Determination of Reducing Sugars, Total Reducing Sugars, Sucrose and Starch

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Determination of Sugar Solutions Color According to ICUMSA / Application

Note Analytical Chemistry
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Application NoteNote
Determination of Sugar Solutions Color

Application
According to ICUMSA
1. Introduction
The measurement of both degrees Brix and solution color are required in sugar manufacturing industries, as a
mean to grade product concentration, quality, or in blending operations. In fact, the formation of color is the result
of specific steps of the manufacturing processes. In sugar refining, the more processing steps raw sugar is
subjected to, the more color is removed: pigmentation of the product reflects the degree of refining to which it has
Multiparameter
been subjected. It helps to think of the process of refining as a series of steps, going from higher-colored raw sugar
with lower market value, to very low-colored, refined white sugar with the highest market value.
Multiparameter
Fig. 1: Sugar refining is a series of steps (left to right), where color and non-sugars are concentrated to the left,
while pure sugar is concentrated to the right. Raw sugar comes into the process to left-of-center, not at one end.
Several scales for degrees Brix and color measurement are used in the food industry. ICUMSA (International
Commission for Uniform Methods of Sugar Analysis) is a world-wide industry body that is concerned with
analytical methods for the sugar industry. Many ICUMSA methods are used for the determination of sugar color in
solution [1-5], and those are summarized in Table 1.
Table 1: ICUMSA methods for determine the color of sugar solutions.
Method Title Year Official

Determination of the solution colour of raw sugars, brown sugars and coloured
GS1/3-7 2011
syrups at pH 7.0
GS2/3-9 The determination of sugar solution colour at pH 7.0 2005
GS2/3-10 The determination of white sugar solution colour 2011
GS2/3-18 The determination of the turbidity of white sugar solutions 2013
GS9/1/2/3-8 The determination of sugar solution colour at pH 7.0 2011
METTLER TOLEDO Page 1 of 17

DE/RE-UV/VIS_01 v5.sg
1.1. Dictionary of sugars
Multiparameter Application Note
Raw sugar is the portion from the initial production process. It has a very high color value. Raw sugar must be
refined and purified prior to consumption.
Brown sugar and natural sugar are purified versions, and the color still has a high value.
Refined and white sugar comes from raw sugar that is refined to extract impurities, and so has a low color value.
This type of sugar is widely consumed among general households and used as raw material in food manufacturing
plants, where moderately-purified sugar is needed, e.g. for drinks, sweetened condensed milk, etc.
Super-refined sugars are those having undergone the refining process as white sugar, but they are purified to even
a greater extent, resulting in a very low color value. The outstanding characteristic is its high purity. Most super-
refined sugars are used in the food & beverage and in pharmaceutical industries.
Molasses is a valuable by-product of sugar processing. It is mainly used as a raw material for the production of
ethanol, liquor, animal feed, etc.
2. Material & methods
2.1. Instruments and accessories
®
Fig. 2: LabX enables the variety of laboratory line instruments to seamlessly integrate into a multi-parameter
platform. The highlighted instrumentation is used in this work.
 RM40 refractometer (51337003), UV5 Spectrophotometer (30254725), S400 pH meter (30046240) and
XP 205 Analytical balance (11106027)
®
 LabX software (30247984)
 Quartz cuvettes 1.0 cm (30258738), 2.0 and 4.0 cm (commercial brand) and 5.0 cm (30258739)
 Cellulose nitrate membrane filters, pore size 0.45 µm, vacuum pump and Büchner funnel

2.2. Samples and reagents
 Different sugar samples
 Neutralizing agents:
o For GS1/3-7: Hydrochloric acid (HCl; aq) (0.1M), Sodium hydroxide (NaOH; aq) (0.1M)
o For GS2/3-9: Triethanolamine (TEA)/Hydrocholoric acid (HCl) pH 7.0 buffer solution (aq)
o For GS9/1/2/3-8: MOPS (3-(N-morpholino)propanesulphonic acid)/Sodium hydroxide (NaOH) pH 7.00

