Waters 996 Operator's Manual

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Waters 996

PD A Detector

Operator’s Guide

34 Maple Street

Milford, MA 01757

053021TP, Revision 0

NOTICE

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 manual 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, the use of this manual.

© 1997, 1993 WATERS CORPORAT ION. PRINTE D IN THE UNITED STATES OF AMERICA. ALL RIG H TS RESERVED. THIS BO OK OR PARTS THERE OF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER.

Millennium, PowerStation, and Waters are registered trademarks and busLAC/E is a trademark of Waters Corporation.

All other trademarks are the sole property of their respective owners.

The quality management system of Waters’ chromatography applications software design and manufacturing facility, Milford, Massachusetts, complies with the International Standard ISO 9001 Quality Managemen t and Quality Assurance Stand ards. Waters’ quality management system is periodically audited by the registering body to ensure compliance.

STOP

Attention:

instrument. When you use the instrument, follow generally accepted procedures for quality control and

methods development. If you observe a change in the retention of a particular compound, in the resolution

between two compounds, or in peak shape, immediately take steps to determine the reason for the changes. Until you determine the cause of a change, do not rely upon the results of the separations.

This is a highly sensitive instrument. Read this user's manual before using the

STOP

Caution:

same type and rating.

Attention:

responsible for compliance could void the user’s authority to operate the equipment.

Attention:

A digital device, pursuant to Part 15 of the FCC rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy, and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference, in which case you must correct the interference at your own expense.

Shielded cables must be used with this unit to ensure compliance with Class A FCC limits.

Note:

Level II category pertains to equipment that receives its electrical power from a local level, such as an electrical wall outlet.

For continued protection against fire hazard, replace fuses with those of the

Changes or modifications to this unit not expressly approved by the party

This equipment has been tested and found to comply with the limits for a Class

The Installation Category (Overvoltage Category) for t his instrument is Level II. The

Canadian Emissions Notice

This digital apparatus does not exceed the Class A limits for radio noise emissions from digital apparatus set forth in the Radio Interference Regulations of the Canadian Departme nt of Communications.

Le présent appareil numérique n’émet pas de bruits radioélectriques dépassant les limites applicables aux appareils numériques de la classe A prescrites dans les règlements sur le brouillage radioélectrique édictés par le Ministère des Communications du Canada.

Symbols Used on the Waters 996 Photodiode Array Detector

Direct current

Alternating current

Protective conductor terminal

Frame or chassis terminal

Caution, risk of electric shock (high voltage)

Caution or refer to manual

Caution, hot surface

Ultraviolet light

How to Use This Guide..................................................................... 10

Chapter 1

Installation ....................................................................................... 14

1.1 Installation Site Requirements .............................................. 14

1.2 Po wer Connections............................................................... 15

1.3 Millennium

1.3.1 Connecting the IEEE-488 Cable................................ 16

1.3.2 Setting the IEEE- 48 8 Addre ss............... ..... ..... .... ..... . 18

1.4 Non-IEEE-488 Communication Connections........................ 19

1.4.1 Connecting Analog Output Cables ............................ 19

1.4.2 Connecting Event Cables .......................................... 20

1.5 Fluidic Connections............................................................... 22

Workstation Connections................................. 16

1.6 Startup/Shutdown ................................................................. 24

Chapter 2

Diagnostics and Calibration ............................................................. 27

2.1 Startup Diagnostics............................................................... 27

2.2 User-Initiated Diagnostics..................................................... 30

2.3 PDA Calibration..................................................................... 31

Table of Content s 5

Chapter 3

Maintenance .................................................................................... 33

3.1 Flow Cell Maintenance.......................................................... 33

3.1.1 Flushing the Flow Cell ............................................... 33

3.1.2 Removing the Flow Cell........... .... .............................. 34

3.1.3 Disassembling and Cleanin g the Flow Cell. ..... .... ..... . 36

3.1.4 Installing the Flow Cell Assembly .............................. 38

3.2 Replacing the Lamp.............................................................. 39

3.2.1 Checking Lamp Usage .. ..... ..... .... ..... ......................... 39

3.2.2 Removing the Lamp....... ..... ..... .... ..... ..... ..... ..... .......... 41

3.3 Replacing the Fuses............................................................. 43

Chapter 4

Principles of the 996 PDA Detector Optics ...................................... 44

4.1 996 Detector Optics .............................................................. 44

4.2 Resolving Spectral Data ....................................................... 46

4.3 Measuring Light at the Photodiode ....................................... 47

4.4 Computing Absorbance Data Points..................................... 50

4.4.1 Calculatin g Absor ban ce........... .... ..... ..... .................... 50

4.4.2 Resolution.............................................. .................... 52

4.4.3 Filterin g Data ................. ..... ....................................... 53

Chapter 5

Spectral Contrast Theory ................................................................. 54

5.1 Comparing Absorbance Spectra........................................... 54

5.2 Representing Spectra as Vectors.......................................... 55

5.2.1 Vectors Derived from Two Wavelengths..................... 56

5.2.2 Vectors Derived from Multiple Wavelengths .............. 56

6 Table of Contents

5.3 Spectral Contrast Angles ...................................................... 57

5.4 Nonidealities ......................................................................... 60

5.4.1 Detector Noise............... ..... ..... .... ..... ..... ..... ..... .......... 60

5.4.2 Photom etric Error ..... .................................. ............... 61

5.4.3 Solvent Changes ...... ..... ............................................ 61

5.4.4 Thresho ld Angl e ... ..... ..... ..... ..... .... ..... ......................... 61

Appendix A

Detector S pecif icatio ns.. ..... ..... ...... ..... ..... ...... .. ...... ..... ..... ...... ..... 63

Appendix B

Spare Par ts ..... ...... ..... ..... ...... ..... ... ..... ..... ...... ..... ..... ...... ..... ..... ... 64

Appendix C

Warranty Informati on ..... ..... ..... ...... ..... ... ..... ..... ...... ..... ..... ...... ..... 65

C.1 Limited Express Warranty..................................................... 65

C.2 Shipments, Damages, Claims, and Returns......................... 69

Appendix D

Mobile P ha se A b so rb a nc e..... .. ... ... .. ................................... ........ 70

Index ................................................................................................ 74

Table of Contents 7

List of Figures

1-1 Waters 996 PDA Detector Dimensions..........................................15

1-2 Detector Rear Panel......................................................................16

1-3 Example of IEEE-488 Cable Connections.....................................17

1-4 Locating the IEEE-488 Address Switches.....................................18

1-5 Analog Output Terminals ...............................................................20

1-6 Event Input/Output Terminal Strip..................................................22

1-7 Compression Screw Assembly......................................................24

1-8 996 Detector Indicator Lights.........................................................25

2-1 996 PDA Detector Indicator Lights ................................................28

3-1 Flow Cell Access Door...................................................................35

3-2 Removing the Flow Cell Assembly ................................................35

3-3 Flow Cell and Fluidic Connections Assemblies.............................36

3-4 Disassembling the Flow Cell..........................................................37

3-5 Lamp Access Door........................................................................40

3-6 Lamp Usage Indicator....................................................................40

3-7 Lamp Power Cord and Mounting Screws.......................................42

3-8 Fuse Block.....................................................................................43

4-1 Optics Assembly Light Path...........................................................45

4-2 Benzene Spectrum at 1.2 nm Resolution......................................47

4-3 Photodiodes Discharged by Light..................................................48

4-4 Absorbance as a Function of Concentration..................................51

5-1 Comparing Spectra of Two Compounds........................................55

5-2 Plotting Vectors for Two Spectra....................................................56

5-3 Spectra with a Large Spectral Contrast Angle...............................58

5-4 Spectra with a Small Spectral Contrast Angle...............................59

5-5 Absorbance Spectra of a Compound at Two Concentrations........ 60

5-6 Effects of pH and Solvent Concentration on the Absorbance

Spectrum of p-Aminobenzoic Acid.................................................62

Table of Contents 8

List of Tables

1-1 Site Requirements ........................................................................14

1-2 Event In (Inject Start) Terminal Specifications on TTL

or Switch Closure.................................................................... 21

1-3 Event Out Terminal Specifications on Contact Closure............ 21

2-1 996 Detector Troubleshooting .......................................................28

4-1 Optics Assembly Components................................................. 45

A-1 996 Detector Specifications..................................................... 63

B-1 Spare Parts............................................................................. 64

C-1 Warranty Periods..................................................................... 68

D-1 Mobile Phase Absorbance Measured Against Air or Water...... 70

Table of Contents 9

How to Use This Guide

Purpose of This Guide

The

Waters 996 PDA Detector Operator’s Guide

maintaining, and troubleshooting the Waters optics and the principles of Spectral Contrast used in the Millennium analyzing the data from the PDA detector. Also included is information on vector analysis, mobile phase absorbance, specifications, and the warranty.

Audience

This guide is intended for individuals who need to install, operate, maintain, and troubleshoot the Waters 996 PDA Detector. It is also intended for users who need to understand the Spectral Contrast principles underlying the processi ng of PDA detector data by Millennium

soft wa r e .

Structure of This Guide

The

Waters 996 PDA Detector Operator’s Guide

Each chapter and appendix page is marked with a tab and a footer to help you quickly access information.

The following table describes the mater ial covered in each chapter and appendix of this guide.

describes the procedures for installing,

996 PDA Detector . It also describes detector

is divided into chapters and appendixes.

software for

Chapter/Appendix Description

Chapter 1, Installation De scr ibes how to install and set up the 996 detector. Chapter 2, Diagnostics

and Calibration Chapter 3,

Maintenance Chapter 4, Princi pl e s of

the 996 PDA Detector Optics

Chapter 5, Spectral Contrast Theory

10 How to Use This Guide

Describes how to troubleshoot the 996 detector.

Describes how to replace the flow cell, the lamp, and the fuse.

Explains the principles involved in resolving spectral data, measuring light at the photodiode, verifying wavelengths, and computing absorbance data.

Describes the calculations used for Spectral Contrast.

Chapter/Appendix Description

Appendix A, Detector

Provides the specifications of the Waters 996 PDA detector.

Specifications Appendix B, Spare

Provides a list of recommended and optional spare par ts.

Parts Appendix C, Warranty

Includes warranty and service information.

Information Appendix D, Mobile

Phase Absorbance

Provides a table of absorbances at several wavelengths for common mobile phases.

Installation

The Waters® 996 Photodiode Array (PDA) Detector operates in any standard laboratory environment. The detector requires electrical power, sample and waste fluidic lines, and the Millennium® communication with chart recorders, data integrators, and other instruments that are not compatible with Millennium software control.

