Waters 2475 Multiwavelength Fluorescence Detector Operator`s Guide

Waters 2475 Multiwavelength Fluorescence Detector Operator`s Guide
2475 Multi λ
Fluorescence Detector
Operator’s Guide
71500247502/Revision F
Copyright © Waters Corporation 2010
All rights reserved
Copyright notice
© 2010 WATERS CORPORATION. PRINTED IN THE UNITED STATES OF
AMERICA AND IN IRELAND. ALL RIGHTS RESERVED. THIS
DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY
FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER.
The information in this document is subject to change without notice and
should not be construed as a commitment by Waters Corporation. Waters
Corporation assumes no responsibility for any errors that may appear in this
document. This document is believed to be complete and accurate at the time
of publication. In no event shall Waters Corporation be liable for incidental or
consequential damages in connection with, or arising from, its use.
Trademarks
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LAC/E, SAT/IN, and “THE SCIENCE OF WHAT’S POSSIBLE.” are
trademarks of Waters Corporation.
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Other registered trademarks or trademarks are the sole property of their
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ii
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Contacting Waters
®
Contact Waters with enhancement requests or technical questions regarding
the use, transportation, removal, or disposal of any Waters product. You can
reach us via the Internet, telephone, or conventional mail.
Waters contact information
Contacting medium
Information
Internet
The Waters Web site includes contact
information for Waters locations worldwide.
Visit www.waters.com.
Telephone and fax
From the USA or Canada, phone 800
252-HPLC, or fax 508 872 1990.
For other locations worldwide, phone and fax
numbers appear in the Waters Web site.
Conventional mail
Waters Corporation
34 Maple Street
Milford, MA 01757
USA
Safety considerations
Some reagents and samples used with Waters instruments and devices can
pose chemical, biological, and radiological hazards. You must know the
potentially hazardous effects of all substances you work with. Always follow
iii
Good Laboratory Practice, and consult your organization’s safety
representative for guidance.
Safety advisories
Consult Appendix A for a comprehensive list of warning and caution
advisories.
Operating this instrument
When operating this instrument, follow standard quality-control (QC)
procedures and the guidelines presented in this section.
Applicable symbols
Symbol
Definition
Manufacturer
Authorized representative of the European
Community
Confirms that a manufactured product complies
with all applicable European Community
directives
Australia C-Tick EMC Compliant
Confirms that a manufactured product complies
with all applicable United States and Canadian
safety requirements
Consult instructions for use
Audience and purpose
This guide is intended for personnel who install, operate, and maintain the
2475 Multi λ Fluorescence detector.
iv
Intended use of the instrument
Waters designed the 2475 Multi λ Fluorescence Detector to analyze samples
in high-performance liquid chromatography (HPLC) applications. The 2475
Multi λ Fluorescence detector is for research use only
Calibrating
To calibrate LC systems, follow acceptable calibration methods using at least
five standards to generate a standard curve. The concentration range for
standards should include the entire range of QC samples, typical specimens,
and atypical specimens.
When calibrating mass spectrometers, consult the calibration section of the
operator’s guide for the instrument you are calibrating. In cases where an
overview and maintenance guide, not operator’s guide, accompanies the
instrument, consult the instrument’s online Help system for calibration
instructions.
Quality-control
Routinely run three QC samples that represent subnormal, normal, and
above-normal levels of a compound. Ensure that QC sample results fall within
an acceptable range, and evaluate precision from day to day and run to run.
Data collected when QC samples are out of range might not be valid. Do not
report these data until you are certain that the instrument performs
satisfactorily.
ISM classification
ISM Classification: ISM Group 1 Class B
This classification has been assigned in accordance with CISPR 11 Industrial
Scientific and Medical (ISM) instruments requirements. Group 1 products
apply to intentionally generated and/or used conductively coupled
radio-frequency energy that is necessary for the internal functioning of the
equipment. Class B products are suitable for use in both commercial and
residential locations and can be directly connected to a low voltage,
power-supply network.
v
EC authorized representative
Waters Corporation (Micromass UK Ltd.)
Floats Road
Wythenshawe
Manchester M23 9LZ
United Kingdom
vi
Telephone:
+44-161-946-2400
Fax:
+44-161-946-2480
Contact:
Quality manager
Table of Contents
Copyright notice ................................................................................................... ii
Trademarks ............................................................................................................ ii
Customer comments ............................................................................................ iii
Contacting Waters ............................................................................................... iii
Safety considerations .......................................................................................... iii
Safety advisories ................................................................................................. iv
Operating this instrument ................................................................................. iv
Applicable symbols ............................................................................................. iv
Audience and purpose......................................................................................... iv
Intended use of the instrument........................................................................... v
Calibrating ........................................................................................................... v
Quality-control ..................................................................................................... v
ISM classification .................................................................................................. v
ISM Classification: ISM Group 1 Class B .......................................................... v
EC authorized representative ........................................................................... vi
1 Theory of Operation .............................................................................. 1-1
Fluorescence theory ......................................................................................... 1-2
Fluorescence detection ....................................................................................
Overview...........................................................................................................
Excitation sources ............................................................................................
Types of light sources ......................................................................................
Excitation wavelength selection .....................................................................
Exciting the sample .........................................................................................
Flow cell............................................................................................................
1-3
1-3
1-3
1-3
1-3
1-4
1-4
Measuring fluorescence .................................................................................. 1-4
Quantitation..................................................................................................... 1-4
Emission wavelength selection ....................................................................... 1-4
Table of Contents
vii
Photomultiplier tube........................................................................................
Scanning ...........................................................................................................
Multichannel operation ...................................................................................
Fluorescence data ............................................................................................
References ........................................................................................................
1-4
1-5
1-5
1-5
1-6
Detector description ........................................................................................ 1-7
Features............................................................................................................ 1-7
Principles of operation .................................................................................... 1-9
Detector optics.................................................................................................. 1-9
Optics assembly light path ............................................................................ 1-10
Photomultiplier (PMT) calibration ............................................................... 1-12
PMT sensitivity.............................................................................................. 1-12
Filtering noise ................................................................................................ 1-12
Electronics ...................................................................................................... 1-13
Wavelength verification and test .................................................................. 1-14
Operational modes .......................................................................................... 1-14
Single-channel mode...................................................................................... 1-14
Multichannel mode ........................................................................................ 1-15
Spectrum scanning ......................................................................................... 1-16
Lamp energy and performance ................................................................... 1-16
Auto-optimize gain .........................................................................................
Method optimization......................................................................................
Example of recommended method development approach..........................
Ensuring gain optimization for each peak of interest .................................
1-17
1-18
1-19
1-20
Startup diagnostic tests ................................................................................ 1-21
Mobile-phase solvent degassing .................................................................. 1-21
Wavelength selection ..................................................................................... 1-22
2 Setting Up the Detector ........................................................................ 2-1
Before you begin ............................................................................................... 2-2
Installing the detector ..................................................................................... 2-3
viii
Table of Contents
Plumbing the detector .....................................................................................
Connecting columns.........................................................................................
Assembling fittings ..........................................................................................
Making tubing connections .............................................................................
2-3
2-3
2-4
2-5
Making signal connections ............................................................................. 2-5
Component connection overview..................................................................... 2-6
Connecting the Ethernet cable........................................................................ 2-7
Choosing signal connections.......................................................................... 2-10
Making I/O signal connections ...................................................................... 2-11
Signal connections ......................................................................................... 2-12
Connecting an Alliance separations module ................................................ 2-14
Connecting RS-232 devices ........................................................................... 2-18
Connecting Ethernet devices......................................................................... 2-20
Connecting other devices ..............................................................................
Required materials ........................................................................................
Connecting cables ..........................................................................................
Connecting a data system using a Bus SAT/IN module ..............................
Connecting a 746 data module ......................................................................
Connecting a chart recorder ..........................................................................
Connecting a 600-series pump ......................................................................
Connecting a 717plus Autosampler ..............................................................
2-23
2-23
2-23
2-24
2-27
2-28
2-29
2-33
Connecting to the electricity source .......................................................... 2-36
3 Using the Detector ................................................................................. 3-1
Starting the detector ........................................................................................
Initializing the detector ...................................................................................
Startup failure .................................................................................................
Idle mode ..........................................................................................................
3-2
3-2
3-3
3-4
Using the operator interface .......................................................................... 3-4
Using the display ............................................................................................. 3-4
Fluorescence and message icons ..................................................................... 3-5
Using the keypad ............................................................................................. 3-9
Navigating the user interface ....................................................................... 3-16
Navigating to and from the home screen...................................................... 3-16
Table of Contents
ix
x
Preparing to start a run ................................................................................
Setting up a run .............................................................................................
Accessing primary and secondary functions ................................................
Operating the trace and scale functions.......................................................
Configuring the detector................................................................................
Configuring event inputs and contact closures ............................................
Setting pulse periods .....................................................................................
Setting the Display Contrast ........................................................................
Displaying system information .....................................................................
Using Online Help .........................................................................................
3-18
3-18
3-19
3-23
3-25
3-25
3-26
3-27
3-28
3-28
Operating the detector ..................................................................................
Two operating modes .....................................................................................
Standalone operation.....................................................................................
Remote control operation for 474 emulation mode via RS-232 ...................
Remote control operation via Ethernet connection using 2475 instrument
control software........................................................................................
Verifying the detector ....................................................................................
Manual wavelength calibration ....................................................................
Normalizing emission units ..........................................................................
Operating the detector in single-channel mode ...........................................
Operating the detector in multichannel mode .............................................
Setting gain and EUFS..................................................................................
3-28
3-29
3-29
3-29
Programming methods and events .............................................................
Storing methods .............................................................................................
Programming timed events ...........................................................................
Programming threshold events.....................................................................
Storing a method............................................................................................
Retrieving a method ......................................................................................
Viewing events within a method...................................................................
Resetting a method ........................................................................................
Clearing events ..............................................................................................
3-43
3-43
3-44
3-47
3-49
3-50
3-50
3-51
3-51
Scanning spectra .............................................................................................
Types of scanning...........................................................................................
Before you begin.............................................................................................
Scanning new spectra ....................................................................................
3-52
3-52
3-52
3-57
Table of Contents
3-34
3-34
3-35
3-36
3-37
3-38
3-40
Parameters used for sample and zero-scans ................................................
Programming a zero-scan..............................................................................
Running a sample scan..................................................................................
Scanning using a static flow cell ...................................................................
3-58
3-59
3-60
3-64
Managing results .............................................................................................
Storing a spectrum.........................................................................................
Getting information about a stored spectrum..............................................
Reviewing a stored spectrum ........................................................................
Creating a difference spectrum (subtracting a spectrum)...........................
Replaying a spectrum ....................................................................................
3-65
3-65
3-66
3-66
3-67
3-67
Conserving lamp life ......................................................................................
Manually extinguishing the lamp.................................................................
Manually lighting the lamp...........................................................................
Using a timed event method to program the lamp ......................................
3-67
3-68
3-69
3-70
Shutting down the detector .......................................................................... 3-70
4 Maintenance Procedures ..................................................................... 4-1
Contacting Waters technical service ............................................................ 4-2
Maintenance considerations .......................................................................... 4-2
Safety and handling......................................................................................... 4-2
Spare parts ....................................................................................................... 4-3
Routine maintenance ....................................................................................... 4-3
Removing the front-left-hand panel cover ...................................................... 4-4
Inspecting, cleaning, and replacing the flow cell .....................................
Flushing and passivating the flow cell ...........................................................
Removing the flow cell assembly ....................................................................
Replacing the flow cell .....................................................................................
4-5
4-5
4-6
4-7
Replacing the lamp ........................................................................................... 4-8
When to replace the lamp................................................................................ 4-8
Removing the lamp .......................................................................................... 4-9
Installing the new lamp................................................................................. 4-12
Recording the new lamp’s serial number ..................................................... 4-13
Table of Contents
xi
Replacing the fuses ......................................................................................... 4-14
Cleaning the instruments exterior ............................................................. 4-15
5 Error Messages, Diagnostic Tests, and Troubleshooting ............. 5-1
Startup error messages ................................................................................... 5-2
Operational error messages ........................................................................... 5-3
User-selected diagnostic tests and settings ................................................ 5-8
Overview of diagnostic tests and settings ...................................................... 5-8
Sample and reference energy diagnostic tests ............................................. 5-11
Raman signal-to-noise test diagnostic test................................................... 5-12
Input and output diagnostic tests and settings ........................................... 5-12
Change-lamp function ................................................................................... 5-15
Testing the keypad ........................................................................................ 5-16
Testing the display ........................................................................................ 5-17
Other diagnostic tests and settings .............................................................. 5-17
Generating test peaks.................................................................................... 5-17
Overriding the optical filter setting .............................................................. 5-18
Reducing PMT sensitivity ............................................................................. 5-19
Troubleshooting ..............................................................................................
Introduction....................................................................................................
Information needed when you contact Waters.............................................
Diagnostic tests..............................................................................................
Power surges ..................................................................................................
Hardware troubleshooting ............................................................................
5-20
5-20
5-20
5-21
5-21
5-21
A Safety Advisories .................................................................................. A-1
Warning symbols ............................................................................................... A-2
Task-specific hazard warnings........................................................................ A-2
Specific warnings ............................................................................................. A-3
Caution symbol .................................................................................................. A-5
Warnings that apply to all Waters instruments ......................................... A-6
Electrical and handling symbols ................................................................. A-12
xii
Table of Contents
Electrical symbols .......................................................................................... A-12
Handling symbols .......................................................................................... A-13
B Specifications ........................................................................................ B-1
C Solvent Considerations ....................................................................... C-1
Introduction ......................................................................................................
Clean solvents ..................................................................................................
Solvent quality .................................................................................................
Preparation checklist.......................................................................................
Water ................................................................................................................
Buffers ..............................................................................................................
Tetrahydrofuran (THF) ...................................................................................
C-2
C-2
C-2
C-2
C-2
C-3
C-3
Solvent miscibility ........................................................................................... C-3
How to use miscibility numbers...................................................................... C-5
Buffered solvents ............................................................................................. C-6
Head height ....................................................................................................... C-6
Solvent viscosity ............................................................................................... C-6
Mobile phase solvent degassing ...................................................................
Gas solubility ...................................................................................................
Solvent degassing methods .............................................................................
Solvent degassing considerations ...................................................................
C-7
C-7
C-8
C-9
Wavelength selection ...................................................................................... C-9
UV cutoffs for common solvents.................................................................... C-10
Index ..................................................................................................... Index-1
Table of Contents
xiii
xiv
Table of Contents
1
Theory of Operation
This chapter explains the theory and technology supporting the
®
operation of the Waters 2475 Multi λ Fluorescence Detector and
describes the instrument’s features.
Contents:
Topic
Page
Fluorescence theory
1-2
Fluorescence detection
1-3
Measuring fluorescence
1-4
Detector description
1-7
Principles of operation
1-9
Operational modes
1-14
Spectrum scanning
1-16
Lamp energy and performance
1-16
Auto-optimize gain
1-17
Startup diagnostic tests
1-21
Mobile-phase solvent degassing
1-21
Wavelength selection
1-22
1-1
Fluorescence theory
Fluorescence occurs when certain molecules absorb light at specific
wavelengths, promoting the molecules to a higher energy state. As they return
to their normal energy states, the “excited” molecules release their absorbed
energy as photons.
Many organic compounds absorb light, but few fluoresce. HPLC systems that
incorporate fluorescence detection effectively identify polyaromatic
hydrocarbons, aflatoxins, vitamins, amino acids, and so on. Chemical
derivatization methods extend fluorescence detection to some nonfluorescing
compounds such as carbamate pesticides.
Fluorescence detection requires both the excitation/emission wavelengths,
leading to a higher degree of sensitivity. As a result, this technique is useful
for analyses requiring low detection limits.
Certain conditions can interfere with a compound’s ability to fluoresce,
diminishing analytical performance:
•
pH changes – The loss or gain of protons and their accompanying
increase or decrease of charge affects an analyte’s electronic structure
and can enhance or degrade fluorescence.
•
Temperature changes – Fluorescence decreases as the sample
temperature increases.
•
Changes in the amount of dissolved oxygen – For some molecules,
fluorescence is quenched (decreased) by the presence of dissolved
oxygen.
Fluorescence detectors can be adapted to measure chemiluminescence, where
a molecule without exposure to any excitation energy emits a low intensity
signal. This type of detection can be accommodated by disabling the light
source or (as in the case of the 2475 detector) enabling a shutter to stop any
excitation light from reaching the flow cell.
The process of fluorescence detection involves an excitation source and the
following processes:
1-2
•
Filtering the source light
•
Exciting the sample with filtered light
•
Collecting and filtering the emitted fluorescence
Theory of Operation
•
Measuring the emitted fluorescence
•
Amplifying the emitted signal
Fluorescence detection
Overview
The scanning fluorescence detector illuminates a sample with a narrow band
of high-intensity light. The detector then measures the low levels of
fluorescence emitted by the sample. The emitted light is filtered, amplified,
and converted to electrical signals that can be recorded and analyzed.
Excitation sources
The typical energy source used for fluorescence detection is a lamp that
provides an intense, stable spectrum of light in the UV and visible ranges. The
resulting fluorescence intensity is directly related to the intensity of the
excitation spectrum. Thus high-sensitivity detectors use the most intense
excitation source available.
Types of light sources
Xenon lamps are the preferred source for general-purpose fluorescence
detectors.
Excitation wavelength selection
The excitation wavelength of choice requires some source-light filtering. In
modern detectors, a monochromator is typically used for the same purpose.
A monochromator is an adjustable device that you use to select wavelengths
over a wide range of the spectrum. A grating monochromator uses a
diffraction grating that passes only a small range, or bandwidth, of
wavelengths. By moving the grating, you can select wavelengths within a
particular range of wavelengths. A grating monochromator also passes
fractions, or orders, of a selected wavelength. For example, if the
monochromator is set to pass light energy at 600 nm, it also passes energy at
the second-order wavelength of 300 nm. A long-pass filter can be used to
absorb the higher-order energy produced by a monochromator. As the
excitation is selected by a monochromator, the emission (radiated energy) can
Fluorescence detection
1-3
also be selected. Detectors with excitation and emission monochromators can
scan holding one monochromator at a constant setting while varying the
setting on the other. This type of operation is necessary when you are
evaluating mixtures or analyzing chemical structures.
Exciting the sample
The broad band of high-intensity light from the lamp passes through a filter or
monochromator, which selects a narrow band of wavelengths. This narrow
band of light is then directed onto the flow cell where it excites the analytes as
they pass through. Excitation wavelengths often correspond with the
absorbance wavelength of the analyte.
Flow cell
The quartz flow cell minimizes the amount of stray light that can affect the
measurement, and it maximizes the fluorescence signal. The sample
compartment is arranged so that the fluorescence energy is collected at an
angle perpendicular to the excitation (lamp) beam. This arrangement
minimizes the effect of Rayleigh scatter on background light levels.
Measuring fluorescence
To measure fluorescence in the flow cell, the detector must balance the need
for high selectivity (to distinguish specific fluorescence wavelengths) with the
need for high sensitivity (to measure low-fluorescence intensities).
Quantitation
Fluorescence is linear at low concentrations but can exhibit nonlinearity at
high concentrations.
Emission wavelength selection
A monochromator is used to select an emission wavelength.
Photomultiplier tube
The photomultiplier tube (PMT) produces a current proportional to the flux of
photons emitted by the molecules in the flow cell.
1-4
Theory of Operation
Scanning
Detectors equipped with excitation and emission monochromators can easily
scan a range of excitation or emission wavelengths. Changing the wavelength
involves changing the monochromator setting. During a scan, the setting on
one monochromator is held constant while the other monochromator scans a
range of wavelengths.
Multichannel operation
Detectors equipped with excitation and emission monochromators can change
the wavelength of the excitation and emission settings. In multichannel
operation, both monochromators move rapidly between the selected
wavelength pairs to produce multiple chromatogram traces. Multiple outputs
can then derive additional information from a single separation.
Fluorescence data
Detectors report their data in units of fluorescence intensity (emission) or
energy. In addition, the 2475 detector reports intensity using normalized
units to compensate for variability between individual detectors and offset any
age-related decrease in lamp intensity. When using normalized units, changes
in gain improve the signal-to-noise ratio but do not change the peak response,
conferring a high degree of bench-to-bench reproducibility of fluorescent
signal measurements.
Emission units and normalization
The 2475 detector offers two types of output units: emission and energy.
Emission units are normalized to a standard water reference, and their
magnitude is as independent of the PMT gain as possible. You can compensate
for changes that normally influence the signal strength of fluorescence
measurements, such as lamp or optics degradations, by periodically
renormalizing to the standard water reference. Renormalizing reduces
variations in fluorescence signal strengths from one detector to another.
The following equation calculates the emission units value (EU) at any time
(t):
EUt = (PMTCountst / Gaint) × (GainRaman / CountsRaman) × 100
where
Measuring fluorescence
1-5
GainRaman and CountsRaman = values from the most recent execution of
the normalize units function
PMTCountst and Gaint = values at the time of data collection
Normalizing the emission units results in a water/Raman signal strength, at
Ex 350 nm/Em 397 nm, of 100 emission units. The xenon spectrum output is
not uniform over the detector’s operating range, and low-UV wavelengths can
degrade faster than normalization wavelengths.
Energy units
The alternative to emission units is energy units, which are similar to those
used by traditional HPLC fluorescence detectors. They directly correlate to
the anode current of the PMT, so they are directly influenced by the gain
setting. All instrumental variables, such as lamp intensity, optics efficiency,
and gain, directly influence the fluorescence emission signal strength. As a
result, energy units are less reliable. Nonetheless, when you must calculate
energy units to conform to established protocols, use the following equation:
EU = PMTCounts × K × (ReferenceCounts0 / ReferenceCountst)
where K scales the maximum detectable fluorescence signal to 10,000 units.
References
Consult the following texts for additional information on fluorescence
detection:
N. Ichinose, G. Schwedt, F. M. Schnepel, and K. Adachi, Fluorometric
Analysis in Biomedical Chemistry, Chapter 5, Wiley-Interscience: New York,
1991.
E. S. Yeung, ed., Detectors for Liquid Chromatography, Chapter 5, Wiley: New
York,1986.
W. R. Seitz, in Treatise on Analytical Chemistry, 2nd ed., P. J. Elving, E. J.
Meehan, I. M. Kolthoff, eds., Part I, Vol. 7, Chapter 4, Wiley: New York, 1981.
J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum: New York,
1983.
1-6
Theory of Operation
S. G. Schulman, Fluorescence and Phosphorescence Spectroscopy:
Physicochemical Principles and Practice, Pergamon Press: New York, 1977.
J. D. Winefordner, S. G. Schulman, and T. C. O’Haver, Luminescence
Spectroscopy in Analytical Chemistry, Wiley-Interscience: New York, 1972.
Detector description
The 2475 Multi λ Fluorescence Detector is a multichannel, tunable,
fluorescence detector designed for high-performance liquid chromatography
(HPLC) applications.
Waters 2475 Multi λ Fluorescence Detector
2475 Detector
Watersore
scence
Multi l Flu
On/off switch
Features
The detector operates from 200 to 900 nm. It uses optics designed with an
enhanced illumination system optimized for LC performance. The following
Detector description
1-7
design features increase the optical throughput and sensitivity, resulting in
an overall increase in the signal-to-noise ratio:
1-8
•
Standalone programmability – Stores up to 10 user-defined programs (or
methods), each consisting of up to 48 programmable, timed events and 2
programmable switches.
•
Single or multichannel mode – Monitors fluorescence at one or more
discrete wavelength pairs.
•
Integral erbium calibration reference – Ensures wavelength accuracy.
•
Automatic second-order filter – Automatically engaged for wavelengths
of 400 nm and greater and removed for wavelengths of 399 nm or less.
•
Spectrum scan and storage display – Supports spectrum scan, display,
and subtraction in addition to standard tunable fluorescence
functionality.
•
Full diagnostic capability – Supports built-in diagnostic tools to optimize
functionality and performance.
•
Data communications and control – By an Empower™ system or
32
Millennium® chromatography workstation (version 3.2 or 4.0).
•
Backward compatibility – Operates as a 474 Detector with an Empower
system or Millennium32 chromatography workstation version, 3.2 or 4.0,
using RS-232, serial communication.
•
Two programmable contact closure outputs – Has two configurable
switches, each of which can accommodate a maximum of +30 V and 1 A.
The switches (SW1 and SW2) can trigger fraction collectors and other
external devices. Time and fluorescence can actuate the switches.
•
Normalized emission units – Enhances unit to unit reproducibility.
•
Idle mode – Closes a shutter to prevent degradation of the optics.
•
Fast Scan mode – Dynamically scans either the emission or excitation
grating through a selectable wavelength range to monitor the
fluorescence of a series of wavelengths. (This feature is available only in
Empower systems through remote Ethernet interface with 2475 ICS
software control.)
Theory of Operation
Principles of operation
To use the detector effectively, you should be familiar with the optical and
electronic design of the detector and the theory and principles of operation.
•
Optics
•
Wavelength verification and test
•
Flow cell
•
Electronics
Detector optics
The optics are based on a pair of tunable monochromators and include the
following parts:
•
Xenon arc lamp
•
Two ellipsoidal mirrors and one parabolic mirror
•
Shutter, wavelength calibration filter, and second-order filter
•
Entrance slits
•
Exit slits
•
Blazed, plane, and concave holographic diffraction gratings
•
Photomultiplier tube (PMT)
•
Waters axially illuminated flow cell
Principles of operation
1-9
The following diagrams show the optics assembly light paths and components.
Excitation monochromator optics assembly
Xenon lamp
Grating
Parabolic
mirror
Filter
wheel
Entrance slit
Ellipsoidal
mirror
Flow cell
Exit slit, excitation
monochromator
Emission monochromator optics assembly
Photomultiplier tube
Exit slit
Ellipsoidal
mirror
Entrance slit
Grating
Flow cell
exit mask
Flow cell
Optics assembly light path
The detector demonstrates superior performance by employing several unique
design elements. Its novel flow cell design minimizes stray background light
1-10
Theory of Operation
and increases the detectability of low-level signals. Keeping the optics simple
tends to minimize loss of signal and maximize throughput.
Light source
The detector uses a high-intensity 150-watt xenon arc lamp as its source. The
emitted light passes through the excitation monochromator to flood the
aperture of the flow cell. The lamp light is collected by an elliptical mirror
behind the lamp, its center of curvature oriented toward the lamp’s bright
spot.
Excitation monochromator
The detector uses a monochromator to select the appropriate excitation
wavelengths defined by its geometry. The grating can rotate quickly,
responding to multiple excitation wavelengths and/or scanning.
Emission monochromator
The light emitted by the sample travels from the top of the flow cell into the
emission optics. The emission optics are positioned at right angles to the
excitation source to minimize the possibility of stray light reaching the PMT.
The emission monochromator selects the appropriate emission wavelengths.
Axially illuminated flow cell
The flow cell design incorporates an axially illuminated, fused-quartz flow
cell.
Axially illuminated flow cell
Emission
energy
Lens
Quartz window
Mirror
Excitation
energy
Mirror
Fluid in
Fluid out
Principles of operation
1-11
The excitation energy is focused on a geometrically matched mirror whose
shape is opposite that of the excitation energy entrance lens. Excitation
energy is reflected along the flow cell axis and back, effectively doubling the
pathlength of the cell. The increased pathlength in turn provides superior
sensitivity compared to traditional fluorescence detectors.
Photomultiplier (PMT) calibration
The sensitivity of the detector is controlled by the gain setting, which
increases the voltage to the PMT to amplify and increase response. The gain is
achieved by controlling the high-voltage supply to the PMT. After the
assembly and alignment of the detector and whenever the PMT or any PC
boards are replaced, Waters personnel calibrate PMTs using an onboard
service diagnostic function.
PMT sensitivity
After calibrating the PMT, you must choose a gain setting for the
photomultiplier tube prior to a chromatographic injection. Saturation, which
occurs when the sample concentration is high, or the experimental mobile
phase has a high background, is always a concern—even when the PMT gain
setting is at the lowest level. For this reason, the 2475 detector’s Auto
Optimize Gain diagnostic function lets you adjust the granularity of the gain.
Filtering noise
The detector uses a digital filter to minimize noise.
Lower-time constant settings produce these effects:
•
Narrow peaks appear with minimal peak distortion and time delay.
•
Very small peaks become harder to discriminate from baseline noise.
•
Less baseline noise is removed.
Higher time constant settings produce these effects:
•
Greatly decreased baseline noise
•
Shortened and broadened peaks
The software includes fast, normal, or slow filtering constants at each data
rate that are appropriate for high-speed or high-sensitivity applications,
respectively.
1-12
Theory of Operation
The following figure shows the relationship between increased time-constant
and response times.
Effect of filter time constant
110
90
0 sec.
Response
70
1 sec.
2 sec.
50
30
10
-10
0
0.5
1
1.5
TP02824
Time (minutes)
Electronics
The electronics consist of the following components:
•
Preamplifier board – Collects and processes the analog input signals
from the PMT and photodiode to the microprocessor for further signal
conditioning. Sample and reference signals are integrated and A/D
conversion is performed simultaneously. This component ensures the
best rejection of common mode noise in the two beams, leading to a quiet
baseline.
•
Personality board – Receives inputs from the preamplifier board and
external events. It also provides control of the optics positioning
subsystems and the lamp power supply.
•
CPU board – Contains the digital signal processor, communication
ports, nonvolatile (battery backed-up) RAM and flashable RAM space in
which the firmware resides.
•
Ethernet communications interface – Enables the detector to
communicate with data system software.
Principles of operation
1-13
•
Lamp power supply – Provides stable xenon lamp operation.
•
DC power supply – Provides voltage for the analog and digital circuitry.
It is the DC power source for the detector.
Wavelength verification and test
The xenon arc lamp and the integral erbium filter exhibit peaks in the
transmission spectrum at known wavelengths. Upon startup, the detector
waits 5 minutes for the xenon lamp to warm and stabilize. The detector
verifies calibration by comparing the locations of these peaks with calibration
data stored in memory. If the results of this verification differ from the stored
calibration by more than ±2.0 nm, the detector displays a wavelength
verification failure message. This message indicates a manual wavelength
calibration is necessary. The detector verifies, rather than recalibrates, to
avoid errors that can occur if the flow cell contains residual materials.
Calibration requires a clean flow cell and transparent mobile phase. You can
initiate a manual wavelength calibration anytime to replace the previous
calibration data with new data.
Tip: The combined wavelength accuracy specification of the detector is
±3.0 nm, but the wavelength accuracy of each grating is held to ±2.0 nm.
When the detector runs continuously, you should perform wavelength
verification weekly by turning it off and then on again, or perform the
calibrate wavelength function through the console. The verification tests
require 5 minutes of lamp warmup time to stabilize the lamp.
Operational modes
The detector operates in single or multichannel mode, allows spectrum
scanning using a flow cell, and provides Difference and MaxPlot functions.
