PIN 220-92340-00

)
RF-5301PC
Personal Fluorescence
Software
Instruction Manual
Revision 1.0
)
§ SI-IIMAOZU
Shimadzu Scientific Instruments, Inc.
7102 Riverwood Drive
Columbia, MD U.S.A. 21046-2502
41 0/381 -1227
Table of Contents
First Edition: April 1995
SHJMADZe SCIENTIFIC iNSTRUMENTS, L'Ic. (SSI) PROVIDES TillS PuBUCATION "As Is" WITHOUT WARRANTY OF ANy
KIND, EITHER EXPRESS OR WPUED, iNCLUDING, BUT NOT LIMITED To, THE WPUED WARRANTIES OF MERCHANT­
ABILITY OR FITNESS FOR A PARTICULAR PuRPOSE. Some states do not allow disclaimer of express or implied
warranties in cenain transactions, therefore this statement may not apply to you.
Any technical inaccuracies, omissions, or typographical errors in this publication will be corrected as soon as
feasible. Changes are periodically made to the information herein and incorporated into new editions of the
publication. SSi may make improvements and/or changes in the produet(s) and/or the prograrn(s) described in
this publication at any time.
It is possible that this publication may contain reference to, or information about, SSi products (instruments
and programs), programming, or services that are not announced in your country. Such references or
information must not be construed to mean that SSi intends to announce such products, programming, or
services in your country.
This document and the software described in it are furnished under license from SSi and may be copied only
in accordance with the terms of such license.
Address any comments to: Shimadzu Scientific Instruments, Inc., 7102 Riverwood Drive, Columbia, MD
21046. SSi may use or distribute any of the information which you supply in any way it believes appropriate
without incurring any obligations whatsoever.
The following registered trademarks are used throughout this document:
IBM, PC DOS and PS/2 are registered trademarks of International Business Machines,
Inc.
Microsoft and MS-DOS are registered trademarks and Windows is a trademark of
Corporation.
PopUp Scan
Thl
is a trademark of Shimadzu Scientific Instruments, Inc., U.S.A.
SpeedBox'" is a registered trademark of Shimadzu Scientific Instruments, Inc., U.S.A.
© Copyright 1995 Shimadzu Scientific Instruments, Inc., U.S.A.
ii
1. IntrOduction 1
2. Installation
) ) 2.1 Inspection "",,, .. ,, " .. " " .. "" .. "." .. """"".""""." "" .. ""."."."." 2-1
2.2 Personal Computer Hardware and Software Requirements "." 2-1
2.3 Hardware Installation ""."".""" "."" " "." 2-2
2.3.1 Personal Computer Requirements " " 2-2
2.3.2 Cable Connections " .. "" 2-2
2.4 Software Installation "" """ " .. 2-2
2.5 Communications and Printer Setup " 2-3
3. RF-5301 PC Tutorial 3-1
3.1 Initialization " " " .. "". " " " " ". ".""" " .. 3-1
3.3 Push Buttons " "" " "" .. 3-2
3.4 Graph Pop-Up Menu " "." " 3-3
3.5 Spectrum Scan (Instrument is ON) " .. "" " " .. ""." .. 3-4
3.6 Quantitative (Instrument is ON) " " " " "."" 3-10
3.7 Time Course (Cell holder is empty) "" " "" "" .. 3-14
4. Software Overview 4-1
4.1 Default Parameters 4-1
4.2 Data Acquisition Modes " " .. " .. " " " " "" ""." 4-1
4.3 Main Menu ""." " "".".. ""."."."."""""." ".".".""."."." 4-2
4.3.1 File Menu " 4-2
4.3.2 Acquire Mode Menu " 4-3
4.3.3 Configure Menu " 4-3
4.3.4 Manipulate Menu " " """ 4-4
4.3.5 Presentation Menu " " .. " "".. " .. " " " 4-5
4.3.6 Help Menu " 4-7
4.4 Status Bar .. " .. " " .. " " " .. """"" .. "" .. " "." .. "" " 4-7
4.5 PopUp Scan™ Push Button "" .. " " " .. "" " 4-8
4.6 SpeedBox® "." "".".. " .. " " "" "." .. " "." .. " 4-9
4.7 The Graph Cursor " " .. """"" " "" ",, 4-9
4.8 Clipboard .. " "". "."." ""."."."" " "."."",," 4-10
4.9 International Support .. "" " " .. "" " """ "." 4-10
5. Menu Commands 5-1
5.1 File Menu " " " " """"""".". 5-1
)
5.1.1 Open " " " "" " " " " 5-1
5.1.2 Save ".""."""" " " " .. ,, 5-3
5.1.3 Save as " " 5-4
5.1.4 Delete " " 5-5
5.1.5 Data Translation " 5-6
5.1.6 Channel 5-7
5.1.7 Exit " 5-11
iii
5.2 Acquire Mode Menu 5-12
Appendices
5.2.1 Spectrum Mode 5-12
5.2.2 Quantitative Mode 5-15
Appendix 1 Fundamentals of Fluorescence Analysis A-3
5.2.3 Time Course Mode 5-19
1.1 What is A-3
5.3 Configure Menu 5-22
1.2 Principles of Fluorescence A-3
5.3.1 Parameters 5-22
\ 1.3 Three Basic Laws of Fluorescence A-4
5.3.2 PopUp Scan Parameters 5-32
5.3.3 PC Configuration 5-33
1.4 Advantages of Fluorescence Analysis A-5
5.3.4 SpeedBox Configuration 5-35
1.4.1 High Selectivity A-5
5.3.5 Instrument 5-37
1.4.2 High Sensitivity A-6
5.3.6 Attachments 5-40 1.5 Considerations for Fluorescence Measurement A-6
5.3.7 Save Parameters 5-41 1.5.1 Sample Temperature Effect A-6
5.3.8 Load Parameters 5-41 1.5.2 Photochemical Reaction of Samples A-6
5.4 Manipulate Menu 5-43 1.5.3 Fluorescence from Impurities A-6
5.4.1 Arithmetic 5-44
1.5.4 Effects of Scattered Light A-7
5.4.2 Transforms 5-45
1.5.5 High Concentration Samples A-8
5.4.3 File/Chnl Calc 5-47
1.5.6 Effects of Cell Contamination A-8
5.4.4 Data Print 5-49
1.5.7 Effect of Dissolved Oxygen A-8
5.4.5 Peak Pick 5-53 1.6 Sample Measurement of a Fluorescence Spectrum A-9
5.4.6 Point Pick 5-56 1.7 Excitation Spectrum A-9
5.4. 7 Peak Area 5-60
1.8 Fluorescence Calibration CUNe A-10
5.4.8 Average (Quantitative Unknown only) 5-62
5.4.9 Working Curve (Quantitative Standard only) 5-62 Appendix 2 Spectrofluorometer Overview A-12
5.5 Presentation Menu 5-63
5.5.1 Channel Status 5-63
D 5.5.2 Graph 5-65
5.5.3 Hide All 5-69
5.5.4 Show SpeedBox 5-70
5.5.5 Plot 5-70
5.5.6 Int/Conc Toggle (Quantitative mode only) 5-74
5.5.7 Display Equation (Quantitative mode only) 5-74
5.6 Graph Pop-Up Menu 5-76
5.6.1 Copy 5-76
5.6.2 Cross Hair 5-76
5.6.3 Fonts 5-76
5.6.4 Limits 5-76
5.6.5 Line Colors 5-76
5.6.6 Options 5-76
5.6.7 Radar 5-76
5.7 Help 5-77
5.7.1 Help for RF-5301 PC 5-77
5.7.2 How to Use Help 5-77
5.7.3 About RF-5301 PC 5-77
6. Troubleshooting 6-1
6.1 Communication Problems 6-1
6.2 Software Operational Error Messages 6-2
6.3 Precautions On Memory Usage 6-8
iv
v
Figure 2-1
Figure 2-2
Figure 2-3
Figure 2-4
Figure 3-1
Figure 3-2
Figure 3-3
Figure 3-4
Figure 3-5
Figure 3-6
Figure 3-7
Figure 3-8
Figure 3-9
Figure 3-10
Figure 3-11
Figure 3-12
Figure 3-13
Figure 3-14
Figure 3-15
Figure 3-16
Figure 3-17
Figure 3-18
Figure 3-19
Figure 3-20
Figure 4-1
Figure 5-1
Figure 5-2
Figure 5-3
Figure 5-4
Figure 5-5
Figure 5-6
Figure 5-7
Figure 5-8
Figure 5-9
Figure 5-10
Figure 5-11
Figure 5-12
Figure 5-13
Figure 5-14
Figure 5-15
Figure 5-16
Figure 5-17
Figure 5-18
List of Figures
PC Configuration Parameters Dialog Box 2-4
Printer Setup Dialog Box 2-4
Save Parameters Dialog Box 2-5
Initialization Window 2-5
Initialization Window 3-1
Application Window of the RF-5301PC Software 3-2
Spectrum Push Button Menu 3-2
Graph Pop-up Menu on Main Screen 3-3
Spectrum Parameters Dialog Box 3-4
File Name Dialog Box 3-5
Spectrum Screen after Data Collection 3-5
Radar Cascading Menu 3-6
Graph Showing Cross Hair 3-7
Plot Layout Window 3-7
Plot Data Dialog Box 3-8
Plot Layout Preview Window 3-9
Quantitative Parameters Dialog Box 3-10
Multipoint Working Curve Dialog Box 3-11
Edit Standard Dialog Box 3-12
Quantitative Screen after Standard is Entered 3-12
Save Channel Dialog Box 3-13
Time Course Parameters Dialog Box 3-14
D
Set Limits Dialog Box 3-15
Time Course Screen after Noise Plot 3- 15
PopUp Scan Window and Menu 4-8
File Menu 5-1
Open Dialog Box 5-2
Save as Dialog Box 5-4
Delete Dialog Box 5-5
Delete this file? Dialog Box 5-5
ASCII / DIF Export Dialog Box 5-6
Save Channel Dialog Box (Spectrum / Time Course modes) .. 5-8
Save Channel Dialog Box (Quantitative mode) 5-8
Erase Channel Dialog Box 5-10
Rename ChanneL Dialog Boxes 5-10
Acquire Mode Menu on the Spectrum Screen 5-12
Spectrum Push Buttons 5-13
Wavelength Selection Dialog Box 5-14
Search Optimal Wavelength Dialog Box 5-14
Quantitative Screen 5-16
Quantitative Push Buttons 5-16
Edit Standard Dialog Box 5-18
Edit Unknown Dialog Box 5-19
Figure 5-19
Figure 5-20
Figure 5-21
Figure 5-22
Figure 5-23
Figure 5-24
Figure 5-25
Figure 5-26
Figure 5-27
Figure 5-28
Figure 5-29
Figure 5-30
Figure 5-31
Figure 5-32
Figure 5-33
Figure 5-34
Figure 5-35
Figure 5-36
Figure 5-37
Figure 5-38
Figure 5-39
Figure 5-40
Figure 5-41
Figure 5-42
Figure 5-43
Figure 5-44
Figure 5-45
Figure 5-46
Figure 5-47
Figure 5-48
Figure 5-49
Figure 5-50
Figure 5-51
Figure 5-52
Figure 5-53
Figure 5-54
Figure 5-55
Figure 5-56
Figure 5-57
Figure 5-58
Figure 5-59
Figure 5-60
Figure 5-61
Figure 5-62
Figure 5-63
Make Standard DiaLog Box 5-19
Time Course Screen 5-20
Time Course Push Button Menu 5-20
Configure Menu 5-22
Spectrum Parameters Dialog Box 5-23
Repeat Scan Parameters Dialog Box 5-24
Repeat Scan Mode Window 5-25
Repeat Scan / Auto File Warning Messages 5-25
Auto File Dialog Box 5-26
Auto File Scan Mode Window 5-27
Quantitative Parameters Dialog Box 5-27
K-Factor Parameters Dialog Box 5-28
Single Point Working Curve Dialog Box 5-28
Multipoint Working Curve Dialog Box 5-29
Time Course Parameters Dialog Box 5-30
PopUp Scan Parameters Dialog Box 5-32
PC Configuration Parameters Dialog Box 5-33
SpeedBox Configuration DiaLog Box 5-34
Recommended SpeedBox Button Settings 5-36
Instrument Parameters DiaLog Box 5-37
SIN Ratio Check 5-38
Attachment Parameters Dialog Box 5-39
Save Parameters Dialog Box 5-40
Load Parameters Dialog Box 5-41
View Window of the Load Parameters Dialog Box 5-41
Delete Windows of the Load Parameters Dialog Box 5-42
Manipulate Menu 5-43
Arithmetic Dialog Box (Spectrum/Time Course modes) 5-44
Arithmetic DiaLog Box (Quantitative mode) 5-44
Spectrum and Time Course Transform Selection Dialog Box 5-45
Derivative Dialog Box 5-46
Quantitative Transform Selection Dialog Box 5-47
File/Channel Calculations (Spectrum / Time Course modes) 5-48
File/Channel Calculations (Quantitative mode) 5-48
Data Print Window 5-50
Data Print Parameters Dialog Box 5-51
Save Data Print Dialog Box 5-51
Activity CaLcuLation Dialog Box 5-52
Peak Pick Window 5-53
Peak Pick Window 5-54
Point Pick Dialog Box 5-57
Point Pick Window 5-58
Formulafor Peak Area 5-60
Peak Area Dialog Box 5-60
Area Output Dialog Box 5-61
vi vii
Figure 5-64 Presentation Menu 5-63
Figure 5-65 Channel Status Dialog Box 5-64
Figure 5-66 Spectrum Parameters Display Window 5-65
Figure 5-67 Spectrum Graph with a Grid 5-66
Figure 5-68 Set Limits Dialog Box 5-66
Figure 5-69 Plot Colors Dialog Box 5-67
Figure 5-70 Custom Colors Dialog Box 5-68
Figure 5-71 Options Dialog Box 5-68
Figure 5-72 Fixed vs. Rounded Tick Marks 5-69
Figure 5-73 Configured SpeedBox 5-70
Figure 5-74 Plot Layout Dialog Box (Spectrum/Time Course modes) 5-71
Figure 5-75 Plot Data Dialog Box (Spectrum / Time Course modes) 5-71
Figure 5-76 Plot Layout Dialog Box (Quantitative mode) 5-73
Figure 5-77 Plot Data Dialog Box (Quantitative mode) 5-73
Figure 5-78 Quantitative Window Showing Equation under the Graph 5-75
Appendices
Figure A-I Principles of Fluorescence A-3
Figure A-2 Spectrum of a Sodium Salicylate Aqueous Solution A-9
Figure A-3 Excitation Spectrum of Sodium Salicylate Aqueous Solution. A-I0
Figure A-4 Working Curves for Aqueous Solution of Diaminostilbene A-II
Figure A-5 Arrangement of the RF-5301PC A-12
List of Tables
Table 6-1 Communications Errors and Remedial Actions 6-1
Table 6-2 Error Messages and Remedial Actions 6-2
Appendices
Table A-I Compounds and Quantum Yield of Fluorescence A-5
Table A-2 Relationships between the Excitation Wavelength and Raman ......
Peak A-8

viii
Introduction
1. Introduction
Thank you for your purchase of the RF-5301 PC Spectrofluorometer.
The software operates under the Microsoft'" Windows'M environment, enabling
ease of use and intuitive operation. It also provides for wide-ranging device
")
support, such as graphics adapters and monitors, computers, and peripheral
devices.
The RF-530IPC software is based on a modular concept, with this main
software package providing initialization and communication functions, data
acquisition, post analysis manipulation, and flexible output.
Data is acquired through three basic modes: Spectrum, Quantitative, and Time
Course, with the software allowing control of all acquisition parameters and
storage formats. Post analysis manipulation includes arithmetic manipulation
and transformation of data, assignment to multiple file memory areas as well
as optimal data presentation on the screen. Flexible output is provided by
enabling a combination of graphs, parameters and text on a single page. On­
line help is available to facilitate operation of this software.
This manual is arranged such that the operator is first guided through the
installation and then briefly through a few typical operation sequences. This
is followed by in-depth descriptions of the various RF-5301PC software
functions. The latter part of the software instruction manual is devoted to
error messages, troubleshooting, and precautions.
It is strongly recommended that the operator proceed with the inspection and
installation of the RF-5301PC system referring closely to the manual to
ensure trouble-free start-up and operation. As part of the software
installation procedure, the user is given the option to view the README. TXT
file. This file may contain information about the software which is more up­
to-date than that provided in this manual. When the file is present, it may also
be viewed after installation using Notepad (see the Microsoft Windows
User's Guide) or it may be printed.
)
RF-5301 PC Instruction Manual 1 - 1
Installation
2. Installation
This chapter lists the minimum hardware and software requirements necessary
to use the RF-530l PC Spectrofluorometer. Also described are the steps to
connect the personal computer to the RF-530 1PC and to install the software.
2.1 Inspection
The following list describes the contents of the RF-5301PC software package.
Please compare the items in this list with the contents of the package received
and report any missing items to your Sales Representative.
Item Q!y,. Description PIN
SIW floppy disk 3.5" 220-92334-13
2 Instruction Manual 220-92340-00
3 RS-232C Communications Cable 220-92172-00
4 Shimadzu Mouse Pad 220-97120-00
5 1 Software License Notice 220-90191-00
6 Warranty Registration Card n/a
2.2 Personal Computer Hardware and Software Requirements
In addition to the RF-5301PC software, the following hardware and software
are either required, recommended, or useful.
Minimum computer hardware requirements
• IBM PC/AT or PS/2 (or Windows 3.1 compatible)
• 640KB Random Access Memory (RAM)
• 1 floppy disk drive, 3.5"
• I hard disk drive with at least 2MB free (40MB hard drive recommended)
• Windows compatible VGA graphics adapter/monitor
• Windows compatible asynchronous serial port
RF-5301 PC Instruction Manual 2 - 1
Installation
Minimum software requirements
• MS or PC Disk Operating System (DOS), Version 3.3
• Microsoft'" Windows™, Version 3.1 or later
Recommended optional accessories
• Mouse-type pointing device
• Math co-processor
• Non-fluorescent cell (lOmm) (PIN 200-34594-03)
2.3 Hardware Installation
2.3.1 Personal Computer Requirements
This software requires that an IBM compatible asynchronous port be
installed in the personal computer. A hardware interrupt must be
assigned to the communication port. By IBM standards, interrupt 4 is
assigned to either COMI or COM3, but not both. Interrupt 3 is assigned
to either COM2 or COM4, but not both. Verify that the personal
computer has a properly configured asynchronous adapter (serial port)
before continuing.
2.3.2 Cable Connections
After the instrument is positioned, the RS-232 interface cable is used to
connect the RF-5301PC to the personal computer (PC). Insert the male
cable connector into the connector labeled RS-232C on the right side of
the RF-5301PC, and secure the cable with the screws. Next, connect one
of the female connectors to the PC serial port (IBM compatibles normally
have two serial ports, COMI and COM2), and secure the cable with the
screws.
2.4 Software Installation
Before installing the RF-5301PC software, it is necessary to first install
Microsoft Windows. Please refer to the appropriate Microsoft Windows
documentation for the installation procedure. The installation procedure for
the RF-5301PC software is performed as described below.
Procedure
1. Insert the RF-5301PC floppy disk into the A: or B: drive.
2. At the C> prompt, type: WIN A[B]:SETUP and press Enter
Installation
or
in the Windows Program Manager, select Run under the File menu,
type a:[b:]\setup and click OK.
3. During installation the user will be visually notified of the progress
through screen messages and the movement of the status bar.
4. During initial installation the user will be prompted for the name of the
licensee (customer name), and the directory in which the program will be
installed; the default directory is C:\RFPC. The program will decompress
files at this point.
5. A dialog box appears asking "Would you like to view the README file
now?" Click Yes or No. (Use the Windows Notepad to read the file at a
later date. See the Microsoft Windows User's Guide for information.)
6. Upon completion of the installation process, a Program Group named
Shimadzu will be created, if not already present, and the RF-5301PC
icon will be placed in it. The user may move the icon to another group if
desired. Refer to the Microsoft Windows User's Manual for more
information.
1) ) 2.5 Communications and Printer Setup
Prior to operating the RF-5301PC software with the RF-5301PC, the
communication port and printer parameters must be configured.
Procedure
1. At the MS-DOS prompt, type: WIN and press the Enter key.
2. When the main Windows screen appears, locate the RF-5301PC icon
which may be found in the Shimadzu program group.
3. Double-click on the RF-5301PC icon to execute the program.
4. When the main screen appears (see Figure 3-2), move the cursor to
Configure on the main menu, and click the left mouse button.
5. Select (click on) PC Configuration from the Configure Menu (see
Section 5.3.3). The dialog box in Figure 2-1 appears. Press the tab
)
key twice to move from the Data Directory item to the Fluorometer
Serial Port item. Use either the arrow keys or the mouse to select the
communication (COM) port (1-4) which is used for communication
between the personal computer and the fluorometer unit.
2-2 RF-5301 PC Instruction Manual
RF-5301PC Instruction Manual 2-3


0 landscape
@ dpi
Printer Resolution
0 100 dpi
rCal!ridges/SIMhh
Printer Memmy
fage Protection:
Memory [MOj:
1011
12
il'J
III
I ps:IO
-­Resource Saving IKDj ­
tIl

o TryeType Screen Fonls Installed
I
Installation
Installation
PC Configuration Parameters 7. To ensure that the communication port and printer(s) selections will be
set whenever the program is started, select Save Parameters under
IC:\RFPC\data\
I Configure in the main menu; indicate the default file name (RFPC),
select Save and select Yes in response to the overwrite warning (see
IC:\RFPC\data\
I
Figure 2-3).
02 03 04
§raphics Printer
IHP laserJet ilion lPT2: It)
Enler File Name: IIi1ii!J -I
ItSetup·:·1 Enler Comment: IThis is the default configuration file.
IBF8·")"'1 1',;,:;t':anC'llr.jt.: ),:.' , ave, ...> ,
..
,'"" ", I; Cancel, J
Figure 2-1 PC Configuration Parameters Dialog Box
Figure 2-3 Save Parameters Dialog Box
6. Next, select the appro
the RF-530IPC syste
list. Click on the Set
item to configure the
priate printer(s) which are currently connected to
m from the Text and/or Graphics Printer drop down
up push button of the Text and/or Graphics Printer
printer(s) settings (see Figure 2-2). (The printer
8. Upon returning to the main screen after validating the above setting(s),
select Configure from the main menu. Select Instrument from the
available selections, and set the fluorometer to ON (see Instrument,
Section 5.3.5). Select OK, and the initialization procedure will begin.
Qata Directory
.f.xport Directory
fluorometer Serial P
Iext Printer
HP laserJet III on lPT
ort @1
2: lot
HP OeskJel 550C Prinler on lPT
HP Plotter on lPT1:
IBM Proorinter Xl on l
I"'" ,"'
.
PT1:
.. "I .OK",
and port designations which appear in the list box were previously
designated using Control Panel; see Text and/or Graphics Printer, Section
5.3.3)
@ffIj;lg4?i4EAIf-mf-i'tf=J:ntfitbTiFMiQ".,i"'ijii
Orientation I
w@Poitrail
Figure 2-2 Printer Setup Dialog Box


