Agilent Technologies 1200 series User manual

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Agilent 1200 Series
Fluorescence Detector
G1321A
User Manual

2 1200 Series FLD User Manual
Notices
© Agilent Technologies, Inc. 2006
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Manual Part Number
G1321-90010
Edition
02/06
Printed in Germany
Agilent Technologies
Hewlett-Packard-Strasse 8
76337 Waldbronn, Germany
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The User Manual G1321-90010 (English)
and its localized versions contain a subset
of the Service Manual and is shipped with
the detector in printed matter.
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contains the complete information about
the Agilent 1200 Series Fluorescence
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1200 Series FLD User Manual 3
In This Guide…
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 gives information on environmental requirements,
physical and performance specifications.
3 Installing the Detector
This chapter describes the installation of the detector.
4 First Steps with the Detector
This chapter guides you how to start the work with the detector.
5 How to optimize the Detector
This chapter provides information on how to optimize the
detector.
6 Troubleshooting and Test Functions
This chapter gives an overview about the troubleshooting and
diagnostic features and the different user interfaces.
7 Maintenance and Repair
This chapter provides general information on maintenance and
repair of the detector.
8 Maintenance
This chapter describes the maintenance of the detector and the
required tests.
9 Parts and Materials for Maintenance
This chapter provides information on parts for maintenance.

4 1200 Series FLD User Manual
A Appendix
This chapter provides safetey and other general information.

1200 Series FLD User Manual 5
Contents
1 Introduction to the Fluorescence Detector
Introduction to the Detector 10
How the Detector Operates 11
Raman Effect 14
Optical Unit 15
Reference System 21
Analytical Information From Primary Data 22
Fluorescence Detection 22
Phosphorescence Detection 23
Processing of Raw Data 24
Electrical Connections 27
Instrument Layout 29
Early Maintenance Feedback (EMF) 30
EMF Counters 30
Using the EMF Counters 32
2 Site Requirements and Specifications
Site Requirements 34
Physical Specifications 36
Performance Specifications 37
3 Installing the Detector
Unpacking the Detector 42
Detector Accessory Kit Contents 43

6 1200 Series FLD User Manual
Optimizing the Stack Configuration 45
Installing the Detector 48
Flow Connections to the Detector 51
4 First Steps with the Detector
Before You Start 56
Optimization Overview 57
Getting Started and Checkout 59
Starting Your Detector 59
Setting the Chromatographic Conditions 60
Observe the maxima via the isoabsorbance plot 62
Method Development 63
Step 1: Check the LC system for impurities 64
Step 2: Optimize limits of detection and selectivity 65
Step 3: Set up routine methods 74
Example: Optimization for Multiple Compounds 78
5 How to optimize the Detector
Optimization Overview 86
Design Features Help Optimization 87
Check Performance Before You Start 87
Finding the Best Wavelengths 88
A Real Example 89
Finding the Best Signal Amplification 90
Changing the Xenon Flash Lamp Frequency 92
Lamp life savings 93
Selecting the Best Response Time 94
Reducing Stray Light 96

1200 Series FLD User Manual 7
6 Troubleshooting and Test Functions
Overview of the Detector’s Indicators and Test Functions 100
Status Indicators 101
Power Supply Indicator 101
Detector Status Indicator 102
User Interfaces 103
Agilent LC Diagnostic Software 104
7 Maintenance and Repair
Introduction into Repairing the Fluorescence Detector 106
Warnings and Cautions 107
Cleaning the Detector 109
Using the ESD Strap 110
8 Maintenance
Overview of Maintenance 112
Exchanging a Flow Cell 113
How to use the Cuvette 116
Flow Cell Flushing 117
Correcting Leaks 118
Replacing Leak Handling System Parts 119
Replacing the Interface Board 120
Replacing the Detector’s Firmware 121
Tests & Calibrations 122
Lamp Intensity Test 123
Wavelength Verification and Calibration 125
Wavelength Calibration Procedure 127