buffer solution (aq)
 Deionized water
3. Measurement
Table 2: Differences in the various ICUMSA methods
20
Cell length pH nD ICUMSA Transmittance
Method Filtration Rounding
(b) in cm regulation (SPS-3) units in %
GS1/3-7 1; 2; 5 HCl/NaOH Yes RDS  250 20  T  80 10
GS2/3-9 4 TEA/HCl Yes RDS*0.989  600 – 1
GS2/3-10 4 – Yes RDS  500 – 1
GS2/3-18 5 – No RDS  500 – 1
GS9/1/2/3-8 1; 2; 4; 5 MOPS Yes –  16 000 15  T  80 1 or 10
3.1. Sample preparation
Weigh quantities of sugar or syrup and water, as shown in the corresponding ICUMSA method tables. Depending
on the color range, sample aliquots and cell path lengths are selected that provide a transmittance in a preferred
range. Dissolve samples by swirling at room temperature.
Depending on the method (see Table 2), pH is measured and adjusted by using buffering or neutralizing agents,
prior to color determination.
Pass the sample solution through an appropriate filter. If necessary, deaerate the filtered solution at room
temperature.
3.2. Procedure
The principles of UV/VIS spectroscopy are described by The Lambert-Beer law: absorbance (A) maintains a linear
relationship with the path length of the instrument (b), the concentration of the analyte (c), and the molar extinction
coefficient of the analyte (a). The subscript  denotes wavelength dependence. This relationship is mathematically
expressed as:

A = a  b  c (Eq.1)
a is also known as absorbancy index of the analyte. The value of the absorbancy index (extinction index) at the
wavelength of 420 nm is multiplied by 1000, and is subsequently reported as ICUMSA color. The resulting values
are designated as ICUMSA Units (IU). If the solution is adjusted to pH 7.0, the units are designated as ICUMSA
Units at pH 7.0 (IU7.0).
Initially measure the refractometric refractive index of the dry substance (RDS) of the solution, using the ICUMSA
method [6]. With the RM40 refractometer, the ICUMSA table is already built-in. Use the RDS to obtain the density of
3 th
the test solution,  in kg/m , from ICUMSA equation [7]. This is a polynomial equation of 6 order, and can be
conveniently programmed as a calculation method function. The concentration of the test solution is described by:
5
c = (RDS)/10 g/mL (Eq.2)
A UV5 spectrophotometer is used to determine absorbance (A420nm) of the sample with water serving as reference
standard for zero absorbance (blank).
From the definition, ICUMSA Color = 1000a420nm, we obtain:
8
ICUMSA Color = (1000A420nm)/(bc) = (10 A420nm)/(bRDS) (Eq. 3)
Finally, color is calculated in ICUMSA units and expressed to the nearest 1 or 10 IU.
3.3. RM40 parameters
Measuring temperature 20.00 °C

Result 1 T[Brix_ICUMSA_nD(nD)]
Result 2 Density calculation via ICUMSA formula [6]
Result 3 Concentration calculation via ICUMSA formula [7]
3.4. UV5 method parameters
Method Fixed wavelength

Path lengths 1.0, 2.0, 4.0, or 5.0 cm
Measurement time 3s
Wavelength 420.0 nm
Background correction 1-point at 720.0 nm
Result 4 ICUMSA color: (A420*1000)/(b*concentration) [IU]

4. Results
4.1. Method GS1/3-7
The method can be applied to raw sugars, affined raw sugars, higher color plantation white sugars and partly
refined and brown sugars. Is designed for sugars having colors greater than 250 ICUMSA units at pH 7.0 (IU 7.0).
Table 3: Optical cells, reagents, settings, range and limits for method GS1/3-7.
20
Cell length nD Transmittance
pH regulation Filtration IU Rounding
(b) in cm (SPS-3) in %
1; 2; 5 HCl/NaOH yes RDS  250 20  T  80 10
20
In Table 3 and Fig. 3 the raw data (nD and A) and the ICUMSA color of five consecutively investigated specimen
of a raw brown sugar are reported.
20
Table 4: Repeatability of refractive index (nD ) and absorbance (A) measurements for raw brown sugar (n=5).
20
Measurement nD A
1 1.38060 0.2016
2 1.38050 0.2018
3 1.38050 0.2029
4 1.38050 0.2014
5 1.38050 0.2018
Mean 1.38050 0.2019
S 0.00004 0.0006

Fig. 3: Repeatability of ICUMSA color results for raw brown sugar measurements (n=5). Dotted lines indicate the
maximum absolute difference allowed for measurement repeatability, as indicated in ICUMSA GS 1/3-7 (110 IU7.0
for this sample and color range).
ICUMSA defines the maximal permitted repeatability as the absolute difference between two results obtained under
repeatability conditions.
In Table 5 and Fig. 4 results of ICUMSA color determination of five consecutively investigated specimen of white-
and brown raw sugars are reported. Due to different solution concentrations, varying cell path lengths are used. For
White 1 and White 2 raw sugar samples, path lengths of 5 and 2 cm are used, respectively. For brown raw
sugars, cuvettes maintaining 1 cm path lengths are used.

