Determination of Sugars in Molasses by HPLC Following

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Determination of sugars in molasses by HPLC following
Solid-Phase Extraction
a b ac
Wanxia Xu , Lei Liang & Mingjun Zhu
a
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou
Higher Education Mega Center, Panyu, Guangzhou, 510006, People’s Republic of China
b
Guangdong Key Laboratory of Sugarcane Improvement and Biorefinery, Guangzhou
Sugarcane Industry Research Institute, Guangzhou, 510316, People’s Republic of China
c
Shenzhen Key Laboratory of Fermentation, Purification and Analysis, Shenzhen
Polytechnic, Shenzhen Guangdong 518055, People’s Republic of China
Accepted author version posted online: 21 Mar 2014.
To cite this article: Wanxia Xu, Lei Liang & Mingjun Zhu (2014): Determination of sugars in molasses by HPLC following Solid-
Phase Extraction, International Journal of Food Properties, DOI: 10.1080/10942912.2013.837064
To link to this article: http://dx.doi.org/10.1080/10942912.2013.837064
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Determination of sugars in molasses by HPLC following

Solid-Phase Extraction
Wanxia Xu1, Lei Liang2, and Mingjun Zhu1,3,*
1
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou
Higher Education Mega Center, Panyu, Guangzhou, 510006, People’s Republic of China
Downloaded by [Baskent Universitesi] at 12:40 20 December 2014
2
Guangdong Key Laboratory of Sugarcane Improvement and Biorefinery, Guangzhou
Sugarcane Industry Research Institute, Guangzhou, 510316, People’s Republic of China
3
Shenzhen Key Laboratory of Fermentation, Purification and Analysis，Shenzhen Polytechnic,
Shenzhen Guangdong 518055, People’s Republic of China
Running head: Determination of sugars in molasses
*Address correspondence to Mingjun Zhu, School of Bioscience and Bioengineering, South

China University of Technology, Guangzhou Higher Education Mega Center, Panyu,
Guangzhou, 510006, People’s Republic of China. E-mail: mjzhu@scut.edu.cn
ABSTRACT
Fructose, glucose, and sucrose in sugarcane molasses are determined simultaneously by high
performance liquid chromatography (HPLC) using maltose as internal standard (IS) with
refractive index detector (RID). The mixture of the diluted samples and IS was purified with
Sep-Pak C 18 solid-phase extraction (SPE) and filtered through a 0.22-μm membrane before
injection. The results showed that the linear ranges for fructose, glucose and sucrose were
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3.30-16.48 g/L, 1.80-9.02 g/L and 5.94-29.70 g/L with the squared correlation coefficients (R2)
being 0.9986, 0.9987 and 0.9955, respectively. The method is simple, quantified, and
time-saving for determination of sugars in sugarcane molasses.
Keywords: solid-phase extraction, high performance liquid chromatography, sugarcane
molasses, sugars, internal standard.

INTRODUCTION
Sugarcane molasses, a by-product from sugarcane refining, is a thick and dark syrup, resulting
from the crystallization and removal of the majority of sucrose from the original juice. The current
total supply of molasses is 300-400 million tons per year in China. In general, molasses contains
about 50% sugar by dry weight, predominantly sucrose, fructose and glucose. Furthermore, unlike
refined sugars, molasses contains trace amounts of vitamins and several minerals. Because of its
unusual properties, molasses is widely used in baking, as an animal feed additive or as a
fermentation feedstock. Therefore, determining accurately the content of fructose, sucrose and
glucose in molasses is important for the development and effective utilization of molasses.
The conventional approaches used to determine sugars are either polarimetry or chemical assay
methods, such as Lane-Eynon method for determining total sugar[1] and DNS method for reducing
sugar.[2] Both the classical and official methods for determining sugars in molasses require
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considerable time, contain inherent errors, and are based on empirically derived constants.[3] The
analysis methods of the oligosaccharides include the near-infrared spectroscopy,[4] the thin layer
chromatography method, gas chromatography (GC)[5] and high performance liquid
chromatography (HPLC).[6, 7] GC has been used to separate carbohydrates employing several types
of derivatization modes. Since the nonvolatility of carbohydrates is caused by polar hydroxyl,
amino, and carboxyl groups, the derivatization of these groups can greatly increase the volatility of
carbohydrates, but the usual challenge to GC is the appearance of multiple peaks in the
chromatogram due to the presence of tautomeric forms of reduced sugars. The GC method is
particularly accurate in determining low sugar concentrations, but sample preparation is time
consuming. The HPLC method can directly determine oligosaccharide with a simple sample
preparation. So HPLC is one of the most promising methods for sugar analysis, due to its
universality, time efficiency, accuracy and selectivity for the quantification of carbohydrates.[8]
Authors such as Nejib et al.[9] and Sims[10] have reported the HPLC method as an established and
preferred method for the determination of individual sugars in carbohydrate mixtures, for its
accuracy and simplicity.
Generally, carbohydrates do not absorb ultraviolet (UV) and visible radiation or emit fluorescence
without suitable prior derivatization, because they possess neither chromophore nor fluophore.
Some carbohydrates absorb near UV radiation in the region of 180-220 nm. Based on
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measurement of the absorption in this range, however, the detection is nonselective. But
carbohydrates can be measured by High Performance Liquid Chromatography-Evaporative Light
Scattering Detector (HPLC-ELSD).[11] While HPLC-ELSD has high sensitivity and can use
gradient elution, it has good reproducibility only at low sugar concentrations. High-performance
anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is one of the
most useful techniques for oligosaccharide determination,[12] but amperometric detection

