UPLC Amino Acid Analysis Solution - waters.com · The Waters® ACQUITY UltraPerformance LC® (UPLC®) system provides separation technology that offers higher throughput, sensitivity,

UPLC Amino Acid Analysis Solution

System Guide

71500129702 / Revision B

Copyright © Waters Corporation 2007All rights reserved

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Copyright notice

© 2007 WATERS CORPORATION. PRINTED IN THE UNITED STATES OF AMERICA AND IRELAND. ALL RIGHTS RESERVED. THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER.

The information in this document is subject to change without notice and should not be construed as a commitment by Waters Corporation. Waters Corporation assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Waters Corporation be liable for incidental or consequential damages in connection with, or arising from, its use.

Trademarks

ACQUITY UPLC, Millennium, UPLC, and Waters are registered trademarks, and AccQ•Fluor, AccQ•Tag, eCord, Empower, and MassLynx are trademarks of Waters Corporation.

PEEK is a trademark of Victrex Corporation.

Teflon is a registered trademark of E. I. DuPont de Nemours and Company.

Other trademarks or registered trademarks are the sole property of their respective owners.

Customer comments

Waters’ Technical Communications department invites you to tell us of any errors you encounter in this document or to suggest ideas for otherwise improving it. Please help us better understand what you expect from our documentation so that we can continuously improve its accuracy and usability.

We seriously consider every customer comment we receive. You can reach us at [email protected].

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Contacting Waters

Contact Waters® with enhancement requests or technical questions regarding the use, transportation, removal, or disposal of any Waters product. You can reach us via the Internet, telephone, or conventional mail.

Waters contact information

Contacting medium Information

Internet The Waters Web site includes phone numbers for Waters locations worldwide. Go to www.waters.com, and click About Waters > Worldwide Offices.

Telephone In the USA or Canada, phone 508 478-2000.

Conventional mail Waters Corporation34 Maple StreetMilford, MA 01757 USA

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Safety considerations

Some reagents and samples used with Waters instruments and devices can pose chemical, biological, and radiological hazards. You must know the potentially hazardous effects of all substances you work with. Always follow Good Laboratory Practice, and consult your organization’s safety representative for guidance.

When you develop methods, follow the “Protocol for the Adoption of Analytical Methods in the Clinical Chemistry Laboratory,” American Journal of Medical Technology, 44, 1, pages 30–37 (1978). This protocol addresses good operating procedures and the techniques necessary to validate system and method performance.

UPLC Amino Acid Analysis Solution system considerations

Intended use

The Waters UPLC® Amino Acid Analysis (AAA) Solution, in conjunction with Waters AccQ•Tag™ Ultra chemistries for amino acid analysis, analyzes protein and peptide hydrolysates (for identification and characterization), cell culture media, and the nutritional composition of foods and feeds. The UPLC AAA Solution is not intended to be used for clinical analysis of physiological amino acids. Coelutions of certain peak pairs are known to exist.

Biological hazardAvoid infection with potentially infectious biological fluids by observing Good Laboratory Practices and consulting your organization’s biohazard safety representative regarding the proper use and handling of such materials. Specific precautions appear in the latest edition of the US National Institutes of Health (NIH) publication, Biosafety in Microbiological and Biomedical Laboratories (BMBL).

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CalibrationTo calibrate methods, follow acceptable calibration methods using at least five standards to generate a standard curve. The concentration range for standards should include the entire range of quality-control samples, typical specimens, and atypical specimens.

Quality controlRoutinely run three quality-control samples that represent subnormal, normal, and above-normal levels of a compound. Ensure that quality-control sample results fall within an acceptable range, and evaluate precision from day to day and run to run. Data collected when quality control samples are out of range might not be valid. Do not report these data until you are certain that the instrument performs satisfactorily.

When analyzing samples from a complex matrix such as soil, tissue, serum/plasma, whole blood, etc., note that the matrix components can adversely affect LC/MS results, enhancing or suppressing ionization. To minimize these matrix effects, Waters recommends you adopt the following measures:

• Prior to the instrumental analysis, use appropriate sample pretreatment such as protein precipitation, liquid/liquid extraction (LLE), or solid phase extraction (SPE) to remove matrix interferences.

• Whenever possible, verify method accuracy and precision using matrix-matched calibrators and QC samples.

• Use one or more internal standard compounds, preferably isotopically labeled analytes.

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IVD authorized representative information

IVD authorized representative

Waters Corporation (Micromass UK Limited) is registered in the United Kingdom with the Medicines and Healthcare Products Regulatory Agency (MHRA) at Market Towers, 1 Nine Elms Lane, London, SW8 5NQ. The reference number is IVD000167.

Waters Corporation (Micromass UK Ltd.)Floats RoadWythenshaweManchester M23 9LZUnited Kingdom

Telephone: +44-161-946-2400

Fax: +44-161-946-2480

Contact: Quality manager

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Table of Contents

1 System Overview .................................................................................... 1-1

Waters UPLC Amino Acid Analysis Solution .............................................. 1-2 ACQUITY UPLC system ................................................................................. 1-2 ACQUITY UPLC TUV Detector...................................................................... 1-3 ACQUITY UPLC PDA Detector...................................................................... 1-3 ACQUITY UPLC FLR Detector ...................................................................... 1-3

Empower software ............................................................................................ 1-3

UPLC Amino Acid Analysis applications kit .............................................. 1-4

Using the AccQ•Tag Ultra method ............................................................... 1-5 Derivatization chemistry overview ................................................................. 1-5

2 System Setup ........................................................................................... 2-1

Installing the 2-µL sample loop ...................................................................... 2-2

Installing the detector tubing ........................................................................ 2-2

Plumbing connections ..................................................................................... 2-3 Installation recommendations for fittings...................................................... 2-3 Plumbing the TUV detector ............................................................................ 2-4 Plumbing the PDA detector............................................................................. 2-5 Plumbing the FLR detector ............................................................................. 2-6 Plumbing the binary solvent manager and sample manager ....................... 2-6 Installing the column for optical detection................................................... 2-12 Installing the column for MS detection ........................................................ 2-13 Connecting to the solvent supply .................................................................. 2-15

Table of Contents ix

Ethernet connections ..................................................................................... 2-16

3 Verifying System Operation ................................................................ 3-1

Preparing the system ....................................................................................... 3-2

Preparing the eluents ...................................................................................... 3-4 Setting up the mobile phase............................................................................ 3-5 Preparing needle wash solvents...................................................................... 3-6

Creating the test methods ............................................................................... 3-7 TUV instrument method ................................................................................. 3-7 PDA instrument method ............................................................................... 3-12 FLR instrument method................................................................................ 3-17

Creating the sample set methods ................................................................ 3-22 TUV sample set method ................................................................................ 3-22 PDA sample set method ................................................................................ 3-23 FLR sample set method................................................................................. 3-23

Preparing the system verification sample ................................................ 3-23

Performing the test ........................................................................................ 3-24

Quantitating and interpreting amino acid analysis data ..................... 3-28 Processing samples ........................................................................................ 3-28

4 Preparing Standards and Samples .................................................... 4-1

Reconstituting the AccQ•Tag Ultra reagent powder ............................... 4-2

Preparing the calibration standard ............................................................. 4-3 Preparing the calibration standard: External method .................................. 4-3 Preparing the calibration standard: Internal method ................................... 4-4 Derivatizing the calibration standard ............................................................ 4-5

Preparing the samples ..................................................................................... 4-6 Determining sample quantity ......................................................................... 4-6 Hydrolyzing the samples ................................................................................. 4-6 Derivatizing the samples................................................................................. 4-7

x Table of Contents

5 Operating the System ............................................................................ 5-1

Preparing the UPLC AAA Solution ............................................................... 5-2 Setting up the mobile phases .......................................................................... 5-2 Preparing the system....................................................................................... 5-2

Setting up Empower software ....................................................................... 5-3 Empower QuickStart Interface ....................................................................... 5-3

Opening Empower and Restoring Projects ................................................ 5-4

Loading, editing, and running a sample set method ................................ 5-6

Processing Data ............................................................................................... 5-10 Batch processing data.................................................................................... 5-10 Reviewing processed data.............................................................................. 5-12 Generating a report ....................................................................................... 5-12

6 Handling Special Samples ................................................................... 6-1

Analyzing protein samples for cysteine and methionine ........................ 6-2 Analyzing cysteine ........................................................................................... 6-2

Analyzing the nutritional content of foods and feeds .............................. 6-3

Analyzing cell culture media ......................................................................... 6-4 Setting up the mobile phase............................................................................ 6-4 Analyzing the samples..................................................................................... 6-5 Representative chromatograms ...................................................................... 6-6

7 Troubleshooting ..................................................................................... 7-1

General troubleshooting principles ............................................................. 7-2

Derivatization troubleshooting ..................................................................... 7-3 Retention time problems ................................................................................. 7-3 Quantitative Problems .................................................................................... 7-4 Derivatization problems .................................................................................. 7-7

Derivatization issues ........................................................................................ 7-9 Insufficient reagent ......................................................................................... 7-9 Sample reconstitution.................................................................................... 7-10

Table of Contents xi

Chromatography troubleshooting .............................................................. 7-11 Eluent delivery............................................................................................... 7-13 System efficiency............................................................................................ 7-13 Column heater ............................................................................................... 7-14

A Safety Advisories .................................................................................. A-1

Warning symbols ............................................................................................... A-2 Task-specific hazard warnings........................................................................ A-2 Warnings that apply to particular instruments, instrument components, and

sample types A-3

Caution symbol .................................................................................................. A-5

Warnings that apply to all Waters instruments ......................................... A-5

Electrical and handling symbols ................................................................. A-13 Electrical symbols .......................................................................................... A-13 Handling symbols .......................................................................................... A-14

B Materials of Construction and Compliant Solvents ..................... B-1

Preventing contamination ............................................................................. B-2

Items exposed to solvent ................................................................................ B-2

Solvents used to prepare mobile phases .................................................... B-3

C Derivatization Guidelines .................................................................. C-1

Introduction ...................................................................................................... C-2

Estimating sample amount ............................................................................ C-3

Estimating the requirement for neutralization ....................................... C-6 Considerations ................................................................................................. C-6

Confirming sufficient reagent ...................................................................... C-7 Calculation terms............................................................................................. C-8

Examples ............................................................................................................ C-9 Example with known composition of sample, concentration in mg/mL........ C-9 Example with unknown sample composition, concentration in mg/mL ..... C-10

xii Table of Contents

Example with unknown sample composition, concentration in mg/tube ... C-11 Example with unknown sample composition, concentration in pmoles/tube.......

C-13 Example with unknown sample composition, concentration in pmoles/µL C-13 Example with unknown sample composition, concentration in µM ........... C-14 Example with unknown sample composition, concentration in %.............. C-16 Example with unknown sample composition, concentration in mg%......... C-17

Index ..................................................................................................... Index-1

Table of Contents xiii

xiv Table of Contents

1 System Overview

The Waters® ACQUITY UltraPerformance LC® (UPLC®) system provides separation technology that offers higher throughput, sensitivity, and peak capacity than traditional HPLC systems. The Waters UPLC Amino Acid Analysis (AAA) Solution combines UPLC separation technology with AccQ•Tag™ Ultra Derivatization Chemistry. The combination enables you to perform protein characterization, cell culture monitoring, and nutritional analysis of foods and feeds in considerably less time than typical HPLC separations require. Using the project templates and custom calculations supplied on the product CD, the UPLC AAA Solution, integrated with the ACQUITY UPLC system, enables you to easily obtain accurate results and exceptional chromatographic resolution.

Use this document in conjunction with the ACQUITY UPLC system documentation and Empower™ data software user documentation.

Contents:

Topic Page

Waters UPLC Amino Acid Analysis Solution 1-2

Empower software 1-3

UPLC Amino Acid Analysis applications kit 1-4

Using the AccQ•Tag Ultra method 1-5

1-1

Waters UPLC Amino Acid Analysis Solution

The UPLC Amino Acid Analysis (AAA) Solution is made up of the following components:

• Binary solvent manager

• Sample manager

• Column heater module

• One of the following detectors:

– Tunable ultraviolet/visible (TUV) detector with flow cell

– PhotoDiode Array (PDA) Detector with flow cell

– Fluorescence (FLR) Detector with flow cell

• UPLC AAA kit:

– AccQ•Tag™ Ultra Chemistry Package, with QC-tested column and appropriate chemistry (eluents, derivatization reagents, and standards)

– Empower project template(s)

– 2-µL sample loop

– Column in-line filter kit

– Tubing kit (0.0025 ID PEEK) - inlet

– Associated manuals

– 150mm column stabilizer

– Total recovery vials with caps

ACQUITY UPLC systemACQUITY UPLC technology uses stationary phase particles measuring 1.7 µm in diameter to improve sensitivity, resolution, and speed. Backpressure, however, increases inversely with the square of the particle diameter.

In another inverse relation, the optimum linear velocity increases as particle size decreases. Combined, these effects mean the pressure needed to maintain optimum linear velocity increases to the third power of the particle size. A first-generation UPLC column, therefore, requires a pressure of about 25 times higher than that required for a 5-µm HPLC column of the same length.

1-2 System Overview

Under isocratic conditions, the UPLC column is three times faster than the HPLC column, and the plate count is three times greater. Performance differences between UPLC and HPLC stand in even greater contrast when you run the columns at the same linear velocity.

ACQUITY UPLC TUV DetectorThe ACQUITY TUV (tunable UV) optical detector is a two-channel, ultraviolet/visible (UV/Vis) absorbance detector and is controlled by Empower software for LC applications.

The 10-mm, path length, light-guide flow cell is intended for high sensitivity chromatography with high peak capacity and has a volume of 500 nL.

The TUV detector operates from 190 to 700 nm.

ACQUITY UPLC PDA DetectorThe ACQUITY PDA (photodiode array) optical detector is an ultraviolet/visible (UV/Vis) light spectrophotometer that operates between 190 and 500 nm and up to 2.0 AU.

The low-volume (500nL) light-guide flow cell and high-sensitivity lamp provide optimum path length and high light throughput.

ACQUITY UPLC FLR DetectorThe ACQUITY FLR detector is a multi-channel, multi-wavelength fluorescence detector with a low-volume, axially illuminated fused quartz flow cell, low-noise electronics, and high-intensity (150 W) mercury-xenon (Hg-Xe) arc lamp.

The detector has an excitation wavelength range of 200 to 890 nm, an emission wavelength range of 210 to 900 nm, and support for high speed data rates up to 80Hz. It also offers 3D scanning capability for easier methods development.

Empower software

The UPLC Amino Acid Analysis Solution system uses Empower software to acquire, process, report, and manage chromatographic information.

The Waters Empower software system includes these elements:

Empower software 1-3

• Control for the ACQUITY UPLC chromatographic instrumentation

• Chromatographic data acquisition and data processing computers

• Empower software, a 32-bit data acquisition and management software application with advanced integrated database architecture

For details about the major subsystems, hardware, and operating system configurations that are supported by Empower software, see the Empower 2 Installation and Configuration Guide.

UPLC Amino Acid Analysis applications kit

The UPLC AAA applications kit includes the AccQ•Tag Ultra Chemistry Package, which you can use to perform up to 250 analyses. The chemistry package includes the following materials:

• AccQ•tag Ultra Reagent Kit

– Waters AccQ•Tag Ultra Borate buffer (1), 5 bottles

– Waters AccQ•Tag Ultra Reagent powder (2A), 5 bottles

– Waters AccQ•Tag Ultra Reagent diluent (2B), 5 bottles

• AccQ•Tag Ultra Amino Acid Analysis Column

The column separates the amino acid derivatives produced by the AccQ•Tag Ultraderivatization reaction. The AccQ•Tag Ultra Column is a high-efficiency ACQUITY UPLC BEH C18, 1.7µm column specifically certified for use with the UPLC AAA Solution. Care and use of the column is described in the ACQUITY UPLC BEH Column Care and Use Instructions document, available on Waters.com.

• AccQ•Tag Ultra Eluent A Concentrate, 950 mL

A premixed concentrated aqueous/organic buffer.

• AccQ•Tag Ultra Eluent B, 950 mL

• Amino Acid Hydrolysate Standard, 10 1-mL ampoules

Each ampoule contains a 2.5 mM mixture of the 17 hydrolysate amino acids, with the exception of cystine (a 1.25 mM mixture).

• 6 × 50-mm sample tubes (for preparing samples and standards)

• 2.0-µL sample loop

• Total recovery vials with caps

1-4 System Overview

• Waters UPLC AAA Solution System CD (includes the UPLC AAA Solution System Guide plus the Empower project templates)

• 150mm column stabilizer

• Tubing kit (0.0025 ID PEEK) - inlet

• Column in-line filter

Tip: Consult the AccQ•Tag Ultra Care and Use manual for storage conditions and lifetime specifications for derivatization and chromatographic supplies.

Using the AccQ•Tag Ultra method

The AccQ•Tag Ultra Method is a precolumn derivatization technique for amino acids. The UPLC AAA Solution, in combination with the AccQ•Tag Ultra Method, enables you to derivatize amino acids, separate the derivatives with reversed-phase UPLC, and quantitate the derivatives based on UV absorbance or fluorescence intensity.

The AAA solution features sub-picomole sensitivity with a high degree of accuracy and ease of use.

Derivatization chemistry overviewThe AccQ•Tag Ultra Method is based on a derivatizing reagent developed specifically for amino acid analysis. Waters AccQ•Tag Ultra Reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, or AQC) is an N-hydroxysuccinimide-activated heterocyclic carbamate, a class of amine-derivatizing compounds.

Caution: To retain the appropriate level of purity, handle all reagents carefully.


Waters AccQ•Tag Ultra reagent

Reagent chemistry

The AccQ•Tag Ultra reagent converts both primary and secondary amino acids to stable derivatives, as illustrated in the figure below. The structure of the derivatizing group is the same for all amino acids, adding both UV absorbance and fluorescent character.

Excess reagent hydrolyzes to yield 6-aminoquinoline (AMQ), a non-interfering by-product.

1-6 System Overview

AccQ•Tag Ultra reaction

Reaction with amino acids

The AccQ•Tag Ultra Reagent reacts rapidly with primary and secondary amino acids to yield highly stable ureas. The resulting derivatives are stable at room temperature for up to 1 week, if evaporation of sample solvent is completely prevented.

Reagent hydrolysis

In a slower reaction, excess reagent hydrolyzes to produce 6-aminoquinoline (AMQ), N-hydroxysuccinimide (NHS) and carbon dioxide (see the figure below). The destruction of excess reagent is completed within 1 minute.

1° or 2°amino acid

Derivatized amino acid

AQC

AMQ NHS

NHS

t1/2 ~ 15 s

t1/2 << 1 s

CO2

H2O


The major hydrolysis product, AMQ, produces a significant peak that is easily resolved chromatographically. NHS and carbon dioxide do not interfere with the analysis.

AccQ•Tag Ultra reaction in the presence of water

1-8 System Overview

2 System Setup

This chapter describes how to make the plumbing and Ethernet connections for the UPLC AAA Solution for use on an ACQUITY UPLC system.

The information presented in this chapter assumes all of the necessary ACQUITY UPLC components are installed, configured, and properly stacked. For details about making signal connections, refer to the ACQUITY documentation.

Contents:

Topic Page

Installing the 2-µL sample loop 2-2

Installing the detector tubing 2-2

Plumbing connections 2-3

Ethernet connections 2-16

2-1

Installing the 2-µL sample loop

The UPLC AAA Solution requires a 2-µL sample loop. The ACQUITY UPLC system uses a 10-µL loop for performance qualification. You must replace the 10-µL loop with the 2-µL loop for amino acid analysis and for the Amino Acid Analysis Performance Qualification (PQ). See Figure “Injection valve connections” on page 2-9.

To install the 2-µL sample loop:

1. Remove the 10-µL loop used to qualify the performance of the ACQUITY UPLC system.

2. Remove the black O-ring from one end of the 2-µL sample loop and place that end firmly in port 1.

3. Finger-tighten the compression screw, and then add a 3/4-turn with the 1/4-inch wrench.

4. Remove the black O-ring from the other end of the 2-µL sample loop and place that end firmly in port 4.

5. Finger-tighten the compression screw, and then add a 3/4-turn with the 1/4-inch wrench.

6. From the ACQUITY console, select Sample Manager and then click Configure > Volumes.

7. In the loop size text box, enter 2.0, and then click OK.

8. Prime the sample syringe for 3 cycles.

9. In the Console, click Maintain > Characterize > Needle and loop volumes. Click Start.

Tip: The measured sample loop must be within 15% of nominal loop volume.

Installing the detector tubing

The UPLC AAA Solution requires the use of low-flow tubing for accurate amino acid analysis. Install part number 430001783, using the procedure outlined in step 3 of “Plumbing the TUV detector” on page 2-4.

2-2 System Setup

Tip: The same tubing is also used for plumbing both the PDA and the FLR detectors. The FLR ships with this tubing kit as standard. Follow the plumbing instructions outlined in each detector’s respective getting started guide to install the low-flow tubing.

Plumbing connections

Once you have properly installed the 2-µL sample loop, you can make the remaining plumbing connections. Compression fittings and ferrules are already fitted to tubing assemblies, but they must be properly set.

Installation recommendations for fittingsThe system uses gold-plated compression screws and two-piece ferrules. See the diagram below for assembly orientation.

Compression screw ferrule assembly

Recommendations:

• To prevent bandspreading, ensure the tube is bottomed in the cone hole before tightening the compression screw.

• For easier accessibility, use long compression screws to attach tubes to the injector and vent valve.

Caution: To prevent contamination, wear particle-free, powder-free, non-latex gloves when plumbing the system.

TubingCompression screwFerrule with locking ring


When tightening system fittings, consult the following table.

Plumbing the TUV detector

Plumbing a detector involves connecting the flow cell and installing the backpressure regulator.

Although the in-line degasser removes most of the gas (air) from solvents, some gas is introduced into the system during partial loop injections. Under pressure, this gas remains in solution. However, because the post-column pressure is normally much lower than the pre-column pressure, the gas can come out of solution and produce an unstable baseline characterized by large, unexpected spikes. The backpressure regulator maintains a minimum post-column pressure of 1724 kPa (17 bar, 250 psi), eliminating post-column outgassing and ensuring a smooth baseline.

To plumb a TUV detector:

1. Open the front panel door of the TUV detector, and install the flow cell so that the 3 thumbscrews align with their holes in the bulkhead.

2. Finger-tighten the thumbscrews.

Installation recommendations for ACQUITY UPLC fittings:

Fitting Recommended tightening

1/4-28 flangeless Snug plus 1/4-turn

10-32 LT135 PEEK™ ferrules Snug plus 1/4-turn; if leaking, tighten another 1/8-turn

10-32 one-piece PEEK Finger-tight

Stainless steel (new) Finger-tight plus 3/4-turn

Stainless steel (re-installed) Finger-tight plus 1/4-turn

Caution: To prevent contamination, wear particle-free, powder-free, non-latex gloves when plumbing the detector.

2-4 System Setup

TUV detector flow cell

3. Install the 0.0025-inch PEEK low flow inlet tubing (part number 430001783) for the UPLC AAA Solution:

a. Remove the inlet tubing from the packaging.

b. Install the PEEK finger-tight fittings onto the column outlet and the detector inlet.

c. Finger-tighten the screws on the end of each fitting.

4. Attach the short length of outlet tubing from the backpressure regulator to the outlet of the flow cell.

5. Route the long end of outlet tubing from the backpressure regulator, through the channel clips along the front, right-hand side of the system, and into a suitable waste container.

Plumbing the PDA detectorIf your system includes a PDA detector, see the ACQUITY UPLC Photodiode Array Detector Getting Started Guide for plumbing instructions.

Flow cell assembly

Outlet tubing

Inlet tubing

Thumbscrew

Backpressure regulator


Plumbing the FLR detectorIf your system includes an FLR detector, see the ACQUITY UPLC Fluorescence Detector Getting Started Guide for plumbing instructions.

Plumbing the binary solvent manager and sample manager

To plumb the binary solvent manager and sample manager:

1. Route the solvent inlet line tubing attached to the in-line degasser and seal wash pump through the channel clips of the sample manager.

2. Continue routing the lines between the column heater module door and hinge, through the detector's clip, and then place the lines in the solvent tray.

Caution: To prevent contamination, wear particle-free, powder-free, non-latex gloves when plumbing the binary solvent manager and sample manager.

2-6 System Setup

Routing solvent inlet lines

3. From the sample manager, route the weak and strong needle wash lines, indicated by the orange and white labels, to their corresponding ports on the in-line degasser in the binary solvent manager. Finger-tighten the knurled nut.

4. Remove the protective tubing cover from the stainless steel pump outlet tubing (indicated by the blue label).

5. Seat the tubing into the binary solvent manager mixer outlet, and tighten the compression fitting using the 1/4-inch and 5/8-inch open-end wrenches.

TP02593

Solvent lines

Clip


Needle wash and pump outlet connections

6. Route the other end of the tubing through the channel clips to the sample manager injector valve.

7. Remove the tubing cover, and seat the tubing into port 5 on the injector valve. Tighten the compression fitting using the 1/4-inch open-end wrench.

TP02480Binary solvent manager

mixer outlet

Strong needle wash

Weak needle wash

2-8 System Setup

Injection valve connections

8. Remove the column heater module cover, and tilt the column tray downward.

9. Install the 150-mm column stabilizer (part number 205000494) in the column tray, using the screws and thermal gasket provided in the kit.

Column stabilizer:

10. Route the injector outlet (long) tube from the stabilizer through the upper notch in the column tray, and then through the channel on the front, right-hand side of the column heater module.

Pump outlet tubing into port 5

Injector outlet/column stabilizer tubing into port 6

Metal clip

Tubing


11. Push down the shutter to hold the tubing in place.

12. Remove the protective cover from the injector outlet tube, and then seat the tube and ferrule in port 6 of the injector valve.

13. Close the column heater tray and then close the cover.

14. Attach a waste line to the barbed fitting of the drain located at the bottom of the binary solvent manager, and route it to a suitable waste container.

Attaching the drain fitting to the binary solvent manager

Caution: To avoid fluid backup, ensure proper drainage of waste:• Place the waste container below the system stack.• Ensure that the drain tubes do not crimp or bend. A crimp or

bend can impede flow to the waste container.• Ensure the exit of the drain tube is not covered by waste

solvent. If necessary, shorten the waste tube so that no portion of it drops below the top of the waste container (see next figure).

TP02479

Drain fitting

2-10 System Setup

Drain tube configuration

15. Attach a waste line to the barbed fitting on the filter drainage assembly (on the lower front side of the sample organizer), and route it to a suitable waste container.

16. Route a waste line from the barbed fitting on the rear of the solvent tray module to a suitable waste container.

Warning: To avoid spills, empty the waste container at regular intervals.

Warning: To avoid releasing solvent vapors into the room, route the in-line degasser’s exhaust tubing in one of the following ways:• To a fume hood or other suitable exhaust system.• To a suitable waste container, ensuring the tubing's discharge end

is at all times above the fluid level.

TP02709Correct Incorrect


17. Close the sample manager door, fluidics drawer, and the binary solvent manager door.

Installing the column for optical detection

To install the column (optical detection):

1. Attach the in-line filter to the column inlet.

2. Open the column heater’s front panel.

3. Tilt the column tray downward.

4. Attach the column inlet filter to the short tube from the stabilizer, and then tighten the special compression fitting using the special fitting wrench tool provided in the application kit.

5. Remove the protective cover from the PEEK tubing from the flow cell inlet.

6. Attach the 0.0025-inch ID inlet tubing (part number 430001783) included in the application kit to the column outlet.

7. Snap a plastic column clip onto the connection between the column end nut and the compression screw, at the column outlet.

8. Position the column inlet end in the cavity of the U-shaped stabilizer, ensuring that the inlet tubing passes behind the positioning clip.

9. Ensure that neither the tubing nor the nut touch the body of the column heater.

10. Tilt the column tray upward to the closed position.

11. Refit the front panel. The panel snaps into place.

12. Examine the seal around the cover, and ensure its correct alignment.

Rationale: This check ensures that the thermal environment is stable.

13. Attach the eCord fob to the receptacle on the side of the column heater module.

Warning: To avoid spills, empty the waste container at regular intervals.

2-12 System Setup

Connecting the eCord:

Installing the column for MS detectionThe UPLC AAA Solution uses mobile phases that are compatible with Electrospray mass spectrometry (MS). This is helpful in identifying unknown species in the sample.

If your system includes a mass spectrometer positioned to the right-hand side of the system stack, swing the column heater module door to the “away” position (that is, swing it rightward). Doing so lets you connect to the mass spectrometer inlet without increasing the tubing length. You must also install the mass spectrometer drip tray to ensure any solvent leaked from the column or column connections is routed into the drip management system.

To install the column (MS detection):

1. Remove any solvent lines that are routed between the column heater door and hinge.

2. Push down on the metal clip at the bottom, left-hand side of the column heater module to release the door, and then swing it fully to the right-hand side.

3. Open the rear cover, and then tilt the column tray downward.

4. Snap the plastic column clips onto the connections between the column end nut and the compression screw.

eCord receptacle

eCord fob


5. Attach the column inlet to the column stabilizer from port 6 of the injector valve.

6. Attach the 0.005-inch ID tubing included with the system to the column outlet.

7. Set the column into the column tray. (Ensure that the column stabilizer from the sample manager is routed through the front notch on the left-hand side of the column tray.)

8. Tilt the column tray upward, to the closed position, and refit the rear cover.

9. Remove the mass spectrometer drip tray from the startup kit.

Installing the mass spectrometer drip tray:

10. Slide the mass spectrometer drip tray into the slot on the side of the column heater chassis so that the drain hole on the left-hand side of the tray is centered over the column heater drain.

11. Attach the eCord fob to the receptacle on the side of the column heater module.

12. Attach the column outlet tubing to the mass spectrometer’s inlet. For more information about connecting to the inlet, consult the instrument’s documentation.

Drain hole

Column heater drain

Tray slot

Tray storage location

2-14 System Setup

Connecting to the solvent supplyThe solvent tray located on top of the system can hold up to 2 L of spilled solvent. You need a suitable waste container to collect spilled solvent from the waste line at the rear of the tray.

To connect the solvent supply:

1. Choose solvent reservoirs that snugly fit the reservoir caps supplied in the startup kit. Waters recommends 1-L reservoirs.

2. Insert the solvent tubing into the solvent bottles in the tray on top of the sample manager or optional detector.

Solvent tubing in bottles:

Warning: To avoid spills, do not place solvent reservoirs on top of thesample organizer.

Caution: To maintain adequate solvent head pressure and ensure proper solvent delivery, position the solvent reservoirs in the solvent tray at the top of the system stack.

Solvent bottles

Solvent tubing

Solvent tray


Ethernet connections

To make Ethernet connections:

1. Unpack and install the preconfigured ACQUITY workstation.

2. Connect one end of one Ethernet cable to the network switch, and then connect its other end to the Ethernet card on the workstation.

Tip: On preconfigured systems, the Ethernet card is identified as the Instrument LAN card.

3. Connect one end of one Ethernet cable to the solvent manager, and then connect its other end to the network switch.

4. Connect one end of one Ethernet cable to the sample manager, and then connect its other end to the network switch.

5. Connect one end of one Ethernet cable to the TUV (or PDA or FLR) detector, and then connect its other end to the network switch.

6. If you are using a sample organizer, connect one end of one Ethernet cable to the sample organizer, and then connect its other end to the network switch.

2-16 System Setup

3 Verifying System Operation

This chapter explains how to clean the system and prepare it for running a test method that will verify the performance of your UPLC Amino Acid Analysis Solution. It also provides examples of quantitating and interpreting amino acid analysis data.

The sample you use to verify system operation is included in the UPLC AAA Kit. It is also provided as part of the optional UPLC AAA Solution Performance Qualification (PQ). Before you begin the verification procedure, your system must be set up and configured as described in the ACQUITY UPLC System Operator’s Guide.

Contents:

Topic Page

Preparing the system 3-2

Preparing the eluents 3-4

Creating the test methods 3-7

Creating the sample set methods 3-22

Preparing the system verification sample 3-23

Performing the test 3-24

Quantitating and interpreting amino acid analysis data 3-28

3-1

Preparing the system

You must first clean the ACQUITY UPLC system before you install the UPLC AAA Solution. Prepare the required solvent mixtures using the procedure outlined below, then use the mixtures to clean the system. The cleaning procedure takes several hours.

Requirement: UPLC AAA systems require stainless steel (part number 289003536) or titanium (part number 289003212) bottle filters.

To prepare the cleaning solvents:

1. Prepare 50:50 (v/v) methanol/water:

a. Measure 500 mL of water into a graduated cylinder.

b. In a separate graduated cylinder, measure 500 mL of methanol.

c. Add the methanol to the water and mix for 5 minutes.

2. Prepare 30:70 (v/v) phosphoric acid/water:

a. Measure 350 mL of water into a graduated cylinder.

b. In a separate graduated cylinder, measure 150 mL of phosphoric acid.

c. Add the phosphoric acid to the water and mix for 5 minutes.

3. Fill a 1-L mobile phase reservoir with 100% water.

4. Fill a 1-L mobile phase reservoir with 100% isopropanol.

Tip: The procedure uses less than 500 mL.

Warning: Remove bottle filters during the acid wash. Do not allow acid to go through the seal wash line.

3-2 Verifying System Operation

To clean the system:

Requirement: Remove the stainless steel (part number 289003536) or titanium (part number 289003212) bottle filters prior to performing the cleaning procedure.

1. Place all lines (A1, A2, B1, B2 seal wash, weak needle wash, and strong needle wash) into 50:50 methanol/water.

2. Prime the solvent lines for 5 minutes each.

3. Prime the seal wash.

4. Prime the wash syringes and sample syringe for 4 cycles.

5. Connect a pressure restrictor in the fluid path after the injector to create approximately 2000 psi backpressure in the system.

