WATERS CORPORATION 2424 Manual

2424 Evaporative Light
Scattering Detector
Operator’s Guide
71500121802/Revision B
Copyright © Waters Corporation 2006−2009
All rights reserved

ii
Copyright notice
© 2006−2009 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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of Waters Corporation.
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Micromass Ltd.
Phillips is a registered trademark of Phillips Screw Company.
Other registered trademarks or trademarks are the sole property of their
owners.

iii
Customer comments
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errors you encounter in this document or to suggest ideas for otherwise
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usability.
We seriously consider every customer comment we receive. You can reach us
at [email protected].

iv
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.
Safety considerations
Some reagents and samples used with Waters instruments and devices can
pose chemical, biological, and radiological hazards. You must know the
potentially hazardous effects of all substances you work with. Always follow
Good Laboratory Practice, and consult your organization’s safety
representative for guidance.
When you develop methods, follow the “Protocol for the Adoption of Analytical
Methods in the Clinical Chemistry Laboratory,” American Journal of Medical
Technology, 44, 1, pages 30–37 (1978). This protocol addresses good operating
procedures and the techniques necessary to validate system and method
performance.
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 [phone].
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

v
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
Audience and purpose
This guide is intended for personnel who install, operate, and maintain 2424
Evaporative Light Scattering (ELS) detectors.
Intended use of the 2424 ELS detector
Waters designed the 2424 ELS detector to analyze and monitor many
compounds.
Symbol Definition
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
This product has been tested to the requirements
of CAN/CSA-C22.2 No. 61010-1, second edition,
including Amendment 1, or a later version of the
same standard incorporating the same level of
testing requirements
ABN 49 065 444 751

vi
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.

vii
EC Authorized Representative
Waters Corporation (Micromass UK Ltd.)
Floats Road
Wythenshawe
Manchester M23 9LZ
United Kingdom
Telephone: [phone]
Fax: [phone]
Contact: Quality manager

viii

Table of Contents ix
Copyright notice ................................................................................................... ii
Trademarks ............................................................................................................ ii
Customer comments ............................................................................................ iii
Contacting Waters ............................................................................................... iv
Safety considerations .......................................................................................... iv
Safety advisories.................................................................................................. v
Operating this instrument .................................................................................. v
Applicable symbols .............................................................................................. v
Audience and purpose.......................................................................................... v
Intended use of the 2424 ELS detector............................................................... v
Calibrating .......................................................................................................... vi
Quality-control .................................................................................................... vi
ISM classification ................................................................................................. vi
ISM Classification: ISM Group 1 Class B ......................................................... vi
EC Authorized Representative ........................................................................ vii
1 2424 ELS Detector Optics Principles ................................................ 1-1
Principles of evaporative light scattering detection ............................... 1-2
Overview........................................................................................................... 1-2
Capabilities ...................................................................................................... 1-2
ELS detection process...................................................................................... 1-2
Detection .......................................................................................................... 1-4
ELS detection limitations................................................................................ 1-6
Detector description ........................................................................................ 1-7
Signal processing and noise calculations........................................................ 1-8
Calibrating the photomultiplier tube (PMT).................................................. 1-8
Filtering noise .................................................................................................. 1-8
Electronics and data acquisition..................................................................... 1-9
Table of Contents

x Table of Contents
Nebulizer .......................................................................................................... 1-9
Optics bench..................................................................................................... 1-9
Temperature control ...................................................................................... 1-10
Startup diagnostics........................................................................................ 1-11
Lamp energy and performance ..................................................................... 1-12
Rear panel ...................................................................................................... 1-13
References ........................................................................................................ 1-13
2 Setting up the Detector ........................................................................ 2-1
Introduction ....................................................................................................... 2-2
Before you begin ............................................................................................... 2-2
Unpacking and inspecting .............................................................................. 2-3
Selecting a site within a laboratory ............................................................. 2-3
Site selection requirements............................................................................. 2-4
Detector dimensions ........................................................................................ 2-5
Power requirements......................................................................................... 2-6
Gas requirements............................................................................................. 2-7
Making the gas supply connection ............................................................... 2-7
Venting the exhaust hose ................................................................................ 2-8
Connecting to the electricity source .......................................................... 2-11
Installing the nebulizer assembly ............................................................... 2-12
Connecting the siphon drain tubing .......................................................... 2-14
Routing the siphon drain tubing down the front of the detector................. 2-14
Routing the siphon drain tubing to the rear of the detector ....................... 2-15
Connecting the drip tray ............................................................................... 2-18
Required materials ........................................................................................ 2-18
Connecting the nebulization gas to the nebulizer .................................. 2-19
Connecting a column or second detector .................................................. 2-19
Required materials ........................................................................................ 2-19