possesses poor stability, and new electrode materials have been constantly explored. High
Performance Liquid Chromatography-Refractive Index Detector (HPLC-RID) today have
achieved considerable improvement in constant velocity and optical element temperature control
for the detection stability. Thus, the HPLC- RID method is widely used in carbohydrate
analysis,[13] due to the reliability of the detection results of glucose, fructose, and sucrose. For the
above reasons, the HPLC-RID method was used in this study, together with an Agilent Zorbax
Carbohydrate column.
Although numerous methods have been published concerning the HPLC determination of
carbohydrates in natural products,[14, 15] the internal standard (IS) method has seldom been used.[16]
An IS preferred over the external standard (ES) because the IS can eliminate the problems
concerning the injection of exact volumes into the liquid chromatography, variations in mobile
phase composition, and potential changes in the ageing of the column. In this study, maltose was
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selected as the IS for the HPLC determination of the sugars because its physical and chemical
characteristics were similar to those of the three sugars and the used molasses contained no
maltose.[3, 17]
For the molasses detection by HPLC, different sample pretreatment procedures of decolorization
and purification were tested. This was necessary because of the presence of pigment and nitrogen
compound mixtures in molasses, which could interfere with the detection and could shorten the
lifespan of chromatographic column.[18] In this study, solid-phase extraction (SPE) was selected to
clean samples, and results were calculated from peak areas automatically generated by a
computing integrator. This work aimed to establish and to apply simple extraction and analytical
conditions to identify and quantify carbohydrates in molasses by HPLC using RID and an
amino-bonded silica column.
MATERIALS AND METHODS
Instrumentation and reagents
The HPLC system consists of a model 1525 Binary pump, a model 7725 manual-sampler, a model
2414 RID (Waters Assoc., Milford, MA, U.S.A) and Agilent Zorbax Carbohydrate column
(250×4.6 mm I.D., 5μm) protected with a guard column (12.5×4.6 mm I.D.). UtimateTM XB-NH 2
column (250×4.6 mm I.D., 5 μm) was from Welch Material Technology (Shanghai) Co., Ltd.
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UV2802SH-type ultraviolet-visible spectrophotometer was purchased from UNICO (shanghai)
Instruments Co., Ltd. (USA). TGL-19G Centrifuge (5 mL) was supplied by Shanghai Anke
company, Ltd. (China).
HPLC-grade acetonitrile (A998-4) was purchased from Thermo Fisher Scientific (U.S.A), and
HPLC-grade methanol was acquired from Kermel Chemical Reagent Co., Ltd. (Tianjin, China).
Water used in the study was double-deionized water (Milli-Q, Millipore Corp., Milford, MA) of
18.2 MΩ/cm resistivity. Molasses was obtained from Zhanjiang Sugarcane Research Center of
Guangzhou Sugarcane Industry Research Institute (Guangdong Province, China). HPLC grade
sugar standards (Sucrose, fructose, glucose and maltose) were supplied by Aladdin Chemistry Co.
Ltd. (China).
Membrane filters (0.22 μm) were purchased from MEMBRANA Company (Germany).
Macroporous resins (Seplite LX-38, LXA-8, LX-17, XDA-8) were acquired from Xi'an LanXiao
Technology Co., Ltd. (China) and resin D101 was from Tianjin Bochum Resin Technology Co.,
Ltd. (China). Waters Sep-Pak C 18 SPE cartridge (WAT020805) was purchased from Waters
Company (Milford, MA, U.S.A). The SPE column was preconditioned by passing it through 2.0
mL of methanol, followed by 4.0 mL of double-deonized water.
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Solutions
Mixed standard stock solution of sucrose, fructose and glucose was prepared by dissolving 2.970 g
of sucrose, 1.648 g of fructose and 0.902 g of glucose in 100 mL water, which was stable for ≥ 6
months at -20°C.The working solutions of sugars were prepared by pipetting 0.4, 0.8, 1.2, 1.6 and
2.0 mL of the above mixed standard stock solution into five 2 mL amber glass volumetric flasks,
and then making up to the mark with water, respectively. Maltose (IS) solution (10.095 g/L) was
prepared by dissolving 2.019 g of maltose in 200 mL deionized water, which was stable for ≥ 6
months at -20°C.
Calibration standard solutions were prepared by pipetting 1.0 mL of the IS stock solutions into five
2.0 mL amber glass volumetric flasks and making up to the mark with 1.0 mL of the above sugars
working solutions separately. Mobile phase (75% acetonitrile in water) was obtained by adding
250 mL deionized water to 750 mL acetonitrile and mixing well by sonication.
Sample preparation
The use of the linear calibration equations was mandatory in the indicated range, and thus the
molasses was required to be diluted by mass ratio as follows: 50 g of molasses was weighed and
poured into a 250 mL glass-stoppered Erlenmeyer flask before being diluted to 150 g with water,
and then mixed uniformly. 100-mL aliquot of solution was used for determination of the Brix (Bx).
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The above diluted molasses (26.7°Bx) was accurately measured (1.0 mL aliquot), poured into a
