are injectedon to the trapping column,washedwithacetonitrile to removeexcess labeling reagents
and then transferred on to the analytical column where they are separated using a gradient of
aqueous ammonium formate in acetonitrile anddetectedwith a fluorescencedetector. Anexternal
calibration curvepreparedwithmaltotriose isused forquantitation, plotting the relative responseof
maltotiose to internal standard against the relative molar concentration of maltotriose to internal
standard. Molar quantities are then converted to weights using the molar mass of the individual
oligosaccharides. These can be determined simultaneously using a mass spectrometer, or can be
determined inadvance for knowngalactooligosaccharide ingredientsusingHPLC-MS.
C.
Apparatus&Materials
(a)
AHighperformance liquid chromatography instrument equippedwith a gradient pump able
to deliver a flow of 1.0mL/min, an online degasser, an autosampler equipped with
refrigerated sample compartment, a temperature controlled column compartment able to
maintain a stable temperature of 23.0 °C ± 1.0 °C, a fluorescence detector, a 2-way 10-port
highpressure switchingvalve: seeplumbing scheme foron line cleanup (Figure2).
(b)
Inlinehighpressure filter 0.5µmwithholder.
(c)
HPLC column: TSKgel Amide-803µm; 4.6mm I.D; 15.0 cm long.
(d)
TSKgel Amide-80; 3µmguard cartridge: 3.2mm I.D; 1.5 cm long.
(e)
Guard cartridgeholder for 3.2mm I.D; 1.5 cm L cartridges
(f)
2-mLmicrotubes, safe lockor screw cap
(g)
Floating rack formicrotubes
(h)
Centrifuge for 2-mL tubesable tooperateat 10000g
(i)
Waterbathsmaintaining 65°C±1.0 °Cand50 °C±1.0 °C
(j)
Vortexmixer
(k)
Mircopipettewith tips (0.1 to1mL)
(l)
Analytical balancewithaprecisionof 0.1mg
(m)Ultrasonicbath
GOS-01
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