the PCA method to group comparable compounds
and distinguish between those which differed.
According to plane 1, the mixture of the three colorants
Figure 12. PCA of the colorants prepared in the laboratory and the raw sugar analysed with its permeates with membranes of 20
kg/mol and 50 kg/mol. Presentation on three principal axis.

10
S3
S1 P20
P50
S2
5
Axis 2 : 31%

0 MA
MB
MC mix
-5
HA
HB
CA
HC
CB
-10
CC

-15
-20 -15 -10 -5 0 5 10 15 20
Axis 1 : 52%
Plane 1

10 HA
HB
HC

5
mix
S1
S2
Axis 3 : 14%

P50
0
S3 P20
MA
CA
MB
-5 CB
MC
CC

-10

-15
-20 -15 -10 -5 0 5 10 15 20
Plane 2 Axis 1 : 52%
(mix) was located between the HADP and the melanoidins.
In plane 2, it was located at the centre of the three col-
orants, although slightly closer to the melanoidins and
HADP than to the caramels. If the three-dimensional repre-
sentation of the mixture (planes 1 and 2) was imagined, it
was clearly located at the centre of the three colorants. On
plane 1, the position of the permeates shows that a filtration
of raw sugar remelt syrup did not indicate any significant
variation in its composition, especially along the second
principal axis (axis 2). The position of the compounds
analysed on plane 2 was particularly interesting. Indeed,
the position of the permeates shows that a filtration of raw
sugar shifted it towards the caramels. This is in line with
findings in the literature; according to Shore et al. (1984)
and Bento and Sa (1998), caramels are amongst the pro-
cessing colorants with the lowest molar masses. The fact
that the P20 permeate was further from raw sugar than P50
on both planes indicates that a smaller pore membrane
removes coloration more efficiently.
The PCA method is capable of distinguishing colorants
just as efficiently as the Hλf vs. Lλf diagram method (Bento,
1995). It can also provide an indication of the efficacy of the
discoloration process of a qualitative (shift of discoloration
by filtration towards caramels) and quantitative (distance
between raw sugar and permeates) nature. The advantage
of this method is that it does not require an arbitrary choice
of a wavelength to separate the Hλf from the Lλf. It takes
account of absorbance at all wavelengths of the UV spectra
without bias. In this way, if two samples are different in
terms of their spectra, a distinction will always be made.
Conclusion
A literature study on different colorants allowed us to clari-
fy the nature and origins of these compounds and to estab-
lish a link with different stages of sugar processing. The
method suggested here for the preparation of these col-
orants is reproducible and provides a clearer understanding
of the kinetics governing their formation. Caramels were
obtained without acidifying the medium, thus proving that
their formation during sugar processing is not impossible. It
should also be remembered that HADP and melanoidins
have the same precursors, that they form at lower tempera-
tures and much more easily under the conditions prevailing
during sugar processing.
The visible spectra of the colorants were almost identical,
whereas the UV spectra intersected and exhibited different
shapes, which is why the UV domain has been used to form
the basis for distinction. Analysing the UV spectra of col-
orants by PCA, as suggested in this paper, has the same
advantages as the method proposed by Bento (1995), but
makes a better distinction between colorants as it takes into
account absorbance at all wavelengths of the UV spectra
without any bias. Considering the advantages of the PCA
method, it will be interesting to apply it to samples collected
at different stages of sugar processing in order to monitor
their evolution. It would also be interesting to compare dif-
ferent discoloration processes. Crystallization tests could
also be conducted on pure sucrose and model colorants, in
order to assess their occlusion into crystals and to determine
their nature by spectral analysis using the PCA method.
This is the edited version of the paper first published in
Industries Alimentaires et Agricoles, 2001, 30:42-51
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