Agilent Technologies 8453 User manual

s1
Agilent 8453 UV-visible
Spectroscopy System
Operator’s Manual

Agilent 8453 UV-visible Spectroscopy System Operator’s Manual
Notices
© Agilent Technologies, Inc. 2002,
2003-2007
No part of this manual may be reproduced in
any form or by any means (including elec-
tronic storage and retrieval or translation
into a foreign language) without prior agree-
ment and written consent from Agilent
Technologies, Inc. as governed by United
States and international copyright laws.
Manual Part Number
G1115-90030
Edition
04/07
Printed in Germany
Agilent Technologies
Hewlett-Packard-Strasse 8
76377 Waldbronn
Warranty
The material contained in this docu-
ment is provided “as is,” and is sub-
ject to being changed, without notice,
in future editions. Further, to the max-
imum extent permitted by applicable
law, Agilent disclaims all warranties,
either express or implied, with regard
to this manual and any information
contained herein, including but not
limited to the implied warranties of
merchantability and fitness for a par-
ticular purpose. Agilent shall not be
liable for errors or for incidental or
consequential damages in connec-
tion with the furnishing, use, or per-
formance of this document or of any
information contained herein. Should
Agilent and the user have a separate
written agreement with warranty
terms covering the material in this
document that conflict with these
terms, the warranty terms in the sep-
arate agreement shall control.
Technology Licenses
The hardware and/or software described in
this document are furnished under a license
and may be used or copied only in accor-
dance with the terms of such license.
Restricted Rights Legend
If software is for use in the performance of a
U.S. Government prime contract or subcon-
tract, Software is delivered and licensed as
“Commercial computer software” as
defined in DFAR 252.227-7014 (June 1995),
or as a “commercial item” as defined in FAR
2.101(a) or as “Restricted computer soft-
ware” as defined in FAR 52.227-19 (June
1987) or any equivalent agency regulation or
contract clause. Use, duplication or disclo-
sure of Software is subject to Agilent Tech-
nologies’ standard commercial license
terms, and non-DOD Departments and
Agencies of the U.S. Government will
receive no greater than Restricted Rights as
defined in FAR 52.227-19(c)(1-2) (June
1987). U.S. Government users will receive
no greater than Limited Rights as defined in
FAR 52.227-14 (June 1987) or DFAR
252.227-7015 (b)(2) (November 1995), as
applicable in any technical data.
Safety Notices
CAUTION
A CAUTION notice denotes a haz-
ard. It calls attention to an operat-
ing procedure, practice, or the like
that, if not correctly performed or
adhered to, could result in damage
to the product or loss of important
data. Do not proceed beyond a
CAUTION notice until the indicated
conditions are fully understood and
met.
WARNING
A WARNING notice denotes a
hazard. It calls attention to an
operating procedure, practice, or
the like that, if not correctly per-
formed or adhered to, could result
in personal injury or death. Do not
proceed beyond a WARNING
notice until the indicated condi-
tions are fully understood and
met.
Microsoft®is a U.S. registered trademark of
Microsoft Corporation.
Software Revision
This handbook is for B.01.xx revisions of the
Agilent ChemStation software, where xx is
a number from 00 through 99 and refers to
minor revisions of the software that do not
affect the technical accuracy of this hand-
book.

Agilent 8453 UV-visible Spectroscopy System Operator’s Manual 3
In This Guide…
To be able to use your new Agilent 8453 UV-visible spectroscopy system
quickly, this book gives you step-wise procedures and examples for basic
operations and tasks.
This book shall not replace the detailed manuals available for installation:
Installing Your UV-visible Spectroscopy System and operation of your
software Understanding Your UV-visible Spectroscopy System nor your
Agilent 8453 Service Manual.
1 Introduction to Your System
In this chapter you will find an introduction to your Agilent 8453
spectrophotmeter and the concept of your Agilent ChemStation software.
2 Installation and Start Up
In this chapter you will find a summary of system installation and start-up of
a measurement session.
3 Good Measurement Practices
Good measurement practices are discussed in this chapter.
4 Using your Agilent 8453 UV-visible Spectroscopy System
Stepwise examples for basic measurements and related tasks are given in this
chapter.