Single-channel mode
The detector defaults to single-channel mode, monitoring a single channel for
an excitation/emission wavelength pair. You can specify the excitation
wavelength between 200 and 890 nm on channel A.
In single-channel mode, the detector automatically engages the second-order
filter for excitation wavelengths of 400 nm and longer and removes it for
wavelengths shorter than 399 nm. The second-order filter is an optical filter
1-14
Theory of Operation
that blocks unwanted ultraviolet (UV) light from reaching the diffraction
grating, which can interfere with fluorescence detection of 400 nm and longer.
Selecting the appropriate sampling rate
A sufficient number of points must fall across a peak to define its shape. For
this reason, the definition between peaks is lost at very low sampling rates.
Empower uses the index of the data point closest to the end time, minus the
index of the data point closest to the start time, to calculate the Points Across
Peak value for each integrated peak in the chromatogram.
Tip: The Points Across Peak value appears in the Peaks table, at the bottom of
the Review Main window. If the Points Across Peak field is not visible,
right-click anywhere in the table, and then click Table Properties. Click the
Columns tab, and then scroll down to find the Points Across Peak field. Clear
the check box, and then click OK.
If the Points Across Peak value for the narrowest peak of interest is less than
25, you must specify a higher sampling rate in the instrument method. If the
value is greater than 50, you should specify a lower sampling rate in the
instrument method.
Set the sampling rate to the lowest value required to achieve 25 or more
points across the narrowest peak. Excessively high sampling rates exhibit
higher noise levels.
Multichannel mode
In multichannel, or multiwavelength mode, the detector monitors two or more
excitation/emission wavelength pairs. The sampling frequency range is
reduced, limiting the use of this mode to a more standard chromatography,
where peaks are not excessively narrow. You can use multiwavelength mode
to obtain additional information about an analyte by running a Difference Plot
or a MaxPlot. The detector allows you to select up to four excitation
wavelengths from 200 to 890 nm and up to four emission wavelengths from
210 to 900 nm. For the best signal-to-noise ratio, set a gain that maximizes
the dynamic range of the electronics. A gain that is too high overloads the
pre-amplifier, resulting in flat-topped peaks and a warning alarm.
MaxPlot
The detector allows you to obtain a MaxPlot in multichannel mode. The
MaxPlot function monitors fluorescence at the selected excitation/emission
Operational modes
1-15
wavelength pairs and plots the maximum fluorescence signal value for each
sample component. The MaxPlot outputs the greater of the fluorescence
values on the selected channels.
Difference plot
The detector allows you to obtain a difference plot in multichannel mode. The
difference plot function monitors fluorescence at user-selected
excitation/emission wavelength pairs and plots the difference in signal value
between them.
Spectrum scanning
You can use the detector as a fluorometer to acquire spectra and store them as
a file. The major difference between this detector and a double-beam
spectrophotometer is that this detector employs only one flow cell rather than
a simultaneous sample and reference flow cell pair. The detector obtains a
fluorescence spectrum by performing the following types of scan on the flow
cell:
•
Zero scan – Characterizes the baseline spectrum of a solvent.
•
Excitation sample scan – Subtracts the zero scan, so the displayed or
charted results are of the sample’s excitation spectra only.
•
Emission sample scan – Subtracts the zero scan, so the displayed or
charted results are of the sample’s emission scan only.
To obtain an excitation or emission spectrum of a sample, run a zero scan
followed by the appropriate sample scan. Typically, a zero scan is run with
pure solvent. A sample scan is usually run with the analyte dissolved in the
same solvent.
Lamp energy and performance
In conventional designs of fluorescence detectors, the signal-to-noise
performance of the instrument is directly proportional to the lamp energy
input to the instrument. Lamp energy input to the detector can be affected by
1-16
•
age and efficiency of the lamp.
•
improperly maintained optics and/or flow cell.
•
normal degradation of optical components (including the PMT).
Theory of Operation
Optical components degrade slowly over time. In conventional fluorescence
detectors, response increases by incrementing the PMT gain. However, the
response of the sample varies with energy throughput. If excitation energy is
degraded, peak response is degraded. If excitation intensity diminishes, peak
response decreases and noise increases.
During normal operation, lamps are commonly replaced when the reference
energy at specific wavelength settings falls below a user-set threshold relative
to initial values. The useful lamp life depends on your requirements for noise
performance.
Tip: You should inspect the detector’s general condition when you replace
lamps.
Predicting when the detector’s performance degrades to an unacceptable level
based solely on reference energy is unsatisfactory. Each user’s analyses will
require different levels of sensitivity. Checking reference energy alone to
evaluate performance assumes that every lamp has the same longevity,
degradation patterns, and spectral output characteristics. To reduce this
uncertainty, Waters designed the detector to operate as independently of lamp
output as possible. After the unit has verified the calibration of the
monochromator, the instrument evaluates the energy levels in a number of
characteristic regions across the spectrum. The integration time of the
front-end electronics is adjusted to maximize the signal within these regions.
The intent is to maintain a high signal-to-noise ratio and operate with a clean
signal. In this way, the instrument’s sensitivity to lamp energy is virtually
eliminated as a major contributor to performance.
Ultimately, the detector’s performance is a function of each unique application
requirements. Signal-to-noise measurements are the best way to evaluate
performance and set the boundaries for acceptable operational sensitivity
limits.
The 2475 detector source lamp is warranted to light and pass startup
diagnostic tests for 2000 hours or 1 year from the date of purchase, whichever
comes first. The detector’s on-board diagnostics allow you to record lamp
usage and report the lamp serial number.
Auto-optimize gain
Proper PMT gain setting maximizes the signal on the internal
analog-to-digital converter without exceeding its potential limit. If you specify
too high a gain, the fluorescence emissions overload the signal collection
Auto-optimize gain
1-17
electronics. Too low a gain reduces sensitivity to emission signals, degrading
signal-to-noise ratios. The detector therefore requires you to specify a gain
setting for the PMT before you inject a sample. However, before the injection,
you cannot know the magnitude of your fluorescence signal. Users
traditionally resolve this difficulty by running several injections to determine
a suitable gain setting, a tedious process, especially when they run timed
event changes in gain and/or wavelength.
The Auto-Optimize Gain diagnostic function runs a trial chromatogram and
displays the ideal gain values. The reported values are based on an algorithm
that ensures a 2× margin against overloading the PMT and its associated
electronics with variations in fluorescence intensity for concentrated samples.
In the case of timed event changes in gain and/or wavelength, the report
reflects adjusted values representing the ideal gain setting for each critical
timed event region. You should incorporate the reported gain values in the
method, including its timed event table, to optimize the method’s
performance.
The detector also monitors the maximum fluorescence signal level throughout
the run. When you use the analog outputs during data collection, it displays a
minimum EUFS value that applies to the entire chromatogram. Like the ideal
gain value, the EUFS value assumes a 2× margin to account for any
variations in fluorescence intensity. Based on this report, you must adjust the
gain values in the method, including its timed event table, to optimize the
performance of the method.
The detector also monitors the maximum fluorescence signal level throughout
the entire run. It recommends a minimum EUFS value, which applies to the
entire chromatogram and appears when you use the analog outputs during
data collection. This value is also computed assuming a 2× margin for error.
Method optimization
You can download a method that includes timed event changes. The timed
event changes that alter gain, excitation wavelength, or emission wavelength
are critical “light condition” changes, the points at which the signal peak
maximum search is renewed. You must therefore enter any timed event gain
changes at strategic points before peaks to improve the detector’s sensitivity
to peaks. The goal is to provide a retention time demarcation point at which a
gain change could be tolerated without disrupting the integration of peaks in
the chromatogram. Before you run the Auto-Optimize Gain diagnostic
function, you must set the initial conditions. Timed events are not absolutely
necessary, but this causes the detector to recommend only one gain value
1-18
Theory of Operation
setting for all peaks in the chromatogram and no segregated peak region
optimization.
Example of recommended method development approach
A method with two timed event changes optimizes the chromatogram shown
below.
Gain optimized chromatogram
Emission
Region 1
Region 2
Region 3
Time
Gain: 10
Ex: 375 nm
Em: 410 nm
Gain: 1000
Ex: 375 nm
Em: 410 nm
Gain: 5
Ex: 395 nm
Em: 440 nm
The first gain setting change occurs at 1.5 minutes, just before the small peak
that is best detected at a gain of 1000. The next change, at 2.0 minutes, is the
required wavelength-pair change. Initial gain setting or conditions are not
critical. The only requirement for the first timed event is that some gain
setting takes place. An initial method table is shown below.
Example of method development
Time (min)
Event
Initial (0.0)
Excitation = 375 nm, Emission = 410 nm, Gain = 100
1.5
Gain = 1
Auto-optimize gain
1-19
Example of method development (Continued)
Time (min)
Event
2.0
Excitation = 375 nm, Emission = 410 nm (no need to change
gain here)
After you run the Auto-Optimize Gain diagnostic function, the detector
displays recommended gain values.
Recommended gain values
EUFS: 2000
event time (min.)
Best gain
0.0 (Initial)
10
1.5
1000
2.0
5
Tips:
•
The previous table contains the best gain values optimized with a 2×
margin for error that holds half its capacity in reserve for unanticipated
fluorescence signal fluctuations.
•
The magnitude of emission units is independent of the gain, so changing
gain does not affect emission unit values. However, when you use
sample energy units, changing the gain does affect the magnitude of the
output signal.
Ensuring gain optimization for each peak of interest
Refer to the figure “Gain optimized chromatogram” on page 1-19. If you use
only one timed event (wavelength pair change at 2 minutes for peaks 3 and 4),
the recommended gain table is as follows.
Recommended gain values with a single timed event change
1-20
EUFS: 2000
event time – min.
Best gain
0.0 (Initial)
10
2.0
5
Theory of Operation
The gain for Region 2 is determined by the maximum signal level in Region 1.
Therefore, a gain of only 10 would be used from time 0.0 to time 2.0, but the
small peak may not be adequately resolved at this setting. If the detector did
find it, the peak area integration would be far less accurate because of higher
baseline noise. Failure to program a gain change at a strategic point in the
chromatogram constitutes a relatively ineffective approach to method
development.
Startup diagnostic tests
The detector runs a series of startup diagnostic tests and posts an error
message if any test fails. The startup diagnostic tests are as follows:
•
Central processing unit (CPU) test
•
Serial communication interface (SCI) test
•
Electrically erasable programmable read-only memory (EEPROM) test
•
RAM test
•
Application program checksum verification
•
Lamp test
•
Photodiode test
•
PMT test
•
Optics test/Wavelength verification
Mobile-phase solvent degassing
Mobile-phase difficulties account for at least 70% of all liquid chromatographic
problems. Using degassed solvents is important, especially at excitation
wavelengths shorter than 220 nm. Bubbles in the flow cell adversely affect
detector performance. Degassing provides
•
reproducible fluorescent response.
•
stable baselines and enhanced sensitivity.
•
reproducible retention times for eluting peaks.
•
reproducible injection volumes for quantitation.
•
stable pump operation.
Startup diagnostic tests
1-21
Wavelength selection
In fluorescence, if the excitation monochromator is set below the UV cutoff of a
mobile-phase component, the solvent absorbs some of the available excitation
light intensity, which in turn reduces the fluorescence emission response for
the sample. For a complete list of UV cutoff ranges for common solvents and
common mixed mobile phases, refer to Appendix C.
Warning: Using incompatible solvents can cause severe damage to
the instrument and injury to the operator.
1-22
Theory of Operation
2
Setting Up the Detector
Contents
Topic
Page
Before you begin
2-2
Installing the detector
2-3
Plumbing the detector
2-3
Making signal connections
2-5
Connecting other devices
2-23
Connecting to the electricity source
2-36
2-1
Before you begin
To install the detector, you must know how to set up and operate laboratory
instruments and computer-controlled devices and how to handle solvents.
Before installing the detector, ensure that
•
it is not situated under a heating or cooling vent.
•
the required components are present.
•
none of the shipping containers or unpacked items are damaged.
Environmental specifications
Attribute
Specification
Operating temperature
4 to 40 °C (39.2 to 104 °F)
Operating humidity
20 to 80%, noncondensing
Shipping and storage temperature
−20 to 80 °C (−4 to 176 °F)
Shipping and storage humidity
0 to 90%, noncondensing
If you discover any damage or discrepancy when you inspect the contents of
the cartons, immediately contact the shipping agent and your local Waters
representative.
Customers in the USA and Canada should report damage and discrepancies to
Waters Technical Service (800 252-4752). Others should phone their local
Waters subsidiary or Waters corporate headquarters in Milford,
Massachusetts (USA), or they may visit http://www.waters.com, and click
Offices.
For complete information on reporting shipping damages and submitting
claims, see the document Waters Licenses, Warranties, and Support Services.
2-2
Setting Up the Detector
Installing the detector
Warning: To avoid injury, Waters recommends that two people lift
the 2475 detector.
Warning: Risk of fire. To avoid overheating, and to provide clearance
for cable connections, make sure there is at least 15.24 cm (6 inches)
of clearance at the rear of the detector.
To install the 2475 detector, place it on a level surface to allow proper function
of the drip management system (drain tube), to which you can connect to a
waste reservoir that diverts solvent leaks from the flow cell.
Plumbing the detector
Caution: To prevent flow cell breakage, do not exceed the flow cell’s
maximum allowable pressure of 1000 kPa (10 bar, 145 psi) and a flow
rate of 5 mL/min.
Connecting columns
Warning: To prevent injury, always observe Good Laboratory Practices
when you handle solvents, change tubing, or operate the system. Know
the physical and chemical properties of the solvents you use. See the
Material Safety Data Sheets for the solvents in use.
The plumbing connections to the detector are at the front of the flow cell
assembly, on it’s right-hand side.
To connect the inlet and outlet tubing:
1.
Attach the compression fitting and ferrule (supplied in the startup kit).
2.
Connect the inlet tubing to the column outlet, ensure the tubing is
seated firmly, and then tighten the compression screw.
Caution: Do not put fluid containers on top of the detector
without a solvent tray to contain spills.
Installing the detector
2-3
3.
®
Connect the Teflon tubing to the flow cell outlet tubing, and route it to
a waste container.
Plumbing connections
Assembling fittings
Slide the compression screw over the tubing end. Follow it with the ferrule
mounted so that its taper faces the end of the tubing.
Compression
screw
Ferrule
Tube
Tubing end (cut
straight and smooth to
achieve maximum
column efficiency)
Distance (determined by each
application, such as union or
column fitting)
2-4
Setting Up the Detector
Making tubing connections
To make tubing connections:
1.
Bottom each tubing end in the column outlet, detector inlet, or detector
outlet fitting.
2.
Seat each ferrule by tightening the compression screw 1/2-turn past
finger-tight.
Tip: To ensure accurate verification during installation, be sure to pump fresh,
degassed, and filtered 100% water through the flow cell before powering it.
Making signal connections
Warning: To avoid electrical shock, power-off and unplug the detector
before making signal connections.
See also: Ethernet Instrument Getting Started Guide.
The following figure shows the rear panel location of the connectors used to
operate the detector with external devices.
Making signal connections
2-5
2475 detector rear panel
Fan vents
Inputs and
outputs
Chassis
ground
Fuse holder
Power input
Component connection overview
Tip: Waters recommends you connect the 2475 detector to other system
components via an Ethernet connection.
The following table summarizes the signal connections needed to connect the
2475 detector to other HPLC system components.
Component connector types
2-6
Connector type
Component
Ethernet connection
Used to connect to a Waters
Empower system using Ethernet.
Analog outputs
• SAT/IN Module
• 746 Data Module (integrator or
data system using the A/D
interface)
• Chart Recorder
Setting Up the Detector
Component connector types (Continued)
Connector type
Component
Event inputs
• System controller (used with the
®
Waters Alliance Separations
Module and the 600-series
solvent delivery system)
• Waters 700 Series or a
non-Waters autosampler
• Waters or other manual injector
RS-232
Allows remote control and direct
data acquisition from an Empower
32
system or a Millennium
workstation (version 3.2 and later)
in 474 emulation mode.
Connecting the Ethernet cable
The Waters instrument communicates with the acquisition computer through
the dedicated local area network (LAN). At the acquisition computer, the
instrument network card provides the communications interface.
You must install the instrument control software driver (ICS) in the
acquisition computer so that the computer can control the instrument.
(Consult the software installation instructions that accompany the
instrument control software for details.)
Single Waters instrument connection
In a single Waters instrument system configuration, the connection hardware
requires only one CAT 5, shielded, Ethernet cross-over cable (startup kit).
Making signal connections
2-7
Single Waters instrument connection
Instrument LAN
network card
Waters
instrument
Acquisition computer
Ethernet
cross-over cable
Multiple Waters instrument connections
In a system configuration that includes many Waters Ethernet instruments,
an Ethernet switch communicates between Waters instruments and the
acquisition computer.
Connection hardware requires one standard, CAT 5, shielded, Ethernet cable
per Waters instrument and a standard, CAT 5, shielded, Ethernet cable
between the network switch and the acquisition computer. See the figure
“Multiple Waters ethernet instrument connections” on page 2-9.
You must install the Waters instrument control software in the acquisition
computer so that the computer can control the Waters instrument. (Consult
the software installation instructions that accompany the driver disk.)
Network installation guidelines
Configurations for multiple Waters instruments use a dedicated local area
network (LAN). See the figure, below. The LAN requires a design based on the
following guidelines:
•
100-base-T, 100-Mbps, CAT 5, shielded, twisted-pair (STP) cable
•
A maximum distance of 100 meters (328 feet)
Tip: You must use a network switch with multiple Ethernet instruments. A
network hub in place of a network switch is not supported.
2-8
Setting Up the Detector
Multiple Waters ethernet instrument connections
Switch
Instrument
network card
TP02075
Workstation
Analog output
100-base-T
ethernet cable
ZQ/EMD
1000
eSAT/IN module
2475
detector
Analog input 1
Analog input 2
Making inject-start signal connections
The Ethernet data system or controller used with the 2475 detector must
receive an inject-start signal from the autosampler or manual injector to
initiate the data collection and time-based programs.
The following table summarizes the inject-start connections for different
system configurations
2475 detector inject-start connections
Inject-start output source
Inject-start input connection
(on the 2475 detector, connector A)
Waters 700 Series Autosampler
Inject Start + / –
Waters Alliance Separations Module Inject Start
Waters manual injector, or
third-party manual injector or
autosampler
Inject Start + / –
Making signal connections
2-9
Tip: For pin-out connections to the 2475 detector, see the figure “I/O signal
inputs and outputs” on page 2-11.
Choosing signal connections
Tip: Connect the 2475 detector to other HPLC system components through an
Ethernet connection.
The rear panel provides two analog connectors and an RS-232
communications port for operating the detector with external devices. You can
connect other instruments to the detector through these connectors to enable
the following signals:
•
Analog outputs – Two attenuated, analog-channel outputs, Detector
Out 1 and Detector Out 2, support 1-V output to external devices or data
systems. For input/output voltage current specifications, see Appendix
B. The 1-V output for channel A and channel B is scaled according to the
EUFS (emission/energy units full scale) setting for each channel. The
detector allows the EUFS to be set individually for the output on each
channel. Volts per EU are calculated for the 1-V output as follows:
Volts out = Fluorescence × 1 V/EUFS
For example, an EUFS setting of 10,000 provides a traditional 0.0001
V/EU output. An EUFS setting of 100,000 provides a 0.00001 V/EU
output, which supports chromatography above 10,000 EU.
2-10
•
Chassis ground stud – Connect the shield from analog connections
here.
•
Switched outputs – You can program two switch contact closures to
turn on, off, toggle, pulse once for a defined duration, or pulse
repetitively for a specified period of time.
•
Event inputs – Four general-purpose TTL contact closures on the
detector’s A (Inputs) terminal support the following functions (see the
table titled “Primary and secondary function (method) parameters” on
page 3-21):
–
Remote or inject start
–
Lamp on/off
–
Chart mark input
–
Auto Zero
Setting Up the Detector
•
RS-232 Interface – The RS-232 connection allows remote control and
32
direct data acquisition from an Empower system or a Millennium
workstation (version 3.2 and later) in 474 emulation mode.
Making I/O signal connections
The rear panel includes two removable connectors that hold the pins for the
I/O signals (see the following figure). These connectors, A and B, are keyed so
that you can insert them only one way.
I/O signal inputs and outputs
B (inputs and outputs)
1
2
3
4
5
6
7
8
9
10
+
−
+
−
+
−
+
−
Detector out 1
Detector out 1
Ground
Detector out 2
Detector out 2
Switch 1
Switch 1
Ground
Switch 2
Switch 2
A (inputs)
1
2
3
4
5
6
7
8
9
10
+
−
+
−
+
−
+
−
Inject start
Inject start
Ground
Lamp on/off
Lamp on/off
Chart mark
Chart mark
Ground
Auto zero
Auto zero
The following table describes each signal available on the I/O connectors. See
Appendix B for the signal’s electrical specifications
I/O signals for the detector
Signal
Description
1
Inject Start
TTL contact closure. Configurable input to initiate
sequencing of time-programmed events. Defines the
start of a run (typically an injection) and resets and
starts the runtime clock at 0.00 minutes. Initial
conditions apply immediately.
Making signal connections
2-11
I/O signals for the detector (Continued)
Signal
Description
Configurable input to allow an external device to turn
the xenon lamp off and on.
1
Lamp On/Off
Configurable input to add a chart mark (at 10% of full
scale) to either or both analog output channels (Detector
Out 1 and Detector Out 2).
1
Chart Mark
Configurable input to Auto Zero both channels (Detector
Out 1 and Detector Out 2).
1
Auto Zero
2
1-V full-scale analog output signal of channel A (scaled to
the current EUFS setting).
Detector Out 2
2
1-V full-scale analog output signal of channel B (scaled to
the current EUFS setting).
Switch 1 (2)
Can be controlled by threshold and timed events.
Switch 2 (2)
Can be controlled by threshold and timed events.
Detector Out 1
1. Inject start, chart mark, Auto Zero, and lamp inputs are configurable. Use the second Configuration screen and set the appropriate parameter to Low (see page 3-25).
2. See page 2-10.
Signal connections
Refer to the signal connection location shown on the silk-screened label on the
rear panel of each instrument.
Tip: To meet the regulatory requirements of immunity from external electrical
disturbances, you must install connection covers over the signal connectors.
2-12
Setting Up the Detector
To make signal connections:
1.
Attach the positive and negative leads of the signal cable to the
connector.
Connector
Signal cable
2.
Slide the clamp (with the bend facing down) into the protective shield.
3.
Insert the clamp and shield (with the bend facing down) into the
connection cover and loosely tighten with one self-tapping screw.
Clamp
Shield
Connection cover
Making signal connections
2-13
4.
Insert the connector with the signal cable into the connection cover,
position the clamp over the cable leads, and then tighten the clamp into
place with the second self-tapping screw.
Cable leads
Clamp
5.
Place the second connection cover over the first cover and snap it into
place.
Signal connector
Connection cover
Connecting an Alliance separations module
You can connect an Alliance separations module to the detector to perform the
following functions (when the detector is not under the control of Empower or
32
Millennium software):
2-14
•
Generate an Auto Zero on injection
•
Generate a chart mark on injection
•
Start a method
Setting Up the Detector
•
Turn the lamp on and off
Generating an auto zero on inject
To auto zero the detector at the start of an injection from an Alliance
separations module:
1.
Make the connections shown in the following table and figure.
Connections for generating an auto zero on inject
2.
Alliance separations module
(B inputs and outputs)
2475 detector (A inputs)
Pin 1 Inject Start
Pin 9 Auto Zero +
Pin 2 Inject Start
Pin 10 Auto Zero –
Configure the auto-zero signal at the detector’s front panel. The default
auto-zero signal is Low (see page 3-25).
Connections for auto zero on injection
Alliance
B (inputs and outputs)
2475 detector
A (inputs)
Red
Inject start
Inject start
Ground
Stop flow
Stop flow
Hold inject 1
Hold inject 1
Hold inject 2
Hold inject 2
Ground
Chart out
Chart out
+
−
+
−
+
−
+
−
+
−
1
2
3
4
5
6
7
8
9
10
11
12
Black
1
2
3
4
5
6
7
8
9
10
+ Inject start
− Inject start
Ground
+ Lamp on/off
− Lamp on/off
+ Chart mark
− Chart mark
Ground
+ Auto zero
− Auto zero
Making signal connections
2-15
Generating a chart mark on inject
To generate a chart mark at the start of an injection from an Alliance
separations module:
1.
Make the connections shown in the following table and figure.
Connections for generating a chart mark on inject
2.
Alliance separations module
(B inputs and outputs)
2475 detector (A inputs)
Pin 1 Inject Start
Pin 6 Chart Mark +
Pin 2 Inject Start
Pin 7 Chart Mark –
Configure the chart mark signal at the detector’s front panel. The
default chart mark signal is Low (see page 3-25).
Connections for chart mark on injection
Alliance
B (inputs and outputs)
2475 detector
A (inputs)
Red
Inject start
Inject start
Ground
Stop flow
Stop flow
Hold inject 1
Hold inject 1
Hold inject 2
Hold inject 2
Ground
Chart out
Chart out
2-16
Setting Up the Detector
+
−
+
−
+
−
+
−
+
−
1
2
3
4
5
6
7
8
9
10
11
12
Black
1
2
3
4
5
6
7
8
9
10
+ Inject start
− Inject start
Ground
+ Lamp on/off
− Lamp on/off
+ Chart mark
− Chart mark
Ground
+ Auto zero
− Auto zero
Starting a method
To enable the detector to start a method when an injection from an Alliance
separations module begins, make the connections shown in the following table
and figure.
Connections for starting a method
Alliance separations module
(B inputs and outputs)
2475 detector (A inputs)
Pin 1 Inject Start
Pin 1 Inject Start +
Pin 2 Inject Start
Pin 2 Inject Start –
Connections for starting a method on injection
Alliance
B (inputs and outputs)
2475 detector
A (inputs)
Red
Inject start
Inject start
Ground
Stop flow
Stop flow
Hold inject 1
Hold inject 1
Hold inject 2
Hold inject 2
Ground
Chart out
Chart out
+
−
+
−
+
−
+
−
+
−
1
2
3
4
5
6
7
8
9
10
11
12
Black
1
2
3
4
5
6
7
8
9
10
+ Inject start
− Inject start
Ground
+ Lamp on/off
− Lamp on/off
+ Chart mark
− Chart mark
Ground
+ Auto zero
− Auto zero
Turning the lamp on or off
To turn the lamp on or off from an Alliance separations module:
1.
Configure the lamp on/off signal at the detector’s front panel by
changing the default lamp configuration parameter from Ignore to High
or Low (see page 3-25).
Making signal connections
2-17
2.
Make the connections shown in the following table and figure.
Connections for turning the detector lamp on or off
Alliance separations module
(A Outputs)
2475 detector (A Inputs)
Pin 1 Switch 1
Pin 4 Lamp On/Off +
Pin 2 Switch 1
Pin 5 Lamp On/Off –
Connections for turning the lamp on or off
Alliance
B (inputs and outputs)
2475 detector
A (inputs)
Red
Inject start
Inject start
Ground
Stop flow
Stop flow
Hold inject 1
Hold inject 1
Hold inject 2
Hold inject 2
Ground
Chart out
Chart out
+ 1
− 2
3
+ 4
− 5
+ 6
− 7
+ 8
− 9
10
+ 11
− 12
Black
1
2
3
4
5
6
7
8
9
10
+ Inject start
− Inject start
Ground
+ Lamp on/off
− Lamp on/off
+ Chart mark
− Chart mark
Ground
+ Auto zero
− Auto zero
Connecting RS-232 devices
Tip: The RS-232 interface connector is used when the detector is in 474
Emulation mode.
The rear panel includes one RS-232 interface connector for digital signal
communications. Use it to connect RS-232 devices, such as an RS-232
32
communications port in an Empower system or Millennium chromatography
2-18
Setting Up the Detector
workstation, to the detector (see the following figure). The RS-232 connector
mates with a standard RS-232 cable.
Caution:
• To avoid possible damage to components, shut down all instruments
already on the RS-232 control bus before you connect an RS-232
interface cable to an additional instrument.
• The maximum total cable length between RS-232 devices in a system
is 65 feet (20 meters). The maximum recommended cable length
between two RS-232 devices is 10 feet (3 meters). Longer total cable
lengths can cause intermittent RS-232 communication failures.
Tip: With the RS-232 cable connected, the detector operates in remote mode.
32
When you connect to an Empower system or Millennium Chromatography
Manager (version 3.2 and later), you must enable the Emulate 474
configuration option for multichannel operation.
IEEE-488 and RS-232 connections in an Empower system
busLAC/E or LAC/E32 Card
Empower
PC
COM or equinox card port
(9-pin)
RS-232 cable
IEEE-488
connector
600-series
pump
717plus
autosampler
2475
detector
Tip: RS-232 communication does not support multichannel mode.
Tip: When connecting the detector to a Waters data system, all detector
parameters not configurable by the data system in use defer to local control.
Making signal connections
2-19
To connect an RS-232 device such as a Waters data system to the detector:
1.
Connect the single receptacle end of the RS-232 cable (supplied with the
detector) to an RS-232 device. Such a device may be an RS-232
communications port or Equinox card in an Empower system or a
Millennium32 chromatography workstation.
2.
Connect the other end of the cable to the RS-232 connector on the
detector’s rear panel.
3.
Ensure all RS-232 cable screws are fastened tightly.
4.
Ensure all input/output connections are correct (see the figure
“IEEE-488 and RS-232 connections in an Empower system” on
page 2-19).
5.
Configure the detector for RS-232 communication, and operate it in
remote mode, as described on page 3-25.
6.
Connect an inject-start cable (see the figure “Connections for starting a
method on injection” on page 2-17).
Connecting Ethernet devices
The detector can be controlled using Ethernet communications when
Empower software build 2154 or build 1154 with Instrument Support Service
Pack 2 (ISSP 2) and the 2475 instrument control software (ICS) are installed.
Connecting the Ethernet cable
The Waters instrument communicates with the acquisition computer through
the dedicated local area network (LAN). At the acquisition computer, the
instrument network card provides the communication interface.
You must install the Waters instrument software driver in the acquisition
computer before the computer can control the Waters instrument. See the
software installation instructions that accompany the instrument control
software.
Single Waters instrument connection
In a single Waters instrument system configuration, the connection hardware
requires only one standard, shielded Ethernet cross-over cable (startup kit).
Refer to the following figure.
2-20
Setting Up the Detector
Single Waters instrument connection
Instrument LAN
network card
Waters
instrument
Acquisition computer
Ethernet
cross-over cable
Multiple Waters instrument connections
In a system configuration with many Waters Ethernet instruments, an
Ethernet switch is required to multiplex the communication between Waters
instruments and the acquisition computer.
Connection hardware requires one standard 100-base-T Ethernet cable per
Waters instrument, and a standard 100-base-T Ethernet cable to connect
between the network switch and the acquisition computer. Refer to the figure
“Multiple Waters ethernet instrument connections” on page 2-22.