10-mL volumetric flask and diluted to the scale mark with water, which was used as the working
molasses solution. Next, 4.0 mL aliquot of the working molasses solution and 4.0 mL aliquot of IS
solution were added to 10-mL centrifuge tubes and mixed thoroughly, followed by the procedures:
(a) Direct injection—The sample-IS solution was filtered through 0.22-μm membranes
before injection.
(b) SPE cleanup—The sample-IS solutions were passed through the activated Sep-Pak C 18
SPE column like this: 8 mL sample-IS solutions was first poured into the activated C 18 SPE
cartridge, followed by inserting the plunger, discarding the initial 3-mL eluate and collecting the
follow-up 3-mL eluate which was passed through 0.22-μm membrane prior to HPLC at the
optimum chromatographic conditions.
(c) Macroporous resin decolorization method—3 mL of the above working molasses
solution was pipetted into 5 mL of centrifugal tubes and mixed with 50 and 100 mg of five
different Macroporous resins, respectively (Seplite LX-38, Seplite LXA-8, Seplite LX-17, Seplite
XDA-8 and D101). Next, the centrifugal tubes were sealed with PARAFILM and incubated at
30°C, 150 rpm for 3 h in the shaking table. After adsorption, 3 mL of liquid supernatant was
harvested by centrifugation for 10 min at 12000 rpm using a TGL-19G centrifuge. Finally, 1 mL of
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the harvested supernatant was used for the analysis of sugars using HPLC, and 2 mL of the
supernatant was used for the absorbance measurements using the UV2802SH-type
ultraviolet-visible spectrophotometer at a wavelength of 560 nm. The decoloration rate (Dec.) on
the samples was calculated as follows:
A 0－A
Dec. = ×100
A0
where A 0 is the original absorbance of the working molasses solution, and A is the absorbance of
the working molasses solution pretreated as described above.
HPLC
The chromatographic separation of sugars (sucrose, fructose and glucose) and the IS method was
achieved with a Waters HPLC system in an isocratic mode. The samples were analyzed on a
Carbohydrate column (250×4.6 mm I.D., 5μm,) equipped with a guard column (12.5×4.6 mm
I.D.). The column and RID temperatures were set at 30 and 35°C, respectively. The mobile phase
was composed of acetonitrile and water (75: 25, v/v), and the flow rate was 1.0 mL/min. The
injection volume was 10 μL. Peak detection and integration were done using Breeze
Chromatographic System (Waters Company, Milford, MA, U.S.A). Maltose was used as an IS and
the internal method was applied for quantification. After the column was equilibrated with mobile
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phase, the flow rate was kept at 1.0 mL/min and 10 μL standard solution was injected to the
chromatographic column. The sample-IS solutions were determined by standards, and the
detection of each sample-IS solution was repeated in triplicate. The column was washed at the end
of each experiment for more than 30 min with the mobile phase.
Method Validation
The calibration curves were prepared for each sugar by injecting the five concentrations of
calibration standard solutions into the HPLC system (10 μL). Linear regression calibration curves
(y=ax+b) based on five points which included 0, represented by the plots of the peak-area ratios of
each sugar to IS multiplied by the IS concentration (y) versus the concentration of the each sugar
calibration standards (x), were constructed using the weighted (1/x2) linear least-squares
regression as the mathematical model.[19] The limits of detection (LOD) was determined by signal
to noise (S/N) ratio method. It was estimated as the minimum concentration of analyte providing a
S/N ratio of 3:1,[20, 21] by injecting a series of diluted solutions with known concentration. The
precision of the HPLC method was assessed for each sugar (sucrose, fructose and glucose) by
repeating five times the analysis of the sample-IS solution prepared as described in “sample
preparation-method (b)”. The precision, reported as RSD, was calculated by the following
formula:
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SD
RSD =
Ar
where A r is the mean peak area ratios of each sugar to IS, and SD is the standard deviation of the
response. To investigate the recovery of the assay, three different concentrations of each sugar
standard solution were added to the molasses sample, and then the IS-sample solutions were
treated as described in “sample preparation-method (b)”. Each sample was injected to HPLC-RI
three times.
Formulas
The capacity factor (k) of the columns was calculated using the following equations:
t R －t M
k=
tM
t R is the time that sample components require from sample injection to the maximum peak,
t M is the retention time of component that is not retained in stationary phase of column. The
recovery was calculated as follows:
Cs — C n
Recovery (%) = Ca × 100
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C s is the concentration of each sugar found in the spiked sample-IS solutions, C n is the
concentration of each sugar in the unprocessed sample-IS solutions, and C a is the concentration of
each sugar added in the spiked sample-IS solutions.
RESULTS AND DISCUSSION
Comparison of two sample pretreatment methods