6. Transfer 1 mL of 50:50 methanol/water to an autosampler vial and place it in position 1:A, 1.

7. Create an instrument method with the following parameters:

• Flow rate = 0.5 mL/min.

• Gradient composition 50% A1:50% B1.

• Full loop injection (2X overfill).

8. Make 30 full loop injections from the vial containing the mobile phase. Set the run time to 0.5 minutes.

Tip: This step takes approximately 30 minutes. Injection to injection takes more time.

9. Repeat steps 1 through 8 using 100% isopropanol as the solvent and the sample.

Restriction: Do not pass effluent through the optical detector for this wash step. Route the restrictor to waste.

10. Repeat steps 1 through 8 using 100% water as the solvent.

11. Remove the solvent reservoir filters.

Warning: Do not allow acid to go through the seal wash line.

Warning: Do not place the seal wash line in acid.

Preparing the system 3-3

12. Repeat steps 1 through 8 using 30:70 (v/v) phosphoric acid/water as the solvent.

13. Repeat steps 1 through 8 using 100% water as the solvent.

14. Reattach the solvent line to the detector.

15. Replace the solvent reservoir filters on all lines.

16. Place the seal wash in 100% water, along with the other lines.

17. Repeat steps 1 through 8 using 50:50 (v/v) methanol/water as the solvent.

For more information about preparing the site and startup testing, see the following documents:

• Site Prep Guide, P/N 715001318

• Installation Checklist, P/N 715001319

• Startup Test, P/N 715001320

Preparing the eluents

Recommendation: Use HPLC-grade solvents to avoid high background contamination, poor signal-to-noise ratio, and loss of sensitivity. Waters recommends using HPLC-grade acetonitrile and HPLC-grade water.

Warning: Always observe Good Laboratory Practices when you use this equipment and when you work with solvents and test solutions. Know the chemical and physical properties of the solvents and test solutions you use. See the Material Safety Data Sheet for each solvent and test solution in use.

Caution: To prevent contamination, do not wash glassware with detergents or with other general glassware. Rinse the glassware with the high purity solvents that will be used.


Setting up the mobile phase

Eluent A1 (Solvent A)

Use Eluent A1 for protein hydrolysates, foods and feeds analysis, and alkylated cysteine analysis.

Prepare Eluent A1 using AccQ•Tag™ Ultra Eluent A concentrate (part number 186003838).

To prepare Eluent A1:

1. Measure 950 mL of water into a 1-L graduated cylinder.

2. In a separate graduated cylinder, measure 50 mL of AccQ•Tag Ultra Eluent A concentrate.

Note: Eluent A concentrate, once opened, must be stored tightly capped at around 4 °C for no longer than 1 month.

3. Add the concentrate to the water and mix thoroughly.

4. Transfer the solution to a 1-L mobile phase reservoir, and label it Eluent A1.

Tip: Dilute Eluent A can be used for 2 to 3 days at room temperature.

Eluent B (Solvent B)

Use Eluent B for protein hydrolysates, foods and feeds analysis, alkylated cysteine analysis, and cell culture analysis.

AccQ•Tag Eluent B is supplied as a working solution (part number 186003839); no additional preparation is required. Eluent B concentrate, once opened, must be stored tightly capped at around 4 °C for no longer than 1 month.

Use AccQ•Tag Eluent B as is in the bottle supplied, or transfer the amount needed to a 1-L mobile phase reservoir and label it Eluent B. Do not place more than 2 to 3 days’ supply of Eluent B on the instrument.

Preparing the eluents 3-5

Preparing needle wash solvents

Weak needle wash solvent

The weak needle wash solvent solution is 5% acetonitrile in water.

To prepare the weak solvent:

1. Fill a 500-mL graduated cylinder with 475 mL of water.

2. In a separate graduated cylinder, measure 25 mL of acetonitrile.

3. Add the acetonitrile to the graduated cylinder containing the water and mix thoroughly.

4. Transfer the solution to a 500-mL mobile phase reservoir. Label it Weak Needle Wash Solvent.

Strong needle wash solvent

The strong needle wash solvent solution is 95% acetonitrile in water.

To prepare the strong solvent:

1. Fill a 500-mL graduated cylinder with 475 mL of acetonitrile.

2. In a separate graduated cylinder, measure 25 mL of water.

3. Add the water to the graduated cylinder containing the acetonitrile, and mix thoroughly.

4. Transfer the solution to a 500-mL mobile phase reservoir. Label it Strong Needle Wash Solvent.

Seal wash solvent

The seal wash solvent solution is 50% acetonitrile in water.

To prepare the seal wash solvent:

1. Fill a 500-mL graduated cylinder with 250 mL of water.

2. In a separate graduated cylinder, measure 250 mL of acetonitrile.


3. Add the acetonitrile to the graduated cylinder containing the water and mix thoroughly.

4. Transfer the solution to a 500-mL mobile phase reservoir. Label it Seal Wash Solvent.

Creating the test methods

The UPLC AAA CD includes a set of projects that cover the TUV, PDA, and FLR detectors and all four sample types for each detector. The projects (methods) are listed in a table at the end of the section.

Recommendation: Restore the projects for the detector in use (as well as the appropriate applications) to the PC.

The methods are locked to protect them from inadvertent modification. You can save the method with a new name so that you can adapt it to the specific requirements of your laboratory.

Use the hydrolysate method for the selected detector to verify system performance.

Tip: The processing method automatically updates retention times. To prevent drift of the times out of the specified window, always create a new method from the standard method supplied with the system.

TUV instrument method

To verify system performance with the TUV detector:

1. Verify the following parameter values are set for the TUV detector:

Parameter Value

Wavelength mode Single Wavelength

Wavelength 260 (nm)

Data mode Absorbance

Sampling rate 10 (points/sec)

Auto Zero on Inject Start enabled

Time constant 0.200 (sec)


2. Verify the instrument method gradient table has these binary solvent manager parameter values:

3. Verify the following chromatographic conditions:

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

• Mobile Phase A: Eluent A1

• Mobile Phase B: Eluent B

4. Verify the following parameter values are set for the sample manager:

Auto Zero on Wavelength Changes

Maintain Baseline

Time (min)

Flow (mL/min) %A %B Curve

1 0.00 0.7 99.9 0.1

2 0.54 0.7 99.9 0.1 6

3 5.74 0.7 90.9 9.1 7

4 7.74 0.7 78.8 21.2 6

5 8.04 0.7 40.4 59.6 6

6 8.05 0.7 10 90 6

7 8.64 0.7 10 90 6

8 8.73 0.7 99.9 0.1 6

9 9.50 0.7 99.9 0.1 6

Parameter Value

Sample Loop Option Partial Loop with Needle Overfill

Weak wash solvent name 5% acetonitrile

Weak wash volume 600 µL

Strong wash solvent name 95% acetonitrile

Strong wash volume 200 µL

Target column temperature 55 °C

Target sample temperature 20 °C

Parameter Value


The following table lists the methods for the TUV detector.

Syringe Draw Rate (Advanced Settings)

30 µL/min

Needle Placement (from bottom)(Advanced Settings)

4.0 mmThis setting is for the recommended Waters total recovery vials. Other sample vials will require a different setting.

Needle Overfill Flush (Advanced Settings)

3.0 µL

TUV methods

Method name Methods

TUV_Hydrolysate_Sep07 Instrument methods• TUV_Hydrol_StartUp_Sep07• TUV_Hydrol_Sep07• TUV_Hydrol_Short_Stop_Sep07• TUV_Hydrol_Long_Stop_Sep07

Processing methods• TUV_Hydrol_General_Sep07• TUV_Hydrol_IntStd_Sep07

Report methods• TUV_Hydrol_General_Sep07

Method set• TUV_Hydrol_StartUp_Sep07• TUV_Hydrol_Sep07• TUV_Hydrol_Short_Stop_Sep07• TUV_Hydrol_Long_Stop_Sep07

Sample set • TUV_Hydrolysate_Sep07

Parameter Value


TUV_Alkylated_Cys_Sep07 Instrument methods• TUV_AlkCys_StartUp_Sep07• TUV_AlkCys_Sep07• TUV_AlkCys_Short_Stop_Sep07• TUV_AlkCys_Long_Stop_Sep07

Processing methods• TUV_AlkCys_General_Sep07• TUV_AlkCys_IntStd_Sep07

Report methods• TUV_AlkCys_General_Sep07

Method set • TUV_AlkCys_StartUp_Sep07• TUV_AlkCys_Sep07• TUV_AlkCys_Short_Stop_Sep07• TUV_AlkCys_Long_Stop_Sep07

Sample set• TUV_Alkylated_Cys_Sep07

TUV methods (Continued)

Method name Methods


TUV_Food_Feed_Sep07 Instrument methods• TUV_Food_Feed_StartUp_Sep07• TUV_Food_Feed_Sep07• TUV_Food_Short_Stop_Sep07• TUV_Food_Long_Stop_Sep07

Processing method• TUV_Food_Feed_General_Sep07• TUV_Food_Feed_IntStd_Sep07

Report method• TUV_Food_Feed_General_Sep07

Method set • TUV_Food_Feed_StartUp_Sep07• TUV_Food_Feed_Sep07• TUV_Food_Short_Stop_Sep07• TUV_Food_Long_Stop_Sep07

Sample set • TUV_Food_Feed_Sep07


Method name Methods


PDA instrument method

To verify system performance with the PDA detector:

1. Verify the following parameter values are set for the PDA detector:

TUV_Cell_Culture_Sep07 Instrument methods• TUV_Cell_Cult_StartUp_Sep07• TUV_Cell_Culture_Sep07• TUV_Cell_Short_Stop_Sep07• TUV_Cell_Long_Stop_Sep07

Processing method• TUV_Cell_Cult_General_Sep07• TUV_Cell_Cult_IntStd_Sep07

Report method• TUV_Cell_Cult_General_Sep07

Method set• TUV_Cell_Cult_StartUp_Sep07• TUV_Cell_Cult_Sep07• TUV_Cell_Short_Stop_Sep07• TUV_Cell_Long_Stop_Sep07

Sample set • TUV_Cell_Culture_Sep07

Parameter Value

3D Data Not Enabled

Resolution 4.8 (nm)

Sampling rate 10 (points/sec)


Exposure time Auto (msec)

2D Channels - Data Mode Absorbance


Method name Methods








2D Channels - Wavelength 260 (nm)

2D Channels - Resolution 4.8 (nm)

Time (min)


1 0.00 0.7 99.9 0.1

2 0.54 0.7 99.9 0.1 6

3 5.74 0.7 90.9 9.1 7

4 7.74 0.7 78.8 21.2 6

5 8.04 0.7 40.4 59.6 6

6 8.05 0.7 10 90 6

7 8.64 0.7 10 90 6

8 8.73 0.7 99.9 0.1 6

9 9.50 0.7 99.9 0.1 6

Parameter Value

Loop option Partial Loop with Needle Overfill






Parameter Value


The following table lists the methods for the PDA detector.



30 µL/min




3.0 µL

PDA methods

Method name Methods

PDA_Hydrolysate_Sep07 Instrument methods• PDA_Hydrol_StartUp_Sep07• PDA_Hydrol_Sep07• PDA_Hydrol_Short_Stop_Sep07• PDA_Hydrol_Long_Stop_Sep07

Processing methods• PDA_Hydrol_General_Sep07• PDA_Hydrol_IntStd_Sep07

Report method• PDA_Hydrol_General_Sep07

Method set • PDA_Hydrol_StartUp_Sep07• PDA_Hydrol_Sep07• PDA_Hydrol_Short_Stop_Sep07• PDA_Hydrol_Long_Stop_Sep07

Sample set • PDA_Hydrolysate_Sep07

Parameter Value


PDA_Alkylated_Cys_Sep07 Instrument methods• PDA_AlkCys_StartUp_Sep07• PDA_AlkCys_Sep07• PDA_AlkCys_Short_Stop_Sep07• PDA_AlkCys_Long_Stop_Sep07

Processing method• PDA_AlkCys_General_Sep07• PDA_AlkCys_IntStd_Sep07

Report method• PDA_AlkCys_General_Sep07

Method set • PDA_AlkCys_StartUp_Sep07• PDA_AlkCys_Sep07• PDA_AlkCys_Short_Stop_Sep07• PDA_AlkCys_Long_Stop_Sep07

Sample set • PDA_Alkylated_Cys_Sep07

PDA methods (Continued)

Method name Methods


PDA_Food_Feed_Sep07 Instrument methods• PDA_Food_Feed_StartUp_Sep07• PDA_Food_Feed_Sep07• PDA_Food_Short_Stop_Sep07• PDA_Food_Long_Stop_Sep07

Processing method• PDA_Food_Feed_General_Sep07• PDA_Food_Feed_IntStd_Sep07

Report method• PDA_Food_Feed_General_Sep07

Method set • PDA_Food_Feed_StartUp_Sep07• PDA_Food_Feed_Sep07• PDA_Food_Short_Stop_Sep07• PDA_Food_Long_Stop_Sep07

Sample set • PDA_Food_Feed_Sep07


Method name Methods


FLR instrument method

To verify system performance with the FLR detector:

1. Verify the following parameter values are set for the FLR detector:

PDA_Cell_Culture_Sep07 Instrument methods• PDA_Cell_Cult_StartUp_Sep07• PDA_Cell_Culture_Sep07• PDA_Cell_Short_Stop_Sep07• PDA_Cell_Long_Stop_Sep07

Processing method• PDA_Cell_Cult_General_Sep07• PDA_Cell_Cult_IntStd_Sep07

Report method• PDA_Cell_Cult_General_Sep07

Method set • PDA_Cell_Cult_StartUp_Sep07• PDA_Cell_Cult_Sep07• PDA_Cell_Short_Stop_Sep07• PDA_Cell_Long_Stop_Sep07

Sample set • PDA_Cell_Culture_Sep07

Parameter Value

Wavelength mode (UV detection) 2D Channels

Wavelength Excitation (λ ex) 266 (nm)

Wavelength Emission (λ em) 473 (nm)

Sampling rate (Data rate) 10 (points/sec)


PMT Gain 1.00


Method name Methods








Data units Emission

Time (min)


1 0.00 0.7 99.9 0.1

2 0.54 0.7 99.9 0.1 6

3 5.74 0.7 90.9 9.1 7

4 7.74 0.7 78.8 21.2 6

5 8.04 0.7 40.4 59.6 6

6 8.05 0.7 10 90 6

7 8.64 0.7 10 90 6

8 8.73 0.7 99.9 0.1 6

9 9.50 0.7 99.9 0.1 6

Parameter Value

Loop option Partial Loop with Needle Overfill







Parameter Value


The following table lists the methods for the FLR detector.


30 µL/min




3.0 µL

FLR methods

Method name Methods

FLR_Hydrolysate_Sep07 Instrument methods• FLR_Hydrol_StartUp_Sep07• FLR_Hydrol_Sep07• FLR_Hydrol_Short_Stop_Sep07• FLR_Hydrol_Long_Stop_Sep07

Processing method• FLR_Hydrol_General_Sep07• FLR_Hydrol_IntStd_Sep07

Report method• FLR_Hydrol_General_Sep07

Method set • FLR_Hydrol_StartUp_Sep07• FLR_Hydrol_Sep07• FLR_Hydrol_Short_Stop_Sep07• FLR_Hydrol_Long_Stop_Sep07

Sample set • FLR_Hydrolysate_Sep07

Parameter Value


FLR_Alkylated_Cys_Sep07 Instrument methods• FLR_AlkCys_StartUp_Sep07• FLR_AlkCys_Sep07• FLR_AlkCys_Short_Stop_Sep07• FLR_AlkCys_Long_Stop_Sep07

Processing method• FLR_AlkCys_General_Sep07• FLR_AlkCys_IntStd_Sep07

Report method• FLR_AlkCys_General_Sep07

Method set • FLR_AlkCys_StartUp_Sep07• FLR_AlkCys_Sep07• FLR_AlkCys_Short_Stop_Sep07• FLR_AlkCys_Long_Stop_Sep07

Sample set • FLR_Alkylated_Cys_Sep07

FLR methods (Continued)

Method name Methods


FLR_Food_Feed_Sep07 Instrument methods• FLR_Food_Feed_StartUp_Sep07• FLR_Food_Feed_Sep07• FLR_Food_Short_Stop_Sep07• FLR_Food_Long_Stop_Sep07

Processing method• FLR_Food_Feed_General_Sep07• FLR_Food_Feed_IntStd_Sep07

Report method• FLR_Food_Feed_General_Sep07

Method set • FLR_Food_Feed_StartUp_Sep07• FLR_Food_Feed_Sep07• FLR_Food_Short_Stop_Sep07• FLR_Food_Long_Stop_Sep07

Sample set • FLR_Food_Feed_Sep07


Method name Methods


Creating the sample set methods

TUV sample set method

To open the TUV sample set method:

1. Open the sample set method “UPLC AAA Reproducibility,” and confirm the following conditions:

• Injection volume is set to 1.0 µL.

• Run time is set to 9.5 minutes.

• Method is set to “TUV_Hydrolysate_Sep07.”