Table of Contents xi
Making signal connections ........................................................................... 2-20
Connecting the Ethernet cable...................................................................... 2-22
Network installation guidelines.................................................................... 2-23
Connecting to other instruments .................................................................. 2-25
Connecting the Waters column heater module ............................................ 2-33
3 Operating the Detector ......................................................................... 3-1
Starting up the detector .................................................................................. 3-2
Initializing the detector................................................................................... 3-2
Using the display ............................................................................................. 3-4
Detector Home and Message screen icons...................................................... 3-5
Using the keypad ............................................................................................... 3-7
Navigating the user interface ...................................................................... 3-13
Navigating to and from the Home screen..................................................... 3-13
Preparing to start a run ................................................................................ 3-15
Primary and secondary functions ................................................................. 3-15
Setting up a run ............................................................................................... 3-17
Setting the nebulizer and drift tube temperature ....................................... 3-18
Setting the gain and gas pressure ................................................................ 3-19
Setting the column heater module temperature.......................................... 3-21
Resetting the stop flow output switch .......................................................... 3-21
Operating the trace and scale functions....................................................... 3-22
Setting the data rate...................................................................................... 3-24
Setting the filter time constant..................................................................... 3-24
Setting the switch output.............................................................................. 3-25
Setting the analog signal output................................................................... 3-25
Setting auto zero options............................................................................... 3-25
Configuring the detector ............................................................................... 3-26
Configuring event inputs............................................................................... 3-27
Configuring stop flow output......................................................................... 3-28
Setting pulse periods ..................................................................................... 3-28
Selecting the type of nebulizer...................................................................... 3-29
Setting the display contrast .......................................................................... 3-29

xii Table of Contents
Displaying system information ..................................................................... 3-30
Using help ...................................................................................................... 3-30
Operating the detector .................................................................................. 3-30
Standalone operation..................................................................................... 3-31
Auto-optimizing gain and LSU-FS ............................................................... 3-31
Programming methods and events ............................................................. 3-34
Overview of methods ..................................................................................... 3-34
Programming timed events ........................................................................... 3-35
Programming threshold events..................................................................... 3-37
Storing a method............................................................................................ 3-38
Retrieving a method ...................................................................................... 3-39
Viewing events within a method................................................................... 3-39
Resetting a method ........................................................................................ 3-39
Clearing events .............................................................................................. 3-40
Conserving lamp life ...................................................................................... 3-41
Changing chromatographic conditions ..................................................... 3-43
Shutting down the detector .......................................................................... 3-44
Periodic maintenance .................................................................................... 3-45
4 Maintaining the Detector ..................................................................... 4-1
Contacting Waters technical service ............................................................ 4-2
Maintenance considerations .......................................................................... 4-2
Safety and handling......................................................................................... 4-2
Spare parts....................................................................................................... 4-3
Replacing the lamp cartridge ........................................................................ 4-3
Replacing the nebulizer .................................................................................. 4-6
Cleaning the nebulizer ultrasonically ......................................................... 4-9
Cleaning the drift tube .................................................................................. 4-12
Servicing the vapor trap ............................................................................... 4-13

Table of Contents xiii
Replacing fuses ................................................................................................ 4-14
Cleaning the instrument’s exterior ............................................................ 4-15
5 Diagnostic Functions and Troubleshooting .................................... 5-1
Error messages .................................................................................................. 5-2
Startup error messages ................................................................................... 5-2
Operational error messages ............................................................................ 5-2
User-selectable diagnostic functions ........................................................... 5-2
Overview........................................................................................................... 5-2
“Sticky diagnostics” tests................................................................................. 5-4
Running diagnostic tests ................................................................................. 5-5
Running the Auto Gain diagnostic function................................................... 5-5
Input and output diagnostic functions ........................................................... 5-6
Lamp, display, and keypad diagnostic functions ........................................... 5-8
Gas and temperature control diagnostic functions...................................... 5-10
Sample and reference energy diagnostic function ....................................... 5-12
Generate Test Peaks diagnostic function ..................................................... 5-13
General troubleshooting ............................................................................... 5-13
Power surges .................................................................................................. 5-13
Detector troubleshooting ............................................................................... 5-14
Power-on confidence check error messages.................................................. 5-16
Operational error messages .......................................................................... 5-17
Chromatography troubleshooting .............................................................. 5-21
Abnormal baseline ......................................................................................... 5-22
Erratic or incorrect retention times.............................................................. 5-27
6 Optimizing Detection and Preparing Solvents ............................... 6-1
Optimizing detector performance ................................................................ 6-2
Optimizing the mobile phase .......................................................................... 6-2
Sample pretreatment....................................................................................... 6-2
Column treatment ........................................................................................... 6-2
Selecting a solvent ............................................................................................ 6-2

