l
Measure, in a 50 mL graduated cylinder, 50 mL of
acetonitrile (ACN) and add them in the 1 L cylinder
with buffer solution.
l
Add water to bring the total volume to 1 L.
l
Leave the cylinder on the stirrer until the solution is
completely homogeneous.
l
Measure the pH with the electrode (7.20 Æ 0.06).
l
Filter by vacuum the solution with a 0.22 μm filter.
(d) λ-DNA Molecular Weight Marker III 0.12–21.2 kb
(Roche; cod. 10528552001): dilute it 20Â with HPLC
eluent before injection.
(e) Sodium azide (NaN 3 ) solution (200 mg/L solution).
(f) Semicarbazide solution (100 mg semicarbazide hydrochloride + 90.5 mg of AcONa anhydrous in 10 mL of
water).
(g) 3-Deoxy-D-manno-oct-2-ulosonic acid (KDO) ammonium salt 40 μg/mL solution (the concentration value is
referred to ammonium salt).
7. Nuclear magnetic resonance (NMR) spectroscopy.
(a) Sodium deuteroxide (NaOD 4 M).
l
Weigh on the balance about 160 mg of NaOH and
transfer in a tube.
l
Add the quantity of deuterium hydroxide (D 2 O)
needed to achieve 4 M solution:
μL D 2 O ¼
mg NaOH
160
 1000
l
Close the tube and mix with vortex mixer. Keep the
tube closed until use in order to limit NaOH
carbonation.
8. 2,4,6-trinitrobenzene sulfonate (TNBS) colorimetric method.
(a) Sodium hydrogen carbonate 4% solution (1 g sodium
hydrogen carbonate dissolved in 25 mL of water).
(b) TNBS 0.1% solution (50 μL of TNBS 1 M solution in
14.6 mL of water).
(c) Adipic acid dihydrazide (ADH) 600 nmol/mL in water.
(d) ADH 100 nmol/mL in water (500 μL of ADH
600 nmol/mL diluted with 2500 μL of water).
9. Reversed-phase high-performance liquid chromatography
(RP-HPLC).
(a) Kinetex C18 column (Phenomenex, 2.6 mm 100A
15,064.6 mm, cod. 00F-4462-E0).
274
Francesca Micoli et al.
Measure, in a 50 mL graduated cylinder, 50 mL of
acetonitrile (ACN) and add them in the 1 L cylinder
with buffer solution.
l
Add water to bring the total volume to 1 L.
l
Leave the cylinder on the stirrer until the solution is
completely homogeneous.
l
Measure the pH with the electrode (7.20 Æ 0.06).
l
Filter by vacuum the solution with a 0.22 μm filter.
(d) λ-DNA Molecular Weight Marker III 0.12–21.2 kb
(Roche; cod. 10528552001): dilute it 20Â with HPLC
eluent before injection.
(e) Sodium azide (NaN 3 ) solution (200 mg/L solution).
(f) Semicarbazide solution (100 mg semicarbazide hydrochloride + 90.5 mg of AcONa anhydrous in 10 mL of
water).
(g) 3-Deoxy-D-manno-oct-2-ulosonic acid (KDO) ammonium salt 40 μg/mL solution (the concentration value is
referred to ammonium salt).
7. Nuclear magnetic resonance (NMR) spectroscopy.
(a) Sodium deuteroxide (NaOD 4 M).
l
Weigh on the balance about 160 mg of NaOH and
transfer in a tube.
l
Add the quantity of deuterium hydroxide (D 2 O)
needed to achieve 4 M solution:
μL D 2 O ¼
mg NaOH
160
 1000
l
Close the tube and mix with vortex mixer. Keep the
tube closed until use in order to limit NaOH
carbonation.
8. 2,4,6-trinitrobenzene sulfonate (TNBS) colorimetric method.
(a) Sodium hydrogen carbonate 4% solution (1 g sodium
hydrogen carbonate dissolved in 25 mL of water).
(b) TNBS 0.1% solution (50 μL of TNBS 1 M solution in
14.6 mL of water).
(c) Adipic acid dihydrazide (ADH) 600 nmol/mL in water.
(d) ADH 100 nmol/mL in water (500 μL of ADH
600 nmol/mL diluted with 2500 μL of water).
9. Reversed-phase high-performance liquid chromatography
(RP-HPLC).
(a) Kinetex C18 column (Phenomenex, 2.6 mm 100A
15,064.6 mm, cod. 00F-4462-E0).
274
Francesca Micoli et al.