09936951A Flexar FL LC Detector Users and Service Guide

LIQUID CHROMATOGRAPHY
FLEXAR
FLUORESCENCE LC DETECTOR
User’s and Service Guide

Release History
Part Number Release Publication Date
09936951 A August 2009
Any comments about the documentation for this product should be addressed to:
PerkinElmer
710 Bridgeport Avenue
Shelton, Connecticut 06484-4794
U.S.A.
Or emailed to: info@perkinelmer.com
Notices
The information contained in this document is subject to change without notice.
Except as specifically set forth in its terms and conditions of sale, PerkinElmer makes no
warranty of any kind with regard to this document, including, but not limited to, the
implied warranties of merchantability and fitness for a particular purpose.
PerkinElmer shall not be liable for errors contained herein for incidental consequential damages in
connection with furnishing, performance or use of this material.
Copyright Information
This document contains proprietary information that is protected by copyright.
All rights are reserved. No part of this publication may be reproduced in any form whatsoever or
translated into any language without the prior, written permission of PerkinElmer, Inc.
Copyright © 2009 PerkinElmer, Inc.
Trademarks
Registered names, trademarks, etc. used in this document, even when not specifically marked as such,
are protected by law.
PerkinElmer is a registered trademark of PerkinElmer, Inc.

Table of Contents
Introduction...................................................................................... 7
Product Warranty ................................................................................. 11
Conventions Used in this Manual ........................................................... 13
Notes, Cautions and Warnings ........................................................ 13
Warning Signs on the Instrument .......................................................... 16
Safety Practices .............................................................................. 17
Overview ............................................................................................. 20
Ignition of Flammable Chemicals..................................................... 21
Explosion of Vapor from Flammable Chemicals ................................. 21
Heavy Instrument.......................................................................... 21
Prevention of Instrument Tipover.................................................... 22
Common Safety Precautions.................................................................. 22
Prior to Use................................................................................... 22
In Use .......................................................................................... 22
During Installation, Maintenance, and Relocation.............................. 22
Xenon Lamp Safety .............................................................................. 23
Injury Due to Xenon Lamp Explosion ............................................... 23
Lamp Replacement ........................................................................ 23
Lamp Installation ........................................................................... 24
Lamp Disposal ............................................................................... 24
Touching Hot Part Could Result in Burns.......................................... 25
Direct Viewing of Xenon Lamp Could Cause Eye Damage .................. 25
Before Turning on Xenon Lamp Again.............................................. 25
General Operating Conditions ................................................................ 26
Environmental Conditions...................................................................... 26
Electrical Safety ................................................................................... 27
ElectroMagnetic Compatibility (EMC) ...................................................... 28
Electric Shock Due to Contact with Inside of Instrument ................... 29
Electric Shock Due to Improper Grounding....................................... 29
WEEE Instructions for PerkinElmer Products ........................................... 30
Decontamination and Cleaning .............................................................. 31
Decontamination ........................................................................... 31
Cleaning the Instrument................................................................. 31
Warning Labels on the Instrument......................................................... 32
Quality Control/Good Laboratory Practices.............................................. 34
Certificate of System Control........................................................... 34
Instrument Performance Verification (IPV) ....................................... 34
Routine Inspection and Suitability Test ............................................ 34
Hazardous Chemicals ..................................................................... 34
Definitions in Warning for Hazardous Chemicals ............................... 35
Accuracy and Precision of Measured Values...................................... 35
Possible Carryover ......................................................................... 35
Safe Handling of Solvents ..................................................................... 36
Disposal of Waste Solution ............................................................. 37
Solvents with Low Boiling Points ..................................................... 37
Buffers.......................................................................................... 37
Inflammation or Injury Due to Toxic or Corrosive Solvent.................. 37
Corrosion ...................................................................................... 37
Solvents with Auto-Ignition Temperature Below 110 °C..................... 38
Air Bubbles.................................................................................... 39
Pressure Buildup............................................................................ 40
Fuses .................................................................................................. 40
System Description......................................................................... 41
Detector Status LEDs............................................................................ 43
LED Functionality........................................................................... 43
Front Power Switch Functionality..................................................... 43
Description of Fluorometry .................................................................... 45
Principle of Fluorometry ................................................................. 45
Advantages of Fluorometry............................................................. 46
Notes on Fluorescence Analysis....................................................... 48
Principle of Operation ........................................................................... 52
Features of Light Source................................................................. 53
Specifications....................................................................................... 54
Installation ..................................................................................... 57
Unpacking ........................................................................................... 59
Checking the Contents ................................................................... 59
Installation Conditions .......................................................................... 60
Installation Space .......................................................................... 60
Environmental Conditions for an Installation Location ....................... 60
Installing ............................................................................................. 62
Remove Transport Screws .............................................................. 62
Tubing Connections ....................................................................... 63
Spare Parts Available ..................................................................... 64
Installing the Maintenance Software ...................................................... 64
Setup .................................................................................................. 64
Setting the Xenon Lamp Lifetime Warning Timer .............................. 64
Operation ........................................................................................ 67
Shutdown Procedure ............................................................................ 70
Flushing the Flow Cell After Use ...................................................... 70
Before Performing Analysis ................................................................... 71
Mobile Phase Selection and Cautions on Handling............................. 72
Characteristics of Organic Solvents.................................................. 73
Cautions on Static Electricity ........................................................... 77
Maintenance ................................................................................... 79
Performance Check .............................................................................. 81
Energy Check of Xe Lamp............................................................... 81
Sensitivity (S/N) Check................................................................... 81
Check Wavelength Accuracy (WL Check) by Using Hg Lamp .............. 81
Leak Sensor Check......................................................................... 83
Periodic Checks.................................................................................... 84
Maintenance and Inspection Methods .................................................... 85
Energy Check of Xe Lamp............................................................... 85
Check of Wavelength Accuracy by Using Hg Lamp ............................ 85
Check of Lamp Logbook ................................................................. 85
Checking and Washing the Flow Cell ...................................................... 86
Disassembly and Cleaning of Flow Cell............................................. 89
Leakage from the Flow Cell............................................................. 91
Cautions on Storage of the Flow Cell ............................................... 91
Lamp Replacement............................................................................... 92
How to Replace the Xenon Lamp..................................................... 92
Positional Adjustment of Xe Lamp.......................................................... 99
Coarse Adjustment Using Chromera ................................................ 99
Fine Adjustment ...........................................................................100
Fuse Replacement ...............................................................................101
Troubleshooting............................................................................ 103
Warnings............................................................................................105
Troubleshooting ..................................................................................106
Appendix A.................................................................................... 109
Maintenance Software Program ............................................................111
Installing the Software ..................................................................111
Operation.....................................................................................116
Description Of Contact Signals..............................................................122
Terminology .......................................................................................123
Index .................................................................................................127
Introduction
8 . Flexar Fluorescence LC Detector User’s and Service Guide
Introduction .9
About this Manual

Be sure to read and understand the following points with regard to this User’s Guide.
¾ Information contained in this document is subject to change without notice for
improvement.
¾ No part of this manual may be reproduced, transmitted or disclosed to a third party in
any form or by any means without the express written permission of PerkinElmer, Inc.
¾ PerkinElmer, Inc. assumes no liability for any direct, indirect, or consequential damages
arising from use not described in this manual.
¾ Utmost care must be exercised when using the instrument.
¾ This document does not provide any warranty or permission for industrial properties or
any rights to grant license lawfully and without infringement.
This instrument is intended for use by persons having a basic knowledge of chemical analysis
procedures. Keep in mind that improper use of analytical instruments, chemicals or samples would
result not only in wrong analytical data but also in consequences adverse to safety.
Please read this instruction manual carefully before attempting operation and acquaint yourself with the
Flexar Fluorescence Detector for its correct use.
The liquid chromatograph system comprises a pump unit for delivering eluents, a sample introduction unit
for injecting samples (autosampler, manual injector), a column unit for separating components of each
sample, a column oven unit for maintaining the column at a constant temperature, and a detector unit for
detecting separated sample components as electric signals. Before using the liquid chromatograph system,
read carefully the instruction manual accompanying each of these units so that you can carry out analysis
properly.
This user’s guide is divided into following chapters:
Chapter 1 Introduction
This chapter contains the product warranty, conventions used in the manual and the
conventions and warnings used in the manual.
Chapter 2 Safety Practices
Important safety information is provided in this chapter.
Chapter 3 System Description
This chapter contains information on the detector, a descriptions of Fluorometry and

instrument specifications.
Chapter 4 Installation
Information on installing and re-installing your instrument should you ever need to move
your system is provided.
Chapter 5 Operation
This chapter provides information on operating the instrument.

Chapter 6 Maintenance
Maintenance and cleaning procedures for the various components of your instrument are
provided.
Chapter 7 Troubleshooting
Troubleshooting information, software and system error messages are provided for the
instrument.
Appendix
How to use the Maintenance Software Program, a Description Of Contact Signals, and
Terminology.
Introduction .11
Product Warranty
This product is warranted to be free from defects in material or workmanship under normal
use within the product specifications indicated in this manual and under conditions given
below.
This warranty is void if the instrument is not used according to the instruction manual.
The manufacturer makes no warranties, either express or implied, except as provided herein,
including without limitation thereof, warranties as to marketability, merchantability, for a
particular purpose or use, or against infringement of any patent.
No oral or written information or advice given by the manufacturer, its dealers, distributors,
agents or employees shall create a warranty or in any way increase the scope of this
warranty.
1. Scope of Warranty:
Any parts that prove to be defective in design or workmanship during the warranty
period will be repaired, adjusted or replaced without charge. A substitute part may
be used for repair, or replacement with an equivalent product may be made instead
of repair. Such system components as a personal computer and printer to be
updated frequently for improvement may not be available in original versions at the
time of replacement.
Note that this warranty does not apply to the instrument after it is discarded, or if
modified by the user or resold without permission from the manufacturer,
consumable parts, and any failure of lifetime-expired parts. The manufacturer
assumes no liability for any damage to data or application software due to any
possible fault or failure of this instrument.
2. Warranty Period:
One year from the date of initial installation (In case a separate warranty document
has been issued, the warranty period indicated in it takes precedence over the above
period)
3. Limitations and Exclusions on Warranty. Note that the following cases are excluded
from the scope of this warranty.
a. Failure due to operation at a place not meeting the installation requirements
specified by the manufacturer.
b. Failure due to power supply voltage/frequency other than specified by the
manufacturer or due to abnormality in power supply.
c. Corrosion or deterioration of the piping due to impurities contained in gas,
compressed air or cooling water supplied by the user.
d. Corrosion of the electric circuits or deterioration of the optical elements due
to highly corrosive atmospheric gas.
e. Failure due to use of software, hardware or spare parts not supplied by the
manufacturer.
f. Failure due to use not described in the manual or improper repair not
approved by the manufacturer.
g. Failure due to maintenance or repair by other than service personnel

qualified by the manufacturer.
h. Failure due to relocation or transport conducted not under the supervision of
the manufacturer after the initial installation of the instrument.
i. Failure due to disassembly, modification or relocation not approved by the
manufacturer.
j. Failure due to acts of God, including fire, earthquake, storm, flood, lightning,
social disturbance, riot, crime, insurrection, terrorism, war (declared or
undeclared), radioactive pollution, contamination with harmful substances,
etc.
k. Failure of the hardware, or damage to the system software, application
software or data due to computer virus infection.
l. After disposal of this instrument, after its resale without prior approval from
the manufacturer, consumable parts, and failure of any part that has
reached the end of its service life.
4. Disclaimer of Warranty
THE MANUFACTURER MAKES NO WARRANTIES, EITHER EXPRESS OR IMPLIED,

EXCEPT AS PROVIDED HEREIN, INCLUDING WITHOUT LIMITATION THEREOF,
WARRANTIES AS TO MARKETABILITY, MERCHANTABILITY, FOR A PARTICULAR
PURPOSE OR USE, OR AGAINST INFRINGEMENT OF ANY PATENT. IN NO EVENT
SHALL THE MANUFACTURER BE LIABLE FOR ANY DIRECT, INCIDENTAL OR
CONSEQUENTIAL DAMAGES OF ANY NATURE, OR LOSSES OR EXPENSES
RESULTING FROM ANY DEFECTIVE PRODUCT OR THE USE OF ANY PRODUCT.
NO ORAL OR WRITTEN INFORMATION OR ADVICE GIVEN BY THE MANUFACTURER,

ITS DEALERS, DISTRIBUTORS, AGENTS OR EMPLOYEES SHALL CREATE A
WARRANTY OR IN ANY WAY INCREASE THE SCOPE OF THIS WARRANTY.
Introduction .13
Conventions Used in this Manual

Normal text is used to provide information and instructions.
Bold text refers to text that is displayed on the screen.
UPPERCASE text, for example, ENTER or ALT, refers to keys on the PC keyboard. ‘+’ is used
to show that you have to press two keys at the same time, for example, ALT+F.
All eight digit numbers are PerkinElmer part numbers unless stated otherwise.
Notes, Cautions and Warnings

Three terms, in the following standard formats, are also used to highlight special
circumstances and warnings.
NOTE: A note indicates additional, significant information that is provided with some procedures.
We use the term CAUTION to inform you about situations that could
CAUTION
result in serious damage to the instrument or other equipment.
Details about these circumstances are in a box like this one.
D Caution (Achtung)
Bedeutet, daß die genannte Anleitung genau befolgt werden muß, um
einen Geräteschaden zu vermeiden.
DK Caution (Bemærk)
Dette betyder, at den nævnte vejledning skal overholdes nøje for at
undgå en beskadigelse af apparatet.
E Caution (Advertencia)
Utilizamos el término CAUTION (ADVERTENCIA) para advertir sobre
situaciones que pueden provocar averías graves en este equipo o en
otros. En recuadros éste se proporciona información sobre este tipo de
circunstancias.
F Caution (Attention)
Nous utilisons le terme CAUTION (ATTENTION) pour signaler les
situations susceptibles de provoquer de graves détériorations de
l'instrument ou d'autre matériel. Les détails sur ces circonstances
figurent dans un encadré semblable à celui-ci.
I Caution (Attenzione)
Con il termine CAUTION (ATTENZIONE) vengono segnalate situazioni
che potrebbero arrecare gravi danni allo strumento o ad altra
apparecchiatura. Troverete informazioni su tali circostanze in un riquadro
come questo.
NL Caution (Opgelet)
Betekent dat de genoemde handleiding nauwkeurig moet worden
opgevolgd, om beschadiging van het instrument te voorkomen.
P Caution (Atenção)
Significa que a instrução referida tem de ser respeitada para evitar a
danificação do aparelho.
Introduction .15
We use the term WARNING to inform you about situations that could
result in personal injury to yourself or other persons. Details about
these circumstances are in a box like this one.
WARNING
Warning (Warnung)
D
Bedeutet, daß es bei Nichtbeachten der genannten Anweisung zu
einer Verletzung des Benutzers kommen kann.
Warning (Advarsel)
DK
Betyder, at brugeren kan blive kvæstet, hvis anvisningen ikke
overholdes.
Warning (Peligro)
E
Utilizamos el término WARNING (PELIGRO) para informarle sobre
situaciones que pueden provocar daños personales a usted o a
otras personas. En los recuadros como éste se proporciona
información sobre este tipo de circunstancias.
Warning (Danger)
F
Nous utilisons la formule WARNING (DANGER) pour avertir des
situations pouvant occasionner des dommages corporels à
l'utilisateur ou à d'autres personnes. Les détails sur ces circonstances
sont données dans un encadré semblable à celui-ci.
Warning (Pericolo)
I
Con il termine WARNING (PERICOLO) vengono segnalate situazioni
che potrebbero provocare incidenti alle persone. Troverete
informazioni su tali circostanze in un riquadro come questo.
Warning (Waarschuwing)
NL
Betekent dat, wanneer de genoemde aanwijzing niet in acht wordt
genomen, dit kan leiden tot verwondingen van de gebruiker.
Warning (Aviso)
P
Significa que a não observância da instrução referida poderá causar
um ferimento ao usuário.
Warning Signs on the Instrument
WARNING
FOR CONTINUED PROTECTION

