9. For the HPLC quantification of sugar degradation products (see
Sect. 3.2 for details), remove an aliquot (0.5–1.0 mL) of each of
the syringe-filtered samples, and place them into the appropriate
HPLC vials for the system to be used.
10. For the HPAEC-PAD quantification of sugars (see Sect. 3.3),
the syringe-filtered samples will need to be diluted due to the
sensitivity of the detection system as care must be taken not to
overload the detector. The precise dilution factor required is
dependent upon the concentration of the sugars within the
sample, which is of course unknown at this stage. However,
typically, 1000Â dilution is first evaluated (see note 5).
11. For achieving a 1000Â dilution for HPAEC-PAD, mix 100 μL
of each of the filtered samples with 9.9 mL of RO water in a
15 mL plastic centrifuge tube or a similar screw-capped test
tube that can be inverted to ensure suitable mixing.
12. Transfer a 1 mL aliquot of this dilution into a suitable HPLC
vial for HPAEC-PAD analysis.
13. If using the more simple colorimetric-based determination of
total reducing sugars, a 100 μL aliquot of each filtered sample is
required (see Sect. 3.4).
3.2 Quantification of
Sugar Degradation
Products by HPLC
1. This protocol utilizes the method described in [8]. The HPLC
system requires UV detection at 270–290 nm.
2. The use of a PDA (photodiode array) variant of UV detection is
highly recommended in order to provide additional spectral data
to further aid the identification of any peaks detected rather than
relying purely on comparison of retention times with those of
authentic standards.
3. The HPLC column used is a C18 Techsphere ODS column
(5 μm, 4.6 mm  250 mm; HPLC Technologies, UK) at ambient temperature and using gradient elution.
4. The mobile phase is a binary mixture of 1% acetic acid (solvent
A) and methanol (solvent B) running at a flow rate of 0.5 mL/
min.
5. The gradient elution ramp is from 20 to 50% methanol over
30 min with a 100% methanol column cleaning phase (for
1 min) and a 9 min re-equilibration period (at 20% methanol)
prior to the next injection.
6. The sample injection volume is 10 μL.
7. Quantification is performed by comparison of peak areas of authentic standards (0.1–1.0 g/L concentration range, dissolved directly
in RO water) including 5-HMF, furoic acid, and furfural (see note
8).
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Emily T. Kostas et al.
Sect. 3.2 for details), remove an aliquot (0.5–1.0 mL) of each of
the syringe-filtered samples, and place them into the appropriate
HPLC vials for the system to be used.
10. For the HPAEC-PAD quantification of sugars (see Sect. 3.3),
the syringe-filtered samples will need to be diluted due to the
sensitivity of the detection system as care must be taken not to
overload the detector. The precise dilution factor required is
dependent upon the concentration of the sugars within the
sample, which is of course unknown at this stage. However,
typically, 1000Â dilution is first evaluated (see note 5).
11. For achieving a 1000Â dilution for HPAEC-PAD, mix 100 μL
of each of the filtered samples with 9.9 mL of RO water in a
15 mL plastic centrifuge tube or a similar screw-capped test
tube that can be inverted to ensure suitable mixing.
12. Transfer a 1 mL aliquot of this dilution into a suitable HPLC
vial for HPAEC-PAD analysis.
13. If using the more simple colorimetric-based determination of
total reducing sugars, a 100 μL aliquot of each filtered sample is
required (see Sect. 3.4).
3.2 Quantification of
Sugar Degradation
Products by HPLC
1. This protocol utilizes the method described in [8]. The HPLC
system requires UV detection at 270–290 nm.
2. The use of a PDA (photodiode array) variant of UV detection is
highly recommended in order to provide additional spectral data
to further aid the identification of any peaks detected rather than
relying purely on comparison of retention times with those of
authentic standards.
3. The HPLC column used is a C18 Techsphere ODS column
(5 μm, 4.6 mm  250 mm; HPLC Technologies, UK) at ambient temperature and using gradient elution.
4. The mobile phase is a binary mixture of 1% acetic acid (solvent
A) and methanol (solvent B) running at a flow rate of 0.5 mL/
min.
5. The gradient elution ramp is from 20 to 50% methanol over
30 min with a 100% methanol column cleaning phase (for
1 min) and a 9 min re-equilibration period (at 20% methanol)
prior to the next injection.
6. The sample injection volume is 10 μL.
7. Quantification is performed by comparison of peak areas of authentic standards (0.1–1.0 g/L concentration range, dissolved directly
in RO water) including 5-HMF, furoic acid, and furfural (see note
8).
188
Emily T. Kostas et al.