4 Agilent 8453 UV-visible Spectroscopy System Operator’s Manual

Agilent 8453 UV-visible Spectroscopy System Operator’s Manual 5
Contents
1 Introduction to Your System 9
Agilent 8453 Spectrophotometer — Overview 10
Optical System Overview 10
Spectrophotometer Description 14
General Purpose Agilent ChemStation Software for UV-visible Spectroscopy —
Overview 18
User Interface Elements 19
Software Structure 23
Standard Mode Tasks 25
Standard Mode Data Processing 28
2 Installation and Start Up 35
Installation Summary for Your Agilent 8453 General Purpose UV-visible
System 36
General 36
Spectrophotometer 36
PC 37
Starting a Measurement Session 38

6 Agilent 8453 UV-visible Spectroscopy System Operator’s Manual
Contents
3 Good Measurement Practices 39
General Considerations 40
Spectrophotometer Design 40
Making Measurements 40
Sample Cell Material 41
Optical Specifications of Cells 42
Apertured Cells 43
Flow Cells 44
Handling and Maintaining Cells 45
Solvents 47
Sample Preparation 48
Photosensitive Samples 49
Stirring and Temperature Control 50
Checklist for Best Results 50
Inserting a Cell 53
4 Using your Agilent 8453 UV-visible Spectroscopy System 55
Starting Your First Measurement Session 56
Starting Your UV-visible Software 58
Measuring Caffeine Absorbance at 273 nm 59
Saving Your Parameters as a Method 62
Retrieving and Printing a Method 64
Saving and Retrieving Data 67
Saving your Samples 67
Saving a Selected Spectrum 69
Retrieving Spectra 71
Deleting Current Spectra 72
Print Preview of Reports 73
Finding the Caffeine Absorbance Maximum 76

Contents
Agilent 8453 UV-visible Spectroscopy System Operator’s Manual 7
Entering your Cell’s Path Length 80
Controlling your Sipper System 81
Using your Multicell Transport 83
Quantitative Analysis using a Calibration with Standards 86
Setup 87
Calibration 89
Analysis 91
How Can I Be Sure That My Agilent 8453 Works Properly? 93
Agilent 8453 Self test 93
How Can I Get a Deeper Understanding of UV-visible Spectroscopy? 96
When Do I Have to Measure a Blank? 98
Index 99

8 Agilent 8453 UV-visible Spectroscopy System Operator’s Manual
Contents

9
Agilent 8453 UV-visible Spectroscopy System
Operator’s Manual
Agilent Technologies
1
Introduction to Your System
Agilent 8453 Spectrophotometer — Overview 10
General Purpose Agilent ChemStation Software for UV-visible
Spectroscopy — Overview 18
Operation of the system is much easier if you understand the implementation
models. The mind-models of data acquisition, data evaluation and data
handling will help you to run the system successfully.
Your spectroscopy system is based on an Agilent 8453 spectrophotometer and
general purpose Agilent ChemStation software for UV-visible spectroscopy
running on a PC with the supported Microsoft operating system(s). These two
components are linked together by a network connection. This type of link is
very flexible: it can be used for a direct connection between the
spectrophotometer and the PC as well as the integration in a enterprise
network with network access from the PC to the spectrophotometer.
The tasks are split between these devices such that the spectrophotometer
acquires and provides absorbance data which are handled by the PC’s
application software. All of the data display, evaluation and longterm storage
is done under software control on the PC.

