Application Note 92

The Determination of Sugars in Molasses by

High-Performance Anion Exchange with
Pulsed Amperometric Detection
INTRODUCTION greater than 12 facilitates the oxidation reaction at the
The accurate measurement of the amount of sugar working electrode and the pulsed potentials keep the
in final molasses is economically important to sugar electrode clean.
mills, because the purchase price of the final molasses Chemists at the Sugar Milling Research Institute
is directly related to its sugar content. have used HPAE-PAD to measure glucose, fructose, and
Historically, gas chromatography (GC) has been sucrose in cane molasses samples.4 J. Thompson refined
used extensively for sugar analysis. While GC results this method with the use of internal and external stan-
are accurate, the analytical process involves a time- dards.5 C. Day-Lewis compared this improved method
consuming derivatization procedure. High-performance with the official GC method and found that the two
liquid chromatography (HPLC) was developed in the methods agreed with respect to precision and accuracy.3
early 1970s, and sugar mill chemists had hopes of using An interlaboratory study using this method has been
this new technique for their sugar analysis. However, completed and approved by the International Commis-
amino-bonded silica columns gave imprecise results for sion of Uniform Methods for Sugar Analysis (ICUMSA).
reducing sugars.1 In final molasses samples, cation Submission to the Association of Official Analytical
exchange columns gave slightly higher glucose and Chemists International (AOAC International) is also
sucrose response and much higher fructose response planned.
when compared with gas chromatography results.2 The
discrepancies were attributed to impurities in the EQUIPMENT
molasses that coeluted with the sugars. The nonspecific Dionex chromatography system consisting of:
refractive index detection used for these analyses could High-Performance Pump
not differentiate between the sugars and other sub- Liquid Chromatography Module
stances present in the molasses.
Pulsed Electrochemical Detector
A high-performance anion exchange method with
Eluent Organizer or Eluent Degas Module
pulsed amperometric detection (HPAE-PAD) has
recently been developed to accurately determine the
sugar concentrations of cane and beet final molasses Dionex PeakNet Chromatography Workstation or
samples.3 Carbohydrates are weak acids with pKas AI-450 Chromatography Workstation
above 11. The use of sodium hydroxide as an eluent
promotes ionization of the carbohydrates to their REAGENTS AND STANDARDS
anionic form. The pellicular resin of the anion ex- Deionized water, 18 MΩ-cm resistance
change column is stable and durable within the 1–14 Sodium hydroxide solution, 50% w/w, low carbonate
pH range. PAD is highly specific when using pulsed Glucose, fructose, lactose, sucrose, and raffinose
potentials optimized for carbohydrates. An eluent pH of
CONDITIONS imately 50 mL of deionized water, transfer it to a
Columns: ™
CarboPac PA1 Analytical (4 x 250 mm) 100 mL volumetric flask, and dilute to the mark with
and guard (4 x 50 mm) deionized water. Dilute 1 mL of this stock cane sample
Expected Operating to the mark of a 100-mL volumetric flask with deion-
Pressure: 8–10 MPa (1200–1500 psi) ized water. Filter this sample through a 0.45-µm filter.
Inj. Volume: 50 µL Make a duplicate sample in a separate container.

Eluent: 150 mM Sodium hydroxide (NaOH)

Beet
Flow Rate: 1 mL/min Weigh 0.7 g of beet sugar in a wide-mouth con-
Detection: Pulsed amperometry, gold working tainer. Add 5 mL of the lactose solution, dissolve in
electrode standard carbohydrate approximately 50 mL of deionized water, transfer it to
settings a 100-mL volumetric flask, and dilute to the mark with
deionized water. Dilute 1 mL of this stock beet sample
Note: See Dionex Technical Note 216 for a discussion of
Pulse Potentials.

PREPARATION OF SOLUTIONS AND REAGENTS

Eluent: 150 mM Sodium Hydroxide Table 1 Cane Molasses
Dilute 7.8 mL of sodium hydroxide solution
Sugars Std C1 Std C2 Std C3
(50% w/w, low carbonate) in 1.0 L of helium sparged
deionized water. Sodium hydroxide pellets are coated Glucose (g) 0.02 0.06 0.10
with a layer of carbonate, and will not produce an accept- Fructose (g) 0.03 0.07 0.11
able eluent.
Sucrose (g) 0.25 0.31 0.37
Lactose Solution (mL) 5.00 5.00 5.00
CALIBRATION STANDARD
Lactose Solution (32g/L)
Lactose is used as the internal standard. In a 250-mL
volumetric flask, dissolve 8 g of lactose in deionized water
Table 2 Beet Molasses
and dilute to the 250-mL mark with deionized water.
Sugars Std B1 Std B2 Std B3
Raffinose Solution (7.05g/L)
Weigh 0.14 g of raffinose and 19.86 g of deionized Sucrose (g) 0.25 0.30 0.35
water into a container. Weight/weight measurement is Raffinose Solution (mL) 1.00 1.50 2.00
used here for precise concentration. Lactose Solution (mL) 5.00 5.00 5.00

