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[ Care and Use Manual ]

AccQTag Ult ra Derivatization Kit

T he AccQTag Ultra Derivatization Kit is a component of the UPLC Amino Acid Analysis Application Solution. T his 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. T he use of the AccQTag 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. T he AccQTag Ultra Derivatization Kit differs from the AccQTag Derivatization Kit (P/N WAT052880). Although the specified chemical composition is the same for both kits, the quality control tests for the AccQTag Ultra Derivatization Kit are executed with UV detection. In contrast, the quality control tests for the AccQTag 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.

AccQTag Ult ra Ov erv ie w

The Waters UPLC Amino Acid Analysis Application Solution is pre-column derivatization and analysis technique. To perform this analysis: 1. Use Waters AccQTag 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 AccQTag Ultra, 2.1 x 100 mm, 1.7 m column. The derivatives are eluted with the specified dilution of Waters AccQTag Ultra Eluent A and Waters AccQTag 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 ent s of Kit

T he Waters AccQTag 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: AccQTag Ultra Borate Buffer - Vial 1. T he buffer is added to the samples to ensure the optimum pH for derivatization. AccQTag Ultra Reagent Powder Vial 2A. T he reagent powder is the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatizing reagent. It is shipped dry for maximum stability. AccQTag Ultra Reagent Diluent Vial 2B. T his diluent, acetonitrile, is used reconstitute the reagent for derivatization.

[ Care and Use Manual ]

Sam p l e P re pa rat ion I. Reconstitute AccQTag Ultra Reagent Powder

a. Preheat a heating block to 55 C. b. Tap vial 2A (AccQTag Ultra Reagent powder) to ensure all reagent is at bottom of vial. c. Draw 1 mL of AccQTag 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

Storage and Stabilit y

The Waters AccQTag Ultra Derivatization Kit may be stored at room temperature for up to one year. Reconstituted AccQTag Ultra Reagent can be stored at room temperature in a dessicator with dry dessicant for up to one week. Note: AccQFluor 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.


Tyr Met Val NVa

dissolves. Do not heat longer than 15 minutes.


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 L of standard and adding it to 960 L of water.
1. 00 1. 50

2. 00


2. 50


a. Transfer the Amino Acid Hydrolysate Standard (P/N WAT088122)

3. 00


3. 50

Arg Gly

II. Prepare 100 pmol/L calibration standard for protein and peptide hydrolysates

4. 00

III. Derivatize standards and samples

a. Use a micropipettor to deliver 70 L of AccQTag Ultra Borate Buffer (vial 1) to a Total Recovery vial (P/N 186000384C). b. Use a micropipettor to add 10 L of the calibration standard or dissolved sample amino acids to the vial. Vortex for several seconds. c. Use a micropipettor to add 20 L of reconstituted AccQTag 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/L.

Minutes Mi nut es

4. 50

5. 00

5. 50

6. 00

6. 50

Derivitization Peak Cys






7. 00

7. 50

Representative Chromatogram: Protein and Peptide Hydrolysates (10 pmol)

Ordering Informat ion

AccQTag Ultra Derivatization Kit, 250 analyses AccQTag Ultra Column, 2.1 x 100 mm, 1.7 m AccQTag Ultra Eluent A Concentrate, 1 L AccQTag Ultra Eluent B, 1 L Amino Acid Hydrolysate Standard, 10 x 1mL Sample Tubes, 6 x 50 mm, 72/pkg Total Recovery Vials, 100/pkg 186003836 186003837 186003838 186003839 WAT088122 WAT007571 186000384C

ILe Leu Phe

8. 00

[ Care and Use Manual ]

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Waters, AccQTag, AccQFluor, Empower, ACQUITY UPLC, UPLC and The Science of Whats Possible are trademarks of Waters Corporation.

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

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