!"#$%&'&()*(+)")(#",(-'&#../#0',1&
'"(2)),(30),4.5&
6&'"7(81*0#.5'91(:",1;(-151.5')"
<'=(>0)$?(@0A(!BB$'.#5')"&(C/1='&5
:",4&50'#$(+#0D15(-191$)B=1"5(E0)4B
-1.(FGGH
Waters Corporation 2000
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
! Nutritional
Labeling of Food Products requires to
list 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
Waters Corporation 2000
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
Waters Corporation 2000
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
Alliance 2690 System
Carbohydrate Column, 3.9mm x 30 cm
75% AcCN / 25% Water
1.4 mL/min at 35°C; BP = 680 psi
Continuous Degassing
20 µL
2410 Refractive Index, 40°C, 128X
0.25% Each Sugar
33 µ RI Units
Fructose
Glucose
Sucrose
Maltose
Lactose
2 .00
3 .00
4 .00
5 .00
6 .00
7 .00
M inute s
8 .00
9 .00
1 0 .00
1 1 .00
1 2 .00
Waters Corporation 2000
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
Alliance System Reproducibility
Five Injection
Analyte
Overlay
0.05% Sugar
Ret Time Peak Area
%RSD
%RSD
1
2
1
2
3
4
5
6.6 µ RI Units
3
Fructose
Glucose
Sucrose
Maltose
Lactose
0.045
0.056
0.078
0.098
0.097
0.37
0.42
0.53
1.06
0.94
4
5
2.00
3.00
4.00
5.00
6.00
7.00
Min u tes
8.00
9.00
10.00
11.00
12.00
Waters Corporation 2000
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
Waters Corporation 2000
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
Minutes
8.00
9.00
10.00
11.00
12.00
Waters Corporation 2000
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
! Sonicate for 15 Minutes
! Cool and Dilute to 100 mL
! Pass through a C18 SepPak Cartridge to
remove lipid, protein, and suspended solids
! Use Filtrate for Analysis
Waters Corporation 2000
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
1
Red Delicious Apple
Bite Size 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
Waters Corporation 2000
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 0.00
9.00
1 1.00
9.50
10.00
1 2.00
1 3.00
1 4.00
Min u tes
Waters Corporation 2000
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
Sucrose = 44.28 ± 0.07%
2 Lactose = 7.43 ± 0.01%
1
2
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
Minutes
Waters Corporation 2000
Analysis of Mono/DiSaccharides
Waters Carbohydrate Analysis Column
2
20 µ RI Units
Five
Injection
Overlay
Powdered Instant Breakfast Drink
1.003 g / 100 mL
1
2
3
4
Label Claim is
20g Sugar
per Serving
Glucose = 0.54 ± 0.02%
Sucrose = 30.35 ± 0.08%
Maltose = 0.79 ± 0.02%
Lactose = 23.43 ± 0.05%
4
LC Analysis gave
20.3 g Sugar
per Serving
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
Waters Corporation 2000
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
Minutes
8.00
9.00
10.00
11.00
12.00
Waters Corporation 2000