Remarks: Perform system equilibration with elution buffer
before starting samples analysis. This equilibration step lasts
70 min if the column system must be equilibrated with a
different eluent; 60 min or more are needed also to warm up
the PDA lamps before the analysis. These steps can be skipped
if the system (HPLC and columns) is already equilibrated with
the buffer and lamps are on. After the last chromatographic
analysis, store the column in 0.02% NaN 3 .
5.
1 H nuclear magnetic resonance (NMR) spectroscopy
(a) Experimental conditions.
l
Sample preparation.
– Dry the OAg (unconjugated or conjugated) sample
(2.5 mg total sugar).
l
Solubilize dried sample in 650 μL D 2 O and transfer
into 5-mm NMR tubes.
System settings.
– Acquisition temperature: 25
C.
– Fid acquisition time: 5 s.
– Relaxation delay: 15 s.
– Number of scans: 64.
(b) OAg identity.
NMR analysis on the liquid state is performed to
confirm the identity of the OAg samples by detecting
typical signals of the OAg chain, confirming the presence
of the characteristic sugars [17–19].
(c) OAg O-acetylation level.
A first
1 H NMR spectrum is recorded to ensure the
absence of impurities at the same chemical shift of the
acetate anion released after de-O-acetylation of the sample
that would interfere with the quantification of the
O-acetyl content. O-acetylation level is quantified by
comparing acetate signal (released after treatment with
NaOD, at 1.91 ppm) and a known signal of the OAg
RU structure (see Note 25).
Record two 1H NMR spectra as described before:
l
the first one in D2O.
l
the second one after de-O-acetylation adding sodium
deuteroxide (NaOD) to a final 200 mM concentration
(35 μL NaOD 4 M directly in the tube after having
recorded the first spectrum) and heat treatment (37
C
for 2 h for complete de-O-acetylation).
O-Antigen Purification and Conjugation
291
before starting samples analysis. This equilibration step lasts
70 min if the column system must be equilibrated with a
different eluent; 60 min or more are needed also to warm up
the PDA lamps before the analysis. These steps can be skipped
if the system (HPLC and columns) is already equilibrated with
the buffer and lamps are on. After the last chromatographic
analysis, store the column in 0.02% NaN 3 .
5.
1 H nuclear magnetic resonance (NMR) spectroscopy
(a) Experimental conditions.
l
Sample preparation.
– Dry the OAg (unconjugated or conjugated) sample
(2.5 mg total sugar).
l
Solubilize dried sample in 650 μL D 2 O and transfer
into 5-mm NMR tubes.
System settings.
– Acquisition temperature: 25
C.
– Fid acquisition time: 5 s.
– Relaxation delay: 15 s.
– Number of scans: 64.
(b) OAg identity.
NMR analysis on the liquid state is performed to
confirm the identity of the OAg samples by detecting
typical signals of the OAg chain, confirming the presence
of the characteristic sugars [17–19].
(c) OAg O-acetylation level.
A first
1 H NMR spectrum is recorded to ensure the
absence of impurities at the same chemical shift of the
acetate anion released after de-O-acetylation of the sample
that would interfere with the quantification of the
O-acetyl content. O-acetylation level is quantified by
comparing acetate signal (released after treatment with
NaOD, at 1.91 ppm) and a known signal of the OAg
RU structure (see Note 25).
Record two 1H NMR spectra as described before:
l
the first one in D2O.
l
the second one after de-O-acetylation adding sodium
deuteroxide (NaOD) to a final 200 mM concentration
(35 μL NaOD 4 M directly in the tube after having
recorded the first spectrum) and heat treatment (37
C
for 2 h for complete de-O-acetylation).
O-Antigen Purification and Conjugation
291