Table 5: Mean values from measured raw sugar samples (n=5) for ICUMSA color along with repeatability data for
both the measured and permitted repeatability. All values are presented in IU7.0.
Max. permitted
Sample Color Measured repeatability
repeatability*
White 1 3280 30 N/A
White 2 3340 30 N/A
Brown 1 3600 20 110
Brown 2 4080 10 300
Brown 3 5310 30 300
* as defined by ICUSMA Method GS1/3-7
Fig. 4: ICUMSA color results for white and brown raw sugars (n=5). Dotted lines indicate the maximum absolute
difference allowed for measurement repeatability, as indicated in ICUMSA GS 1/3-7 for sample and color range.

4.2. Method GS2/3-9
This method can be applied to all crystalline, powdered white sugars and very pure syrups of color values up to
600 IU7.0.
20
pH regulation Filtration IU Rounding
4 TEA/HCl yes RDS*0.989  600 – 1
In Table 7, results of ICUMSA color for two white sugars are reported. Sugars are dissolved in TEA/HCl buffer
solutions adjusted to a pH of 7.0. The concentration of sample solids in solution is calculated from the RDS
measurements. To allow for concentration of TEA/HCl buffer in the test solution, measured RDS values must be
multiplied by 0.989, in order to express the "corrected RDS". For this measurement, a cuvette maintaining a 5 cm
path lengths was used.
Table 7: Mean values from measured white sugar samples (n=5): ICUMSA color along with data for both the
measured and permitted repeatability. All values in IU7.0.
Max. permitted
repeatability*
White 1 386 3 49
White 2 362 0 44
* as defined by ICUSMA Method GS2/3-9

4.3. Methods GS2/3-10, GS2/3-18
GS1/3-10 is the method used for determining white sugar solution color values for all crystalline or powdered white
sugars, and very pure syrups. GS2/3-18 is the method for determining turbidity in white sugar, and is a derived of
ICUMSA method GS1/3-10. Turbidity is calculated as the difference in color of white sugar solutions before and
after filtration. For both methods, a pH adjustment is not needed. Resulting values are designated as ICUMSA
Units (IU).
20
Cell length pH nD Transmittance
Method Filtration IU Rounding
(b) in cm regulation (SPS-3) in %
GS2/3-10 4 – yes RDS  500 – 1
GS2/3-18 5 – no RDS  500 – 1
Fig. 5: ICUMSA color repeatability results for crystalline white sugar measurements (n=5). Dotted lines indicate the
maximum absolute difference allowed for measurement repeatability, as indicated in ICUMSA GS 2/3-10 (3 IU).

4.4. Method GS9/1/2/3-8
This method is applicable for plantation white sugars, raw sugars, affined raw sugars and very low-color sugars. It
is designed for all sugars in the solution color value up to 16 000 ICUMSA Units at pH 7.0 (IU7.0).
Table 9: Optical cells, reagents, settings, range and limits for method GS9/1/2/3-8.
20
pH regulation Filtration IU Rounding*
1; 2; 4; 5 MOPS yes –  16 000 15  T  80 10 or 1
* for results over 1000 IU7.0, nearest 10 IU7.0; for results under 1000 IU7.0, nearest 1 IU7.0.
In Table 10 and Fig. 6, results for ICUMSA color of five consecutively-investigated specimen of different white-,
brown- and raw sugars are reported. Due to differences in solution concentrations, varying cell path lengths were
used. For white 1, 2 and 3 sugars, a 5 cm path length was used. For brown 1 and 2 sugars, cuvettes maintaining
a 2 cm path length were used. For raw 1 and 2 sugars, cuvettes with 1 cm path length were used.
Table 10: Mean values (n=5) from measured white, brown and raw sugar samples: ICUMSA color, measured and
permitted repeatability. All values in IU7.0.
Max. permitted
repeatability*
White 1 3397 32 339
White 2 3183 33 339
White 3 3264 34 323
Brown 1 3673 12 348
Brown 2 3732 39 348
Raw 1 3070 10 220
Raw 2 3590 10 220
Raw 3 3850 10 220
* as defined by ICUSMA Method GS9/1/2/3-8

Fig. 6: ICUMSA color results for different white, brown and raw sugars (n=5). Dotted lines indicate the maximum
absolute difference allowed for measurement repeatability, as indicated in ICUMSA GS9/1/2/3-8 for sample and
color range.

