Agilent Technologies 1260 Infinity User manual

Agilent Technologies
Agilent 1260 Infinity
Fluorescence Detector
User Manual

Agilent 1260 FLD User Manual
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Agilent 1260 FLD User Manual 3
In This Guide
In This Guide
This manual covers the Agilent 1260 Infinity Fluorescence Detector (G1321B).
1 Introduction to the Fluorescence Detector
This chapter gives an introduction to the detector, instrument overview and
internal connectors.
2 Site Requirements and Specifications
This chapter provides information on environmental requirements, physical
and performance specifications.
3 Installing the Module
This chapter gives information about the preferred stack setup for your system
and the installation of the module.
4 Using the Fluorescence Detector
This chapter guides you how to start the work with the detector.
5 Optimizing the Detector
This chapter provides information on how to optimize the detector.
6 Troubleshooting and Diagnostics
This chapter gives an overview about the troubleshooting and diagnostic
features and the different user interfaces.
7 Error Information
This chapter describes the meaning of error messages, and provides
information on probable causes and suggested actions how to recover from
error conditions.

4 Agilent 1260 FLD User Manual
In This Guide
8TestFunctions
This chapter describes the detector’s built in test functions.
9 Maintenance
This chapter provides general information on maintenance of the detector.
10 Parts for Maintenance
This chapter provides information on parts for maintenance.
11 Identifying Cables
This chapter provides information on cables used with the 1290 series of
HPLC modules.
12 Appendix
This chapter provides safetey and other general information.

Agilent 1260 FLD User Manual 5
Contents
Contents
1 Introduction to the Fluorescence Detector 9
Introduction to the Detector 10
How the Detector Operates 11
Raman Effect 14
Optical Unit 15
Analytical Information From Primary Data 23
Early Maintenance Feedback 28
Instrument Layout 29
Electrical Connections 30
Interfaces 32
Setting the 8-bit Configuration Switch 38
2 Site Requirements and Specifications 45
Site Requirements 46
Physical Specifications 49
Performance Specifications 50
3 Installing the Module 53
Unpacking the Module 54
Optimizing the Stack Configuration 56
Installing the Module 61
Flow Connections to the Module 64
4 Using the Fluorescence Detector 69
Before You Start 70
Getting Started and Checkout 71
Method Development 75
Example: Optimization for Multiple Compounds 93

6 Agilent 1260 FLD User Manual
Contents
5 Optimizing the Detector 103
Optimization Overview 104
Design Features Help Optimization 106
Finding the Best Wavelengths 107
Finding the Best Signal Amplification 109
Changing the Xenon Flash Lamp Frequency 115
Selecting the Best Response Time 117
Reducing Stray Light 120
6 Troubleshooting and Diagnostics 123
Overview of the Module’s Indicators and Test Functions 124
Status Indicators 125
User Interfaces 127
Agilent Lab Advisor Software 128
7 Error Information 129
What Are Error Messages 130
General Error Messages 131
Detector Error Messages 138
8 Test Functions 147
Diagram of Light Path 148
Lamp Intensity Test 149
Dark-Current Test 151
Excitation and Emission Grating Resistance History 153
Raman ASTM Signal-to-Noise Test 155
Using the Built-in Test Chromatogram 162
Wavelength Verification and Calibration 164
Wavelength Accuracy Test 167
Wavelength Calibration Procedure 173
D/A Converter (DAC) Test 178
Diagnostic Signals 180

Agilent 1260 FLD User Manual 7
Contents
9 Maintenance 181
Introduction to Maintenance 182
Warnings and Cautions 183
Overview of Maintenance 185
Cleaning the Module 186
Exchanging a Flow Cell 187
How to use the Cuvette 190
Flow Cell Flushing 191
Correcting Leaks 192
Replacing Leak Handling System Parts 193
Replacing the Interface Board 194
Replacing Module Firmware 195
Tests and Calibrations 196
10 Parts for Maintenance 197
Overview of Maintenance Parts 198
Cuvette Kit 199
Accessory Kit 200
11 Identifying Cables 203
Cable Overview 204
Analog Cables 206
Remote Cables 208
BCD Cables 211
CAN/LAN Cables 213
External Contact Cable 214
Agilent Module to PC 215
12 Appendix 217
General Safety Information 218
The Waste Electrical and Electronic Equipment (WEEE) Directive
(2002/96/EC) 221
Lithium Batteries Information 222
Radio Interference 223
Sound Emission 224
UV-Radiation (UV-lamps only) 225
Solvent Information 226
Agilent Technologies on Internet 228

