Manual Spectroflourometer softwareRF5301PC

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
1. IntrOduction 1~1
2. Installation 2~1
) ) 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
First Edition: April 1995 3.1 Initialization " " " .. "". " " " " ". ".""" " .. 3-1
3.3 Push Buttons " "" " "" .. 3-2
SHJMADZe SCIENTIFIC iNSTRUMENTS, L'Ic. (SSI) PROVIDES TillS PuBUCATION "As Is" WITHOUT WARRANTY OF ANy 3.4 Graph Pop-Up Menu " "." " 3-3
KIND, EITHER EXPRESS OR WPUED, iNCLUDING, BUT NOT LIMITED To, THE WPUED WARRANTIES OF MERCHANT
3.5 Spectrum Scan (Instrument is ON) " .. "" " " .. ""." .. 3-4
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. 3.6 Quantitative (Instrument is ON) " " " " "."" 3-10
3.7 Time Course (Cell holder is empty) "" " "" "" .. 3-14
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 4. Software Overview 4-1
publication. SSi may make improvements and/or changes in the produet(s) and/or the prograrn(s) described in 4.1 Default Parameters 4-1
this publication at any time.

4.2 Data Acquisition Modes " " .. " .. " " " " "" ""." 4-1
It is possible that this publication may contain reference to, or information about, SSi products (instruments 4.3 Main Menu ""." " ""." .. ""."."."."""""." ".".".""."."." 4-2
and programs), programming, or services that are not announced in your country. Such references or 4.3.1 File Menu " 4-2
information must not be construed to mean that SSi intends to announce such products, programming, or 4.3.2 Acquire Mode Menu " 4-3
services in your country. 4.3.3 Configure Menu " 4-3
4.3.4 Manipulate Menu " " """ 4-4
This document and the software described in it are furnished under license from SSi and may be copied only
4.3.5 Presentation Menu " " .. " "" .. " .. " " " 4-5
in accordance with the terms of such license.

4.3.6 Help Menu " 4-7
Address any comments to: Shimadzu Scientific Instruments, Inc., 7102 Riverwood Drive, Columbia, MD 4.4 Status Bar .. " .. " " .. " " " .. """"" .. "" .. " "." .. "" " 4-7
21046. SSi may use or distribute any of the information which you supply in any way it believes appropriate 4.5 PopUp Scan™ Push Button "" .. " " " .. "" " 4-8
without incurring any obligations whatsoever. 4.6 SpeedBox® "." ""." .. " .. " " "" "." .. " "." .. " 4-9
The following registered trademarks are used throughout this document:

4.7 The Graph Cursor " " .. """"" " "" ",, 4-9
4.8 Clipboard .. " "". "."." ""."."."" " "."."",," 4-10
IBM, PC DOS and PS/2 are registered trademarks of International Business Machines, 4.9 International Support .. "" " " .. "" " """ "." 4-10
Inc.
5. Menu Commands 5-1
Microsoft and MS-DOS are registered trademarks and Windows is a trademark of

~icrosoft Corporation.
5.1 File Menu " " " " """"""".". 5-1
) 5.1.1 Open " " " "" " " " " 5-1
PopUp Scan Thl is a trademark of Shimadzu Scientific Instruments, Inc., U.S.A. 5.1.2 Save ".""."""" " " " .. ,, 5-3
5.1.3 Save as " " 5-4
Speed Box'" is a registered trademark of Shimadzu Scientific Instruments, Inc., U.S.A. 5.1.4 Delete " " 5-5
5.1.5 Data Translation " 5-6
5.1.6 Channel 5-7
© Copyright 1995 Shimadzu Scientific Instruments, Inc., U.S.A.

5.1.7 Exit " 5-11
ii
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 Fluore~cence? A-3
5.3 Configure Menu 5-22
5.3.1 Parameters 5-22
1.2 Principles of Fluorescence A-3
5.3.2 PopUp Scan Parameters 5-32

\ 1.3 Three Basic Laws of Fluorescence A-4
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
5.5.2
5.5.3
Channel Status
Graph
Hide All
5-63
5-65
5-69
5.5.4 Show Speed Box 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
List of Figures Figure 5-19

Make Standard DiaLog Box 5-19
Figure 5-20
Time Course Screen 5-20
Figure 2-1
PC Configuration Parameters Dialog Box 2-4
Figure 5-21
Time Course Push Button Menu 5-20
Figure 2-2
Printer Setup Dialog Box 2-4
Figure 5-22
Configure Menu 5-22
Figure 2-3
Save Parameters Dialog Box 2-5
Figure 5-23
Spectrum Parameters Dialog Box 5-23
Figure 2-4
Initialization Window 2-5
Figure 5-24
Repeat Scan Parameters Dialog Box 5-24
Figure 3-1
Initialization Window 3-1
Figure 5-25
Repeat Scan Mode Window 5-25
Figure 3-2
Application Window of the RF-5301PC Software 3-2
Figure 5-26
Repeat Scan / Auto File Warning Messages 5-25
Figure 3-3
Spectrum Push Button Menu 3-2
Figure 5-27
Auto File Dialog Box 5-26
Figure 3-4
Graph Pop-up Menu on Main Screen 3-3
Figure 5-28
Auto File Scan Mode Window 5-27
Figure 3-5
Spectrum Parameters Dialog Box 3-4
Figure 5-29
Quantitative Parameters Dialog Box 5-27
Figure 3-6
File Name Dialog Box 3-5
Figure 5-30
K-Factor Parameters Dialog Box 5-28
Figure 3-7
Spectrum Screen after Data Collection 3-5
Figure 5-31
Single Point Working Curve Dialog Box 5-28
Figure 3-8
Radar Cascading Menu 3-6
Figure 5-32
Multipoint Working Curve Dialog Box 5-29
Figure 3-9
Graph Showing Cross Hair 3-7
Figure 5-33
Time Course Parameters Dialog Box 5-30
Figure 3-10
Plot Layout Window 3-7
Figure 5-34
PopUp Scan Parameters Dialog Box 5-32
Figure 3-11
Plot Data Dialog Box 3-8
Figure 5-35
PC Configuration Parameters Dialog Box 5-33
Figure 3-12
Plot Layout Preview Window 3-9
Figure 5-36
SpeedBox Configuration DiaLog Box 5-34
Figure 3-13
Quantitative Parameters Dialog Box 3-10
Figure 5-37
Recommended SpeedBox Button Settings 5-36
Figure 3-14
Multipoint Working Curve Dialog Box 3-11
Figure 5-38
Instrument Parameters DiaLog Box 5-37
Figure 3-15
Edit Standard Dialog Box 3-12
Figure 5-39
SIN Ratio Check 5-38
Figure 3-16
Quantitative Screen after Standard is Entered 3-12
Figure 5-40
Attachment Parameters Dialog Box 5-39
Figure 3-17
Figure 3-18
Save Channel Dialog Box

Time Course Parameters Dialog Box
3-13
3-14
D Figure 5-41
Figure 5-42
Save Parameters Dialog Box

Load Parameters Dialog Box
5-40
5-41
Figure 3-19
Set Limits Dialog Box 3-15
Figure 5-43
View Window of the Load Parameters Dialog Box 5-41
Figure 3-20
Time Course Screen after Noise Plot 3- 15
Figure 5-44
Delete Windows of the Load Parameters Dialog Box 5-42
Figure 4-1
PopUp Scan Window and Menu 4-8
Figure 5-45
Manipulate Menu 5-43
Figure 5-1
File Menu 5-1
Figure 5-46
Arithmetic Dialog Box (Spectrum/Time Course modes) 5-44
Figure 5-2
Open Dialog Box 5-2
Figure 5-47
Arithmetic DiaLog Box (Quantitative mode) 5-44
Figure 5-3
Save as Dialog Box 5-4
Figure 5-48
Spectrum and Time Course Transform Selection Dialog Box 5-45
Figure 5-4
Delete Dialog Box 5-5
Figure 5-49
Derivative Dialog Box 5-46
Figure 5-5
Delete this file? Dialog Box 5-5
Figure 5-50
Quantitative Transform Selection Dialog Box 5-47
Figure 5-6
ASCII / DIF Export Dialog Box 5-6
Figure 5-51
File/Channel Calculations (Spectrum / Time Course modes) 5-48
Figure 5-7
Save Channel Dialog Box (Spectrum / Time Course modes) .. 5-8
Figure 5-52
File/Channel Calculations (Quantitative mode) 5-48
Figure 5-8
Save Channel Dialog Box (Quantitative mode) 5-8
Figure 5-53
Data Print Window 5-50
Figure 5-9
Erase Channel Dialog Box 5-10
Figure 5-54
Data Print Parameters Dialog Box 5-51
Figure 5-10
Rename ChanneL Dialog Boxes 5-10
Figure 5-55
Save Data Print Dialog Box 5-51
Figure 5-11
Acquire Mode Menu on the Spectrum Screen 5-12
Figure 5-56
Activity CaLcuLation Dialog Box 5-52
Figure 5-12
Spectrum Push Buttons 5-13
Figure 5-57
Peak Pick Window 5-53
Figure 5-13
Wavelength Selection Dialog Box 5-14
Figure 5-58
Peak Pick Window 5-54
Figure 5-14
Search Optimal Wavelength Dialog Box 5-14
Figure 5-59
Point Pick Dialog Box 5-57
Figure 5-15
Quantitative Screen 5-16
Figure 5-60
Point Pick Window 5-58
Figure 5-16
Quantitative Push Buttons 5-16
Figure 5-61
Formulafor Peak Area 5-60
Figure 5-17
Edit Standard Dialog Box 5-18
Figure 5-62
Peak Area Dialog Box 5-60
Figure 5-18
Edit Unknown Dialog Box 5-19
Figure 5-63
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 Installation
Minimum software requirements or

• MS or PC Disk Operating System (DOS), Version 3.3 in the Windows Program Manager, select Run under the File menu,
type a:[b:]\setup and click OK.
• Microsoft'" Windows™, Version 3.1 or later
3. During installation the user will be visually notified of the progress
Recommended optional accessories through screen messages and the movement of the status bar.
• Mouse-type pointing device
4. During initial installation the user will be prompted for the name of the
• Math co-processor licensee (customer name), and the directory in which the program will be
installed; the default directory is C:\RFPC. The program will decompress
• Non-fluorescent cell (lOmm) (PIN 200-34594-03) files at this point.
5. A dialog box appears asking "Would you like to view the README file
2.3 Hardware Installation 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.)
2.3.1 Personal Computer Requirements
This software requires that an IBM compatible asynchronous port be 6. Upon completion of the installation process, a Program Group named
installed in the personal computer. A hardware interrupt must be Shimadzu will be created, if not already present, and the RF-5301PC
assigned to the communication port. By IBM standards, interrupt 4 is icon will be placed in it. The user may move the icon to another group if
assigned to either COMI or COM3, but not both. Interrupt 3 is assigned desired. Refer to the Microsoft Windows User's Manual for more
to either COM2 or COM4, but not both. Verify that the personal information.
computer has a properly configured asynchronous adapter (serial port)
before continuing. 1) ) 2.5 Communications and Printer Setup
2.3.2 Cable Connections Prior to operating the RF-5301PC software with the RF-5301PC, the
After the instrument is positioned, the RS-232 interface cable is used to communication port and printer parameters must be configured.
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 Procedure
the RF-5301PC, and secure the cable with the screws. Next, connect one 1. At the MS-DOS prompt, type: WIN and press the Enter key.
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 2. When the main Windows screen appears, locate the RF-5301PC icon
screws. which may be found in the Shimadzu program group.
3. Double-click on the RF-5301PC icon to execute the program.

2.4 Software Installation
4. When the main screen appears (see Figure 3-2), move the cursor to
Before installing the RF-5301PC software, it is necessary to first install Configure on the main menu, and click the left mouse button.
Microsoft Windows. Please refer to the appropriate Microsoft Windows
documentation for the installation procedure. The installation procedure for 5. Select (click on) PC Configuration from the Configure Menu (see
the RF-5301PC software is performed as described below. ) 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
Procedure
Serial Port item. Use either the arrow keys or the mouse to select the
communication (COM) port (1-4) which is used for communication
1. Insert the RF-5301PC floppy disk into the A: or B: drive. between the personal computer and the fluorometer unit.
2. At the C> prompt, type: WIN A[B]:SETUP and press Enter
2-2 RF-5301 PC Instruction Manual RF-5301PC Instruction Manual 2-3

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
Qata Directory 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
.f.xport Directory IC:\RFPC\data\ I Figure 2-3).
fluorometer Serial Port @1 02 03 04
Iext Printer §raphics Printer
HP laserJet III on lPT 2: lot IHP laserJet ilion lPT2: It)

Enler File Name: IIi1ii!J -I
.
HP OeskJel 550C Prinler on lPT
ItSetup·:·1 Enler Comment: IThis is the default configuration file.
HP Plotter on lPT1:
IBM Proorinter Xl on l PT1:
I"'" . "I
,"' .OK", I; Cancel, J IBF8·")"'1
),:.' , ave, ...> 1',,;,:;t. ':anC'l",lr.jt.:
,'"" --~
Figure 2-1 PC Configuration Parameters Dialog Box Figure 2-3 Save Parameters Dialog Box
6. Next, select the appro priate printer(s) which are currently connected to 8. Upon returning to the main screen after validating the above setting(s),
the RF-530IPC syste m from the Text and/or Graphics Printer drop down select Configure from the main menu. Select Instrument from the
list. Click on the Set up push button of the Text and/or Graphics Printer available selections, and set the fluorometer to ON (see Instrument,
item to configure the printer(s) settings (see Figure 2-2). (The printer Section 5.3.5). Select OK, and the initialization procedure will begin.
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)
D Instrument: RF-5300
~§i @ffIj;lg4?i4 EA If-mf-i'tf=J:ntfitbTiFMiQ".,i"'ijii Serial Number: 00000000000001

ROM Version: 0.90
Orientation
111lk.~Jf@llWI
I
w
~ 0
@Poitrail
landscape
~ROM
~RAM
~opies: ~ [~+J i'l EEPROM

Printer Resolution I rCal!ridges/SIMhh 1F'."';o..."D'%. '7'j
'.'.
*,~!!l~.~~-:";j"';f
~ Excitation Slit
@ ~oo dpi 0 100 dpi @) Emission Slit
~1.
~",)~ ~
@) Excitation Monochromator
Printer Memmy
110~ [ill Emission Monochromator
fage Protection: 1011 il'J o Baseline
Memory [MOj: 12 III
- Resource Saving IKDj - [JTesting ~ Passed ~ Failed
PCL~ I ps:IO tIl o TryeType Screen Fonls Installed
Figure 2-4 Initialization Window
Figure 2-2 Printer Setup Dialog Box
)
2-4 RF-5301 PC Instruction Manual RF-5301 PC Instruction Manual 2-5

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
~RAM
~ EEPROM
@l Excitation Slit
~ Emission Slit
tl Excitalion Monochromator
Biil Emission Monochromator
) o Baseline
I!J Testing ~ Passed • Failed
Figure 3-i initialization Window

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' :":J~,
F;fe ~cquire Mode l;;onfigure Manipulate eresentation tlelp
) Pop-up Menu, Section 5.6)
IlIjl
eresentation t!elp
Title
Bar
Main
Menu
>
~
c
~
<;
./ / / /
Push
Buttons
~ I) )
SpeedBoxCi> Status Bar Lamp Window AutolRepeat Mode Window fluorometer Window
Figure 3-2 Application Window of the RF-5301 PC Software
Figure 3-4 Graph Pop-up Menu on Main Screen

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.
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
Comment box by tabbing to it. Select Save to save the data in the next
Spectrum Type: Excitation available Spectrum channel. When Discard is selected, the data is erased
EM Wavelength: 400 from memory.
EX Wavelength Range: 375 (Start); 475 (End)
Recording Range: -10.000 (Low); 500.0 (High)
Scan Speed: Fast Enter File N~me: c---~
Sampling Interval: 1.0

Entet Comment
EX / EM Slit Width:
Sensitivity:
10 /10
High
I I
Response Time: 0.02 I. s.y~ I I 01&",,"' I
Repeat Scan / Auto File: No setting required at this time.
Figure 3-6 File Name Dialog Box
,""'1 ....
o Emission o Synchronous I
Spectrum Iype:
EM Wavelength:
@ Excitation
1
400 I nm D l::!elp
EX Wavelength Range: ~tart 375

1 I .End
1475 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):
Sensitivity:
E~
@High
[10 !I
o Low
EM 110 iii I U
Res1\onse Time (sec): 10 . 02 ~
~ :
I~.~e~~!.~~~.;;.:;
w_'_"~"__"""'1 I'. /8utofile...
. ·.. ] I'
., 01( .". "') I.. ' Cancel' ..!
4000 420.0 440.0 4600 475.0
Figure 3-5 Spectrum Parameters Dialog Box Wavelength (nm)
~r---=-
3) Select OK to validate the parameters. The main screen reappears. As the

parameters are sent to the instrument, the Fluorometer Window I) ) r
indicates Fluorometer Setup. The EX and the EM wavelengths and the
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-5301 PC Instruction Manual 3-5

Tutorial Tutorial
9) To see the cross hair (X, Y) coordinates of the mouse cursor position,
NOTE: The RF-5301 PC software maintains the Spectrum and Time
press the right mouse button. When the Graph Pop-up menu appears,
Course data in channels. A channel is an area in the personal
click on Cross Hair and then Display. As the mouse is moved, the X,Y
computer's memory (RAM) which is used to temporarily store all of
coordinates can be seen along the axes of the graph referencing the
the information relevant to a particular scan. The Spectrum and Time
intersection of the cross hair (see Figure 3-9). To remove the cross hair,
Course acquisition modes each contain ten channels, numbered 0-9.
reselect the Graph Pop-up menu and click on Display.
Data acquired from the fluorometer is held in the next available
channel; it is not yet saved to disk. (Channel Status under the Ille
Presentation menu shows the saved status of any active channel.) A file Acquire Mode .configure Manipulate eresentation tlelp
channel's data can be saved to disk only when the channel is assigned 30.001 )
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 20 000)
: J\
18.400
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).
10.000
7) From the Presentation menu under Graph, select Radar (Both Axes) or
press Ctrl+R (see Figure 3-8).
0.000
350.0 360.0 400.0 420.0
,..tilt
[ife Acquire Mode l.;onfigure
Figure 3-9 Graph Showing Cross Hair
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).
i
t ...-.
GUlph PaHlms. File Channel/F. Name au.dr.ntls]
~~O 0 1
Screen 01 020304
~::~.,~::::, '~;"'" ,;,,,~'j
350.0 360.0 380.0 400.0 420.0 440.0 ~50.0 61 0 0 0 I 0 01 020304
Wevelet1gth(nm)
.(; I 0 0 0 01 0203 04
I
['-'-"~"~''''''''~-'-.~;'-''''";, 01 0203 04
Q I 0 0 0 I
Figure 3-8 Radar Cascading Menu ) I. 'Printj I, :c.nce,:j 1·~.View, I I.' Sel~p J
[ill]
[ill]
8) 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 Figure 3-10 Plot Layout Window
without a mouse, as described in Limits, Section 5.5.2.4) Use Ctrl+R to
return to the enlarged screen.

Tutorial 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 ""I
lS.U':"
Parameter Channel dialog box and the third column is associated with
the Open File dialog box.)
.1rn1I~lf'-
O:R~A.~! OJ
0, 0;'
0,; 01
OJ OJ
O~ 01
I2l Jicreen o Spcctra Qnly rirtN~: 3Y2t32
Graph nllc Crnttd: 15:59 081111').1

D~n. O,i9ifl~1
[ I ~."."'.
~
b~_ftt. U-S301
~<tr_ Tvpe: 1M
3(u lugt: 350.011111 to .SO.Onm
EX W:no"kflqt\: 350.0 11M
:I r.. xis n~~!)nt 3~lit<:h:lO
Slit Widt...: IX:5.0flm !M:5.()_
.VPfjCr : I 1~.522 I
1~,c~~~11
3(~ ~"d: Vtty r~:;1;
~.~it"'il:~': ";91,
Jt<;~OII~,,';_:Avt<)
:!IIOJntr: M_:oI,Opet>
",nowtr: [ .1%(1~
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
Figure 3-12 Plot Layout Preview Window

containing data.)
13) Select Preview in the Plot Layout box to view the layout as it will 14) Click anywhere on the screen or press any key to return to the Plot
appear on the hard copy (see Figure 3-12). Layout dialog box.
15) 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)

Qrder of Curve ..... C> 1sl 02nd @::Jr;f
1) Select Quantitative from the Acquire Mode menu. The Quantitative
Zero Interception.. OYes @No
Parameters dialog box automatically appears (see Figure 3-13).
(Whenever the acquisition mode is changed, the Parameters dialog box
appears.) ~ ICancel I
2) Set the parameters as follows: t ')
Figure 3-14 Multipoint Working Curve Dialog Box
Method: Multipoint Working Curve
3) Select OK to validate the parameter settings. The Fluorometer Window
(3rd order, no zero intercept) shows the Fluorometer Setup and indicates slewing of the EX
Enter values into the dialog box which wavelength to 350nm and the EM wavelength to 425nm.
appears on the screen and click OK
(see Figure 3-14). 4) Prepare at least four Standard samples with expected intensity values
EX / EM Wavelength:
350/425 between 0.00 and 800.00 (see NOTE which/ollows). Skip to Step 5 when
not entering "known" Standards.
EX / EM Slit Width:
10/ 10
Concentration Units:
nm
Concentration Range:
0.000 (Low); 100.000 (High) NOTE: Instead of preparing Standards at this time, a "known"
Standard curve may be entered. Press the Enter key to access the Edit
Recording Range:
0.000 (Low); 1000.00 (High)
Standard dialog box (see Figure 3-15). Enter the Concentration
Sensitivity:
High
(Cone.), tab to the Intensity (Int.) field, enter the Intensity value, then
Auto Scan Mode:
Unchecked
click on OK, or press the Enter key, to record the Standard. Continue
Response Time:
0.02 to press the Enter key from the Quantitative table to access the Edit
Repetitions:
1 ( dialog box until the Standard values listed below are complete.
Cone. Int.
Quantitative Parameters Standard 1: 1.00 3.00
Method: !;oncentration Standard 2: 5.00 15.00
Standard 3: 13.00 116.00
IMUlti Point Working Curve
~ Units: Inm 1 Standard 4: 25.00 230.00
E~ Wavelength: 1350 . 0 1
Range: I 0.000 1
to 1100.000 I Standard 5: 34.00 375.00
Standard 6: 43.00 490.00
EM Wavelength: 1 4 25.0
I 50.00 620.00
Recording Range Standard 7:
Slit Width (nm): EX ~ Standard 8: 70.00 825.00
EM 110 ~
bOW 1 0.000 I .!::!igh 11000.00 1 Standard 9: 75.00 850.00
Standard 10: 80.00 875.00
~ensitivity: @High o Low Response lime: 10.02 0 Standard 11: 85.00 885.00
o Auto Scan Mode Bepetitions:
~
I ~O.(· .
'X <
I I Cancel" I 1;- )
Figure 3-13 Quantitative Parameters Dialog Box
3 - 10 RF-5301PC Instruction Manual RF-530 1PC Instruction Manual 3 - 11

Tutorial Tutorial
9) The Unknown data can now be read. Select the Unknown push button or
Standard Number the F2 key.
Concentration I 0.00000 I 10) Place an Unknown in the sample compartment and select the Read push
button (or the F7 key) for each Unknown to be read.
Int. I 0.000 I 11) When reading the Unknowns is complete, select Save Channel from the
" ) Channel cascading menu under the File menu. The Save Channel dialog
I ilK . 1 I C3n~1 I I QeleJel 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
Figure 3-15 Edit Standard Dialog Box 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
5) With the sample compartment empty, or with a cuvette containing solvent box by tabbing to it. Click on the Unknown check box and enter a file
in the sample holder, select the Auto Zero push button at the bottom of name and comment as above (.qnt will be the extension). Select OK to
the screen (or the F5 key) to zero the instrument. save the files.
6) Place the first Standard into the sample compartment. Select the Read
NOTE: Using the Save as function does not allow for comments to be
push button or press the F7 key, and the Edit Standard dialog box
entered.
appears.
7) Enter the concentration value for the first Standard and click on OK or
press the Enter key.
8) Repeat steps 6 and 7 for the second, third and fourth Standards. If the I:8J ~tandard 0 Clear after Save
Standard curve was entered from the table as des~ribed in the previous
File Name: ITESTSTD I
NOTE, the screen will look similar to Figure 3-16.
flil. Comment:
Elle Acquire Mode l;onfigurc Manipul8te fre.entation tjelp
SlMd¥d 1_ _
1iXlJOOO
Standard
Humber Cone. I nt.
1
2
1.0000
5.0000
3.000
15.000
8OO.iXlJ
I:8J !M~~~~~:~j o Clear after Save
3 13.000 116.000 > 600iXlJ
File Name: IUNKTST I
4
5
25.000
34.000
230.000
315.000 i
b 43.000 490.000 1: 4OO.iXlJ
1 50.000 620.000 Comment:
8 10.000 825.000
9
10
11
15.000
80.000
85.000
850.000
815.000
885.000
I I
20 iXlJ 4O.iXlJ 60.iXlJ 80.000 l00.iXlJ
rm
Int = k3 C'3 • k2 C'2 t kl C • kO

k3 ·0.00229 k2 - 0.260
·.· 8"'·""'"
El . OK
,""
. Status
.,., ~
I,·~.;~.··I)
,.
kl = 4.868 kO ='4.607
Chi-Square: 21.73510
Figure 3-17 Save Channel Dialog Box
•
St
)
Figure 3-16 Quantitative Screen after Standard is Entered
3 - 12 RF-530 1PC Instruction Manual RF-530 1PC Instruction Manual 3 - 13

Tutorial Tutorial
3.7 Time Course (Cell holder is empty) 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
1) Select Time Course from the Acquire Mode menu. The Time Course available Time Course channel.
Parameters dialog box automatically appears for review (see Figure 3-18).
(Whenever the acquisition mode is changed, the Parameters dialog box 7) Select Limits from the Graph cascading menu under the Presentation
appears.) menu (see Figure 3-19).
)
2) Set the parameters as follows: Graph Limits
'Y A x i s - - - - - - - , X Axi.------,
EX / EM Wavelength: 350/397
Ma~ IIIlIiIllD I "'-ax: LI6"'0"'0."'0_----'
Recording Range: -100.000 (Low); 1000.00 (High)
EX / EM Slit Width 10/ 10 Min: I ·21.345 I Min [Wii~0 I
Sensitivity: High
Response Time: Auto I", (lie. -I F:: .!lese' I Ir Cancel I
Reaction Time: 60.0
Figure 3-19 Set Limits Dialog Box
Timing Mode: Auto
Units: Seconds 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
E~ Wavelength:
~
~
I Recording Range the Enter key. The screen will look similar to that shown in Figure 3-20.
Low: 1-100.000 I tligh: 11000.00 I ...
EM Wavelength: ~ -, ;'.11111; !,~I
file Acquire ~ode konfigure Manipulate .eresent81ion tielp
Slit Width (nmJ: EX 110 I:!J EM 110m
.5.ensitivity: @[~!~:~: 0 Low RJ;sponse Time:

=-- Ii)
IAuto
,Timing MOde~ .qnH~~

Beaction Time: ~ I @ AUlD 0 Manual I o HnIH"
r~cquisinOH n~tr::
~ o Mi"u!c!< f
1:
Nump.e~ H! rtf.: .H:Hng~~,; 3001
1 I @ Seconds
, .,
li;:;~,K:>il 1;:ci7~t~i'J(J
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. Figure 3-20 Time Course Screen after Noise Plot
4) Select the Auto Zero push button to zero the instrument.
This completes the tutorial portion of the instruction manual. Since the tutorial is
5) Select Start to plot the noise level graph. ) 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
6) When the plot is completed. the File Name dialog box appears. In the
RF-530 I PC software.
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
3 - 14 RF-530 1PC 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 Software Overview
4.3 Main Menu 4.3.2 Acquire Mode Menu

This menu enables selection of the data acquisition modes which include:
4.3.1 File Menu
The File menu contains items concerned with channel and disk file Spectrum Mode
management. (In Quantitative mode, the standard data and unknown data Establishes a mode in which a spectrum is acquired over a designated
are maintained in separate channels.) The menu items include: wavelength range.
Open Quantitative Mode

Used to load previously acquired and stored data into channels. A file's Establishes a mode in which the relative fluorescence intensity at fixed
parameters may be previewed before loading. excitation and emission wavelengths is measured for individual samples.
These values are used to calculate concentration by means of a standard
Save curve.
Performs a quick save to disk of the data contained in the channels.
Time Course Mode
Save As Establishes a mode in which the relative fluorescence intensity is
Saves a channel to disk while changing the file name and/or directory measured at fixed excitation and emission wavelengths over time.
associated with the channel.
4.3.3 Configure Menu
Delete This menu contains commands for setting parameters related to data
Deletes a file from a specified directory. acquisition, device communication, and instrument settings. It also
includes menu items for loading and saving configuration files, for
Data Translation SpeedBox configuration, control of attachments, and for system
ASCII / DIF Export: Converts a data file into ASCII (American communication and control.
Standard Code for Information Interchange) or DIF (Data Interchange
Format) format. Parameters
Allows setting of all parameters related to the current acquisition mode.
Channel
Save Channel
PopUp Scan Parameters
Saves individual channels to disk. Allows setting of the acquisition parameters for the PopUp Scan feature.
Erase Channel PC Configuration

Deletes the data from one or more channels. Designates the default data and export directories and sets parameters for
communication between the personal computer and the fluorometer as
Rename Channel well as printers. (Reminder: The printer/plotter must also be configured
Modifies the file name and/or comment assigned to a particular correctly in the Windows Control Panel.)
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. Speed Box Configuration
Allows the user to select functions for the SpeedBox, and also the style in
Exit which the SpeedBox will be displayed.
Exits the RF-5301PC program.

Instrument Peak Pick

Enables monitoring and control of the communications status between the A tabulation function which designates the peaks on the graph of
personal computer and the RF-5301 PC fluorometer unit, i.e., settings for Spectrum or Time Course data and lists them in a window. Threshold
Fluorometer, -HV Control, PMT Protect, and Auto Shutter; adjustments adjustment, valley designation (minimum value), replotting and printout
for Dark Level Correction and Lamp Alignment; SIN Ratio Check; and in either tabular form or graphics plot are possible. The table may be
information on the instrument serial number, ROM version, and elapsed stored as a text file or copied to the clipboard.
lamp usage time.
Point Pick
Attachments A tabulation function which allows X-axis designation of up to 15 points
Allows configuration of optional instrument accessories. 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
Save Parameters and Load Parameters replotted, tables or graphs printed, and data copied to the clipboard.
For saving or loading a particular configuration file. When a Frequently used X-axis values may be saved and recalled.
configuration file is saved, all current parameters in all modes are saved
to disk in the directory in which the program is installed. Peak Area
Calculates the area under a curve. Up to four different areas may be
4.3.4 Manipulate Menu graphically selected. The results and graphs may be printed or sent to the
clipboard. Frequently used Peak Area configurations may be saved and
The Manipulate menu contains functions which enable various methods
recalled.
of manipulating data for display or printout. The menu items include:
Average
Arithmetic
In Quantitative mode, averages the relative fluorescence intensities for all
Performs scalar operations (+, -, *, /) on the ordinate axis of Spectrum,
Quantitative, or Time Course data. ) unknown quantitative data that have the same ID number.
Working Curve
Transforms
In Quantitative mode, allows setting the curve fitting parameters (order of
Performs function operations (smoothing, derivative, reciprocal,
curve, zero intercept) for the standard curve.
logarithm) on the ordinate axis of Spectrum or Time Course data; and
reciprocal and logarithm functions on Quantitative data.
4.3.5 Presentation Menu
File/Chnl Calc This menu consists of items which determine the presentation format of a
Performs vector operations (+,-, *,/) on the ordinate axis of Spectrum, data file on the screen or as a printout. The menu items include:
Quantitative, or Time Course data.
Channel Status
Data Print A window displaying a list of all occupied channels and their status with
A tabulation function in which all the measured data points for a respect to graph line color, whether the channel is currently displayed,
particular channel are displayed in a window. Data may be adjusted, and whether it has been saved to disk. Individual channel acquisition
saved in a text file, output to a printer/plotter, or copied to the clipboard. parameters may be viewed and the display status changed.
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., Graph
the activity) of the time course line. Copy
Copies the main graph to the clipboard.
Display Grid
An ON/OFF toggle for displaying the graph with a grid pattern.

Fonts 4.3.6 Help Menu

Allows changes of fonts, and font types, styles and sizes for the The Help selections offer a summary of the RF-5301PC software. Help is
coordinate values, axis labels and headings. especially useful when you require information quickly or when the
instruction manual is not available.
Limits
Allows you to change the X- and Y-axis limits of the graph on the Help for RF-5301 PC
screen, or to zoom in or out on an area without using the mouse. Contains the on-line help information pertaining to specific RF-5301PC
features.
Line Colors
Assigns a chosen color, type of line, and annotation type for each of How to Use Help
the ten channels in Spectrum and Time Course modes, and for Contains a general explanation of how the help system works.
Standard, Unknown and Standard curve in Quantitative.
About RF-5301 PC
Options
Contains the version number of the RF-5301PC software as well as
Allows selection of various grid lines, grid colors, line types, and license and serial number information.
background colors as well as rounded or minor tick marks.
Radar
4.4 Status Bar
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 The Status Bar contains windows which display information about the status of
axis, or Both Axes. the optical bench or about a current reading. The following is a description of
the windows in the Status Bar:
Hide All
This is an ON/OFF toggle for hiding and re-displaying all of the graph
I) )
Lamp Window
data on the screen. This window displays the xenon lamp's current status. When the instrument is
attached and communication has been initiated, the xenon lamp status is
Show Speed Box displayed as ON or OFF in this window.
This is an ON/OFF toggle for hiding and re-displaying the SpeedBox.
Scan Mode Window
Plot Information is displayed in this window about the current scanning mode of
Enables layout of a combination of graph overlays, parameters and text operation. The operation modes are Repeat Scan and Auto File in Spectrum
files for the four quadrants of a page. The results can be previewed on mode, and Auto Scan Mode in Quantitative mode. When Repeat Scan is
the screen and output to the printer/plotter. 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.
Int.lConc Toggle (Quantitative only) The same information is displayed for Auto File except instead of Repeat, AF
Toggles the Y-axis of the unknown graph between intensity and is displayed. When Auto Scan Mode is selected from the Quantitative
concentration. Parameters dialog box, Scan Mode is displayed in this window.
Equation Display (Quantitative only) Fluorometer Window

This is an ON/OFF toggle for hiding and re-displaying quantitative This window displays the current excitation and emission wavelength settings
coefficients and constants. and the real-time relati ve fluorescence intensity at those wavelengths. It also
displays various status messages concerning the initialization, setup, slewing,
etc.

4.5 PopUp Scan ™ Push Button NOTE: Closing the PopUp Scan window or starting another PopUp
Scan will discard (without warning) the previous PopUp Scan.
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.
4.6 SpeedBox®
The window, as shown in Figure 4-1, has a menu offering the following
selections: The SpeedBox is a quick accelerator device which uses buttons to represent
commonly used menu commands. The SpeedBox may be configured to the
Radar individual user's preference via the SpeedBox Configuration dialog box (see
The Radar function adjusts the graph limits so that data will be completely SpeedBox Configuration, Section 5.3.4). The user is able to select a button to
visible on the graph. This option may be selected from the menu or clirectly represent a specific command and place this item in the SpeedBox. The
from the keyboard (when the pop-up window is active) by pressing Alt+R. 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
Set Limits SpeedBox is remembered as well as whether it was visible.
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 4.7 The Graph Cursor
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 The mouse cursor changes to a cross hair when moved over a graph. When
graph limits. 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
Hadar! Set Limits... liave ...

I) )
display the Graph Pop-up menu (see Graph Pop-up Menu, Section 5.6).
Use the following steps to zoom in on any area of the graph:
28.276 -Move the cross hair to one corner of the area of interest.
25.000
-Press and hold down the left mouse button.
> 20.000
""c
if) -Drag the cross hair to the diagonally opposite corner.
Q)
15.000
:f;
-Release the left mouse button.
10.000
The graph will be redrawn with the selected area at full scale.
4.290
380.0 400.0 420.0 440.0 450.0
Wavelength (nm)
To find the X, Y coordinates of any position in the graph:
Figure 4-1 PopUp Scan Window and Menu
-Press the right mouse button to bring up the Graph Pop-up menu.
Save Select Cross Hair and Display.
The Save option allows the scan to be saved in a file which may later be The Y-axis coordinate of the cursor location is displayed beside the Y-axis,
loaded while in Spectrum acquisition mode. A dialog box appears for and the X-axis coordinate of the cross hair location will be displayed below the
entering a file name (an .spc extension will be used) and any desired comment. X-axis. To turn off this display, select Cross Hair, Display.

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

before loading it.
5-2 RF-5301 PC Instruction Manual RF-530 1PC Instruction Manual 5-3

5.1.3 Save as 5.1.4 Delete

This command is used to save a channel or table to a new file or directory The Delete command can be used to remove one or more files from the
and can be selected from the File menu or by pressing Ctrl+A from the disk. When this command is selected, the Delete dialog box appears (see
main window. This command renames the channel in memory as well as Figure 5-4).
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). Delete
File Name: J2ireclories:
- Save As I"." e:\rlpe\data
I _ iii r=--:---------c=<
File Name: 1!irectories: Be:\
~
ImmJ ,!
c:\rfp c\d ata
Brlpe
e data
32'2.81)(;
fH~,Bnr.
8oc:\ ~
8 rfpe
ab.so£
hatch1 r,l.soc ~data
iluorl.soc
kbs<lmn2.slJc list Files 01 Jype: Olives:
ml.soc
marvOO.snc I II '",") l1! IIiiI e: ms-dos_6 I
marv{l"l.snc
~
List Files of Iype: Driyes: .channel Figure 5-4 Delete Dialog Box
Ispectrum r·spe) I Ira e: ms-dos 6 II IChannelO I
NOTE: Use caution when selecting items to be deleted as deletion is
Figure 5-3 Save as Dialog Box 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).
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. Delete this file?
When the Save as dialog box appears, it displays a list of the files in the File: Selected: 001
default data directory (see PC Configuration, Section 5.3.3). To save a KBSAMP2.SPC Deleted: 000
file to another directory and/or drive, select the drive from the Drives Directory:
C:\RFPC\DATA\
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). Figure 5-5 Delete this file? Dialog Box

This dialog box displays each file before deletion while offering the After selecting the channels to be saved in ASCII format, select OK to
following options: 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
Delete: Delete this file; continue to next file in selection. extension.
Skip: Do not delete this file; continue to next file in selection. When ASCII Export is selected from Quantitative mode, a Standard
and/or Unknown selection dialog box appears. Check the desired
Cancel: Return to the Delete dialog box.
check boxes and select OK to proceed. Since it is often useful to have
Delete All: Delete all remaining files in selection; return to Delete the same file name (with different extensions, i.e., .std and .qnt) for
dialog box. 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 Delete dialog box appears with a list of the files in the default data The converted file is saved to disk in the export directory with an .asc
directory (see PC Configuration, Section 5.3.3). To choose files from extension.
another directory and/or drive, select the drive from the Drives combo
box and select the directory from the Directories list box. 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
NOTE: The Delete command does not remove data from the computer's is:
RAM (see Erase Channel, Section 5.1.6.2).
X,Y<CR><LF>
5.1.5 Data Translation X,Y<CR><LF>
5.1.5.1 ASCII Export
This option is selected from the Data Translation cascading menu. It ) <CR> and <LF> are the codes for a carriage return and line feed.
saves the data in a channel(s) to an ASCII (American Standard Code 5.1.5.2 DIF Export
for Information Interchange) file(s). When ASCII Export is selected
This option is selected from the Data Translation cascading menu.
in Spectrum or Time Course modes, the channel selection dialog box
This command is similar to ASCII Export, except that the converted
appears (see Figure 5-6).
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
Oi!!.§~~ O S·
data will be the Sample ID values; for Standard Quantitative data, the
"
01KBSAMP2 Oh X-axis data will be the concentration values. The DIF files are saved
O? Of in the export directory.
0) O~
01 Oh 5.1.6 Channel
OAJI
5.1.6.1 Save Channel
This option is found in the Channel cascading menu and is selected
I@~~fl Ifo.~il If§i~t~~il for saving any combination of the named channels to the data
directory. When selected, the Save Channel dialog box appears as
Figure 5-6 ASCII / DIF Export Dialog Box shown in Figure 5-7 (Spectrum and Time Course) or Figure 5-8
(Quantitative).

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
OI£lA.MAN 0'.,
memory after data has been saved to disk. This selection is useful
01 KBSAMP2 Of,
when preparing to collect another set of data.
O? Oi.
OJ. O~ The Status push button, when selected, displays the Channel Status
OJ Ol dialog box, which is the same dialog box displayed when the Channel
Status option is selected within the Presentation menu (see Channel
o £;11 Status, Section 5.5.1).
IIf~JI
., l~ilfim~1
:~~~.<- IJI1f;'
t. .~.'m.
The Cancel push button causes the software to ignore any selections
and return to the main screen. Pressing the Esc key has the same
Figure 5-7 Save Channel Dialog Box (Spectrum / Time Course modes) effect as Cancel.
When in Spectrum or Time Course acquisition mode, select any 5.1.6.2 Erase Channel
combination of channels to be saved using the mouse or by entering The Erase Channel command, found in the Channel cascading menu,
the number(s) corresponding to the underlined number(s). Select All clears data from the computer's RAM. Select the channel(s) to be
to designate all the channels for saving. (Select "All" again to erased and select OK to continue. In Spectrum or Time Course
deselect all the channels.) Only channels where a file name has been modes, when any data in the channels has not been saved, the message
entered are saved. Channels containing no data are dimmed and "Warning! Some data has NOT been saved! Do you wish to
cannot be selected. 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.
(Z]~t8ndardl o Clear after Save
File Name:
c In Quantitative mode, select the Standard and/or Unknown check
boxes to indicate the table to erase. When any data in the channels/
Comment: tables has not been saved, the message "Warning! Standard has
_ _ _I NOT been saved! Do you wish to continue?" is issued. Select Yes
Ov.nb;nY!i o ~~~t::~! al1.n $;jYt:
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.
File Name: I I
Comment
I 1
PTOO'
:JlJ:,,>?', J;7 ~1tll~tJ
I~J~.;;.:~m.l; 1!~;i!1
i~~~A
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.
5-8 RF-5301PC Instruction Manual RF-5301 PC Instruction Manual 5-9

Change the information as desired and select Save or press the Enter
key to implement the changes. Select Discard to cancel the command
O.Q RAMAN' 0 '. and return to the main window.
01 KBSAMP2 0 !'
0, Dr In Quantitative mode, the file name entry dialog box is displayed,
0:; 0"
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).
o1 O~'.
Then press the Enter key or select OK.
o All
Clear the file name field and select OK to assign No Name to the data,
l<oli!f;1 I(~~'n~f! I:~;;;ru5j 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
This option exits the program and returns to Windows Program Manager.
The Rename Channel command, found in the Channel cascading
When data has not been saved, a warning is displayed and you may elect
menu, is used to modify the file name and/or comment assigned to a
to return and save the data, or exit and lose the data. Any data that has
particular channel. This causes the channel's "data saved" status to be
been assigned a file name, but has not been saved, is saved automatically
set to "not saved." A name may also be assigned to a No Name
at this time. Exit can be selected from the File menu or by pressing
channel. In Quantitative mode, use Rename Channel to assign file
Alt+F4 from the main window.
names to the Standard and Unknown channels so that the file will be
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.
@>:p~ 0"
° 1 KBSAMPZ 0.,
Enler File Name:
IIi1mm I
0, Of
0' 0" Eoler Comment:
I
Romon Peok I
Ol 0"
...~/,
~
)~~we'.,..,
"", Ihl(;.~j
IY;~ oj Ic..·~('l
)
Figure 5-JO Rename Channel Dialog Boxes
5 - 10 RF-530 1PC Instruction Manual RF-5301 PC Instruction Manual 5 - 11

5.2 Acquire Mode Menu 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
The mode in which data is acquired is selected from the Acquire Mode menu, with the keyboard via the function keys F2-F8.
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.
F2 R F4 F5 F6 F7 F8
, I . ' 1111 i'1 I~·,...
Manipulate Eresentation !ielp

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.
l'
f
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
360.0 370.0
Wavelength (nm)
380.0 390.0 397.0
) 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:
Figure 5-11 Acquire Mode Menu on the Spectrum Screen
Read - The monochromators slew to the new wavelength values and
5.2.1 Spectrum Mode the intensity reading in the dialog box is updated. In order to
Under the Acquire Mode menu, use Spectrum (noted with a check mark) keep these wavelength values and update the parameters, Set
to acquire a spectrum over a designated wavelength range. When must be selected.
Spectrum is the acquisition mode upon starting the program, select Set - The monochromators slew to the new wavelength values.
Parameters from the Configure menu to set the parameters for real-time These values are displayed in the Fluorometer Window and
data acquisition (see Spectrum Parameters, Section 5.3.1.1). (When stored in the parameter entry form (PEF) as the fixed
Spectrum is selected while another acquisition mode is active, the wavelengths. The dialog box is closed.
Spectrum Parameters dialog box (see Figure 5-23] appears Cancel - Resets the original wavelength values and exits the dialog
automatically.) The Spectrum screen is similar to Figure 5-11. To turn box.
the instrument ON, either select Instrument from the Configure menu
and set the Fluorometer, -HV Control and PMT Protect to ON (see Figure NOTE: When the wavelengths are set to the desired values, select the
5-38), or click on the Start push button. 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

4) Auto Zero push button

This function zeroes the fluorometer unit at the designated excitation
E;><: Wavelength EM Wavelength
and emission wavelengths. Select with the mouse or press the FS key.
@ill ~ This function does not change the AID (analog voltage converted to
1"',IfJ·;.~i1 [tll!!!!,l."l digital signals) calibrated signal; it merely subtracts the reading from
all subsequent readings to correct for small changes, such as drift due
Intensity: Ci 137 I to thermal effects.
!:'ti\lli:adT:1 1!(1lf!~JryM'1 rmi9t~El~ 5) PopUp Scan push button

Select with the mouse or press F6 key to perform a scan using the
Figure 5-13 Wavelength Selection Dialog Box 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
2) Search Optimal WL (wavelength) push button a sample without changing the current Spectrum parameters. The
This search function is used to find the optimal excitation and emission PopUp Scan contains a menu bar which includes: Radar (changes the
wavelengths for a sample. Select with the mouse or press the F3 key to graph scale), Set Limits, and Save. The PopUp Scan uses a parameter
display the dialog box. Enter the EX / EM start and end wavelengths, set similar to those used for Spectrum mode scanning. The PopUp
and the excitation wavelength interval (valid range is 10 - 99nm) on Scan parameters are used exclusively by the PopUp Scan feature. The
the excitation side. Pressing the Search push button finds the excitation current instrument settings (those set in Parameters) are retransmitted
wavelength and emission wavelength which gives the greatest to the instrument after completion of the PopUp Scan. Press the Stop
fluorescent intensity, while eliminating peaks due to scattered push button to interrupt the scan.
excitation light, second-order light, and Raman bands, and displays the
6) Start push button
results in the Search Optimal Wavelength dialog box. The Save
Select with the mouse or press the F7 key to start the scan. When the
function saves the optimal wavelengths into the fixed wavelengths of
the Parameters dialog box. The optical bench is set to the wavelengths I) ) scan is completed, the Enter File Name dialog box appears (see
Tutorial, Section 3.5, Figure 3-6). When Save is selected after entering
which are reflected in the Status Bar.
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
Search Optimal Wavelength
Auto File mode is used. When Discard is selected, the data is erased.
E"" Range: ~ to ~ When communication between the computer and the instrument is OFF,
this push button may be used to establish communications.
EM Range: ~ to §O
I 7) Stop push button
EX !nterval: ~ Select with the mouse or press the F8 key to interrupt the scan. The
Optimal Wavelengths:
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
Excitation: [-=-=::J nm
to discontinue communication with the instrument.

Emission: c::=:::J nm
5.2.2 Quantitative Mode

Select Quantitative from the Acquire Mode menu (noted with a check
Figure 5-14 Search Optimal Wavelength Dialog Box mark) to collect Quantitative data. When Quantitative is selected upon
starting the program, it is necessary to select Parameters from the
3) Shutter push button Configure menu to change the parameters for real-time data acquisition.
Select this push button with the mouse or press the F4 key to open and When Quantitative is selected while another acquisition mode is in effect,
close the shutter (the shutter position is visually displayed on the push the Quantitative Parameters dialog box (see Figure 5-29) automatically
button). appears. The Quantitative screen appears as shown in Figure 5-15.
5 - 14 RF-5301 PC Instruction Manual RF-5301 PC Instruction Manual 5 - 15

1'5 •• ~t ... 3) Search Optimal WL (wavelength) push button

file Acquire Mode ~onfigure Manipulate fresentation tlelp
This search function is used to find the optimal excitation and emission
Seq. Sa~ple
wavelength for a sample. Select with the mouse or press the F3 key to
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
10 1S 17
seq • 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.
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.
F4
6) PopUp Scan push button
Fl F2 f3 F5 F6 F7 F8
Select with the mouse or press F6 key to perform a survey scan. The
Figure 5-16 Quantitative Push Buttons 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
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
2) Unknown push button so that the concentration value may be entered. When the
Select the Unknown push button (when not dimmed) with the mouse or communication status between the computer and the instrument is OFF,
press the F2 key to display Unknown data and graph for acquisition, this push button may be used to establish communications.
manipulation or editing of Unknown data.

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 10 1111 I
used to discontinue communication with the instrument.
In1. I 71.217J
The Standard (when using multipoint quantitative method) or Unknown
is displayed in both a tabular and graphic format on the Quantitative
I~,!,nd"rd'i
Cone. 0.00000
acquisition screen. The Standard and Unknown push buttons described
above are used to change the display (as well as the acquisition and
Figure 5-18 Edit Unknown Dialog Box
manipulation) between Standard and Unknown data. Use the mouse or
arrow keys to change the currently selected (highlighted) item in the data Select the Standard button and enter the value for the concentration in
table. Note that the Read push button replaces the relative fluorescence the Make Std dialog box which appears (see Figure 5-19). The selection
intensity of the current selection and makes the next item in the table the is removed from the Unknown table and appended to the end of the
current selection. Double-clicking a selection with the mouse or pressing Standard table.
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 Concentration: I 1.000000 I
modify) values for concentration and intensity. Use Delete to remove the
entry from the Standard table.
1~~i!lI~j¥1
Standard Number 2
) Figure 5-19 Make Standard Dialog Box
5.2.3 Time Course Mode

Concentration 1-,·HuwHl I
Select Time Course from the Acquire Mode menu (noted with a check
Int. 1203.366 I 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
,>"~
1.····°_ 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
Figure 5-17 Edit Standard Dialog Box another acquisition mode is in effect, the Time Course Parameters
dialog box (see Figure 5-33, Section 5.3.1.3) automatically appears.
The Edit Unknown dialog box (see Figure 5-18) is used to enter values After the parameters are selected, the Time Course screen appears as
for Sample ID and intensity. Select Delete to remove the entry from the shown in Figure 5-20. The Time Course screen contains eight push
Unknown data table. Use the Standard button to create a Standard from buttons at the bottom of the screen, any of which may be selected by
an Unknown value in the following manner: highlight the desired clicking with the mouse, or with the keyboard via the function keys FI
selection in the Unknown table using the arrow keys and press the Enter F8.
key, or double-click on the selection with the mouse.
NOTE: Sample IDs may consist of a maximum of eight numeric characters only.

,1;'111"; 3) Shutter push button (see Spectrum)

Ble Acquire Mode .c.onfigure Manipulate Eresentation tlelp Select with the mouse or press the F4 key.
1000.000"-----~~---..---~-~-""Cl=~~=-:cc_-----:cc_-_,
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.
~
c The scan is displayed in a pop-up window (see Figure 4-1 in Software
:i:
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
-100.000' ,.. ~ <" -, instrument after completion of the PopUp Scan.
0.00 2000 4000 60.00
TIll'le (sec)
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.
Figure 5-20 Time Course Screen
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.

5.3 Configure Menu

Spectrum Iype: @:E~!i~.i!o;; 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
configuration files. When the Configure menu is selected, the screen appears
EX Wavelength Range: Jitart ~ .End 1770 I
as shown in Figure 5-22. Recording Range: bOW ~ !::!igh 150.000 I
1·-'11
SJ;anning Speed:
lil::::E
I~j'r1K(j3ncef,1
~ Figure 5-23 Spectrum Parameters Dialog Box
~
1:
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
400.0 6CJ.0 770.0 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.

Recording Range • ..'... iT......
These entries correspond to the ordinate scale limits of the PC's graph.
The allowable range is -100.00 - 1000.00.
Bepetitions 0 12-100)
Repeat Time Interval ~ Seconds

Scan Speed
The actual scanning speed at which data is collected can be set at file Name II [1-6 Characters)
Survey, Super, Very Fast, Fast, Medium, Slow and Very Slow.
IfIiislttil~.1
Sampling Interval
This sets the interval at which data is acquired by the instrument. Figure 5-24 Repeat Scan Parameters Dialog Box
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 Enter the number of repetitions to be performed and the time interval
interval is fixed at 2.0 nm. When Super or Very Fast is selected, the (in seconds) between the starts of two consecutive scans. When the
sampling interval is fixed at 1.0 nm. Note that selecting a small time required for one cycle is longer than the time interval, the next
sampling interval and a large wavelength range will affect the scan starts immediately when the wavelength is reset to the start
performance of this software. (See Precautions on Memory Usage, wavelength. Enter up to six characters for the file name to save the
Section 6.3 in Troubleshooting, Chapter 6.) acquired data to disk.
EX I EM Slit Width When Enable is selected, the Scan Mode window on the Status Bar,
The allowable excitation slit width settings are 1.5,3,5, 10, 15,20, to the left of the Fluorometer Window, begins tracking the number of
6HH (half-height). The allowable emission slit width settings are 1.5, scans. The Status Bar displays the word Repeat and the designated
3,5, 10, 15,20, Close. file name, accompanied by two digits representing the sequence
Sensitivity
I) ) 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
The sensitivity of the detector can be set at High or Low (with High entered; see Figure 5-25.) The Scan Mode Window remains active
providing approximately 50 times the sensitivity of Low). 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
Response Time directory with an .spc extension. A tone signals the completion of all
This function sets the response time of the instrument. Possible of the scans.
selections are 0.02, 0.03, 0.1, 0.25, 0.5, 2.0, 4.0, 8.0, and Auto.
When Disable is selected, the Repeat Scan Mode is turned off. Also,
Repeat Scan use Disable to exit from the Repeat Scan mode of data collection when
Repeat Scan allows automatic consecuti ve scanning of 2 to 100 scans it has been previously set.
on the basis of a set time interval. When the Repeat Scan button is
selected, the dialog box shown in Figure 5-24 appears.
SoloW
W'l"i"""··"'wm mYj
"'fTxer~plr-' r-Repea~:JC"EST~'- r--E>t35UtrIM15(ro---"2:~

) Figure 5-25 Repeat Scan Mode Window

When the scan mode is set to Repeat Scan, attempting to set Auto File When Enable is selected, the Scan Mode window appears to the left
will result in a warning. Repeat Scan must first be cancelled before of the Fluorometer Window on the main screen. The Status Bar
Auto File is set. This is also true when attempting to set Repeat Scan displays AF (for Auto File) and the designated file name accompanied
when Auto File has already been set. by two digits representing the number of the scan to be performed
next. (Thus, for the first scan, the Status Bar would display AF
I~~f ,tmmttm 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
Mode is set to Repeat Scan! Mode is set to Auto File! 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
l:tiP!.,,] 1""""-'
10K, ·].· 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
Figure 5-26 Repeat Scan I Auto File Warning Messages Window), the next set of Auto File scans begins at zero.
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
r~rll*l
p~1IpIL:&J e
St
the Auto File mode, select the Start push button on the main screen. Ctx35D.O"EM:450.O:----:2.11~:
When the file name which is entered already exists, it will be Figure 5-28 Auto File Scan Mode Window
overwritten without warning.
Upon completion of each scan, the data file is saved to disk in the data
directory with an .spc extension.
Auto File
Auto File allows a maximum of 100 scans to be performed in
) 5.3.1.2 Quantitative Parameters
succession and automatically saves them in the designated file (stored The Quantitative Parameters dialog box is shown in Figure 5-29. A
to disk) after each scan. The data is also displayed in real-time on the description of the parameter functions follows:
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. fOur.
Method:
.(;oncentration
IMulti Point Working Curve
IJ Units: ~
~umber of Sc.ns ~ (1-100) E~ Wavelength: 1350.0 I Range: 1 0.000 1 to 1100.000 I
EM Wavelength: 1 425 . 0
file N.me ITEST I (1-6 characters)
I
Slit 't!'idth (nm]: EX 110 II Recording Range
IW.-'E'ni;ble'!ifii) Ilij2isabie..'.<.'j
...... , ..... :. -';"·"1"'1" EM 110 II o.
bOW ~ tjigh 11000.00 I
Figure 5-27 Auto File Dialog Box Sensitivity: @ High 0 Low Response lime: 10 . 02 il
o ~uto Scan Mode Bepetilions: II ~
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 Ijf$'1jRir~; Irt~~!!)>>
file name. Figure 5-29 Quantitative Parameters Dialog Box
5 .. 26 RF-530 1PC Instruction Manual RF-530 1PC Instruction Manual 5 - 27

Method NOTE: Using Multipoint Working Curve: The concentration values

This function establishes how the concentration is determined. for the Unknown are displayed as 0.000 until there is a valid working
Selections include raw data, K-factor, single point working curve and curve. When enough standards have been entered (and read) to solve
multipoint working curve. 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
Select Raw Data Measurement when no quantitation is required. the Unknown concentrations are calculated. The working curve is
drawn over the concentration range (+ I 0%) of the standards. When
Select K-Factor to enter the coefficients of a known equation for a
using 2nd and 3rd order polynomials to calculate Unknown sample
standard curve. The dialog box as shown in Figure 5-30 is used to
concentrations, there may be multiple solutions (sample concentrations)
enter the equation (1 st order) for a known working curve. Select OK
to the working curve for any given intensity. In this case, the
to return to the Quantitative Parameters or Cancel to select another
following algorithm is used to determine the best solution
method.
(concentration of the Unknown): 1) When the concentration value is
less than or equal to the largest Standard and greater than or equal to
'. III( l i11II :rrrT.1Trr'" ~
the smallest Standard, the value is considered to be correct; 2) When
[Cone) = Kl • [UNK Int) + KO none of the solutions satisfy the criteria in No.1, the concentration
Kl = IIiIiIiIiIil I K.!! = I 0.0000 I 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.
Figure 5-30 K-Factor Parameters Dialog Box
Select Single Point Working Curve when only one Standard is used.
When the dialog box appears, enter the concentration of the Standard
I) )
Qrder of Curve o 1st 02nd @:~rcj"
(see Figure 5-31). Select OK to return to the Quantitative Parameters
or Cancel to select another method. Note that this method assumes Zero Interception o Yes ~'No
that the blank (zero concentration) has a relative fluorescence
intensity of zero.
0"
Figure 5-32 Multipoint Working Curve Dialog Box

-S.TD Concentration I 1.0000
EX Wavelength
1:~·;m.1
...,an~.,
Enter the excitation wavelength at which data will be acquired.
Allowable entries are 0, and 220 nm to 900 nm.
EM Wavelength
Figure 5-31 Single Point Working Curve Dialog Box Enter the emission wavelength at which data will be acquired.
Allowable entries are 0, and 220 nm to 900nm.
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
)
EX I EM Slit Width
polynomial) and whether to force the curve through zero (see Figure
The allowable excitation slit width settings are 1.5,3,5, 10, 15,20,
5-32). Select OK to return to the Quantitative Parameters or Cancel to
6HH (half-height). The allowable emission slit width settings are 1.5,
select another method.
3,5,10, 15,20, Close.

Concentration
Range values are used to set the initial scale of the Standard curve X Recording Range
axis and the Unknown concentration Y-axis. The displayed standard E~ Wavelength: 1350 ,0
curve is calculated only for the concentration range. The
bOW: 1-100.0 t!igh: 11000.00 I
EM Wavelength: 1397.0 1
concentration units are used as headings for various tables and labels
for the appropriate graph axes. Slit Width (nm): EX 11 0 ~ EM 110 ~
-Sensitivity:@HighOLowR.!;.sponseTime: I=A=ut'---o--~
Recording Range
These entries correspond to the ordinate scale limits of the PC's graph.
Timing Mode (lnits ..
The allowable range is -100.0 to 1000.0. Beaction Time: I 60.00
• . 't·10ft rn,.il i e: "fJ?
@ Au!o 0 Manual o HnUh
u.<....
Sensitivity
""CjjWSI 1
o Minutt's
The sensitivity of the detector can be set at High or Low (with High Num1l.er oi Headings: I JUG i @ Seconds
providing approximately 50 times the sensitivity of Low).
Auto Scan Mode

Auto Scan Mode automatically starts PopUp Scan prior to every Read Figure 5-33 Time Course Parameters Dialog Box
allowing the user to preview the spectrum of a sample. When this is
selected, the Status Bar will display Scan Mode. EX Wavelength
This is the excitation wavelength at which data will be acquired.
Response Time Allowable entries are 0, and 220 - 900nm.
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. ] )
EM Wavelength
This is the emission wavelength at which data will be acquired.
Repetitions Allowable entries are 0, and 220 - 900nm.
This function sets the number of Unknown replicates to be measured.
For example, select 3 to record readings in triplicate (see Data Print Recording Range
and Average in the Manipulate menu). These entries correspond to the ordinate scale limits of the PC's graph.
The allowable range is -100.00 - 1000.00.
5.3.1.3 Time Course Parameters
When Time Course is selected as the acquisition mode, the Time EX I EM Slit Width
Course Parameters dialog box is displayed as shown in Figure 5-33. The allowable excitation slit width settings are 1.5,3,5,10, 15,20,
A description of the parameter functions follows: 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.

Timing Mode (Acquisition Rate I Number of Readings I Units) ·'1]11111
When Auto is selected, enter the total Reaction Time. The time unit
is always seconds. The Acquisition Rate and Number of Readings
Spectrum Iype: C' Excitation @ [g~is~i~~] o Synchronous
-----,
are calculated automatically. Any channels whose X-axis values are EX 'f!'avelength: 1350 1 nm
not currently in seconds are converted to seconds. EM Wavelength Range: Jitart 1380 .End (450 1
When Manual is selected, enter the time Unit, the Acquisition Rate Recording Range: Low I 0.000 tiigh [1000.00 1
(minimum of 0.1 seconds), and the Number of Readings (maximum
of 12,000 readings). The Reaction Time is calculated on the basis of
S.!;anning Speed: IFast IIi
the entered values. Any channels that are already loaded will have Sampling !nterval (nm): 11.0 II!I
their X values (time unit) converted to the time units entered in this E~ 1:!1
dialog box. When a file is loaded into a channel through the Open
Slit Width (nm): 115 EM
~
Sensitivity: @ High 0 Low
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 Resl!0nse Time (sec): IAuto II
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).
Figure 5-34 PopUp Scan Parameters Dialog Box
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, 5.3.3 PC Configuration
directly after they are read. This function allow the user to designate the default data and export
directories, and the parameters for communication between the personal
5.3.2 PopUp Scan Parameters
j) ) computer and the fluorometer, as well as printer information. When this
option is selected, the dialog box shown in Figure 5-35 appears.
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. "III"iTiW:roT roT' ........
The software sends these parameters to the instrument upon
commencement of the PopUp Scan while saving the setup for the Qata Directory 1C:\RFPC\dala\ 1
currently selected acquisition mode. After the PopUp Scan has been
completed, the current instrument settings (along with any related .Export Directory IC:\RFPC\data\ I
parameters) are restored.
fluorometer Serial Port @1 02 03 04
Iext Prinler-----------, !'iraphics Printer
HP LaserJet III on LPT2: ~ IHP LaserJet III on LPT2: III

.
HP DeskJel 550C Printer on LPT
"
~
HI-' I-'Iotter on LPT1:
IBM Proprinter XL on LPT1:
)
1;;W;iJg~F"f;ril
Figure 5-35 PC Configuration Parameters Dialog Box

Data Directory The Control Panel program can be run by making the program an icon
Type the name of the directory to which data files are saved and loaded, (see the Microsoft Windows User's Guide: Using Control Panel). Use the
making sure that the directory entered already exists. Strict MS-DOS Setup push button (or Control Panel) to change the page orientation,
protocol must be followed in the directory assignment (i.e., directory graphics resolution and other printer features before outputting to the
name must start with a drive designation and end with "\", as in printer or plotter.
C:\RFPC\DATA\).
5.3.4 SpeedBox Configuration
Export Directory The SpeedBox Configuration dialog box (Figure 5-36) allows the user to
Type the name of the directory to which export files (ASCII or DIF) will select the functions to be placed in the SpeedBox, as well as the style in
be saved, making sure that the directory entered already exists. Strict which the SpeedBox is displayed. All selections are automatic, on
MS-DOS protocol must be followed in the directory assignment (i.e., command; there is no undo command.
directory name must start with a drive designation and end with "\", as in
C: \RFPC\EXPORT\).
SpeedBox (RJ Configuration
I!em to Change:
Fluorometer Serial Port
Select the port number (1 - 4) to be used for the fluorometer unit.
1111 Arithmetic ~
NOTE: This software requires that a hardware interrupt be assigned Command: ~tyle:
to the communication port. By IBM standards, interrupt 4 is assigned
to either COMI or COM3, but not both. Interrupt 3 is assigned to a
R.
Arithmetic
ASCII Export
i+
N~
either COM2 or COM4, but not both. Please be certain of the lBJ Attachments
employed hardware configuration before selecting this value. When [II
) Average
selecting a nonexistent port and ignoring the ensuing WARNING, R Both Axes
Channel Status
automatic initialization of the fluorometer unit is turned off.
III Ii¥j Copy
Data Print ''f,
Text and Graphics Printer Figure 5-36 SpeedBox Configuration Dialog Box
Selection of the text and graphics printer is contingent upon which
printers have been installed in Windows (multiple printers may be Item to Change
installed). Click on the drop-down list in either the Text or Graphics This drop-down list contains the functions currently assigned to the
selection box to designate the printer(s) to be used (see Figure 5-35). SpeedBox; selecting an item allows for additions, changes or removal
The Text Printer is used for all tabular output using Print commands. from the SpeedBox.
The Graphics Printer is used for all graphics and graphics/table
combinations. (Note that a plotter device may usually be selected as a Button
text printer.) 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
The same printers appear in both the Text and the Graphics Printer list
current SpeedBox item. Available buttons and recommended SpeedBox
boxes. Highlight the printer to be used and select OK to confirm the
settings are shown in Figure 5-37.
designation. When the text and/or graphics printer designation is
changed using this dialog box, it may be necessary to run Control Panel
Command
to adjust the printer port assignments.
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

Add
~ Arithmetic ~ Help ~ PopUp Scan TlJ Parameters
Select both a Button and a Command and click on Add to add a new
item to the SpeedBox. ~ ASCII Export liJ Hide All ~ Quantitative Mode
LiI Attachments ~ Instrument [jl Radal Both A e,
Insert ~ Average [II Int/Conc Toggle lJ Radar X Axis
This option inserts a new item (select a combination of Button and ~ Channel Status fl) Limits ~ Radar Y Axis
Command) into the SpeedBox after the currently selected item in the ~ Copy (Graph) LIJ Line Colols ~.~ Rename Channel
Item to Change combo box. ~ Data Print [j] Load Parameters ~ Save
~ Delete ~ Open
,
[1;j1 Save as
Change ~"!r;+":
DIF Export ~ Options II Save Channel
This option is used to change a selected SpeedBox item. Select an Item ~
'Rl::" Display Equation [ft Parameters [11 Save Parameters
to Change, then select a new Button and/or Command and click on the [II [IJ ~
•
Display Grid PC Configuration Spectrum Mode
Change button to reflect the change.
Erase Channel ~J.~
Peak Area [Ij SpeedBox® Configuration
Remove
[0
','. Exit [it
:'?',
Peak Pick [B
%. Time Course Mode
This function is used to remove an item from the Item to Change list. ~ File/Channel Calc [iii Plot [II
/xc:< Transforms
Select an item, click on Remove, and the item is immediately deleted [II Fonts ~ POint Pick [II Working Curve
from the SpeedBox.

Figure 5-37 Recommended SpeedBox Button Settings
Exit
5.3.5 Instrument
Select this option to close the SpeedBox Configuration dialog box and
save all changes. 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
Style
This list shows the eight different styles of the SpeedBox: vertical, parameters. When Instrument is selected from the Configure menu, the
horizontal, single or double row, and top or side caption. Click on a style dialog box shown in Figure 5-38 appears.
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.
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

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
)
I;:#"itfrint ." "I
360.0 3800 400.0 4200 440.0 450.0
minimized in Windows operation to run another program, the VVavelength (nrn)
communication with the fluorometer unit will be suspended. Noise
1.000
-HV Control ~
0.500 Irr;~~Ph "'J
~ 0.000
This function turns the Negative High Voltage control ON and OFF. The "
1:
·0500
xenon lamp used in the spectrofluorometer provides strong light intensity ·1.000
but has inherent instability. The negative high voltage supplied to the 000 20000 400.00 600.00
Time (sec)
photomultiplier is controlled to compensate for fluctuation of the light
source. Instrument Serial Number: !A401932000010151
Peak Height: 1 12.556 1 SIN Ratio: 1235.572
PMT Protect
When the Photomultiplier Tube Protect is ON and the sample Figure 5-39 SIN Ratio Check
compartment is opened, the excitation slit is automatically closed to
protect the photomultiplier from light saturation. When PMT Protect is Press the Stop push button or Esc key to interrupt the SIN Ratio check;
OFF, the excitation slit remains in the current position and the press Exit and then Perform in the Instrument dialog box to restart the
photomultiplier tube could be saturated with light. The setting defaults to check.
OFF every time the program is started.
]) ) Press Print to obtain a hard copy of the report which is automatically
Auto Shutter sent to the graphics printer specified in PC Configuration Parameters (see
Section 5.3.3).
This feature enables the automatic operation of the shutter when various
functions are performed, i.e., Read. This feature protects very sensitive
Select the Spc Graph push button to switch between the Spectrum and
samples from being damaged by blocking illumination of the sample Time Course graph selections. This automatically brings up the Graph
before the reading is taken.
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
Dark Level Correction
3.5, #8). Note: Ctrl+R is disabled in the SIN Ratio Check.
This button performs a Dark Level Correction on the instrument by
closing the shutter, taking a reading, and performing an internal Lamp Align
calibration of the instrument. This process may take a few seconds.
This function allows for proper positioning of a newly installed lamp in
the instrument as well as resetting of the Lamp Time Used value.
SIN Ratio Check
This function is used to check the performance of the instrument by Instrument Serial Number
running a Spectrum scan (for peak height; takes a few seconds) and a The fourteen digit number of the instrument appears here. The serial
Noise scan (for the signal/noise ratio; takes approximately ten minutes). number is set at the factory and cannot be changed by the user.
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 Instrument ROM Version
cell in the holder. Click either OK to automatically begin the check or
The current revision level of the ROM installed in the instrument.
Cancel to return to the Instrument dialog box.

Lamp Time Used Sip Button

The value displayed is the total number of hours the lamp has been on. It Use the Sip push button to verify that the sample will reach the flow cell
is recommended that the lamp be replaced after 500 hours of use. 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). 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
Sip SpJ;.ed @[F-~sl' 0 Medium 0 Slow
current parameters to disk in the directory in which the program is
Sip lime ~ [99,9 to -99.9 sec) installed. The file will have a .cfg extension. To change the program
£,urge Time ~ [99.9 to -99.9 sec) default configuration, use the file name RFPC.
Qwell Time ~ (0 to 99.9 sec)
[!]
III".
Number of Binses (0 to 4)
Enter File Name: 1mB I
t· i" ~&"",,,,,,, 11i$""""'~''''''1
?E\%9K ,i';r
Enter Comment: This is the default configuration file.
Figure 5-40 Attachment Parameters Dialog Box
For the sipper, set the following parameters within the designated ranges: IPi!r""">'0'
(~. ~.~~Y!h<>.
Sip Speed
(Fast, Medium, Slow) Along with sip time, determines how much of a Figure 5-41 Save Parameters Dialog Box
sample is sipped using the sipper peristaltic pump.
5.3.8 Load Parameters
Sip Time This function is used to load a configuration file previously saved with
(99.9 - -99.9 sec.) (length of time for sipping) Positive/negative times the Save Parameters command. When selected, a list box containing all
determine the direction of the sipper motor (see Purge Time). of the configuration files appears as shown in Figure 5-42.
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.