You must install the Waters instrument control software in the acquisition
computer before the computer can control the Waters instrument. See the
software installation instructions that accompany the software instrument
driver disk.
Network installation guidelines
Configurations for multiple Waters instruments use a LAN (see the following
figure). This dedicated LAN requires a design based on the following
guidelines:
•
100-base-T, 100-Mbps shielded twisted-pair (STP) cable
•
A maximum distance of 100 meters (328 feet)
Tip: You must use a network switch if you are using multiple Ethernet
instruments. Use of a network hub in place of a network switch is not
supported.
Making signal connections
2-21
Multiple Waters ethernet instrument connections
Switch
Instrument
network card
TP02075
Analog output
Workstation
eSAT/IN module
ZQ/EMD
1000
2475
detector
100-base-T
ethernet cable
Analog input 1
Analog input 2
Making inject-start signal connections
When you are using an Ethernet data system with the 2475 detector, the data
system or controller must receive an inject-start signal from the autosampler
or manual injector to initiate the data collection and time-based programs.
The following table summarizes the inject-start connections for different
system configurations.
2475 detector inject-start connections
Inject-start output source
Inject-start input connection
(on the 2475 detector, connector A)
Waters 700 Series Autosampler
Inject Start + / –
Waters Alliance Separations Module Inject Start
Waters manual injector, or
third-party manual injector or
autosampler
Inject Start + / –
Tip: For pin-out connections to the 2475 detector, see the figure “I/O signal
inputs and outputs” on page 2-11.
2-22
Setting Up the Detector
Connecting other devices
You can connect many devices to the detector, including these:
•
Empower PC using the bus SAT/IN™ module
•
Millennium32 chromatography workstation using the bus SAT/IN
module
•
Waters 746 Data Module
•
Chart recorder
•
Waters 600-series pump
•
Waters 717plus Autosampler
•
Waters Fraction Collector II or III
Required materials
To connect cables to the terminals on the detector’s rear panel, you need the
following tools:
•
Small flat-blade screwdriver (startup kit)
•
Electrical insulation stripping tool
Connecting cables
To connect the cables from other HPLC system devices to the A and B
terminals on the detector’s rear panel:
1.
Remove terminal A or B (see the figure “I/O signal inputs and outputs”
on page 2-11).
2.
Unscrew the connecting pin terminal.
3.
Using the stripping tool, strip the wire about 1/8-inch from the end.
4.
Insert the stripped wire into the appropriate connector.
5.
Tighten the screw until the wire is held securely in place.
6.
Reinsert the terminal.
7.
Press the terminal firmly to ensure that it is inserted fully.
Connecting other devices
2-23
Connecting a data system using a Bus SAT/IN module
You can acquire data from the detector with the Empower system or the
Millennium32 chromatography workstation using the Bus SAT/IN module
instead of the RS-232 bus (see page 2-18). This method requires connections
between the detector and a satellite interface (SAT/IN) module.
The bus SAT/IN module translates analog signals from the detector into
32
digital form. It then transmits them to the busLAC/E™ or LAC/E card
installed in an Empower system or Millennium32 chromatography
workstation.
Bus SAT/IN module (front panel)
To connect an Empower system or Millennium32 chromatography
workstation to the detector:
1.
Connect the Bus SAT/IN module to the busLAC/E or LAC/E32 card in an
32
Empower system or Millennium computer according to the
instructions in the Waters Bus SAT/IN Module Installation Guide.
Caution:
• Do not turn start the Bus SAT/IN module until you perform all
procedures in the Waters Bus SAT/IN Module Installation
Guide. Improper startup can damage the unit and void the
warranty.
• The Bus SAT/IN module does not have a power switch. To
prevent damage to the module, always disconnect the power
cord at the wall outlet or the power supply before attaching or
removing the power connection to the module.
2.
2-24
Connect the Bus SAT/IN module to the B (Inputs and Outputs) terminal
on the detector’s rear panel.
Setting Up the Detector
a.
Using the electrical insulation stripping tool, strip about 1/8 inch
from one end of the Bus SAT/IN connector, exposing the white and
black wires.
b.
For channel A (see the figure “Connecting the Bus SAT/IN module
channel 1 to the detector” on page 2-26 and the figure “I/O signal
inputs and outputs” on page 2-11):
Connect the white wire to pin 1 on B (Detector Out 1 + [+1 V]).
Connect the black wire to pin 2 on B (Detector Out 1 – [–1 V]).
c.
For channel B (see the figure “Connecting the Bus SAT/IN module
channel 2 to the detector” on page 2-26 and the figure “I/O signal
inputs and outputs” on page 2-11):
Connect the white wire to pin 4 on B (Detector Out 2 + [+1 V]).
Connect the black wire to pin 5 on B (Detector Out 2 – [–1 V]).
d.
3.
Connect the other end of the cable to the channel 1 or channel 2
connector on the front panel of the Bus SAT/IN module.
Configure the serial port for the Bus SAT/IN module as described in the
32
Empower Software Getting Started Guide or the Millennium Software
Getting Started Guide.
Caution: To minimize the chance of creating a ground loop that
can adversely affect measurement, connect the shield of the cable
to the chassis ground at one end only.
Connecting other devices
2-25
Connecting the Bus SAT/IN module channel 1 to the detector
Bus SAT/IN module
2475 detector
B (inputs and outputs)
1
2
3
4
5
6
7
8
9
10
+ Detector out 1
− Detector out 1
Ground
+ Detector out 2
− Detector out 2
+ Switch 1
− Switch 1
Ground
+ Switch 2
− Switch 2
Connecting the Bus SAT/IN module channel 2 to the detector
Bus SAT/IN module
2475 detector
B (inputs and outputs)
1
2
3
4
5
6
7
8
9
2-26
Setting Up the Detector
+ Detector out 1
− Detector out 1
Ground
+ Detector out 2
− Detector out 2
+ Switch 1
− Switch 1
Ground
+ Switch 2
Detector connections to the Bus SAT/IN module
Bus SAT/IN connector
2475 detector (B outputs)
Channel 1 or 2
Pin 1 Detector Out 1 + (white)
Pin 2 Detector Out 1 – (black)
Channel 1 or 2
Pin 4 Detector Out 2 + (white)
Pin 5 Detector Out 2 – (black)
Connecting a 746 data module
You can connect a Waters 746 data module to the detector using the analog
output connector on the detector’s rear panel. The analog connector provides
1 V output, which is scaled to the EUFS sensitivity setting and the voltage
offset setting.
Tip: To prevent oversaturation of the signal from the detector to the data
module, do not exceed the input voltage rating of the data module.
To send the analog output signal from the detector to the data module, use the
cable provided in the detector’s startup kit to make the connections shown in
the following table and figure.
Detector inputs and 746 terminals
746 terminals
2475 detector (B inputs and outputs)
+
Pin 1 Detector Out 1 + (red)
–
Pin 2 Detector Out 1 – (black)
+
Pin 4 Detector Out 2 + (red)
–
Pin 5 Detector Out 2 – (black)
Tip: To minimize the chance of creating a ground loop that can adversely
affect measurement, connect the shield of the cable to the chassis ground at
one end only.
Connecting other devices
2-27
Connecting a 746 data module to the detector
2475 detector
B (inputs and outputs)
1
2
3
4
5
6
7
8
9
10
Red
Black
+ –
746 data module
terminals
+ Detector out 1
− Detector out 1
Ground
+ Detector out 2
− Detector out 2
+ Switch 1
− Switch 1
Ground
+ Switch 2
− Switch 2
Connecting a chart recorder
Recorder signal
The A and B terminals on the detector’s rear panel provide 1-V analog output
signals that you can send to a chart recorder. To send a 1-V signal from the
detector to a chart recorder, use the cable in the startup kit to make the
connections in the following table and figure.
Detector inputs and chart recorder terminals
Chart recorder
terminals
2475 detector (B inputs and outputs)
+
Pin 1 Detector Out 1 + (1 V)
–
Pin 2 Detector Out 1 – (1 V)
+
Pin 4 Detector Out 2 + (1 V)
–
Pin 5 Detector Out 2 – (GND)
Tip: To minimize the chance of creating a ground loop that can adversely
affect measurement, connect the shield of the cable to the chassis ground at
one end only.
2-28
Setting Up the Detector
Connecting a chart recorder to the detector
2475 detector
B (inputs and outputs)
Red
Black
+
–
1
2
3
4
5
6
7
8
9
10
+ Detector out 1
− Detector out 1
Ground
+ Detector out 2
− Detector out 2
+ Switch 1
− Switch 1
Ground
+ Switch 2
− Switch 2
Chart recorder
terminals
Chart marks
You can also generate a chart mark from the detector’s front panel whenever
you perform these actions:
•
Press Chart Mark on the detector’s keypad.
•
You program a timed event to generate a chart mark.
•
A chart mark signal is generated at the chart mark inputs on the analog
connector.
Connecting a 600-series pump
To connect a 600-series pump:
1.
Place the detector on a level surface.
2.
Make the plumbing connections described on page 2-3.
Connecting other devices
2-29
Lamp on/off connections
To make lamp on/off connections:
1.
Make the lamp on/off connections shown in the following table and
figure with a signal cable.
2.
Configure the lamp on/off signal at the detector’s front panel by
changing the default from Ignore to High or Low (see page 3-25).
3.
Using the signal cable, make the lamp on/off connections from the pump
controller to the detector shown in the following table and figure.
Detector inputs and 600-series pump terminal connections
600-series pump terminal
2475 detector (A inputs)
S1, S2, S3, or S4
Pin 4 Lamp On/Off +
GND (any one of four)
Pin 5 Lamp On/Off –
Lamp on/off connections for the 600-series pump
2475 detector
A (inputs and outputs)
2-30
Setting Up the Detector
S4
S2
S1
S3
AUX.
+12V
GND
HOLD
STOP
FLOW
PRESSURE
Red
SWITCHES
GND
GND
_
_
CHART
GND
+
+
CHART
PRESSURE
INJECT
600-series pump
Black
1
2
3
4
5
6
7
8
9
10
+ Inject start 1
− Inject start 1
Ground
+ Lamp on/off
− Lamp on/off
+ Chart mark
− Chart mark
Ground
+ Auto zero
− Auto zero
Auto-zero connections
To make auto-zero connections:
1.
Make the connections shown in the following table and figure with a
signal cable.
2.
Program the pump to provide a pulse output on the applicable switch
(S1, S2, or S4) at the beginning of each run (see the Waters 600E
Multisolvent Delivery System User’s Guide).
Auto-zero connections for the 600-series pump
600-series pump terminal
2475 detector (A inputs)
S1, S2, S3, or S4
Pin 9 Auto Zero +
GND (any one of four)
Pin 10 Auto Zero –
Auto-zero connections for the 600-series pump
2475 detector
A (inputs and outputs)
HOLD
S1
S2
S3
GND
GND
GND
AUX.
+12V
S4
STOP
FLOW
_
_
CHART
SWITCHES
GND
+
PRESSURE+
CHART
INJECT
600-series pump
PRESSURE
Red
1
2
3
4
5
6
7
8
9
10
+ Inject start 1
− Inject start 1
Ground
+ Lamp on/off
− Lamp on/off
+ Chart mark
− Chart mark
Ground
+ Auto zero
− Auto zero
Black
Chart-mark connections
To make chart-mark connections:
1.
Make the connections shown in the following table and figure using a
signal cable.
Connecting other devices
2-31
2.
Program the pump to provide a pulse output on the selected switch at
the beginning of each run. See the Waters 600E Multisolvent Delivery
System User’s Guide.
Chart-mark connections for the 600-series pump
600-series pump terminal
2475 detector (A inputs)
S1, S2, S3, or S4
Pin 6 Chart Mark +
GND (any one of four)
Pin 7 Chart Mark –
Chart-mark connections for the 600-series pump
2475 detector
A (inputs and outputs)
S4
S2
S1
S3
AUX.
+12V
GND
HOLD
STOP
FLOW
SWITCHES
GND
_
_
GND
CHART
GND
+
PRESSURE+
CHART
INJECT
600-series pump
Red
PRESSURE
Black
1
2
3
4
5
6
7
8
9
10
+ Inject start 1
− Inject start 1
Ground
+ Lamp on/off
− Lamp on/off
+ Chart mark
− Chart mark
Ground
+ Auto zero
− Auto zero
Inject-start connections
When the detector is connected to an Empower system or Millennium32
chromatography workstation, the inject-start connections allow it to initiate
data acquisition.
To make inject-start connections:
1.
2-32
Make the connections shown in the following table and figure with a
signal cable.
Setting Up the Detector
2.
Program the pump to provide a pulse output on the selected switch at
the beginning of each run (see the Waters 600E Multisolvent Delivery
System User’s Guide).
Inject-start connections for the 600-series pump
600-series pump terminal
2475 detector (A inputs)
S1, S2, S3, S4 , or inject
Pin 1 Inject Start +
GND (any one of four)
Pin 2 Inject Start –
1
1. You may also connect the pump’s Inject terminal to pin 1, Inject Start +, on the
detector and the pump’s Inject Ground terminal to the detector’s pin 2, Inject
Start –.
Inject-start connections for the 600-series pump
2475 detector
A (inputs and outputs)
S4
S3
S2
AUX.
S1
GND
+12V
HOLD
GND
_
_
GND
CHART
Red
SWITCHES
STOP
FLOW
+
+
GND
CHART
PRESSURE
INJECT
600-series pump
PRESSURE
Black
1
2
3
4
5
6
7
8
9
10
+ Inject start 1
− Inject start 1
Ground
+ Lamp on/off
− Lamp on/off
+ Chart mark
− Chart mark
Ground
+ Auto zero
− Auto zero
Connecting a 717plus Autosampler
The Waters 717plus Autosampler signals the start of an injection through a
contact closure signal on its inject-start terminals. You can use this contact
closure signal to command the detector to auto zero at the start of an injection.
Connecting other devices
2-33
Auto-zero connections
To auto zero the detector at the start of an injection, make the connections in
shown in the following table and figure.
Auto-zero connections for the 717plus autosampler
717plus autosampler terminal
2475 detector (A inputs)
Inject Start + (any one of many paired Pin 9 Auto Zero +
with –)
Inject Start – (any one of many paired Pin 10 Auto Zero –
with +)
The following figure illustrates the connections between the detector and
autosampler. Use any available pair of inject-start terminals on the
autosampler.
Auto-zero connections for the 717plus autosampler
2475 detector
A (inputs and outputs)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
717plus Autosampler
2-34
Red
Black
Setting Up the Detector
1
2
3
4
5
6
7
8
9
10
+ Inject start 1
− Inject start 1
Ground
+ Lamp on/off
− Lamp on/off
+ Chart mark
− Chart mark
Ground
+ Auto zero
− Auto zero
Inject-start connections
To program the start of an active method, connect the autosampler’s injectstart terminals to the detector’s inject-start inputs (see the following table and
figure).
Inject-start connections for the 717plus Autosampler
717plus Autosampler terminal
2475 detector (A inputs)
Inject Start + (any one of many paired with Pin 1 Inject Start +
+)
Inject Start – (any one of many paired with Pin 2 Inject Start –
–)
The following figure illustrates the connections between the detector and the
autosampler. Use any available pair of inject-start terminals on the
autosampler.
Inject-start connections for the 717plus Autosampler
2475 detector
A (inputs and outputs)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
717plus Autosampler
Red
Black
1
2
3
4
5
6
7
8
9
10
+ Inject start 1
− Inject start 1
Ground
+ Lamp on/off
− Lamp on/off
+ Chart mark
− Chart mark
Ground
+ Auto zero
− Auto zero
Connecting other devices
2-35
Connecting to the electricity source
The 2475 detector requires a separate, grounded electricity source. The
ground connection in the electrical outlet must be common and connected near
the system.
Warning: To avoid electrical shock:
• Use power cord SVT-type in the United States and HAR-type or
better in Europe. For other countries, contact your local Waters
distributor.
• Power-off and unplug the detector before performing any
maintenance operation on the instrument.
• Connect the detector to a common ground.
To connect to the electricity source:
Tip: Use a line conditioner or an uninterruptible power supply (UPS) for
optimum long-term input voltage stability.
1.
Place the Off/On (
) switch in the Off (
) position.
2.
Connect the female end of the power cord to the receptacle on the rear
panel of the detector.
3.
Connect the male end of the power cord to a suitable wall outlet.
Tip: Do not turn the detector on at this time.
2-36
Setting Up the Detector
3
Using the Detector
After you install the detector, you must set it up and operate it as a
standalone instrument or as part of a data system.
•
As a standalone instrument – You can use the detector as a
standalone detector with a system such as the Waters Alliance
system or with any pump, injector, chart recorder, or integrator.
You can program the detector’s front panel unless it is controlled by
32
a data system such as Waters Empower or Millennium software.
•
As part of an Empower system – You can configure the detector for
use in an Empower system. To do so, follow the instructions in the
Empower Online Help to set parameters for controlling the detector.
•
As part of a Millennium chromatography workstation – You can
32
configure the detector for use with a Millennium chromatography
workstation, version 3.2 and later. To do so, follow the instructions
32
in the Millennium Online Help to set parameters for controlling
the detector. In Millennium32 Chromatography Manager, version
3.2 and later, the detector is configured in 474 Emulation mode and
recognized by Millennium as a 474 detector.
32
Contents:
Topic
Page
Starting the detector
3-2
Using the operator interface
3-4
Preparing to start a run
3-18
Operating the detector
3-28
Programming methods and events
3-43
Scanning spectra
3-52
Managing results
3-65
Conserving lamp life
3-67
Shutting down the detector
3-70
3-1
Starting the detector
Warning:
• Always observe Good Laboratory Practices when you use this
equipment and when you work with solvents and test solutions.
Know the chemical and physical properties of the solvents and test
solutions you use. See the Material Safety Data Sheet for each
solvent and test solution in use.
• Using incompatible solvents can cause severe damage to the
instrument and injury to the operator. Refer to Appendix C for more
information.
Warning: Explosion hazard. The flash point is the lowest temperature at
which a flame can propagate through the vapor of a combustible
material to its liquid surface. A chemical’s flash point is determined by
the vapor pressure of the liquid. Only when a sufficiently high
concentration is reached can a solvent vapor support combustion.
Initializing the detector
Before you start the detector, be sure the power cord is properly installed at
the detector’s rear panel and plugged into the power source.
To start the detector, press the on/off switch on its front, lower-right-hand
corner (see the figure “Waters 2475 Multi λ Fluorescence Detector” on
page 1-7).
The detector beeps three times, displays the message “Booting System...
Please Wait (Service Keypad Inputs Accessible for 6 sec.),” and runs a series of
startup diagnostic tests.
Tip: Service keypad inputs are coded for use only by Waters service engineers
for troubleshooting purposes.
The detector displays the following messages:
3-2
1.
Initializing grating
2.
Initializing system
3.
Lighting lamp
4.
Warmup time left (counts down from five minutes)
Using the Detector
2475 detector startup screen
5.
Homing optical filter
6.
Searching for Zero Order Peaks
7.
Finding erbium calibration peaks
8.
Restoring last setup
When initialization is complete, the 2475 detector displays the Fluorescence
home screen. See page 3-9 and page 3-16 for more information.
Fluorescence home screen
Tip: For normal use, allow the detector to warm at least 60 minutes before
operating.
Startup failure
If one or more of the internal startup checks fails, the detector beeps and
displays an error message. For serious errors, the detector displays the word
“Error” in brackets (<Error>) in place of the runtime emission units on the
home screen.
“Startup error messages” on page 5-2 lists startup diagnostics failures, error
messages, and recommended recovery actions. The table titled “Error
messages preventing operation” on page 5-4 displays operational error
messages and suggested corrective actions. “Hardware troubleshooting” on
page 5-21 displays hardware-related causes of startup diagnostics failure and
corrective actions.
Starting the detector
3-3
Idle mode
When the detector is successfully started, it defaults to idle mode (see the
figure “2475 detector idle mode screen” on page 3-4). When it is not
performing any function requiring the shutter to be open (local methods,
scans, noise test, and so on), the shutter is closed, and the detector remains in
idle mode with the lamp lit. The Closed Shutter limits unnecessary UV light
from reaching the detector’s optics bench.
2475 detector idle mode screen
Using the operator interface
Using the display
The detector’s operator interface includes a 128 × 64 bitmap graphic display
and a 24-key membrane keypad. The home screen appears as shown in below.
Finding parameters in the fluorescence home screen
Emission/energy
units
Units indicator
Channel selector
Lamp on/off
Shift on/off
Single/multiwavelength
Gain
Keypad lock/unlock
Excitation
wavelength
Local method #/Remote Control
Run time (minutes)
Emission
wavelength
3-4
Using the Detector
Next screen
Sensitivity
Sticky diagnostics
on/off
You can display the home screen anytime by pressing HOME. When you first
use the detector, the home screen shows factory-set defaults. Afterward, it
shows the settings displayed before the detector was last shut down. The
home screen continues to change as the run continues.
In real time, the detector monitors fluorescence in terms of emission or energy
units of one or more wavelength pairs. Simultaneously, you can modify all
parameters in the table titled “2475 detector home and message screen icons”
on page 3-5. Press the A/B key to toggle between home screens for channels A
and B.
Fluorescence and message icons
The Fluorescence screens and message screens display the icons or fields
shown on page 3-4 and described in the following table. For a list of ranges
and defaults for the function icons and fields in the following table, see the
table titled “Primary and secondary function (method) parameters” on
page 3-21.
Caution: Changing the sensitivity (EUFS) setting affects the 1-V output.
For example, setting sensitivity to 500 EUFS on a 1-V output gives
500 EU/V and a 250 EU signal gives 0.5-V.
2475 detector home and message screen icons
Icon or field
Description
Function
Excitation
wavelength
Selects the digital fluorescence
wavelength pair monitored on the
selected channel. In single-channel
mode, you cannot independently
control a wavelength pair on
channel B.
Emission
wavelength
Selects the digital fluorescence
wavelength pair monitored on the
selected channel. In single-channel
mode, you cannot independently
control a wavelength pair on
channel B.
Using the operator interface
3-5
2475 detector home and message screen icons (Continued)
Icon or field
Description
Function
Gain
Selects the PMT gain setting.
Sensitivity (EUFS) Selects the chart sensitivity in
emission or sample energy units full
scale (EUFS) for the selected channel
(digital data is not affected).
Numerical
field (0.00)
Channel selector
Changes the channel when you press
A/B. The selected channel overlaps the
other channel.
Channel On
Displays the ON A or ON B icon for the
channel on which a timed or threshold
event is programmed.
Channel trace
When you press TRACE, displays the
fluorescence intensity, also known as
emission, for the channel indicated (A
or B).
Fluorescence in
emission units or
sample energy
units
Displays current normalized emission
units or sample energy units for the
selected channel that are not
normalized.
The displayed units depend on the
output units selected on the second
screen of the operator interface.
emission units Units indication
Displays the data units selection.
energy units
Lamp on
3-6
Using the Detector
Indicates the lamp is on.
2475 detector home and message screen icons (Continued)
Icon or field
Description
Function
Lamp off
Indicates the lamp is off.
Shift off
Blank = Shift off
Shift on
Indicates the shift is on for one key
press.
Single wavelength
Indicates the detector is operating in
single-channel mode.
Multiwavelength
Indicates the detector is operating in
dual-channel mode.
Triple wavelength
Indicates the detector is operating in
triple-channel mode. Icon only appears
if instrument is under control of an
external data system (remote mode
only).
Quad wavelength
Indicates the detector is operating in
quadruple-channel mode. Icon only
appears if instrument is under control
of an external data system (remote
mode only).
3D wavelength
Indicates the detector is operating in
3D-scan mode. Icon only appears if
instrument is under control of an
external data system (remote mode
only).
Using the operator interface
3-7
2475 detector home and message screen icons (Continued)
Icon or field
Numerical
field (0.00)
3-8
Using the Detector
Description
Function
Keypad unlock
Indicates unrestricted keypad entry.
Keypad lock
Indicates parameter changes are not
allowed; instrument is under control of
an external data system (remote mode
only).
Sticky diagnostic
on
Indicates a sticky diagnostic setting is
active. See page 5-8, for an explanation
of sticky diagnostic settings.
Local method
number
Indicates that the 2475 detector is not
controlled by a data system. It displays
either a cursive “m” and the current
method number or an asterisk (*),
which indicates current conditions are
not stored as a method.
RS-232 control
Indicates that the 2475 detector is
controlled by a data system, and
displays a remote control icon.
Ethernet control
Indicates that the 2475 detector is
controlled by a data system, and
displays a remote control icon.
Run time (minutes) Displays the time elapsed since you
pressed Run, or since an inject-start
signal was received.
Next
Indicates that pressing Next displays
additional screens.
Message screen
icon.
Indicates an error message.
2475 detector home and message screen icons (Continued)
Icon or field
Description
Function
Message screen
icon.
Indicates a question.
Message screen
icon.
Indicates a warning message.
Message screen
icon.
Indicates information is being
displayed.
Message screen
icon.
Indicates that you should standby.
Using the keypad
The keypad (see the figure “2475 detector keypad” on page 3-11) consists of 24
keys, which provide these functions:
•
Full numeric entry – 10 digits and a decimal point.
•
Global functions – Enter, Shift, CE (Clear Entry), Next, and ? (Help).
•
Navigation – and (used for navigation only; pressing can also
move you to the previous column, to the subsequent column). On
screens with a scrollable list, these keys move the highlight upward
(toward the beginning of the list) or downward (toward the end).
•
A/B – Toggles between channels A and B.
•
Direct access to specific screens – HOME, METHOD, CONFIGURE,
DIAG (Diagnostics), TRACE, and SCAN.
•
Primary functions – Chart Mark, Auto Zero, and Run/Stop. Primary
function keys take effect immediately, with no further entry required.
•
Secondary functions – SCAN, λ/λλ (single or multichannel), Reset
(clock), Lamp, Lock, Calibrate, System Information, Contrast, Previous,
Cancel, +/–, and Clear Field. Secondary function keys require you to
Using the operator interface
3-9
enter information into parameter fields, and then press Enter to actuate
the specified functions.
Keys that appear in all-uppercase letters (HOME, METHOD, CONFIGURE,
DIAG, TRACE, and SCAN) display a function, directly, from most screens.
Select a numerical entry on a list or menu as follows:
•
For numerical entries 1 through 9 on lists or menus, enter the number
corresponding to a desired item, and then press Enter.
•
For the number 10, select 0, and then press Enter.
•
To go to the end of a list, press •.
•
For entries numbered 11 or 12, press the
item on the list, and then press Enter.
or
key to scroll to a desired
Tip: The and keys do not incrementally increase or decrease field entries.
To change field entries, use the numerical keypad.
3-10
Using the Detector
2475 detector keypad
?
SCAN
λ/λλ
Reset
HOME
Chart Mark
Auto Zero
Run/Stop
METHOD
Lamp
Lock
Calibrate
A/B
1
2
3
CONFIGURE
System Info
DIAG
4
5
7
8
9
Cancel
+/−
.
Clear Field
Contrast
Previous
6
Next
Scale
TRACE
Shift
0
Enter
CE
The following table explains the functions of the primary and secondary keys.
To initiate a secondary function, press Shift and then the key.
2475 detector keypad description
Description
Key
?
HOME
Unshifted
After pressing shift
HOME – Displays the home
screen, which displays icons,
excitation and emission
wavelengths, EUFS, and Gain
fields.
? – Displays
context-sensitive Help,
when available.
Using the operator interface
3-11
2475 detector keypad description (Continued)
Description
Key
Unshifted
SCAN
Chart Mark
λ/λλ
Auto Zero
Reset
Run/Stop
After pressing shift
Chart Mark – Causes a
SCAN – Displays the list of
momentary pulse to the analog options for generating and
output (A and B, depending on manipulating spectra.
specified settings). This key has
no effect if the chart mark
function is disabled on both
1
channels.
Auto Zero – Sets the
fluorescence offset so that the
analog output (A and B,
depending on specified
settings) reads 0 EU. This key
has no effect if Auto Zero is
disabled on both channels. You
can enable or disable Auto Zero
from the fourth home screen
(see the figure “Secondary
functions of the home screen”
1
on page 3-17).
λ/λλ – From the home
screen, use this key to
toggle between single and
multichannel modes. The
current mode is indicated
by an icon on the display.
Run/Stop – Starts or stops
(freezes) the run clock and
initiates scans. The elapsed
time appears near the
lower-right-hand side of the
home screen. The shutter opens
when you press Run while the
idle mode function is enabled.
Reset – Resets the detector
run clock to zero minutes
and returns the detector to
initial conditions for a
current method. Closes the
shutter and displays Idle
Mode when the idle mode is
enabled.
and – On screens with entry fields (edit, check box, or
list), the active field has a thick border. You can use the
arrow keys to make a different field active. ( moves up or
leftward; moves down or right.) On screens with a
scrollable list, these keys move the highlight upward (toward
the beginning of the list) or downward (toward the end).
Other screens can have special instructions for using the
and keys (for example, the display contrast screen).
3-12
Using the Detector
2475 detector keypad description (Continued)
Description
Key
Previous
Next
METHOD
A/B
CONFIGURE
Unshifted
After pressing shift
Next – Displays a screen with
additional options related to
the current screen. Repeatedly
pressing this key always
returns the display to the
screen you started with. On
most screens where this key is
active, the NEXT arrow
appears in the
lower-right-hand corner.
Previous – When the Next
key is available, Previous
navigates through the
screens in the reverse order.
A/B – On screens that have the
A/B icon in the upper-left-hand
corner, this key toggles
between channel A and
channel B parameters.
METHOD – Displays the
list of options for creating
and clearing timed and
threshold events and
storing, retrieving, and
resetting methods.
DIAG – Displays the list of
diagnostic routines.
CONFIGURE – Displays
the first Configuration
screen.
TRACE – Displays the
fluorescence monitor trace for
channel A or B.
Scale – When the
wavelength trace or
spectrum screen is visible,
use this function to modify
the display range in the X
(time or wavelength) and Y
(fluorescence) dimensions.
DIAG
Scale
TRACE
Shift
Shift – Enables the shifted functions (identified by the text at
the top of most keys). The shifted state is temporary (lasting
for one keystroke only) and resets after the next keystroke.
Using the operator interface
3-13
2475 detector keypad description (Continued)
Description
Key
0-9
Unshifted
After pressing shift
0-9 – Enters the corresponding
number into the current field.
Also positions the cursor at the
corresponding entry in a list
(0 = tenth item). Selects the
corresponding number from a
list.
0-9 – See the descriptions
that follow for specific,
shifted, numeric keys.
1 – See 0-9 above.
Lamp – Displays the lamp
use statistics for the
currently installed lamp
and allows you to turn the
lamp on or off. The state of
the lamp is indicated by an
icon on the home screen.
2 – See 0-9 above.
Lock – When you are
viewing the home screen,
enables or disables the
keypad lock feature. Use
the lock to prevent
inadvertent changes to
detector settings. The
current lock state is
indicated by an icon on the
home screen.
