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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
7.
CALIBRATION & CALCULATIONS
Use the instrument software to plot a six point standard curve of “Instrument response for
maltotriose / Instrument response for laminaritriose” against the “concentration of maltotriose” in
the standard (in µmol/mL). Fit a linear model to the data without forcing through zero.
Use the standard curve to calculate the molar concentration (in µmol/mL) of each oligosaccharide
in the chromatogram (C
m
) without enzyme treatment, and calculate the total oligosaccharides in
that sample as follows:-
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE