Waters ACQUITY UPC2 Operating manual

ACQUITY UPC2
Photodiode Array Detector
Overview and Maintenance Guide
Revision A
Copyright © Waters Corporation 2012
All rights reserved

ii
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Table of Contents iii
Copyright notice ...................................................................................................................... ii
Overview  
Detector optics .................................................................................................................... 1
Calculating absorbance....................................................................................................... 4
Flow cell operating principles ............................................................................................ 6
Detector capabilities ......................................................................................................... 16
Preparing the detector ......................................................................................................... 17
Installing the detector .......................................................................................................... 18
Plumbing the detector .......................................................................................................... 21
Installing the multi-detector drip tray ............................................................................... 24
Making Ethernet connections ............................................................................................. 25
I/O signal connector.......................................................................................................... 26
Connecting to the electricity source ................................................................................... 26
Starting the detector ............................................................................................................ 27
Monitoring detector LEDs................................................................................................ 29
About the detector control panel....................................................................................... 30
Using a cuvette ...................................................................................................................... 31
Shutting down the detector ................................................................................................. 34
Maintaining the detector ..................................................................................................... 34
Contacting Waters technical service ................................................................................. 34
Maintenance considerations.............................................................................................. 35
Proper operating procedures ............................................................................................. 36
Maintaining the leak sensor .............................................................................................. 37
Replacing the detector’s leak sensor................................................................................. 42
Maintaining the flow cell.................................................................................................. 44
Replacing the lamp ........................................................................................................... 49
Reading lamp energy ........................................................................................................ 52
Replacing the fuses ........................................................................................................... 52
Table of Contents

iv Table of Contents
Cleaning the instrument’s exterior.................................................................................... 53
Spectral contrast theory ...................................................................................................... 54
Comparing absorbance spectra ......................................................................................... 54
Representing spectra as vectors ........................................................................................ 55
Spectral contrast angles .................................................................................................... 57
Undesirable effects ........................................................................................................... 60
Error messages and troubleshooting .................................................................................. 64
Startup error messages...................................................................................................... 64
Error messages preventing operation................................................................................ 67
Detector troubleshooting .................................................................................................. 70
Specifications ........................................................................................................................ 72
ACQUITY UPC2PDA detector specifications ................................................................ 72
Solvent considerations ......................................................................................................... 74
Solvent miscibility ............................................................................................................ 75
UV cutoffs for common solvents...................................................................................... 75

Overview 1
Overview
The Waters ACQUITY UltraPerformance Convergence Chromatography
(ACQUITY UPC2™) photodiode array (PDA) detector is an ultraviolet- and
visible-wavelength (UV/Vis) spectrophotometer designed for use in the
ACQUITY UPC2system. Empower™ or MassLynx™ software can control the
detector for LC/MS and LC applications.
With a photodiode array of 512 photodiodes and an optical resolution of
1.2 nm, the detector operates within the range 190 to 800 nm.
To use the detector’s operating software effectively, you must understand the
principles that underlie operation of its optics and electronics.
Detector optics
The light path through the optics assembly of the detector is shown in the
following figure.

2
Optics assembly light path:
The following table describes the optics assembly components.
Optics assembly components:
Component Function
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.
TP02819
Grating
Photodiode
array
Spectrograph
mirror and
mask
Slit (50-µm)
Flow cell
Window
Filter Flag
Lamp and
lamp optics
M1 mirror
Order filter

Overview 3
Flow cell Houses the segment of the flow path (containing eluent
and sample) through which the polychromatic light
beam passes.
Grating Blazed, holographic diffraction grating that disperses
light into bands of wavelengths and focuses them onto
the plane of the photodiode array.
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.
M1 mirror Off-axis, ellipsoidal mirror that projects light from the
lamp into the flow cell.
Order filter Reduces the contribution of second-order diffraction of
UV light (less than 370 nm) to the light intensity
observed at visible wavelengths (greater than 370 nm).
Photodiode array An array of 512-pixel photodiodes arranged linearly.
The diode width (50-μm), together with a 50-μm slit,
yield single wavelength resolution of 1.2 nm.
Slit Determines wavelength resolution and intensity of
light striking the photodiodes. The width of the slit is
50 μm.
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.
Window Used to help minimize air infiltration into the lamp
housing.
Optics assembly components: (Continued)
Component Function

4
Calculating absorbance
The detector computes absorbance by subtracting the dark current (see “Dark
current” on page 14) 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= lc
where
A= dimensionless quantity measured in absorbance units
= 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.

