Waters 2996 Operator's Manual

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Waters 2996

PDA Detector

Operator’s Guide

34 Maple Stre

Milford, MA 01 75

71500023202, Revision

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 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, the use of this docum ent.

© 2001 WATERS CORPORATION. PRINTED IN THE UNITED STATES OF AMERICA. ALL RIGHTS RESERVED. THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER.

Millennium, PIC, and Waters are register ed t rad emar ks, and busLAC/E and PowerStation are trademarks of Waters Corporation.

Micromass is a registered trademark, and MassLynx is a trademark of Micromass Ltd. All other trademarks or registered trademarks are the sole property of their respective

owners.

STOP

Note: 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 determine the reason for the changes. Until you determine the cause of a change, do not rely on the separation results.

Note: The Installation Category (Overvoltage Category) for this instrument is Level II. The Level II Category pertains to equipment that receives its electrical power from a local level, such as an electrical wall outlet.

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.

Atención: cualquier cambio o modificación realizado a esta unidad que no haya sido expresamente aprobado por la parte responsable del cumplimiento puede anular la autorización de la que goza el usuario para utilizar el equipo.

Caution: 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 Tefzel tubing that has been severely stressed or kinked.

• Do not use Tefzel tubing with tetrahydrofuran (THF) or concentrated nitric or sulfuric acids.

• Be aware that methylene chloride and dimethyl sulfoxide cause Tefzel tubing to swell, which greatly reduces the rupture pressure of the tubing.

Attention : soyez très prudent en travaillant avec des tuyaux de polymères sous

pression :

• Portez toujours des lunettes de protection quand vous vous trouvez à proximité de tuyaux de polymères.

• Eteignez toutes les flammes se trouvant à proximité.

• N'utilisez pas de tuyau de Tefzel fortement abîmé ou déformé.

• N'utilisez pas de tuyau de Tefzel avec de l'acide sulfurique ou nitrique, ou du tétrahydrofurane (THT).

• Sachez que le chlorure de méthylène et le sulfoxyde de diméthyle peuvent provoquer le gonflement des tuyaux de Tefzel, diminuant ainsi fortement 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 Tefzel-Schläuche verwenden, die stark geknickt oder überbeansprucht sind.

• Tefzel-Schläuche nicht für Tetrahydrofuran (THF) oder konzentrierte Salpeter- oder Schwefelsäure verwenden.

• Durch Methylenchlorid und Dimethylsulfoxid können Tefzel-Schläuche quellen; dadurch wird der Berstdruck des Schlauches erheblich reduziert.

Precauzione: prestare attenzione durante le operazioni con i tubi di polimero sotto

pressione:

• Indossare sempre occhiali da lavoro protettivi nei pressi di tubi di polimero pressurizzati.

• Estinguere ogni fonte di ignizione circostante.

• Non utilizzare tubi Tefzel soggetti a sollecitazioni eccessive o incurvati.

• Non utilizzare tubi Tefzel contenenti tetraidrofurano (THF) o acido solforico o nitrico concentrato.

• Tenere presente che il cloruro di metilene e il dimetilsolfossido provocano rigonfiamento nei tubi Tefzel, che riducono notevolmente il limite di pressione di rottura dei tubi stessi.

Advertencia: manipular con precaución los tubos de polimero bajo presión:

• Protegerse siempre los ojos a proximidad de tubos de polimero bajo presión.

• Apagar todas las llamas que estén a proximidad.

• No utilizar tubos Tefzel que hayan sufrido tensiones extremas o hayan sido doblados.

• No utilizar tubos Tefzel con tetrahidrofurano o ácidos nítrico o sulfúrico concentrados.

• No olvidar que el cloruro de metileno y el óxido de azufre dimetilo inflan los tubos Tefzel lo que reduce en gran medida la presión de ruptura de los tubos.

Caution: 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.

Precauzione: 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.

Caution: 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.

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

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

Caution: To avoid possible electrical shock, power off the instrument and disconnect the power cord before servicing the instrument.

Attention : Afin d’éviter toute possibilité de commotion électrique, mettez hors tension l’instrument et 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 abgeschaltet und vom Netz getrennt werden.

Precauzione: per evitare il rischio di scossa elettrica, spegnere lo strumento e scollegare il cavo di alimentazione prima di svolgere la manutenzione dello strumento.

Precaución: para evitar choques eléctricos, apague el instrumento y desenchufe el cable de alimentación antes de realizar cualquier reparación en el instrumento.

Commonly Used Symbols

Direct current Courant continu Gleichstrom Corrente continua Corriente continua

Alternating current Courant alternatif Wechselstrom Corrente alternata Corriente alterna

Protective conductor terminal Borne du conducteur de protection Schutzleiteranschluss Terminale di conduttore con protezione Borne del conductor de tierra

Frame or chassis term inal Borne du cadre ou du châssis Rahmen- oder Chassisanschluss Terminale di struttura o telaio Borne de la estructura o del chasis

Caution or refer to manual Attention ou reportez-vous au guide Vorsicht, oder lesen Sie das Handbuch Prestare attenzione o fare riferimento alla guida Actúe con precaución o consulte la guía

Caution, hot surface or high temperature Attention, surface chaude ou température élevée Vorsicht, heiße Oberfläche oder hohe Temperatur Precauzione, superficie calda o elevata temperatura Precaución, superficie caliente o temperatura elevada

Commonly Used Symbols (Continued)

Caution, risk of electric shock (high voltage) Attention, risque de commotion électrique (haute tension) Vorsicht, Elektroschockgefahr (Hochspannung) Precauzione, rischio di scossa elettrica (alta tensione) Precaución, peligro de descarga eléctrica (alta tensión)

Caution, risk of needle-stick puncture Attention, risques de perfor at io n de la tail le d’u n e aigu il le Vorsicht, Gefahr einer Spritzenpunktierun g Precauzione, rischio di puntura con ago Precaución, riesgo de punción con aguja

Caution, ultraviolet light

Attention, rayonnement ultrviolet Vorsicht, Ultraviolettes Licht Precauzione, luce ultravioletta Precaución, emisiones de luz ultravioleta

2996 PDA Detector Information

Intended Use

The Waters® 2996 PDA Detector can be used for in-vitro diagnostic testing to analyze many compounds, including diagnostic indicators and therapeutically monitored compounds. 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 prot ocol covers good operating procedures and techniques necessary to validate system and method performance.

Biological Hazard

When you analyze physiological fluids, take all necessary precautions and treat all specimens as potentially infectious. Precautions are outlined in “CDC Guidelines on Specimen Handling,” CDC – NIH Manual, 1984.

Calibration

Follow acceptable methods of calibration with pure standards to calibrate methods. Use a minimum of five standards to generate a standard curve. The concentration range should cover the entire range of quality-control samples, typical specimens, and atypical specimens.

Quality Control

Routinely run three quality-control samples. Quality-control samples should represent subnormal, normal, and above-normal levels of a compound. Ensure that quality-control sample results are with in an ac cept ab le range, and evaluate precision from day to day and run to run. Data collected when qua lity- contr ol samp les are out of range may not be val id. Do not report this data until you ensure that chromatographic system performance is acceptable.

General Maintenance

To clean the outside of the Waters 2996 PDA Detector, use only a soft lint-free paper or cloth dampened with mild soap and water.

