Care and use manual, Iii. contents of kit, Iv. sample preparation – Waters AccQ-Tag Ultra Derivatization Kit User Manual
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[ CARE AND USE MANUAL ]
HILIC Quality Control Reference Material
III. CONTENTS OF KIT
The AccQ•Tag Ultra Derivatization Kit contains five sets of the
derivatizing reagents used for the UPLC Amino Acid Analysis
Application Solution. Each set of reagents includes one vial each of:
■
■
AccQ•Tag Ultra Borate Buffer – Vial 1. The buffer is added to
the samples to ensure the optimum pH for derivatization.
■
■
AccQ•Tag Ultra Reagent Powder – Vial 2A. The reagent powder
is the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
(AQC) derivatizing reagent. It is shipped dry for maximum
stability.
■
■
AccQ•Tag Ultra Reagent Diluent – Vial 2B. This diluent,
acetonitrile, is used reconstitute the reagent for derivatization.
IV. SAMPLE PREPARATION
I. Reconstitute AccQ•Tag Ultra Reagent Powder.
a. Preheat a heating block to 55 ˚C.
b. Tap vial 2A (AccQ•Tag Ultra Reagent powder) to ensure all
reagent is at bottom of vial.
c. Draw 1 mL of AccQ•Tag Ultra Reagent Diluent from vial 2B.
d. Transfer to vial 2A.
e. Vortex for 10 seconds.
f. Heat on top of heating block, vortex occasionally, until the
powder dissolves. Do not heat longer than 15 minutes.
II. Prepare 100 pmol/μL calibration standard for protein and
peptide hydrolysates.
a. Transfer the Amino Acid Hydrolysate Standard
(P/N WAT088122) from the supplied ampule to a vial
appropriate for storage in a freezer.
b. Dilute Amino Acid Hydrolysate Standard to appropriate
concentration by taking 40 μL of standard and adding it to
960 μL of water.
III. Derivatize standards and samples.
a. Use a micropipettor to deliver 70 μL of AccQ•Tag Ultra
Borate Buffer (Vial 1) to a Total Recovery Vial
(P/N 186000384C).
b. Use a micropipettor to add 10 μL of the calibration standard
or dissolved sample amino acids to the vial. Vortex for
several seconds.
c. Use a micropipettor to add 20 μL of reconstituted AccQ•Tag
Reagent to the vial.
d. Cap vial and vortex for several seconds.
e. Let stand for one minute at room temperature.
f. Heat in heating block for 10 minutes at 55 ˚C.
g. Remove from heating block with forceps and place in
instrument for analysis. The resulting standard has a
concentration of 10 pmol/μL.
Representative Chromatogram: Protein and Peptide
Hydrolysates (10 pmol)
AM
Q
NH
3
Hi
s
Arg Gl
y
Asp
Gl
u
Th
r
Al
a
Pro
Se
r
Derivitization Peak
Cy
s
Ly
s
Ty
r
Me
t
Va
l
NV
a
IL
e
Le
u
Ph
e
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0 min