10 Agilent 8453 UV-visible Spectroscopy System Operator’s Manual
1 Introduction to Your System
Agilent 8453 Spectrophotometer — Overview
Agilent 8453 Spectrophotometer — Overview
This section gives an overview of the optical system and explains the
spectrophotometer front and back panels. It also explains the layout and
construction of the spectrophotometer including the electronic and
mechanical assemblies inside the spectrophotometer.
Optical System Overview
Optical System
The optical system of the spectrophotometer is shown in Figure 1. Its
radiation source is a combination of a deuterium-discharge lamp for the
ultraviolet (UV) wavelength range and a tungsten lamp for the visible and
short wave near-infrared (SWNIR) wavelength range. The image of the
filament of the tungsten lamp is focused on the discharge aperture of the
deuterium lamp by means of a special rear-access lamp design which allows
both light sources to be optically combined and share a common axis to the
source lens. The source lens forms a single, collimated beam of light. The beam
passes through the shutter/stray-light correction filter area then through the
sample to the spectrograph lens and slit. In the spectrograph light is dispersed
onto the diode array by a holographic grating. This allows simultaneous access
to all wavelength information. The result is a fundamental increase in the rate
at which spectra can be acquired.

Agilent 8453 UV-visible Spectroscopy System Operator’s Manual 11
Introduction to Your System 1
Agilent 8453 Spectrophotometer — Overview
•Lamps
The light source for the UV wavelength range is a deuterium lamp with a
shine-through aperture. As a result of plasma discharge in a low pressure
deuterium gas, the lamp emits light over the 190 nm to approximately
800 nm wavelength range. The light source for the visible and SWNIR
wavelength range is a low-noise tungsten lamp. This lamp emits light over
the 370 nm to 1100 nm wavelength range.
•Source Lens
The source lens receives the light from both lamps and collimates it. The
collimated beam passes through the sample (if one is present) in the sample
compartment.
•Shutter
The shutter is electromechanically actuated. It opens and allows light to
pass through the sample for measurements. Between sample measurements
it closes to limit exposure of sample to light. If the measurement rate is very
fast, you can command the shutter to remain open (Agilent ChemStation
software) or it stays open automatically (handheld controller software).
Figure 1 Optical System of Spectrophotometer
Tungsten
lamp
Source lens
Deuterium
lamp
Shutter/stray
light filter
Cuvette
Photo diode
array
Source lens
Slit
Grating

12 Agilent 8453 UV-visible Spectroscopy System Operator’s Manual
1 Introduction to Your System
Agilent 8453 Spectrophotometer — Overview
•Stray-Light Correction Filter
In a standard measurement sequence, reference or sample intensity spectra
are measured without and then with the stray-light filter in the light beam.
Without the filter the intensity spectrum over the whole wavelength range
from 190–1100 nm is measured. The stray-light filter is a blocking filter
with 50 % blocking at 420 nm.
With this filter in place any light measured below 400 nm is stray light only.
This stray-light intensity is then subtracted from the first spectrum to give a
stray-light corrected spectrum. Depending on the software, you can switch
off the stray light correction (Agilent ChemStation software) in case you
want to do very fast repetitive scans or it is switched off automatically.
•Sample Compartment
The spectrophotometer has an open sample compartment for easier access
to sample cells. Because of the optical design a cover for the sample area is
not required. The spectrophotometer is supplied with a single-cell holder
already installed in the sample compartment. This can be replaced with the
Peltier temperature control accessory, the thermostattable cell holder, the
long path cell holder or the multicell transport. All of these optional cell
holders mount in the sample compartment using the same quick, simple
mounting system. An optical filter wheel is also available for use with the
spectrophotometer and most of the accessories.
•Spectrograph
The spectrograph housing material is ceramic to reduce thermal effects to a
minimum. It main components of the spectrograph are the lens, the slit, the
grating and the photo diode array with front-end electronics. The mean
sampling interval of the diode array is about 0.9 nm over the wavelength
range 190 nm to 1100 nm. The nominal spectral slit width is 1 nm.
•Spectrograph Lens
The spectrograph lens is the first of the parts which are collectively known
as the spectrograph. It is mounted on the housing of the spectrograph. The
spectrograph lens refocuses the collimated light beam after it has passed
through the sample.