Molasses Calibration Standards

Dissolve and dilute each of the standard sets in Table 1
(Cane Molasses) and each of the standard sets in Table 2 to the mark of a 100-mL volumetric flask with deion-
(Beet Molasses) in individual 100-mL volumetric flasks, ized water. Filter this sample through a 0.45-µm filter.
with deionized water to the 100-mL mark. Dilute 1 mL of Make a duplicate sample in a separate container.
each stock standard to the 100-mL mark with deionized
water in another 100-mL volumetric flask. EXPERIMENTAL SETUP
The same protocol applies for both cane and beet
SAMPLE PREPARATION samples. The three calibration standards are initially
Cane run in sequence and sucrose is checked for linearity.
Weigh 1.0 g of cane sugar in a wide-mouth container. For the sample runs, the middle calibration standard is
Add 5 mL of the lactose solution, dissolve in approxi- run first, followed by the two duplicates of a molasses
 EXAMPLE 1: Example of calculations for determining sucrose in final cane molasses

Calibration Mass for Sucrose in C2 run before molasses sample ..... 0.3100 g RRF of sucrose before sample:
Calibration Mass for Sucrose in C2 run after molasses sample ........ 0.3100 g 0.3100 161321 = 2.36 {1}
x
Calibration Height for Sucrose in C2 run before molasses sample ..... 132444 132444 0.160
Calibration Height for Sucrose in C2 run after molasses sample ........ 132438
RRF of sucrose after sample:
Calibration Mass for Lactose in C2 run before molasses sample ....... 0.160 g 0.3100 161830 = 2.37 {1}
x
Calibration Mass for Lactose in C2 run after molasses sample .......... 0.160 g 132438 0.160
Calibration Height for Lactose in C2 run before molasses sample ..... 161321
Calibration Height for Lactose in C2 run after molasses sample ........ 161830 Average of the RRFs:
2.36 + 2.37 = 2.36 {2}
2
Mass of molasses sample .................................................................. 1.0725 g Percentage of sucrose in the molasses sample:
Height for Sucrose in molasses sample .............................................. 144579 144579 x 0.160 100 = 30.8% {3}
Mass for Lactose in molasses sample ................................................. 0.160 g x 2.36 x
165183 1.0725
Height for Lactose in molasses sample ............................................... 165183

sample, followed by another middle calibration stan- Step 1: Determine the average of the RRFs for sucrose
dard, followed by the two duplicates of a different from the standards immediately before and after the
molasses sample, etc. This pattern is repeated until all final molasses sample by using the following
molasses samples have been run. The middle calibration equation:
standards that bracket the molasses sample runs are
used to determine the relative response factors (see the RRFave = RRF1 + RRF2 {2}
“Calculations” section ). 2

CALCULATIONS where: RRFave = Average RRF for sucrose

Relative Response Factors (RRFs) are calculated for RRF1 = RRF of sucrose before the cane
each sugar (i.e., glucose, fructose and sucrose for cane; molasses sample
and sucrose and raffinose for beet) of each calibration RRF2 = RRF of sucrose after the cane
standard that is run. For example, the RRF of sucrose in molasses sample
a standard can be calculated by using the following
equation: Step 2: Determine the percentage of sucrose in the final
cane molasses sample by using the following
Msuc-std Hlac-std {1} equation:
RRF = x
Hsuc-std Mlac-std
%suc = Hsuc-smp x Mlac-smp x RRFave x 100 {3}
where: RRF = Relative Response Factor Hlac-smp Mmol-smp
Msuc-std = Mass of sucrose standard (g)
Hsuc-std = Peak height of sucrose standard where: %suc = Percentage of sucrose in the sample
Mlac-std = Mass of internal lactose standard (g) Hsuc-smp = Peak height for sucrose in the sample
Hlac-std = Peak height of lactose standard Hlac-smp = Peak height for lactose in the sample
Mlac-smp = Mass of internal lactose standard (g)
The sugar percentages in each final molasses sample RRFave = Average RRF taken from Step 1
can then be calculated in two steps (see Example 1). Mmol-smp = Mass of molasses (g)
Relative Standard Deviation (RSD) should not be For further details concerning pulse sequences used
greater than 1% for duplicates of sucrose, should be in pulsed amperometric detection, refer to Dionex
less than 2% for duplicates of glucose and fructose, and Technical Note 21.6 To drive the oxidation reaction at
should be less than 6% for duplicates of raffinose. If the the working electrode of the detector, the eluent pH
RSD is not within these guidelines, the sample should should be greater than 12. For further details concerning
be reinjected or prepared again. carbohydrate determination, refer to Dionex Technical
Note 20.7
RESULTS AND DISCUSSION
The CarboPac PA1 column reproducibly separates
the sugar components of cane and beet final molasses. Peaks: 1. Glucose 4.39 %
3
PAD, with pulse potentials optimized for carbohydrates, 2. Fructose 6.67
8 2 3. Lactose Standard
permits detection of the sugars in final molasses sam- 4. Sucrose 30.8
4
ples without interferences from coeluting components,
if any. 1

Figure 1 shows the separation of the sugars in cane

final molasses and the internal standard, lactose. The
elution order is glucose, fructose, lactose, then sucrose. µA

After performing the experiment, the percentage of each

sugar is calculated (see Example 1). The expected
ranges for cane final molasses are 2–10% for glucose,
3–11% for fructose, and 25–37% for sucrose. The
percentages determined for this sample, as shown in 3
Figure 1, are 4.39% for glucose, 6.67% for fructose, and
0 2 4 6 8 10 12 14
30.8% for sucrose.
Minutes
Figure 2 shows the separation of the sugars in beet
final molasses and the internal standard, lactose. The
elution order is lactose, sucrose, then raffinose. The Figure 1 Sugar cane sample prepared and run by this method.
percentage of each sugar is calculated (as in Example 1).
The expected ranges for beet final molasses are 35–50%
for sucrose and 1–2% for raffinose. The percentages
Peaks: 1. Lactose Standard
determined for this sample were 47.7% for sucrose and 2. Sucrose 47.7 %
2.10% for raffinose. Because of the low concentration 3. Raffinose 2.10

of raffinose in beet molasses and the high dilution factor 9

1

required for optimal sucrose analysis, the raffinose 2

percentage is less precise than the sucrose percentage.

When necessary, the raffinose can be determined
separately using the first 1:100 dilution, rather than the
second 1:100 dilution. µA

HPAE-PAD methodology uses pellicular anion

exchange resin technology coupled with selective
amperometric detection. The monosaccharides will
elute first, followed by disaccharides and then trisaccha- 3
3
rides. Pulsed amperometric detection uses a repeating
sequence of three potentials, which are applied for specific 0 5 10 15 20
durations. Using the pulsed conditions in Technical Note Minutes

21,6 detection is optimized for carbohydrates.

Figure 2 Sugar beet sample prepared and run by this method.
PRECAUTIONS
Metal should be eliminated from the eluent flow
path, including the injection valve, prior to the column.
Metal contamination of the analytical column can result
in poor peak efficiency and/or symmetry, which may
lead to poor reproducibility.

REFERENCES
1. Kort, M.J. High-Pressure Liquid Chromatography,
SMRI Annual Report for 1979–1980, 1980, 13–14.
2. Day-Lewis, C.M.J.; Schäffler, K.J. HPLC of Sugars:
The Analytical Technique for the 1990’s?, Proceed-
ings of the Fourth African Sugar Technologists
Association, Durban, Natal, South Africa, 1990, 64,
174–178.
3. Day-Lewis, C.M.J.; Schäffler, K.J. Analysis of
Sugars in Final Molasses by Ion Chromatography,
Proceedings of the South African Sugar Technolo-
gists Association, Durban, Natal, South Africa,
June 1992, 131–135.
4. Morel du Boil, P.G.; Schäffler, K.J. Ion Chromatog-
raphy: A Comparison Between Anion and Cation
Exchange HPLC for Carbohydrates, Proceedings of
the 1990 Sugar Processing Research Conference,
Sugar Processing Research Inc., San Francisco,
California, USA, May 29–June 1, 1990, 397–412.
5. Thompson, J.C. Methods for the Determination of
Carbohydrates by Ion Chromatography, Proceedings
of the 1990 Sugar Processing Research Conference,
Sugar Processing Research Inc., San Francisco,
California, USA, May 29–June 1, 1990, 381–396.
6. Dionex Technical Note 21: “Optimal Settings for
Pulsed Amperometric Detection of Carbohydrates
using the Dionex Pulsed Electrochemical Detector
(PED-2) and the Pulsed Amperometric Detector
(PAD-2)”.
7. Dionex Technical Note 20: “Analysis of Carbohy-
drates by Anion Exchange Chromatography with
Pulsed Amperometric Detection”.
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