Page 1
LS 55
User’s Guide
Page 2
Release History
Part Number Release Publication Date
09934436 C March 2007
Any comments about the documentation for this product should be addressed to:
User Assistance
PerkinElmer Ltd.
Chalfont Road
Seer Green
Beaconsfield
Bucks
HP9 2FX
United Kingdom
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 © 2007 PerkinElmer, Inc.
Produced in the UK.
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.
FL WinLab is a trademark of PerkinElmer, Inc.
Windows is a trademark of the Microsoft Corporation.
Posidriv is a registered trademark of the Phillips Screw Company.
Page 3
LS 55 User’s Guide . 3
Contents
Contents.................................................................................................... 3
Warnings and Safety Information...................................................... 7
Safety Information ..................................................................................... 8
Environmental conditions for safe operation ..........................................8
Warning Labels on the Instrument ............................................................ 10
Further assistance.............................................................................. 11
Introduction ..................................................................................... 13
About this Manual .................................................................................... 14
Conventions ...................................................................................... 14
Definitions......................................................................................... 17
Specifications of the LS 55........................................................................ 18
Unpacking and Installation .............................................................. 21
Introduction............................................................................................. 22
Shipping kit list ........................................................................................ 24
Lifting the LS 55 Luminescence Spectrometer ............................................ 25
Removal of the shipping clamps................................................................ 26
Electrical Connections............................................................................... 29
Connection to electrical mains supply.................................................. 29
Accessory connectors......................................................................... 29
Rear panel connectors........................................................................ 30
Switching on the LS 55............................................................................. 34
Accessories: Installation and Use .................................................... 37
Single Position Cellholder (standard) ......................................................... 38
Description ........................................................................................ 38
Installation and maintenance.............................................................. 38
Stirred Single Position Cellholder ............................................................... 39
Description ........................................................................................ 39
Installation and maintenance.............................................................. 39
Operation.......................................................................................... 39
Stirred Four-Position Cellchanger .............................................................. 40
Description ........................................................................................ 40
Installation and maintenance.............................................................. 40
Biokinetics Accessory................................................................................ 41
Description ........................................................................................ 41
Installation and maintenance.............................................................. 41
Operation.......................................................................................... 42
Screw-fitting Flowcell ............................................................................... 43
Description ........................................................................................ 43
Installation and maintenance.............................................................. 44
Operation.......................................................................................... 44
Installing Thermostatting Tubing to Cellholders.......................................... 45
The four-position cellchanger ............................................................. 47
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4 . LS 55 User’s Guide
Using the Septum Injector ........................................................................ 48
Using Stirrer Bars ..................................................................................... 50
Minimum volumes with various cuvette types............................................. 51
Semi-micro Cuvette and Holder ................................................................. 52
Description ........................................................................................ 52
Installation ........................................................................................52
Stirred Semi-micro Cuvette .......................................................................53
Description ........................................................................................ 53
Operation ..........................................................................................54
Care of Optical Mirrors Inside Cellholder Accessories ..................................55
Resistance to Solvents ..............................................................................56
Sipper...................................................................................................... 57
Description ........................................................................................ 57
Installation and maintenance ..............................................................58
Operation ..........................................................................................60
LC Flowcell............................................................................................... 63
Description ........................................................................................ 63
Installation ........................................................................................63
Operation ..........................................................................................65
Front Surface Accessory............................................................................ 66
Description ........................................................................................ 66
Installation and maintenance ..............................................................67
Operation ..........................................................................................70
Using Powders ...................................................................................71
Plate Reader ............................................................................................ 73
Description ........................................................................................ 73
Installation ........................................................................................73
Operation ..........................................................................................80
The Well Plate Reader application ....................................................... 80
The TLC Scan application.................................................................... 81
Measuring TLC plates, gels or other flat samples.................................. 82
Red-sensitive Detector.............................................................................. 83
Description ........................................................................................ 83
Installation ........................................................................................84
Total Emission Accessory ..........................................................................85
Description ........................................................................................ 85
Installation ........................................................................................85
Operation ..........................................................................................86
External Fibre Optic Accessory ..................................................................87
Description ........................................................................................ 87
Installation ........................................................................................88
Operation ..........................................................................................92
The Fast Filter Accessory ..........................................................................94
Description ........................................................................................ 94
Installation ........................................................................................96
Operation ........................................................................................ 104
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LS 55 User’s Guide . 5
The Polarizer Accessory .......................................................................... 105
Description ...................................................................................... 105
Installation ...................................................................................... 106
Bit Switches..................................................................................... 110
Operation........................................................................................ 110
The Low Temperature Accessory............................................................. 111
Description ...................................................................................... 111
Installation ...................................................................................... 112
Filling the accessory with liquid nitrogen ........................................... 117
Operation........................................................................................ 118
Removing the accessory................................................................... 119
Accessory spares.................................................................................... 120
RS232 Commands ..........................................................................121
Introduction........................................................................................... 122
Section 1- RS232C Communications ........................................................ 125
Monochromator Drive....................................................................... 127
Plate Reader Driver.......................................................................... 127
Source Control and Photomultiplier Control........................................ 128
Slit and Filter Motors ........................................................................ 128
Analogue Output.............................................................................. 128
Sample Acquisition Accessories......................................................... 129
Fast Filter Accessory (FFA) ............................................................... 129
Section 2 - Command Formats ................................................................ 131
Monochromator Scan Control Commands .......................................... 133
Time Drive Commands ..................................................................... 136
TLC Scan Commands ....................................................................... 139
Data Collection/Processing Set Up Commands ................................... 142
Data Output Control Commands ....................................................... 147
Motor Control .................................................................................. 148
Calibration Commands ..................................................................... 151
Miscellaneous Commands................................................................. 152
Plug-in Accessory Control Commands................................................ 155
Well Plate Reader (WPR) Commands ................................................ 157
Fast Kinetics Application (FKA) Commands ........................................ 158
Fast Filter Accessory (FFA) Commands .............................................. 161
System Description and Maintenance ............................................ 163
The Optical System ................................................................................ 164
The Electronics System and Signal Handling ............................................ 166
Instrument Modes............................................................................ 167
Signal Handling Diagram .................................................................. 170
Fuses .................................................................................................... 171
Other Fuses..................................................................................... 172
Removing the Main Cover....................................................................... 173
Refitting the main cover ................................................................... 175
Removing the Main Optical Cover............................................................ 176
Refitting the optical cover................................................................. 178
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6 . LS 55 User’s Guide
Customizing filter wheels ........................................................................179
Changing the Lamp ................................................................................ 181
Preparing the LS 55 for transport ............................................................188
Inserting the clamps ........................................................................ 188
Lifting the LS 55 Luminescence Spectrometer .................................... 191
Removing the clamps ....................................................................... 191
Cleaning the LS 55 .................................................................................192
Cleaning the air filter .............................................................................. 193
Changing the Instrument Operating Voltage ............................................ 194
Changing the mains fuse .................................................................. 196
The mains voltage plate ...................................................................197
Index ..............................................................................................199
Index..................................................................................................... 200
Page 7
Warnings and Safety
Information
Page 8
8 . LS 55 User’s Guide
Safety Information
The LS 55 Luminescence Spectrometer has been designed and tested in accordance
with PerkinElmer Specifications and IEC 61010-1, 'Safety requirements for electrical
equipment for measurement, control and laboratory use'.
This apparatus is protected in accordance with IEC Class 1 rating. This manual
contains information and warnings that must be followed by the operator to ensure
safe operation and to retain the instrument in a safe condition.
The instrument has been designed for indoor use and will operate correctly under
the following conditions:
Ambient temperature 15 °C to 35 °C
Relative humidity 75% maximum, non-condensing
Environmental conditions for safe operation
The LS 55 has been designed to be safe under the following conditions:
• Indoor use.
• Altitude up to 2000 m.
• Temperature range 5 °C to 40 °C.
• Maximum relative humidity 80% for temperatures up to 31 °C, decreasing
linearly to 50% relative humidity at 40 °C.
• Mains voltage fluctuations not to exceed 10% of the nominal voltage.
• An IEC Installation Category II (Overvoltage Category II) classification, suitable
for connection to local level power supplies.
• An IEC Pollution Degree 2 classification: normally only non-conductive pollution
occurs, occasionally, however, a temporary conductivity caused by
condensation must be expected.
• An IEC Insulation Class 1 rating for external circuits – only connect equipment
that meets the requirements of IEC 61010-1, IEC 60950 or equivalent
standards.
Page 9
Warnings and Safety Information . 9
This equipment must be earthed (grounded).
WARNING
Any interruption of the protective conductor, inside or outside the instrument, or
disconnection of the protective earth terminal is likely to make the instrument
dangerous.
When the instrument is connected to the mains supply, terminals may be
hazardous when live and the opening of covers or the removal of parts (except
those to which access can be gained by hand) is likely to expose live parts. Any
adjustment, maintenance and repair of the opened operating instrument must be
carried out only by a skilled person who is aware of the hazards involved (a
RESPONSIBLE BODY). The instrument must be disconnected from all voltage
sources before it is opened for any adjustment, replacement, maintenance or
repair.
Capacitors inside the instrument may still be charged even if the instrument has
been disconnected from all voltage sources. Only fuses with the required current
and voltage rating and of the specified type are to be used for replacement.
Makeshift fuses must NOT be used and fuse holders must not be short-circuited.
Whenever it is likely that the protection has been impaired, the instrument must be
made inoperative and secured against any unauthorized operation. The protection
is likely to be impaired, for example, when the instrument:
• Shows visible damage;
• Fails to perform the intended measurement;
• Has been subjected to prolonged storage under unfavorable conditions;
• Has been subjected to severe transport stresses.
NOTE: Using the equipment in a manner not specified in this User’s Guide may also
impair the protection provided by the equipment.
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10 . LS 55 User’s Guide
Warning Labels on the Instrument
Figure 1 Caution label
When the Caution label (
risk of danger’. Refer to this User’s Guide to find out the nature of the potential
hazard and any actions which have to be taken.
There are three Caution labels on the instrument:
• In the sample area – where the symbol denotes that there is a possibility of
eye damage, and you must not stare into the beam in the sample
compartment.
• Near the mains inlet fuse – where the symbol denotes that the operator
replaceable fuse (see
approved fuse.
• By the left hand screw that fixes the top cover (at the front left of the
instrument) – where the symbol denotes that removing this cover may expose
hazardous voltages. Any adjustment, maintenance, or repair of the opened,
operating instrument, must be performed by a skilled person who is aware of
the hazards involved (a RESPONSIBLE BODY).
Figure 1) is attached to the instrument it means ‘Caution,
Fuses
on page 171) is not an IEC fuse, but a CSA
Figure 2 High voltage label
The high voltage label (see
access to hazardous voltages.
Figure 2) means that removing this cover will give
Page 11
Warnings and Safety Information . 11
There are two High Voltage labels on the instrument:
• Inside the instrument on top of the source cover.
• Inside the instrument on top of the monochromator cover (on the right side of
the instrument).
Both these labels mean that the instrument must be switched off, and the mains
supply lead removed from its socket before the cover is removed. Any adjustment,
maintenance, or repair of the opened, operating instrument, must be performed by
a skilled person who is aware of the hazards involved (a RESPONSIBLE BODY).
Further assistance
For technical assistance, please contact your local PerkinElmer office or agent, or
the address at the front of this manual.
Page 12
12 . LS 55 User’s Guide
Page 13
Introduction
Page 14
14 . LS 55 User’s Guide
About this Manual
This LS 55 User's Guide describes specifications and the installation of the
LS 55 Luminescence spectrometer and its accessories.
Information about the FL WinLab Software package can be found in the
FL WinLab Software User's Guide
Conventions
The following conventions are used in this manual:
• Normal text is used to provide information and instructions.
Bold text refers to text that is displayed on the PC screen.
•
• UPPERCASE text, for example ENTER or ALT, refers to keys on the PC
keyboard. '
time, for example,
• All eight character ‘numbers’, for example L2250140, are PerkinElmer part
numbers unless stated otherwise.
Notes, warnings and cautions
Three terms, in the following standard formats, are also used to highlight special
circumstances and warnings.
+' is used to show that you have to press two keys at the same
ALT+F.
, which is delivered with the instrument.
NOTE: A note indicates additional, significant information that is provided with
some procedures.
Page 15
WARNING
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.
D
DK
E
F
I
NL
P
Warning (Warnung)
Bedeutet, daß es bei Nichtbeachten der genannten Anweisung
zu einer Verletzung des Benutzers kommen kann.
Warning (Advarsel)
Betyder, at brugeren kan blive kvæstet, hvis anvisningen ikke
overholdes.
Warning (Peligro)
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)
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)
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)
Betekent dat, wanneer de genoemde aanwijzing niet in acht
wordt genomen, dit kan leiden tot verwondingen van de
gebruiker.
Warning (Aviso)
Significa que a não observância da instrução referida poderá
causar um ferimento ao usuário.
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16 . LS 55 User’s Guide
CAUTION
DK
NL
D
E
F
P
I
We use the term CAUTION to inform you about situations that
could result in serious damage to the instrument or other
equipment. Details about these circumstances are in a box like
this one.
Caution (Achtung)
Bedeutet, daß die genannte Anleitung genau befolgt werden
muß, um einen Geräteschaden zu vermeiden.
Caution (Bemærk)
Dette betyder, at den nævnte vejledning skal overholdes nøje
for at undgå en beskadigelse af apparatet.
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.
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.
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.
Caution (Opgelet)
Betekent dat de genoemde handleiding nauwkeurig moet
worden opgevolgd, om beschadiging van het instrument
te voorkomen.
Caution (Atenção)
Significa que a instrução referida tem de ser respeitada para
evitar a danificação do aparelho.
Page 17
Introduction . 17
Definitions
OPERATOR: Person operating the equipment for its intended purpose.
RESPONSIBLE BODY: Individual or group responsible for the use and
maintenance of the equipment and for ensuring that the OPERATORS are
adequately trained.
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18 . LS 55 User’s Guide
Specifications of the LS 55
Principle: Computer controlled ratioing luminescence spectrometer with the
capability of measuring fluorescence, phosphorescence, chemiluminescence and
bioluminescence.
Source: Xenon discharge lamp, equivalent to 20 kW for 8 μs duration. Pulse width
at half height <10 μs.
Sample detector: Gated photomultiplier with modified S5 response for operation up
to around 650 nm. Red-sensitive R928 photomultiplier can be optionally fitted for
operation up to 900 nm.
Reference detector: Photodiode for operation up to around 900 nm.
Monochromators: Monk-Gillieson type monochromators cover the following ranges:
• Excitation 200-800 nm with zero order selectable.
• Emission 200-650 nm with standard photomultiplier with zero order
selectable, 200-900 nm with optional R928 photomultiplier.
Synchronous scanning is available with constant wavelength or constant energy
difference.
Wavelength accuracy: +
Wavelength reproducibility: +
Spectral bandpass: The excitation slits (2.5-15.0 nm) and emission slits
(2.5-20.0 nm) can be varied and selected in 0.1 nm increments.
Phosphorescence mode: Delay and gate times can be varied to measure
phosphorescence. Minimum total period 13.0 ms (50 Hz)
Scanning speed: Scanning speed can be selected in increments of 1 nm for
10-1500 nm/min. Time-dependent data can also be collected.
Emission filters: Computer selectable cut-off (high-pass) filters at 290, 350, 390,
430 and 515 nm, a blank (to act as shutter), a 1% T attenuator and clear beam
position.
Sensitivity: Signal to noise is 500:1 r.m.s., using the Raman band of water with
excitation at 350 nm, excitation and emission bandpass 10 nm.
Standard cellholder: A single position water thermostattable holder for 10 mm
cuvettes.
1.0 nm
0.5 nm
Page 19
Supply Voltages: 100 V / 120 V / 220 V / 230 V
Frequency: 50 / 60 Hz
Power: 150 VA
Introduction . 19
Page 20
20 . LS 55 User’s Guide
Page 21
Unpacking and Installation
Page 22
22 . LS 55 User’s Guide
Introduction
The Model LS 55 Luminescence Spectrometer consists of a spectrometer unit
connected to a personal computer (PC). At the front of the Model LS 55 is the
sample compartment, which is hinged on the underside. The compartment can be
opened by using the finger grip on top of the compartment, as shown in
to pull the cover down.
Figure 3 The LS 55
The sample cells are located in the sample compartment; a range of sampling
accessories can be installed in this area. Also within the sampling area are two
sockets used for controlling accessories that are electrically operated.
Figure 3,
Figure 4 Inside the sample compartment
Accessory
control
sockets
Page 23
Unpacking and Installation . 23
The Model LS 55 has several other sockets on the rear panel. The electrical mains
socket is located on the lower right hand side (viewed from the back) of the rear
panel. On the left hand side of the rear panel are an RS232C socket and a terminal
block.
Figure 5 The rear of the LS 55
CAUTION
Do not obstruct the air filter and cooling vents on the rear of
the instrument.