Agilent 8453 UV-visible Spectroscopy System Operator’s Manual 13
Introduction to Your System 1
Agilent 8453 Spectrophotometer — Overview
•Slit
The slit is a narrow aperture in a plate located at the focus of the
spectrograph lens. It is exactly the size of one of the photo diodes in the
photo diode array. By limiting the size of the incoming light beam, the slit
makes sure that each band of wavelengths is projected onto only the
appropriate photodiode.
•Grating
The combination of dispersion and spectral imaging is accomplished by
using a concave holographic grating. The grating disperses the light onto
the diode array at an angle linear proportional to the wavelength.
•Diode Array
The photodiode array is the heart of the spectrograph. It is a series of 1024
individual photodiodes and control circuits etched onto a semiconductor
chip. With a wavelength range from 190 nm to 1100 nm the sampling
interval is about 0.9 nm.

14 Agilent 8453 UV-visible Spectroscopy System Operator’s Manual
1 Introduction to Your System
Agilent 8453 Spectrophotometer — Overview
Spectrophotometer Description
Your spectrophotometer is very easy to use. It has a line power indicator, a
status indicator and some push buttons. All electrical connections are made at
the rear of the spectrophotometer.
Front View
The front view of the spectrophotometer is shown in Figure 2. Notice that the
sample compartment is open. Unlike conventional spectrophotometers the
Agilent 8453 does not suffer from ambient false light. The open sample area
makes it easier to access for cuvette handling and to connect tubing to a flow
cell or thermostattable cell holder. The spectrophotometer is shipped with the
standard single-cell holder. Standard and accessory cell holders can be
removed and replaced in seconds with few or no tools.
The line power switch is located at the lower-left part of the
spectrophotometer. Pressing it in turns on the spectrophotometer. It stays
pressed in and shows a green light when the spectrophotometer is turned on.
When the line power switch stands out and the green light is off, the
spectrophotometer is turned off.
Figure 2 Front View of Spectrophotometer
Indicator
Pushbuttons
Line power switch
with green light

Agilent 8453 UV-visible Spectroscopy System Operator’s Manual 15
Introduction to Your System 1
Agilent 8453 Spectrophotometer — Overview
On the front panel of the spectrophotometer is a status indicator which will
display different colors depending of the actual condition of the
spectrophotometer.
•Green—the spectrophotometer is ready to measure.
•Green, blinking—the spectrophotometer is measuring.
•Yellow—the spectrophotometer is in not-ready state, for example, turning
one of the lamps on or if both lamps are switched off.
•Red—error condition, that is, the spectrophotometer does not pass one of
the self-tests which are run when the spectrophotometer is turned on or an
error occurred during operation. In this case the UV-visible operating
software gives a detailed error message and possible explanations are in
the online help system and in your Service Manual Chapter 3 “Diagnostics
and Troubleshooting”.
•Red, blinking—error condition of the spectrophotometer processor system.
Because in this case there is no communication with the computer there
will be no error message. The online help system and your Service Manual
Chapter 3 “Diagnostics and Troubleshooting” give more information about
troubleshooting.
The four measure push buttons on the front panel cause the following actions
to be performed and the resulting data being sent to the computer. The push
button functionality is controller by the UV-visible ChemStation software and
adjusted to the current measurement task.
•BLANK—the spectrophotometer takes a blank measurement. This comprises
a reference measurement that is used in all subsequent sample
measurements until a new blank measurement is taken. Following the
reference measurement an additional baseline spectrum is measured and
displayed on the PC.
•SAMPLE—the spectrophotometer takes a sample measurement or starts a
series of measurements. This depends on the parameters set in your
software.
•STANDARD—the spectrophotometer takes a measurement of a standard.
Additional information such as concentration and so on, have to be entered
in the operating software.
•STOP—the spectrophotometer and/or software aborts any ongoing activity
and returns to a to ready state.