1.1 Instal lation Site Requirements

Install the Waters 996 PDA Detector (Figure 1-1) at a site that meets the specifications listed in Table 1-1

Table 1-1 Site Requirements

Ambient temperature 4 to 40° C (39 to 104° F) Relativ e hum idi ty 20 to 80 percent,

Workstation. Optional connections on the detector rear panel allow

Factor Specification

noncondensing

Bench space Width: 11.5 in. (29 cm)

Bench support Capable of supporting

Clearance At least 4 in. (10 cm) on

14 Installation

Depth: 24 in. (61 cm) Height: 8.125 in. (22 cm)

31.5 pounds (14.3 kg)

the back and left sides for ventilation

11.5 in. (29 c m )

WATERS 996

Photodiode Array

8.5 in.

(22 cm)

24 in.

(61 cm)

Figure 1-1 Waters 996 PDA Detector Dimensions

1.2 Power Connections

Ensure that power connections for the 996 PDA Detector are made according to the procedures that follow.

LAMP

Sample Inlet

Sam ple Out let

Drain Line

Operating Voltage

The 996 PDA Detector has a universal input power supply that requires no voltage adjustment. The electrical power requirements for the Waters 996 PDA Detector are:

Voltage range:

•

Frequency range:

•

95 to 240 Vac (±10%)

50 to 60 Hz (±3 Hz)

Fuses

The Waters 996 PDA Detector is shipped with fuses rated for North American operation. If you operate the Waters 996 PDA Detector in another location, install the IEC-rated fuses (supplied in the Waters 996 Detector Startup Kit) in the fuse holder in the rear of the detector (refer to Section 3.3,

Replacing the Fuses).

Power Connections 15

Connecting the Power Cord

Connect one end of the 996 detector power cord to the rear panel power receptacle (Figure 1-2

) and the other end to a power outlet.

Power Cord

Receptacle

TP01452

Figure 1-2 Detector Rear Panel

1.3 Millennium32 Workstation Connections

The 996 detector requires signal connections to the Millennium32 Workstation over the IEEE-488 bus. All detector control and data acquisition communications take place over the IEEE-488 bus.

If an inject start signal is not available over the IEEE-488 bus, you must provide a

Note:

signal at the Event In terminals on the 996 detector rear panel (see Section 1.4.2,

Connecting Event Cables).

1.3.1 Connecting the IEEE-488 Cable

If the 996 detector is to be rack-mounted or stacked on other instruments, use the

Note:

right-angle adaptor included in the Star t up Kit when you make the IEEE-488 connection.

16 Installation

To connect the 996 detector to a Millennium32 Workstation:

1. Con nect one end of the IEEE-488 cable to the IEEE-488 receptacle on the rear panel of the 996 detector. Connect the other end of the cable (stackable connector for daisy-chaining additional instruments) to the IEEE-488 connector on any of the other instruments in your chromatographic system (Figure 1-3

Waters

IEEE-488

Cable

2690

IEEE-488 Cabl e

Wate r s 996

Detector

TP01544

IEEE-488

Connector

Millennium

busLAC/E Card

(on Workstati on)

Wate rs

Separations Module

Figure 1-3 Example of IEEE-488 Cable Connections

The order in which you connect IEEE-488 devices to the busLAC/E card on

Note:

the workstation is not important. For example, you can connect the 2690 separations module before or after the 996 detector.

2. Use another IEEE-488 cable to connect to the stackable connector on the first instrument and the IEEE-488 connector on another instrument.

3. Repeat step 2 for each IEEE-488 i nst rument in your chromatographic system, up to a maximum of 14 IEEE-488 instruments.

Keep in mind cable-length limitations when you set up your system. For a list

Note:

of IEEE-488 interface guidelines, refer to the Millennium

System

Installation/Configuration Guide, Section 2.3.1, Connecting IEEE-488 Devices.

4. Ensure that all IEEE-488 cable screws are fastened finger-tight.

Millennium32 Workstation Connections 17

1.3.2 Setting the IEEE-488 Address

To set the IEEE-488 address for the 996 detector:

1. Use a small screwdriver (or similar device) to set the DIP switches on the detector rear panel (Figure 1-4

) to the IEEE-488 address of the 996 detector. The address

must be a number from 2 to 29 and must be unique within your network. Refer to the

Millennium32 System Installation/Configuration Guide,

Section 2.3.1,

Connecting IEEE-488 Devices, for the correct IEEE-488 DIP switch settings.

IEEE-488 Cable

Connection

12345

IEEE-488

Address

Switches

– OPEN –

IEE 488 ADDRESS

2. To instruct the 996 detector to accept the new IEEE-488 address, power off, then power on the detector (see Section 1.6,

18 Installation

TP01457

Figure 1-4 Locating the IEEE-488 Address Switches

Startup/S hutdown).

1.4 Non-IEEE-488 Communication Connections

Non-IEEE-488 communication connections on the 996 detector include:

Analog Ou t pu ts

STOP

• absorbance-unit analog output channels to integrators, chart recorders, or other components.

Event Inputs and Outputs

• signals to and from other instruments.

You make all non-IEEE-488 communication cable connections to the 996 detector at the rear panel (see Figure 1-2

Attention:

disturbances that may affect the performance of this instrument, do not use cables longer than 9.8 feet (3 meters) when you make connections to the screw-type barrier terminal strips. In addition, ensure that you always connect the shield of each cable to chassis ground at one instrument only.

To me et the regulator y requirem ents of immunity from external electrical

– The 996 detector provides two, unattenuated, 1 volt-per

– The 996 detector sends and receives contact closure

1.4.1 Connecting Analog Output Cables

The values of the analog output signals generated by the 996 detector are specified by parameter valu es set from the Millen niu m “Waters 996 Detector Properties” topic in the

Required Materials

• One small, flat-blade screwdriver

Workstation. For details, refer to the

Millennium32 Online Help

Find tab.

• One electrical insulation stripping tool

• Analog signal cables (from Startup Kit)

Procedure

To connect the Waters 996 detector to a device that receives analog output signals from the 996 detector

1. Pull off the Analog output term inal str ip from the 996 detector rear panel (Figure 1-5

). This step simplifies the following steps.

Non-IEEE-488 Communication Connections 19

Removable An a l og O utput

Term inal Strip

Analog Ou t 1 –

IEE 488 ADDRESS

123456

Analog Out 2

–

Figure 1-5 Analog Output Terminals

2. Insert the bare wires at one end of an analog signal cable into the positive (+) and negative (–) terminals of Analog Out 1 (see Fi gure 1-5 screws to secure the + and – wires.

3. Connect the other end of the analog signal cable to the appropriate analog input terminal on the external device, being sure to maintain negative-to-negative and positive-to-positive continuity.

4. Reinstall the Analog Output strip.

1.4.2 Connecting Event Cables

The 996 detector has four terminal strip connections for contact closure signals:

TP01456

). Tighten the two

• Two input (inject start) signal terminals

• Two output (programmable event table) signal terminals

If an inject start signal is not avail a ble over the IEEE-488 bus, you must provide a signal at an Event In terminal on the 996 detector rear panel. Manual injectors such as the Rheodyne 7725i provide a cable that connects the injector to an Event In terminal on the 996 detector rear panel.

20 Installation

The values of the event output signals generated by the 996 detector are specified by parameter values set from the Millennium 996 Detector Properties” topic in the

Workstation. For details, refer to the “Waters

Millenn iu m32 Online Help

Find tab.

Electrical Specifications

Before you connect an external device to an event input or output terminal, refer to the electrical specifications in Table 1-2

Table 1-2 Event In (Inject Start) Ter m in al Specifications on TTL or Switch Closure

Parameter Specification

Low trigger <1.8 V High trigger >3.0 V Protected to ±30 V Minimum pulse width 30 msec Maximum current 5 mA

Table 1-3 Event Out Ter minal Spec ifications on Contact Closure

Parameter Specification

and Table 1-3.

Maximum power 10 W Maximum current 0.5 A at 20 V Maximum voltage 24 V RMS

Attention:

STOP

electrical connections as outlined in this section.

Required Materials

• Small flat-blade screwdriver

• One electrical insulation stripping tool

• Event signal cables (in Startup Kit)

To avoid damage to the 996 detector electronics, be sure you make the proper

Non-IEEE-488 Communication Connections 21

Making Event Input/Output Connections

To connect the 996 detector to an external event input or output device:

1. Pull off the Event Input/Output terminal strip from the rear panel (Figure 1-6 simplifies the following steps.

Removable Event

Input/Output

Terminal Strip

Event Out 2

Event Out 1

Event

–

In 2

1234567890

–

Event

In 1

IEE 488 ADDRESS

TP01455

Figure 1-6 Event Input/Output Terminal Strip

). This

2. Insert the bare wires at one end of the event signal cable into the positive (+) and negative (–) slots of the appropriate event input or output terminal (see

Figure 1-6

). Tighten the two screws to secure the +and – wires.

3. Connect the other end of the event signal cable to the appropriate event input or event output terminal on the external device.

4. Reinstall the Event Input/Output strip.

1.5 Fluidic Connections

Caution:

handing solvents. Refer to the Material Safety Data Sheets for the solvents in use.

22 Installation

To avoid chemical hazards, always observe safe laboratory practices when

Required Materials

• 5/16-inch open-end wrench

• 0.009-inch (0.23 mm) I.D. stainless steel tubing (in Startup Kit)

• Stainless steel tubing cutter or scribing file

• Pliers, plastic-covered, or with cloth

• Compression screw assembles, three

Procedure

To make fluidic connections to the 996 detector:

1. Measure the lengths of tubing needed to connect:

• The column outlet to the 996 detector inlet.

Be sure that you keep the length of this tubing as short as possible to

Note:

prevent band broadening.

• The 996 detector outlet to a waste collection bottle.

Ensure the length of this tubing is at least 1 to 2 feet (30 to 60 cm) to

Note:

prevent air bubbles from forming in the flow cell.

2. Cut the two lengths of tubing as follows:

• Use a Waters 1/16-inch stainless steel tubing cutter or a file with a cutting edge to scribe the circumference of the tubing at the desired break point.

• Grasp the tubing on both sides of the scribed mark with cloth- or plastic-covered pliers (to prevent marring the surface), then gently work the tubing back and forth until it separates.

• File the tubing ends smooth and straight to minimize dead volume and band broadening.