3 – See 0-9 above.
Calibrate – Initiates the
wavelength calibration
routine.
4 – See 0-9 above.
System Info – Displays
system information
including software version,
checksum, and instrument
serial number.
Lamp
1
Lock
2
Calibrate
3
System Info
4
3-14
Using the Detector
2475 detector keypad description (Continued)
Description
Key
Unshifted
After pressing shift
6 – See 0-9 above.
Contrast – Use to adjust
contrast (viewing angle) in
the liquid crystal display.
0 – See 0-9 above.
Cancel – In some modes,
Cancel backs out of a
prompt without completing
the task. The word Cancel
appears as a cue in the
lower-right-hand border of
the message.
Contrast
6
Cancel
0
+/–
•
Clear Field
CE
Enter
• – Enters a decimal point. Also +/– – Some edit fields accept
positions the cursor at the last negative number entry. Use
entry in a list.
this function to invert the
sign of the number in the
active field.
CE – Clears an editing change, Clear Field – Blanks the
and returns the contents of a
current entry field before
field to its previous value. Sets you specify a new value.
the value to a unique word for
some fields. For example, in the
voltage offset diagnostic, you
can enter either a numeric
offset value or press CE to
change it to OFF.
Enter – Completes the entry in an editable field. Also
advances the active field, as if had been pressed (except
after editing the wavelength on the home screen). Press
Enter to acknowledge error messages and other prompts. In
these cases, the word Enter appears as a cue in the
lower-right-hand border of the message.
1. Chart Mark and Auto Zero do not affect the output with reference energy selected.
Using the operator interface
3-15
Navigating the user interface
Press Enter or and to navigate among editable fields. A thick border
appears around the active field. When you complete an entry, press Enter to
advance to the next active field.
If you make an error, press CE (Clear Entry) to undo any changes and return
to the active entry field.
An active field containing a list has a number to the right of the field within
the thick border. To display a list, press Enter, and then perform one of these
actions:
•
Press the corresponding number key to select an item immediately.
•
Use
and
to scroll through the list, and then press Enter.
If you know the number corresponding to a choice, you can press that number
without pressing Enter first.
The and keys do not incrementally increase or decrease numerical field
entries. To change field entries, use the numerical keypad.
Navigating to and from the home screen
Pressing HOME displays the home screen from most other screens. From the
home screen, you can choose several secondary functions. To move to the home
screen’s secondary function screens, press Next. The secondary functions
include these:
•
Filter type
•
Analog output specifications
•
Time constant
•
Data units selection
•
Voltage offset
•
Chart polarity
•
Enable/disable several inputs
•
Enable/disable external events
The values and settings you specific in the secondary function fields become
part of a current method’s conditions and are retained when you store the
method (see page 3-43).
3-16
Using the Detector
When you press Next, the detector displays three additional home screens,
labeled “2 of 4”, “3 of 4”, and “4 of 4” (see the figure “Secondary functions of the
home screen” on page 3-17).
Secondary functions of the home screen
Press Next
Fluorescence home screen
Press Next
auto zero-on-inject, gain, and λ
changes.
filter type, analog out, and time
constant (available for both λ and
λλ)
Press Next
Press Next
data units selection, voltage offset,
and chart polarity (available for
both λ and λλ)
Using the operator interface
3-17
Preparing to start a run
You must set up a run before you make fluorescence measurements. To start a
run, you can press Run/Stop or trigger the detector’s operation via the injectstart terminals on the rear panel. When you start a run, the shutter opens
automatically and the detector performs an auto-zero function (when the
function is enabled).
You must select Auto Zero-On-Inject for the detector to automatically auto
zero (see page 3-19).
32
While under Empower or Millennium control (see page 3-29), the shutter
closes when the detector finishes a run, and the run timer stops and then
resets. If you run the detector manually, you can close the shutter by pressing
Reset or waiting until the run ends.
To avoid the automatic opening and closing of the shutter, you can disable the
idle mode by checking the Selection box in the Configuration screen (see
page 3-4).
Setting up a run
After you press HOME to return to the home screen and select a channel mode
(λ or λλ), you can set up the detector for a run. Before beginning a run,
however, you must select the channel mode, and program the following
parameters:
•
Operating wavelength pairs
Tip: The emission λ setting (eλ) must be at least 10 nm above the
excitation λ setting (xλ).
•
Gain
•
Output sensitivity, EUFS
•
Filter type
•
Time constant
•
Analog output type
•
Data units
If you want to perform other functions during a run, you must program other
parameters. “Accessing primary and secondary functions” on page 3-19 and
the table titled “Primary and secondary function (method) parameters” on
3-18
Using the Detector
page 3-21 describe the functions, fields, screen number, display units,
allowable ranges, and default settings for the home screen and its secondary
function screens.
Accessing primary and secondary functions
You can access the primary and secondary functions from the home screen or
by pressing Next.
•
Excitation Wavelength – Defines the operating excitation (xλ)
wavelength for the channel.
•
Emission Wavelength – Defines the operating emission (eλ) wavelength
for the channel.
•
EUFS (emission/sample energy units full scale) – Defines the
relationship between the fluorescence signal response (EU) and the
analog output voltage. The output voltage reaches full scale when
fluorescence attains the EUFS value.
Caution: Changing the sensitivity (EUFS) setting affects only the
1-V output. The digital output at the RS-232 connector remains
unchanged.
•
Gain – This setting controls the full-scale sensitivity of the detector by
defining the PMT gain factor from 1 to 1000. Each gain setting must
have a linear relationship with the actual fluorescence signal.
•
Filter type – Defines your choice of noise filter (Hamming filter is the
default).
•
Data units – Defines data units.
•
Emission – The standard chromatography mode of the detector, which
normalizes the output to a water standard in emission units. Emission
units are measurements of light that are independent of PMT gain.
Through normal use, the optics in any detector age and, therefore,
change, resulting in measurement variation over time. By using
emission units, you can eliminate optics deterioration as a variable in
your measurements. When emission units are used, measurements
taken on different 2475 detectors are fully compatible with one another.
•
Energy – Energy units do not have the normalization advantage. Those
traditional units of measurement conform to currently established test
methods. However, the results depend greatly on PMT gain.
Preparing to start a run
3-19
•
•
Analog out (single λ pair)
•
Emission – Fluorescence output corresponding to the data units
selected.
•
Reference Energy – Charts the lamp energy from the reference
photodiode located in the excitation optic bench. Reference scaling is
fixed at 10,000 units per volt.
•
Output Off– Output set to zero volts.
Analog out (multi λλ) – In addition to the selections for single λ, you can
chart the same parameters on the other channel at a different excitation
and emission wavelength pair, and you can chart the following
parameters:
•
MaxPlot – Charts the fluorescence of multiple compounds with
different fluorescence values at different excitation and emission
wavelength pairs on a single data channel. Scaling for MaxPlot is
the same as for Fluorescence, except that the charted fluorescence is
the larger of the fluorescences measured on channels A, B, C, and D.
The detector uses the EUFS, data offset, and voltage offset of the
selected fluorescence channel regardless of which channel is larger.
Volts out = Larger fluorescence (A or B) × 1 V/EUFS (of selected
channel)
•
Difference Plot (A-B and B-A) – Charts the difference in
fluorescences at two different wavelength pairs. The scaling for the
difference plot is identical to emission or sample energy selections,
except that the charted fluorescence is the difference in value of the
two fluorescences measured on channels A and B. The detector uses
the EUFS (of the selected channel), emission offset, and voltage
offset of the selected channel for scaling.
Volts out = Fluorescence difference (A – B or B – A) × 1 V / EUFS
3-20
•
Time constant – Adjusts the noise filter (time constant) to achieve the
optimum signal-to-noise ratio without changing the sensitivity setting
(see page 1-12).
•
Voltage offset – Adjusts the charted analog output signal. Specified in
millivolts, voltage offset adjusts the 1-V signal by the entered value.
This is useful for making minor adjustments, and for nulling any offset
between the detector and a connected external data system.
•
Chart polarity – Inverts the chromatogram on the analog output.
Entering the plus symbol (+) produces a normal chromatogram; entering
Using the Detector
the minus symbol (–) produces an inverted chromatogram at the analog
output channel.
•
Auto Zero-On-Inject – Selected by default, this parameter specifies the
auto-zero behavior each time the detector receives an inject-start signal.
You can disable this parameter by pressing any numerical key to clear
this field for either or both channels.
•
Auto Zero on λ and gain changes – This function produces an auto zero
each time a wavelength change or gain change is requested. If you
disable it, significant changes in measured fluorescence can occur after
each wavelength change. Selecting “to zero” sets the signal level to zero.
Selecting “to baseline” maintains the previous baseline level when the
gain or wavelength change is made. “To baseline” is the default setting.
•
Enable keypad & event-in chart mark – Selected by default, this
parameter results in a chart mark each time one is requested. You can
disable this parameter by pressing any numerical key, clearing this field
for either or both channels. Chart marks work with analog channels
only.
Primary and secondary function (method) parameters
Function
Screen
Type
Units
Range
Default
xλ
(Excitation
wavelength)
home
Numeric
nm
Integer 200 to 350 nm
890 nm
eλ
(Emission
wavelength)
home
Numeric
nm
Integer 210 to 397 nm
900 nm
Tip: The emission λ setting must always be at least 10 nm above the
excitation λsetting.
Gain
1
Numeric
Emission 0 to 1,000
or
energy
units
0
EUFS
1
Numeric
EUFS
1 to 100,000
10,000
Filter type
2 (of 4)
Choice
None
• Hamming
• RC
• None
Hamming
Preparing to start a run
3-21
Primary and secondary function (method) parameters (Continued)
3-22
Function
Screen
Type
Units
Range
Default
Analog out
(single λ)
2 (of 4)
Choice
None
• Emission
A
• Reference
energy A
• Output off
Emission A
Analog out
(multi λλ)
2 (of 4)
Choice
None
• Emission
Emission A
A
• MaxPlot A,
B, C, D
• Diff (A-B)
• Diff (B-A)
• Reference
energy A
• Output off
Time
constant
2 (of 4)
Numeric
sec
• Hamming
(λ): 0.1 to
5.0
• Hamming
(λλ): 1 to
50
• RC(λ): 0.1
to 99
• RC(λλ): 1
to 99
• 0 to
disable
filtering
1.5
Data units
3 (of 4)
Choice
None
• Emission
• Energy
Emission
Voltage offset 3 (of 4)
Numeric
mV
Integer –1000 0
to +1000
Chart
polarity
Choice
None
+
–
Using the Detector
3 (of 4)
+
Primary and secondary function (method) parameters (Continued)
Function
Screen
Type
Units
Range
Default
Auto Zero on
inject
4 (of 4)
Check
box
None
Checked
not checked
Checked
Auto Zero on
λ changes
4 (of 4)
Choice
None
• To
baseline
• To zero
• Disable
To baseline
Operating the trace and scale functions
The trace function displays a fluorescence signal for the last n minutes (up to
and including 60) of detector operation.
•
Press TRACE to display fluorescence acquired over the last 30 minutes
by default. The trace is updated once every 20 seconds.
•
Select Scale (Shift, TRACE) to display the scaled trace, which shows T1,
the ending time (-30 for the last 30 minutes), by default.
You can change the ending time, specifying any number from 3 through
60. You can use the scale function to zoom in on a particular section of
the trace.
Displaying the scale parameters
To display the scale parameters:
1.
Press Scale.
2.
Press Next to display T2 (starting time). The default is 0.
3.
Press Next again to display EU1 (starting or low fluorescence). The
default is auto.
4.
Press Next again to display EU2 (ending or high fluorescence). The
default is auto.
By entering appropriate times and fluorescence numbers in the four
scaling parameter boxes, you can zoom in on one section of an ongoing
fluorescence trace.
•
For EU1 and EU2, press CE to reset to auto.
Preparing to start a run
3-23
•
T1 represents the left-hand side of the trace, or ending time (default
is -30).
•
T2 represents the right-hand side of the trace, or starting time
(default is 0).
The following figure shows a 60-minute trace of continuous injections of
salicylic acid and naproxin with the excitation wavelength set to 240 nm and
emission to 355 nm.
Scaled trace of continuous injections with T1 changed to -60
The following figure shows a 4-minute scaled trace (or zoom) of the 60 minutes
of continuous injections shown in the previous figure. T1 is changed to -4. T2
is changed to 0. EU1 and EU2 remain as auto.
Scaled trace for 4 minutes changing T1 to -4
The following figure shows a 60-minute trace on channel A scaled to the last
15 minutes. T1 is changed to -15.
Scaled trace changing T1 to -15
3-24
Using the Detector
As you modify the output using the scale function, the trace function
continues to display the 2475 detector output in real time on either or both
channels.
Configuring the detector
You can configure the detector to emulate the Waters 474 detector
communication protocol in the Configuration screens. Select CONFIGURE
(Shift, DIAG). The first of three Configuration screens appears. Then select
474 emulation.
Tip: Other functions, such as specifying event inputs and enabling pulse
periods, are also available in the Configuration screens.
Configuration screens
Configuration screen 1 of 3
Configuration screen 2 of 3
Configuration screen 3 of 3
Disabling shutter idle mode
You can disable the shutter idle mode in the first Configuration screen. When
selected, the shutter does not close to protect the detector’s optics after the
completion of a run to protect the detector’s optics (see page 3-4).
Configuring event inputs and contact closures
You can also use CONFIGURE to edit event input settings and specify
switched output settings. Use Enter and the numeric keypad or and
select the appropriate entry.
to
The second Configuration screen includes four editable entry fields.
•
Inject – You can specify an inject-start input to signal the start of a run,
an event that resets the run-time clock and applies initial method
conditions immediately:
Preparing to start a run
3-25
•
•
•
–
High – Starts run when contact closure changes from off (open)
to on (closed).
–
Low – (Default) starts run when contact closure changes from
on (closed) to off (open).
–
Ignore – No response to inject-start input.
Chart mark – You can specify a chart mark input to create a chart mark
on channel A and/or channel B. To determine the response of the
channel, enable the chart mark function (see the table titled “Primary
and secondary function (method) parameters” on page 3-21 and the
figure “Secondary functions of the home screen” on page 3-17):
–
High – Creates a chart mark(s) when contact closure changes
from off (open) to on (closed).
–
Low – (Default) creates a chart mark(s) when contact closure
changes from on (closed) to off (open).
–
Ignore – No response to chart-mark input.
Auto Zero – You can configure the auto-zero input to auto zero
fluorescence readings on channel A and/or channel B. To determine the
response of the channel, enable the auto-zero function (see the table
titled “Primary and secondary function (method) parameters” on
page 3-21 and the figure “Secondary functions of the home screen” on
page 3-17):
–
High – auto zeroes the channel when contact closure changes
from off (open) to on (closed).
–
Low – (Default) auto zeroes the channel when contact closure
changes from on (closed) to off (open).
–
Ignore – No response to auto-zero input.
Lamp – You can configure the Lamp input level to turn the xenon lamp
on or off from an external device:
–
High – Turns lamp on when contact closure is on (closed).
–
Low – Turns lamp on when contact closure is off (open).
–
Ignore – (Default) does not respond to Lamp input.
Setting pulse periods
In the third Configuration screen (see the figure “Configuration screens” on
page 3-25), you set the pulse width or activate a rectangular wave on SW1 or
3-26
Using the Detector
SW2. The figure “Setting the pulse period or signal width on SW1 or SW2” on
page 3-27 shows a single pulse and a rectangular wave.
•
Single pulse (in seconds) – If SW1 or SW2 is programmed to generate a
pulse as a timed or threshold event, then this field specifies the period of
the signal (single pulse width; range 0.1 to 60 seconds).
•
Rectangular wave (in seconds) – If SW1 or SW2 is programmed to
initiate a rectangular wave as a timed or threshold event, then this field
specifies the period of the signal (the width of one pulse period in a
rectangular wave or pulse train; range 0.2 to 60 seconds).
Setting the pulse period or signal width on SW1 or SW2
n seconds
Single pulse
n seconds
Rectangular wave
Setting the Display Contrast
Using the contrast function, you adjust the contrast of the detector display
screen. When you select Contrast (Shift, 6), the Display Contrast screen
appears. Use and to adjust the contrast of the display, and then press
Enter.
Display Contrast screen
Preparing to start a run
3-27
Displaying system information
Select System Info (Shift, 4) for information about the detector, including
(where applicable) the serial number, software version number with
checksum, and version date. Press Enter to return to the home screen.
Example of a System Info screen
Tip: The 2475 detector release notes also reflect the checksum and version.
Using Online Help
The detector has limited context-sensitive Help. When you select “?” (Shift,
HOME) from a point in the program associated with a Help screen, the screen
appears. If Online Help is not available, selecting “?” effects no response. Press
Enter to return to the home screen.
Example of a Help screen
Operating the detector
3-28
•
If you are operating the detector under the control of an external data
system, you can program any parameters not controlled by the external
data system at the detector’s front panel before the external system
takes control.
•
To prevent resorption of dissolved oxygen, sparge or run the solvent
degasser continuously when operating the detector (see Appendix C).
Using the Detector
Two operating modes
You can use the detector in either single-channel or multichannel mode over a
range of 200 to 900 nm. The detector defaults to its mode of operation when
last shut down.
When the detector is operating in single-channel mode, you can configure
analog outputs on channel B. In single-channel mode, the detector tracks a
single wavelength on both channels A and B. You can use channel B for these
purposes:
•
Track fluorescence (EU) at an alternate EUFS.
•
Monitor sample or reference energy outputs.
•
Set a different time-constant.
The table titled “Primary and secondary function (method) parameters” on
page 3-21 includes more information on operating parameters adapted in
single or multichannel mode. When an Empower or Millennium32 data system
controls the detector, it functions as a Waters 474 detector (see page 3-29).
Standalone operation
When using the detector as a standalone instrument, you can store up to 10
methods, each containing up to 48 timed and 2 threshold events (see
page 3-43). An asterisk in the method number field on the home screen
indicates conditions at the time of a run, not a stored method.
Remote control operation for 474 emulation mode via RS-232
The remote control icon appears on the home screen (see the figure “Finding
parameters in the fluorescence home screen” on page 3-4 and the table titled
“2475 detector home and message screen icons” on page 3-5) when an external
32
data system controls the detector. Under Empower or Millennium control for
474 emulation, the detector uses the RS-232 connector (see page 2-18), and an
“R” appears within the remote icon, which itself appears on the busLAC/E
data system’s configuration screen as a 474 detector when the Emulate 474
option is enabled and operating in single-channel mode.
To connect the detector to an HPLC system, see page 2-3. To connect the
detector to an external system, see page 2-5.
Operating the detector
3-29
Instrument setup
To set up the instrument:
1.
On the configuration screen (Shift, DIAG), configure the detector to
operate in 474 Emulation mode.
2.
Connect the detector via a standard RS-232 cable to any available COM
32
port on a Millennium PC.
3.
In the operating system’s device manager, check to ensure the COM port
you connected the detector to is available.
Tip: The same parameters apply to a 474 detector.
PC configuration for remote control
Parameter
Value
Baud rate
4,800
Stop bits
2
Parity
None
Data length
8 bits
Flow control
Xon/Xoff
Method parameters
The initial conditions for the method are specified on the General tab of the
32
fluorescence method editor in Empower or Millennium software.
Tip: The 2475 detector and the 474 detector interpret some method
parameters differently.
Example of method parameters
3-30
Parameter
Value
Excitation λ (nm)
350
Emission λ (nm)
397
Bandwidth (nm)
18
Filter Type
Digital
Filter Response
10
Using the Detector
Example of method parameters (Continued)
Parameter
Value
Lamp Off Time (hrs)
1.0
Sampling Rate
2
Offset (mv)
0
Gain
1
Attenuation
64
Auto Zero
Auto
Polarity
+
•
Excitation (xλ or Ex) and Emission (eλ or Em) λ (nm) – Wavelength
setpoints for the two monochromators. The detector’s excitation range is
200 to 890 nm and emission range is 210 to 900 nm. You must set the
emission wavelength at least 10 nm above the excitation wavelength.
Tip: If any of these conditions are ignored, a detector error occurs, and
the sample set can be suspended.
•
Bandwidth (nm) – Not an adjustable parameter on the detector, so the
value in this field is ignored. However, the static bandwidth of a detector
is 20 nm, so an entry of 18 nm in this field would document a value close
to the actual value in the method (for sample detail archival purposes in
Empower or Millennium32 software).
•
Filter Type – Either a digital (Hamming) or RC filter. The digital filter
tends to yield peaks with far less distortion than does a standard RC
filter. The RC filter serves the need of those who want to follow
established measurement conventions.
•
Filter Response – Time-constant setting of the filter (3, 5, 10, 20, and
40). The 474 detector interprets these numbers in terms of seconds.
However, values of this magnitude are too high for chromatography, so
the 2475 detector interprets these inputs at one tenth the specified
value.
Operating the detector
3-31
When you select RC as the filter type, only three response choices are
possible (Fast, Std., and Slow). These are the time-constants associated
with those selections:
–
Fast = 0.5 sec.
–
Std. = 1.5 sec.
–
Slow = 4.0 sec.
Digital time constant response settings
32
•
Empower or Millennium selection
2475 time constant (sec.)
3
0.3
5
0.5
10
1.0
20
2.0
40
4.0
Lamp Off Time (hrs) – Determines how long after the start of an
injection the lamp turns off. The timer resets at the beginning of every
injection. Typically, you can set this function to a value that is
comfortably longer than the runtime of any injection. For example, if
your longest runtime is 30 minutes, choose a Lamp Off Time selection of
2 hours. By leaving 90 minutes of idle “lamp on” time, you can easily
start another sample set without having to wait for the lamp to warm
again.
Tip: It is best to shut the lamp off only when all runs are completed. You
should program the lamp to turn off (or turn off the lamp manually) only
when you do not expect to use it for at least four hours.
3-32
•
Sampling Rate – Number of data points per second that the detector
32
transmits to Empower or Millennium software.
•
Offset – Level of offset, in millivolts, applied to the channel A analog
output only. It does not affect the digital data transmitted to Empower
32
or Millennium software over RS-232.
•
Gain – Gain value applied to the PMT. The choices are 1, 10, 100, and
1000.
•
Attenuation – Analogous to EUFS. Values from the 474 detector are
translated according to the following table. This parameter affects only
Using the Detector
the data on the channel A analog output, not the data output to
32
Empower or Millennium software through the RS-232 connection.
474 and 2475 attenuation values
474 attenuation constants
2475 translation to EUFS
S (short)
1
1
1
2
10
4
50
8
100
16
500
32
1,000
64
5,000
128
10,000
256
100,000
•
Auto Zero – Corresponds directly with enabling (Auto) and disabling
(Manual) the automatic Auto Zero on injections, gain, or wavelength
changes. You change these settings via the boxes on page 4 of the 2475
operator interface. If you select manual, the detector performs an Auto
Zero only when directly commanded to do so, either as a timed event,
front panel button, or rear panel (terminal block) contact closure. If you
select auto, it performs an Auto Zero at the start of any injection or when
the wavelength or gain is changed.
•
Polarity – Defines the polarity of the analog output only.
Operation details
•
Inject Start – The detector is configured with a switch-closure signal
input to the chart mark terminal on the rear panel. A signal input starts
injection run timer. You can also attach the wire to the inject start
terminal on the rear panel.
•
Compatibility with the Waters 474 detector – Separation methods for
the 2475 and 474 detectors are compatible except for their gain values.
See page 3-19, “Emission (A & B)” and “Sample Energy (A & B).” The
higher sensitivity of the 2475 detector’s flow cell produces significantly
higher signals. Thus gain values optimized for the 474 detector can yield
Operating the detector
3-33
saturated, highly distorted measurements when you apply them to the
2475 detector. You must therefore reduce the 2475 detector’s gain
setting as much as tenfold when using a method developed for the 474
detector. A gain setting of 1 suffices in most cases because of the
2475 detector’s favorable signal-to-noise ratio.
32
•
Sample Energy or Emission Units – Empower or Millennium software
can accept either sample energy or emission units. You can edit the data
field Channel Description when creating an Empower or Millennium
instrument method to record the units used, selecting the units from the
Analog Out field on the second of the four home screens.
•
Run Time – The run time is not synchronized with that of Empower or
32
Millennium software. However, the run time (and therefore
programmed timed events) is synchronized with the recorded time axis
32
of the chromatogram in Empower and Millennium software.
Remote control operation via Ethernet connection using 2475
instrument control software
This mode of operation uses the Ethernet connection and appears in the
Empower software Configuration window as a 2475 detector. The remote
control also icon appears on the detector’s home screen (see the figure
“Fluorescence home screen” on page 3-3) with an “E” at it’s center (see the
table titled “2475 detector home and message screen icons” on page 3-5). For
this mode of operation, you must disable the Emulate 474 option from the
Empower Configuration window.
Verifying the detector
Perform tests to verify wavelength accuracy (see page 3-35) and optimize
emission units (see page 3-36). Doing so ensures no components in the flow
cell interfere with the erbium lines at 379 and 522 nm. Completing these
procedures successfully ensures that the detector’s optics and electronics
function properly.
Tip: Before you pump solvent or mobile phase through the system, flush the
lines with filtered, degassed, and sparged HPLC-grade water. Then pump the
mobile phase, provided you encounter no miscibility problems, at 1 mL/min for
15 minutes, minimum.
3-34
Using the Detector
Manual wavelength calibration
You can calibrate the detector manually from the keypad by pressing the
manual calibration key at any time during detector operation or when
calibration errors arise during startup. You need not restart the detector after
a successful wavelength calibration.
Tip: Before you pump solvent or mobile phase through the system, flush the
lines with filtered, degassed, and sparged HPLC-grade water and continue to
pump at 1 mL/min.
To manually calibrate the wavelength:
1.
Select Calibrate (Shift, 3) from the keypad.
Wavelength calibration message
2.
Ensure the flow cell is prepared, and then press Enter.
The detector cycles through the calibration procedure and momentarily
displays a series of initialization messages similar to those displayed at
startup. If calibration is successful, the detector beeps three times.
If the maximum error is greater than 2.0 nm, the detector displays the
maximum error of the farthest calibration shift from the previous
calibration.
Calibration successful message
3.
Press Enter. A “Calibration complete” message appears momentarily.
Other messages, such as “Optimizing system performance” and
“Restoring last setup”, can appear before the home screen reappears.
Operating the detector
3-35
4.
If calibration is not successful, repeat it.
5.
If calibration is still not successful, shut down the detector and restart it
(see Chapter 5).
6.
Run the detector normalization diagnostic test (see page 3-36).
Requirement: If this test yields a failing result, repeat it.
Normalizing emission units
You can select either emission or energy units in the Output field of page 2
(see page 3-19). If you select emission units, normalize to a standard water
reference on a monthly basis to ensure that measured signal strengths are as
consistent as possible with those measured by other 2475 detectors.
Tip: Before you pump solvent or mobile phase through the system, flush the
lines with filtered, degassed, and sparged HPLC-grade water. Then pump the
mobile phase, provided you encounter no miscibility problems, at 1 mL/min for
15 minutes, minimum.
Tip: Normalize emission units weekly.
To normalize emission units:
1.
Power-on the detector, and allow it to warm and stabilize for at least 1
hour.
2.
Run clean, degassed water through the flow cell at 1 mL/min (or a flow
rate sufficient to prevent the formation of air bubbles).
3.
Press DIAG, and then press 1 Normalize Units.
The detector adjusts the PMT gain, and it sets the excitation
monochromator wavelength to 350 nm. The emission monochromator
scans from 390 to 405 nm to find the Raman signal for water (397 nm),
minimizing the occurrence of wavelength accuracy errors from distorting
the normalization constants. After the emission monochromator finds
this signal peak, PMT gain is optimized, and the normalization
constants appear.
3-36
Using the Detector
Normalization values at completion
The values are used in the emission units formula described on page 1-14. As
the lamp and optics age, the Raman Gain will gradually increase to a
maximum of 1000, and the Raman Counts value can decrease. If the Raman
signal for water occurs within 3 nm of 397 nm, the normalized units are
embedded in the detector’s memory. If the signal fails to occur within that
range, the detector does not save the values but instead preserves the prior
constants.
Tip: If the Raman signal is not within 3 nm of 397 nm, usually something
other than pure water is in the flow cell or the flow cell is dirty.
Operating the detector in single-channel mode
The detector is optimized for single-channel (λ) operation, the default
operating mode.
To specify single-channel mode when the detector is in multichannel mode:
1.
From the home screen, select l/λλ (Shift, Auto Zero).
Result: The detector displays an appropriate message while switching to
single-channel operation.
2.
Enter the wavelength, gain, and sensitivity on the home screen. Also
enter any secondary parameters, timed events, or threshold events (see
the figure “Secondary functions of the home screen” on page 3-17 and
the table titled “Primary and secondary function (method) parameters”
on page 3-21 through the table titled “Threshold events “To”
parameters” on page 3-48).
Tip: Changing the sensitivity (EUFS) setting affects the 1-V output.
3.
To select a second sensitivity setting while in single-channel mode, press
A/B, and enter the appropriate EUFS on the channel B screen.
Tip: A single channel is tracked on channel A, leaving channel B
available for monitoring emission with an alternate EUFS setting. You
Operating the detector
3-37
can also use the Energy setting on channel B while making the primary
fluorescence measurement specified by a EUFS on channel A. For
example, while operating in single-channel mode, you can set an EUFS
of 500 on the second channel, providing a different scaling factor on the
channel B 1-V output.
The detector engages the second-order filter for all excitation wavelengths
greater than 400 nm.
You can configure the detector to show the measurement results in either
emission units or energy units (see page 3-19).
Operating the detector in multichannel mode
You can operate the detector with expanded chart-out selections in
multichannel (λλ) mode, which offers the following functions:
•
Emission (A and B)
•
Sample energy (A and B)
•
Reference energy (A and B)
•
MaxPlot
•
Difference (A-B) or (B-A)
For details about these functions, see page 3-19 and the table titled “Primary
and secondary function (method) parameters” on page 3-21.
Changing from single to multichannel mode
To change from single to multichannel mode:
1.
From the home screen in single-channel mode (λ), select λ/λλ (Shift,
Auto Zero).
This key toggles between single and multichannel mode, and the
detector displays a message indicating it is setting up multichannel
operation.
3-38
2.
Specify the excitation wavelength to monitor in the xλ field, and then
press Enter.
3.
Specify the emission wavelength to monitor in the eλ field, and then
press Enter.
Using the Detector
4.
Specify the other operating parameters and any timed or threshold
events, if desired.
5.
Specify the desired gain.
6.
Press A/B to switch channels. The home screen for the other channel
appears.
7.
Specify the operating parameters for the second excitation and emission
wavelength pair monitor, the gain, and any timed and threshold events,
if desired.
For more information on operating the detector in single-channel mode, see
page 3-37.
For more information on programming timed and threshold events, see
page 3-43.
Tip: In multichannel mode, do not set the gain setting for each channel
individually.
Obtaining a MaxPlot or difference plot
You can obtain a MaxPlot or difference plot by monitoring fluorescence at two
selected wavelengths while plotting the maximum fluorescence for each
sample component. Be sure the detector is operating in multichannel mode.
To obtain a MaxPlot or difference plot:
1.
From the home screen, press Next.
Screen 2 of 4 appears (see the figure “Secondary functions of the home
screen” on page 3-17).
2.
Select the filter type (Hamming is the default) in the first field, and
press Enter.
3.
In the second field (analog out), make the appropriate selection:
•
3, maxplot A,B
•
4, difference A-B
•
5, difference B-A
4.
Press Enter to select the MaxPlot function.
5.
Return to the home screen by pressing HOME.
Operating the detector
3-39
Setting gain and EUFS
Choosing a gain setting for the PMT before injecting sample is a necessary
part of fluorescence measurement with detectors that use PMTs. For the best
signal-to-noise ratio, set a gain that maximizes the dynamic range of the
electronics (see page 1-17). A gain that is too high overloads the pre-amplifier,
and a value of –9999.9 appears in the Emission/Energy units field.
Fluorescence
Gain set too high (fluorescence pinned to –9999.9 EU)
Minutes
Gain set too high alarm message
The value of -9999.9 EU in the Emission/Energy Units field and the alarm
help differentiate between too high a gain setting and too low an EUFS
setting. When the EUFS is set too low, flat-topped peaks appear, because the
upper limit of the output range is exceeded.
Tip: An EUFS over-range situation occurs only when you use the analog
outputs.
3-40
Using the Detector
Fluorescence
EUFS set too low (flat-topped peak)
Minutes
Auto-optimizing gain and EUFS
The auto-optimize gain diagnostic test allows the detector to run a single trial
chromatogram and suggests the ideal gain values to optimize the dynamic
range of the signal collection electronics. You must program a separation
method for the trial chromatogram. This can be done by entering a method via
the keypad or by retrieving a previously created method from a stored memory
location.
32
If you are using Empower or Millennium software, you must enter the
method into the 474 Fluorescence Detector Instrument method editor. The
method is then downloaded to the 2475 detector when you make an injection.
After you program the method into the 2475 detector (or Empower or
32
Millennium editor), press DIAG, and then press 3 Auto-Optimize Gain.
Selecting the auto-optimize gain diagnostic test
Operating the detector
3-41
Selecting Auto-Optimize Gain prepares the diagnostic to be executed on the
next injection. The sticky diagnostic (wrench) icon appears on the home
screen, and <Auto Gain> appears in the emission field.
Auto-optimize gain diagnostic test is implemented
Auto Gain diagnostic
Sticky diagnostic tool
You can start the injection after you arm the diagnostic with a start pulse
trigger from an injector input to the inject event terminal on the detector rear
panel. You can also press Run/Stop on the front panel as the sample is injected
into the fluid stream.
Tip: You must synchronize the start trigger with the chromatography so that
the timed events occur at proper times relative to the peaks.
Select Make Injection if you are running under Empower or Millennium32
control, or start the injection through other devices (such as an Alliance 2695
Separations Module).
Tip: The gain setting appears as 1 during the auto-optimize gain run.
Gain set to 1 automatically during the auto-optimize gain run
The detector runs the programmed timed events and displays the ideal gain
table at the end of the run (see the next figure). Use the arrow keys to advance
through the table.
The run timer is automatically stopped and reset when running under
32
Empower or Millennium control. In standalone mode, the detector runs for
an unspecified duration, so you must stop the run by pressing Run/Stop, and
then select Reset (Shift, Stop) on the detector’s front panel.
3-42
Using the Detector
Results in the auto-optimize gain table
Once you clear the display by pressing home, the auto-optimize gain
diagnostic test is automatically disengaged. With the proper gains and EUFS
values specified in the method, the chromatogram is on scale.
Fluorescence
Gain and EUFS optimally set
Minutes
Programming methods and events
Storing methods
You can store and retrieve up to ten methods. The detector designates stored
methods as the numerals 1 through 10. If you are using a stored method
during operation, the method number appears on the home screen (see the
figure “Finding parameters in the fluorescence home screen” on page 3-4). An
asterisk in the method number icon (see the table titled “2475 detector home
Programming methods and events
3-43
and message screen icons” on page 3-5) indicates that conditions are not
stored.
If you edit a parameter such as wavelength or EUFS, you are editing the
current conditions (Method *). You may store the method in one of the 10
available method storage slots, or you can replace the current method with
one previously stored. When you retrieve a previously stored method, you
replace the existing method conditions with those of the stored method.
The method number displayed on the home screen is that of the retrieved
method until you make a change. Any parameter change (for example,
wavelength or EUFS) alters conditions so that the original recalled method is
no longer in effect, causing the method number to change to an asterisk.
On startup, the operating parameters at the time the detector was last shut
down are restored. However, any timed events or thresholds associated with
the method are deactivated when power is restored. Thus, on startup, an
asterisk always appears inside the method icon on the home screen.
When the detector is operating under remote control by Empower or
32
Millennium software, the remote icon appears (see the table titled “2475
detector home and message screen icons” on page 3-5).
Programming timed events
You can program up to 48 timed events to the nearest 0.01 minute. As you
enter timed events, each new event appears at the end of the timed event list.
You can specify a time that is not in sequence with the events specified
previously, and the timed event list is sorted automatically when you press
Next. The following table lists the twelve timed events.
Timed event parameters
Number
Event
Units
Range or
default
Specify
channel
1
Excitation
wavelength
nm
200 to 890
Yes
2
Emission
wavelength
nm
210 to 900
Yes
Tip: The emission λ setting must always be at least 10 nm above the
excitation λ setting.
3-44
Using the Detector
Timed event parameters (Continued)
Number
Event
Units
3
Time
constant
Seconds
4
Gain
5
Sensitivity
6
Range or
default
Specify
channel
0: Disable
Yes
filter
Hamming: (λ)
0.1 to 5.0,
(λλ) 1 to 50
RC: (λ) 0.1 to
99 sec
RC: (λλ) 1 to
99 sec
0 to 1000
Yes
EUFS
1 to 100,000
Yes
Chart mark
(10% of full
scale)
Does not
apply
Does not
apply
Yes
7
Polarity
1. –
2. +
+
Yes
8
Auto Zero
Does not
apply
Does not
apply
Yes
9
Lamp
1. Off
2. On
Off
No
10
Switch 1
1.
2.
3.
4.
High
Low
Pulse
Rect wave
Low
No
11
Switch 2
1.
2.
3.
4.
High
Low
Pulse
Rect wave
Low
No
Programming methods and events
3-45
Timed event parameters (Continued)
Number
Event
Units
12
Threshold
EU
Range or
default
Specify
channel
–100.0 to
1100.0 EUs
or variable,
depending on
output
selection
Yes
To program a new timed event:
1.
Select METHOD (Shift, A/B).
Method list
2.
From the method list, select 1 Timed events.
An active field for specifying the time of the event appears.
3.
Specify the time for the event.
Tip: When you begin, additional fields appear.
Timed events screen
4.
3-46
Press Enter. To advance to the Set field (Events list), press
Using the Detector
.
5.
Press Enter again to display the list. If you know the event number,
simply press it (see the table titled “Timed event parameters” on
page 3-44).
6.
Enter the appropriate selection in the To field if the field appears.
Tip: If you want the same event programmed on both channels, you
must enter two events, one for channel A and one for channel B.
7.
Press A/B to set the threshold on the other channel.
Tip: ON A or ON B indicates the channel on which the event is
programmed. You can program all or some events on channel A and all
or some events on channel B. Event programming is time-based, not
channel-specific.
8.
Press Next to advance to a new timed event. To delete a timed event,
press CE when the time field is active to change it to OFF.
9.
Press HOME to return to the home screen, then press Run/Stop to start
the method.
10. Select Reset (Shift, Run/Stop) to reset the run clock to 0.
Tip: If the detector is configured with the 717plus Autosampler or other
external device, the inject-start signal programmed from that device
starts the method.
Caution: When you are working in real time, under current
conditions (method *), a power failure or shutdown causes loss of
all timed or threshold events if you do not store them as a method
(see page 3-49).
Programming threshold events
You can program threshold events on channel A and channel B to control the
switch contact closure outputs as when using a fraction collector. Program the
switch to change when the programmed output (fluorescence/EU, energy, etc.)
on the channel is above a specified threshold. The following table lists the
contact closure switches you can program.
Programming methods and events
3-47
Threshold events “Set” parameters
Number
Event
1
Set switch 1
2
Set switch 2
Below the specified threshold, program the switch parameters as in the table
below.
Threshold events “To” parameters
Number
Set to
Below threshold
switch state
1
On
Off
2
Off
On
3
Pulse
Off
4
Rect wave (rectangular Off
wave)
To define the pulse period, or frequency, of a wave, see page 3-25.
To program a threshold event:
1.
Select METHOD (Shift, A/B) on the keypad. The Method list appears.
2.
From the method list, select 2 Threshold events.
An active field (EU) for entering the threshold appears. When you begin
to enter a number in the EU field, additional fields appear.
Tip: The threshold event allows you to specify a threshold value (EU)
that triggers a switch if the fluorescence signal intensity rises above the
value you entered.
3-48
Using the Detector
Threshold events screen
3.
Press Enter to advance to the Set field, or press
the three fields.
and
to move among
4.
When the Set field is active, press Enter to display the threshold events
list, or press the number corresponding to the event you are
programming (see the table titled “Threshold events “Set” parameters”
on page 3-48).
5.
When the To field is active, press Enter to display the options shown in
the table titled “Threshold events “To” parameters” on page 3-48.
As an alternative, press the number corresponding to the threshold
parameter you are programming.
6.
Press A/B to set the threshold on the other channel, and repeat the
procedure.
Storing a method
A method consists of all programmable parameters shown on the home and
associated screens as well as timed and threshold events.
You can store the current method by selecting a location numbered 1 through
10.
To store a method:
1.
Select METHOD (Shift, A/B).
2.
From the method list, select 4 Store method *. A method number field
appears.
Tip: No warning message appears when the method number you specify
in the method number box is already assigned a previously stored
method. When you enter a number and press Enter, the current method
conditions are stored, overwriting any previous method stored in the
same slot.
Programming methods and events
3-49
Specifying a method number
3.
Specify a number from 1 through 10 and press Enter.
A brief message (“Storing * as method n”) appears, and then your
method number appears within the method icon. This method remains
active until you retrieve another method, or reset the detector to default
conditions (Method *).
Retrieving a method
To retrieve a method:
1.
Select METHOD (Shift, A/B).
2.
From the method list, select 3 Retrieve a method.
The last method number stored or retrieved appears in the method
number slot box.
3.
Specify the number of the method you wish to retrieve and press Enter.
A brief message (“Retrieving method n”) appears, and then the method
number you specified appears within the method number icon (see the
table titled “2475 detector home and message screen icons” on page 3-5).
Viewing events within a method
To view events within a method:
1.
Retrieve the method (see page 3-50).
2.
Press 1 to view the Timed events or 2 to view the Threshold events.
Tip: If you change a timed or threshold event within a method, the
asterisk appears (Method *), indicating that the method (*) is no longer
the same as the stored method you retrieved in step 1. You can store the
method containing the altered event(s) in the same storage slot.
3-50
Using the Detector
Resetting a method
Resetting a stored method is a two-step process. First you reset the current
conditions to the defaults; then you save the defaults in a storage location.
To reset a method:
1.
Select METHOD (Shift, A/B).
2.
From the method list, select 5 Reset method *.
A message screen asks if you want to set the current conditions to the
factory defaults. The table titled “Primary and secondary function
(method) parameters” on page 3-21 lists the parameter default settings.
Pressing Enter at this point causes these actions in the software:
•
All timed events are deleted.
•
All threshold events are disabled.
•
All other operating parameters (xλ, eλ, ΕUFS, etc.) are set to
defaults.
If you select Cancel (Shift, 0), the Method list appears.
Tip: To prevent loss of the current conditions before you clear the
method, store them in a storage slot. When you clear the storage slots,
you can restore the previous conditions.
3.
Press 4 Store method, and enter a storage location number.
4.
To clear other stored methods, repeat step 2.
5.
When you press HOME, the method number icon has an asterisk.
Clearing events
To clear all active timed or threshold events:
1.
Select METHOD (Shift, A/B).
2.
From the method list, select 6 Clear events.
You are asked whether you want to clear all active events. Pressing
Enter at this point causes these actions in the software:
•
All timed and threshold events in the method are cleared.
•
All other operating parameters of the method (λ, EUFS, etc.) are
unaffected.
Programming methods and events
3-51
If you select Cancel (Shift, 0), the Method list appears.
3.
When you press HOME, the method number icon has an asterisk.
Scanning spectra
Types of scanning
The detector can collect sample scans for either excitation and emission
fluorescence spectra. A zero-scan is desirable initially.
•
Zero-scan – A reference scan that characterizes the fluorescence
spectrum of a solvent in the flow cell.
•
Sample scan – An excitation or emission scan of an analyte in solvent
(after subtracting the zero-scan of the solvent) to provide the actual
spectrum of the sample.
The detector can measure the spectrum of a sample using the flow cell (see
page 3-64) for scanning procedures.
Before you begin
Before you run a spectrum scan, you need to specify the following parameters:
3-52
•
λ1 – Starting wavelength. Scanning begins at this wavelength.
•
λ2 – Ending wavelength. Scanning ends at this wavelength.
•
Gain – The gain setting for the PMT. Sometimes, you must increase this
for low sample concentrations.
•
Scan Type – The type of scan (excitation or emission).
•
λother – The wavelength setting of the stationary monochromator.
•
Pace – Rate of scanning in nm/min. The pace value determines how fast
a scan runs and acquires data. The scan data are acquired at the highest
Using the Detector
possible resolution for the specified pace. As shown by the following
table, the higher the pace value, the poorer the resolution.
Pace and sampling resolution examples
Pace (nm/min)
Emission sampling
resolution (nm)
Excitation sampling
resolution (nm)
100 and less
0.7
0.9
200
1.4
1.8
400
2.8
3.6
The following figure shows two emission scans of anthracene. At a pace
of 1000 nm/min, the second scan (right) shows a reduced number of
points scanned. Thus the resolution is diminished relative to that in the
original scan (left) at a pace of 100 nm/min.
Scans of anthracene at 100 nm/min and 1000 nm/min
Pace = 100 nm/min
Pace = 1000 nm/min
•
Tick marks – Generates tick marks at a specified wavelength interval,
which helps to interpret charted data. The figure “Scan of water without
tick marks” on page 3-54 shows a scan of water standard in a cuvette
from 390 to 455 nm at a pace of 200 nm/min without tick marks. The
figure “Scan of water with tick marks” on page 3-54 shows the same scan
with tick marks every 20 nm.
•
EUFS – The sensitivity setting for scaling the charted spectrum.
Scanning spectra
3-53
Emission units
Scan of water without tick marks
Wavelength (nm)
Emission units
Scan of water with tick marks
Wavelength (nm)
Enter scanning parameters when you select the type of scan, zero or sample.
Use the detector’s scan function to run a new zero or sample scan, store,
review, subtract, obtain scan information, and replay.
When you select a sample or zero-scan, the detector displays three additional
screens. You can change all parameter values on these screens, including
starting and ending wavelengths and the pace.
3-54
Using the Detector
Zero and sample scan screens
Zero-scan (screen 1 of 4)
Sample scan (screen 1 of 4)
(screens 2, 3, and 4 not shown)
Zero-scan
Zero-scan
(screen 2 of 4)
(screen 3 of 4)
Zero-scan (screen 4 of 4)
When you select a sample scan after performing a zero-scan, the detector
displays one additional screen, labeled 2 of 2. Note that you cannot change the
starting parameter value, ending wavelength parameter value, or the pace
parameter value.
Scanning spectra
3-55
Sample scan screens after a zero-scan
Sample scan
(screen 1 of 2)
Sample scan (screen 2 of 2)
When you run the zero-scan, you specify the starting and ending wavelengths,
other wavelength, pace, tick marks, and sensitivity for the zero-scan and
subsequent sample scans. You must try to run sample scans within 15
minutes of the baseline zero-scan.
Tip: The most recent zero-scan performed or retrieved remains current until
you perform or retrieve another or clear the zero-scan. The zero-scan must
suit the subsequent sample scans you perform. The sample scan uses the
starting and ending wavelength values and the pace value of the most recent
zero-scan. Only when these parameter values are identical for both the zero
and sample scans can you then subtract the zero-scan.
During a zero-scan, data are charted out via the detector analog channel A. At
the same time, reference energy is charted out via channel B, at the EU
specified on channel A.
During a sample scan, data are charted out via the detector analog channel A
using the specified EUFS setting. At the same time, reference energy is
charted out via channel B.
3-56
Using the Detector
Scanning new spectra
To scan new spectra:
1.
Select SCAN (Shift, Chart Mark).
Scan list
2.
From the scan list, select 1 New scan, or use
the list.
and
to scroll through
The detector displays the first of four parameter screens.
3.
Press Next to advance through the New scan parameter screens.
4.
On the first New scan screen, specify the type of scan:
•
For a sample scan, press 1, or press Enter to display the list.
•
For a zero-scan, press 2, or press Enter to display the list.
The detector displays three additional screens. All parameters appear on
the first New scan screen for both a zero and a sample scan. You can
return to screen 1 to review the parameter values for either scan type by
pressing Next from the Run screen.
Tip: You can press Run/Stop from any New scan screen.
5.
Press Run/Stop.
Scanning spectra
3-57
Parameters used for sample and zero-scans
The following table provides the defaults and ranges for all parameters for the
sample and zero-scans.
Sample and zero-scan parameters
3-58
Range or
default
Parameter
Screen
Units
Type
1
n/a
Sample scan: 1
Zero-scan: 2
Default: 1
λ range
2
nm
Range: 200 to
900 nm
Default: 200 or
210 nm
Pace
3
nm/min
Range: 30 to
1000 nm/min
Default: 100
nm/min
EUFS
3
EU
Range: 1 to
100,000
Default: Last
number entered
Tick mark
4
(Mark each nm)
nm
Range: 10 to
100
Default: Last
number entered
Gain
2
n/a
Range: 1 to
1,000
λ other
2
nm
Default: 200 or
210 nm
Monochromator 2
scan type
n/a
Excitation or
emission
Using the Detector
Programming a zero-scan
To program a zero-scan:
1.
Select SCAN (Shift, Chart Mark).
2.
Press 1 New Scan, then press 2 Zero Scan.
3.
Press Next.
The second zero-scan parameter screen appears.
4.
Specify the zero-scan parameters:
a.
Select the type of zero-scan.
b.
Specify the starting wavelength for the zero-scan, and then press
Enter.
c.
Specify the ending wavelength for the zero-scan, and then press
Enter.
d.
Specify the wavelength for the stationary monochromator, and then
press Enter.
e.
Press Next.
5.
Specify the Gain value, and then press Enter.
6.
Specify the EUFS value, and then press Enter.
7.
Specify a value in the Pace field for the rate at which the detector scans
the specified wavelength range.
Tip: The default is 100 nm/min, and the allowable range is from 30 to
1000 nm. The figure “Scans of anthracene at 100 nm/min and 1000
nm/min” on page 3-53 shows two emission scans of anthracene, one at
100 nm/min and one at 1000 nm/min. The higher the number you enter
in the Pace field, the lower the resolution of the scan.
8.
Press Next. The fourth zero-scan parameter screen appears.
9.
Specify a number from 10 through 100 nm if you want to specify tick
marks, and then press Enter (see the figure “Scan of water without tick
marks” on page 3-54 and the figure “Scan of water with tick marks” on
page 3-54). Press CE if you want to disable tick marks.
Scanning spectra
3-59
10. Press Run/Stop to start the zero-scan, or press Next, to return to the
first zero-scan parameter screen to review your parameter value, and
then press Run/Stop.
The Scanning screen displays a progress bar and instantaneous energy
(EU).
Zero-scan progress bar
When the detector completes the zero-scan, the software returns to
displaying the scan list.
Running a sample scan
To run a sample scan:
Caution: Ensure your sample and mobile phase are degassed.
Tip: Run a zero-scan before running the sample scan. To ensure identical flow
cell and solvent conditions, run the sample scan for the corresponding
zero-scan within 15 minutes of running the zero-scan.
1.
Set up and run the zero (or reference) scan (see page 3-59).
2.
Return to the first New scan screen, and press 1 Sample Scan.
The parameters appear for wavelength range, EUFS, Pace, and
wavelength for stationary monochromator (tick mark) you entered for
the zero-scan.
3.
Press Next to advance to the second sample scan screen.
Tip: If desired, you may change the entry in the Mark field.
4.
3-60
Press Run/Stop to run the sample scan. A brief message (“Initializing”)
appears, and the Scanning screen shows the progress of the scan, in
nanometers.
Using the Detector
A progress bar reports the degree of fluorescence, in emission or energy
units (EU).
Sample scan progress bar
Tip: When you perform a zero-scan before a sample scan, the detector
indicates that the zero-scan is being subtracted from the sample scan in
progress.
Subtracting the zero-scan progress bar
After the scan is complete, the detector displays the sample scan
graphically.
Graph of sample anthracene scan
5.
Press Next to display the parameters for up to three of the highest peaks
scanned within the specified range.
Scanning spectra
3-61
Highest peaks of sample anthracene scan
6.
Press Next to return to the graph.
7.
Select Scale (Shift, TRACE) to change the scale and zoom in on one
section (artifact) of the spectrum. The scale of the spectrum is affected
by the EUFS setting. You can alter the following four scaling
parameters:
8.
•
λ1 – Minimum wavelength displayed.
•
λ2 – Maximum wavelength displayed.
•
EU1 – Minimum fluorescence displayed. (The default is auto.)
•
EU2 – Maximum fluorescence displayed. (The default is auto.)
Press Next to advance through the four scaling parameters. The
following figure shows the sample from the figure “Graph of sample
anthracene scan” on page 3-61 after scaling the wavelength parameters
to 225 and 420 nm.
Anthracene scan with λ2 changed to 420 nm
9.
If you change one or more scaling parameters, press Enter to reformat
the graph.
10. Press Next to display the properties of the highest peaks of the scaled
scan.
3-62
Using the Detector
Highest peaks from the scaled anthracene scan
11. Press Next to return to the sample scan display.
To display use of the software’s scaling function, the figure “Three scans
of anthracene in acetonitrile” on page 3-64 shows a series of scans of
anthracene dissolved in acetonitrile. The zero-scan is not displayed.
For the scaling parameters, EU1 and EU2, the default is auto. You can
change the EU parameter based on the fluorescence of the spectrum. To
return the default to auto, press CE.
12. Select SCAN (Shift, Chart Mark) after you finish manipulating the
sample scan graphical display. The scan list appears. To store the scan,
see page 3-65.
Scanning spectra
3-63
Three scans of anthracene in acetonitrile
Sample emission scan 350
to 460 nm
Excitation=249 nm
Anthracene
Zoom of sample emission scan
350 to 440 nm
20 to 35 EU
Excitation=249 nm
Anthracene
λ2 changed to 440 nm
Zoom of sample emission scan
360 to 420 nm
Excitation=249 nm
Anthracene
λ1 changed to 360 nm
λ2 changed to 420 nm
EU1 and EU2 on auto
Scanning using a static flow cell
To scan using a static flow cell:
3-64
1.
Use a syringe to fill the flow cell with the mobile phase or solvent in
which your sample is dissolved.
2.
Run a zero-scan (see page 3-57).
3.
Use a syringe to fill the flow cell with the analyte.
4.
Run a sample scan, keeping the pressure below 145 psi to avoid
overpressuring the flow cell.
5.
Use the functions storage, review, subtract and review, and replay to
compare scanned data.
Using the Detector
Managing results
In standalone mode, having run a spectrum, you can store it for later review,
subtraction, or playback. You can store up to five spectra (see page 3-65). You
can the retrieve the spectrum for purposes of reviewing it from one of the five
storage slots by selecting the review function from the scan list (see
page 3-66). If you store more than one spectrum, you can create a difference
spectrum (see page 3-67).
Tip: The current spectrum is the one being subtracted from; the stored
spectrum, whose slot number you enter, is the spectrum being subtracted.
You can replay the current spectrum or a stored one in real time using the
real-time replay function on the scan list. The detector plays the selected
spectrum in real time, both on the detector display and out the analog
Detector Output 1 to the chart or data collection system. Once you retrieve a
spectrum for playback, the detector displays it graphically.
Storing a spectrum
To store a spectrum:
1.
Select SCAN (Shift, Chart Mark) from the graph of a sample scan.
2.
Press 2 Store last scan.
Tip: When you select store last scan, you are storing the zero-scan and
the sample scan as a pair.
Slot number box
3.
In the slot-number box, specify a number from 1 through 5.
4.
Press Enter to store the last sample scan, paired with its zero-scan.
Managing results
3-65
Getting information about a stored spectrum
To get information about a stored spectrum
1.
Select SCAN (Shift, Chart Mark).
2.
Press 3 Get scan info.
A slot number box appears, its default, Last (for the most recently stored
spectrum).
3.
Press Enter for information about the last stored spectrum.
As an alternative, press the number (1 to 5) of the stored spectrum about
which you want information, and then press Enter. A screen containing
the following information appears:
4.
•
Storage slot number – Of the selected scan (or “Last scan”)
•
Scan type – Displays the chosen type of scan performed
•
λ range – Displays the wavelength range for the selected spectrum
•
λ other – The wavelength setting of the stationary monochromator
•
Pace – Displays the pace for the selected spectrum
•
Gain – The gain setting for the PMT (it can sometimes be necessary
to increase the gain value when working with for low sample
concentrations)
Press Enter to return to the scan list.
Reviewing a stored spectrum
To review a stored spectrum:
1.
Select SCAN (Shift, Chart Mark).
2.
Press 4 Review.
Tip: Selecting Review retrieves both the zero-scan and sample scan.
3.
Specify the storage slot number (1 through 5) of the spectrum you want
to review, and then press Enter.
Tip: “Retrieving spectrum n” appears, and then the stored spectrum
appears. You can view the stored spectrum graphically, and adjust the
wavelength and EU ranges, if necessary. You can also run a new sample
scan based on the retrieved zero-scan.
3-66
Using the Detector
Creating a difference spectrum (subtracting a spectrum)
To create a difference spectrum:
1.
Select SCAN (Shift, Chart Mark).
2.
Press 5 Subtract & Review.
Tip: To subtract one spectrum from another, the starting and ending
wavelengths (λ1 and λ2) and the pace of both spectra must be identical.
3.
Specify the storage slot number (1 through 5) of the spectrum you want
to subtract from the current (or retrieved) spectrum, and then press
Enter.
The message “Subtracting spectrum n” appears. The detector reviews
and subtracts the spectrum specified from the current spectrum and,
after a brief wait, displays the difference spectrum. You can store the
results in one of the five storage slots.
Replaying a spectrum
To replay a spectrum:
1.
Select SCAN (Shift, Chart Mark).
2.
Press 6 Real-time replay.
3.
Specify the storage slot number (1 through 5) of the spectrum you want
to play back, and then press Enter.
Tip: The default is the last spectrum acquired.
After a pause to retrieve the selected spectrum, the detector plays the
spectrum on the analog connection, and then the spectrum graph
appears.
Conserving lamp life
To conserve the lamp without shutting down the detector, you can leave the
instrument on and extinguish the xenon lamp in these ways:
•
Manually
•
By programming a timed event
•
By using the external contact closure
Conserving lamp life
3-67
If the detector is operating under remote control, you can program the
controller to extinguish the lamp without using the detector’s front panel.
Tip: You should extinguish the lamp only if the lamp will remain unlit more
than 4 hours.
Use the Lamp key to light and extinguish the lamp manually. When the lamp
is unlit, the home screen displays the words “Lamp off,” and an “X” is
superimposed on the lamp icon.
Select the Lamp key (Shift, 1) to extinguish or light the lamp manually and to
display the lamp’s usage statistics.
Manually extinguishing the lamp
To manually extinguish the lamp:
1.
Select Lamp (Shift, 1). The lamp control screen appears.
Lamp control screen
2.
3-68
Select Lamp (Shift, 1) again to extinguish it. The home screen appears
with an X through the lamp indicator icon and the words “Lamp off”.
Using the Detector
Lamp off/on sequence
Lamp off indicator
Lamp on indicator
Manually lighting the lamp
To manually light the lamp:
1.
Select Lamp (Shift, 1).
The lamp control screen appears with 0 hours and 00 minutes in the
“Lamp has been on” field.
2.
Select Lamp (Shift, 1) again to light the lamp.
Tip: When the lamp is lit, the “X” no longer covers the lamp icon on the
home screen.
Conserving lamp life
3-69
Using a timed event method to program the lamp
You can conserve lamp life by programming it to light and extinguish (for
example, overnight) using a timed event method. To program the lamp, select
Timed events in the Method list, or program it through one of the external
contact closures. See “Programming methods and events” on page 3-43 and
the table titled “Timed event parameters” on page 3-44 for more information
on programming the lamp to turn on or off using a timed event. See
“Configuring event inputs and contact closures” on page 3-25 for more
information on programming the lamp through the external contact closure.
Shutting down the detector
Before you shut down the detector, you must remove any buffered mobile
phase present in the fluid path.
Caution: To avoid damaging to your column, remove it before you
perform the following procedure.
To shut down the detector:
3-70
1.
Remove buffered mobile phase from the fluid path, replace it with 100%
HPLC-quality water, and flush the system for 10 minutes at 3 mL/min.
2.
Replace the water mobile phase with a solution of 90:10 methanol/water,
and flush the system for 10 minutes at 2 mL/min.
3.
Follow the manufacturer’s recommended procedures for injector purging
and pump-priming.
4.
To shut down the detector, press the on/off switch on its front,
lower-right-hand corner.
Using the Detector
4
Maintenance Procedures
Contents:
Topic
Page
Contacting Waters technical service
4-2
Maintenance considerations
4-2
Routine maintenance
4-3
Inspecting, cleaning, and replacing the flow cell
4-5
Replacing the lamp
4-8
Replacing the fuses
4-14
Cleaning the instruments exterior
4-15
4-1
Contacting Waters technical service
If you are located in the USA or Canada, report malfunctions or other
problems to Waters Technical Service (800 252-4752). Otherwise, phone the
Waters corporate headquarters in Milford, Massachusetts (USA), or contact
your local Waters subsidiary. Waters’ site includes phone numbers and e-mail
addresses for Waters locations worldwide. Visit www.waters.com, and click
About Waters > Worldwide Offices.
When you contact Waters, be prepared to provide this information:
•
Nature of the symptom
•
Instrument serial number
•
Solvent(s)
•
Method parameters (sensitivity and wavelength)
•
Type and serial number of column(s)
•
Sample type
•
Empower software version and serial number
For complete information on reporting shipping damages and submitting
claims, see the document Waters Licenses, Warranties, and Support Services.