16 Agilent 8453 UV-visible Spectroscopy System Operator’s Manual
1 Introduction to Your System
Agilent 8453 Spectrophotometer — Overview
Rear View
All connections are made at the rear of the spectrophotometer, see Figure 3.
•The multicell connector allows you to connect a multicell transport.
•The GPIO (general-purpose input/output) connector allow you to control a
sipper and autosampler or other accessories depending on the software you
are using.
•The remote connector is currently not used by Agilent instrument control
software. It can be used e.g. to synchronize instruments.
•The RS232C connector may be used to control the spectrophotometer from
a computer through RS232 connection, using appropriate software. The
Multiple Instrument Firmware Tool provided can be used with RS232
communication. The UV-visible ChemStation software currently supports
LAN and GPIB communication only.
Figure 3 Rear View of Spectrophotometer
Line connector
GPIB connector
and switches
Multicell transport
connector
GP-IO connector
RS 232 connector
Remote connector
Two CAN
connectors
MIO slot for
LAN interface

Agilent 8453 UV-visible Spectroscopy System Operator’s Manual 17
Introduction to Your System 1
Agilent 8453 Spectrophotometer — Overview
•The GPIB connector is used to connect the spectrophotometer with a
computer. The 8-bit configuration switch module next to the GPIB
connector determines the GPIB address of your spectrophotometer. The
switches are preset to a default address recognized by the operating
software from Agilent.
Only a single controller must be connected to the spectrophotometer at a
time.
•The MIO board slot is reserved for a LAN interface board.
•The accessory board slot is reserved for future use.
•The power input socket does not have a voltage selector because the power
supply has wide-ranging capability, for more information see your Service
Manual Chapter 1 “Specifications”. There are no externally accessible
fuses, because automatic electronic fuses are implemented in the power
supply. The security lever at the power input socket prevents that the
spectrophotometer cover is taken off when line power is still connected.
Side of the Spectrophotometer
On the right side of the spectrophotometer is a double door for access to the
lamps. For exchanging lamps both doors, the plastic door and the sheet-metal
door must be opened. Safety light switches are automatically turning off the
lamps when the inner sheet metal door is opened.

18 Agilent 8453 UV-visible Spectroscopy System Operator’s Manual
1 Introduction to Your System
General Purpose Agilent ChemStation Software for UV-visible Spectroscopy — Overview
General Purpose Agilent ChemStation Software for UV-visible
Spectroscopy — Overview
This section gives an overview of the elements of the user interface
implemented with your Agilent ChemStation software and the data analysis
concept behind. It explains how data are processed and what the advantages
of this processing are on a practical point of view.

Agilent 8453 UV-visible Spectroscopy System Operator’s Manual 19
Introduction to Your System 1
General Purpose Agilent ChemStation Software for UV-visible Spectroscopy — Overview
User Interface Elements
Your general purpose Agilent ChemStation software for UV-visible
spectroscopy facilitates operation of your diode-array-based UV-visible
spectrophotometer in daily routine operation. The focus of this software is on
ease-of-use and ease-of-learning. A graphical user interface visualizes the
spectrophotometer operation and usage. This user interface consists of a
number of elements as described in the following sections.
Menu bar
Tool bar
Analysis panel
Instrument panel
Method name
Mode switch
Tabular results
Spectra

20 Agilent 8453 UV-visible Spectroscopy System Operator’s Manual
1 Introduction to Your System
General Purpose Agilent ChemStation Software for UV-visible Spectroscopy — Overview
Menu
The more traditional menu interface at the top of the Agilent ChemStation
window allows to control all operations.When you choose an item from the
menu bar a list of commands and submenus is displayed. An operation is
performed by choosing a command (mouse click or ENTER key).
Toolbar
The tool bar below the menu bar shows buttons with symbols, icons, which
allow direct access to basic operations such as printing result reports, loading
methods, and saving methods and data.
Side Panels
The panels on the left side are the analysis panel and instrument panel. The
size and position of these panels are fixed but are a function of your display’s
resolution. The minimum resolution is 600 × 800 pixels.
Analysis Panel
The analysis panel gives you a graphical visualization of the current context in
which you are working. In addition it provides access to the setup dialog of
your actual task by means of the Setup button.