REPLACE ONLY WITH SAME FUSE
TYPE AND RATING
Power On/Off push button
Alternating current
Safety Practices
18. Flexar Fluorescence LC Detector User’s and Service Guide
Overview
This chapter describes the general safety practices and precautions that must be observed
when operating the Flexar Fluorescence LC Detector.
This advice is intended to supplement, not supersede, the normal safety codes in the user’s
country. It is also a supplement to the PerkinElmer standard Safety and Health Policy. The
information provided does not cover every safety procedure that should be practiced.
Ultimately, maintenance of a safe laboratory environment is the responsibility of the analyst
and the analyst’s organization.
Please consult all manuals supplied with the Flexar Fluorescence Detector and accessories
before you start working with the instrument. Carefully read the safety information in this
chapter and in the other manuals supplied. When setting up the instrument or performing
analyses or maintenance procedures, strictly follow the instructions provided.
We use the term CAUTION to inform you about situations that could
CAUTION
result in serious damage to the instrument or other equipment.
Details about these circumstances are in a box like this one. Be sure
that all instrument operators read and understand the
precautions listed below.
• External input/output are for contact closure. Never apply voltage to the External
input/output.
• As the detector is readily affected by the ambient temperature, use it in places where
there is little wind or change in the ambient temperature. Do not use the detector
near any source of vibration, electrical noise, or in places where corrosive gas and a
lot of dust exist.
• Do not connect any tube other than the provided “OUT tube” to the outlet tubing of
the detector. Put the exit end of the “OUT tube” in the waste liquid bottle and do not
apply back pressure.
• When connecting this detector to other detectors in series, put it last.
• If eluent freezes inside the detector, the flow line might break and damage the
detector. If there is any chance of eluent freezing during stoppage or storage of the
detector, withdraw the eluent completely from the flow line of the detector.
• When using any eluent containing a salt of high concentration, make sure that the
flow line is washed with water thoroughly after use. Failure to do so may result in
the plugging of the line causing the detector to cease operation.
• If the detector is not to be used for more than one week, then, prior to storage,
wash the flow line with pure water or acetone, and store in isopropanol or 50%
solution of isopropanol and HPLC grade water.
• Do not use any eluent which might corrode the material, such as stainless steel, that
it comes in contact with. Use of such eluent might cause a baseline drift and damage
the detector.
• Use a dry cloth to wipe the detector. Do not use water or alcohol. Use of such liquids
may damage the detector or erase characters or color on the panel.
NOTE: Do not connect any cord to the output signal terminal other than the attached signal cord.
Safety Practices . 21
NOTE: Use a thoroughly degassed eluent. It is recommended that a degassing device be used to
degas the eluent as it is easy to use and permits continuous degassing.
NOTE: If an eluent is replaced with another eluent in which it is insoluble, after having withdrawn
the old eluent from the line, wash the line with a solvent which is soluble in both eluents, and
then fill the line with the new eluent. For example, if water is replaced with chloroform, wash
the line with isopropyl before filling the line with chloroform.
NOTE: When replacing an eluent containing salt with an eluent containing organic solvent, wash the
line with pure water and then isopropyl before filling the line with the organic- solvent-
containing eluent. In contrast when replacing an eluent containing organic solvent with an
eluent containing salt , wash the line with isopropyl first and then pure water before filling
the line with salt-containing-eluent.
We use the term WARNING to inform you about situations that

could result in personal injury to yourself or other persons. Be
sure that all instrument operators read and understand the
precautions listed below. It is advisable to post a copy of the
WARNING precautions near or on the instrument shelf.
Ignition of Flammable Chemicals

• This instrument is not explosion-proof. In unattended operation, do not use organic
solvents having an ignition point below 70 °C.
• Beware of ignition hazard when using flammable chemicals such as organic solvents.
a. Do not bring a heat or flame source near the instrument.
b. Well-ventilate the laboratory room where the instrument is used.
c. Always check the following conditions. If an abnormality is found, stop
operation immediately and unplug the power cord from the supply.
¾ Leakage of solvent or waste solution.
¾ Leakage of solvent inside the instrument.
• When using flammable chemicals, be careful about possible ignition due to static
electricity. To prevent the build-up of static electricity, use a conductive container for
waste solution and provide proper grounding connection to it.
Explosion of Vapor from Flammable Chemicals

If a flammable chemical such as organic solvent leaks from the flow path of the instrument
and its vapor concentration reaches the explosion limit, it could cause spontaneous
combustion with dangerously explosive results.
When using a flammable and readily volatile chemical, be sure to check for leakage from the
instrument flow path and ventilate the laboratory room adequately.
Heavy Instrument
This instrument weighs 29 kg.
When carrying this instrument, exercise care not to incur injury by dropping it accidentally.
Be sure to hold the front and rear parts of the instrument securely when moving it.
Prevention of Instrument Tipover

Personal injury could be incurred if the instrument tips over. When installing the instrument,
provide a proper means for preventing the instrument from tipping over accidentally.
Common Safety Precautions
Prior to Use
• Before using the instrument, be sure to read this user’s guide carefully to attain a full
understanding of its operations.
• Keep this user’s guide handy nearby so it can be referred to whenever needed.
• Be sure to observe the procedures specified in this user’s guide.
• Be sure to understand and follow all the safety instructions given in this user’s guide.
• Be sure to observe all the hazard warnings attached to the instrument or provided in
this user’s guide. Failure to do so could result in personal injury or damage to the
instrument.
• Be sure to follow all the methods of use instructed in this user’s guide for proper
application of the product.
• Absolutely avoid modifying the product, using non-specified parts, or removing safety
devices as it could be hazardous.
• Do not perform any operation or action other than described in this user’s guide. If
you have any trouble in the instrument, notify the nearest sales representative or
service office of PerkinElmer.
• When using chemicals for the instrument, be sure to provide proper ventilation of the
room. Inadequate ventilation could endanger human health.
• Keep in mind that the hazard warnings in the manuals or on the product cannot
cover every possible case, as it is impossible to predict and evaluate all
circumstances beforehand. Always be alert and use your common sense.
In Use
If an abnormality such as unusual noise, odor, fuming, or gas leakage occurs during
operation of the instrument, immediately disconnect power to the instrument, and
take proper safety measures as required. Then, contact your PerkinElmer service
representative.
During Installation, Maintenance, and Relocation

• At the time of delivery, installation of the instrument shall be carried out by or under
supervision of qualified service personnel of the manufacturer or its authorized
service agent for ensuring safety and high accuracy in operation of the instrument. It
is not permitted for the user to carry out installation.
• After completion of installation, check that all the standard parts are provided. If the
instrument is made active with any one of the standard parts not equipped, a failure
could occur which may result in a hazardous condition.
NOTE: If any item is missing or damaged or if you have any questions, please notify PerkinElmer.
• The maintenance and checkup procedures to be performed by the user are only
those described in the manual. When performing the maintenance and checkup
procedures described in the manual, attain a clear understanding of them.
• Do not perform other maintenance and checkup procedures to avoid jeopardizing

safety and causing troubles in the instrument.
• After installation, do not relocate the instrument. If the instrument is relocated,

vibration or impact applied during relocation could cause a malfunction in the optical
components that have been adjusted precisely.
• If any warning/caution label has become illegible due to deterioration with age or it has
been damaged due to any cause, contact your PerkinElmer service representative.
Xenon Lamp Safety
Injury Due to Xenon Lamp Explosion

Replace the xenon lamp with a new one before its cumulative turn-on time exceeds the
guaranteed useful lifetime.
With the lapse of the guaranteed useful lifetime, the bulb wall darkens due to evaporation of
electrode material and adhesion of scattered substances, resulting in a decrease in heat
dissipation. This may increase temperature (pressure) inside the bulb excessively
to cause explosion.
Lamp Replacement
Replace the xenon lamp with a new one before its cumulative turn-on time exceeds the
guaranteed useful lifetime.
P/N Part Name Limit

N2922082 150 W xenon lamp 500 hours
Before removing the xenon lamp for replacement, turn off the xenon
lamp (turn off power to the instrument and unplug the power cord)
and then wait for at least one hour until the xenon lamp becomes
sufficiently cool to reduce its internal pressure to the normal safety
WARNING level.
If a strong shock or impact is applied to the xenon lamp or if the

surface of its quartz glass part is scratched, it may explode and
scatter glass pieces, resulting in personal injury.
WARNING
Be sure to wear proper protective gear such as safety goggles, safety

mask, thick long sleeves, and gloves when handling the xenon lamp.
WARNING
Lamp Installation
• When loosening or tightening the retaining nut for the xenon lamp, be careful not to
apply excessive force to its glass bulb part. Never hold and turn the glass bulb part
for loosening/tightening.
• Do not touch the quartz glass part of the xenon lamp with bare hands.
If the quartz glass part of the xenon lamp is contaminated with dust or fingerprints,
wipe it using a gauze sheet or absorbent cotton cloth slightly moistened with
isopropyl alcohol.
If the xenon lamp is turned on with dust or fingerprints left on the surface of the
quartz glass part, it may cause contamination burn-in to decrease the mechanical
strength of the glass part, resulting in explosion of the xenon lamp.
• Be sure to mount the xenon lamp in the specified direction. If the mounting direction
(polarity) is wrong, the cathode will be consumed significantly to which will prevent
turn-on of the lamp.
Mount the lamp so that the ‘+’ (anode) mark on it will be positioned at the support
metal of the lamp holder. If the lamp with its cathode consumed excessively is used
continuously, pressure inside the lamp bulb may become too high, causing possible
explosion. To prevent this, replace the lamp with a new one immediately if its
cathode has been consumed substantially.
• If the nut on the lamp base and wiring part is loose, the contact resistance between
them will increase due to poor electrical contact. This could generate a large amount
of heat which will make the lamp extremely hot, resulting in possible explosion.
To prevent this, be sure to tighten the nut securely.
Lamp Disposal
The xenon lamp is filled with high-pressure gas (approximately 1 Mpa at room
temperature, approximately 4 Mpa under operating condition), and this high-
pressure gas still remains in the xenon lamp after it is demounted for replacement.
For disposal of the used xenon lamp, wrap it with a thick cloth (e.g., triple-folded
cotton cloth) completely and crush its glass part with a hammer or the like. Then,
discard the xenon lamp properly as a dangerous waste item.
If the xenon lamp is discarded without being crushed, it could explode due to
possible impact at the time of disposal, scattering glass pieces which may cause
personal injury. (See Maintenance)
Touching Hot Part Could Result in Burns

The xenon lamp and its housing become very hot during operation and remain hot for a
while after operation.
Touching the hot xenon lamp or its housing could result in severe burns. Before proceeding
to lamp replacement, turn off the power switch, unplug the power cord and then wait for at
least one hour until the lamp and its housing cool down sufficiently. (See Maintenance)
Direct Viewing of Xenon Lamp Could Cause Eye Damage
The xenon lamp radiates intense ultraviolet light when it is on. If you
look at the illuminating xenon lamp during coarse adjustment of its
position, your eyes could be damaged.
WARNING Do not look at the xenon lamp directly when it is lit. Be sure to wear
tinted safety glasses to prevent possible eye damage.
Before Turning on Xenon Lamp Again

After the xenon lamp is turned off, it remains very hot for a while. Under this condition, it is
not allowed to turn on the xenon lamp again for measurement. Before turning on the xenon
lamp again, wait at least five minutes.
General Operating Conditions

Only use the Flexar Fluorescence Detector indoors and under the following conditions:
Temperature 10 oC to 35 oC
Relative Humidity 25% to 80% relative humidity (non-condensing)
If possible, avoid any adjustment, maintenance and repair of the opened, operating instrument.
If any adjustment, maintenance and repair of the opened instrument are necessary, this must be
done by a skilled person who is aware of the hazard involved. Whenever it is likely that the Flexar
is unsafe, make it inoperative. The Flexar may be unsafe if it:
• shows visible damage
• has been subjected to prolonged storage in unfavorable conditions
• has been subjected to severe transport stresses
Environmental Conditions
Normal Operating Conditions:

• Indoors use.
• The location must be free of smoke, dust, corrosive fumes, direct
sunlight and excessive vibration.
• Ambient temperature: +10°C to +35°C (+50°F to +95°F).
• Relative humidity: 25% to 80%, without condensation.
• Altitude: in the range 0 m to 2,000 m (sea level to 6,562 feet).
• Mains fluctuations not exceeding +/- 10% of the nominal voltage.
Safe Operating Conditions:

• Indoor use.
• Ambient temperature: of +5oC to +40oC (+41°F to +104°F).
• Altitude: 0 m to 2000 m.
Storage Conditions:
• Ambient temperature: –20°C to +60°C (–4°F to +140°F).
• Altitude: in the range 0 m to 12,000 m (sea level to 39,370 feet).
If the equipment is used in a manner not specified herein, the protection

provided by the equipment may be impaired.
WARNING
Electrical Safety
The Flexar Fluorescence Detector has been designed to protect the operator from potential
electrical hazards. This section describes some recommended electrical safety practices.
Lethal voltages are present at certain areas within the

instrument. Installation and internal maintenance of the
instrument should only be performed by a PerkinElmer Service
Representative or similarly authorized and trained person.
WARNING When the instrument is connected to line power, opening the instrument
covers is likely to expose live parts. Even when the power switch is off,
high voltages can still be present. Capacitors inside the instrument may
still be charged even if the instrument has been disconnected from all
voltage sources.
The instrument must be correctly connected to a suitable electrical supply. The supply must
have a correctly installed protective conductor (earth ground) and must be installed or
checked by a qualified electrician before connecting the instrument.
Any interruption of the protective conductor (earth ground) inside or

outside the instrument or disconnection of the protective conductor
terminal is likely to make the instrument dangerous. Intentional
interruption is prohibited.
WARNING
When working with the instrument:
• Connect the instrument to a correctly installed line power outlet that has a protective
conductor connection (earth ground).
• Do not operate the instrument with any covers or internal parts removed.
• Do not attempt to make internal adjustments or replacements except as directed in

the manuals.
• Disconnect the instrument from all voltage sources before opening it for any
adjustment, replacement, maintenance, or repair. If afterwards, the opened
instrument must be operated for further adjustment, maintenance, or repair, this
must only be done by a PerkinElmer Service Representative.
• Whenever it is possible that the instrument is no longer electrically safe for use,
make the instrument inoperative and secure it against any unauthorized or
unintentional operation. The electrical safety of the instrument is likely to be
impaired if, for example, the instrument shows visible damage; has been subjected
to prolonged storage under unfavorable conditions; or has been subjected to severe
stress during transportation.
• Service on electrical components should be performed only by a qualified

