Remark: As with the samples, the calibration curve
solutions must also be prepared just prior to the assay.
l
Filter the solutions through a 0.2 μm nylon filter into
different HPLC vials labeling them with content
standard name.
(b) Samples preparation.
l
Weigh in an Eppendorf tube about 5 mg (total sugar)
of sample and dissolve it in the suitable quantity of
water/ACN 50% in order to obtain a 20 mg/mL
(total sugar) solution.
Water=ACN 50%to be added mL
ð
Þ
: sample weight mg
ð ÞÂpurity %
ð Þ=20:
l
For each sample:
– Label one Eppendorf tube with “sample name,” transfer
100 μL of sample and 100 μL of water/ACN 50% performing
a factor 2 dilution.
– Filter the samples through a 0.2 μm nylon filter into different
HPLC vials, labeling them with content sample name.
(c) System setting.
l
Column System: HPLC in line filter—Kinetex C18 column
connected in series.
l
Column compartment: 30
C.
l
Autosampler compartment: 4
C.
l
UV detector: wavelength set at 195 nm.
l
Chromatographic conditions:
– Separation performed in isocratic condition: eluent 50%
ACN and 50% water (mixture generated by
HPLC pump).
– Flow rate: 1 mL/min.
– Run time: 8 min.
– Sample injection volume: 25 μL.
– Remarks: After the last chromatographic analysis store
the column in 65% ACN and 35% water.
(d) Calculation of free active ester groups.
Quantify the amount of free SIDEA using the calibration
curve built with the peak areas of SIDEA standard at 195 nm.
Calculate percentage of free active ester groups as molar
ratio % of free ester groups (see Note 28) divided by total
active ester groups (determined by A260 after ammonia
treatment).
296
Francesca Micoli et al.
solutions must also be prepared just prior to the assay.
l
Filter the solutions through a 0.2 μm nylon filter into
different HPLC vials labeling them with content
standard name.
(b) Samples preparation.
l
Weigh in an Eppendorf tube about 5 mg (total sugar)
of sample and dissolve it in the suitable quantity of
water/ACN 50% in order to obtain a 20 mg/mL
(total sugar) solution.
Water=ACN 50%to be added mL
ð
Þ
: sample weight mg
ð ÞÂpurity %
ð Þ=20:
l
For each sample:
– Label one Eppendorf tube with “sample name,” transfer
100 μL of sample and 100 μL of water/ACN 50% performing
a factor 2 dilution.
– Filter the samples through a 0.2 μm nylon filter into different
HPLC vials, labeling them with content sample name.
(c) System setting.
l
Column System: HPLC in line filter—Kinetex C18 column
connected in series.
l
Column compartment: 30
C.
l
Autosampler compartment: 4
C.
l
UV detector: wavelength set at 195 nm.
l
Chromatographic conditions:
– Separation performed in isocratic condition: eluent 50%
ACN and 50% water (mixture generated by
HPLC pump).
– Flow rate: 1 mL/min.
– Run time: 8 min.
– Sample injection volume: 25 μL.
– Remarks: After the last chromatographic analysis store
the column in 65% ACN and 35% water.
(d) Calculation of free active ester groups.
Quantify the amount of free SIDEA using the calibration
curve built with the peak areas of SIDEA standard at 195 nm.
Calculate percentage of free active ester groups as molar
ratio % of free ester groups (see Note 28) divided by total
active ester groups (determined by A260 after ammonia
treatment).
296
Francesca Micoli et al.