1F'."';o..."D'%. '.'. '7'j


D
)
Instrument: RF-5300
Serial Number: 00000000000001
ROM Version: 0.90


i'l EEPROM
Excitation Slit
@) Emission Slit
@) Excitation Monochromator
[ill Emission Monochromator
o Baseline
[JTesting Passed Failed
Figure 2-4 Initialization Window
RF-5301PC Instruction Manual 2-5
2-4 RF-5301 PC Instruction Manual
Tutorial
3. RF-5301 PC Tutorial
The procedures in this chapter provide direct and rapid introduction to the RF­
5301 PC software by guiding the user through the typical Spectrum,
Quantitative, and Time Course sequences of the data acquisition modes.
)
- Turn on the computer, monitor, and the RF-5301PC.
- At the MS-DOS prompt,
type: WIN and press the Enter key to start Microsoft Windows.
Double-click on the RF-530XPC icon in the Program Manager window.
• or, at the MS-DOS prompt in the RFPC directory (C:\RFPC),
type: WIN RFPC and press Enter.
3.1 Initialization
When the RF-5301PC program is executed, an initialization procedure begins
which requires approximately 30 seconds to complete. A window appears
with a list of items being tested. The status is shown next to each item. The
yellow square indicates testing, the green circle indicates passed, and the red
diamond indicates a failure (see Figure 3-i).
Instrument: RF-5300
Serial Number: 00000000000001
ROM Version: 0.90
CD ROM
~ R A M
~ EEPROM
@l Excitation Slit
~ Emission Slit
tl Excitalion Monochromator
Biil Emission Monochromator
)
o Baseline
I!J Testing ~ Passed • Failed
Figure 3-i initialization Window
RF-5301 PC Instruction Manual 3 - 1
Tutorial Tutorial
3.2 The Application Window 3.4 Graph Pop-up Menu
The application window displays the main screen of the RF-5301PC software. The Graph Pop-up Menu is accessed by pressing on the right mouse button
Various application software functions are accessed from the window using when the cursor is inside the graph area. The menu appears on the screen
either the mouse or the keyboard. where the mouse cursor is placed. Click on the main screen with the left
mouse button to remove the Graph Pop-up menu. (For more detail, see Graph
,1&1IlIIi'
)
Pop-up Menu, Section 5.6) F;fe Mode l;;onfigure Manipulate eresentation tlelp
IlIjl
eresentation t!elp
Title
Bar
Main
Menu


c
<;

Push
I) )
Buttons
Figure 3-2 Application Window of the RF-5301PC Software
3.3 Push Buttons
The RF-5301PC screen contains 7 to 8 push buttons at the bottom. Anyone of
the push buttons may be selected by clicking once with the left mouse button,
or with the keyboard-via the function keys from right to left-beginning with
the F8 key and ending with the Fl key, respectively.
./ / / /
SpeedBoxCi> Status Bar Lamp Window AutolRepeat Mode Window fluorometer Window
Figure 3-4 Graph Pop-up Menu on Main Screen
F2 F3 F4 F5 F6 F7 F8 )
Figure 3-3 Spectrum Push Buttons
3-2 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 3-3
Tutorial Tutorial
3.5 Spectrum Scan (Instrument is ON)
5) Click on the Start push button at the lower right of the screen. As the
RF-5301PC performs the Spectrum scan, the Fluorometer Window
I) Select Spectrum from the Acquire Mode menu after the displays the wavelength values and the intensity readings (see Figure 3-7).
spectrofluorometer initialization is completed.
6) When the scan is completed, the File Name dialog box appears (see
2) Select Parameters from the Configure menu to display the Spectrum Figure 3-6). In the box adjacent to Enter File Name, type in a file name
Parameters dialog box (see Figure 3-5). Set the parameters as follows ) of up to eight characters. Do not type in an extension, as this is assigned
by tabbing through the box: by default, in this case .spc. A comment may be typed into the Enter
Spectrum Type:
EM Wavelength:
Excitation
400
Comment box by tabbing to it. Select Save to save the data in the next
available Spectrum channel. When Discard is selected, the data is erased
from memory.
EX Wavelength Range: 375 (Start); 475 (End)
Recording Range: -10.000 (Low); 500.0 (High)
Scan Speed: Fast
Sampling Interval: 1.0
EX / EM Slit Width: 10 /10
Sensitivity: High
Response Time: 0.02
Repeat Scan / Auto File: No setting required at this time.
Spectrum Iype: @ Excitation o Emission o Synchronous I
D
EM Wavelength: 400 Inm
1
EX Wavelength Range:
1
375 .End
1
475
I I
Recording Range: low
1-10.000 I !::!igh 1500.000 I
S.!;anning Speed: IFast
rn
Sampling jnterval (nml:
11.0 [!I
Slit Width (nm):
[10 !I
EM 10 I
1 iii
Sensitivity: @High o Low
Res1\onse Time (sec): 02
1
0
.

I I' . ] I' .". "') I ' .!
. /8utofile... ·.. ., 01( .. Cancel' .
Figure 3-5 Spectrum Parameters Dialog Box
3) Select OK to validate the parameters. The main screen reappears. As the
I) )
parameters are sent to the instrument, the Fluorometer Window
indicates Fluorometer Setup. The EX and the EM wavelengths and the
Enter File
Entet Comment
I I
I. I I 01&",,"' I
Figure 3-6 File Name Dialog Box
,""'1 ....
475.0
:
4600
l::!elp
420.0 440.0
Wavelength (nm)

r
4000
U
Figure 3-7 Spectrum Screen after Data Collection
intensity reading are displayed in the Fluorometer Window when the
setup is completed.
4) Insert a cuvette containing distilled water into the sample holder.
3-4 RF-S301PC Instruction Manual RF-5301PC Instruction Manual 3-5
Tutorial
Tutorial
NOTE: The RF-5301 PC software maintains the Spectrum and Time
Course data in channels. A channel is an area in the personal
computer's memory (RAM) which is used to temporarily store all of
the information relevant to a particular scan. The Spectrum and Time
Course acquisition modes each contain ten channels, numbered 0-9.
Data acquired from the fluorometer is held in the next available
channel; it is not yet saved to disk. (Channel Status under the
Presentation menu shows the saved status of any active channel.) A
channel's data can be saved to disk only when the channel is assigned
a file name, and the user specifically selects Save or Save as from the
File menu or Save Channel from the Channel cascading menu under
the File menu. Also, data is automatically saved to disk when a new
acquisition mode is selected or when the program is exited after afile
name has been assigned. When no file name is specified for a channel,
there is a warning and an opportunity to assign file name(s) (see
Rename Channel, Section 5.1.6).
7)
8)
From the Presentation menu under Graph, select Radar (Both Axes) or
press Ctrl+R (see Figure 3-8).
,..tilt
440.0 420.0 400.0
Wevelet1gth(nm)
380.0

350.0 360.0

i
t
[ife Acquire Mode l.;onfigure
,
Figure 3-8 Radar Cascading Menu
To zoom in on a portion of the screen plot, use the left mouse button to
anchor a point and drag a box over that portion. (This is also possible
without a mouse, as described in Limits, Section 5.5.2.4) Use Ctrl+R to
return to the enlarged screen.
RF-5301PC Instruction Manual
9)
To see the cross hair (X, Y) coordinates of the mouse cursor position,
press the right mouse button. When the Graph Pop-up menu appears,
click on Cross Hair and then Display. As the mouse is moved, the X,Y
coordinates can be seen along the axes of the graph referencing the
intersection of the cross hair (see Figure 3-9). To remove the cross hair,
reselect the Graph Pop-up menu and click on Display.
file Acquire Mode
: J
\
Ille
.configure Manipulate eresentation tlelp
420.0 400.0
360.0
Figure 3-9 Graph Showing Cross Hair
) 30.001
20 000 )
18.400


10.000
0.000
350.0
10) To obtain a hard copy printout, choose the Presentation menu, then
select Plot to bring up the Plot Layout window (see Figure 3-10).
...-.
GUlph PaHlms.

61 0 0
File
0
0
1
I
Channel/F. Name
Screen
0
au.dr.ntls]
01 020304
01 020304
.(; I 0 0 0
I
01 0203 04
)
Q I
0 0
I. 'Printj
0
I
I, :c.nce,:j I I.'
01
J
0203
[ill]
[ill]
04
Figure 3-10 Plot Layout Window
RF-5301 PC Instruction Manual
3-6
3-7
Tutorial
11) Under the Graph column, click on the first box in row A to bring up the
Plot Data dialog box (see Figure 3-11). (If A is already checked,
uncheck it and check again.) (The second column brings up the
Parameter Channel dialog box and the third column is associated with
the Open File dialog box.)

OJ
0, 0;'
0,; 01
OJ OJ
01
I2l Jicreen o Spcctra Qnly
Graph nllc
[ I

:I r...xis
.VPfjCr: I 1 I

",nowtr: [
Figure 3-11 Plot Data Dialog Box
D
12) Select OK. When the Plot Layout box reappears, click in the box in row
B under Params and click in boxes 3 and 4 in row B under Quadrants
(see Figure 3-10). (At this point in the tutorial, there is only one channel
containing data.)
13) Select Preview in the Plot Layout box to view the layout as it will
14)
appear on the hard copy (see Figure 3-12).
15)
Tutorial
""I
lS.U':"
3Y2t32
Crnttd: 15:59 081111').1

U-S301
Tvpe: 1M
3(u lugt: 350.011111 to .SO.Onm
EX W:no"kflqt\: 350.0 11M

Slit Widt...: IX:5.0flm !M:5.()_
Vtty
";91,

:!IIOJntr: M_:oI,Opet>
Figure 3-12 Plot Layout Preview Window
Click anywhere on the screen or press any key to return to the Plot
Layout dialog box.
Select Print to begin the printout (only after the printer is connected and
configured); otherwise, select Cancel.
3-8 RF-5301PC Instruction Manual RF-530 1PC Instruction Manual 3-9
Tutorial Tutorial
3.6 Quantitative (Instrument is ON)
1) Select Quantitative from the Acquire Mode menu. The Quantitative
Parameters dialog box automatically appears (see Figure 3-13).
(Whenever the acquisition mode is changed, the Parameters dialog box
Qrder of Curve..... C> 1sl 02nd @::Jr;f
Zero Interception.. OYes @No

ICancel I
Figure 3-14 Multipoint Working Curve Dialog Box
3) Select OK to validate the parameter settings. The Fluorometer Window
shows the Fluorometer Setup and indicates slewing of the EX
wavelength to 350nm and the EM wavelength to 425nm.
4) Prepare at least four Standard samples with expected intensity values
between 0.00 and 800.00 (see NOTE which/ollows). Skip to Step 5 when
not entering "known" Standards.
NOTE: Instead of preparing Standards at this time, a "known"
Standard curve may be entered. Press the Enter key to access the Edit
Standard dialog box (see Figure 3-15). Enter the Concentration
(Cone.), tab to the Intensity (Int.) field, enter the Intensity value, then
click on OK, or press the Enter key, to record the Standard. Continue
to press the Enter key from the Quantitative table to access the Edit
dialog box until the Standard values listed below are complete.
Cone. Int.
Standard 1: 1.00 3.00
Standard 2: 5.00 15.00
Standard 3: 13.00 116.00
Standard 4: 25.00 230.00
Standard 5: 34.00 375.00
Standard 6: 43.00 490.00
Standard 7: 50.00 620.00
Standard 8: 70.00 825.00
Standard 9: 75.00 850.00
Standard 10: 80.00 875.00
Standard 11: 85.00 885.00
appears.)
2) Set the parameters as follows:
Method:
EX / EM Wavelength:
EX / EM Slit Width:
Concentration Units:
Concentration Range:
Recording Range:
Sensitivity:
Auto Scan Mode:
Response Time:
Repetitions:
t
')
(
1;- )
Multipoint Working Curve
(3rd order, no zero intercept)
Enter values into the dialog box which
appears on the screen and click OK
(see Figure 3-14).
350/425
10/ 10
nm
0.000 (Low); 100.000 (High)
0.000 (Low); 1000.00 (High)
High
Unchecked
0.02
1
Quantitative Parameters
!;oncentration Method:
IMUlti Point Working Curve Units: Inm

Range:
I 0.000 Wavelength:
1
350
.
0
1
EM Wavelength: 14 25.0
I
Recording Range
Slit Width (nm): EX
bOW 10.000
I
EM 110
@High o Low Response lime:
o Auto Scan Mode Bepetitions:
I
. I
'X < I
Cancel" I
1
1
to
1100.000 I
.!::!igh 11000.00 1
1
0
.
02
0

Figure 3-13 Quantitative Parameters Dialog Box
3 - 10 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 3 - 11
Tutorial
5)
6)
7)
8)
Standard Number
Concentration I 0.00000 I
Int. I 0.000 I
I ilK .. 1I I IQeleJel
Figure 3-15 Edit Standard Dialog Box
With the sample compartment empty, or with a cuvette containing solvent
in the sample holder, select the Auto Zero push button at the bottom of
the screen (or the F5 key) to zero the instrument.
Place the first Standard into the sample compartment. Select the Read
push button or press the F7 key, and the Edit Standard dialog box
appears.
Enter the concentration value for the first Standard and click on OK or
press the Enter key.
Repeat steps 6 and 7 for the second, third and fourth Standards. If the
Standard curve was entered from the table as in the previous
NOTE, the screen will look similar to Figure 3-16.
flil.
Elle Acquire Mode l;onfigurc Manipul8te fre.entation tjelp
Tutorial
9) The Unknown data can now be read. Select the Unknown push button or
the F2 key.
10) Place an Unknown in the sample compartment and select the Read push
button (or the F7 key) for each Unknown to be read.
11) When reading the Unknowns is complete, select Save Channel from the
" )
Channel cascading menu under the File menu. The Save Channel dialog
box opens, as shown in Figure 3-17. Click on the Standard check box.
In the box adjacent to File Name, type in a file name consisting of up to
eight characters. Do not type in an extension, as this is assigned by
default (in this case .std). A comment may be typed into the Comment
box by tabbing to it. Click on the Unknown check box and enter a file
name and comment as above (.qnt will be the extension). Select OK to
save the files.
NOTE: Using the Save as function does not allow for comments to be
entered.
I:8J 0 Clear after Save
File Name: ITESTSTD I
Comment:
1__­
o Clear after Save
I:8J
File Name: IUNKTST I
Comment:
I I
. OK .Status
,. ·.· 8"'·""'"

El
.,., ,""
Figure 3-17 Save Channel Dialog Box
)
SlMd¥d
1iXlJOOO
8OO.iXlJ
> 600iXlJ
i
1: 4OO.iXlJ
4O.iXlJ 60.iXlJ 80.000 l00.iXlJ 20 iXlJ
rm
Int = k3 C'3 • k2 C'2 t kl C • kO
k3 ·0.00229 k2 - 0.260
kl =4.868 kO ='4.607
Chi-Square: 21.73510
St

Standard
Humber Cone. I nt.
1 1.0000 3.000
2 5.0000 15.000
3 13.000 116.000
4 25.000 230.000
5 34.000 315.000
b 43.000 490.000
1 50.000 620.000
8 10.000 825.000
9 15.000 850.000
10 80.000 815.000
11 85.000 885.000
Figure 3-16 Quantitative Screen after Standard is Entered
3 - 12 RF-5301PC Instruction Manual RF-530 1PC Instruction Manual 3 - 13
Tutorial
3.7 Time Course (Cell holder is empty)
1) Select Time Course from the Acquire Mode menu. The Time Course
Parameters dialog box automatically appears for review (see Figure 3-18).
(Whenever the acquisition mode is changed, the Parameters dialog box
appears.)
2) Set the parameters as follows:
EX / EM Wavelength: 350/397
Recording Range: -100.000 (Low); 1000.00 (High)
EX / EM Slit Width 10/ 10
Sensitivity: High
Response Time: Auto
Reaction Time: 60.0
Timing Mode: Auto
Units: Seconds
I Recording Range
Wavelength:
Low: 1-100.000 I tligh: 11000.00 I
EM Wavelength:
Slit Width (nmJ: EX 110 I:!J EM 110m
=--­
.5.ensitivity: 0 Low RJ;sponse Time: IAuto Ii)
,Timing
Beaction Time:

I @ AUlD 0 Manual I o HnIH"


o Mi"u!c!<
H! rtf.: 3001
1 I @ Seconds
,

I
Figure 3-18 Time Course Parameters Dialog Box
3) Select OK to validate the parameter settings. The Fluorometer Window
shows the Fluorometer Setup and indicates slewing of the EX
wavelength to 350nm and the EM wavelength to 397nm.
4) Select the Auto Zero push button to zero the instrument.
5) Select Start to plot the noise level graph.
6) When the plot is completed. the File Name dialog box appears. In the
box adjacent to Enter File Name, type a file name consisting of up to 8
characters. Do not type an extension, as this is assigned by default (in
Tutorial
this case .tmc). A comment may be typed in the Enter Comment box by
tabbing the cursor to this box. Select Save to save the data in the next
available Time Course channel.
7) Select Limits from the Graph cascading menu under the Presentation
menu (see Figure 3-19).
)
Graph Limits
X Axi.------, 'Y Axis-------,
M IIIlIiIllD I "'-ax: LI6"'0"'0."'0_----'
Min I Min: I·21.345 I
I", (lie. -I F::: .!lese' I Ir Cancel I
Figure 3-19 Set Limits Dialog Box
Enter -5.0 and 5.0 for the Y-axis minimum and maximum values,
respectively. (See Limits, Section 5.5.2.4; The reset values originate
from those entered in the Parameters dialog box.) Select OK or press
the Enter key. The screen will look similar to that shown in Figure 3-20.
...
file Acquire konfigure Manipulate .eresent81ion tielp
-, ;'.11111;
f
1:
.,
This completes the tutorial portion of the instruction manual. Since the tutorial is
)
meant only as a brief hands-on guide through the basic procedures, please refer to
the following chapters for in-depth instruction on the many features available in the
RF-530 I PC software.
Figure 3-20 Time Course Screen after Noise Plot
3 - 14 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 3 - 15
Software Overview
4. Software Overview
This section describes critical features of the RF-530IPC software and gives
brief descriptions of each of the RF-530IPC menu items.
4.1 Default Parameters
When the program is executed the RFPC.CFG configuration file is read, and
the saved parameters become the current configuration parameters. Select
Configure to make any changes to these parameters (see Configure, Section
5.3). Different sets of parameters may be saved in different files. Any of these
configuration files may be loaded to replace the current default parameters.
When the program is executed, a specific configuration file may be loaded by
typing in the name of that file (e.g., WIN RFPC FlLENAME.CFG).
4.2 Data Acquisition Modes
The three modes in which data can be acquired are Spectrum, Quantitative
and Time Course. They are selected from the Acquire Mode menu. In
Spectrum and Time Course acquisition modes, up to 10 scans may be saved in
temporary memory channels at one time. In Quantitative acquisition mode, up
to 20 standards and 400 unknowns may be saved at one time.
When changing acquisition modes, the channels that contain a file name and
which have not already been saved will be saved to disk. A warning will be
issued for those channels without a file name, reminding you that not all data
has been saved. When this is ignored, the data will be lost. To save any of the
No Name data channels, it is necessary to first provide a file name (see File
menu, Rename Channel, Section 5.1.6.3) before changing acquisition modes.
When a file has been modified, it is no longer saved and it will be re-saved
when changing acquisition modes or exiting the program. A warning will be
issued before any disk file is overwritten (modified). To avoid overwriting
the file, it is necessary to use either Rename Channel or Erase Channel in
the File menu to change the file name or to delete the modified data.
When the acquisition mode is changed, the currently loaded configuration file
is updated to contain the new acquisition mode. Upon exit the program saves
the last-used acquisition mode, and returns to that mode when next executed.
The parameters, however, must explicitly be saved via Save Parameters.
RF-5301PC Instruction Manual 4 - 1
Software Overview
4.3 Main Menu
4.3.1 File Menu
The File menu contains items concerned with channel and disk file
management. (In Quantitative mode, the standard data and unknown data
are maintained in separate channels.) The menu items include:
Open
Used to load previously acquired and stored data into channels. A file's
parameters may be previewed before loading.
Save
Performs a quick save to disk of the data contained in the channels.
Save As
Saves a channel to disk while changing the file name and/or directory
associated with the channel.
Delete
Deletes a file from a specified directory.
Data Translation
ASCII / DIF Export: Converts a data file into ASCII (American
Standard Code for Information Interchange) or DIF (Data Interchange
Format) format.
Channel
Save Channel
Saves individual channels to disk.
Erase Channel
Deletes the data from one or more channels.
Rename Channel
Modifies the file name and/or comment assigned to a particular
channel, or assigns a file name to a channel that originally was not
assigned a name. Data is not saved to the disk at this time.
Exit
Exits the RF-5301PC program.
Software Overview
4.3.2 Acquire Mode Menu
This menu enables selection of the data acquisition modes which include:
Spectrum Mode
Establishes a mode in which a spectrum is acquired over a designated
wavelength range.
Quantitative Mode
Establishes a mode in which the relative fluorescence intensity at fixed
excitation and emission wavelengths is measured for individual samples.
These values are used to calculate concentration by means of a standard
curve.
Time Course Mode
Establishes a mode in which the relative fluorescence intensity is
measured at fixed excitation and emission wavelengths over time.
4.3.3 Configure Menu
This menu contains commands for setting parameters related to data
acquisition, device communication, and instrument settings. It also
includes menu items for loading and saving configuration files, for
SpeedBox configuration, control of attachments, and for system
communication and control.
Parameters
Allows setting of all parameters related to the current acquisition mode.
PopUp Scan Parameters
Allows setting of the acquisition parameters for the PopUp Scan feature.
PC Configuration
Designates the default data and export directories and sets parameters for
communication between the personal computer and the fluorometer as
well as printers. (Reminder: The printer/plotter must also be configured
correctly in the Windows Control Panel.)
SpeedBox Configuration
Allows the user to select functions for the SpeedBox, and also the style in
which the SpeedBox will be displayed.
4-2 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 4-3
Software Overview Software Overview
Instrument
Enables monitoring and control of the communications status between the
personal computer and the RF-5301 PC fluorometer unit, i.e., settings for
Fluorometer, -HV Control, PMT Protect, and Auto Shutter; adjustments
for Dark Level Correction and Lamp Alignment; SIN Ratio Check; and
information on the instrument serial number, ROM version, and elapsed
lamp usage time.
Attachments
Allows configuration of optional instrument accessories.
Save Parameters and Load Parameters
For saving or loading a particular configuration file. When a
configuration file is saved, all current parameters in all modes are saved
to disk in the directory in which the program is installed.
4.3.4 Manipulate Menu
The Manipulate menu contains functions which enable various methods
of manipulating data for display or printout. The menu items include:
Arithmetic
Performs scalar operations (+, -, *, /) on the ordinate axis of Spectrum,
Quantitative, or Time Course data.
Transforms
Performs function operations (smoothing, derivative, reciprocal,
logarithm) on the ordinate axis of Spectrum or Time Course data; and
reciprocal and logarithm functions on Quantitative data.
File/Chnl Calc
Performs vector operations (+,-, *,/) on the ordinate axis of Spectrum,
Quantitative, or Time Course data.
Data Print
A tabulation function in which all the measured data points for a
particular channel are displayed in a window. Data may be adjusted,
saved in a text file, output to a printer/plotter, or copied to the clipboard.
In Quantitative mode, Data Print is used primarily to print reports. In
Time Course mode, Data Print may be used to calculate the slope (i.e.,
the activity) of the time course line.
Peak Pick
A tabulation function which designates the peaks on the graph of
Spectrum or Time Course data and lists them in a window. Threshold
adjustment, valley designation (minimum value), replotting and printout
in either tabular form or graphics plot are possible. The table may be
stored as a text file or copied to the clipboard.
Point Pick
A tabulation function which allows X-axis designation of up to 15 points
of Spectrum or Time Course data for display on the graph and listing in a
window. Values may be changed, tables saved in text files, graphs
replotted, tables or graphs printed, and data copied to the clipboard.
Frequently used X-axis values may be saved and recalled.
Peak Area
Calculates the area under a curve. Up to four different areas may be
graphically selected. The results and graphs may be printed or sent to the
clipboard. Frequently used Peak Area configurations may be saved and
recalled.
Average
In Quantitative mode, averages the relative fluorescence intensities for all
)
unknown quantitative data that have the same ID number.
Working Curve
In Quantitative mode, allows setting the curve fitting parameters (order of
curve, zero intercept) for the standard curve.
4.3.5 Presentation Menu
This menu consists of items which determine the presentation format of a
data file on the screen or as a printout. The menu items include:
Channel Status
A window displaying a list of all occupied channels and their status with
respect to graph line color, whether the channel is currently displayed,
and whether it has been saved to disk. Individual channel acquisition
parameters may be viewed and the display status changed.
Graph
Copy
Copies the main graph to the clipboard.
Display Grid
An ON/OFF toggle for displaying the graph with a grid pattern.
4-4 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 4-5
Software Overview Software Overview
Fonts
Allows changes of fonts, and font types, styles and sizes for the
coordinate values, axis labels and headings.
Limits
Allows you to change the X- and Y-axis limits of the graph on the
screen, or to zoom in or out on an area without using the mouse.
Line Colors
Assigns a chosen color, type of line, and annotation type for each of
the ten channels in Spectrum and Time Course modes, and for
Standard, Unknown and Standard curve in Quantitative.
Options
Allows selection of various grid lines, grid colors, line types, and
background colors as well as rounded or minor tick marks.
Radar
Automatically sets the graph limits so that all data in all displayed
channels are visible (on scale) in the graph. Available for X- or Y­
axis, or Both Axes.
Hide All
This is an ON/OFF toggle for hiding and re-displaying all of the graph
data on the screen.
Show SpeedBox
This is an ON/OFF toggle for hiding and re-displaying the SpeedBox.
Plot
Enables layout of a combination of graph overlays, parameters and text
files for the four quadrants of a page. The results can be previewed on
the screen and output to the printer/plotter.
Int.lConc Toggle (Quantitative only)
Toggles the Y-axis of the unknown graph between intensity and
concentration.
Equation Display (Quantitative only)
This is an ON/OFF toggle for hiding and re-displaying quantitative
coefficients and constants.
4.3.6 Help Menu
The Help selections offer a summary of the RF-5301PC software. Help is
especially useful when you require information quickly or when the
instruction manual is not available.
Help for RF-5301 PC
Contains the on-line help information pertaining to specific RF-5301PC
features.
How to Use Help
Contains a general explanation of how the help system works.
About RF-5301 PC
Contains the version number of the RF-5301PC software as well as
license and serial number information.
4.4 Status Bar
The Status Bar contains windows which display information about the status of
the optical bench or about a current reading. The following is a description of
the windows in the Status Bar:
I)
)
Lamp Window
This window displays the xenon lamp's current status. When the instrument is
attached and communication has been initiated, the xenon lamp status is
displayed as ON or OFF in this window.
Scan Mode Window
Information is displayed in this window about the current scanning mode of
operation. The operation modes are Repeat Scan and Auto File in Spectrum
mode, and Auto Scan Mode in Quantitative mode. When Repeat Scan is
enabled this window displays Repeat and the designated file name with an
additional two digits representing the number of the scan to be performed next.
The same information is displayed for Auto File except instead of Repeat, AF
is displayed. When Auto Scan Mode is selected from the Quantitative
Parameters dialog box, Scan Mode is displayed in this window.
Fluorometer Window
This window displays the current excitation and emission wavelength settings
and the real-time relati ve fluorescence intensity at those wavelengths. It also
displays various status messages concerning the initialization, setup, slewing,
etc.
4-6 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 4-7
Software Overview
4.5 PopUp Scan™ Push Button
The PopUp Scan feature allows a sample to be scanned without changing the
current acquisition mode or parameters. The scan is displayed in a pop-up
window which may be moved, sized, or closed.
The window, as shown in Figure 4-1, has a menu offering the following
selections:
Radar
The Radar function adjusts the graph limits so that data will be completely
visible on the graph. This option may be selected from the menu or clirectly
from the keyboard (when the pop-up window is active) by pressing Alt+R.
Set Limits
This option allows changing the X- and Y-axis limits of the graph in the pop­
up window, or zooming in on an area without using the mouse. When the
Limits clialog box appears on the screen (see Limits, Section 5.5.2.4), enter
maximum and minimum values for the X- and Y-axes and select OK to update
the graph axes. Select Cancel to exit the dialog box without changing the
graph limits.
Hadar! Set Limits... liave...
28.276
25.000
20.000