PerkinElmer service representative or a similarly trained and authorized person.
• Be sure the power cord is the correct one for your laboratory. The line cord used
must meet the national safety agency’s guidelines for the particular country of use.
• Some power cords available from PerkinElmer are illustrated below:
North America Old British Standard

NEMA-5-15 BS 546
09988986 India
09991423
Europe British Standard

CEE 7 BS 1363
"Schuko" United Kingdom
09991415 09991414
Europe Australia
Switzerland ETSA S/86
09991413 09991417
Europe Israel
Italy 09991424
09991422
Denmark Japan
09991416 09991425
China
09290348
ElectroMagnetic Compatibility (EMC)

Europe: All information concerning EMC standards is in the Declaration of Conformity, and
these standards may change as the European Union adds new requirements.
PerkinElmer instruments have been designed and manufactured, having regard to the state
of the art, to ensure that:
a. the electromagnetic disturbance generated does not exceed the level above which radio
and telecommunications equipment or other equipment cannot operate as intended;
b. it has a level of immunity to the electromagnetic disturbance to be expected in its
intended use which allows it to operate without unacceptable degradation of its intended
use.
Electric Shock Due to Contact with Inside of Instrument

• The xenon lamp is supplied with high voltage of 30 kV, which could cause an electric
shock to result in serious or fatal injury. Before removing the light source cover for
replacement of the light source lamp, be sure to turn off the power switch and
unplug the power cord before proceeding.
• When replacing the fuse with a new one, be careful not to receive an electric shock.
To prevent this, be sure turn off the power switch and unplug the power cord before
proceeding to fuse replacement.
Electric Shock Due to Improper Grounding

To prevent an electric shock hazard, a properly grounded power supply outlet must be
provided for the instrument.
Be sure to use the grounded three prong power cable, which is supplied as a standard
accessory for the instrument. The use of a different type of power cable may result in an
electric shock hazard. Connect the three prong power cable to a grounded three prong power
outlet.
WEEE Instructions for PerkinElmer Products
or
A label with a crossed-out wheeled bin symbol and a rectangular bar indicates that the
product is covered by the Waste Electrical and Electronic Equipment (WEEE) Directive and is
not to be disposed of as unsorted municipal waste. Any products marked with this symbol
must be collected separately, according to the regulatory guidelines in your area.
The objectives of this program are to preserve, protect and improve the quality of the
environment, protect human health, and utilize natural resources prudently and rationally.
Specific treatment of WEEE is indispensable in order to avoid the dispersion of pollutants into
the recycled material or waste stream. Such treatment is the most effective means of
protecting the customer’s environment.
Requirements for waste collection reuse, recycling, and recovery programs vary by regulatory
authority at your location. Contact your local responsible body (e.g., your laboratory
manager) or authorized representative for information regarding applicable disposal
regulations. Contact PerkinElmer at the web site listed below for information specific to
PerkinElmer products.
Web address: http://las.perkinelmer.com/OneSource/Environmental-directives.htm
Customer Care: 1-800-762-4000 (inside the USA)

(+1) 203-925-4602 (outside the USA)
0800 40 858 (Brussels)
0800 90 66 42 (Monza)
Products from other manufacturers may also form a part of your PerkinElmer system. These
other producers are directly responsible for the collection and processing of their own waste
products under the terms of the WEEE Directive. Please contact these producers directly
before discarding any of their products.
Consult the PerkinElmer web site (above) for producer names and web addresses.
Decontamination and Cleaning

Before using any cleaning or decontamination methods except those specified by
PerkinElmer, users should check with PerkinElmer that the proposed method will not damage
the equipment.
Decontamination
Customers wishing to return instrumentation and/or associated materials to
PerkinElmer for repair, maintenance, warranty or trade-in purposes are advised that
all returned goods must be certified as clean and free from contamination.
The customer’s responsible body is required to follow the "Equipment Decontamination

Procedure" and complete the “Certificate of Decontamination”. These documents are
available on the PerkinElmer public website:
http://las.perkinelmer.com/OneSource/decontamination.htm
If you do not have access to the internet and are located in the U.S., call toll free at 1-800-
762-4000 or (+1) 203-925-4602, 8:30 a.m. – 7 p.m. EST and speak to Customer
Support.
In Canada, call toll free 800-561-4646 and speak to Customer Support.
If you are located outside of the United States or Canada, please call your local PerkinElmer
sales office for more information.
Cleaning the Instrument

Exterior surfaces may be cleaned with a soft cloth, dampened with a mild detergent and
water solution. Do not use abrasive cleaners or solvents.
Warning Labels on the Instrument

The warning labels shown below are attached on the Flexar Fluorescence Detector.
Read the warning labels carefully, and check the instructions on them to attain a clear
understanding with reference to actual parts. Periodically check the appearances of these
warning labels to see if they are clean to allow easy reading over a safe distance. If any one
of the warning labels becomes illegible due to deterioration, contact PerkinElmer.
WARNING
Beware of electric shock!
Can cause death or serious
injury.
Provide proper grounding
connection.
Removal of this cover from the
instrument is permitted only to
qualified service personnel.
Figure 1 Warning label on the right side of the instrument

Figure 2 Warning labels on the front of the instrument

Quality Control/Good Laboratory Practices

The user should develop appropriate quality control procedures for the instrument to ensure
suitability for its intended use. These procedures typically consist of periodic performance
verifications and routine inspections and suitability tests.
Certificate of System Control

Each instrument is carefully built and tested in a controlled system in accordance with the
requirements specified in its applicable PerkinElmer Final Assembly and Test Specification.
Each instrument is certified to meet its functional and performance specification upon release
to shipment. The integrity of this quality system is routinely audited and certified.
Instrument Performance Verification (IPV)

To maintain functional performance, PerkinElmer recommends a yearly Instrument
Performance Verification (IPV) of the instrument and its other system components by a
PerkinElmer Service Engineer to ensure its operation within published specifications. These
tests consist of measuring the most important characteristics. Certification is available for
regulatory compliance. Contact your local PerkinElmer Sales and Service office.
Routine Inspection and Suitability Test

Prior to any sample analysis, a system suitability test, which closely resembles the intended
assay, should be performed to ensure that the LC system is operating within established
criteria (e.g., peak resolution, peak asymmetry, precision, retention time, column plate count,
pressure limits, signal/noise ratio, etc.)
While the Universal Test Mix (UTM) P/N 00890893 can be used for a system check, we
recommend that you develop a separate system suitability test and acceptance criteria for
each of your assays.
Hazardous Chemicals
Before using mobile phase solvents, you should be thoroughly familiar with all hazards and
safe handling practices. Observe the manufacturer’s recommendations for use, storage and
disposal. These recommendations are normally provided in the material safety data sheets
(MSDS) supplied with the solvents.
Some chemicals used with this instrument may be hazardous or may

become hazardous after completion of an analysis. The responsible body
(e.g., Lab Manager) must take the necessary precautions to ensure that
the surrounding workplace and instrument operators are not exposed to
WARNING hazardous levels of toxic substances (chemical or biological) as defined
in the applicable Material Safety Data Sheets (MSDS) or OSHA, ACGIH,
or COSHH documents. Venting for fumes and disposal of waste must be
in accordance with all national, state and local health and safety
regulations and laws.
Definitions in Warning for Hazardous Chemicals
Responsible body: “Individual or group responsible for the use and maintenance of
equipment, and for ensuring that operators are adequately
trained.” [per IEC 61010-1]
Operator: “Person operating equipment for its intended purpose.” [per IEC
61010-1, ]
OSHA: Occupational Safety and Health Administration (United States)
ACGIH: American Conference of Governmental Industrial Hygienists
COSHH: Control of Substances Hazardous to Health (United Kingdom)
Accuracy and Precision of Measured Values

Carry out control sample measurements to ensure that the performance of the instrument is
normal.
Possible Carryover
When examining the results of measurements, take into account the possibility of carryover.
Since fluorocarbon resin and quartz materials are used for the wetted parts of piping,
particular attention should be paid when analyzing the samples that are likely to be adsorbed
by these materials.
Safe Handling of Solvents
Give careful attention to the hazards associated with the

solvents you are using. Refer to the safety data sheets
provided by the manufacturer. For example, Material Safety
Data Sheets (MSDS) in the USA.
WARNING
• Wear appropriate eye protection at all times when handling chemicals. Use safety
glasses with side shields, goggles, or full-face shields, according to the types of
chemicals you will be handling.
• Wear suitable protective clothing, including gloves that are specifically resistant to
the chemicals being handled.
• Always use clean solvents. Solvents which have been distilled in glass (HPLC Grade)
are recommended.
• Filter the solvents and buffers through a 0.5-micron medium as an additional
precaution.
• Degas all aqueous and most organic solvents prior to use.
• Store flammable solvents or solvents which may form hazardous by-products when
the instrument is shut down, by following the recommended shutdown procedure.
• Check compatibility of solvent(s) with the type of column(s) being used.
• Know the relative polarity and miscibility of the solvents being used.
• Fluorocarbon resin and quartz materials are used for the wetted parts of the
instrument. Never use solvents that would corrode these materials.
• This instrument has internal parts made of materials that could be corroded by
strongly acidic solutions, strongly alkaline solutions or organic solvents. Take care not
to spill these corrosive solutions or solvents into the inside of the instrument. If a
corrosive solution or solvent spills inside, wipe it off immediately.
• For use of the liquid leak sensor, follow the instructions given below.
a. Turn on the leak sensor function when feeding a solvent.
b. The leak sensor function may remain inactive in cases where the amount of
leakage is too small.
Furthermore, leakage of a highly volatile solvent may not be detected. Be sure to
check for liquid leakage regularly to prevent a possible serious accident
regardless of whether the leak sensor is activated or not.
c. In routine inspection of the instrument, visually check for liquid leakage before
attempting measurements.
d. In periodic inspection of the instrument, check that the leak sensor works normally.
Referring to the instruction manual accompanying each module unit of the liquid
chromatograph system, set up the automatic liquid feed stop function (such as the
pressure limiter function of the pump unit) to prevent a possible problem.
Disposal of Waste Solution

Be sure to collect the waste solvent and treat it for proper disposal in accordance with the
relevant laws and regulations regarding water pollution control and sewage treatment.
Improper treatment of waste solution may result in environmental pollution and could also
lead to a penalty.
Solvents with Low Boiling Points

Do not use liquids that have a boiling point less than 30 °C.
Do not use carbon disulfide or other solvents which have an auto-

ignition temperature below 110 °C.
WARNING
Buffers
Exercise care when using buffers in conjunction with organic solvents. NEVER LEAVE BUFFERS
IN THE SYSTEM OVERNIGHT. Buffers left in the system can form salt crystals which may cause
premature injector valve failure and plug the transfer tubing and sample needle. To remove
buffers, flush the system with water followed by methanol or isopropanol. Remember to
change the flush solvent from methanol or isopropanol to water before using buffers.
Inflammation or Injury Due to Toxic or Corrosive Solvent

When using a toxic or corrosive solvent, be careful not to incur a physical inflammation or
injury. For details of the properties of each solvent and how to handle it, refer to the relevant
Material Safety Data Sheets (MSDS). Be sure to handle each solvent properly.
• Wear proper protective clothes (e.g., safety goggles) so that a solvent will not
come into direct contact with the skin.
• Ventilate the laboratory room adequately to prevent accidental inhalation of

harmful solvent vapor.
Corrosion
All parts of the instrument that contact mobile phase are made of stainless steel, KelF, and
quartz. Some of these materials are extremely sensitive to acid chlorides. If you have
questions about your mobile phase or flush solvent, contact a PerkinElmer representative.
Refer to the table below.
Solvents Which May Corrode an LC Instrument
Aqua Regia (80% HCl, 20% HNO3) Bromine

Hydrogen Peroxide Freon 12 (wet)
Anhydrous Chloride Chlorinated solvents
Dichloromethane Hydrofluorosilicic Acid (20%)
Sulfuric Acid (Conc.) Hydrobromic Acid (20%)
Hydrochloric Acid (20% and 37%) Ferric Chloride
Hydrofluoric Acid (20%, 50%, and 75%) Ferrous Chloride
Copper Chloride Mercuric Chloride (Dilute)
If you have questions about your mobile phase, contact a PerkinElmer representative. Refer
to the table below:
Aqua Regia and sulfuric acid are known to dissolve titanium.
WARNING
Solvents with Auto-Ignition Temperature Below 110 °C

Certain solvents have a temperature at which they are combustible upon contact with a
heated surface, even in the absence of a spark or other source of ignition. A list of common
solvents with their auto-ignition temperatures is shown below.
Do not use carbon disulfide or other solvents which have an auto-

ignition temperature below 110 °C.
WARNING
Auto-Ignition Temperatures of Common LC Solvents.