Maintenance considerations
Safety and handling
Observe these warning and caution advisories when you perform maintenance
operations on your detector.
Warning: To prevent injury, always observe Good Laboratory
Practices when you handle solvents, change tubing, or operate the
system. Know the physical and chemical properties of the solvents
you use. See the Material Safety Data Sheets for the solvents in use.
4-2
Maintenance Procedures
Warning: Avoid electric shock:
• Do not open the detector cover. The components within are not
user-serviceable.
• Power-off and unplug the detector before performing any
maintenance operation on the instrument.
Caution: To avoid damaging electrical parts, never disconnect an
electrical assembly while power is applied to the detector. To
interrupt power to the detector, set the power switch to Off, and then
unplug the power cord from the AC outlet. Wait 10 seconds thereafter
before you disconnect an assembly.
Warning: Using incompatible solvents can cause severe damage to
the instrument and injury to the operator. Refer to Appendix C for
more information.
Spare parts
Replace only the component parts mentioned in this document. See the
Waters Quality Parts Locator on the Waters Web site’s Service/Support page.
Routine maintenance
For sustained optimal performance, the 2475 detector requires minimal
routine maintenance:
1.
Replace the HPLC system’s solvent reservoir filters regularly.
2.
Filter and degas solvents to prolong column life, reduce pressure
fluctuations, and decrease baseline noise.
3.
Flush buffered mobile phases from the detector with HPLC-grade water
followed by a 5 to 10% methanol solution each time the detector is shut
down.
Tip: Flushing prevents the following problems:
•
Plugging of the solvent lines and flow cell
•
Damage to various components
•
Microbial growth
Routine maintenance
4-3
Removing the front-left-hand panel cover
Caution:
• Because no user-serviceable parts are housed inside the detector,
never remove the top cover. However, some procedures require you
to remove the front-left-hand panel cover.
• To maintain optimum system performance, be sure to refit the
front-left-hand front panel cover before resuming normal
operation.
To remove the front-left-hand panel cover:
1.
Pull the bottom of the cover away from the detector gently, all the while
holding on to the cover’s top.
2.
Withdraw the top of the cover gently, and store the cover nearby.
2475 detector with the front-left-hand panel cover removed
!
4-4
Maintenance Procedures
Inspecting, cleaning, and replacing the flow cell
A dirty flow cell can cause baseline noise, decreased sample energy levels,
calibration failure, and other problems. This section provides information
about the following procedures:
•
Flushing the flow cell
•
Removing and cleaning the flow cell
•
Replacing the flow cell
Flushing and passivating the flow cell
Flush and passivate the flow cell when you suspect that it is dirty.
To flush and passivate the flow cell:
1.
Discontinue the flow of mobile phase.
2.
Remove the column.
3.
Attach tubing from the detector to the injector outlet (from which the
column has been removed).
4.
Flush the mobile phase from the detector with a miscible solvent and
water (unless the mobile phase is miscible with water).
5.
Flush the detector with HPLC-quality water to remove contaminants
from the flow path.
6.
Pump 6 N nitric acid through the flow cell to clean and remove any
accumulated oxides from the internal pathways (passivation).
7.
Flush with HPLC-quality water until the cell is pH-neutral.
8.
Reattach the column.
9.
Resume mobile phase flow.
Requirement: Use an intermediary solvent if you are using a mobile
phase that is not miscible with water.
Inspecting, cleaning, and replacing the flow cell
4-5
Removing the flow cell assembly
To remove the flow cell assembly:
1.
Shut down the detector.
2.
Flush and dry the flow cell (see page 4-5), and then disconnect and cap
the inlet and outlet LC tubing.
3.
Remove the front-left-hand panel cover.
4.
Use a 1/4-inch, flat-blade screwdriver to loosen the three captive screws
on the flow cell assembly front plate.
Unscrewing the flow cell assembly’s captive screws
Captive screws
4-6
Maintenance Procedures
5.
Pull the assembly gently toward you, tilting the bottom of the cell
upward to avoid disturbing the cell mask.
2475 detector flow cell assembly
6.
Place the flow cell assembly on a flat clean surface.
Replacing the flow cell
The detector is shipped with a standard analytical flow cell installed. Replace
the flow cell when it becomes damaged.
Before you begin:
1.
Unpack and inspect the new flow cell.
2.
Shut down the detector.
3.
Remove the front-left-hand panel cover.
4.
Disconnect the detector inlet/outlet tubing from the main column
connection and cap.
To replace the flow cell:
1.
Use a 1/4-inch flat-blade screwdriver to loosen the three captive screws
on the flow cell assembly front plate (see the figure “Unscrewing the flow
cell assembly’s captive screws” on page 4-6).
2.
Pull the assembly gently toward you.
Inspecting, cleaning, and replacing the flow cell
4-7
3.
Insert the new flow cell assembly into the detector.
4.
Tighten the captive screws.
5.
Confirm that the flow cell seats properly.
6.
Reconnect the inlet/outlet tubing.
7.
Start the detector.
8.
Calibrate (see page 3-35) and normalize (see page 3-36) the detector.
Replacing the lamp
This section describes the procedure for removing and replacing the xenon
lamp.
The 2475 detector source lamp is warranted to light and pass startup
diagnostic tests for 2000 hours or 1 year from the date of purchase, whichever
comes first.
Warning: Always use protective eye wear when replacing a lamp.
Tip: Always perform the procedure on page 3-34 each time you change the
lamp.
When to replace the lamp
Replace the lamp when either of these conditions applies:
•
It fails to ignite on startup.
•
The lamp’s energy level causes a decrease in sensitivity to the point
where the baseline is too noisy for your LC application.
Performance requirements and permitted tolerances vary from application to
application. If the lamp no longer provides an adequate signal-to-noise ratio
for your specific application, replace it.
4-8
Maintenance Procedures
Removing the lamp
Warning: The lamp housing becomes extremely hot during lamp
operation. To prevent burn injuries,
• allow the lamp to cool for 60 minutes before removing it.
• keep the lamp in the housing when handling the lamp.
Warning: To avoid eye injury from ultraviolet radiation exposure,
• power-off the detector before changing the lamp.
• wear eye protection that filters ultraviolet light.
• keep the lamp in the housing during operation.
To remove the lamp:
1.
Power-off the detector and disconnect the power cable from the rear
panel.
Warning: The lamp and lamp housing can be hot. Wait 60
minutes after powering off the detector for these components to
cool before touching them.
2.
Allow the lamp to cool for at least 60 minutes.
3.
Remove the front-left-hand panel cover.
4.
Open the lamp access door with a small flat-blade screwdriver.
Caution: Do not grasp the connector by the wire. Doing so could
damage the connector or cable.
5.
Disconnect electrical connections to the lamp:
a.
Pull the top connector straight out, gently.
Replacing the lamp
4-9
b.
Pinch the locking mechanism on the bottom connector before pulling
it out.
Unplugging the lamp assembly
4-10
Maintenance Procedures
6.
Loosen the two captive screws on the lamp housing.
Warning:
• To avoid injury, always keep the lamp facing away from you
when removing it.
• Lamp gas is under positive pressure. To prevent shattering
the glass, use care when disposing of the lamp. Waters
suggests that you adequately cushion an old lamp by
containing it in the packaging of its replacement before you
dispose of it.
7.
Gently pull the lamp out.
Removing the lamp assembly
Replacing the lamp
4-11
Installing the new lamp
Warning: To avoid exposing your eyes to harmful ultraviolet
radiation, wear eye protection that filters ultraviolet light, and
keep the lamp in the housing during operation.
Caution: Do not touch the glass bulb on the new lamp. Dirt or skin oils
on the bulb affect detector operation. If the lamp needs cleaning, gently
clean the bulb with ethanol and lens tissue. Do not use any abrasive
tissue or apply excessive pressure.
Before you begin:
1.
Unpack the lamp from its packing material.
2.
Record the serial number, which is located on a label attached to the
lamp’s connector wire, following the procedure on page 4-13.
Warning: Make sure electrical power to the detector is off and the
power cord is disconnected.
To install the new lamp:
1.
Position the lamp cartridge, and insert it in the housing.
Tip: No additional alignment is required.
2.
Push the lamp forward gently, until it bottoms into position.
3.
Tighten the two captive screws.
4.
Reconnect the lamp’s power connectors.
Tip: The bottom connector locks into place.
5.
Close and secure the lamp access door.
6.
Connect the power cord, and start the detector.
Allow at least 60 minutes for the lamp to warm before resuming
operations.
7.
4-12
Record the new lamp’s serial number (see the next section).
Maintenance Procedures
Recording the new lamp’s serial number
Tip: If you do not record a new lamp’s serial number, the date of the previous
lamp installation remains in the detector’s memory, voiding the new lamp’s
warranty.
Using the detector software, you can record and store the serial number and
date of installation of a new lamp so that you can monitor the age of the lamp
and its number of ignitions.
To record the new lamp serial number:
1.
Once the unit warms, press DIAG.
2.
Press 4 Lamp, display & keypad.
3.
Press 1 Change lamp.
Tip: Be sure to enter the lamp’s serial number and not its part number
when performing this procedure.
4.
Specify the serial number of the new lamp in the active field.
Tip: This field accepts numeric entries only.
Change lamp screen
5.
Press Enter to store the serial number and move to the “date installed”
field.
6.
Select the current month from the list.
7.
Press Enter twice to update the month and move to the day field.
8.
Specify the number for the day of the month you installed the lamp, and
then press Enter to enter the date and move to the year field.
9.
Specify the year (last two digits only), and then press Enter.
10. Press HOME.
The “OK to store” message appears.
Replacing the lamp
4-13
Lamp serial number OK to store message
11. Press Enter if the serial number and date of installation of the new lamp
are correct.
12. Press Enter at the confirmation message.
13. Perform a manual wavelength calibration (see page 3-35).
14. Perform a normalization procedure (see page 3-36).
Replacing the fuses
Warning: To avoid electric shock, power-off and unplug the 2475
detector before examining the fuses. For continued protection
against fire, replace fuses with those of the same type and rating
only.
The detector requires two 100 to 240 VAC, 50 to 60-Hz, F 3.15-A, 250-V
(fast-blow), 5 × 20 mm (IEC) fuses.
Suspect a fuse is open or otherwise defective when
•
the detector fails to power-on.
•
the display is blank.
•
the fans do not operate.
To replace the fuses:
Replace both fuses, even when only one is open or otherwise defective.
4-14
1.
Power-off the detector and disconnect the power cord from the power
entry module.
2.
Insert the tip of a small flat-blade screwdriver into the fuse holder slot
on the rear panel of the detector. With minimum pressure, pull on the
spring-loaded fuse holder and remove it from the rear panel.
Maintenance Procedures
Removing and replacing the rear panel fuses
Flat-blade
screwdriver
Fuse holder
3.
Remove and discard the old fuses.
4.
Make sure that the ratings of the new fuses suit your requirements.
5.
Insert the new fuses into the fuse holder.
6.
Insert the fuse holder into the receptacle, and gently push until it locks
into position on the rear panel.
7.
Start the detector.
Cleaning the instruments exterior
Use a soft cloth, dampened with water, to clean the outside of the detector.
Cleaning the instruments exterior
4-15
4-16
Maintenance Procedures
5
Error Messages, Diagnostic
Tests, and Troubleshooting
The detector provides both user and service diagnostics to troubleshoot
system problems.
Only qualified Waters Service personnel can access the service
diagnostics.
•
Error messages – Startup, calibration, and other error messages
and recommended actions for correcting the errors.
•
Diagnostic tests – User diagnostic tests for troubleshooting and
configuring the detector.
Contents:
Topic
Page
Startup error messages
5-2
Operational error messages
5-3
User-selected diagnostic tests and settings
5-8
Troubleshooting
5-20
5-1
Startup error messages
Startup diagnostic tests verify the proper operation of the electronics. If one or
more of the internal diagnostic tests fails, the detector beeps and displays an
error message. For serious errors, <Error> appears on the home screen
instead of the runtime.
Tip: To prevent errors at startup, be sure the flow cell contains degassed
transparent solvent (methanol or water) flowing at 1 ml/min, and the
front-left-hand panel cover is attached securely.
The following table lists the startup messages with descriptions and corrective
actions in alphabetical order. “Wavelength verification and test” on page 1-14,
“Verifying the detector” on page 3-34, and “Manual wavelength calibration” on
page 3-35 have verification and calibration information and procedures.
Startup error messages
5-2
Error message
Description
Corrective action
Calibration differs: n.n
nm
At startup, the unit
performs a complete
verification and
measures all
calibration points. New
calibration points are
compared to stored
points from the most
recent manual
calibration. This
message appears when
any point differs by
more than 2.0 nm.
• Cycle power (shut
down, wait 10
seconds, then
restart).
• Perform manual
calibration.
• Contact Waters
Technical Service.
Calibration not found
Stored calibration data
not valid.
Perform manual
calibration procedure.
Calibration
unsuccessful: Peak out
of range n.n nm
Results of a calibration
operation outside of
specification. Unit uses
previously stored
calibration.
• Flush flow cell with
water.
• Replace lamp.
Error Messages, Diagnostic Tests, and Troubleshooting
Startup error messages (Continued)
Error message
Description
Corrective action
Lamp external input
conflict
A timed event or front
panel action attempts
to change the lamp
state in conflict with
enabled lamp input
contact closure.
• Check contact
closure status.
• Check timed events.
• Cycle power.
Lamp failure
Lamp indicates Off
• Check lamp icon.
when it should indicate • Cycle power.
On
• Replace lamp.
Lamp lighting failure
The lamp failed to
ignite
• Cycle power.
• Check lamp power
connection.
• Replace lamp.
Peak not found:
Erbium n nm
No local maximum in
the erbium filter
calibration range
• Flush flow cell with
water.
• Replace lamp.
Operational error messages
During initialization, calibration, and operation, the home screen may
sometimes displays “<Error>”. This type of error is usually catastrophic,
preventing further operation, halting the fluorescence output, and suspending
the display. In most cases, you can cycle power (shut down, wait 10 seconds,
then restart) to correct the error. However, if the error persists, contact
Waters Technical Service (see page 4-2).
Catastrophic error display on the home screen
Catastrophic
error
If a catastrophic error appears
Operational error messages
5-3
1.
Ensure that the flow cell is clean.
2.
Ensure that the lamp access door is shut securely.
3.
Recycle power to the detector.
4.
If the catastrophic error persists, contact Waters Technical Service (see
page 4-2).
The following table has error messages that prevent operation, listed in
alphabetical order.
Error messages preventing operation
Error message
5-4
Description
Corrective action
Communication failure: A/D communication
Reference A/D
test failed.
1. Power-off and
power-on again.
2. If the problem
persists, call Waters
Technical Service.
Communication failure: A/D communication
Sample A/D
test failed.
1. Power-off and
power-on again.
2. If the problem
persists, call Waters
Technical Service.
Configuration not
found
Stored configuration
data is invalid.
Power-off and power-on
again.
Dark current too high:
nnnnnnn
The dark energy level is 1. Power-off and
above 1000000.
power-on again.
2. If the problem
persists, call Waters
Technical Service.
Dark current too low: 0
The dark energy level
equals 0.
Error Messages, Diagnostic Tests, and Troubleshooting
1. Power-off and
power-on again.
2. If the problem
persists, call Waters
Technical Service.
Error messages preventing operation (Continued)
Error message
Description
Corrective action
Electronic A/D failure
Lamp optimization is
adjusted at the
minimum level.
Power-off and power-on
again.
Data acquisition via
1. Power-off and
A/D converters is
power-on again.
interrupt-driven. If
2. If the problem
interrupt is too long,
persists, call Waters
problem with data
Technical Service.
acquisition is indicated.
Filter initialization
Unit sensors cannot
failure: Erbium position find erbium filter
position.
1. Ensure solvent is
flowing.
2. If the problem
persists, power-off
and power-on again.
3. If the problem
persists, call Waters
Technical Service.
Filter initialization
failure: Order filter
position
1. Ensure solvent is
flowing.
2. If the problem
persists, power-off
and power-on again.
3. If the problem
persists, call Waters
Technical Service.
Unit sensors cannot
find the order filter
position.
Filter initialization
Unit sensors observe
failure: No filters found transition to dark
before homing the
optical filter.
1. Ensure solvent is
flowing.
2. If the problem
persists, power-off
and power-on again.
3. If the problem
persists, call Waters
Technical Service.
Operational error messages
5-5
Error messages preventing operation (Continued)
5-6
Error message
Description
Corrective action
Filter initialization
failure: No reference
energy
Unit sensors cannot
find any light energy
before homing the
optical filter.
1. Ensure solvent is
flowing.
2. If the problem
persists, power-off
and power-on again.
3. If the problem
persists, call Waters
Technical Service.
Filter initialization
failure: No response
Unit sensors cannot
identify any dark
regions.
1. Ensure solvent is
flowing.
2. If the problem
persists, power-off
and power-on again.
3. If the problem
persists, call Waters
Technical Service.
Filter initialization
failure: Shutter
position
Unit sensors cannot
find the shutter
position.
1. Ensure solvent is
flowing.
2. If the problem
persists, power-off
and power-on again.
3. If the problem
persists, call Waters
Technical Service.
Grating initialization
failure: Backlash too
high
The difference between 1. Ensure solvent is
the forward and reverse
flowing.
peak positions of
2. If the problem
special features is
persists, power-off
greater than 1 step.
and power-on again.
3. If the problem
persists, call Waters
Technical Service.
Error Messages, Diagnostic Tests, and Troubleshooting
Error messages preventing operation (Continued)
Error message
Description
Corrective action
Grating initialization
Search for the home
failure: No home sensor sensor failed.
1. Power-off and
power-on again.
2. If the problem
persists, call Waters
Technical Service.
Hardware failure: lamp The lamp failed to
relay cannot open!
extinguish when the
door or thermal switch
were opened.
Close the lamp door,
and contact Waters
Technical Service.
Lamp data not found
Stored lamp data are
not valid.
Power-off and power-on
again.
Lamp is disabled
The lamp door or
thermal switch is open.
1. Close the lamp door.
2. Remove any cooling
vent obstructions.
3. If the problem
persists, call Waters
Technical Service.
Method not found
Stored method data is
not valid.
Power-off and power-on
again.
PMT not calibrated
No valid gain settings
Call Waters Technical
are saved in the
Service.
battery-backed memory
of the CPU.
Sample signal is
saturated. Gain set too
high.
Emission signal has
overloaded PMT
electronics.
1. Reduce gain.
2. Reduce sample
concentration or
background
fluorescence.
3. Use PMT sensitivity
diagnostic test.
Scan not found
Stored scan data are
not valid.
Power-off and power-on
again.
Operational error messages
5-7
Error messages preventing operation (Continued)
Error message
Description
Corrective action
System cannot respond
Error occurs while unit 1. Power-off and
is positioning next
power-on again.
wavelength or changing 2. If the problem
modes during
persists, call Waters
initialization or
Technical Service.
calibration.
System not calibrated
The calibration read
from nonvolatile
memory is not valid.
1. Power-off and
power-on again.
2. If the problem
persists, call Waters
Technical Service.
Units not normalized
No valid normalization
constants are saved in
the battery-backed
memory of the CPU.
Run the normalization
function (see
page 3-36).
User-selected diagnostic tests and settings
Tip: The detector uses both user-selectable and service diagnostic tests and
settings. Only qualified Waters Service personnel can access service
diagnostic tests.
Overview of diagnostic tests and settings
You may operate several diagnostic tests and settings for use in
troubleshooting the detector and verifying proper functioning of the detector’s
electronics and optics.
Performing tests and changing settings
To perform the tests and change settings:
1.
5-8
Press DIAG on the keypad. The Diagnostics list appears.
Error Messages, Diagnostic Tests, and Troubleshooting
Test and settings list
2.
Use or to select the test you want to run or setting you want to
change, and press Enter, or press a number from 1 through 8. Choices
that display other choices are indicated by >> (see the table titled “2475
detector diagnostic tests and settings” on page 5-10).
Sticky diagnostic settings
Sticky diagnostic settings remain in effect until you disable them. When a
sticky diagnostic test is active, the home screen displays a wrench icon. If no
tests are active, the wrench icon does not appear on the home screen.
Home screen showing active sticky diagnostic settings
Wrench icon
•
To disable a specific sticky diagnostic setting, reset it to the default
settings.
•
To disable all active sticky diagnostic settings, press DIAG, then press 4
Reset diagnostics.
•
When you shut down the detector, all sticky diagnostic settings are
disabled.
You can select the following sticky diagnostic settings:
•
Auto-optimize gain
•
Fix EU [Fix (set) fluorescence input]
•
Fix voltage [Fix (set) voltage outputs]
•
Generate test peaks
User-selected diagnostic tests and settings
5-9
•
Optical filter override
Tip: Sticky diagnostic settings remain in effect even after you exit the
diagnostic function and can affect results. Press 4 Reset diagnostics from the
test list or shut down the detector to clear changes to the voltage output or
fluorescence input or to make a manual optical filter change.
The following table lists the diagnostic tests and settings by number with a
brief description.
2475 detector diagnostic tests and settings
Diagnostic
Description
1 Normalize Units
Normalizes the emission units of the detector to
100 EU using a standard clean water reference.
2 Raman S/N Test
Runs the 15 minute signal-to-noise test for
water.
3 Auto-Optimize Gain
Displays a table of recommended gain settings
for a method based on a trial sample injection.
4 Reset diagnostics
Resets all diagnostic tests to defaults. Disables
sticky diagnostic tests and removes the wrench
icon.
5 Sample & ref energy
Allows you to view sample and reference energy
(displayed in nanoAmps) on channel A or B.
6 Input & output >>
List of diagnostic tests that control four contact
closure inputs and two switch outputs:
1 Auto-zero offsets
2 Fix EU
3 Fix voltage
4 Contact closures & events
5 Previous choices <<
7 Lamp, display &
keypad >>
5-10
List of diagnostic tests for the lamp, display, and
keypad functions:
1 Change lamp
2 Test keypad
3 Test display
4 Previous choices <<
Error Messages, Diagnostic Tests, and Troubleshooting
2475 detector diagnostic tests and settings (Continued)
Diagnostic
Description
8 Other diagnostics >>
Tests that allow you to generate test peaks to
determine wavelength accuracy or override the
default filter setting:
1 Generate test peaks
2 Optical filter override
3 Previous choices <<
9 Service
Diagnostic tests used by Waters service
personnel.
Normalize units setting
See page 1-5 and page 3-36.
Auto-optimize gain test
See page 1-17 and page 3-40.
Sample and reference energy diagnostic tests
The sample and reference energy tests plot the output of the analog channels,
to examine noise fluctuations, and to compare with the EU time trace. The
current sample and reference energy readings appear in energy units from 1
to 10,000.
Sample and reference energy tests
To perform sample and reference energy tests:
1.
Press DIAG, then press 5 Sample & ref energy.
User-selected diagnostic tests and settings
5-11
2.
Specify a new wavelength number to change the wavelength, and then
press Enter. When the new wavelength shifts to the left, the
corresponding sample and reference energies appear.
3.
If you are operating the detector in multichannel mode, press A/B to
view sample and reference energy on the other wavelength.
Raman signal-to-noise test diagnostic test
The Raman signal-to-noise test evaluates the detector’s signal-to-noise
performance. Before running the test, run the normalize units test (see
page 3-36). When you start the signal-to-noise test, the detector sets the
excitation wavelength to 350 nm, it sets the emission wavelength and gain to
the values stored during the normalize units test.
Clean, degassed water must be flowing through the flow cell for this test.
To perform the Raman signal-to-noise diagnostic test:
1.
Press DIAG, then press 2 Raman S/N Test.
2.
Press Enter to confirm the presence of clean, degassed water.
3.
Wait 15 minutes for the detector to display the results.
Raman S/N test screen
Input and output diagnostic tests and settings
Use the input and output tests and settings for these purposes:
5-12
•
Display and reset the auto-zero offsets.
•
Fix (set) EU.
•
Fix (set) the voltage on the 1-V output.
•
Monitor contact closures and toggle event switches.
•
Generate test peaks.
Error Messages, Diagnostic Tests, and Troubleshooting
•
Override the optical filter.
To perform one of the input and output tests or change a setting, press 6 Input
& output. A list of four diagnostic tests and settings appears.
Input and output diagnostic tests and settings
Displaying auto-zero offsets
To display auto-zero offsets:
1.
From the Input & output list, press 1 Auto zero offsets.
Auto-zero offsets screen
2.
Select Cancel (Shift, 0) if you want to clear the offset on both channels to
zero.
Setting the fixed EU value
This function sets the voltages on the analog output channels based on the
current EUFS setting.
To set the fixed EU value:
From the Input & output list, press 2 Fix EU to set a fixed fluorescence value
for channel A or channel B. The allowable range is from –100.0 to +1000 EU.
You can also specify sensitivity in EUFS. The allowable range is from 10 to
100,000 EUFS.
User-selected diagnostic tests and settings
5-13
Fix EU screen
Setting fixed voltage output
This function drives the voltage on the selected analog channel (A or B).
To set the fixed voltage output:
From the Input & output list, press 3 Fix voltage to select a voltage for the
analog output. You can select a voltage for both output channels (range -0.10
to +1.10-V).
Fix voltage screen
Monitoring contact closures and setting switches
To monitor contact closures and setting switches:
1.
5-14
From the Input & output list, press 4 Contact closures & events to
monitor the four contact closure inputs and to control the two switch
outputs.
Error Messages, Diagnostic Tests, and Troubleshooting
Contact closures & events screen
You can monitor the state of the contact closure inputs in real time. A
solid (filled in) circle indicates the contact closure is closed (ON = High).
An open (empty) circle indicates the contact closure is open (OFF = Low).
2.
For the outputs (SW1 and SW2)
a.
Press Enter to display the active switch (surrounded by a
dotted-line border).
b.
Press any numerical key to change the status (from ON to OFF, or
vice versa).
c.
Press Enter to select the second switch.
Change-lamp function
Whenever you change the lamp, use this function to enter a new serial
number and installation date. See page 4-12 and page 4-13 for a complete
explanation of the lamp replacement procedure.
Tip: If you do not record the new lamp’s serial number using the procedure on
page 4-12 and page 4-13, the date of the previous lamp installation remains in
the detector memory, and the lamp warranty is voided.
Caution: Interrupt power to the detector before you replace the lamp.
To enter a serial number and date:
1.
Press DIAG, and then press 7 Lamp, display & keypad >>.
2.
Press 1 Change lamp.
User-selected diagnostic tests and settings
5-15
Change lamp screen
3.
On the Change Lamp screen, specify the serial number, and then press
Enter.
Tip: Be sure to specify the lamp’s serial number and not its part number.
4.
Specify the date of installation for the new lamp, and then press Enter.
Testing the keypad
To test the keypad:
1.
Press DIAG, and then press 7 Lamp, display & keypad >>.
2.
Press 2 Test keypad.
Keypad test
3.
Press any key to begin the test, and then press each key until you try all
of them.
Tip: If the keypad is operating properly, each key location is filled in and
then cleared with a second press of the key. If any key does not respond
when pressed, contact your Waters service representative.
4.
5-16
Press Enter twice to exit the keypad test.
Error Messages, Diagnostic Tests, and Troubleshooting
Testing the display
To test the display:
1.
Press DIAG, then press 7 Lamp, display & keypad >>.
2.
Press 3 Test display.
The display fills from top to bottom and right to left, and then returns to
the Lamp, display & keypad list. If the display does not completely fill
horizontally or vertically, contact your Waters service representative.
3.
Press 4 to return to the diagnostics list.
Other diagnostic tests and settings
The Other diagnostic tests and settings screen provides three additional
diagnostic functions:
•
Generate test peaks – Generates test peaks to calibrate a chart recorder
or data system.
•
Manually override the optical filter – Selects a filter different from the
one used in the detector’s normal operating mode.
•
PMT Sensitivity – Reduces PMT sensitivity by a factor of 10 or 100 to
avoid saturation with highly fluorescent samples and mobile phases.
Press DIAG, and then press 8 Other diagnostics. You can generate test peaks
(see page 5-17) or override the optical filter (see page 5-18).
Other diagnostic tests and settings
Generating test peaks
The generate test peaks function changes the first entry on the list to “Disable
test peaks.”
User-selected diagnostic tests and settings
5-17
To generate test peaks:
1.
Press DIAG, then press 8 Other diagnostics.
2.
Press 1 Generate test peaks to generate test peak.
Tip: Every 100 seconds, the detector generates a 100-EU peak with a
standard deviation of 10 seconds on the trace, chart, or data system
display. The amplitude of the test peaks is affected by your choice of
filter time constant. The gain is automatically set to 1000.
Generate test peaks message
Tip: You must manually disable the Generate test peaks function to stop
it.
3.
Press 1 Disable test peaks to stop generating test peaks.
Overriding the optical filter setting
The detector normally operates with the filter in the Automatic position. Use
this function to override the default setting.
To override the optical filter setting:
1.
Press DIAG, then press 8 Other diagnostics.
2.
Press 2 Optical filter override to manually override the detector’s
automatic filter choice. The Optical Filter screen appears.
Optical filter setting screen
5-18
Error Messages, Diagnostic Tests, and Troubleshooting
3.
Press Enter.
Automatic
1
Second Order
2
None
3
Erbium
4
Shutter
5
4.
In the list of filters, press a number to the corresponding filter, or leave
the default filter (Automatic) on.
5.
Press DIAG, and then press 1, or select Automatic for the default filter.
Reducing PMT sensitivity
The detector’s design increases the limit of detection, but in some
circumstances the photomultiplier tube can be overloaded by large
fluorescence signals. You can reduce the sensitivity of the photomultiplier
tube by a constant factor while preserving the available linear gain range of 1
to 1000. This feature is intended for use when samples or mobile phase
solvents exhibit high fluorescence emissions that saturate the PMT signal but
reducing concentration is not possible. The PMT sensitivity is divided by a
user selectable factor of 10 or 100.
Unlike the ordinary PMT gain range of 1 to 1000, the reduction in PMT
sensitivity is not linear, and this function should not be used as a substitute
for ordinary gain changes.
To reduce PMT sensitivity:
1.
Press DIAG, and then press 8 Other diagnostics.
2.
Press 3 PMT Sensitivity override to reduce the sensitivity by a specified
factor. The PMT Sensitivity screen appears.
User-selected diagnostic tests and settings
5-19
PMT sensitivity screen
3.
Click the Low Sensitivity Mode checkbox.
4.
Enter a reduction value of 10 or 100.
Troubleshooting
Introduction
This section provides some causes of errors and recommended troubleshooting
actions. Keep in mind that the source of apparent detector problems lie within
the chromatography or your other instruments as well as with the detector.
Most detector problems are relatively easy to correct. If you are unable to
correct a problem or failed condition after running the user diagnostics
applicable to the problem, contact Waters Technical Service (see page 4-2).
