Waters AccQ-Tag Ultra Derivatization Kit User Manual
[ CARE AND USE MANUAL ]
AccQ•Tag Ultra Derivatization Kit
CONTENTS
I. INTRODUCTION
II. ACCQ•TAG ULTRA OVERVIEW
III. CONTENTS OF KIT
IV. SAMPLE PREPARATION
V. STORAGE AND STABILITY
I. INTRODUCTION
The AccQ•Tag™ Ultra Derivatization Kit is a component of the UPLC®
Amino Acid Analysis Application Solution. This application solution is
an integrated combination of instrumentation, derivatization chemistry,
separation column and eluents, methods, and software. Analysts are
assured of accurate and precise amino acid analyses with the complete
application solution. The use of the AccQ•Tag Ultra Derivatization Kit
without the rest of the application solution is not supported as an amino
acid analysis method because there are significant risks of inaccurate
qualitative and quantitative analysis.
The AccQ•Tag Ultra Derivatization Kit differs from the AccQ•Tag
Derivatization Kit (P/N WAT052880). Although the specified chemical
composition is the same for both kits, the quality control tests for The
AccQ•Tag Ultra Derivatization Kit are executed with UV detection. In
contrast, the quality control tests for The AccQ•Tag Derivatization Kit are
executed with fluorescence detection. Substituting one kit for the other
may result in unexpected interferences that may compromise sensitivity or
introduce ambiguity into peak identification.
II. ACCQ•TAG ULTRA OVERVIEW
The UPLC Amino Acid Analysis Application Solution is pre-column
derivatization and analysis technique. To perform this analysis:
1. Use AccQ•Tag Ultra Reagent Powder (US Patent 5,296,599 and
European Patent EP 0 533 200 B1) to derivatize the amino acids.
2. Separate the derivatives using the properly configured ACQUITY
UPLC System.
3. The reversed phase separation occurs on the Waters AccQ•Tag Ultra,
2.1 x 100 mm, 1.7 μm column. The derivatives are eluted with the
specified dilution of Waters AccQ•Tag Ultra Eluent A and Waters
AccQ•Tag Ultra Eluent B.
4. Quantitate the derivatives with UV detection.
5. Process the data using Empower® software.
For complete details of the instrument configuration and of the separation
and analysis methods, consult the UPLC Amino Acid Analysis Application
Solution System Guide.
[ CARE AND USE MANUAL ]
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.
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)
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.
Pro
Lys
t
Tyr
NVa
Val
Me
Cys
Derivitization Peak
ILe
Leu
Phe
AMQ
Thr
Ala
Glu
Asp
Arg
Gly
3
NH
1.01.5 2.02.5 3.03.5 4.04.5 5.05.5 6.06.5 7.07.5 8.0 min
Ser
His
2
HILIC Quality Control Reference Material
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