Auto-Ignition
Solvent
Temperature (°C)
Carbon Disulfide 100
Diethyl Ether 180
Cyclohexane 260
Hexane 261
Petroleum Ether (naphtha) 288
Pentane 309
Tetrahydrofuran 321
Dioxane 366
Propanol 404
iso-Octane 418
Ethanol 423
Isopropyl Ether 443
Dimethylformamide 445
Methanol 446
Isopropanol 455
Methylethylketone 474
Acetonitrile 524
Ethyl Acetate 524
Toluene 536
Acetone 538
Benzene 562
Air Bubbles
To prevent air from entering the system, and to ensure that pressure fluctuations do not
occur, observe the following precautions:
• Ensure that the pump's solvent inlet filter is below the solvent level in the solvent
reservoir.
• If the pump has not been used for an extended period of time, remove air bubbles by
connecting a priming syringe to the drain valve on the pump, opening the drain valve,
and fast-flushing the system to prime the pump. After priming the pump, close the
drain valve completely.
• If bubbles are observed in the flow cell, degas the solvent and add back-pressure device
P/N 09907126.
Pressure Buildup
Over time, you may observe a gradual increase in the system operating pressure. If you
observe pressure readings greater than 3.45 MPa (500 psi) above the normal operating
pressure of your analysis, the following items may need attention:
• If you are injecting “clean” samples and there is no other apparent cause for the
pressure build-up, remove, disassemble, and clean the flow cell.
• If you are injecting “dirty” samples, the flow cell may be clogged or the column packing
material may have retained contaminated particulates. Remove and clean the flow cell,
and replace the column.
• The column end fitting or column frit may be plugged. Replace the end fitting or frit to
relieve excess pressure.
• In-line filters may be plugged. Replace the filter element.
Fuses
Disconnect the instrument from its power source if you need to

replace the fuses. For protection against fire hazard, replace only with
the same type and rating of fuses.
WARNING
System Description
System Description . 43
Detector Status LEDs

The Flexar Fluorescence LC Detector is controlled by Chromera. In addition, the detector
status is displayed on the front panel through the following indicator lights. The three LED’s
indicate; instrument Power On status, Instrument Ready status, and lamp status.
The main Power On/Off Switch is on the LED panel. Push and hold the Power On/Off
Switch on the LED panel until you see the power on LED turn green.
Power On Ready/Error Xe Lamp Main Power On/Off

LED LED LED Switch
Green Green/Red Green Single pole Momentary
LED Functionality
Power On LED Instrument Status
OFF OFF
OFF Standby
ON (green) ON
Ready/Error LED Instrument Status

OFF OFF / Standby
ON (green) Ready/Running – No Error
ON (red blinking) Error
Xe Lamp LED Lamp Status

OFF Lamp OFF
ON (green) Lamp ON
Front Power Switch Functionality

Power Switch Instrument Status Action
OFF OFF Remains OFF
Switch to ON OFF Instrument set to Standby
ON Standby Remains in Standby
ON Standby Waken instrument
ON ON Remains ON
ON Don’t care Instrument set to Standby
The Rear Panel
4 Marker In
(option)
5 Recorder 1 E-Line
(option)
6 Processor
(option) 2 Lamp Off
In
3 Auto Zero
In
Figure 3 Rear Panel
No. Indication Function Remarks

1 E-Line Ports Connects the USB to the FL detector
2 LAMP OFF IN The lamp is turned off by means of a
contact signal input.
3 AUTO ZERO IN The auto zero function is activated by
means of contact signal input.
4 MARKER IN The marker function is activated by The marker function is started
(option) means of contact signal input. when the short-circuit signal of
more than one second is input.
5 RECORDER Analog output for recorder The absorbance value data is
(option) The absorbance value at each point of output.
time
Stored spectral data
6 PROCESSOR Analog output for data processor The absorbance value data is
(option) (integrator) output.
The absorbance value at each point of
time
Stored spectral data
Description of Fluorometry
Principle of Fluorometry
Figure 4 Typical Organic Molecular Energy Level Diagram

Figure 4 shows the energy level transitions of an organic molecule for the various processes
involved in absorption, fluorescence and phosphorescence.
When light strikes an organic molecule in the ground state, it absorbs radiation of specific
wavelengths and several excited states are populated. A part of the excitation (absorbed)
energy is lost in vibrational relaxation, i.e. radiationless transition to the lowest vibrational
level in the excited state.
The molecule can return to the ground state by;
1. Emitting radiation (Fluorescence)
2. Undergoing a radiationless transition to populate the triplet state. The triplet state can
emit radiation (Phosphorescence). Generally phosphorescence persists for 10-4 seconds
or longer due to the selection rule imposed on the triplet-to-singlet transition. In
contrast, fluorescence takes place over a period of 10-8 to 10-9 seconds.
3. Going through a radiationless transition to return directly to the ground state.
Since a part of the radiation absorbed by the substance is lost as vibrational energy; the
energy emitted by the excited state is less than that absorbed by the compound (i.e. the
fluorescence wavelength is longer than the excitation wavelength, *Stokes’ Law).
The ratio of the number of photons emitted during fluorescence to the number of photons
absorbed is called the quantum efficiency of fluorescence (Fluorescence Yield). If two
compounds absorb the same number of photons, the fluorescence intensity of the compound
with the larger fluorescence quantum yield will be greater than that from a compound with a
lower fluorescence quantum yield.
When a dilute sample is used, the intensity of fluorescence is expressed by:
F = KIocλεϕ
F : Fluorescence intensity
K : Instrumental constant
Io : Intensity of exciting radiation
c : Concentration of the compound of interest
λ : Optical path length of cell
ε : Absorbtivity of substance
ϕ : Quantum efficiency of substance
Advantages of Fluorometry
Fluorescence can provide a significantly greater degree of sensitivity than absorbance
measurements. This increase in sensitivity is due to the fact that in fluorescence, the signal
due to the compound of interest is measured relative to the fluorescence of the blank (which
is zero). In contrast, absorbance measurements compare the transmittance of the solution
of the compound of interest relative to the transmittance of the blank. As the concentration
of the solution falls, the transmittance of the sample and the blank become more similar.
In this discussion, we will consider the detection of a sample that has a transmittance of 99%
(relative to the blank). If we assume that the inaccuracy of the % transmittance
measurement is 0.1%, the reliability of the observed data is as follows:
Percent transmittance of blank 100.0 ± 0.1%

Percent transmittance of sample 99.0 ± 0.1%
Difference (proportional to concentration of sample) 1.0 ± 0.2%
In this example, the uncertainty in the concentration measurement is ±20%. In contrast, in

fluorometry, a difference in the signal is directly proportional to the concentration of sample.
An analysis of the error in the measurement is as follows:
Output signal level at measurement of sample 100 ± 0.1

Value corresponding to blank
0 ± 0.1
Difference(proportional to concentration of sample) 100 ± 0.2
The error in the measurement using fluorescence is considerably smaller than that obtained
using absorbance, thus fluorometry is very advantageous for analyzing samples with a low
concentration of the compound(s) of interest. If a compound exhibits a strong fluorescence
signal, it is likely that fluorescence can detect concentrations that are two orders of
magnitude lower than absorbance.
A graphical description of why fluorescence can provide better sensitivity than absorbance is
presented in Figure 5. In this figure, the signal IS is used to represent the difference
between the intensity of the incident beam Io and the intensity of the transmitted beam It in
absorptiometry. The detection limit is the point where the difference between It and Io is
equivalent to the noise level. In contrast, when fluorometry is used, the observed signal IF is
directly proportional to the concentration and the background has a fluorescence of zero.
When a small signal is observed it is compared to a very small signal (since the blank does
not fluoresce) and is readily amplified for detection.
In addition, since the fluorescence emission wavelength is different from the excitation
wavelength (incident beam wavelength), scattering due to the excitation radiation is
negligible.
Figure 5 Comparison of Absorptiometry and Fluorometry

In addition to providing high sensitivity, fluorescence detection can provide a fluorescence

spectrum and an excitation spectrum (which is very similar to the absorbance spectrum).
These may be useful in identifying the eluent.
If the eluent contains two compounds, selection of the appropriate excitation and emission
wavelengths may be used to provide qualitative and quantitative information about the
components in the mixture. This point is described in Figure 6. An attempt to quantitate
compound B in a mixture of A and B using absorbance will not be successful because the
absorbance spectra of the two compounds overlap at all wavelengths (Figure 6 (a)).
In contrast, if the fluorescence spectra do not overlap (Figure 6 (b)), quantitative analysis of
each compound can be made by selecting the appropriate fluorescence emission wavelength
(even if the absorption wavelengths of two components are the same).
Absorption spectra Excitation spectra Fluorescence spectra
Component A Component A Component B
Component B
(a) Absorptiometry (b) Fluorometry

Figure 6 Spectroscopic Measurement of a Sample Containing Two Components
Comparison of Analytical Information

Attainable with Absorptiometry and Fluorometry
Absorptiometry Fluorometry
Absorption spectra only • Excitation spectra

(corresponding to excitation spectra)
• Fluorescence spectra
Notes on Fluorescence Analysis

Raman Scattering
When fluorescence is measured, two additional peaks may appear in the spectrum. The
Rayleigh peak appears at the excitation wavelength and is due to scattered light, while the
Raman peak appears at longer wavelength than the excitation. The position of the Raman
band is dependent on the excitation wavelength, while the position of the fluorescence is
independent of the excitation wavelength.
The Rayleigh peak and the Raman peak will occur even if the eluent does not contain any
compound that fluoresces. If there is any doubt whether an observed peak is a Raman peak,
a Rayleigh peak or a peak due to the fluorescence from the compound of interest, simply
change the excitation wavelength slightly.

If the peak is due to the Raman Effect or the Rayleigh Effect, the observed wavelength will
shift. If the peak is due to fluorescence from the compound of interest the wavelength will
not change (although the peak height may change).
The Raman Effect is moderately strong when water is the solvent, but is considerably weaker
for other solvents that are commonly used in HPLC.
80
Relative intensity (%) 60
40
Raman scattering
20
0
350 400 (nm)
Excitation wavelength
Figure 7 Raman Spectrum of Water
Raman Spectral Peak Position at Each Excitation Wavelength
(Excitation Carbon
Water Ethanol Cyclohexane Chloroform
Wavelength) Tetrachloride
Excitation 248 271 267 267 — —
wavelength, and 313 350 344 344 320 346
Raman peak 365 416 405 408 375 410
position (nm) 405 469 459 458 418 461
436 511 500 499 450 502
Notes on Handling Samples That Contain a High Concentration of the

Compound of Interest
When the concentration of the compound of interest is relatively high, the concentration
reported by fluorescence detection may be lower than the actual concentration. This
phenomenon is due to the "Inner Filter effect" which is depicted in Figure 8.
In this example, the concentration of the compound of interest is sufficiently high whereby
molecules that are close to the entrance slit absorb essentially all of the excitation radiation.
These molecules emit, but are not in the region that is defined by the emission slit so that
the fluorescence is not observed. Molecules that are in the region defined by the emission
(fluorescence) slits are not excited by the incoming radiation (since it was already absorbed).
The example described above is an extreme situation. Typically if the absorbance of the
sample is greater than 0.05, the inner filter effect may reduce the observed fluorescence
intensity.
To determine if the inner filter effect is reducing the fluorescence intensity, it is suggested
that the sample be diluted and the intensity measured again. If the reduction in the
fluorescence intensity matches the dilution, the inner filter effect is not in effect.
Excitation beam
Fluorescence
Fluorescence reflected
Figure 8 Sample Having Extremely High Concentration
Another potential error that may occur when fluorescence detection is used is the re-
absorption of fluorescence (self-absorption of fluorescence). An example of this
phenomenon is shown in Figure 9. Re-absorption of fluorescence occurs when the tail of the
shorter-wavelength side of fluorescence spectrum overlaps the tail of the long-wavelength
side of the excitation spectrum. When this phenomenon occurs, the fluorescence spectrum
appears to be somewhat shifted toward the longer-wavelength.
This phenomenon will very rarely cause a substantial error in quantitation when fluorescence
detection is used, and should not be a cause for concern.
Excitation Fluorescence
spectrum spectrum
Relative intensity
Wavelength
Fluorescence re-absorbed
Figure 9 Re-absorption of Fluorescence
Second-order and Third-order Radiation
A diffraction grating that is set to transmit radiation of a specific wavelength (e.g. 250
nm) will also transmit multiples of the radiation (e.g. 500 nm). When a fluorescence
spectrum is collected, the analyst should be aware of the second order (and third order)
phenomena and take care that peaks are not misidentified.
For instance, if the excitation wavelength is 240 nm, the second-order and third-order
excitation occur at 480 and 720 nm, respectively. To eliminate second and third order
radiation, it is simply necessary to place a short-wavelength cutoff filter in the path of
fluorescing radiation (before the fluorescence monochromator).
An Example of a Fluorescence Spectrum
Figure 10 provides an example of a fluorescence spectrum, which contains the various

types of peaks indicated above. In most cases, the fluorescence of the compound of
interest lies closer to the excitation than shown in Figure 9, and overlapping of peak of
interest and the second order radiation is rarely a problem. Typically, the peak for the
compound of interest might correspond to peak *D, and the analyst might have to
correct for the Raman peak (e.g. use a shorter excitation wavelength).
*A
*E
*D
*B
Relative intensity
*C
EX EX × 2 Wavelength
*A Scattering of excitation beam

*B Raman scattering due to eluent
*C Fluorescence of impurity
*D Fluorescence of compound of interest
*E Second-order scattering of excitation beam
Figure 10 Measurement Example of Fluorescence Spectrum
Temperature Dependency of Fluorescence Intensity
In most cases, the fluorescence intensity decreases by 1 to 2% as the temperature

of sample increases by 1 °C. It should be noted, however, that in some biochemical
samples, the fluorescence intensity decreases by approx. 10% as the temperature of
sample increases by 1 °C (especially if the fluorescence is due to the indole ring
system).
Principle of Operation
Excitation Diffusion plate

Plane mirror monochromator
Sample (Flow cell) Monitor detector
(Photodiode)
Xenon Measuring detector

lamp (Photomultiplier)
Emission monochromator
Xenon lamp
(Fluorescence
starter monochromator)
Xenon lamp Photomultiplier high

ballast voltage circuit
Sample Monitor detector

fluorescence amplifier
amplifier
Operational
circuit
Recorder
or integrator
Electrical connection
Optical path
Figure 11 Functional Block Diagram
The beam from the xenon lamp is converged and strikes the excitation monochromator that
disperses the radiation via the large-aperture concave grating. Only radiation of the specified
wavelength is allowed to pass through the exit slit. After the exit slit, the excitation radiation
strikes the sample.
The fluorescence (emitted radiation) enters the emission monochromator, where it is

dispersed by the monochromator. The selected radiation strikes the measuring detector
(photomultiplier).
The monitor detector (photodiode) measures the intensity of the excitation beam as a
function of time.
Features of Light Source

The light source thermally dissociates ozone that is formed from the ultraviolet radiation via
the heat generated by the xenon lamp itself. This allows the use of a fused-silica xenon lamp
that produces intense radiation even in the short wavelength region.
As a safety measure against failure or stoppage of the cooling fan, a thermal sensor works to
extinguish the xenon lamp in about 15 minutes if the fan stops during analysis at a room
temperature of 27 °C, or in about 20 minutes if the fan was stopped from the beginning, in
order to prevent failure or damage of the instrument.
Specifications
Optical system Ratio photometry; intensity of transmitted beam is

monitored
Light source Xe lamp (150 W); Hg lamp (for checking wavelength)
Measuring wavelength range 200 to 850 nm/Zero order

Excitation wavelength
Measuring wavelength range 250 to 900 nm/Zero order

emission wavelength
Photomultiplier must be changed at emission wavelength greater than
731 nm
Spectral bandwidth 15 nm
excitation side
Spectral bandwidth emission 15 nm, 30 nm (variable)

side
With this instrument, spectrum bandwidth is defined by half-value width.
Therefore, in order to separate a spectrum completely, a wavelength
interval of 30 nm or more is required.
Wavelength setting By communication (E-line)
Wavelength accuracy ±3 nm
Wavelength repeatability ±0.5 nm
Recorder output (option) 10 mV full-scale; full scale range 1 to 1000 settable in

increments of 1
Processor output (option) 1 V full-scale; -40 to 1000 mV
Response Changeable in 7 steps corresponding to time constants of

0.05/0.1/0.5/1.0/2.0/4.0/8.0 seconds
Sensitivity With emission-side spectral bandwidth set at 15 nm; S/N

ratio for Raman peak of water 700 or more (Baseline
method); S/N ratio for Raman peak of water 525 or more
(Tangent method)
With emission-side spectral bandwidth set at 30 nm; S/N
ratio for Raman peak of water 900 or more (Baseline
method); Wavelength: 350 nm, Time constant: 2.0 s
Auto Zero range 0 to 1000
Offset range 0 to 1000 (settable in steps of 1)

Spectrum memory Memorized contents: Excitation and fluorescence spectra

Number of spectra memorized: 4
Measuring wavelength range: Changeable in measurement
condition setting
Marker (option) Marker output of approx. 0.6 mV at recorder terminal

