Waters AccQ-Fluor Reagent Kit - Manual (Page 3)

[ Care and Use ManUal ]
AccQ•Fluor Reagent Kit 3
vI. usIng Waters accQ•Fluor reagent
Note: The Waters AccQ•Fluor Reagent Kit is not for In vitro diagnostic
use. The Waters AccQ•Fluor Reagent Kit is for research use only.
Use reconstituted AccQ•Fluor Reagent to convert samples and
standards to highly stable fluorescent
derivatives. This section describes the procedures for:
– Derivatizing a calibration standard
– Derivatizing a hydrolyzed sample
Note: Pyrolyze all glassware al 500 °C for at least 4 hours.
vII. derIvatIzIng the calIbratIon standard
The calibration standard contains 100 pmol/μl of each amino
acid (except cystine, at 50 pmol/μl). To derivatize the calibration
standard:
1. Preheat a heating block to 55 °C.
2. Use a clean syringe to deliver 10 μl of calibration standard to
the bottom of a clean sample tube.
3. Use a micropipette to add 70 μl of AccQ•Fluor Borate Buffer
(Vial 1) to the sample tube. Vortex briefly.
4. Use a micropipette with a clean tip to add 20 μl of reconstituted
AccQ•Fluor Reagent to the sample tube. Vortex immediately for
several seconds.
5. Allow to incubate for 1 minute at room temperature.
6. Use a 9-Inch Pasteur pipette to transfer the contents of the
sample tube to the bottom of an autosampler vial limited
volume Insert (LVI). Cap with a siliconelined septum.
7. Heat the vial in a heating block for 10 minutes at 55 °C.
Note: Heating converts a minor side product of Tyr to the major
mono-derivatized compound. Conversion occurs more slowly at room
temperature, with a half life of approximately 1 hour.
A 5 μl injection of derivatized standard contains 50 pmol of each
amino acid derivative (except) cystine at 25 pmol.
Derivitizing a Sample
Before derivatizing a vacuum-dried sample, you must reconstitute it.
vIII. reconstItutIng the hydrolyzed saMPle
To reconstitute the sample:
1. Prepare a 20 mM HCI solution by adding 10 μL constant-boiling
(6N) HCI to 3 ml HPLC grade water in a clean autosampler vial.
2. Add 20 μl of 20 mM constant-boiling HCI to the sample.
3. Vortex thoroughly.
IX. derIvatIzIng the saMPle
To derivatize the reconstituted sample:
I . Preheat a heating block to 55 °C.
2. Add 60 μl of AccQ•Fluor Borate Buffer to the reconstituted
sample in the 6 x 50 mm sample tube and vortex.
3. Add 20 μl of AccQ•Fluor reagent. Vortex immediately for
several seconds.
4. Allow to incubate for one minute at room temperature.
5. Transfer the contents of the tube to an autosampler vial low
volume insert (LVI). Cap with a silicone-lined septum.
6. Heat the vial In a heating block for 10 minutes at 55 °C.
X. storIng derIvatIves
Amino acid derivatives may be stored at room temperature for up to
one week if tightly sealed and protected from evaporation.