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24 . LS 55 User’s Guide
Shipping kit list
Part Number Description
L2255133 Communications cable, LS 45 to PC
L2255142 Analog signal cable
OC973134 Fuse, 250 V T2 A Slo-Blo
OC962348 Fuse, 250 V, 1 AT
04969185 Fuse, 250 V, T100 mA Slo-Blo
04970839 Fuse, 250 V, 2 A Slo-Blo
04967940 Fuse, 250 V, 500 mA Slo-Blo
04960999 Hex key (5/64 AF, for lamp change)
04961071 Spare septa (x3, for septum injector)
04967778 Screwdriver 3/16 inch Blade
04969221 Screwdriver No. 2 Posidriv
L2251286 + L2251287 Septum injector assembly
L2251188 Mains Label 100 V / 220 V
Page 25
Unpacking and Installation . 25
Lifting the LS 55 Luminescence Spectrometer
Consult local codes of practice issued by safety advisors before
attempting to lift the spectrometer.
WARNING
As the LS 55 Luminescence Spectrometer weighs approximately 49 kg
(approximately 59 kg with packaging), we recommend that the spectrometer is
lifted by 2 adults, and that it is lifted by the base of the instrument.
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26 . LS 55 User’s Guide
Removal of the shipping clamps
Two clamps are used during shipping of the LS 55 to prevent damage to the
monochromators. The clamps are located inside the instrument main cover, and
must be removed before switching on the LS 55.
CAUTION
To remove the shipping clamps proceed as follows:
1. Position the LS 55 on the front of the bench so that it overhangs the bench by
approximately 8 cm.
2. Undo the two captive screws positioned in recesses underneath the front of
the instrument.
There is one on the left side and one on the right side, as shown in
Figure 6 The cover retaining screws
Do not switch on the LS 55 before removing the clamps, or the
calibration of one or both monochromators will almost
certainly be compromised, resulting in a wavelength failure of
14 nm. This will have to be recalibrated by a service engineer,
and is not covered by warranty!
Figure 6.
3. Lift the main cover and rest it on the stay, as shown in
The shipping clamps are now accessible.
Figure 7.
Page 27
Unpacking and Installation . 27
Figure 7 Inside the LS 55
4. Remove the two screws securing one of the clamp plates, as shown in
Figure 8.
Figure 8 Shipping clamp screws
5. Lift out the plate.
6. Remove the hexagonal bolt, as shown in
Figure 9 Removing the shipping bolt
Figure 9.
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28 . LS 55 User’s Guide
7. Turn the clamp plate through 180 degrees so that the hole in the plate no
longer lines up with the hole where the bolt was, then replace it, securing it
with the two screws removed in step 4, as shown in Figure 10.
Figure 10 Replacing the plate
8. Repeat steps 4 to 7 for the second clamp.
9. Store the two hexagonal clamps in their fixing points on the left side of the
instrument, as shown in
Figure 11.
Figure 11 Storing the bolts
10. Lower the instrument cover, close the sample compartment and secure the
cover by retightening the two screws loosened in step 2.
11. Slide the instrument back on the bench so that it does not protrude.
Page 29
Unpacking and Installation . 29
Electrical Connections
Connection to electrical mains supply
The mains socket is located on the instrument rear panel, as shown in Figure 12.
Ensure that the voltage plate displays the correct voltage setting for the mains
supply to be used before plugging the mains lead into the socket. If the correct
voltage is not displayed, refer to
194.
page
Figure 12 Mains connection and voltage plate
Changing the Instrument Operating Voltage
Accessory connectors
Inside the sample area door are two electrical control sockets for accessories, as
shown in
socket has 19 pins, so it is not possible to incorrectly connect accessory control
plugs. Connecting specific accessories is described in the relevant sections of this
manual.
Figure 13. Note that the left-hand socket has 12 pins and the right-hand
on
Figure 13 Accessory electrical sockets
NOTE: Remove the two dust covers before connecting accessories.
Page 30
30 . LS 55 User’s Guide
Rear panel connectors
On the left-hand side of the rear panel (viewed from the back) is a socket labeled
RS232C Controller and an eight tag terminal block, as shown in
Figure 14.
Figure 14 Rear panel electrical connections
Page 31
Unpacking and Installation . 31
The RS232C connection
The RS232C connection is used to communicate with your PC. Connect your LS 55
to your PC as follows:
1. Attach the anti-surge protector to the RS232C Controller socket of the
LS 55, securing it with the built-in screws.
2. Attach the RS232C cable, supplied, to the anti-surge protector.
3. Attach the 9-pin socket of the RS232C cable to an RS232C socket on the PC.
Note which RS232C socket on the PC was used, as this information must be
entered during installation of the FL WinLab software.
The Terminal block
The terminal block is used to provide analog signal output from the LS 55, to
initiate remote starts and to provide logic status signals for use with intelligent
external systems.
To make a connection in the terminal block:
1. Insert a small screwdriver into the bottom of the terminal block and push
upwards.
2. Feed the connecting wire into the hole at the top of the terminal block.
3. Remove the screwdriver.
The wire is now firmly clamped.
Analog signal outputs
External signal integration devices, chart recorders, etc., can be connected to the
analog outputs. One of the connecting wires (in an unbalanced system having one
ground and one signal wire this must be the ground wire) must be connected to the
OVA connection. The other wire can be connected to the required output, 1V, 10V
or 10mV.
Remote starts and event marking
Some FL WinLab software applications can be remotely started on receiving a
contact closure between the Remote Start and OVA connections on the terminal
block. For example, if a liquid chromatograph or stopped-flow rapid mixing device is
used, then the FL WinLab TimeDrive applications can be automatically started by
the device.
Page 32
32 . LS 55 User’s Guide
Timed event marks can be recorded by supplying a contact closure between the
OVA and the Event Mark connections, or by using the EVENT button on the
Biokinetic accessory. All FL WinLab software applications which display a Show
Timed Events option will respond to the contact closure/button press.
Scan Busy
This is a logic level signal, and follows the status of the LS 55:
• during data collection the Scan Busy line is set high.
• on ending the data collection run the logic level reverts to zero.
This function is particularly critical when an external device is attached and the
instrument can be started by a Remote Start event. The external device should
then monitor the Scan Busy line and not send out a further start signal until the
Scan Busy line shows that data collection is complete.
Since this signal is a logic level, devices requiring a contact closure can be
connected via a small solid-state relay (with a switching diode for anti-bounce).
Optimal connection to external autosamplers
Some commercially available autosamplers can be programmed to send and receive
contact closure signals. Connecting such an autosampler to the LS 55 is
straightforward since the LS 55 can be connected via the Remote Start connectors
Remote starts and event marking
(see
establishes one-way communication and cannot be considered optimal since the
control system is not closed.
on page 31). This connection, however, only
Better control can be achieved by making a closed control loop. Use the Remote
Start to control the LS 55’s measurement and the Scan Busy to control the
autosampler. A typical application of such a control system is shown in
Figure 15.
Page 33
Unpacking and Installation . 33
Figure 15 Autosampler control system
NOTE: The use of a solid state relay and anti-bounce signal diode provides the
autosampler with status information from the LS 55.
Such a system, if programmed properly in the autosampler, is considered ´closed´
since the LS 55 will wait for the autosampler signal before performing a
measurement, then the autosampler will wait until the LS 55 has finished being
´busy´ before taking the next sample.
Page 34
34 . LS 55 User’s Guide
Switching on the LS 55
The instrument is switched on and off using the switch on the left-hand side panel
of the instrument, as shown in
Figure 16 The power switch
Figure 16.
Make sure that the power switch on the left side of the
instrument is not obstructed. Leave approximately 200 mm
gap to allow easy access in case of emergency.
WARNING
When the instrument is switched on, the neon lamp located on the front lower lefthand side of the instrument is lit.
The LS 55 takes about 1 minute to initialize and should be switched on before the
PC. The procedure for switching on is as follows:
1. Before switching on any of the units, ensure that the PC is connected to the
LS 55 via the RS232C cable and that both the LS 55 and PC are connected to
the electrical mains supply.
As a precautionary measure, a mains surge filter can be used to protect the
PC (the LS 55 is very robust and not damaged by typical mains surges). This
filter should be used between the mains supply and all components of the LS
55 PC system including the PC, LS 55 and printer.
2. Switch on the LS 55 using the power switch on the lower left-hand side panel.
The neon lamp on the lower left-hand side of the LS 55 will light, indicating
that the instrument is on.
Page 35
Unpacking and Installation . 35
3. Switch on the PC.
4. When Windows has loaded, start the FL WinLab software.
NOTE: If the instrument has not yet finished initializing, this will be shown on the
bottom-right side of FL WinLab Application dialogs. This will automatically
reset to Online when the system is ready. At this point, data collection can
proceed.
Page 36
36 . LS 55 User’s Guide
Page 37
Accessories: Installation and Use
Page 38
38 . LS 55 User’s Guide
Single Position Cellholder (standard)
Figure 17 Single position cellholder
Description
The standard cellholder (L2250140) delivered with the LS 55 accepts 12.5 mm
square (10 mm pathlength) cuvettes, and can be thermostatted using circulated
water (as detailed in
45).
page
Installing Thermostatting Tubing to Cellholders
on
Installation and maintenance
This cellholder needs no alignment or setting up.