Table of Contents xi

Preface................................................................................................................ xix

Chapter 1 Installation

........................................................................................................... 1

1.1 Installation Site Requirements .......................................................................... 1

1.2 Power Connections............................................................................................ 2

1.3 Millennium32 Workstation Connections ........................................................... 3

1.3.1 Connecting the IEEE-488 Cable.......................................................... 4

1.3.2 Setting the IEEE-488 Address ............................................................ 5

1.4 Non-IEEE-488 Communication Connections................................................... 6

1.4.1 Connecting Analog Output Cables ...................................................... 6

1.4.2 Connecting Event Cables .................................................................... 7

1.5 Fluidic Connections........................................................................................... 9

1.6 Starting Up and Shutting Down the Detector.................................................. 11

Chapter 2 Diagnostics and Calibration

.......................................................................... 15

2.1 Diagnostics...................................................................................................... 15

2.2 User-Initiated Diagnostics............................................................................... 17

2.3 PDA Calibration.............................................................................................. 18

Chapter 3 Maintenance

...................................................................................................... 19

3.1 Flow Cell Maintenance ................................................................................... 19

3.1.1 Flushing the Flow Cell....................................................................... 20

3.1.2 Removing the Flow Cell ................................................................... 20

3.1.3 Disassembling and Cleaning the Flow Cell ...................................... 22

3.1.4 Installing the Flow Cell Assembly .................................................... 24

Table of Contents

xii Table of Contents

3.2 Replacing the Lamp ........................................................................................ 25

3.3 Replacing the Fuses......................................................................................... 27

Chapter 4 Principles of the 2996 PDA Detector Optics

.............................................. 29

4.1 2996 Detector Optics....................................................................................... 29

4.2 Resolving Spectral Data.................................................................................. 31

4.3 Measuring Light at the Photodiode................................................................. 32

4.4 Computing Absorbance Data Points............................................................... 35

4.4.1 Calculating Absorbance..................................................................... 35

4.4.2 Resolution ......................................................................................... 38

4.4.3 Filtering Data .................................................................................... 38

Chapter 5 Spectral Contrast Theory

.............................................................................. 41

5.1 Comparing Absorbance Spectra...................................................................... 41

5.2 Representing Spectra as Vectors ..................................................................... 42

5.2.1 Vectors Derived from Two Wavelengths .......................................... 43

5.2.2 Vectors Derived from Multiple Wavelengths ................................... 43

5.3 Spectral Contrast Angles................................................................................. 44

5.4 Undesirable Effects ......................................................................................... 47

5.4.1 Detector Noise ................................................................................... 47

5.4.2 Photometric Error ............................................................................. 48

5.4.3 Solvent Changes ............................................................................... 48

5.4.4 Threshold Angle ............................................................................... 48

Table of Contents xiii

Appendix A Detector Specifications

................................................................................... 51

Appendix B Spare Parts

........................................................................................................ 53

Appendix C Mobile Phase Absorbance

.............................................................................. 55

Index ................................................................................................................... 59

xiv Table of Contents

List of Figures xv

1-1 Waters 2996 PDA Detector Dimensions.................................................... 2

1-2 Detector Rear Panel ................................................................................... 3

1-3 Example of IEEE-488 Cable Connections................................................. 4

1-4 Locating the IEEE-488 Switches............................................................... 5

1-5 Analog Out Terminal Strip......................................................................... 7

1-6 Event Input/Output Terminal Strip ............................................................ 8

1-7 Compression Screw Assembly ................................................................ 11

1-8 2996 Detector Indicator Lights................................................................ 12

3-1 Removing the Flow Cell Assembly......................................................... 21

3-2 Flow Cell and Fluidic Connections Assemblies...................................... 22

3-3 Disassembling the Flow Cell................................................................... 23

3-4 Lamp Power Connector and Mounting Screws....................................... 26

3-5 Fuse Block ............................................................................................... 27

4-1 Optics Assembly Light Path.................................................................... 30

4-2 Benzene Spectrum at 1.2 nm Resolution................................................. 32

4-3 Photodiodes Discharged by Light............................................................ 33

4-4 Absorbance as a Function of Concentration............................................ 36

5-1 Comparing Spectra of Two Compounds.................................................. 42

5-2 Plotting Vectors for Two Spectra............................................................. 43

5-3 Spectra with a Large Spectral Contrast Angle......................................... 45

5-4 Spectra with a Small Spectral Contrast Angle......................................... 46

5-5 Absorbance Spectra of a Compound at Two Concentrations .................. 47

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

p-Aminobenzoic Acid ............................................................................. 49

List of Figures

xvi List of Figures

List of Tables xvii

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

1-2 Event In Terminal Specifications on TTL or Switch Closure................... 9

1-3 Event Out Terminal Specifications on Contact Closure ........................... 9

1-4 LED Indicator Sequences During Startup............................................... 12

2-1 2996 Detector Troubleshooting .............................................................. 15

4-1 Optics Assembly Components................................................................ 30

A-1 2996 Detector Specifications.................................................................. 51

B-1 Spare Parts.............................................................................................. 53

C-1 Mobile Phase Absorbance Measured Against Air or Water................... 55

List of Tables

xviii List of Tables

xix

Preface

The Waters 2996 PDA Detector Operator’s Guide describes the p rocedures for installi ng, maintaining, and troubleshooting the Waters

2996 PDA Detector. It also describes

detector optics and th e principle s of Spectral Contrast used in the Millenni um®

software for analyzing the data from the PDA detector. Also included is information on vector analysis, mobile phase absorbance, specifications.

This guide is intended for individuals who need to install, operate, maintain, and troubleshoot the 2996 PDA Detector. It is also intended for users who need to understand the Spectral Contrast principles underlying the processing of PDA Detector data by Millennium

software.

Organization

This guide contains the following:

Chapter 1 describes how to install and set up the 2996 Detector. Chapter 2 describes how to troubleshoot the 2996 Detect or. Chapter 3 describes how to replace the flow cell, the lamp, and the fuse. Chapter 4 explains the principles involved in resolving spectral data, measuring light

at the photodiode, verifying wavelengths, and computing absorbance data. Chapter 5 describes the calculations used for Spectral Contrast. Appendix A provides the specifications of the 2996 PDA Detector. Appendix B lists recommended spare parts. Appendix C provides a table of absorbances at several wavelengths for common

mobile phases.

Related Documentation

W at ers Li censes , Warranties, and Supp ort: Provi des software lic ense and warranty information, describes training and extended support, and tells how Waters handles shipments, damages, claims, and returns.

Millennium

Help: Describes al l Millennium32 windows, menus, menu selections,

and dialog boxes. Also includes reference information and procedures for performing

all tasks required to use the Millennium32 software. Included as part of the Millennium

Millennium

software.

Software Ge tting Started Guide : Provides an introduction to the

System. Describes the basics of how to use Millennium32 software to acquire data, develop a processing method, review results, and print a report. Also covers basics for managing projects and configuring systems.

Millennium

to use Millennium

PDA Software Ge tting Started Guide : Describes the basics of how

PDA software to dev elop a PDA proces sing method an d to review

PDA results.

Millennium

System Installation an d Configu ration Gui de: Describes

software installation, including the stand-alone workstation, PowerStation™ system, and client/server system. Discusses how to configure the computer and chromatographic instruments as part of the Millennium covers the installation, config uration, a nd use of ac quisition s e rvers such as the LAC/E

module, the busLAC/E card, and interface cards used to com municate with

serial instruments Water s 29 96 PD A De tector Qual ificati on Workbook: Describes qualification

procedures for the 2996 PDA Detector.

Documentation Conventions

The following conventions may be used in this guide:

Convention Usage

system . Also

Bold Bold indicates user action such as keys to press, menu selections,

Italic Italic indicat es infor mation that you s upply such as varia bles. It also

Courier

Courier Bold

and commands. For example, “Click Next to go to the next page.”

indicates emphasis and document titles. For example, “Replace file_name with the actual name of your file.”

Courier indicates examples of source code and system output. For example, “The

SVRMGR> prompt appears.”

Courier bold indicates characters that you type or keys you press in examples of source code. For example, “At the enter

set password oracle to access Oracle.”

LSNRCTL> prompt,

Convention Usage

Keys The word key refers to a computer key on the keypad or keyboard.

Screen keys refer to the key s on the instrument located im mediately

below the screen. For example, “The A/B screen key on the 2414 Detector displays the selected channel.”

… Three periods indicate that more of the same type of item can

optionally follow. For example, “You can store filename1, filename2, … in each folder.”

> A right arrow between menu options indicates you should choose

each option in sequence. For example, “Select File > Exit” me ans you should selec t File from the menu bar, then select Exit from the File menu.

Notes

Notes call out information that is helpful to the operator. For example:

Note: Record your result before you proceed to the next step.

Attentions

Attentions provide information about preventing possible damage to the system or equipment. For example:

Attention: To avoid damaging the detector flow cell, do not touch the flow cell

STOP

Cautions

window.

Cautions provide information essential to the safety of the operator. For example:

Caution: To avoid possible burns, turn off the lamp at least 15 minutes before removing it for replacement or adjustment.

Caution: To avoid possible electrical shock and injury, always turn off the detector and unplug the power cord before performing maintenance procedures.

Caution: To avoid chemical or electrical hazards, always observe safe laboratory practices when operating the system.

xxi

xxii

Installation Site Requirements 1

Chapter 1 Installation

The Waters® 2996 Photodiode Array (PDA) Detector operates in any standard laboratory environment. The detector requires electrical power, sample and wast e fluidic lines, and either the Millennium®

or MassLynx™ workstations. Optional connections on the detector rear panel allow communication with chart recorders, data integrators, and other instruments that are not compatible with Millennium

or MassLynx software control.

1.1 Installation Site Requirements

Install the 2996 PDA Detector at a site that meets the specifications in Table 1-1 and Figure 1-1.

Table 1-1 Site Requirements

Factor Specification

Ambient temperature 4 to 40 °C (39 to 104 °F), not to vary more

than 1 °C per hour (to prevent drift) Relative humidity 20 to 80%, noncondensing Bench space Width: 11.5 in. (29 cm)

Depth: 24 in. (61 cm)

Height: 8.5 in. (22 cm) Bench support Capable of supporting 31.5 pounds (14.3 kg) Clearance At least 4 in. (10 cm) on the back side for

ventilation Power Grounded ac, 100 to 240 Vac

11.5 in. (29 cm)

WATERS 996

Photodiode Array

8.5 in.

(22 cm)

24 in.

(61 cm)

Sample Outlet

Figure 1-1 Waters 2996 PDA Detector Dimensions

1.2 Power Connections

Ensure that power connections for the 2996 PDA Detector are made according to the procedures that follow.

Operating Voltage

The 2996 PDA Detector has a universal input power supply that requires no voltage adjustmen t. The electrical power requiremen ts for the Waters 2996 PDA De tector are:

LAMP

Sample Inlet

Drain Line

• Voltage range: 100 to 240 Vac

• Total power: 100 VA

• Frequency range: 50 to 60 Hz

Fuses

The 2996 PDA Detector is shipped with fuses rated for North American operation. If you operate the 2996 P DA Detect or in anot her loca tion, install t he IEC-rat ed fuses (supplie d in

2Installation

Millennium32 Workstation Connections 3

the Waters 2996 Detector St ar tup Kit ) in the fu se h older in the rear of the de tecto r (re fer t o Section 3.3, Replacing the Fuses).

Connecting the Power Cord

Connect one end of the 2996 Detector power cord to the rear panel power receptacle (Figure 1-2) and the other end to a power outlet.

Figure 1-2 Detector Rear Panel

1.3 Millennium32 Workstation Connections

The 2996 Detector requires signal connections to the Millennium32 Work station over the IEEE-488 bus. All detector control and data acquisition communications take place over the IEEE-488 bus.

Note: If an inject start signal is not available over the IEEE-488 bus, you must provide a signal at the Event In 1 terminals on the 2996 Detector rear panel (Section 1.4.2, Connecting Event Cables).

TP01452

AC Input

1.3.1 Connecting the IEEE-488 Cable

To connect the 2996 Detector to a Millennium32 Workstation:

1. Connect one end of the IEEE-488 ca ble to t he IEEE-488 recepta cle on t he rear panel of the 2996 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

IEEE-488

Connector

Millennium

busLAC/E Card

(on Workstation)

Waters

Alliance

Separations Module

IEEE-488 Cable

Waters 2996

Detector

Note: The order in which you connect IEEE-488 devices to the busLAC/E card on the workstation is not important. For example, you can connect the 2690 Separations Module before or after the 2996 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 instrument in your chromatographic system, up to a maximum of 14 IEEE-488 instruments.

Note: Keep in mind cable-length limitations when you set up your system. For a list of IEEE-488 interface guidelines, refer to the Millennium Configuration Guide.

4Installation

TP01544

Figure 1-3 Example of IEEE-488 Cable Connections

System Installation and

Millennium32 Workstation Connections 5

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

Cable Lengths

The maximum length of all cables connecting instruments to one busLAC/E is 2 meters multiplied by the numb er of devices (or 20 mete rs, whichever is smaller).

The maximum length of cable between devices is 4 meters.

Note: The maximum number of devices to be connected together is 14.

1.3.2 Setting the IEEE-488 Address

To set the IEEE-488 address for the 2996 Detector: Use a small screwdriver (or similar device) to set the IEEE-488 switches on the detector

rear panel (Figure 1-4) to the IEEE-488 address of the 2996 Detec tor. The address must be a number from 2 to 29 and must be different than that of any other component connected to your acquisition server.

Refer to the Millennium

System Installation and Configuration Guide for the correct

IEEE-488 GPIB switch settings.

Figure 1-4 Locating the IEEE-488 Switches

IEE 488 ADDRESS

12345

 OPEN 

TP01457

IEEE-488 Address Switches

IEEE-488 Cable Connection

1.4 Non-IEEE-488 Communication Connections

Non-IEEE-488 communication connections on the 2996 Detector include:

• Analog Outputs – The 2996 Detect or provides two, unattenuated, 1 volt-per

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

• Event Inputs and Outputs – The 2996 Detector sends and receives contact closure

signals to and from other instruments.

You make all non-I EEE-488 c ommunicat ion ca ble c onne ction s to t he 299 6 Detec tor a t the rear panel on the analog/event terminal (Figure 1-2).

Attention: To meet the regulatory requirements of immunity from external electrical

STOP

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.

1.4.1 Connecting Analog Output Cables

The values of the analog output signals generated by the 2996 Detector are specified by parameter values set from the Millennium

Millennium

Help.

Workstation. For details, refer to the

Required Materials

• One small, flat-blade screwdriver

• One electrical insulation stripping tool

• Analog signal cables (included with the Waters 2996 Detector Startup Kit)

Procedure

To connect the 2996 Detector to a device that receives analog output signals:

1. Pull off the ana log out te rminal strip from the 2996 Detec tor rear pa nel (Figure 1-5). This step simplifies the following steps.

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

6Installation

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 Ana log Output Terminal strip.

Removable Analog Output

Terminal Strip

Analog Out 1

123456

Analog Out 2

Figure 1-5 Analog Out Terminal Strip

IEE 488 ADDRESS

TP01456

1.4.2 Connecting Event Cables

The 2996 Detector has four terminal strip connections for contact closure signals:

• Two input signal terminals (generally used for inject start)

• Two output (programmable event table) signal terminals

If an inject start s ignal is not ava ilable over the IEEE-488 bus , you must provide a signal at Event In 1 terminal on the 2996 Detector rear panel. Manual injectors such as the Rheodyne 7725i provide a cable that connects the injector to an Event In terminal on the 2996 Detector rear panel.

The values of the event output signals generated by the 2996 Detector are specified by parameter values set from the Millennium

Millennium

Help.

Workstation. For details, refer to the

Non-IEEE-488 Communication Connections 7

Required Materials

• Small flat-blade screwdriver

• One electrical insulation stripping tool

• Event signal cables (included in the Waters 2996 Detector Startup Kit)

Procedure

To connect the 2996 Detector to an external event input or output device: Pull off the event terminal strip from the rear panel (Figure 1-6). This simplifies the

following steps:

1. 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 (Figure 1-6). Tighten the two screws to secure the + and – wires.

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

3. Reinstall the event terminal strip.

Removable Event

Input/Output

Terminal Strip

8Installation

Event Out 2

Event Out 1

Event



In 2

1234567890

 +

Event In 1

IEE 488 ADDRESS

Figure 1-6 Event Input/Output Terminal Strip

TP01455

Fluidic Connections 9

Electrical Specifications

Before you connect an external device to an event input or output terminal, refer to the electrical specifications in Table 1-2 and Table 1-3.

1.5 Fluidic Connections

Required Materials

• 5/16-inch open-end wrench

• 0.009-inch (0.23 m m) ID stainless steel tubing (included in the 2996 Detector

Startup Kit)

Table 1-2 Event In Terminal 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 (this may lim it compatibility

with valves that require a quick pulse)

Maximum current 5 mA

Table 1-3 Event Out Terminal Specifications on Contact Closure

Parameter Specification

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

STOP

Attention: To avoid damage to the 2996 Detector electronics, be sure you make the proper electrical connections as outlined in this section.

Caution: To avoid chemical hazards, always observe safe laboratory practices when handling solvents. Refer to the Material Safety Data Sheets for the solvents in use.

• Stainless st eel tubing cutter or scri bing file

• Pliers, plastic- covered, or with cloth

• Compression screw assemblies, three

Procedure

To make fluidic connections to the 2996 Detector:

1. Measure the lengths of tubing needed to connect:

• The column outlet to the 2996 Detector inlet

Note: Keep the length of this tubing as short as possible to prevent band broadening.

• The 2996 Detector outlet to a waste collection bottle

Note: Ensure the length of this tubing is at least 1 to 2 feet (30 to 60 cm) to prevent air bubbles from forming in the flow cell.

2. Cut the two lengths of tubing as follows: a. Use a Waters 1/16-inch stain less stee l tubing cu tter o r a file with a cu tting edge to

scribe the circumference of the tubing at the desired break point.

b. Grasp the tubing on both sides of the sc ri bed mark with cl ot h- or pl ast ic-covered

pliers (to prevent marring the surface), then gently work the tubing back and forth until it separates.

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

3. Assem ble a compre ssion fittin g (Figure 1-7 ) at both ends of the col umn outlet line and at one end of the detector outlet line.

10 Installation

broadening.

Compression

Screw

Ferrule

Tubing

Distance Determined by

the Union or Column Fitting

TP01139

Figure 1-7 Compression Screw Assembly

End Must be Straight

and Smooth to Prevent

Dead Volume

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

5. Seat the other end of the tubi ng in the fitt ing of the detector inlet, then tighten the compression screw as in step 4.

6. Seat the end of the detect or ou tl et tu bing wi th t he compress ion f itting in the detect or outlet fitting, then tighten the compression screw about 3/4-turn past finger-tight. Insert the other end of the tubing in the waste container.

STOP

Attention: To avoid damage to the flow cell, avoid pressures approaching its maximum allowable pressure, 1000 psi (70 kg/cm

1.6 Starting Up and Shutting Down the Detector

The entire startup procedure takes less than 1 minute. Once completed, you should allow the 2996 Detector to warm up for at least 1 hour before running an analysis. Follow the procedures in this section to ensure reliable detector performance.

Starting Up the Detector

To start up the 2996 Detector:

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 Millennium

Starting Up and Shutting Down the Detector 11

Help.

12 Installation

Note: Use only thoroughly degassed HPLC-grade solvents. Gas in the mobile phase may form bubbles in the flow cell and cause the detector to fail the Reference 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) to the 1 (On) position.

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

• If both lights remain illuminated, the detector passed the internal diagnostics.

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

Chapter 2, Diagnostics and Calibration.

5. Wait 1 hour for the 2996 Detector to stabilize before acquiring data.

Figure 1-8 2996 Detector Indicator Lights

Table 1-4 LED Indicator Sequences During Startup

Lamp LED Status LED Indicates Troubleshooting

OFF OFF No power or CPU

failure.

Check ac source and main fuse. Contact Waters Technical Service.

TP01460

LAM

WATERS 2996

Photodiode Array Detector

Lamp Indicator

Status Indicator

On/Off Switch

Starting Up and Shutting Down the Detector 13

Shutting Down the Detector

To shut down the 2996 Detector:

1. If the mobile phase contains buffers, set the solvent delivery syst em 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.

OFF FLASHING The 2996 is running

power-on self-tests.

FLASHING FLASHING 2996 has failed one of

the self-tests.

Contact Waters Technical Service.

OFF FLASHING The 2996 is running

Confidence tests.

ON FLASHING Lamp start was

successful. The 2996 is starting calibration.

ON FLASHING for more

than 30 seconds

The 2996 may not have calibrated correctly.

Could be air bubble in flow cell (Table 2-1). Contact Waters Technical Service.

ON ON Calibration was

successful.

Table 1-4 LED Indicator Sequences During Startup (Continued)

Lamp LED Status LED Indicates Troubleshooting

14 Installation

Diagnostics 15

Chapter 2 Diagnostics and Calibration

The Waters 2996 Photodiode Array Detector automatically runs a series of internal diagnostics upon sta rt up. The indica tor LEDs on the front of the detec tor and message s at the Millennium

Wor kstation show the results of the start up interna l diagnostics

(Figure 1-8). 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

Workstation. Additional information about the performance of the detector is also available through the PDA Calibration window, accessed from Run Samples in the Millennium

software.

If you encounter a problem that you cannot troubleshoot (Section 2.1, Diagnostics), contact Waters T e chnical Servi ce at (800) 25 2-4752, U.S. and Canadian customers only. Other customers, cal l you r l ocal Waters sub sidiary or your local Waters Technical Service Representative in Milford, Massachusetts (U.S.A.) for assistance.

2.1 Diagnostics

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

Table 2-1 2996 Detector Troubleshooting

Symptom Possible Cause Corrective Action

Both LEDs off No power 1. Check line cord connections.

2. Check outlet for power.

Blown fuse Replace fuse (Section 3.3, Replacing

the Fuses).

Status light bl inks and lamp light is off

The 2996 is running Confidence tests.

16 Diagnostics and Calibration

Status light bl inks and lamp light on

Failed startup diagnostics

Reseat and check alignment of flow cell.

Flush the flow cell (Section 3.1.1, Flushing the Flow Cell).

Insuffic ie nt energy reaching photodiode array because of air bubble, or dirty flow cell can cause shutter diagnostic to fail

Flush the flow cell (Section 3.1.1, Flushing the Flow Cell).

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

Weak lamp Replace lamp (Section 3.1.2,

Removing the Flow Cell).

Shutter failure message

Shutter failed Run the Shutter diagnostic. For details,

see Millennium

Help.

Detector not responding to Millennium

Workstation

Detector not connected to busLAC/E or to LAC/E

Acquisition Server in t he Millennium

Workstation

Check IEEE-488 cable connections, tighten connectors.

Incorrect IEEE-488 address

1. Ensure that the 2996 Detector IEEE-488 address is unique and within the range 2 to 29 (see the

Millennium

System Instal lation

and Configuration Guide).

2. Rescan the IEEE-488 bus. For details, see Millennium

Help.

Table 2-1 2996 Detector Troubleshooting (Continued)

Symptom Possible Cause Corrective Action

User-Initiated Diagnostics 17

2.2 User-Initiated Diagnostics

Note: The system administrator can restrict access to the 2996 Detector diagnostics by disabling user access to Run Samples. For details, refer to the Millennium

Help.

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

• Internal Tests – Tests run by the instrument firmware that help you determine the

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

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

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

You can run all user -initia ted diagnos tics from Run Sampl es in the Mill ennium

software.

For more information on Run Samples and PDA diagnostics, refer to

the Millennium

Help.

Change in reference spectrum

Mobile phase contain s gas or is contaminated

Prepare fresh mobile phase and degas thoroughly.

Air bubbles trapped in flow cell

Reseat and check alignment of flow cell.

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

T o prevent ai r bubbles, check that there is a 1- to 2-foot (30- to 60-cm) length of 0.009-inch (0.23-mm) ID tubing connected to the detector waste outlet.

Solvent in drain line Leak from flow cell

gasket

Rebuild flow cell with a new gasket (Section 3.1.3, Disassembling and Cleaning the Flow Cell).

Leak from flow cell fittings

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

Table 2-1 2996 Detector Troubleshooting (Continued)

Symptom Possible Cause Corrective Action

18 Diagnostics and Calibration

2.3 PDA Calibration

You can adjust, or calibrate, the 2996 Detector to ensure that wavelength readings are accurate. Recalibrate the 2996 Detector only if the Wavelength Accuracy diagnostic (in the Internal Diagnostics tests) fails. Calibration allows you to correct small errors in wavelength which may be caused by aging optics or excessive vibration.

You calibrate the 2996 Detector using the PDA Calibration window, which you access from Run Samples 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.

Note: The system administrator can restrict access to the PDA Calibration window by disabling access to Run Samples.

Note: Ensure that the flow cell is clean before you check calibration (Section 3.1.1, Flushing the Flow Cell).

To prepare for calibration:

1. Set the pump t o deli ver 1 mL/min of degassed methan ol fo r 10 minut es. If metha nol

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.

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

For information on pe rforming calibration, refer to the M illen nium32 Help.

STOP

Attention: Recalibrating the wavelength requires that spectral libraries be reentered.

Flow Cell Maintenance 19

Chapter 3 Maintenance

This chapter covers main tenanc e of the Waters 2996 Photo diode Ar ray De tect or flow cel l, lamp, and fuse.

3.1 Flow Cell Maintenance

The flow ce ll requires m aintenance w hen:

• The reference spectrum changes.

• The cell fluid leaks out of the drain tube.

• The Lamp diagnostic (in the Millennium

PDA Diagnostics window) fails and the

lamp status light is on (Table 2-1).

• The 2996 Detector causes high backpressure.

Note: Conditions other than a dirty flow cell may cause decreased lamp intensity. For more information, refer to Chapter 2, Diagnostics and Calibration.

Flow cell maintenance consists of:

• Flushing the flow cell

• Removing the flow cell

• Disassembling and cleaning the flow cell

• Installing the flow cel l assembly

Caution: To avoid possible electric shock, do not remove the 2996 Detector power supply covers. The power supply does not contain user-serviceable components.

20 Maintenance

3.1.1 Flushing the Flow Cell

Required Materials

• HPLC-grade water

• HPLC-grade methanol

If the flow cell requires cleaning, first try flushing it with solvent.

Procedure

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 for 10 minutes at 1 mL/min, then switch to a low-su rface-tension solvent s uch as metha nol.

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

3. Test the lamp energy by perf orming the Lamp diagnostic test. For details, refer to

the Millennium

Help.

If the lamp diagn ost ic fails and the lamp has not been used more than 2000 hours or 1 year (whichever comes first), disassemble the flow cell and clean the flow cell components using the procedure described in Section 3.1.2, Removing the Flow Cell.

3.1.2 Removing the Flow Cell

Note: You do not need to shut down the 2996 Detector to remove and replace the flow cell.

Required Materials

• 5/16-inch open-end wrench

• Phillips screwdriver

• Powder-fr ee gloves

STOP

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

Flow Cell Maintenance 21

Procedure

To remove the 2996 Detector flow cell:

1. Set the flow to 0.0 mL/min.

2. Power off the solvent delivery system or pump to avoid exposure to chemicals.

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

4. Lift up the 2996 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).

Figure 3-1 Removing the Flow Cell Assembly

6. Use the Phillips screwdr iver to loosen the thr ee thumbs crew s that hold the flo w

cell assembly to the optics bench and the thumbscrew that secures the bracket holding the flui dic co nne ction s, then detach th e bra cket .

7. Pull the flow cell assembly gently toward you to remove it from the detector

(Figure 3-2).

Caution: To avoid the possibility of leaking mobile phase, do not disconnect the inlet or outlet fluidic lines while there is pressure in the chromatographic system. Always vent your system before disconnecting fluidic lines.

TP01462

Thumbscrews

Holding Flow

Cell Assembly

and Bracket

Fluidic Connections (Inside Bracket)

Thumbscrews

Holding Flow

Cell Assembly

and Bracket

Flow Cell Body

Lens Holder

Assembly

Fluidic

Connections

Figure 3-2 Flow Cell and Fluidic Connections Assemblies

Bracket

TP01463

3.1.3 Disassembling and Cleaning the Flow Cell

Attention: The lens surface finish and the alignment of the lenses are critical to the

STOP

performance of the 2996 Detector. Be careful not to touch or damage the lenses and the lens holders.

Attention: To prevent lens contamination, use powder-free gloves when disassembling, inspecting, cleaning, or replacing parts within the flow cell or when removing or replacing the flow cell within its assembly.

Required Materials

• TORX™ T10 screwdriver

• Small, flat-blade screwdriver

• Lens tissue or nonparticulating swab

22 Maintenance

Flow Cell Maintenance 23

• HPLC-grade methanol

• Belleville spring washer

• Flow cell gasket

• Powder-fr ee gloves

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 3-3).

Figure 3-3 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.

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

STOP

Attention: Solvents other than methanol may damage a disassembled flow cell. In normal use, the gasket protects the lens holder from solvents.

TP01464

Gasket

Flow Cell Disk

Lens Assembly

Flow Cell Disk

Belleville

Spring

Washer

Lens Assembly

Flow Cell

Body

Spring

Washer

Slot for

Removing

Lens

Assembly

Screws

24 Maintenance

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.

Reassembling the Flow Cell

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

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 (with the concave 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, the n tighten each screw 1/4-turn. If a torque screwdriver is available, tighten the screws to 16 in-oz (0.113 N-m).

6. Check for leaks. If you find any leaks, repeat step 5.

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

3.1.4 Installing the Flow Cell Assembly

To in stall the flow cell assembly:

1. While you hold the flow cell assembly in a vertical orientation (Figure 3-2), insert

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

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

3. Hand-tighten the thumbscrews.

4. Reconnect the fluidic lines.

STOP

Attention: Be careful not to overtighten the screws.

STOP

Attention: The alignment of the flow cell in the optics bench is critical to detector operation. Be careful not to damage the flow cell body.

Replacing the Lamp 25

5. Replace the front cover.

6. Flush the flow cell (Section 3.1.1, Flushing the Flow Cell).

3.2 Replacing the Lamp

Replace the lamp in the 2996 Detector when either of the following conditions exists:

• The sampling rate requires an excessively long exposure time (more than 100 milliseconds).

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

Note: An improperly inserted flow cell could appear to be a problem with the lamp.

Note: If lamp intensity is low, but the lamp has not been used for 2000 hours, you may be

able to increase lamp intensity by cleaning the flow cell (Section 3.1, Flow Cell Maintenance).

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

Caution: To avoid electrical hazards and exposure to UV light, turn off the power and disconnect the power cord before you begin this procedure.

Caution: 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.

STOP

Attention: Do not touch the lamp glass while unpacking or inserting the lamp. Touching lamp glass damages the lamp and reduces life expectancy.

STOP

Attention: To prevent lamp glass contamination, use powder-free gloves when removing or replacing the lamp.

Caution: The lamp may be very hot! Please allow it to cool for at least 15 minutes before you attempt to remove it.

Required Materials

• Slotted head screwdriver

• Powder-fr ee gloves

Caution: To avoid electrical hazards when you perform the following procedure, power off the 2996 Detector and disconnect the power cord.

Procedure

To replace the la mp:

1. Power off the 2996 Detector, disconnec t t he power cord, and allow the lamp to cool

for at least 15 minutes.

Caution: To avoid the possibility of contacting hot surfaces, wait at least 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 lamp secure panel.

Alignment

Notch

Mounting

Screws

Figure 3-4 Lamp Power Connector and Mounting Screws

26 Maintenance

Lamp

Lamp Power Connector

Lamp Usage Indicator

TP01466

Replacing the Fuses 27

4. Use a slotted head screwdriver to unscrew the two mounting screws.

5. Grip the metal base of the lamp, pull the lamp out, and set it aside. Do not pull the

wires to remove the lamp.

6. Carefully unpack the replacement lamp.

7. While wearing powder-free gloves and holding the lamp by its base, position the

lamp so that the not ch in the base aligns with the pos it ioning pin in the optics bench.

8. Insert the lamp and secure it with the two screws. Make sure that the lamp base is

flush against the lamp housing.

9. Reconnect the lamp power connector (Figure 3-4).

10. Secure the lamp access panel.

11. Install the front panel cover.

12. Reconnect the power cord and power on the 2996 Detector.

3.3 Replacing the Fuses

Replace the fuses unde r the conditions indicated in the troub les hooting table (Section 2.1, Diagnostics). The 2996 Detector requires two 2 A, 250 V fuses (5 mm × 20 mm).

To replace the two fuses in the 2996 Detector:

1. Power off the 2996 Detector and remove the power cord.

2. Locate the fuse block below the power cord plug (Figure 3-5) on the rear panel.

Figure 3-5 Fuse Block

Caution: To avoid electrical hazards, power off the 2996 Detector and disconnect the power cord before you perform the following procedure.

Squeeze Side Clips to

Access Fuses

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

4. Remo ve the fuses from the block, then ins tall the new fuses.

5. Position 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 2996 Detector.

28 Maintenance

2996 Detector Optics 29

Chapter 4 Principles of the 2996 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 2996 PDA Detector.

4.1 2996 Detector Optics

The 2996 Detector is an ultraviolet/visible light (UV/Vis) spectrophotometer with:

• 512 photodiodes

• Optical resolution of 1.2 nm per diode

• Operating wavelength range from 190 to 800 nm

The light path through the optics assembly of the 2996 Detector is shown in Figure 4-1.

30 Principles of the 2996 PDA Detector Optics

Figure 4-1 Optics Assembly Light Path

Table 4-1 describes the optics assembly components in the 2996 Detector.

Table 4-1 Optics Assembly Components

Component Function

Lamp and lamp optics

Focuses light from the deuterium so urce lamp via a mirror through a beamsplitter to the flow cell.

Beamspl itter and reference diode

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

Flow cell assembly

Houses the segment of the flow path (conta ining eluent and sa mple) 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.

Reference

Diode

Beamsplitter

Assembly

50-µm Aperture

Lamp and

Lamp Optics

Spectrographic

Mirror and Mask

Shutter

Assembly

Flow Cell

Assembly

Grating

Photodiode

Array

Resolving Spectral Data 31

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 apertu re width determines:

• Attainable wavelength bandwidth at the photodiode array

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

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 shows an absorbance spectrum of benzene obtained from the 2996 Detector using the 50-µm aperture. In this spectrum, the wavelength resolution is sufficient to resolve five principal benzene absorption peaks.

Spectrograph mirror and mask

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

Aperture Controls wavelength resolution and intensity of light striking the

photodiodes. The width of the aperture is 50 µm.

Shutter assembly Prevents light from reaching the photodiode array except during

sampling and calibration. For details on the dark current, see Section 4.4.1, Calculating Absorbance.

Grating Disperses the light into bands of wavelengths and focuses those

wavelength bands onto the plane of the photodiode array.

Second-order filter

Reduces the contribution of second-order reflection of UV light (less than 370 nm) to the light intensity observed at visible wavelengths (greater than 370 nm).

Photodiode array An array of 512 photodiodes arranged li nearl y. The diode width and

spacing provide a single wavelength resolution of 1.2 nm.

Table 4-1 Optics Assembly Components (Continued)

Component Function

32 Principles of the 2996 PDA Detector Optics

Figure 4-2 Benzene Spectrum at 1.2 nm Resolution

4.3 Measuring Light at the Photodiode

The 2996 Photodiode Array Det ector mea sures the amount of light s triking the photodi ode array to determine the absorbance of the sample in the flow c ell.

The array consists of 512 photodiodes arranged in a row. Each photodiode acts as a capacitor by holding a fixed amount of charge.

Light striking a photodiode discharges the diode (Figure 4-3). The magnitude of the discharge depends on the amount of light striking the photodiode.

Absorbance

Sample in flow cell

absorbs at specific

wavelengths.

Deuterium

Lamp

Grating

Light from grating

dispersed into 1.2-nm

wavelength beams

continuously

discharges diodes.

Mirror

Figure 4-3 Photodiodes Discharged by Light

Flow

Cell

The 2996 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 2996 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 2996 Detector requires less than 10 msec to sequentially read all of t h e d iod es in the array and process the data. The minimum exposure time is 10 msec. You can set exposure time from 10 to 500 msec.

For example, if an exposure time is set to 50 milliseconds, the 2996 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

Measuring Light at the Photodiode 33

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

510 photodiodes

4. Waits approximately 45 msec before beginning the recharge-and-reading sequence

with diode 1 after all diodes have been recharged and read.

You set the exposure time parameter in the General tab of the 2996 PDA Instrument Method Editor. You can specify either Auto Expo sure or Expo sur e T i me. For det ails, refe r to the Millennium

Help.

Note: For best signal-to-noise performance, adjust the wavelength range optimize autoexposure computations. For details, refer to the Millennium

Help.

Using the Auto Exposure Parameter

The Auto Exposure time parameter allows the 2996 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 using a single Deuterium light source from 190 to 800 nm. To minimize detector noise, Auto Exposure adjusts the exposure time from 50 to 90% 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 2996 Detector:

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

• Limits the exposure so that no diode within the given wavelength range discharges more than 90%

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

The Auto Exposure time setting may not support certain combinations of sampling rates, wavelength ranges, or filter time-constant settings required for your analysis. If this is the case, you can set the exposure time manuall y to adjus t the exposure time from expe riment to experim ent.

Using the Exposure Time Parameter

The Exposure Time parameter enables you to manually set the length of tim e the photodiodes are exposed to light before they are read. The supported range is 10 to 500 msec.

34 Principles of the 2996 PDA Detector Optics

Computing Absorbance Data Points 35

Note: Changing exposure times within a set of samples can cause changes in baseline noise.

Be aware that increasing the Exposure Time parameter has the potential to saturate the photodiodes. A longer exposure time may cause the 2996 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 the next topic “Optimizing the Signal-to-Noise Ratio”).

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 (Appendix C, Mobile Phase Absorbance). Setting the bandwidth to higher values can improve signal-to-noise ratios.

4.4 Computing Absorbance Data Points

The 2996 Detector calculates absorbance values before transmitting the data to the Millennium

database. To calculate absorbance, the 2996 Detector:

• Computes the absorb ance at each diode using the dark current and reference spectrum (Section 4.4.1, Calculating Absorbance)

• Averages the absorbances at a particular wavelength as specified in the spectra per second sample rate and reports the average as a single data point (Section 4.4.2, Resolution)

• Can apply a filter (Section 4.4.3, Filtering Data)

4.4.1 Calculating Absorbance

The detector computes absorbance by subtracting the dark current and referenc e s pec tr um from the acquired spectrum. Absorbance is based on the principles of Beer’s Law.

36 Principles of the 2996 PDA Detector Optics

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 is expressed as:

A = εlc where: A = absorbance

ε = molar absorptivi ty

l = path length (1.0 cm in the 2996 Detector normal flow cell) c = molar concentration

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). The absorbance of mobile phase can reduce the linear range by the amounts shown in Appendix C, Mobile Phase Absorbance.

Figure 4-4 Absorbance as a Function of Concentration

Ideal

Actual

Absorbance

Working Range

Background Absorbance

Concentration

Dark Current

Photodiodes los e char ge over t ime even when t hey are not exposed t o light. Th e amount of charge lost is called dark c urrent.

At the start of a chromatographic run, the 2996 Detector closes the shutter to take a dark current reading for each d iode. Th e shutt er cl os es after th e exposu re ti me is c alcul ated an d 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.

Reference Spectrum

Immediately after the dark current measurement and before any components are eluted, the 2996 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.

Note: For best results, the reference spectrum should be representative of the initial mobile phase.