Besides saccharides, sugarcane molasses contain pigments, nitrogen compounds and inorganic
ions such as potassium, calcium, sodium, and magnesium. Pigments in sugarcane molasses
include polyphenols plant pigments and pigments generated in the course of production, such as
pseudo-melanin caused by reducing sugar decomposition, the reaction between reducing sugar and
amino acid, and caramel pigments resulting from charred sucrose in the process of heating.
Nitrogenous compounds encompass proteins, amino acids and amide. The presence of the
substances influences the separation of sugars, and particularly the pigment substances can even
pollute chromatographic column easily, reducing separation degree and lifespans of the column.[14,
18]
Despite little difference in saccharide peak shapes between pretreated and unpretreated
samples, the long-term accumulation of the pigments in the chromatographic column can reduce
the column lifespan. So it is necessary to pretreat the sample before injection.
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The macroporous resin decolorization method and the Sep-Pak C 18 SPE column purification
method were used to pretreat sugarcane molasses samples in this study. The decolorizing effect of
XDA-8 resin on molasses samples was more significant than that of the other resins (data not
shown). However, the sugar loss of the macroporous resin method was more significant than that
of the SPE column purification method, which affecting the accuracy of the subsequent
measurement (see Table 1).

As shown in Table 1, Dec. of these two methods were both about 87%, but the total sugar loss for
the macroporous resin method (11.32%) was about 17-fold higher than that of SPE method
(0.69%). The sample-IS solutions were passed through the Sep-Pak C 18 cartridges that had been
primed with 4.0 mL of methanol and 8.0 mL of water. The substances including pigments and
polyphenols in the sample were loaded onto the Sep-Pak C 18 cartridge, but the sugars were not
absorbed due to their stronger hydrophilicity, indicating that substances including pigments,
polyphenol and lipid can be removed. In summary, the SPE cleanup method is of higher
discoloration efficiency, lower sugar loss and better chromatographic peak separation. Therefore,
SPE was used to remove the interferents of molasses samples in this study.
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Selection of Chromatography column
Chromatographic separation column is the core of the HPLC method, which directly influences
the component resolution and analysis results. Agilent Zorbax Carbohydrate column and
UtimateTM XB-NH 2 column were compared on the separation and determination of carbohydrate
material. As shown in Table 2, the retention time of the three sugars on the Carbohydrate column
increased gradually, suggesting the moderate interval and good separation effect, while the
retention time of fructose and glucose on XB-NH 2 column was too close to be well separated. As
to capacity factor (k) of the columns, the larger the capacity factor, the greater capacity of the
component that stationary phase fixes, which indicates that the components flow more slowly
from the column and has the longer retention time. If the difference between the capacity factors of
two components on the same column is greater, the difference between retention times of the two
components on the same column may be significant, leading to better separation of the two
components. As shown in Table 2, capacity factors (k) of the three sugars on the Carbohydrate
column are all larger than that of the three sugars on the XB-NH 2 column. Moreover, when
performed with common amino based columns (XB-NH 2 column), it often results in the formation
of a Schiff base (reaction between the sugar and the amine group of the stationary phase), which
might have a significant influence on the stationary phase of the chromatography (loss of amine
group and cannot be regenerated). The sugar that is bound to the surface of the stationary phase
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cannot be eluted from the column, thereby affecting quantification and shortening the lifespan of
column. In addition, the pressure of the XB-NH 2 column was relatively higher than that of the
Carbohydrate column, which may decrease the lifespan of the XB-NH 2 column with a long-term
use. For the above reasons, the Carbohydrate column was selected to separate and detect sugars in
the present study.