if)
""
c
Q)
15.000
:f;
10.000
4.290
380.0 400.0 420.0
Wavelength (nm)
440.0 450.0
Figure 4-1 PopUp Scan Window and Menu
Save
The Save option allows the scan to be saved in a file which may later be
loaded while in Spectrum acquisition mode. A dialog box appears for
entering a file name (an .spc extension will be used) and any desired comment.
RF-5301 PC Instruction Manual
Software Overview
NOTE: Closing the PopUp Scan window or starting another PopUp
Scan will discard (without warning) the previous PopUp Scan.
4.6 SpeedBox®
The SpeedBox is a quick accelerator device which uses buttons to represent
commonly used menu commands. The SpeedBox may be configured to the
individual user's preference via the SpeedBox Configuration dialog box (see
SpeedBox Configuration, Section 5.3.4). The user is able to select a button to
represent a specific command and place this item in the SpeedBox. The
SpeedBox may be placed anywhere on the screen, and may be displayed in one
of eight different styles. When the software is exited, the last position of the
SpeedBox is remembered as well as whether it was visible.
4.7 The Graph Cursor
The mouse cursor changes to a cross hair when moved over a graph. When
this cursor is within the graph border, the left mouse button may be used to
zoom in on a portion of the graph and the right mouse button may be used to
display the Graph Pop-up menu (see Graph Pop-up Menu, Section 5.6).
I)
)
Use the following steps to zoom in on any area of the graph:
-Move the cross hair to one corner of the area of interest.
-Press and hold down the left mouse button.
-Drag the cross hair to the diagonally opposite corner.
-Release the left mouse button.
The graph will be redrawn with the selected area at full scale.
To find the X, Y coordinates of any position in the graph:
-Press the right mouse button to bring up the Graph Pop-up menu.
Select Cross Hair and Display.
The Y-axis coordinate of the cursor location is displayed beside the Y-axis,
and the X-axis coordinate of the cross hair location will be displayed below the
X-axis. To turn off this display, select Cross Hair, Display.
RF-5301 PC Instruction Manual 4-8 4-9
Software Overview
4.8 Clipboard
The RF-5301 PC software supports Microsoft Windows clipboard as a method
of transferring text and graphics to and from other Windows applications. The
contents of a window can be copied to the clipboard via the main menu items,
i.e., Copy is available under Edit in Data Print, Peak Pick, and Point Pick;
under Graph in Peak Area; in Graph under the Presentation menu; and the
Graph Pop-up menu. These menu commands are discussed in more detail in
Menu Commands, Chapter 5.
4.9 International Support
The RF-530 1PC software supports Windows international settings for date,
time, and floating point numbers. Dates are always formatted with leading
zeros and a two-digit year.
)
4 - 10 RF-5301 PC Instruction Manual
Menu Commands
5. Menu Commands
This chapter describes the options controlled through the menus at the top of
the screen, from left to right. When a menu option appears dimmed rather than
black, it cannot be selected until the appropriate condition is satisfied, i.e.,
until a file is loaded/saved, fluorometer is turned ON, proper acquisition mode
selected, etc.
5.1 File Menu
The File menu consists of commands to manage channels, files and file format.
When the File menu is selected, the screen appears as shown in the following
figure.
Save :;!s"
Qelete ...
Data Iranslation
.!:;hannel
E1!;it
.g
:l;
360.0 380.0 400.0 420.0 440.0 450.0
Wavelength (nm)
Figure 5-1 File Menu
5.1.1 Open
This command opens an existing data file and loads it into memory. It
can be selected via the File menu or by pressing Ctrl+O from the main
window. When this command is selected, the Open dialog box appears
(see Figure 5-2).
RF-5301PC Instruction Manual 5 - 1
Menu Commands Menu Commands
I. Open
File Qirectories:
IT. SPC c:\rfpc\data
I Qpt:" .... .1

aaz.spc •••••••1 I
r:'
ab.spc
batchl nl .spc
f1uorl.spc
I<bsamp2.spc
ml.spc 00
List Files of Iype: Drives:
ISpectrum r·spc) III IIEB c: ms-dos 6 [I
Figure 5-2 Open Dialog Box
To load a file, either double-click on the file name, type the file name
into the File Name edit box, or select it from the file list and press the
Open push button. The selected file is loaded into the next available
channel. In Quantitative mode, the file is loaded into the appropriate
data table (Standard or Unknown) and replaces any existing data in that
table. Several files can be loaded before clicking on Exit.
NOTE: In Quantitative mode, when loading Unknown files without
a Standard loaded, the method is changed to the method in which the
Unknown file was collected (see Parameters, Section 5.3. J). Also, in
Quantitative mode, the List Files of Type combo box is used to select
between Standard and Unknown files. Note, only Unknown files are
available in Raw Data mode.
When the Open dialog box appears, it displays a list of the files in the
default data directory (see PC Configuration, Section 5.3.3). To load
files from another directory and/or drive, select the new drive from the
Drives combo box and select the new directory from the Directories list
box. Please see the Microsoft Windows User's Guide for more
information on using the common dialog boxes such as Open.
The Exit push button closes the dialog box and returns the main screen to
view.
The Status push button displays the Channel Status dialog box, which is
the same dialog box displayed when the Channel Status command is
selected from the Presentation menu (see Channel Status, Section 5.5. J).
Select the Params option to preview a file's data acquisition parameters
before loading it.
RF-5301PC Instruction Manual
After loading the file in Spectrum or Time Course modes, the data will
not be visible when the main graph's current axis limits do not coincide
or overlap with the scanning parameters of the data file. To display the
data, use the Radar command (or press Ctrl+R) in the Presentation menu
under Graph (see Radar, Section 5.5.2.7).
NOTE: In Quantitative mode, the acquisition parameters and the
equation for the working curve are saved for both Standard and
Unknown data. When an Unknown file (.qnt) is loaded from the disk,
the parameters and equation for the working curve are loaded only
when there is no Standard curve currently in memory. It is therefore
possible to recalculate the concentrations based on the working curve
that was present at the time the data was saved (Load Unknown
without a Standard). When a Standard data file is loaded, the
Quantitati ve Parameters are updated to those that were saved with the
Standard curve. The user must verify that the data in the Standard
and Unknown tables are compatible, i.e., data was acquired with the
same reference and Auto Zero settings as well as slit width and
wavelength settings.
When the Acquisition mode is changed or the program is exited, the
Standard and Unknown data are saved to disk when they have file
J) ) names (see Save as and Rename Channel). When neither Standard
nor Unknown data have a file name, a warning is issued allowing
return to the Main menu where Save as may be selected. Note that
when any changes are made to the data (via edit, read or manipulate),
the data is marked as Not Saved. Saving the file will overwrite any
existing disk file with the same name (select Save, change the
Acquisition mode, or exit the program). A warning is issued so that
the original data may be preserved before the file is overwritten. To
preserve the disk file (avoid overwriting it), enter a new file name
using Rename Channel or Save as or use Erase Channel to clear
(discard) the data from memory.
5.1.2 Save
This command performs a quick save to disk for all channels which have
a file name. Files that were loaded using Open are saved to their original
directories. However, newly acquired data is saved in the Data Directory.
)
For channels with *No Name*, the Save as dialog box appears. The
Save command can be selected from the File menu or by pressing Ctrl+S
from the main window.
RF-530 1PC Instruction Manual 5-2 5-3
Menu Commands Menu Commands
5.1.3 Save as 5.1.4
This command is used to save a channel or table to a new file or directory
and can be selected from the File menu or by pressing Ctrl+A from the
main window. This command renames the channel in memory as well as
saves it to disk and/or into a new directory. When this command is
selected, the Save as dialog box appears (see Figure 5-3).
- Save As
File Name: 1!irectories:
ImmJ ,!
32'2.81)(;

ab.so£
hatch1 r,l.soc
iluorl.soc
kbs<lmn2.slJc
ml.soc
marvOO.snc
marv{l"l.snc
List Files of Iype:
Ispectrum r·spe) I Ira e: ms-dos 6 II
c:\rfpc\data
.channel
IChannelO I
8oc:\

8 rfpe


Driyes:
Figure 5-3 Save as Dialog Box
In Quantitative mode, the List Files of Type option is used to select
between Standard and Unknown files. Note that only Unknown files are
available in Raw Data mode.
When the Save as dialog box appears, it displays a list of the files in the
default data directory (see PC Configuration, Section 5.3.3). To save a
file to another directory and/or drive, select the drive from the Drives
combo box and select the directory from the Directories list box.
The Status button displays the Channel Status dialog box, which is the
same dialog box displayed when the Channel Status command is
selected in the Presentation menu (see Channel Status, Section 5.5.1).
Delete
The Delete command can be used to remove one or more files from the
disk. When this command is selected, the Delete dialog box appears (see
Figure 5-4).
Delete
File Name:
I"."
J2ireclories:
e:\rlpe\data
I iii r=--:---------c=< _
list Files 01 Jype:
I II '",") l1!
Be:\

Brlpe
e data
Olives:
IIiiI e: ms-dos_6 I
Figure 5-4 Delete Dialog Box
NOTE: Use caution when selecting items to be deleted as deletion is
permanent.
j) )
To save a file, select the channel to save using the Channel combo box;
To delete a file, type in a file name and press the Delete push button, or
the channel's file name is automatically placed in the File Name edit box.
double-click on a file name in the list box. To delete multiple files, click
When the channel does not have a file name, the File Name edit box is
on the first file to delete, hold the left mouse button down and drag the
blank and the Save as push button is disabled. Type in a new file name,
mouse to the last file to delete, and then press the Delete push button.
when appropriate, and press the Save as push button, or double-click on a
Individual files may be added to, or removed from, the selection
file name in the File Name edit box. When the file already exists, a
(highlighted) list by holding the Ctrl key while clicking on a file name.
warning message appears asking for confirmation to overwrite the
selected file.
When the Delete push button is pressed or a file name is double-clicked,
the Delete this file? message box appears (see Figure 5-5).
Delete this file?
File:
Selected: 001
KBSAMP2.SPC
Deleted: 000
Directory:
C:\RFPC\DATA\
Figure 5-5 Delete this file? Dialog Box
5-4 RF-5301 PC Instruction Manual RF-530 1PC Instruction Manual 5-5
Menu Commands
This dialog box displays each file before deletion while offering the
following options:
Delete: Delete this file; continue to next file in selection.
Skip: Do not delete this file; continue to next file in selection.
Cancel: Return to the Delete dialog box.
Delete All: Delete all remaining files in selection; return to Delete
dialog box.
The Delete dialog box appears with a list of the files in the default data
directory (see PC Configuration, Section 5.3.3). To choose files from
another directory and/or drive, select the drive from the Drives combo
box and select the directory from the Directories list box.
NOTE: The Delete command does not remove data from the computer's
RAM (see Erase Channel, Section 5.1.6.2).
5.1.5 Data Translation
5.1.5.1 ASCII Export
This option is selected from the Data Translation cascading menu. It
saves the data in a channel(s) to an ASCII (American Standard Code
for Information Interchange) file(s). When ASCII Export is selected
in Spectrum or Time Course modes, the channel selection dialog box
appears (see Figure 5-6).
O


"
01KBSAMP2 Oh
O? Of
0)
01 Oh
OAJI

Figure 5-6 ASCII / DIF Export Dialog Box
Menu Commands
After selecting the channels to be saved in ASCII format, select OK to
complete the operation. The converted file is saved to disk in the
export directory and has the same name as the channel with an .asc
extension.
When ASCII Export is selected from Quantitative mode, a Standard
and/or Unknown selection dialog box appears. Check the desired
check boxes and select OK to proceed. Since it is often useful to have
the same file name (with different extensions, i.e., .std and .qnt) for
Standards and Unknowns, a dialog box appears so that the file name
for the ASCII file may be entered. Enter the file name and select OK.
The converted file is saved to disk in the export directory with an .asc
extension.
The data remains unchanged in the channel. The descriptive
information about the file is removed, and only ordinate and abscissa
information remains in the file. The general format of the ASCII file
is:
X,Y<CR><LF>
X,Y<CR><LF>
)
<CR> and <LF> are the codes for a carriage return and line feed.
5.1.5.2 DIF Export
This option is selected from the Data Translation cascading menu.
This command is similar to ASCII Export, except that the converted
file is in DIF (Data Interchange Format) format and the file name has
a .dif extension. The X-axis data is not translated for Spectrum and
Time Course data (although enough information is included so that the
X-axis may be generated). For Unknown Quantitative data, the X-axis
data will be the Sample ID values; for Standard Quantitative data, the
X-axis data will be the concentration values. The DIF files are saved
in the export directory.
5.1.6 Channel
5.1.6.1 Save Channel
This option is found in the Channel cascading menu and is selected
for saving any combination of the named channels to the data
directory. When selected, the Save Channel dialog box appears as
shown in Figure 5-7 (Spectrum and Time Course) or Figure 5-8
(Quantitative).
5-6 RF-5301PC Instruction Manual RF-5301 PC Instruction Manual 5-7
Menu Commands
OI£lA.
MAN 0'.,
01 KBSAMP2 Of,
O? Oi.
OJ.
OJ Ol
o£;11
t. ., IJI1f;'
Figure 5-7 Save Channel Dialog Box (Spectrum / Time Course modes)
When in Spectrum or Time Course acquisition mode, select any
combination of channels to be saved using the mouse or by entering
the number(s) corresponding to the underlined number(s). Select All
to designate all the channels for saving. (Select "All" again to
deselect all the channels.) Only channels where a file name has been
entered are saved. Channels containing no data are dimmed and
cannot be selected.
o Clear after Save
File Name:
c
Comment:
___I
Ov.nb;nY!i o al1.n $;jYt:
File Name:
I I
Comment
I 1
PTOO'7
:JlJ:,,>?', J; ..
Figure 5-8 Save Channel Dialog Box (Quantitative mode)
When a channel has the expression *No Name* in place of the file
name, its data is not saved to disk even though it may be selected. To
save these channels, they must first be named using the Rename
Channel option in the File menu (see Rename Channel, Section
5.1.6.3), or select Save As, rename the channel, and save it to disk.
RF-5301PC Instruction Manual
Menu Commands
When in Quantitative mode, select the Standard and/or Unknown
check boxes. Enter a file name and comment (when desired) for the
item selected. Then select OK or press the Enter key. Select the
Clear after Save check box to remove the data from the computer's
memory after data has been saved to disk. This selection is useful
when preparing to collect another set of data.
The Status push button, when selected, displays the Channel Status
dialog box, which is the same dialog box displayed when the Channel
Status option is selected within the Presentation menu (see Channel
Status, Section 5.5.1).
The Cancel push button causes the software to ignore any selections
and return to the main screen. Pressing the Esc key has the same
effect as Cancel.
5.1.6.2 Erase Channel
The Erase Channel command, found in the Channel cascading menu,
clears data from the computer's RAM. Select the channel(s) to be
erased and select OK to continue. In Spectrum or Time Course
modes, when any data in the channels has not been saved, the message
"Warning! Some data has NOT been saved! Do you wish to
continue?" is issued. Select Yes to discard the data or No to cancel
the operation. Select Save, Save as, or Save Channel from the File
menu to save the data to disk.
In Quantitative mode, select the Standard and/or Unknown check
boxes to indicate the table to erase. When any data in the channels/
tables has not been saved, the message "Warning! Standard has
NOT been saved! Do you wish to continue?" is issued. Select Yes
to discard the data. To preserve the data, select No and select Save,
Save as, or Save Channel from the File menu to save the data to disk.
RF-5301 PC Instruction Manual
5-8
5-9
Menu Commands
O.Q RAMAN' 0 '.
01 KBSAMP2 0 !'
0, Dr
0:; 0"
o 1
o All
l<oli!f;1 I I
Menu Commands
Change the information as desired and select Save or press the Enter
key to implement the changes. Select Discard to cancel the command
and return to the main window.
In Quantitative mode, the file name entry dialog box is displayed,
similar to that in Figure 5-8. Select the Standard and/or Unknown
check boxes and enter or change the file name(s) and comment(s).
Then press the Enter key or select OK.
Clear the file name field and select OK to assign No Name to the data,
so that the data will not be saved when changing acquisition modes or
exiting the program.
Figure 5-9 Erase Channel Dialog Box
5.1.7 Exit
5.1.6.3 Rename Channel
The Rename Channel command, found in the Channel cascading
menu, is used to modify the file name and/or comment assigned to a
particular channel. This causes the channel's "data saved" status to be
set to "not saved." A name may also be assigned to a No Name
channel. In Quantitative mode, use Rename Channel to assign file
names to the Standard and Unknown channels so that the file will be
This option exits the program and returns to Windows Program Manager.
When data has not been saved, a warning is displayed and you may elect
to return and save the data, or exit and lose the data. Any data that has
been assigned a file name, but has not been saved, is saved automatically
at this time. Exit can be selected from the File menu or by pressing
Alt+F4 from the main window.
automatically saved when the program is exited.
When Rename Channel is selected in Spectrum and Time Course
I\ )
modes, the channel selection dialog box is displayed. Select the
channel to be renamed or for which the comment will be changed.
Then press the Enter key or select OK. The Rename Channel dialog
box appears (see Figure 5-JO) with the current file name and
comments contained in the edit boxes.

