INSTRUCTION MANUAL

HITACHI FLUORESCENCE SPECTROPHOTOMETER

FL SOLUTIONS PROGRAM
(OPERATION MANUAL)

Hitachi High-Technologies Corporation

3rd Edition, November 2001

1st Edition, 2001
Copyright Hitachi High-Technologies Corporation 2001. All rights reserved. Printed in Japan. Part No. 251-9067-2 TF-TF (GT-LT)
PREFACE
Thank you very much for purchasing the Hitachi
High-Technologies Corporation fluorescence spectrophotometer.

This instrument is intended for use by persons having a

basic knowledge of chemical analysis. Remember that
improper use of analytical instruments, chemicals or
samples would result not only in wrong analytical data but
also in consequences adverse to safety.

Carefully read this instruction manual before attempting

operation. For proper use of the fluorescence
spectrophotometer, please acquaint yourself with it.

ABOUT THIS MANUAL

The Hitachi High-Technologies Corporation fluorescence

spectrophotometer is accompanied by the following two
instruction manuals:

F-2500/F-4500 Fluorescence Spectrophotometer Instruction

Manual - Maintenance
Hitachi High-Technologies Corporation Fluorescence
Spectrophotometer Instruction Manual - FL Solutions Program
- Operation

The cautionary instructions concerning safety are contained in

the F-2500/F-4500 Fluorescence Spectrophotometer Instruction
Manual - Maintenance. Be sure to read them before attempting
operation.

This instruction manual describes the operational procedures for

the FL Solutions program formulated for the Hitachi High-
Technologies Corporation fluorescence spectrophotometer.
Be sure to read through this instruction manual before attempting
operation.

-1-
-2-
IMPORTANT
Precautions on Electromagnetic Wave Interference

1. Possible Electromagnetic Wave Interference Caused by

The Instrument

When the instrument is used in a residential area or an

adjacent area thereto, it may cause interference to radio
and television reception.
To prevent this, use the specified system connection cables
in strict accordance with the instruction manual.
The instrument is designed to minimize possible
electromagnetic wave interference caused by it if the
specified cables are connected properly.
However, there is no guarantee that electromagnetic wave
interference will not be caused by the instrument.
If the instrument does cause interference to radio or
television reception, which can be determined by turning
the instrument off and on, the user is encouraged to try to
correct the interference by one or more of the following
measures:
Increase separation between the instrument and the
radio/TV receiver.
Connect the instrument to an outlet on a circuit different
from that to which the radio/TV receiver is connected.

2. Possible Electromagnetic Wave Interference Affecting

the Instrument

If the instrument is used near an intense electromagnetic

source, interference noise may be given to the instrument
to incur an adverse effect on its performance or
functionality.
To present this, use the specified system connection cables
in strict accordance with the instruction manual.
The instrument is designed to minimize possible
electromagnetic wave interference affecting it if the
specified cables are connected properly.
However, there is no guarantee that electromagnetic wave
interference will not occur in the instrument. If the
instrument does incur electromagnetic wave interference,
which can be determined by turning on and off possible
sources of electromagnetic interference nearby, the user is
encouraged to try to correct the interference by one or more
of the following measures:

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 Reorient the instrument.
Increase separation between the instrument and
possible sources of electromagnetic interference.
Increase separation between the power cable of the
instrument and possible sources of electromagnetic
interference.
Connect the instrument to an outlet on a circuit different
from that to which possible sources of electromagnetic
interference are connected.
Check that any other device connected with the
instrument is not affected by electromagnetic
interference.

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WARRANTY ON PRODUCT
The Hitachi fluorescence spectrophotometer is warranted to be
free from defects in material or workmanship under normal use
within the product specifications indicated in this manual and
under conditions given below. This warranty is void if the
instrument is not used in accordance with the instruction manual.

(1) Scope of Warranty

(a) Any parts which prove to be defective in design or
workmanship during the warranty period will be
repaired, adjusted or replaced without charge.
(b) A substitute part may be used for repair, or
replacement with an equivalent product may be made
instead of repair.
(c) Such system components as a personal computer and
printer to be updated frequently for improvement may
not be available in original versions at the time of
replacement.

(2) Warranty Period

One year from the date of initial installation
(Excluding consumables such as a xenon lamp)

(3) Availability of Technical Support Service

Technical support service for the instrument is available
within regular working hours on workdays predetermined by
us.

(4) Limitations and Exclusions on Warranty

Note that the following cases are excluded from the scope
of this warranty.
(a) Failure due to operation at a place not meeting the
installation requirements specified by us.
(b) Failure due to power supply voltage/frequency other
than specified by us or due to abnormality in power
supply.
(c) Corrosion or deterioration of the tubing due to
impurities contained in reagent, gas, air or cooling
water supplied by the user.
(d) Corrosion of the electric circuits or deterioration of the
optical elements due to highly corrosive atmospheric
gas.

-5-
(e) Failure due to use of hardware, software or spare parts
other than specified by us.
(f) Failure due to improper handling or maintenance by
the user.
(g) Failure due to maintenance or repair by a service
agent not approved or authorized by us.
(h) After disposal of the instrument, or after its resale
without our approval.
(i) Failure due to relocation or transport after initial
installation.
(j) Failure due to disassembly, modification or relocation
not approved by us.
(k) Consumables, and failure of parts that have reached
the end of specified useful life.
(l) Failure of parts excluded from the warranty in the
instruction manual or other documents.
(m) Failure due to acts of God, including fire, earthquake,
storm, flood, lightning, social disturbance, riot, crime,
insurrection, war (declared or undeclared), radioactive
pollution, contamination with harmful substances, etc.
(n) Failure of the hardware, or damage to the system
software, application software, data or hard disk due to
computer virus infection.
(o) Failure of the personal computer connected with the
instrument, or damage to the system software,
application software, data or hard disk due to power
interruption or momentary power voltage drop caused
by lightning or the like.
(p) Failure of the personal computer connected with the
instrument, or damage to the system software,
application software, data or hard disk due to
disconnection of main power to the personal computer
without taking the specified normal shutdown
procedure.

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DISCLAIMER OF WARRANTY
(1) WE MAKE NO WARRANTIES, EITHER EXPRESS OR
IMPLIED, EXCEPT AS PROVIDED HEREIN, INCLUDING
WITHOUT LIMITATION THEREOF, WARRANTIES AS TO
MARKETABILITY, MERCHANTABILITY, FOR A
PARTICULAR PURPOSE OR USE, OR AGAINST
INFRINGEMENT OF ANY PATENT. IN NO EVENT
SHALL WE BE LIABLE FOR ANY DIRECT, INCIDENTAL
OR CONSEQUENTIAL DAMAGES OF ANY NATURE, OR
LOSSES OR EXPENSES RESULTING FROM ANY
DEFECTIVE PRODUCT OR THE USE OF ANY PRODUCT.

(2) NO ORAL OR WRITTEN INFORMATION OR ADVICE

GIVEN BY US, OUR DEALERS, DISTRIBUTORS,
AGENTS OR EMPLOYEES SHALL CREATE A
WARRANTY OR IN ANY WAY INCREASE THE SCOPE
OF THIS WARRANTY.

Installation, Relocation and After-sale Technical Service

Installation of the instrument shall be carried out by or under

supervision of our qualified service personnel or our authorized
service agent.
Before installation of the instrument, the customer shall make
preparations for satisfying the installation requirements in
accordance with this manual.
When relocation of the instrument becomes necessary after
initial installation (delivery), please notify our local sales
representative or service office nearest you.

Technical Seminars and Training for Customers

For the customers to acquire in-depth understanding of the

analytical instruments, technical seminars and customer training
courses are available at our or your site. For further information,
contact our local sales representative.
(The technical seminars and customer training courses are
available at charge.)

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OTHER PRECAUTIONS
Handling of Chemicals and Samples

(1) The user is responsible for following relevant legal

standards and regulations in the handling, storage and
discarding of chemicals and samples used in analytical
operations of the instrument.

(2) Reagents, standard solutions and accuracy-control

samples shall be handled, stored and discarded as
instructed by the respective suppliers.

Notice on This Instruction Manual

(1) The information contained in this manual is subject to

change without notice for product improvement.

(2) This manual is copyrighted by Hitachi High-Technologies

Corporation with all rights reserved.

(3) No part of this manual may be reproduced or transmitted in

any form or by any means without our express written
permission.

Trademark Acknowledgments

Microsoft, Windows, Microsoft Excel, Microsoft Word, and

Windows 98 are registered trademarks of Microsoft Corporation
USA.
All other brand names and product names used in this manual
are trademarks, registered trademarks, or trade names of their
respective holders.

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 SAFETY SUMMARY
PRECAUTIONS ON SAFETY

Before using the Hitachi fluorescence spectrophotometer, be

sure to read the following safety instructions carefully.

General Safety Guidelines

Follow all the operating procedures provided in this manual.

Installation and maintenance of this instrument shall be
carried out by service personnel qualified therefore.
Be sure to observe the warnings indicated on the product and
in the instruction manual. Failure to do so could result in
personal injury or damage to the product.
The hazard warnings which appear on the warning labels on
the product or in the manual have one of the following alert
headings consisting of an alert symbol and signal word
DANGER, WARNING, or CAUTION.

DANGER : Indicates an imminently hazardous

situation which, if not avoided, will result
in death or serious injury.
(This warning does not apply to this
product.)
WARNING : Indicates a potentially hazardous
situation which, if not avoided, can result
in death or serious injury.
CAUTION : Indicates a hazardous situation which, if
not avoided, will or can result in minor or
moderate injury, or serious damage to
the product.
: The alert symbol shown at left precedes
every signal word for hazard warnings,
and appears in safety-related
descriptions in the manual.

"NOTE" and "NOTICE" are heading words which do not concern

personal safety directly.

SAFETY - 1
 SAFETY SUMMARY
General Safety Guidelines (Continued)

NOTICE : Used to indicate an instruction for preventing

damage to the product.
NOTE : Used to indicate an instruction for ensuring
correct use of the product and accurate analysis
therewith.

Do not modify the instrument, replace parts that are not user-
serviceable, use non-specified parts, nor remove safety
devices, as it could be hazardous.
Installation at delivery, maintenance and relocation should be
referred to service personnel qualified by us.
Do not perform any operation or action other than described
in this manual. When in doubt, please contact our local
sales representative or service office nearest you.
When using a chemical for analytical operation, be sure to
provide proper ventilation in the laboratory room as per local
requirements. Inadequate ventilation could endanger your
health.
Keep in mind that the hazard warnings in this manual or on
the product cannot cover every possible case, as it is
impossible to predict and evaluate all circumstances
beforehand.
Be alert and use your common sense.

SAFETY - 2
 SAFETY SUMMARY
Safety Notices on Fluorescence Spectrophotometer

Electricity

Make sure that the fluorescence spectrophotometer is

supplied with power of 100V AC, 1 kVA or more (50 Hz or
60 Hz). If the power voltage fluctuates or noise is applied to
the power line, the spectrophotometer may be adversely
affected, resulting in any trouble in it.
Be sure to provide grounding connection together with power
line connection. The grounding resistance should be 100
ohms or less (conforming to Class-D grounding installation of
electric facility technical standards). Check that the
grounding connection is proper. If the grounding connection
is improper, the spectrophotometer may become vulnerable to
external noise or stray voltage may be produced in it, resulting
in an electric shock hazard.
The high voltage circuit is used inside the spectrophotometer.
To prevent electric shock, do not open covers during
operation unless otherwise necessary.

Source of Heat/Flame

Do not smoke or hold a flame near the fluorescence

spectrophotometer.

Data Backup

Data stored on the hard disk may become unusable due to a

system failure, wrong operation, computer virus infection, etc.
To ensure data integrity in case of accidental damage to the hard
disk, periodically make backup copies of hard disk files onto
floppy disks.
To prevent an erroneous operation on the hard disk, always
reserve approx. 100 MB of free space as a working area for
application software.

SAFETY - 3
 SAFETY SUMMARY
Protection against Computer Viruses

If any program/data is damaged suddenly or an unexpected

operation/screen is encountered, the personal computer is
suspected of being infected by a computer virus.
Computer viruses are malicious programs that sneak into
personal computers to cause misbehavior or damage to data.
And, a program designed to offer protection against computer
viruses is called a vaccine program.

Possible causes of virus infection are:

Downloading a virus-laden program through communication.
Using a floppy disk or other storage medium infected by a
virus.

Note also that once any personal computer is infected by a virus,

it may spread to other computers via communication or storage
medium.
Never use a program or storage medium that is suspected
of containing a virus.
If there is a possibility of virus infection, check for a virus using a
vaccine program. Note, however, that some kinds of vaccine
programs cannot eradicate particular viruses. So, be sure to
make a backup of hard disk files beforehand.
The user is requested to prepare a vaccine program and carry
out virus removal on his or her own responsibility.

Power Interruption

On occurrence of momentary power voltage drop due to power

interruption or lightning, the personal computer may become
faulty or the system software, application software or data may
be damaged. For protection against momentary power voltage
drop, it is advisable to use an AC uninterruptible power supply
unit (stated according to the Japanese Electronic Industry
Development Association guidelines for protection against
momentary power voltage drop in personal computers).

SAFETY - 4
 SAFETY SUMMARY
Turning On/Off Personal Computer

Do not turn off power to the personal computer while the hard
disk or floppy disk drive is active. If power to the personal
computer is turned off while the hard disk or floppy disk is being
accessed, the personal computer may become faulty or
data/software stored in it may be damaged.
Before turning off power to the personal computer, be sure to
quit the fluorescence spectrophotometer control/data processing
program (FL Solutions program) first and then take the shut-
down procedure using the system software.

SAFETY - 5
FOREWORD
This instruction manual is intended for the users of the Hitachi
fluorescence spectrophotometer. It describes the installation,
maintenance, operation and measurement procedures.
The fluorescence spectrophotometer is designed for the
purpose of fluorescence analysis. Never use this instrument
for any other purposes.
The fluorescence spectrophotometer is intended for use by
persons having a basic knowledge of chemical analysis.
Other persons should use them in the presence of a person
familiar with chemical analysis. Remember that improper
use of analytical instruments, chemicals or samples would
result not only in wrong analytical data but also in
consequences adverse to safety.
Before attempting installation/maintenance of the
fluorescence spectrophotometer, read the instruction manual
carefully and fully understand the cautionary instructions for
the sake of safety.
Keep this manual handy for easy reference when necessary.
The information contained in this manual is subject to change
without prior notice.

FOREWORD -
1
 CONTENTS

PREFACE .........................................................................................................................1

ABOUT THIS MANUAL ...................................................................................................1

IMPORTANT ....................................................................................................................3
Precautions on Electromagnetic Wave Interference ......3
1. Possible Electromagnetic Wave Interference Caused
by the Instrument .............................................................3
2. Possible Electromagnetic Wave Interference Affecting
the Instrument ..................................................................3

WARRANTY ON PRODUCT ............................................................................................5

Installation, Relocation and After-sale Technical Service ..........................................7

Technical Seminars and Training for Customers ........................................................7

OTHER PRECAUTIONS ..................................................................................................8

Handling of Chemicals and Samples ...................................8
Notice on This Instruction Manual ........................................8
Trademark Acknowledgments ..............................................8

SAFETY SUMMARY.................................................................................... SAFETY-1

PRECAUTIONS ON SAFETY ............................... SAFETY-1
General Safety Guidelines ............................... SAFETY-1
Safety Notices on Fluorescence
Spectrophotometer ................................................. SAFETY-3
Electricity ............................................................... SAFETY-3
Source of Heat/Flame ........................................... SAFETY-3
Data Backup .......................................................... SAFETY-3
Protection against Computer Viruses .................... SAFETY-4
Power Interruption ................................................. SAFETY-4
Turning On/Off Personal Computer ....................... SAFETY-5

FOREWORD ...............................................................................................FOREWORD-1

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1. FUNCTIONS AND SCREEN COMPOSITION ...................................................... 1-1
1.1 Functions ............................................................. 1-1
1.2 Installing the FL Solutions Program .................. 1-5
1.3 Startup of FL Solutions Program ....................... 1-9
1.4 Screen Composition ............................................ 1-13
1.4.1 Basics of Window Operation ..................... 1-13
1.4.2 Kinds of Windows ...................................... 1-17
1.4.3 Data Processing Window .......................... 1-18
1.4.4 Explanation of Tool Buttons ...................... 1-21
1.5 Functions of Function Keys ............................... 1-26
1.6 Explanation of Monitor Window ......................... 1-27
1.7 Input of Spectrophotometer Serial No. .............. 1-30
1.8 Window Setting .................................................... 1-31
1.9 Saving a Data File ................................................ 1-39
1.10 Entry of File Name for Saving ............................ 1-40
1.11 Photometer Menu Commands ............................ 1-42
1.12 Utility Menu Commands ...................................... 1-43
1.13 Termination of FL Solutions Program ............... 1-44

2. WAVELENGTH SCAN .......................................................................................... 2-1

2.1 Flow of Operation ................................................ 2-1
2.2 Creating an Analysis Method ............................. 2-2
2.3 Defining Your Samples ....................................... 2-15
2.4 Prescan ................................................................. 2-17
2.5 Conducting a Wavelength Scan ......................... 2-18
2.6 Data Printout ........................................................ 2-20

3. TIME SCAN ........................................................................................................... 3-1

3.1 Flow of Operation ................................................ 3-1
3.2 Creating an Analysis Method ............................. 3-2
3.3 Defining Your Samples ....................................... 3-14
3.4 Conducting a Time Scan ..................................... 3-16
3.5 Data Printout ........................................................ 3-18
3.6 Calculating the Phosphorescence Life ............. 3-20

4. PHOTOMETRY ..................................................................................................... 4-1

4.1 Flow of Operation ................................................ 4-1
4.2 Creating an Analysis Method ............................. 4-2
4.3 Defining Your Samples ....................................... 4-21
4.4 Conducting Measurement .................................. 4-22
4.4.1 Manual Input of Standard Data ................. 4-26
4.4.2 Statistical Calculation ................................ 4-27
4.5 Printing Data ........................................................ 4-28
4.6 Remeasuring Standard Data and
Sample Data ......................................................... 4-30
4.7 Manual Input of Standard Data ........................... 4-31
4.8 Measurement Using Saved
Calibration Curve .................................................. 4-32

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5. 3-D SCAN................................................................................................................ 5-1
5.1 Flow of Operation ................................................ 5-1
5.2 Creating an Analysis Method ............................. 5-2
5.3 Defining Your Samples ....................................... 5-13
5.4 Conducting Measurement .................................. 5-14
5.5 Displaying a Birds-Eye View ............................. 5-16
5.6 Data Printout ........................................................ 5-17

6. DATA PROCESSING ............................................................................................ 6-1

6.1 Loading of Data ................................................... 6-1
6.2 Spectrum Tracing ................................................ 6-3
6.3 Scale Change ....................................................... 6-5
6.4 Change of Graph Axis ......................................... 6-6
6.5 Change of Spectral Line ..................................... 6-7
6.6 Detection of Spectral Peaks ............................... 6-9
6.7 Indication of Peak Wavelength on Spectrum .... 6-10
6.8 Smoothing of Spectrum ...................................... 6-12
6.9 Derivative Operation on Spectrum .................... 6-13
6.10 Overlaid Display of Spectra ................................ 6-14
6.11 Arithmetic Operation between Spectra ............. 6-17
6.12 Area Calculation .................................................. 6-20
6.13 Transfer of Data to Microsoft(R) Excel .............. 6-23
6.14 Pasting of Spectra to Microsoft(R) Word .......... 6-25
6.15 Conversion of Data to ASCII Text File ............... 6-27
6.16 Renaming Your Sample ...................................... 6-29
6.17 Printing of Wavelength and
Photometric Values under Display .................... 6-30
6.18 Reference to Spectrum Measured in
Photometry Mode ................................................ 6-33

7. MORE CONVENIENT OPERATING METHODS (APPLICATION PART) ............ 7-1

7.1 For Always Starting Up with
Same Analysis Method ....................................... 7-1
7.2 Online/Offline Setting of Photometer ................. 7-3
7.3 Check of Data File Information ........................... 7-4
7.4 Saving of Analysis Method ................................. 7-7
7.5 Even Log .............................................................. 7-8
7.6 Check of Program Version ................................. 7-10
7.7 Saving of Sample Table ...................................... 7-11
7.8 Change of Window Display ................................ 7-12
7.9 Check of Photometric Value at
Specified Wavelength ........................................... 7-15
7.10 Collective Printing of Files ................................. 7-16

- iii
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8. MEASUREMENT USING OPTIONAL ACCESSORIES ........................................ 8-1
8.1 Use of Sample Sipper .......................................... 8-1
8.1.1 When Not Using a Sample Table .............. 8-1
8.1.2 When Using a Sample Table .................... 8-4
8.1.3 In Wavelength Scan or
Time Scan Mode ....................................... 8-8
8.2 Use of AS-3000 Intelligent Autosampler ........... 8-9
8.2.1 When Not Using a Sample Table .............. 8-9
8.2.2 When Using a Sample Table .................... 8-9
8.3 For Analog Output ............................................... 8-10

APPENDIX ................................................................................................................... A-1

INDEX ...................................................................................................................INDEX-1

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 1.1

1. FUNCTIONS AND SCREEN COMPOSITION

1.1 Functions

In this program, the following measurement modes are available:

Wavelength scan
Time scan
Photometry
3-D Scan
(3-D Scan program (option) required for Model F-2500)

Wavelength Scan
(1) Photometric value data can be printed at the specified
wavelength intervals or at up to 12 specified wavelengths.

(2) The function keys provide functions unique to the

wavelength scan mode.
Measurement: [F4], Stop: [F6]

(3) Wavelength scan measurement can be repeated max. 99

times. And, a repetition period is settable.