2. Run 2 blank gradients before injecting the sample.

FLR_Cell_Culture_Sep07 Instrument methods• FLR_Cell_Cult_StartUp_Sep07• FLR_Cell_Culture_Sep07• FLR_Cell_Short_Stop_Sep07• FLR_Cell_Long_Stop_Sep07

Processing method• FLR_Cell_Cult_General_Sep07• FLR_Cell_Cult_IntStd_Sep07

Report method• FLR_Cell_Cult_General_Sep07

Method set • FLR_Cell_Cult_StartUp_Sep07• FLR_Cell_Cult_Sep07• FLR_Cell_Short_Stop_Sep07• FLR_Cell_Long_Stop_Sep07

Sample set • FLR_Cell_Culture_Sep07


Method name Methods


PDA sample set method

To open the PDA sample set method:




• Method is set to “PDA_Hydrolysate_Sep07.”


FLR sample set method

To open the FLR sample set method:




• Method is set to “FLR_Hydrolysate_Sep07.”


Preparing the system verification sample

Proper pipetting is essential to ensure the quality of the samples. Adhere to your company’s procedures for proper pipetting techniques.

To prepare the system verification sample (50 pmol/µL):

1. Use a micropipettor to deliver the following components to a total recovery vial:

a. Deliver 70 µL of AccQ•Tag Ultra Borate buffer (reagent 1).

b. Add 10 µL of 500 pmol/µL Standard (see Chapter 4 for instructions on preparing standards).

c. Add 20 µL of reconstituted AccQ•Tag Ultra reagent.

Preparing the system verification sample 3-23

2. Cap the vial and vortex mix for several seconds.

3. Let stand 1 minute at room temperature.

4. Heat in a heating block for 10 minutes at 55 °C.

5. Remove from heating block with forceps.

Performing the test

When the system is prepared, the test methods are verified, and the samples are derivatized, you can perform the test.

See Chapter 4 for instructions on preparing standards, and Chapter 5 for preparing the UPLC AAA Solution system.

To perform the test:

1. In Run Samples, start the run by opening the project.

2. Select the “UPLC AAA Reproducibility” sample set, and then select Run and Report.

3. When the sample set is complete, enter the appropriate results in the table below.

4. Review the gradient performance report. Compare the chromatogram on the report to one of the sample chromatograms below, depending on the type of detector you are using.

Sample set results

ComponentPeak Retention Time Mean Value

Standard Deviation

Acceptable Standard Deviation

histidine < 0.116

alanine < 0.116

phenylalanine < 0.116


Representative TUV chromatogram: Protein and peptide hydrolysate standards (50 pmol)

This chromatogram shows the elution position of norvaline. This amino acid is added to the standard and to samples as an internal standard. It is not present in the Waters amino acid hydrolysate standard.


Representative PDA chromatogram: Protein and peptide hydrolysate standards (50 pmol)

AM

Q

NH

3

His

Ser Arg

Gly A

sp

Glu Th

r

Ala

Pro

Der

ivP

eak

Cys

Lys

Tyr

Met Val Ile

Leu P

he

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.110

0.120

0.130

0.140

0.150

0.160

0.170

0.180

0.190

0.200

0.210

0.220

0.230

0.240

0.250

0.260

0.270

0.280

Minutes1.50 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

AM

Q

NH

3

His

Ser Arg

Gly A

sp

Glu Th

r

Ala

Pro

Der

ivP

eak

Cys

Lys

Tyr

Met Val Ile

Leu P

he

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.110

0.120

0.130

0.140

0.150

0.160

0.170

0.180

0.190

0.200

0.210

0.220

0.230

0.240

0.250

0.260

0.270

0.280

Minutes1.50 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

AM

Q

NH

3

His

Ser Arg

Gly A

sp

Glu Th

r

Ala

Pro

Der

ivP

eak

Cys

Lys

Tyr

Met Val Ile

Leu P

he

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.110

0.120

0.130

0.140

0.150

0.160

0.170

0.180

0.190

0.200

0.210

0.220

0.230

0.240

0.250

0.260

0.270

0.280

Minutes1.50 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

AM

Q

NH

3

His

Ser Arg

Gly A

sp

Glu Th

r

Ala

Pro

Der

ivP

eak

Cys

Lys

Tyr

Met Val Ile

Leu P

he

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.110

0.120

0.130

0.140

0.150

0.160

0.170

0.180

0.190

0.200

0.210

0.220

0.230

0.240

0.250

0.260

0.270

0.280

Minutes1.50 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


Representative FLR chromatogram: Protein and peptide hydrolysate standards (50 pmol)

AM

Q

NH

3

His

Ser

Arg

Gly

Asp

Glu

Th

r

Ala

Pro

Cys

Lys

Tyr

Met

Val

IleL

euP

he

EU

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

1400.00

1600.00

1800.00

2000.00

2200.00

2400.00

2600.00

2800.00

3000.00

3200.00

3400.00

Minutes1.50 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

AM

Q

NH

3

His

Ser

Arg

Gly

Asp

Glu

Th

r

Ala

Pro

Cys

Lys

Tyr

Met

Val

IleL

euP

he

EU

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

1400.00

1600.00

1800.00

2000.00

2200.00

2400.00

2600.00

2800.00

3000.00

3200.00

3400.00

Minutes1.50 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


Quantitating and interpreting amino acid analysis data

Processing samples

To process samples:

1. Bring the data into Review.

2. Open the processing method for the type of detector you are using:

• For TUV, open “TUV_Hydrol_General_Sep07.”

• For PDA, open “PDA_Hydrol_General_Sep07.”

• For FLR, open “FLR_Hydrol_General_Sep07.”

3. Integrate and quantitate.

4. Check the retention times against the representative chromatogram. Update retention times as necessary in the processing method.


4 Preparing Standards and Samples

The AccQ•Tag Ultra Chemistry Package contains the items you need for up to 250 derivatizations. Follow the procedures in this chapter to prepare the standards and samples you will use to perform the analyses.

Contents:

Topic Page

Reconstituting the AccQ•Tag Ultra reagent powder 4-2

Preparing the calibration standard 4-3

Preparing the samples 4-6

4-1

Reconstituting the AccQ•Tag Ultra reagent powder

The AccQ•Tag Ultra Reagent Kit includes preformulated reagents tested to ensure minimum amino acid contamination. Reconstituted AccQ•Tag Ultra reagent is approximately 10 mM AccQ•Tag Ultra reagent in acetonitrile.

To reconstitute the AccQ•Tag Ultra reagent:

1. Preheat a heating block to 55 °C.

2. Tap vial 2A lightly before opening to ensure all AccQ•Tag Ultra reagent powder is at the bottom of the vial.

3. Rinse a clean micropipettor by drawing and discarding 1 mL of AccQ•Tag Ultra reagent diluent from vial 2B. Repeat 2 times.

4. Draw 1.0 mL from vial 2B and transfer it to the AccQ•Tag Ultra reagent powder in vial 2A. Cap the vial tightly.

5. Vortex mix for 10 seconds.

6. Heat vial 2A on top of the heating block until the AccQ•Tag Ultra reagent powder is dissolved. Do not heat the reagent for longer than 10 minutes.

Tip: You can store the reconstituted AccQ•Tag Ultra reagent in a desiccator at room temperature for up to 1 week. Because the chance of moisture contamination increases with each opening of the vial, do not use any one particular vial for more than 3 derivatization experiments.

Warning: Acetonitrile is flammable and toxic. Refer to the Material Safety Data Sheet.

Caution: AccQ•Tag Ultra reagent reacts with atmospheric moisture. Seal the container tightly when not in use. Do not refrigerate. Do not use discolored reagent, especially if it is yellow or green.

4-2 Preparing Standards and Samples

Preparing the calibration standard

This section describes methods for preparing external and internal calibration standards and the procedure for derivatizing the calibration standard.

Use the calibration standard to calculate protein/peptide compositions in hydrolyzed samples, mole-percent values for the individual amino acids in a hydrolysis sample, and the absolute amounts injected.

Required Equipment

• Adjustable micropipettors (1, 10, 20, 200, and 1000 µL) and tips

• Vortex mixer

• Waters Total Recovery vials

• Heating block

• Mill-Q® or other 18-megaohm water

Preparing the calibration standard: External methodThe Waters AccQ•Tag Chemistry Package includes the Waters Amino Acid Hydrolysate Standard. The standard mixture contains a 2.5 mM concentration of each of the dissolved 0.1 N HCl hydrolysate amino acids, with the exception of cystine (1.25 mM).

Tip: Ensure that all glassware is clean. For high-sensitivity analyses, pyrolyze all glassware at 500 °C for at least 4 hours.

To prepare the calibration standard, mix 100 µL Waters Amino Acid Hydrolysate Standard with 900 µL Milli-Q or other 18-megaohm water in a total recovery vial. The dilute calibration standard contains 250 pmol/µL of each amino acid with the exception of cystine (125 pmol/µL, which is equivalent to 250 pmol/µL cysteine).


Store the standards following these guidelines:

• Store the dilute calibration standard at -20 °C for up to 1 month.

• Transfer the remaining Amino Acid Hydrolysate standard from the ampoule to a vial with a cap for storage. Store the remaining Hydrolysate standard for up to 3 months at -20 °C. Protect the standard from dessication and freezer burn during storage.

• Store unopened Amino Acid Hydrolysate Standard ampoules for up to 1 year at 4 °C.

Protect all standards from contamination and evaporation.

Preparing the calibration standard: Internal methodUse an internal standard with your calibration standard to correct for volumetric errors introduced during sample preparation. Use an amino acid that is stable and elutes at a known unique retention time. The recommended internal standard is norvaline. An acceptable alternative is α-aminobutyric acid.

Preparing the internal standard stock solution

Use the internal standard stock solution to prepare these items:

• Calibration standard with an internal standard

• Internal standard solution that you add to the sample

To prepare a 2.5 mM internal standard stock solution of norvaline, add 2.94 mg of norvaline to 10 mL 0.1 N HCl.

To prepare a 2.5 mM internal standard stock solution of α-aminobutyric acid, add 2.58 mg of α-aminobutyric acid to 10 mL 0.1 N HCl.

Store the internal standard stock solution at -20 °C for up to 6 months.

Preparing calibration standard with internal standard

To prepare a calibration standard with an internal standard, combine 100 µL internal standard stock solution, 100-µL Waters Amino Acid Hydrolysate Standard Mixture, and 800 µL Milli-Q or other 18-megaohm water in a clean autosampler vial.


The calibration standard with the internal standard contains 250 pmol/µL of each amino acid with the exception of cystine (125 pmol/µL, which is equivalent to 250 pmol/µL cysteine).

Store the calibration standard with internal standard at -20 °C for up to 1 month. Cap the vial tightly and protect it from dessication.

Derivatizing the calibration standardDerivatization converts the amino acids in the standard into highly stable derivatives.

To derivatize the calibration standard:


2. Use a micropipettor to add 70 µL of AccQ•Tag Ultra Borate buffer (reagent 1) to a clean total recovery vial.

3. Use a micropipettor to deliver 10 µL of calibration standard to the vial. Vortex mix briefly.

4. Use a micropipettor with a clean tip to add 20 µL of reconstituted AccQ•Tag Ultra reagent to the sample tube. Vortex mix immediately for several seconds.

5. Let stand for 1 minute at room temperature.

6. Heat the vial in a heating block or oven for 10 minutes at 55 °C.

Tip: Heating converts a minor side product of tyrosine (Tyr) to the major mono-derivatized compound. Conversion occurs more slowly at room temperature, with a half-life of approximately 1 hour.

A 1-µL injection of derivatized standard contains 50 pmol of each amino acid derivative (except cystine, at 25 pmol).

Store derivatives at room temperature for up to 1 week. Seal the vials tightly with non-punctured septa to prevent evaporation of liquid during storage.


Preparing the samples

To prepare a sample for amino acid analysis with the AccQ•Tag Ultra method:

1. Determine the quantity of sample you need.

2. Hydrolyze the sample to its component amino acids (if required).

3. Derivatize the amino acids with AccQ•Tag Ultra reagent.

Consult Appendix C for a detailed discussion of sample amounts.

The sample preparation procedures are the most common entry points for amino acid contamination. Tips for minimizing background contamination are listed at the end of “Hydrolyzing the samples” on page 4-6.

Determining sample quantityConsult Appendix C for a detailed discussion of sample amounts.

Hydrolyzing the samplesThe UPLC AAA Solution is compatible with all of the common hydrolysis procedures used for purified proteins as well as for food and feed samples. It is necessary to follow the guidelines in Appendix A to ensure that the amount of sample falls in the linear range of the assay, and that there is optimal pH for the reaction.

Background contamination

Background contamination can easily contribute 10 pmol or more of certain amino acids to the sample. Glycine, Serine, Aspartate, and Glutamate tend to be the most abundant contaminants.

See also: For more detailed information on how to minimize contamination, refer to Controlling Contamination in LC/MS Systems.

Potential sources of contamination include

• dirty glassware.

• laboratory animals.

• tobacco smoke.


• airborne dust.

• fingerprints.

• kimwipes.

For best results, follow these guidelines:

• Handle samples in a clean, low-traffic work area.

• Use clean, talc-free gloves and clean forceps.

• Use disposable glassware where possible.

• Pyrolyze all sample tubes and reaction vials by heating at 500 °C for 4 hours.

• Consider the vapor-phase acid hydrolysis procedure for pure proteins. Use 1-ml ampoules of high purity HCl (6N).

• Check background contributions with blank derivatizations and appropriate controls.

• Use an empty sample tube as a control hydrolysis blank.

• Be aware of potential contamination for pipettes, disposable tips, syringes, and forceps. Avoid cross-contamination of sample from calibration standards.

Derivatizing the samplesDerivatization converts the amino acids in the sample solution into stable amino acid derivatives.

Reconstituting the hydrolyzed sample

To reconstitute dry sample:

1. Prepare a 100 mM HCl solution by adding 50 µL constant-boiling (6 N) HCl to 2.5 mL Milli-Q or other 18-megaohm water to a clean autosampler vial.

2. Dissolve the sample in a volume estimated from the guidelines in Appendix C.

3. Vortex mix thoroughly.


To reconstitute the sample to contain an internal standard:

1. Prepare a 100 mM HCl solution by adding 50 µL of constant-boiling (6 N) HCl to 2.5 mL Milli-Q or other 18-megaohm water to a clean autosampler vial.

2. Prepare an internal standard solution by adding 20 µL of 2.5 mM internal standard stock solution to 980 mL 20 µM HCl.

See also: “Preparing the internal standard stock solution” on page 4-4.

3. Dissolve the sample in a volume estimated from the guidelines in Appendix C.

4. Vortex mix thoroughly.

Derivatizing a sample

To derivatize a sample:


2. Add sample, AccQ•Tag Ultra Borate Buffer, and neutralization reagent to the total recovery vial. The vial must be tightly sealed throughout the derivatization.

Tips:

• If convenient, you can conduct the derivatization in the hydrolysis tube.

• You can change the volume of Borate to adjust for sample neutralization, as described in Appendix C.

3. Add 20 µL of AccQ•Tag Ultra reagent. Vortex mix immediately for several seconds.

4. Wait 1 minute.

Result: After 1 minute, derivatization is complete and the excess reagent is hydrolyzed to AMQ, terminating the derivatization reaction.

5. Transfer the contents of the tube to a total recovery vial (if necessary).

6. Heat the vial in a heating block for 10 minutes at 55 °C.

Tip: Heating converts a minor side product of tyrosine to the major mono-derivatized compound. Conversion occurs more slowly at room temperature.


Derivatizing a blank

Ensure all glassware is clean. Pyrolyze sample tubes at 500 °C for at least 4 hours.

To derivatize a blank:

1. Place 80 µL of AccQ•Tag Ultra Borate buffer in a vial.

2. Add 20 µL AccQ•Tag Ultra reagent, and vortex mix.

3. Wait 1 minute for excess reagent to hydrolyze to AMQ.

4. Heat the vial for 10 minutes at 55 °C.



5 Operating the System

This chapter describes how to prepare the system and set up Empower software to perform a calibration and to start acquiring data.

Contents:

Topic Page

Preparing the UPLC AAA Solution 5-2

Setting up Empower software 5-3

Opening Empower and Restoring Projects 5-4

Loading, editing, and running a sample set method 5-6

Processing Data 5-10

5-1

Preparing the UPLC AAA Solution

Setting up the mobile phasesPrepare the eluents as described in “Preparing the eluents” on page 3-4.

Preparing the system

To prepare the system:

1. Install the 2-µL sample loop.

2. Configure the sample loop volume to be 2.0 µL, and then characterize the loop and needle volume.

3. Install the column with an in-line filter on the system, using column clips to ensure uniform heat dissipation. Follow these guidelines:

• Install the column stabilizer, along with the thermal gasket provided, by securing it with the screws provided. Install the stabilizer and gasket at the side of the column heater to ensure proper solvent pre-heating. This region is marked by a notch in the tray.

• Set the column heater temperature to 55 °C.

