Waters AccQ-Fluor Reagent Kit User Manual

[ Care and Use ManUal ]

ACCQ•Fluor reagent kIt

I. IntroduCtIon

The Waters AccQ•Fluor™ Reagent Kit contains five sets of the deriva-

tizing reagents used to perform the AccQ•Tag™ Amino Acid Analysis

Method. A set consists of one vial each of:

– AccQ•Fluor Borate Buffer - Vial 1. You add the buffer to the sam-

ples to generate the proper pH environment for the derivatizatlon.

– AccQ•Fluor Reagent Powder - Vial 2A. The reagent powder Is the

6-aminoquinolyl-N-hydroxysuccinimidyI carbamate (AQC) derivatiz-

ing reagent. It Is shipped dry for maximum stability.

– AccQ•Fluor Reagent Diluent - Vial 2B. Use the diluent(acetonitrile)

to reconstitute the reagent for derivatization.

The Waters AccQ•Fluor Reagent Kit (Figure 1) contains sufficient

reagent for up to 250 analyses.

Contents

I. IntroduCtIon

II. Waters accQ•tag Method overvIeW

III. reagent PurIty

Iv. storage

v. reconstItutIng accQ•Fluor reagent

vI. usIng Waters accQ•Fluor reagent

vII. derIvatIzIng the calIbratIon standard

vIII. reconstItutIng the hydrolyzed saMPle

IX. derIvatIzIng the saMPle

X. storIng derIvatIves

Figure 1: AccQ•Fluor Reagent Kit

AccQ•Fluor Reagent Kit 1

XI. sPecIFIcatIons

XII. technIcal lIterature

XIII. orderIng InForMatIon

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II. Waters accQ•tag Method overvIeW

The AccQ•Tag Method is a precolumn derivatization and analysis

technique for peptide and protein hydrolysate amino acids. To

perform the AccQ-Tag Method:

1. Use Waters AccQ•Fluor Reagent (US Patent 5,296,599

and European Patent EP 0 533 200 B1) to derivatize the

amino acids.

2. Separate the derivatives by reversed-phase HPLC.

3. Quantitate the derivatives by fluorescence detection.

III. reagent PurIty

The Waters AccQ•Fluor Reagent Kit is manufactured in Waters’- ISO

9001-2006 certified facilities under strict guidelines to ensure the

lowest possible amino acid background. To retain this level of purity,

handle all reagents carefully.

6. Heat Vial 2A on top of heating block, vortex occasionally, until

the powder dissolves. Do not heat longer than 10 minutes.

Reconstituted AccQ•Fluor Reagent is approximately 10 mM

AQC in acetontrile.

Note: You can store the reconstituted AccQ•Tag reagent in a desiccator

at room temperature for up to one week. The AccQ•Tag reagent reacts

with atmospheric moisture so seal the container tightly when not in

use and DO NOT refrigerate.

Reconstituted AccQ•Fluor Reagent is approximately 10 mM AQC in

acetontrile.

IV. storage

If unopened, the AccQ•Fluor Reagent Kit may be stored at room

temperature for up to one year.

v. reconstItutIng accQ•Fluor reagent

To reconstitute the AccQ•Fluor Reagent (Figure 2):

1. Preheat a heating block to 55 °C.

2. Tap Vial 2A lightly before opening to ensure all AccQ•Fluor

Reagent Powder is at the bottom of the vial.

3. Rinse a clean micropipette by drawing and discarding 1 ml of

AccQ•Fluor Reagent Diluent from Vial 2B.

WARNING: Acetonitrile is flammable and toxic. Refer to the Material

Safety Data Sheet.

4. Transfer 1.0 ml of AccQ•Fluor Reagent Diluent From Vial 2B to

the AccQ•Fluor Reagent Powder In Vial 2A.

Figure 2: Reconstituting AccQ•fluor Reagent

storage

Store reconstituted AccQ•Fluor Reagent at room temperature in a

desiccator for up to one week.

Note: AccQ•Fluor reagent reacts with atmospheric moisture. Seal the

container tightly when not in use.

5. Cap Vial 2A lightly and vortex for 10 seconds.

AccQ•Fluor Reagent Kit 2

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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. T his 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.

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. 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.

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.

AccQ•Fluor Reagent Kit 3

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XI. sPecIFIcatIons

Waters manufactures the AccQ•Fluor Reagent Kit to exacting analyti-

cal specifications to minimize amino acid background. A 10 μL injec-

tion of a property prepared derivatizatlon blank (20 μL reconstituted

AccQ•Fluor Reagent plus 80 μL. AccQ•Fluor Borate Buffer) contains

less than 2 pmol of any of the hydrolysate amino acids (less than 4

pmol Cys).

Ensure all glassware is clean. For high sensitivity analyses, pyrolyze

all sample tubes at 500 °C for at least 4 hours.

XII. technIcal lIterature

Cohen, S.A. and D.P. Michaud, (1993) “Synthesis of a Fluorescent

Derivatizing Reagent, 6-Aminoquinolyl-N- Hydroxysuccinimidyl Car-

bamate, and its Application for the Analysis of Hydrolysale Amino

Acids via High Performance Liquid Chromatography,” Anal. Biochem.

211, pp 279.287.

XIII. orderIng InForMatIon

Item Part Number

AccQ•Fluor Reagent Kit WAT052880

AccQ•Tag Chemistry Package WAT052875

AccQ•Tag Column, 3.9 x 150 mm WAT052885

AccQ•Tag Eluent A Concentrate, 950 ml WAT052890

AccQ•Tag EIuent B, 950 ml WAT052895

Amino Acid Hydrolysale Standard WA T0 8 8122

Aulosampler Vials, 4 ml, Glass, 48/pkg WAT 072710

Caps for 4 ml Aulosampler Vials, 144/pkg W AT 0 7 9 711

Septa for 4 mL Aulosampler Vials, 1440/pkg WAT073005

150 μL Low Volume Inserts, 144/pkg WAT072704

Springs for Low Volume Inserts, 144/pkg WAT072708

Sample tubes, 6 x 50 mm, 72/pkg WAT00757I

Cohen, S.A., De Antonis, K., and D.P. Michaud, (1993) “Compositional

Protein Analysis Using 6-Aminoquinolyl-N-Hydroxysuccinimidyl

Carbamate, a Novel Derivatization Reagent,” Techniques In Protein

Chemistry IV, pp 289.298, (R.H. Angeletil, Ed.) Academic Press, San

Diego, CA.

AccQ•Fluor Reagent Kit 4

[ Care and Use ManUal ]

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© 2008 Waters Corporation. Waters, The Science of

What’s Possible, AccQ•Tag, and AccQ•Fluor are
trademarks of Waters Corporation.

May 2008 WAT0052881 Rev 3

Waters Corporation

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

AccQ•Fluor Reagent Kit 5