l
To each vial containing standards or samples add 150 μL
TFA 8 M (final concentration 2 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 4 h.
l
After this time, 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:
20 min, NaOH 18 mM (36% of eluent NaOH 50 mM)
10 min NaOH 28 mM, AcONa 100 mM (36% NaOH
50 mM; 10% AcONa 1 M with NaOH 100 mM).
20 min NaOH 18 mM (36% of eluent NaOH 50 mM).
– Remark: after the last chromatographic analysis is completed, store the column in 18 mM NaOH solution.
l
Per each sugar standard calculate a linear regression
between concentration and the peak area and calculate on
it the concentration of each sugar monomer in the samples
(Fig. 4).
3. HPAEC-PAD for GlcNAc (see Note 22).
(a) Calibration curve standard solutions.
O-Antigen Purification and Conjugation
285
To each vial containing standards or samples add 150 μL
TFA 8 M (final concentration 2 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 4 h.
l
After this time, 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:
20 min, NaOH 18 mM (36% of eluent NaOH 50 mM)
10 min NaOH 28 mM, AcONa 100 mM (36% NaOH
50 mM; 10% AcONa 1 M with NaOH 100 mM).
20 min NaOH 18 mM (36% of eluent NaOH 50 mM).
– Remark: after the last chromatographic analysis is completed, store the column in 18 mM NaOH solution.
l
Per each sugar standard calculate a linear regression
between concentration and the peak area and calculate on
it the concentration of each sugar monomer in the samples
(Fig. 4).
3. HPAEC-PAD for GlcNAc (see Note 22).
(a) Calibration curve standard solutions.
O-Antigen Purification and Conjugation
285