3. Assemble a compression fitting (as shown in Figure 1-7 column outlet line and at one end of the detector outlet line.

) at both ends of the

Fluidic Connec tions 23

Tubing

Compression

Screw

Ferrule

End Must Be Straight

and Smoot h to Prevent

Dead Volume

TP01139

Figure 1-7 Compression Screw Assembly

4. Bottom one end of the column outlet tubing in the fitting seat of the column outlet, then tighten the compress ion screw about 3/4-turn past finger-tight ( using the 5/16-inch open-end wrench).

5. Bottom the other end of the tubing in the fitting seat of the detector inlet, then tighten the compression screw as in step 4.

6. Bottom one end of the detector outlet tubing with the compression fitting in the fitting seat of the detector outlet, then tighten the compression screw about 3/4-turn past finger-tight. Insert the other end of the tubing in t he waste container.

To avoid damage to the flow cell, avoid pressures approaching the maximum

STOP

Attention:

pressure for the analytical flow cell, 1000 psi (70 kg/cm

1.6 Startup/Shutdown

Follow the procedures in this section to ensure reliable detector performance.

Distan ce D etermi n e d by

the Union or Column Fitting

Startup

To star t up the 996 detecto r :

1. In your instrument method, set the solvent delivery system or pump to deliver 1 mL/min of degassed mobile phase. For details, refer to the “Waters 2690 Separations Module Proper ties” or the “Waters 600 Properties” topic in the

Millennium32 Online Help

24 Installation

Find tab.

Use only thoroughly degassed HPLC-grade solvents. Gas in the mobile

Note:

phase may form bubbl es in the fl ow cell and cause the detector to fail the Ref erence Energy diagnostic.

2. Flush the detector for 10 minutes or until no bubbles appear in the outlet line.

3. Press the 0/1 (Off/On) switch on the front panel of the detector (Figure 1-8

(On) position.

) to the

4. Observe the Lamp and Status indicator lights on the front panel of the detector (Figure 1-8

• If both lights remain illu mi nated , the detector pas sed the internal diagnostic s.

• If either indicator light blinks or is off, refer to the troubleshooting tables in

Chapter 2,

Diagnostics and Calibration.

WATERS 996

Photodiode Array Detector

On/Off Switch

LAMP

Status

Indicator

Lamp

Indicator

TP01460

Figure 1-8 996 Detector Indicator Lights

5. Wait 1 hour for the deuterium lamp to stabilize before you attempt to acquire data at low absorbances.

Startup/Shutdown 25

Shutdown

To shut down the 996 detector:

1. If the mobile phase contains buffers, set the solvent delivery system or pump to deliver 1 mL/min of HPLC-grade water for 10 minutes. Otherwise, set the solvent delivery system or pump to deliver 1 mL/min of degassed methanol for 10 minutes.

2. Press the 0/1 (Off/On) Switch on the front panel of the detector to the 0 (Off) position.

26 Installation

Diagnostics and Calibration

The Waters 996 Photodiode Array Detector automatically runs a series of internal diagnostics upon start up. The indicator lights on the front of the detector and messages at the Millennium (Figure 2-1

Workstation show the results of the start up internal diagnosti cs

If you need to determine the cause of a problem during operation of the detector, you can run the same internal diagnostics from the Millennium information about the performance of the detector is also available through the PDA Calibration window, accessed from QuickSet in the Millennium

If you encounter a problem that you cannot troubleshoot (see Section 2.1,

Diagnostics), contact Waters Technical Service at (800) 252-4752,

customers only

Technical Service Representative, or call Wat ers c orporate headquarters for assistance at 1-508-478-2000 (U.S.).

. Other customers, call your local Waters subsidiary or your local Waters

2.1 Startup Diagnostics

Refer to Table 2-1 to troubleshoot problems encountered during startup diagnostics and during detector operation.

Workstation. Additional

software .

Startup

U.S. and Canadian

Startup Diagnostics 27

WATERS 996

Photodiode Array Detector

Figure 2-1 996 PDA Detector Indicator Lights

Table 2-1 996 Detector Troubleshooting

Symptom Possible Cause Corrective Action

On/Off Switch

LAMP

Status

Indicator

Lamp

Indicator

TP01460

Status light off No power. 1. Check line cord connections.

Status light blinks and lamp light off

28 Diagnostics and Calibration

2. Check outlet for power.

Blown fuse. Replace fuse (see Section 3.3,

Replacing the Fuses).

Detector is st ill

Wait for diagnostics to end. performing diagnostics

Fail ed startup diagnostics.

1. Check that lamp door is secure.

2. Replace lamp. If replacing the lamp fails to correct the problem, contact Waters Technical Service.

Table 2-1 996 Detector Troubleshooting (Continued)

Symptom Possible Cause Corrective Action

Status light blinks and lamp light on

Shutter failure message.

Detector not responding to Millennium Workstation

Fail ed startup diagnostics.

Insufficient energy reaching photodiode array because of air bubble or dirty flow cell can cause shutter diagnostic to fail.

Weak lamp. Replace lamp (see Section 3.2,

Shutter failure. Run the Shutter diagnostic. For

Detector not connected to busLAC/E or to

LAC/E server in the Millennium Workstation

acquisition

Flush the flow cell (see Section 3.1.1,

Flushing the Flow Cell).

Flush the flow cell (see S ec tion 3.1.1,

Flushing the Flow Cell).

To prevent air bubbles from forming, check that there is a 1- to 2-f oot (30- to 60-cm) length of 0.009-inch (0.23-mm) I.D. tubing connected to the detector waste outlet.

Replacing the Lamp).

details, refer to the “PDA Diagnostics Window” topic in the

Online Help

Check IEEE-488 cable connections, tighten connectors.

Find tab.

Millennium32

Incorrect IEEE-488 address.

1. Ensure that the 996 detector IEEE-488 addre ss is unique and within the range 2 to 29 (see the

Millennium

System

Insta l latio n /Confi gurat i o n Guide

2. Rescan the IEEE-488 bus. For details, see the “Scanning the bus/LAC/E Card for Serial Instruments” topi c in the

Millennium32 Online H elp

Startup Diagnostics 29

Find tab.

Table 2-1 996 Detector Troubleshooting (Continued)

Symptom Possible Cause Corrective Action

Change in reference spectrum

Solvent in drain line Leak from flow cell

Mobile phase contains gas or is contaminated.

Air bubbles trapped in flow cell.

gasket.

Leak from flow cell fittings.

2.2 User-Initiated Diagnostics

Prepare fresh mobile phase and degas thoroughly.

Flush the flow cell, or apply slight backpressure on the detector waste outlet.

To prevent air bubbles, check that there is a 1- to 2-foot (30- to 60-cm) length of 0.009-inch (0.23-mm) I.D. tubing connected to the detector waste outlet.

Rebuild flow cell with a new gasket (see Section 3.1.3,

Cleaning the Flow Cell).

Check fittings for overtightening or undertightening , and replace fittings if necessary.

Disassembling and

There are two types of user-initiated PDA diagnostic tests:

Internal Tests

• source of a malfunction. These tests do not require connections to external devices.

Interactive Tests

• communications between the detector and connected external devices. These tests require connections to pump flow and/or test equipment.

You cannot perform diagnostics on a 996 detector while it is acquiring data.

Note:

The system administrator can restrict access to the 996 detector diagnostics by

Note:

disabling user access to Quick Set. For details, refer to the “User Type Properties Dialog Box” topic in the Millennium

30 Diagnostics and Calibration

– Tests run by the instrument firm ware that help you determine the

– Tests that check analog output and event input/output signal

Online Help Find tab.

You can run all user-initiated dia gnos tics from Qui ckSet in the Millen nium32 software. For more information on QuickSet and PDA diagnostics, refer to the “PDA Diagnostics Window” topic in the

Millennium32 Online Help

Find tab.

If you encounter a problem that you cannot troubleshoot (see Section 2.1,

Diagnostics), contact Waters Technical Service at (800) 252-4752,

customers only

Technical Service Representative, or call Wat ers c orporate headquarters for assistance at 1-508-478-2000 (U.S.).

. Other customers, call your local Waters subsidiary or your local Waters

2.3 PDA Calibration

You can adjust, or calibrate, the 996 detector to ensure that wavelength readings are accurate. Recalibrate the 996 detector Internal Diagnostics tests) fails.

You calibrate the 996 detector using the PDA Calibration window, which you access from QuickSet and which allows you to:

• View the effects of exposure time on photodiode saturation for a given wavelength range.

• Verify the wavelength location of the deuterium spectrum Balmer lines (486.0 nm and

656.1 nm).

• Recalibrate to set the 486-nm peak at the proper wavelength.

• Ensure precise data for library matching.

Startup

U.S. and Canadian

only

if the Wavelength Accuracy diagnostic (in the

The system administrator can restrict access to the PDA Calibration window.

Note:

Recalibrating the wavelength requires that spectral libraries be reentered.

Required Materials

• HPLC-grade methanol

• HPLC-grade water

Preparing for Calibration

Ensure that the flow cell is clean before you check calibration. (See Section 3.1.1,

Note:

Flushing th e F low Cell .)

PDA Calibration 31

To prepare for calibration:

1. Set the pump to deliver 1 mL/min of degassed methanol for 10 minutes. If methanol is not miscible with the previous solvent, flush with a miscible sovent before switching to methanol.

2. If you have been using buffers, flush with HPLC-quality water at 1 mL/min for 10 minutes, then switch to methanol for 10 minutes.

Ensure that the solvent is miscible with the previous mobile phase.

Note:

For information on performing calibration, refer to the “PDA Calibration Window” topic in

Millennium32 Online Help

the

Find tab.

32 Diagnostics and Calibration

Maintenance

This chapter covers maintenance of the Waters 996 Photodiode Array Detector flow cell, lamp, and fuse.

Caution:

supply covers. The power supply does not contain user-serviceable component s.

To avoid the po ssibility of elec tric shock, do no t rem ove the 996 de tec t or power

3.1 Fl ow Cell Maintenance

The flow cell requires maintenance when:

• The reference spectrum changes.

• The Lamp diagnostic (in the Millennium lamp status light i s on (see Table 2-1

• The 996 detector causes high backpressure.

Conditions other than a dirty flow cell may cause decreased lamp intensity . F or

Note:

more information, refer to Chapter 2,

Flow cell maintenance consists of:

• Flushing the flow cell

• Removing the flow cell

• Cleaning the flow cell

• Installing the flow cell a ssembly

Diagnostics and Calibration.

PDA Diagnostics window) fails, and the

3.1.1 Flushing the Flow Cell

Required Materials

• HPLC grade water

• HPLC grade methanol If the flow cell requires cleaning, the first cleaning method to try is flushing the flow cell

with solvent.