Overview 5
Absorbance as a function of concentration:
Concentration
Absorbance
Ideal
Actual
Working range
Background absorbance

6
Flow cell operating principles
The Waters TaperSlit™ flow cell used in the detector renders the detector
baseline essentially insensitive to changes in mobile phase refractive index
(RI). RI changes occur during gradient separations or result from temperature
or pump-induced pressure fluctuations.
To achieve RI immunity, a combination of a spherical mirror, a lens at the
entrance of the flow cell, and a taper to the internal bore of the flow cell
prevents light rays from striking the internal walls of the flow cell. The
Waters TaperSlit flow cell, so-called because of the shape of the flow cell exit
face, matches the shape of the spectrograph slit. Compared with a
conventional flow cell with a cylindrical shape, the detector achieves higher
light throughput for a given spectral resolution with the TaperSlit cell design.
Comparison of flow cell characteristics:
Resolving spectral data
Together with photodiode spacing, the detector’s 50-μm-wide slit 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.
Window
Window
UV light
Conventional flow cell:
TaperSlit analytical flow cell:
Window
Lens
UV light

Overview 7
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:
Measuring light at the photodiode array
The photodiode array detector measures the amount of light striking the
photodiode array to determine the absorbance of the sample in the flow cell.
The array consists of 512 photodiodes arranged in a row. Each photodiode acts
as a capacitor by holding a fixed amount of charge.
Light striking a photodiode discharges the diode. The magnitude of the
discharge depends on the amount of light striking the photodiode.
Absorbance
nm

8
Photodiodes discharged by light:
The detector measures the amount of current required to recharge each
photodiode. The current is proportional to the amount of light transmitted
through the flow cell over the interval specified by the diode exposure time.
Exposure time
The detector recharges each diode and reads the recharging current, one diode
at a time. The interval between two readings of an individual diode is the
exposure time. The detector requires less than 5 msec to sequentially read all
of the diodes in the array and process the data. The minimum exposure time is
5 msec. You can set exposure time from 5 to 500 msec. For example, if an
exposure time is set to 50 milliseconds, the detector performs as follows:
1. Recharges diode 1, and reads the current required to recharge diode 1.
2. Recharges diode 2, and reads the current required to recharge diode 2.
3. Sequentially recharges and reads the current required to recharge all
the remaining 510 photodiodes.
4. Waits approximately 45 msec before beginning the
recharge-and-reading sequence, with diode 1, after all diodes are
recharged and read.
Flow cell
Deuterium lamp
Light from grating
dispersed onto
diodes.
Sample in flow cell
absorbs at specific
wavelengths.
Grating
Mirror
Slit

Overview 9
You specify the exposure time in the General tab of the PDA Instrument
Method Editor: Auto Exposure or Exposure Time. For details, refer to the
Empower or MassLynx online Help.
Tip: For best signal-to-noise performance, adjust the wavelength range to
optimize autoexposure computations. For details, refer to the Empower or
MassLynx online Help.
Using Auto Exposure
Use the Auto Exposure function to calculate the optimum exposure time
needed to recharge the diodes, based on lamp energy, lamp spectrum, mobile
phase absorbance, and the chosen wavelength range using a single deuterium
light source of 190 to 800 nm. To minimize detector noise, Auto Exposure
adjusts the exposure time to approximately 85% of full scale for the diode
generating the highest signal within the selected wavelength range.
With Auto Exposure enabled, the detector performs as follows:
• Produces the highest signals possible consistent with not saturating due
to overexposure
• Calculates exposure time at the start of a sample set based on maximum
light intensity within the selected wavelength range
• Limits the exposure so that no diode within the given wavelength range
discharges more than approximately 85%
• Provides settings for an optimal signal-to-noise ratio and dynamic range
for each run
For certain combinations of sampling rates, wavelength ranges, or filter-time
constants, the Auto Exposure time setting does not always optimize
performance. If this is the case, you can set the exposure time manually, in the
instrument editor.
Using the Exposure Time function
Specify an exposure time when you want to manually set the length of time
the photodiodes are exposed to light before they are read. The supported range
is5to500msec.
Note: Changing exposure times within a set of samples can cause changes in
baseline noise. Increasing the exposure time can saturate the photodiodes and
cause the detector to lose signal at certain wavelengths. To avoid signal loss,
select an exposure time-value that provides settings for an optimum

10
signal-to-noise ratio over the wavelength range of your analysis (see the next
section, “Optimizing the signal-to-noise ratio”).
Optimizing the signal-to-noise ratio
To optimize signal-to-noise ratios, choose an acquisition wavelength range
that includes only the wavelengths of interest. It is also important that the
range be one in which the mobile phase absorbs only minimally. You can also
improve the signal-to-noise ratio by increasing the spectral resolution value.
For example, you can choose to operate at 3.6 nm instead of at 1.2 nm
resolution. The signlal-to-noise ratio is also impacted by the filter-time
constant and the sampling rate.
Filtering data
On the General tab of the PDA Instrument Method Editor you can apply an
optional noise filter (via the Digital Filtering parameter) to the data acquired.
See also: The Empower or MassLynx online Help.
The detector uses a Hamming filter to minimize noise. The filter is a digital
finite-impulse-response filter that creates peak-height degradation and
enhances the filtering of high frequency noise.
The behavior of the filter depends on the filter-time constant you select.
Increasing the filter-time constant reduces baseline noise, improving
signal-to-noise. However, increasing the filter-time constant too much will
artificially broaden the peak and reduce chromatographic resolution.
You can choose among these options when programming a filtering time: Fast,
Slow, Normal, or Other. If you select a fast, slow, or normal filtering time, you
need not specify a value, because the he filtering constant is determined by
the data rate. If you select the Other option, you can specify a value.
Nevertheless, the value you enter is rounded up or down to a value based on
the data rate. Selecting Other and entering a value of 0.0 disables all filtering.
The following table lists the digital filter settings for the allowable data rates.
Digital filter settings for data rates:
Data
Rate Slow Normal Fast
1 4.000 2.000 1.000
2 2.000 1.000 0.500

Overview 11
Lower filter-time constant settings produce these effects:
• Narrow peaks, with minimal peak distortion and time delay
• Very small peaks may become more difficult to discriminate from
baseline noise
• Less baseline noise is removed
Higher filter-time constant settings produce these effects:
• Greatly decrease baseline noise
• Shorten and broaden peaks
For the highest resolution, select an appropriate sampling rate for your
separation and choose a fast filter-time constant. For the highest sensitivity,
select an appropriate sampling rate for your separation and choose a normal
filter-time constant.
The following figure shows the relationship between increased filter-time
constant and absorbance.
5 0.800 0.400 0.200
10 0.400 0.200 0.100
20 0.200 0.100 0.050
40 0.100 0.050 0.025
80 0.050 0.025 0.0125
Digital filter settings for data rates: (Continued)
Data
Rate Slow Normal Fast

12
Filter-time constant comparison:
Tip: Although the peak shape shows some distortion and the signal output is
delayed with different filter-time constants, the peak area remains the same.
Selecting the appropriate sampling rate
A sufficient number of points must fall across a peak to define its shape. Thus,
at very low sampling rates, the definition between peaks is lost. Empower
software 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.
The Points Across Peak value
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.
0 sec
1 sec
2 sec
Time (minutes)
Absorbance

Overview 13
If the Points Across Peak value for the narrowest peak of interest is less than
15, to improve peak integration reproducibility, specify a higher sampling rate
in the instrument method. If the value is greater than 100, specify a lower
sampling rate.
Set the sampling rate to the lowest value required to achieve 25 to 50 points
across the narrowest peak. Excessively high sampling rates increase baseline
noise and lead to very large data files.
Median baseline filter
The median baseline filter enhances the detector's baseline stability by
decreasing the baseline's curvature, facilitating the development of
integration methods. The filter's primary purpose is to reduce the effects of
mobile phase gradient separations that demonstrate gradual compositional
changes. Note that it should not be applied in cases where abrupt gradient
changes, such as steps, are evident.
Generally, the filter does not significantly change peak area, peak height,
width or retention times. Nevertheless, it can create baseline distortions
around very wide peaks, and these distortions can affect peak area. Therefore,
the filter is not recommended for situations where peak widths (measured at
5% height) are greater than 5% of run time.
In the ACQUITY UPC2PDA detector, the filter works with 2D channels only.
It cannot be applied to 3D or extracted 2D channels. When the MBF data
mode is selected for a channel, the presentation of the data in the real-time
data display plot is delayed by a percentage (~25%) of the run time. A
countdown clock in the instrument control panel indicates the length of the
delay.