Optimization of mobile phase
Due to polar groups contained in the sugar molecules, solvents with strong polarity (such as water,
methanol and acetonitrile) should be chosen as mobile phase. It has been shown that a single
solvent cannot achieve ideal separation of the sugars, and thus a mixed solvent is necessary for
mobile phase. Wei et al. reported that the content of water in mobile phase had a great effect on the
retention capacity of the carbohydrates, and the carbohydrates would be eluted more quickly with
increasing content of water in mobile phase.[22] In the present work, mobile phase proportion was
optimized by choosing acetonitrile and water at the ratios of 60: 40, 65: 35, 70: 30, 75: 25, and 80:
20 (v/v), respectively. As a result, the resolution (the degree of separation of two adjacent peaks in
chromatogram) was improved and the retention time was extended with increasing acetonitrile
concentration. The acetonitrile-water ratio of 80: 20 exhibited the optimum separation with the
longest retention time, but the ratio of 70: 30 was unable to separate fructose and glucose well. In
contrast, the ratio of 75: 25 compromised on the analysis time and separation degree between
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fructose and glucose. Hence, the acetonitrile-water ratio of 75: 25 (v/v) was chosen for further
experiments.
Chromatographic separation
Under the chromatographic conditions, a good separation could be achieved among sucrose,
fructose and glucose (see Table 2). The retention time of fructose, glucose, sucrose, and maltose in
mixed standard solutions were 8.62, 9.70, 13.22 and 15.74 min, respectively. The retention time of
fructose, glucose, sucrose, and maltose in the molasses-IS samples were 8.69, 9.76, 13.29 and
15.83 min, respectively. Comparing the HPLC sugar profiles of samples with the commercial
standards available, the fructose, glucose and sucrose have been identified. The chromatograms of
standard mixture and molasses samples are shown in Figures 1 and 2.
Linear range and limit of detection
The calibration curves were consistently linear from 5.94 to 29.70 g/L for sucrose, 3.30-16.48 g/L
for fructose, and 1.80-9.02 g/L for glucose. The mean squared correlation coefficients (R2) were all
generally≥ 0.995. LODs of three carbohydrates were investigated and the results were shown in
Table 3. Three carbohydrates in the range exhibit an excellent linear relationship. The studies on
LODs were also reported by other workers using HPLC. Kakita et al.[8] analyzed monosaccharides
and oligosaccharides by HPLC with postcolumn fluorescence derivatizatioin, and the LODs of
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glucose and maltohexaose were 1.78 and 2.59 pmol, respectively. Scotter et al.[18] developed a
selective and rapid procedure by HPLC and PDA for the determination of coumarin in foodstuffs
with LOD ranging from 0.05 to 2.5 mg/kg. Wei et al.[22] reported LODs were between 0.2 and 1.2
μg for different carbohydrates in drinks by HPLC with ELSD.
Precision and recovery

