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Sample preparation:
IFT: Weigh accurately 800mg IFT (containing± 3.5%GOS) in a volumetric flask of
50mL
AddMilli-Q until ¾ of the flask is filled
Add 750 µl Carrez reagent 1 and homogenize thoroughly
Add 750 µl Carrez reagent 2 and homogenize thoroughly
Adjust the volume until 50mL and homogenize
Fill an Eppendorf tubewith sample and centrifuge for 10minutes at 13000
rpm.
Prepare for the samples vials for the fractionation on the Dionex ICS-5000
After centrifugation filtrate the supernatant through a 0.2 µmmicrofilter and
add to the vials.
Run a sequence withmethod “fractionation detector on” at least 3 times a
carrez treated, lactose standard of 60 ppm to stabilize the column. Check if
retention time for lactose is stable. If so, use the retention time of the end of
the lactose peak of the, 60 ppm carrez treated standard, to start collecting
the GOS fraction. (usually around 3.1min)
In the sequence switch to programmethod “fractionation detector off”.
(aquire only pressure data).
When in the sequence is switched to the programmethod “fractionation cell
off”, disconnect the tubing of the Dionex ICS-5000which goes into the
detector. Monitor the time on screen and discard the collected fraction up to
3.1min (end lactose peak).
Collect the fractions of the samples from 3.1minutes to 7.2minutes in a 15
mL centrifuge tube.
OPTIONAL:
After collecting the samples connect the tubing to the detector
again and run the lactose standard and samples againwith the program
method “fractionation detector on” to check the stability of the retention
times.
Prepare aCarbograph column:
By vacuum elution. Make sure that the flow of the eluents is constant and at a
rate of 1 to 2 drops per second. Do not let the column bed get dry. Close the
tap exactlywhen the eluent level reaches the top of the column bed
1.5mL 80% ACN+ 0.1% TFA »waste
1.5mL Milli-Q »waste
Total fraction »waste
1.5mL Milli-Q »waste
Remove the SPE column from the vac-elute. Add 1.5mL 25% ACN and
collect the fraction in a 2mL Eppendorf tube by using pressure from a
pipet to get a flowrate of 1 or 2 drops per second. Carefully (watch out
for bubbles) press the last of the liquid into the tube.
Dry the collected sample in the Eppendorf concentrator under vacuum at
45°C until all liquid is evaporated (±4 hours) or overnight at room
temperature. When evaporating at 45°C reduce the temperature to room
temperature at the end when only a small amount of ACN is present.
Warm up thewaterbath to 60°C
Dissolve the dried residue in 500 µl 40mM phosphate buffer pH 6.0with 30
µg/mL Ribose
GOS-02
FORWORKINGGROUP/ERPUSEONLY
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