Maintenance consists of routine cleaning of the optical mirrors built into the
cellholder block, see
55) and cleaning the base and pressure bar if spillages occur (see
page
to Solvents
on page 56).
Care of Optical Mirrors Inside Cellholder Accessories
Resistance
on
Page 39
Accessories: Installation and Use . 39
Stirred Single Position Cellholder
Figure 18 Stirred single position cellholder
Description
The single position stirred cellholder (L2250141) accepts 12.5 mm square
(10 mm pathlength) cuvettes, and can be thermostatted using circulated water (as
detailed in
Additionally, a stirrer bar can be inserted into the cuvette to ensure that cells,
proteins, etc., remain in suspension. Stirring is also essential for accurate
temperature work. Without stirring, temperature gradients of 1 °C can build up
within the cuvette. Inert polymer-coated bars are delivered with the accessory (see
Using Stirrer Bars
Installing Thermostatting Tubing to Cellholders
on page 50).
on page 45).
Installation and maintenance
This cellholder needs no alignment or setting up.
Maintenance consists of routine cleaning of the optical mirrors built into the
cellholder block, see
55) and cleaning the base and pressure bar if spillages occur (see
Solvents
on page 56).
Care of Optical Mirrors Inside Cellholder Accessories
on page
Resistance to
Operation
The stirrer status (Low, High or Off) can be set by clicking the accessory icon in
the LS 55 Status application, as discussed in
Using Stirrer Bars
on page 50.
Page 40
40 . LS 55 User’s Guide
Stirred Four-Position Cellchanger
Figure 19 Stirred four-position cellchanger
Description
The four-position stirred cellchanger (L2250134) accepts 12.5 mm square
(10 mm pathlength) cuvettes, and can be thermostatted using circulated water (as
detailed in
Installing Thermostatting Tubing to Cellholders
on page 45).
Additionally, a stirrer bar can be inserted into the cuvette to ensure that cells,
proteins, etc., remain in suspension. Stirring is also essential for accurate
temperature work. Without stirring, temperature gradients of 1 °C can build up
within the cuvette. Inert polymer-coated bars are delivered with the accessory (see
Using Stirrer Bars
can be stirred continuously.
on page 50). Each position has its own stirrer, so each cuvette
Installation and maintenance
This cellholder needs no alignment or setting up.
Maintenance consists of routine cleaning of the optical mirrors built into the
cellholder block (see
55) and cleaning the base and pressure bar if spillages occur (see
page
to Solvents
on page 56).
Care of Optical Mirrors Inside Cellholder Accessories
on
Resistance
Page 41
Accessories: Installation and Use . 41
Biokinetics Accessory
Figure 20 Biokinetics accessory
Description
The Biokinetics accessory (L2250145) is a single position cellholder, which accepts
12.5 mm square (10 mm pathlength) cuvettes, and can be thermostatted using
circulated water (as detailed in
45).
page
Installing Thermostatting Tubing to Cellholders
on
Additionally, a stirrer bar can be inserted into the cuvette to ensure that cells,
proteins, etc., remain in suspension. Stirring is also essential for accurate
temperature work. Without stirring, temperature gradients of 1 °C can build up
within the cuvette. Inert polymer-coated bars are delivered with the accessory (see
Using Stirrer Bars
on page 50).
Installation and maintenance
This cellholder needs no alignment or setting up.
Maintenance consists of routine cleaning of the optical mirrors built into the
cellholder block (see
55) and cleaning the base and pressure bar if spillages occur (see
Solvents
on page 56).
Care of Optical Mirrors Inside Cellholder Accessories
on page
Resistance to
Page 42
42 . LS 55 User’s Guide
Operation
The most important feature of the Biokinetics accessory is the temperature sensor.
This is located in the block of the cellholder, and is used to report the temperature
of the sample.
NOTE: The sensor should be calibrated using the FL WinLab LS 55 Status
application.
FL WinLab applications which display graphical data (Scan, TimeDrive, etc.)
automatically record the temperature at the start of data collection in the result
dataset header.
The Read application collects intensity, polarization or anisotropy data and saves
this as a spreadsheet. If the Biokinetics accessory is fitted, then the temperature is
saved simultaneously.
Another important feature is the event marker button on the front plate of the
accessory. This is intended for identification of times when reagents are added to
the cuvette. In most FL WinLab applications that collect time-dependent data, an
option Show Timed Events is available. If this option is enabled, when you push
the Event button, the Timed Event trace is modified to include a marker.
Page 43
Accessories: Installation and Use . 43
Screw-fitting Flowcell
Figure 21 Screw-fitting flowcell
Description
The screw-fitting flowcell (B0631133) is intended for automated flow-through, for
example using an autosampler.
NOTE: The flowcell is not intended for HPLC, or other high resolution applications
(see instead the
The flowcell has three main advantages:
LC Flowcell
on page 63).
• The screw fittings allow tight connections to be made with autosamplers or
automated flow systems where high sample throughput requires high reliability
due to the unattended nature of data collection. Use the PerkinElmer Flange
toolkit, Part number B3000001.
• The internal volume of the flowcell (the internal chamber has dimensions
3x3x10 mm, a volume of 90 μL) is almost perfectly matched to the light beam
geometry of the LS 55 (the measured volume is 3x3x9 mm, a volume of 81μL).
This means that this flowcell does not suffer from the sensitivity losses
normally associated with flowcells, but gives excellent sensitivity, equivalent to
a normal cuvette.
• The flowcell fits into a standard cuvette holder, allowing the flowcell to be
thermostatted (as detailed in
45).
page
Installing Thermostatting Tubing to Cellholders
on
Page 44
44 . LS 55 User’s Guide
Installation and maintenance
Flowcells are marked with an arrow that indicates the inlet side, as the reagent
stream on the inlet side is brought to the bottom of the flowcell, assisting in debubbling.
This inlet side should be routed to the sample tube, or to the sampling needle of
the autosampler. The outlet side should be routed to the peristaltic pump. An
example is shown in
Figure 22 Connecting the flowcell
Figure 22.
The system should be cleaned regularly with the solvent used in the analysis. If
the system is to be left overnight or longer without being used, then the system
should ideally be cleaned with de-ionized water, then with ethanol, then pumped to
dryness.
Operation
The inlet and outlet tubing should be installed with screw fittings using the
PerkinElmer Flange tool (B3000001). This enables you to fit a wide range of screw
fittings to various diameter tubing. Ideally, PTFE or FEP tubing should be used, as
this is chemically inert.
NOTE: The internal diameter of the tubing is important, too small a diameter could
lead to inefficient pumping and cavitation, producing air bubbles. Too large
a diameter will lead to large sample volumes. Typical internal diameter is
0.7-1.0 mm.
Page 45
Accessories: Installation and Use . 45
Installing Thermostatting Tubing to Cellholders
All cuvette holders supplied for use with the LS 55 are thermostattable using
circulating water:
• The cuvette holders are made watertight with a sealant that is guaranteed up
to 60 °C.
• Higher temperatures may make the sealant leak, in which case you should
replace it with a suitable sealant with higher temperature specification.
• At temperatures below 15 °C, condensation may occur on the surfaces of the
cuvette. If this is the case, either purge the sample area with dry air, or lead
dry air onto the cuvette surfaces using tubing.
To fit the thermostatting tubing:
1. Route the tubing into the sample area and onto the two tubing ports under
the sample area door, as shown in
Figure 23.
Tubing ports
Figure 23 Tubing ports
2. The tubing is then brought around the outside of the accessory bracket, as
shown in Figure 24.
Page 46
46 . LS 55 User’s Guide
Figure 24 Routing the tubing
3. Attach the tubing to the cellholder nozzles and secure with cable ties or
clamps, as shown in Figure 25.
Figure 25 Attaching the tubing to the cellholder
Page 47
Accessories: Installation and Use . 47
The four-position cellchanger
Installing tubing for the four-position cellchanger is slightly more complicated:
1. Disconnect the cellchanger, so it can be turned manually.
2. Turn the cellchanger to appear as shown in
Figure 26 Positioning the cellchanger
3. Install the tubing.
This will ensure that the tubing will only turn through a maximum of
135 degrees during operation.
CAUTION
If the tubing is installed with the cellchanger at position 1, the
tubing turns through 270 degrees, putting greater strain on
the stepper motor, leading to potential step failures.
Figure 26.
Page 48
48 . LS 55 User’s Guide
Using the Septum Injector
Figure 27 The septum injector
The LS 55 is delivered with a septum injector accessory. This locates on top of the
sample area door, and guides a syringe needle into the cuvette.