8 Agilent 1260 FLD User Manual
Contents

9
Agilent 1260 FLD User Manual
Agilent Technologies
1
Introduction to the Fluorescence
Detector
Introduction to the Detector 10
How the Detector Operates 11
Raman Effect 14
Optical Unit 15
Reference System 22
Analytical Information From Primary Data 23
Fluorescence Detection 23
Phosphorescence Detection 24
Processing of Raw Data 24
Early Maintenance Feedback 28
Instrument Layout 29
Electrical Connections 30
Serial Number Information 31
Electrical Connections 30
Interfaces 32
Overview Interfaces 34
Setting the 8-bit Configuration Switch 38
Communication Settings for RS-232C 41
Special Settings 43
This chapter gives an introduction to the detector, instrument overview and
internal connectors.

10 Agilent 1260 FLD User Manual
1Introduction to the Fluorescence Detector
Introduction to the Detector
Introduction to the Detector
The detector is designed for highest optical performance, GLP compliance and
easy maintenance. It includes the following features:
•flash lamp for highest intensity and lowest detection limit
•multi-wavelength mode for on-line spectra
•spectra acquisition and simultaneous multi-signal detection
•optional cuvette is available and can be used for off-line measurements
•easy front access to flow cell for fast replacement and
•built-in wavelength accuracy verification.
For specifications, see “Performance Specifications” on page 50
Figure 1 The Agilent 1260 Infinity Fluorescence Detector

Agilent 1260 FLD User Manual 11
Introduction to the Fluorescence Detector 1
How the Detector Operates
How the Detector Operates
Luminescence Detection
Luminescence, the emission of light, occurs when molecules change from an
excited state to their ground state. Molecules can be excited by different forms
of energy, each with its own excitation process. For example, when the
excitation energy is light, the process is called photoluminescence.
In basic cases, the emission of light is the reverse of absorption, see Figure 2
on page 11. With sodium vapor, for example, the absorption and emission
spectra are a single line at the same wavelength. The absorption and emission
spectra of organic molecules in solution produce bands instead of lines.
Figure 2 Absorption of Light Versus Emission of Light
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12 Agilent 1260 FLD User Manual
1Introduction to the Fluorescence Detector
How the Detector Operates
When a more complex molecule transforms from its ground energy state into
an excited state, the absorbed energy is distributed into various vibrational
and rotational sub-levels. When this, same molecule returns to the ground
state, this vibrational and rotational energy is first lost by relaxation without
any radiation. Then the molecule transforms from this energy level to one of
the vibrational and rotational sub-levels of its ground state, emitting light, see
Figure 3 on page 12. The characteristic maxima of absorption for a substance
is its λEX, and for emission its λEM.
Figure 3 Relationship of Excitation and Emission Wavelengths
Photoluminescence is the collective name for two phenomena, fluorescence
and phosphorescence, which differ from each other in one characteristic
way--the delay of emission after excitation. If a molecule emits light 10-9 to
10-5 seconds after it was illuminated then the process was fluorescence. If a
molecule emits light longer than 10-3 seconds after illumination then the
process was phosphorescence.
Phosphorescence is a longer process because one of the electrons involved in
the excitation changes its spin, during a collision with a molecule of solvent,
for example. The excited molecule is now in a so-called triplet state, T, see
Figure 4 on page 13.
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Agilent 1260 FLD User Manual 13
Introduction to the Fluorescence Detector 1
How the Detector Operates
Figure 4 Phosphorescence Energy Transitions
The molecule must change its spin back again before it can return to its
ground state. Since the chance of colliding with another molecule with the
necessary spin for change is slight, the molecule remains in its triplet state for
some time. During the second spin change the molecule loses more energy by
relaxing without radiation. The light which is emitted during phosphorescence
therefore has less energy and is at a longer wavelength than fluorescence.
Formula: E = h x l-1
In this equation:
E is energy
h is Planck's constant
lis the wavelength
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14 Agilent 1260 FLD User Manual
1Introduction to the Fluorescence Detector
Raman Effect
Raman Effect
The Raman effect arises when the incident light excites molecules in the
sample which subsequently scatter the light. While most of this scattered light
is at the same wavelength as the incident light, some is scattered at a different
wavelength. This inelastically scattered light is called Raman scatter. It results
from the molecule changing it's molecular motions.
Figure 5 Raman
The energy difference between the incident light (Ei) and the Raman scattered
light (Es) is equal to the energy involved in changing the molecule's vibrational
state (i.e. getting the molecule to vibrate, Ev). This energy difference is called
the Raman shift.
Ev= Ei- Es
Several different Raman shifted signals will often be observed; each being
associated with different vibrational or rotational motions of molecules in the
sample. The particular molecule and its environment will determine what
Raman signals will be observed (if any).
A plot of Raman intensity versus Raman shift is a Raman spectrum.
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Agilent 1260 FLD User Manual 15
Introduction to the Fluorescence Detector 1
Optical Unit
Optical Unit
All the elements of the optical system, shown in Figure 6 on page 16, including
Xenon flash lamp, excitation condenser, excitation slit, mirror, excitation
grating, flow cell, emission condenser, cut-off filter, emission slit, emission
grating and photo-multiplier tube are housed in the metal casting inside the
detector compartment. The fluorescence detector has grating/grating optics,
enabling the selection of both excitation and emission wavelengths. The flow
cell can be accessed from the front of the fluorescence detector.

