I.
Chromatography
The solutions prepared in section H are ready to be injected on the chromatographic system using
the conditionsdescribedbelow:-
Column:
TSKgel Amide-80; 3µm; 4.6mm x150mm
Trapping column
TSKgel Amide-80Guard cartridge; 3µm; 3.2mm x15mm
Columnoven temperature.
23 °C±1 °C
Injectionvolume:
20µL
FluoresenceDetector:
Excitationwavelength: 330nm Emissionwavelength: 420nm
Mobilephase:
A: Acetonitrile100%
B:Ammonium formate50mmol/L; pH4.4 (r)
Table3: Elutionprogram
Time
(min)
Flow
(mL/min)
Mobilephase
6ports valve
position
Comment
%A
%B
0
1.0
98
2
6-1 (load)
Inject (10or 20) µL
Sample loadingon trapping col.
4.0
1.0
98
2
6-1 (Load)
Auto zero, Start acquisition
7.5
1.0
98
2
1-2 (Analyse)
Switchvalvepos. 1-2
8.0
1.0
84
16
1-2 (Analyse)
16.0
1.0
84
16
1-2 (Analyse)
Elution
50.0
1.0
61
39
1-2 (Analyse)
51.0
0.80
20
80
1-2 (Analyse)
Columnwash
54.0
0.80
20
80
1-2 (Analyse)
Columnwash
55.0
0.80
90
10
1-2 (Analyse)
Equilibration
61.0
1.0
90
10
1-2 (Analyse)
Equilibration
Before startingananalysis allow the chromatographic system toequilibrate for at least 15minunder
the initial conditions. Make sure the base line and pressure of the system are stable before starting
the analysis. Before starting a series of analysis, make at least one injection of a known profile (ref.
Sample or standards). Check retention times, separation and comparewithprevious runs. Check the
linear response of your fluorimeter by measuring the stability of the response factor of different
maltotriose concentrations covering the whole scale of your detector. Fluorimeters are generally
linear over thewholemeasuring range.
GOS-01
FORWORKINGGROUP/ERPUSEONLY
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