Flow Cell Maintenance 33

To flush the flow cell:

1. Select a solvent compatible with the samples and mobile phases that you have been using. If you have been using buffers, flush with HPLC-grade water f or 10 minutes at 1 mL/min, then switch to a low-surface-tension solvent such as methanol.

STOP

Attention:

2. Set p ump flow to 1 mL/min, then run the pump 10 minutes.

3. Test the lamp energy by performing the Lamp diagnostic tes t. For details, refer to the “PDA Diagnostics Window” topic in the

If the lamp diagnostic fails and the lamp has not been used more than 1000 hours, disassemble the flow cell and clean the flow cell components using the procedure described in Section 3.1.2,

Ensure that the solvent is miscible with the previous mobile phase.

3.1.2 Removing the Flow Cell

You do not need to shut down the 996 detector to remove and replace the flow cell.

Note:

Required Materials

• 5/16-inch open-end wrench

• Phillips screwdriver

Procedure

To remove the 996 detector flow cell:

1. Set the flow to

2. Power off the solvent delivery system or pump.

0.0

mL/min.

Millennium

Removing the Flow Cell.

Online Help

Find tab.

Caution:

inlet or outlet fluidic lines while there is pressure in the chromatographic system. Always vent your system before disconnecting fluidic lines.

3. Use the 5/16-inch wrench to disconnect the fluidic lines at the front of the detector.

4. Lift up the 996 detector front cover and pull the front cover from the detector chassis.

5. Open the flow cell access door by pulling the black thumbtab, then pull the door gently toward you (Figure 3-1

34 Maintenance

To avoid the possibility of leaking mobile phase, do not disconnect the

Flow C ell Access

Door

Thumbtabs

LAMP STATUS

TP01461

Figure 3-1 Flow Cell Access Door

6. Use th e P h illips s crewd river to lo osen the two thu m b scr ews tha t ho ld t h e f low cell assembly to the optics bench and the thumbscrew that secures the bracket holding the fluidic connections, then detach the bracket (Figure 3-2

Thumbs c rews

Holding Flow

Cell Assembly

and Bracket

Fluidic Connec ti ons

(Inside Bracket)

TP01462

Figure 3-2 Removing the Flow Cell Assembly

Flow Cell Maintenance 35

7. Pull the flow cell assembly and fluidic connection bracket gently toward you to remove it from the detector (Figure 3-3

Flow Cell Body

Lens Holder

Assembly

Fluidic

Connections

Figure 3-3 Flow Cell and Fluidic Connections Assemblies

3.1.3 Disassembling and Cleaning the Flow Cell

The lens surface finish and the alignment of the lenses are critical to the performance of the 996 detector. Be careful not to touch or damage the lenses and the lens holders.

STOP

Attention:

inspecting, cleaning, or replacing parts within the flow cell or when removing or replacing the flow cell within its assembly.

Attention:

inspecting, cleaning, or replacing parts within the flow cell or when removing or replacing parts within the flow cell or when removing or replacing the flow cell within its subassembly.

To prevent contamination, use powder-free gloves when disassembling,

Bracket

TP01463

36 Maintenance

Required Materials

• TORX T10 screwdriver

• Small, flat-blade screwdriver

• Lens tissue or nonparticulating swab

• HPLC-grade methanol

• Belleville spring washer

• Flow cell gas ket

Procedure

To disassemble and clean the flow cell (and lenses):

1. Use the TORX T10 screwdriver to remove the three screws that secure one of the lens holder assemblies (Figure 3-4

Screws

Flow Cell Disk

Lens Assembl y

Flow Cell

Body

Spring

Washer

Gasket

Slot for

Removing

Lens

Assembly

Lens Assembly

Flow Cell Disk

Belleville

Spring

Washer

TP01464

Figure 3-4 Disassembling the Flow Cell

2. Use the small, flat-blade screwdriver to gently pry the lens assembly from the flow cell body at the slots.

Flow Cell Maintenance 37

Attention:

STOP

norma l use, the gasket protects the lens holder from solvents.

3. Use a lens tissue or a nonpar ticulating swab to wipe the lens with methanol.

4. Remove and discard the gasket.

5. Repeat steps 1 through 4 to remove, disassemble, and clean the other lens holder assembly.

6. Use methanol and a nonparticulating swab to clean the flow cell body.

Solvents other than methanol may damage a disassembled flow cell. In

Reassembling the Flow Cell

To reassemble the flow cell (see Figure 3-4):

1. Insert a replacement gasket into one side of the flow cell body.

2. Align the screw holes of the lens assembly with the holes in the flow cell body.

3. Place the new Belleville spring washers (wit h t h e c o ncave side facing out) onto the lens assembly.

4. Place the flow cell disk over the lens assembly.

5. Insert the three screws using the TORX T10 driver to gradually tighten each screw, alternating between the screws in a clockwise pattern. Tighten until the screws meet the flow cell disk, then tighten each screw 1/4-turn. If a torque screwdriver is available, tighten the screws to 16 in-oz (0.113 N-m).

STOP

Attention:

6. Repeat steps 1 through 5 to reassemble the other side of the flow cell.

Be careful not to overtighten the screws.

3.1.4 Installing the Flow Cell Assembly

Attention:

STOP

38 Maintenance

operation. Be careful not to damage the flow cell body.

To install the flow cell assembly:

1. While you hold the flow cell assembly in a vertical orientation (see Figure 3-3

2. Gently push the front of the assembly until it seats on the front alignment pins.

3. Hand-tighten the thumbscrews.

4. Secure the fluidic connection bracket.

5. Reconnect the fluidic lines.

The alignment of the flow cell in the optics bench is critical to detector

the assembly into the optics bench. Note that the flow cell is self-aligning and uses the guide pins on the optics bench.

), insert

6. Replace the front cover.

7. Flush the flow cell (refer to Section 3.1.1,

3.2 Replacing the Lamp

Replace the lamp in the 996 detector when either of the following conditions exists:

• Intensity is low enough that sensitivity is not sufficient for your method.

• The sampling rate requires an exposure time shorter than the minimum exposure time you can set with the current lamp.

Before you replace the detector lamp, check the lamp usage indicator located to the front of the lamp retainer, as described in Section 3.2.1,

3.2.1 Checking Lamp Usage

The Waters 996 detector lamp is designed to provide adequate energy for more than 1000 hours of operation. You can monitor lamp usage by checking the lamp usage indicator, a mercury column with a scale of 0 to 10, where 10 represents 1000 hours. As the lamp ages, the bubble in the mercury column moves toward the 10.

Caution:

disconnect the power cord before you begin this procedure. Note that the lamp and housing are extremely hot. To avoid the possibility of contacting hot surfaces, allow the lamp to cool for 15 minutes before you handle the lamp assembly or surfaces close to the lamp.

To avoid electrical hazards and exposure to UV light, turn off the power and

Flushing the Flow Cell).

Checking Lamp Usage.

Procedure

To inspect the lamp usage indicator:

1. Power off the 996 detector, remove the pow er cord, and allow the lamp to cool for at least 15 minutes.

2. Lift up the front panel cover and pull it away from the chassis.

3. Open the lamp access door by pulling t he t humbtab, then pulling the door toward you (Figure 3-5

Replacing the Lamp 39

Thumbtabs

LAMP

STATUS

Figure 3-5 Lamp Access Door

Lamp

Access Door

TP01461

4. Examine the lamp usage indicator (Figure 3-6

Figure 3-6 Lamp Usage Indicator

Lamp Us age

Indicator

TP01466

40 Maintenance

If lamp intensity is low , but the lamp has not been used for 1000 hours, you

Note:

may be able to increase lamp intensity by cleaning the flow cell (see Section 3.1,

Flow Cell Maintenance).

Absorbance by the mobile phase also affects the apparent lamp intensity. For example, acetonitrile is more transparent than methanol at wavelengths under 220 nm.

3.2.2 Removing the Lamp

Attention:

STOP

lamp glass damages the lamp and reduces life expectancy.

Attention:

TOP

the lamp.

Required Materials

TORX T20 screwdriver

Caution:

the 99 6 d etect or and disc onne ct the power cord.

Procedure

To replace the lamp in the 996 detector:

1. Power off the 996 detector, remove the power cord, and allow the lamp to cool for at least 15 minutes.

Caution:

15 minutes after powering off the detector before you handle the lamp.

2. Lift up the front panel cover and pull it away from the chassis.

3. Open the lamp access door by pulling the thumbtab, then pull the door toward you (see Figure 3-5

Do not touch the lamp glass while unpacking or inserting the lamp. Touching

To prevent contamination, use powder-free gloves when removing or replacing

To avoid electrical hazards when you perform the following procedure, power off

To avoid the possibility of contacting hot surfaces, wait at least

4. Disconnect the lamp power connector (Figure 3-7

Replacing the Lamp 41

Lamp Alignment Notch

Lamp Mounting

Screws

Figure 3-7 Lamp Power Cord and Mounting Screws

5. Use the TORX T20 screwdriver to unscrew the two T20 lamp mounting screws.

6. Grip the metal base of the lamp, pull the lamp out, and set it aside.

7. Carefully unpack the replacement lamp.

8. While wearing powder-free gloves and holding the lamp by its base, orient the lamp so that the notch in the base aligns with the positioning pin in the optics bench.

Lamp Power

Connector

TP01467

9. Insert the lamp and secure it with the two T20 screws. Make sure that the lamp base is flush against the lamp housing.

10. Reconnect the lamp power connector (see Figure 3-7

11. Close the lamp door and secure it with the thumbtab.

12. Install the front panel cover.

13. Reconnect the power cord and power on the 996 detector.

42 Maintenance

3.3 Replacing the Fuses

Replace the fuses under the conditions indicated in the troubleshooting table (see Section

2.1, Start up Diagnostics). The 996 detector requires two 4 A, 250 V fuses (5 mm ×20

mm).

Caution:

cord before you perform the following procedure.

To avoid electrical hazards, power off the 996 detector and disconnect the power

Procedure

To replace the two fuses in the 996 detector:

1. Power off the 996 detector and remove the power cord.

2. Locate the fuse block above the power cord plug (Figure 3-8

Squeeze Side C li p s

to Access Fuses

Figure 3-8 Fuse Block

3. Squeeze the two side clips on the fuse block while you pull out the block.

4. Remove the fuses from the block, then install the new fuses.

) on the rear panel.

5. Orient the fuse block with the small tab pointing down, then push in the block until the side clips engage.

6. Connect the power cord, then power on the 996 detector.

Replacing the Fuses 43

4 Pr incipl es of the 996 PDA Detector Optics

To use the Millennium32 PDA software effectively, you must be familiar with the principles

of operation of the optics and electronics of the Waters 996 PDA Detector.