Tables 4 and 5 summarize the precision and recovery validation of molasses samples as described
above. The RSDs of precision experiments of the three carbohydrates were 4.81%, 4.42% and
3.43%, respectively, each of which was less than 5.0%, indicating that the method was precise.
Suitable amounts of the carbohydrate standards were added to the molasses samples of known
carbohydrate content, the mixtures were analyzed as described in the “sample
preparation-method (b)”. The standard addition recoveries were 100.33%, 100.30% and
96.40%, respectively. RSDs were less than 5.0%, indicating that this method was accurate. Wei et
al.[22] analyzed carbohydrates in drinks by HPLC with a dynamically modified amino column and
the recoveries of carbohydrates were between 95.8% and 103.0%. Sanchez-Mata et al.[23]
determined monosaccharide, disaccharide, and oligosaccharide in Legumes by HPLC using
amino-bonded silica column with recovery percentages between 93.33% and 101.45%.
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Reproducibility and analysis of the samples
The reproducibility of our HPLC method was tested by repeating the analysis of one sugarcane
molasses sample four times. The sample pretreatment method followed SPE cleanup of sample
preparation procedures. The results are presented in Table 6. At the same time, we also adopted
“sample preparation-method (a)”, and the results are shown in Table 7. Tables 6 and Table 7
show the RSDs range from 0.57% to 2.28% and 0.97% to 4.13%, respectively, which are less than
5.0%, indicating that the methods possess good reproducibility. The difference detected between
the two methods indicates that the SPE absorbs less sugar. Therefore, the SPE-HPLC method is
appropriate for the quantification of sugars in sugarcane molasses.
CONCLUSION
SPE-HPLC with IS method was successfully developed for the separation and determination of
fructose, glucose and sucrose in sugarcane molasses. The sample preparation of the the established
method was simple and easy to perform, and the results were accurate and quantified. The present
investigation suggests that SPE-HPLC with IS method is of high practical value for the
determination of sugars in molasses.
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ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China [grant numbers
51078147, 51278200]; Guangdong Natural Science Foundation [grant number
S2012010010380]; the Fundamental Research Funds for the Central Universities, SCUT [grant
number 2012ZM0081]; and the Guangdong Provincial Science and Technology Program [grant
numbers 2012B091100163, 2011B090400033, and 2010A010500005].
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Nutrition 2010, 65, 38-43.
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High-Performance Liquid Chromatography Using an Amino-Bonded Silica Column. Jouranl of
Agricultural and Food Chemistry 1998, 46, 3648-3652.

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Captions
List of Figures
Figure 1 HPLC-RID chromatogram of a standard mixture of three carbohydrates with a maltose
internal standard.
Determination conditions: mobile phase of acetonitrile and water (75: 25, v/v), flow rate 1.0
mL/min, column temperature 30 °C, and RID temperature 35 °C.
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Figure 2 HPLC-RID chromatogram of three carbohydrates in molasses samples with a maltose
internal standard.
Determination conditions: mobile phase of acetonitrile and water (75: 25, v/v), flow rate 1.0
mL/min; column temperature 30 °C, and RID temperature 35 °C.
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Table 1 Effect of sample pretreatment on sugars in molasses.
Rate of decoloration
Rate of total sugar loss（
Pretreatment method
%）
（%）
Macroporous resin (XDA-8 resin) 86.54 11.32

C 18 SPE column (Sep-Pak) 87.19 0.69
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Table 2 Comparison of two columns on the sugar separationa.
Agilent Zorbax Carbohydrate UtimateTM XB-NH 2

Chromatographic column
column column
Fructose 8.62 7.19

Retention time
Glucose 9.70 7.87

(min)
Sucrose 13.22 11.17
Fructose 1.51 1.39
Capacity factor Glucose 1.83 1.68
Sucrose 2.85 1.66
Column pressure
847 1012
(psi)
a
Both determination conditions: mobile phase of acetonitrile and water (75: 25, v/v), flow rate
1.0 mL/min, column temperature 30 °C, RID temperature 35 °C.
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Table 3 Parameters of quantitative analysis for three carbohydrates.
Regression Linear range Detection

The squared
Component
limits
equation correlation coefficient (g/L)
（R2） (g/L)
Fructose y=1.11x+0.117 0.9986 3.30-16.48 0.16
Glucose y=1.11x-0.0719 0.9994 1.80-9.02 0.04
Sucrose y=1.27x-0.396 0.9954 5.94-29.70 0.08
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Table 4 The precision for fructose, glucose and sucrose (n=5)a.
Peak area ratios of each sugar to IS