(corresponding to about 6 graduations on chart)
Backup memories Full-scale range

Spectrum Measuring range
PMT VOLT
OFFSET value
Time program Number of programs: 9

Settable time: Up to 600 minutes increments of 0.1 minute
Number of steps storable: 100 steps for a total of 9 files
Programmable parameters: Measurement wavelength; (Ex
and Em); Baseline processing; PMT gain
External Output terminals Analog output for processor: 1 V full scale

(option)
Analog output for recorder: 10 mV full scale
External I/O contact Time Program Start

terminals Error Input/Output
Busy Output
Auto Zero Input
Lamp Off Input
Marker Input
Flow cell capacity Standard cell 12 µL (irradiated capacity)
Flow cell withstand pressure 1.0 MPa
Operating temperature range 4 to 35 °C (non-condensing)
Operating humidity range 25 to 85% RH (non-condensing)
Power requirement 100-115/220-240 V AC/50 or 60 Hz
Power consumption 450 VA
Dimensions 340(W) mm× 510(D) mm × 300(H) mm
Weight 29 kg
Installation
Installation . 59
Unpacking
This instrument weighs 29 kg. When carrying this instrument, exercise

care not to incur injury by dropping it accidentally. Be sure to hold the
front and rear parts of the instrument securely when moving it.
WARNING
Carefully unpack the items in the shipping case. Take out module units and standard
accessories. Be sure to place them on a sturdy and level desk or bench.
NOTE: Customers must not attempt installation of the instrument at delivery. To insure safe and
correct use of the instrument, its installation must be carried out your PerkinElmer service
representative.
Before installation of this instrument the customer shall make preparations for satisfying the
installation requirements in accordance with this manual.
If relocation of this instrument becomes necessary after the initial installation (at delivery),
please contact your PerkinElmer service representative.
Checking the Contents

After unpacking, check the delivered contents against the packing list furnished with the
instrument. If anything is missing or damaged, or if you have any question, please contact
PerkinElmer.
Contents:
User’s Guide on CD
Xenon Lamp
E-Line Cable
Signal Cord
E-DIO Cable
Installation Conditions
For installation of this instrument, confirm the following conditions:
Power Supply:
Power voltage: 100 to 115/220 to 240 V AC
Fluctuation: Within ±10% of the rated voltage
Frequency:
50 or 60 Hz
Fluctuation: Within ±0.5 Hz of the rated frequency
Power consumption:
450 VA or more in this instrument only
1050 VA or more in total system
In an example of a total system arrangement, an organizer, Flexar pump, Flexar
autosampler, Flexar column oven and Flexar FL detector are included.
The AC power source must be rated to supply the power requirements of the LC
system and any peripheral equipment.
Lab outlet:
The AC power source should be available near the planned location of the LC system.
Electric Shock due to Improper Grounding
To prevent an electric shock hazard, provide a proper grounding

connection.
Be sure to use the grounded three-prong power cord which is supplied

WARNING with the instrument. The use of a different type of power cord may
result in an electric shock hazard. Connect the three-prong power cord
to a grounded three-prong power outlet.
Installation Space
Prepare a level, sturdy installation table capable of bearing a load of at least 150 kg.
Check and prepare an installation place satisfying the space requirements for your actual LC
system and peripheral equipment to be used in combination.
Environmental Conditions for an Installation Location

When selecting an installation location for this instrument, be sure to check the following
conditions:
1. Operating Ambient Temperature: 10 to 35 °C

Temperature variations during measurement should be minimized.
2. Operating Ambient Humidity: 25 to 85% Relative Humidity (non-condensing)

Installation . 61
3. Atmosphere:
a. Well ventilated.
b. Free from acidic or alkaline gases that could corrode metals significantly.
c. Free from vapors of solvents (e.g., benzene, thinner) that could affect the paint
coating on the instrument.
4. Other Requirements
a. Not exposed to direct sunlight. Exposure to direct sunlight could result in
deteriorated performance or discoloration of the instrument.
Avoid installing the instrument near a window of the laboratory room.
b. Not exposed to drafts directly.
c. Free from intense vibrations and shocks.
d. Apart from a heat source such as a gas burner, electric heater, or oven.
e. Apart from a strong magnetism source such as an electric welder, high-frequency
electric furnace, or pole transformer.
f. Free from dust and dirt.
Contamination with dust and dirt could deteriorate the performance of the
instrument.
g. No abrupt fluctuations in power voltage.
Fluctuations in power voltage could cause noise in analytical data.
h. Do not connect the instrument to a power line used by an electric device that is not
equipped with a noise suppressor circuit (e.g., stirrer, vibrator).
Installing
Remove Transport Screws

1. Remove three screws from the bottom face.
Use a Phillips screwdriver to remove transport screws, *A, *B and *C
Transport screw *C (for lamp house)
Transport screw *A Transport screw *B

(for main body) (for main body)
Figure 12 Location of Transport Screws on the Bottom Panel
2. Remove the lamp house from the instrument. (See Maintenance.)
3. Use a Phillips screwdriver to remove the transport screw *D, *E and Transport TY RAP
*F (Figure 13) from the lamp house.
4. Mount the xenon lamp and place it into the unit. (See Maintenance.)
5. Adjust the position of the xenon lamp.

Please adjust VERTICAL first because the lamp position is shifted downward for fixation
of the transport screw. (See Maintenance.)
Installation . 63
NOTE: After removing transport screws, store them in a safe place so you will not lose them.
Figure 13 Transport Screw Removal/Xe Lamp Attachment
Tubing Connections
In the Flexar Fluorescence Detector, connect tubing from the column to the flow cell inlet
and direct the flow cell outlet to the solvent waste reservoir. Typically, connect tubing from
the pump to the autosampler, column and then to the Fluorescence detector depending on
your system. Hold the tubing in place using the tube management clips on each of the four
corners of the front fascia.
Flow Cell
Inlet
Flow Cell
Outlet
Outlet
Tubing
Inlet
Tubing
Tube Management
Clips
Figure 14 Connecting Tubes

Spare Parts Available

The fuse and xenon lamp are the only replacement parts and consumables for this
instrument. It is suggested that you stock a few fuses and lamps in the laboratory to
minimize down time.
Replacement Parts
Part No. Description
09991667 Fuse - 5 Amp 250 V Slo-Blow
N2922082 Xenon lamp – 150 W
Installing the Maintenance Software

Refer to Appendix A for instructions on how to install the Maintenance Software.
Setup
¾ Turn ON the Flexar Fluorescence LC Detector by pressing the power switch on the front
panel.
At the same time, a test for self-diagnosis (initialization) is carried out. The current
channel setting LED and the Xe LAMP LED will light in green.
The sequence of wavelength drive (check of reference point for drive), lighting of the
lamp and auto zeroing is performed.
Setting the Xenon Lamp Lifetime Warning Timer

The xenon lamp life time timer value is settable through the Maintenance Software.
Injury due to Xenon Lamp Explosion

Replace the xenon lamp with a new one before its cumulative turn-on
time exceeds the guaranteed useful lifetime.
With the lapse of the guaranteed useful lifetime, the bulb wall darkens
WARNING due to evaporation of electrode material and adhesion of scattered
substances, resulting in a decrease in heat dissipation. This may increase
temperature (pressure) inside the bulb excessively to cause explosion.
This setting activates the following function. If the total operation time of the Xe lamp is
longer than the standard lifetime upon turning on the lamp, an error message is indicated to
notify that the time point of lamp replacement is reached.
Installation . 65
1. Start the Maintenance Software program and click the Maintenance button on the
initial screen to display the Maintenance screen.
2. Click the Exchange button on the Maintenance page to display the Xe Lamp
Exchange Alarm.
3. Check the part number of your Xe lamp and input the correct value listed below.
Part No. Part Name Guaranteed Life Limit Allowable Input
N2922082 150W Xe lamp 150 hours 500 hours 500 or less
NOTE: Enter a number about 20 hours less than the allowable limit.
The Default value is 500 (This detector unit has been shipped with N2922082 lamp
attached.)
Operation
Operation . 69
Setting Operating Parameters

You set the Flexar Fluorescence Detector operating parameters in Chromera. Before
setting the parameters in Chromera, make sure the detector’s power switch on the front
panel is ON (indicated by the green LED).
• Allow the detector to warm up at least 1 hour for stabilization of the xenon lamp and
accuracy of measurement.
• Most Flexar Fluorescence Detector settings that you would need to make are made in
Chromera.
• There are some additional settings you may need to make using the Maintenance
Software. See Appendix A
1. To autozero simply click Autozero in the Chromera Control Panel.
2. The output value should be zeroed when zero point adjustment is required or when the
data baseline deviates from the recorder zero point.
3. Set the Excitation wavelength and Emission wavelength in Chromera.
4. Set the Offset in Chromera.

The OFFSET is a set input signal amount that is added to the actually measured value
for output. The range is from 0 to 1000 in increments of 1 step.
0
-10 mV
(a) Offset is not used (b) Offset is used
5. Set the Sampling Rate in Chromera.
6. Set the Sensitivity in Chromera.

PMT VOLT (photomultiplier voltage)
7. Set the Slit Width in Chromera.

A bandwidth value of the emission monochromator (also called the slit width) is
settable. Standard is 15 nm and wide setting is 30 nm.
8. Set the xenon lamp ON or OFF in the Chromera Control Panel.

NOTE: Shortly after xenon lamp is turned off, it cannot be turned on again, since it is still at a high
temperature. Therefore, measurement is impossible. For re-measurement, wait at least 5
minutes before restarting the lamp.
Shutdown Procedure
Turn OFF the detector’s power switch to shut it down. The set measurement conditions are
retained by the memory even when the power is switched off. When power is turned on
again, the conditions that were in place when power was turned off will be restored.
Flushing the Flow Cell After Use

Make sure to flush the flow cell with distilled water after using any buffers or salts in the
mobile phase. Failure to carry out flushing may lead to clogging and/or breakage of the flow
cell.
Operation . 71
Before Performing Analysis

This section describes the basic items to be careful about before starting analysis.

1. This instrument is not explosion-proof. In unattended operation, do
not use organic solvents having an ignition point below 70 °C.
WARNING 2. Beware of ignition hazard when using flammable chemicals such as
organic solvents.
b. Well-ventilate the laboratory room where the instrument is used.
c. Always check the following conditions. If an abnormality is
found, stop operation immediately.
Leakage of solvent or waste solution.
Leakage of solvent inside the instrument.
3. When using flammable chemicals, be careful about possible ignition
due to static electricity. To prevent the build-up of static electricity,
use a conductive container for waste solution and provide proper
grounding connection to it.
If a flammable chemical such as organic solvent leaks from the flow

path of the instrument and its vapor concentration reaches the explosion
WARNING limit, it could cause spontaneous combustion with dangerously explosive
results.
When using a flammable and readily volatile chemical, be sure to check
for leakage from the instrument flow path and ventilate the laboratory
room adequately.
Inflammation or Injury due to Toxic or Corrosive Solvent
When using a toxic or corrosive solvent, be careful not to incur a

physical inflammation or injury.
WARNING For details of the properties of each solvent and how to handle it, refer
to the relevant Material Safety Data Sheets (MSDS). Be sure to handle
each solvent properly.
• Wear proper protective clothes (e.g., safety goggles) so that a

solvent will not come into direct contact with the skin.
• Ventilate the laboratory room adequately to prevent accidental

inhalation of harmful solvent vapor.
Mobile Phase Selection and Cautions on Handling

1. Be careful about restriction on use due to characteristics and temperature of mobile
phase to be used. (See the Table containing the Characteristics of Organic Solvents)
2. When performing high sensitivity analysis with a UV detector, use solvents whose UV
absorption characteristics are especially small. Use solvents for liquid chromatograph
commercially available.
Absorption characteristics of special grade reagents such as methanol, ethanol and
acetonitrile generally and widely used are greatly different in the short wavelength
range, depending on the manufacturing lot and the manufacturer.
Be careful especially when performing high sensitivity analysis.
3. Degas the mobile phase before use.

It is recommended to use a degasser because organic solvents have an especially high
content of air and a poor baseline may result.
A malfunction of the check valve and loss of column efficiency may also occur.
Degasser is especially required for mixing solvents for gradient use. Degas the solvents
before use. See the section in this chapter on Degassing Mobile Phase.
4. When using mobile phase, since the connections of the tubing for the pump and
separation column use stainless steel, solvents or reagents that may corrode stainless
steel should be avoided.
For example, solutions which contain halogen ions such as HCl, KCl, NaCl, NH4Cl.
Reagents which may be restricted are shown in Table 5-1; use the table as a rough
standard.
Operation . 73
Reagents for Mobile Phase that may be Restricted
Use is Use is Possible if Use is Possible if 10% Reagents Unsuitable

Possible 50% or Lower or Lower for SUS316
Phosphoric Acetic acid Disodium phosphate Ammonium chloride
acid
Ammonium citrate Ammonium formate Potassium chloride
Sodium
Ammonium nitrate Ammonium perchlorate Sodium chloride
phosphate
Citric acid Ammonium phosphate Tolufluoroacetic acid
Butyric acid Boric acid
Sodium nitrate Formic acid Note 1:
(up to pH3) Max. concentration of
above reagents should
Hydrochloric acid
be 5-6%.
(up to pH3)
Note 2:
Potassium nitrate
When the above
Sodium reagents have been used
hydrogencarbonate necessarily, wash all the
flow paths with distilled
Sodium carbonate
water completely without
fail.
Do not use reagents that corrode SUS316 though they are not shown in the table above. If
viscosity of solvent is high, pumping is impossible. Be careful.
Characteristics of Organic Solvents

Characteristics of organic solvents as mobile phase for liquid chromatograph are shown in
Characteristics of Organic Solvents Table below. Use it for reference.
This instrument is not explosion-proof. In unattended operation, do not

use organic solvents having an ignition point below 70 °C.
WARNING
Beware of ignition hazard when using flammable chemicals such as
organic solvents.
1. Do not bring a heat or flame source near the instrument.
2. Well-ventilate the laboratory room where the instrument is used.
3. Always check the following conditions. If an abnormality is found,
stop operation immediately.
Leakage of solvent or waste solution.
Leakage of solvent inside the instrument.
4. When using flammable chemicals, be careful about possible ignition
due to static electricity. To prevent the build-up of static electricity,
use a conductive container for waste solution and provide proper
grounding connection to it.
If a flammable chemical such as organic solvent leaks from the flow

path of the instrument and its vapor concentration reaches the explosion
WARNING limit, it could cause spontaneous combustion with dangerously explosive
results.
When using a flammable and readily volatile chemical, be sure to check

for leakage from the instrument flow path and ventilate the laboratory
room adequately.
Inflammation or Injury due to Toxic or Corrosive Solvent
When using a toxic or corrosive solvent, be careful not to incur a

physical inflammation or injury.
WARNING For details of the properties of each solvent and how to handle it, refer
to the relevant Material Safety Data Sheets (MSDS). Be sure to handle
each solvent properly.
Wear proper protective clothes (e.g., safety goggles) so that a
solvent will not come into direct contact with the skin.
Ventilate the laboratory room adequately to prevent accidental
inhalation of harmful solvent vapor.
Operation . 75
Characteristics of Organic Solvents
Ignition Point Vapor Specific