4.1 996 Detector Optics

The 996 detector is an ultraviolet/visible light (UV/Vis) spectrophotometer with:

• 512 diodes

• Optical resolution of 1.2 nm per diode

• Operating wavelength range from 190 nm to 800 nm

The light path through the optics assembly of the 996 detector is shown in Figure 4-1

44 Principles of the 996 PDA Detector Optics

Photodiode

Array

50-mm

Aperture

Assembly

Spectrographic

Mirror and Mask

Shutter

Flow Cell

Assembly

Lamp and

Lamp Optics

Figure 4-1 Optics Assembly Light Path

Grating

Beamsplitter

Assembly

Reference

Diode

Table 4-1

describes the optics assembly components in the 996 detector.

Table 4-1 Optics Assembly Components

Component Function

Lamp and lamp optics

Beamsplitter and reference diode

Focuses light from the deuterium source lamp through a beamsplitter to the flow cell.

Reflects par t of the light back to a reference diode, which measures the intensity of the light emitted by the lamp. The detector uses this measurement to keep the lamp output constant.

996 Detector Optics 45

Table 4-1 Optics Assembly Components (Continued)

Component Function

Flow cell assembly

Spectrograph mirror and mask

Aperture Controls wavelength resolution and intensity of light striking the

Shutter assembly Prevents light from reaching the photodiode array except during

Grating Disperses the light into bands of wavelengths and focuses those

Second-order filter

Houses the segment of the flow path (containing eluent and sample) through which the polychromatic light beam passes. This arrangement of optical components, with the flow cell positioned between the lamp and the grating, is commonly called reversed optics.

The mirror focuses light transmitted through the flow cell onto the aperture at the entrance to the spectrographic portion of the optics. The mirror mask defines the beam of light focused on the spectrograph mirror.

photodiodes. The width of the aperture is 50 µm.

sampling and calibration. For details on the dark current, see

Section 4.4.1,

wavelength bands onto the plane of the photodiode array. Reduces the contribution of second-order reflection of UV light

(less than 350 nm) to the light intensity observed at visible wavelengths (greater than 350 nm).

Calculating Absorbance.

Photodiode array An array of 512 diodes arranged linearly. The diode width and

spacing provide a single wavelength resolution of 1.2 nm.

4.2 Resolving Spectral Data

The ability to distinguish similar spectra depends on photodiode spacing and the

bandwidth of the light striking the photodiode. The bandwidth of the light striking the

photodiodes depends on the aperture width.

The aperture width determines:

• Attainable wavelength bandwidth at the photodiode array

• Intensity of the light reaching the photodiode array (optical throughput)

46 Principles of the 996 PDA Detector Optics

The aperture creates a narrow beam that reflects from the grating to the photodiode array.

The wavelength that strikes a particular diode depends on the angle of reflection from the

grating.

Figure 4-2

using the standard 50-µm aper ture. In this spectrum, the wavelength resolution is

sufficient to resolve five principal benzene absorption peaks.

shows an absorbance spectrum of benzene obtained from the 996 detector

Absorbance

Figure 4-2 Benzene Spectrum at 1.2 nm Resolution

4.3 Measuring Light at the Photodiode

The Waters 996 Photodiode Array Detector measures the amount of light striking the

photodiode array to determine the absorbance of the sample in the flow cell.

The array consists of 512 photodiodes arranged in a row. Each photodi ode acts as a

capacitor by holding a fixed amount of charge.

Light striking a photodiode discharges the diode (Figure 4-3

discharge depends on the amount of light striking the photodiode.

Measuring Light at the Photodiode 47

). The magnitude of the

Light from gra ti ng

dispersed in to 1.2 nm

wav elength beams

continuously

discharges diodes.

Grating

Sample in flow cell absorbs at spe cific

wav elengths

100%

Charge

0 %

Deuterium Lamp

Flow

Cell

Mirror

Figure 4-3 Photodiodes Discharged by Light

The 996 detector measures the amount of current required to recharge each photodiode.

The current is proportional to the amount of light transmitted through the flow cell over the

interval specified by the diode exposure time.

Exposure Time

The 996 detector recharges each diode and reads the recharging current one diode at a

time. The interval between two readings of an individual diode is the exposure time. The

996 detector requires 11 msec to sequentially read all of the diodes in the array. The

minimum exposure time is 11 msec. You can set exposure time from 11 to 500 msec.

For example, if an exposure time is set to 50 milliseconds, the Waters 996 detector:

1. Recharges diode 1 and reads the current required to recharge diode 1.

2. Recharges diode 2 and reads the current required to recharge diode 2.

3. Sequentially recharges and reads the current required to recharge all the remaining 510 photodiodes.

48 Principles of the 996 PDA Detector Optics

4. After all of the diodes have been recharged and read (11 msec), the detector waits 39 msec before beginning the recharge-and-reading sequence with diode 1.

You set the exposure time parameter in the General tab of the 996 PDA Instrument Method Editor. You can specify either Auto Exposure or Exposure Time. For details, refer to the “Waters 996 PDA Detector Properties” topic in the tab.

Millennium32 Online Help

Using the Auto Exposure Parameter

The Auto Exposure time parameter allows the 996 detector optics to calculate the optimum exposure time needed to recharge the diodes based on lamp energy, lamp spectrum, mobile phase absorbance, and the chosen wavelength range. To minimize detector noise, Auto Exposure adjusts the exposure time to 80 to 90 percent of full scale.

The Auto Exposure time setting ensures that the photodiodes are:

• Not saturating due to overexposure

• Operating above the range of normal, dark current discharge

With auto exposure enabled, the 996 detector:

• Calculates exposure time at the start of a run based on maximum light intensity within the wavelength range

• Limits the exposure so that no diode within the given wavelength range is discharged more than 80%

Find

• Provides proper settings for signal-to-noise and dynamic range for each run

The Auto Exposure time setting may not support certain sampling rates or wavelength ranges required for your analysis. If this is the case, you can set the exposure time manually to adjust the exposure time from experiment to experiment.

Using the Exposure Time Parameter

The Exposure Time parameter enables you to manually set the length of time the photodiodes are exposed to light before they are read. The supported range is 11 to 500 msec.

Be aware that increasing the Exposure Time parameter has the potential to saturate the photodiodes. A longer exposure time may cause the 996 detector to lose the signal at certain wavelengths because of diode saturation. When specifying the Exposure Time, select a value that provides settings for an optimum signal-to-noise ratio over the wavelength range of your analysis (see “

Optimizing the Signal-to-Noise Ratio” below).

Measuring Light at the Photodiode 49

Optimizing the Signal-to-Noise Ratio

To optimize signal-to-noise ratios, choose an acquisition wavelength range that includes only the wavelengths of interest and over which the mobile phase absorbs minimally (see

Appendix D,

Mobile Phase Absorbance).

4.4 Computing Absorbance Data Points

The 996 detector calculates absorbance values before transmitting the data to the Millenn iu m

database. To calculate absorbance, the 996 detector:

• Computes the absorbance at each diode using the dark current and reference spectrum (see Section 4.4.1,

• Aver ages the absorbances at a particular wavelength as specified in the spectra per second sample rate and repor ts the average as a single data point (see Section

4.4.2, Resolution).

• Can apply a filter that acts like an analog filter (see Section 4.4.3,

4.4.1 Calculating Absorbance

The 996 detector computes absorbance by subtracting the dark current and reference spectrum from the acquired spectrum . Absorbanc e is based on the pr inciple s of Beer's Law.

Beer’s Law

The relationship between the quantity of light of a particular wavelength arriving at the photodiode and the concentration of the sample passing through the flow cell is described by the Beer-Lambert Law (commonly called Beer’s Law). Beer’ s Law i s ex pressed as

Where:

Calculating Absorbance).

Filtering Data).

= absorbance

= molar absorptivity

= path length (1.0 cm in the 996 detector normal flow cell)

= molar concentration

50 Principles of the 996 PDA Detector Optics

Beer’s Law applies only to well-equilibrated dilute solutions. It assumes that the refractive index of the sample remains constant, that the light is monochromatic, and that no stray light reaches the detector element. As concentration increases, the chemical and instrumental requirements of Beer's law may be violated, resulting in a deviation from (absorbance versus concentration) linearity (Figure 4-4 can reduce the linear range by the amounts shown in Appendix D,

Absorbance.

Absorbance

). The absorbance of mobile phase

Mobile Phase

Ideal

Actual

Working Ran ge

AbsorbanceBackground

Concentrati on

Figure 4-4 Absorbance as a Function of Concentration

Dark Current

Photodiodes lose charge over time even when they are not exposed to light. The amount of charge lost is called

At the start of a chromatographic run, the 996 detecto r closes the shutter to take a dark current reading for each diode. The shutter closes after the exposure time is calculated and stays closed for the same interval as the exposure time.

The detector subtracts the dark current values from the current values recorded during absorbance measurements for both the sample and the reference spectra.

dark current

Computing Absorbance Data Points 51

Refer ence Spectrum

Immediately after the dark current measurement and before any components are eluted, the 996 detector records a reference spectrum. The reference spectrum is a measure of lamp intensity and mobile phase absorbance over the interval specified in the exposure time taken with the shutter open.

For best results, the reference spectrum should be representative of the initial

Note:

mobile phase.

For extremely long exposure times, the dark current and reference spectrum

Note:

readings may take several minutes to finish.

Absorbance

The 996 detector calculates the absorbance for each diode at the end of each exposure time using the following equation:

Sn Dn

–()

Absorbance

where:

= Signal obtained during sample analysis

= Signal obtained during the dark test

= Signal obtained from the reference spectrum

= Diode number

log=

-------------------------

Rn Dn

–()

4.4.2 Resolution

The data reported by the 996 detector to the Millennium32 database can be the average of a number of data points. After calculating absorbance, the detector averages absorbance values based on:

• Spectral resolution

• Sample rate

Averaging Spectral Data Based on Resolution

Spectral resolution (or bandwidth) is the wavelength interval (in nanometers) between data points in an acquired spectrum. The minimal resolution of the 996 detector is 1.2 nm. Spectral resolution with the 996 detector is always a multiple of 1.2 nm. For example, the 996 detector averages three diodes for each reported wavelength when the spectral resolution is set in the Millennium

52 Principles of the 996 PDA Detector Optics

software to 3.6 nm.

Averaging Chromatographic Data Based On Sample Rate

Sample rate is the number of data points per second reported to the Millennium32 database. The number of times the photodiodes are read during the sample rate interval is dependent on the exposure time.

