10 min NaOH 28 mM, AcONa 100 mM (36%
NaOH 50 mM; 10% AcONa 1 M with
NaOH 100 mM).
20 min NaOH 10 mM (20% of eluent NaOH
50 mM).
– Remark: after the last chromatographic analysis is
completed, store the column in 18 mM NaOH
solution.
l
Calculate a linear regression for GlcNAc standard
between concentration and the peak area and calculate on it the concentration of the GlcNAc in the
samples.
4. HPLC-SEC.
HPLC-SEC analysis is used to estimate the molecular size
distribution of OAg samples. Samples are run, without any pretreatment, diluted with water at 100–200 μg/mL OAg
concentration.
1. System setting.
(a) Column System: TSK gel G3000 PWXL column with a
TSK gel PWXL guard column.
(b) Calculate void and bed volume calibration injecting
λ-DNA and NaN3, respectively. For Kd determination,
the following equation was used: Kd ¼ (T e À T 0 )/
(T t À T 0 ) where: T e ¼ elution time of the analyte, T 0 ¼ elution time of the biggest fragment of λ-DNA and T t ¼ elution time of NaN3.
(c) Column compartment: 30
C.
(d) Autosampler compartment: 4
C.
(e) Detector: OAg peaks by differential refractive index
(dRI). UV detection at 214 and 260 nm for following
impurity reduction during the purification steps.
2. Chromatographic conditions:
(a) Mobile phase: 0.1 M NaCl, 0.1 M NaH 2 PO 4 , 5% ACN,
pH 7.2.
(b) Flow rate: 0.5 mL/min.
(c) Run time: isocratic for 30 min.
(d) Sample injection volume: 80 μL.
(see Note 24)
HPLC-SEC analysis is used to estimate the molecular size
distribution of the glycoconjugate, in comparison with free OAg
and free protein. Samples are run usually diluted in PBS at
50–100 μg/mL protein concentration.
O-Antigen Purification and Conjugation
287
NaOH 50 mM; 10% AcONa 1 M with
NaOH 100 mM).
20 min NaOH 10 mM (20% of eluent NaOH
50 mM).
– Remark: after the last chromatographic analysis is
completed, store the column in 18 mM NaOH
solution.
l
Calculate a linear regression for GlcNAc standard
between concentration and the peak area and calculate on it the concentration of the GlcNAc in the
samples.
4. HPLC-SEC.
HPLC-SEC analysis is used to estimate the molecular size
distribution of OAg samples. Samples are run, without any pretreatment, diluted with water at 100–200 μg/mL OAg
concentration.
1. System setting.
(a) Column System: TSK gel G3000 PWXL column with a
TSK gel PWXL guard column.
(b) Calculate void and bed volume calibration injecting
λ-DNA and NaN3, respectively. For Kd determination,
the following equation was used: Kd ¼ (T e À T 0 )/
(T t À T 0 ) where: T e ¼ elution time of the analyte, T 0 ¼ elution time of the biggest fragment of λ-DNA and T t ¼ elution time of NaN3.
(c) Column compartment: 30
C.
(d) Autosampler compartment: 4
C.
(e) Detector: OAg peaks by differential refractive index
(dRI). UV detection at 214 and 260 nm for following
impurity reduction during the purification steps.
2. Chromatographic conditions:
(a) Mobile phase: 0.1 M NaCl, 0.1 M NaH 2 PO 4 , 5% ACN,
pH 7.2.
(b) Flow rate: 0.5 mL/min.
(c) Run time: isocratic for 30 min.
(d) Sample injection volume: 80 μL.
(see Note 24)
HPLC-SEC analysis is used to estimate the molecular size
distribution of the glycoconjugate, in comparison with free OAg
and free protein. Samples are run usually diluted in PBS at
50–100 μg/mL protein concentration.
O-Antigen Purification and Conjugation
287