4. Install Eluent A1 on line A1 and Eluent B on lines B1 and B2.

5. Install the weak needle wash line into a 5:95 acetonitrile/water solution.

6. Install the strong needle wash line into a 95:5 acetonitrile/water solution.

7. Install the seal wash line into a 50:50 acetonitrile/water solution.

8. Prime all solvent lines for 5 minutes.

9. Prime wash/sample syringes for 4 cycles.

10. From the ACQUITY Console, characterize the needle and loop volume.

11. Set the column to equilibrate in 50:50 A1/B1 at 0.2 mL/min at 55 °C. Equilibrate for 30 minutes.

12. Place the samples in the ACQUITY Sample Manager.

5-2 Operating the System

Setting up Empower software

The UPLC AAA Solution requires that Empower software be fully installed prior to running any analyses. If Empower software is not installed, use the Empower CD to install and configure Empower software on your system.

The following information provides a direct method to begin using the UPLC AAA Solution. For a complete discussion of Empower functions and options, see the Empower software documentation that accompanies the system.

Empower QuickStart InterfaceQuickStart, a streamlined Empower interface, runs samples easily and simply for everyday tasks. The QuickStart Project window contains three main viewing areas:

• Navigation bar – A vertical panel on the left-hand side of the main window that you use to move among the tasks and functions: Run Samples, Browse Project (Sample Queue, Control Panel), View Data, View Method (Method Set, Instrument, and Processing) and View Acquisition. These tasks and functions are directly related to acquiring, processing, reviewing and reporting chromatographic data. “Show Me…,” the sixth tool, provides interactive help.

• Work area: – A window to the right-hand side of the navigation bar, this window displays what you select in the navigation bar. Here you set up data collection, view data, process data and preview reports.

• View acquisition: – A display area below the work area in which you can view the system status, the current acquisition and related parameters. The left-hand side of the area contains tools to access functions associated with acquisitions. To the right-hand side, the chromatogram pane includes tools for display options and access to the Review window with the current data.

Setting up Empower software 5-3

QuickStart Project Window

Opening Empower and Restoring Projects

The UPLC AAA Solution ships with a system CD containing predefined Empower projects and test methods. See the Read Me file on the CD for the file descriptions.

Perform these tasks before starting the Empower software application:

• Power on the chromatographic instruments, and wait until their internal diagnostic tests are complete.

• Power on printers and other peripherals, and then power on the computer.

To start Empower:

1. Double-click the Empower Login icon.

Viewacquisition

WorkareaNavigation

bar


2. At the Login dialog box, enter User Name and Password. (The defaults are “system” and “manager” respectively.)

Recommendation: The system default specifies whether the QuickStart or Pro interface opens. This procedure uses the QuickStart interface. If Pro is the current default, click the Advanced>> button at the Login screen to select QuickStart as the interface.

Tip: Your system administrator can set the System Defaults as QuickStart so that you do not have to choose an interface.

3. Select a project and system, and then click OK.

Rule: Only projects and chromatographic systems to which you have access appear in the lists. Your UPLC Amino Acid Analysis chromatographic system must have been configured at installation. If no system has been configured, consult your system administrator.

4. Load the UPLC Amino Acid Analysis System Solution CD into the drive. In the QuickStart project window, select Manage > Restore Project.

5. At the Restore Project Wizard screen, click the browse button, and then specify the path to the project folder(s) for the detector in your system and the applications that will be used in your laboratory. Click Next.

Tip: If you are restoring a project that already exists, you are prompted to create a new project name. Also, if you are restoring to Empower build 2154, the following message appears. Click OK to continue.

Opening Empower and Restoring Projects 5-5

6. When the Restore Display box says “The import completed successfully”, click Finish.

7. Click Manage > Change Project/System. Select “Switch Project/System in this window”, select an appropriate project and System name from the available options, and then click OK to load the newly restored project into QuickStart.

Loading, editing, and running a sample set method

An example Sample Set Method is provided in each project. You can edit its table to match the intended batch of standards and samples.

A sample set method is a reusable set of instructions that provides the Empower software with information about how to acquire data. This information includes vial positions, injection volumes, sample names, method sets, and sample type custom fields. A sample set method can also include instructions for printing summary reports, clearing reports, performing custom calculations with summary functions, and so on.

A sample set is the bound group of data files that results from running a sample set method.

To load, edit, and run the sample set method:

1. Open the example Sample Set Method provided by clicking this icon

in the work area of the QuickStart window or by selecting File > New Sample Set > Using Sample Set Method.

Tip: For future analyses, open a previously modified Sample Set Method. Select an appropriate method, and then click Open to load the method.

The example sample set method contains this information:


a. A 30-minute condition column line that equilibrates the column isocratically, at 0.2 mL/min in 50:50 A1/B1, at the method temperature.

b. Two condition column lines that run the gradient method used for sample analysis to further prepare the column for the analysis.

Recommendation: The two condition column lines are essential for sample injection reproducibility. Keep them in your sample set method.

c. A gradient blank sample injection that tests the quality of the mobile phases and sample preparation solvents.

d. A reagent blank sample injection that tests the quality of the derivatization reagent.

e. A 25 pmole standard injection used for quantifying the samples.

f. A sample injection.

g. A 15-minute condition column line that prepares the system for short-term shutdown. This method turns the detector lamp and column heater off, and it reduces the flow rate to 0.05 mL/min, in 50:50 A1/B1. (If long-term storage is needed at the end of the analysis, place the A2 line in 100% water and the B2 line in 100% acetonitrile, and change the method set to the long-stop method. After flushing the system for 15 minutes, this method turns off the detector lamp, column heater, and pump flow.)


Example sample set method

2. Before beginning any modifications to the sample set method, save it with a new name, to assure that desired information is not overwritten.

3. You can edit a sample set method before running by clicking the desired field and changing its value or adding or removing rows from the sample set. Enter the names of any additional samples or standards specifying parameters for each, as follows:


• To delete a row, right-click it, and then select Delete Row(s).

• To add a row, right-click it, and then select Add Row or Insert Row.

– Add Row adds the row to the bottom of the table. If the row above contains a sample, the new row is identical except that the Empower software increases the vial number incrementally.

– Insert Row inserts the row wherever the cursor is. The new row is a copy of the row above. Be certain that the sample name, method set, run time, and so on are correct.

Requirement: Empower software increases by increments the vial number for the new row but does not do so for any samples below the new row. You must therefore renumber those rows.

4. Once edits are complete, save the changes to the sample set method.

Requirement: Upon restoring projects from the CD, the methods are locked to prevent from overwriting. Therefore, you must save the sample set methods under a different name.

5. In QuickStart, select Run and Report from the drop-down list at the top of the work area.

6. Start running the sample set by clicking the icon in the view acquisition area of the Quick Start window or by selecting Inject > Run from the toolbar at the top of the window. Name your sample set, select the run mode, and then click Run.


Processing Data

After an analysis ends, you can process the data for a sample.

To process data manually:

1. Click the Browse Project tab in the navigation bar. In the project table of the work area, click the Sample Sets tab.

2. Click the line number of the sample set you want to process, and then click View > Channels.

3. In the Channels table, double-click the sample to review the data for processing. That data file opens in the work area, in the format specified by the processing method, as a chromatogram, results table, or other presentation. Select the desired view from the Window menu of the View Data window.

4. In the QuickStart window, click File > Open > Processing Method, and then select the desired processing method. Click Open.

5. Click (Integrate) and (Quantitate), or click (Calibrate), for processing standards, to process your samples.

6. Click File > Save > Result to save the processed data.

Requirement: When you change the processing method, you must save it as a new processing method before you can save a result.

Batch processing dataThere are two methods for batch processing:

• Sample set – When you process a sample set, Empower software processes the data files according to the instructions in the sample set method.

• Channels – When you process individual data files from the Channels table, even if you select all the files in a sample set, Empower software processes the files according to the instructions in the method set or processing method.


To batch process data:

Tip: The Empower software processes data files in the order in which you select them. When selecting files from the Channel view table, remember to select standards before unknowns.

1. Click the Browse Project tab in the navigation bar. In the project table in the work area, click the Sample Sets tab.

2. Click the line number of the sample set you want to process, and then click Tools > Process.

The Background Processing and Reporting dialog has three options.

• The “Use acquisition method set” option processes the sample set with the method set used to collect the data.

• The “Use the specified method set” option requires you to choose a method set from the drop-down set.

• The “Use specified processing method” option requires you to choose a processing method from the drop-down list.

Requirement: Systems with a PDA or fluorescence detector can acquire either 2D or 3D data. The methods provided on the system CD specify only a 2D channel. The “Use specified processing method” option processes data acquired with these methods. When you create additional 3D methods, you must also create a method set that defines the extracted wavelength and the processing method.

When you make any changes to the processing method, you must add the new processing method to the method set. Adding the new processing method to the method set ensures that Empower uses the new method for batch processing.

3. In the QuickStart Background Processing and Reporting window, be sure Process remains enabled, with a check mark in the box, and select “Use specified method set”. Choose the desired method set from the drop-down menu. Select Clear Calibration when a calibration curve for this processing method exists and you want to generate a new one. Otherwise, standard points are added to the existing calibration curve. Click OK.

Tip: Processing a sample set generates a result set. Empower software gives the result set the same name as the sample set. The Oracle relational database assigns unique identifiers for the sets, individual


injections, channels and results with date and time stamps for each to ensure uniqueness.

The only way to generate a result set is by processing a sample set. If you select all the individual channels of a sample set and batch process them, you do not generate a result set.

Reviewing processed data

To review result sets:

1. Click the Browse Project tab in the navigation bar. In the project table of the work area, click the Result Sets tab.

2. Click the line number of the result set you want to review.

3. Click Tools > Review.

Result: The Review window opens. The Result set tree appears on the left-hand side.

• The tree lists the results in the result set showing data channels of multiple injections from the same vial.

• The result highlighted in the tree corresponds to the chromatogram.

4. Use the 2D Channels tab at the bottom of the View Data window to select and view processed data channels.

5. View the Peaks table, which contains individual peak information such as retention time, area, amount, concentration, and so on for the selected channel.

Tip: No 3D channels appear with the provided AAA methods.

Generating a report

To generate a report:

1. Click the Browse Project tab in the Navigation Bar. In the Results table of the work area, click the line number of the result for which you want to generate a report.

2. Click Tools > Preview.

3. Select “Use the Following Report Method.”


4. Choose either the General report provided or another report that has been created, and then click OK.

5. If the presentation of the report is satisfactory, click File > Print.



6 Handling Special Samples

Contents:

Topic Page

Analyzing protein samples for cysteine and methionine 6-2

Analyzing the nutritional content of foods and feeds 6-3

Analyzing cell culture media 6-4

6-1

Analyzing protein samples for cysteine and methionine

Analyzing cysteineCysteine and cystine are analyzed as derivatives formed by reduction and alkylation of the protein before hydrolysis. Many protocols are available in the open literature.

The UPLC AAA Solution provides an example method for two of the most common derivatives. Carboxymethyl cysteine is produced by alkylation with iodoacetic acid or iodoacetamide. Reaction with vinylpyridine yields pyridylethylcysteine.

To analyze these samples, select the method name containing “_AlkCys_” for the type of detector you are using.

To analyze cysteine:

1. Open the sample set method and confirm the following conditions:




3. Open the sample set in Run Samples, select the sample set, and then select Run and Report.

4. When the sample set is complete, review the gradient performance report. Compare the chromatogram on the report to the sample chromatogram below.

Tip: The representative chromatogram shows the elution position of cystine, CM-cysteine, and PE-cysteine. Only one of these compounds can appear in any sample.

6-2 Handling Special Samples

Representative chromatogram: alkylated cysteine analysis (10 pmol)

Note: The chromatogram shows the elution position of carboxymethylcysteine (CM Cys) and pyridylethylcysteine as well as norvaline. These amino acids are not present in the Waters hydrolysate standard and must be added separately.

Analyzing the nutritional content of foods and feeds

Samples of foods and feeds are often analyzed using two different sample preparation methods. Most amino acids give accurate results following direct acid hydrolysis. The sulfur amino acids, however, are best converted to the stable cysteic acid and methionine sulfone using performic acid oxidation.

To analyze both sample types, select the method name containing “_Food_Feed_” for the type of detector you are using.

The representative chromatogram shows the elution position of cysteic acid and methionine sulfone. These compounds can never occur in the same sample as methionine and cystine. The chromatogram also shows the elution of taurine and the alternative internal standards alpha aminobutyric acid and norvaline.

Analyzing the nutritional content of foods and feeds 6-3

Representative chromatogram: foods and feeds analysis (10 pmol)

Note: The chromatogram shows the elution position of several amino acids that are not present in the Waters hydrolysate standard. Cysteic acid, taurine, methionine sulfone, alpha aminobutyric acid, and norvaline must be added separately.

Analyzing cell culture media

Cell culture media is typically analyzed for free amino acids. No hydrolysis is required, but many additional amino acids are included.

Setting up the mobile phaseThe method for cell culture media uses the same flow rate and gradient table as other samples. The temperature is 60 °C rather than 55 °C. Use Eluent A2.

Prepare Eluent A2 using AccQ•Tag Ultra Eluent A concentrate (part number 186003838).


To prepare Eluent A2:

1. Measure 900 mL of water into a 1-L graduated cylinder.

2. In a separate graduated cylinder, measure 100 mL of AccQ•Tag Ultra Eluent A concentrate.

3. Add the concentrate to the water, and then mix for 5 minutes.

4. Transfer the solution to a 1-L mobile phase reservoir and label it Eluent A2.

5. Place Eluent A2 on BSM Line A1. This is required for the methods provided.

Analyzing the samplesTo analyze these samples, use the method name containing “_CellCult_” for the type of detector you are using.

Since the standard for cell culture analysis contains many additional amino acids, it must be added separately.


Representative chromatograms

10 pmol cell culture standard with recommended TUV detector settings

AM

Q

NH

3H

yPro

His A

sn Tau S

erG

lnA

rg Gly A

sp

Glu T

hr

Ala

GA

BA

Pro

HyL

ys1

HyL

ys2

AA

BA

Orn

Der

ivP

eak

Cys

Lys

Tyr

Met V

alN

Va

IleL

eu Ph

eT

rp

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

0.075

0.080

0.085

0.090

0.095

0.100

Minutes1.00 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

AM

Q

NH

3H

yPro

His A

sn Tau S

erG

lnA

rg Gly A

sp

Glu T

hr

Ala

GA

BA

Pro

HyL

ys1

HyL

ys2

AA

BA

Orn

Der

ivP

eak

Cys

Lys

Tyr

Met V

alN

Va

IleL

eu Ph

eT

rp

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

0.075

0.080

0.085

0.090

0.095

0.100

Minutes1.00 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


10 pmol cell culture standard with recommended PDA detector settings

AM

Q

NH

3

His A

sn

Tau Ser

Gln

Arg G

ly Asp

Glu T

hr

Ala GA

BA

Pro

HyL

ys1

HyL

ys2

AA

BA

Orn

Der

ivP

eak

Cys

Lys

Tyr

Met V

alN

Va

IleL

eu Ph

e Trp

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

0.075

0.080

Minutes1.00 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

HyP

ro

AM

Q

NH

3

His A

sn

Tau Ser

Gln

Arg G

ly Asp

Glu T

hr

Ala GA

BA

Pro

HyL

ys1

HyL

ys2

AA

BA

Orn

Der

ivP

eak

Cys

Lys

Tyr

Met V

alN

Va

IleL

eu Ph

e Trp

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

0.075

0.080

Minutes1.00 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

HyP

ro


10 pmol cell culture standard with recommended FLR detector settings

AM

Q

NH

3H

yPro H

is

Asn

Tau

Ser

Gln

Arg

Gly Asp

Glu

Th

r

Ala

GA

BA

Pro

HyL

ys1

HyL

ys2

AA

BA

Orn

Der

ivP

eak

Cys

Lys

Tyr

Met

Val NV

a

IleL

euP

he

Trp

EU

0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

400.00

450.00

500.00

550.00

600.00

650.00

700.00

750.00

800.00

850.00

900.00

Minutes1.00 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

*

* Tryptophan peak not detected


7 Troubleshooting

This chapter provides information for troubleshooting the UPLC AAA Solution. It includes a checklist of symptoms and recommended corrective actions. More detailed text follows each troubleshooting table.

Tip: For troubleshooting specific to the ACQUITY UPLC System or Empower software, see the documentation that came with each product.

Contents:

Topic Page

General troubleshooting principles 7-2

Derivatization troubleshooting 7-3

Derivatization issues 7-9

Chromatography troubleshooting 7-11

7-1

General troubleshooting principles

See also: Controlling Contamination in LC/MS Systems.

Refer to Appendix D of the ACQUITY UPLC System Operator’s Guide for information on solvent compatibility with ACQUITY UPLC.

Follow these general principles for troubleshooting the Waters UPLC AAA Solution.