°1 KBSAMPZ
0"
0.,
0, Of
0' 0"
Ol
0"
oj Ic..
Enler File Name:
IIi1mm I
Eoler Comment:
IRomon Peok I

...
..,
"",

)
Figure 5-JO Rename Channel Dialog Boxes
RF-5301 PC Instruction Manual 5 - 11
5 - 10 RF-5301PC Instruction Manual
Menu Commands Menu Commands
5.2 Acquire Mode Menu
The mode in which data is acquired is selected from the Acquire Mode menu,
which includes discrete modes of data acquisition. A check mark precedes the
active mode in the menu. All subsequent selections in the program will
correspond to the currently active acquisition mode. When the Acquire Mode
menu is selected, the screen appears as shown in Figure 5-11.
I ~ · , . . .
397.0 390.0 370.0 380.0
Wavelength (nm)
,I.'1111 i'1
Manipulate Eresentation !ielp
360.0

l'
f
Figure 5-11 Acquire Mode Menu on the Spectrum Screen
5.2.1 Spectrum Mode
Under the Acquire Mode menu, use Spectrum (noted with a check mark)
to acquire a spectrum over a designated wavelength range. When
Spectrum is the acquisition mode upon starting the program, select
Parameters from the Configure menu to set the parameters for real-time
data acquisition (see Spectrum Parameters, Section 5.3.1.1). (When
Spectrum is selected while another acquisition mode is active, the
Spectrum Parameters dialog box (see Figure 5-23] appears
automatically.) The Spectrum screen is similar to Figure 5-11. To turn
the instrument ON, either select Instrument from the Configure menu
and set the Fluorometer, -HV Control and PMT Protect to ON (see Figure
5-38), or click on the Start push button.
The Spectrum screen contains seven push buttons at the bottom of the
screen. Anyone of the push buttons may be selected with the mouse, or
with the keyboard via the function keys F2-F8.
F2 R F4 F5 F6 F7 F8
Figure 5-12 Spectrum Push Buttons
NOTE: The push buttons at the bottom of the screen are mapped to
the function keys from right to left beginning with the F8 key (all
modes) and ending with the F2 key in Spectrum mode, and the F I key
in Quantitative and Time Course modes.
Following are descriptions of the various push button's functions:
1) Go To WL (wavelength) push button
This button allows you to set the excitation and emission wavelengths.
Select with the mouse or press the F2 key to display the Wavelength
)
Selection dialog box (see Figure 5-13). You may manually enter new
EX and/or EM wavelength values or change them by using the scroll
bars below the respective edit boxes. During scrolling the edit boxes
reflect the new wavelengths. The valid wavelengths are 0, and 220 ­
900nm. The push buttons are explained below:
Read - The monochromators slew to the new wavelength values and
the intensity reading in the dialog box is updated. In order to
keep these wavelength values and update the parameters, Set
must be selected.
Set - The monochromators slew to the new wavelength values.
These values are displayed in the Fluorometer Window and
stored in the parameter entry form (PEF) as the fixed
wavelengths. The dialog box is closed.
Cancel - Resets the original wavelength values and exits the dialog
box.
NOTE: When the wavelengths are set to the desired values, select the
Read push button to display the fluorescence intensity reading at the
new wavelength settings. The Fluorometer Window displays "Hold"
because the intensity is currently displayed in the dialog box. When
the dialog box is closed, the Fluorometer Window displays the new EX
and EM wavelength values and the intensity.
5 - 12 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 5 - 13
Menu Commands Menu Commands
EM Wavelength E;><: Wavelength
@ill

[tll!!!!,l."l
Intensity: Ci 137 I
!:'ti\lli:adT:1
Figure 5-13 Wavelength Selection Dialog Box
2) Search Optimal WL (wavelength) push button
This search function is used to find the optimal excitation and emission
wavelengths for a sample. Select with the mouse or press the F3 key to
display the dialog box. Enter the EX / EM start and end wavelengths,
and the excitation wavelength interval (valid range is 10 - 99nm) on
the excitation side. Pressing the Search push button finds the excitation
wavelength and emission wavelength which gives the greatest
fluorescent intensity, while eliminating peaks due to scattered
excitation light, second-order light, and Raman bands, and displays the
results in the Search Optimal Wavelength dialog box. The Save
function saves the optimal wavelengths into the fixed wavelengths of
the Parameters dialog box. The optical bench is set to the wavelengths I)
)
which are reflected in the Status Bar.
Search Optimal Wavelength
E"" Range: to

EM Range: to §O

I
EX !nterval:

Optimal Wavelengths:
Excitation: [-=-=::J nm
Emission:
c::=:::J
nm
Figure 5-14 Search Optimal Wavelength Dialog Box
3) Shutter push button
Select this push button with the mouse or press the F4 key to open and
close the shutter (the shutter position is visually displayed on the push
button).
4) Auto Zero push button
This function zeroes the fluorometer unit at the designated excitation
and emission wavelengths. Select with the mouse or press the FS key.
This function does not change the AID (analog voltage converted to
digital signals) calibrated signal; it merely subtracts the reading from
all subsequent readings to correct for small changes, such as drift due
to thermal effects.
5) PopUp Scan push button
Select with the mouse or press F6 key to perform a scan using the
PopUp Scan parameters. The scan is displayed in a pop-up window
(see Software Overview, Section 4.5, Figure 4-1) allowing you to view
a sample without changing the current Spectrum parameters. The
PopUp Scan contains a menu bar which includes: Radar (changes the
graph scale), Set Limits, and Save. The PopUp Scan uses a parameter
set similar to those used for Spectrum mode scanning. The PopUp
Scan parameters are used exclusively by the PopUp Scan feature. The
current instrument settings (those set in Parameters) are retransmitted
to the instrument after completion of the PopUp Scan. Press the Stop
push button to interrupt the scan.
6) Start push button
Select with the mouse or press the F7 key to start the scan. When the
scan is completed, the Enter File Name dialog box appears (see
Tutorial, Section 3.5, Figure 3-6). When Save is selected after entering
or not entering a file name, the data is saved to the next available
channel. It is not stored to disk at this time unless the Repeat Scan or
Auto File mode is used. When Discard is selected, the data is erased.
When communication between the computer and the instrument is OFF,
this push button may be used to establish communications.
7) Stop push button
Select with the mouse or press the F8 key to interrupt the scan. The
data collected to this point can be saved to a channel; the remainder of
the scan range has a value of zero. This push button may also be used
to discontinue communication with the instrument.
5.2.2 Quantitative Mode
Select Quantitative from the Acquire Mode menu (noted with a check
mark) to collect Quantitative data. When Quantitative is selected upon
starting the program, it is necessary to select Parameters from the
Configure menu to change the parameters for real-time data acquisition.
When Quantitative is selected while another acquisition mode is in effect,
the Quantitative Parameters dialog box (see Figure 5-29) automatically
appears. The Quantitative screen appears as shown in Figure 5-15.
RF-5301 PC Instruction Manual RF-5301 PC Instruction Manual 5 - 14 5 - 15
Menu Commands Menu Commands
1'5•• ... 3) Search Optimal WL (wavelength) push button
file Acquire Mode
10 1S 17
Manipulate fresentation tlelp
This search function is used to find the optimal excitation and emission
wavelength for a sample. Select with the mouse or press the F3 key to
Seq.
A ID Cone. Int.
display the dialog box. Enter the EX / EM start and end wavelengths,
and the excitation wavelength interval (valid range is 10 - 99nm) on

the excitation side. Pressing the Search push button locates the
excitation wavelength and emission wavelength that gives the greatest
fluorescent intensity, while eliminating peaks due to scattered
excitation light, second-order light, and Raman bands, and displays the
results in the Search Optimal Wavelength box. The Save function

saves the optimal wavelengths into the fixed wavelengths of the
Parameters dialog box. The optical bench is set to the wavelengths
which are reflected in the Fluorometer Window.
seq •
4) Shutter push button
This push button is used to open and close the shutter and visually
displays the shutter position. Select with the mouse or press the F4
key.
Figure 5-15 Quantitative Screen
5) Auto Zero push button
The Quantitative screen contains eight push buttons at the bottom of the Select with the mouse or press the FS key. This function zeroes the
screen. Any push button may be selected with the mouse, or with the fluorometer unit at the designated excitation and emission wavelengths.
keyboard via the function keys F1-F8. This function does not change the AID (analog voltage converted to
)
digital signals) calibrated signal; it merely subtracts the reading from
all subsequent readings to correct for small changes, such as drift due
to thermal effects.
6) PopUp Scan push button
Select with the mouse or press F6 key to perform a survey scan. The
scan is displayed in a pop-up window (see Figure 4-1 in Software
Figure 5-16 Quantitative Push Buttons
Overview) to view a sample without changing to Spectrum mode. The
PopUp Scan uses a parameter set (found under the PopUp Scan
NOTE: The push buttons at the bottom of the screen are mapped to the Parameters) similar to those used for Spectrum mode scanning. The
function keys from right to left beginning with the F8 key and ending current instrument settings (in Parameters) are retransmitted to the
with the F1 key, respectively. instrument after completion of the PopUp Scan.
7) Read push button
Following are descriptions of the various push button's functions:
Select with the mouse or press the F7 key to read the current value
(item highlighted in the table will be replaced) from the fluorometer
1) Standard push button
into the Standard or Unknown data table (see also Auto Scan Mode in
Select the Standard push button (when not dimmed) with the mouse or
Quantitative parameters, Section 5.3.1.2). Note that when acquiring
press the Fl key to display Standard data and graph for acquisition,
Standard data, and when no concentration has been previously entered,
manipulation, or editing of Standard data.
the Edit Standard dialog box (see Figure 5-17) automatically appears
so that the concentration value may be entered. When the
2) Unknown push button
communication status between the computer and the instrument is OFF,
Select the Unknown push button (when not dimmed) with the mouse or
this push button may be used to establish communications.
press the F2 key to display Unknown data and graph for acquisition,
manipulation or editing of Unknown data.
F8 F7 F6 F5 F4 f3 F2 Fl
5 - 16 RF-530 1PC Instruction Manual RF-5301 PC Instruction Manual 5 - 17
Menu Commands
Menu Commands
8) Stop push button
Select with the mouse or press the F8 key to interrupt the PopUp Scan
(see the Stop push button in Spectrum). This push button may also be
used to discontinue communication with the instrument.
The Standard (when using multipoint quantitative method) or Unknown
is displayed in both a tabular and graphic format on the Quantitative
acquisition screen. The Standard and Unknown push buttons described
above are used to change the display (as well as the acquisition and
manipulation) between Standard and Unknown data. Use the mouse or
arrow keys to change the currently selected (highlighted) item in the data
table. Note that the Read push button replaces the relative fluorescence
intensity of the current selection and makes the next item in the table the
current selection. Double-clicking a selection with the mouse or pressing
the Enter key displays the Edit Standard or Edit Unknown dialog box
allowing the values in the table to be entered manually.
The Edit Standard dialog box (see Figure 5-17) is used to enter (or
modify) values for concentration and intensity. Use Delete to remove the
entry from the Standard table.
Standard Number 2
Concentration 1-,·HuwHl I
Int. 1203.366 I

1.····°_
Figure 5-17 Edit Standard Dialog Box
The Edit Unknown dialog box (see Figure 5-18) is used to enter values
for Sample ID and intensity. Select Delete to remove the entry from the
Unknown data table. Use the Standard button to create a Standard from
an Unknown value in the following manner: highlight the desired
selection in the Unknown table using the arrow keys and press the Enter
key, or double-click on the selection with the mouse.
NOTE: Sample IDs may consist of a maximumof eight numeric characters only.
10
1111 I
In1. I 71.217J
Cone. 0.00000

Figure 5-18 Edit Unknown Dialog Box
Select the Standard button and enter the value for the concentration in
the Make Std dialog box which appears (see Figure 5-19). The selection
is removed from the Unknown table and appended to the end of the
Standard table.
Concentration: I 1.000000 I

Figure 5-19 Make Standard Dialog Box
)
5.2.3 Time Course Mode
Select Time Course from the Acquire Mode menu (noted with a check
mark) to acquire relative fluorescence intensities at fixed excitation and
emission wavelengths over a period of time. Upon starting the program,
when Time Course is selected, it is necessary to select Parameters from
the Configure menu to set the parameters as the next step in real-time
data acquisition in this mode. When Time Course is selected while
another acquisition mode is in effect, the Time Course Parameters
dialog box (see Figure 5-33, Section 5.3.1.3) automatically appears.
After the parameters are selected, the Time Course screen appears as
shown in Figure 5-20. The Time Course screen contains eight push
buttons at the bottom of the screen, any of which may be selected by
clicking with the mouse, or with the keyboard via the function keys FI ­
F8.
RF-5301PC Instruction Manual 5 - 19
5 - 18 RF-5301PC Instruction Manual
Menu Commands Menu Commands
60.00 4000 2000
-100.000' ,.. <" -,
0.00
>

c
:i:
1000.000"
,1;'111";
Ble Acquire Mode .c.onfigure Manipulate Eresentation tlelp
TIll'le (sec)
Figure 5-20 Time Course Screen
3) Shutter push button (see Spectrum)
Select with the mouse or press the F4 key.
4) Auto Zero push button (see Spectrum)
Select with the mouse or press the FS key.
5) PopUp Scan push button
Select with the mouse or press the F6 key to perform a survey scan.
The scan is displayed in a pop-up window (see Figure 4-1 in Software
Overview) to view a sample without changing to Spectrum mode. The
PopUp Scan uses a parameter set (found under the PopUp Scan
Parameters) similar to those used during Spectrum mode. The current
instrument settings (those set in Parameters) are retransmitted to the
instrument after completion of the PopUp Scan.
6) Start push button
Select with the mouse or press the F7 key to start the scan. When the
communication status between the computer and the instrument is OFF,
this push button may be used to establish communications.
7) Stop push button
Select with the mouse or press the F8 key to interrupt the scan. This
NOTE: The push buttons at the bottom of the screen are mapped to
push button may also be used to discontinue communication with the
the function keys from right to left beginning with the F8 key and
instrument.
ending with the FI key, respectively.
]) )
FI F2 F3 F4 F5 F6 F7 F8
Figure 5-21 Time Course Push Buttons
Following are descriptions of the various push button's functions:
1) Cell Blank push button
When this push button is selected (use mouse or press the Fl key), the
fluorometer unit takes a reading and subtracts this reading from each of
the data points in the scan. This cell blank reading remains valid until
the acquisition mode is changed or until the Auto Zero push button is
selected.
2) Go To WL (wavelength) push button (see Spectrum)
Select with the mouse or press the F2 key.
3) Search Optimal WL (wavelength) push button (see Spectrum)
Select with the mouse or press the F3 key.
5 - 20 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 5 - 21
Menu Commands Menu Commands
770.0 6CJ.0 400.0


1:
5.3 Configure Menu
Spectrum Iype: 0 Emission <;, Synchronous
The options in this menu are used to set parameters related to data acquisition,
EM Yiavelength: nm
device communications, instrument settings, and saving and loading
EX Wavelength Range: Jitart .End
I 1770
configuration files. When the Configure menu is selected, the screen appears
Recording Range: bOW !::!igh 150.000 I
as shown in Figure 5-22.
1·-'11
SJ;anning Speed:
lil::::E
I
Figure 5-23 Spectrum Parameters Dialog Box
Spectrum Type
This selection refers to the type of spectrum to be scanned: excitation,
emission or synchronous. When excitation is selected, the emission
grating is in a fixed position and when emission is selected, the
excitation grating is in a fixed position. When synchronous is
selected, both gratings move simultaneously. The emission range is
V'.'avelength (r.:n)
) selected such that it starts at a wavelength higher than the starting
excitation wavelength. This offset (dA.) is maintained during the
synchronous scan. For example, when the excitation wavelength
range is selected from 300 to 500nm, the emission wavelength range
Figure 5-22 Configure Menu
might start at 305nm. Synchronous scanning can be used to eliminate
the Rayleigh and Raman bands in a spectrum. When the excitation
5.3.1 Parameters
scanning range is out of the monochromator's range, a warning
Selection of the Spectrum, Quantitative or Time Course acquisition mode
message, "EX Wavelength Too High or EM Range Too Large for
determines which parameter dialog box appears when the Parameters
Synchronous Scan," appears. Select a new EX starting wavelength to
option is selected. The Parameters dialog box automatically appears
start the scan or reduce the EM wavelength range.
whenever the acquisition mode is changed in the Acquire Mode menu. It
can also be displayed using the accelerator key strokes Ctrl+P while
EX I EM Wavelength
viewing the main screen. This field allows the excitation and emission wavelengths to be set. It
is dependent upon the Spectrum type. The allowable settings are 0,
5.3.1.1 Spectrum Parameters and 220 - 900nm.
The Spectrum parameters dialog box appears as shown in Figure 5-23.
A description of the parameter functions follows:
EX I EM Wavelength Range
)
These are the starting and ending wavelength values for Spectrum
scans. The allowable range is 220 - 900nm. The minimum scanning
range is 25nm.
5 - 22 RF-5301PC Instruction Manual
RF-5301PC Instruction Manual 5 - 23
Menu Commands Menu Commands
Recording Range
These entries correspond to the ordinate scale limits of the PC's graph.
The allowable range is -100.00 - 1000.00.
Scan Speed
The actual scanning speed at which data is collected can be set at
Survey, Super, Very Fast, Fast, Medium, Slow and Very Slow.
Sampling Interval
This sets the interval at which data is acquired by the instrument.
When the interval selected is 0.2nm, a reading is taken at every 0.2nm
for the specified range. When Survey Scan is selected, the sample
interval is fixed at 2.0 nm. When Super or Very Fast is selected, the
sampling interval is fixed at 1.0 nm. Note that selecting a small
sampling interval and a large wavelength range will affect the
performance of this software. (See Precautions on Memory Usage,
Section 6.3 in Troubleshooting, Chapter 6.)
EX I EM Slit Width
The allowable excitation slit width settings are 1.5,3,5, 10, 15,20,
6HH (half-height). The allowable emission slit width settings are 1.5,
3,5, 10, 15,20, Close.
I)
)
Sensitivity
The sensitivity of the detector can be set at High or Low (with High
providing approximately 50 times the sensitivity of Low).
Response Time
This function sets the response time of the instrument. Possible
selections are 0.02, 0.03, 0.1, 0.25, 0.5, 2.0, 4.0, 8.0, and Auto.
Repeat Scan
Repeat Scan allows automatic consecuti ve scanning of 2 to 100 scans
on the basis of a set time interval. When the Repeat Scan button is
selected, the dialog box shown in Figure 5-24 appears.
)
• ..'... iT......
Bepetitions 0 12-100)
Repeat Time Interval Seconds
file Name II [1-6 Characters)

Figure 5-24 Repeat Scan Parameters Dialog Box
Enter the number of repetitions to be performed and the time interval
(in seconds) between the starts of two consecutive scans. When the
time required for one cycle is longer than the time interval, the next
scan starts immediately when the wavelength is reset to the start
wavelength. Enter up to six characters for the file name to save the
acquired data to disk.
When Enable is selected, the Scan Mode window on the Status Bar,
to the left of the Fluorometer Window, begins tracking the number of
scans. The Status Bar displays the word Repeat and the designated
file name, accompanied by two digits representing the sequence
number of the scan to be performed next. (Thus, for the first scan, the
window would display Repeat - TestOO if Test was the file name
entered; see Figure 5-25.) The Scan Mode Window remains active
until Disable is selected in the Repeat Scan Parameters dialog box.
Upon completion of each scan, the data file is saved to disk in the data
directory with an .spc extension. A tone signals the completion of all
of the scans.
When Disable is selected, the Repeat Scan Mode is turned off. Also,
use Disable to exit from the Repeat Scan mode of data collection when
it has been previously set.

SoloW
W'l"i"""··"'wm
mYj
Figure 5-25 Repeat Scan Mode Window
5 - 24 RF-5301PC Instruction Manual 5 - 25
RF-5301PC Instruction Manual
Menu Commands Menu Commands
When the scan mode is set to Repeat Scan, attempting to set Auto File
will result in a warning. Repeat Scan must first be cancelled before
Auto File is set. This is also true when attempting to set Repeat Scan
when Auto File has already been set.
,tmmttm
Mode is set to Repeat Scan! Mode is set to Auto File!
l:tiP!.,,] 1""""-'·].· 10K,
Figure 5-26 Repeat Scan I Auto File Warning Messages
NOTE (for Repeat Scan and Auto File): Each scan is saved to the
disk, not to a channel. The file must be loaded at a later time in order
to perform any post-analysis operations. To perform the next scan in
the Auto File mode, select the Start push button on the main screen.
When the file name which is entered already exists, it will be
overwritten without warning.
Auto File
Auto File allows a maximum of 100 scans to be performed in
succession and automatically saves them in the designated file (stored
to disk) after each scan. The data is also displayed in real-time on the
video display, but will not be stored in channels. When the Auto File
button is selected, the dialog box shown in Figure 5-27 appears.
of Sc.ns (1-100)
file N.me ITEST I (1-6 characters)
IW.-'E'ni;ble'!ifii) Ilij2isabie..'.<.'j
......, ..... :. -';"·"1"'1"
Figure 5-27 Auto File Dialog Box
Enter the number of scans to be performed in Auto File mode and the
file name for the scans. When Disable is selected, the mode is not set
to Auto File as Disable exits from the Auto File mode and clears the
file name.
When Enable is selected, the Scan Mode window appears to the left
of the Fluorometer Window on the main screen. The Status Bar
displays AF (for Auto File) and the designated file name accompanied
by two digits representing the number of the scan to be performed
next. (Thus, for the first scan, the Status Bar would display AF ­
TestOO if Test was the file name entered; see Figure 5-28.) The Scan
Mode Window remains active until the specified number of scans has
been performed or until Disable is selected in the Auto File dialog
box. When the file name from the previous set of Auto File runs is
changed, or the number of Auto File scans is set to less than the
current number of completed scans (as displayed in the Scan Mode
Window), the next set of Auto File scans begins at zero.

e

St

Figure 5-28 Auto File Scan Mode Window
Upon completion of each scan, the data file is saved to disk in the data
directory with an .spc extension.
)
5.3.1.2 Quantitative Parameters
The Quantitative Parameters dialog box is shown in Figure 5-29. A
description of the parameter functions follows:
fOur.
Method:
IMulti Point Working Curve IJ
Wavelength: 1350.0 I
EM Wavelength: I 1 425.0
Slit 't!'idth (nm]: EX 110 II
EM 110 o. II
Sensitivity: @ High 0 Low
o Scan Mode

Figure 5-29 Quantitative Parameters Dialog Box
.(;oncentration
Units:
Range: 1 0.000 1 to 1100.000 I
Recording Range
bOW tjigh 11000.00 I
Response lime:
1
0
.
02
il
Bepetilions:
II
5 .. 26 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 5 - 27
Menu Commands
Method
This function establishes how the concentration is determined.
Selections include raw data, K-factor, single point working curve and
multipoint working curve.
Select Raw Data Measurement when no quantitation is required.
Select K-Factor to enter the coefficients of a known equation for a
standard curve. The dialog box as shown in Figure 5-30 is used to
enter the equation (1 st order) for a known working curve. Select OK
to return to the Quantitative Parameters or Cancel to select another
method.
'. III( l i11II :rrrT.1Trr'"
[Cone) = Kl • [UNK Int) + KO
Kl = IIiIiIiIiIil I K.!! = I 0.0000 I
Figure 5-30 K-Factor Parameters Dialog Box
Select Single Point Working Curve when only one Standard is used.
I)
)
When the dialog box appears, enter the concentration of the Standard
(see Figure 5-31). Select OK to return to the Quantitative Parameters
or Cancel to select another method. Note that this method assumes
that the blank (zero concentration) has a relative fluorescence
intensity of zero.
0"
-S.TD Concentration I 1.0000
...
Figure 5-31 Single Point Working Curve Dialog Box
Select Multipoint Working Curve to use up to twenty standards. In
)
the dialog box, choose the order of the curve (1st, 2nd, or 3rd order
polynomial) and whether to force the curve through zero (see Figure
5-32). Select OK to return to the Quantitative Parameters or Cancel to
select another method.
Menu Commands
NOTE: Using Multipoint Working Curve: The concentration values
for the Unknown are displayed as 0.000 until there is a valid working
curve. When enough standards have been entered (and read) to solve
for the curve fitting parameters, the equation for the working curve
(and the curve if the Standard graph is on the screen) is displayed and
the Unknown concentrations are calculated. The working curve is
drawn over the concentration range (+ I 0%) of the standards. When
using 2nd and 3rd order polynomials to calculate Unknown sample
concentrations, there may be multiple solutions (sample concentrations)
to the working curve for any given intensity. In this case, the
following algorithm is used to determine the best solution
(concentration of the Unknown): 1) When the concentration value is
less than or equal to the largest Standard and greater than or equal to
the smallest Standard, the value is considered to be correct; 2) When
none of the solutions satisfy the criteria in No.1, the concentration
value that is closest to the smallest Standard concentration is considered
to be correct; 3) When a 2nd order calibration curve is selected and
the intensity of an Unknown is out of the range of the Standard sample,
the concentration may not be calculated correctly. In this case the
concentration is listed as 0.000.
Qrder of Curve o 1st 02nd
Zero Interception o Yes
Figure 5-32 Multipoint Working Curve Dialog Box
EX Wavelength
Enter the excitation wavelength at which data will be acquired.
Allowable entries are 0, and 220 nm to 900 nm.
EM Wavelength
Enter the emission wavelength at which data will be acquired.
Allowable entries are 0, and 220 nm to 900nm.
EX I EM Slit Width
The allowable excitation slit width settings are 1.5,3,5, 10, 15,20,
6HH (half-height). The allowable emission slit width settings are 1.5,
3,5,10, 15,20, Close.
5 - 28 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 5 - 29
Menu Commands Menu Commands
Concentration
Range values are used to set the initial scale of the Standard curve X­
axis and the Unknown concentration Y-axis. The displayed standard
curve is calculated only for the concentration range. The
concentration units are used as headings for various tables and labels
for the appropriate graph axes.
Recording Range
These entries correspond to the ordinate scale limits of the PC's graph.
The allowable range is -100.0 to 1000.0.
Sensitivity
The sensitivity of the detector can be set at High or Low (with High
providing approximately 50 times the sensitivity of Low).
Auto Scan Mode
Auto Scan Mode automatically starts PopUp Scan prior to every Read
allowing the user to preview the spectrum of a sample. When this is
selected, the Status Bar will display Scan Mode.
Response Time
This function sets the response time of the instrument. Possible
selections are 0.02, 0.03, 0.1, 0.25, 0.5, 2.0, 4.0, 8.0, and Auto.
Repetitions
This function sets the number of Unknown replicates to be measured.
For example, select 3 to record readings in triplicate (see Data Print
and Average in the Manipulate menu).
5.3.1.3 Time Course Parameters
When Time Course is selected as the acquisition mode, the Time
Course Parameters dialog box is displayed as shown in Figure 5-33.
A description of the parameter functions follows:
]
)
Wavelength: 1350 ,0
Recording Range
bOW: 1-100.0 t!igh: 11000.00 I
EM Wavelength: 1397.0 1
Slit Width (nm): EX 11 0 EM 110
-Sensitivity:@HighOLowR.!;.sponseTime:
Timing Mode
(lnits ..
Beaction Time: I 60.00
@ Au!o 0 Manual o HnUh
• . 't· n i 1 "fJ? ""CjjWSI 10ft r ,.il e: u.<....
o Minutt's
Num1l.er oi Headings: IJUG i
@ Seconds
Figure 5-33 Time Course Parameters Dialog Box
EX Wavelength
This is the excitation wavelength at which data will be acquired.
Allowable entries are 0, and 220 - 900nm.
EM Wavelength
This is the emission wavelength at which data will be acquired.
Allowable entries are 0, and 220 - 900nm.
Recording Range
These entries correspond to the ordinate scale limits of the PC's graph.
The allowable range is -100.00 - 1000.00.
EX I EM Slit Width
The allowable excitation slit width settings are 1.5,3,5,10, 15,20,
6HH (half-height). The allowable emission slit width settings are 1.5,
3,5,10, 15,20, Close.
Sensitivity
The sensitivity of the detector can be set at High or Low (with High
providing approximately 50 times the sensitivity of Low).
Response Time
This function sets the response time of the instrument. Possible
selections are 0.02, 0.03, 0.1, 0.25, 0.5, 2.0, 4.0, 8.0, and Auto.
5 - 30 RF-5301PC Instruction Manual RF-530 1PC Instruction Manual 5 - 31
Menu Commands Menu Commands
Timing Mode (Acquisition Rate I Number of Readings I Units)
When Auto is selected, enter the total Reaction Time. The time unit
is always seconds. The Acquisition Rate and Number of Readings
are calculated automatically. Any channels whose X-axis values are
not currently in seconds are converted to seconds.
When Manual is selected, enter the time Unit, the Acquisition Rate
(minimum of 0.1 seconds), and the Number of Readings (maximum
of 12,000 readings). The Reaction Time is calculated on the basis of
the entered values. Any channels that are already loaded will have
their X values (time unit) converted to the time units entered in this
dialog box. When a file is loaded into a channel through the Open
option in the File menu, this data is also converted to the current time
units displayed in this dialog box. Note that selecting a small
acquisition rate and a large reaction time will affect the performance
of this software (See "Precautions on Memory Usage," Section 6.3 in
Troubleshooting, Chapter 6).
Note: In the Time Course mode, the instrument is used as the time­
base controller; data points are sent to the computer in real-time,
directly after they are read.
5.3.2 PopUp Scan Parameters
These parameters (see Figure 5-34) are similar to those in the Spectrum
mode, with the absence of the Auto File and Repeat Scan capabilities.
The software sends these parameters to the instrument upon
commencement of the PopUp Scan while saving the setup for the
currently selected acquisition mode. After the PopUp Scan has been
completed, the current instrument settings (along with any related
parameters) are restored.
5.3.3
j) )
)
·'1]11111
Spectrum Iype:
C' Excitation @ o Synchronous
-----,
EX 'f!'avelength: 1350 1nm
EM Wavelength Range: Jitart 1380 .End
(450 1
Recording Range: Low I 0.000 tiigh [1000.00 1
S.!;anning Speed: IFast IIi
Sampling !nterval (nm): 11.0 II!I
Slit Width (nm): 115 1:!1 EM