Time Scan
(1) Kinetics, or rate calculation will be performed.

(2) Photometric value data can be printed at the specified time

intervals or at up to 12 specified time points.

Photometry
(1) Quantification method is selectable among specified
wavelength, peak area, peak height, peak ratio and
derivative methods. Up to 20 standards can be measured.
A calibration curve is prepared by drawing polygonal lines
through each point of standard concentrations or a
regression calibration curve is prepared by least square fit.
It is also possible to measure up to 20 standards and
prepare a calibration curve based on an average value of
standard concentrations.

(2) A calibration curve can be redrawn by re-measuring a

specific standard. In a calibration curve graph on the
screen window, a value at each point can be read out
using the cursor.

1-1
1.1 Functions

(3) A calibration curve can be printed.

A calibration curve can be drawn by inputting its factor
value.

(4) Measurement data and a calibration curve are displayed

on the same screen window. It is therefore possible to
carry out measurement while observing the calibration
curve.

(5) According to the coefficient of determination calculated

automatically, judgment can be made on a degree of
matching between standard values and a calibration curve.
Two/three-wavelength calculation is permitted in
preparation of a calibration curve.

(6) Statistical calculation is possible.

Mean value of measured data and standard deviation (SD)
can be calculated.

(7) Upper/lower limit judgment will be performed.

(8) Spectrum display under photometry

In the data measured by a quatification method; peak
height, peak area, peak ratio or derivative, a spectrum due
to wavelength scan is displayable.

3-D Scan
(1) Spectral calculation can be performed using the measured
data.

(2) A peak table can be displayed.

(3) A birds-eye view/synchronous spectrum can be displayed

and printed.

(4) Printing conditions are selectable.

In use of the DDE function, the wavelength interval and
start/end wavelengths of data list can be selected.

1-2
 1.1

Secondary Processing of Data

Measured data can be saved, and a variety of processings can
be carried out on the saved data.

(1) Automatic reading of data (spectrum tracing)

By placing the cursor on the currently displayed spectral
graph, a photometric value at the cursor position can be
read out.

(2) Peak detection

Peaks and valleys of the currently displayed spectrum are
detected automatically.

(3) Change of scale

A measured spectrum can be enlarged or reduced by
changing the ordinate and abscissa scales.

(4) Smoothing
A measured spectrum can be smoothed for elimination of
noise components.

(5) Derivative
Spectral derivative is obtainable. It is permitted to specify
an order of derivative, that of smoothing and a count value
of data.

(6) Spectrum calculation

Fundamental arithmetic operations can be made between
spectra or a spectrum can be multiplied with a factor.

(7) Area
The area value of the specified peak will be calculated.
The average area value is also calculable among peaks.

(8) File conversion

Measurement data can be converted into an ASCII text file
(*.txt) or JCAMP-DX file (*.dx). It is also possible to save
a spectrum and calibration curve graph in a Windows meta
file (*.wmf).

(9) Display/print font setting

The font, style and size of characters to be displayed and
printed can be changed as desired.

1-3
1.1 Functions

(10) Data acquisition

Photometric value data on the data processing window and
monitor window can be acquired. Acquisition is allowed
within 100 data.

(11) Printing items collectively specifiable

Printing items on overlaid spectra can be specified
collectively.

(12) Specification of printing items

Items in a peak table can be selected and printed.

(13) Portrait or Landscape printing settable

Printing can be made Portrait or Landscape.

(14) Collective spectrum printing

Printing can be carried out just by specifying a file.

(15) Selection of spectrum to be displayed

The spectrum to be displayed for overlaying is selectable.

(16) Automatic saving mode ON/OFF settable

ON/OFF can be set in the sample name/comment input
window.
Automatic saving is effective in all the modes that use a
sample table.

(17) Data in each mode can be transferred to Microsoft Excel.

(This is valid when Microsoft Excel 97 has been installed
in PC.)

(18) After copying spectral data into commercial software such

as Microsoft Word, its scale, line type, color, etc. are
changeable on the software.

(19) The report generator (separately available, P/N 250-0455)

can be used.

1-4
 1.2

1.2 Installing the FL Solutions Program

For using the FL Solutions program, it must be installed using

the attached system disks.
Installation of the FL Solutions program is required when using it
at the first time (not required later for each operation). In the
following installation session, it is assumed that:
The floppy disk drive is assigned as drive A.
The destination of installation is assigned as drive C.

(1) Using the mouse, click the Start button of the Windows 98
and select Settings - Control Panel. The Control Panel
window will then appear as shown in Fig. 1-1.

Fig. 1-1

(2) In the Control Panel window, double-click the Add/Remove

Programs icon. The Add/Remove Programs dialog box
will then appear as shown in Fig. 1-2. In this dialog box,
click Install.

Fig. 1-2

1-5
1.2 Installing the FL Solutions Program

(3) Then, when the dialog box shown in Fig. 1-3 appears, push
in the first one of the attached system disks and click Next.

Fig. 1-3

(4) Then, when the dialog shown in Fig. 1-4 appears, click
Finish.

Fig. 1-4

Installation process will then begin. Take steps according

to the instruction messages.

1-6
 1.2

(5) On completion of the installation process, restart the

personal computer.

(6) For startup of the FL Solutions program, refer to 1.3.

NOTES: 1. Monitor resolution must be set at 800 600 at

minimum. Otherwise, each window of the FL
Solutions program may not be displayed fully.
2. When the large font size of display is selected,
words may not be indicated normally on each
window. In this case, font size should be changed
to the small one. For change, select Control
Panel - Display Properties - Settings - Font Size.

1-7
1.2 Installing the FL Solutions Program

Reference:
Re-installation of FL Solutions Program

For re-installing this program, remove it first and then install it in

the following procedure.
Select Start - Settings - Control Panel. In the Control Panel
window, double-click the Add/Remove Programs icon, and the
dialog box shown in Fig. 1-5 will then appear.

Fig. 1-5

In this dialog box, select FL Solutions and click the Add/

Remove button. When it is removed, click OK.
Thereafter, install the FL Solutions program again.

1-8
 1.3

1.3 Startup of FL Solutions Program

To start up the FL Solutions program, take the following

procedure.

(1) Connect the communication cable (standard accessory)

between the fluorescence spectrophotometer and the
personal computer. Then, turn on power to the
spectrophotometer and the computer in this order.

(2) The Windows 98 starts.

(3) Click the Start button. Point to Programs and Hitachi

Applications and select FL Solutions. (See Fig. 1-6.)
Or double-click the FL Solutions program icon.
If the FL Solutions program is set as a startup application
in the Windows 98, the program automatically starts when
turning on power supply of the personal computer.
This setting can be selected at installation of the program.

Icon of FL Solutions program

Fig. 1-6

1-9
1.3 Startup of FL Solutions Program

(4) The fluorescence spectrophotometer connected to the

communication COM port of computer is now recognized
and the window shown in Fig. 1-7 is displayed. Then, the
spectrophotometer is initialized.

Fig. 1-7

(5) A gauge of initialization is displayed as shown in Fig. 1-8,

so its progress can be visually checked.

Fig. 1-8

1 - 10
 1.3

NOTE: If the spectrophotometer is not connected with

the COM port or its power is not turned on, the
monitor window (F-2500 Fluorescence
Spectrophotometer on COM1 indicated on its
title bar) is not presented. In this status,
functions for other than measurement are usable.
For measurement, first confirm connection of the
cable, and then turn on power supply of the
spectrophotometer. Next, select the
Instruments (I) command from the Tools (T)
menu. When selecting and connecting the
spectrophotometer, the window shown in Fig. 1-8
is presented.

In execution of initialization, self-diagnosis is performed for

the following items:
ROM
RAM
Wavelength drive system
Xenon lamp turn-on
EEPROM
Slit mechanism
Chopper
Backup battery voltage (only with Model F-4500)
Contents of wavelength and slit calibration values

If an abnormality is found in the self-diagnosis, the relevant

error message will be displayed. In this case, a corrective
measure should be taken, referring to 4.7 of the
maintenance edition.
If necessary, notify our local repair service office.

Fig. 1-9

1 - 11
1.3 Startup of FL Solutions Program

(6) On completion of initialization, the window shown in

Fig. 1-10 is displayed.

Fig. 1-10

1 - 12
 1.4.1

1.4 Screen Composition

1.4.1 Basics of Window This manual contains multiple methods for executing a function.
Operation For implementing each method, explanation is given using the
naming described below.

(1) Menu bar and Command

Menu bar is a bar on which menus such as File (F) and

Edit (E) are arranged. Commands are contained in each
menu.
In the case shown in Fig. 1-11, an expression of Scan (S)
command of Spectrophotometer (S) menu is used.
A menu or command is selectable by 2 methods.
Move the mouse pointer to a menu and click on it.
Key in the alphabetic letter after a menu name with the
Alt key held down. Then, key in the alphabetic letter
after a command name.

Menu bar

Command

Fig. 1-11 Menu Bar and Command

1 - 13
1.4 Screen Composition

(2) Toolbar and Tool button

Toolbar is a row of tool buttons such as and tool

buttons provide shortcuts for common menu commands.
In using , it is expressed as button of
Measurement toolbar or (analytical condition)
button.
For details of each button, refer to 1.4.4.

Toolbars

Fig. 1-12 Toolbar

1 - 14
 1.4.1

(3) Dialog box

When you click the button of Measurement toolbar, a

small window as shown in Fig. 1-13 is presented.
This window is called a dialog box.

Fig. 1-13 Dialog Box

(4) Tab

Tabs are used in the Analysis Method window or Property

window.
Click a tab for changing display. Each tab contains setting
items. The display shown in Fig. 1-14 is expressed as
Instrument tab.

Tabs

Fig. 1-14

1 - 15
1.4 Screen Composition

(5) Spin button

This button is used for inputting a numeral. Click

or for setting a numeral. However, a value below
decimal point cannot be set. Such a value must be input
with the keyboard.

Combo box

Spin button

Fig. 1-15 Spin Button and Combo Box

(6) Combo box

Selection is allowed among the predetermined conditions.

Click for selecting the desired condition.

(7) Check box

For turning on a check box, put a check mark in it by

clicking.
The check box is then set in the status.
For turning off the check box, click it again to remove the
check mark.

Check box

Fig. 1-16 Check Box

1 - 16
 1.4.2

1.4.2 Kinds of Windows The FL Solutions program have two kinds of windows; monitor
window and data processing window.
Set the monitor window active for measurement, or the data
processing window for data processing.
The toolbar differs between these windows.

Fig. 1-17 Monitor Window

Fig. 1-18 Data Processing Window

1 - 17
1.4 Screen Composition

1.4.3 Data Processing The items displayable in each measurement mode are shown
Window below.

(1) Wavelength Scan

A spectrum alone can be displayed or both spectrum and

peak table can be displayed as shown in Fig. 1-19.
Display of the peak table can be turned ON/OFF from the
View menu.

Spectrum

Peak table

Fig. 1-19 Wavelength Scan

(2) Time Scan

A spectrum alone can be displayed or both spectrum and

kinetics result table can be displayed as shown in Fig. 1-20.
Display of the kinetics result table or peak table can be
turned ON/OFF from the View menu.

Spectrum

Peak table
Fig. 1-20 Time Scan (kinetics)

1 - 18
 1.4.3

Spectrum

Peak table

Fig. 1-21 Time Scan (peak table)

(3) Photometry

The items shown in Fig. 1-22 are selectively displayed

depending on measurement method.

On measurement starting with calibration curve

Standard data Calibration

curve

Sample data Calibration

curve data

Fig. 1-22

1 - 19
1.4 Screen Composition

On generation of calibration curve through factor input

Sample data Calibration

curve factor

Fig. 1-23

On display of photometric value at each wavelength in

multi-wavelength mode

Sample data

Fig. 1-24

1 - 20
 1.4.4

1.4.4 Explanation of The FL Solutions program provides five kinds of toolbars as

Tool Buttons shown below. You can select a command by pulling down each
menu from the menu bar. But for quick and easy operation, it is
advisable to use these tool buttons.

Standard Toolbar
Has buttons related to printing and file handling.

Instrument Toolbar (displayed in monitor window)

Has buttons related to spectrophotometer control such as
wavelength drive and baseline measurement.

Measurement Toolbar (displayed in monitor window)

Has buttons related to measurement such as analysis method
setting and sample name input.

Spectrum Toolbar (displayed in data processing window)

Has buttons related to spectral display such as auto scaling,
ruled line indication and spectrum tracing.

1 - 21
1.4 Screen Composition

Processing Toolbar (displayed in data processing window)

Has buttons for smoothing, differentiation, etc.

These five toolbars are independent and each of them can be

located at a desired position as shown in Fig. 1-25 (monitor
window).

Fig. 1-25 An Example of Toolbar Arrangement

1 - 22
 1.4.4

The following tables present the functional descriptions of the

buttons on each toolbar.
The function keys on the keyboard can also be utilized. For the
functional assignment to each function key, refer to 1.5.

Table 1-1 Standard Toolbar

Button Function
Creates a new analysis method/sample table.

Opens data files.

Saves the current file.

Stores a selected character string or graphic item into

the clipboard.
Pastes the contents of the clipboard.

Opens the print window.

Indicates the present status of the spectrophotometer

connected.
Indicates the information (version number, etc.) on the
FL Solutions program.
Calls up the help window.

Table 1-2 Measurement Toolbar

Button Function
Opens the Analysis Method window.

Displays the sample name/comment name entry

window.
Displays the sample table for sample name/comment
name input.
For manual input of standard sample values.

Starts measurement.

Stops measurement.

Closes the shutter.

Opens the shutter.

1 - 23
1.4 Screen Composition

Table 1-3 Spectrum Toolbar

Button Function
Restores the scale to its original condition.

Indicates ruled lines.

Indicates a birds-eye view.

For display from the rear.

For display from the right side.

Selects a spectrum to be displayed.

Indicates the trace cursor.

For display in a contour map.

For display from the front.

For display from the left.

Automatically sets the scale.

Table 1-4 Instrument Toolbar

Button Function
Performs wavelength drive.

Carries out automatic pre-scanning.

Conducts zero-point adjustment.

Enlarges the vertical-axis double.

Reduces the vertical-axis to half.

Restores the scale to its original condition.

Adjusts the vertical-axis display automatically so that

you can view the entire spectral graph.
Indicates ruled lines.

1 - 24
 1.4.4

Table 1-5 Processing Toolbar

Button Function
Opens the Spectrum Properties window.

Outputs a report of data file.

Performs peak detection.

Performs differentiation.

Performs peak detection in 3-dimensional measured

data.
Performs area calculation.

Performs fundamental arithmetic calculations between

spectra.
Calculates the life of phosphorescence.

Performs smoothing.

Resets data.

Performs fundamental arithmetic calculations of 3-

dimensional spectra.

1 - 25
1.5 Functions of Function Keys

1.5 Functions of Function Keys

The function keys on the keyboard provides the following

functions.
Each function can be directly executed.

Wavelength
Key No. Time Scan Photometry 3-D Scan
Scan
[F4] Measurement Measurement Sample measurement Measurement
[F5] Blank measurement
[F6] Stop Stop Stop Stop
[F7] Interrupting
measurement
[F8] Remeasurement
[F9] End

1 - 26
 1.6

1.6 Explanation of Monitor Window

The monitor window contains the following information.

(2)
(1)
(3)
(4)
(5)
(6)

(7)

(8)

(9)

(10)
(11)
(12)

(13)

(14)

Fig. 1-26 Monitor Window

(1) Sample name indication

A sample name is indicated. For its input, use the sample

table or sample name/comment input window.

(2) Current photometric value indication

The current photometric value is indicated.

(3) EX

The current excitation wavelength is indicated.

(4) EM

The current emission wavelength is indicated.

1 - 27
1.6 Explanation of Monitor Window

(5) EX slit

The current excitation slit width is indicated.

(6) EM slit

The current emission slit width is indicated.

(7) Shutter

Shutter opening or closing is indicated.

(8) Lamp

ON/OFF state of the light source lamp is indicated.

(9) Shutter control

The active state of shutter control is indicated.

(10) Corrected spectra

Setting of spectrum correction is indicated.

(11) Sample

The number assigned to the sample under measurement is

indicated.

(12) Replicate count

A count in repeated measurement is indicated.

(13) Message indication

An information message such as Ready, Scanning or

Parameter is presented.
When the message Ready is indicated, it is allowed to
press (measurement) button.

(14) Error message indication

An error message such as Xe Lamp Error! is presented.

If any error message appears, take a proper
countermeasure referring to 4.7 of the maintenance edition.

1 - 28
 1.6

Table 1-6 Possible Error Messages

Message Indication
ROM Status Error! A ROM error has occurred.
RAM Status Error! A RAM error has occurred.
Xe Lamp Error! The Xe lamp is not lit.
Momentary Power Power supply of the spectrophotometer was
Failure! cut off. Restart the spectrophotometer for
initialization.
Shutter Initialize Shutter position cannot be detected.
Error
Zero Adjustment The spectrophotometer is abnormal.
Error
Auto Prescan Error Peak cannot be detected.
Common Prescan Peak cannot be detected.
Error
EX Wavelength The initial position of the wavelength drive
Initialize Error screw cannot be detected.
EM Wavelength The initial position of the wavelength drive
Initialize Error screw cannot be detected.
Battery Voltage Line voltage has dropped.
Low
EEPROM Error An EEPROM error has occurred.
H16 Program Error A program error of H16 has occurred.
Chopper Error Chopper position cannot be detected.

1 - 29
1.7 Input of Spectrophotometer Serial No.

1.7 Input of Spectrophotometer Serial No.

Select the Instruments (I) command from the Tools (T) menu or
click the button. The dialog box shown in Fig. 1-27 opens.

Fig. 1-27

Click the Instrument area and then Serial Number. The dialog
box shown in Fig. 1-28 will open. In this box, input the serial No.
of your spectrophotometer.

Fig. 1-28

After input, click OK.

The serial No. input here is saved as an analytical condition item
into the data file and can be printed. In connection with the
F-4500 series (F-4500 or F-4510), the relevant
spectrophotometer needs to be selected. Click Instrument
Name. In the dialog box shown below, select the model of your
spectrophotometer.

Fig. 1-29 Instrument Name

1 - 30
 1.8

1.8 Window Setting

The information to be displayed upon starting the FL Solutions

program or opening the data processing window can be selected
and unnecessary one can be excluded from display.
Select Options (O) command from the Tools (T) menu.
In the Options dialog box, the following settings can be made.
After your setting, click OK.
The set functions are validated when starting the monitor window
again or opening the data processing window.

Fig. 1-30 Options Dialog Box

This box has the following tabs.

Display tab
Fonts tab
Processing tab
Colors tab
Startup tab
Sensitivity Slits tab

1 - 31
1.8 Window Setting

(1) Display Tab

Items to be displayed when opening a saved data file or

data immediately after measurement can be set here.
Set the number of data columns to be displayed.
The items other than the number of display columns to be
set here can also be set by a tool button or View menu after
measurement.

Fig. 1-31 Display Tab

(a) Trace
Put a check mark in this box, and spectrum can be
traced when the data processing window is open.

(b) Ruled line

The contents of monitor and data processing windows
can be displayed with ruled lines.
1) Wide
2) Intermediate
3) Narrow

(c) Wavelength scan

Items to be displayed when opening the data
processing window in the wavelength scan mode can
be set.
1) Show graph only
2) Show graph plus peak table

1 - 32
 1.8

(d) Time scan

Items to be displayed when opening the data
processing window in the time scan mode can be set.
1) Show graph only
2) Show graph plus kinetics data
3) Show graph plus peak table

(e) 3-dimensional (effective for Model F-2500 when the

3-D Scan program (option) is installed)
Items to be displayed when opening the data
processing window in the time scan mode can be set.
1) Show contour plus EXEM spectrum
2) Show contour, EXEM spectrum plus peak table

(f) Intracellular calcium (effective when the intracellular

calcium measurement program (option) is installed)
Items to be displayed when opening the data
processing window in the intracellular calcium mode
can be set.
1) Show graph only
2) Show graph plus kinetics data
3) Show graph plus average data
4) Show graph plus markers

(g) Intracellular calcium spectrum (effective when the

intracellular calcium measurement program (option) is
installed)
Items to be displayed when opening the data
processing window in the intracellular calcium mode
can be set.
1) Show fluorescence only
2) Show fluorescence plus ratio
3) Show fluorescence plus conc.
4) Show fluorescence, ratio plus conc.

(h) Fluorescence polarization (effective when the

fluorescence polarization measurement program
(option) is installed)
Items to be displayed when opening the data
processing window in the fluorescence polarization
mode can be set.
1) Show graph only
2) Show graph plus kinetic data
3) Show graph plus markers

1 - 33
1.8 Window Setting

(i) Fluorescence polarization spectrum (effective when

the fluorescence polarization measurement program
(option) is installed)
Items to be displayed when opening the data
processing window in the fluorescence polarization
mode can be set.
1) Show fluorescence only
2) Show fluorescence plus polarization
3) Show fluorescence plus anisotropy
4) Show fluorescence, polarization plus anisotropy

(2) Fonts Tab

Display font, character size, etc. can be set.

Printer font, character size, etc. are also settable.

Fig. 1-32 Fonts Tab

(a) Display Font

Font, character size, etc. in the spectrum area under
display can be set.

1 - 34
 1.8

(b) Printer Font

Printer font is settable.
Clicking Select (S) presents the following dialog box.

Fig. 1-33 Font Specification

Set Font, Font style and Size.

(3) Processing Tab

Fig. 1-34 Processing Tab

1 - 35
1.8 Window Setting

(a) Area integration method

1) Rectangular
2) Trapezoid
3) Romberg

(b) Calibration
A calibration curve formula to be used for the
Photometry mode can be set.
1) Data = f(Conc)
2) Conc = f(Data)

(c) Peak table

Inclusion of area calculation item in the peak table can
be selected or avoided.