• Define problems in detail:

– Complete failure of analysis

– Peaks correctly identified

– Quantitative problems (bad precision, or bad accuracy)

• Run the standard exactly as it was run at installation and as described above:

– Use a fresh, tested column

– Use bottled, tested eluents

• Adhere to these guidelines when judging the result for standard runs:

– Retention times must be the same on replicate injections

– Retention times must match those specified for the method

– Peak areas must be the same

– Peak areas must match the results acquired on installation

– Compare the area ratios with the installation result

Warning: Always observe Good Laboratory Practices when troubleshooting. Know the chemical and physical properties of the solvents and test solutions you use. See the Material Safety Data Sheet for each solvent and test solution in use.

7-2 Troubleshooting

Derivatization troubleshooting

Retention time problemsThese are the most common sources of retention time problems:

• Wrong methods

• Installation and placement of column stabilizer

• Wrong flow path configuration

• Pump pressure fluctuations

• Leaks

• Wrong solvents

• Wrong needle wash solvents

Retention time problems

Problem Possible solution or item to check

Times are randomly early or late from run to run

1. Verify there is no check valve or pump seal failure.2. Examine the pressure trace.3. Use static and dynamic leak tests to identify the

component for replacement.

Times are drifting over a series of runs

1. Equilibration is not complete – follow the standard sample set.

2. Column is contaminated – test with a new column.3. There is a progressively failing component or a

developing leak.

Times reproducibility is early or late, relative to the standard method

• If all peaks are affected to approximately the same extent, then there is a leak or the column is incorrectly placed in the column heater.

• If early eluting peaks are affected more than late peaks, then either a pump is failing or Eluent A was improperly diluted.

• If late eluting peaks are affected more than early peaks, then Pump B is failing.


Quantitative ProblemsSee Appendix C for a detailed discussion of sample amount and other derivatization guidelines.

Loss of resolution If all peaks are wider, check for the following problems:• Column life exhausted• Detector cell leak• Fitting leakIf only early peaks are wider, but late peaks are normal, check for one of these problems:• Injection volume too large• Too high organic concentration in sample (this

often occurs with a pipetting error of too much reagent or too little borate, both of which give a higher acetonitrile concentration)

• Voided column, damaged in-line filter, or injector malfunction

If only histidine is affected, the problem can indicate• a marginal manifestation of early peaks being

wider and late peaks being normal.• a need to repeat the system cleaning used at

startup.

Quantitative problems


pH too low for derivatization – only unprotonated amines react

• Yields of Asp, Glu, Lys, and Ala are most affected• Typically observe peak for mono-derivatized lysine

between histidine and serine

Retention time problems (Continued)


7-4 Troubleshooting

Insufficient reagent excess for the amount of sample

• AMQ peak is smaller than observed with 25pmole/µL standard



Reagent degradation

• AMQ peak will be the same as with a 25pmole/µL standard



Problems that appear related to insufficient reagent

• Pipetting errors• Inadequate mixing

Problems with the sample

• Amino acids not fully in solution• Inaccurate estimate of sample concentration

Characteristics of acid hydrolysis:• Decomposition of tryptophan and cysteine/cystine• Reduced recoveries expected for tyrosine,

threonine, and serine due to partial decomposition • Reduced recoveries expected for Ile and Val due to

slow release• Poor recovery expected for Met due to oxidation • Asn and Gln hydrolyzing to Asp and GluMany of amino acid hydrolysis procedures described in this document were designed primarily to improve recoveries of sensitive or hard-to-release amino acids. None give perfect results for all amino acids.

Quantitative problems (Continued)



Quantitative effects of sample diluent – Usually manifests as an increased proportion or amount of early eluting peaks and reduced proportion or amount of later eluting peaks. Check the following:• If organic concentration of derivatized sample is

low, the more hydrophobic derivatives will come out of solution, reducing peak size in analysis. This may occur if too little reagent is added.

• If the sample diluent is evaporating, both the volume and acetonitrile concentration will be lower. The early eluting peaks will be larger as they are concentrated by evaporation, and the late eluting peaks will be smaller because they will precipitate with the reduced acetonitrile concentration. This phenomenon is observed with incompletely sealed vials, pre-slit septa, or with reinjection from a punctured vial after a few hours.

Contamination, due to one of the following:• Laboratory environments are rich in amino acids

and proteins• Glycine can come from many sources• Serine is most abundant on skin• Aspartate and glutamate come from paper• Proline and hydroxylproline come from skin

Quantitative problems (Continued)


7-6 Troubleshooting

Derivatization problems

Derivatization problems and solutions

Symptom Possible Cause Solution

No reagent peak present, no amino acid peaks present

No reagent added Add reagent, reinject.

No injection made Check autosampler and injector, repair as needed.

Detector failure Check detector, repair as needed.

No reagent peak present, amino acid peaks are present

Too much sample Use less sample.

Insufficient reagent Use less sample.

Severely degraded reagent

Reconstitute fresh reagent.

Low values in samples for Asp and/or Glu, reagent peak <80% of reagent peak in a derivatized blank



Degraded reagent Reconstitute fresh reagent.

Early peaks split (especially His)

Excessive reagent Prepare derivatized sample again, keeping reagent less than 20% of the final sample composition.

Peaks broad in first half of chromatography; second half OK

Too much reagent Prepare derivatized sample again, keeping reagent to 20% of the final sample composition.

Mono-derivatized Lys peak(s) present, Lys values low, reagent peak <80% of the reagent peak in a derivatization blank




Mono-derivatized Lys peak(s) present, Lys values low, reagent peak >80% of the reagent peak in a derivatization blank

Degraded reagent Reconstitute fresh reagent

Inadequate mixing Ensure reagent mixes well with sample, vortex mix immediately after reagent addition.

Low values in samples for Arg, His, and/or Lys; reagent peak normal size

Improper sample reconstitution

Use 100 mM HCl for samples, vortex thoroughly.

Low values in samples for Asp and/or Glu; reagent peak normal size

Sample not buffered properly because salts are present

• Desalt sample• Limit amount of

sample present• Neutralize as

described in Appendix C.

Excess HCl from hydrolysis step

Remove HCl prior to derivatization.

Derivatization poor for most or all amino acids, sample is yellow after reagent addition

Sample not buffered properly because salts are present, pH acidic

• Desalt sample or limit amount present

• Neutralize as described in Appendix C.

Excess HCl from hydrolysis step

• Remove prior to derivatization

• Neutralize as described in Appendix C.

Derivatization agent is too old

Prepare AccQ•Tag Ultra again per instructions.

Derivatization problems and solutions(Continued)


7-8 Troubleshooting

Derivatization issues

Insufficient reagentConsult Appendix C for derivatization guidelines. Appropriate excess of reagent in a sample should produce an AMQ peak with an area that exceeds 80% of the AMQ peak in a derivatization blank.

The effect of insufficient reagent is greatest for Asp, Glu, and Lys. Lys, which is normally derivatized at two sites (the α-amino group and the ε-amino group on the side chain) can yield one or both of the possible mono-derivatized isomers if there is too little reagent or too much sample. These monoderivatives give a peak between histidine and serine.

Problems that appear related to insufficient reagent include

• too much sample.

• pipetting errors.

• degraded reagent.

• inadequate mixing.

Too much sample

Consult Appendix C for derivatization guidelines.

Pipetting errors

Delivering too little reagent produces the same symptoms as having too much sample. Too much buffer reduces organic concentration, causing precipitation of most hydrophobic derivatives.

Degraded reagent

Degraded reagent can produce a normal-size AMQ reagent peak, but amino acid yields can be low and you can observe one or both of the mono-Lys peaks. The cause can be hydrolysis of reagent due to improper storage. Severely degraded reagent produces no AMQ peak at all.

Derivatization issues 7-9

Inadequate mixing

Mix each sample immediately after reagent addition. Do not wait until after adding reagent to the entire sample group. Inadequate mixing after reagent addition can cause the reagent to hydrolyze prior to contacting the entire sample solution.

Sample reconstitutionAfter hydrolysis, basic amino acids can have a strong affinity for the sample tube glass. To ensure good recovery, use 100 mM HCl to reconstitute the sample.

Buffers

The UPLC AAA Solution is compatible with a wide variety of common buffer salts and detergents. However, HCl hydrolysis in the presence of some salts produces nonvolatile acids, which cannot be adequately buffered by the derivatization buffer.

To ensure adequate derivatization, the sample pH must be between 8.2 and 10.

• If the pH is below 6, addition of reagent can result in a yellow color, and derivatization will be poor. Desalt the sample or increase the amount of buffer to overcome the buffering capacity of the nonvolatile acids.

• If you increase buffer volume, increase the amount of reagent to keep the reagent at a 20% (by volume) concentration. The reagent will also be colored and destroyed if the pH is very high, for example, after alkaline hydrolysis. Follow the neutralization guidelines in Appendix C.

7-10 Troubleshooting

Chromatography troubleshooting

Before referring to the troubleshooting tables below, follow these guidelines:

• Record retention times of key component peaks: His, Ala, Phe, Arg, Gly, Cys, and Lys.

• Note area(s) of resolution difficulties.

• Start with testing the obvious things, and proceed to more complex testing, if required:

– Check column heater operation

– Check system backpressure

– Check flow rate with a graduated cylinder

– Check gradient accuracy

– Perform a column efficiency test, if indicated

– Ensure proper placement of column stabilizer

The table below summarizes the most common retention problems.

General retention problems


Retention times increased for all peaks

Gradient wrong Check gradient table.

Low delivery of strong eluent

Check flow rate and solvent composition.

Column temperature low

Check column oven operation.

Check position of column stabilizer.

Eluent delivery low Check flow rate.

Leaks Check all fittings.

Retention times increased for peaks eluting before Cys

Improper placement of column stabilizer

Check placement of column stabilizer.

Column temperature too low



Retention times decreased for all peaks

Excessive delivery of strong eluent

Check pump performance.

Column temperature too high


Poor retention time reproducibility

Inconsistent pump operation

Check backpressure, prime pump.

All peaks elute at or near the void volume

Eluents switched Correct as needed.

All peaks broad Poor column efficiency Check column, replace as needed.

Excessive system bandspread

Diagnose and reduce bandspread caused by injector, tubing connections, and/or detector cell.

Peaks broad in first half of chromatogram, second half OK

Poor column efficiency Check column, replace as needed.

Excessive system bandspread

Diagnose and reduce bandspread caused by injector, tubing connections, and/or detector cell.

Excessive injection volume

Limit injections to 1.0 µL.

Insufficient column equilibration

Use specified method run time to provide extra equilibration.

Too high organic in sample

Follow protocol exactly.

Early peaks split (especially His)

Excessive injection volume

Limit injections to 1.0 µL.

General retention problems(Continued)



The table below summarizes problems with specific peak pairs.

Eluent deliveryInconsistent pump operation usually results in poor retention time reproducibility and can have several causes including inconsistent or complete loss of pump prime (air in pump heads), check valve problems, or pump primary seal failure. Check the system backpressure to ascertain the cause. Low flow rates can be an indicator of failure.

System efficiencySystem efficiency has the largest impact on closely eluting peak pairs such as Arg/Gly, Cys/Lys, and Ile/Leu. Low efficiency can be a result of column aging or deterioration, or of chromatographic component failure. In general, bandspread occurring before the column (injectors, pre-column fittings) affects early peaks (up to Gly most). Post-column bandspread (for example, detector-related) affects all peaks. Ensure the low-flow TUV inlet tubing is connected from the column outlet to the flow cell inlet. Loss of column efficiency can affect all peaks or early peaks only.

Problems with specific peak pairs


Arg/Gly resolution low Improper position of column stabilizer

Check position of column stabilizer

Column temperature wrong

Check column heater operation

Wrong dilution of Eluent A concentrate

Prepare Eluent A again, from concentrate solution

Cys/Lys resolution low Improper position of column stabilizer

Check position of column stabilizer

Column temperature wrong

Check column heater operation

Wrong dilution of Eluent A concentrate

Prepare Eluent A again, from concentrate solution


Column heaterEnsure the heater is operating and set at the correct temperature. Small changes in temperature have the greatest effect on Arg/Gly and Cys/Lys resolution. Lowering the set temperature can improve Arg/Gly resolution but can decrease resolution of Cys/Lys. Ensure the ambient temperature fluctuations do not exceed +5 °C.

The largest source of temperature problems is the placement of the stabilizer and the column within the heater. Readjust their placement as described in Chapter 2.


A Safety Advisories

Waters instruments display hazard symbols designed to alert you to the hidden dangers of operating and maintaining the instruments. Their corresponding user guides also include the hazard symbols, with accompanying text statements describing the hazards and telling you how to avoid them. This appendix presents all the safety symbols and statements that apply to the entire line of Waters products.

Contents

Topic Page

Warning symbols A-2

Caution symbol A-5

Warnings that apply to all Waters instruments A-5

Electrical and handling symbols A-13

A-1

Warning symbols

Warning symbols alert you to the risk of death, injury, or seriously adverse physiological reactions associated with an instrument’s use or misuse. Heed all warnings when you install, repair, and operate Waters instruments. Waters assumes no liability for the failure of those who install, repair, or operate its instruments to comply with any safety precaution.

Task-specific hazard warningsThe following warning symbols alert you to risks that can arise when you operate or maintain an instrument or instrument component. Such risks include burn injuries, electric shocks, ultraviolet radiation exposures, and others.

When the following symbols appear in a manual’s narratives or procedures, their accompanying text identifies the specific risk and explains how to avoid it.

Warning: (General risk of danger. When this symbol appears on an instrument, consult the instrument’s user documentation for important safety-related information before you use the instrument.)

Warning: (Risk of burn injury from contacting hot surfaces.)

Warning: (Risk of electric shock.)

Warning: (Risk of fire.)

Warning: (Risk of needle puncture.)

Warning: (Risk of injury caused by moving machinery.)

Warning: (Risk of exposure to ultraviolet radiation.)

Warning: (Risk of contacting corrosive substances.)

Warning: (Risk of exposure to a toxic substance.)

A-2 Safety Advisories

Warnings that apply to particular instruments, instrument components, and sample types

The following warnings can appear in the user manuals of particular instruments and on labels affixed to them or their component parts.

Burst warning

This warning applies to Waters instruments fitted with nonmetallic tubing.

Mass spectrometer flammable solvents warning

This warning applies to instruments operated with flammable solvents.

Warning: (Risk of personal exposure to laser radiation.)

Warning: (Risk of exposure to biological agents that can pose a serious health threat.)

Warning: Pressurized nonmetallic, or polymer, tubing can burst. Observe these precautions when working around such tubing:• Wear eye protection.• Extinguish all nearby flames.• Do not use tubing that is, or has been, stressed or kinked.• Do not expose nonmetallic tubing to incompatible compounds like

tetrahydrofuran (THF) and nitric or sulfuric acids.• Be aware that some compounds, like methylene chloride and

dimethyl sulfoxide, can cause nonmetallic tubing to swell, which significantly reduces the pressure at which the tubing can rupture.

Warning: Where significant quantities of flammable solvents are involved, a continuous flow of nitrogen into the ion source is required to prevent possible ignition in that enclosed space. Ensure that the nitrogen supply pressure never falls below 400 kPa (4 bar, 58 psi) during an analysis in which flammable solvents are used. Also ensure a gas-fail connection is connected to the LC system so that the LC solvent flow stops if the nitrogen supply fails.

Warning symbols A-3

Mass spectrometer shock hazard

This warning applies to all Waters mass spectrometers.

This warning applies to certain instruments when they are in Operate mode.

Biohazard warning

This warning applies to Waters instruments that can be used to process material that might contain biohazards: substances that contain biological agents capable of producing harmful effects in humans.

Warning: To avoid electric shock, do not remove the mass spectrometer’s protective panels. The components they cover are not user-serviceable.

Warning: High voltages can be present at certain external surfaces of the mass spectrometer when the instrument is in Operate mode. To avoid non-lethal electric shock, make sure the instrument is in Standby mode before touching areas marked with this high voltage warning symbol.

Warning: Waters's instruments and software can be used to analyze or process potentially infectious human-sourced products, inactivated microorganisms, and other biological materials. To avoid infection with these agents, assume that all biological fluids are infectious, observe good laboratory practices and, consult your organization’s biohazard safety representative regarding their proper use and handling. Specific precautions appear in the latest edition of the US National Institutes of Health (NIH) publication, Biosafety in Microbiological and Biomedical Laboratories (BMBL).


Chemical hazard warning

This warning applies to Waters instruments that can process corrosive, toxic, flammable, or other types of hazardous material.

Caution symbol

The caution symbol signifies that an instrument’s use or misuse can damage the instrument or compromise a sample’s integrity. The following symbol and its associated statement are typical of the kind that alert you to the risk of damaging the instrument or sample.

Warnings that apply to all Waters instruments

When operating this device, follow standard quality control procedures and the equipment guidelines in this section.

Warning: Waters instruments can be used to analyze or process potentially hazardous substances. To avoid injury with any of these materials, familiarize yourself with the materials and their hazards, observe Good Laboratory Practices (GLP), and consult your organization’s safety representative regarding proper use and handling. Guidelines are provided in the latest edition of the National Research Council's publication, Prudent Practices in the Laboratory: Handling and Disposal of Chemicals.

Caution: To avoid damage, do not use abrasives or solvents to clean the instrument’s case.

Caution symbol A-5

Attention: Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user’s authority to operate the equipment.