8 1200 Series FLD User Manual
Preparation of the Glycogen Calibration Sample 127
Preparation of the Flow Cell 128
Wavelength Calibration 129
9 Parts and Materials for Maintenance
Overview of Maintenance Parts 132
Cuvette Kit 133
Spare Parts 134
Accessory Kit 135
A Appendix
General Safety Information 138
Lithium Batteries Information 141
Radio Interference 142
Sound Emission 143
UV-Radiation (UV-lamps only) 144
Solvent Information 145
Agilent Technologies on Internet 147
Index

9
Agilent 1200 Series Fluorescence Detector
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
Analytical Information From Primary Data 22
Electrical Connections 27
Instrument Layout 29
Early Maintenance Feedback (EMF) 30
This chapter gives an introduction to the detector, instrument overview and
internal connectors.

10 1200 Series FLD User Manual
1 Introduction to the Fluorescence 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 37
Figure 1 The Agilent 1200 Series Fluorescence Detector.

1200 Series FLD User Manual 11
Introduction to the Fluorescence Detector 1
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.
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

12 1200 Series FLD User Manual
1 Introduction to the Fluorescence Detector
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. The characteristic maxima of absorption for a substance is its λEX,
and for emission its λEM.
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.
Figure 3 Relationship of Excitation and Emission Wavelengths

1200 Series FLD User Manual 13
Introduction to the Fluorescence Detector 1
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.
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
Figure 4 Phosphorescence Energy Transitions

14 1200 Series FLD User Manual
1 Introduction to the Fluorescence Detector
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.
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.
Figure 5 Raman
Raman Scatter
(new wavelength)
Raleigh Scatter
(same wavelength
as incident light)
Sample
Incident Light
Scattered Light

1200 Series FLD User Manual 15
Introduction to the Fluorescence Detector 1
Optical Unit
All the elements of the optical system, shown in Figure 6 on page 15, 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.
Figure 6 Optical Unit
Xenon Flash lamp
Flash lamp board
EM Grating
assembly
Photo Multiplier
Tube
Slit EM
EX Grating
assembly
Flow Cell
Condenser EM
Mirror
Condenser EX
Slit EX
Trigger Pack
Cutoff filter
REF Diode
Diffuser

16 1200 Series FLD User Manual
1 Introduction to the Fluorescence Detector
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. 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.
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)
300 400 500 600 700 800 900
Wavelenght [nm]
Relative
Intensity

1200 Series FLD User Manual 17
Introduction to the Fluorescence Detector 1
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.
Figure 8 Mirror Assembly
Mirror
Grating (EX) inside

18 1200 Series FLD User Manual
1 Introduction to the Fluorescence Detector
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 illustrates the light path at the surface of
the 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.
Figure 9 Dispersion of Light by a Grating

1200 Series FLD User Manual 19
Introduction to the Fluorescence Detector 1
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.
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 18.
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.
Figure 10 Cross-Section of Flow Cell

20 1200 Series FLD User Manual
1 Introduction to the Fluorescence Detector
On the photocathode, Figure 11, incident photons generate electrons. These
electrons are accelerated by an electrical field between several arc-shaped
dynodes. Depending on the voltage difference between any pair of dynodes, an
incident electron may spark-off further electrons which accelerate onto the
next dynode. An avalanche effect results: finally so many electrons are
generated that a current can be measured. The amplification is a function of
the voltage at the dynodes and is microprocessor controlled. You can set the
amplification using the PMTGAIN function.
This type of so-called side-on photo-multiplier is compact ensuring fast
response, conserving the advantages of the short optical path shown in
Figure 6 on page 15.
PMTs are designed for specific wavelength ranges. The standard PMT offers
optimum sensitivity from 200 to 600 nm. In the higher wavelength range a
red-sensitive PMT can improve performance. For additional PMT types refer
to“Spare Parts” on page 134.
Figure 11 Photo-multiplier Tube
incident light