Polarity Viscosity Refractive UV Cut Flash Boiling
Lower Upper Density Gravity
E2 (Al2O3) (cP20 °C) Index off (nm) Point (°C) °C Point (°C)
Limit Limit (Air: 1) (Water: 1)
Fluoroalkanes -0.25 1.25

n-Pentane 0.00 0.23 1.358 210 < -40 308.9 1.5 7.8 2.5 36.1 0.6
Hexane 0.00 1.375 210 -21.7 233.9 1.2 7.5 3.0 68.9 0.7
Isooctane 0.01 1.404 210
Petroleum ether 0.01 0.3 210
n-Decane 0.04 0.92 1.412 46.1 207.8 0.8 5.4 4.9 173.9 0.7
Cyclohexane 0.04 1.00 1.427 210 -20 260 1.3 8 2.9 81.7 0.8
Cyclopentane 0.05 0.47 1.406 210
Diisobutylene 0.06 1.411 210
i-Pentene 0.08 1.371 -17.8 272.8 1.5 8.7 2.4 30 0.7
Carbon disulfide 0.15 0.37 1.626 380 -30 100 1.3 44 2.6 46.1 1.3
Carbon tetrachloride 0.18 0.97 1.466 265
Amyl chloride 0.26 0.43 1.413 225 12.8 343.3 1.6 8.6 3.7 106.1 0.9
Butyl chloride 0.26 1.436 220 -9.4 460 1.8 10.1 3.2 0.9
0.81† o-17.2 463.9 1.0 6.0 144.4
Xylene 0.26 0.61† ~ 1.50 290 m-25 527.8 1.1 7.0 3.7 138.9 0.9
0.64† p-25 528.9 1.1 7.0 138.3
i-Propyl ether 0.28 1.368 220 -27.8 443.3 1.4 21 3.5 68.9 0.7
i-Propyl chloride 0.29 0.37 1.378 225 -32.2 593.3 2.8 10.7 2.7 35 0.9
Toluene 0.29 0.33 1.496 285 4.4 536.1 1.4 6.7 3.1 110.6 0.9
n-Propyl chloride 0.30 0.59 1.389 225 < -17.8 2.6 11.1 2.7 46.1 0.9
Chlorobenzene 0.30 0.35 1.525 32.2 637.8 1.3 7.1 3.9 132.2 1.1
Benzene 0.32 0.80 1.501 280 -11.1 562.2 1.4 7.1 2.8 80 0.9
Ethyl bromide 0.37 0.65 1.424 511.1 6.7 11.3 3.8 37.8 1.4
Ethyl ether 0.38 1.353 220 -45 180 1.9 48 2.6 35 0.7
Ethyl sulfide 0.38 0.23 1.442 290
Chloroform 0.40 0.45 1.443 245
Methylene chloride 0.42 0.57 1.424 245 -50 518.9 3.8 15.4 2.2 38.5 0.9
Methyl i-butyl ketone 0.43 0.44 1.394 330
Tetrahydrofurane 0.45 1.408 220 -14.4 321.1 2 11.8 2.5 66.1 0.9
Ethylene dichloride 0.49 1.445 230 13.3 412.3 6.2 16 3.4 83.9 1.3
Methyl ethyl ketone 0.51 0.79 1.381 330 -6.1 515.6 1.8 10 2.5 80 0.8
i-Nitropropane 0.53 1.400 380 48.9 420.6 2.6 3.1 131.1 1.0
Acetone 0.56 1.359 220 -17.8 537.8 2.6 12.8 2.0 56.7 0.8
Dioxane 0.56 0.32 1.422 260 12.2 180 2.0 22 3.0 101.1 1.0
Ethyl acetate 0.58 1.54 1.370 260 4.4 460 1.8 8 3.5 90 0.9
Methyl acetate 0.60 0.45 1.362 210 -10 501.7 3.1 16 2.6 60 0.9
Amyl alcohol 0.61 0.37 1.410 32.8 300 1.2 10.0 3.0 137.8 0.8
Dimethyl sulfoxide 0.62 4.1
Aniline 0.62 2.24 1.586 70 617.2 1.3 3.2 184.4 1.0
Dimethyl amine 0.63 4.4 1.387 275 < -17.8 312.2 1.8 10.1 2.5 56.7 0.7
Nitromethane 0.64 0.38 1.394 380 35 418.3 7.3 2.1 101.1 1.1
Acetonitrile 0.65 0.67 1.344 210 5.6 1.4 81.7 0.8
Pyridine 0.71 0.37 1.510 305 20 1.8 12.4 2.7 115 1.0
Butyl cellosolve 0.74 0.94 220
i-Propanol n-Propanol 0.82 1.38 210 11.7 398.9 2.0 12 2.1 82.8 0.8
Ethanol 0.88 2.3 1.361 210 12.8 422.8 4.3 19 1.6 78.3 0.8
Methanol 0.95 1.20 1.329 210 11.1 463.9 7.3 36 1.1 63.9 0.8
Ethylene glycol 1.11 0.60 1.427 210 111.1 412.8 3.2 197.2 1.1
Acetic acid Large 19.9 1.372
Water Large 1.26 1.333
Salts & buffer Larger
* L.R.Snydar, Dekker. “Principles of Adsorption Chromatography”

†
The Handbook of Chemistry, 2nd ed., The Chemical Society of Japan
Solvent with low boiling point as shown in a box cannot be used.

Warnings for Using Organic Solvents as Mobile Phase
1. When using organic solvents, be careful about UV cut off wavelength.

Light cannot be transmitted in the range shorter than this
WARNING wavelength. For example:
The cut off wavelength of chloroform is 245 nm.
Chloroform cannot be used in the range shorter than 245 nm
(e.g. 210 nm).
2. Boiling point of mobile phase.

Solvents of low boiling point cannot be used as mobile phase. Use
solvents whose boiling point is 55 °C or higher.
Solvents of low boiling point are not only dangerous but also
generate bubbles when sucked by pump so that normal pumping will
be impossible.
3. When performing high sensitivity analysis with UV detector, use

solvents which absorb especially small amount of UV light.
Since special grade reagents such as methanol and acetonitrile
generally used absorb the shorter wavelengths a lot, use reagents for
liquid chromatograph commercially available.
Acetonitrile (special grade) Condition

Photometer: U-3000
Slit : 2 nm
Acetonitrile (for HPLC) Blank : Air
Figure 15 Absorption Spectra of Organic Solvents

Operation . 77
Cautions on Static Electricity
Ignition of Flammable Chemicals by Static Electricity
When using flammable chemicals, be careful about possible ignition due

to static electricity. To prevent the build-up of static electricity, use a
WARNING conductive container for waste solution and provide proper grounding
connection to it.
Since flammable organic solvents are used for the high speed liquid chromatograph, be
careful about heat and fire.
Since thin tubes are used for the liquid chromatograph, static electricity is easily generated.
If charged liquid is collected into an insulated waste container, for example, and an electric
discharge occurs, flammable material if present may catch fire.
Maintenance
Maintenance . 81
Performance Check
Check the performance when the instrument has been installed.
NOTE: First adjust the xenon lamp position and then check the performance. Positional adjustment
of the lamp is necessary for an accurate performance check.
Energy Check of Xe Lamp

Check the energy value of the Xe lamp at a wavelength of 254 nm (see Operation).
NOTE: When the energy value of the Xe lamp has decreased by 50% or more in comparison with
the value at the start of use, the useful life of the lamp is considered to have expired. Use
the decrease in energy value plus baseline fluctuation (data variation or noise) in judging the
lifetime of the lamp.
Sensitivity (S/N) Check

The S/N ratio of the Raman spectrum of water is used to measure the sensitivity of the
fluorescence detector. Run this check using the Maintenance Software, S/N(Raman of
Water), as described in Appendix A.
Check Wavelength Accuracy (WL Check) by Using Hg

Lamp
NOTE: First adjust the xenon lamp position and then check the wavelength accuracy. An accurate
wavelength check cannot be made unless the lamp position is adjusted.
Wavelength accuracy is checked by using the bright line at 254 nm of the Hg lamp. It passes
when the wavelength error is within (±3 nm, or fails when the error exceeds 3 nm. Run this
check using the Maintenance Software, WL Check, as described in Appendix A.
1. Run the Maintenance Software and click the Maintenance button on the initial
screen to display the Maintenance screen.
2. To test the wavelength accuracy, click on the WL Check… button.

A box displaying the last set of values measured displays.
3. Click on Check Start to execute the routine using the internal Hg lamp.
After the values are measured (it takes about 5 to 10 min) the results will be displayed
in the table.
¾ If the difference is within +/- 3 nm, then the specifications have been met. If the
difference is > 3 nm, then a correction must be applied.
• Click on the Correction … button. The correction factors will automatically be
determined and applied, and new values will be displayed in the table.
You can view the individual scans that were obtained by clicking on the Show
Spectrum … button.
Maintenance 83
Leak Sensor Check

1. Perform an operational check of leak sensor by dripping water onto the leak sensor.
Drip water Leak sensor
2. Confirm that a leak error is detected.
Observe the following precautions before using the leak sensor which
detects a liquid leak.
CAUTION
1. While a solvent is used, the leak sensor function should be kept
active.
2. The leak sensor function of this instrument may not work when
liquid leaks in a small amount.
Leakage of a highly volatile solvent may not be detectable either.
Even when the leak sensor function is activated, you should be
careful not to overlook liquid leakage for avoiding a serious
situation.
3. As a routine checkup, visually check for any liquid leak before
measurement.
4. As a periodic checkup, make sure the leak sensor works normally.
5. Cleaning the Leak Sensor

When separating the drain receiver from the instrument for cleaning of the leak sensor,
etc., keep the leading end of sensor in contact with the drain receiver.
NOTE: Mount the leak sensor at the correct position. If the sensor is mounted at a wrong position, it
may not work properly or an error may occur.
Periodic Checks
Check Item Frequency of Check Procedure
Check
Lighting of lamp Daily Check that Xe is indicated on LEDs.
Liquid leakage Daily Visually check that the liquid is not leaking
from the eluent bottle, tubing and waste
bottle.
Fluorescence intensity Daily Autozero the detector and check if FL

check indication is zeroed.
Lifetime of lamp Monthly 1) Check lamp for service life by energy

check, or by cumulative lamp turn-on
time.
2) Check if baseline drifts considerably (or if

baseline fluctuates or noise becomes
high).
Leak sensor Monthly 1) Drip water onto the leak sensor and
confirm the occurrence of an error.
2) After confirmation, wipe off the water.
Wavelength accuracy Yearly Check wavelength by using bright line of Hg

lamp at 254 nm.
Maintenance 85
Maintenance and Inspection Methods

NOTE: First adjust the xenon lamp position (see Maintenance), then make an energy check of the
xenon lamp and a wavelength accuracy check using the Hg lamp. Correct measurement
results will not be obtained unless the lamp position is adjusted.
Energy Check of Xe Lamp

Check the energy level of the Xe lamp at a wavelength of 254 nm.
NOTE: When the energy level of the Xe lamp has decreased by 50% or more in comparison with the
level at initial use, the useful life of the lamp is considered to have ended. The lamp life can
be judged by the energy level, or by baseline fluctuation (fluctuation of data and/or noise).
Check of Wavelength Accuracy by Using Hg Lamp

NOTE: First adjust the xenon lamp position and then check the wavelength accuracy. An accurate
wavelength check cannot be made unless the lamp position is adjusted.
Wavelength accuracy is checked by using the bright line at 254 nm of the Hg lamp. It passes
when the wavelength error is within (±3 nm, or fails when the error exceeds 3 nm. Run this
check using the Maintenance Software, WL Check, as described in Appendix A.
NOTE: Wavelength calibration is necessary when it fails. See the procedure for the calibration, later
in this chapter.
Check of Lamp Logbook

The cumulative turn-on time, number of lightings, and last replacement date for the Xe and
Hg lamps can be checked via the GLP function.
Checking and Washing the Flow Cell

This instrument is not explosion-proof. During unmanned
operation, do not use organic solvents having an ignition
WARNING point below 70 °C.
Beware of ignition hazard when using flammable chemicals such as
organic solvents.
b. Well-ventilate the laboratory room where the instrument is
used.
c. Always check the following conditions of the flow path, etc.
If an abnormality is found, stop operation immediately.
Leakage of solvent or waste solution
Leakage of solvent inside the instrument
When using flammable chemicals, be careful about possible
ignition due to static electricity. Particularly when using non-
conductive chemicals, employ a conductive vessel made of metal or the
like and provide grounding connection correctly.
If a flammable chemical such as organic solvent leaks from the

flow path of the instrument and its vapor concentration
WARNING exceeds the explosion limit, it may cause spontaneous
combustion with dangerously explosive results.
When using a flammable and readily volatile chemical, check

for leakage from the instrument flow path and ventilate the
laboratory room adequately.
If the inside of the flow cell is contaminated, it must be cleaned. Prepare the following items
for the cleaning.
• Glass syringe with capacity of about 10 mL
• Washing solution
Maintenance 87
1. Remove the front magnetized panel by gripping the left and right sides and pulling it
straight off.
Figure 16 Front Panel Removed
2. Loosen the retaining screw of the flow cell unit (see the figure below).
Flow Cell Unit

(P/N 8906923)
Retaining Screw
Figure 17 Flow Cell Location
3. Pull the flow cell unit straight out.
4. Check the inside of the cell visually to see if it is contaminated with any foreign
substance (oil, salt, buffer, etc.).
5. Check the outside of the cell for any leakage.
6. If contamination is observed inside the cell, it must be cleaned with an appropriate

solution. If an aqueous solvent has been used, distilled water should be used for
washing. And if an organic solvent has been used, then isopropyl alcohol should be used
for washing. Connect the syringe to the tip of the drain tube, and inject the washing
solution into the cell for cleaning (see the Figure 18).
NOTE: This flow cell unit uses apart similar to the one used in the fluorescence detector. Be careful
not to mount the part of the flow cell unit to the detector and vice versa.
Figure 18 Injection by Syringe
6. Replace the washing solution with the eluent that is used for separation, and repeat the
procedure in step (5).
7. If air bubbles are observed in the flow cell, supply solution into the cell with the pump at
a flow rate within 1.0 mL/min and a pressure within 1.0 MPa. While the solution is being
supplied into the flow cell, pinch the end of the drain tube with your fingers for a few
seconds to increase the pressure inside the cell, then release your fingers quickly. Repeat
this procedure a few more times until all the air bubbles have been removed.
Figure 19 Removing Air Bubbles
NOTE: Applying too much pressure may damage the flow cell. Set the pressure limiter of the pump
at 1.0 MPa max before removing air bubbles.
Maintenance 89
Disassembly and Cleaning of Flow Cell

If contamination is not removed by just flushing the interior of the flow cell, then
disassemble and clean the cell in the procedure below.
To disassemble the Flow Cell:
Figure 20 Flow Cell Unit
1. Loosen retaining screws 1 and 2 and detach the shielding plate.
2. Detach the cell window.
3. Remove the cell clamp.
4. Loosen the set screws.
5. Take out the flow cell.
Figure 21 Disassembly of Flow Cell Unit