For ex ample, if e xposure time is 25 msec, and sample rate is 1 second, then readings per data point are

1000 msec

------------------------- 40= 25 msec

The readings are averaged and reported as a single data point.

Combining Spectral Resolution and Sample Rate

Spectral resolution and sample rate have opposite effects on noise and spectral detail. Increasing the value of the spectral resolution parameter and decreasing the number of spectra per second decrease the size of the data file.

The data storage rate is based on wavelength range, spectral resolution, and

Note:

sample rate, which are set in the General tab of the 996 PDA Instrument Method Editor. For details, ref er to the “Waters 996 PDA Detector Properties” topic in the Millennium

Online Help Find tab.

4.4.3 Filtering Data

Use the Channel 1 tab of the 996 PDA Instrument Method Editor (for details, refer to the “Waters 996 Detector Properties” topic in the an optional noise filter (the Filter Response parameter) to the dat a sent to the Mi llennium software database. A noise filter of 1 second is the default value, which provides a good signal-to-noise ratio for most chromatographic separations.

Note the following with regard to filtering data:

• The noise filter is similar in function to an analog RC filter.

• The filter calculates a data point that is a modified rolling average for a wavelength over a number of readings.

• The filter values are comparable to the effects of a 1-, 2-, or 3-second RC filter.

Millennium32 Online Help

Computing Absorbance Data Points 53

Find tab) to apply

Spectral Contrast Theory

This chapter explains the theory behind the Spectral Contrast technique, which is used to compare UV/Vis absorbance spectra collected by the 996 detector. Spectral Contrast makes use of the fact that different compounds have differently shaped absorbance spectra. This chapter describes how Spectral Contrast represents absorbance spectra as vectors. When applied to the UV/Vis absorbance data collected by the 996 detector, the Spectral Contrast technique determines whether differences between spectra are due to the presence of multiple compounds in the same peaks (coelution) or due to nonideal conditions such as noise, photometric error, or solvent effects.

5.1 Comparing Absorbance Spec tra

The shape of an absorbance spectrum is determined by the relative absorbance at different wavelengths. The shape of a compound’s absorbance spectrum is a characteristic of that compound at the solvent and pH conditions under which the absorbance spectrum is measured.

Figure 5-1

absorbance at 245 nm to the absorbance at 257 nm is approximately 2.2 for compound A and 0.7 for compound B.

The absorbance ratios of two wavele ngth pairs is a limited spectral comparison. For more information, you need to compare the absorbance ratios of multiple wavelength pairs.

54 Spectral Contrast Theory

shows the absorbance spectra for the two compounds, A and B. The ratio of the

Compound A:

245 nm

257 nm

Compound A

Compound B:

Compound B

Figure 5-1 Comparing Spectra of Two Compounds

5.2 Representing Spectra as Vectors

245

-------------- 2.2= Ab

257

245

-------------- 0.7= Ab

257

The Spectral Contrast technique uses vectors to quantify differences in the shapes of spectra. Spectral Contrast converts baseline-corrected spectra to vectors and then compares the vectors. Spectral vectors have two properties:

Length

•

Direction

•

– Proportiona l to analyte concentration.

– Determined by the relative absorbance of the analyte at all wavelengths

(its absorbance spectrum). Direction is independent of concentration.

Vector direction contributes to the identification of a compound, since the direction is a function of the absorbance spectrum of the compound. The ability of spectral vectors to differentiate compounds depends on the resolution of spectral features. As both wavelength range and spectral resolution increase, the precision of a spectral vector for the resultant spectrum increases. A vector derived from the Waters 996 PDA Detector can include absorbances in any range between 190 nm and 800 nm with a spectral resolution of 1.2 nm.

Representing Spectra as Vectors 55

5.2.1 Vectors Derived from Two Wavelengths

The Spectral Contrast algorithm uses vectors to characterize spectra (Figure 5-2). To understand the vector principle, consider two vectors (Figure 5-2 depicted in Figure 5-1

AU at 257 nm

AU at 245 nm

Figure 5-2 Plotting Vectors for Two Spectra

) based on the spectra

The axes in Figure 5-2 the absorbance ratio shown in Figure 5-1 intersection of the absorbance values (for Compound A) at the two wavelengths represented by each axis. The other vector is similarly derived for the spectrum of Compound B.

The vector for Compound B points in a direction different from that of the vector for Compound A. The difference in direction, which reflects the difference in the absorbance ratios of the two compounds at wavelengths 245 nm and 257 nm, is called the Spectral Contrast Angle. A Spectral Contrast angle (e.g., θ in Figure 5-2 indicates a shape difference between spectra (see Section 5.3, The length of the vector is proportional to the concentration.

are in absorbance units at the two wavelengths used to calculate

. The head of the vector for Compound A is at the

5.2.2 Vectors Derived from Multiple W av elengths

When absorbance ratios are limited to two wavelengths, the chance that two different spectra will have the same absorbance ratio is much greater than if comparison is made using absorbance ratios at many wavelengths. Therefore, the Spectral Contrast technique uses absorbances from multiple wavelengths to form a vector in an

space, where

56 Spectral Contrast Theory

is the number of wavelengths from the spectrum.

)

greater than zero

Spectral Contrast Angles).

-dimensional vector

To compare two spectra, the Spectral Contrast technique forms a vector for each spectrum

in an compute the angle between the two vectors.

Just as in the 2-wavelength comparison, a Spectral Contrast angle of zero in

match. Conversely, if any comparison of ratios does not match, then the corresponding vectors point in different directions.

-dimensional space. The two spectral vectors are compared mathematically to

-dimensional space means that all ratios of absorbances at corresponding wavelengths

5.3 Spectral Contrast Angles

Spectra that have the same shape have vectors that point in the same direction. Spectra that have different shapes have vectors that point in different directions. The angle between the two vectors of any two spectra, the Spectral Contrast angle, quantifies the magnitude of the shape difference between the spectra. The the difference in direction between the spectral vectors of two spectra.

A Spectral Contrast angle can vary from 0 degrees to 90 degrees. A Spectral Contrast angle near 0 degrees indicates little shape difference between the compared spectra. Matching a spectrum to itself produces a Spectral Contrast angle of exactly 0 degrees. The maximum Spectral Contrast angle, 90 degrees, indicates that the two spectra do not overlap at any wavelength.

Spectral Co ntrast an gle

To illustrate the relationship between Spectral Contrast angle and spectral shape differences, consider the pairs of spectra shown in Figure 5-3

, Figure 5-4, and Figure 5-5.

Spectra with Different Shapes

In Figure 5-3, the absorbance spectra of two compounds, A and B, are distinctly different, and therefore, have a large Spectral Contrast angle (62.3°).

Spectral Contrast Angles 57

Compound B

Normalized Absorbance

Figure 5-3 Spectra with a Large Spectral Contrast Angle

Spectra with Similar Shapes

Spectral Contrast Angle: 62.3°

Compound A

Wavelength (nm)

In Figure 5-4, the absorbance spec tra of two compounds, A and B, are similar, and therefore, have a small Spectral Contrast angle (3.0°).

58 Spectral Contrast Theory

Spectral Cont rast Angle: 3.0

Compound A

Compound B

Normalized Absorba nce

Wavelength (nm)

Figure 5-4 Spectra with a Small Spectral Contrast Angle

Differences Between Spectra of the Same Compound

Small but significant differences between absorbance spectra can occur because of factors other than those due to the absorbance properties of different compounds. For

same

example, multiple spectra of the of detector noise, photometric error, high sample concentration, or variations in solvent conditions. The spectra in Figure 5-5 the shape of an absorbance spectrum of one compound. This effect is most likely to occur at low concentrations where the signal-to-noise ratio is lo w . Note that the Spectral Contrast angle between these absorbance spectra of the same compound is 3.4°.

compound may exhibit slight differences because

, for example, show how instrument noise can affect

Spectral Contrast Angles 59

Normalized Spectra of a Compound at Different

Concentratio ns

Nor m alized Ab so r b ance

Figure 5-5 Absorbance Spectra of a Compound at Two Concentrations

5.4 Nonidealities

Shape differences between absorbance spectra can be caused by one or more of the following nonideal phenomena:

• Detector noise

• Photometric error caused by high sample concentration

Spectral Contrast Angle: 3.4

Wavelength (nm)

• V ariation in solvent composition

These sources of spectral variation can cause chemically pure, baseline-resolved peaks to exhibit a small level of spectral inhomogeneity. You c an assess the significance of spectral inhomogeneity by comparing a Spectral Contrast angle to a Section 5.4.4).

5.4.1 Detector Noise

Statistical and thermal variations add electrical noise to the absorbance measurem ent s made by the 996 detector. The noise manifests itself as fluctuations in the baseline, known

baseline noise

as thermal variations can be predicted from the instrument noise in the baseline region of a chromatogram.

60 Spectral Contrast Theory

. The magnitude of any absorbance differences caused by statistical and

Threshold angle

(see

5.4.2 Photometric Error

At high absorbances (generally greater than 1 AU), a combination of effects can produce slight departures (about 1%) from Beer’s Law due to photometric error. Although photometric errors at this level may have a negligible effect on quantitation, they can be a significant source of spectral inhomogeneity . To minimize the effects of photometric error for all Spectral Contrast operations, the maximum spectral absorbance of a compound should be less than 1 AU. Keep in mind that the absorbance of the mobile phase reduces the working linear dynamic range by the amount of mobile phase absorbance at each wavel ength. For e xamples of mobile phase absorbance, see Appendix D,

Absorbance.

For more information about the effects of the photometric error curve, refer to

Note:

Principles of Instrumental Analysis, third edition, by Douglas A. Skoog, Saunders College Publishing,1985, pp 168–172.

5.4.3 Solvent Changes

As long as solvent concentration and composition do not change (isocratic operation), the background absorbance, if any, by the solvent remains constant. A change, however, in solvent pH or composition, such as occurs in gradient operation, can affect the intrinsic spectral shape of a compound, as shown in Figure 5-6

5.4.4 Threshold Angle

Mobile Phase

In addition to computing Spectral Contrast angles, the Spectral Contrast technique also computes a Threshold angle. The Threshold angle is the maximum Spectral Contrast angle between spectra that can be attributed to nonideal phenomena.

Comparison of a Spectral Contrast angle to its Threshold angle can assist in determining if the shape difference between spectra is genuine, that is, generated by mixtures that are dissimilar. In general, a Spectral Contrast angle less than its Threshold angle indicates that shape differences can be attributed to nonideal phenomena alone, and that there is no evidence for genuine differences between the spectra. A Spectral Contrast angle greater than its Threshold angle indicates that the shape differences are due to genuine differences between the spectra.