To install the septum injector:
1. Place a septum into the injector barrel (L2251286) and push the cap
(L2251287) into the barrel, trapping the septum, as shown in Figure 28.
Figure 28 Assembling the septum injector
Page 49
Accessories: Installation and Use . 49
2. Check that the cap fits securely.
If not, the septum is probably too thick to allow the cap to grip the barrel
adequately, and should be replaced.
3. Remove the blanking plug from the LS 55 sample area door, as shown in
Figure 29.
Blanking plug
Figure 29 The blanking plug
4. Insert the septum injector.
NOTE: The distance between the top of the cuvette and the bottom of the injector
barrel avoids the thermostatting tubing fouling the injector when the door is
closed. For this reason, a short syringe needle will not reach the cuvette,
and there is no guarantee that injected reagents will enter the cuvette. A
minimum syringe needle length of 70 mm is required to ensure that the
needle enters the cuvette.
Page 50
50 . LS 55 User’s Guide
Using Stirrer Bars
Several accessories feature built-in stirring mechanisms.
It is intended that stirring is performed in a cuvette supplied by PerkinElmer. These
cuvettes are completely optically flat on all surfaces, including the base, allowing
the stirrer bar to freely rotate, whereas certain disposable cuvettes, made of
polystyrene or a similar polymer, have curved internal bases from the molding
process. This can cause severe physical disturbance of the rotation of the stirrer,
such that the stirrer cannot rotate at all or it rotates very erratically. In this case, it
is best to use a flat, round stirrer with a cross or vane built into the top. This type
of stirrer is more stable, and can rotate on an uneven surface.
PerkinElmer cuvette Polymer cuvette Special stirrer
Figure 30 Stirrers in cuvettes
The stirrer is controlled using the FL WinLab LS 55 Status application, by clicking
the accessory icon. Three speeds can be set:
• Off - no stirring.
• Low - intended for keeping sensitive cells in suspension without physical
damage.
• High - intended for keeping the sample suspended (or the temperature
homogeneous) where the sample will not be physically damaged by the
grinding action between the stirrer and the bottom of the cuvette.
NOTE: High speed is not intended for rapid mixing within the cuvette as stirrer
speeds high enough to effect rapid mixing often cause a vortex to be
created, producing bubbles in the light beam and high and erratic
background levels.
Page 51
Accessories: Installation and Use . 51
Minimum volumes with various cuvette types
It is often desirable to minimize the volume of reagents used in the cuvette. This
can be done using a smaller internal pathlength cuvette, with or without a stirring
chamber underneath.
It should also be noted that cuvettes do not have to be totally filled, the liquid only
has to cover the entire height of the light beam.
NOTE: However, failure to cover the height of the beam will cause light to be
scattered off the underside of the meniscus, producing very high
background signals.
The excitation light beam is 9 mm high in the center of the cuvette. The center of
the excitation beam is 13.5 mm from the base of the cuvette:
Figure 31 Position of the beam through a cuvette
2
In a 1 cm
2 cm (2 ml of liquid) in order to avoid light scattering by the meniscus.
Some users place a spacer block (up to 8 mm thick, at this thickness allowing
800 μL less volume) under the cuvette to raise it, eliminating the unmeasured
volume beneath the light beam. In principle this is acceptable, the notable
exception being that if a stirrer is being used, then the spacer would reduce the
magnetic coupling between the stirrer coils and stirrer bar to almost zero and the
stirrer bar will not turn.
cuvette, for example, the liquid has to fill the cuvette to a level of
Page 52
52 . LS 55 User’s Guide
Semi-micro Cuvette and Holder
Figure 32 The semi-micro cuvette assembly
Description
The semi-micro cuvette and holder (L2250139) enables you to measure smaller
samples than with a standard cuvette. In spite of the decreased volume, however,
sensitivity is not compromised since the LS 55 has a measured volume of
3 x 3 x 9 mm, which is smaller than the semi-micro cuvette's internal 5 mm
pathlength.
Installation
The holder is simply inserted into a standard cellholder, as shown in Figure 33.
NOTE: The lip at the top, for removal of the holder, must be aligned away from
you so that the lip does not contact the thermostatting nozzles, which
would stop the holder from sitting properly in the cellholder.
Figure 33 Inserting the semi-micro cuvette
Page 53
Accessories: Installation and Use . 53
Stirred Semi-micro Cuvette
Figure 34 The stirred semi-micro cuvette
Description
Micro and semi-micro cuvettes, which allow the user to measure smaller sample
volumes than standard 1cm pathlength cuvettes, typically have disadvantages in
that stirring and thermostatting are difficult to achieve.
In the case of stirring, this is either because the internal dimensions of the
microcuvette are too small or because the small size of the stirrer for cuvettes with
5 mm pathlength do not function efficiently.
Thermostatting is difficult due to the use of a semi-micro cuvette adaptor, which
fits into the standard cuvette holder and accepts the smaller cuvette. These
adaptors are normally manufactured from polymer material, which is an effective
thermal insulator.
The stirred semi-micro cuvette (B0631132) overcomes both of these problems:
• The outside dimensions are the same as for a 1 cm cuvette, giving good
contact with the cellholder for thermostatting (as detailed in
Thermostatting Tubing to Cellholders
are smaller for decreased sample volume.
• At the base of the cuvette there is a chamber for insertion of a standard stirrer.
on page 45), but the internal dimensions
Installing
Page 54
54 . LS 55 User’s Guide
Figure 35 Cross-section of the stirred semi-micro cuvette
Operation
The stirrer is controlled using the LS 55 Status application in the FL WinLab
software, as discussed in
Using Stirrer Bars
on page 50.
Page 55
Accessories: Installation and Use . 55
Care of Optical Mirrors Inside Cellholder Accessories
The mirrors built into cellholder accessories are used to reflect unabsorbed
excitation light back into the sample to give a second pass at exciting the sample.
The emission mirror collects a portion of light emitted away from the instrument
and reflects it back into the instrument.
Both mirrors together provide a 2.5-fold increase in signal, so in the worst case if
both mirrors are totally corroded, they will not reflect light and the signal will
decrease by a factor of 2.5-fold.
Additionally, if fluorescent material is allowed to build up on the mirrors, then
background levels will increase significantly.
To prevent these possibilities, the mirrors should be regularly cleaned with
methanol or a non-corrosive solvent.
Page 56
56 . LS 55 User’s Guide
Resistance to Solvents
The base of the cuvette holder block is manufactured from Delrin, a reasonably
inert polymer. This is done to effect thermal isolation from the baseplate of the
accessory for thermostatting. Although Delrin is resistant to most solvents, it can
still be attacked by particularly aggressive solvents and acids such as 0.1 M
perchloric acid.
The pressure bar which presses the cuvette back into the mirrored corner of the
cuvette is also manufactured from Delrin.
To prevent damage, spillages should be washed with non-corrosive solvent or
methanol.
If the base becomes attacked, the consequences are not severe unless it is totally
dissolved, in which case the cuvette can fall through the base of the holder onto
the metal baseplate. If the pressure bar is attacked, then the cuvette will not be
located as firmly on the rear surfaces of the cellholder, possibly leading to
unreproducible signals.
Page 57
Accessories: Installation and Use . 57
Sipper
Figure 36 The Sipper accessory
Description
The Sipper accessory (L2250135) is used to increase sample throughput, either by
itself or in conjunction with an external autosampler.
The Sipper consists of a flow cell coupled to a peristaltic pump. When used without
an autosampler, sampling is initiated by putting the sample container under the
tube, lifting the container so that the tube goes into the sample and then raising it
further until a microswitch is activated which starts the pump, as shown in
Figure 37. The sample is then pumped into the flowcell and measured.
Figure 37 Using the Sipper
Page 58
58 . LS 55 User’s Guide
The measured volume of the sipper flowcell is approximately 1.5 x 1.5 x 10 mm.
The maximum measured volume of the LS 55 (with wide open excitation and
emission slits) is approximately 3 x 3 x 10 mm, so the absolute sensitivity when
using the Sipper accessory would be around a factor of 4 times less sensitive than
when using a cuvette. In spite of this loss, the Sipper accessory represents a much
more sensitive option for high sampling throughput than for example a plate
reader, where sensitivity would be at least a factor of 20 less sensitive (even for
dedicated plate reader instruments).
Installation and maintenance
The Sipper accessory must be aligned before use, to ensure that the center of the
flowcell sits correctly at the optical focus of the LS 55. Installation is carried out as
follows:
1. If the tubing is clamped in the switch lever, loosen the tubing clamp screw, as
shown in Figure 38, and pull out the tubing.
Figure 38 The front of the Sipper
2. Loosen the two front plate fixing screws, as shown in
the front plate.
3. Loosen the two mount plate locking screws, as shown in Figure 39.
This enables the mount plate to move freely during alignment.
Figure 38, and remove
Page 59
Accessories: Installation and Use . 59
Figure 39 The mount plate
4. Fix the Sipper to the LS 55 accessory bracket and lock it into place by
pressing the two quick release fasteners.
5. Insert the accessory cable into the right-hand accessory socket inside the
sample area.
6. Ensure that the tubing is connected, and that the peristaltic pump tubing is
correctly held in the pump cam/roller assembly.
7. Turn the pressure block pin carrier until the pressure pin latches into its
bracket, as shown in
Figure 40.
Figure 40 Pressure pin and adjuster screw
8. Tighten the adjuster screw, as shown in
applied to the pump tubing to enable the sipper to pump efficiently.
NOTE: When the sipper is correctly installed it needs no priming, as it will pump
from dry.
9. Pump a fluorescent sample into the Sipper.
Figure 40, until sufficient pressure is
Page 60
60 . LS 55 User’s Guide
10. Start a TimeDrive using suitable wavelengths for the fluorescent dye used,
using slit widths of 10 nm (excitation) and 5 nm (emission).
Duration should be 2-3 minutes.
11. During data collection, adjust the two (1/8") Hex screws shown in
Figure 41 until the maximum signal is observed.
Figure 41 Adjustment screws
12. Remove the accessory from the LS 55 sample area and tighten the locking
screws shown in
Figure 39.
13. Re-fit the front plate, as shown in
14. Re-insert and re-clamp the tubing using the tubing clamp screw, as shown in
Figure 38.
Figure 38.
Operation
The Sipper accessory can be controlled manually using the LS 55 Status application.
The Scan and Concentration applications include full sipper control. The Sipper
parameters (pump time, wait time, purge time and direction) are set in the
application so that when the Sipper is started by pushing the Sipper arm upwards,
sampling and measurement are automatically triggered.
Manual control
1. Start the LS 55 Status application from the FL WinLab software.
Page 61
Accessories: Installation and Use . 61
2. Click the Sipper icon in the LS 55 Status application.
3. Select whether to Pump Forwards or to Pump Reverse, enter the required
Pump Time (in seconds) and click Sip.
Scanning spectra
1. In the Scan application, select the kinetic scan option.
Page 62
62 . LS 55 User’s Guide
2. Set up the kinetic scan parameters panel as required.
Now, when you start a scan by clicking on the green traffic light, the system will
wait for the Sipper switch to be activated. The sample will then be pumped into the
flowcell and the spectrum measured. The system will wait again for the Sipper
switch to be activated for the next spectrum. When all spectra have been
measured, a 3D file will be created which can be viewed by selecting 3D Viewer on
the Data Handling menu in FL WinLab.
Routine quantitation with the Concentration application
1. In the Concentration application, select Setup parameters and set up the
sipper panel as required.
The example here will sample for 6 seconds, wait 1 second for de-bubbling,
measure the intensity then return the sample for 9 seconds.
3. Select User info and set up the Options panel as required.
Measurement of references and unknown samples is performed as normal, except
that the system will wait for the user to insert the sipper inlet tube into the sample
vial and activate the sipper switch.
Page 63
Accessories: Installation and Use . 63
LC Flowcell
Figure 42 The LC Flowcell
Description
The LC Flowcell accessory (L2250138) enables you to collect chromatographic data
using the LS 55. The flowcell has internal dimensions of 1.5 x 1.5 x
10 mm (measured volume approximately 20 microlitres) and is manufactured from
synthetic fused silica, giving optical performance down to 200 nm.
Inlet and outlet tubes are made of an inert polymer material (FEP) which ensures
biocompatibility.
NOTE: FEP tubing has low structural strength and thus poor resistance to back
pressure, so care should be taken when placing the LS 55 with LC Flowcell
into a series of detectors. The LS 55 should be located at the end of the
chain to reduce back pressure.
Installation
The LC Flowcell accessory must be aligned before use, to ensure that the center of
the flowcell sits correctly at the optical focus of the LS 55:
1. Loosen the two front plate fixing screws and remove the front plate, gently
feeding the inlet and outlet tubes through the grommets on the front plate, as
shown in
Figure 43.
Page 64
64 . LS 55 User’s Guide
Figure 43 Fixing screws and tubing
2. Loosen the two mount plate locking (1/8") hex screws.
This enables the flowcell mount plate to move freely during alignment.
Figure 44 Mount plate locking screws
3. Affix the LC Flowcell accessory to the LS 55 and lock it into place by pressing
the two quick release fasteners.
4. Connect the syringe supplied to the inlet tube.
5. Pump a fluorescent sample into the flowcell until the liquid emerges from the
outlet tube, checking by eye that there are no air bubbles trapped in the LC
Flowcell.
6. Start a TimeDrive using suitable wavelengths for the fluorescent dye used,
using slit widths of 10 nm (excitation) and 5 nm (emission).
Duration should be 2-3 minutes.
7. During data collection, adjust the two (1/8") Hex screws shown in
Figure 45 until the maximum signal is observed.
Page 65
Accessories: Installation and Use . 65
Figure 45 Adjustment screws
8. Remove the accessory from the LS 55 sample area and tighten the locking
screws shown in Figure 44.
9. Carefully feed the inlet and outlet tubes through the grommets on the front
cover plate then re-secure the front cover plate using the two screws, as
shown in
Figure 43.
Operation
Data collection for the LC Flowcell is made using the TimeDrive application. Data
collection can be synchronized with a signal from an HPLC pump using contacts on
the LS 55’s rear panel (see
Remote starts and event marking
on page 31).
Page 66
66 . LS 55 User’s Guide
Front Surface Accessory
Figure 46 The Front Surface accessory
Description
The Front Surface accessory (52123130) is used for the measurement of powders
and flat samples such as papers, leaves, polymers, etc. It is also used in the life
sciences for turbid samples where the sample cannot be diluted but gives severe
quantitative problems due to light scattering.
The Front Surface accessory must be aligned to ensure correct sensitivity. Failure to
do this could lead to a large loss of sensitivity. Furthermore, the process of
alignment optimizes sensitivity to luminescence signals and minimizes the effect of
light scattering.
Page 67
Accessories: Installation and Use . 67
Installation and maintenance
1. Remove the two screws that hold the fascia and front panel, as shown in
Figure 47.
Figure 47 Front bolts
2. Refit the front plate without the fascia, as shown in
Figure 48 Front plate without fascia
3. Loosen the two upper clamping screws, as shown in
Figure 48.
Figure 49.
Page 68
68 . LS 55 User’s Guide
Figure 49 Upper clamping screws
4. Loosen the three screws on the underside of the accessory, as shown in
Figure 50.
Figure 50 The screws underneath the accessory
5. Fit the accessory to the LS 55 using the two cam-lock fasteners.
6. Mount a flat fluorescent sample in the Front Surface accessory (see
Operation
on page 70).
A business card is useful for this purpose as most are highly fluorescent, and
the card is strong enough to remain flat during clamping.
7. Start a TimeDrive from the FL WinLab software, using suitable parameters.
For a business card, these would be:
Excitation wavelength 300 nm
Emission wavelength 550 nm
Slits 10/2.5 nm or 10/5 nm (Excitation/Emission)
Page 69
Accessories: Installation and Use . 69
8. Using the LS 55 Status application, click the emission monochromator icon
and select emission filter = 1% T attenuator.
9. Using a flat screwdriver, turn the two adjustment screws shown in
Figure 51, to optimize the signal.
During the run it may be necessary to click the Autoexpand Y-axis toolbar
button if the signal goes off-scale.
Figure 51 Adjustment screws
NOTE: If the signal exceeds 999.999, then the run will have to be stopped and
more attenuation or narrower emission slit width used.
10. When the maximal signal has been reached, stop the run and remove the
accessory from the LS 55.
11. Tighten the two upper locking screws, as shown in Figure 49, and the three
locking screws underneath the accessory, as shown in
The alignment will now remain constant.
12. Remove the two screws from the front plate and refit the front plate with
fascia, as shown in Figure 47.
13. Re-fit the accessory to the LS 55 using the two cam-lock fasteners.
Figure 50.
Page 70
70 . LS 55 User’s Guide
Operation
There are two adjustable parts in the Front Surface accessory, these allow the
measurement of samples with varying thickness.
The plunger plate is spring loaded, the stroke of the spring action is controlled by a
metal ring, locked by a horizontal screw, as shown in
is pulled back to allow insertion of the sample.
Figure 52 The plunger
Coarse movement of the plunger plate is achieved by moving the plunger base
itself. This moves linearly and is locked by a large, vertical screw.
Figure 52. The plunger plate
1. Unlock the vertical screw, as shown in
back.
2. Insert the sample so that it rests against the measurement baffle.
3. Push the plunger plate forwards so that some pressure is applied to the
plunger plate to hold the sample in position.
4. Lock the vertical screw.
You replace the sample by pulling back the plunger plate.
Figure 52, and pull the plunger plate
Page 71
Accessories: Installation and Use . 71
Using Powders
The powder accessory can be used to measure powders or flat samples which are
too small to be held in the accessory’s window.
The sample should be as finely powdered and as homogenous as possible to avoid
surface structure effects. Also, the vertical excitation light beam image is
9 mm high and 3 mm wide, so the sample should fill the center of the window at
least to fill this area (if in doubt, set the excitation monochromator to a visible
wavelength such as 550 nm and observe the relative locations of the light area and
the sample).
For the measurement of oils or very viscous samples, the sample can be
sandwiched between two silica windows.
1. Unscrew the base plate from the Powder holder and add the sample onto the
silica window, as shown in
Figure 53.
Figure 53 The powder accessory
2. Replace the cap and screw it home until the sample is held tightly.
3. Place the powder holder into the Front Surface accessory checking that the
location shoulder fits into the window of the accessory’s measurement baffle.
If the shoulder does not fit, then the hole in the baffle should be enlarged
suitably with a round file.
Page 72
72 . LS 55 User’s Guide
Figure 54 Using the powder accessory
Page 73
Accessories: Installation and Use . 73
Plate Reader
Figure 55 The Plate Reader
Description
The Plate Reader accessory (L2250140) attaches to the front of the LS 55 and can
be used to measure microplates or scan any flat sample up to a measurement area
of 128 mm x 96 mm.
Measurement of microplates can use a wavelength program of up to 20 wavelength
parameter sets, in addition the measurement can be automatically repeated for
kinetics.
Microplate measurement results in the creation of a data spreadsheet. Flat sample
measurement creates a 3D datafile that can be viewed using the 3D Viewer
included with FL WinLab.
Installation
Before use, the Plate Reader must be correctly installed. This involves optical
alignment of the Plate Reader accessory and the creation of a Plate Format file, so
that the FL WinLab software can measure in the correct locations on the microplate.
In order to perform the optical alignment, a small (~4 cm square) flat mirror and a
dentist’s mirror should be used.
1. Open the LS 55 top cover, as described in
page
173.
Removing the Main Cover
on
Page 74
74 . LS 55 User’s Guide
2. Switch off the HT supply to the photomultiplier, as shown in Figure 56.
Figure 56 HT switch for the photomultiplier
3. Open the sample area accessory cover and remove the existing sampling
accessory by pressing on the 2 quick release fasteners.
4. Undo the three screws which attach the sample accessory bracket to the
LS 55, as shown in Figure 57.
Page 75
Accessories: Installation and Use . 75
Figure 57 Sampling accessory bracket screws
5. Unscrew the two fixing screws and remove the optical assembly, as shown in
Figure 58.
This is so that the sharp square edges at the front of the optical assembly
cannot accidentally break the LS 55´s optical windows during fitting.
Figure 58 Optical assembly screws
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76 . LS 55 User’s Guide
6. Fix the plate reader body to the LS 55 by aligning the steel dowels, as shown
in
Figure 59.
Figure 59 Locating dowels
7. Tighten the three screws that secure the Plate Reader to the LS 55, as shown
in Figure 60.
Figure 60 Plate Reader screws
8. Attach the optical assembly to the Plate Reader and tighten the two screws
which secure it, as shown in
Figure 61.
Page 77
Accessories: Installation and Use . 77
Figure 61 Attaching the optical assembly
9. Using the LS 55 Status application, set the excitation wavelength to 0 (zero
order, white light) to assist in alignment.
10. Insert the dentist’s mirror between the LS 55 front plate and the optical
assembly.
The idea is to observe the excitation fibre to ensure that the excitation light
falls directly on the fibre bundle, optimising light throughput.
11. Loosen the excitation fibre locking screw, as shown in
Figure 62 Excitation fibre screws
Figure 62.
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78 . LS 55 User’s Guide
12. Rotate the mirror via the adjustment screw, as shown in Figure 62, until the
light image is centralized on the fibre bundle, as shown in
Figure 63 The end of the fibre bundle as seen in the dentist’s mirror
Figure 63.
13. Tighten the locking screw.
14. Open the LS 55 main cover as detailed in
173.
page
15. Remove the main optical cover as detailed in
Cover
on page 176.
16. Switch off the room lighting.
This enables you to see the emission entrance slit easier.
17. Set the excitation wavelength to zero order.
This produces white light.
18. Set the emission slit width to 10 nm.
19. Place a plane mirror beneath the fibre optic sampling end.
This reflects the white light back into the instrument, for alignment purposes.
20. Observe the emission entrance slit though the open aperture in the emission
filter wheel, as shown in
Figure 64.
Removing the Main Cover
Removing the Main Optical
on
Page 79
Accessories: Installation and Use . 79
Figure 64 Position of the emission entrance slit
21. Loosen the emission fibre locking screw, as shown in
Figure 65 Emission fibre screws
22. Turn the adjustment screw until the white light is centralized on the emission
slit inside the LS 55, as shown in
Figure 66.
Figure 65.
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80 . LS 55 User’s Guide
Figure 66 Aligning the emission fibre
23. Tighten the emission locking screw.
NOTE: The Plate Reader can now be interchanged with the standard sample holder
without need for further alignment.
Operation
Data can be collected from the Plate reader in one of three ways:
• Automatically driving the Plate Reader within a microplate-like matrix coupled
with the collection of a wavelength program. This is done using the FL WinLab
Well Plate Reader application.
• Automatically driving the Plate Reader in a continuous mode over a flat surface,
generating a 3D plot of intensity vs. Distance (mm) vs. Distance (mm). This is
done using the FL WinLab TLC Scan application.
• By manually driving the fibre optic to a discrete position followed by running
any FL WinLab application (for example Scan). This is done within the FL
WinLab Well Plate Reader or TLC Scan applications, depending on the type of
sample.
The Well Plate Reader application
The Well Plate Reader application is used to collect data from samples with a
discrete format of rows and columns like a microplate.
To drive the Plate Reader to a well, from the
mouse button on a well. The Plate Reader will be driven to this position (in
Figure 67, to well B1) and the intensity measured and displayed.
Setup plate tab click with the right
Page 81
Accessories: Installation and Use . 81
Figure 67 The Well Plate Reader application
NOTE: The Plate Reader will remain at this position until it is sent to a different
position, it is sent to Park/Datum position, or a Plate Reader run is started.
By opening the Scan application, for example, spectra can be collected from
the microplate.
The TLC Scan application
Unlike the Well Plate Reader application, where the accessory is sent to one of a
series of discrete positions, the TLC Scan application enables you to send the Plate
Reader to any position.
This is done from the Setup parameters tab, by moving the mouse over the
sample area image until the desired position (shown as X and Y in millimeters on
the top left of the tab) and clicking with the right mouse button on the sample area
image.
The Plate Reader is sent to the position (in
the plate holder platten, 10 mm from the top edge).
Figure 68 The TLC scan application
Figure 68, 20 mm from the left side of
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82 . LS 55 User’s Guide
Measuring TLC plates, gels or other flat samples
The Plate Reader accessory is shipped with an anodized aluminium plate
(L2251251) that is fitted with 2 clips (L2251249) for holding the sample flat to
ensure baseline and signal value consistency, as shown in Figure 69.
Figure 69 Flat sample holder
This is inserted in place of the microplate in the Plate Reader, the clips are
positioned so that they hold the sample securely but do not restrict the movement
of the plate reader during measurement.
NOTE: Position the clips so that the fibre optic probe does not come into contact
with the clips during movement, or a stepper motor error message will
appear, and the run will be aborted.
Page 83
Accessories: Installation and Use . 83
Red-sensitive Detector
Figure 70 The Red-sensitive detector
Description
The red-sensitive photomultiplier (52124966) is fitted for the collection of emission
data above 630 nm, above which point the standard photomultiplier has little or no
sensitivity, as shown in
Figure 71.
Figure 71 Photomultiplier sensitivity
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84 . LS 55 User’s Guide
Installation
The photomultiplier installation procedure must be performed by a PerkinElmer
Service Engineer.
NOTE: In order to obtain corrected emission spectra, the correction factor
spectrum will have to be collected specifically for the red-sensitive
photomultiplier. Data obtained using the standard photomultiplier is not
applicable.
Page 85
Accessories: Installation and Use . 85
Total Emission Accessory
Figure 72 The Total Emission accessory
Description
Gratings used in monochromators have relatively poor transmission of white light.
When absolute sensitivity is preferred over spectral resolution, using the zero order
position (where the grating transmits white light) gives only small increases in
sensitivity. The Total Emission accessory (L2250101) is a mirror which replaces the
emission grating in the beam, giving a 10 to 15-fold increase in sensitivity.
Installation
The accessory must be installed in the factory, or by a service engineer, since
alignment is required.
CAUTION
Incorrect installation of the accessory can lead to irreparable
damage to the emission grating.
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86 . LS 55 User’s Guide
Operation
When the Total Emission accessory is fitted, it can be selected in the LS 55 Status
application's monochromator dialog. The emission grating is then driven out of the
beam and is replaced by the mirror.
Select an emission filter to avoid excitation light being scattered directly onto the
sample photomultiplier. If this not done, a warning will be displayed.
NOTE: The use of the Total Emission accessory is strongly recommended for bio-
and chemi-luminescence measurements.
Page 87
Accessories: Installation and Use . 87
External Fibre Optic Accessory
Figure 73 The external fibre optic accessory
Description
The external fibre optic accessory (L2250144) consists of a 1 meter-long fibre optic
attached to a sample area holder, and can be used for remote sampling of
luminescent samples. The transmission characteristics of silica allow excitation
down to approximately 260 nm. At the sampling end, the two fibre bundles are
hemispherical, as shown in
Figure 74.
Figure 74 The end of the fibre optic accessory
The numerical aperture (around 0.2) of the material produces an
emergence/acceptance angle of between 22-25 degrees, as shown in Figure 75.
Figure 75 The emergence/acceptance angle
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88 . LS 55 User’s Guide
Installation
The accessory needs to be optically aligned to ensure that excitation light passes
into the fibre and that emission light from the fibre is correctly passed into the
emission monochromator.
CAUTION
1. Remove the top cover as described in
173.
page
2. Turn off the voltage to the photomultiplier, as shown in
Take care not to bend the fibre to a radius of less than 10 cm,
as this may cause damage to the fibres.
Removing the Main Cover
on
Figure 76.
Figure 76 Photomultiplier switch
3. Loosen the two knurled screws underneath the accessory, as shown in
Figure 77.
Page 89
Accessories: Installation and Use . 89
Figure 77 Accessory screws
4. Attach the sample area accessory to the LS 55.
5. Insert the sampling end of the fibre into the right side holder of the sample
area accessory and tighten the grub screw, as shown in
Figure 78.
Figure 78 Right holder grub screw
6. Using the FL WinLab LS 55 Status application, set the excitation wavelength
to 0 (white light) to give good visibility during alignment.
7. Position the right side holder so that white light enters the fibre and emerges
from the two free ends of the fibre optic.
8. Tighten the knurled locking screw underneath the accessory so the right-hand
fibre holder is locked in place.
9. Insert one of the free fibre optic ends into the left side holder, as shown in
Figure 79.
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90 . LS 55 User’s Guide
Figure 79 Inserting into the left holder
10. Turn the fibre until it is vertical and tighten the grub screw, as shown in
Figure 80.
Figure 80 Left holder grub screw
11. Open the central optical cover inside the LS 55 and observe the emission
entrance slit through the open position of the emission filter wheel, as shown
Figure 81.
in
Figure 81 Viewing the emission slit
Page 91
Accessories: Installation and Use . 91
12. Move the left fibre holder until the light is central on the slit, as shown in
Figure 82.
Figure 82 Aligning the light
13. Tighten the knurled screw under the left fibre holder, to lock it.
14. Remove the sampling fibre end from the right fibre holder.
15. Loosen the grub screw in the right fibre holder and insert the free fibre end,
as shown in
Figure 83.
Figure 83 Inserting the fibre end
16. Rotate the fibre until it is vertical then tighten the grub screw (
Figure 84 Right holder grub screw
Figure 84).
Page 92
92 . LS 55 User’s Guide
17. Rotate the right fibre until the excitation light beam image is centralized on
the optical fibres.
18. Loosen the grub screw again and move the fibre into and out of the holder
(keeping the fibre bundle vertical) to optimize the focus.
19. Tighten the knurled screw under the base plate to lock the fibre.
20. Replace the LS 55 central optical cover.
21. Turn on the photomultiplier voltage.
22. Close the main cover.
Operation
The ~22o angle of emergence of light from the sampling end means that the
further the sampling end is from the sample, the larger the excitation image will be,
as shown in
Figure 85.
Figure 85 Sampling positions
However, if the sampling end contacts the sample, very little light will be accepted
by the emission fibre. Optimal sensitivity is achieved with a 1-2 mm distance
between the fibre optic and the sample.
Improving resolution further can be achieved by masking the sampling end of the
fibre to eliminate the outer parts of the semicircles from measurement:
Page 93
Figure 86 Masking the outer areas
Accessories: Installation and Use . 93
Page 94
94 . LS 55 User’s Guide
The Fast Filter Accessory
Figure 87 The Fast Filter accessory
Description
The introduction of a range of cell permeable fluorescent probes that bind to
intracellular ions, enables cell biologists to study the role of these ions as cellular
messengers. Changes in the fluorescent properties of these probes when they
chelate to intracellular ions reflect increasing or decreasing concentrations of free
ions inside the cell. The spectral properties of some common probes are shown in
the table:
Page 95
Accessories: Installation and Use . 95
Probe Target ion Excitation wavelength
Bound Free
FURA-2 Ca++ 340 nm 380 nm 510 nm 510 nm
INDO-1 Ca++ 355 nm 355 nm 405 nm 480 nm
Mag-FURA-2 Mg++ 340 nm 380 nm 510 nm 510 nm
Mag-INDO-1 Mg++ 355 nm 355 nm 405 nm 480 nm
BCECF pH 495 nm 440 nm 530 nm 530 nm
SNARF-1 pH 514 nm 514 nm 580 nm 640 nm
An example of the spectral dependence on ion binding of a fluorescent probe is
given by the binding of calcium ions to FURA-2, as shown in
Emission wavelength
Bound Free
Figure 88.
Figure 88 Binding of calcium ions to FURA-2
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96 . LS 55 User’s Guide
Installation
The Fast Filter accessory (L2250019) is normally fitted in the factory or is installed
by a Service engineer. This involves among other things the installation of the Fast
Filter controller circuit board and the base plinths for the Fast Filter drive units. In
normal use, the only user access would be to change filters if required. It may be,
however, that the user has a set of standard polarizers and one or two Fast Filter
wheels. In order to change between Fast Filter operation and standard polarizers,
the accessories must be exchanged. It is this process that is described here.
There are two parts to the installation of the Fast Filter accessory, these are the
physical installation and the software installation.
Installing filters into the filter wheels
1. Lay the Fast Filter motor unit carefully onto a flat surface.
2. Undo the central screw, as shown in
Figure 89 The central screw
3. Remove the 2 filter brackets.
4. Attach the 2 required filter brackets to the Fast Filter motor, as shown in
Figure 90.
NOTE: The filter brackets have a peg and slot system on one side and a lapped
joint on the other, so they only fit one way round onto the motor assembly.
Figure 89.
Page 97
Figure 90 Assembling the filter brackets
Accessories: Installation and Use . 97
The positioning of the filters is predefined:
• For polarizer filters, the vertical filter MUST be located in position 1 or 2.
• For ratio filters, the top part of the ratio (for example, the 340 nm filter for
FURA-2) MUST be located in position 1 or 2.
• The positions for the excitation wheel are shown in
Figure 91.
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98 . LS 55 User’s Guide
Figure 91 Positions for the excitation wheel
• The positions for the emission wheel are shown in Figure 92.
Figure 92 Positions for the emission wheel
Installing the excitation Fast Filter into the LS 55
1. Remove the LS 55 main cover, as described in
page
173.
Removing the Main Cover
on
Page 99
Accessories: Installation and Use . 99
2. Remove the central optical cover as described in
Cover on page
3. Unplug the excitation filter wheel cable, as shown in
Figure 93 Unplugging the cable
4. Undo the two screws and remove the excitation filter wheel, as shown in
Figure 94.
NOTE: Take care not to touch any of the optical surfaces or mirrors.
176.
Removing the Main Optical
Figure 93.
Figure 94 Excitation filter wheel screws
Page 100
100 . LS 55 User’s Guide
5. Gently pry off the cable clamp to allow access for the Fast Filter plinth.
6. Locate the Fast Filter plinth onto the LS 55 baseplate, first aligning the pin on
the baseplate with the hole in the plinth.
7. Secure the plinth using the two long hex screws.
8. Fit the Fast Filter wheel to the plinth, first aligning the pin on the plinth with
the hole on the Fast Filter wheel motor assembly.
9. Secure using the 2 long screws fixed to the motor assembly, as shown in
Figure 95.
Figure 95 Fixing the filter wheel to the plinth
10. Attach the ribbon cable to the Fast Filter motor assembly.
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