To wash the Flow Cell:
1. Wash the flow cell in running water. If the contamination cannot be removed with
running water, then use an ultrasonic cleaner. In this case be sure to put the flow cell
in a paper or plastic container and then place it in the ultrasonic cleaner, so as to avoid
scratching the cell.
2. Wipe the surface of the flow cell with gauze that is moistened with acetone or ethanol.
NOTE: Be careful not to scratch the flow cell surface when disassembling it.
NOTE: Make sure to put the flow cell in a paper or plastic container when cleaning it. The use of a
glass container may scratch the cell or preclude obtaining the desired performance from the
instrument.
Reassemble the Flow Cell
1. Assemble the flow cell into the cell holder in the orientation shown in Figure 22 and fix it
with the cell clamp.
2. Tighten the upper and lower retaining screws 1 and 2.
Figure 22 Assembly of Flow Cell Unit
3. Attach the cell window and shielding plate.
4. After assembling the flow cell in the order reverse to disassembly, flow a liquid through
the unit and make sure there is no leakage.
NOTE: Be careful not to scratch the flow cell surface when assembling it.
NOTE: Take care about the orientation of the polished surfaces of the flow cell when putting it into
the cell holder (see Figure 22). A mistake in the orientation may preclude obtaining the
proper performance from the instrument.
NOTE: Tighten the setscrew 1 fully with hand, and then tighten the setscrew 2 about 30° manually
or tighten the setscrew 1 further 30° with a wrench.
Maintenance 91
Leakage from the Flow Cell

It is important to check for any liquid leakage from the flow cell unit prior to analysis. If a
leak is found, stop the pump immediately and remove the flow cell unit. Take the following
procedure to remedy the leak.
Figure 23 Inlet and Outlet Tube
1. Supply liquid using the pump, and check that liquid comes out of the OUTLET tube of
the flow cell.
2. If liquid does not come out of the tube, it indicates the tube is clogged. Replace the
OUTLET tube with a new one.
3. If a leakage occurs although liquid comes out of the tube normally, it may be that the
upper nipple is not securely tightened. Try tightening another 10 to 20° lightly with the
furnished spanner.
4. Wipe up any liquid spilt on the outside of the flow cell.
5. Before returning the flow cell unit into position, supply liquid with the pump and make
sure there is no more leakage.
NOTE: It is important that the OUTLET tube of the flow cell be checked first for clogging. If the
upper nipple is found to be loose and is retightened with the wrench without checking the
OUTLET tube, then the flow cell may be damaged or broken.
NOTE: If leakage is found in the sample compartment or on the optical base inside it, wipe up the
liquid thoroughly and then clean with a cloth moistened with water.
Cautions on Storage of the Flow Cell

If the flow cell will be left unused for more than a few days, clean the flow cell interior
thoroughly by pumping distilled water or alcohol through it and then store it in a safe place.
If the period of nonuse is less than a few days, the inside of the cell may be filled completely
with a solvent such as isopropyl alcohol and then stored.
Lamp Replacement
Injury Due to Xenon Lamp Explosion
Before removing the xenon lamp for replacement, turn off the xenon
lamp (turn off power to the instrument) and then wait for at least one
WARNING hour until the xenon lamp becomes sufficiently cool to reduce its internal
pressure the normal safety level.
The xenon lamp and its housing become very hot during operation and
remain hot for a while after operation. Touching the hot xenon lamp or
its housing could result in severe burns. Before proceeding to lamp
WARNING replacement, turn off the power switch and then wait for at least one
hour until the lamp and its housing cool down sufficiently.
How to Replace the Xenon Lamp
When replacing the xenon lamp immediately after the use, the lamp must be cool to the
touch. Once the Xe lamp has been cooled adequately, you can replace it.
1. Turn off the Xenon Lamp and allow it to cool.
NOTE: After the xenon lamp is off, the fan continues to cool down the lamp. Wait for at least 30
minutes in before turning off the power supply.
Electric Shock due to Contact with Inside of Instrument
The xenon lamp is supplied with high voltage of 30 kV, which could
cause an electric shock to result in serious or fatal injury.
WARNING
Before removing the light source cover for replacement of the light
source lamp, be sure to turn off the power switch.
Injury due to Xenon Lamp Explosion
If a strong shock or impact is applied to the xenon lamp or if the surface

of its quartz glass part is scratched, it may explode and scatter glass
WARNING pieces, resulting in personal injury. Be sure to wear proper protective
gear such as safety goggles, safety mask, thick long sleeves, and gloves
when handling the xenon lamp.
Maintenance 93
2. Turn OFF the power switch and unplug the power cord.
3. Loosen six retaining screws securing the light source cover, and remove the cover (see
Figure 24).
Retaining
Screws
Figure 24 Removal of Light Source Cover
4. Put on safety goggles before performing the following steps.
5. Detach the connector between lamp house and main body, and loosen the lamp house
retaining screw.
Connector
Flow Cell Unit
Flow Cell
Retaining Screw
Lamp House
Grip
Retaining Screw
Figure 25 Connector and Retaining Screw Location

6. Gently grip and lift the lamp house as you pull it toward you to remove the lamp house
from the instrument body.
Lamp House
Grip
Figure 26 Removing the Lamp House
Figure 27 Lamp House after Removal

Maintenance 95
Injury due to Xenon Lamp Explosion (at disposal)

The xenon lamp is filled with high-pressure gas (approx. 1 MPa at room
temperature, approx. 4 MPa under operating condition), and this high-pressure
gas still remains in the xenon lamp after it is demounted for replacement.
WARNING
For disposal of the used xenon lamp, wrap it with a thick cloth (e.g., triple-folded
cotton cloth) completely and crush its glass part with a hammer or the like. Then,
discard the xenon lamp as a dangerous waste item properly. If the xenon lamp is
discarded without being crushed, it could explode due to possible impact at the
time of disposal, scattering glass pieces to cause personal injury.
Injury due to Xenon Lamp Explosion (at mounting)
When loosening or tightening the retaining nut for the xenon lamp, be
careful not to apply excessive force to its glass bulb part. Never hold and
WARNING turn the glass bulb part for loosening/tightening.
7. Remove the old xenon lamp by loosening the cathode nut and removing the cathode
lead plate (see the following figure).
Figure 28 Xenon Lamp Connections

Do not touch the quartz glass part of the xenon lamp with bare hands.
WARNING If the quartz glass part of the xenon lamp is contaminated with dust or
fingerprints, wipe it using a gauze sheet or absorbent cotton cloth slightly
moistened with high-quality alcohol.
If the xenon lamp is turned on with dust or fingerprints left on the surface of
the quartz glass part, it may cause contamination burn-in to decrease the
mechanical strength of the glass part, resulting in explosion of the xenon
lamp.
Be sure to mount the xenon lamp in the specified direction. If the mounting
direction (polarity) is wrong, the cathode will be consumed significantly to
WARNING disable turn-on of the lamp.
Mount the lamp so that the '+' (anode) mark on it will be positioned at the
support metal of the lamp holder. If the lamp with its cathode consumed
excessively is used continuously, pressure inside the lamp bulb may become
too high, causing possible explosion. To prevent this, replace the lamp with a
new one immediately if its cathode has been consumed substantially.
If the nut on the lamp base and wiring part is loose, the contact resistance
between them will increase due to poor contacting. This could generate a
WARNING large amount of heat to make the lamp extremely hot, resulting in possible
explosion. To prevent this, be sure to tighten the nut securely.
8. Mount and attach a new xenon lamp in place.
9. Attach the cathode lead plate to the threaded part of the cathode, and secure the lead
plate with the cathode nut (Figure 29). Mount the lamp so that the protrusion on the
bulb faces the instrument front panel (Figure 30).
10. Be careful not to touch the lamp bulb. Note that a thin metal wire is attached near the
center of the bulb, which facilitates ignition of the lamp. Take care not to break this wire
when handling the lamp.
11. Also, if the thin metal wire is not located on the side of the lamp having the small
protrusion, slide the wire so it is properly positioned there.
Maintenance 97
Figure 29 The Xenon Lamp
Figure 30 Orientation of Lamp Bulb Protrusion

12. As shown in Figure 29, put the anode terminal (marked “+”) of the lamp into the metal
fixture on the ceramic plate. Then, using the anode nut, mount the lamp so that the
protrusion on the bulb is positioned as shown in Figure 30. Refer to Figure 24 for a view
of the lamp when assembled.
NOTE: Mount the xenon lamp so that the protrusion on the bulb faces the instrument front panel
(Figure 27). A mistake in the orientation may cause an increased light loss and preclude
obtaining the desired performance from the instrument.
13. After mounting the Xe lamp in the lamp house, assemble it into the main body (Figure 25).
Raise the lamp house and insert it into the main body, then lower it and press it downward
into place. The lamp house will not move if installed correctly. Finally, secure the lamp
house with retaining screws and connect its connector with the main body.
14. When installation of the lamp house is finished, close the light source cover and turn ON
the power (check that the lamp comes on).
Now proceed to Positional Adjustment of the Xe Lamp.

Maintenance 99
Positional Adjustment of Xe Lamp
Direct Gazing at Illuminating Xenon Lamp Could Cause Eye Damage

The xenon lamp radiates intense ultraviolet light when it is on.
If you look at the illuminating xenon lamp during coarse adjustment of its
WARNING position, your eyes could be damaged.
Do not look at the xenon lamp directly when it is lit. Be sure to wear tinted
safety glasses to prevent possible eye damage.
The position of the beam slightly differs from one lamp to another, so it is necessary to
adjust the lamp position before the detector is first used or when the lamp is replaced. Use
the three adjusting screws on the right side of the instrument for the adjustment.
HORIZONTAL: For adjusting horizontal position
FOCUS: For adjusting the focal point of light source
VERTICAL: For adjusting vertical position
Lamp adjustment involves coarse adjustment, in which the image of the light beam is
monitored and fine adjustment, in which detailed adjustment is made while observing the
data. Coarse adjustment is required at first.
Horizontal
Focus
Vertical
Figure 31 Lamp Positional Adjustment
Coarse Adjustment Using Chromera

Set the Excitation wavelength at 550 nm, and make the adjustment while observing the
image of the light beam.
1. Launch Chromera and select the Run Time Manual Control.

2. In the present status, pull out the flow cell unit (see Figure 25).
3. Set an Excitation wavelength to 550 nm.

The Emission wavelength will not be set here. Leave the present setting as it is.
4. Place a paper strip at the Excitation beam exit center of the sample compartment as
shown in the following figure and observe the condition of the excitation beam.
5. Repeat the VERTICAL, HORIZONTAL and FOCUS adjustments in this order little by little
until the excitation beam (rainbow color) becomes brightest. The coarse adjustment of
the light source position is thus completed. Please adjust VERTICAL first in installation
because the lamp position is shifted downward for fixation of the transport screw.
Observe the excitation

beam on paper strip
Figure 32 Observation of Excitation Beam
Fine Adjustment
Following the coarse adjustment, set the Excitation wavelength at 350 nm make a fine
adjustment while observing the data.
1. Re-mount the flow cell unit.
2. Launch Chromera and select the Run Time Manual Control
3. Set an Excitation wavelength of 350 nm
4. Emission wavelength will not be set here. Leave the present setting as it is.
5. In Chromera status monitor the energy, turn the FOCUS, HORIZONTAL and VERTICAL
adjusting screws gradually in a repetitive manner while watching the Excitation value on
the screen until this value is maximum. When a maximum value is reached, the fine
adjustment of the lamp position is completed.
Maintenance 101
Fuse Replacement
Electric Shock due to Contact with Inside of Instrument
When replacing the fuse with a new one, be careful not to receive an
electric shock. To prevent this, be sure turn off the power switch and
WARNING unplug the power cord before proceeding to fuse replacement.
Prior to fuse replacement, make sure the power switch of the instrument is OFF and the
power cord is unplugged.
1. Hold the tabs on both sides of the fuse holder and detach the holder.
Figure 33 Fuse Replacement
2. Replace the blown fuse(s) with new one(s), then reinsert the fuse holder into the power
entry module.
Troubleshooting
Troubleshooting . 105
Warnings
Electric Shock in Contact with Inside of Instrument
The xenon lamp utilizes a power supply of 30 kV, so there is a danger of

serious or fatal injury due to electric shock
WARNING
Before removing the light source cover for replacement of the light
source lamp in a detector, turn OFF the power switch and unplug the
power cable.
The xenon lamp remains hot for a while even after power-off and can
severely burn you if touched. Before replacement of the lamp, turn off
WARNING the power and unplug the power cable. Then wait at least two hours for
the lamp to cool down.
Upon detaching the light source cover, the xenon lamp is automatically
turned off by a safety mechanism. Avoid releasing this safety
mechanism.
Directly Gazing at Illuminated Xenon Lamp Could Cause Eye

Damage
The xenon lamp radiates intense ultraviolet light when it is on. Do not
WARNING look at the xenon lamp directly when it is lit. Wear tinted safety glasses
to prevent possible eye damage.
NOTE: If the instrument becomes faulty, contact the dealer from whom you purchased the
instrument or the nearest PerkinElmer service engineer. Repairs must be left to the service
personnel who have undergone technical training since there are potential hazards.
106 Flexar Fluorescence LC Detector User’s and Service Guide
Troubleshooting Guide
Symptom Cause Judgment/Check Remedy
Self-diagnosis The power cord plug Visual check Plug in the power cord
(initialization) is not is not securely securely.
performed when the plugged in.
POWER switch is
turned on.
The fuse is blown. Check the fuse for Replace the fuse with a new
continuity. one.
The light source Visual check Retighten the retaining

cover is not properly screws of the light source
attached. (The cover cover.
activates a safety
interlock switch.)
Excessive noise The flow cell is Visual check Clean the flow cell.
contaminated.
The mobile phase is Spike noise occurs. Degas the mobile phase
not sufficiently thoroughly.
degassed.
The mobile phase Check the mobile Purify the mobile phase, or
contains impurities. phase. replace it with mobile phase
of a higher quality.
The gain level of the Check the gain level Adjust the gain level of the
recorder is too high. of the recorder. recorder.
Check the pump.
The pump is faulty. Check to ensure the
operation of the
pump is smooth.
Check for noise when
the pump is stopped.
The xenon lamp has Check the Xe lamp Replace the xenon lamp
reached the end of logbook data. with a new one.
its useful life.
Excessive drift The warm-up period ____________ Wait until the instrument
is not sufficient. becomes stable (at least 1
hour).
Mobile phase is Check each fitting. Retighten the offending

leaking from a fitting. fitting.
An impurity is eluting Stop feeding liquid Wait until elution is

from the column. from the pump. completed, or replace the
column with a new one.
Troubleshooting 107
Symptom Cause Judgment/Check Remedy
There is a significant ____________ Control the temperature

change in the variation.
ambient
temperature.
The mobile phase is Check the mobile Purify the mobile phase, or
not sufficiently pure. phase solvent. replace it with one of a
higher quality.
Poor S/N ratio Flow cell Visual check Clean the flow cell.
contaminated
Xenon lamp off- Check lamp energy Adjust lamp position.

position level
Error in flow cell Visual check Correctly mount flow cell.

mounting
Flow cell surface Visual check Replace flow cell.

scratched
Appendix A
Appendix A 111
Maintenance Software Program

The Maintenance Software may be used for three reasons:
1. To test the performance of the FL Detector for…

• Wavelength Accuracy
• Raman Signal-to-Noise
2. To obtain a stop-flow excitation or emission scan.
3. To set the number of hours to sound the alarm to remind you to exchange the lamp.
The procedures to install and use this program are detailed in this document.
Installing the Maintenance Software

NOTE: Before installing the Maintenance Software, be sure that the USB board is installed in the FL
Detector, and that the black E-Line cable which supplies power to the USB board is
connected.
1. Power down the Flexar FL Detector.

