Waters ACQUITY UPLC Operating manual

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ACQUITY UPLC
Photodiode Array and
eλPhotodiode Array
Detector
Operator’s Overview and Maintenance Guide
Revision A
Copyright © Waters Corporation 2010
All rights reserved

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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
ACQUITY, ACQUITY UPLC, UPLC, Waters PIC, and Waters are registered
trademarks, and Empower, MassLynx, and “THE SCIENCE OF WHAT’S
POSSIBLE.” are trademarks of Waters Corporation.
PEEK is a trademark of Victrex Corporation.
Teflon is a registered trademark of E. I. du Pont de Nemours and Company.
Triton is a trademark of Union Carbide Corporation.
Other registered trademarks or trademarks are the sole property of their
owners.

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Customer comments
Waters’ Technical Communications department invites you to tell us of any
errors you encounter in this document or to suggest ideas for otherwise
improving it. Please help us better understand what you expect from our
documentation so that we can continuously improve its accuracy and
usability.
We seriously consider every customer comment we receive. You can reach us
at [email protected].
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
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

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Good Laboratory Practice, and consult your organization’s safety
representative for guidance.
Considerations specific to the ACQUITY PDA/eλPDA detector
High voltage hazard
Safety advisories
Consult the Safety Advisories section on page 80 for a comprehensive list of
warning and caution advisories.
Warning: To avoid electric shock, do not remove the PDA/eλPDA
detector’s protective panels. The components within are not
user-serviceable.

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Operating the ACQUITY PDA/eλPDA detector
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
ACQUITY PDA/eλPDA detectors. It gives an overview of the instrument’s
technology and operation.
Intended use of the ACQUITY PDA/eλPDA detector
The Waters ACQUITY PDA/eλPDA detector is for research use only and is not
intended for use in diagnostic applications.
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

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Calibrating
To calibrate LC systems, follow acceptable calibration methods using at least
five standards to generate a standard curve. The concentration range for
standards must 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.

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

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Table of Contents ix
Copyright notice ................................................................................................... ii
Trademarks ............................................................................................................ ii
Customer comments ............................................................................................ iii
Contacting Waters ............................................................................................... iii
Safety considerations .......................................................................................... iii
Considerations specific to the ACQUITY PDA/eλPDA detector ...................... iv
Safety advisories................................................................................................. iv
Operating the ACQUITY PDA/eλPDA detector .............................................. v
Applicable symbols .............................................................................................. v
Audience and purpose.......................................................................................... v
Intended use of the ACQUITY PDA/eλPDA detector ........................................ v
Calibrating .......................................................................................................... vi
Quality-control .................................................................................................... vi
ISM classification ................................................................................................. vi
ISM Classification: ISM Group 1 Class B ......................................................... vi
EC authorized representative .......................................................................... vii
Overview .................................................................................................................... 1
Detector optics ...................................................................................................... 1
Calculating absorbance ....................................................................................... 4
Operating principles of the light-guiding flow cell ..................................... 5
Resolving spectral data ....................................................................................... 8
Measuring light at the photodiode array............................................................ 9
Computing absorbance data points .................................................................. 12
Table of Contents

x Table of Contents
Flow cell options ................................................................................................ 17
Before you begin ................................................................................................ 18
Installing the detector ...................................................................................... 19
Plumbing the detector ...................................................................................... 21
Installing the multi-detector drip tray ............................................................. 25
Making Ethernet connections ......................................................................... 27
I/O signal connector ........................................................................................... 27
Connecting to the electricity source ............................................................. 28
Starting the detector ......................................................................................... 29
Monitoring detector LEDs................................................................................. 31
About the detector control panel....................................................................... 31
Shutting down the detector ............................................................................. 34
Shutting down for less than 24 hours............................................................... 34
Shutting down for more than 24 hours............................................................. 34
Maintaining the detector ................................................................................. 36
Contacting Waters technical service................................................................. 36
Maintenance considerations.............................................................................. 37
Proper operating procedures ............................................................................. 37
Maintaining the leak sensor ............................................................................. 39
Replacing the detector’s leak sensor................................................................. 43
Maintaining the flow cell................................................................................... 45
Replacing the lamp ............................................................................................ 56
Replacing the fuses ............................................................................................ 59
Cleaning the instrument’s exterior................................................................... 60
Spectral contrast theory .................................................................................. 61
Comparing absorbance spectra ......................................................................... 61
Representing spectra as vectors........................................................................ 62
Spectral contrast angles .................................................................................... 64
Undesirable effects ............................................................................................ 67
Error messages and troubleshooting ............................................................ 71
Startup error messages ..................................................................................... 71