4.5. The Effect of pH
It is well known that absorbance values of sugar solutions are pH-dependent. In turn, this can modify the ICUMSA
Color [8], especially at high color values.
To illustrate this observation, an experiment on brown sugar samples was performed to determine subsequent
ICUMSA color as function of differing pH of the solutions. After sugar dissolution, a pH of 6.43 was obtained. As
described in method GS1/3-7, the appropriate pH adjustment is performed with the addition of either HCl or NaOH.
The upper diagram in Fig. 7 demonstrates the pH-dependence of the ICUMSA color profile of a brown sugar
solution. The relationship is sigmoidal in nature: an absorbance increase occurs when the pH value increased,
whereas the reverse trend is observed by lowering pH. An interpolation of data points is carried out using a
generalized logistic functional form. The latter is used for illustrative purposes, and serves to calculate the first
derivative. The lower diagram of Fig. 7 depicts the first derivative of the respective color by pH of 0.1 units.
The absolute value of ICUMSA color for this brown sugar solution at pH 7.0 was 610 IU7.0. At a pH of 6.43, just
after sugar dissolution and without adjusting pH, a color value of 525 IU was obtained. The difference between the
two results amounted to 85 IU. Analysis of the first derivative indicated that, at around pH 7  0.1 units, a
corresponding difference of 18.2 IU was observed. Based on these two observations, the following conclusions
were reached: pH values substantially impact measurements, and accurate determination of pH is essential to
repeatable and reproducible data. Thus, ICUMSA color values are inherently sensitive to even small variations and
errors in pH determination or pH solution adjustment.

Fig. 7: The upper diagram depicts brown sugar solution ICUMSA color as function of pH. The lower diagram reports
first derivative values as a function of 0.1 pH.
It has been reported that results from the lower color range up to about 60 IU are nearly equal in both pH corrected
and non-corrected methods [9], and are well-correlated. The results obtained for higher color ranges show greater
deviations between both methods, and those results obtained without pH adjustment were much lower. Therefore,
for color values over 60 IU, solution color is pH-dependent. If a sugar has a solution color less than 60–65 IU,
then the simpler, more eco–friendly method, GS2/3-10, can be used. Irrespective of the sugar solution color
measured, the recommendation is to always state the method used in determining the color value.

4.6. The relationship between path length and linearity
For optimal measurements that comply with the Lambert–Beer law, absorbance must be in the linear range, and is
therefore dependent on concentration of the analyte path length and instrument performance.
Absorbance A of a sample is calculated via transmittance, and is expressed as -log10T. This is why some ICUMSA
methods specify working with a given path length or transmittance interval. For low IU sugars, cell lengths no
smaller than 4 or 5 cm are recommended (GS2/3-9, GS2/3-10 and GS2/3-18). However, a fundamental question
still remains: how to achieve results within the range of linearity for a given path length and instrument selected to
measure a specific sample concentration? One solution available to resolve this challenge is to simply measure a
series of the same sample with varying concentrations.
Absorbance values for sugar solutions are measured using a fixed path length and a series of diluted samples, as
shown in Fig. 8. The initial stock concentration used is indicated as c0/1, and successive 50-50% solution–water
dilutions were measured sequentially. As shown in the graph, all sample concentrations tested returned measured
absorbance values within the range of linearity of the path length and instrument used.
According to the linear relationship described by The Lambert–Beer law, the absorbancy index a is an intrinsic
property of the absorbing species, and this value does not change unless the chemical nature of the species
changes. Hence, decreasing the concentration will have the same resultant effect as increasing path length. Thus,
in the linear range, dividing c0 by a factor, corresponds to multiply the path length b0 by the same amount, as
represented in the alternative x–axis (top of the diagram).

Fig. 8: Lambert–Beer linearity: measured values are in the linear range of absorbance.
For this experiment, c0 corresponds to 0.5859 g/mL, and b0 is 1.0 cm. Fig. 8 demonstrates absorbance values
with a very high degree of linearity across large concentration and path length ranges. By varying these two
parameters, sample concentrations can be maintained within the range of linearity for the instrument, as described
by the Lambert–Beer relation. This is further indicated by the linear interpolation, y = 1.090x - 0.01, where the
2
slope and offset are close to unity and zero respectively. The correlation coefficient (R ) approaches 1.000. These
data support the use of standard-sized cuvettes (1.0 cm) as sufficient for determining ICUMSA colors. Using larger
path lengths does not improve quality of the results.

