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