(d) Smooth
The initial values of data processing function
parameters can be set. However, this setting will not
be reflected on the parameters of Analysis Method
window.

(e) Derivative
The parameters of smoothing in an analysis method or
to be carried out after measurement can be set.

(4) Colors Tab

The color of the spectrum to be overlaid is settable here.

Color 1 (bottom) is the color of the first spectrum.

Fig. 1-35 Colors Tab

1 - 36
 1.8

Click Select. The dialog box shown in Fig. 1-36 is then

presented. A basic color or custom color is selectable.
If there is no basic color you want, click Define Custom
Colors (D).

Fig. 1-36 Color Setting

The dialog box shown in Fig. 1-37 will appear. Select hue,
saturation, luminance, etc. and click Add to Custom
Colors (A). Your color is then added to the custom colors.
So select it and click OK.

Fig. 1-37 Addition of Color

1 - 37
1.8 Window Setting

(4) Startup Tab

Analysis method to be taken upon startup of the FL

Solutions program is settable here.

Fig. 1-38 Startup

(a) Restore last method used

The method used last will be brought in.

(b) Auto Parameter File

The instrument will start with the fixed method brought
in. Click the model name of your instrument.

Fig. 1-39

Specify a file and click Open (O).

1 - 38
 1.9

1.9 Saving a Data File

Data can or cannot be saved depending on setting. So perform

setting with reference to the following table. Setting is any of
patterns to is recommended.

Use sample table Sample Name/Comment Input

Setting
ON/OFF (Use sample table OFF)

Automatic saving ON OFF

Analysis Method - Monitor tab ON OFF ON OFF ON OFF

Open data processing

window after data acquisition
Result Display
Automatic saving
Manual saving Possible Impossible
Pattern

Read and symbols in the Result - Display row as follows.

: Icons or data processing window is open immediately after
measurement.
: The data processing window is not open immediately after
measurement. In patterns and , data is saved.
So the relevant data file can be opened later.

1 - 39
1.10 Entry of File Name for Saving

1.10 Entry of File Name for Saving

File name needs to be set before measurement. Select the

Sample Table (S) command in the Edit menu (E), and the dialog
box shown in Fig. 1-40 will open. Input a sample name and
comment. Then, bring the cursor to the file name column and
click the button at (1).

(1)

Fig. 1-40

The dialog box shown in Fig. 1-41 will appear. Enter your
choice for Save In and specify File Name.

Fig. 1-41

1 - 40
 1.10

After input, click Save.

If the file name field remains blank, a file name such as
[Link] is automatically given.
Once a measurement has been performed, you may perform the
next ones with no change in setting (using the same sample
table). In this case, file names Sample [Link], Sample
[Link] and so on will be given. However, when there is no
file name in a loaded analysis method file, file names will be
given using the analysis method name as follows; Method .uds,
Method [Link] and Method [Link].
Enterable number of characters
Sample name : 20 characters
Comment : 255 characters

NOTE: If the specified storage medium (such as floppy disk or

MO) does not have an adequate free area for new
storage, the relevant file will be saved in the FL
Solutions folder. This also occurs when the specified
folder does not have an adequate free area.

1 - 41
1.11 Photometer Menu Commands

1.11 Photometer Menu Commands

The following commands are available in the Spectrophotometer

menu.
Set Wavelength
Zero Adjust
Auto Pre-scan
Shutter Open/Close

(1) Set Wavelength

This command sets the excitation/emission wavelength to a

specified wavelength.
Click the Set Wavelength command or button, and the
dialog box shown below will come up.

(2) Auto Pre-scan

This command automatically searches for an optimum

excitation/emission wavelength for the sample under
measurement. Other peaks than the fluorescence peak
(such as peaks of excitation scattered light and Raman
spectrum) are ignored. On completion of automatic pre-
scanning, the optimum excitation/emission wavelength is
indicated on the monitor window (in the current wavelength
indication field detailed in 1.6).

(3) Zero Adjust

This command automatically closes the shutter and

measures a zero point in detection. When the zero point
is changed, e.g., after the photomultiplier voltage is
changed, automatic zero-point adjustment is carried out.

(4) Shutter Open/Close

This command is available for opening/closing the

excitation beam shutter. The current state is indicated on
the right side of the monitor window.

1 - 42
 1.12

1.12 Utility Menu Commands

The Utility menu contains the following commands:

Instrumental Response
Analog Output Gain
EX Power Monitor
Sens. Check
Lamp Usage
Lamp Off (only with Model F-2500)

(1) Instrumental Response

When you use this function. You need the Spectral

correction accessories kit (P/N 251-0126).

(2) Analog Output Gain

Use this command for setting a data value corresponding to

1 V of analog output. For details, refer to 8.4.

(3) EX Power Monitor

Use this command for checking output of the monitor

detector. When the EX Power Monitor is turned on
(clicked), monitor value data is displayed regardless of
measurement condition.

(4) Sens. Check

Use this command for checking sensitivity. You can check

sensitivity/drift with this command. For details, refer to the
maintenance edition.

(5) Lamp Usage

Use this command for checking/resetting the usage period

of the xenon lamp. A cumulative turn-on time of the xenon
lamp is indicated. After lamp replacement, reset this
indication. For details, refer to the maintenance edition.

(6) Lamp Off (only with Model F-2500)

Use this command for turning off the xenon lamp.

For turning on the xenon lamp again, restarting the
spectrophotometer is necessary.

1 - 43
1.13 Termination of FL Solutions Program

1.13 Termination of FL Solutions Program

After completion of measurement, shut down the instrument in

the following procedure.

(1) From the File (F) menu, select the Exit (X) command.

Fig. 1-42

Select Yes, and the FL Solutions program will then be

terminated.

(2) Turn off the power switch of the spectrophotometer main

unit.

(3) Click the Start button of the Windows 98 and select the
Shut Down (U) item. In the Shut Down Windows dialog
box, select Shut down and click OK.

(4) Turn off power to the personal computer and the CRT
display monitor.
(At step (3), the personal computer may be turned off in
some settings.)

(5) Turn off power to the printer.

1 - 44
 2.1

2. WAVELENGTH SCAN

2.1 Flow of Operation

This section explains the condition setting and operating method

for wavelength scan.
Measurement is made in the following procedure.

Start

Method setting
Click button.
(See page 2-2.) Scan range
Data mode
Scan speed

Sample name/comment input Click button.

(See page 2-15.)

Pre-scan
Click button.
(See page 2-16.)

Measurement
Click button.
(See page 2-16.)

Data processing
(Refer to Section 6.) Spectrum tracing
Peak detection, etc.

Print
Click button.
(See page 2-19.)

End

Fig. 2-1 Operational Flow (wavelength scan)

2-1
2.2 Creating an Analysis Method

2.2 Creating an Analysis Method

Set the analytical conditions (method) prior to measurement.

From the Edit menu, select the Method command or click the
button. The dialog box shown in Fig. 2-2 will appear.

Fig. 2-2 Analysis Method

The following tabs are provided on the Analysis Method window

for the wavelength scan mode. These tabs are explained on
the subsequent pages.
General
Instrument
Monitor
Processing
Report

2-2
 2.2

(1) General

The parameters shown in Fig. 2-3 are provided under the

General tab.

Fig. 2-3 General Tab

(a) Measurement (mode)

Select a measurement mode from among the four
listed below.
Wavelength scan
Time scan
Photometry
3-D Scan (displayed when the 3-D Scan program
(option) is installed in combination with Model
F-2500)

Here, select Wavelength scan. For Photometry,

Time scan and 3-D Scan, refer to Sections 3, 4 and 5.

(b) Operator
Enter an operator name.

(c) Instrument
The model of the connected instrument is indicated.

(d) Use sample table (Set measurement sample)

Apply a check mark here when a sample table is used.

2-3
2.2 Creating an Analysis Method

(e) Sampling
Standard
When sample is exchanged manually.

(f) Comments
Enter a description or notes on measuring conditions.

(g) Load
Click this button to read out saved analytical conditions.
The file opening window appears upon clicking, so
select an analytical conditions file.

(h) Save
By clicking this button after setting the parameters for
all five tabs, the analytical conditions are saved.
Clicking this button overwrites parameters on the
existing ones.

(i) Save As
Click this button for renaming the set conditions for
saving. The Save As window will appear, so enter a
file name.

(2) Instrument

When you click the Instrument tab, the window in Fig. 2-4
appears.

Fig. 2-4 Instrument Tab

2-4
 2.2

(a) Scan mode

Select one of the following scan modes. In case of
Synchronous, excitation wavelength appears on the
abscissa of the graph.
Excitation
Emission
Synchronous

(b) Data mode

Select one of the following data modes.
The Luminescence mode is for measuring
luminescence in the biological/chemical field and only
the sample side signal is acquired without performing
ratio photometry. The Phosphorescence mode is for
acquiring only phosphorescence after extinction of
excitation beam while turning a chopper on the
excitation side.
Fluorescence
Luminescence
Phosphorescence (with Model F-4500)

(c) EM WL
This is the start wavelength for wavelength scan on the
excitation side. Input the shorter wavelength side of
measuring wavelength range.

Model Input Range

F-4500 200 to 900 nm or 200 to 890 nm in
Synchronous scan mode
F-2500 220 to 800 nm or 220 to 790 nm in
Synchronous scan mode

(d) EX Start WL
This is the start wavelength for wavelength scan on the
excitation side. Input the shorter wavelength side of
measuring wavelength range.

Model Input Range

F-4500 200 to 890 nm
F-2500 220 to 790 nm

2-5
2.2 Creating an Analysis Method

(e) EX End WL
This is the end wavelength for wavelength scan on the
excitation side. Input the longer wavelength side of
measuring wavelength range.

Model Input Range

F-4500 210 to 900 nm
F-2500 230 to 800 nm

(f) EX WL
An excitation wavelength can be input. It is the end
wavelength for wavelength scan on the excitation side.
Input the longer wavelength side of measuring
wavelength range.

Model Input Range

F-4500 200 to 900 nm
F-2500 220 to 800 nm

(g) EM Start WL
This is the start wavelength for wavelength scan on the
emission side. Input the shorter wavelength side of
measuring wavelength range.

Model Input Range

F-4500 200 to 890 nm
F-2500 220 to 790 nm

(h) EM End WL
This is the end wavelength for wavelength scan on the
emission side. Input the longer wavelength side of
measuring wavelength range.

Model Input Range

F-4500 210 to 900 nm
F-2500 230 to 800 nm

2-6
 2.2

(i) Scan speed

Set a wavelength scan speed. (Unit: nm/min)

F-4500 F-2500
30000 3000
12000 1500
2400 300
1200 60
300 15
60
15

For the Phosphorescence data mode, select one of the

following values.
240 nm/min
60 nm/min
15 nm/min

(j) Delay
After pressing the Measure button, measurement is
started following the delay time set here. It is used for
temperature stabilization, etc. In repeat measurement,
it is the time until the start of the first measurement.
The delay time is invalid for the second and
subsequent measurements.
Input range: 0 to 9999 s

(k) EX Slit
Select a slit width for the excitation side. Unit: nm

F-4500 F-2500
1.0 2.5
2.5 5.0
5.0 10.0
10.0 20.0
20.0

2-7
2.2 Creating an Analysis Method

(l) EM Slit
Select a slit width for the emission side. Unit: nm

F-4500 F-2500
1.0 2.5
2.5 5.0
5.0 10.0
10.0 20.0
20.0

(m) PMT Voltage

A function for controlling the voltage of the
photomultiplier detector (photomultiplier tube).
Select one of the following values. Changing from
400 V to 700 V increases the data value about two
digits and changing from 700 V to 950 V increases it
about 1 digit.
950 V (only with F-4500)
700 V
400 V

(n) Response
Select a response time from the following.

F-4500 F-2500
0.0004 0.04
0.01 0.08
0.05 0.4
0.1 2
0.5 8
2.0 Auto
8.0
Auto

By selecting Auto, a response time is set automatically

according to scan speed. Select Auto usually.

2-8
 2.2

(o) Corrected spectra

A function for determining the spectrum inherent to a
sample by correcting the photometer wavelength
characteristic using the saved instrument parameters,
following measurement with the instrument parameters
for photometer control. If there are no instrument
parameters for the wavelength range to be measured,
only the wavelength range that allows correction is
scanned. And if there is no wavelength area
measurable, an error will occur.
For using this function, a Spectra Correction Parts Set
(P/N 251-0126) is required. The Instrument
Response command is provided in the Utility menu for
operating this function.
When this setting is at ON, Corrected spectra
appears on the monitor window. (See Fig. 1-26 in
1.6.)

(p) Shutter control (only with F-4500)

The shutter can be automatically closed in other than
measurement for suppressing sample deterioration
due to the energy of excitation beam and opened when
measurement starts. When you put a check mark at
the head, the shutter will close and open at start of
measurement. The shutter will close again when
measuring wavelength begins returning to the start
wavelength after measurement in the wavelength scan
mode.

(q) Replicates
Set the number of repeat measurements. Input range
is 1 to 99. An average spectrum can also be
determined in repeat measurement. Setting is
available on the Processing tab.

(r) Cycle time

Set a repetition interval. Input range is 0.0 to
180.0 min.

2-9
2.2 Creating an Analysis Method

(3) Monitor

When selecting the Monitor tab, the window in Fig. 2-5

appears.

Fig. 2-5 Monitor Tab

Assign the following parameters.

(a) Y-Axis Max (maximum)

Input a maximum value for Y-axis on the monitor
window.

(b) Y-Axis Min (minimum)

Input a minimum value for Y-axis on the monitor
window.

(c) Open data processing window after data acquisition

Select whether or not to conduct data processing after
sample measurement. When selected (check mark is
applied), an icon is displayed for the data processing
window at the end of measurement. And by opening
this icon, data processing such as peak detection is
available.

2 - 10
 2.2

NOTE: When the remaining memory capacity of PC

in operation is very small (system resource
is less than 20%), the data processing
window will not open even if this item is
turned ON. Either close the presently
opened data processing window or restart
the FL Solutions program.

(d) Print report after data acquisition

Select whether or not to print a report after sample
measurement. When selected (check mark is
applied), printing will be made automatically after
measurement. The items set under the Report tab
will be printed. When this item is not selected, data
processing can be made by reading out a file via File -
Open.

(e) Overlay
Spectra can be overlaid in the monitor window.
This setting cannot be selected for repeat
measurement.

(4) Processing

On selection of the Processing tab, the window in Fig. 2-6

will be displayed.

Fig. 2-6 Processing Tab

2 - 11
2.2 Creating an Analysis Method

(a) CAT (Computing for Averaging Transient)

Used for obtaining an average spectrum in repeat
measurement. This is especially effective for
measuring samples with which the emission intensity
varies with time.

(b) Processing choices

A list of data processing (Savitsky-Golay, Mean
smooth, Median smooth, Derivative) is shown.
Select a data processing item, and click the rightward-
pointing arrow key between the Processing choices
and Processing steps display fields. Then, the
selected method appears in the Processing steps field.

(c) Processing steps

The processing sequence is displayed. To delete a
processing method, first select the method, and then
click the leftward-pointing arrow key between the
Processing choices and Processing steps display
fields. Then, the selected method disappears from
the Processing steps field.
Also, in case you want to carry out any smoothing in
Processing steps, double-click the relevant item and
the smoothing parameters (order, data points, etc.) can
be changed.

(d) Peak Finding

Select a peak detection method indicated in the data
processing window.

(e) Threshold
Set a detection limit for the photometric value axis of
peak and valley. Peaks or valleys below the setting
will not be detected. Input range is 0.001 to 1000.

(f) Sensitivity
Select the number of data points in the horizontal axis
direction (abscissa). Select a sensitivity of 1 for
detecting sharp peaks. Or select 8 for detecting
broad peaks.
1
2
4
8

2 - 12
 2.2

(5) Report

Select the Report tab, and the window in Fig. 2-7 will
appear.

Fig. 2-7 Report Tab

This window is used to select an output method and items

of measurement result.

(a) Output
1) Print Report
A report will be printed. For printing method,
refer to 2.6.
2) Use Microsoft Excel
Data will be transferred to Microsoft Excel.
For transfer method, refer to 5.13.
3) Use print generator sheet
Select this method for using the report generator
(option). For usage, refer to the instruction
manual of the report generator.

(b) Orientation
Selectable when Report is chosen for Print Method.
Select a printing orientation. Either Portrait or
Landscape orientation is selectable. When the
Landscape orientation is selected, date and method
can be printed.

2 - 13
2.2 Creating an Analysis Method

(c) Printable (transferable) items

1) Include date
2) Include method
3) Include graph
4) Include data listing
Constant
Data will be printed at fixed intervals between 2
wavelength points. Input Data interval, Start
wavelength and End wavelength.
Select data
Data at the specified wavelength will be printed.
Up to 12 wavelengths can be specified.
5) Include peak table
Select output items in the peak table.
Peak WL/Peak data
Start WL/End WL
Valley WL/Valley data
Peak Area

(d) Printer Font

Click this button for changing the font to be used in the
report.

2 - 14
 2.3

2.3 Defining Your Samples

When Use Sample Table has been set for the Analysis Method,
the samples must be defined before measurement.

(1) Open the monitor window and click the button.

(2) When the Sample Table window in Fig. 2-8 appears, fill in
the blank section. For instance, if there are five samples,
click the Insert button five times.

Fig. 2-8 Sample Table Window

(3) Input the Sample, Comments and File Name for each
sample. The sample names input here appear on the
monitor window also. And the sample names input here
are not changeable.
Maximum number of characters to be input
Sample : 20 characters
Comments : 255 characters

2 - 15
2.3 Defining Your Samples

(4) When input is finished, check the entries and then click the
OK button.
If a sample table is not used, then click the button.
The window in Fig. 2-9 will appear. Make entries for
Sample and Comment. For automatic saving, put a check
mark in the Auto File box and specify a file name for
saving.

Fig. 2-9

2 - 16
 2.4

2.4 Prescan

Select Pre-scan command from the Spectrophotometer menu or

click the (pre-scan) button.
This function executes high-speed scan under the set measuring
conditions, detects the peak wavelength and moves the system
to that wavelength. Also, the upper limit photometric value is
automatically set so that the peak-wavelength value becomes
approximately 70% of that upper limit value.
Scan ranges are as follows.

Excitation spectra : Start wavelength to emission

wavelength - 20 nm or shorter
wavelength side of end wavelength

Emission spectra : Excitation wavelength or longer

wavelength side of start wavelength +
20 nm to end wavelength

Synchronous spectra : Start wavelength to end wavelength

After prescan, the upper limit photometric value is automatically

set.

2 - 17
2.5 Conducting a Wavelength Scan

2.5 Conducting a Wavelength Scan

(1) There are two methods of starting the scan.

(a) Select the Measure command from the

Spectrophotometer menu.

(b) Click the (measurement) button on the

measurement toolbar.
For halting the measurement midway, click the
button on the toolbar.

(2) Upon clicking the button, the window shown in

Fig. 2-10 appears when using a sample table. Now click
Yes.

Fig. 2-10

(3) The dialog box as in Fig. 2-11 will open. Set each sample
and click OK.

Fig. 2-11

2 - 18
 2.5

(4) When automatic saving is set, data will be saved after

completion of measurement. Upon opening its file, a data
processing window will appear as in Fig. 2-12.
Unless automatic saving is set, select the Save As
command from the File menu and save data in file.

(a) Spectrum

(b) Peak table

Fig. 2-12 Data Processing Window (wavelength scan)

(a) Spectrum
The spectrum of a measured sample is traced.

(b) Peak table

Peak wavelength, peak height and valley wavelength
are displayed.

2 - 19
2.6 Data Printout

2.6 Data Printout

Printout can be provided even when the function for automatic

printout after measurement is not used. The following two
procedures are available.

(1) Select the Print command from the File menu. Or click the
print button on the toolbar. The field active on the
data processing window (e.g., (a) in Fig. 2-12) will be
printed. What is displayed by clicking the Print Preview
command in the File menu will be printed.

Fig. 2-13

2 - 20
 2.6

(2) Select the Report command in the Data menu. Or click

the button on the toolbar. The Print Preview window
will appear, so click Print. Select the items to be printed
at the Report tab under Properties.

Fig. 2-14

NOTE: If characters overflow from the print frame in

repeat measurement or in overlaying, then change
the printing orientation from vertical to horizontal.

2 - 21
 3.1

3. TIME SCAN

3.1 Flow of Operation

This section explains the parameter setting and operating

method for time scan.
Measurement is performed in a flow shown below.

Start

Method setting
Click button.
(See page 3-2.) Measuring
wavelength
Scan time

Sample name/comment input Click button.

(See page 3-13.)

Measurement
Click button.
(See page 3-15.)

Data processing
(Refer to Section 6.) Rate calculation
Peak detection
Spectrum tracing
Spectral calculation

Print
Click button.
(See page 3-17.)

End

Fig. 3-1 Operational Flow (time scan)

3-1
3.2 Creating an Analysis Method

3.2 Creating an Analysis Method

Set the analytical conditions (method) prior to measurement.

From the Edit menu, select the Method command or click the
button. The dialog box shown in Fig. 3-2 will appear.

Fig. 3-2 Analysis Method

The following tabs are provided on the Analysis Method window

for the time scan mode. These tabs are explained on the
subsequent pages.
General
Instrument
Monitor
Processing
Report

3-2
 3.2

(1) General

The parameters shown in Fig. 3-3 are provided under the

General tab.