Note: For extremely long exposure times, the dark current and reference spectrum readings may take several seconds to finish.

Absorbance

The 2996 Detector calculates the absorbance for each diode at the end of each exposure time using the following equation:

Sn Dn–()

Absorbance

where:

S = obtained during sample analysis D = obtained during the dark current test R = obtained from the reference spectrum n = diode number

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

log=

Rn Dn–()

Computing Absorbance Data Points 37

4.4.2 Resolution

The data reported by t he 2996 Detec tor to the Mill ennium32 database can be th e 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 2996 Detector is

1.2 nm. For example, in 3D mode, the 2996 Detector averages three adjacent diodes for each reported wavelength when the spectral resolution is set in the Millennium to 3.6 nm. In 2D mode, absorbance values are computed based on the bandwidth setting. 2D mode is supported in Millennium

Averaging Chromatographic Data Based On Sample Rate

Sample rate i s the nu mber of da ta point s per se cond r eported to the Mil lennium32 database. The number of ti mes the p hotodio des are read d uring the sample r ate i nterv al is de pendent on the exposure tim e. For example, if exposure time is 25 msec, and sample rate is 1 sec, then readings per data point are:

software versions 4.0 or higher.

1000 msec

------------------------25 msec

40=

software

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.

Note: The data storage rate is based on wavelength range, spectral resolution, and sample rate, which are set in the General tab of the 2996 PDA Instrument Method Editor. For details, refer to the Millennium

Help.

4.4.3 Filtering Data

Use the General tab of the 2996 PDA Instrument Method Editor (for details, refer to the

Millennium

38 Principles of the 2996 PDA Detector Optics

Help) to apply an optional noise filter (the Filter Response parameter) to

the data sent to the Millennium32 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 fo llowing with regard to fi ltering data:

• The noise filter is a digital (low pass) filter.

• The filte r 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 0.1- to 3-second RC filter.

Computing Absorbance Data Points 39

40 Principles of the 2996 PDA Detector Optics

Comparing Absorbance Spectra 41

Chapter 5 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 2996 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 2996 Detector, the Spectral Contrast technique determine s whether di ffe rences bet ween spe ctra are due t o 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 Spectra

The shape of an absor bance sp ectru m is det ermine d by the re la tive a bsorban ce at di f fere nt 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 shows the absorbance spectra for the two compounds, A and B. The ratio of t he 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 rati os of two wavel en gth pairs is a limited spectral comparison. For more information, you need to compare the absorbance ratios of multiple wavelength pairs.

Compound A:

245 nm

257 nm

Compound A

Figure 5-1 Comparing Spectra of Two Compounds

Compound B:

Compound B

245

----------- ---- 2 . 2= Ab

257

245

---------- ----- 0 . 7= Ab

257

5.2 Representing Spectra as Vectors

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

• Length – Proportional to analyte concentration.

• Direction – Determined by the relative absorbance of the analyte at all wavelengths (its absorbance spectrum). Direction is independent of concentration for peaks that are less than 1.0 AU across the collected wavelength range.

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 spec tral resolut ion increase , the precision of a spectral vecto r for the resultant spectrum increases. A vector derived from the 2996 PDA Detector can include absorbances in any range from 190 to 800 nm. To enhance spectral sensitivity, set the bench resolution to 1.2 nm.

42 Spectral Contrast Theory

Representing Spectra as Vectors 43

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) based on the spectra depicted in Figure 5-1.

Figure 5-2 Plotting Vectors for Two Spectra

The axes in Figure 5-2 are in absorbance units at the two wavelengths used to calculate the absorbance ratio shown in Figure 5-1. The head of the vector for Compound A is at the 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 Spec tral Cont rast angle (e .g., θ in Figure 5-2) greater than zero ind icates a shape difference between spectra (Section 5.3, Spectral Contrast Angles). The length of the vector is proportional to the concentration.

5.2.2 Vectors Derived from Multiple Wavelengths

When absorbance ratios are limited to two wavelengths, the chance that two different spectra will have the same absorbance ratio is much gr eater than if comparison is made using absorbance ratios at many wavelengths. Therefore, the Spectral Contrast technique

AU at 257 nm

AU at 245 nm

44 Spectral Contrast Theory

uses absorbances from multiple wavelengths to form a vector in an n-dimensional vector space, where n is the number of wav elengths from the spectrum.

To compare two spectra, the Spectral Contrast techni que for ms a vector for each sp ectru m in an n-dimensional space. The two spectral vectors are compared mathematically to compute the angle between the two vectors.

Just as in the two-wavelength comparison, a Spectral Contrast angle of zero in n-dimensional space means that all ratios of absorbances at corresponding wavelengths match. Conversely, if any comparison of ratios doe s not mat ch, the corresponding vectors point in different directions.

5.3 Spectral Contrast Angles

Spectra that have the same sha pe have vectors tha t point in the same dire ction. Spectra th at have differ ent shapes have vector s that poi nt in dif ferent directi ons. The angl e between t he two vectors of any two spectra, the Spectral Contrast angle, quantifies the magnitude of the shape difference between the spectra. The Spectral Contrast angle is the difference in direction between the spectral vectors of two spectra.

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

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 distinctl y di fferent, and therefore, have a large Spectral Contrast angle.

Spectral Contrast Angles 45

Figure 5-3 Spectra with a Large Spectral Contrast Angle

Spectra with Similar Shapes

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

Normalized Absorbance

Wavelength (nm)

Compound A

Compound B

Spectral Contrast Angle: 62.3°

46 Spectral Contrast Theory

Figure 5-4 Spectra with a Small Spectral Contrast Angle

Differences Between Spectra of the Same Compound

Small but significa nt dif fe rence s between a bsor bance sp ectra can occur because of facto rs other than those due to the absorbance properties of different compounds. For example, multiple spectra of the same compound may exhibit slight differences because of det ect or noise, photometri c error, high sample concentration, or variations in solvent conditions. The spectra in F igure 5-5, for example, show how inst rument n ois e can a f fect the sha pe o f an absorbance spectrum of one compound. This effect is most likely to occur at low concentratio ns wher e the signa l-to -no ise r atio is l ow. Note that th e S pectra l Cont rast angle between these absorbance spectra of the same compound is 3.4°.

Normalized Absorbance

Wavelength (nm)

Compound A

Compound B

Spectral Contrast Angle: 3.0°

Undesirable Effects 47

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

5.4 Undesirable Effects

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

• Detector noise

• Photometric error caused by high sample concentration

• Variation in solvent co mposition

These sources of spectral variation can cause chemically pure, baseline-resolved peaks to exhibit a sma ll leve l of s pectra l in homogenei ty. You can assess the signi fica nce of s pectr al inhomogeneity by comparing a Spectral Contrast angle to a Threshold angle (Section 5.4.4).

5.4.1 Detector Noise

Statistical and thermal variations add electrical noise to the absorbance measurements made by the 2996 Detector. The noise manifests itself as fluctuations in the baseline, known as baseline noise. The magnitude of any absorbance differences caused by statistical an d thermal varia tions can be pre dicted from the i nstrument nois e in the base line region of a chromatogram.

Normalized Absorbance

Wavelength (nm)

Spectral Contrast Angle: 3.4°

Normalized Spectra of a Compound at Different

Concentrations

48 Spectral Contrast Theory

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 thi s le vel may have a neg li gib le effect on quantitation, they can be a significant sour ce of spe ctr al inh omogen eity. To minimize the effects of pho tometr ic 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 wavelength. For examples of mobile phase absorbance, see Appendix C, Mobile Phase Absorbance.

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

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 absorba nce, if any, by the solvent remains con stant. 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

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

Comparison of a Spectr al Contras t angle to its Thr eshold angle can assi st in dete rmining if the shape difference between spectra is genuine, that is, generated by mixtures that are dissimilar. In general, a Spectral Contrast angle le ss than its Thres hold angle indicat es 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 Thresh old angle indi cates that the sha pe differences are due to ge nuine dif fer ences between the spectra. When automating the spectra contrast comparison, the maximum absorbance of the spectra must not exceed 1 AU.

