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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.

AccQ•TAg UlT rA Ov erv iew

The Waters UPLC Amino Acid Analysis Application Solution is pre-column

derivatization and analysis technique. To perform this analysis:

1. Use Waters 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 Waters

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.

cOnT enTs Of K iTThe Waters 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.

AccQ•TAg UlT rA DerivAT izAT iOn KiT

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2

sAmple p repArAT iOn

I. Reconstitute AccQ•Tag Ultra Reagent Powdera. 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 samplesa. Use a micropipettor to deliver 70 µL of AccQ•Tag Ultra Borate Buf-

fer (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.

sTOrAge AnD sTAbil iT y• The Waters AccQ•Tag Ultra Derivatization Kit may be stored at

room temperature for up to one year.

• Reconstituted AccQ•Tag Ultra Reagent can be stored at room

temperature in a dessicator with dry dessicant for up to one week.

Note: AccQ•Fluor™ Ultra Reagent reacts with atmospheric water. Seal the

vial tightly. Do not refrigerate because of the risk of condensation.

• Derivatized amino acid samples may be stored at room temperature

for up to one week.

OrDering infOrmAT iOn

AccQ•Tag Ultra Derivatization Kit, 250 analyses 186003836

AccQ•Tag Ultra Column, 2.1 x 100 mm, 1.7 µm 186003837

AccQ•Tag Ultra Eluent A Concentrate, 1 L 186003838

AccQ•Tag Ultra Eluent B, 1 L 186003839

Amino Acid Hydrolysate Standard, 10 x 1mL WAT088122

Sample Tubes, 6 x 50 mm, 72/pkg WAT007571

Total Recovery Vials, 100/pkg 186000384C

Representative Chromatogram: Protein and Peptide Hydrolysates (10 pmol)

AMQ

NH3 His

Arg

Gly Asp Gl

u Thr

Ala

Pro

Ser

Derivitization Peak Cys

Lys

Tyr

Met Val

NVa

ILe

Leu

Phe

M inutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Minutes

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Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters, AccQ•Tag, AccQ•Fluor, Empower, ACQUITY UPLC, UPLC and The Science of What’s Possible are trademarks of Waters Corporation.

©2012 Waters Corporation. 715001331 Rev D 10/12 VW-PDF

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