xiv Table of Contents
Solvent degassing ............................................................................................. 6-7
Solvent degassing methods ............................................................................. 6-7
Solvent degassing considerations ................................................................... 6-8
Optimization protocol ...................................................................................... 6-9
Nebulizer gas pressure .................................................................................... 6-9
Nebulizer temperature .................................................................................... 6-9
Drift tube temperature .................................................................................. 6-10
Selecting the optimum temperature............................................................. 6-10
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-5
Electrical and handling symbols ................................................................. A-12
Electrical symbols .......................................................................................... A-12
Handling symbols .......................................................................................... A-13
B Specifications ........................................................................................ B-1
2424 ELS detector specifications ................................................................. B-1
Index ..................................................................................................... Index-1

1-1
12424 ELS Detector Optics
Principles
To use the 2424 ELS detector effectively, you must understand the principles
that underlie operation of the detector’s optics and electronics.
Contents:
Topic Page
Principles of evaporative light scattering detection 1-2
Detector description 1-7
References 1-13

1-2 2424 ELS Detector Optics Principles
Principles of evaporative light scattering detection
Overview
Evaporative light scattering (ELS) detection works by nebulizing the solvent
flow from a liquid chromatography (LC) system and entraining the resultant
droplets in a gas stream. Mobile phase is then evaporated from the droplets.
When an analyte is less volatile than the mobile phase, it remains in the gas
stream as a “dry” solute particle and flows to the ELS detector. Once there,
the particles scatter the light beam. The amount of scattered light is measured
and bears a relationship to the concentration of material eluting.
Capabilities
The 2424 ELS detector is compatible with virtually all modes of
chromatography including flow injection analysis. The detector responds to all
compounds that are, relative to their mobile phase, sufficiently nonvolatile at
the conditions of analysis. Applications for ELS detection include
combinatorial libraries of small molecules, natural product extracts and
libraries, food products, and related materials. For detecting compounds that
exhibit little to no UV/Vis response and do not ionize well for mass
spectrometry, the ELS detector complements HPLC for analyzing sugars,
antibiotics, antivirals, lipids, phospholipids, biomolecules, and natural
products. You can use ELS detection in a system that includes a mass
spectrometer and absorbance detector, applying it as a qualitative tool to
demonstrate the purity or complexity of a sample. Quantitation can be
achieved by carrying out a calibration plot, as explained later in this guide.
Note, however, that the curve will not be linear because ELS detectors give a
non-linear response.
ELS detection performs well in isocratic or gradient elution with a wide
variety of mobile phases and additives. Waters recommends using mass
spectrometry-compatible volatile mobile-phase modifiers.
ELS detection process
The three separate regions of an ELS detector are nebulization, desolvation,
and detection. In all ELS detectors, these three regions are positioned so that
the chromatographic effluent is nebulized and mobile phase is evaporated so

Principles of evaporative light scattering detection 1-3
that dry solute particles, consisting only of analytes, reach the light source for
scattering.
Low temperature nebulization
In the detector’s nebulization region, the chromatographic effluent is
transformed into a fine aerosol. A concentric tube, or flow-type nebulizer,
mixes chromatographic effluent with a carrier gas (usually nitrogen)
developing a series of droplets that forms the aerosol that enters a
narrow-orifice drift tube.
Nebulization region and drift tube (representative)
The concentric flow nebulizer allows you to control the carrier gas flow versus
the chromatographic effluent flow rate. High gas flow produces small droplets,
requiring less heat to evaporate the solvent. Conversely, low gas flow produces
large droplets, requiring more heat to evaporate the solvent.
Desolvation
In the desolvation region, the mobile phase evaporates, leaving dried solute
particles in the drift tube.
As the aerosol drops exiting the nebulizer pass through the drift tube, they
become smaller. The carrier gas sweeps the dried, aerosolized solute particles
along to the instrument’s detection region.
Evaporation occurs as a function of time, temperature, and pressure of the
carrier gas. It is therefore important to use HPLC mobile phases that easily
and quickly evaporate and desolvate. Solvents of fairly low boiling point and
low viscosity are best. They include the more commonly used HPLC mobile
phases: water, acetonitrile, methanol, ethanol, and THF. Viscous and
high-boiling solvents might fail to fully separate from the analyte molecules or
Nebulization region Drift tube

