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GOA L
To successfully determine sugar content
in a variety of fruit juices with minimum
sample preparation, rapid analysis time,
and cost effectiveness.
BAC KG ROU N D
The healthier option of consuming pure fruit
juice instead of soft drinks containing sugar
has been promoted in recent years due to
studies linking the latter with obesity, coronary
problems, and the development of Type 2
Diabetes. Additionally, certain juices such as
pomegranate have been reported to provide
health benefits due to their antioxidant content.
These products, therefore, often command
premium prices.
To ensure quality and satisfy regulatory
agencies, the fruit juice industry must test
these products to meet certain standards. One
indicator is sugar content. Fructose, sucrose,
and glucose are important analytes in fruit juice
analysis. Their content and relative ratio vary
and serve as markers for various cultivars.
HPLC with evaporative light scattering
(ELS) detection has long been a recognized
technique for these analyses. Columns for sugar
separations have used amine-based chemistry.
However, this can lead to the formation of Schiff
bases that can shorten column lifetime and lead
to inaccurate sugar quantification.
Five food sugars are rapidly analyzed and
quantified using the robust XBridge™ BEH Amide
XP Column chemistry with proven Alliance® HPLC
System technology.
Waters® proprietary amide column chemistry prevents the formation of Schiff
bases, eliminating the aforementioned problems. This study shows the
versatility of the Waters XBridge BEH Amide XP Column for sugar analysis.
Figure 1. Alliance HPLC System with ELS detector.
Mark E. Benvenuti and Jennifer Burgess
Determination of Food Sugars in Fruit Juice Using Evaporative Light Scattering Detection and BEH Amide Column Chemistry
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
T H E SO LU T IO N
Various fruit juices were purchased in local
markets. Samples of these juices were centrifuged
at a high speed to remove pulp and other particulate
matter. A portion of the supernatant of each juice
was diluted with a 1:50 water/acetone mixture.
This was the only sample preparation required.
The analyses were performed on an Alliance HPLC
System equipped with a 2424 Evaporative Light
Scattering Detector. ELS detection is appropriate
here as the target analytes are non-chromophoric
excluding the use of UV and fluorescence
detection. ELS also allows gradient elution enabling
the resolution of related compounds such as
other sugars, sugar alcohols (sorbitol), and higher
oligomers. An XBridge BEH Amide XP 4.6 x 100 mm,
2.5 µm Column was used. Gradient elution was used
with a binary mobile phase consisting of water and
acetone modified with triethyl amine (TEA). Samples
were quantified against a six-point calibration curve
generated by running dilutions of a stock sugar
mixture. Figure 2 shows the separation of four
food sugars and sorbitol, a sugar alcohol found in
certain cultivars that can be used as an adulteration
marker. A profile of several fruit juices is shown in
Figure 3. The slight presence of sorbitol in apple
juice is noteworthy.
SUMMA RY
XBridge BEH Amide column chemistry provided
an analysis time of less than 7.5 minutes for
the analysis of sugars in fruit juice. This method
enabled the accurate quantification of sugars in
fruit juices using the robust XBridge BEH Amide
XP column chemistry with proven Alliance HPLC System technology for accurate
solvent delivery, repeatable injection performance, the ability to create non-
linear gradients, and simple ELS detection, meeting the goals of simple sample
preparation, rapid analysis time, and cost effectiveness.
Waters and Alliance are registered trademarks of Waters Corporation. XBridge and T he Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2012 Waters Corporation. Produced in the U.S.A.December 2012 720004548EN TC-PDF
Figure 2. ELSD chromatogram showing a gradient separation of the five food sugars.
Figure 3. Sugar profiles of four fruit juices. Calculated amounts of different sugars are labeled (the 50x dilution in sample preparation has been taken into account).
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
1- Fructose- 0.22 g/100 mL 2- Sorbitol- 0.21 g/100 mL 3- Glucose- 0.21 g/100 mL 4- Sucrose- 0.21 g/100 mL 5- Maltose- 0.22 g/100 mL
2000
LSU
5
4
3
2
1
Minutes2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4
Minutes2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4
Minutes2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4
Minutes2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4
1- Fructose- 4.67 g/100 mL 2- Glucose- 5.00 g/100 mL 3- Sucrose- 3.66 g/100 mL
1- Fructose- 7.82 g/100 mL 2- Glucose- 7.46 g/100 mL 3- Sucrose- Not Detected
1- Fructose- 6.73 g/100 mL *- Sorbitol- 0.45 g/100 mL 2- Glucose- 1.77 g/100 mL 3- Sucrose- 1.81 g/100 mL
*
1- Fructose- 2.47 g/100 mL 2- Glucose- 2.62 g/100 mL 3- Sucrose- 4.94 g/100 mL
Pineapple Juice White Grape Juice
Apple Juice Orange Juice
1000
LSU
1000
LSU
10
00 L
SU
1000
LSU
1
3
2 1
2
1 3
2
3
2
1