4.7. Miscellany
Software associated with instrumentation is programmed to process the results algebraically from concentrations to
ICUMSA color calculations. Results are generated fully automatically, avoiding tedious data transcription and
manual calculations. RDS and  are also automatically retrieved, either by table lookup or using a polynome.
Depending on the ICUMSA method, aliquots of sugar and water will differ, and must be within specified tolerances.
The same is true for pH, transmittance and cell length. Repeatability thresholds are also tabulated. Automatic
parameter selection, tolerances control and documentation are operations carried out by software associated with
the instrumentation.
4.8. An infrastructure for quality control
Harvesting sugarcane or sugar beet is a seasonal activity, and therefore occurs during a defined and limited time of
the year. Often very large harvest quantities are involved. The juncture between these two factors poses major
challenges to sugar manufacturers, since the incoming raw sugar must be rapidly processed. Effective
TM
automatization solutions like InMotion and SC30 autosampler can be used to increase testing throughput.
Additionally, software support is also available to process the flow of results for quality control purposes. Statistical
evaluations can be routinely used to test results against a target value or tolerance, generating automatic Pass/Fail
reports.
5. Conclusions
ICUMSA color of sugar solutions is calculated as function of sugar concentration and UV/VIS absorbance: refractive
index of the solution and absorption at 420 nm are measured. Final results are given in ICUMSA Units (IU or IU7.0,
rounded or not), and calculated from the sugar concentration, the absorption value and the path length used for
®
measurement. The sugar concentration is determined using Brix value and specific ICUMSA formulae. With LabX ,
the laboratory Software for full user guidance and automatic data handling, the data access method function can be
used to transfer all results to a calculation method. Thus, the ICUMSA color is fully calculated automatically within
®
LabX .
®
Using LabX PC Software, limits can be easily set for ICMUMSA color, with easy implementation of automatic
tolerance checks of measurement results. In the result overview, customized text and background color codes can
be used for easy identification of sugars for pass/fail tolerance checks.
For all investigated sugar solutions, repeatability of the measurements was excellent and well within the maximally-
tolerated repeatability criteria. In summary, we establish a highly reproducible method, and offering fully automated
ICUMSA color calculations.

References
[1] ICUMSA Method GS1/3-7 Determination of the Solution Colour of Raw Sugars, Brown Sugars and Coloured
Syrups at pH 7.0 – Official (2011)
[2] ICUMSA Method GS2/3-9 The determination of sugar solution colour at pH 7.0 – Official (2005)
[3] ICUMSA Method GS2/3-10 The determination of white sugar solution colour – Official (2011)
[4] ICUMSA Method GS2/3-18 The determination of the turbidity of white sugar solutions – Official (2013)
[5] ICUMSA Method GS9/1/2/3-8 The determination of sugar solution colour at pH 7.0 – Official (2011)
[6] ICUMSA Specification and Standards SPS-3 Refractometry and Tables – Official (2000)
[7] ICUMSA Specification and Standards SPS-4 Densimetry and Tables – Official (1998)
[8] K. Sing et al., Recent developments in white sugar solution colorimetry, Sugar Industry (3), 2013:159–163.
[9] H. Puke, C. Lakenbrink, A treatise on ICUMSA solution colour – Part 2: Studies and conclusions, Sugar
Industry (3), 2013:153–158.
Disclaimer
This report presents one or more examples of analysis. The experiments were conducted with the utmost care using
the instruments specified in the description of each application.
The results have been evaluated according to the current state of our knowledge. This does not however absolve
you from personally testing the suitability of the examples for your own methods, instruments and purposes. Since
the transfer and use of an application is beyond our control, we cannot of course accept any responsibility.
When chemicals, solvents and gases are used, the general safety rules and the instructions given by the
manufacturer or supplier must be observed.
Further information
www.mt.com/DERE
www.mt.com/UVVIS
https://youtu.be/lZWj_Y9PrWg Video-URL

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Determination of Sugar Solutions Color According to ICUMSA / Application

Chapter · January 2018

The user has requested enhancement of the downloaded file.

Determination of Sugar Solutions Color

Table 1: ICUMSA methods for determine the color of sugar solutions.

Method Title Year Official

METTLER TOLEDO Page 1 of 17

2. Material & methods

2.1. Instruments and accessories

METTLER TOLEDO Page 2 of 17

 Different sugar samples

o For GS9/1/2/3-8: MOPS (3-(N-morpholino)propanesulphonic acid)/Sodium hydroxide (NaOH) pH 7.00

Table 2: Differences in the various ICUMSA methods

3.1. Sample preparation

METTLER TOLEDO Page 3 of 17

From the definition, ICUMSA Color = 1000a420nm, we obtain:

3.3. RM40 parameters

Measuring temperature 20.00 °C

3.4. UV5 method parameters

Method Fixed wavelength

METTLER TOLEDO Page 4 of 17

4.1. Method GS1/3-7

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4.8. An infrastructure for quality control

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