Important: Toute modification sur cette unité n’ayant pas été expressément approuvée par l’autorité responsable de la conformité à la réglementation peut annuler le droit de l’utilisateur à exploiter l’équipement.

Achtung: Jedwede Änderungen oder Modifikationen an dem Gerät ohne die ausdrückliche Genehmigung der für die ordnungsgemäße Funktionstüchtigkeit verantwortlichen Personen kann zum Entzug der Bedienungsbefugnis des Systems führen.

Avvertenza: eventuali modifiche o alterazioni apportate a questa unità e non espressamente approvate da un ente responsabile per la conformità annulleranno l’autorità dell’utente ad operare l’apparecchiatura.

Atencion: cualquier cambio o modificación efectuado en esta unidad que no haya sido expresamente aprobado por la parte responsable del cumplimiento puede anular la autorización del usuario para utilizar el equipo.


Warning: Use caution when working with any polymer tubing under pressure:• Always wear eye protection when near pressurized polymer tubing.• Extinguish all nearby flames.• Do not use tubing that has been severely stressed or kinked.• Do not use nonmetallic tubing with tetrahydrofuran (THF) or concentrated

nitric or sulfuric acids.• Be aware that methylene chloride and dimethyl sulfoxide cause

nonmetallic tubing to swell, which greatly reduces the rupture pressure of the tubing.

Attention: Manipulez les tubes en polymère sous pression avec precaution:• Portez systématiquement des lunettes de protection lorsque vous vous

trouvez à proximité de tubes en polymère pressurisés.• Eteignez toute flamme se trouvant à proximité de l’instrument.• Evitez d'utiliser des tubes sévèrement déformés ou endommagés.• Evitez d'utiliser des tubes non métalliques avec du tétrahydrofurane

(THF) ou de l'acide sulfurique ou nitrique concentré.• Sachez que le chlorure de méthylène et le diméthylesulfoxyde entraînent le

gonflement des tuyaux non métalliques, ce qui réduit considérablement leur pression de rupture.

Vorsicht: Bei der Arbeit mit Polymerschläuchen unter Druck ist besondere Vorsicht angebracht:• In der Nähe von unter Druck stehenden Polymerschläuchen stets

Schutzbrille tragen.• Alle offenen Flammen in der Nähe löschen.• Keine Schläuche verwenden, die stark geknickt oder überbeansprucht

sind.• Nichtmetallische Schläuche nicht für Tetrahydrofuran (THF) oder

konzentrierte Salpeter- oder Schwefelsäure verwenden.• Durch Methylenchlorid und Dimethylsulfoxid können nichtmetallische

Schläuche quellen; dadurch wird der Berstdruck des Schlauches erheblich reduziert.


Attenzione: prestare attenzione durante l’utilizzo dei tubi di polimero pressurizzati:• Indossare sempre occhiali da lavoro protettivi nei pressi di tubi di polimero

pressurizzati.• Estinguere ogni fonte di ignizione circostante.• Non utilizzare tubi soggetti che hanno subito sollecitazioni eccessive o son

stati incurvati.• Non utilizzare tubi non metallici con tetraidrofurano (THF) o acido

solforico o nitrico concentrato.• Tenere presente che il cloruro di metilene e il dimetilsolfossido provocano

rigonfiamento nei tubi non metallici, riducendo notevolmente la resistenza alla rottura dei tubi stessi.

Advertencia: se recomienda precaución cuando se trabaje con tubos de polímero sometidos a presión:• El usuario deberá protegerse siempre los ojos cuando trabaje cerca de

tubos de polímero sometidos a presión.• Si hubiera alguna llama las proximidades.• No se debe trabajar con tubos que se hayan doblado o sometido a altas

presiones.• Es necesario utilizar tubos de metal cuando se trabaje con

tetrahidrofurano (THF) o ácidos nítrico o sulfúrico concentrados.• Hay que tener en cuenta que el cloruro de metileno y el sulfóxido de

dimetilo dilatan los tubos no metálicos, lo que reduce la presión de ruptura de los tubos.



Warning: The user shall be made aware that if the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired.

Attention: L’utilisateur doit être informé que si le matériel est utilisé d’une façon non spécifiée par le fabricant, la protection assurée par le matériel risque d’être défectueuses.

Vorsicht: Der Benutzer wird darauf aufmerksam gemacht, dass bei unsachgemäßer Verwenddung des Gerätes unter Umständen nicht ordnungsgemäß funktionieren.

Attenzione: l’utente deve essere al corrente del fatto che, se l’apparecchiatura viene usta in un modo specificato dal produttore, la protezione fornita dall’apparecchiatura potrà essere invalidata.

Advertencia: el usuario deberá saber que si el equipo se utiliza de forma distinta a la especificada por el fabricante, las medidas de protección del equipo podrían ser insuficientes.


Warning: To protect against fire hazard, replace fuses with those of the same type and rating.

Attention: Remplacez toujours les fusibles par d’autres du même type et de la même puissance afin d’éviter tout risque d’incendie.

Vorsicht: Zum Schutz gegen Feuergefahr die Sicherungen nur mit Sicherungen des gleichen Typs und Nennwertes ersetzen.

Attenzione: per una buona protezione contro i rischi di incendio, sostituire i fusibili con altri dello stesso tipo e amperaggio.

Advertencia: sustituya los fusibles por otros del mismo tipo y características para evitar el riesgo de incendio.


Warning: To avoid possible electrical shock, disconnect the power cord before servicing the instrument.

Attention: Afin d’éviter toute possibilité de commotion électrique, débranchez le cordon d’alimentation de la prise avant d’effectuer la maintenance de l’instrument.

Vorsicht: Zur Vermeidung von Stromschlägen sollte das Gerät vor der Wartung vom Netz getrennt werden.

Attenzione: per evitare il rischio di scossa elettrica, scollegare il cavo di alimentazione prima di svolgere la manutenzione dello strumento.

Precaución: para evitar descargas eléctricas, desenchufe el cable de alimentación del instrumento antes de realizar cualquier reparación.


Electrical and handling symbols

Electrical symbolsThese can appear in instrument user manuals and on the instrument’s front or rear panels.

Electrical power on

Electrical power off

Standby

Direct current

Alternating current

Protective conductor terminal

Frame, or chassis, terminal

Fuse

Recycle symbol: Do not dispose in municipal waste.

Electrical and handling symbols A-13

Handling symbolsThese handling symbols and their associated text can appear on labels affixed to the outer packaging of Waters instrument and component shipments.

Keep upright!

Keep dry!

Fragile!

Use no hooks!


B Materials of Construction and Compliant Solvents

Warning: To confirm the integrity of the source exhaust system, you must address any safety issues raised by the contents of this Appendix.

Topic Page

Preventing contamination B-2

Items exposed to solvent B-2

Solvents used to prepare mobile phases B-3

B-1

Preventing contamination

For information on preventing contamination, refer to Controlling Contamination in LC/MS Systems (part number 715001307). You can find this document on http://www.waters.com; click Services and Support and then Support Center.

Items exposed to solvent

The items that appear in the following table can be exposed to solvent. You must evaluate the safety issues if the solvents used in your application differ from the solvents normally used with these items. See “Solvents used to prepare mobile phases” on page B-3 for details about the most common ingredients used to prepare mobile phases.


Item Material

APPI lamp drive assembly:

Mounting shaft Stainless steel

Repeller electrode Stainless steel

Insulator PEEK

Lamp window Magnesium fluoride

Autotune vials HDPE

Corona discharge pin mounting contact

PEEK

Gas exhaust port Aluminium

Gas tubes FEP

Ion block Stainless steel

Ion block support PEEK

Isolation valve Gold-plated aluminium/bronze

O-rings Viton® or PTFE-encapsulated Viton

Probe adjuster bellows PTFE/Viton

Probe adjuster assembly Anodized aluminium, glass filled acetal, and stainless steel

B-2 Materials of Construction and Compliant Solvents

Solvents used to prepare mobile phases

The following lists the most common ingredients used to prepare mobile phases for reverse-phase LC/MS (API):

• Water

• Methanol

• Acetonitrile

• Formic acid (<0.1%)

• Acetic acid (<0.1%)

• Trifluoroacetic acid (<0.1%)

• Ammonium acetate (<10 mM)

• Ammonium formate (<10 mM)

These solvents are not expected to cause any problems with the materials identified in “Items exposed to solvent” on page B-2.

Probe shaft PEEK

Push-in gas fittings Nickel/brass

Solvent waste/leak management Tygon tubing

Source enclosure Alochromed Aluminium

Source enclosure view port Toughened plate glass

Waste bottle Polypropylene

Waste bottle push-in fittings NBR, SST, PBT, and POM


Item Material

Solvents used to prepare mobile phases B-3

B-4 Materials of Construction and Compliant Solvents

C Derivatization Guidelines

The AccQ•Tag™ derivatization reagent reacts rapidly, on a msec time scale, with primary and secondary amine groups. This appendix describes the steps you can use to systematically control all the reaction related factors, except contamination. It also describes experiments for unknown samples.

Contents:

Topic Page

Introduction C-2

Estimating sample amount C-3

Estimating the requirement for neutralization C-6

Confirming sufficient reagent C-7

Examples C-9

C-1

Introduction

The AccQ•Tag™ derivatization reagent reacts rapidly, on a msec time scale, with primary and secondary amine groups. The excess reagent reacts more slowly with water, hydrolyzing in a few tens of seconds.

The following are possible actions that can occur during derivatization which will give inaccurate amino acid analysis:

• The reagent only reacts with unprotonated amines so the pH must be between pH 8 and 10.

• There must be sufficient excess of reagent to drive the derivatization reaction to completion.

• The organic concentration of the derivatization cocktail must be high enough to keep the reagent and the derivatives in solution but not so high as to distort the chromatography.

• The pH must not be so low or so high that the reagent is destroyed.

• The amino acid concentration must be above the required sensitivity limits.

• The sample must not be contaminated with amino acids, with proteins, or with other environmental amines.

Follow the procedures in this appendix to systematically control all the reaction related factors (except contamination). In all cases, the objective is to bring the experiment into the range of conditions that are likely to work. The method has a working linear dynamic range that exceeds three orders of magnitude, thus the estimated conditions here need only be approximations.

Steps in designing an amino acid analysis:

1. Estimate the minimum amount of a given sample required for good quantitation without exceeding the limits of the method.

2. Estimate the requirement for neutralization.

3. Confirm that sufficient reagent is present.

C-2 Derivatization Guidelines

Estimating sample amount

The following are typical descriptions of sample concentrations:

• mg

• pmoles-nmoles-µmoles

• mg/mL

• pmoles-nmoles-µmoles/µL-mL;

• µM (micromolar, i.e., micromoles/liter)

• %

• mg%

The typical description of amino acid analyzer sensitivity and linear range is pmoles on column.

To estimate the sample amount:

1. Start with known characteristics of UPLC Amino Acid Analysis Solution, adhering to the following guidelines:

• Limit of quantitation is 50fmoles on column of a given amino acid.

• Best quantitation is easy to achieve at 1pmole or higher on column.

• Upper limit is defined by reagent excess, not detector properties.

• Recommended total volume of derivatization is 100µL.

• Maximum injection volume is 1µL.

2. The amounts used in describing an amino acid analysis chromatogram refer to the quantity of each individual peak. This must be related to the total amount derivatized and to concentration of the starting amino acid solution (either standard or sample). Use the following table as a guide.

Determining amounts for amino acid analysis

pmoles on column[pmoles/µL in final derivatized sample]

pmoles derivatized

Starting Concentration of Amino Acid Solution[10µL derivatized]

0.05 5 0.5pmoles/µL

0.50 50 5.0pmoles/µL

1.00 100 10.0pmoles/µL


3. Translate the sample concentration into pmoles of amino acid, considering the following proportionality guidelines:

a. The sample concentration is usually protein concentration.

b. The total amount of protein is the sum of the amounts of all the amino acids.

c. Hydrolysis of a protein yields sixteen different amino acids.

d. For a first approximation, divide the amount of protein by 16 to obtain the amount of each individual amino acid.

e. The amino acids are never present in equal amounts, so the least abundant amino acid will be less than 1/16 the total amount in the sample. It is the least abundant amino acid that defines the lower limit of concentration.

f. If the amino acid composition is known for the specific sample, divide the number of residues of the least abundant amino acid by the number of total amino acid residues. (Exclude the labile amino acids tryptophan, cysteine, methionine from these calculations.) Multiply the amount of protein by this factor to estimate the amount of the least abundant amino acid.

g. In the absence of other information, estimate that the least abundant amino acid will be 2-4% of the total amino acids. Multiply the amount of protein by 0.03 to estimate the amount of the least abundant amino acid.

4. Perform a sample calculation, using the following criteria and formulas:

a. Sample is 1.0mg/mL protein.

b. Estimate least abundant amino acid as 0.03mg/mL.

c. Convert to moles (the average molecular weight of an amino acid residue in a protein is 110).

10.00 1000 100.0pmoles/µL

25.00 2500 250.0pmoles/µL

50.00 5000 500.0pmoles/µL

Determining amounts for amino acid analysis (Continued)


d. Calculate volume to give 1 pmol on column. Since the standard target injection is 1µL out 100µL total volume, take 100pmoles of the estimated least abundant amino acid.

e. Since 0.37µL is hard to measure, dilute the sample in a convenient way. Since we prefer to pipet 10µL of sample into the reaction vial, we can divide 10µL desired by 0.37mL required. This gives a 27-fold dilution. If we can accurately pipet 5µL of sample, we would dilute with 27 times as much 0.1M HCl. In this example, 5µL of sample + 135µL of 0.1M HCl would give 10µL measured into the reaction vial.

5. Calculate using other units, as follows:

a. pmoles-nmoles-mmoles/µL: In the above calculation, a mass/volume was converted to a molar amount/volume. If the sample is already described in those terms, the calculation is simpler. Now, however, the number of moles of amino acid/mole of protein is used rather than the average weight of an amino acid. If the molecular weight of the protein is known, the number of amino acids will be known. One mole of protein represents that number of moles of amino acids. Typically, a few hundred moles of amino acids make up one mole of protein. If the protein is unknown, assume that the molecular weight is 55,000 and that there are 500 total amino acids. This will be an error of no more than a factor of 3 high or low. Again for an unknown, assume that the least abundant amino acid is about 3% of the total.

b. µM: The same considerations as in section c.i. The large numbers involved in µMole/L

c. % and mg%: These are the same approach as in the first example, but it is necessary to more exactly define the units. % is usually grams/100grams or grams/100mL. mg% is an inexact usage frequently found in protein descriptions because the molecular weight is so high. It usually mg/100gm.

LpmolesL

ml

mg

gm

mole

pmoles

gmAA

moleAA

mL

mg µµ

/27010

1

10

110

101.1

110333

12

2

2

=××××

×× −

Lpmoles

Lpmoles µµ

37.0270

1100 =×


Estimating the requirement for neutralization

The AccQ•Tag™ derivatization chemistry requires non-protonated amines at pH 8-10. Very acid samples should be neutralized without driving the pH too high or leaving it too low.

Considerations

Note these considerations when estimating neutralization requirements:

1. Almost all users know the solution in which their sample is dissolved.

2. Almost all users run the same type of samples all the time so the samples are always in the same solution.

3. If the amino acid solution is dissolved in 0.1M HCl, 10-20µL of sample can be used in the derivatization cocktail without risk of too low pH. The cocktail is then (10µL Sample : 70 µL Borate : 20µL Reagent) or (20µL Sample : 60 µL Borate : 20µL Reagent). It is still necessary to ensure that the available reagent is not exceeded as described below.

4. If the amino acid solution is in a higher concentration of acid, it should be neutralized with an equal volume of sodium hydroxide at the same concentration. This may be done in bulk separately from the derivatization, or it may be combined with the derivatization step.

a. 500µL of Sample in 6M HCL mixed with 500µL 6M NaOH. Then follow step 3 above.

b. In the reaction vial: mix 10µL sample in 6M HCL, 10µL 6M NaOH, 60µL Borate, and 20µL reagent.

5. The above principle is generalized to any concentration or any acid. Match equal volumes of equal concentrations.


Confirming sufficient reagent

The AccQ•Tag derivatization chemistry is like any derivatization reaction. There must be an excess of reagent over derivatizable groups. An 2-5 fold molar excess of AccQ•Tag reagent over total derivatizable amines is the usual target.

To confirm sufficient reagent:

1. The standard AccQ•Tag reagent vial contains 3-4mg of reagent. This approximates 10-14 µmoles of reagent. This is dissolved in 1mL of acetonitrile, and 20µL is used for each 100µL derivatization reaction. Each reaction vessel contains, therefore, 210-280 nmoles of derivatization reagent.

2. Ideally, each reaction vial should contain no more than 40-140nmoles of total amines.

3. Referring to the table above, we describe an amino acid sample in terms of number of pmoles on column for a given amino acid. There are 17 derivatizable components in the standard. The injection is 1/100 of the amount derivatized. Multiply, therefore, by 1700 to estimate the total derivatizable amine.