Information needed when you contact Waters
To expedite your request for service, keep the following information at hand
when you call Waters Technical Service:
5-20
•
2475 detector serial number
•
Problem symptom(s)
•
Operating wavelength pair(s)
•
EUFS or measurement range
•
Flow rate
•
Filter setting
•
Type of column
•
Operating pressure
•
Solvent(s)
Error Messages, Diagnostic Tests, and Troubleshooting
•
System configuration (other components)
Tip: The detector may be configured as part of an Alliance system with an
Empower system, a Millennium32 chromatography workstation, other Waters
HPLC components, or a non-Waters product.
Diagnostic tests
The detector performs some user-selected diagnostic tests to help you
troubleshoot basic system problems. “Overview of diagnostic tests and
settings” on page 5-8 has diagnostic descriptions and instructions on how to
use them. The table titled “2475 detector diagnostic tests and settings” on
page 5-10 and the table titled “General hardware troubleshooting” on
page 5-21 describe error messages that can appear onscreen as you start up or
operate the detector, and it suggests corrective actions.
Power surges
Power surges, line spikes, and transient energy sources can adversely affect
detector operations. Ensure that the electrical supply is properly grounded
and free from any of these conditions.
Hardware troubleshooting
The following table contains general hardware troubleshooting.
General hardware troubleshooting
Symptom
Possible cause
Corrective action
Analog output incorrect EUFS setting changed
Reset the EUFS
setting.
Calibration or energy
error on startup
Flow cell has an air
bubble or UV absorber
Flush the flow cell.
Output units selection
is incorrect
Check output mode
(home screen 2).
Troubleshooting
5-21
General hardware troubleshooting (Continued)
Symptom
Possible cause
Corrective action
Detector inoperative
Open (blown) fuse
Ensure the front panel
display is operational.
Replace the AC rear
panel fuses, if
necessary.
No power at outlet
Check outlet by
connecting an electrical
device known to be in
working order and see
if it operates.
Broken electrical
connection
Check electrical
connections.
Open (blown) fuse
Check and, if
necessary, replace
fuse(s).
Bad LCD or control
board
Call Waters Technical
Service.
Faulty EPROMs or bad
LCD control board
Call Waters Technical
Service.
Front panel display
fails to illuminate
Front panel displays
odd characters
5-22
Keypad not functioning Defective keypad
1. Power-off and
power-on again.
2. Run the keypad
diagnostic test.
3. If the problem
persists, call Waters
Technical Service.
No sample and
reference energy
Lamp life expired
1. Attempt to reignite
by selecting Lamp
(Shift, 1).
2. Replace the lamp.
Lamp off
1. Check the lamp icon.
2. Run the Sample &
ref energy diagnostic
test.
Error Messages, Diagnostic Tests, and Troubleshooting
General hardware troubleshooting (Continued)
Symptom
Possible cause
Corrective action
RS-232 problems
Disabled RS-232
configuration
Set the Configuration
screen correctly.
Bad RS-232 cable
Check and, if
necessary, replace the
RS-232 cable.
Faulty lamp
Replace the lamp.
Lamp not plugged in
Plug in the lamp
connector.
Xenon lamp does not
light
Bad lamp power supply Contact Waters
Technical Service.
Lamp switch off
Check the rear panel
connections.
Troubleshooting
5-23
5-24
Error Messages, Diagnostic Tests, and Troubleshooting
A
Safety Advisories
Waters instruments display hazard symbols designed to alert you to the
hidden dangers of operating and maintaining the instruments. Their
corresponding user guides also include the hazard symbols, with
accompanying text statements describing the hazards and telling you
how to avoid them. This appendix presents all the safety symbols and
statements that apply to the entire line of Waters products.
Contents
Topic
Page
Warning symbols
A-2
Caution symbol
A-5
Warnings that apply to all Waters instruments
A-6
Electrical and handling symbols
A-12
A-1
Warning symbols
Warning symbols alert you to the risk of death, injury, or seriously adverse
physiological reactions associated with an instrument’s use or misuse. Heed
all warnings when you install, repair, and operate Waters instruments.
Waters assumes no liability for the failure of those who install, repair, or
operate its instruments to comply with any safety precaution.
Task-specific hazard warnings
The following warning symbols alert you to risks that can arise when you
operate or maintain an instrument or instrument component. Such risks
include burn injuries, electric shocks, ultraviolet radiation exposures, and
others.
When the following symbols appear in a manual’s narratives or procedures,
their accompanying text identifies the specific risk and explains how to avoid
it.
Warning: (General risk of danger. When this symbol appears on an
instrument, consult the instrument’s user documentation for important
safety-related information before you use the instrument.)
Warning: (Risk of burn injury from contacting hot surfaces.)
Warning: (Risk of electric shock.)
Warning: (Risk of fire.)
Warning: (Risk of sharp-point puncture injury.)
Warning: (Risk of hand crush injury.)
Warning: (Risk of exposure to ultraviolet radiation.)
Warning: (Risk of contacting corrosive substances.)
Warning: (Risk of exposure to a toxic substance.)
Warning: (Risk of personal exposure to laser radiation.)
A-2
Safety Advisories
Warning: (Risk of exposure to biological agents that can pose a serious
health threat.)
Warning: (Risk of tipping.)
Warning: (Risk of explosion.)
Warning: (Risk of eye injury.)
Specific warnings
The following warnings can appear in the user manuals of particular
instruments and on labels affixed to them or their component parts.
Burst warning
This warning applies to Waters instruments fitted with nonmetallic tubing.
Warning: Pressurized nonmetallic, or polymer, tubing can burst.
Observe these precautions when working around such tubing:
• Wear eye protection.
• Extinguish all nearby flames.
• Do not use tubing that is, or has been, stressed or kinked.
• Do not expose nonmetallic tubing to incompatible compounds like
tetrahydrofuran (THF) and nitric or sulfuric acids.
• Be aware that some compounds, like methylene chloride and
dimethyl sulfoxide, can cause nonmetallic tubing to swell, which
significantly reduces the pressure at which the tubing can rupture.
Warning symbols
A-3
Mass spectrometer flammable solvents warning
This warning applies to instruments operated with flammable solvents.
Warning: Where significant quantities of flammable solvents are
involved, a continuous flow of nitrogen into the ion source is required to
prevent possible ignition in that enclosed space.
Ensure that the nitrogen supply pressure never falls below 690 kPa
(6.9 bar, 100 psi) during an analysis in which flammable solvents are
used. Also ensure a gas-fail connection is connected to the LC system so
that the LC solvent flow stops if the nitrogen supply fails.
Mass spectrometer shock hazard
This warning applies to all Waters mass spectrometers.
Warning: To avoid electric shock, do not remove the mass spectrometer’s
protective panels. The components they cover are not user-serviceable.
This warning applies to certain instruments when they are in Operate mode.
Warning: High voltages can be present at certain external surfaces of
the mass spectrometer when the instrument is in Operate mode. To
avoid non-lethal electric shock, make sure the instrument is in Standby
mode before touching areas marked with this high voltage warning
symbol.
A-4
Safety Advisories
Biohazard warning
This warning applies to Waters instruments that can be used to process
material that might contain biohazards: substances that contain biological
agents capable of producing harmful effects in humans.
Warning: Waters instruments and software can be used to analyze or
process potentially infectious human-sourced products, inactivated
microorganisms, and other biological materials. To avoid infection with
these agents, assume that all biological fluids are infectious, observe
Good Laboratory Practices, and consult your organization’s biohazard
safety representative regarding their proper use and handling. Specific
precautions appear in the latest edition of the US National Institutes of
Health (NIH) publication, Biosafety in Microbiological and Biomedical
Laboratories (BMBL).
Chemical hazard warning
This warning applies to Waters instruments that can process corrosive, toxic,
flammable, or other types of hazardous material.
Warning: Waters instruments can be used to analyze or
process potentially hazardous substances. To avoid injury
with any of these materials, familiarize yourself with the
materials and their hazards, observe Good Laboratory
Practices (GLP), and consult your organization’s safety
representative regarding proper use and handling.
Guidelines are provided in the latest edition of the National
Research Council's publication, Prudent Practices in the
Laboratory: Handling and Disposal of Chemicals.
Caution symbol
The caution symbol signifies that an instrument’s use or misuse can damage
the instrument or compromise a sample’s integrity. The following symbol and
its associated statement are typical of the kind that alert you to the risk of
damaging the instrument or sample.
Caution: To avoid damage, do not use abrasives or solvents to clean the
instrument’s case.
Caution symbol
A-5
Warnings that apply to all Waters instruments
When operating this device, follow standard quality control procedures and
the equipment guidelines in this section.
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: qualsiasi modifica o alterazione apportata a questa unità e non
espressamente autorizzata dai responsabili per la conformità fa decadere il
diritto all'utilizzo dell'apparecchiatura da parte dell'utente.
Atencion: cualquier cambio o modificación efectuado en esta unidad que no
haya sido expresamente aprobado por la parte responsable del cumplimiento
puede anular la autorización del usuario para utilizar el equipo.
注意:未經有關法規認證部門允許對本設備進行的改變或修改,可能會使使用者喪失操作該設
備的權利。
注意:未经有关法规认证部门明确允许对本设备进行的改变或改装,可能会使使用者丧失操
作该设备的合法性。
주의: 규정 준수를 책임지는 당사자의 명백한 승인 없이 이 장치를 개조 또는 변경할 경우,
이 장치를 운용할 수 있는 사용자 권한의 효력을 상실할 수 있습니다.
注意:規制機関から明確な承認を受けずに本装置の変更や改造を行うと、本装置のユー
ザーとしての承認が無効になる可能性があります。
A-6
Safety Advisories
Warning: Use caution when working with any polymer tubing under pressure:
• Always wear eye protection when near pressurized polymer tubing.
• Extinguish all nearby flames.
• Do not use tubing that has been severely stressed or kinked.
• Do not use nonmetallic tubing with tetrahydrofuran (THF) or concentrated
nitric or sulfuric acids.
• Be aware that methylene chloride and dimethyl sulfoxide cause nonmetallic
tubing to swell, which greatly reduces the rupture pressure of the tubing.
Attention: Manipulez les tubes en polymère sous pression avec precaution:
• Portez systématiquement des lunettes de protection lorsque vous vous
trouvez à proximité de tubes en polymère pressurisés.
• Eteignez toute flamme se trouvant à proximité de l’instrument.
• Evitez d'utiliser des tubes sévèrement déformés ou endommagés.
• Evitez d'utiliser des tubes non métalliques avec du tétrahydrofurane (THF)
ou de l'acide sulfurique ou nitrique concentré.
• Sachez que le chlorure de méthylène et le diméthylesulfoxyde entraînent le
gonflement des tuyaux non métalliques, ce qui réduit considérablement leur
pression de rupture.
Vorsicht: Bei der Arbeit mit Polymerschläuchen unter Druck ist besondere
Vorsicht angebracht:
• In der Nähe von unter Druck stehenden Polymerschläuchen stets
Schutzbrille tragen.
• Alle offenen Flammen in der Nähe löschen.
• Keine Schläuche verwenden, die stark geknickt oder überbeansprucht sind.
• Nichtmetallische Schläuche nicht für Tetrahydrofuran (THF) oder
konzentrierte Salpeter- oder Schwefelsäure verwenden.
• Durch Methylenchlorid und Dimethylsulfoxid können nichtmetallische
Schläuche quellen; dadurch wird der Berstdruck des Schlauches erheblich
reduziert.
Warnings that apply to all Waters instruments
A-7
Attenzione: fare attenzione quando si utilizzano tubi in materiale polimerico
sotto pressione:
• Indossare sempre occhiali da lavoro protettivi nei pressi di tubi di polimero
pressurizzati.
• Spegnere tutte le fiamme vive nell'ambiente circostante.
• Non utilizzare tubi eccessivamente logorati o piegati.
• Non utilizzare tubi non metallici con tetraidrofurano (THF) o acido solforico
o nitrico concentrati.
• Tenere presente che il cloruro di metilene e il dimetilsolfossido provocano
rigonfiamenti nei tubi non metallici, riducendo notevolmente la pressione di
rottura dei tubi stessi.
Advertencia: se recomienda precaución cuando se trabaje con tubos de
polímero sometidos a presión:
• El usuario deberá protegerse siempre los ojos cuando trabaje cerca de tubos
de polímero sometidos a presión.
• Si hubiera alguna llama las proximidades.
• No se debe trabajar con tubos que se hayan doblado o sometido a altas
presiones.
• Es necesario utilizar tubos de metal cuando se trabaje con tetrahidrofurano
(THF) o ácidos nítrico o sulfúrico concentrados.
• Hay que tener en cuenta que el cloruro de metileno y el sulfóxido de dimetilo
dilatan los tubos no metálicos, lo que reduce la presión de ruptura de los
tubos.
警告:當在有壓力的情況下使用聚合物管線時,小心注意以下幾點。
•
•
•
•
•
A-8
當接近有壓力的聚合物管線時一定要戴防護眼鏡。
熄滅附近所有的火焰。
不要使用已經被壓癟或嚴重彎曲管線。
不要在非金屬管線中使用四氫呋喃或濃硝酸或濃硫酸。
要了解使用二氯甲烷及二甲基亞楓會導致非金屬管線膨脹,大大降低管線的耐壓能力。
Safety Advisories
警告:当有压力的情况下使用管线时,小心注意以下几点:
• 当接近有压力的聚合物管线时一定要戴防护眼镜。
• 熄灭附近所有的火焰。
• 不要使用已经被压瘪或严重弯曲的管线。
• 不要在非金属管线中使用四氢呋喃或浓硝酸或浓硫酸。
• 要了解使用二氯甲烷及二甲基亚枫会导致非金属管线膨胀,大大降低管线的耐压能力。
경고: 가압 폴리머 튜브로 작업할 경우에는 주의하십시오.
• 가압 폴리머 튜브 근처에서는 항상 보호 안경을 착용하십시오.
• 근처의 화기를 모두 끄십시오.
• 심하게 변형되거나 꼬인 튜브는 사용하지 마십시오.
• 비금속(Nonmetallic) 튜브를 테트라히드로푸란(Tetrahydrofuran: THF) 또는
농축 질산 또는 황산과 함께 사용하지 마십시오.
• 염화 메틸렌(Methylene chloride) 및 디메틸술폭시드(Dimethyl sulfoxide)는
비금속 튜브를 부풀려 튜브의 파열 압력을 크게 감소시킬 수 있으므로 유의하십시오.
警告:圧力のかかったポリマーチューブを扱うときは、注意してください。
• 加圧されたポリマーチューブの付近では、必ず保護メガネを着用してください。
• 近くにある火を消してください。
• 著しく変形した、または折れ曲がったチューブは使用しないでください。
• 非金属チューブには、テトラヒドロフラン(THF)や高濃度の硝酸または硫酸などを流
さないでください。
• 塩化メチレンやジメチルスルホキシドは、非金属チューブの膨張を引き起こす場合が
あり、その場合、チューブは極めて低い圧力で破裂します。
Warnings that apply to all Waters instruments
A-9
Warning: The user shall be made aware that if the equipment is used in a
manner not specified by the manufacturer, the protection provided by the
equipment may be impaired.
Attention: L’utilisateur doit être informé que si le matériel est utilisé d’une
façon non spécifiée par le fabricant, la protection assurée par le matériel risque
d’être défectueuses.
Vorsicht: Der Benutzer wird darauf aufmerksam gemacht, dass bei
unsachgemäßer Verwenddung des Gerätes die eingebauten
Sicherheitseinrichtungen unter Umständen nicht ordnungsgemäß
funktionieren.
Attenzione: si rende noto all'utente che l'eventuale utilizzo
dell'apparecchiatura secondo modalità non previste dal produttore può
compromettere la protezione offerta dall'apparecchiatura.
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.
警告:使用者必須非常清楚如果設備不是按照製造廠商指定的方式使用,那麼該設備所提供
的保護將被消弱。
警告:使用者必须非常清楚如果设备不是按照制造厂商指定的方式使用,那么该设备所提供
的保护将被削弱。
경고: 제조업체가 명시하지 않은 방식으로 장비를 사용할 경우 장비가 제공하는 보호 수단이
제대로 작동하지 않을 수 있다는 점을 사용자에게 반드시 인식시켜야 합니다.
警告: ユーザーは、製造元により指定されていない方法で機器を使用すると、機器が提供
している保証が無効になる可能性があることに注意して下さい。
A-10
Safety Advisories
Warning: To protect against fire, replace fuses with those of the type
and rating printed on panels adjacent to instrument fuse covers.
Attention: pour éviter tout risque d'incendie, remplacez toujours les
fusibles par d'autres du type et de la puissance indiqués sur le panneau
à proximité du couvercle de la boite à fusible de l'instrument.
Vorsicht: Zum Schutz gegen Feuer die Sicherungen nur mit
Sicherungen ersetzen, deren Typ und Nennwert auf den Tafeln neben
den Sicherungsabdeckungen des Geräts gedruckt sind.
Attenzione: per garantire protezione contro gli incendi, sostituire i
fusibili con altri dello stesso tipo aventi le caratteristiche indicate sui
pannelli adiacenti alla copertura fusibili dello strumento.
Advertencia: Para evitar incendios, sustituir los fusibles por aquellos
del tipo y características impresos en los paneles adyacentes a las
cubiertas de los fusibles del instrumento.
警告 : 為了避免火災,更換保險絲時,請使用與儀器保險絲蓋旁面板上所印刷之相同類
型與規格的保險絲。
警告 : 为了避免火灾,应更换与仪器保险丝盖旁边面板上印刷的类型和规格相同的
保险丝。
경고: 화재의 위험을 막으려면 기기 퓨즈 커버에 가까운 패널에 인쇄된 것과 동일한
타입 및 정격의 제품으로 퓨즈를 교체하십시오.
警告 : 火災予防のために、ヒューズ交換では機器ヒューズカバー脇のパネルに記
載されているタイプおよび定格のヒューズをご使用ください。
Warnings that apply to all Waters instruments
A-11
Electrical and handling symbols
Electrical symbols
These can appear in instrument user manuals and on the instrument’s front
or rear panels.
Electrical power on
Electrical power off
Standby
Direct current
Alternating current
Protective conductor terminal
Frame, or chassis, terminal
Fuse
Recycle symbol: Do not dispose in municipal waste.
A-12
Safety Advisories
Handling symbols
These handling symbols and their associated text can appear on labels affixed
to the outer packaging of Waters instrument and component shipments.
Keep upright!
Keep dry!
Fragile!
Use no hooks!
Electrical and handling symbols
A-13
A-14
Safety Advisories
B
Specifications
Physical specifications
Attribute
Specification
Height
20.8 cm (8.2 inches)
Depth
50.3 cm (19.8 inches)
Width
28.4 cm (11.2 inches)
Weight
13.61 kg (30 pounds)
Environmental specifications
Attribute
Specification
Operating temperature
4 to 40 °C (39.2 to 104 °F)
Operating humidity
20 to 80%, noncondensing
Shipping and storage temperature
–40 to 70 °C (–40 to 158 °F)
Shipping and storage humidity
20 to 80%, noncondensing
Electrical specifications
Attribute
Specification
Class I
a
Protection class
b
Overvoltage category
II
Pollution degree
2
Moisture protectiond
Normal (IPXO)
c
Line voltages, nominal
Grounded AC, 120 V, 240 V, ±10%
B-1
Electrical specifications (Continued)
Attribute
Specification
Altitude
2000 m (6561.6 feet)
Line frequency
50/60 Hz
Fuse ratings
Two fuses: 100 to 240 VAC, 50 to 60
Hz
F 3.15 A, 250 V FAST BLO, 5 × 20
mm (IEC)
Power consumption
280 VA (nominal)
Two attenuated analog output
channels: 1 VFS
Attenuation range: 1 to 100,000
EUFS
1V output range: –0.1 to +1.1 V
Two event outputs
Type: Contact closure
Voltage: +30 V
Current: 1 A
Four event inputs
Input voltage: +30 V maximum
100 ms (minimum period)
a. Protection Class I – The insulating scheme used in the instrument to protect from electrical shock. Class I identifies a single level of insulation between live parts (wires) and
exposed conductive parts (metal panels), in which the exposed conductive parts are connected to a grounding system. In turn, this grounding system is connected to the third pin
(ground pin) on the electrical power cord plug.
b. Overvoltage Category II – Pertains to instruments that receive their electrical power
from a local level such as an electrical wall outlet.
c. Pollution Degree 2 – A measure of pollution on electrical circuits, which may produce a
reduction of dielectric strength or surface resistivity. Degree 2 refers only to normally
nonconductive pollution. Occasionally, however, expect a temporary conductivity caused
by condensation.
d. Moisture Protection – Normal (IPXO) – IPXO means that no Ingress Protection
against any type of dripping or sprayed water exists. The X is a placeholder that identifies protection against dust, if applicable.
Performance specifications
B-2
Attribute
Specification
Wavelength range
Ex: 200 to 890 nm
Em: 210 to 900 nm
Bandwidth
20 nm (maximum)
Wavelength accuracy
+3 nm
Specifications
Performance specifications (Continued)
Attribute
Specification
Wavelength
repeatability
+0.25 nm
Sensitivity, single
channel
Ex: 350 nm
Em: 397 nm
(Signal-to-noise ratio of water Raman peak ≥1000.
Hamming filter TC = 1.5 sec)
Sensitivity setting
range
1 to 100,000 EUFS
Filter setting range
Single-channel:
0.1 to 5.0 seconds, Hamming (default)
0.1 to 99 seconds, RC
Multichannel:
1 to 50 seconds, Hamming (default)
1 to 99 seconds, RC
Optical Component Specifications
Lamp source
Xenon arc lamp (150 W)
Flow cell
Axial Illuminated Flow Cell design
Cell volume
(illuminated)
8 µL (standard analytical)
Pressure limit
145 psi (flow rate not to exceed 5 mL/min)
Materials
316 stainless steel, fused silica, Teflon
®
B-3
B-4
Specifications
C
Solvent Considerations
Contents:
Topic
Page
Introduction
C-2
Solvent miscibility
C-3
Buffered solvents
C-6
Head height
C-6
Solvent viscosity
C-6
Mobile phase solvent degassing
C-7
Wavelength selection
C-9
C-1
Introduction
Warning: To avoid chemical hazards, always observe safe laboratory
practices when operating your system.
Clean solvents
Clean solvents provide
•
reproducible results
•
operation with minimal instrument maintenance
A dirty solvent can cause
•
baseline noise and drift
•
blockage of the solvent filters with particulate matter
Solvent quality
Use HPLC-grade solvents to ensure the best possible results. Filter through
0.22-µm filters before use. Solvents distilled in glass generally maintain their
purity from lot to lot; use them to ensure the best possible results.
Preparation checklist
The following solvent preparation guidelines help to ensure stable baselines
and good resolution:
•
Filter solvents with a 0.22-µm filter.
•
Degas and/or sparge the solvent.
•
Stir the solvent.
•
Keep solvent in a place free from drafts and shock.
Water
Use water only from a high-quality water purification system. If the water
system does not provide filtered water, filter it through a 0.22-µm membrane
filter before use.
C-2
Solvent Considerations
Buffers
When you use buffers, dissolve salts first, adjust the pH, then filter to remove
insoluble material.
Tetrahydrofuran (THF)
When using unstabilized THF, ensure that your solvent is fresh. Previously
opened bottles of THF contain peroxide contaminants, which cause baseline
drift.
Warning: THF contaminants (peroxides) are potentially explosive
when concentrated or taken to dryness.
Solvent miscibility
Before you change solvents, consult the following table to determine the
miscibility of the solvents to be used. When you change solvents, be aware
that:
•
Changes involving two miscible solvents may be made directly. Changes
involving two solvents that are not totally miscible (for example, from
chloroform to water), require an intermediate solvent (such as
isopropanol).
•
Temperature affects solvent miscibility. If you are running a
high-temperature application, consider the effect of the higher
temperature on solvent solubility.
•
Buffers dissolved in water may precipitate when mixed with organic
solvents.
When you switch from a strong buffer to an organic solvent, flush the buffer
out of the system with distilled water before you add the organic solvent.
Solvent miscibility
Polarity
Solvent
index
Viscosity
CP, 20 °C
Boiling
point °C
(1 atm)
Miscibility
number
(M)
λ Cutoff
(nm)
–0.3
N-decane
0.92
174.1
29
––
–0.4
Iso-octane
0.50
99.2
29
210
Solvent miscibility
C-3
Solvent miscibility (Continued)
C-4
Polarity
Solvent
index
Viscosity
CP, 20 °C
Boiling
point °C
(1 atm)
Miscibility
number
(M)
λ Cutoff
(nm)
0.0
N-hexane
0.313
68.7
29
––
0.0
Cyclohexane
0.98
80.7
28
210
1.7
Butyl ether
0.70
142.2
26
––
1.8
Triethylamine
0.38
89.5
26
––
2.2
Isopropyl ether
0.33
68.3
––
220
2.3
Toluene
0.59
100.6
23
285
2.4
P-xylene
0.70
138.0
24
290
3.0
Benzene
0.65
80.1
21
280
3.3
Benzyl ether
5.33
288.3
––
––
3.4
Methylene chloride
0.44
39.8
20
245
3.7
Ethylene chloride
0.79
83.5
20
––
3.9
Butyl alcohol
3.00
117.7
––-
––
3.9
Butanol
3.01
177.7
15
––
4.2
Tetrahydrofuran
0.55
66.0
17
220
4.3
Ethyl acetate
0.47
77.1
19
260
4.3
1-propanol
2.30
97.2
15
210
4.3
2-propanol
2.35
117.7
15
––-
4.4
Methyl acetate
0.45
56.3
15, 17
260
4.5
Methyl ethyl ketone 0.43
80.0
17
330
4.5
Cyclohexanone
2.24
155.7
28
210
4.5
Nitrobenzene
2.03
210.8
14, 20
––
4.6
Benzonitrile
1.22
191.1
15, 19
––
4.8
Dioxane
1.54
101.3
17
220
5.2
Ethanol
1.20
78.3
14
210
5.3
Pyridine
0.94
115.3
16
305
5.3
Nitroethane
0.68
114.0
––
––
5.4
Acetone
0.32
56.3
15, 17
330
5.5
Benzyl alcohol
5.80
205.5
13
––
Solvent Considerations
Solvent miscibility (Continued)
Polarity
Solvent
index
Viscosity
CP, 20 °C
Boiling
point °C
(1 atm)
Miscibility
number
(M)
λ Cutoff
(nm)
5.7
Methoxyethanol
1.72
124.6
13
––
6.2
Acetonitrile
0.37
81.6
11, 17
210
6.2
Acetic acid
1.26
117.9
14
––
6.4
Dimethylformamide 0.90
153.0
12
––
6.5
Dimethylsulfoxide
2.24
189.0
9
––
6.6
Methanol
0.60
64.7
12
210
7.3
Formamide
3.76
210.5
3
––
9.0
Water
1.00
100.0
––
––
How to use miscibility numbers
Use miscibility numbers (M-numbers) to predict the miscibility of a liquid
with a standard solvent (see the table titled “Solvent miscibility” on page C-3).
To predict the miscibility of two liquids, subtract the smaller M-number value
from the larger M-number value.
•
If the difference between the two M-numbers is 15 or less, the two
liquids are miscible in all proportions at 15 °C.
•
A difference of 16 indicates a critical solution temperature from 25 to
75 °C, with 50 °C as the optimal temperature.
•
If the difference is 17 or greater, the liquids are immiscible, or their
critical solution temperature is above 75 °C.
Some solvents prove immiscible with solvents at either end of the lipophilicity
scale. These solvents receive a dual M-number:
•
The first number, always lower than 16, indicates the degree of
miscibility with highly lipophilic solvents.
•
The second number applies to the opposite end of the scale. A large
difference between these two numbers indicates a limited range of
miscibility.
Solvent miscibility
C-5
For example, some fluorocarbons are immiscible with all the standard
solvents and have M-numbers of 0, 32. Two liquids with dual M-numbers are
usually miscible with each other.
A liquid is classified in the M-number system by testing for miscibility with a
sequence of standard solvents. A correction term of 15 units is then either
added or subtracted from the cutoff point for miscibility.
Buffered solvents
When using a buffer, use a good quality reagent and filter it through a
0.22-µm filter.
Do not leave the buffer stored in the system after use. Flush all fluidic
pathways with HPLC-quality water before shutting the system down and
leave distilled water in the system (flush with 90% HPLC-quality water:10%
methanol for shutdowns scheduled to be more than one day). Use a minimum
of 15 mL for sparge-equipped units, and a minimum of 45 mL for inline
vacuum degasser-equipped units. Some modern systems, such as Waters
®
Alliance , may require volumes lower than this, depending on inline degasser
volumes and slow-rate operation limits.
Head height
Position the solvent reservoirs at a level above the HPLC equipment or on top
of the pump or detector (with adequate spill protection).
Warning: Since the 2475 detector contains a high voltage power
source, all solvents should be isolated from the detector.
Solvent viscosity
Generally, viscosity is not important when you are operating with a single
solvent or under low pressure. However, when you are running a gradient, the
viscosity changes that occur as the solvents are mixed in different proportions
can result in pressure changes during the run. For example, a 1:1 mixture of
water and methanol produces twice the pressure of either water or methanol
alone.
C-6
Solvent Considerations
If the extent to which the pressure changes will affect the analysis is not
known, monitor the pressure during the run using the Chart Out terminal.
Mobile phase solvent degassing
Mobile phase difficulties account for 70% or more of all liquid
chromatographic problems. Using degassed solvents is important, especially
at excitation wavelengths below 220 nm. Degassing provides these benefits:
•
Stable baselines and enhanced sensitivity
•
Reproducible retention times for eluting peaks
•
Reproducible injection volumes for quantitation
•
Stable pump operation
This section explains the solubility of gases, solvent degassing methods, and
solvent degassing considerations.
Gas solubility
Only a finite amount of gas can be dissolved in a given volume of liquid. This
amount depends on:
•
The chemical affinity of the gas for the liquid
•
The temperature of the liquid
•
The pressure applied to the liquid
Changes in the composition, temperature, or pressure of the mobile phase can
all lead to outgassing.
Effects of intermolecular forces
Nonpolar gases (N2, O2, CO2, He) are more soluble in nonpolar solvents than
in polar solvents. Generally, a gas is most soluble in a solvent with
intermolecular attractive forces similar to those in the gas (like dissolves like).
Effects of temperature
Temperature affects the solubility of gases. If the heat of solution is
exothermic, the solubility of the gas decreases when you heat the solvent. If
the heat of solution is endothermic, the solubility increases when you heat the
solvent. For example, the solubility of He in H2O decreases with an increase in
Mobile phase solvent degassing
C-7
temperature, but the solubility of He in benzene increases with an increase in
temperature.
Effects of partial pressure
The mass of gas dissolved in a given volume of solvent is proportional to the
partial pressure of the gas in the vapor phase of the solvent. If you decrease
the partial pressure of the gas, the amount of that gas in solution also
decreases.