1-4 2424 ELS Detector Optics Principles
species before the detection step. This adds to the background noise and
decreases the analyte signal response, which causes low sensitivity (slope of
the calibration plot) and high limits of detection (LOD). The evaporated HPLC
solvents are condensed and captured in the recommended solvent trap and
exhaust routing. Nevertheless, small amounts of residual can persist, and
these should be exhausted into a fume hood to prevent their escape into the
laboratory.
Detection
The analyte particles enter the detection region where a light source impinges
on the particles. The light is thus scattered and focused onto a photomultiplier
tube (PMT) where its intensity is measured.
The size (diameter) of the analyte particles determines how the light is
scattered. The detector measures the intensity of the scattered light at 60°
relative to the excitation beam to minimize polarization effects and stray
light. Particles of different sizes exhibit different angular distributions of the
scattered light, and particles whose sizes and shapes vary have different
light-scattering cross sections. In general, larger particles scatter more light,
yielding more intense signals and peak responses.
A photomultiplier tube (PMT) converts the scattered light signal to a voltage
that can be recorded and analyzed. The stronger the scattering, the more
intense the final signal on the ELS detection chromatogram. The scattered
light is a rough measure of the mass of material represented by a
chromatographic peak. To some degree, this “mass” response can be
compound-independent. However, many factors can also affect the mass
response, particularly the density of the analyte in a small dried particle. For
example, a popped kernel of corn has a lower density than the unpopped
kernel from which it originated. Yet, because it is larger, in most cases it
would scatter more light. You should also remember that the output of an ELS
detector has no direct relation to the molecular weight of an analyte.
Types of light scattering
The three possible regimes of light scattering are
• Rayleigh
•Mie
• refraction-reflection

Principles of evaporative light scattering detection 1-5
Light scattering direction
For a nebulizer that produces an average droplet diameter of D0, the diameter
of an average, resulting dry analyte particle is
D0= Average liquid droplet diameter
c= Concentration of the analyte
p= Density of the dry analyte
For any given analyte peak, the response of an ELS detector can be that of all
three light scattering regimes. The light-scattering type depends on the size of
the particles going through the light beam. The ratio of particle diameter, D,
to the incident wavelength,
λ , or , defines the type of scattering that results.
• Rayleigh scattering occurs for the smallest particles where <0.1. The
scattered light from a particle is proportional to D6, and consequently
the scattered signal is proportional to c2.
• Mie scattering occurs for particles where >0.1, but <1.0. The scattered
light is proportional to D4, and the scattered signal is proportional to c4/3.
Rayleigh scattering Mie scattering Refraction-reflection scattering
Direction of incident light
DD
0cp⁄()
13/
=where
D
λ
----
D
λ
----
D
λ
----

1-6 2424 ELS Detector Optics Principles
• Refraction-reflection scattering occurs for particles where >1.0. The
scattered light is proportional to D2, and the scattered signal is
proportional to c2/3.
• As a chromatographic peak elutes from a column, the concentration of
the analyte it represents changes. Concentration goes from near-zero at
the baseline to a maximum that corresponds to column efficiency,
injection volume, retention time, and concentration of the sample when
injected. From the maximum level, the concentration then returns to
near-zero. If the concentration is high enough, the diameter of a dry
analyte particle can vary through all three scattering regimes—
Rayleigh, Mie, and refraction-reflection scattering. It is this variance
that prevents linearity in ELS detection calibration plots over more than
one order of magnitude.
ELS detection limitations
Consider these limitations when implementing global ELS detection
separation methods:
• ELS detection lacks linearity over wide concentration ranges. When you
use the detector for assays, you may need to experiment with a variety of
“best fits” using linear, quadratic, and log-log responses for the
compounds of interest. You might also need to establish groupings for
expected concentration ranges.
• ELS detection is a destructive technique; the analyte is sacrificed to
generate the scattering particles. Ideally, therefore, the ELS detector
should be the final detector in a series. Alternatively, you can place the
ELS detector upstream of others, provided you split the column effluent
so that the ELS detector receives its own stream from the LC.
• Any particle can interfere with the sample signal, including particulates
in poor-grade chromatographic solvents because the detector responds
equally to all particulates. This lack of selectivity can cause problematic
background noise.
• The detector’s sensitivity to the particulates increases noise and,
consequently, signal-to-noise variation for a given method arising from
differences in the quality of mobile phases. Moreover, stationary phase
components can leach from the column and contribute particulates to
the sample flow.
D
λ
----