8. Vortex each tube vigorously every 5–10 min. Vortex samples
carefully to ensure all solids remain at the bottom of the tube
and immersed in acid.
9. Place a rubber stopper snugly in the top of the glass tube. Place
a tear-away aluminum crimp cap on top of the rubber stopper.
Crimp the caps tightly, and vortex to mix the contents thoroughly. Tubes may also be inverted to mix, ensure that the
solids remain in the liquid, and do not end up stuck to the
rubber stopper.
10. Place the tubes in a rack suitable for autoclaving. Be sure to
place the rack in a tray for secondary containment. Secondary
containment should contain any tubes and their contents if
they were to break during the autoclave cycle.
11. After completion of the autoclave cycle, open the autoclave door,
but allow tubes to cool for approximately 15 min before removing
them from the autoclave. Once the samples are removed from the
autoclave, allow the samples to cool for 30 min to 1 h or until
tubes reach room temperature. Use caution when handling hot
pressure tubes after removal from the autoclave. The pressurized
tubes are a possible explosion hazard if not cooled properly.
12. Hydrolyzed samples for spectrophotometric and liquid chromatographic analysis should be prepared by separating the
solids from the hydrolysate. Acidic solid-free hydrolysate may
be stored in a refrigerator (4
C) for up to 24 h before being
analyzed. If longer storage is necessary, store the hydrolysate
(separated from the solids) in a freezer (À20
C) for up to
7 days. The sample should be filtered through a 0.2 μm nylon
filter (use the disposable syringes and compatible filters) OR
centrifuged to remove ALL solids.
13. If using a digital dry block for heating, regularly check the
temperature of each block using a certified thermometer as
the heating coils used in these systems can fail over time.
14. Create a six-point calibration with the 0.25 mg/mL glucose
stock solution using the dilutions outlined in Table 1. Prepare
dilutions directly into the glass reaction vials (13 Â 100 mm or
equivalent). Prepare the CVS at the center of the calibration
range: 75 μL CVS glucose stock solution (0.25 mg/mL) and
425 μL water. Prepare the CVS dilution directly into the glass
reaction vial. Prepare sample dilutions directly in the glass
reaction vials: start with a 1:10 (50 μL hydrolysate) or greater
dilution of the filtered/centrifuged acidic hydrolysate. Dilute
the sample to a total volume of 0.5 mL. If the sample is known
to be high in carbohydrates, start with a 1:20 dilution. The
goal is to have the sample absorbance fall in the middle of the
calibration curve. DO NOT use neutralized hydrolysate as the
soluble salts leftover from the neutralization will affect the
198
S. Van Wychen and L.M.L. Laurens
carefully to ensure all solids remain at the bottom of the tube
and immersed in acid.
9. Place a rubber stopper snugly in the top of the glass tube. Place
a tear-away aluminum crimp cap on top of the rubber stopper.
Crimp the caps tightly, and vortex to mix the contents thoroughly. Tubes may also be inverted to mix, ensure that the
solids remain in the liquid, and do not end up stuck to the
rubber stopper.
10. Place the tubes in a rack suitable for autoclaving. Be sure to
place the rack in a tray for secondary containment. Secondary
containment should contain any tubes and their contents if
they were to break during the autoclave cycle.
11. After completion of the autoclave cycle, open the autoclave door,
but allow tubes to cool for approximately 15 min before removing
them from the autoclave. Once the samples are removed from the
autoclave, allow the samples to cool for 30 min to 1 h or until
tubes reach room temperature. Use caution when handling hot
pressure tubes after removal from the autoclave. The pressurized
tubes are a possible explosion hazard if not cooled properly.
12. Hydrolyzed samples for spectrophotometric and liquid chromatographic analysis should be prepared by separating the
solids from the hydrolysate. Acidic solid-free hydrolysate may
be stored in a refrigerator (4
C) for up to 24 h before being
analyzed. If longer storage is necessary, store the hydrolysate
(separated from the solids) in a freezer (À20
C) for up to
7 days. The sample should be filtered through a 0.2 μm nylon
filter (use the disposable syringes and compatible filters) OR
centrifuged to remove ALL solids.
13. If using a digital dry block for heating, regularly check the
temperature of each block using a certified thermometer as
the heating coils used in these systems can fail over time.
14. Create a six-point calibration with the 0.25 mg/mL glucose
stock solution using the dilutions outlined in Table 1. Prepare
dilutions directly into the glass reaction vials (13 Â 100 mm or
equivalent). Prepare the CVS at the center of the calibration
range: 75 μL CVS glucose stock solution (0.25 mg/mL) and
425 μL water. Prepare the CVS dilution directly into the glass
reaction vial. Prepare sample dilutions directly in the glass
reaction vials: start with a 1:10 (50 μL hydrolysate) or greater
dilution of the filtered/centrifuged acidic hydrolysate. Dilute
the sample to a total volume of 0.5 mL. If the sample is known
to be high in carbohydrates, start with a 1:20 dilution. The
goal is to have the sample absorbance fall in the middle of the
calibration curve. DO NOT use neutralized hydrolysate as the
soluble salts leftover from the neutralization will affect the
198
S. Van Wychen and L.M.L. Laurens