RSD
Component
(%)
1 2 3 4 5
Fructose 0.5439 0.6113 0.6031 0.6074 0.6083 4.81

Glucose 0.2679 0.2912 0.2942 0.3016 0.2847 4.42
Sucrose 1.2236 1.3091 1.2882 1.3132 1.3422 3.43
a
n is the number of samples analyzed
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Table 5 Results of spiked recovery test (n=3)a.
Added Detected Average

Amount
Recovery RSD
Component of sample amount amount recovery
(%) (%)
(g/L) (g/L) (g/L) (%)
1.441 4.138 101.26
Fructose 2.667 2.641 5.356 101.82 100.33 2.10
4.490 7.063 97.91
0.674 2.040 95.35
Glucose 1.397 1.226 2.651 102.31 100.30 4.30
2.084 3.548 103.23
Sucrose 5.535 2.787 8.148 94.46 96.40
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5.029 10.455 97.81 1.80
8.549 13.824 96.94
a
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Table 6 Results of sample determination by SPE (n=4)a.
Average
Component RSD
1 2 3 4 concentration
(g/L) (%)
(g/L)
Fructose 2.684 2.647 2.667 2.671 2.667 0.57

Glucose 1.389 1.402 1.436 1.359 1.397 2.28
Sucrose 5.520 5.437 5.536 5.651 5.536 1.59
a
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Table 7 Results of sample determination by direct injection (n=3)a.
Average
Component RSD
1 2 3 concentration
(g/L) (%)
(g/L)
Fructose 2.795 2.741 2.58 2.705 4.13

Glucose 1.401 1.412 1.39 1.401 0.97
Sucrose 5.417 5.470 5.811 5.566 3.85
a
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Determination of Sugars in Molasses by HPLC Following

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Determination of Sugars in Molasses by HPLC Following

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International Journal of Food Properties

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Determination of sugars in molasses by HPLC following

Shenzhen Guangdong 518055, People’s Republic of China

Running head: Determination of sugars in molasses

*Address correspondence to Mingjun Zhu, School of Bioscience and Bioengineering, South

time-saving for determination of sugars in sugarcane molasses.

Keywords: solid-phase extraction, high performance liquid chromatography, sugarcane

molasses, sugars, internal standard.

unusual properties, molasses is widely used in baking, as an animal feed additive or as a

chromatography method, gas chromatography (GC)[5] and high performance liquid

of derivatization modes. Since the nonvolatility of carbohydrates is caused by polar hydroxyl,

accuracy and simplicity.

carbohydrates can be measured by High Performance Liquid Chromatography-Evaporative Light

anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is one of the

most useful techniques for oligosaccharide determination,[12] but amperometric detection

Performance Liquid Chromatography-Refractive Index Detector (HPLC-RID) today have

amino-bonded silica column.

MATERIALS AND METHODS

Instrumentation and reagents

UV2802SH-type ultraviolet-visible spectrophotometer was purchased from UNICO (shanghai)

company, Ltd. (China).

mL of methanol, followed by 4.0 mL of double-deonized water.

250 mL deionized water to 750 mL acetonitrile and mixing well by sonication.

optimum chromatographic conditions.

(c) Macroporous resin decolorization method—3 mL of the above working molasses

ultraviolet-visible spectrophotometer at a wavelength of 560 nm. The decoloration rate (Dec.) on

the samples was calculated as follows:

the working molasses solution pretreated as described above.

recovery was calculated as follows:

each sugar added in the spiked sample-IS solutions.

RESULTS AND DISCUSSION

Comparison of two sample pretreatment methods

the column lifespan. So it is necessary to pretreat the sample before injection.

measurement (see Table 1).

Selection of Chromatography column

the present study.

Optimization of mobile phase

standard mixture and molasses samples are shown in Figures 1 and 2.

Linear range and limit of detection

μg for different carbohydrates in drinks by HPLC with ELSD.

Precision and recovery

carbohydrate content, the mixtures were analyzed as described in the “sample

determined monosaccharide, disaccharide, and oligosaccharide in Legumes by HPLC using

Reproducibility and analysis of the samples

appropriate for the quantification of sugars in sugarcane molasses.

determination of sugars in molasses.

51078147, 51278200]; Guangdong Natural Science Foundation [grant number

numbers 2012B091100163, 2011B090400033, and 2010A010500005].

Company, New Delhi 1977, 9-13.

Chemistry 1959, 31, 426-428.

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