Simple Sugar Analysis by HPLC Using
Refractive Index Detection
Jim Krol
Market Development
Waters Corporation
© 2000 Waters Corp
Introduction to sugar analysis application. This work was performed on a Waters
Alliance HPLC System consisting of a 2690 Separations Module, 2410 Differential
Refractive Index Detector, and Millennium32 chromatography software.
5
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
1
Red Delicious Apple
9.24g / 100 mL
47 µRI Units
Five
Chromatogram
Overlay
1 Fructose = 5.16 ± 0.01%
2 Glucose = 2.31 ± 0.01%
3 Sucrose = 0.09 ± 0.05%
2
3
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
M in u te s
© 2000 Waters Corp
Analysis of mono/disaccharide's; fructose, glucose, and sucrose in an apple. The
excellent reproducibility of the 5 overlay chromatograms demonstrates the
exceptional flow characteristics of the of the 2690 Separations Module.
6
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
! Nutritional
Labeling of Food Products requires
listing sugar and total carbohydrate content
! Common
•
•
Sugars defined as
Monosaccharides: Fructose and Glucose
Disaccharides: Sucrose, Maltose, Lactose
! AOAC
HPLC Methods Recommend the use of
Propyl Amine functional columns for analysis of
mono and disaccharides in food products
© 2000 Waters Corp
This slide presents the guidelines for sugar determination according to the
Nutritional Labeling & Education Act passed by the U.S. Congress. Note that
labeling is not required until the levels are quite high (0.5g total sugar per serving
size) and that only the mono-saccharides fructose, glucose and the di-saccharides
sucrose, maltose and lactose need to be labeled. These five sugars are the most
common in food systems.
In the Official Methods of Analysis of AOAC International (Association of
Official Analytical Chemists), the HPLC Methods recommend the use of an
analytical column containing a silica stationary phase with a propyl amine
functionality.
7
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
0.25% Each Sugar
33 µ RI Units
Fructose
Glucose
Column :
Mobile Phase:
Flow Rate:
Detection:
Injection:
Carbohydrate, 3.9mm x 30 cm
75% AcCN / 25% Water
1.4 mL/min at 35°C; BP = 680 psi
2410 Refractive Index, 40°C, 128X
20 µL
Sucrose
Maltose
Lactose
2 .0 0
3 .0 0
4 .0 0
5 .0 0
6 .0 0
7 .0 0
M inute s
8 .0 0
9 .0 0
1 0 .0 0
1 1 .0 0
1 2 .0 0
© 2000 Waters Corp
The Waters Carbohydrate Analysis Column incorporates propyl amine
functionality. Baseline resolution of the mono/disaccharides is obtained using the
chromatographic conditions described above. Also available, the Waters High
Performance Carbohydrate Analysis Column incorporates the familiar chemistry
and selectivity of propyl amine functionality bonded onto the highly efficient
support of 4 micron Nova Pak silica. This material is packed into a new column
format. The Waters stainless steel cartridge column provides the ability to use an
integrated guard column to prolong the life of the analytical column. This design
also lowers the column replacement cost by incorporating reusable end fittings.
8
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
Alliance System Reproducibility
Five Injection
Analyte
Overlay
0.05% Each Sugar
1
2
1
2
3
4
5
6.6 µ RI Units
3
Fructose
Glucose
Sucrose
Maltose
Lactose
4
2 .0 0
3 .0 0
4 .0 0
5 .0 0
6 .0 0
7 .0 0
8 .0 0
Ret Time Peak Area
%RSD
%RSD
0.045
0.056
0.078
0.098
0.097
0.37
0.42
0.53
1.06
0.94
5
9 .0 0
1 0 .0 0
1 1 .0 0
1 2 .0 0
M in u tes
© 2000 Waters Corp
The Alliance HPLC System demonstrates excellent reproducibility for analysis of
mono/disaccharides based upon retention time and peak area %RSD (percent
relative standard deviation).
9
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
Response Linearity and Precision
Glucose
Sucrose
Maltose
Lactose
Linearity, r2
0.9998
0.9981
0.9999
0.9999
0.9997
0.50%
0.49
0.49
0.56
0.53
0.98
0.25%
0.15
0.17
0.31
0.35
1.27
0.10%
0.56
0.65
0.68
0.43
0.82
0.05%
0.37
0.42
0.53
1.06
0.94
0.01%
1.87
6.46
2.44
4.31
6.68
0.005%
9.65
10.56
8.71
7.23
10.97
% Sugar Concentration
Fructose
0. 005%
Data as Peak Area %RSD over 5 Injections
© 2000 Waters Corp
Excellent linearity and precision is also obtained. The precision decreases at the
0.005% sugar concentration because we are approaching the method detection limit
making it more difficult to integrate the smaller peaks. This is demonstrated in the
next slide.
