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Single Lab Validation of a Method for the Determination of β‐
Galactooligosaccharides in Infant Formula & Adult Nutritionals / Sean Austin (NRC,
Lausanne)
25 Jul 2016
CONFIDENTIAL
∙ This document may not be reproduced or disclosed to third parties without prior authorization
Page 10 / 35
2.2.6
Chromatographic Conditions
Analyse the prepared samples and standards on a UHPLC system equipped with a FLD under the
following conditions:
FLD: Excitation λ: 330 nm Emission λ: 420 nm
Trap Column: Acquity BEH Amide (2.1 mm x 5 mm; 1.7 µm) or equivalent (see Figure 1 for
plumbing)
Column: Acquity UPLC BEH Glycan (2.1 mm x 150 mm; 1.7 µm) or equivalent
Column temperature: 25°C ± 1 °C
Injection volume: 3 µL
Eluent A: Acetonitrile
Eluent B: Ammonium formate 0.1 mol/L, pH 4.4
Gradient:
Time
(min)
Flow
(mL/min)
%A
(Acetonitrile)
%B
(Formate)
Switching
Valve
Comment
0
0.6
97
3
6‐1
Inject sample on trap column
5.0
0.6
97
3
6‐1
End of sample wash, start acquisition
5.5
0.6
88
12
1‐2
Switch valve, start analytical gradient
12.5
0.6
88
12
1‐2
22.5
0.6
83
17
1‐2
26.5
0.6
83
17
1‐2
45.0
0.6
68
32
1‐2
End of analytical gradient
45.1
0.2
68
32
1‐2
Reduce flow
45.5
0.2
30
70
1‐2
Start column wash
55.5
0.2
30
70
1‐2
End column wash
57.0
0.2
88
12
1‐2
Start analytical column equilibration
62.0
0.2
88
12
1‐2
72.0
0.6
88
12
1‐2
End analytical column equilibration
72.5
0.6
97
3
6‐1
Switch valve, equilibrate trap column
73.5
0.6
97
3
6‐1
End
Figure 1: Configuration of 2-way 6-port switching valve
VALIDATION REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE