horiba FluoroMax-3 User manual

FluoroMax-3 v. 3.1 (3 Mar 2006)
i
FluoroMax®-3 & FluoroMax®-P
with FluorEssence™
Operation Manual
http://www.jobinyvon.com
Rev. 3.1
USA: HORIBA Jobin Yvon Inc., 3880 Park Avenue, Edison, NJ 08820-3012, Toll-Free: +1-866-jobinyvon
Tel: [phone], Fax: [phone], E-mail: [email protected], www.jobinyvon.com
France: HORIBA Jobin Yvon S.A.S., 16-18, rue du Canal, 91165 Longjumeau Cedex,
Tel: [phone]
, Fax: [phone], www.jobinyvon.fr
Japan: HORIBA Ltd., JY Optical Sales Dept, Higashi-Kanda, Daiji Building, 1-7-8 Higashi-Kanda
Chiyoda-ku, Tokyo 101-0031, Tel: [phone], www.jyhoriba.jp
Germany: [phone] Italy: [phone] UK: [phone]
China: [phone]
(All HORIBA Jobin Yvon companies were formerly known as Jobin Yvon)

FluoroMax-3 v. 3.1 (3 Mar 2006)
ii
Copyright © 2001–2006 by HORIBA Jobin Yvon
Inc.
All rights reserved. No part of this work may be
reproduced, stored, in a retrieval system, or
transmitted in any form by any means, including
electronic or mechanical, photocopying and
recording, without prior written permission from
HORIBA Jobin Yvon Inc. Requests for permission
should be requested in writing. Origin®is a registered
trademark of OriginLab Corporation. Alconox®is a
registered trademark of Alconox, Inc. Ludox®is a
registered trademark of W.R. Grace and Co. Teflon®
is a registered trademark of E.I. du Pont de Nemours
and Company.
Information in this manual is subject to change
without notice, and does not represent a commitment
on the part of the vendor.
April 2006
Part Number 81038

FluoroMax-3 v. 3.1 (3 Mar 2006)
iii
Table of Contents
0: Introduction ................................................................................................ 0-1
About the FluoroMax®-3 and FluoroMax®-P...........................................................................................0-1
Chapter overview....................................................................................................................................0-2
Disclaimer...............................................................................................................................................0-3
Safety summary......................................................................................................................................0-5
Risks of ultraviolet exposure...................................................................................................................0-7
Additional risks of xenon lamps............................................................................................................0-10
1: Requirements & Installation ............................................................................ 1-1
Safety-training requirements ..................................................................................................................1-1
Surface requirements.............................................................................................................................1-2
Environmental requirements...................................................................................................................1-3
Electrical requirements...........................................................................................................................1-4
Unpacking and Installation .....................................................................................................................1-5
Software emulation.................................................................................................................................1-9
2: System Description....................................................................................... 2-1
Introduction.............................................................................................................................................2-1
Basic theory of operation........................................................................................................................2-1
Optical layout..........................................................................................................................................2-2
3: System Operation ......................................................................................... 3-1
Introduction.............................................................................................................................................3-1
Controls and indicators...........................................................................................................................3-1
Turning on the system............................................................................................................................3-2
Checking system performance...............................................................................................................3-3
4: Data Acquisition........................................................................................... 4-1
Experiment Menu button ........................................................................................................................4-2
Previous Experiment Setup button.........................................................................................................4-4
Auto Run Previous Experiment button ...................................................................................................4-5
Make Overlay File button........................................................................................................................4-6
3D Scan to 3D Profile button..................................................................................................................4-7
Get Peak Information button...................................................................................................................4-9
Running an unknown sample...............................................................................................................4-10
5: Optimizing Data ........................................................................................... 5-1
Cuvette preparation................................................................................................................................5-1
Sample preparation ................................................................................................................................5-2
Running a scan on a sample..................................................................................................................5-4
Measuring the Gfactor...........................................................................................................................5-6
Improving the signal-to-noise ratio .........................................................................................................5-8
Correcting data.....................................................................................................................................5-14
6: Maintenance............................................................................................... 6-1
Introduction.............................................................................................................................................6-1
Lamp replacement..................................................................................................................................6-1
Electronics..............................................................................................................................................6-9
7: Troubleshooting........................................................................................... 7-1
Chart.......................................................................................................................................................7-1
Checking the FluoroMax®’s BIOS...........................................................................................................7-3
Using diagnostic spectra ........................................................................................................................7-5
Further assistance… ............................................................................................................................7-10

FluoroMax-3 v. 3.1 (3 Mar 2006)
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8: Producing Correction Factors ........................................................................... 8-1
Introduction.............................................................................................................................................8-1
Generating emission correction factors..................................................................................................8-2
Calculating emission correction factors..................................................................................................8-3
Calculating excitation correction factors.................................................................................................8-5
Using correction-factor files..................................................................................................................8-10
9: FluoroMax®-P Phosphorimeter Operation ............................................................ 9-1
Introduction.............................................................................................................................................9-1
Theory of operation ................................................................................................................................9-2
Applications for the phosphorimeter.......................................................................................................9-5
Operation of the phosphorimeter............................................................................................................9-8
Processing phosphorimeter data..........................................................................................................9-10
Lamp replacement................................................................................................................................9-11
10: Automated Polarizers..................................................................................10-1
Introduction...........................................................................................................................................10-1
Installation.............................................................................................................................................10-5
Alignment..............................................................................................................................................10-6
Using automated polarizers................................................................................................................10-14
Maintenance.......................................................................................................................................10-19
Troubleshooting..................................................................................................................................10-20
11: Technical Specifications ..............................................................................11-1
Spectrofluorometer system...................................................................................................................11-2
Minimum computer requirements.........................................................................................................11-4
Software................................................................................................................................................11-4
12: Components & Accessories...........................................................................12-1
Itemized list of FluoroMax®-3 accessories ...........................................................................................12-2
Model 1940 Absorption/Transmission Accessory................................................................................12-3
FL-1013 Liquid Nitrogen Dewar Assembly...........................................................................................12-6
Model 1908MOD Scatter Block Assembly ...........................................................................................12-7
Model 1908 Standard Lamp Assembly ................................................................................................12-7
Sample cells .........................................................................................................................................12-8
F-3000 Fiber Optic Mount.....................................................................................................................12-9
Model 1938 Cut-On Filter...................................................................................................................12-10
Model 1939 Cut-On Filter...................................................................................................................12-10
FL-1010 Cut-On Filter Holder.............................................................................................................12-11
FL-1011 Four-Position Thermostatted Cell Holder ............................................................................12-12
FL-1012 Dual-Position Thermostatted Cell Holder ............................................................................12-14
Model 1933 Solid Sample Holder.......................................................................................................12-16
F-3005/6 Autotitration Injector............................................................................................................12-18
Microscope Interface..........................................................................................................................12-19
Model 1905-OFR 150-W Xenon Lamp...............................................................................................12-20
F-3004 Sample Heater/Cooler Peltier Thermocouple Drive ..............................................................12-21
FM-2005 Phosphorimeter upgrade ....................................................................................................12-22
MicroMax 384 Microwell Plate Reader...............................................................................................12-23
Fl-1044 L-Format Polarizer.................................................................................................................12-24
FL-1015 Injector Port..........................................................................................................................12-25
Quantum-Yield accessory ..................................................................................................................12-26
SFA-20/SPEX Stopped-flow accessory .............................................................................................12-27
FM-2013 TCSPC upgrade..................................................................................................................12-28
F-1001/1 Temperature Bath...............................................................................................................12-29
Model TRIG-15/25 External Trigger Accessory .................................................................................12-30
FM-2007 Windows for the FluoroMax®-3 sample compartment ........................................................12-31
13: Glossary .................................................................................................13-1
14: Bibliography ............................................................................................14-1

FluoroMax-3 v. 3.1 (3 Mar 2006)
v
15: Declaration of Conformity........................................................................ 15-1
16: Index .................................................................................................... 16-1

FluoroMax-3 v. 3.1 (3 Mar 2006)
vi

FluoroMax-3 v. 3.1 (3 Mar 2006) Introduction
0-1
Note: Keep this and the other reference
manuals near the system.
0: Introduction
About the FluoroMax®-3 and FluoroMax®-P
Both the FluoroMax®-3 and FluoroMax®-P are self-contained, fully automated spectro-
fluorometer systems. Data output is viewed on a PC, while printouts may be obtained
via an optional plotter or printer. All FluoroMax®-3 and FluoroMax®-P functions are
under the control of FluorEssence™ spectroscopy software. The main parts of the
FluoroMax®-3 and FluoroMax®-P spectrofluorometer systems are:
•State-of-the-art optical components
•A personal computer
•FluorEssence™ for Windows™, the driving software.
The difference between the FluoroMax®-3 and FluoroMax®-P is that the FluoroMax®-P
contains a phosphorimeter for phosphorescence measurements. This manual explains
how to operate and maintain a FluoroMax®-3 and FluoroMax®-P spectrofluorometer.
The manual also describes measurements and tests essential to obtain accurate data. For
a complete discussion of the almost limitless power provided by FluorEssence™, refer
to the FluorEssence™ User’s Guide (especially regarding software installation) and the
on-line help for Origin®and FluorEssence™, which accompany the system.

FluoroMax-3 v. 3.1 (3 Mar 2006) Introduction
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Chapter overview
1: Requirements & Installation Power and environmental requirements;
select the best spot for the instrument.
2: System Description How the FluoroMax®-3 and -P work.
3: System Operation Operation of the spectrofluorometer system,
and calibration instructions.
4: Data Acquisition How to use the special FluorEssence™
buttons to acquire and plot data; how to
determine peaks in an unknown sample.
5: Optimizing Data Acquisition Hints for improving the signal-to-noise ratio,
instructions for obtaining corrected data, and
other information useful for optimizing data
and ensuring reproducibility.
6: System Maintenance Routine maintenance procedures such as
replacing the lamp.
7: Troubleshooting Potential sources of problems, their most
probable causes, and possible solutions.
8: Producing Correction Factors How to correct for variation in the system’s
sensitivity across the spectral range.
9: FluoroMax®-P Phosphorimeter Opera-
tion
Theory, operation, applications, and
troubleshooting of the phosphorimeter,
available on the FluoroMax®-P system.
10: Automated Polarizers Installation, operation, and troubleshooting
of the optional automated polarizers.
11: Technical Specifications Instrument specifications and computer
requirements.
12: Components & Accessories Accessories available for the FluoroMax®-3,
and how to use them.
13: Glossary Some useful technical terms related to
fluorescence spectroscopy.
14: Bibliography Other important sources of information.
15: Declaration of Conformity
16: Index

FluoroMax-3 v. 3.1 (3 Mar 2006) Introduction
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Disclaimer
By setting up or starting to use any HORIBA Jobin Yvon product, you are accepting
the following terms:
You are responsible for understanding the information contained in this document. You
should not rely on this information as absolute or all-encompassing; there may be local
issues (in your environment) not addressed in this document that you may need to ad-
dress, and there may be issues or procedures discussed that may not apply to your situa-
tion.
If you do not follow the instructions or procedures contained in this document, you are
responsible for yourself and your actions and all resulting consequences. If you rely on
the information contained in this document, you are responsible for:
•Adhering to safety procedures
•Following all precautions
•Referring to additional safety documentation, such as Material Safety Data Sheets
(MSDS), when advised
As a condition of purchase, you agree to use safe operating procedures in the use of all
products supplied by HORIBA Jobin Yvon, including those specified in the MSDS
provided with any chemicals and all warning and cautionary notices, and to use all
safety devices and guards when operating equipment. You agree to indemnify and hold
HORIBA Jobin Yvon harmless from any liability or obligation arising from your use or
misuse of any such products, including, without limitation, to persons injured directly
or indirectly in connection with your use or operation of the products. The foregoing
indemnification shall in no event be deemed to have expanded HORIBA Jobin Yvon’s
liability for the products.
HORIBA Jobin Yvon products are not intended for any general cosmetic, drug, food, or
household application, but may be used for analytical measurements or research in
these fields. A condition of HORIBA Jobin Yvon’s acceptance of a purchase order is
that only qualified individuals, trained and familiar with procedures suitable for the
products ordered, will handle them. Training and maintenance procedures may be pur-
chased from HORIBA Jobin Yvon at an additional cost. HORIBA Jobin Yvon cannot
be held responsible for actions your employer or contractor may take without proper
training.
Due to HORIBA Jobin Yvon’s efforts to continuously improve our products, all speci-
fications, dimensions, internal workings, and operating procedures are subject to
change without notice. All specifications and measurements are approximate, based on
a standard configuration; results may vary with the application and environment. Any
software manufactured by HORIBA Jobin Yvon is also under constant development
and subject to change without notice.
Any warranties and remedies with respect to our products are limited to those provided
in writing as to a particular product. In no event shall HORIBA Jobin Yvon be held li-

FluoroMax-3 v. 3.1 (3 Mar 2006) Introduction
0-4
able for any special, incidental, indirect or consequential damages of any kind, or any
damages whatsoever resulting from loss of use, loss of data, or loss of profits, arising
out of or in connection with our products or the use or possession thereof. HORIBA
Jobin Yvon is also in no event liable for damages on any theory of liability arising out
of, or in connection with, the use or performance of our hardware or software, regard-
less of whether you have been advised of the possibility of damage.

FluoroMax-3 v. 3.1 (3 Mar 2006) Introduction
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Safety summary
The following general safety precautions must be observed during all phases of opera-
tion of this instrument. Failure to comply with these precautions or with specific warn-
ings elsewhere in this manual violates safety standards of design, manufacture and in-
tended use of instrument. HORIBA Jobin Yvon assumes no liability for the customer’s
failure to comply with these requirements. Certain symbols are used throughout the text
for special conditions when operating the instruments:
A WARNING notice denotes a hazard. It calls
attention to an operating procedure, practice, or
similar that, if incorrectly performed or ad-
hered to, could result in personal injury or
death. Do not proceed beyond a WARNING
notice until the indicated conditions are fully
understood and met. HORIBA Jobin Yvon Inc.
is not responsible for damage arising out of
improper use of the equipment.
A CAUTION notice denotes a hazard. It calls
attention to an operating procedure, practice, or
similar that, if incorrectly performed or ad-
hered to, could result in damage to the product.
Do not proceed beyond a CAUTION notice
until the indicated conditions are fully under-
stood and met. HORIBA Jobin Yvon Inc. is not
responsible for damage arising out of improper
use of the equipment.
Ultraviolet light! Wear protective goggles, full-
face shield, skin-protection clothing, and UV-
blocking gloves. Do not stare into light.
Intense ultraviolet, visible, or infrared light!
Wear light-protective goggles, full-face shield,
skin-protection clothing, and light-blocking
gloves. Do not stare into light.
Extreme cold! Cryogenic materials must al-
ways be handled with care. Wear protective
goggles, full-face shield, skin-protection cloth-
ing, and insulated gloves.
Caution:
Caution:
Caution:
Caution:
Warning:

FluoroMax-3 v. 3.1 (3 Mar 2006) Introduction
0-6
Explosion hazard! Wear explosion-proof gog-
gles, full-face shield, skin-protection clothing,
and protective gloves.
Risk of electric shock! This symbol warns the
user that uninsulated voltage within the unit
may have sufficient magnitude to cause electric
shock.
Danger to fingers! This symbol warns the user
that the equipment is heavy, and can crush or
injure the hand if precautions are not taken.
This symbol cautions the user that excessive
humidity, if present, can damage certain
equipment.
Hot! This symbol warns the user that hot equi-
pment may be present, and could create a risk
of fire or burns.
Read this manual before using or servicing the
instrument.
Wear protective gloves.
Warning:
Caution:
Warning:
Warning:
Warning:

FluoroMax-3 v. 3.1 (3 Mar 2006) Introduction
0-7
Wear appropriate safety goggles to protect the
eyes.
Wear an appropriate face-shield to protect the
face.
General information is given concerning opera-
tion of the equipment.
Note:

FluoroMax-3 v. 3.1 (3 Mar 2006) Introduction
0-8
Risks of ultraviolet exposure
Do not aim the UV light at anyone.
Do not look directly into the light.
Always wear protective goggles, full-face shield and skin protection clothing and
gloves when using the light source.
•Light is subdivided into visible light, ranging from 400 nm (violet) to 700 nm (red);
longer infrared, “above red” or > 700nm, also called heat; and shorter ultraviolet ra-
diation (UVR), “below violet” or < 400nm. UVR is further subdivided into UV-A
or near-UV (320–400 nm), also called black (invisible) light; UV-B or mid-UV
(290–320 nm), which is more skin penetrating; and UV-C or far-UV (< 290 nm).
•Health effects of exposure to UV light are familiar to anyone who has had sunburn.
However, the UV light level around some UV equipment greatly exceeds the level
found in nature. Acute (short-term) effects include redness or ulceration of the skin.
At high levels of exposure, these burns can be serious. For chronic exposures, there
is also a cumulative risk of harm. This risk depends upon the amount of exposure
during your lifetime. The long-term risks for large cumulative exposure include
premature aging of the skin, wrinkles and, most seriously, skin cancer and cataract.
•Damage to vision is likely following exposure to high-intensity UV radiation. In
adults, more than 99% of UV radiation is absorbed by the anterior structures of the
eye. UVR can contribute to the development of age-related cataract, pterygium,
photodermatitis, and cancer of the skin around the eye. It may also contribute to
age-related macular degeneration. Like the skin, the covering of the eye or the cor-
nea, is epithelial tissue. The danger to the eye is enhanced by the fact that light can
enter from all angles around the eye and not only in the direction of vision. This is
especially true while working in a dark environment, as the pupil is wide open. The
lens can also be damaged, but because the cornea acts as a filter, the chances are re-
Caution: This instrument is used in conjunction with ul-
traviolet light. Exposure to these radiations, even re-
flected or diffused, can result in serious, and sometimes
irreversible, eye and skin injuries.
Overexposure to ultraviolet rays threatens human
health by causing:
•Immediate painful sunburn
•Skin cancer
•Eye damage
•Immune-system suppression
•Premature aging

FluoroMax-3 v. 3.1 (3 Mar 2006) Introduction
0-9
duced. This should not lessen the concern over lens damage however, because cata-
racts are the direct result of lens damage.
Burns to the eyes are usually more painful and serious than a burn to the skin. Make
sure your eye protection is appropriate for this work. NORMAL EYEGLASSES OR
CONTACTS OFFER VERY LIMITED PROTECTION!
Training
For the use of UV sources, new users must be trained by another member of the labora-
tory who, in the opinion of the member of staff in charge of the department, is suffi-
ciently competent to give instruction on the correct procedure. Newly trained users
should be overseen for some time by a competent person.
Warning: UV exposures are not immediately felt. The
user may not realize the hazard until it is too late and
the damage is done.

FluoroMax-3 v. 3.1 (3 Mar 2006) Introduction
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Additional risks of xenon lamps
Among the dangers associated with xenon lamps
are:
•Burns caused by contact with a hot xenon lamp.
•Fire ignited by hot xenon lamp.
•Interaction of other nearby chemicals with intense ultraviolet, visible, or infrared
radiation.
•Damage caused to apparatus placed close to the xenon lamp.
•Explosion or mechanical failure of the xenon lamp.
Visible radiation
Any very bright visible light source will cause a human aversion response: we either
blink or turn our head away. Although we may see a retinal afterimage (which can last
for several minutes), the aversion response time (about 0.25 seconds) normally protects
our vision. This aversion response should be trusted and obeyed. NEVER STARE AT
ANY BRIGHT LIGHT-SOURCE FOR AN EXTENDED PERIOD. Overriding the
aversion response by forcing yourself to look at a bright light-source may result in per-
manent injury to the retina. This type of injury can occur during a single prolonged ex-
posure. Excessive exposure to visible light can result in skin and eye damage.
Visible light sources that are not bright enough to cause retinal burns are not necessar-
ily safe to view for an extended period. In fact, any sufficiently bright visible light
source viewed for an extended period will eventually cause degradation of both night
and color vision. Appropriate protective filters are needed for any light source that
causes viewing discomfort when viewed for an extended period of time. For these rea-
sons, prolonged viewing of bright light sources should be limited by the use of appro-
priate filters.
The blue-light wavelengths (400–500 nm) present a unique hazard to the retina by caus-
ing photochemical effects similar to those found in UV-radiation exposure.
Infrared radiation
Infrared (or heat) radiation is defined as having a wavelength between 780 nm and 1
mm. Specific biological effectiveness “bands” have been defined by the CIE (Commis-
sion International de l’Eclairage or International Commission on Illumination) as fol-
lows:
•IR-A (near IR) (780–1400 nm)
•IR-B (mid IR) (1400– 3000 nm)
Warning: Xenon lamps
are dangerous.
Please read the fol-
lowing precautions.

FluoroMax-3 v. 3.1 (3 Mar 2006) Introduction
0-11
•IR-C (far IR) (3000 nm–1 mm)
The skin and eyes absorb infrared radiation (IR) as heat. Workers normally notice ex-
cessive exposure through heat sensation and pain. Infrared radiation in the IR-A that
enters the human eye will reach (and can be focused upon) the sensitive cells of the ret-
ina. For high irradiance sources in the IR-A, the retina is the part of the eye that is at
risk. For sources in the IR-B and IR-C, both the skin and the cornea may be at risk from
“flash burns.” In addition, the heat deposited in the cornea may be conducted to the lens
of the eye. This heating of the lens is believed to be the cause of so called “glass-
blowers’ ” cataracts because the heat transfer may cause clouding of the lens.
•Retinal IR Hazards (780 to 1400 nm): possible retinal lesions from acute high ir-
radiance exposures to small dimension sources.
•Lens IR Hazards (1400 to 1900 nm): possible cataract induction from chronic lower
irradiance exposures.
•Corneal IR Hazards (1900 nm to 1 mm): possible flashburns from acute high irradi-
ance exposures.
Who is likely to be injured? The user and anyone exposed to the radiation or xenon
lamp shards as a result of faulty procedures. Injuries may be slight to severe.

FluoroMax-3 v. 3.1 (3 Mar 2006) Introduction
0-12

FluoroMax-3 v. 3.1 (3 Mar 2006) Requirements & Installation
1-1
1: Requirements & Installation
Safety-training requirements
Every user of the FluoroMax®-3 and FluoroMax®-P must know general and specific
safety procedures before operating the instrument. For example, proper training in-
cludes (but is not limited to):
•Understanding the risks of exposure to ultraviolet, visible, and infrared light, and
how to avoid unsafe exposures to these types of radiation
•Handling xenon-lamp bulbs, and their dangers
•Safe handling for all chemicals and other samples used in the instrument
Safety-training may be purchased from HORIBA Jobin Yvon. Contact your Spex®
Fluorescence Representative or the Fluorescence Service Department for details.

FluoroMax-3 v. 3.1 (3 Mar 2006) Requirements & Installation
1-2
Surface requirements
•A sturdy table- or bench-top.
•Surface must hold 90 kg (200 lbs.).
•Surface should be about 27" × 72" (69 cm × 183 cm) to hold spectrofluorometer,
computer, and accessories comfortably.
•Overhead clearance should be at least 36" (91 cm).