Solvent degassing methods
This section describes the solvent degassing techniques that will help you
attain a stable baseline. Degassing your solvent also improves reproducibility
and pump performance. You can use either of the following methods to degas
solvents:
•
Sparging with helium
•
Vacuum degassing
Sparging
Sparging removes gases from solution by displacing dissolved gases in the
solvent with a less soluble gas, usually helium. Well-sparged solvent improves
pump performance. Helium sparging brings the solvent to a state of
equilibrium, which can be maintained by slow sparging or by keeping a
blanket of helium over the solvent. Blanketing inhibits resorption of
atmospheric gases.
Sparging can change the composition of mixed solvents.
Vacuum degassing
The in-line vacuum degasser operates on the principle of Henry’s law to
remove dissolved gases from the solvent. Henry’s law states that the mole
fraction of a gas dissolved in liquid is proportional to the partial pressure of
that gas in the vapor phase above the liquid. If the partial pressure of a gas on
the surface of the liquid is reduced, for example, by evacuation, then a
proportional amount of that gas comes out of solution.
Vacuum degassing can change the composition of mixed solvents.
C-8
Solvent Considerations
Solvent degassing considerations
Select the most efficient degassing operation for your application. To remove
dissolved gas quickly, consider sparging or vacuum degassing.
Sparging
In a detector, helium sparging gives stable baselines and better sensitivity
than sonication and prevents resorption of atmospheric gases. Use this
method to retard oxidation when you are using THF or other peroxide-forming
solvents.
Vacuum degassing
The longer the solvent is exposed to the vacuum, the more dissolved gases are
removed. Two factors affect the amount of time the solvent is exposed to the
vacuum:
•
Flow rate – At low flow rates, most of the dissolved gas is removed as the
solvent passes through the vacuum chamber. At higher flow rates, lesser
amounts of gas per unit volume of solvent are removed.
•
Surface area of the degassing membrane – The length of the degassing
membrane is fixed in each vacuum chamber. To increase the length of
membrane, you can connect two or more vacuum chambers in series.
The inline degasser is available as an option or factory-installed in the Waters
Alliance System.
Wavelength selection
In fluorescence, if the excitation monochromator is set below the UV cutoff of a
mobile phase component, the solvent will absorb some of the available
excitation light intensity. This will reduce the fluorescence emission response
for the sample.
This section includes UV cutoff ranges for:
•
Common solvents
•
Common mixed mobile phases
•
Chromophores
Wavelength selection
C-9
UV cutoffs for common solvents
The following table shows the UV cutoff (the wavelength at which the
absorbance of the solvent is equal to 1 AU for some common chromatographic
solvents). Operating at an excitation wavelength near or below the cutoff
increases baseline noise due to the solvent’s ability to absorb excitation light
energy.
UV cutoff wavelengths for common chromatographic solvents
C-10
Solvent
UV Cutoff (nm)
Solvent
UV Cutoff (nm)
1-Nitropropane
380
Ethylene glycol
210
2-Butoxyethanol
220
Iso-octane
215
Acetone
330
Isopropanol
205
Acetonitrile
190
Isopropyl
chloride
225
Amyl alcohol
210
Isopropyl ether
220
Amyl chloride
225
Methanol
205
Benzene
280
Methyl acetate
260
Carbon disulfide
380
Methyl ethyl
ketone
330
Carbon
tetrachloride
265
Methyl isobutyl
ketone
334
Chloroform
245
Methylene
chloride
233
Cyclohexane
200
n-Pentane
190
Cyclopentane
200
n-Propanol
210
Diethyl amine
275
n-Propyl chloride
225
Dioxane
215
Nitromethane
380
Ethanol
210
Petroleum ether
210
Ethyl acetate
256
Pyridine
330
Ethyl ether
220
Tetrahydrofuran
230
Ethyl sulfide
290
Toluene
285
Ethylene
dichloride
230
Xylene
290
Solvent Considerations
Mixed mobile phases
The following table contains approximate wavelength cutoffs for some other
solvents, buffers, detergents, and mobile phases. The solvent concentrations
represented are those most commonly used. If you want to use a different
concentration, you can determine approximate fluorescence using Beer’s law,
because fluorescence is proportional to concentration.
Wavelength cutoffs for different mobile phases
Mobile phase
UV Cutoff
(nm)
Mobile phase
UV Cutoff
(nm)
Acetic acid, 1%
230
Sodium chloride, 1 M
207
Ammonium acetate,
10 mM
205
Sodium citrate, 10 mM
225
Ammonium bicarbonate,
10 mM
190
Sodium dodecyl sulfate
190
BRIJ 35, 0.1%
190
Sodium formate, 10
mM
200
CHAPS, 0.1%
215
Triethyl amine, 1%
235
Diammonium phosphate, 205
50 mM
Trifluoracetic acid,
0.1%
190
EDTA, disodium, 1 mM
190
TRIS HCl, 20 mM, pH
7.0,
pH 8.0
202, 212
HEPES, 10 mM, pH 7.6
225
Triton-X™ 100, 0.1%
240
Hydrochloric acid, 0.1%
190
Waters PIC Reagent
A, 1 vial/liter
200
MES, 10 mM, pH 6.0
215
Waters PIC Reagent
B-6, 1 vial/liter
225
Potassium phosphate,
monobasic, 10 mM
dibasic, 10 mM
190
190
Sodium acetate, 10 mM
205
®
Waters PIC Reagent
190
B-6, low UV, 1 vial/liter
Waters PIC Reagent
D-4, 1 vial/liter
190
Wavelength selection
C-11
Wavelength selection for chromophore detection
Certain functional groups found in most compounds absorb light selectively.
These groups, known as chromophores, and their behavior can be used to
categorize the detection of sample molecules. The following table lists some
common chromophores and their detection wavelengths (λmax), as well as the
molar absorptivity (εmax) of each group1. Use this information as a guide to
select the optimal operating wavelength for a particular analysis. Because of
the diversity possible within a given sample, scanning over a range of
wavelengths can be necessary to determine the best wavelength for a
particular analysis.
Wavelength selection for chromophore detection
Chromophore
Chemical
Configuration
λmax ∈max
(nm) (L/m/cm)
Ether
—O—
185
1000
Thioether
—S—
194
4600
Amine
—NH2
195
2800
Thiol
—SH
195
1400
Disulfide
—S—S—
194
5500
Bromide
—Br
208
300
Iodide
—I
260
400
Nitrile
—C≡N
160
—
Acetylide
—C≡C—
175–
180
6000
Sulfone
—SO2 —
180
—
Oxime
—NOH
190
5000
Ethylene
—C=C—
190
8000
Ketone
>C=O
195
1000
Thioketone
>C=S
205
strong
Esters
—COOR
205
50
λmax
(nm)
∈max
(L/m/cm)
215
1600
255
400
270–28 18–30
5
1. Willard, H. H. and others. Instrumental Methods of Analysis, 6th ed. Litton Educational Publishing,
Inc., 1981. Reprinted by permission of Wadsworth Publishing Co., Belmont, California, 94002.
C-12
Solvent Considerations
Wavelength selection for chromophore detection (Continued)
Chromophore
Chemical
Configuration
λmax ∈max
(nm) (L/m/cm)
λmax
(nm)
∈max
(L/m/cm)
Aldehyde
—CHO
210
strong
280–30 11–18
0
Carboxyl
—COOH
200–
210
50–70
Sulfoxide
>S→O
210
1500
Nitro
—NO2
210
strong
Nitrite
—ONO
220–
230
1000–2000 300–40 10
0
Azo
—N=N—
285–
400
3–25
Nitroso
—N=O
302
100
Nitrate
—ONO2
270
12
(shou
lder)
Allene
—(C=C)2—
(acyclic)
210–
230
21,000
Allene
—(C=C)3—
260
35,000
Allene
—(C=C)4—
300
52,000
Allene
—(C=C)5—
330
118,000
Allene
—(C=C)2—
(alicyclic)
230–
260
3000–8000
Ethylenic/
Acetylenic
C=C—C≡C
219
6,500
Ethylenic/Amido
C=C—C=N
220
23,000
Ethylenic/
Carbonyl
C=C—C=O
210–
250
10,000–20,
000
Ethylenic/Nitro
C=C—NO2
229
9,500
Wavelength selection
C-13
C-14
Solvent Considerations
Index
Symbols
+/− key 3-15
? key 3-11, 3-28
• key 3-15
Numerics
2475 detector
operating under remote control
3-44
setting up 3-18
start up procedures 3-2
600 Series Pump. See Waters 600
Series Pump
A
A/B key 3-5, 3-13
accessing secondary functions 3-16
activating a pulse or rectangular wave
3-26
active method 3-50
adjusting
analog signal 3-20
contrast 3-27
advancing to the next field 3-15
Alliance Separations Module
generating a chart mark from 2-16
generating Auto Zero on inject from
2-15
starting a method from 2-17
turning the 2475 lamp on or off
from 2-17
analog outputs
channel outputs 2-10
connections 2-23
multiwavelength signal 3-22
other parameters 3-20
signal adjusting 3-20
single wavelength signal 3-22
single-channel 3-20
analog signals 2-6
anthracene 3-53, 3-64
audience and purpose iv
Auto Zero key 3-12
automatic second-order filter 1-8, 1-9
auto-optimize gain 1-17
Auto-Optimize Gain diagnostic 3-41
auto-zero
configuring 3-26
connections to 600 Series pump
2-31
connections to 717plus
Autosampler 2-34
function 3-12, 3-21
offset diagnostic setting 5-10, 5-13
on inject parameter 3-21, 3-23
on wavelength changes 3-21
on wavelength changes parameter
3-23
timed event parameter 3-45
B
bandwidth specification B-2
benefits of degassing C-7, C-8
biohazard warning A-5
buffered solvents C-6
burst warning A-3
Bus SAT/IN module, connecting 2-24
C
Calibrate key 3-14, 3-35
calibrating, photomultiplier tube
(PMT) 1-12
calibration
errors during startup 3-35
Index-1
manual 3-14, 3-35
Cancel key 3-15
caution symbol A-5
CE key 3-15
change lamp diagnostic test 5-10
change lamp function 5-16
changing
channel, from multi to single 3-38
channel, from single to multi 3-38
channels 3-13
contrast 3-15
filter type 3-16
modes 3-38
scale on a fluorescence trace 3-13
time constant 3-16
wavelength, from multi to single
3-12
wavelength, from single to multi
3-12
channel
A and B outputs 2-10
changing 3-5, 3-13, 3-38
on 3-6
selector 3-6
chart mark
configuring event inputs 3-26
enabling 3-21
generating 2-29, 3-12
generating from the Alliance
Separations Module 2-16
Waters 600 Series Pump
connections 2-31
Chart Mark key 3-12
chart polarity
function 3-16, 3-20
parameter 3-22
chart recorder, connecting 2-28–2-29
charting
difference plot 3-20
Index-2
MaxPlot function 3-20
chemical hazard warning A-5
chemiluminescence 1-2
chromatography, fluorescence 1-2
cleaning the flow cell 4-5
cleaning, detector exterior 4-15
Clear Field key 3-15
clearing
editing changes 3-15
events 3-51
column connections 2-3
CONFIGURE key 3-13, 3-25
configuring
auto-zero event input 3-26
detector 3-13
event inputs 3-25
lamp signal 2-30
connecting
Alliance Separations Module 2-14
Alliance system 2-14
Bus SAT/IN module 2-24
chart recorder 2-28–2-29
column 2-3
electricity source 2-36
Ethernet cable 2-7, 2-20
external devices 2-10
inject-start 2-9, 2-22
Millennium chromatography
workstation 2-24
multiple Waters instruments 2-8,
2-21
other equipment 2-23–2-35
signal cables 2-5
single Waters instrument 2-7, 2-20
Waters 600 Series Pump 2-29–2-33
Waters 717plus Autosampler
2-33–2-35
Waters 746 data module 2-27–2-28
connections, signal 2-13
conserving lamp life 3-67
contact closures
and events diagnostic setting 5-10
configuring event inputs 3-25
monitoring 5-14
contacting Waters Technical Service
4-2, 5-20
context-sensitive Help 3-28
contrast
adjusting 3-27
changing 3-15
function 3-27
Contrast key 3-15
controlling
from data systems 3-1
from older external data systems
3-29
cooling off the lamp 4-9
cover, removing 4-4
CPU board 1-13
current method conditions 3-16, 3-44,
3-51
D
damage
to the flow cell 4-7
damage, reporting 4-2
data system control 3-1
data units 3-19
data units selection 3-16
DC power supply 1-14
decimal point key 3-15
degassing
benefits 1-21, C-7, C-8
considerations C-9
deleting a timed event 3-47
design
electronic 1-9
optical 1-9
detector
exterior, cleaning 4-15
overview 1-7
setup 2-2
DIAG key 3-13
I
diagnostic settings
auto-zero offset 5-10, 5-13
contact closures and events 5-10
fix (set) EU 5-10
fix (set) voltage 5-10
input and output 5-12
optical filter override 5-11
procedure 5-8–5-20
reset 5-10
setting a fixed voltage output 5-14
sticky 5-9
user-selected 5-8–5-20
diagnostic tests
change lamp 5-10
failure 3-3, 5-2
generating test peaks 5-11
input and output 5-10, 5-12
keypad 3-13
keypad test 5-10
lamp, display, and keypad 5-10
procedure 5-8–5-20
reducing PMT sensitivity 5-19
sample and reference energy 5-10,
5-11
service 5-11
startup 1-21, 3-2
sticky 3-8
test display 5-10, 5-17
test keypad 5-16
user-selected 5-8–5-20
using 5-1–5-23
difference plot 1-16, 3-20
diffraction grating 1-9
dirty flow cell 4-5
disabling
Index-3
external events 3-16
inputs 3-16
disassembling the flow cell 4-8
display
diagnostic test 5-10, 5-17
fluorescence trace 3-13
lamp use statistics 3-14
options 3-13
system information 3-28
test 5-10
testing 5-17
E
EC Authorized Representative vi
electrical
specifications B-1
electrical symbols A-12
electricity source, connecting 2-36
electronics 1-13
emission 3-19, 3-20
monochromator 1-11
monochromator optics 1-10
units 1-5
wavelength selection 1-4
wavelength, field 3-5
enabling
chart mark 3-21
chart mark event inputs 3-26
external events 3-16
inputs 3-16
ending wavelength 3-52
energy sources, excitation 1-3
energy units 1-6, 3-19
Enter key 3-15
entering negative numbers 3-15
entrance slits 1-9
environmental specifications 2-2, B-1
equipment guidelines iv, A-6
erbium
Index-4
filter 1-8
scan 3-53
error messages 5-1–5-23
errors
calibration 3-35
startup 3-35, 5-2
Ethernet
communications interface 1-13
Ethernet cable, connecting 2-7, 2-20
EU 1-5
EUFS
defined 3-19
function 3-19
parameter 3-21
sensitivity 3-53
event inputs
auto-zero 3-26
chart mark 3-26
configuring 3-25
functions 2-10
inject-start 3-25
lamp 3-26
excitation
energy sources 1-3
light sources 1-3
monochromator 1-11
monochromator optics 1-10
wavelength selection 1-3
exit slits 1-9
external events
disabling 3-16
enabling 3-16
F
failure of startup diagnostic tests 3-3
fields, emission wavelength 3-5
filter type
changing 3-16
function 3-19
parameter 3-21
filters
changing the filter type 3-16
erbium 1-8
filter setting specification B-3
optical override 5-11, 5-18
second-order 1-8, 1-9
time constant 3-20
types of 3-19
fix (set) EU diagnostic setting 5-10
fix (set) voltage diagnostic setting 5-10
flammable solvents A-4
flow cell 1-4, 1-9, 1-11
cleaning 4-5
damaged 4-7
dirty 4-5
disassembling 4-8
flushing 4-5
inspecting 4-5
passivating 4-5
reassembling 4-8
replacing 4-7
replacing parts 4-5
flow cell assembly, removing 4-6
fluorescence 1-2
chromatography 1-2
difference plot 3-20
MaxPlot function 3-20
normalized units 1-5
process of detection 1-2
threshold events 3-46
threshold timed event parameter
3-46
trace 3-13
Fluorescence screen
display 3-11
icons 3-5
flushing, flow cell 4-5
front-left-hand panel cover 4-4
functions
analog outputs, single-channel 3-20
auto-zero on wavelength changes
3-21
I
change lamp 5-16
generating test peaks 5-17
MaxPlot 3-20, 3-39
optical filter override 5-18
primary 3-19
secondary 3-19
time constant 3-20
zoom 3-23
fuses, replacing 4-14
G
gas solubility C-7–C-8
gases, sparging C-8
generating
Auto Zero on inject from the
Alliance Separations
Module 2-15
chart mark from the Alliance
Separations Module 2-16
chart marks 2-29, 3-12
spectra 3-12
test peak diagnostic test 5-11
test peaks 5-17
tick marks 3-53
grating
diffraction 1-9
monochromator 1-3
H
handling symbols A-13
Help key 3-11, 3-28
HOME key 3-5, 3-11, 3-16
Home screen 3-4
home screen 3-4
navigating from 3-16
secondary pages 3-19
Index-5
I
I/O signals 2-11
icons
channel on 3-6
channel selector 3-6
keypad lock 3-8
keypad unlock 3-8
lamp off 3-7
lamp on 3-6
local/remote control 3-8
method number 3-8, 3-44
next 3-8
run time 3-8
shift 3-7
sticky diagnostic tests 3-8
table of 3-5
wrench 3-8
idle mode 1-8
initial method conditions 3-12, 3-25,
3-50
initializing the detector 3-2
initiating a scan 3-12
inject signal 3-25
inject-start
connection 2-9, 2-22
signal 2-9, 2-22
Waters 600 Series Pump
connections 2-32
Waters 717plus Autosampler
connections 2-35
input and output diagnostic
settings 5-12
tests 5-10, 5-12
inputs
disabling 3-16
enabling 3-16
signals 2-11
inspecting, flow cells 4-5
installation
Index-6
columns 2-3
network guidelines 2-8, 2-21
installing a new lamp 4-12
intended use v
inverting the chart 3-16, 3-20
ISM classification v
K
keypad
+/− key 3-15
? key 3-11, 3-28
• key 3-15
A/B key 3-5, 3-13
Auto Zero key 3-12
Calibrate key 3-14, 3-35
Cancel key 3-15
CE key 3-15
Chart Mark key 3-12
Clear Field key 3-15
CONFIGURE key 3-13, 3-25
Contrast key 3-15
decimal point key 3-15
description 3-11
DIAG key 3-13
Enter key 3-15
functions 3-9, 3-11
Help key 3-11, 3-28
HOME key 3-11
λ/λλ key 3-12, 3-37, 3-38
Lamp key 3-14
lamp, display, and keypad
diagnostic tests 5-10
Lock key 3-14
locking 3-14
METHOD key 3-13, 3-46
Next key 3-13
numerical keys 3-14
Previous key 3-13
Reset key 3-12
Run/Stop key 3-12
Scale key 3-13, 3-23
SCAN key 3-12, 3-54
Shift key 3-13
System Info key 3-14
test 5-16
test diagnostic test 5-10
TRACE key 3-13, 3-23
up/down arrow keys 3-12
using 3-9
keypad lock icon 3-8
keypad unlock icon 3-8
lamp on/off connections to 600 Series
Pump 2-30
light filters 1-3
light sources, excitation 1-3
I
line spikes 5-21
local/remote control icon 3-8
lock icon 3-8
Lock key 3-14
locking the keypad 3-14
long-pass filter 1-3
loss of current method conditions 3-51
L
maintenance
considerations 4-2
routine 4-3
manual calibration 3-14, 3-35
manual wavelength calibration 1-14
mass spectrometer shock hazard A-4
MaxPlot 1-15
MaxPlot function
charting 3-20
obtaining 3-39
method
active 3-50
current conditions 3-16
initial conditions 3-25, 3-50
list 3-13
method * 3-44, 3-50
preventing loss of current
conditions 3-51
programming 3-43–3-52
resetting a stored 3-51
retrieving 3-50
storing 3-44, 3-49
viewing events within 3-50
METHOD key 3-13, 3-46
method number icon 3-8, 3-44
method optimization 1-18
λ/λλ key 3-12, 3-37, 3-38
lamp
change 5-16
change lamp diagnostic test 5-10
configuring lamp event inputs 3-26
conserving lamp life 3-67
cooling time 4-9
energy 1-16
installing 4-12
lamp, display, and keypad
diagnostic tests 5-10
new 4-12
performance 1-16
removing 4-9
replacing 4-8
serial number 4-13
timed event parameter 3-45
turning off 3-67–3-70
turning on or off 3-14
turning on or off from the Alliance
2-17
use statistics 3-14
warranty 4-13
Lamp key 3-14
lamp off icon 3-7
lamp on icon 3-6
M
Index-7
mirrors, ellipsoidal and parabolic 1-9
miscibility of solvents C-3–C-6
mode
idle 1-8
multichannel 1-15
multiwavelength 1-15
single-channel 1-14
modes, changing 3-38
monitoring contact closures 5-14
monochromator
emission 1-11
excitation 1-11
overview 1-3
moving to the last entry in a list 3-15
multichannel
mode 1-15
operation 1-5
multichannel mode, changing to single
3-38
multiple Waters instruments,
connecting 2-8, 2-21
multiwavelength mode 1-15
description 3-12
key 3-12
N
navigating
from the home screen 3-16
in reverse order 3-13
the user interface 3-16
negative number entry 3-15
network, installation guidelines 2-8,
2-21
new timed event 3-46
Next arrow 3-13
Next icon 3-8
Next key 3-13
noise
adjusting filter 3-20
Index-8
filters 3-19
specifications B-3
noise, filtering 1-12
normalized units of fluorescence 1-5
numerical keys 3-14
O
obtaining
MaxPlot 3-39
stored spectrum information 3-66
offset
auto-zero offset diagnostic setting
5-10
voltage 3-16
operating
as a stand-alone instrument 3-29
in single wavelength mode 3-38
in single-channel mode 3-37
modes 3-29
under remote control 3-44
operation, theory and principles of 1-1
optical
and electronic design 1-9
component specifications B-3
filter override 5-18
filter override diagnostic setting
5-11
optics 1-9
optics assembly
emission monochromator 1-10
excitation monochromator 1-10
light path 1-10
optimization, method 1-18
other equipment, connecting 2-23–2-35
output
connections 2-10
signals 2-11
output off 3-20
overriding, optical filter setting 5-18
P
pace 3-52
parameters
analog out (multiwavelength) 3-22
analog out (single wavelength) 3-22
auto-zero on inject 3-21, 3-23
auto-zero on wavelength changes
3-23
auto-zero timed event 3-45
chart polarity 3-22
EUFS 3-21
filter type 3-21
fluorescence threshold timed event
3-46
lamp timed event 3-45
polarity timed event 3-45
primary 3-21
sample scan 3-55–3-56
secondary 3-21
sensitivity timed event 3-45
SW1 timed event 3-45
SW2 timed event 3-45
time constant 3-22
time constant timed event 3-45
timed event 3-44
voltage offset 3-22
wavelength 3-21
wavelength timed event 3-44
zero-scan 3-54
passivating
flow cell 4-5
peaks, generating test 5-11, 5-17
performance specifications B-2
performing verification procedures
3-34
personality board 1-13
photomultiplier tube 1-4, 1-9
calibrating 1-12
gain setting 1-17
sensitivity 1-12
physical specifications B-1
plus/minus key 3-15
PMT 1-4, 1-9
I
calibration 1-12
gain setting 1-17
sensitivity 1-12
sensitivity, reducing 5-19
polarity timed event parameter 3-45
polarity, chart 3-16, 3-20
power
supply 1-14
surges 5-21
power cord 2-36
powering off 3-70
preamplifier board 1-13
preventing loss of current method
conditions 3-51
Previous key 3-13
primary functions 3-19, 3-21
principles of operation 1-1
programming
switches 3-26
threshold events 3-47
timed events and methods
3-43–3-52
pulse periods, setting 3-26
purpose and audience iv
Q
quantitation 1-4
R
rear panel
illustration 2-10
signal connections 2-10
reassembling the flow cell 4-8
recalling the Fluorescence screen 3-5
recording new lamp’s serial number
4-13
Index-9
rectangular wave signal 3-26
reducing PMT sensitivity 5-19
reference energy 3-20, 3-56
remote control 3-44
removing
flow cell assembly 4-6
front-left-hand panel cover 4-4
lamp 4-9
repeatability specifications B-3
replacing
flow cell 4-7
flow cell parts 4-5
front-left-hand panel cover 4-4
fuses 4-14
lamp 4-8
replaying a spectrum 3-67
reservoirs, positioning C-6
reset diagnostic settings 5-10
Reset key 3-12
resetting
run clock 3-12
stored method 3-51
retrieving a method 3-50
returning to initial conditions 3-12
reviewing a scan 3-66
run clock, stopping 3-12
run time icon 3-8
Run/Stop key 3-12
running, new scan 3-57–3-63
S
safety advisories A-1
safety considerations, maintenance 4-2
sample and reference energy diagnostic
tests 5-10, 5-11
sample energy 3-56
sample scan
procedure 3-55–3-56
screens 3-55
Index-10
when to run 3-56
sample, exciting 1-4
Scale key 3-13, 3-23
scale, zooming 3-62
scaling factor 3-13
SCAN key 3-12, 3-54
scanning
anthracene 3-53, 3-64
erbium 3-53
EUFS 3-53
initiating 3-12
new spectra 3-57–3-63
pace 3-52
reference energy 3-56
replaying a spectrum 3-67
reviewing a scan 3-66
sample energy 3-56
sample scan 3-55–3-56
screens 3-55
sensitivity 3-53
spectra 3-52–3-64
storing a scan 3-65
subtracting 3-67
tick marks 3-53
timing 3-56
zero-scan 3-54
screen, Fluorescence (home) 3-4
secondary functions 3-16, 3-19, 3-21
secondary pages 3-19
second-order filter 1-8, 1-9
selectivity 1-4
sensitivity 1-4
EUFS parameter 3-21
scanning 3-53
setting specification B-3
timed event parameter 3-45
serial number, lamp 4-13
service
contacting Waters 5-20
diagnostic tests 5-11
set EU diagnostic setting 5-10
set voltage diagnostic setting 5-10
setting
a fixed voltage output 5-14
pulse periods 3-26
switches 5-14
the detector up to run 3-18
shift icon 3-7
Shift key 3-13
shutting down the detector 3-70
signal
cables, connecting 2-5
connections 2-5
input 2-11
output 2-11
start of run 3-25
signal connections
inject-start 2-9, 2-22
making 2-13
single pulse signal 3-26
single Waters instrument, connecting
2-7, 2-20
single wavelength mode
key 3-12
operating in 3-38
single wavelength pair mode 3-12
single-channel mode 1-14
changing to multi 3-38
operating in 3-37
parameters 3-20
slits, entrance 1-9
slits, exit 1-9
solvent
buffered solvents C-6
guidelines C-2
miscibility C-3–C-6
reservoirs C-6
UV cutoff C-9–C-11
viscosity considerations C-6
spare parts 4-3
sparging, overview C-8
specifications
I
bandwidth B-2
electrical B-1
environmental B-1
filter setting B-3
noise B-3
optical component B-3
performance B-2
physical B-1
repeatability B-3
sensitivity setting B-3
wavelength accuracy B-2
wavelength range B-2
specifications, environmental 2-2
spectra
generating 3-12
new 3-57–3-63
obtaining information about 3-66
replaying 3-67
reviewing 3-66
scanning 3-52–3-64
storing 3-65
subtracting 3-67
spectrum, scanning 1-16
stand-alone operation 3-1, 3-29
starting
detector 3-2
methods from the Alliance 2-17
run 3-25
run clock 3-12
wavelength 3-52
startup
diagnostic test failure 3-3
diagnostic tests 3-2
errors 3-35, 5-2
kit 2-27, 2-28
Index-11
startup diagnostic tests 1-21
sticky diagnostic settings 5-9
sticky diagnostic tests 3-8
stopping the run clock 3-12
stored spectra
obtaining information 3-66
reviewing information 3-66
storing
methods 3-44, 3-49
spectra 3-65
subtracting a spectrum 3-67
switch 1 timed event parameter 3-45
switch 2 timed event parameter 3-45
switched outputs 2-10
switches
programming 3-26
setting 5-14
symbols
caution A-5
electrical A-12
handling A-13
warning A-2
system
displaying information 3-14
information 3-28
System Info key 3-14
T
Technical Service 5-20
test display diagnostic test 5-10
test keypad diagnostic test 5-16
test peaks, generating 5-17
theory of operation 1-1
threshold events
clearing 3-51
programming 3-47
tick marks, generating 3-53
time constant
changing 3-16
Index-12
function 3-20
parameter 3-22
timed event parameter 3-45
timed events
and methods 3-43–3-52
clearing 3-51
deleting 3-47
description 3-44
lamp parameters 3-45
parameters 3-44, 3-46
auto-zero 3-45
polarity 3-45
sensitivity 3-45
time constant 3-45
wavelength 3-44
programming 3-43–3-52
programming a new event 3-46
switch 1 parameters 3-45
switch 2 parameters 3-45
toggling between channels 3-5
TRACE key 3-13, 3-23
transient energy 5-21
troubleshooting
contacting Waters 5-20
diagnostic tests 5-1–5-23
hardware 5-21
turning lamp on or off
from an external device 3-26
from front panel 3-14
to conserve lamp life 3-67–3-70
U
unlock icon 3-8
up/down arrow keys 3-12
user interface 3-16
user-selected diagnostic
settings 5-8–5-20
tests 5-8–5-20
using
2475 detector with older external
data systems 3-29
A/B key 3-5
diagnostic tests 5-1–5-23
input and output diagnostic
settings 5-12
input and output diagnostic tests
5-12
keypad 3-9
sample and reference energy
diagnostic tests 5-11
scale function to zoom 3-23
V
vacuum degassing. See degassing
verifying
detector 3-34
viewing events within a method 3-50
voiding the warranty 4-13
voltage offset
function 3-16, 3-20
parameter 3-22
W
warm up time 3-3
warning symbols A-2, A-6
warranty
lamp 4-13
voiding 4-13
Waters 600 Series Pump
auto-zero connections 2-31
chart mark connections 2-31
configuring the detector lamp
signal 2-30
connecting 2-29–2-33
inject-start connections 2-32
lamp on/off connections 2-30
Waters 717plus Autosampler
auto-zero connections 2-34
connecting 2-33–2-35
inject- start connections 2-35
Waters 746 data module, connecting
2-27–2-28
Waters Technical Service, contacting
I
4-2, 5-20
wavelength
accuracy specifications B-2
calibration 3-14, 3-35
changes, auto-zero on 3-21
changing 3-12
ending 3-52
parameter 3-21
range specifications B-2
selection 1-22, C-9–C-11
emission 1-4
excitation 1-3
starting 3-52
timed event parameter 3-44
verification failure message 1-14
wrench icon 3-8
X
xenon lamp
installing 4-12
optics 1-9
Z
zero-scan
parameters 3-54
screens 3-55
zoom function 3-23, 3-62
Index-13
Index-14
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