Absorbance

Effect of pH

pH 6.9

pH 5.1

pH 3.1

Wavelength (nm)

Effect of Concentration

Absorbance

Note position of Maxima can be shifted.

Figure 5-6 Effects of pH and Solvent Concentration

on the Absorbance Spectrum of p-Aminobenzoic Acid

Wavelength (nm)

Undesirable Effects 49

50 Spectral Contrast Theory

Detector Specifications 51

Appendix A Detector Specifications

Table A-1 lists the 2996 PDA Detector specifications.

Table A-1 2996 Detector Specifications

Item Specification

Dimensions Width: 11.5 in. (29 cm)

Depth: 24 in. (61 cm)

Height: 8.5 in. (22 cm) Weight 31.5 lbs (14.3 kg) Wavelength range 190 to 800 nm Wavelength accuracy ±1 nm Linearity range

*Per ASTM 685-79

5% at 2.0 AU, propylparaben, at 256 nm Spectral resolution 1.2 nm Baseline noise ±1.5 × 10

–5

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

Drift 1 × 10

–3

AU/hour at 254 nm (after warmup)

∆T ≤ 1°C per hour Flow cells

Standard Semi-preparative Variable path flow cell Microbore Inert Autopurification

Pathlength (mm): Tubing (ID):

10 0.009 in.

3 0.040 in.

0.15 to 3 0.004 in.

3 0.005 in.

10 0.010 in.

0.5 0.009 in. (A Inlet)

0.020 in. (P Inlet)

0.040 in. (Common Outlet)

52 Detector Specifications

Spare Parts 53

Appendix B Spare Parts

The spare parts listed in Table B-1 are recommended for customer installation. Damage incurred by performing unauthorized work on your 2996 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 Flow cell, Autopurification 289000612 Variable pathlength flow cell WAT057664 Gasket, flow cell (2) WAT057924 Belleville washer (2) WAT057925 Lens mount and lens (2) WAT057923 Semi-prep lens kit WAT057968 Deuterium lamp WAT052586 (PM Kit) Fuse, fast 4A, 250 V (5 × 20 mm) WAT057337 Waters

Erbium Perchlorate Wavelength

Accuracy Solution

WAT042885

Waters Absorbance Detector Linearity Solution

WAT042881

54 Spare Parts

Mobile Phase Absorbance 55

Appendix C Mobile Phase Absorbance

This appendix lists 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 wavelen gths. Suc h a mobile ph ase ensure s that any absorbanc e is due only to the sample. Absorbance by the mobile phase also reduces the linear dynamic range of the detector by the am ount 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 in Table C-1.

Table C-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 0.050.030.020.010.01<0.01————

Methanol (not degassed)

2.06 1.00 0.53 0.37 0.24 0.11 0.05 0.02 <0.01 —

Methanol (degassed)

1.91 0.76 0.35 0.21 0.15 0.06 0.02 <0.01 — —

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)

2.44 2.57 2.31 1.80 1.54 0.94 0.42 0.21 0.09 0.05

Unstablized Tetrahydrofuran (THF, old)

>2.5 >2.5 >2.5 >2.5 >2.5 >2.5 >2.5 >2.5 2.5 1.45

Table C-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%

Trifluoroacetic acid

Diammonium phosphate, 50 mM

Triethylamine, 1%

Buffers and Salts

Ammonium acetate, 10 mM

Ammonium bicarbonate, 10 mM

0.110.02<0.01———————

<0.01—————————

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

1.850.670.150.02<0.01—————

2.33 2.42 2.50 2.45 2.37 1.96 0.50 0.12 0.04 <0.01

1.88 0.94 0.53 0.29 0.15 0.02 <0.01 — — —

0.410.100.01<0.01——————

EDT A, disodium, 1 mM

HEPES, 10 mM, pH 7.6

MES, 10 mM, pH 6.0

56 Mobile Phase Absorbance

0.11 0.07 0.06 0.04 0.03 0.03 0.02 0.02 0.02 0.02

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 — — —

Table C-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————————

0.530.160.050.01<0.01—————

1.85 0.96 0.52 0.30 0.15 0.03 <0.01 — — —

2.001.670.400.10<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

1.99 0.75 0.19 0.06 0.02 0.01 0.01 0.01 0.01 <0.01

1.400.770.280.100.04<0.01————

1.801.901.110.430.13<0.01————

Mobile Phase Absorbance 57

Table C-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

Wat er s P IC® 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

J 35, 1% 0.06 0.03 0.02 0.02 0.02 0.01 <0.01 — — —

BRI

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% 0.020.01<0.01———————

Triton X-100, 0.1%

Tween™

0.1%

20,

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

58 Mobile Phase Absorbance

Index 59

I N D E X

Absorbance

maximum 48 mobile phase 55 photometric error 48 solvent change effects 49 Waters 2996 calculations 35, 37

Acquisition

Auto Exposure parameter 34

Exposure Time parameter 34 Analog output specifications 6, 7 Aperture width 31 Auto Exposure parameter 34

Beer’s law 36, 48

Calibration 18 Column, connecting 10 Compression fittings 10 Connections

column 10

events 8

fluidic 10

non-IEEE-488 6

rear panel 3

terminal strip 8 Contact closures 6 Contacting Waters Te chni ca l Service 15, 17 Conventions, documentation x x

Dark current 37 Data acquisition

Auto Exposure parameter 3 4

Exposure Time parameter 34 Derived vectors 43, 44 Diagnostics 17 Documentation convention s xx

Electrical connections 2 Events

connections 6, 8

electrical specifications 9

terminal strip connections 7 Exposure Time parameter 3 4

Fittings 10 Flow cell

cleaning 23

exploded view 23

flushing 20

maintenance 19

removing 20 Fluid

connecting lines 10

fittings 10 Fuses

IEC-rated 2

maintenance 27

replacement 27

Index

60 Index

I N D E X

IEC-rated fuses 2 Inputs 6, 8, 9 Installation

electrical 2 fluidic 10 site selection 1

Instrument method

Auto Exposure parameter 34 Exposure Time parameter 34

Lamp

hardware theory 30 replacement 25, 26

Maintenance

flow cell 19 fuse 27 lamp 25

PDA detector 19–28 Match Angle, photometric error effe cts 48 Maximum absorbance 48 Millennium Chromatography Manager,

connections 3

Mobile phase

absorbances 55

wavelengths 55

Noise effects 47 Nonidealities 47 Non-IEEE-488 connections 6

Outputs 6, 8, 9

Parts, spare 53 Photodiode array 32 Photometric error 48 Power connections 2 Purity Angle, photometric error effects 4 8

Rear panel connections 3 Reference spectrum 37

Shutdown, procedure 13 Solvent A ngle, photometric error effects 48 Solvent changes 48 Spare parts 53 Specifications

analog output 6, 7 event inputs 9 event outputs 9 Waters 2996 51

Spectra

derived vectors 43, 44 spectral shape differences 47 vectors 42

Spectral Contrast

derived vectors 43, 44 spectral shape differences 47 theory 4 1–49 vectors 42

Spectral resolution 31

Index 61

I N D E X

Spectrum match, spectral shape differences

Start up, procedure 11

Terminal strip

connections 7, 8

diagram 8 Threshold angle 47 Troubleshooting 15–18 Tubing, cutting 10

Undesirable effects, shape differences 47

Vectors

derived from multiple wavelengths 44

derived from two wavelengths 43

spectra, representing 42

spectral contrast 4 2

Waters 2996

absorbance calculations 35, 37

aperture width 31

dark current 37

detector optics, overview 29–31

hardware theory 29–39

photodiode array overview 32

reference spectrum 37

spare parts 53

specifications 51

spectral resolution 31

Waters Technical Ser vi ce, contacting 15, 17 Wavelength

accuracy 18 derived vectors 43, 44 mobile phase absorbances 55

I N D E X

62 Index

Waters 2996 Operator's Manual

Specifications and Main Features

Frequently Asked Questions

User Manual