10
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
Low Concentration Detection
Lactose
Fructose
Sucrose
Maltose
0.001% Sugar
200 ng each
0.8 µ RI Units
Glucose
0.005% Sugar
1 µg each
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
Minutes
© 2000 Waters Corp
The method detection limit for the mono/disaccharides is 0.0001% or 200 ng of
each sugar on column. However, note that labeling is not required until the levels
are quite high (0.5g total sugar per serving size).
11
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
Sample Preparation
! Prepare
approximately a 1% Solution of the food
matrix
•
•
•
•
•
Add 80 mL of Warm (80°C) DI Water
Sonicate for 15 Minutes
Cool and Dilute to 100 mL
Pass through a C18 Sep-Pak Cartridge to remove
lipid, protein, and suspended solids
Use Filtrate for Analysis
© 2000 Waters Corp
Start with a sample concentration of 1 gram to 100 mL.
This is the sample preparation procedure utilized for all of the food products shown
in this section.
12
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
1
Five
Injection
Overlay
1
2
3
4
2
3 .0 0
4 .0 0
5 .0 0
6 .0 0
4
3
Unk
6.50
2 .0 0
Fructose = 35.30 ± 0.02%
Glucose = 31.34 ± 0.02%
Sucrose = 0.46 ± 0.01%
Maltose = 1.26 ± 0.02%
Unk
1.2 µ RI Units
3.5 µ RI Units
1.09g Honey / 100 mL
7 .0 0
7.00
7.50
8 .0 0
8.00
9 .0 0
8.50
1 0 .0 0
9.00
1 1 .0 0
9.50
10 .0 0
1 2 .0 0
1 3 .0 0
1 4 .0 0
M in u tes
© 2000 Waters Corp
The following slides demonstrate the different sugar profiles obtained from four
food products. Approximately 300 injections were performed on the same column
to demonstrate its ruggedness and reproducibility.
Natural honey contains high concentrations of fructose and glucose. Adulteration of
honey can be determined by monitoring the fructose/glucose ratio. High fructose
corn syrup can be added to provide sweetness at less expense.
13
Analysis of Mono/DiSaccharide
Waters Carbohydrate Analysis Column
1
Milk Chocolate Candy Bar
0.552g / 100 mL
16.4 µ RI Units
Three
Injection
Overlay
1 Sucrose = 44.28 ± 0.07%
2 Lactose = 7.43 ± 0.01%
2
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
Minutes
© 2000 Waters Corp
Sucrose and lactose (high in calories) are added to milk chocolate to make it sweet.
14
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
2
Powdered Instant Breakfast Drink
1.003 g / 100 mL
Five
Injection
Overlay
20 µ RI Units
1
2
3
4
Glucose = 0.54 ± 0.02%
Sucrose = 30.35 ± 0.08%
Maltose = 0.79 ± 0.02%
Lactose = 23.43 ± 0.05%
4
3
1
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
Minutes
© 2000 Waters Corp
This breakfast drink is marketed as providing high energy and nutritional value.
Note that it also contains high levels of sucrose and maltose. Therefore, it is also
high in calories!
15
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
1
Liquid Diet Breakfast Drink
0.998 g / 100 mL
1 Fructose = 2.66 ± 0.02%
2 Glucose = 0.10 ± 0.001%
3 Sucrose = 3.70 ± 0.03%
4 Lactose = 3.51 ± 0.03%
3
3.2 µ RI Units
Five
Injection
Overlay
2
2.00
3.00
4.00
5.00
4
6.00
7.00
8.00
9.00
10.00
11.00
12.00
Minutes
© 2000 Waters Corp
Note the contrast in this diet breakfast drink. This product contains significantly
lower concentrations of sucrose and lactose so it also has a lot less calories! Less
expensive high fructose corn syrup is added to make the product taste good.
16
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
! Waters
Carbohydrate Analysis Column is a 10 µm
Silica Based Propyl Amine Column
! Current
•
•
•
•
•
AOAC LC Methods
977.20 Honey
980.13 Chocolate
982.14 Presweetened Cereal
984.17 Licorice Extracts
984.22 Purity of Lactose
© 2000 Waters Corp
Currently there are official AOAC methods for these analytes which incorporates
HPLC. It should be noted that they all use propyl amine chemistry bonded to silica
stationary phases and that they are very specific to the matrix. Also note that the
newest of these methods is from 1984 and are still viable method today (2nd and
3rd digits of the method number represent the year of acceptance). The reasons for
the matrix specificity are sample preparation issues and potential matrix
interference issues. Both of these issues can be minimized using the column
chemistry from Waters.
There are a variety of separation mechanisms and chemistries for the HPLC
determination of sugars. Anion exchange, cation exchange, liquid/liquid partition
and size exclusion represent a few useful chemistries. However, with the five "food
sugars" commonly analyzed by food chemists, the propyl amine chemistry provides
the desired separation.
17