Sensitivity: @ High 0 Low
Resl!0nse Time (sec): IAuto II
Figure 5-34 PopUp Scan Parameters Dialog Box
PC Configuration
This function allow the user to designate the default data and export
directories, and the parameters for communication between the personal
computer and the fluorometer, as well as printer information. When this
option is selected, the dialog box shown in Figure 5-35 appears.
"III"iTiW:roTroT' ........
1C:\RFPC\dala\
IC:\RFPC\data\
Qata Directory
.Export Directory
fluorometer Serial Port @1 02 03 04
Iext Prinler-----------,
HP LaserJet III on LPT2:
HP DeskJel 550C Printer on LPT
. "
HI-' I-'Iotter on LPT1:
IBM Proprinter XL on LPT1:


!'iraphics Printer
IHP LaserJet III on LPT2:
1
I
III
Figure 5-35 PC Configuration Parameters Dialog Box
5 - 32 RF-530 1PC Instruction Manual
RF-5301 PC Instruction Manual 5 - 33
Menu Commands
Data Directory
Type the name of the directory to which data files are saved and loaded,
making sure that the directory entered already exists. Strict MS-DOS
protocol must be followed in the directory assignment (i.e., directory
name must start with a drive designation and end with "\", as in
C:\RFPC\DATA\).
Export Directory
Type the name of the directory to which export files (ASCII or DIF) will
be saved, making sure that the directory entered already exists. Strict
MS-DOS protocol must be followed in the directory assignment (i.e.,
directory name must start with a drive designation and end with "\", as in
C: \RFPC\EXPORT\).
Fluorometer Serial Port
Select the port number (1 - 4) to be used for the fluorometer unit.
NOTE: This software requires that a hardware interrupt be assigned
to the communication port. By IBM standards, interrupt 4 is assigned
to either COMI or COM3, but not both. Interrupt 3 is assigned to
either COM2 or COM4, but not both. Please be certain of the
employed hardware configuration before selecting this value. When
selecting a nonexistent port and ignoring the ensuing WARNING,
automatic initialization of the fluorometer unit is turned off.
Text and Graphics Printer
Selection of the text and graphics printer is contingent upon which
printers have been installed in Windows (multiple printers may be
installed). Click on the drop-down list in either the Text or Graphics
selection box to designate the printer(s) to be used (see Figure 5-35).
The Text Printer is used for all tabular output using Print commands.
The Graphics Printer is used for all graphics and graphics/table
combinations. (Note that a plotter device may usually be selected as a
text printer.)
The same printers appear in both the Text and the Graphics Printer list
boxes. Highlight the printer to be used and select OK to confirm the
designation. When the text and/or graphics printer designation is
changed using this dialog box, it may be necessary to run Control Panel
to adjust the printer port assignments.
Menu Commands
The Control Panel program can be run by making the program an icon
(see the Microsoft Windows User's Guide: Using Control Panel). Use the
Setup push button (or Control Panel) to change the page orientation,
graphics resolution and other printer features before outputting to the
printer or plotter.
5.3.4 SpeedBox Configuration
The SpeedBox Configuration dialog box (Figure 5-36) allows the user to
select the functions to be placed in the SpeedBox, as well as the style in
which the SpeedBox is displayed. All selections are automatic, on
command; there is no undo command.
SpeedBox (RJ Configuration
I!em to Change:
1111 ~ Arithmetic
Command: ~ t y l e :
i+
N ~
Arithmetic
ASCII Export R.
lBJ
Attachments
[II Average
)
Both Axes
R Channel Status
Copy
III Ii¥j
Data Print ''f,
a
Figure 5-36 SpeedBox Configuration Dialog Box
Item to Change
This drop-down list contains the functions currently assigned to the
SpeedBox; selecting an item allows for additions, changes or removal
from the SpeedBox.
Button
A button, when combined with a command, is added to the SpeedBox
with the Add or Insert commands. A selected button may also change a
current SpeedBox item. Available buttons and recommended SpeedBox
settings are shown in Figure 5-37.
Command
A command, when selected in combination with a button, can be used to
add, insert, or change an item in the SpeedBox.
5 - 34 RF-5301PC Instruction Manual
RF-5301 PC Instruction Manual 5 - 35
Menu Commands Menu Commands
Add
Select both a Button and a Command and click on Add to add a new
item to the SpeedBox.
Insert
This option inserts a new item (select a combination of Button and
Command) into the SpeedBox after the currently selected item in the
Item to Change combo box.
Change
This option is used to change a selected SpeedBox item. Select an Item
to Change, then select a new Button and/or Command and click on the
Change button to reflect the change.
Remove
This function is used to remove an item from the Item to Change list.
Select an item, click on Remove, and the item is immediately deleted
from the SpeedBox.
Exit
Select this option to close the SpeedBox Configuration dialog box and
save all changes.
Style
This list shows the eight different styles of the SpeedBox: vertical,
horizontal, single or double row, and top or side caption. Click on a style
to select it. The new style takes effect after the Exit push button is
pressed. When no selection is made, the previous style remains in effect.
~
~
LiI
~
~
~
~
~
~
"!r;+":
'Rl::"

~
[II
[0
','.
~
[II
Arithmetic
ASCII Export
Attachments
Average
Channel Status
Copy (Graph)
Data Print
Delete
DIF Export
Display Equation
Display Grid
Erase Channel
Exit
File/Channel Calc
Fonts
~ Help ~ PopUp Scan
TlJ
Parameters
liJ Hide All ~ Quantitative Mode
~ Instrument
[jl
Radal Both A e,
[II
Int/Conc Toggle lJ Radar X Axis
fl)
Limits ~ Radar Y Axis
LIJ
Line Colols
~
. ~
Rename Channel
[j]
Load Parameters ~ Save
~ Open
[1;j1 ,
Save as
~
[ft
Options
Parameters
II
[11
Save Channel
Save Parameters
[IJ
PC Configuration ~ Spectrum Mode
~
J . ~
Peak Area
[Ij
SpeedBox® Configuration
[it
:'?',
Peak Pick
[B %.
Time Course Mode
[iii
Plot
[II /xc:<
Transforms
~ POint Pick
[II
Working Curve
Figure 5-37 Recommended SpeedBox Button Settings
5.3.5 Instrument
Various monitoring and control functions of the fluorometer unit as well
)
as communication with the personal computer are performed through this
dialog box. This option also allows for setting the fluorometer
parameters. When Instrument is selected from the Configure menu, the
dialog box shown in Figure 5-38 appears.
fluorometer:
@ : 2 ~ j o Off
-!:IV Control: @On o Off
EMT Protect: @On o Off
Auto Shutter: 0 On @ Off
Dark Level Correction:
SIN Ratio Test:
Lamp Align:
Instrument Serial Number: 00000000000001
Instrument ROM Version: 0.90
Lamp Time Used: 91
Figure 5-38 Instrument Parameters Dialog Box
5 - 36 RF-5301PC Instruction Manual RF-530 1PC Instruction Manual 5 - 37
Menu Commands Menu Commands
The instrument functions are as follows:
Fluorometer
This indicates and controls the communication status between the
computer and fluorometer. Select ON to enable fluorometer
communication, and OFF to disable it. When the program has been
minimized in Windows operation to run another program, the
communication with the fluorometer unit will be suspended.
-HV Control
This function turns the Negative High Voltage control ON and OFF. The
xenon lamp used in the spectrofluorometer provides strong light intensity
but has inherent instability. The negative high voltage supplied to the
photomultiplier is controlled to compensate for fluctuation of the light
source.
PMT Protect
When the Photomultiplier Tube Protect is ON and the sample
compartment is opened, the excitation slit is automatically closed to
protect the photomultiplier from light saturation. When PMT Protect is
OFF, the excitation slit remains in the current position and the
photomultiplier tube could be saturated with light. The setting defaults to
OFF every time the program is started.
Auto Shutter
This feature enables the automatic operation of the shutter when various
functions are performed, i.e., Read. This feature protects very sensitive
samples from being damaged by blocking illumination of the sample
before the reading is taken.
Dark Level Correction
This button performs a Dark Level Correction on the instrument by
closing the shutter, taking a reading, and performing an internal
calibration of the instrument. This process may take a few seconds.
SIN Ratio Check
This function is used to check the performance of the instrument by
running a Spectrum scan (for peak height; takes a few seconds) and a
Noise scan (for the signal/noise ratio; takes approximately ten minutes).
When Perform is pressed in the instrument dialog box, a message appears
which reads: SIN Ratio Check; Fill a cell with distilled water; Set the
cell in the holder. Click either OK to automatically begin the check or
Cancel to return to the Instrument dialog box.
)
])
)
440.0 450.0 400.0 4200 3800 360.0
I;:#"itfrint ." "I
VVavelength (nrn)
Noise
1.000
I r r ; ~ ~ P h "'J 0.500
~
~ 0.000
1: "
·0500
·1.000
000 20000 400.00 600.00
Time (sec)
Instrument Serial Number: !A401932000010151
Peak Height: 1 12.556 1 SIN Ratio: 1235.572
Figure 5-39 SIN Ratio Check
Press the Stop push button or Esc key to interrupt the SIN Ratio check;
press Exit and then Perform in the Instrument dialog box to restart the
check.
Press Print to obtain a hard copy of the report which is automatically
sent to the graphics printer specified in PC Configuration Parameters (see
Section 5.3.3).
Select the Spc Graph push button to switch between the Spectrum and
Time Course graph selections. This automatically brings up the Graph
Pop-up menu (see Tutorial, Section 3.4). A portion of either graph can
be enlarged by zooming in on it (see Tutorial, Spectrum Scan, Section
3.5, #8). Note: Ctrl+R is disabled in the SIN Ratio Check.
Lamp Align
This function allows for proper positioning of a newly installed lamp in
the instrument as well as resetting of the Lamp Time Used value.
Instrument Serial Number
The fourteen digit number of the instrument appears here. The serial
number is set at the factory and cannot be changed by the user.
Instrument ROM Version
The current revision level of the ROM installed in the instrument.
5 - 38 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 5 - 39
Menu Commands Menu Commands
Lamp Time Used
The value displayed is the total number of hours the lamp has been on.
is recommended that the lamp be replaced after 500 hours of use.
It
Sip Button
Use the Sip push button to verify that the sample will reach the flow cell
in the time entered for Sip Time.
5.3.6 Attachments 5.3.7 Save Parameters
Select Attachment Parameters to set the sipper attachment parameters
) This function saves all the current parameters (including Spectrum,
in the dialog box which appears (see Figure 5-40).
Sip SpJ;.ed 0 Medium 0 Slow
Sip lime [99,9 to -99.9 sec)
£,urge Time [99.9 to -99.9 sec)
Qwell Time (0 to 99.9 sec)
Number of Binses [!] (0 to 4)

t· i" ?E\%9
K
,i';r
III".
Figure 5-40 Attachment Parameters Dialog Box
For the sipper, set the following parameters within the designated ranges:
Sip Speed
(Fast, Medium, Slow) Along with sip time, determines how much of a
sample is sipped using the sipper peristaltic pump.
Quantitative and Time Course) to a new configuration file. When
selected, the dialog box shown in Figure 5-41 appears. Type in a file
name (without extension) of up to eight characters. A comment may also
be typed into the Enter Comment text box. Select Save to save the
current parameters to disk in the directory in which the program is
installed. The file will have a .cfg extension. To change the program
default configuration, use the file name RFPC.
Enter File Name: 1mB I
Enter Comment:
This is the default configuration file.
IPi!r""">'0'

Figure 5-41 Save Parameters Dialog Box
Sip Time
(99.9 - -99.9 sec.) (length of time for sipping) Positive/negative times
determine the direction of the sipper motor (see Purge Time).
Purge Time
(99.9 - -99.9 sec.) (length of time for purging) When a negative time is
designated, the sample can be recovered. When a positive value is
entered, the sample will be flushed to the waste container.
Dwell Time
(0 - 99.9 sec.) Wait time between completion of sipping and taking a
reading.
Number of Rinses
(0 - 4) (number of rinses before each scan) The same sequence will
occur for the rinse except that no reading will be taken.
)
5.3.8 Load Parameters
This function is used to load a configuration file previously saved with
the Save Parameters command. When selected, a list box containing all
of the configuration files appears as shown in Figure 5-42.
5 - 40 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 5 - 41
Menu Commands
Menu Commands
...I.r.T7i11"iTil••••••••1
You are about to DELETE a file from disk!
@
ARE YOU SURE?
rlpc.ctg
sample.dg

IF
1"""".... '!lif).
Figure 5-44 Delete Windows of the Load Parameters Dialog Box
5.4 Manipulate Menu
The Manipulate menu contains functions which enable various methods of
manipulating data for display or printout. When the Manipulate menu is
selected, the screen appears as shown in Figure 5-45.
mm
(J) Default Conligurl!llion File Can Not be Deleted!
Ii oK")
Figure 5-42 Load Parameters Dialog Box
Move the cursor to any of the file names in the list box, and select Load.
This changes the current parameters to the values contained in the file.
The mode of data collection does not change. Select View to open a
window displaying the information entered in the Save Parameters
dialog box: file name, user comment, and time and date created (see
Figure 5-43).
j) )
The first three options, Arithmetic, Transforms, and File/Chnl Calc, perform
mathematical operations on the ordinate axis of the data. The operation is
identical for both Spectrum and Time Course data.
rtpc.ctg
This is the default configuration file.
Created: 18:27 12/15/94

W<1UsfuW?¥ft
Figure 5-43 View Window of the Load Parameters Dialog Box
When Delete is selected, a warning message appears indicating that the
highlighted file is about to be deleted (see Figure 5-44). When YES is
selected in response to the inquiry "Are You Sure?," the file is deleted
from the directory. Select NO to cancel the command. The RFPC.CFG
file cannot be deleted as it is the default configuration file.
Figure 5-45 Manipulate Menu
In Spectrum or Time Course modes, the first three procedures require the
selection of a source channel, an operation and a destination channel. The
source is the channel number of the data undergoing analysis; the destination
is the channel number where the result is stored. When the designated
destination channel already contains data (for example, the source channel),
the data in the channel will be overwritten (there will be a warning and
file
f
1:
5 - 42 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 5 - 43
Menu Commands
opportunity to cancel or proceed) and the file name for the channel will
remain as it was. When a new destination channel (default) is selected, a file
name and comment for that channel may be entered as usual.
In Quantitative mode, the currently displayed data (Standard or Unknown) is
used for the operation (as both the source and destination) of the first three
functions.
WARNING: In Quantitative mode, the Arithmetic, Transforms,
File/Chnl Calc and Average functions replace the currently displayed
data with the result of the operation.
The results of all calculations are automatically graphed, although it may be
necessary to change the graph scale (see Radar, Section 5.5.2.7) for the results
to be visible.
5.4.1 Arithmetic
Arithmetic operations are scalar operations (+, -, *, /) of the general form
S op C = D, where S is the source channel, C is a constant, D is the result
destination channel and 'op' is addition, SUbtraction, multiplication, or
division. (In Quantitative mode, S = D, that is, the source is replaced with
the result.) The operations are often useful for purposes of display, Le.,
offset a spectrum, scale a derivative, etc. When the Arithmetic option is
selected while in Spectrum or Time Course mode, the following dialog
box appears.
,Source

01
07
0)
O.
00
O.
0,
0;:
0,
,Qper8tion
@+Add
0- Sublroet
a-MUltiply
O/Qivide
I I
B
. ',. '" , ." ii
Oestinalion­
00
01
@2
0
0
Os
0&
Or
Ov
Os

Menu Commands
Select the operation, source and destination channels, enter the constant
and select OK to proceed with the calculations. Select Cancel to
terminate the process or select the Status push button to review the
contents of the channels.
When the Arithmetic option is selected while in Quantitative mode, the
following dialog box appears.
Qperators--­

0- .subtract
I.(;onstant
I
I
I
I
O'Multiply
O/Qivide
I- ;; ()K I
Figure 5-47 Arithmetic Dialog Box (Quantitative mode)
The data in the currently displayed table (Standard or Unknown) will be
replaced with the result of the operation. When operating on the
) Unknown data table, the concentration values (except raw data) are
recalculated using the new (calculated) intensity values. Select the
operation, enter the constant and select OK to proceed with the
calculations. Select Cancel to terminate the process.
5.4.2 Transforms
Transforms are function operations of the general form D=f(S), where S
is the source channel, D is the destination channel and f( ) is the
transform function. (In Quantitative mode, S=D, that is, the source is
replaced with the result). The transform functions available in Spectrum
and Time Course modes are:
Derivative (0 through 4th order, where 0 is smoothing)
Reciprocal (IN)
Logarithm (Base 10)
The transform functions available in Quantitative mode are:
Reciprocal (IN)
Logarithm (Base 10)
Figure 5-46 Arithmetic Dialog Box (Spectrum !Time Course modes)
5 - 44 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 5 - 45
Menu Commands
When the Transforms option is selected while in Spectrum or Time
Course mode, the dialog box shown in Figure 5-48 appears.
IE "tl! "",l"
Menu Commands
The smoothing and differentiation calculation is based on a convolution
function using 17 data points. The derivative wavelength and time
difference values are selectable in four steps, depending on the sampling
interval. Although a larger dX (delta X) will provide decreased noise, it
Iransform Destination
00
@ Qerivative
01
[Smoothing)
2 07
03 03 o 1/'i
04 04
0'., O"i
o !"ogarithm
Of; 06
Of 01
OB Os
o Os

sillllJ,illl Ilflm:I LA:;'Al!'.;:'
Figure 5-48 Spectrum and Time Course Transform Selection Dialog Box
Select the source and destination channels, and the transform function,
and then select OK to perform the calculations. Select Cancel to
terminate the process or select the Status push button to review the
contents of the channels.
When the Derivative (Smoothing) function is selected, another dialog
box appears (see Figure 5-49) so that the order of the derivative and the
size of the X value (Delta Lambda for Spectrum, Delta T for Time
Course) may also be designated.
Delta Lambda-- Qrder

@ 10.000
I
lllJWXllWj
.'w
tfM
o lsI
o 20.000
o 2nd
o
o .:Ird
o 80.000
O.1lh
Figure 5-49 Derivative Dialog Box
5 - 46 RF-5301PC Instruction Manual
will detract from spectrum resolution. Accordingly, select values which
will provide the best compromise between noise and resolution.
Calculation of the derivative value requires the data points before and
after the center wavelength, which means that the derivative or smoothed
spectrum cannot be calculated at both ends of the wavelength range. The
original spectrum has a unit of intensity as its ordinate value. However,
the derivative units are defined as follows:
Derivative Spectrum Time Course
1st derivative Intensity/nm Intensity/time
2nd derivative Intensity/nm
2
Intensity/time
2
3rd derivative Intensity/nm
3
Intensity/time
3
4th derivative Intensity/nm
4
Intensity/time
4
Note that, in the Time Course mode, the unit of time may be hours,
minutes or seconds, depending on the value selected in the parameters.
)
When the Transforms option is selected while in Quantitative mode, the
dialog box shown in Figure 5-50 appears.
Il;lr.
Iransform---------,
@:j'Ti
o Logarithm
Figure 5-50 Quantitative Transform Selection Dialog Box
Select the appropriate Transforms function, and select OK to perform the
calculations. Select Cancel to terminate the process.
RF-5301PC Instruction Manual 5 - 47
Menu Commands Menu Commands
5.4.3 File/Chnl Calc
When the File/Chnl Calc option is selected while in Spectrum or Time
Course mode, the dialog box shown in Figure 5-51 appears.
File or Channel Calculations are vector operations (+, -, *, /) of the
general form A op B = C where A and B are the source channel numbers,
C is the destination channel number (result) and op, the vector operation
addition, subtraction, multiplication or division.
Note that Band C cannot be the same channel number. The range
(wavelength or time) or pitch (sampling interval or acquisition rate) of
channels A and B need not be the same since any necessary interpolation
is performed using a cubic spline algorithm. The result (channel C) has
the same acquisition parameters (including range and pitch) as the first
source channel (channel A).
After choosing A, B, C, and the operation, select OK to perform the
calculations. Select Cancel to terminate the process or select the Status
Figure 5-52
push button to review the contents of the channels.
Table + testqnt > Table
Qperators
@ + Add O'Mulliply
G' ­ Subtract C'/ Qivide
zz.qnt
samkbl.qnt
zzz.qnt
tim.qnt
Ii "'oKP.:]
.
File/Channel Calculations (Quantitative mode)
The data in the currently displayed table (Standard or Unknown) will be
A
@[o:
01
02
OJ
0;.
0';
00
01
On
Oq
Qp
@+
C)­
O'
0/
A op B = C
B
00

