l
Chromatographic conditions:
– Eluent: 0.1 M NaCl, 0.1 M NaH 2 PO 4 , 5% ACN,
pH 7.2 (isocratic conditions).
– Flow rate: 0.5 mL/min.
– Run time: 35 min.
– Sample injection volume: 80 μL.
Remark: Perform system equilibration with elution
buffer before starting the 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 is completed,
store the system in NaN 3 0.02% preservative solution.
l
Quantification of reactive carbonyl groups (nmol/
mL) in the samples.
– Correct the peak area corresponding to OAg in
samples derivatized with semicarbazide by subtracting the area of the corresponding blank.
– Quantify the amount of KDO using the calibration
curve built with the peak areas of derivatized KDO
standard at 252 nm (Fig. 5).
7. Amino Groups Determination by Colorimetric TNBS
Method [16].
(a) Calibration curve standard solutions.
l
In 5 mL glass tubes, prepare in duplicate the ADH
dilutions starting from 100 nmol/mL, as reported in
Table 5.
(b) Sample preparation.
l
Dilute the sample with water to a suitable concentration to fit the calibration curve. Prepare two 5 mL
tubes containing 500 μL of the diluted sample.
(c) Sample/standard treatment.
l
To each tube containing standards and samples, add
500 μL of sodium hydrogen carbonate 4% solution and
vortex each tube for few seconds.
l
To each tube, add then 500 μL of TNBS 0.1% solution
and vortex each tube for few seconds.
l
Cover tubes with aluminum foil and incubate in a
preheated thermostatic bath at 40
C for 2 h.
O-Antigen Purification and Conjugation
293
Chromatographic conditions:
– Eluent: 0.1 M NaCl, 0.1 M NaH 2 PO 4 , 5% ACN,
pH 7.2 (isocratic conditions).
– Flow rate: 0.5 mL/min.
– Run time: 35 min.
– Sample injection volume: 80 μL.
Remark: Perform system equilibration with elution
buffer before starting the 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 is completed,
store the system in NaN 3 0.02% preservative solution.
l
Quantification of reactive carbonyl groups (nmol/
mL) in the samples.
– Correct the peak area corresponding to OAg in
samples derivatized with semicarbazide by subtracting the area of the corresponding blank.
– Quantify the amount of KDO using the calibration
curve built with the peak areas of derivatized KDO
standard at 252 nm (Fig. 5).
7. Amino Groups Determination by Colorimetric TNBS
Method [16].
(a) Calibration curve standard solutions.
l
In 5 mL glass tubes, prepare in duplicate the ADH
dilutions starting from 100 nmol/mL, as reported in
Table 5.
(b) Sample preparation.
l
Dilute the sample with water to a suitable concentration to fit the calibration curve. Prepare two 5 mL
tubes containing 500 μL of the diluted sample.
(c) Sample/standard treatment.
l
To each tube containing standards and samples, add
500 μL of sodium hydrogen carbonate 4% solution and
vortex each tube for few seconds.
l
To each tube, add then 500 μL of TNBS 0.1% solution
and vortex each tube for few seconds.
l
Cover tubes with aluminum foil and incubate in a
preheated thermostatic bath at 40
C for 2 h.
O-Antigen Purification and Conjugation
293