Table of Contents xi
Error messages preventing operation............................................................... 74
Detector troubleshooting ................................................................................... 77
Safety advisories ................................................................................................ 80
Warning symbols ............................................................................................... 80
Caution symbol .................................................................................................. 83
Warnings that apply to all Waters instruments .............................................. 84
Electrical and handling symbols....................................................................... 90
Specifications ...................................................................................................... 92
ACQUITY UPLC PDA detector specifications ................................................. 92
ACQUITY UPLC eλPDA detector specifications ............................................. 95
Solvent considerations ..................................................................................... 98
Introduction........................................................................................................ 98
Solvent miscibility ........................................................................................... 100
Wavelength selection....................................................................................... 102
Mobile phase absorbance ............................................................................... 105

xii Table of Contents

Overview 1
Overview
The Waters ACQUITY UPLC®photodiode array (PDA) detector and
extendable λphotodiode array (eλPDA) detector are ultraviolet/visible
(UV/Vis) spectrophotometers designed for use in the family of ACQUITY
UPLC®Systems, such ACQUITY UPLC H-Class or bioACQUITY. The
detectors, controlled by Empower™, MassLynx™, or third-party software for
both LC/MS and LC applications, operate as integral parts of the system.
With a photodiode array of 512 photodiodes and an optical resolution of
1.2 nm, the detectors operate within a range of between 190 and 500 nm for
the PDA and between 190 and 800 nm for the eλPDA.
To use the detector’s operating software effectively, you must understand the
principles that underlie operation of the detector’s optics and electronics.
Detector optics
The light path through the optics assembly of the detector is shown in the
following figure.

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Optics assembly light path
The following table describes the optics assembly components.
Optics assembly components
Component Function
Thermal switch Disconnects power to the lamp if the temperature rises
to an unacceptable level.
M1 mirror Off-axis, ellipsoidal mirror that projects light from the
lamp into the flow cell.
Lamp and lamp
optics
Focuses light from the high-brightness deuterium (D2)
source lamp and, via a mirror, redirects the light
through a beam splitter and then to the flow cell.
Window Used to help minimize air infiltration into the lamp
housing.
TP02522
Grating
Photodiode
array
Spectrograph
mirror and
mask
100-µm slit (PDA)
50-µm slit (eλPDA)
190 nm
Flow cellWindow Filter FlagLamp and
lamp optics
500 nm (PDA)
800 nm (eλPDA)
Thermal
switch
M1 mirror
Order filter

Detector optics 3
Filter flag Influences the light entering the flow cell. These are
the flag settings:
• Shutter – Prevents light from entering the flow cell.
In the shutter position, dark counts are measured at
each pixel and subsequently subtracted from
observed signal counts to give true signal counts.
• Open – Allows light to pass into the flow cell. It is
the normal setting when performing runs.
• Erbium – Inserts an erbium filter into the light
beam that allows the wavelength calibration to be
checked or updated.
• UV blocking filter – Inserts a UV blocking filter into
the light beam that minimizes light with
wavelengths shorter than, approximately, 210 nm.
Flow cell Houses the segment of the flow path (containing eluent
and sample) through which the polychromatic light
beam passes.
Shunt
(not pictured)
Diagnostic tool used in place of the light-guiding flow
cell to emulate light transmission without fluid flow.
Spectrograph
mirror and mask
The mirror focuses light transmitted through the flow
cell onto the slit at the entrance to the spectrographic
portion of the optics. The mirror mask defines the size
of the beam at the grating.
Slit Determines wavelength resolution and intensity of
light striking the photodiodes. The width of the slit is
100 µm (for the PDA) or 50 µm (for the eλPDA).
Grating Blazed, holographic diffraction grating that disperses
light into bands of wavelengths and focuses them onto
the plane of the photodiode array.
Order filter Reduces the contribution of second-order diffraction of
UV light (less than 340 nm for PDA, or 370 nm for
eλPDA) to the light intensity observed at visible
wavelengths (greater than 340 nm for PDA, or 370 nm
for eλPDA).
Optics assembly components (Continued)
Component Function

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Calculating absorbance
The detector computes absorbance by subtracting the dark current (see “Dark
current” on page 12) and reference spectrum from the acquired spectrum.
Absorbance is based on the principles of Beer’s law.
Beer’s law
The relationship between the quantity of light of a particular wavelength
arriving at the photodiode and the concentration of the sample passing
through the flow cell is described by the Beer-Lambert law (commonly called
Beer’s law). Beer’s law is expressed as A= εlc where
A= dimensionless quantity measured in absorbance units
ε = constant of proportionality, known as the molar extinction coefficient
l= path length, in centimeters (1.0 cm in the detector’s normal flow cell)
c= concentration, in moles per liter
Beer’s law applies only to well-equilibrated dilute solutions. It assumes that
the refractive index of the sample remains constant, that the light is
monochromatic, and that no stray light reaches the detector element. As
concentration increases, the chemical and instrumental requirements of
Beer’s law can be violated, resulting in a deviation from (absorbance versus
concentration) linearity. The absorbance of mobile phase can reduce the linear
range by the amounts shown in “Mobile phase absorbance” on page 105.
Photodiode array An array of 512-pixel photodiodes arranged linearly.
The diode width (50-µm), together with a 100-µm slit
(for the PDA) or 50-µm slit (for the eλPDA), yield single
wavelength resolution of 1.2 nm.
Optics assembly components (Continued)
Component Function