Nonidealities 61

Effect of pH

-Aminobenzoic Acid

p - Aminobenzoic Acid

pH 3.1 pH 5.1 p H 6.9

COOH

pH 6.9

COO–

COOH

Absorbance

pH 3.1

200 220 240 260 280 300 320 340

Effect of Acetonitrile Concentration

Effect of Acetonitrile Concentration p - Aminobenzoic Acid

-Aminobenzoic Acid

2–10% Acetonitrile

Absorbance

Wa vel ength

pH 5.1

COOH

200 220 240 260 280 300 320 340

Figure 5-6 Effects of pH and Solvent Concentration

on the Absorbance Spectrum of

62 Spectral Contrast Theory

Wavelength (n m )

-Aminobenzoic Acid

Append ix A Detector Specifications

Table A-1 lists the Waters 996 PDA Detector specifications.

Table A-1 996 Detector Specifications

Item Specification

Dimensions 11.5 × 22 inches (29 × 56 cm) Weight 31 lbs (14.3 kg) Wavelength range 190 to 800 nm Wavelength accuracy ±1 nm Linearity range

Spectral resolution 1.2 nm Baseline noise ±1.5 × 10

Drift 1 × 10

5% at 2.0 AU, propylparaben, at 258 nm

–5

AU peak-to-peak, dry, at 254 nm

–3

AU/hour

at 254 nm (after warmup)

Flow cells Standard Semi-preparative Microbore Inert

Per ASTM 685-79

Pathlength (mm): Tubing (I.D . )

10 0.009 in

3 0.040 in 3 0.005 in

10 0.010 in

Appendix A 63

Append ix B Spare Parts

The s par e parts lis ted in Table B-1 are those parts recommended for customer installation. Damage incurred by performing unauthorized work on your 996 detector may invalidate certain warranties.

Table B-1 Spare Parts

Item Part Number

Flow cell, standard WAT057919 Flow cell, semi-preparative WAT057463 Flow cell, microbore WAT057462 Flow cell, inert WAT057461 Gasket, flow cell ( 2 ) WAT057924 Belleville wash er (2) WAT05 7925 Lens mount and lens (2) WAT057923 Semi-prep lens kit WAT057968 Deuterium lamp WAT057760 Fuse, 4 A (5 × 20 mm) WAT057337 Waters Erbium Perchlorat e Wavelength

Accuracy Solution

WAT042885

Waters Absorbance Detector Linearity Solution

Waters 996 PDA Detector Qualification Workbooks

WAT042881

WA T509-01

Appendix B 64

Append ix C Warranty Information

This appendix includes information on:

• Limited express warranty

• Shipments, damages, claims, and returns

C.1 Limited Express Warranty

Waters® Corporation provides this limited express warranty (the Warranty) to protect customers from nonconformity in the product workmanship and materials. The Warranty covers all new products manufactured by Waters.

Waters warrants that all products that it sells are of good quality and workmanship. The products are fit for their intended purpose(s) when used strictly in accordance with Waters’ instructions for use during the applicable warranty period.

Limited Warranty

Waters Corporation warrants that the Waters 996 PDA Detector is a Class I medical device under 21 CFR 862.2260, as now in effect, and is for general purpose use and is not for use in clinical diagnostic procedures, and that during the Warranty period, the performance of all components of the Waters 996 PDA Detector [other than Third-Party Components (non-Waters named)], will not deviate materially from the Specifications for such detectors. Warranties, if any, that may be applicable to Third-Party Components shall be provided by the respective manufacturers or suppliers of such Third-Party Components, and Waters Corporation shall use reasonable efforts to assist Customer in securing the benefits of any such warranties.

Exclusions

The foregoing warranty does not apply to any material deviation from the Specifications by any component of the Waters 996 PDA Detector that results from (a) use of the Waters 996 PDA Detector for any purpose other than general purpose use and sp ecifically excluding use of the Waters 996 PDA Detector in clinical diagnostic procedures, or use of the Waters 996 PDA Detector for investigational use with or without confirmation of diagnosis by another, medically established diagnostic product or procedure, (b) errors or defects in any

Appendix C 65

Third-Party Component, (c) modification of the Waters 996 PDA Detector by anyone other than Waters Corporation, (d) failure by Customer to install any Standard Enhancement in accordance with an update procedure, release of firmware or any operating system release, (e) any willful or negligent action or omission of Customer, (f) any misuse or incorrect use of the Waters 996 PDA Detector, (g) any malfunction of any information system or instrument with which the Waters 996 PDA Detector may be connected, or (h) failure to establish or maintain the operating environment for the W aters 996 PDA Detector in accordance with the operator’s manual.

Exclusive Remedy

In the event of any failure of the Waters 996 PDA Detector to perform, in any material respect, in accordance with the warranty set forth herein, the only liability of Waters Corporation to Customer, and Customer’s sole and exclusive remedy, shall be the use, by Waters Corporation, of commercially reasonable efforts to correct for such deviations, in Waters Corporation’s sole discretion, replacement of the purchased Waters 996 PDA Detector, or refund of all amounts theretofore paid by Customer to Waters Corporation for the Waters 996 PDA Detector.

Disclaimers

THE LIMITED W ARRANTY SET FO RTH HEREIN IS EXCLUSIVE AND IN LIEU OF, AND CUST OMER HER EBY W AI VES , ALL O THER R EPRES ENTATIONS, W ARRANT IES AN D GU AR ANTEES, EXPRESS OR IMPLIED, IN C LUDING WITHOUT LIMITATION ANY IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS OF THE WATERS 996 PDA DETECTOR FOR A PARTICULAR PURPOSE, IN CL U D ING FITNESS FOR USE IN CLINICAL DIAGNOSTIC PROCEDURES OR FOR INVESTIGATIONAL USE WITH OR WITHOUT CONFIRMATION OF DIAGNOSIS BY ANOTHER MEDICALLY ESTABLISHED DIAGNOSTIC PRODUCT OR PROCEDURE, OR NONINFRINGEMENT, AND ANY WARRANTIES ARISING OU T OF CO UR S E OF DEAL IN G OR COU RSE OF PERFORMANCE. CUSTOMER EXPRESSLY ACKNOWLEDGES THAT BECAUSE OF THE COMPLEX NATURE OF THE WATERS 996 PDA DETECTOR AND ITS MANUFACTURE, WATERS CORPORATION CANNOT AND DOES NOT W ARRANT THAT THE OPERATION OF THE WATERS 996 PDA DETE CTOR WILL BE WITH OUT DEFECT. CUSTOMER EXPRESSLY ACKNO WLEDGES THAT CUSTOMER IS SOLELY RESPONSIBLE FOR USE OF THE WATERS 996 PDA DETECTOR IN CLINCIAL DIAGNOSTIC PROCEDURES OR FOR INVESTIGATIONAL USE WITH OR WITHOUT CONFIRMATION OF DIAG N O SIS BY AN OTHER MEDICALLY ESTABLISHED DIAGNOSTIC PRODUCT OR PROCEDURE.

66 Appendix C

Wa rr anty Service

Warranty service is performed at no charge and at Waters’ option in one of three ways:

• A service representative is dispatched to the customer facility.

• The product is repaired at a Waters repair facility.

• Replacement part s with appropriate installation instructions are sent to the customer.

Nonconforming products or parts are repaired, replaced with new or like-new parts, or refunded in the amount of the purchase price, when the product is returned. Warranty service is performed only if the customer notifies Waters during the applicable warranty period.

Unless otherwise agreed at the time of sale, warranty service is not provided by dispatching a service represent ative when the equipment has been removed from the initial installation location to a new location outside the home country of the selling company.

Warranty service is provided during business hours (8 Friday). Service is not available when Waters offices are closed in observance of legal holidays.

to 5PM, EST, Monday through

Wa rr anty Service Exceptions

Warranty service is not performed on:

• Any product or part that has been repaired by others, improperly installed, altered, or damaged in any way.

• Products or parts identified prior to sale as not manufactured by Waters. In such cases, the warranty of the original manufacturer applies.

• Products that malfunction because the customer has failed to perform maintenance, calibration checks, or observe good operating procedures.

• Products that malfunction due to the use of unapproved parts and operating supplies.

Repair or replacement is not made:

• For expendable items such as gaskets, windows, lenses, and fuses, if such items were operable at the time of initial use.

• Because of decomposition due to chemical action.

• For used equipment.

• Because of poor facilities, operating conditions, or utilities.

Appendix C 67

Wa rr anty Period

The warranty period begins when the product is installed or, in the case of a customer installation, 15 days after shipment from Waters.

In no case does the warranty period extend beyond 15 months from date of shipment. If an item is replaced during its warranty period, the replacement par t is warranted for the balance of the original warranty period. Table C-1 applicable components.

Table C-1 Warranty Periods

Item Warranty

Waters 996 PDA Detector 1 year Deuterium lamp 1000 hours

summarizes the warranty periods for

Gaskets Windows Lenses Fuses Plunger seals Tubing and fittings

Not warranted items

Lamp Replacement Warranty

The Waters 996 PDA Detector deuterium lamp is warranted to light and pass powerup verification tests for 1000 hours.

68 Appendix C

C.2 Shipments, Damages, Claims, and Returns

Shipments

As all shipments are made Free On Board (FOB) shipping point, we suggest insurance be authorized on all shipments. Instruments and major component s are packed and shipped via surface, unless otherwise required. Supplies and/or replacement parts are packed and shipped via United Parcel Service (UPS), UPS Blue, air parcel post, or parcel post unless otherwise requested.

Damages

The Interstate Commerce Commission has held that carriers are as responsible for concealed damage as for visible damage in transit. Unpack shipment promptly after receipt as there may be concealed damage even though no evidence of it is apparent. When concealed damage is discovered, cease further unpacking of the unit involved and request immediate inspection by local agent or carrier and secure written report of his findings to support claim. This request must be made within 15 days of receipt. Otherwise, the claim will not be honored by the carrier. Do not return damaged goods to the factory without first securing an inspection report and contacting Waters for a return merchandise authorization number (RMA).

Claims

After a damage inspection report is secured, Waters cooperates fully in supplying replacements and handling of a claim which may be initiated by either party.

Returns

No returns may be made without prior notification and authorization. If for any reason it is necessary to return material to Waters, please contact Waters Customer Service or your nearest Waters subsidiary or representative for a return merchandise authorization (RMA) number and forwarding address.