02
)3
04
0"
.,
Or,
01
Ou
09
ki
00
0:
@2
03
04
0"
.,
Or,
0,
Oil
09
I
j) )
5.4.4
replaced by the result of the selected operation with the selected file.
The data in the selected file is treated sequentially only, that is, the ID
value in Unknown files or the concentration in Standard files is ignored.
When operating on the Unknown data table, the concentration values
(except raw data) are recalculated using the new (calculated) intensity
values.
After selecting the desired file and operation, select OK to perform the
calculations. Select Cancel to terminate the process.
Data Print
Data Print will output a selected channel's data to the screen in a
scrollable window, save it to a disk file (text), output the data to a printer,
or copy it to the Clipboard. When the Data Print option is selected, the
Data Print channel selection dialog box appears (similar to the Save

':{{""'."' ... :,," Channel dialog box in Figure 5-7, Save Channel, Section 5.1.6.1). Select
the desired data channel, click OK and the Data Print scrollable window
Figure 5-51 File/Channel Calculations (Spectrum / Time Course modes) appears, as shown in Figure 5-53. When there is only one channel
) loaded, the channel selection dialog box is not displayed. The Data Print
When the File/Chnl Calc option is selected while in Quantitative mode,
window for Channel 0 is automatically displayed.
the dialog box shown in Figure 5-52 appears.
5 - 48 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 5 - 49
Menu Commands Menu Commands
The functions available with this window are provided through the menu
bar options described in the following text. Numerous Data Print
windows may be displayed simultaneously. These windows may be
in the specified display range. When OK is selected, the table is updated
with the new values. For Time Course data, the range entered in Params
is used for the Activity Calculations.
scrolled, moved or sized using various function keys or the mouse as
described in the Microsoft Windows User's Manual.
Created: 11 :21 08/31/94
Data: Original
InstrulTlent: RF -5301
Spectrulll Tl}pe: Dl
Scan Range: 350.0nlll to 450.0nlll
IJ:'rn'I'
Enter -S.tart X Value:
le
ilil
8U I
Enter .End X Value:

Enter Skip factor:
o
"' , li,\A'of;;=1 .. ",
Figure 5-53 Data Print Window
Wauelength (nlll)
350.0
350.2
350.4
350.6
350.8
351.0
Intensitl}
4.574
4.408
4.337
4.245
4.212
4.280
350. OnIII
0.2
EX:5.0nlll EM:5.0nlTl
MediulIl
High
Auto
Manual, Open
EX
Salllple Pitch:
Slit Width:
Scan Speed:
Sensitiuitl} :
Response Tillle:
Shutter:
IJ
)
Figure 5-54 Data Print Parameters Dialog Box
Copy
Copies only the highlighted text to the Clipboard. To highlight text,
click on the first line to be selected, hold the mouse button down and
drag to the last line. Release the mouse button and select Copy from
the Edit menu. The highlighted text is placed in the Clipboard.
Copy All
Sends all of the text to the Clipboard.
Edit
This menu allows data to be copied to the Clipboard.
In the Quantitative acquisition mode, Data Print is used primarily for
printing reports. The Standard or Unknown data, as well as the
parameters, are automatically printed when Printer is selected. When
any replicate Sample IDs exist in the data, the mean of the replicates is
displayed (and printed) below the last of the replicate IDs.
Disk...
To save the currently displayed data to disk in a text file, select Disk
from the menu bar. The Text File dialog box appears, as shown in Figure
5-55.
When a channel is selected for Data Print in the Time Course mode, the
Text File
unit for the time values is the one currently set in the Time Course
Parameters dialog box.
Enter File Name: It:m,mm,
Params...
I
To view a specific range of points, select the Params option in the menu
bar of the Data Print window. The Data Print Parameters dialog box
appears, as shown in Figure 5-54. Enter the Start and End X Values and
a Skip Factor, which designates the point interval between data readings
Figure 5-55 Save Data Print Dialog Box
5 - 50 RF·5301PC Instruction Manual RF-530 1PC Instruction Manual 5·51
Menu Commands
When a file name is associated with the displayed data of the selected
channel, (the name of a file that was loaded or the name that was entered
after the scan was completed), it is displayed in the Text File Dialog Box.
Select OK to use this file name or enter a new name. This file is saved in
the data directory with a .txt extension and can be retrieved and plotted
by selecting the Plot option of the Presentation Menu or incorporated into
a third party text processing software.
Printer
Select Printer from the menu bar to automatically output the currently
displayed data to a printer.
Act. Calc. (Activity Calculation)
When the displayed data was acquired in the Time Course mode, it is
possible to calculate the Slope, Activity (slope times a multiplication
factor) and the Correlation Coefficient (estimate of closeness offit to a
linear model) over the range of data displayed (i.e., designated in
Params) by selecting Act. Calc. from the menu bar. The window shown
in Figure 5-56 appears.
.Mull. Factor:
IIllIiIiIll
Slope: 0.0200
Activity: 0.0200
Corr. Coefficient: 0.9751
Figure 5-56 Activity Calculation Dialog Box
When the Correlation Coefficient is not sufficiently close to 1 in value,
select OK and designate a more linear range using the Params option as
described above. Confirm the relative linearity by once again selecting
the Act. Calc. option. Enter an appropriate multiplication factor and
press the Enter key, or click on Recalc. to calculate (by the least squares
method) and display the Activity. In this way, it is possible to consider
the Slope over a range of Time Course data as the (linear) reaction rate,
and the Activity of the reactants as the rate multiplied by a constant.
Select Recalc. to perform the calculation again with another constant and
view the resultant activity.
Menu Commands
into a third party text processing software. When Save is selected, enter
the file name (without extension) and select OK. Select OK to close the
Activity Calculation window. To close the Data Print Window, select the
control menu box at the upper left comer of the window and select Close,
or double-click the control menu box.
5.4.5 Peak Pick
This option numbers the peaks of selected channels on the graph and
also lists them in a window. When the Peak Pick option is selected, a
channel selection dialog box appears. Choose the channel for the Peak
Pick operation and select OK. When only one channel is loaded, the
program defaults to channel 0 and no channel selection is necessary.
The graph is redrawn with each of the selected channel peaks numbered
(see Figure 5-57), and a scrollable window appears containing the list of
peaks, as shown in Figure 5-58 (also in lower left of Figure 5-57). This
procedure may be performed for any combination of the available
channels. A Peak Pick scrollable window appears for each of the
channels selected (change the size to suit). (NOTE: It is possible to select
the same channel more than once to display peaks and/or valleys using
the various Options.)
)
Figure 5-57 Peak Pick Window
5 - 52
Select Save to save the results to disk in a text file (.txt) in the data
directory. This file can be retrieved and plotted by selecting the Plot
option of the Presentation menu (see Plot, Section 5.5.5) or incorporated
RF-530 1PC Instruction Manual
Options
The Options menu in the Peak Pick window contains the following items
for modifying peak selection and display (see Figure 5-58).
RF-5301 PC Instruction Manual 5 - 53
Menu Commands Menu Commands
Find Valleys
Select Find Valleys and a check mark appears next to the Find
Valleys option, indicating that the values displayed on the graph are
minima (valleys) instead of maxima (peaks). Select Find Valleys
again (when it is checked) to remove the check mark, and the
displayed values are again maxima (peaks).
m.
Qptions fdit
I
Outj!ut Beplot
:r.­
Batch 1 1
Created: 15 :33 118/31/94
Data: Original
RF-53111
Type: EM
Scan Range: to
EX Wauelength: 41111.
Pitch: 1.11
Slit Width:
Scan Speed: Uery Fast
Sensi tiui ty: High
Response 11.5 Seconds
Shutter: Manual, Open
Ho. Wauelength Intensity
1 424.11 -19.288
2 466.11 21.473
3 514.11 -5.763
Figure 5-58 Peak Pick Window
Change Threshold
This option allows you to enter a new value for the threshold which
determines peak selection. In general, a larger threshold decreases the
number of peaks found, while smaller threshold values result in more
peaks. (See the following discussion of Peak Pick algorithm.)
Peak Pick Algorithm: A four point successive comparison method is
used to define a peak as at least four consecuti ve increases in value
followed by at least four consecuti ve decreases in data. The top of the
peak is the point before the decreases begin. In Find Valleys, the
situation is reversed (4 + decreases, bottom, 4 + increases).
The threshold is defined as the distance between a peak point and a
line connecting the two adjacent valley points (or vice versa for
valleys). When the threshold value of the peak is less than the
threshold value entry (0.001 is default), the peak is rejected. The
threshold value can be adjusted to filter out unwanted peaks or noise.
The reproducibility of the peak detection algorithm may be estimated
as 1/10 to 1/1 00 of the peak width at half the height of the absorption
band. There may be slight scanning errors for very sharp bands. In
such cases, it is necessary to compare the ordinate values step-by-step
around the peak wavelength in order to define the precise peak
wavelength (see Data Print, Section 5.4.4). In a Peak Pick window,
each designated peak is tabulated with a number (No.), the value for
the X-axis (Wavelength or Time) and the value for the Y-axis
(Intensity).
Mark Graph
When the graph is redrawn on the first selection of Peak Pick, the
positions of the peaks are automatically marked on the graph by
number. When the graph is redrawn after changing the limits using
Limits on the Graph Pop-up menu or Radar (see Limits, Section
5.5.2.4 and Radar, Section 5.5.2.7), the peaks (or valleys) are no
longer numbered. Selecting Mark Graph replaces the numbers on
the graph for the selected channel.
Edit
This menu allows data to be copied to the Clipboard.
)
Copy
Copies only the highlighted text to the Clipboard. To highlight text,
click on the first line to be selected, hold the mouse button down and
drag to the last line. Release the mouse button and select Copy from
the Edit menu. The highlighted text is placed in the Clipboard.
Copy All
Sends all of the text to the Clipboard.
Output
The Output menu contains items for handling output of Peak Pick data.
Print Table
Select Print Table to automatically print the Peak Pick data in tabular
form with the text printer selected in the PC Configuration dialog box.
Save Table
This option creates a tabular text file (with .txt extension) and saves it
to disk in the data directory. Select Save Table and enter a file name
(without extension) in the Save .txt File dialog box. The file that is
created may be used in Plot (see Section 5.5.5) or in a word processor
or editor.
5 - 54 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 5 - 55
Menu Commands Menu Commands
Graphics Plot
Select Graphics Plot to automatically plot the Peak Pick graphic data
of a selected channel with the printer selected in the PC Configuration
dialog box. The plot will print the marked peaks (and/or valleys) and
contain a list of the peaks as well as the acquisition parameters.
Replot
Select Replot to redraw the graph and clear it of all marks (including
Point Pick marks, which may be used in combination with Peak Pick; see
Point Pick).
5.4.6 Point Pick
The Point Pick option allows designation of up to 15 points on the X­
axis. The designated points are marked on the graph and the points and
corresponding Y-axis values are listed in a window. When this command
is selected and more than one channel has data, a channel selection dialog
box appears. Select the channel on which Point Pick will be performed.
When only Channel 0 has data, the Point Pick dialog box immediately
appears displaying the graph and a vertical marker line for using the
mouse to select the points (see Figure 5-59). (In Time Course, the
marker line may be on the far left at 0.0 and hard to distinguish between
the graph border.)
To use the mouse to select the points, move the mouse over the marker
line until the cursor turns into a horizontal double arrow, hold down the
left mouse button, drag the marker line to the point you want to record
and release the mouse button. The marker always snaps to the nearest
valid data point and the cursor moves to the next edit box. Thus it is easy
to enter all 15 points using the mouse. For better resolution, zoom in
using the left mouse button (see Zoom in Graph Cursor, Section 4.6). To
return the graph to its original scale, use the Graph Pop-up menu (see
Section 5.6) or use one of the Radar functions (select Radar under the
Graph push button). NOTE: When the X-axis value entered is out of the
range, the maximum or minimum value is substituted for that entry.
)
Point Pick
1"-- ,;Qpen >!
]

,.,
c
Q)
1/ Ii'l\Pb .1

450.0 500.0 5500 600.0
Wavelength (nm)
£loints
1 1-45-8.0-1 2 I478.0 I 3 I517.0 I 4 I560.0 I 5 II
6 I I 7 I I 8 I I 9 I I 10 1====
I
12
1 I
13
1 I
14
1 I
15
1 11 1 __
Figure 5-59 Point Pick Dialog Box
Press the Open push button to load a Point Pick template file previously
saved to disk using Save as.
Press the Save as push button to save the points just selected into a
template file for later recall.
Press Graph to open the Graph Pop-up Menu (see Section 5.6).
Press Cancel to close the operation with no changes.
Select OK to close the dialog box. The points are marked on the main
graph and the Point Pick window is displayed (Figure 5-60) listing the
channel's acquisition parameters and the selected points. This window
may be moved and resized for convenient viewing of the data.
5 - 56 RF-530 1PC Instruction Manual RF-5301PC Instruction Manual 5 - 57
Menu Commands Menu Commands
Qptions .Edit OutQut Beplot!
Raman Peak
Created: 11:21 08/31/94
Data: Original
Instrument: RF -5301
Spectrum Type: EM
Scan Range: 350.0nm to 450.0nm
EX Wauelength: 350.0nm
Sample Pitch: 0.2
Slit Il1dth: EX:5.0nm EM:5.0nm
Scan Speed: Medium
Sensitiuity: High
Response Time: Auto
Shutter: 11anual, Open
Ho. Wauelength (nm) Intensity
1 364.2 4.530
2 369.0 5.099
3 372.8 8.464
4 376.2 15.054
5 378.8 18.159
6 383.0 13.963
7 385.8 9.788
8 390.2 7.470
9 398.2 7.836
10 411.4 7.976
Figure 5-60 Point Pick Window
The Point Pick window menu bar contains the following commands:
Options
The Options menu contains items for modifying point selection and
display.
Change Values
This re-displays the Point Pick dialog box shown in Figure 5-59.
Mark Graph
When the graph is redrawn after the first selection of Point Pick, the
positions of the points are automatically marked by number on the
graph. When the graph is redrawn after changing the limits, the points
are not marked (see Limits, Section 5.5.2.4, and Radar, Section
5.5.2.7). Select Mark Graph to replace the marks on the graph for
the selected channel.
Edit
The Edit menu allows data to be copied to the Clipboard.
Copy
Copies only the highlighted text to the Clipboard. To highlight text,
click on the first line to be selected, hold the mouse button down and
drag to the last line to be highlighted. Release the mouse button and
select Copy to place the highlighted text in the Clipboard.
Copy All
Sends all of the text to the Clipboard.
Output
The Output menu contains items for handling the output of Point Pick
data.
Print Table
Select Print Table to automatically print the Point Pick data in tabular
form by the text printer specified in the PC Configuration dialog box.
Save Table
This option creates a tabular text file (with . txt extension) and saves it
to disk in the data directory. After selecting Save Table, enter a file
name (without extension) in the Save .txt File dialog box. The
created file may be used in Plot (see Section 5.5.5) or in a word
processor or editor.
)) J
Graphics Plot
Select Graphics Plot to graphically plot the Point Pick data of a
selected channel by the graphics printer specified in the PC
Configuration dialog box. The plot will print with the marked points
and a list of the points as well as the acquisition parameters.
Replot
Select Replot to redraw the graph and clear it of all marks (including
Peak Pick marks, which may be used in combination with Point Pick; see
Peak Pick, above).
RF-5301PC Instruction Manual 5 - 59 5 - 58 RF-5301PC Instruction Manual
Menu Commands Menu Commands
5.4.7 Peak Area The color and pattern used to highlight the area under the curve for each
Region can be changed by clicking on the appropriate Region box, which
Select this command to calculate the area for up to four separate regions
is located just to the left of the Start edit box. Pressing Alt and the
of a scan and output the results (plot and/or table) to the printer or the
region number (1, 2, 3, or 4) also selects the Region box.
Clipboard. The Peak Area dialog box appears (see Figure 5-62) when
this command is selected. The area under the curve is calculated using
The Result field is calculated with the following formula:
the following formula:
Result =(Area * FactorlDivisor).
D [ il Ae-P
The Factor is applied to all four regions and is set by pressing the
a+I3=(.L D[i])*P
Parameters push button, to bring up the Peak Area Parameters dialog
t=A
s
box (selecting Baseline to Zero calculates all areas using the X-axis as a
baseline). The Divisor is set for each individual region by entering a
13 = * (Ds + De) * (Ae - As)
value in the Divisor edit box on the appropriate row.
-+1 I+-
A
e P As
The Reset button restores Start, End, Divisor, Factor, and Baseline to
Zero to match the currently displayed area(s) and result(s).
Figure 5-61 Formula for Peak Area
Press Graph to bring up the Graph Pop-up Menu (see Section 5.6).
The Output button sends a plot and/or table to the printer or Clipboard,
and saves the table to a text file.
a
13
1 11111538.0 1 1586.0 11.000 I 4673.91 B 4673.918
Z I§S!j 1 400.0 I 1400.0 1 11.000 1 0.000 0.000
J 'Btl! 413.0 I I428.0 I 11.000 I -267.613 -267.613
! lmll !! 1 1 I
Region Start
450.0
End
500.0
W.velength (nm)
Divisor
550.0
Area
604.0
Result

I)
)
Figure 5-62 Peak Area Dialog Box
@
o Save table to text file
o Graphics Plot
o Copy graph to clipboard
o Copy table to clipboard
I M
Figure 5-63 Area Output Dialog Box
To calculate the area under a curve for a Region, enter the starting and
The Open push button can be used to recall a template file that was
ending points in the Start and End edit boxes. Enter the divisor (default
previously saved using the Save as push button. All the values for Start,
value is 1.0) in the Divisor edit box and press the Recalc push button.
End, Divisor, Factor, Baseline to Zero, color and brush types for the
The Start and End values can also be graphically selected: move the
areas, and the graph limits are replaced with those from the template file.
mouse over the left marker until the cursor turns into a horizontal double
After opening a template file the name of the file is appended to the title
arrow, hold down the left mouse button and drag the marker to the
of the Peak Area dialog box. The template file blank.pka is provided to
desired starting point. Follow the same procedure to mark the end point
clear all values in the dialog box. When changing the default values
using the right marker. Press Recalc; the area under the curve for that
contained in this file, set the desired colors, patterns, and parameters and
region is displayed and the Area and Result are calculated.
select the Save as push button.
5 - 60 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 5 - 61
Menu Commands Menu Commands
The Save as push button saves the current settings (Start, End, graph
limits, etc.) to a template file for later recall using the Open push button.
5.4.8 Average (Quantitative Unknown only)
The Average option is used in conjunction with the Repetitions selection
in the Quantitative parameters.
The relative fluorescence intensities of any replicate 10 numbers are
replaced with the average of all the relative fluorescence intensities with
the same 10 number. This selection replaces the data in the Unknown
data table with a table containing no replicate 10 numbers where the
relative fluorescence intensities of (previously) replicate 10 numbers are
the mean values.
For display of both the original data and the averages, see Data Print,
Section 5.4.4.
5.4.9 Working Curve (Quantitative Standard only)
Use this command to modify the order of the polynomial fitting for the
Standard data in the Multipoint Working Curve method. This option
allows changing the Y intercept and the order of the standard curve (l st ­
3rd order polynomial). These selections are also available in Parameters
after choosing the Multipoint Working Curve (see Figure 5-32, Section
5.3.1.2).
A minimum number of standards must be present for each order fit. As
enough standards are collected to fit the data to the selected polynomial,
the equation is calculated for review. Additional standards will prompt
for recalculation of the calibration curve.
Polynomial Order Minimum Number of Standards
No Zero Interception Zero Interception
1st 2
2nd 3 2
3rd 4 3
The Working Curve option is used to change the curve fitting
parameters for the Standard data. Refer to Multipoint Working Curve
under the Method drop-down box in the Quantitative Parameters. Note
that this selection is not in effect when the method is not set to
Multipoint Working Curve.
5 - 62 RF-5301PC Instruction Manual
5.5 Presentation Menu
The Presentation menu consists of items which monitor and control the
presentation format of data files on the screen and determine their printout
format (see Figure 5-64).
~ ... ,:j ...
600.0 550.0 500.0 450.0
50.

~
f:
file Acquire Mode .(;onfigure
c : : : : ~ (
Figure 5-64 Presentation Menu
5.5.1 Channel Status
This option is used for monitoring the screen presentation status of the
occupied channels. The Channel Status window is accessed via the
Presentation menu or directly from the keyboard by entering Ctrl+C.
Wavelength (ron)
I}
)
When Channel Status is selected in Spectrum or Time Course mode, the
window shown in Figure 5-65 appears. In Quantitative mode, the Channel
Status dialog box displays whether the Standard and Unknown have been
saved to disk.
RF-5301 PC Instruction Manual 5 - 63
Menu Commands Menu Commands
Line Channel File Hame Displayed Saved
:r.


Figure 5-65 Channel Status Dialog Box
For each occupied channel, the window displays the following information
from left to right:
Line: Color and type
Channel: Channel number (0 - 9)
File Name: Shows the file name (or *No Name*) associated with the
channel
I)
)
Displayed: Y / N indicates whether a channel is currently displayed on
the graph
Saved: Y / N indicates whether the data has been saved to disk
The Displayed status may be changed for any channel by selecting the channel
(using up/down arrow keys or mouse) and selecting the Display On/Off push
button. The same operation may be performed by double-clicking the desired
channel.
The acquisition parameters of the data in any of the channels (see Figure 5-66)
may be viewed by highlighting the channel (using up/down arrow keys or
mouse) and selecting the Parameters push button.
File: BATCH1 N1 Batch 1 Sample Number 1
Instrument Type: RF-5301
Created: 15:33 08131194 Data: Original
Spec1rum Type: EM EX Wavelength: 400 nm
EM Wavelength Range: 400 to 600 nm
Sample pitch: 1.0
Scan Speed: Very Fast Sensitivity: High
Slit Width: EX: 10.0 nm EM: 5.0 nm
Response Time: 0.5 sec
Shutter Mode: Manual Shutter Position: Open
Figure 5-66 Spectrum Parameters Display Window
After reviewing the parameters, select OK to return to the Channel Status
window.
Select OK in the Channel Status window to return to the main screen. The
graph is updated to reflect any changes made with regard to displayed
channels.
5.5.2 Graph
5.5.2.1 Copy
Use this command to automatically copy the main graph to the
Clipboard. The bitmap sent to the Clipboard is the actual size of the
main graph. To send a smaller size graph, resize the main screen so
that the graph is the appropriate size, and select Copy. Any other
windows (i.e., Data Print, etc.) or parts of other windows that are over
the main graph area also become part of the bitmap sent to the
Clipboard. (See the Microsoft Windows User's Guide for information
on the Clipboard and the Copy command.)
5.5.2.2 Display Grid
Select Display Grid from the Presentation menu to toggle the display
of a grid pattern on the graph (see Figure 5-67).
5 - 64 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 5 - 65
••••••••
••••••••
Menu Commands Menu Commands
Select Cancel to exit the dialog box without changing the graph
limits.
5.5.2.5 Line Colors
The Line Colors option allows assignment of a particular color and
type of line for each of the ten channels to suit the type of monitor or
printer/plotter and personal taste of the user (see Figure 5-69).
Colors
Data: !!.asic CoiOls:
Ii.'

L .:. _] Channell wj
••
h:'$'<iI Channel 2
1*;ffiH Channel 3 .1Iilil ••••••
Wt;;@'1 Channel ..
1:!Phd Channel 5
L:¥1wl Channel 6
Channel 7 +
•••••
Line and Annotation llpes: CoiOls:
Figure 5-67 Spectrum Graph with a Grid
ttFf#iflI#%$?f1 fiI:lfill ffi"Fil DDDDDDDD
FFJ r;i'9 If,;'J • !NEI IiZ7iI WFlj­
DDDDDDDU
5.5.2.3 Fonts
Ifil84F,jif.
"
Allows selection of font, font style, size, effects and color for the
coordinate values, axes labels and headings.
])
) Figure 5-69 Plot Colors Dialog Box
5.5.2.4 Linnits
To change the line characteristics, select a channel number from the
This option allows you to change the X- and Y-axis limits of the graph
Data list box to highlight the channel. Then select the color and line
on the screen, or zoom in or out on an area, without using the mouse
440.0 450.0
(see Figure 5-68).
I Graph Limits

['­
Mali: IIImII 1 }of.ax: 1600.0
Min: I -21.345 I Min: 1400.0
1
I

Figure 5-68 Limits Dialog Box
type to be associated with the designated channel from the Basic
Colors or Custom Colors and Line and Annotation Types selection
boxes.
Note: The color and line type selected for channel 0 is used for the
PopUp Scan. Line color and line type for the Standard and Unknown
data points and the Standard Curve may be selected in the Colors
dialog box.
When a black and white printer is used (dot matrix or laser printers),
line patterns can be used to discriminate between scans.
Enter maximum and minimum values for the X- and Y-axes and select
OK to update the graph axes. Select Reset to scale the graph to the
acquisition parameters currently set in the Parameters dialog box in
the Configure menu. When the minimum value is greater than the
maximum, they are automatically switched.
To add a Custom Color, select the Define Custom Colors push
button. The Custom Color Selector dialog box appears as in Figure 5-70.
Define the color either by directly entering the numeric value or by
using the mouse to specify the color coordinates. When the setting is
correct, select Add Color. Multiple custom colors may be added at
5 - 66 RF-530 1PC Instruction Manual RF-5301PC Instruction Manual 5 - 67
Menu Commands Menu Commands
one time-each consecutive selection will be placed one box to the
right. When the last box is reached, the first color will be overwritten.
Select Close to end the color definition.
.!led:


ColorlSQ.lid
hum: BIQe:
Figure 5-70 Custom Colors Dialog Box
5.5.2.6 Options
Select Options to change the grid lines, type and its colors, line type,
and the background color, as well as enable or disable Rounded and/or
Minor Tick marks.
1'- Options
Grid bines:
I
[gJ Bounded Tick Marks
Grid Color: D Minor Tick Marks
IBJ Teal II
Line Iype:
0 0 11ed 1- I
f!.ackground Color:

PH£t:1 Silver
1-------­
Figure 5-71 Options Dialog Box
Select the Rounded Tick Marks check box to create tick marks at
evenly calculated intervals on the graph (i.e., 20.0, 40.0, 60.0, and
80.0). Minor Tick Marks are smaller marks evenly spaced between
the rounded marks. When Rounded Tick Marks is not enabled (not
checked), the tick marks are placed at fixed locations on the graph.
Refer to Figure 5-72 for a visual comparison.
764. 2 764.27"T'---,-------,----,-------,
600.00
400.00 389.65
200.00
15.03iL1 ..LI__---"--__---'---__--'---_-----l

37.567 58.456 79.34 37.567 50.000 60.000 70.000 79.34
Fixed Rounded
Figure 5-72 Fixed vs. Rounded Tick Marks
) Select OK to save changes.
Select Cancel to terminate the changes and close the dialog box.
Select Reset to restore the line characteristics to the values last saved.
5.5.2.7 Radar
The Radar function adjusts the graph limits so that the entire range of
the chosen axis data of all displayed channels is visible on the graph.
Radar may be applied to the X- or Y-axis independently, or radar may
be applied to both axes simultaneously. This option may be selected
from Graph under the Presentation menu or from the Graph Pop-up
Menu. Or, apply radar to both axes from the keyboard using Ctrl+R.
5.5.3 Hide All
Select Hide All from the Presentation menu to clear the graph of all
displayed data. (It will not delete any data.) The data is still available in
the respective channels. A check appears next to the Hide All option to
indicate that it is selected. Select Hide All, when it is checked, to display
all currently loaded data on the graph. This function is useful when the
screen is cluttered with previous data that cannot yet be stored or erased.
The screen will be clear for the next acquisition channel.
5 - 68 RF-5301 PC Instruction Manual RF-5301PC Instruction Manual 5 - 69
Menu Commands Menu Commands
5.5.4 Show SpeedBox
Select Show SpeedBox from the Presentation menu to display or hide the
SpeedBox. The SpeedBox is an accelerator device which uses picture
buttons to represent commonly used menu commands. Figure 5-73
shows a configured SpeedBox. The SpeedBox contains a caption bar that
can be used to move the SpeedBox around the screen just like a window.
(See the Microsoft Windows User's Guide for more information on
moving windows.)
Figure 5-73 Configured SpeedBox
The SpeedBox control menu has three selections, Move, Close, and
About; select Move to move the SpeedBox around the screen via the
keyboard; Close hides the SpeedBox; About displays the About
SpeedBox dialog box. The SpeedBox is configured using SpeedBox
Configuration under the Configure menu (see Section 5.3.4). The
SpeedBox may also be configured by double-clicking on its caption bar.
The RF-530IPC software remembers the last position of the SpeedBox
when the program is exited or the SpeedBox is hidden.
5.5.5 Plot
This option enables layout of a combination of graph overlays,
parameters, and text files on the four quadrants of a page for output to a
graphics printer. Before selecting Plot, confirm that the graphics printer
has been installed and is selected in the PC Configuration dialog box.
When Plot is selected while in Spectrum or Time Course modes, the
Plot Layout dialog box appears, as shown in Figure 5-74.
Graph Params. File
·'W,!WiITl
Channel/F. Name Quadrant[s)
I
[8]
f!1
0
0
[8]
0
0
I
I
Screen
0
01
rzJ1
0203 04
[8]2 [8]j 04
I
QI
0
0
0
0
0
0
I
I
01
0,
0203 04
0203 04
tffiJ 3 4
Figure 5-74 Plot Layout Dialog Box (Spectrum I Time Course modes)
A maximum of four items (Rows A - D in the dialog box) may be chosen for a
single printout, one for each quadrant of the page. Beginning with Row A,
select either Graph, Params. (parameters), or File using the check boxes in
the selection box adjacent to A.
When Graph is selected, the Plot Data dialog box appears, as shown in Figure
5-75. Select one or more channels of data to be plotted in the conventional X,
I) ) Y format, or select Screen to plot the entire contents of the graph as it appears
on the screen. A title may be added to this graph by entering text in the Graph
Title box, and the Y-axis range may be modified by entering new upper and/or
lower values.
·Jri'flI:roU".
O!lM 05
OJ Dr,;
O!
OJ Ofl
O.H
(8) [ I Spectra Qnly
Graph Iille

fAxis Rimgt
(,1(""''': I <D.WI I
Lm.<fer: I
Figure 5-75 Plot Data Dialog Box (Spectrum I Time Course modes)
RF-5301PC Instruction Manual 5 - 71
5 -70 RF-530 1PC Instruction Manual
Menu Commands Menu Commands
The Spectra Only option in the dialog box may be checked in the case of
overlaying data only in a graph which has already been printed. This
option requires a printer which allows multiple printouts on a single page.
Select OK to close the Plot Data dialog box and update the ChannellF.
Name column in Row A in the Plot Layout dialog box.
NOTE: When more than one data channel is selected, the X-axis
range is taken from the acquisition parameters of the lowest channel
number selected, or from the screen when selected. Note also that the
Y-axis range given in the Plot Data dialog box is not used when Screen
is selected.
Next, select the appropriate quadrants in the Quadrant(s) column of Row
A to determine the portion of the page to be occupied by the plot. (Refer
to the quadrant layout designation at the bottom of the Quadrant(s)
column.) Either one, two or four quadrants may be selected for one lot.
Select the Preview push button to view the layout on the screen as it will
appear when printed. Click on the Preview screen or press any key to
return to the Plot Layout dialog box.
To include data acquisition parameters in the printout, assuming that at
least one quadrant is available, select Params. as the second item (Row
B) to bring up the Parameter Channel dialog box. Only one channel's
parameters may be selected per item. Select OK to close the dialog box
and update the ChannellF. Name column in Row B of the Plot Layout
dialog box.
Select an available quadrant from the Quadrant(s) column as described
above.
Any text file (.txt) may also be included as another item in the page
format when at least one quadrant remains available. These include Data
Print, Peak Pick, Point Pick, Activity Calculation, or any file created in a
word processing or editor program (such as Microsoft Notepad).
Select File in the next available row, and then select a file name (only one
per item) from the Open dialog box which appears. The default directory
is the data directory entered in the PC Configuration dialog box. Select
OK to close the box and update the ChannellF. Name column in the next
row of the Plot Layout dialog box.
When Plot is selected while in Quantitative mode, a Plot Layout dialog
box similar to the one described above appears (see Figure 5-76).
@it layout
Std. iinY. Params. File
AI[gJ 0001
12
1
000
[gJ1
fCO=O 00[
QIO 0001
'I tffiJ
Figure 5-76 Plot Layout Dialog Box (Quantitative mode)
As above, a maximum of four items (Rows A - D in the dialog box) may
be chosen for a single printout, one for each quadrant of the page.
Beginning with Row A, select either Std. (Standard), Unk. (Unknown),
Params. (parameters) or File, using the check boxes in the selection box
adjacent to A.
)
When Std or Unk is selected, the Plot Data dialog box appears as shown
in Figure 5-77. Select Table to display the data in a tabular format and
enter a title when desired. Note that any portion of the table that does not
fit in the selected area of the page will not be displayed. Select Graph to
display the data in a graphic format. Enter a title (when desired) and the
desired limits for the X- and Y-axes.
Format------------,
o Iable
Tit!e:
Max Min

I 100.00 II 0.00 I
't Axis: 11000.000 II 0.000 I

Il'B6;e't:"1 I'cancel I
Figure 5-77 Plot Data Dialog Box (Quantitative mode)
Format Quadrantls)
Graph
[gJ1 02 03 04
aa.M 01 [gJ203 04
Std. Params. 01 o 2 04
01 020304
5 - 72 RF-5301PC Instruction Manual RF-5301PC Instruction Manual 5 -73
Figure 5-78 Quantitative Window Showing Equation under the Graph
Menu Commands Menu Commands
Select Params. to display the acquisition parameters on the printed page.
NOTE: Chi is the index representation of the variance of the measured
Choose Standard or Unknown to display the file name and comment of
standard values from the calibrated points defined by the following
the Standard or Unknown with the parameter display. (All other
formula:
parameters are the same for both the Standard and Unknown).
The Setup push button allows the user to configure the selected printer's
options, i.e., print quality, printout orientation, landscape/portrait, etc.
( Std(C) _f(C) )2
Chi =
f(C) I
After ascertaining the acceptability of the overall page format using the
Preview function, select Print in the Plot Layout dialog box to output the
result to the graphics printer. When Print is selected, a Cancel Printing where Std (C) is the standard intensity value of the concentration C, and
dialog box is displayed while the output is being produced. The software fCc) is the calculated intensity value by the calibration curve f ( ).
is free to operate and perform other functions when the Cancel Printing
dialog box disappears. When the Print Manager is enabled, the printer
:'! . ~ )
,1·'11";
continues in the background until the assigned plot is finished.
[ile Acquire Mode l;onfigure Manipuia!e Eresenta!ian tlelp
When Cancel is selected, the printout is cancelled and the dialog box is
closed.
Note: In some cases the use of the Print Manager may not be desired
(i.e., with networks). To disable Windows Print Manager, please refer
to the Microsoft Windows User's Guide.
)
5.5.6 Int/Cone Toggle (Quantitative mode only)
Select Int/Conc Toggle from the Presentation menu to toggle the units
used for the Y-axis of the Unknown display between the intensity and
concentration units. Each time this is selected, the Unknown graph
Y-axis will change to the alternate unit. Note that the state of the screen
Y-axis (intensity or concentration units) is used for graphs printed using
Plot.
5.5.7 Display Equation (Quantitative mode only)
Select Display Equation from the Presentation menu to display or hide
the equation below the graph.
)
Standard
Humber Cone. Int.
1 56.000 2.251
'" 3 0.0000 3.443
4 0.0000 3.154
5
Standard

~
~
1:
2O.00J <O.OOJ 61.600
mgll
Cone - klint' kO
k1 = -11 ~ . 5
kO = 378.2
Chi-Square: O . 2 ~ 2 0 8
5 -74 RF-5301PC Instruction Manual RF-530 1PC Instruction Manual 5 - 75
Menu Commands
5.6 Graph Pop-up Menu
The Graph Pop-up Menu is accessed by pressing on the right mouse button
when the cursor is inside the graph area. The menu appears on the screen
wherever the mouse cursor is placed. Select any Graph Pop-up menu function
to display a dialog box for that function. Clicking on the main screen with the
left mouse button removes the Graph Pop-up menu.
5.6.1 Copy
Use this command to automatically copy the main graph to the Clipboard.
(See Section 5.5.2.1)
5.6.2 Cross Hair
Click on Cross Hair and then Display. As the mouse is moved, the X,Y
coordinates can be seen along the axes of the graph referencing the
intersection of the cross hair (see Figure 3-9, Tutorial). To remove the
cross hair, reselect the Graph Pop-up menu and click on Display.
Select Lock to lock the cross hair in a selected place on the graph.
5.6.3 Fonts
Allows selection of font, font style, size, effect and color for the
coordinate values, axes labels and headings.
5.6.4 Limits
This option allows changing of the X- and Y-axis limits of the graph on
the screen, or zooming in on an area without using the mouse (see Section
5.5.2.4).
5.6.5 Line Colors
The Line Colors option allows assignment of a particular color and type
of line for each of the ten channels to suit the type of monitor or printer/
plotter and personal taste of the user (see Section 5.5.2.5).
5.6.6 Options
Select Options to change the grid line, type, colors and line type. The
background color may also be changed as well as enabling or disabling of
Rounded and/or Minor Tick marks. (See Section 5.5.2.6)
5.6.7 Radar
The Radar function adjusts the graph limits so that the chosen axis data
of all displayed channels are completely visible on the graph. (See
Section 5.5.2.7.)
Menu Commands
5.7 Help
The Help selections offer a summary of the RF-530l PC software. Help is
especially useful when you require information quickly or when the instruction
manual is not available.
5.7.1 Help for RF-5301 PC
Select this menu item to open the Windows Help program and load the
RF-5301 PC Help file. The initial Help window displays the acquisition
modes for which help is available. When selecting one of these, help on
any menu item is available. The user may also select Search to locate
information on a specific topic. Please refer to the Microsoft Windows
User's Guide for more information on the Windows Help System.
5.7.2 How to Use Help
Select this item to access information on the Windows Help system. This
may be used as a quick reference in addition to the Microsoft Windows
User's Guide.
5.7.3 About RF-5301 PC
Select About RF-5301PC from the Help menu to see the program
version, the software licensee, and the software serial number. To exit
this dialog box, select OK.
II
)
5 - 76 RF-5301PC Instruction Manual RF-530 1PC Instruction Manual 5·77
--
Troubleshooting
6. Troubleshooting
6.1 Communication Problems
Communication failures are often due to oversights in the instrument
preparation or neglect of periodic maintenance. These are generally easily
overcome and corrected. If the instrument has power but communications
cannot be established with the instrument (when OFF is displayed in the
Fluorometer Window), check the table below for possible solutions.
Table 6-1 Communications Errors and Remedial Actions
Items to be checked
RS-232C serial cable
Computer RS-232C port
Remedial Action
Fasten the connectors securely on the terminal at the
fluorometer unit and at the computer. Select Instrument
under the Configure menu. Select the ON radio button to
establish communications with the fluorometer unit.
: Select OK; Fluorometer Setup should be displayed in the
, Fluorometer Window.
- - _ . _ ~ - -
Check the documentation for the computer to see that the
port is being addressed properly (dip switch, jumpers,
setup, etc.) Select PC Configuration under the Configure
menu. Designate the proper port number under
. Fluorometer Serial Port. Establish communication with
the fluorometer unit as outlined above. If successful, store
these parameters by selecting Save Parameters under the
Configure menu. Save these parameters (as file name
RFPC.CFG) to create the default configuration file used
when the program is initiated.
RF-5301 PC Instruction Manual 6 - 1
i
Troubleshooting
6.2 Software Operational Error Messages
Table 6-2a Error Messages and Remedial Actions
IVIessage
AutoZero Level Over
Bad or Missing
Configuration File!
Cannot Establish
Communcations!
Cannot Set -HV mode to
0\1
Co d Checksu
mman m
Communcations
Initialization Failure!
Communications Open
Failure!
Remedial Action
The instrument attempted to perfonm an AutoZero and the
· signal level was too high for this operation. Verify that
there is no sample in the cell holder and repeat the last
operation. Separate the excitation and emission
wavelengths when they are too close; lower the sensitMty
setting; or decrease the bandwidth.
The configuration file is conrupted or is not in the proper
directory. The default configuration file is used at
initialization. This may be copied into the correct directory
• from the original master disk.
I
i PC has ost connection with fluorometer unit. Can be
caused by loose or broken cable. Verify proper cable
oonnections and continuity.
Xenon lamp is OFF. Negatwe high voltage cannot be
tumed on when the lamp is not lit.
There was a problem sending or receiving data to or from
E r1 the instrument. Please try your last operation again. If
rro. this problem persists, please contact your Service
1
! Windows was unable to initialize the specified
Asynchronous Communcations Port for some unknown
reason. Try rebooting PC. If the port still fails, check for
hardware problems.
Windows was unable to open the specified Asynchronous
Communications Port. This can be caused by another
program (i.e., Windows Tenminal) having oontrol of the
COM Port. Another cause oould be a Fatal Windows
Error (one which returns the PC to MS-DOS). This will
force any programs attached to the COM Port to exit
without detaching from the port. If Windows receives a
· Fatal Error, always reboot the PC. If you select a
I Fluorometer Serial Port in the PC Configuration Parameter
! Entry Fonm that does not exist, this error message will be
· issued.
Troubleshooting
Table 6-2b Error Messages and Remedial Actions (continued)
Message
DCB Initialization Failure!
Default configuration file
cannot be deleted!
Directory Does Not Exist!
Error Converting Channel
Time Units! Delete
Unnecessary Channels!
Error Creating Temporary
)
File!
Error Opening Rle!
Error Reading Rle!
Emission Monochromator
Slip
Excitation Monochromator
Slip
Remedial Action
Windows was unable to create a Device Control Block for
the specified Asynchronous Communications Port. This is
usually caused by a corrupted configuration (.CFG) file.
Default configuration file cannot be deleted as it is
required for the software to load.
The directory entered must exist on the disk. See MKDIR
in the MS-DOS manual.
· When the time units are changed in Parameters under the
· Configure menu, all current channels are changed to
those units for consistency. This message will appear
when there is not enough memory available. Delete any
channels not needed at this time to make room for new
i data.
: A temporary file is written to disk for Peak Pick and Point
· Pick operations. If the file -TEMP_PK.TXT exists in the
: data directory, delete it. Also see Rle Creation Error.
I same as Rle Creation Error!
· Generally, this is a hardware problem. The disk file has
been corrupted, the door of the disk drive is open, etc.
Consult the computer and MS-DOS documentation.
The emission monochromator of the fluorometer unit has
slipped while moving. Discontinue communications with
the instrument by turning off the Fluorometer Window,
and power the instrument OFF and ON. If the problem
persists, consult the hardware section of the manual or
contact your Service Representative.
The excitation monochromator of the fluorometer unit has
slipped while moving. Discontinue communications with
the instrument by turning off the Fluorometer Window,
and power the instrument OFF and ON. If the problem
persists, consult the hardware section of the manual or
contact your Service Representative.
Communications Time Out! Same as Cannot Establish Communications!
6-2 RF-5301 PC Instruction Manual RF-530 1PC Instruction Manual 6-3
- - --------
Troubleshooting
Table 6-2c Error Messages and Remedial Actions (continued)
Troubleshooting
Table 6-2d Error Messages and Remedial Actions (continued)
Message Remedial Action
The configuration file in question (those with a .CFG
extension) cannot be loaded because it is not the right
Invalid Configuration File! type for the instrument being used. This may occur when
a completely foreign file type was stored with the .CFG
extension.
Fluorometer unit was unable to interpret the instruction
Invalid Fluorometer
sent by the PC. This may be caused by a corrupted .CFG
Commard
file or by a loose or broken communications cable.
A value was input in a dialog box that was outside the
Invalid Entry! acceptable limits or of an invalid fonmat. Enter the correct
value and proceed.
Invalid Numerical The lower limit of the recording range must be lower than
Relationship! the higher limit.
Xenon lamp illumination time is abnonmal. Please reset
Lamp Time is Invalid ! the lamp time to zero (0). If it occurs again, call your
: Service Representative.
II
)
1- -- --I- -,-- - -­
: Other programs are conflicting with Microsoft Windows
Main Initialization Error! ! and are not compatible. Discontinue the operation of
; other loaded software running under Windows.
;...-----_._-­
Main Window Creation i
i See Main Initialization Error.
Error!
i
1 Not When this error
Memory Error!
even when memory is freed up, reboot the system.
, No More Channels , All RAM channels for scans are occupied. Select Erase
Available! Channel under the File menu.
Printer must be installed through Windows using the
No Printers installed! Control Panel or when installing Windows via SETUP.
See the Microsoft Windows User's Guide.
The Average function found no sample IDs with the same
No replicate IDs found.
, value, therefore nothing was averaged.
Not a Valid Configuration The selected configuration file is for a different application.
File for Current j Use unit's software.
RF-530 1PC Instruction Manual
Message
File Creation Error!
File will not be displayed
entirely. Do you want to
continue?
File Write Error!
General Communications
Error!
Remedial Action
An attempt has been made to write a file to the disk which
violates DOS protocol (e.g., a directory that is nonexistent
or a full disk) or computer has encountered a bad sector
or improperly formatted disk. Check the PC Configuration
and consult the computer and DOS manuals. Check that
the disk drive door is properly closed when using floppy
disks.
When printing a text file on a graphics printer, the file may
, be too large to fit within the defined graphics area. When
Continue is selected, the text will be trimmed and not fUlly
printed. When cancelled, the text file may be edited (e.g.,
editing the file with Microsoft Notepad) to make it smaller
to fit into the graphics space. The printer resolution can
also be redefined in the Microsoft Control program to
produce small fonts and allow the file to fit into the
allowable graphics range.
I ' --------­
, Same as File Creation Error.
Same as Transmit or Receive Error!

Graphics Initialization Error! See Main Initialization Error.
Windows has run out of graphics memory. Windows must
Graphics Memory Error!
be restarted.
Graphics Printer Error!
Graphics Printer has NOT
been Selected!
Incorrect File Type!
Same as Text Printer Error.
, The proper printer must be selected in the software as
I well as in the Microsoft Windows Control Panel program.
Save to disk under Save Parameters.
The file in question cannot be loaded because it is not
valid for the mode of operation or analysis.
RF-5301PC Instruction Manual 6-4 6-5
Troubleshooting Troubleshooting
Table 6-2e Error Messages and Remedial Actions (continued) Table 6-2/ Error Messages and Remedial Actions (continued)
Message
Not Enough Memory for
Calculations!
Not Enough Memory for
Operation!
Not Enough Memory for
Peak Pick!
Optimal Wavelengths Not
Found
Options Are Not Attached
Reaction time may not
exceed 100,000
Scan Interrupted by EOT
Slit Origin Not Found
Some data has NOT been
saved to disk! Do you wish
to continue?
Table will not fit in area
selected.
Remedial Action
Not enough computer RAM space free to perform the
designated operation. Restart Windows.
Same as Not Enough Memory for Calculations!
Same as Not Enough Memory for Calculations!
. The Optimal Wavelength Search could not locate the EX
and EM wavelengths. The fluorescence peak was not
detected during a search. Please adjust the parameters
and try again.
The instrument option is not responding. Please check
the attachments for proper cable connection to the
instrument.
In Time Course mode, the total time (Acquisition Rate x
Number of Readings) may not exceed 100,000. When
using manual timing mode, reduce either Acquisition Rate
or Number of Readings.
The instrument interrupted a scan that was being
performed on the computer. Turn the Fluorometer
Window OFF and turn the instrument OFFand ON. When
the problem persists, refer to the instruction manual or
contact your Service Representative.
A motor in the instrument is not working properly. Please
turn the instrument OFF and ON and try again. If the
problem persists, please contact your Service
Representative.
Changing the mode of data acquisition or exiting the
program may leave data in channels which have not been
stored to disk. When permanent storage to disk is
necessary, answer NO to cancel the request to change
the acquisition mode or exit the program.
When printing data tables via Plot, data which does not fit
into the allotted quadrant(s) is not printed. To fit the table
on one page, change the graphics printer to Portrait
orientation (see PC Configuration) or select more
quadrants. Otherwise, use Data Print under the
Manipulate menu for large data tables.
Message
Text Printer Error!
Text Printer has NOT been
Selected!
This is NOT a Valid
Configuration File!
Too Many Timers for Opt.
Bench!
)
Too Many Timers,
Time/Date Update
Disabled!
Transmit or Receive Error
Unknown Communication
Error Status!
Value Out of Range
Wavelength Origin Not
Found
Window Creation Error!
Remedial Action
1) The printer driver is not installed. Install the proper
printer drivers in Windows Control Panel.
2) The printer is not online. See that the printer is online
and ready to accept commands from the computer.
3) The printer was not initialized by the computer when it
was turned on. Turn the computer OFF and ON and
reinitialize.
4) An improper connection was made. Verify that the
correct interface port (COM1, LPT1, etc.) is selected to
correspond to the hardware configuration.
Same as Text Printer Error!
The selected configuration file is not compatible with this
software.
Other programs may be running that use the clock.
Discontinue other applications using the timer.
Same as Too Many Timers for Opt. Bench!
PC has lost connection with fluorometer unit. Can be
caused by loose or broken cable. May also be caused by
running other programs while communicating with the
optical bench.
Same as Transmit or Receive Error.
Refer to the section in this manual which lists the
acceptable range for the parameter entered.
A motor in the instrument is not working properly. Please
turn the instrument OFF and ON and try again. If the
problem persists, please contact your Service
Representative.
. A window is requested to appear but the command cannot
I be carried out. In almost all cases this is due to a
: shortage of memory. See memory-related items above.
6-6 RF-530 1PC Instruction Manual RF-530 1PC Instruction Manual 6-7
Troubleshooting
6.3 Precautions On Memory Usage
When the available memory (RAM) for program operation becomes slow,
performance of Microsoft Windows shows a tendency to degrade and disk
drive activity increases. To avoid decreased performance due to insufficient
RAM, the following precautions should be taken to maintain adequate RAM
for efficient operation.
1) When the employed applications require large data files, load only a few
data files at a time into the channels.
2) Refrain from running too many Windows applications when data is
loaded into many channels or the loaded data files are very large.
3) When increased disk drive activity is noticed, this may be taken as a
warning to save data and erase channels or close some of the other
applications.
)
RF-5301PC Instruction Manual 6-8
Appendices
Appendix 1 Fundamentals of Fluorescence
Analysis
1.1 What is Fluorescence?
The phenomenon in which certain kinds of substances emit light upon
absorbtion of various levels of light energy, without involving heat generation,
is called luminescence. Luminescence which is emitted on exposure to
ultraviolet or visible rays is called photo luminescence.
Fluorescence and phosphorescence, representative of photoluminescence,
possess hues different from the reflected or transparent color of a substance,
emitting longer wavelengths of light than incident light. Familiar examples are
the green color observed in a red ink in the daylight (eosin aqueous solution)
and a pale color in shirts.
1.2 Principles of Fluorescence
This section explains the principles of fluorescence using an organic
compound as an example. When a molecule in the ground state So is exposed
)
to light, the kinetic energy of the electrons in the molecule is altered, moving
the molecule into the excited state SI' with a higher energy level as shown in
Figure A-I.
Non-rad'
S,
T.
Light abso
,(
./
~
~ 'Ion-radiation transition
Fluorescence
rption
Phosphorescence
So
Figure A-J Principles of Fluorescence
RF-5301PC Instruction Manual A - 3
Appendices Appendices
However, the excited state soon changes back to the ground state as the
molecule is deactivated by radiating the energy in the form of heat or light.
The light which the molecule emits as it returns to the ground state So is called
fluorescence.
Since part of the energy of the absorbed light has been lost as vibration or
heat energy, the emitted light is of a longer wavelength than the light to which
the molecule was originally exposed (Stokes law).
The light which the molecule emits as it returns from the triplet state T[ to the
ground state So is called phosphorescence. Phosphorescence has a lifetime
longer than 10-
4
seconds because of the need for spin transformation.
1.3 Three Basic Laws of Fluorescence
Law 1: In order for a substance to fluoresce, light absorption must first take
place.
Law 2: Generally, fluorescence has a longer wavelength than excitation
light.
Law 3: The quantum yield of fluorescence (Q) is determined by the
frequency of non-radiative transfer of the absorbed energy to heat or
others.
ne
Q
ne + nf
where ne Frequency of light emission
and nf Frequency of non-radiative transition
Law 1 dictates that the task of testing an unknown sample begins with
measurement of its absorption spectrum with a relatively higher concentration.
When there is no absorption at all, then it is considered that fluorescence is not
emitted even when the sample is excited at the wavelength. Conversely,
fluorescence is emitted most intensely when the sample is excited with the
absorption peak wavelength.
Law 2 indicates that, since part of the energy of the light absorbed is lost as
heat or vibration, the residual radiation energy is reduced, shifting the
wavelength of the fluorescence to the red. Hence, the task of measuring the
fluorescence spectrum is simplified by scanning only the wavelengths longer
than the excitation light.
Law 3 uses the quantum yield of fluorescence (Q) to indicate what proportion
of the energy absorbed is radiated as fluorescence. The higher the value of Q,
the easier the substance produces fluorescence. Table A-I lists the quantum
yields of typical fluorescent substances.
Table A-l Compounds and Quantum Yield of Fluorescence
]') )
Compound Solution Quantum Yield
Fluorescein 0.1 N-NaOH 0.92
Eosin 0.1N-NaOH 0.19
Rhodamine B Ethanol 0.97
Riboflavin Aqueous solution, pH7 0.26
Anthracene Ethanol 0.3
Naphthalene Ethanol 0.12
Indole Water 0,45
Chlorophyll a Ether 0.32
Chlorophyll b Ether 0.12
1.4 Advantages of Fluorescence Analysis
1.4.1 High Selectivity
When multiple substances are intermixed in a single sample, selective
fluorescence measurement of a particular substance is possible without
removing substances which do not fluoresce.
Further, even when several fluorescent substances are intermixed in a
sample, measurement can still distinguish among them by setting their
wavelengths in an appropriate manner when they vary in excitation or
emission wavelengths.
A-4 RF-5301PC Instruction Manual RF-5301 PC Instruction Manual A-5
Appendices
1.4.2 High Sensitivity
Fluorescence analysis may be 100 to 1,000 times more sensitive than
absorptiometry, allowing measurement of ultra micro-quantity.
1.5 Considerations for Fluorescence Measurement
1.5.1 Sample Temperature Effect
In many samples, each rise of 1°C in sample temperature is said to
produce a loss of I - 2% in fluorescence intensity, though this is
dependent on the type of sample. Certain biochemical samples reportedly
produce a change of some 10% in fluorescence intensity in response to a
temperature change of 1°C. Temperature-dependent samples need to be
tested in the constant temperature cell holder.
1.5.2 Photochemical Reaction of Samples
Exposure to excitation light causes certain samples to produce a
photochemical reaction, resulting in a change of fluorescence intensity.
Testing of such samples may benefit from regulating the shutter to
expose the sample to excitation light only for the duration of
measurement. Other techniques available would be to increase the
spectrum scan speed to the maximum or to narrow the bandwidth of the
excitation light.
1.5.3 Fluorescence from Impurities
Peaks caused by fluorescent compounds other than the component of
interest during fluorescence spectrum measurement are called
fluorescence from impurities. Fluorescence from impurities are
associated with CD scattered light and its secondary light, ClJ Raman
scattered light of the solvent, and (J) fluorescence from the solvent or cell.
CD and ClJ are discussed later in Section 1.5.4. For (J), commercially
available grades of reagents often detect fluorescence caused by the
presence of impurities in the solvent. Remember that high-sensitivity
testing in the ultraviolet region is particularly susceptible to the effects of
solvent fluorescence. Fluorescence-free solvents are available for
fluorescence analysis use. To remove concern over the possible effects
of solvent fluorescence, either use these commercial solvents or refine
your own solvent.
Appendices
Generally, quartz cells will produce weak fluorescence when they are
excited at around 260nm because of impurities (aluminum) inherent in
the cells. Use of a fluorescence-free cell that contains artificial quartz (PI
N 200-34594-03) is recommended for exciting traces of samples at
around 260nm.
1.5.4 Effects of Scattered Light
In fluorescence testing, peaks caused by scattered light and Raman
scattering may be observed in addition to the fluorescence components of
primary interest. Scattered light is associated with the scattering of
excitation light by solvent entering the emission monochromator.
Scattered light is manifest particularly in the testing of solid samples.
These peaks are readily distinguished because they appear at the
wavelength of the excitation light.
Depending on the characteristics of the grating monochromator, scattered
light may also appear in the wavelength regions two and three times the
excitation light wavelength as second order and third order light,
respectively. With an excitation light wavelength of 220nm, for example,
second order light appears at 440nm and third order light appears at
660nm. A short wavelength cutoff filter is inserted at the emission side
to remove such light.
)
Further, when the excitation light wavelength is set to visible, light
having half the wavelength is also emitted from the excitation
monochromator. With an excitation light wavelength of 450nm, for
example, light of 225nm may also be observed. A short wavelength cut­
off filter is inserted at the excitation side to remove such light. An
optional the filter set is available when second order or third order light is
of concern.
Raman scattering appears at wavelengths longerthan the excitation
wavelength and may be confused with sample fluorescence. Raman
scattering is distinguishable because it remains essentially unchanged in
intensity with changes of the sample concentration, and also because
changes of excitation light wavelength vary the position of the peaks
caused by Raman scattering but not the peak position of fluorescence.
Table A-2 summarizes the relationships between the excitation light
wavelength and Raman peak.
A-6 RF-5301PC Instruction Manual RF-5301PC Instruction Manual A-7
Appendices Appendices
Table A-2 Relationships between the Excitation Wavelength and Raman Peak
1.6 Sample Measurement of a Fluorescence Spectrum
Solvents and Raman Peak Wavelengths (nm)
Water Ethanol Cyclohexane Carbon Tetrachloride Chloroform
248 271 267 267
Excitation
light
wavelength
(nm)
313
365
405
436
350
416
469
511
344
409
459
500
344
408
458
499
320
375
418
450
346
410
461
502
1.5.5 High Concentration Samples
Samples with high concentrations can be a source of errors. Because
spectrofluorometers are designed to detect fluorescence emitted from the
center of the cell, the excitation light would be absorbed on the front face
of the cell when the sample concentration is too high. The failure of the
excitation light to fully reach the center of the cell causes a loss of
apparent fluorescence intensity. Further, that portion of fluorescence
emitted from the center of the cell having a shorter wavelength is
reabsorbed by the sample in the cell, making the spectrum look as if it
were shifted toward the longer wavelength side.
Generally, samples with absorbances up to 0.02 (in a 10 mm cell) in the
ultraviolet region are said to be free from these phenomena.
1.5.6 Effects of Cell Contamination
In fluorescence analysis, the slightest smear on the cell could affect
measurement accuracy. Especially, when the sample is left in the cell,
the solvent could evaporate and adhere to the cell wall as a persistent
smear. In testing an extremely dilute sample, dirt on the external, as well
as internal, walls of the cell should be of concern. When a sample
solution contacts the external walls of the cell, wipe it off completely
with tissue paper (free of bleaching agents) before inserting the cell in the
cell holder.
1.5.7 Effect of Dissolved Oxygen
Generally, oxygen dissolved in a solvent exerts a marked fluorescence
extinction effect on certain samples (quenching). When quenching by
dissolved oxygen is not negligible, solvent degassing is required. Solvent
degassing is conveniently accomplished by bubbling dry nitrogen into the
solvent or decompressing the solvent under a vacuum.
Figure A-2 shows a fluorescence spectrum of an aqueous solution of sodium
salicylate. Generally, the fluorescence spectrum of a dilute solution provides a
record of the fluorescence of the sample as well as contributions from
Rayleigh and Raman scatter, second-order light, and solvent impurities.
Raman scattered light of water; fluorescence of solvent impurities, sodium
salicylate; and second-order scattered light are shown in the spectrum
displayed in Figure A-2.
Excitation scattered light (282nm)
100
Raman spectrum of solvent
Relative Fluorescence of
intensity sodium salicylate
50
Fluorescence
of impurities
)
contained in solvent ~ , =:':(-L / / / / ~ " !
300 400 500 600
Figure A-2 Spectrum of a Sodium Salicylate Aqueous Solution
1.7 Excitation Spectrum
Figure A-3 shows the excitation spectrum of a sodium salicylate aqueous
solution. Excitation at the peak of 302nm is found to maximize fluorescence
with the highest excitation efficiency. The peak of 405nm is caused by a
scattering of the excitation light.
Since the peak of the excitation spectrum and that of the absorption spectrum
essentially correspond, the peak wavelength of the excitation spectrum of a
particular sample can be estimated by analogy when its absorption maximum
wavelength is known. In precise terms, because it is a corrected excitation
spectrum that is analogous to the absorption spectrum, the apparent peak of the
excitation spectrum does not completely match that of the absorption
spectrum.
A-8 RF-5301PC Instruction Manual RF-530 1PC Instruction Manual A-9
Appendices Appendices
302nm (peak wavelength) Since the absorbance is proportional to the concentration C, this equation can
be transformed by integration to
100
B (A. ') = KC
Relative
intensity
Hence, the calibration curve that is proportional to the concentration C is a
straight line. As the concentration of the sample rises, however, the
50
calibration curve would be curved as previously explained in High
Concentration Samples, Section 1.5.5. Figure A-4 shows calibration curves
Scattered light generated with a diaminostilbene aqueous solution.
100 100
Relative
Relative
200 300 400
fluorescence
fluorescence
Wavelength (nm)
intensity
intensity
50 50
Figure A-3 Excitation Spectrum of Sodium Salicylate Aqueous Solution
1.8 Fluorescence Calibration Curve
o 20 40 60 80 100
The relationship between the intensity and concentration of fluorescence
emitted from a point in a cell can be stated as:
1) )
o 2
(x 1O-'fl g/ml) (flg/ml)
Concentration Concentration
db (A ') =~ E A F A ( A ') KAbA I
47tn Figure A-4 Working Curves for Aqueous Solutions of Diaminostilbene
where
References:
db (A ') Intensity of the fluorescence observed at wavelength A'
"Principles, Application, and Equipment Structure of Fluorescence Analysis,"
n Refractive index Shimadzu Fluorescence Analysis Course, Shimadzu Corporation
Reflective coefficient
EA Intensity of the excitation light at wavelength
H(A') True fluorescence intensity at wavelength Ain the
spectrum emitted by the excitation light at wavelength A'
K Absorbance at wavelength
dA' Bandwidth at wavelength A'
4 6 8 10
A - 10 RF-5301PC Instruction Manual RF-5301PC Instruction Manual A-11
Appendices Index
Appendix 2 Spectrofluorometer Overview Index
A spectrofluorometer is an instrument designed to expose a sample to
excitation light and measure the resultant fluorescence emitted from the
sample for qualitative and quantitative analysis. Figure A-5 illustrates the
arrangement of a spectrofluorometer using the RF-5301PC as an example.
(
Emission

monochromator
2
Figure A-5 Arrangement afthe RF-5301PC
The excitation monochromator CD disperses light from a light source to
isolate the excitation light. A large aperture grating, measuring 55mm by
55mm, is used in the excitation monochromator to collect as much light as
possible, because the brighter the excitation light is, the more sensitive the
unit becomes. The grating can be rotated by a motor so that simple key
operations can set the excitation light at the desired wavelength.
A cell loaded with a sample is set into the cell holder ell. The emission
monochromator (j) disperses the fluorescence emitted from the sample for
measurement by a photomultiplier. The fluorescence monochromator also
uses a grating as large as that of the excitation monochromator to collect as
much light as possible.
The xenon lamp generally used in spectrofluorometers provides the
advantages of exceptionally high light intensity and a continuous emission
spectrum. But its fluctuated illumination would magnify the signal noise
unless it is compensated. Moreover, because the emission spectrum of the
lamp, spectral sensitivity of the photomultiplier, etc.-collectively known as
instrument functions-are not uniform, the spectrum tends to be distorted.
These problems are alleviated by monitoring part of the excitation light with
the monitor photomultiplier @ and feeding it back to the fluorescence
photomultiplier (3). (This scheme of measurement is called light source
compensation. )
Symbols
-HV Control 5-38
A
Acquire Mode Menu 4-3,5-12
Acquisition Rate 5-32
Activity Calculation 5-52
Area under a curve 5-61
Arithmetic 4-4, 5-44
ASCII Export 4-2, 5-6
Attachments 4-4, 5-40
Auto File 5-26
Auto Scan Mode 5-30
Auto Shutter 5-38
Auto Timing Mode 5-32
Auto Zero 5-15
Average 4-5, 5-62
B
Baseline to Zero 5-61
1) )
c
Cable Connections 2-2
Calibration Curve A-1O
Cell Blank 5-20
Cell Contamination A-8
Change Threshold 5-54
Change Values 5-59
Channel 3-6, 5-7
Calculations 5-48
Erase 5-9
Rename 4-2, 5-10
Save 5-7
Status 4-5, 5-2, 5-4, 5-9, 5-63
Clear after Save 5-9
Clipboard 4-10, 5-59
Colors 4-6, 5-67, 5-76
Communication Problems 6-1
Concentration 5-17, 5-28, A-8
Range 5-30
Units 5-30
Configuration Files 4-1
Configure Menu 4-3, 5-22
Copy 5-59, 5-62
Copy All 5-59
Correlation Coefficient 5-52
Cross Hair 4-9, 5-76
Custom Colors 5-67
D
Dark Level Correction 5-38
Data Acquisition Modes 4-1
Data Directory 5-2, 5-34
Data Interchange Format 5-7
Data Print 4-4, 5-49
Data Translation 5-6
Default Parameters 4-1
Delete 5-5
Derivative 4-4, 5-45
DIP Export 5-7
Directory
Default 4-3
Export 5-34
Display Equation 5-74
Display Grid 4-5, 5-65
Dwell Time 5-40
E
Edit 5-51, 5-59
Standard 5-17
Unknown 5-18
Emission Wavelength 5-29
Erase Channel 5-9
Errors 6-1
Software 6-2
Excitation Spectrum A-9
Excitation Wavelength 5-29
F
File Menu 4-2, 5-1
File/Chnl Calc 4-4, 5-47
Find Valleys 5-54
A - 12 RF-5301 PC Instruction Manual RF-5301PC Instruction Manual 1-1
Index
Fluorescence A-3
Measurement A-6
Principles A-3
Spectrum A-9
Fluorescence intensity. See Intensity
Fluorometer
Serial Port 5-34
Fluorometer Communication 5-38
Fonts 5-66
G
Go To WL 5-13
Graph 5-65
Graph Cursor 4-9
Graph Pop-up Menu 5-39, 5-76
Graphics Plot 5-56
H
Hide All 4-6, 5-69
I
ill numbers 5-62
Impurities A-6
Instrument 4-4
Instrument Parameters 5-37
Int/Conc Toggle 4-6, 5-74
Intensity
fluorescence 5-13
International Support 4-10
K
K-Factor 5-28
L
Lamp Align 5-39
Lamp Time 5-40
Line Colors 5-67, 5-76
List Files of Type 5-4
Load Parameters 4-4, 5-41
Logarithm 5-45
M
Manipulate Menu 4-4, 5-43
Manual Timing Mode 5-32
Mark Graph 5-55
Menu
Configure 5-22
File 5-1
Help 5-77
Manipulate 5-43
Presentation 5-63
Method of Quantification 5-28
Minor Tick Marks 5-68
Mode
Quantitative 5-15
Spectrum 4-3, 5-12
Time Course 4-3, 5-19
Multi-Point WorlGng Curve 5-28
N
No Name 5-3, 5-8
Number of Readings 5-32
Number of Rinses 5-40
o
Open 5-1
Optimal Wavelength 5-14
Options 5-59
Output 5-55
p
Parameters 4-3
Attachment 5-40
Default 4-1
Instrument 5-37
Load 4-4, 5-41
Peak Area 5-61
PopUp Scan 5-32
Quantitative 5-27
Save 4-4, 5-41
Spectrum 5-22
Time Course 5-30
PC Configuration 4-3, 5-33
Peak Area 4-5, 5-60
Parameters 5-62
Peak Pick 4-5, 5-53
Algorithm 5-54
Plot 4-6, 5-70
PMT Protect 5-38
Point Pick 4-5, 5-56
RF-5301PC Instruction Manual
Index
PopUp Scan
Parameters 4-3, 5-32
PushButton 5-15
Presentation Menu 4-5
Print Table 5-55
Printer
Text and Graphics 5-34
Printer Setup 2-3
Purge Time 5-40
Push Button
AutoZero 5-15
Go to WL 5-13
PopUp Scan 5-15
Search Optimal WL 5-14
Shutter 5-14
Standard 5-16
Start 5-15
Stop 5-15
Unknown 5-16
Q
Quadrants 5-72
Quantitative Mode 4-1, 4-3, 5-2, 5-15
1) Quantitative Parameters 5-27
Quick save 5-3
R
Radar 4-6, 5-69
Raman Light 5-14
Raman Scattering A-7
Raw Data Measurement 5-28
Raw Data Mode 5-2
Reaction A-6
Reaction Time 5-32
Read Push Button 5-17
Reciprocal 5-45
Recording Range 5-24
Rename Channel 4-2, 5-10
Repeat Scan 5-24
Repetitions 5-30
)
Replot 5-56
Response Time 5-24
Rounded Tick Marks 5-69
S
SIN Ratio Check 5-38
Sampling Interval 5-24
Save 4-8
Parameters 4-4, 5-41
Save As 5-4
Save Channel 5-7
Save Table 5-55
Scan Speed 5-24
Scattered Excitation Light 5-14
Scattered Light A-7
Search Optimal WL 5-14
Selectivity A-5
Sensitivity 5-24, A-6
Set Limits 3-6, 4-8
Show SpeedBox 5-70
Shutter
Auto Shutter 5-38
Push button 5-14
Single Point WorlGng Curve 5-28
Sip Button 5-41
Sip Speed 5-40
Sip Time 5-40
Slit Width 5-24
Smoothing 5-45
Software Errors 6-2
Software Installation 2-2
Spectrofluorometer A-12
Spectrum, Excitation A-9
Spectrum Mode 4-1, 4-3, 5-12
Spectrum Parameters 5-22
Spectrum Type 5-23
SpeedBox Configuration 4-3, 5-35
SpeedBox® 4-9
Standard Push Button 5-16
Start Push Button 5-15
Status Bar 5-25
Stop Push Button 5-15
Survey Scan 5-21
T
Temperature A-6
Text and Graphics Printer 5-34
Threshold 4-5, 5-54
Tick Marks 5-68
RF-5301PC Instruction Manual 1-2 1-3
Index
Time Course 5-7, 5-10, 5-19
Parameters 5-30
Time Course Mode 4-1, 4-3
Timing Mode
Manual 5-32
Transforms 4-4, 5-45
Translate
DIP 4-2
U
Unknown Push Button 5-16
v
Version
Instrument ROM 5-39
W
Warning 5-26, 5-43
Wavelength
Emission 5-29
Excitation 5-29
Range 5-23
Wavelength Selection
Read Push Button 5-13
Set Push Button 5-13
Working Curve 4-5
Multi-Point 5-28
Single Point 5-28
Z
Zoom 4-9
)
RF-5301PC Instruction Manual 1-4

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