14
Computing absorbance
The detector calculates absorbance values before transmitting the data to the
Empower or MassLynx database. It does so as follows:
• Computes the absorbance at each diode using the dark current and
reference spectrum (see “Calculating absorbance” on page 4).
• Averages the absorbances at a particular wavelength, as specified in the
spectra-per-second sample rate, and reports the average as a single data
point (see “Resolution” on page 15).
• Also, the detector can apply a filter when calculating absorbance (see
“Filtering data” on page 10).
Dark current
Photodiodes produce thermally excited charge even when not exposed to
light. The amount of thermally excited charge produced is called dark
current.
When a dark current update is necessary, the detector closes the shutter
to take a dark-current reading for each diode. The shutter closes after
the exposure time is calculated and stays closed for the same interval as
the exposure time.
The detector subtracts the dark-current values from the current values
recorded during absorbance measurements for both the sample and the
reference spectra.
Reference spectrum
Immediately after the dark-current measurement, and before any
components elute, the detector records a reference spectrum. The
reference spectrum is a measure of lamp intensity and mobile phase
absorbance that ideally represents the initial mobile phase. With the
shutter open, the reference spectrum is determined over the interval
specified in the exposure time.
For extremely long exposure times, the dark current and reference
spectrum readings can take several seconds to finish.

Overview 15
Absorbance
The detector calculates the absorbance for each diode at the end of each
exposure time using the following equation:
where
S = obtained during sample analysis
D= obtained during the dark current test
R = obtained from the reference spectrum
n= diode number
Resolution
The data the detector report to the Empower or MassLynx database can be the
average of a number of data points. After calculating absorbance, the detector
averages absorbance values based on spectral resolution and sample rate.
Averaging spectral data based on resolution
Spectral resolution, or bandwidth, is the wavelength interval. in
nanometers, between data points in an acquired spectrum. The
detector’s lowest resolution setting is 1.2 nm. For example, in 3D-mode,
the detector averages three adjacent diodes for each reported
wavelength when you specify the spectral resolution as 3.6 nm. In 2D
mode, absorbance values are computed based on the bandwidth setting.
Averaging chromatographic data based on sample rate
Sample rate is the number of data points acquired per second. The
number of times the photodiodes are read during the sample rate
interval depends on the exposure time. For example, if exposure time is
25 msec, and sample rate is 20 Hz, then readings per data point are
calculated as follows:
The software then averages the readings and reports the average as a
single data point.
Absorbancen
Sn Dn–
Rn Dn–
-------------------------
log=
1 sec
20 samples
-------------------------- 1 exposure
25 msec
--------------------------
1000 msec
1 sec
--------------------------
2exposures
sample
------------------------
=

16
Detector capabilities
The detectors, whose capabilities are described in the table below, operate at
wavelengths ranging from 190 to 800 nm and can sample up to 80 data points
per second.
Detector capabilities:
Capability Description
Full, three-dimensional spectrum
data
Enables collecting the full spectral range
throughout the chromatogram.
Individual 2D channels Monitor absorbance of one through eight
discrete wavelengths.
Wavelength verification reference
filter
Ensures wavelength accuracy.
Fixed, second-order filter Filters UV wavelengths above 370 nm.
Full diagnostic capability Supports built-in diagnostic tools, to
optimize functionality and performance.
One contact closure output The detector has one configurable
switch, which can accommodate a
maximum of +30 VDC, 1.2-A current
carrying capacity, and 0.5-A current
switching. The switch can trigger
fraction collectors and other external
devices and it can activate according to
time, absorbance threshold, or ratio
criteria.
Wavelength compensation Defines a region of the spectrum for use
as a reference, suppressing baseline
wander caused by refractive index or
other dynamics.