Appendix C 69

Append ix D Mobile Phase Absorban ce

This appendix provides a list of the absorbances at several wavelengths for commonly used mobile phases. Choose your mobile phase carefully to reduce baseline noise.

The best mobile phase for your application is one that is transparent at the chosen detection wavelengths. Such a mobile phase ensures that any absorbance is due only to the sample. Absorbance by the mobile phase also reduces the linear dynamic range of the detector by the amount of absorbance that is autozeroed out. Wavelength, pH and concentration of the mobile phase will affect its absorbance. Examples of several mobile phases are provided below.

Table D-1 Mobile Phase Absorbance Measured Against Air or Water

Absorbance at Specified Wavelength (nm)

200 205 210 215 220 230 240 250 260 280

Solvents

Acetonitrile 0.05 0.03 0.02 0.01 0.01 <0.01 — — — — Methanol (not

degassed) Methanol

(degassed) Isopropanol 1.80 0.68 0.34 0.24 0.19 0. 08 0.04 0.03 0.02 0.02 Unstablized

Tetrahydrofuran (THF, fresh)

Unstablized Tetrahydr o fu ran (THF, old)

70 Appendix D

2.06 1.00 0.53 0.37 0.24 0.11 0.05 0.02 <0.01 —

1.91 0.76 0.35 0.21 0.15 0. 06 0.02 <0.01 — —

2.44 2.57 2.31 1.80 1.54 0.94 0.42 0.21 0.09 0.05

>2.5 >2.5 >2.5 >2.5 >2.5 >2.5 >2.5 >2.5 2.5 1. 45

Table D-1 Mobile Phase Absorbance Measured Against Air or Water (Continued)

Absorbance at Specified Wavelength (nm)

200 205 210 215 220 230 240 250 260 280

Acids and Bases

Acetic acid, 1% 2.61 2.63 2.61 2. 43 2.17 0. 87 0.14 0. 01 <0.01 — Hydrochloric

acid, 0.1% Phosphoric

acid, 0.1% Triufluoroacetic

acid Diammonium

phosphate, 50 mM

Triethylamine, 1%2.33 2.42 2.50 2.45 2.37 1. 96 0.50 0.12 0.04 <0.01

Buffers and Salts

Ammonium acetate, 10 mM

Ammonium bicarbonate, 10 mM

EDTA, disodium, 1 mM

0.11 0.02 <0.01 — — — — — — —

<0.01 — — — — — — — — —

1.20 0.78 0.54 0.34 0.22 0.06 <0.02 <0.01 — —

1.85 0.67 0.15 0.02 <0.01 — — — — —

1.88 0.94 0.53 0.29 0.15 0.02 <0 .01 — — —

0.41 0.10 0.01 <0.01 — — — — — —

0.11 0.07 0.06 0.04 0.03 0.03 0.02 0.02 0.02 0.02

HEPES, 10 mM, pH 7.6

MES, 10 mM, pH 6.0

2.45 2.50 2.37 2.08 1.50 0.29 0.03 <0.01 — —

2.42 2.38 1.89 0.90 0.45 0.06 <0.01 — — —

Appendix D 71

Table D-1 Mobile Phase Absorbance Measured Against Air or Water (Continued)

Absorbance at Specified Wavelength (nm)

200 205 210 215 220 230 240 250 260 280

Potassium phosphate, monobasic (KH 10 mM

Potassium phosphate, dibasic, (K 10 mM

Sodium acetate, 10 mM

Sodium chloride, 1 M

Sodium citrate, 10 mM

Sodium formate, 10 mM

2PO4

HPO4),

0.03 <0.01 — — — — — — — —

0.53 0.16 0.05 0.01 <0.01 — — — — —

1.85 0.96 0.52 0.30 0.15 0.03 <0.01 — — —

2.00 1.67 0.40 0.10 <0.01 — — — — —

2.48 2.84 2.31 2.02 1.49 0.54 0.12 0.03 0.02 0.01

1.00 0.73 0.53 0.33 0.20 0.03 <0.01 — — —

Sodium phosphate, 100 mM, pH 6.8

Tris HCl, 20 mM, pH 7.0

Tris HCl, 20 mM, pH 8.0

72 Appendix D

1.99 0.75 0.19 0.06 0.02 0. 01 0. 01 0.01 0.01 <0.01

1.40 0.77 0.28 0.10 0.04 <0 .01 — — — —

1.80 1.90 1.11 0.43 0.13 <0 .01 — — — —

Table D-1 Mobile Phase Absorbance Measured Against Air or Water (Continued)

Absorbance at Specified Wavelength (nm)

200 205 210 215 220 230 240 250 260 280

Waters PIC Reagents

PIC A, 1 vial/L 0. 67 0.29 0. 13 0.05 0. 03 0.02 0.02 0. 02 0.02 <0 .01 PIC B6, 1 vial/L 2.46 2.50 2.42 2.25 1.83 0.63 0.07 <0.01 — — PIC B6, low UV,

1 vial/L PIC D4, 1 vial/L 0.03 0.03 0.03 0.03 0.02 0. 02 0. 02 0. 02 0. 02 0. 01

Detergents

BRI J 35, 1% 0.06 0.03 0. 02 0. 02 0.02 0.01 <0.01 — — — CHAPS, 0.1% 2.40 2.32 1.48 0.80 0.40 0.08 0.04 0.02 0.02 0.01 SDS, 0.1% 0.02 0.01 <0.01 — — — — — — — Triton™ X-100,

0.1% Tween™ 20,

0.1%

0.01 <0.01 — — — — — — — —

2.48 2.50 2.43 2.42 2.37 2. 37 0.50 0.25 0.67 1.42

0.21 0.14 0.11 0.10 0.09 0. 06 0.05 0.04 0.04 0.03

Appendix D 73

Index

Absorbance

maximum mobile phase photometric error solvent change effects Waters 996 calculations

Acquisition

Auto Exposure parameter

Exposure Time parameter Address, setting Analog output specifications Aperture width Auto Exposure parameter

Beer’s law

50, 61

50, 52

19, 21

Damage, warranty Dark current Data acquisition

Auto Exposure parameter

Exposure Time parameter Derived vectors Diagnostics DIP switch, setting

56, 56

Electrical connections Events

connections

electrical specifications

terminal strip connections Exception to service warranty Exclusive remedy Exposure Time par ameter

19, 22, 22

I N D E X

Calibration Claims, warranty Column, connecting Compression fittings Connections

column events fluidic non-IEEE-488 rear panel

terminal strip Contact closures Contacting Waters T e chnical Service

27, 31

Fittings Flow cell

Fluid

23, 23, 23

access cleaning exploded view flushing maintenance removing

connecting lines fittings

Index 74

Fuses

IEC-ra te d maintenance replacement

IEC-rated f u ses IEEE-488 address, setting Inputs Installation

Instrument me thod

19, 21, 22, 22

electrical fluidic site selection

Auto Exposure parameter Exposure Time parameter

14, 14

Mobile phase

absorbances wavelengths

Network address Noise effects Nonidealities Non-IEEE-488 connections

18 60 60

Outputs

19, 21, 22, 22

I N D

E X

Lamp

hardware theory replacement replacement warranty

usage indicator Liability Limited express warranty

39, 41

Maintenance

flow cell

fuse

lamp

PDA detector Match Angle, photometric error effects Maximum absorbance Millennium³² Chromatography Manager,

connections

33–43

Parts, spare Photodiode array Photometric error Power connections Purity Angle, photometric error effects

61, 61

Rear panel connections Reference spectrum Returns, warranty

Service

exceptions to warranty warranty

Setting

DIP switch IEEE-488 address

Shipments

Index 75

N D E X

Shutdo wn, procedure Solvent Angle, photometric error effects Solvent changes Spare parts Specifications

analog output

event inputs

event outputs

Waters 996 Spectra

derived vectors

spectral shape differences

vectors Spectral Contrast

derived vectors

spectral shape differences

theory

vectors Spectral resolution Spectrum match, spectral shape

Start up, procedure

54–62

differences

19, 21

56, 56

Terminal strip

connections

diagram Threshold angle Troubleshooting Tubing, cutting

20, 22

27–32

Warranty

claim s damages disclaimers exclusions information lamp replacement limited period returns ser vi c e service exceptions

Waters 996

absorbance calculations aperture width dark current detector optics, overview hardware theory photodiode array overview reference spectrum spare parts specifications

spectral resolution Waters liability Waters Technical Service, contacting Wa velength

accur acy

derived vectors

mobile phase absorbances

65–68

50, 52

44–46

44–53

56, 56

27, 31

Vectors

derived from multiple wavelengths derived from two wavelengths spectra, representing spectral contrast

76 Index

Waters 996 Operator's Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Table of Contents

List of Figures

List of Tables

How to Use This Guide

Installation

1.1 Instal lation Site Requirements

1.2 Power Connections

1.3 Millennium32 Workstation Connections

1.3.2 Setting the IEEE-488 Address

1.4 Non-IEEE-488 Communication Connections

1.4.1 Connecting Analog Output Cables

1.4.2 Connecting Event Cables

1.5 Fluidic Connections

1.6 Startup/Shutdown

Diagnostics and Calibration

2.1 Startup Diagnostics

2.2 User-Initiated Diagnostics

2.3 PDA Calibration

Maintenance

3.1 Fl ow Cell Maintenance

3.1.1 Flushing the Flow Cell

3.1.2 Removing the Flow Cell

3.1.3 Disassembling and Cleaning the Flow Cell

3.1.4 Installing the Flow Cell Assembly

3.2 Replacing the Lamp

3.2.1 Checking Lamp Usage

3.2.2 Removing the Lamp

3.3 Replacing the Fuses

4 Pr incipl es of the 996 PDA Detector Optics

4.1 996 Detector Optics

4.2 Resolving Spectral Data

4.3 Measuring Light at the Photodiode

4.4 Computing Absorbance Data Points

4.4.1 Calculating Absorbance

4.4.2 Resolution

4.4.3 Filtering Data

Spectral Contrast Theory

5.1 Comparing Absorbance Spec tra

5.2 Representing Spectra as Vectors

5.2.1 Vectors Derived from Two Wavelengths

5.2.2 Vectors Derived from Multiple W av elengths

5.3 Spectral Contrast Angles

5.4 Nonidealities

5.4.1 Detector Noise

5.4.2 Photometric Error

5.4.3 Solvent Changes

5.4.4 Threshold Angle

Append ix A Detector Specifications

Append ix B Spare Parts

Append ix C Warranty Information

C.1 Limited Express Warranty

C.2 Shipments, Damages, Claims, and Returns

Append ix D Mobile Phase Absorban ce

Index