16 Agilent 1260 FLD User Manual
1Introduction to the Fluorescence Detector
Optical Unit
Figure 6 Optical Unit
The radiation source is a xenon flash-lamp. The 3 µs flash produces a
continuous spectrum of light from 200 nm to 900 nm. The light output
distribution can be expressed as a percentage in 100 nm intervals, see
Figure 7 on page 17. The lamp can be used for some 1000 hours depending on
the sensitivity requirements. You can economize during automatic operation
using keyboard setpoints, so the lamp flashes during your analysis only. The
lamp can be used until it no longer ignites, but the noise level may increase
with usage.
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Agilent 1260 FLD User Manual 17
Introduction to the Fluorescence Detector 1
Optical Unit
UV degradation, especially below 250 nm is significantly higher compared to
Visible wavelength range. Generally the "LAMP ON during run" - setting or
using "economy mode" will increase lamp life by a magnitude.
Figure 7 Lamp Energy Distribution (vendor data)
The radiation emitted by the lamp is dispersed and reflected by the excitation
monochromator grating onto the cell entrance slit.
The holographic concave grating is the main part of the monochromator,
dispersing and reflecting the incident light. The surface contains many minute
grooves, 1200 of them per millimeter. The grating carries a blaze to show
improved performance in the visible range.
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18 Agilent 1260 FLD User Manual
1Introduction to the Fluorescence Detector
Optical Unit
Figure 8 Mirror Assembly
The geometry of the grooves is optimized to reflect almost all of the incident
light, in the 1st order and disperse it with about 70 % efficiency in the
ultra-violet range. Most of the remaining 30 % of the light is reflected at zero
order, with no dispersion. Figure 9 on page 19 illustrates the light path at the
surface of the grating.
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Agilent 1260 FLD User Manual 19
Introduction to the Fluorescence Detector 1
Optical Unit
Figure 9 Dispersion of Light by a Grating
The grating is turned using a 3-phase brushless DC motor, the position of the
grating determining the wavelength or wavelength range of the light falling
onto the flow cell. The grating can be programmed to change its position and
therefore the wavelength during a run.
For spectra acquisition and multi-wavelength detection, the grating rotates at
4000 rpm.
The excitation and emission gratings are similar in design, but have different
blaze wavelengths. The excitation grating reflects most 1st order light in the
ultra-violet range around 250 nm, whereas the emission grating reflects better
in the visible range around 400 nm.
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20 Agilent 1260 FLD User Manual
1Introduction to the Fluorescence Detector
Optical Unit
The flow cell is a solid quartz body with a maximum back pressure of 20 bar.
Excessive back pressure will result in destruction of the cell. Operating the
detector close to waste with low back pressure is recommended. A slit is
integrated to the quartz body.
Figure 10 Cross-Section of Flow Cell
The luminescence from the sample in the flow cell is collected at right angles
to the incident light by a second lens, and passes through a second slit. Before
the luminescence reaches the emission monochromator, a cut-off filter
removes light below a certain wavelength, to reduce noise from 1st order
scatter and 2nd order stray light, see Figure 9 on page 19.
The selected wavelength of light is reflected onto the slit in the wall of the
photo-multiplier compartment of the optical unit. The bandwidth of the
emitted light is 20 nm.