Fig. 3-3 General Tab

(a) Measurement (mode)

Select a measurement mode from among the four
listed below.
Wavelength scan
Time scan
Photometry
3-D Scan (displayed when the 3-D Scan program
(option) is installed in combination with Model
F-2500)

Here, select Time scan. For Wavelength scan,

Photometry and 3-D Scan, refer to Sections 2, 4 and 5.

(b) Operator
Enter an operator name.

(c) Instrument
The model of the connected instrument is indicated.

(d) Use sample table (Set measurement sample)

Apply a check mark here when a sample table is used.

3-3
3.2 Creating an Analysis Method

(e) Sampling
Standard
When sample is exchanged manually.

(f) Comments
Enter a description or notes on measuring conditions.

(g) Load
Click this button to read out saved analytical conditions.
The file opening window appears upon clicking, so
select an analytical conditions file.

(h) Save
By clicking this button after setting the parameters for
all five tabs, the analytical conditions are saved.
Clicking this button overwrites parameters on the
existing ones.

(i) Save As
Click this button for renaming the set conditions for
saving. The Save As window will appear, so enter a
file name.

(2) Instrument

This window appears when Time scan is specified for

Measurement under the General tab on the Analysis
Method window. When you click the Instrument tab, the
window in Fig. 3-4 appears.

Fig. 3-4 Instrument Tab

3-4
 3.2

(a) Data mode

Select one of the following data modes.
The Luminescence mode is for measuring
luminescence in the biological/chemical field and only
the sample side signal is acquired without performing
ratio photometry.
If phosphorescence life is shorter than 20 ms,
Phosphorescence short life should be selected.
In this case, unit time and measuring time are
automatically set to ms and 20 ms.
Fluorescence
Luminescence
Phosphorescence (only with Model F-4500)
Phosphorescence Life Time (only with Model
F-4500)
Phosphorescence Life Time (Short) (only with
Model F-4500)

(b) EX WL
Input an excitation wavelength.

Model Input Range

F-4500 0, 200 to 900 nm
F-2500 0, 200 to 800 nm

(c) EM WL
Input an emission wavelength.

Model Input Range

F-4500 0, 200 to 900 nm
F-2500 0, 200 to 800 nm

(d) Time unit (only with Model F-4500)

Set a time unit.
s
ms

3-5
3.2 Creating an Analysis Method

(e) Scan time

Set a scan time.

Model Input Range

F-4500 10 to 3600 s
1000 to 9999 ms
F-2500 10 to 9000 s

According to the set scan time, data sampling interval

is set as listed below.

Model F-4500

Measuring Time Sampling Interval

(s) (ms)
~3 1
~7 2
~ 18 5
~ 36 10
~ 72 20
~ 180 50
~ 360 100
~ 720 200
~ 1800 500
~ 3600 1000

In the Phosphorescence mode, the minimum sampling

interval is 50 ms. It is fixed at 40 s in
phosphorescence short-life measurement.

Model F-2500

Measuring Time Sampling Interval

(s) (ms)
~ 120 100
~ 240 200
~ 600 500
~ 1200 1000
~ 2400 2000
~ 6000 5000
~ 9000 10000

3-6
 3.2

(f) Delay
After pressing the Measure button, measurement is
started following the delay time set here. It is used for
temperature stabilization, etc. In repeat measurement,
it is the time until the start of the first measurement.
The delay time is invalid for the second and
subsequent measurements.
Input range: 0 to 9999 s

(g) EX Slit
Select a slit width for the excitation side. Unit: nm

F-4500 F-2500
1.0 2.5
2.5 5.0
5.0 10.0
10.0 20.0
20.0

(h) EM Slit
Select a slit width for the emission side. Unit: nm

F-4500 F-2500
1.0 2.5
2.5 5.0
5.0 10.0
10.0 20.0
20.0

(i) PMT Voltage

A function for controlling the voltage of the
photomultiplier detector (photomultiplier tube).
Select one of the following values. Changing from
400 V to 700 V increases the data value about two
digits and changing from 700 V to 950 V increases it
about 1 digit.
950 V (only with F-4500)
700 V
400 V

3-7
3.2 Creating an Analysis Method

(j) Response
Select a response time from the following.

F-4500 F-2500
0.04 0.04
0.01 0.08
0.05 0.4
0.1 2
0.5 8
2.0 Auto
8.0
Auto

By selecting Auto, a response time is set automatically

according to scan speed. Select Auto usually.

(k) Shutter control (only with F-4500)

The shutter can be automatically closed in other than
measurement for minimizing sample deterioration due
to the energy of excitation beam and opened when
measurement starts. When you put a check mark at
the head, the shutter will close and open at start of
measurement. The shutter will close again when
measuring wavelength begins returning to the start
wavelength after measurement in the time scan mode.

(l) Replicates
Set the number of repeat measurements. Input range
is 1 to 99.

(m) Cycle time

Set a repetition interval. Input range is 0.0 to
180.0 min.

3-8
 3.2

(3) Monitor

When selecting the Monitor tab, the window in Fig. 3-5

appears.

Fig. 3-5 Monitor Tab

Assign the following parameters.

(a) Y-Axis Max (maximum)

Input a maximum value for Y-axis on the monitor
window.

(b) Y-Axis Min (minimum)

Input a minimum value for Y-axis on the monitor
window.

(c) X-Axis Max (maximum)

Input a maximum value for X-axis on the monitor
window.

(d) X-Axis Min (minimum)

Input a minimum value for X-axis on the monitor
window.

NOTE: X-Axis Max and Min correspond to a display

interval on the monitor window.
For example, when Max and Min are 60 and
30, display on the window is made within 30
to 60 s, 60 to 90 s, 90 to 120 s and so on.

3-9
3.2 Creating an Analysis Method

(f) Open data processing window after data acquisition

Select whether or not to conduct data processing after
sample measurement. When selected (check mark is
applied), data is automatically saved after
measurement.

NOTE: When the remaining memory capacity of PC

in operation is very small (system resource is
less than 20%), the data processing window
will not open even if this item is turned ON.
Either close the presently opened data
processing window or restart the FL
Solutions program.

(g) Print report after data acquisition

Select whether or not to print a report after sample
measurement. When selected (check mark is
applied), printing will be made automatically after
measurement. The items set under the Report tab
will be printed.

(h) Overlay
Spectra can be overlaid in the monitor window.
This setting cannot be selected for repeat
measurement.

(4) Processing

On selection of the Processing tab, the window in Fig. 3-6

will be displayed.

Fig. 3-6 Processing Tab

3 - 10
 3.2

(a) Processing choices

A list of data processing (Savitsky-Golay smooth,
Mean smooth, Median smooth, Derivative) is shown.
Select a data processing item, and click the rightward-
pointing arrow key between the Processing choices
and Processing steps display fields. Then, the
selected method appears in the Processing steps field.

(b) Processing steps

The processing sequence is displayed. To delete a
processing method, first select the method, and then
click the leftward-pointing arrow key between the
Processing choices and Processing steps display
fields. Then, the selected method disappears from
the Processing steps field.
Also, in case you want to carry out any smoothing in
Processing steps, double-click the relevant item and
the smoothing parameters (order, data points, etc.) can
be changed.

(c) Peak Finding

Set threshold and sensitivity values for peak detection.

(d) Kinetics
Input a start time, end time and K-factor for kinetics
(rate calculation).

3 - 11
3.2 Creating an Analysis Method

(5) Report

Select the Report tab, and the window in Fig. 3-7 will
appear.

Fig. 3-7 Report Tab

This window is used to select an output method and items

of measurement result.

(a) Output
1) Print Report
A report will be printed. For printing method,
refer to 3.6.
2) Use Microsoft Excel
Data will be transferred to Microsoft Excel.
For transfer method, refer to 6.13.
3) Use print generator sheet
Select this method for using the report generator
(option). For usage, refer to the instruction
manual of the report generator.

(b) Print format

Selectable when Report is chosen for Print Method.
Select a printing orientation. Either Portrait or
Landscape orientation is selectable. When the
Landscape orientation is selected, date and method
can be printed.

3 - 12
 3.2

(c) Printable (transferable) items

1) Include data
The date and time of analysis can be printed.
Printing time will always be printed.
2) Include method
3) Include graph
4) Include data listing
Constant
Data will be printed at fixed intervals between 2
wavelength points. Input Data interval, Start
wavelength and End wavelength.
Select data
Data at the specified wavelength will be printed.
Up to 12 wavelengths can be specified.
5) Include peak table
Select output items in the peak table.
Peak time/Peak data
Start time/End time
Valley time/Valley data
Peak Area
6) Include kinetics

(d) Printer Font

Click this button for changing the font to be used in the
report.

3 - 13
3.3 Defining Your Samples

3.3 Defining Your Samples

When Use Sample Table has been set for the Analysis Method,
the samples must be defined before measurement.

(1) Open the monitor window and click the button.

(2) When the Sample Table window in Fig. 3-8 appears, fill in
the blank section. For instance, if there are five samples,
click the Insert button five times.

Fig. 3-8 Sample Table Window

(3) Input the Sample, Comments and File Name for each
sample. The sample names input here appear on the
monitor window also.
Maximum number of characters to be input
Sample : 20 characters
Comments : 255 characters

3 - 14
 3.3

(4) When input is finished, check the entries and then click the
OK button.
If a sample table is not used, then click the button.
The window in Fig. 3-9 will appear. Make entries for
Sample and Comment. For automatic saving, put a check
mark in the Auto file saving after measurement box and
specify a file name for saving.

Fig. 3-9

3 - 15
3.4 Conducting a Time Scan

3.4 Conducting a Time Scan

(1) There are two methods of starting the scan.

(a) Select the Measure command from the

Spectrophotometer menu.

(b) Click the (measurement) button on the

measurement toolbar.

For halting the measurement midway, click the button

on the toolbar.

(2) Upon clicking the button, the window shown in

Fig. 3-10 appears when using a sample table. Now click
Yes.

Fig. 3-10

(3) The dialog box as in Fig. 3-11 will open. Set each sample
and click OK.

Fig. 3-11

3 - 16
 3.4

(4) When automatic saving is set, data will be saved after

completion of measurement. Upon opening its file, a data
processing window will appear as in Fig. 3-12.
Although a peak table can be displayed, simultaneous
display of rate calculation (kinetics) result and peak table
cannot be selected. Fig. 3-12 shows a spectrum and the
result of rate calculation. This is selectable from the View
menu. Unless automatic saving is set, select the Save As
command from the File menu and save data in file.

(a) Spectrum

(b) Result of rate

calculation

Fig. 3-12 Data Processing Window (time scan)

(a) Spectrum
The spectrum of a measured sample is traced.

(b) Result of rate calculation

The results of rate calculation (gradient, activity, R, R2)
are displayed. Start time and end time are settable by
numeric input or clicking Set with the line cursor
aligned. After setting, click Update. For rate
calculation, refer to APPENDIX B.

(c) Peak table

Peak wavelength, valley wavelength, peak start/end
wavelengths and peak area are displayed.

3 - 17
3.5 Data Printout

3.5 Data Printout

Printout can be provided even when the function for automatic

printout after measurement is not used. The following two
procedures are available.

(1) Select the Print command from the File menu. Or click the
(print) button on the toolbar. The field active on the
data processing window (profiled in blue when clicked, field
(a) in Fig. 3-12) will be printed. The contents, that are
displayed by selecting the Print Preview command from the
File menu as shown in Fig. 3-13, will be printed.

Fig. 3-13

3 - 18
 3.5

(2) Select the Report command in the Data menu. Or click

the (report) button on the toolbar. The Print Preview
window will appear as shown in Fig. 3-14. Then, click
Print. Select the items to be printed at the Report tab
under Properties on the Edit menu.

Fig. 3-14

NOTE: If characters overflow from the print frame in

repeat measurement or in overlaying, then
change the printing orientation from Portrait to
Landscape.

3 - 19
3.6 Calculating the Phosphorescence Life

3.6 Calculating the Phosphorescence Life

This is the function for calculating phosphorescence life from the

measurement result (phosphorescence attenuation curve).
Replacing data value at T1 with I0, I0/e time (T2) is found, and T1,
T2 and (phosphorescence life) = T2 - T1 are displayed.
Select the phosphorescence life command from the data (D)
menu or click the button on the Processing toolbar.
A window as in Fig. 3-15 will appear.

Fig. 3-15

Move the line cursor to the start point (T1) of calculation by the
mouse or arrow keys [] and [] on the keyboard. Right-click
the mouse at the start point. Or input a numeral in the T1 field
and click Calculate. The result of calculation will be displayed.

Fig. 3-16

3 - 20
 3.6

For printout of the calculation result, click Print. For printout of

the spectrum and calculation result, apply a check mark to With
spectrum record and click Print.
For scale expansion, specify the area to be enlarged with the
mouse.
Draw a rectangle with the mouse button held down as shown in
Fig. 3-17.

Fig. 3-17

When releasing the mouse button, the specified area will be

enlarged as shown in Fig. 3-18.

Fig. 3-18

Double-clicking on the spectrum selects auto scale.

3 - 21
 4.1

4. PHOTOMETRY

4.1 Flow of Operation

This section explains the method of determining sample

concentration with a calibration curve prepared. Following is
the operational flow.

Start

Method setting Click button.

(See page 4-2.) Photometric method
Measuring
wavelength
Standard table

Sample name/comment input Click button.

(See page 4-18.)

Measurement Click (measurement)

button.
(See page 4-19.)
Measurement of standard
and sample

Print
Click button.
(See page 4-24.)

End

Fig. 4-1 Operational Flow (photometry)

4-1
4.2 Creating an Analysis Method

4.2 Creating an Analysis Method

Set the analytical conditions (method) prior to measurement.

From the Edit menu, select the Method command or click the
button. The dialog box shown in Fig. 4-2 will appear.

Fig. 4-2 Analysis Method

The following tabs are provided on the Analysis Method window

for the photometry mode. These tabs are explained on the
subsequent pages.
General
Quantitation
Instrument
Standards
Monitor
Report

4-2
 4.2

(1) General

The parameters shown in Fig. 4-3 are provided under the

General tab.

Fig. 4-3 General Tab

(a) Measurement (mode)

Select a measurement mode from among the four
listed below.
Wavelength scan
Time scan
Photometry
3-D Scan (displayed when the 3-D Scan program
(option) is installed in combination with Model
F-2500)
Here, select Photometry. For Wavelength scan,
Time scan and 3-D Scan, refer to Sections 2, 3 and 5.

(b) Operator
Enter an operator name.

(c) Instrument
The model of the connected instrument is indicated.

(d) Use sample table (Set measurement sample)

Apply a check mark here when a sample table is used.

4-3
4.2 Creating an Analysis Method

(e) Sampling
Standard
When sample is exchanged manually.

(f) Comments
Enter a description or notes on measuring conditions.

(g) Load
Click this button to read out saved analytical conditions.
The file opening window appears upon clicking, so
select an analytical conditions file.

(h) Save
By clicking this button after setting the parameters for
all six tabs, the analytical conditions are saved.
Clicking this button displays the Save As window in
creation of a new analytical conditions file. So input a
file name.

(i) Save As
Click this button for renaming the set conditions for
saving.

(2) Quantitation

Upon selecting the Quantitation tab, the window shown in

Fig. 4-4 appears.

Fig. 4-4 Quantitation Tab

4-4
 4.2

(a) Quantitation type

Select the method of creating a calibration curve.
Wavelength, Peak area, Peak height or Derivative
method is selectable.
Spectra measured by the Peak area, Peak height and
Derivative method can be checked.

1) Wavelength
Upon selecting None for calibration curve type in
this quantitation method, up to 6 wavelengths can
be set. When any other than None is selected
for quantitation type, up to 3 wavelengths can be
set.
(i) One wavelength calculation
This is the most generally used method.
As shown in Fig. 4-5 (a), one wavelength is
specified, and the concentration is
determined from the data I1 at that time.
(ii) Two wavelength calculation
As shown in Fig. 4-5 (b), assuming I1 and I2
are the absorbance values at two different
wavelengths, concentration is calculated
through determination of I by the equation
below.
I = I2 - I1
(iii) Three wavelength calculation
As shown in Fig. 4-5 (c), assuming I1, I2 and
I3 are the data values at three different
wavelengths, concentration is calculated
through determination of I by the equation
below. Photometry is made in the order of 1,
2, 3.
1 - 2 I3 + 2 - 3 I1
I = I2 -
1-3

But the following relation must hold:

1 < 2 < 3 or 1 > 2 > 3

4-5
4.2 Creating an Analysis Method

I2 I2
I1
I

I I I3

I1

WL 1 WL 1 WL 2 I1 WL 1 WL 2 WL 3

1 - 2 I3 + 2 - 3 I1
I = I1 I = I2 - I1 I = I2 -
1-3

(a) One wavelength (b) Two wavelength (c) Three wavelength

calculation calculation calculation

Fig. 4-5 Calculation Method

2) Peak area
Input the peak wavelength and its accuracy.
Measurement is made in the wavelength range
specified at the Instrument tab.
Peak wavelength is obtained and a calibration
curve is prepared using the peak area of relevant
peak. Select an integration method among
Rectangular, Trapezoid and Romberg. Also set
a threshold value and sensitivity for defining the
peak.

Fig. 4-6 Quantitation Tab (peak area)

4-6
 4.2

3) Peak height
Input the peak wavelength and its accuracy.
Measurement is made in the wavelength range
specified at the Instrument tab. Peak is
determined within the accuracy set here and
calculation is made in the height of that peak.

Fig. 4-7 Quantitation Tab (peak height)

4) Derivative
Input a peak wavelength (or wavelength to be
used for calibration curve preparation).
Measurement is made in the wavelength range
specified at the Instrument tab and a calibration
curve is created using the derivative value at the
peak wavelength.
Set the derivative order, smoothing order and
data points as derivative parameters.

Fig. 4-8 Quantitation Tab (derivative)

4-7
4.2 Creating an Analysis Method

5) Ratio
Upon selecting Ratio, an input column for three
factors (K0, K1, K2) and a setting column for
calculation method (+, -, *) are displayed.

NOTE: By this quantitation method, a

calibration curve cannot be created.

Fig. 4-9 Quantitation Tab (peak ratio)

Calculation is made by the following equation.

K1 (A (WL1) A (WL3))
RATIO = #K0
K2 (A (WL2) A (WL3))

# (calculation method): +, -, *
WL1, 2, 3 : Specified wavelengths
WL3 : Background correcting wavelength
A( ) : Measured value at specified
wavelength

(b) Calibration type

Set a calibration type. When selecting factor input
(when standard calibration curve and its factors are
already known), put a mark in the check box and enter
values for A0, A1, A2 and A3.

4-8
 4.2

1) None
Mode in which calibration curve is not used.
When this mode is selected with Wavelength set
for Quantification type, multi-wavelength
measurement at up to six wavelengths is possible.
However, no spectrum can be displayed.
2) 1st order
This specifies the most generally used calibration
curve. A linear calibration curve is to be created.
The formula of calibration curve is as follows.

x = A1y + A0

Here, x is sample concentration and y is sample

data.
Data

Ko Conc

Fig. 4-10 1st Order

4-9
4.2 Creating an Analysis Method

3) 2nd order
A calibration curve is created using a quadratic
curve. The formula of calibration curve is as
follows.

x = A2y2 + A1y + A0

Here, x is sample concentration and y is sample

data.

Data

Ko Conc

Fig. 4-11 2nd Order

4 - 10
 4.2

4) 3rd order
A calibration curve is created using a cubic curve.
The formula of calibration curve is as follows.

x = A3y3 + A2y2 + A1y + A0

Here, x is sample concentration and y is sample

data.

Data

Ko Conc

Fig. 4-12 3rd Order

4 - 11
4.2 Creating an Analysis Method

5) Segmented
A calibration curve created by connecting
standard samples linearly according to the
measured values of respective samples or input
data.

STD 7
Data

STD 6

STD 5 For the part which

exceeds the measuring
range, extend the line
STD 4 just before it.

STD 3

Max. No. of standards: 20

STD 2

STD 1

Conc

Fig. 4-13 Segmented

NOTE: A proper calibration curve can be

created when the photometric value
increases or decreases monotonously
versus the concentration value. With a
monotonic increase, the calibration
curve is created with standard 1 (STD1)
as origin. And with a monotonic
decrease, the calibration curve is
created with STD1 plotted on the
photometric value axis and STD5 on the
concentration axis. In these examples,
the number of standards is set at 5.

4 - 12
 4.2

STD 5
STD 4

Data
STD 3

STD 2

STD 1
Conc

Calibration curve with monotonous increase

STD 1
STD 2
STD 3
Data

STD 4

STD 5

Conc

Calibration curve with monotonous decrease

(c) Number of wavelengths

When Calibration type is None, up to six
wavelengths are selectable, and with other calibration
types, 1 to 3 wavelengths are selectable.

(d) Concentration unit

Input a concentration (in mg/mL for example) from the
keyboard.

(e) Manual calibration

Put a check mark in this box for creating a calibration
curve using factors. The number of factors (A0, A1, A2,
A3) varies with the calibration type selected.

4 - 13
4.2 Creating an Analysis Method

(f) Force curve through zero

By putting a check mark in this box, a calibration curve
is created so that its factor A0 passes through 0
automatically.

(g) Digit after decimal point

Set the number of digits after decimal point for the
calculated concentration of the measured sample.
The concentration value is rounded off to the set
number of digits.
Input range: 0 to 3

(h) Lower concentration limit/Upper concentration limit

This is used to set a normal value range for the
concentration of the measured sample. If the
concentration is higher than the upper limit set here,
H will appear in the Avg Conc column, and if lower
than the lower limit set here, L will appear in the Avg
Conc column.

(3) Instrument

When you click the Instrument tab, the window in Fig. 4-14
appears.

Fig. 4-14 Instrument Tab

4 - 14
 4.2

(a) Data mode

Select one of the following data modes.
The Luminescence mode is for measuring
luminescence in the biological/chemical field and only
the sample side signal is acquired without performing
ratio photometry.
Fluorescence
Luminescence
Phosphorescence

(b) Wavelength mode

Select a wavelength for creating a calibration curve.
EX WL Fixed
EM WL Fixed
Both WL Fixed

(c) Wavelength (1 to 6)
Input the number of wavelengths specified at the
Quantitation tab.

Model Input Range

F-4500 0, 200 to 900 nm
F-2500 0, 220 to 800 nm

(d) EX Slit
Select a slit width for the excitation side. Unit: nm

F-4500 F-2500
1.0 2.5
2.5 5.0
5.0 10.0
10.0 20.0
20.0

(e) EM Slit
Select a slit width for the emission side. Unit: nm

F-4500 F-2500
1.0 2.5
2.5 5.0
5.0 10.0
10.0 20.0
20.0

4 - 15
4.2 Creating an Analysis Method

(f) PMT Voltage

A function for controlling the voltage of the
photomultiplier detector (photomultiplier tube).
Select one of the following values. Changing from
400 V to 700 V increases the data value about two
digits and changing from 700 V to 950 V increases it
about 1 digit.
950 V (only with F-4500)
700 V
400 V

(g) Auto statistic cal. number

Calculation is made for mean value, SD (standard
deviation) and CV (coefficient of variation). Set the
number of samples for this purpose.
Input range: 2 to 4000

(h) Replicates
Set the number of measurements.
Input range: 1 to 20

(i) Integration time

A function for obtaining data averaged over the
specified time for the purpose of acquiring stabilized
data.
F-4500: 0.1 to 10.0 s
F-2500: 1, 2, 5, 10 s

(j) Delay
After pressing the Measure button, measurement is
started following the delay time set here. It is used for
temperature stabilization, etc. In repeat measurement,
it is the time until the start of the first measurement.
The delay time is invalid for the second and
subsequent measurements.
Input range: 0 to 9999 s

4 - 16
 4.2

(4) Standards

Clicking the Standards tab opens the window shown in Fig.

4-16.

Fig. 4-16 Standards Tab

(a) Sample table

Sample table gives a list of standards for sample
measurement or calibration curve preparation.
This table contains the items listed below.

NOTE: The size of the sample table columns (cells)

is changeable. Bring the cursor to a
boundary line between left and right table
headers, and it changes into the one shown
below.

Now click the mouse button on the line and

drag it to the left or right to obtain a suitable
column size.

1) No.
This is the number given to each sample.
2) Sample
Enter a sample name in up to 20 characters.
3) Comments
Enter comments on each sample.
4) Concentration
Enter a sample concentration.

4 - 17
4.2 Creating an Analysis Method

(b) Update
By clicking this button, sample numbers are set by the
entered number of samples. The displayed sample
names, comments, etc. are all cleared.

(c) Insert
When the initial screen is opened, the Insert button
becomes active. Click this button to insert data at the
end of the sample list.

(d) Delete
Click the column of the sample No. to be deleted, and
it becomes active. Now click the Delete button and
the item is deleted.

(5) Monitor

When selecting the Monitor tab, the window in Fig. 4-17

appears.

Fig. 4-17 Monitor Tab

Assign the following parameters.

(a) Y-Axis Max (maximum)

Input a maximum value for Y-axis on the monitor
window.

(b) Y-Axis Min (minimum)

Input a minimum value for Y-axis on the monitor
window.

4 - 18
 4.2

(c) Open data processing window after data acquisition

Select whether or not to conduct data processing after
sample measurement. When selected (check mark is
applied), the data is saved automatically after
measurement and its file is displayed as an icon on the
window. And by opening this icon, data processing
such as peak detection is available. When this
function is not selected, data processing can be
achieved by reading out the file via File-Open.

(d) Print report after data acquisition

Select whether or not to print a report after sample
measurement. When selected (check mark is
applied), printing will be made automatically after
measurement. The items set under the Report tab
will be printed.

(6) Report

Select the Report tab, and the window in Fig. 4-18 will
appear.

Fig. 4-18 Report Tab

On this window, select the items to be contained in an analysis

report.

4 - 19
4.2 Creating an Analysis Method

(7) Output

(a) Print Report

A report will be printed. For printing method, refer to
4.5.

(b) Use Microsoft Excel

Data will be transferred to Microsoft Excel.
For transfer method, refer to 6.13.

(c) Use print generator

Select this method for using the report generator
(option). For usage, refer to the instruction manual of
the report generator.

<Printable (transferable) items>

Include date
Include method
Include calibration curve
Include standards data
Include calibration data
Include sample data

(8) Printer Font

Click this button for changing the font to be used in the

report.

4 - 20
 4.3

4.3 Defining Your Samples

When Use Sample Table has been set for the Analysis Method,
the samples must be defined before measurement.

(1) Open the monitor window and select the Sample table
command in the Edit menu or click the button.

(2) When the Sample Table window in Fig. 4-19 appears, fill in
the blank section. For instance, if there are five samples,
click the Insert button five times. Or enter 5 for Number
of Samples and click the Update button.

Fig. 4-19 Sample Table Window

(3) Input the sample name of each sample and comments on it.
Since sample names cannot be changed after
measurement, input them here.
Maximum number of characters to be input
Sample : 20 characters
Comments : 255 characters

(4) Input the name of the data file after measurement.

(5) When input is finished, check the entries and then click the
OK button.

4 - 21
4.4 Conducting Measurement

4.4 Conducting Measurement

First, create a working curve, and then proceed to sample

measurement.

(1) Set the analysis method, and the window in Fig. 4-20 will
appear provided a sample table is not used.

Fig. 4-20

When a sample table is used, the window in Fig. 4-21 will

appear.

Fig. 4-21

4 - 22
 4.4

(2) There are two methods of starting measurement.

(a) Select the Measure command from the

Spectrophotometer menu.

(b) Click the (measurement) button on the

measurement toolbar.

(3) Standards will be measured. When finished, the window

in Fig. 4-22 will appear. So click the OK button.

Fig. 4-22

(4) Now measure the samples. If a sample table is not used,

click the Sample button. To interrupt the measurement for
other samples, click the Interrupt button.
The window in Fig. 4-23 will appear, so enter a sample No.

Fig. 4-23

Click the OK button and measurement starts.

When a sample table is used, a message box is displayed,
so conduct operation according to the messages.

(5) When sample measurement is finished and a sample table

is not used, click the End button. From the File menu,
select the Save As command and save the data.
When a sample table is used, the data will be saved
automatically.

4 - 23
4.4 Conducting Measurement

(6) For conducting remeasurement after completing a series of

measurements, click the data area to be remeasured as
shown in Fig. 4-24 and then click the Remeasure button.
For saving data after remeasurement, click the End button
or button. The procedure is the same when a sample
table is used. Both standards and samples can be
remeasured.

Fig. 4-24

(7) For conducting a blank measurement, click the Blank

button.

(8) Data is saved on completion of measurement. When its

file is opened, a data processing window shown in Fig. 4-25
appears.
(d) Calibration curve data

(a) Standard data

(c) Sample data

(b) Calibration curve

Fig. 4-25 Data Processing Window (photometry)

4 - 24
 4.4

(a) Standard data

The measurement results of standards used for
preparing a calibration curve are displayed.

Fig. 4-25-1 Standard Data

(b) Calibration curve

A calibration curve obtained from standard data is
displayed. The curve can also be traced.
Concentration and photometric value are indicated at
the top left of this field.

(c) Sample data

Sample data such as photometric value (Abs),
concentration (Conc), mean value (Avg Conc),
standard deviation (SD), coefficient of variation (CV)
and upper/lower limit judgment results (HIGH/LOW)
are displayed.

Fig. 4-25-2 Sample Data

4 - 25
4.4 Conducting Measurement

(d) Calibration curve data

Data such as determination coefficient and factors (A0,
A1, A2, A3) of calibration curve are displayed. And the
calibration type can be changed.

Fig. 4-25-3 Calibration Curve Data

4.4.1 Manual Input of The standard data can be input manually. This function is
Standard Data usable after measurement of standards. Either select Standard
from the Edit menu of monitor window or click the button.
A window as in Fig. 4-26 will appear. So enter values in the
Data column. After entry, click the OK or Update button.
Then, the calibration curve is recalculated and displayed.

Fig. 4-26

4 - 26
 4.4.2

4.4.2 Statistical Statistical calculation is performed for the measured sample.

Calculation The results are displayed in the Avg Conc column of the data
processing window as shown in Fig. 4-25-2.

Xi
MEAN =
N

SD
CV = 100
MEAN

X i ( X i ) N
2 2
SD =
N 1

Here, N = number of standards

MEAN = mean value
SD = standard deviation
CV = coefficient of variation (%)

NOTE: ***** is indicated for SD and CV values when they are

nearly 0.

4 - 27
4.5 Printing Data

4.5 Printing Data

Printout can be provided even when the function for automatic

printout after measurement is not used. The following two
procedures are available.

(1) Select the Print command from the File menu. Or click the
print button on the toolbar. The field active on the
data processing window (e.g., (b) in Fig. 4-25) will be
printed. What is displayed by clicking the Print Preview
command in the File menu will be printed.

Fig. 4-27

4 - 28
 4.5

(2) Select the Report command in the Data menu. Or click

the button on the toolbar. The Print Preview window
will appear. So click the Print button. Select the items to
be printed at the Report tab with the Properties command
chosen in the Edit menu. The measuring parameters will
also be printed.

Fig. 4-28

4 - 29
4.6 Remeasuring Standard Data and Sample Data

4.6 Remeasuring Standard Data and Sample Data

Standard data and sample data can be remeasured at the mid-

point of a series of measurements or after it.
For remeasurement, implement the following procedure.

(1) Open the monitor window after completion of measurement.

(2) Click the data to be remeasured.

Fig. 4-37

(3) Click Remeasure or select the Remeasure (E) command

from the Spectrophotometer (S) menu. The dialog box in
Fig. 4-38 will open.

Fig. 4-38

(4) Click OK, and measurement will start. When a standard is

remeasured, the calibration curve will be recalculated and
displayed.

(5) Click the button, and the data will be saved.

4 - 30
 4.7

4.7 Manual Input of Standard Data

The standard data can be input manually. This function is

usable after measurement of standards. Either select Standard
(T) from the Edit menu of monitor window or click the button.
A window as in Fig. 4-39 will appear. So enter values in the
Data column. After entry, click the OK or Update button.
Then, the calibration curve is recalculated and displayed.

Fig. 4-39

4 - 31
4.8 Measurement Using Saved Calibration Curve

4.8 Measurement Using Saved Calibration Curve

Call a calibration curve in the following way.

(1) Set measurement mode in Photometry.

(2) From the File (F) menu, select the calibration curve load
command. A window as shown below will appear.
Select the file of the calibration curve to be used.

Fig. 4-40

(3) When selecting the file, the window as shown in Fig. 4-41
will appear. The sample data section is blank.

Fig. 4-41

4 - 32
 4.8

(4) Edit a sample table.

(5) Set a blank sample to carry out auto zero.

From the Spectrophotometer (S) menu, select the Auto
zero (Z) command. Or click the button.

(6) From the Spectrophotometer (S) menu, select Scan (S)

command. Or click the button on the toolbar.

(7) Set and measure a sample.

4 - 33
 5.1

5. 3-D SCAN

5.1 Flow of Operation

This section explains the parameter setting and operating

method for 3-D Scan.
For using the 3-D Scan mode in the Model F-2500, the optional
3-D Scan program needs to be installed.
Measurement is made in the following procedure.

Start

Method setting Click button.

(See page 5-2.) Scan range
Data mode
Scan speed

Sample name/comment input Click button.

(See page 5-13.)

Pre-scan
Click button.
(See page 5-14.)

Measurement Click button.

(See page 5-14.)

Data processing Spectrum tracing

Peak detection
(Refer to Section 6.) Spectral calculation

Print
Click button.
(See page 5-17.)

End

Fig. 5-1 Operational Flow (3-D Scan)

5-1
5.2 Creating an Analysis Method

5.2 Creating an Analysis Method

Set the analytical conditions (method) prior to measurement.

(1) From the Edit menu, select the Method command or click
the button. The dialog box shown in Fig. 5-2 will
appear.

Fig. 5-2 Analysis Method

(2) Set the analysis method on this window. For details of

each tab, refer to 5.1.2 onward.
The following tabs are provided on the Analysis Method
window for the 3-D Scan mode.
These tabs are explained on the subsequent pages.
General
Instrument
Monitor
Processing
Report

5-2
 5.2

(3) General

The parameters shown in Fig. 5-3 are provided under the

General tab.

Fig. 5-3 General Tab

(a) Measurement (mode)

Select a measurement mode from among the four
listed below.
Wavelength scan
Time scan
Photometry
3-D Scan
Here, select 3-D Scan. For Wavelength scan, time
scan and Photometry, refer to Sections 2, 3 and 4.

(b) Operator
Enter an operator name.

(c) Instrument
The model of the connected instrument is indicated.

(d) Sampling
Standard
When sample is exchanged manually.

(e) Comments
Enter a description or notes on measuring conditions.

5-3
5.2 Creating an Analysis Method

(f) Load
Click this button to read out saved analytical conditions.
The file opening window appears upon clicking, so
select an analytical conditions file.

(g) Save
By clicking this button after setting the parameters for
all five tabs, the analytical conditions are saved.
Clicking this button overwrites parameters on the
existing ones.

(h) Save As
Click this button for renaming the set conditions for
saving. The Save As window will appear, so enter a
file name.

(4) Instrument

This window appears when 3-D Scan is selected for the

Measurement under the General tab of the Analysis
Method window. When you click the Instrument tab, the
window in Fig. 5-4 appears.

Fig. 5-4 Instrument Tab

5-4
 5.2

(a) Data mode

Select either of the following data modes.
The Phosphorescence mode is for acquiring only
phosphorescence after extinction of excitation beam
while turning a chopper on the excitation side.
Fluorescence
Phosphorescence (with Model F-4500)

(b) EX Start WL
This is the start wavelength for wavelength scan on the
excitation side. Input the shorter wavelength side of
measuring wavelength range.

Model Input Range

F-4500 0, 200 to 850 nm
F-2500 0, 200 to 750 nm

(c) EX End WL
This is the end wavelength for wavelength scan on the
excitation side. Input the longer wavelength side of
measuring wavelength range.

Model Input Range

F-4500 0, 200 to 900 nm
F-2500 0, 200 to 800 nm

(d) EX Sampling interval

In the 3-dimensional measurement mode, emission
spectrum measurement is repeated while shifting the
excitation wavelength. Therefore, a shorter sampling
interval on the excitation side will result in a longer
measurement time.
Input range: 1 to 50 nm

(e) EM Start WL
This is the start wavelength for wavelength scan on the
emission side. Input the shorter wavelength side of
measuring wavelength range.

Model Input Range

F-4500 0, 200 to 850 nm
F-2500 0, 200 to 750 nm

5-5
5.2 Creating an Analysis Method

(f) EM End WL
This is the end wavelength for wavelength scan on the
emission side. Input the longer wavelength side of
measuring wavelength range.

Model Input Range

F-4500 0, 200 to 900 nm
F-2500 0, 200 to 800 nm

(g) EM Sampling interval

In the 3-D Scan mode, emission spectrum
measurement is repeated while shifting the excitation
wavelength. Therefore, a shorter sampling interval on
the emission side will result in a longer measurement
time.
Input range: 1 to 10 nm

(h) Scan speed

Set a scan speed. (Unit: nm/min)

F-4500 F-2500
30000 3000
12000 1500
2400 300
1200 60
300 15
60
15

(i) EX Slit
Select a slit width for the excitation side. Unit: nm

F-4500 F-2500
1.0 2.5
2.5 5.0
5.0 10.0
10.0 20.0
20.0

5-6
 5.2

(j) EM Slit
Select a slit width for the emission side. Unit: nm

F-4500 F-2500
1.0 2.5
2.5 5.0
5.0 10.0
10.0 20.0
20.0

(k) PMT Voltage

A function for controlling the voltage of the
photomultiplier detector (photomultiplier tube).
Select one of the following values. Changing from
400 V to 700 V increases the data value about two
digits.
950 V (only with F-4500)
700 V
400 V

(l) Response
Select a response time from the following.

F-4500 F-2500
0.04 0.04
0.01 0.08
0.05 0.4
0.1 2
0.5 8
2.0 Auto
8.0
Auto

By selecting Auto, a response time is set automatically

according to scan speed. Select Auto usually.

5-7
5.2 Creating an Analysis Method

(m) Corrected spectra

A function for determining the spectrum inherent to a
sample by correcting the photometer wavelength
characteristic using the saved instrument parameters,
following measurement with the instrument parameters
for photometer control. If there are no instrument
parameters for the wavelength range to be measured,
only the wavelength range that allows correction is
scanned. And if there is no wavelength area
measurable, an error will occur.
For using this function, a Spectra Correction Parts Set
(P/N 251-0126) is required. The Instrument
Response command is provided in the Utility menu for
operating this function. When this setting is at ON,
Corrected spectra appears on the monitor window.
(See Fig. 1-26 in 1.6.)

(n) Shutter control (only with F-4500)

The shutter can be automatically closed in other than
measurement for suppressing sample deterioration
due to the energy of excitation beam and opened when
measurement starts. When you put a check mark at
the head, the shutter will close and open at start of
measurement. The shutter will close again when
measuring wavelength begins returning to the start
wavelength after measurement in the wavelength scan
mode.

5-8
 5.2

(5) Monitor

When selecting the Monitor tab, the window in Fig. 5-5

appears.

Fig. 5-5 Monitor Tab

Assign the following parameters.

(a) Y-Axis Max (maximum)

Input a maximum value for Y-axis on the monitor
window.

(b) Y-Axis Min (minimum)

Input a minimum value for Y-axis on the monitor
window.

(c) Contour interval

Function for setting intervals of drawing contour lines.
Input range: 0.01 to 1000

(d) Open data processing window after data acquisition

Select whether or not to conduct data processing after
sample measurement. When selected (check mark is
applied), an icon is displayed for the data processing
window at the end of measurement. And by opening
this icon, data processing such as peak detection is
available.

5-9
5.2 Creating an Analysis Method

NOTE: When the remaining memory capacity of PC

in operation is very small (system resource is
less than 20%), the data processing window
will not open even if this item is turned ON.
Either close the presently opened data
processing window or restart the FL
Solutions program.

(e) Print report after data acquisition

Select whether or not to print a report after sample
measurement. When selected (check mark is
applied), printing will be made automatically after
measurement. The items set under the Report tab
will be printed. When this item is not selected, data
processing can be made by reading out a file via File-
Open.

(6) Processing

On selection of the Processing tab, the window in Fig. 5-6

will be displayed.

Fig. 5-6 Processing Tab

(a) Peak Finding

Select a peak detection method indicated in the data
processing window.

5 - 10
 5.2

(b) Threshold
Set a detection limit for the photometric value axis of
peak and valley. Peaks or valleys below the setting
will not be detected. Input range is 0.001 to 1000.

(c) Sensitivity
Select the number of data points in the horizontal axis
direction (abscissa). Select a sensitivity of 1 for
detecting sharp peaks. Or select 8 for detecting
broad peaks.
1
2
4
8

(7) Report

Select the Report tab, and the window in Fig. 5-7 will
appear.

Fig. 5-7 Report Tab

(8) Output

(a) Print Report

A report will be printed. For printing method, refer to
5.7.

(b) Use Microsoft Excel

Data will be transferred to Microsoft Excel.
For transfer method, refer to 6.13.

5 - 11
5.2 Creating an Analysis Method

(c) Use print generator sheet

Select this method for using the report generator
(option). For usage, refer to the instruction manual of
the report generator.

(9) Orientation
Selectable when Report is chosen for Print Method.
Select a printing orientation. Either Portrait or Landscape
orientation is selectable. When the Landscape orientation
is selected, date and method can be printed.

(a) Printable (transferable) items

1) Include date
2) Include instrument
3) Include graph
Contour
Birds-eye view
Contour and birds-eye view
Contour and Spectrum
4) Include peak table

(b) Printer Font

Click this button for changing the font to be used in the
report.

5 - 12
 5.3

5.3 Defining Your Samples

Samples must be defined before measurement.

Click the button. The dialog box in Fig. 5-8 will appear.

Fig. 5-8

Input for Sample and Comments. For automatic saving, put a

check mark in the Auto File box and specify a file name.
To specify a file name, click Select.

5 - 13
5.4 Conducting Measurement

5.4 Conducting Measurement

(1) There are two methods of starting the measurement.

(a) Select the Measure command from the

Spectrophotometer menu.

(b) Click the (measurement) button on the

measurement toolbar.
For halting the measurement midway, click the
button on the toolbar.

(2) When automatic saving is set, data will be saved after

completion of measurement. Unless automatic saving is
set, select the Save As command from the File menu and
save data in file. Upon opening its file, a data processing
window will appear as in Fig. 5-9.

(b) Excitation spectrum (d) Peak table

(a) Contour lines

(c) Emission spectrum

Fig. 5-9 Data Processing Window (3-D Scan)

(a) Contour lines

From these contour lines, excitation and emission
spectra can be read out.
As the cursor is moved, the cursor-specified excitation
and emission spectra are displayed in (b) and (c).

5 - 14
 5.4

(b) Excitation spectrum

An excitation spectrum at the cursor position in (a) is
displayed. Wavelength and photometric value can be
read by selecting the trace command from the View
menu or clicking the button.

(c) Emission spectrum

An emission spectrum at the cursor position in (a) is
displayed. Wavelength and photometric value can be
read by selecting the trace command from the View
menu or clicking the button.