4. A “25pmole” standard is a total of 42.5 nmoles of amines, safely within the limits of reagent excess.

5. For a protein sample, use the weight of sample and the average weight of an amino acid to estimate the required excess. In the 1mg/ml example above:

In this case, the estimate of the minimum required for sensitivity led to an estimate equal to about 1/3 of a µL of sample so the total amount of amine should be well within the limits of required reagent excess.

LnmolesL

ml

mg

gm

mole

nmoles

gmAA

moleAA

mL

mg µµ

/1.910

1

10

110

101.1

1133

9

2=×××

××

nmolesdiluteLdiluteL

stockL

stockL

nmoles3.310

140

51.9 =×× µµ

µµ

total amines in reaction vial

Confirming sufficient reagent C-7

Calculation terms

1 g = 103 mg = 106 µg

1 L = 103 mL = 106 µL

1 mole = 103 mmole = 106 µmole = 109 nmole = 1012 pmole

When working with protein samples of unknown composition, use the following guidelines for sample estimation calculations:

• Average MW of a protein =

• Average protein contains 500 amino acids

• Average MW of an amino acid =

• Estimate 3% of total protein amount when determining amount of the least abundant amino acid (LAAA) in the protein

• % Solutions are a representation of g/100g or g/100mL

• mg% solutions represent mg/100g or mg/100mL (density is assumed to be = 1 if not provided)

L

ginMWmole =1

pL

pmole

nL

nmole

L

mole

mL

mmole

L

moleSolutionM

111111 =====

µµ

mol

g55000

mol

g110


Examples

Example with known composition of sample, concentration in mg/mL

• 1.37 mg/mL Bovine Serum Albumin in 6M HCl

• MW 66430

a Due to partial or complete destruction during hydrolysis, do not consider as least abundant for estimat-ing sensitivity requirementbDue to conversion to Glutamic acid and Aspartic Acid during hydrolysis, add the acid and the amide together first when identifying the least abundant

Estimation of Sample amount

Calculation for least abundant amino acid in solution (Ile 15)

Ile in solution

Amino Acid residues for BSA

Ala 48 aCys 35 Asp 41 Glu 58

Phe 30 Gly 17 His 16 Ile 15

Lys 60 Leu 65 aMet 5 bAsn 14

Pro 28 bGln 21 Arg 26 Ser 32

Thr 34 Val 38 aTrp 3 Tyr 21

%47.2100607

15 =×residuesAATotal

residuesIle

mL

mg

mL

mg 034.00247.0

37.1 =×

LIlepmolesL

ml

mg

gm

mole

pmoles

Ilegm

Ilemole

BSAmL

Ilemg µµ

/30010

1

10

110

113

1

1

104.333

122

=××××× −

Lpmoles

Lpmoles µµ

33.0300

1100 =× dilutionx

requiredL

desiredL30

33.0

10 =µµ

withstockdilutetoHClMLpipettoL 1.0150305 µµ =×

Examples C-9

HClMr 29.0

moles

pH Consideration

Final acid concentration of dilution

Because the resulting acid concentration is greater than 0.1 M HCl, an equal volume of 0.3 M NaOH will need to be added to derivatization cocktail.

Borate volume must be adjusted accordingly. Therefore, 10µL sample and 10µL 0.3 M NaOH should be mixed with 60µL Borate buffer before adding the derivatizing reagent.

Estimation of Reagent Excess

Calculation of total amino acids in reaction vessel

4.0 nmol is well below the 140 nmol limit

Example with unknown sample composition, concentration in mg/mL

3.5 mg/mL in 4M NaOH


least abundant AA in solution (LAAA)

oL

HClmoles

DiluteTotalL

HClmolesHClmoles

DiluteTotalL

L

molesL

L

molesL

29.0

155

)1530(

155

)1.0

150()6

5(

µµ

µµµ

µµµµ

µµµ

=+=×+×

ndilutionLdilutionL

stockL

L

ml

mg

gm

mole

nmoles

gmAA

moleAA

BSAmL

mg0.410

155

5

10

1

10

110

101.1

137.133

9

2=×××××

×× µ

µµ

µ

mL

mg

mL

mg 105.003.0

5.3 =×

LLAAApmolesL

ml

mg

gm

mole

pmoles

gm

mole

solutionmL

LAAAmg µµ

/95510

1

10

110

110

1

1

105.033

12

=××××

Lpmoles

Lpmoles µµ

105.0955

1100 =× dilutionx

requiredL

desiredL96

105.010 =

µµ



nmoles3.3=

pH Consideration

1. Sample dilution requires 5 µL of 4 M NaOH

2. Sample is diluted with 480 µL of 0.1 M HCl

480 µL 0.1 M HCl = 48 µL 1 M HCl = 12 µL 4 M HCl

3. Take the difference of acid and base amounts:

12 µL HCl – 5 µL NaOH

4. Sample now contains the equivalent of 7 µL of 4 M HCl in excess

Because the resulting acid concentration is less than 0.1 M HCl, it is not necessary to make any further adjustments to pH prior to derivatization.




Example with unknown sample composition, concentration in mg/tube

1.2 mg protein/tube

HClMdilutionTotalL

HClML058.0

485

47 =×µµ

dilutionLdilutionL

stockL

L

ml

mg

gm

mole

nmoles

gmAA

moleAA

stockmL

mg10

4855

101

10110

101.115.3

33

9

2×××××

×× µ

µµ

µ

Examples C-11


Since this volume is larger than we would care to dilute with, a starting stock concentration can be prepared in 1000 µL



tube

proteinnmoles

mole

nmoles

mg

g

g

mole

tube

proteinmg 8.2110

10

1

55000

2.1 9

3=×××

tube

AAnmoles

protein

AA

tube

proteinnmoles 109005008.21 =×

tube

LAAAnmoles

tube

AAnmoles 32703.0

10900 =×

tubetoaddtoHClMLpmoles

L

nmole

pmoles

tube

nmoles1.032700

10

10327 3

µµ =××

L

proteinpmoles

nmole

pmoles

L

proteinnmoles

µµ8.2110

1000

8.21 3

=×

L

AApmoles

protein

AA

L

proteinpmoles

µµ109005008.21 =×

L

LAAApmoles

L

AApmoles

µµ327

03.010900 =×

Lpmoles

Lpmoles µµ

31.0327

1100 =× dilutionx

requiredL

desiredL32

31.0

10 =µµ



nmoles


Example with unknown sample composition, concentration in pmoles/tube

28 pmoles protein/tube





Example with unknown sample composition, concentration in pmoles/µL

1 mL of 1.5 pmoles protein/µL in 0.1 M HCl

dilutionLdilutionL

stockL

L

ml

mg

gm

mole

nmoles

gmAA

moleAA

stockmL

mg3.310

165

5

10

1

10

110

101.1

12.133

9

2=×××××

×× µ

µµ

µ

tube

AApmoles

protein

AA

tube

proteinpmoles 1400050028 =×

tube

LAAApmoles

tube

AApmoles 42003.0

14000 =×

tubetoaddtoHClML

L

desiredpmolesLAAApmoles

1.04210420 µ

µ

=

nmolesprotein

AAL

pmoles

nmole

L

pmoles3.3

50010

10

1

42

283

=××× µµ

Examples C-13

Estimation of Sample amount:

Estimation of Reagent Excess:



Example with unknown sample composition, concentration in µM1 mL of 4 µM protein solution in 6 M HCl

L

AApmoles

protein

AA

L

proteinpmoles

µµ7505005.1 =×

L

LAAApmoles

L

AApmoles

µµ5.22

03.0750 =×

Lpmoles

Lpmoles µµ

44.45.22

1100 =× dilutionx

requiredL

desiredL25.2

44.4

10 =µ

µ

withstockdilutetoHClMLpipettoL 1.05.2225.210 µµ =×

nmolesdilutionLprotein

AA

dilutionL

stockL

pmoles

nmole

stockL

proteinpmoles3.210

500

5.32

10

10

15.13

=×××× µµµ

µ


HClM94.0


pH Consideration


Because the resulting acid concentration is greater than 0.1 M HCl, an equal volume of 1 M NaOH will need to be added to derivatization cocktail.

Borate volume must be adjusted accordingly. Therefore, 10µL sample and 10µL 1 M NaOH should be mixed with 60µL Borate buffer before adding the derivatizing reagent.




L

proteinpmoles

mole

pmoles

L

L

L

molesM

µµµµµ 410

10

144

6

6=××=

L

AApmoles

protein

AA

L

proteinpmoles

µµ20005004 =×

L

LAAApmoles

L

AApmoles

µµ60

03.02000 =×

Lpmoles

Lpmoles µµ

67.160

1100 =× dilutionx

requiredL

desiredL6

67.1

10 =µµ


orL

HClmoles

DiluteTotalL

HClmolesHClmoles

DiluteTotalL

L

molesL

L

molesL

94.0

70

)660(

70

)1.0

60()6

10(

µµ

µµµ

µµµµ

µµµ

=+=×+×

nmolesdilutionLprotein

AA

dilutionL

stockL

pmoles

nmole

stockL

proteinpmoles9.210

500

70

10

10

143

=×××× µµµ

µ

Examples C-15

HClMor 11.0

nmoles

Example with unknown sample composition, concentration in %0.5% protein solution in 1.5 M HCl



pH Consideration


Because the resulting acid concentration is roughly equivalent to 0.1 M HCl, it is not necessary to adjust with volume of base prior to derivatization.




mL

proteinmg

g

mg

mL

g 510

100

5.0%5.0

3

=×=

mL

mg

mL

mg 15.003.0

5 =×

LLAAApmolesL

ml

mg

gm

mole

pmoles

gm

mole

solutionmL

LAAAmg µµ

/136410

1

10

110

110

1

1

15.033

12

=××××

Lpmoles

Lpmoles µµ

07.01364

1100 =× dilutionx

requiredL

desiredL136

07.0

10 =µ

µ


L

HClmoles

DiluteTotalL

HClmolesHClmoles

DiluteTotalL

L

molesL

L

molesL

11.0

685

)685.7(

685

)1.0

680()5.1

5(

µµ

µµµ

µµµµ

µµµ

=+=×+×

dilutionLdilutionL

stockL

L

ml

mg

gm

mole

nmoles

gmAA

moleAA

stockmL

mg3.310

685

5

10

1

10

110

101.1

1533

9

2=×××××

×× µ

µµ

µ


Example with unknown sample composition, concentration in mg%

36 mg% protein solution in 4 M NaOH

Estimation of Sample amount:


pH Consideration

1. Sample dilution requires 5 µL of 4 M NaOH

2. Sample is diluted with 50 µL of 0.1 M HCl

50 µL 0.1 M HCl = 5 µL 1 M HCl = 1.25 µL 4 M HCl

3. Take the difference of acid and base amounts:

5 µL NaOH – 1.25 µL HCl

4. Sample now contains the equivalent of 3.75 µL 4 M NaOH in excess

Because the resulting base concentration is greater than 0.1 M NaOH, an equal volume of 0.25 M HCl should be added to derivatization cocktail.

mL

proteinmg

mL

mgmg

36.0

100

36%36 ==

mL

mg

mL

mg 011.003.0

36.0 =×

LLAAApmolesL

ml

mg

gm

mole

pmoles

gm

mole

solutionmL

LAAAmg µµ

/10010

1

10

110

110

1

1

011.033

12

=××××

Lpmoles

Lpmoles µµ

1100

1100 =× dilutionx

requiredL

desiredL10

1

10 =µµ


NaOHMdilutionTotalL

NaOHML27.0

55

475.3 =×µµ

Examples C-17

nmoles

Borate volume must be adjusted accordingly. Therefore, 10µL sample and 10µL 0.25 M HCl should be mixed with 60µL Borate buffer before adding the derivatizing reagent




dilutionLdilutionL

stockL

L

ml

mg

gm

mole

nmoles

gmAA

moleAA

stockmL

mg3.310

685

5

10

1

10

110

101.1

1533

9

2=×××××

×× µ

µµ

µ


Index

AAccQ•Tag Ultra Chemistry

columns 1-4description 1-5eluent A 1-4eluent B 1-4

AccQ•Tag Ultra Reagentdescription 1-5–1-8hydrolysis 1-7kit 1-4reconstituting 4-2storing 4-2

amino acid hydrolysate standard 1-4, 3-23, 4-3, 4-4

AMQ 1-6, 1-7analysis

cysteine 6-2AQC, see AccQ•Tag Ultra Reagent

Bbackpressure regulator 2-5

explanation 2-4bandspreading, preventing 2-3binary solvent manager, plumbing 2-6biohazard warning A-4burst warning A-3

Ccalibration standard, preparing

derivatized 4-5external 4-3internal 4-4–4-5

caution symbol A-5chemical hazard warning A-5chromatograms

alkylated cysteine analysis 6-3foods and feeds analysis 6-4

protein and peptide hydrolysate 3-25, 3-26, 3-27

cleaning the system 3-2, 3-3column

connecting eCord 2-12installing

MS detection 2-13optical detection 2-12

compliant solvents B-1compression screw ferrule assembly

2-3connecting

eCord 2-12, 2-14solvent supply 2-15

connections, Ethernet 2-16construction materials B-1container, waste 2-11contamination, preventing B-2cysteine, analyzing 6-2

Dderivatization chemistry 1-5derivatizing

blanks 4-9samples 4-7–4-8

drain tube configuration 2-11drip tray, installing 2-14

EeCord, connecting 2-12, 2-14electrical symbols A-13Eluent A1, preparing 3-5Eluent B1, preparing 3-5Empower software

for use with UPLC AAA 1-3setting up for UPLC AAA Solution

5-3

Index-1

with UPLC AAA Solution 1-1equipment guidelines 5Ethernet connections 2-16exhaust hood 2-11

Ffittings, tightening recommendations

2-3flammable solvents A-3flow cell, TUV detector 2-5FLR detector

description 1-3fume hood 2-11

Hhandling symbols A-14

Iinjector valve cartridge, connecting 2-9installing

columnMS detection 2-13optical detection 2-12

MS drip tray 2-14instrument method, creating

for FLR 3-17for PDA 3-12for TUV 3-7

internal standard, preparing 4-4–4-5

Llow-flow tubing, installing 2-2, 2-5

Mmass spectrometer shock hazard A-4materials of construction B-1mixer outlet, pictured 2-8mobile phase

setting up 3-5, 5-2, 6-4solvents used in preparation of B-3

MS drip tray, installing 2-14

Nneedle wash connections 2-8

PPDA detector

description 1-3pipetting techniques 3-23plumbing

binary solvent manager 2-6connections 2-3sample manager 2-6TUV detector 2-4

preparingeluent A1 3-5eluent B1 3-5samples 4-6–4-8solvents 3-2strong needle wash solvent 3-6system 3-2weak needle wash solvent 3-6

preventing contamination B-2processing samples 3-28

Rreagent, AccQ•Tag Ultra

degraded 7-9description 1-5–1-8hydrolysis 1-7insufficient 7-9–7-10reconsituting 4-2reconstituting 4-2storing 4-2

regulator, backpressure 2-5representative chromatograms

alkylated cysteine analysis 6-3foods and feeds analysis 6-4protein and peptide hydrolysate

3-25, 3-26, 3-27

Index-2

Ssafety advisories A-1sample loop, installing 2-2sample manager, plumbing 2-6sample set method, creating 3-22sample set results, entering for test

3-24samples

derivatizing 4-7–4-8preparing 4-6–4-8processing for analysis 3-28

solventinlet lines, routing 2-7supply, connecting 2-15tray 2-15

solventscompliant B-1exposure of SQ detector

components to B-2preparing 3-2strong needle wash 3-6use in mobile phases B-3weak needle wash 3-6

SQ detectorcompliant solvents B-1materials of construction B-1

standardamino acid hydrolysate 1-4, 3-23,

4-3, 4-4preparing external calibration 4-3preparing internal calibration

4-4–4-5storing

AccQ•Tag Ultra Reagent 4-2calibration standard with an

internal standard 4-5derivatized calibration standards

4-5internal standard stock solution 4-4

I

strong needle wash connection 2-8symbols

caution A-5electrical A-13handling A-14warning A-2

system cleaning 3-3system efficiency 7-13system preparation 3-2system verification sample, preparing

3-23

Ttest methods, creating 3-7tightening recommendations, fittings

2-3troubleshooting

chromatography 7-11–7-14derivatization 7-2–7-10insufficient reagent 7-9–7-10specific peak pairs 7-13

Troubleshooting principles 7-2TUV detector

description 1-3pictured 2-5plumbing 2-4

UUPLC AAA Solution

intended use i-vkit 1-2preparing solvents 3-2preparing the system 3-2sample loop 1-4, 2-2system CD 1-5system components 1-2system overview 1-1with AccQ•Tag Ultra Chemistry

1-1with Empower data software 1-1

Index-3

UPLC Amino Acid Analysis Solutionsee UPLC AAA Solution

Wwarning symbols A-2, A-5waste container 2-11weak needle wash connection 2-8

Index-4

Documents

UPLC Amino Acid Analysis Solution - waters.com · The Waters® ACQUITY UltraPerformance LC® (UPLC®) system provides separation technology that offers higher throughput, sensitivity,