l
In 2 mL screw cap vials, prepare in duplicate the dilutions starting from the 11.25 μg/mL GlcNAc standard
(Table 3):
(b) Sample preparation.
l
Dilute the sample with water to a suitable concentration to fit the calibration curve.
l
Prepare three (in triplicate) 2 mL screw cap vials containing 450 μL of the diluted sample.
(c) Sample/standard treatment.
l
To each vial containing standards and samples add
150 μL TFA 4 M (final concentration 1 M) (see
Note 23), close them and vortex each vial for few
seconds.
l
Place all the vials in a rigid cardboard rack and incubate in a preheated oven at 100
C for 6 h.
l
Allow all the tubes to cool at 2–8
C for 30 min.
l
Remove the caps and dry the samples/standards overnight in centrifugal evaporator at RT in order to
remove solvent/TFA.
l
Redissolve the content of each vial adding 450 μL of
water and vortex accurately for few seconds.
l
Filter the content of each vial in the sampler polypropylene vials using 0.45 nylon syringe (4 mm) filters.
(d) Analysis setting for Thermo Dionex ICS series
instrument.
l
Column System: Dionex CarboPac PA10 guard—
CarboPac PA10 columns connected in series.
l
Column/detector compartment: 35
C.
l
Autosampler compartment: 10
C.
l
Electrochemical detector equipped with gold working electrode, potential sets with standard carbohydrate waveform.
l
Chromatographic conditions:
– Sample injection volume: 25 μL, full loop mode.
– Run time: 50 min.
– Flow rate: 1 mL/min.
– Eluent program:
Gradient from NaOH 10 mM to 18 mM in 20 min
(from 20% to 36% of NaOH 50 mM eluent).
286
Francesca Micoli et al.
In 2 mL screw cap vials, prepare in duplicate the dilutions starting from the 11.25 μg/mL GlcNAc standard
(Table 3):
(b) Sample preparation.
l
Dilute the sample with water to a suitable concentration to fit the calibration curve.
l
Prepare three (in triplicate) 2 mL screw cap vials containing 450 μL of the diluted sample.
(c) Sample/standard treatment.
l
To each vial containing standards and samples add
150 μL TFA 4 M (final concentration 1 M) (see
Note 23), close them and vortex each vial for few
seconds.
l
Place all the vials in a rigid cardboard rack and incubate in a preheated oven at 100
C for 6 h.
l
Allow all the tubes to cool at 2–8
C for 30 min.
l
Remove the caps and dry the samples/standards overnight in centrifugal evaporator at RT in order to
remove solvent/TFA.
l
Redissolve the content of each vial adding 450 μL of
water and vortex accurately for few seconds.
l
Filter the content of each vial in the sampler polypropylene vials using 0.45 nylon syringe (4 mm) filters.
(d) Analysis setting for Thermo Dionex ICS series
instrument.
l
Column System: Dionex CarboPac PA10 guard—
CarboPac PA10 columns connected in series.
l
Column/detector compartment: 35
C.
l
Autosampler compartment: 10
C.
l
Electrochemical detector equipped with gold working electrode, potential sets with standard carbohydrate waveform.
l
Chromatographic conditions:
– Sample injection volume: 25 μL, full loop mode.
– Run time: 50 min.
– Flow rate: 1 mL/min.
– Eluent program:
Gradient from NaOH 10 mM to 18 mM in 20 min
(from 20% to 36% of NaOH 50 mM eluent).
286
Francesca Micoli et al.