(d) Peak table

Peak wavelength and peak height are indicated.
For setting threshold and sensitivity values, click the
button.

5 - 15
5.5 Displaying a Birds-Eye View

5.5 Displaying a Birds-Eye View

3-D Scan data can be displayed in a birds-eye view.

Select the Birds-eye view command from the View menu or click
the (birds-eye view) button on the Spectrum toolbar.

Fig. 5-10

Viewing direction is changeable by clicking (from front),

(from rear), (from left) or (from right).
Clicking (contour line diagram) displays a contour line
diagram.

5 - 16
 5.6

5.6 Data Printout

Printout can be provided even when the function for automatic

printout after measurement is not used. The following two
procedures are available.

(1) Select the Print command from the File menu. Or click the
print button on the toolbar. The field active on the
data processing window (e.g., (a) in Fig. 5-9) will be printed.
The contents of display upon clicking the Print Preview
command in the File menu will be printed.

Fig. 5-11

5 - 17
5.6 Data Printout

(2) Select the Report command in the Data menu. Or click

the button on the toolbar. The Print Preview window
will appear, so click Print. Select the items to be printed
at the Report tab under Properties.

Fig. 5-12

5 - 18
 6.1

6. DATA PROCESSING

This section explains the operation method for data processing

after measurement.

6.1 Loading of Data

Load saved data as follows.

Either click the (Open) button or select the Open command
from the File menu. A window as in Fig. 6-1 will appear.

Fig. 6-1

Select the file type of data file in Files of type where file types
are listed. Specify a desired file and click Open, and the data
file will open.
The following types are included in Files of type.
Wavelength scan files (*.fds)
Time scan files (*.fdt)
Photometry files (*.fdq)
3-D Scan files (*.fd3)
Method files (*.flm)
Sample table files (*.fls)
Report files (*.flr)

6-1
6.1 Loading of Data

Processed data files (*.fdp)

F-4500 wavelength scan files (*.fds)
F-4500 time scan files (*.fdt)
F-4500 photometry files (*.fdq)
F-4500 3-D Scan files (*.fcd)
All files (*.*)

The window (Fig. 6-1) has four tool buttons. Table 6-1 lists the
tool buttons and their functions.

Table 6-1 File-Open Window Buttons

Button Function
Displays a folder of one level higher.

Creates a new folder.

Lists the file names included in selected

folder.
Displays file details such as file name,
size and type.

NOTE: When the remaining memory capacity of PC in

operation is very small (system resource is less than
20%), the data processing window will not open.
Either close the presently opened data processing
window or restart the FL Solutions program.

6-2
 6.2

6.2 Spectrum Tracing

This function is used to read the photometric value at a certain

wavelength on the measured spectrum. First, bring the cursor
onto the displayed spectrum, then right-click the mouse and a
window as in Fig. 6-2 appears.

Fig. 6-2

Put a check mark before Trace or click the button. A line

cursor appears on the spectrum. The wavelength and
photometric value at the cursor position are indicated at the
location (a) in Fig. 6-3.

(a)

Fig. 6-3

6-3
6.2 Spectrum Tracing

The line cursor can be moved in the following two ways.

(1) Movement using mouse

Move the line cursor with the mouse to the position for
readout.

(2) Movement using arrow keys on the keyboard

Click the [] or [] key and the line cursor moves in the

corresponding direction.

6-4
 6.3

6.3 Scale Change

The X and Y-axis scales of data can be changed.

Click the (properties) button and select the Scale tab.
Or right-click on the spectrum and select Scale change.

Fig. 6-4 Scale Tab

Enter values in the Maximum and Minimum columns for the X

and Y axes. The initial values are those that have been saved.
The above window contains the following setting items.

(1) X Axis
Enter minimum and maximum values for the X axis.

(2) Y Axis
Enter minimum and maximum values for the Y axis.

(3) Default Range

System returns to initial values of X and Y axes.

Scale can be expanded with the mouse. Specify an area to be

expanded. Holding the mouse button held down, draw a
rectangle, and then release the button. Scale will be expanded.
For returning to the original scale, click the Default Range button
or the button on the toolbar.

6-5
6.4 Change of Graph Axis

6.4 Change of Graph Axis

The horizontal axis of a graph is changeable on a spectrum.

Click the (properties) button and select the Scale tab.

Fig. 6-5 Scale Tab

(1) X Axis mode

X-axis is indicated in either unit below.

(a) nm
Indicated in wavelength (nm).

(b) Kcm-1
Indicated in wavenumber (Kcm-1, K = 1000).
x = 10000 (x: Kcm-1, : nm)

6-6
 6.5

6.5 Change of Spectral Line

The line and color of a spectrum can be changed. Select the

Lines/Points tab of Spectrum Properties. Upon clicking this tab,
the window below appears.

Fig. 6-6 Lines/Points Tab

The following setting items are contained in this window.

(1) Graph

Select a spectrum for setting lines/points.

(2) Display mode

Data can be indicated in the following modes.

Lines
Points
Lines & Points

(3) Line pattern

Specify the kind of line such as dotted line or solid line.

(4) Point style

Select the point style to be used in graph printing or display.

6-7
6.5 Change of Spectral Line

(5) Line color

Use of the Color window enables you to change the color of

spectral lines or points. There are 48 basic colors.
By clicking the Define Custom Colors button, a smaller
window is added on the right side of the Color window as
shown in Fig. 6-7. And by clicking Define Custom Colors
again, 16 colors are added. These colors are usable or
the spectral lines and points. For defining the colors,
entries can be made for Hue, Sat (saturation) and Lum
(luminance), Red, Green and Blue. When the definition is
finished, click the Add to Custom Colors button. Then,
select the desired ones from the defined colors.

Fig. 6-7

6-8
 6.6

6.6 Detection of Spectral Peaks

The spectral peaks and valleys are detectable automatically.

Select the Find Peaks command from the Data menu or click
button, and a window as in Fig. 6-8 appears.
Set Integration method, Threshold and Sensitivity.

Fig. 6-8

(1) Integration method

The following three methods are available and any one may
be selected for the peak wavelength. But the area
calculation result indicated in the peak table will vary with
the selected method.
Rectangular
Trapezoid
Romberg

For details of these methods, refer to APPENDIX D.

(2) Threshold

Set a detection limit for the photometric value axis of peaks

and valleys. Peaks and valleys below the set value will
not be detected.
Input range: 0.001 to 1000

(3) Sensitivity

Select the number of data points in X axis direction.

For detection of sharp peaks, select a sensitivity level of
1, and for broad peaks, select 8.
1
2
4
8

6-9
6.7 Indication of Peak Wavelength on Spectrum

6.7 Indication of Peak Wavelength on Spectrum

Select the Properties (O) command from the Edit (E) menu or
click the (properties) button on the Processing toolbar.
The window in Fig. 6-9 will open.

Fig. 6-9 Display Tab

In the Comments under this tab, the following settings can be

made.

(1) None

Only a spectrum is displayed.

(2) Peak Number

Peak No. is indicated on a spectrum. This number is the

one indicated in the peak table.

(3) Peak Apex

Peak wavelength is indicated on a spectrum.

This wavelength is the one indicated in the peak table.
It appears as shown in Fig. 5-9 and can be printed.

(4) Valley No.

Valley No. is indicated on a spectrum. This number is the

one indicated in the peak table.

6 - 10
 6.7

(5) Valley wavelength

Valley wavelength is indicated on a spectrum.

This wavelength is the one indicated in the peak table.
It can be printed.

(6) Peak and Valley Number

Peak and valley Nos. are indicated on a spectrum.

These numbers are the ones indicated in the peak table.

(7) Peak and valley wavelength

Peak and valley wavelengths are indicated on a spectrum.

These wavelengths are the ones indicated in the peak table.
They can be printed.

Fig. 6-10

6 - 11
6.8 Smoothing of Spectrum

6.8 Smoothing of Spectrum

Select the Smooth (S) command from the Data (D) menu or click
the button. The window in Fig. 6-11 will open.

Fig. 6-11

Set the following parameters on this window.

(1) Function type

Select a smoothing method from the following three.

Savitsky-Golay
Mean
Median

(2) Smoothing order

Set a smoothing order.

Input range: 2 to 4

(3) Number of points

Set the number of points to be used in calculation.

Input range: 5 to 101, Initial value: 7

(4) Number of times

Set the number of smoothing operations.

Input range: 1 to 100
For returning to the original status (before calculation), click
.

6 - 12
 6.9

6.9 Derivative Operation on Spectrum

Select the Derivative (D) command from the Data (D) menu or
click the button. The window in Fig. 6-12 will open.

Fig. 6-12

(1) Derivative order

Set a derivative order.

Input range: 1 to 4

(2) Smoothing order

Set a smoothing order.

Input range: 2 to 4

(3) Number of points

Set the number of points to be used in calculation.

Input range: 5 to 101, Initial value: 15

(4) Overlay derivative

Spectra before and after derivative operation are overlaid

for display. Note that the value on Y axis is not the one
after derivative operation.
For returning to the original status (before calculation), click
.

6 - 13
6.10 Overlaid Display of Spectra

6.10 Overlaid Display of Spectra

This function is convenient for comparison of two or more

spectra.
There are 3 methods selectable for this function.

(1) Select the Overlay (O) command from the Data (D) menu.

Fig. 6-13

Click the Insert button and a list of files that allow overlay is
displayed. Select a file name and click the Open button.
The selected sample name is added and displayed as in
Fig. 6-14. Now click the Close button.

Fig. 6-14

6 - 14
 6.10

(2) Open a file to be overlaid to display its data.

Fig. 6-15

Drag the blue profile of the displayed spectrum so that the

mouse pointer comes onto the target spectrum. When the
mouse pointer changes as shown in Fig. 6-16, drop the
blue profile.

Fig. 6-16

(3) Open a file as in (2) above. Click the spectrum to be

overlaid and press Ctrl+C on the keyboard or select the
Copy (c) command from the Edit (E) menu. Then click the
target spectrum and paste the first spectrum on it by Ctrl+V.
The spectra are now overlaid.

6 - 15
6.10 Overlaid Display of Spectra

(a) Deletion from Overlaid Display

Select the Overlay command from the Processing
menu. In case you want to delete the spectrum
having sample name Filter 02, select the sample
name and click Delete.

Fig. 6-17

(b) Display of Selected Spectrum Alone from Overlaid

Spectra
Click (Display spectrum) on the Spectrum toolbar.

To be displayed

Not to be displayed

Fig. 6-18

Click the spectrum not to be displayed.

Click OK. In this case, spectra Nos. 1 and 2 will be
displayed. Printout of this data covers not the spectra,
but the sample names, analytical conditions, etc.
After file saving in the current state, 4 spectra will be
displayed upon loading the file again.

6 - 16
 6.11

6.11 Arithmetic Operation between Spectra

Shown here is an example of obtaining the difference between

two overlaid spectra.
Display the spectrum for arithmetic operation. Now, click the
Arithmetic (A) command on the Data (D) menu or click the
(spectral calculation) on the Processing toolbar. A window as in
Fig. 6-19 will open.
(4) Spectral calculation
(3) Factor input ON/OFF

(2) Spectrum

(1) Sample name

Fig. 6-19

(1) The sample name of the file open in the window (file name
in parentheses) is indicated. Select the file for arithmetic
operation.

(2) The spectrum selected in (1) is displayed here.

(3) A factor for the spectrum selected in (1) is settable.

For multiplying the spectrum by a factor, click Calculate
here.

(4) Apply a check mark here for arithmetic operation between

two spectra.
As a result, the window as in Fig. 6-20 appears.

6 - 17
6.11 Arithmetic Operation between Spectra

(7) Factor input (8) Mathematical operator

(6) Spectrum

(5) Sample name

Fig. 6-20

(5) Another sample name (file name in parentheses) is

indicated. Select the file for arithmetic operation.

(6) The spectrum selected in (5) is displayed.

(7) Set the factor for the spectrum selected in (5).

(8) Select a mathematical operator for the two spectra.

+: Addition, -: Subtraction, *: Multiplication, /: Division

After setting, click Calculate. The result of arithmetic operation

is displayed as shown in Fig. 6-21.

6 - 18
 6.11

Result of arithmetic operation

Fig. 6-21 After Arithmetic Operation

Click Save As, and the result of calculation can be saved in file.

6 - 19
6.12 Area Calculation

6.12 Area Calculation

An area value is obtainable by integration in the specified range

of the presently displayed spectrum.
Select the Integration (I) command from the Data (D) menu or
click the (area calculation) button on the Processing toolbar.
The window as in Fig. 6-22 will appear.

Fig. 6-22

Click Start and input the range for area calculation. Or move
and right-click the cursor, and a value will be entered. In the
same way, enter the end wavelength. After entry, click
Calculate. The result will be displayed.
For printout, click Print. In order to print a spectrum and the
result of calculation, apply a check mark to With spectrum
record and click Print.
When clicking the OK button after calculation, its result will be
saved. Operation will be reset by closing the data processing
window or clicking the button on the Processing toolbar.
Clicking Cancel closes the window.

6 - 20
 6.12

For scale expansion, specify the area to be expanded with the

mouse. Draw a rectangle with the mouse button held down.

Fig. 6-23

Then, release the button, and the specified area will be

expanded as shown in Fig. 6-24.

Fig. 6-24

Double clicking the right mouse button returns the scale to the
original one.

6 - 21
6.12 Area Calculation

The principle of calculation is shown below.

D2

Dn - 1 D1

Dn

2
Section end Specified range Section start
wavelength wavelength

S1 Sa
Sc
Sb
S2 Sd
WL1 WL2 WL1 WL2

Fig.6-25

The elements (D1 to Dn) of a spectrum are subjected to the

following calculation for obtaining the area value.

S3 = {D2 + D3 + Dn-2 + Dn-1 + D1 + Dn}/2

S2 = (D1 + Dn) { (n-1)}
(D1 + Dn) (Section start wavelength (WL1))
(Section end wavelength (WL2))
S1 = S3 S2
D1 : Spectral data at section start wavelength
Dn : Spectral data at section end wavelength
: Sampling interval

In the time scan mode, change as follows.

Start wavelength (WL1) end time (T2)
End wavelength (WL2) start time (T1)

6 - 22
 6.13

6.13 Transfer of Data to Microsoft(R) Excel

Measured data can be transferred to a worksheet of Microsoft(R)

Excel.
Procedure is given below.

(1) Click the (properties) button and select the Report tab.
A window as in Fig. 6-26 will open. In Print Method, select
Use Microsoft(R) Excel. Also select the data to be
transferred (peak table and data list in case of wavelength
scan). Then, click OK.

Fig. 6-26

(2) Click the (Report) button and the Microsoft(R) Excel

will automatically start to read the data. The subsequent
procedure concerns the Microsoft(R) Excel operation.

Fig. 6-27

6 - 23
6.13 Transfer of Data to Microsoft(R) Excel

(3) Data can be saved with extension code xls of Microsoft

Excel.
Select File - Save Copy As and enter a file name.

Fig. 6-28

(4) Upon closing the Microsoft Excel, the window in Fig. 6-29
appears. Now close this window. The original data
processing window will appear again.

Fig. 6-29

6 - 24
 6.14

6.14 Pasting of Spectra to Microsoft(R) Word

In report preparation, spectra can be pasted as they are.

Make the spectral data active (click the relevant spectrum) and
then select the Copy command from the Edit menu. Now open
the Microsoft(R) Word and select the Paste command from the
Edit menu. The data can be pasted to a Microsoft(R) Word text.
Peak tables and/or kinetics data can also be pasted in the same
way. By double-clicking the pasted spectrum shown in
Fig. 6-30, functions such as scale change and calculation result
display are applicable. The spectrum can be selected as a
meta-file by the Paste Special (S) command with a form chosen.
In the same way, peak tables and/or kinetics data can also be
pasted.

Fig. 6-30

When you double-click the pasted spectrum, you can edit it.
Right-clicking on it permits editing such as scale change.
A change made here will be brought into a Microsoft(R) Word
text.

NOTE: he above editing in the Microsoft(R) Word can be made

with a PC in which the Microsoft(R) Word and FL
Solutions program are installed.

6 - 25
6.14 Pasting of Spectra to Microsoft(R) Word

For pasting a spectrum to the Microsoft(R) Excel, specify

Paste Special. The dialog box in Fig. 6-31 will appear.

Fig. 6-31

Click OK will paste the spectrum.

6 - 26
 6.15

6.15 Conversion of Data to ASCII Text File

A file conversion function is provided for processing of data with

other spreadsheet software. Conversion can be made to the
following three data files.
JCAMP-DX file (*.dx)
ASCII text file (*.txt)
Graph meta file (*.wmf)

NOTES: 1. Date/time, analytical conditions and data list (raw

data) will be converted into a JCAMP-DX file
2. The items selected at the Report tab of
Properties will be converted into an ASCII text
file.

(1) Open the data processing window.

(2) Click the Save As (A) command in the File (F) menu.

(3) A window as in Fig. 6-32 will open.

Select ASCII text files (*.txt) at Save as type.
And specify the folder for saving.

Fig. 6-32

6 - 27
6.15 Conversion of Data to ASCII Text File

(4) Enter a file name and click Save.

Fig. 6-33

File conversion is effective for a data file in any measurement

mode. The selected data is separated by tab.

6 - 28
 6.16

6.16 Renaming Your Sample

After measurement, the sample name of saved data can be

changed.
Proceed s follows.

(1) Open the data file for renaming.

(2) Select the Properties (O) command from the Edit (E) menu
or click the button.

(3) A window as in Fig. 6-34 will open. Click the Info tab.

Fig. 6-34

(4) Input a new sample name for Sample name and click OK.

NOTE: This function is effective in the wavelength scan, time

scan and 3-D Scan modes.
For saving existing data in a different sample name,
select the Save As (A) command from the File (F)
menu.

6 - 29
6.17 Printing of Wavelength and Photometric Values under Display

6.17 Printing of Wavelength and Photometric Values under Display

This program provides a function for acquiring data such as

presently displayed wavelength and photometric values, and
traced value on the data processing window. These data can
be printed.
Select the Data read (R) command from the Tools (T) menu.
A window as in Fig. 6-35 will appear.

Fig. 6-35

For reading presently displayed wavelength and photometric

values:
Move the mouse pointer onto the monitor window and right-click
it there. A window as in Fig. 6-36 will be presented.

Fig. 6-36

6 - 30
 6.17

Select Present data read and the photometric value on the

monitor window will be read on the window shown in Fig. 6-35.
Display becomes as shown in Fig. 6-37.

Fig. 6-37

For reading data from data processing window

Display the window for data readout as instructed above and
move the mouse pointer onto date processing window.
Right-click the mouse there, and a window as in Fig. 6-38 will
open.

Fig. 6-38

6 - 31
6.17 Printing of Wavelength and Photometric Values under Display

When you select Trace value read, a window as in Fig. 6-39

will appear.

Fig. 6-39

In reading data from the data processing window, sample name

information is displayed. Sample names can be edited.
Click the sample name column, and input is allowed.
The relevant data can be printed. From the File (F) menu,
select the Print (P) command or click the (Print) button.
Data can be deleted by the following method. Click the data
value and select Delete. For deletion of all data, select Delete
All.

6 - 32
 6.18

6.18 Reference to Spectrum Measured in Photometry Mode

When the quantification method is peak area, peak height or

derivative in the photometry mode, wavelength scan will be
carried out and according to its result, a calibration curve will be
prepared. The spectrum measured in this case can be
displayed and printed.
Open the data file. Right-click the numerical part of standard
data or sample data and select Spectrum reference. Or click
a numeral and select the Spectrum reference (R) command from
the Data (D) menu.

Fig. 6-40

6 - 33
6.18 Reference to Spectrum Measured in Photometry Mode

A spectrum appears as shown in Fig. 6-41. Spectrum tracing

and scale change are possible. The spectrum can be printed
by selecting the Print (P) command from the File (F) menu or
click the button.

Fig. 6-41

6 - 34
 7.1

7. MORE CONVENIENT OPERATING METHODS

(APPLICATION PART)

7.1 For Always Starting Up with Same Analysis Method

The FL Solutions program can be started with the analysis

method specified. Select Tools - Options as in Fig. 7-1.

Fig. 7-1 Tools Menu

Now select the Startup tab as in Fig. 7-2.

Select Use fixed method and click Set F-2500 Method when
the Model F-2500 is used or Set F-4500 Method when the
Model F-4500 is used.

Fig. 7-2 Startup Tab

7-1
7.1 For Always Starting Up with Same Analysis Method

A window as in Fig. 7-3 will open. Set your method file here.

Fig. 7-3

This function is validated when the FL Solutions program is

restarted.

7-2
 7.2

7.2 Online/Offline Setting of Photometer

Connection with the photometer can be set offline. And this

connection can be made again.
Select the Instrument command from the Tools menu or click the
button. The window in Fig. 7-4 appears. This window is
for connection with the photometer.

Fig. 7-4

Click the instrument name and then the Cut button. The offline
status will be set. For online setting, click the Connect button.
Initialization will be carried out and the monitor window will
appear.

7-3
7.3 Check of Data File Information

7.3 Check of Data File Information

You can check the conditions under which specific data was
measured.
For instance, when you want to know the analytical conditions or
measurement date of a data file when opening it, specify and
right-click the file and select Properties as shown in Fig. 7-5.

Fig. 7-5

The window in Fig. 7-6 will open. Information can be checked

by clicking the Info and Scan tabs.

Fig. 7-6

7-4
 7.3

On selection of Quick Viewer, a window appears as shown in

Fig. 7-7. So a spectrum can be checked.

Fig. 7-7

Quick Viewer is usable when extension code is *.fds, *.fdt or

*.fd3. The same operation as above can be performed by using
Explorer.
The information on the open data such as analytical conditions
can be checked.

Select the Properties (O) command from the Edit (E) menu or
click the button. The Info tab contains the date of
measurement and the instrument used, and the Scan tab
contains measuring conditions such as measuring wavelength,
scan speed and slit width.

Fig. 7-8 Info Tab

7-5
7.3 Check of Data File Information

Fig. 7-9 Scan Tab

When multiple data are displayed, click the drop-down list box of
sample names and select the sample name of the desired data.

7-6
 7.4

7.4 Saving of Analysis Method

The set analysis method can be saved as a file. So instead of

setting the method each time, you can just call out the file for
setting. Select the Method command from the Edit menu on
monitor window or click the button.

Fig. 7-10

When the setting is finished, click Save As and enter a file name.
For calling out the saved file, click Load and select the file.

7-7
7.5 Even Log

7.5 Even Log

The date/time of lamp exchange and the occurrence of troubles

can be recorded in a log. Select Tools - Event Log.

Fig. 7-11

The window in Fig. 7-12 shows the occurrence time of

communication errors, etc., and user notes. For displaying only
errors or only user notes, put a check mark at either item.

Fig. 7-12 Event Log Tab

7-8
 7.5

For adding a note, select the User Notes tab, put an explanation
in the Description field and click the Add Note button.
Maximum number of input characters: 127 characters

Fig. 7-13 User Notes Tab

7-9
7.6 Check of Program Version

7.6 Check of Program Version

The version of the program currently being used can be checked.

Select the About FL Solutions (A) command from the Help menu.

Fig. 7-14

A window as in Fig. 7-15 will open. So check the program code.

Fig. 7-15

7 - 10
 7.7

7.7 Saving of Sample Table

Sample tables can be saved.

This is convenient for editing the sample table under the same
name each time.

Fig. 7-16 Sample Table

After editing the sample table, click Save As and the window in
Fig. 7-16 will appear. Enter a file name. For calling out the
saved file (*.fls), click Load and select the file.

Fig. 7-17

7 - 11
7.8 Change of Window Display

7.8 Change of Window Display

When selecting the View (V) menu, display/non-display of

toolbars and method of displaying multiple spectra can be
selected.

Display/
non-display
setting

Monitor window Data processing window

Fig. 7-20 View Menu Commands

Scale Y Axis Up : Y axis scale is doubled for

display.

Scale Y Axis Down : Y axis scale is contracted by half

for display.

Reset Y Axis, Reset Axes : The scale set under the Monitor
tab is taken.

Autoscale Y Axis, Autoscale : Scale is adjusted to spectrum

size for display.

7 - 12
 7.8

The method of displaying multiple spectra is selectable.

Overlay

Fig. 7-21

Stack

Fig. 7-22

7 - 13
7.8 Change of Window Display

Tile

Fig. 7-23

NOTE: When six, seven or more spectra are displayed, there

is a possibility of the spectra becoming too small, or not
all the traced values can be displayed. In this case,
use the Overlay (O) command of Data (D) menu to
delete unnecessary spectra.

7 - 14
 7.9

7.9 Check of Photometric Value at Specified Wavelength

This is a function for shifting to the specified wavelength. It is

usable for checking the optical axis, etc.
Open the monitor window and select the Set Wavelength (W)
command from the Spectrophotometer (S) menu or click the
(Set Wavelength) button.
A window as in Fig. 7-24 will open.

Fig. 7-24

Input target wavelength values and click OK.

Wavelength is now driven to the specified one and a photometric
value is read on the monitor window. (Refer to (2) in 1.6.)

Wavelength drive to zero-order light

For checking the optical axis, etc. in the zero-order light or
moving the instrument, wavelength needs to be driven to the
zero-order light. Open the window shown in Fig. 7-24 and
set wavelength at 0. Then click OK, and wavelength will be
adjusted to the zero-order light.

NOTE: For termination with wavelength kept at the zero-

order light, select Close the monitor window, but
keep the lamp operating? in shutting down the
application (terminating the program). Close the
monitor window, but keep the lamp operating? must
also be selected for turning off the photometer
power supply with the wavelength position kept
unchanged.

7 - 15
7.10 Collective Printing of Files

7.10 Collective Printing of Files

Wavelength scan data files (*.uds) and time scan data files
(*.udt) can be collectively printed.
From the File (F) menu, select the collective spectrum recording
(L) command. A window as in Fig. 7-25 will appear.

Fig. 7-25

Click Select file.

Fig. 7-26

7 - 16
 7.10

Specify the folder of a file to be printed at Look In and specify

the file. Click Open (O).

Fig. 7-27

After check, click Print. Printing starts. The items to be

printed are the saved ones. A maximum of 100 files can be
specified.

7 - 17
 8.1.1

8. MEASUREMENT USING OPTIONAL ACCESSORIES

For the cable connection method for each accessory, refer to the
instruction manual attached to the accessory. Explanation is
given here on operations after setting of your analysis method,
which is required as instructed in Sections 2 to 5.

8.1 Use of Sample Sipper

Hook up the sample sipper to the spectrophotometer.

The parameters for the sipper are settable on its front panel.
Explained here is the procedure for creating a calibration curve
and measuring an unknown sample in the Photometry mode.

8.1.1 When Not Using A message box appears in the standard measurement session,
a Sample Table so operate in accordance with the messages. A message box
does not appear in the sample measurement session.
Measurement can be started just by pressing the sipping lever of
the sample sipper.

NOTES: 1. When using the sample sipper, remeasurement,

interrupt measurement and blank measurement
cannot be made. But remeasurement of
standards is possible.
2. When repeat measurement is set, the standards
will be measured by the set number of times.
Samples will be measured only once.

Select the Measure command from the Spectrophotometer menu

or click the (measurement) button on the toolbar, and the
window in Fig. 8-1 will open.

Fig. 8-1

8-1
8.1 Use of Sample Sipper

Press the sipping lever and measurement starts. A message

box for the second measurement now appears.
For carrying out remeasurement or for skipping the second
standard measurement, select the relevant item with the mouse
(put a bullet in its radio button) and then click Yes.
When clicking No, the current measurement sequence will be
terminated.
For restarting measurement, click the button.
Measurement then starts from the 1st standard.

Fig. 8-2

On completion of standard measurement, the window in Fig. 8-3

will open.

Fig. 8-3

8-2
 8.1.1

By clicking OK with the mouse, a calibration curve is drawn and

then samples can be measured. When the window in Fig. 8-4
appears, you should press the sipping lever.
Measurement results appear at the area indicated by (1) in
Fig. 8-5.
By clicking Cancel, the measurement is terminated, and the
standards data is not saved.
Now select Repeat the last measurement and click OK.
Set the final standard in place and press the sipping lever.
When repeat measurement has been set, measurement is
started from the 1st one ([Link].1) for that standard.

Fig. 8-4

(1)

Fig. 8-5

When measurement is finished, click the End button, and the

data will be saved.

8-3
8.1 Use of Sample Sipper

For remeasuring any of the standards after their measurements,

click its data as shown in Fig. 8-6. Then click the Remeasure
button.

Fig. 8-6

A message box will open as in Fig. 8-7, so press the sipping

lever.

Fig. 8-7

When a standard is remeasured, the calibration curve is

recalculated and sample data is also recalculated based on the
curve and then displayed. To saved the recalculated data, click
the End button or the (save) button on the toolbar.

8.1.2 When Using a A message box is displayed in both standard and sample
Sample Table measurement sessions. So operate according to the messages.
When the message box appears, press the sipping lever of the
sample sipper.

NOTE: When the sample sipper is used, blank measurement

cannot be made.

8-4
 8.1.2

Select the Measure command from the Spectrophotometer menu

or click the (measurement) button on the toolbar, and the
window in Fig. 8-8 will open.

Fig. 8-8

Press the sipping lever and measurement starts.

For measurement of the 2nd and subsequent standards, a
message box as in Fig. 8-9 will open. For remeasurement or
for skipping measurement of the next standard, select the
relevant item with the mouse (put a bullet in its radio button) and
then click Yes. When clicking No, the current measurement
sequence will be terminated.
For restarting measurement, click the button.
Measurement then starts from the 1st standard.

Fig. 8-9

8-5
8.1 Use of Sample Sipper

On completion of standard measurement, the window in Fig.

8-10 will open.

Fig. 8-10

By clicking OK with the mouse, a calibration curve is drawn and

then samples can be measured.
By clicking Cancel, the measurement is terminated, and the
standards data is not saved.
For remeasurement of the final standard, select Repeat the last
measurement and click OK. Set the final standard in place and
press the sipping lever. When repeat measurement has been
set, measurement is started from the 1st one ([Link].1) for that
standard.

In the case of sample measurement also, a message box as in

Fig. 8-11 will appear. Press the sipping lever, and
measurement of the 1st sample starts.

Fig. 8-11

8-6
 8.1.2

For measurement of the 2nd sample, a message box as in

Fig. 8-12 opens.

Fig. 8-12

Press the sipping lever for measuring the 2nd sample.

For skipping this sample (and measuring the 3rd sample), select
Skip this sample? and click the Yes button with the mouse.
For remeasurement, select Repeat the last measurement? and
click Yes button with the mouse. When repeat measurement
has been set, measurement is started from the 1st one
([Link].1) for the 1st sample.
In case of interrupt measurement, select Insert a new sample
into the sequence and click the Yes button with the mouse.
A message box as in Fig. 8-13 opens, so click the OK button.

Fig. 8-13

8-7
8.1 Use of Sample Sipper

For remeasurement after a series of measurements, click the

data to be remeasured as in Fig. 8-14. Then click the
Remeasure button.

Fig. 8-14

A message box will open as in Fig. 8-15. Press the sipping

lever, and measurement starts. When remeasuring a standard,
the calibration curve is recalculated and sample data is also
recalculated based on the curve and then displayed. To save
the recalculated data, click the (Save) button.

Fig. 8-15

8.1.3 In Wavelength (1) When not Using Sample Table

Scan or Time
Scan Mode Set your analysis method, and when the monitor window
appears, press the sipping lever. Measurement starts.

(2) When Using Sample Table

Select the Measure command from the Spectrophotometer

menu or click the (measurement) button on the toolbar.
A message box opens. Follow the messages given.

8-8
 8.2.1

8.2 Use of AS-3000 Intelligent Autosampler

Set the rack parameters, injection volume and other items with
the AS-3000 intelligent autosampler. For details, refer to the
instruction manual attached to the AS-3000.

8.2.1 When Not Using Select the Measure command from the Spectrophotometer menu
a Sample Table or click the (measurement) button on the toolbar.
Then, press the START key on the AS-3000 intelligent
autosampler. Measurement will start.

NOTE: In measurement without using a sample table,

remeasurement, interrupt measurement and blank
measurement cannot be done.

8.2.2 When Using a NOTE: In measurement when using a sample table,

Sample Table remeasurement, interrupt measurement and blank
measurement cannot be done. But remeasurement of
standards is possible. The standard to be
remeasured should be set in the initial test tube
position.

For measurement after completion of settings:

(1) Select the Measure command from the Spectrophotometer

menu in the FL Solutions program or click the
(measurement) button on the toolbar.
The window in Fig. 8-16 opens.

Fig. 8-16 For Start from Calibration Curve Creation

(2) Press the START key on the AS-3000.

(3) Measurement is started.

8-9
8.3 For Analog Output

8.3 For Analog Output

For connection, use the analog output terminal. Select the

Analog Output Gain command from the Utility menu.

Fig. 8-17

This is a setting of data value corresponding to analog output of

1 V.
Analog output value (V) becomes as follows.

Model F-4500
Low : (Data value - photometric value lower limit)/1024
High : (Data value - photometric value lower limit)/8192

Model F-2500
Low : (Data value)/1024
High : (Data value)/8192

8 - 10
 APPENDIX A DETAILS ON QUANTITATION

A.1 Foreword
In the Photometry mode of the F-2500 spectrophotometer, there
are four calibration types.

Linear working curve

Quadratic working curve
Cubic working curve
Multiple segment working curve

These are explained in more detail below.

A.2 Linear Working Curve (1st order)

Regression line is determined via least squares method from a

maximum of 20 data. The calculation formula is as follows :
x = A1 y + A0
1
yixi yi xi
n xi yi
A1 = , A0 = A1
1 n n
y i ( y i )
2 2

n
where, x : Concentration of each sample (input value)
y : Data on each sample (measured value)
n : Number of samples

A.3 Quadratic Working Curve (2nd order)

A quadratic curve is determined via least squares method from a

maximum of 20 data. The calculation formula is as follows :
x = A2 y2 + A1 y + A0

A2 =
( ) ( )
S y 2 x S( yy) S( yx)S yy 2
S(yy) = y i2
( y i ) 2
S( yy)S( y y ) S( yy )}
2 2 2
2 n

S( yx)S( y y ) S( y x)S( yy )
2 2 2 2
yi xi
A1 = S(yx) = y i x i
S( yy)S( y y ) S( yy )}
2 2 2
2 n

2 2
xi xi yi yi yi
A0 = A1 A2 S(yy2) = y i3
n n n n
2
yi xi
S(y2x) = y i2 x i
n

( y )
2
2
2 2 i
S(y y ) = y i4
n

A -
1
 where, x : Concentration of each sample (input value)
y : Data on each sample (measured value)
n : Number of samples

A.4 Cubic Working Curve (3rd order)

A cubic curve is determined via least squares method from a

maximum of 20 data. The calculation formula is as follows :
X = A3 Y3 + A2 y2 + A1 y + A0
where, x : Concentration of each sample (input value)
y : Data on each sample (measured value)
n : Number of samples

A.5 Multiple Segment Working Curve (Segmented)

The calibration curve is apt to bend when measuring turbid

samples or the like. Use of a quantification program allows
correcting the calibration curve by using up to 20 standards.
Figure A-1 shows an example of curve correction. For the part
which exceeds the measuring range of the standards, simply
extend the line as it is.

Data STD6
STD5

STD4

STD3

STD2

STD1

CONC

Fig. A-1 Correction of Curve Bending

A -
2
<Notes on Preparation of Multiple Segment Working Curve>

(1) A correct calibration curve can be created only when the

measured value increases or decreases monotonically
versus the concentration value. Especially when the
inclination is negative-going, be sure to measure a blank
having a data value larger than the other standards.
A curve not showing a monotonic increase will appear as in
Fig. A-2. And when the data value of the blank is small,
regardless of a negative-going inclination, the curve will
appear as in Fig. A-3.

Data
STD5
STD6
STD4

STD3

STD2

STD1

CONC

Fig. A-2 Example of Curve without Monotonic Increase

(unsuitable curve)

Data
STD1
STD2
STD3

STD4

STD5

CONC

Fig. A-3 Example of Negative-going Curve where

Measured Value of Blank is Small
(unsuitable curve)

A -
3
 (2) Remeasurement of Standards

The standards can be remeasured after once preparing a

calibration curve. The curve prepared in such case will
appear as in Fig. A-4.

Data

STD6
STD5
Redrawn
calibration curve STD4

STD3

Remeasured
STD1 STD2
Initially
measured STD1

STD1

CONC

Fig. A-4 Calibration Curve when Standards are Remeasured

A -
4
 APPENDIX B DETAILS OF RATE ANALYSIS FUNCTION

B.1 Foreword

Rate analysis is used in the analysis of enzyme reactions. It is

utilized for clinical and biochemical tests by reagent
manufacturers, hospitals and so on. A computer is used to
calculate the concentration from the variation in data per unit
time, and the result is displayed and printed out.

B.2 Calculation Method

A timing chart for rate analysis is shown in Fig. B-1. Data is

acquired when the initial delay time has elapsed after pressing
the Measure button. A regression line is determined from this
data via the least squares method, and the gradient and activity
value are calculated. The calculation formula is as follows.

Data Data : A0, A1, A2, A3, A4

A0
A1
A2
A3
A4
A5
A6
Td : Initial delay time
A7 Tm : Measurement time
Tc : Sampling interval
Tt : Calculation time

Time
Td Tc
Start of Tt
measurement
Tm

Fig. B-1

A -
5
 Prepare a regression line via least squares method from the
measured data, and obtain a determination coefficient.
y = ax + b
where,
xi yi
xiyi a * ( x i )
a= n b = yi
2 ( x i ) 2 n
xi
n
xi : Time (sec) of each data
yi : Value of each data
n : Number of samples

The determination coefficient CD becomes as follows :

CD =
(n x i y i x i y i )2
{n x 2
i
2
}{
( x i ) n y i2 ( y i )
2
}
Gradient (variation per minute)
a
Di = = 60a (/min)
Tk

Activity
Ci = k Di

R (determination coefficient)
(n x i y i x i y i )
2

R= CD =

2
{
i ( x i ) } n y i ( y i ) }
n x 2 2 2

R2
(n x i y i x i y i )
2

R2 = (R)2 =

2
{
i ( x i ) } n y i ( y i ) }
n x 2 2 2

NOTE : If the range for rate calculation does not coincide with
the actual measured data range, then use only the
measured data within that range for the calculation.

A -
6
 APPENDIX C DETERMINATION COEFFICIENT OF
CALIBRATION CURVE

C.1 Calculation of Determination Coefficient

The determination coefficient and other factors are calculated via

the following formula.

Data

A3
A2

A1

C1 C2 C3 Conc
Cstd1 Cstd2 Cstd3

An : Photometric or average value of standards

Cn : Concentration on approximation curve
versus A
Cstdn : Concentration of standard (input value)
N : Number of standards

DIFF : DIFFn = Cn - Cstdn

DIFF
RD : RDn = 100
A
An
A=
N
DIFFn
t : tn =
2
DIFF
N1

( ) (C n C stdn )
2 2
Cn C
Determination coefficient : R= ,
( )
2
Cn C

R2 = (R)2

A -
7
C.2 Usage of Determination Coefficient

The determination coefficient indicates the goodness of fit of the

measured standards and the prepared calibration curve.
The closer this value is to 1, the better the fit of the measured
values and calibration curve. If the value is far from 1, then
the standards must be remeasured or the calibration curve mode
must be changed. Examples of determination coefficients upon
changing the calibration curve mode are given below.

x: standard measurement
points
Data When calibration curve type is
set to linear,
determination coefficient < 1

Conc

Data
When calibration curve type is
set to quadratic,
determination coefficient 1

Conc

With the above standard data, it can be seen that a quadratic

curve provides a better result. A calibration curve, which was
formerly judged through skill or experience, can thus be judged
more easily through numerical means.

A -
8
 APPENDIX D INTEGRATION METHOD
D.1 Foreword

The integration methods used in the FL Solutions program are

explained next.

D.2 Integration Methods

The following three methods are available in the FL Solutions

program.

Rectangular
Trapezoid
Romberg

D.2.1 Rectangular The rectangular method is the simplest one among the above
Method three. Since one sampling cycle is equivalent to the width of
each sectional area, the total of all data points including peaks
approximates the area to be obtained.
The object area shown in Fig. D-1 is the total of the rectangular
sections obtained from linear approximation of the curve drawn
via the data points. If a peak has few data points, the
approximation will be a rough estimate.

Fig. D-1

A -
9
D.2.2 Trapezoid This method is a further improvement on peak area calculation.
Method The section of each sampling cycle is indicated by a rectangle on
which a triangle is formed. The object area is the sum of all
these areas.

Fig. D-2

By taking just one rectangle, the area of the rectangular part Ir is

expressed by the following formula.

Ir = f1x
f2 where, x : Sampling interval
f1 : Height on left side
f1
of rectangle

Fig. D-3

A triangular part is added to this rectangle. Assuming the area

of this component is IT and the area of the triangular part is It, we
obtain the following :
f2 f1 f +f
IT = It + Ir = x + f1x = 1 2 x
2 2
Considering this with respect to the whole area, we obtain :

A -
10
 f1 f
IT = + f2 +K+ fn1 + n x
2 2

A -
11
D.2.3 Romberg The Romberg method is the most accurate of the three methods
Method discussed here. The trapezoid method provides different step
sizes (sampling intervals) for area determination with high
accuracy. In this method based on the sum of errors, two
different step sizes for individual cases are available.
However, unlike the conventional (classic) method of continuous
approximation, an arbitrary decrease in step size is not allowed
(as in case of an increase in the X-axis direction) for the purpose
of accurate integration. This is because the data points that
define the spectrum are handled as average data. Still, an
increase in step size can be made using two or four factors if this
is necessary for applying the Romberg method. The Romberg
method will take the following form.

(1) Integration is made via trapezoid method per data point.

(2) Integration is made via trapezoid method per two data

points.

(3) By combining the above two results, the following formula is

obtained.

I n I 2n
IR = In +
3
where, IR = Integration by Romberg method
In = Trapezoid integration per data point
I2n = Trapezoid integration per 2 data points

A -
12
 APPENDIX E DESCRIPTION OF FLUOROMETRY
E.1 Description of Fluorometry

Excitation

Fluorescence/
Stable stage phosphorescence
Unstable stage
(Ground state) (Excited state)

Radiationless Radiationless
transition transition

3
2
1
Excited state V = 0

Excited triplet state

Light Light
Excitation light

3
2
1
Ground state V = 0
Absorption Fluorescence Phosphorescence

Fig. E-1 Typical Organic Molecular Energy Level

Figure E-1 illustrates the energy level transitions in an organic

molecule in processes of light absorption and emission.
When light strikes an organic molecule in the ground state, it
absorbs radiation of certain specific wavelengths to jump to an
excited state. A part of the excitation (absorbed) energy is lost
on vibration relaxation, i.e., radiationless transition to the lowest
vibrational level takes place in the excited state.

A -
12
And, eventually the molecule returns to the ground state while
emitting fluorescent radiation. Also, if radiationless transition to
the triplet state takes place, then phosphorescence is emitted
during triplet-to-singlet transition (from the excited triplet state to
the ground singlet state). Generally phosphorescence persists
for 10-4 sec or longer due to the selection rule imposed on the
triplet-to-singlet transition. In contrast, fluorescence persists for
a period of 10-8 to 10-9 sec in most cases.

