l
Under chemical hood add the required quantity of
water using a suitable graduated cylinder and mix the
bottle in order to dissolve the phenol.
(b) 0.5 mg/mL glucose (Glc) solution.
l
Weight on analytical balance about 22 mg of Glc
monohydrate, transfer it into a glass bottle.
l
Calculate the quantity of water needed to obtain a
0.5 mg/mL Glc solution using the following
formula:
Water to add (mL) ¼ Glc weight (mg) Â 1.82.
l
Add the required quantity of water using a suitable
graduated cylinder and mix the bottle in order to
dissolve Glc.
3. High-performance anion exchange chromatography coupled
with pulsed amperometric detection (HPAEC-PAD).
(a) Rhamnose (Rha), galactose (Gal), glucose (Glc), mannose
(Man) sugars mix (neutral sugar) standard solution
11.25 μg/mL each.
l
In different 2 mL vials, weigh accurately about 9 mg of
each standard and dissolve them in a suitable amount of
water added by weighting on balance, in order to
obtain solutions at 4.5 mg/mL. To calculate the
amount of water and sugar to be added to each vial
use the following formulas:
μg sugar ¼ weight μg  purity% Â
no hydrated sugar MW
hydrated sugar MW
(see Note 4).
(If the sugar is not in the hydrated form the last
ratio present in the formula is equal to 1)
μL water ¼
μg sugar
4:5
l
Weigh an empty 500 mL glass bottle on the technical balance
and transfer 1 mL of each sugar solution prepared in previous
section; then add water up to achieve a total weight of 400 g in
order to obtain a solution with a concentration of 11.25 μg/mL
for each standard.
(b) N-acetylglucosamine (GlcNAc) standard solution 11.25 μg/
mL.
l
In a 2 mL vial weigh about 9 mg of GlcNAc and dissolve it
in a suitable amount of water added by weighting on
balance, in order to obtain a solution at 4.5 mg/mL. To
O-Antigen Purification and Conjugation
271
Under chemical hood add the required quantity of
water using a suitable graduated cylinder and mix the
bottle in order to dissolve the phenol.
(b) 0.5 mg/mL glucose (Glc) solution.
l
Weight on analytical balance about 22 mg of Glc
monohydrate, transfer it into a glass bottle.
l
Calculate the quantity of water needed to obtain a
0.5 mg/mL Glc solution using the following
formula:
Water to add (mL) ¼ Glc weight (mg) Â 1.82.
l
Add the required quantity of water using a suitable
graduated cylinder and mix the bottle in order to
dissolve Glc.
3. High-performance anion exchange chromatography coupled
with pulsed amperometric detection (HPAEC-PAD).
(a) Rhamnose (Rha), galactose (Gal), glucose (Glc), mannose
(Man) sugars mix (neutral sugar) standard solution
11.25 μg/mL each.
l
In different 2 mL vials, weigh accurately about 9 mg of
each standard and dissolve them in a suitable amount of
water added by weighting on balance, in order to
obtain solutions at 4.5 mg/mL. To calculate the
amount of water and sugar to be added to each vial
use the following formulas:
μg sugar ¼ weight μg  purity% Â
no hydrated sugar MW
hydrated sugar MW
(see Note 4).
(If the sugar is not in the hydrated form the last
ratio present in the formula is equal to 1)
μL water ¼
μg sugar
4:5
l
Weigh an empty 500 mL glass bottle on the technical balance
and transfer 1 mL of each sugar solution prepared in previous
section; then add water up to achieve a total weight of 400 g in
order to obtain a solution with a concentration of 11.25 μg/mL
for each standard.
(b) N-acetylglucosamine (GlcNAc) standard solution 11.25 μg/
mL.
l
In a 2 mL vial weigh about 9 mg of GlcNAc and dissolve it
in a suitable amount of water added by weighting on
balance, in order to obtain a solution at 4.5 mg/mL. To
O-Antigen Purification and Conjugation
271