Connect the cable from the USB port on the back of the detector to a USB port on the
computer.
USB Cable on the back of the

detector that connects to the
computer USB
E-Line Cable on the back of

the detector that connects the
USB Board to FL Detector
2. Insert the CD (P/N 09936761) into the computer CD drive.

3. Double-click on the PKI_FL_Maintenance_Install folder and the following screen

displays:
4. Double-click on the Disk 1 folder and the following screen displays.
5. Double-click on the setup.exe file. The InstallShield Wizard opens.

Appendix A 113
6. Click Next to continue.
7. Click Next to install the software in the default folder c:\FLMaintenance which will
automatically be created, or if desired, you can change the folder location.
8. Click Install to begin the installation of the software.

A Setup Status screen will appear and show the installation progress bar.
9. Click Finish when the installation is complete. Then Reboot the computer.
Note: If Chromera is installed, the Fluorescence drivers for the USB port should be installed as part
of the Chromera installation. These drivers will be installed specifically for whichever USB
port the cable is connected to on the computer. If this cable is disconnected and
reconnected to a different USB port on the computer at a later time, the drivers will need to
be installed again. Skip to Operation.
10. Turn on the FL Detector. Immediately, Windows will detect that new hardware is
present and the Found New Hardware Wizard will open.
11. Click on No, not this time then click Next to continue.
12. Click on Install from a list or specific location (Advanced) and then Next.
13. From the next screen, click on Include this location in the search.
14. Click Next.

Windows will now search for and install the driver (this may take several minutes).
Appendix A 115
Upon completion, the following screen appears.
15. Click on Finish to complete the installation.

Operation
1. Open the C:\FLMaintenance\PKITools\HIMAINTE folder.
2. To launch the software, double-click the maintenance.exe file.

The opening screen appears.
3. If the serial number of the instrument is displayed in the middle of this screen, then
communications have been established. If the serial number is not displayed, then click
the Connect button.
Appendix A 117
4. Once communications are established, click on the Maintenance button.

The Maintenance screen displays.
From this screen:
¾ the Status of the instrument is read
¾ some instrument real time parameters may be changed (e.g., Lamp on/off, PMT
Voltage, the Em bandwidth)
¾ performance functions such as AutoZero, wavelength (WL) Check and the

signal-to-noise (S/N) of Raman band of water, may be measured
¾ an excitation or emission spectrum may be obtained.
Note: If printed copies are desired for the results obtained from any of the tests performed in the
Maintenance Software, a screen capture program, or simultaneously holding down the Alt
and PrintScreen keys, may be used to capture the image. The captured image can them be
pasted into another program (e.g., WordPad), and printed.
5. To test the wavelength accuracy, click on the WL Check… button.

A box displaying the last set of values measured displays.
6. Click on Check Start to execute the routine using the internal Hg lamp.
After the values are measured (it takes about 5 to 10 min) the results will be displayed
in the table.
¾ If the difference is within +/- 3 nm, then the specifications have been met. If the
difference is
> 3 nm, then a correction must be applied.
• Click on the Correction … button. The correction factors will automatically be
determined and applied, and new values will be displayed in the table.
You can view the individual scans that were obtained by clicking on the Show
Spectrum … button.
Appendix A 119
7. To measure the S/N ratio for the Raman band of water:
a. Fill the flow cell with degassed HPLC grade water.

b. From the main screen, click on the S/N(Raman of Water)… button.
c. The S/N ratio may be determined using either the standard Em band width (15
nm) or the wide Em bandwidth (30 nm); the default is the standard. The signal
test may be performed by clicking on the appropriate S/N Ratio button.
d. The results will be displayed:
e. You can display the Raman band by clicking on the Show Spectrum … button.
f. Click on the Noise Check… button to perform the noise measurement and
calculation.
g. The results are displayed:
The specifications for the S/N measurement results are: Tangent >545
8. Click the Get Spectrum button on the Maintenance page of this software program to
obtain either an excitation or emission FL scan of whatever is in the flow cell.
Appendix A 121
9. Click the Exchange button on the Maintenance page of this software program to
choose between the standard (500 hours) and extended lifetime (1000 hours) lamp
lifetime. When this number of hours is reached, an alarm will sound.
10. When done, click the Close button on the Maintenance page, then click the Close
button on the Maintenance Software main page.
Description Of Contact Signals

The following two types of contact signals are used.
1. Contact Signals (START, ERROR, BUSY) Entering the E-Line Connector
These are contact signals having input/output functions that are used in common for
each unit. These signals are respectively formed from:
the START (IN/OUT), ERROR (IN/OUT) and BUSY (IN/OUT) lines.
2. Individual Contact Signals Used by Each Unit

These are contact signals that are input and output via 3-pin connector. Their names and
functions vary with the unit.
A name that indicates the function is given at each connector.
e.g., output contact signal: EVENT of pump
e.g., input contact signal: LAMP OFF IN of detector
Appendix A 123
Terminology
¾ Auto zero [A/Z]

Absorbance is electrically adjusted to zero to cancel insignificant parts of data. In
common analytical practice, perform auto zero adjustment immediately before or
after the start of operation.
¾ Auto zero in
The external contact signal can be input for auto-zero adjustment.
¾ Battery backup
This function is incorporated to retain measurement parameters and other analytical
conditions even after power is turned off.
¾ Diffusion plate
This quartz plate is used for diffusion of excitation beam.
¾ Drain port
Equipped for allowing solution leaked from the flow cell/joint to flow to an external
waste bottle.
¾ E-Line
Digital network line dedicated for analytical system.
¾ Emission (Fluorescence) intensity
Unit of measure of fluorescence (relative intensity).
¾ Emission (Fluorescence) wavelength
A particular wavelength of fluorescence emission caused by excitation
¾ Ex (Excitation) wavelength
Used for exciting a sample.
¾ Flow cell
Contains a sample for measurement.
¾ Fluorescence
A kind of luminescence, which is emitted from a sample when a stimulating
excitation beam is given to it.
¾ Full scale
Used to determine the magnitude of output to such an external device as data
processor.
¾ GLP
The confidence function is used to check the status of instrument.
¾ Initial screen
After power on, the initial screen appears upon completion of initialization.
¾ Lamp energy
Represents the intensity of lamp radiation. This value is used as an approximate
value of lamp service life.
¾ Lamp house
Equipped for holding the light source lamp. This part becomes extremely hot when
the lamp is lit. Be careful not to incur burns by touching it.
¾ Lamp-off in
The external contact signal can be input for turning off the light source lamp.
¾ Lamp turn-on count
Indicates the number of times the light source lamp has been turned on.
¾ Lamp turn-on cumulative time
Indicates a cumulative total time of lamp turn-on periods.
¾ Log book
Means the information containing records of instrument operating conditions.
¾ Marker in
The external contact signal can be input to provide marking on record.
¾ Offset
On output, a preset input signal value is added to an actually measured value. This
function is particularly useful when the baseline varies on the negative side.
¾ Output range
Used to determine the magnitude of output signal to such an external device as data
processor.
¾ Photomultiplier
Used to detect fluorescence emission.
¾ Raman scattering
In Raman effect, a kind of scattered light appears on the side of wavelength slightly
longer than excitation wavelength.
¾ Raman spectrum
Scattered light spectrum that occurs due to Raman effect.
¾ RECORDER
Used for analog output to a recorder.
¾ Scan
Means a sequential shifting operation of monochromator wavelength for spectral
measurement.
¾ Scattered light
Includes Raman scattered light, 2nd-order light, 3rd-order light, etc. In fluorescence
analysis, particular attention should be paid to scattered light.
¾ 2nd-order light
A spectral peak (a kind of scattered light) which appears at a wavelength of 2 times
the level of excitation intensity. In a common arrangement, a filter is used to remove
or cut off the 2nd-order scattered light.
Appendix A 125
¾ Stokes law
This law states that the wavelength of a fluorescent emission is always longer than
the excitation wavelength.
¾ 3rd-order light
A spectral peak (a kind of scattered light) which appears at a wavelength of 3 times
the level of excitation intensity. In a common arrangement, a filter is used to remove
or cut off the 3rd-order scattered light.
¾ Time program
The time program is used to adjust such measurement parameters as detection
wavelength according to measurement time.
¾ Utility
Used to set up operating conditions of the instrument.
¾ Xenon lamp
The xenon lamp is used for fluorescence measurement.
Index
A L
Air Bubbles ......................................................... 39 Labels
Assembling ......................................................... 62 warning signs ................................................. 16
WEEE Instructions .......................................... 30
Laboratory Practices
B GLP ............................................................... 34
Buffers ............................................................... 37 Lamp Logbook
check ............................................................. 85
Leakage
C flow cell ......................................................... 91
LEDs .................................................................. 43
Check
lamp logbook ................................................. 85
Chemicals M
Definitions of Warnings................................... 35
Hazardous...................................................... 34 Maintenance
Cleaning the Instrument ...................................... 31 Hg lamp wavelength check.............................. 85
Conventions performance check ......................................... 81
Notes, cautions and warnings.......................... 13 periodic checks............................................... 84
text ............................................................... 13 Xe energy check ............................................. 81
Messages
troubleshooting ............................................ 106
D
Decontamination................................................. 31 O
Operating Conditions ........................................... 26
E Operation
entering parameters ....................................... 69
Electric Shock shutdown procedure ....................................... 70
safety warnings .............................................. 29 turning on power ............................................ 64
Electrical Safety .................................................. 27
ElectroMagnetic Compatibility .............................. 28
Electromagnic Wave Interference P
precautions .................................................... 31
Environmental Conditions .................................... 26 Parameters
operation ....................................................... 69
Performance Check
F maintenance .................................................. 81
Periodic Checks
Flammable Chemicals maintenance .................................................. 84
explosion ....................................................... 21 Power
safety warnings .............................................. 21 turning on ...................................................... 64
Flow Cell Power Switch, front ............................................. 43
flushing ......................................................... 70 Precautions......................................................... 22
leakage.......................................................... 91 Operating Pressure.............................................. 40
storage .......................................................... 91
Fuse
replacment....................................................101 Q
Quality Control .................................................... 34
G
General Operating Conditions .............................. 26 R
Replacing
H fuse ............................................................. 101
Hg Lamp Wavelength Check

maintenance .................................................. 85 S
Safety
I Suitability test ................................................ 34
Safety Information .............................................. 22
Installation electrical safety .............................................. 27
conditions ...................................................... 60 environmental conditions ................................ 26
IPV .................................................................... 34 Shutdown ........................................................... 70
Solvents ............................................................. 36
Auto-Ignition Temperature .............................. 38
Low boiling points........................................... 37
128 . Flexar User’s Guide
safety warnings .............................................. 37

Spare Parts......................................................... 64
W
Static Electricity Warnings
cautions......................................................... 77 Hazardous Chemical........................................ 35
Storage Warranty ............................................................ 11
flow cell ......................................................... 91
X
T
Xe Lamp Energy Check
Troubleshooting Messages..................................106 maintenance .................................................. 81
Tubing Arrangements.......................................... 63 Xenon Lamp Explosion
safety warnings .............................................. 23
U
Unpacking .......................................................... 59
PerkinElmer Life and Analytical Sciences
710 Bridgeport Avenue
Shelton, CT 06484-4794, U.S.A.
Internet: http://www.perkinelmer.com
email: info@perkinelmer.com
PerkinElmer is a registered trademark of PerkinElmer, Inc.

09936951A Flexar FL LC Detector Users and Service Guide

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09936951A Flexar FL LC Detector Users and Service Guide

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LIQUID CHROMATOGRAPHY

User’s and Service Guide

Part Number Release Publication Date

09936951 A August 2009

Or emailed to: info@perkinelmer.com

Copyright © 2009 PerkinElmer, Inc.

PerkinElmer is a registered trademark of PerkinElmer, Inc.

About this Manual

Chapter 2 Safety Practices

Important safety information is provided in this chapter.

Chapter 3 System Description

This chapter contains information on the detector, a descriptions of Fluorometry and

This chapter provides information on operating the instrument.

g. Failure due to maintenance or repair by other than service personnel

THE MANUFACTURER MAKES NO WARRANTIES, EITHER EXPRESS OR IMPLIED,

NO ORAL OR WRITTEN INFORMATION OR ADVICE GIVEN BY THE MANUFACTURER,

Conventions Used in this Manual

Bold text refers to text that is displayed on the screen.

Notes, Cautions and Warnings

Warning Signs on the Instrument

FOR CONTINUED PROTECTION

Power On/Off push button

We use the term WARNING to inform you about situations that

Ignition of Flammable Chemicals

Explosion of Vapor from Flammable Chemicals

Prevention of Instrument Tipover

Common Safety Precautions

During Installation, Maintenance, and Relocation

• Do not perform other maintenance and checkup procedures to avoid jeopardizing

• After installation, do not relocate the instrument. If the instrument is relocated,

Xenon Lamp Safety

Injury Due to Xenon Lamp Explosion

P/N Part Name Limit

If a strong shock or impact is applied to the xenon lamp or if the

Be sure to wear proper protective gear such as safety goggles, safety

To prevent this, be sure to tighten the nut securely.

Touching Hot Part Could Result in Burns

Direct Viewing of Xenon Lamp Could Cause Eye Damage

Before Turning on Xenon Lamp Again

General Operating Conditions

Normal Operating Conditions:

Safe Operating Conditions:

If the equipment is used in a manner not specified herein, the protection

Lethal voltages are present at certain areas within the

Any interruption of the protective conductor (earth ground) inside or

When working with the instrument:

• Do not attempt to make internal adjustments or replacements except as directed in

• Service on electrical components should be performed only by a qualified

• Some power cords available from PerkinElmer are illustrated below:

North America Old British Standard

Europe British Standard

ElectroMagnetic Compatibility (EMC)

Electric Shock Due to Contact with Inside of Instrument

Electric Shock Due to Improper Grounding

WEEE Instructions for PerkinElmer Products

Web address: http://las.perkinelmer.com/OneSource/Environmental-directives.htm

Customer Care: 1-800-762-4000 (inside the USA)

Decontamination and Cleaning

The customer’s responsible body is required to follow the "Equipment Decontamination

In Canada, call toll free 800-561-4646 and speak to Customer Support.

Cleaning the Instrument

Warning Labels on the Instrument

Figure 1 Warning label on the right side of the instrument

Figure 2 Warning labels on the front of the instrument