As mentioned above, part of the radiation absorbed by the

substance is lost as vibration energy, etc.; therefore, the
fluorescence wavelength emitted from it is longer than the
excitation wavelength (Stokes law).
The ratio of the number of photons emitted during fluorescence
to the number of photons absorbed is called the quantum
efficiency of fluorescence. The larger the quantum efficiency a
substance has, the more fluorescence it emits. Also, the
intensity of florescence emitted from a substance is proportional
to the quantity of light absorbed by it. When a dilute solution
sample is measured, the intensity of fluorescence is expressed
by
F = KI0cl
where,
F : Intensity of fluorescence
K : Instrumental constant
I0 : Intensity of exciting radiation
c : Concentration of substance
l : Cell path length (distance in the cell through which the
radiation travels)
: Absorptivity of substance
: Quantum efficiency of substance

A -
13
E.2 Advantages of Fluorometry

As contrasted with fluorometry, absorptiometry for a

low-concentration sample is explained in the following :
A sample having 99% transmittance to blank is taken as an
example. In the absorbance measurement of such a substance,
inaccuracies must always be taken into consideration.
Here, the inaccuracy is assumed to be 0.1%.
Since it has an effect on both the blank and sample,
Percent transmittance of blank 100.0 0.1%
Percent transmittance of sample 99.0 0.1%
Difference (proportional to 1.0 0.2%
concentration of sample)

In this example, the uncertainty in concentration measurement is

20%. While in the fluorometry, a difference from zero level
corresponds to the concentration of sample.
Accordingly, measurement accuracy is as follows :
Output signal level at measurement of sample 100 0.1
Value corresponding to blank 0 0.1
Difference (proportional to 100 0.2
concentration of sample)

As can be seen from the above, the fluorometry is very

advantageous for analyzing a low-concentration sample since its
uncertainty is in most cases theoretically independent of the
concentration of sample. Although actual practice may involve
some error factors which increase as the concentration of
sample becomes extremely low, the fluorometry is capable of
measuring low concentration with an accuracy 100 times higher
than in absorptiometry.
Figure E-2 is an explanatory illustration of the foregoing
description. In absorptiometry, a difference between the
quantity of incident radiation Io and the quantity of transmitted
radiation is represented by signal Is. A level at which the signal
Is becomes almost equal to a noise level is used as a detection
limit.
In fluorometry, however, since the quantity of fluorescence IF
itself is represented as a signal, just a small amount of
fluorescence can be amplified electrically for enabling detection.
Still more, since a fluorescence wavelength of a substance is
different from its excitation wavelength (incident light
wavelength), the fluorescence wavelength is not readily affected
by the exciting radiation, thereby contributing to ensuring high
sensitivity.

A -
14
High Low High Low
concentration concentration concentration concentration

Amplified
Amplified

(a) Absorptiometry (b) Fluorometry

Fig. E-2 Comparison between Absorptiometry and Fluorometry

In addition to high sensitivity, fluorometry is advantageous in that

more information is attainable. A fluorescence spectrum is also
available besides an excitation spectrum which corresponds to
an absorption spectrum in absorptiometry.
The two kinds of wavelengths can be selected as desired, and a
fluorescence spectrum can be recorded using a properly
selected excitation wavelength (or vice versa). Thus,
quantitative and qualitative analyses can be made for a sample
containing plural components.
Figure E-3 shows a simplified spectral graph of measurement of
a sample containing multiple components. In absorptiometry,
since only the absorption spectrum can be measured, two or
more component wavelengths are presented. If the absorption
wavelengths are similar to each other, each component cannot
be separated in measurement. In fluorometry, even if the
absorption wavelengths are similar, a difference in fluorescence
makes it possible to select each fluorescence wavelength
properly. Thus, each component can be separated in
measurement.

Absorption spectra Excitation spectra Fluorescence spectra

Component A
Component A Component B

Component B

(a) Absorptiometry (b) Fluorometry

Fig. E-3 Measurement of Mult-Component Sample

A -
15
 Table E-1 compares information attainable in absorptiometry and
that in fluorometry.

Table E-1 Comparison of Information Attainable in

Absorptiometry and Fluorometry
Absorptiometry Fluorometry

Absorption spectrum only Excitation spectrum

(Corresponding to excitation Fluorescence spectrum
spectrum in fluorometry)

E.3 Notes of Fluorescence Analysis Measurement

E.3.1 Temperature For most kinds of samples, an increase of 1C in the

Dependency of temperature of sample causes the intensity of fluorescence to
Fluorescence decrease by 1 to 2%. It is also reported that for some kinds of
Intensity biochemical samples, the intensity of fluorescence decreases as
much as 10% as the temperature increases by 1C.
When analyzing a sample having a temperature-dependent
property, it is advisable to use the constant-temperature cell
holder (P/N 650-0150). Constant-temperature measurement
can be carried out by circulating constant-temperature water
through this cell holder.

E.3.2 Chemical Some kinds of samples may be susceptible to a chemical

Change in change due to exciting radiation. In analysis of such a sample,
Sample due to keep the shutter closed to cut off an excitation beam until
Radiation measurement is started, and then open the shutter immediately
before measurement. If any chemical change due to exciting
radiation is observed still, determine a signal level at the start
time through extrapolation according to variation in signal level.

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16
E.3.3 Raman In fluorescence measurement, spectra having different natures
Scattering from that of fluorescence may be observed. These are called
Rayleigh scattering spectrum and Raman scattering spectra; the
former appearing at the same wavelength position as the
excitation spectrum, and the latter appearing at the
longer-wavelength side near Rayleigh scattering.
In a fluorescence spectrum, when the excitation wavelength is
shifted, only the peak height is changed while the peak
wavelength position remains intact. In a Raman scattering
spectrum, when the excitation wavelength is shifted, the peak
wavelength position is also changed accordingly. Both the
Rayleigh scattering and Raman scattering are caused by a
solvent which may be contained in the sample.
When examining the spectral plot, be careful not to mistake
these scattering effects of the fluorescence peak of interest.
Table E-3 presents the Raman spectral peak position at each
excitation wavelength for the purpose of reference.

Relative intensity

Raman scattering

Excitation wavelength

Fig. E-4 Raman Spectrum of Water

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17
 Table E-3 Raman Peak Positions at Respective Excitation
Wavelengths

(Excitation Carbon
Water Ethanol Cyclohexane Chloroform
wavelength) Tetrachloride
Excitation 248 271 267 267
wavelength 313 350 344 344 320 346
and
Raman 365 416 405 408 375 410
peak 405 469 459 458 418 461
position
(nm) 436 511 500 499 450 502

E.3.4 Handling of In measurement of a high-concentration sample, a variety of

High- error factors may be involved. The most significant error factor
Concentration consists in that an excitation beam is absorbed at the entrance of
Samples a cell to prevent a sufficient level of excitation at the center of
cell.
Figure E-5 illustrates an extreme case of this condition.
Although fluorescence is emitted in the vicinity of the entrance
for the excitation beam, it is not taken into the emission
monochromator.

Excitation beam

Fluorescence

Fluorescence is reflected here.

Fig. E-5 Sample Having an Extremely High Concentration

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18
 If only the incident point of excitation beam is bright, it is
necessary to dilute the sample properly for measurement.
The second significant error factor consists in extinction due to
concentration. This condition is caused by preventing activation
through interaction of molecules.
The third significant error factor consists in re-absorption of
fluorescence. As shown in Figure E-6, this condition occurs
due to overlapping between the shot-wavelength side of
fluorescence spectrum and the long-wavelength side of
excitation spectrum. Therefore, it seems that the fluorescence
spectrum has been shifted toward the long-wavelength side to
some extent.
In measurement of an ordinary kind of sample, however, this
condition will not impede quantitative determination significantly.

Excitation spectrum
Fluorescence spectrum

Relative intensity

Wavelength
Re-absorption occurs here.

Fig. E-6 Explanatory Illustration of Re-absorption

In any case, if there is a possibility of a measurement error due

to high concentration of a sample, dilute the sample properly or
carry out surface fluorescence measurement using a solid
sample holder.

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E.3.5 Second-Order Where the excitation and emission wavelengths are plotted near
Scattered each other, care should be exercised not to mistake the Raman
Radiation and Rayleigh scattering for the fluorescence spectrum as
mentioned in E.3.3. Where the excitation and emission
wavelengths are plotted apart from each other, care should be
exercised not to mistake the second-order and third-order
scattered radiations for the fluorescence spectrum.
The second-order scattered radiation appears at a wavelength
two times longer than the excitation wavelength, and the
third-order scattered radiation occurs at a wavelength three
times longer.
For instance, if the excitation wavelength is 240 nm, the
second-order and third-order radiations take place at 480 and
720 nm, respectively. For eliminating these scattered radiations,
insert a short-wavelength cutoff filter in the path of fluorescing
radiation (before the emission monochromator). It is advisable
to use the filter set (P/N 650-0157) which is available as an
optional accessory.

E.3.6 Contamination Since the fluorescence spectrophotometer provides high

of Cell sensitivity, just a slight amount of contamination on a cell may
have an adverse effect on results of measurement. To prevent
this, treat the cell properly after its use.
Do not leave the cell containing say sample. In evaporation of a
solvent, a residue of sample may adhere to the wall of the cell to
cause contamination.
In measurement of a very dilute sample, contamination on the
inner and outer walls of the cell may cause a problem. If a
droplet of sample solution is put on the outer wall of the cell in
sample injection into it, wipe off the cell with tissue paper and
then set it on the cell holder.

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E.3.7 Measurement Figure E-7 shows a measurement example of fluorescence
Example of spectrum.
Fluorescence
Spectrum

(1)

(4) (5)
(2)
Relative intensity

(3)

EX EX 2 Wavelength
(1) Scattering of exciting radiation
(2) Raman spectrum of solvent
(3) Fluorescence of impurities, solvent, etc.
(4) Fluorescence of sample
(5) Second-order spectrum of exciting radiation

Fig. E-7 Measurement Example of Fluorescence Spectrum

As shown in Figure E-7, other peaks than a fluorescence peak of

sample appear in fluorescence spectral measurement.
With reference to this example, it is necessary to identify a
fluorescence peal of sample.

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 APPENDIX F MEASUREMENT OF INSTRUMENTAL
RESPONSE (CORRECTED SPECTRA)

Spectrum correction is performed to enable measuring a true

spectrum by eliminating instrumental response such as
wavelength characteristics of the monochromator or detectors.
The measurement of instrumental response is needed to perform
spectrum correction. Instrumental Response is the function to
measure and save the instrumental response.

F.1 Measurement of Instrumental Response on Excitation Side

This is the function to obtain the instrumental response on the

excitation side such as wavelength characteristics of the
excitation monochromator using Rhodamine B as a standard
(quantum counter).
The instrumental response is automatically read with a single
wavelength scan operation. A spectrum is correctable within a
range of 200 to 600 nm.

(1) Handling of Rhodamine B

Pour Rhodamine B into a triangular cell in the procedure

illustrated in Fig. F-1. The triangular cell filled with
Rhodamine B should in principle be stored at a dark place.

Syringe F649090

Rhodamine B

Cut the supplied Suck the solution Open the cover Fill the cell with the
ampoule of into a syringe. of triangular cell solution in a volume
Rhoda mine B and pour the at least half the
with a cutter. solution into it. capacity and close
the cover.
Fig. F-1 Handling of Rhodamine B

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22
(2) Operating Procedures

(a) Set PMT Voltage to 400 V and Data Mode to

Fluorescence. Perform Zero Adjust to calibrate
the zero point.

(b) Set the triangular cell filled with Rhodamine B into the
cell holder in the sample compartment. (See Fig.
F-2).
The triangular cell must be set in the direction shown
in Fig. F-3.

Triangular cell with metal

fitting 650 to 1640.
(filled with Rhodamine B)

C915758

Fig. F-2 Setting of Rhodamine B

Excitation light

Emission

Metal part

Triangular cell

Fig. F-3 Setting Direction of Triangular Cell (Top view)

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23
(c) Select EX of Instrumental Response: Utility.
EM wavelength is set at 640 nm and the instrument
starts EX wavelength scan (from 198.6 nm to
601.4 nm).
At this stage, the instrument is automatically set in the
following conditions.

Slit (EX/EM) ...........5.0/20.0 nm

Scan speed............60 nm/min
Scan mode.............Fluorescence
PMT Voltage ..........400 V
Response...............Auto

(b) Upon completing the measurement, all of measuring

conditions are set to those set before.
Now, instrumental response on the excitation side has
been measured and stored. Take out the triangular
cell filled with Rhodamine B.

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24
F.2 Measurement of Instrumental Response on Emission Side

This is the function to obtain the instrumental response on the

emission side such as wavelength characteristics of the emission
monochromator and detector (photomultiplier).
The instrumental response on the emission side is determined by
measuring a combination of instrumental response on both the
excitation and emission sides by synchronous wavelength scan,
and dividing it by the instrumental response on the excitation side
preliminarily measured. An emission spectrum is correctable
within a range of 200 to 600 nm.

(1) Operating Procedures

(a) Set PMT Voltage to 400 V and Data Mode to

Fluorescence.
Perform Zero Adjust to calibrate the zero point.

(b) Set the diffuser into the cell holder in the sample
compartment. (See Fig. F-4).

Diffuser
650-1576

C915759

Fig. F-4 Setting of Diffuser

NOTE: Measurement of instrumental response on the

excitation side must be completed before
executing a measurement of instrumental
response on the emission side.

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25
(c) Select EM (200 to 600 nm) of Instrumental
Response: Utility.
EM wavelength is set at 198.6 nm and the instrument
starts synchronous wavelength scan (from 198.6 nm
to 601.4 nm).
At this stage, the instrument is automatically set in the
following conditions.

Slit (EX/EM) ......... 5.0/20.0 nm

Scan speed.......... 60 nm/min
Scan mode .......... Fluorescence
PMT Voltage ........ 400 V
Response ............ Auto

(d) Upon completing the measurement, all of measuring

conditions are set to those set before.
Now, instrumental response on the emission side has
been measured and stored. Take out the diffuser.

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26
F.3 Measurement of Instrumental Response on the Long Wavelength Range
of Emission Side (only with Model F-4500)

When a corrected spectrum in the long wavelength area of

600 nm is required on the emission side, the beam of a lamp for
which the wavelength characteristics are known is allowed to be
irradiated to the emission optics, and the instrumental response
in the long wavelength range on the emission side is read for the
correction.
As a light source, a sub standard light source (P/N 250-0124 or
250-0125) is used. The correction range by it is 500 to 900 nm
and, when combined with an instrumental response in the short
wavelength range, a spectrum correction is available over the
entire wavelength range (200 to 900 nm) on the emission side.

(1) Mounting and Positioning Sub Standard Light Source

Refer to the instruction manual for the installation of the sub

standard light source (optional accessory P/N 250-0124 or
250-0125).

(2) Operating Procedures

(a) Close the cover of the sample compartment.

(b) Set PMT Voltage to 400 V.

Perform Zero Adjust to calibrate the zero point.

(c) Turn ON the power switch of the sub standard light

source and wait for approx. 5 minutes until the lamp
stabilizes.

(d) Select EM (500 to 900 nm) of Instrumental

Response: Utility.
The instrument starts EM wavelength scan (from
498.6 nm to 901.4 nm).
At this stage, the instrument is automatically set in the
following conditions.

Slit (EM) ............... 5.0 nm

Scan speed.......... 60 nm/min
Scan Mode .......... Luminescence
PMT Voltage ........ 400 V
Response ............ Auto

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27
(e) Upon completing the measurement, all of measuring
conditions are set to those set before.
Now, instrumental response on the emission side has
been measured and stored. Remove the sub
standard light source.

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28
 INDEX

A
ASCII text file ............................................................................................................. 1-3, 6-25
Activity ....................................................................................................................... 3-16, A-6
Analog output gain ................................................................................................... 1-41, 8-10
Auto pre-scan ................................................................................................................... 1-40
Autoscale Y Axis ............................................................................................................... 7-10

B
Birds-eye view ........................................................................................................... 1-2, 5-11
Blank measurement ............................................................................................ 4-21, 8-1, 8-9

C
CAT (Computing for Averaging Transient) ....................................................................... 2-11
Calibration curve data .............................................................................................. 4-21, 4-22
Calibration curve graph ..................................................................................................... 4-22
Calibration type ............................................................................................................ 4-7, A-1
Concentration unit ............................................................................................................. 4-10
Contour ...................................................................................................................... 5-11, 5-9
Corrected spectra ........................................................................................1-26, 2-9, 5-8, A-22

D
Data menu .......................................................................................................... 2-5, 3-4, 4-12
Delay ............................................................................................................ 2-7, 3-6, 4-13, 5-6
Derivative ................................................................................................................... 4-6, 6-12
Description of fluorometry................................................................................................... A-12
Details of quantitation ......................................................................................................... A-1
Details of rate analysis function .......................................................................................... A-5
Determination coefficient ................................................................................... 4-22, A-6, A-7
Digit after decimal point .................................................................................................... 4-11
Display tab ........................................................................................................................ 1-30

E
Error message .................................................................................................................. 1-27
Event Log ............................................................................................................................ 7-7
Ex power monitor .............................................................................................................. 1-41

INDEX -
1
F
Factor input ......................................................................................................................... 4-7
Fonts tab ........................................................................................................................... 1-32

G
General tab .................................................................................................... 2-3, 3-2, 4-2, 5-3
Gradient ..................................................................................................................... 3-16, A-6

H
Help menu .......................................................................................................................... 7-8

I
Info tab .............................................................................................................................. 6-26
Installation ........................................................................................................................... 1-5
Instrument tab .............................................................................................. 2-4, 3-4, 4-11, 5-4
Instrument toolbar .................................................................................................... 1-20, 1-22
Integration method ....................................................................................................... 6-8, A-9
Integration time ................................................................................................................. 4-13
Interrupt measurement ................................................................................................ 8-1, 8-9

J
JCAMP-DX file ........................................................................................................... 1-3, 6-25

K
K factor ............................................................................................................................. 3-10

L
Lamp Off ........................................................................................................................... 1-41
Line points tab .................................................................................................................... 6-6
Luminescence ..................................................................................................... 2-5, 3-4, 4-12

M
Measurement mode ....................................................................................... 4-3, 5-3, 2-3, 3-3
Measurement toolbar ........................................................................................................ 1-20
Monitor tab ................................................................................................. 2-10, 3-8, 4-15, 5-8

INDEX -
2
N
Number of wavelengths...................................................................................................... 4-10

P
Peak area ........................................................................................................................... 4-5
Peak detection ......................................................................................... 1-3, 2-12, 3-10, 5-10
Peak height ......................................................................................................................... 4-6
Peak table .................................................................. 1-16, 2-18, 2-14, 3-12, 3-16, 5-11, 5-15
Photometry ......................................................................................................... 1-1, 4-1, 7-18
Pre-scan ........................................................................................................................... 2-16
Print Preview .......................................................................................... 2-19, 3-17, 4-24, 5-17
Processing tab ................................................................................................ 2-11, 3-10, 5-10
Processing toolbar ............................................................................................................ 1-21

Q
Quantitation tab .................................................................................................................. 4-4
Quantitation type ................................................................................................................. 4-4

R
Ratio ..................................................................................................................................... 4-7
Remeasure ................................................................................................................ 4-25, 8-4
Replicates ........................................................................................................... 2-9, 3-8, 4-13
Report tab .............................................................................................. 2-13, 3-11, 4-16, 5-10
Response .............................................................................................................. 2-8, 3-7, 5-7
Result of rate calculation .................................................................................................. 3-16

S
Sample data ...................................................................................................................... 4-21
Sample sipper ..................................................................................................................... 8-1
Sample table ............................................................................................ 2-15, 3-13, 4-18, 7-9
Saved calibration curve .................................................................................................... 4-27
Scale Y Axis Down ........................................................................................................... 7-10
Scale Y Axis Up ................................................................................................................ 7-10
Scale tab ...................................................................................................................... 6-4, 6-5
Scan speed .................................................................................................................. 2-7, 5-6
Scan tab .............................................................................................................................. 7-3
Sens. check ...................................................................................................................... 1-41
Shutter closing .................................................................................................................. 1-40
Shutter control ............................................................................................. 1-26, 2-9, 3-8, 5-8
Shutter opening ................................................................................................................ 1-40
Smoothing .................................................................................................................. 1-3, 6-11

INDEX -
3
Spectral calculation ........................................................................................................... 6-16
Spectrophotometer menu ................................................................................................. 1-40
Spectrum toolbar ..................................................................................................... 1-20, 1-22
Stack ................................................................................................................................. 7-11
Standard ........................................................................................................................... 4-21
Standard deviation ............................................................................................ 1-2, 4-13, 4-23
Standard toolbar ...................................................................................................... 1-19, 1-21
Standards tab ................................................................................................................... 4-14
Statistical calculation ................................................................................................. 1-2, 4-23

T
Threshold ..........................................................................................2-12, 3-10, 4-5, 5-10, 6-8
Tile .................................................................................................................................... 7-11
Time scan ........................................................................................................... 1-1, 4-1, 1-17
Toolbar .............................................................................................................................. 1-19

U
User Notes .......................................................................................................................... 7-7

V
View menu ........................................................................................................................ 7-10

W
Wavelength drive system .................................................................................................. 1-11
Wavelength scan ................................................................................................ 1-16, 2-1, 6-1
Wavenumber ...................................................................................................................... 6-5

X
Xenon lamp ....................................................................................................................... 1-11

Z
Zero adjust ........................................................................................................................ 1-40

INDEX -
4