Operating principles of the light-guiding flow cell 5
Absorbance as a function of concentration
Operating principles of the light-guiding flow cell
Small-bore, high-capacity columns like those used in UPLC produce
small-volume peaks. To avoid bandspreading and maintain concentration, the
volume of a detector’s flow cell must be correspondingly small. A good rule of
thumb is to hold the volume to 1/10th or less than the peak volume. To achieve
the required volume reduction with conventional absorbance detector flow
cells, the pathlength must be reduced, to avoid a significant decrease in light
throughput. Reduced pathlength results in less analytical sensitivity, as
predicted by Beer’s law, yet high light levels are necessary to preserve a high
signal-to-noise ratio.
Using a small-volume, light-guiding flow cell designed with optimum
pathlength and high light throughput, resolves the problem. Such a flow cell is
analogous to an optical fiber, where the core is the fluid sample and the
cladding is Teflon®AF, a unique, chemically inert, amorphous fluoropolymer
made by DuPont. The refractive index of Teflon AF is lower than that of water
or other HPLC mobile phases. Light rays entering the liquid core, within the
cone half-angle, α, are internally reflected when they meet the Teflon AF
boundary. These rays are transmitted through the flow cell, theoretically
without loss, except for absorption by the sample.
Concentration
Absorbance
Ideal
Actual
Working range
Background absorbance

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Light transmission through a light-guiding flow cell
This information complements the foregoing illustration:
• The core of the light guide is the fluid sample, with refractive index n1.
• The cladding is a Teflon AF tube, with refractive index n2.Index n2< n1.
• The cross-sectional area of the tube is A and the length d. Cell volume =
Ad.
In the preceding figure, two rays of light are shown reflecting from the
core-cladding interface. In a flow cell, the number of “bounces” depends on the
length of the Teflon AF tube, its inside diameter (lumen), and the ray angle,
“α”. The light beam (which represents the energy transmitted through the
cell) is comprised of many such rays, up to a maximum whose angle is
theoretically set by the refractive index of the core and cladding. In the
ACQUITY UPLC PDA detector, this angle is mechanically controlled by
components external to the flow cell so that the variation in refractive index
arising from different mobile phases does not materially influence the
efficiency of the transmitted energy.
The following schematic diagram of the flow cell shows the light-guiding
portion of the cell inside the cell assembly.
Rays of light
Cladding (Teflon AF)Core (sample fluid)
α

Operating principles of the light-guiding flow cell 7
Light-guiding portion of flow cell
The sample fluid is introduced and removed from the flow cell via PEEK™
tubing. Probe radiation from the lamp housing is focused onto the input face of
the optical fiber that forms one end of the flow cell. Light travels down this
optical fiber until it encounters the fluid channel defined by the internal
diameter of the Teflon AF tube. The light then exits the optical fiber and
enters the fluid-filled Teflon AF tube. As the light passes through this tube, it
interacts with the sample stream. Any absorption by the fluid reduces the
light intensity. The reduction is subsequently converted to absorbance. The
light exits the flow cell through a fused silica window where it projects onto
the slit of the spectrograph. A concave grating then disperses and projects the
light onto the photodiode array.
Tip: Unlike other flow cell designs, in which light beams do not strike the
internal walls of the cell, light-guiding relies on internal reflections from the
walls of the Teflon AF tubing. Consequently, you must maintain flow cell
cleanliness by following the recommended procedures described in
“Maintaining the detector” on page 36. With such care, the instrument and
flow cell will provide continuous, sensitive detection.
Requirement: To ensure the cell’s proper alignment and calibration, fill the
flow cell with flowing solvent before you power-on the detector. An empty flow
cell causes calibration error. Refer to the recommended procedures described
in “Maintaining the detector” on page 36 for more information
Window
Tef lon AF
Fluid out
Light in
Fluid in
Light out

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Resolving spectral data
Together with photodiode spacing, the detector’s 100-µm-wide slit
(50-µm-wide slit for eλPDA) determines the intensity and bandwidth of the
light that strikes the photodiode array. Variations in intensity and bandwidth
provide the means to distinguish among similar spectra.
The grating images the slit onto the photodiode array. The angle of diffraction
from the grating determines the wavelength that strikes a particular
photodiode in the array.
The following figure shows an absorbance spectrum of benzene. Note that the
wavelength resolution is sufficient to resolve five principal absorption peaks.
Benzene spectrum at 1.2 nm resolution
Absorbance
nm