,~.;:<' I.r.T7i11"iTil• • • • • • • •
... 1
'~ mm
rlpc.ctg
@ You are about to DELETE a file from disk!
ARE YOU SURE? (J) Default Conligurl!llion File Can Not be Deleted!
sample.dg
li;'1;;~(f'ql Ii oK")
IF :i~S!Jl 1;'t!~!J
1"""".... '!lif).
;i1l~le\'{1': 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.
Figure 5-42 Load Parameters Dialog Box The first three options, Arithmetic, Transforms, and File/Chnl Calc, perform
mathematical operations on the ordinate axis of the data. The operation is
Move the cursor to any of the file names in the list box, and select Load. identical for both Spectrum and Time Course data.
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 file
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) )
rtpc.ctg f
1:
This is the default configuration file.
Created: 18:27 12/15/94
1~~fZ¥1
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 Figure 5-45 Manipulate Menu
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 In Spectrum or Time Course modes, the first three procedures require the
from the directory. Select NO to cancel the command. The RFPC.CFG selection of a source channel, an operation and a destination channel. The
file cannot be deleted as it is the default configuration file. 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
5 - 42 RF-5301 PC Instruction Manual RF-5301PC Instruction Manual 5 - 43

opportunity to cancel or proceed) and the file name for the channel will Select the operation, source and destination channels, enter the constant
remain as it was. When a new destination channel (default) is selected, a file and select OK to proceed with the calculations. Select Cancel to
name and comment for that channel may be entered as usual. terminate the process or select the Status push button to review the
contents of the channels.
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 When the Arithmetic option is selected while in Quantitative mode, the
functions. following dialog box appears.
WARNING: In Quantitative mode, the Arithmetic, Transforms,

File/Chnl Calc and Average functions replace the currently displayed Qperators--
I.(;onstant I
data with the result of the operation. ~i'Addi I I
0- .subtract
I
The results of all calculations are automatically graphed, although it may be
O'Multiply
necessary to change the graph scale (see Radar, Section 5.5.2.7) for the results
to be visible. O/Qivide
I- ;; ()K -j~ I~'~;;~t" I
5.4.1 Arithmetic
Arithmetic operations are scalar operations (+, -, *, /) of the general form Figure 5-47 Arithmetic Dialog Box (Quantitative mode)
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 The data in the currently displayed table (Standard or Unknown) will be
division. (In Quantitative mode, S = D, that is, the source is replaced with replaced with the result of the operation. When operating on the
the result.) The operations are often useful for purposes of display, Le., ) Unknown data table, the concentration values (except raw data) are
offset a spectrum, scale a derivative, etc. When the Arithmetic option is recalculated using the new (calculated) intensity values. Select the
selected while in Spectrum or Time Course mode, the following dialog operation, enter the constant and select OK to proceed with the
box appears. calculations. Select Cancel to terminate the process.
5.4.2 Transforms
,Source ,Qper8tion Oestinalion
Transforms are function operations of the general form D=f(S), where S
@[~I @+Add 00 is the source channel, D is the destination channel and f( ) is the
01 0- Sublroet 01
transform function. (In Quantitative mode, S=D, that is, the source is
07 @2
a-MUltiply replaced with the result). The transform functions available in Spectrum
0) 0
O.
O/Qivide
0
and Time Course modes are:

00 Os
Derivative (0 through 4th order, where 0 is smoothing)
O. 0&
0, Or
0;: I I Ov
Reciprocal (IN)
0, Os
Logarithm (Base 10)
F;;w~d;;; 1R"!i~FI
~. .',. '" , ." ii !iil:i:~~!!iii The transform functions available in Quantitative mode are:
Figure 5-46 Arithmetic Dialog Box (Spectrum !Time Course modes) Reciprocal (IN)
Logarithm (Base 10)

When the Transforms option is selected while in Spectrum or Time The smoothing and differentiation calculation is based on a convolution
Course mode, the dialog box shown in Figure 5-48 appears. function using 17 data points. The derivative wavelength and time
difference values are selectable in four steps, depending on the sampling
IE "tl! "",l" interval. Although a larger dX (delta X) will provide decreased noise, it
will detract from spectrum resolution. Accordingly, select values which
r'~~
Iransform Destination
will provide the best compromise between noise and resolution.
00
@ Qerivative
[Smoothing)
01 Calculation of the derivative value requires the data points before and
07 I~ 2 after the center wavelength, which means that the derivative or smoothed
03 o 1/'i 03 spectrum cannot be calculated at both ends of the wavelength range. The
04 04 original spectrum has a unit of intensity as its ordinate value. However,
0'., O"i the derivative units are defined as follows:
o !"ogarithm
Of; 06
Derivative Spectrum Time Course
Of 01
OB Os 1st derivative Intensity/nm Intensity/time
o ~l Os 2nd derivative Intensity/nm 2
Intensity/time2
3rd derivative Intensity/nm 3 Intensity/time 3
~0F"Ilm~
sillllJ,illl II=~I
't_~. Ilflm:I
LA:;'Al!'.;:' 4th derivative Intensity/nm 4 Intensity/time 4
Figure 5-48 Spectrum and Time Course Transform Selection Dialog Box
Note that, in the Time Course mode, the unit of time may be hours,
Select the source and destination channels, and the transform function,
and then select OK to perform the calculations. Select Cancel to ) minutes or seconds, depending on the value selected in the parameters.
terminate the process or select the Status push button to review the When the Transforms option is selected while in Quantitative mode, the
contents of the channels. dialog box shown in Figure 5-50 appears.
When the Derivative (Smoothing) function is selected, another dialog Il;lr.

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 Iransform - - - - - - - - - ,
Course) may also be designated. @:j'Ti
o Logarithm
Qrder Delta Lambda--
@!g,,;~ti!~i~g! @ 10.000
o lsI IlllJWXllWj
.'w tfM
o 20.000
o 2nd o ~O.OOO
o .:Ird Figure 5-50 Quantitative Transform Selection Dialog Box
o 80.000
O.1lh Select the appropriate Transforms function, and select OK to perform the
calculations. Select Cancel to terminate the process.
Figure 5-49 Derivative Dialog Box

5.4.3 File/Chnl Calc

When the File/Chnl Calc option is selected while in Spectrum or Time
Table + testqnt > Table
Course mode, the dialog box shown in Figure 5-51 appears.
Qperators
File or Channel Calculations are vector operations (+, -, *, /) of the

@ + Add O'Mulliply
general form A op B = C where A and B are the source channel numbers,

G' Subtract C'/ Qivide
C is the destination channel number (result) and op, the vector operation
addition, subtraction, multiplication or division.
zz.qnt
samkbl.qnt
Ii "'oK P.:]
Note that Band C cannot be the same channel number. The range zzz.qnt
tim.qnt
(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).
&~~I.
~ .
After choosing A, B, C, and the operation, 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. Figure 5-52 File/Channel Calculations (Quantitative mode)
The data in the currently displayed table (Standard or Unknown) will be

replaced by the result of the selected operation with the selected file.
A op B = C
The data in the selected file is treated sequentially only, that is, the ID
A Qp B
ki I j) ) value in Unknown files or the concentration in Standard files is ignored.
@[o: 00 00 When operating on the Unknown data table, the concentration values
01 @+ ~1 0: (except raw data) are recalculated using the new (calculated) intensity
02 02 @2 values.
OJ C)
)3 03
0;. 04 04 After selecting the desired file and operation, select OK to perform the
calculations. Select Cancel to terminate the process.
0'; O'
0"., 0".,
00 Or, Or,
5.4.4 Data Print
01 01 0,
0/ Data Print will output a selected channel's data to the screen in a
On Ou Oil
Oq
scrollable window, save it to a disk file (text), output the data to a printer,
09 09
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
Iw~ty~:1
:,,"
':{{""'."' . . . 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.

The functions available with this window are provided through the menu in the specified display range. When OK is selected, the table is updated
bar options described in the following text. Numerous Data Print with the new values. For Time Course data, the range entered in Params
windows may be displayed simultaneously. These windows may be 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. IJ:'rn'I'
Enter -S.tart X Value:

le ilil 8U I
Enter .End X Value:
~
Created:
Data:
11 :21
Original
08/31/94 Enter Skip factor:
o
InstrulTlent:
Spectrulll Tl}pe:
RF -5301
Dl
li,\A'of;;=1
"' .. , ", If?Cllll~";:il
Scan Range: 350.0nlll to 450.0nlll
EX I~auelength: 350. On III Figure 5-54 Data Print Parameters Dialog Box
Salllple Pitch: 0.2
Slit Width: EX:5.0nlll EM:5.0nlTl
Scan Speed: MediulIl Edit
Sensitiuitl} : High This menu allows data to be copied to the Clipboard.
Response Tillle: Auto
Shutter: Manual, Open
Copy
Wauelength (nlll) Intensitl} Copies only the highlighted text to the Clipboard. To highlight text,
350.0 4.574
350.2
350.4
4.408
4.337 I J ) 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
350.6 4.245 the Edit menu. The highlighted text is placed in the Clipboard.
350.8 4.212
351.0 4.280
Copy All
Figure 5-53 Data Print Window Sends all of the text to the Clipboard.
In the Quantitative acquisition mode, Data Print is used primarily for
Disk...
printing reports. The Standard or Unknown data, as well as the
To save the currently displayed data to disk in a text file, select Disk
parameters, are automatically printed when Printer is selected. When
from the menu bar. The Text File dialog box appears, as shown in Figure
any replicate Sample IDs exist in the data, the mean of the replicates is
5-55.
displayed (and printed) below the last of the replicate IDs.
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!f;!!:Of(i:~rtl
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 Figure 5-55 Save Data Print Dialog Box
a Skip Factor, which designates the point interval between data readings
5 - 50 RF·5301PC Instruction Manual RF-530 1PC Instruction Manual 5·51

When a file name is associated with the displayed data of the selected into a third party text processing software. When Save is selected, enter
channel, (the name of a file that was loaded or the name that was entered the file name (without extension) and select OK. Select OK to close the
after the scan was completed), it is displayed in the Text File Dialog Box. Activity Calculation window. To close the Data Print Window, select the
Select OK to use this file name or enter a new name. This file is saved in control menu box at the upper left comer of the window and select Close,
the data directory with a .txt extension and can be retrieved and plotted or double-click the control menu box.
by selecting the Plot option of the Presentation Menu or incorporated into
a third party text processing software. 5.4.5 Peak Pick
This option numbers the peaks of selected channels on the graph and
Printer
also lists them in a window. When the Peak Pick option is selected, a
Select Printer from the menu bar to automatically output the currently channel selection dialog box appears. Choose the channel for the Peak
displayed data to a printer. Pick operation and select OK. When only one channel is loaded, the
program defaults to channel 0 and no channel selection is necessary.
Act. Calc. (Activity Calculation)
When the displayed data was acquired in the Time Course mode, it is The graph is redrawn with each of the selected channel peaks numbered
possible to calculate the Slope, Activity (slope times a multiplication (see Figure 5-57), and a scrollable window appears containing the list of
factor) and the Correlation Coefficient (estimate of closeness offit to a peaks, as shown in Figure 5-58 (also in lower left of Figure 5-57). This
linear model) over the range of data displayed (i.e., designated in procedure may be performed for any combination of the available
Params) by selecting Act. Calc. from the menu bar. The window shown channels. A Peak Pick scrollable window appears for each of the
in Figure 5-56 appears. 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.)
.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. Figure 5-57 Peak Pick Window
Select Save to save the results to disk in a text file (.txt) in the data Options
directory. This file can be retrieved and plotted by selecting the Plot The Options menu in the Peak Pick window contains the following items
option of the Presentation menu (see Plot, Section 5.5.5) or incorporated for modifying peak selection and display (see Figure 5-58).

Find Valleys The reproducibility of the peak detection algorithm may be estimated
Select Find Valleys and a check mark appears next to the Find as 1/10 to 1/1 00 of the peak width at half the height of the absorption
Valleys option, indicating that the values displayed on the graph are band. There may be slight scanning errors for very sharp bands. In
minima (valleys) instead of maxima (peaks). Select Find Valleys such cases, it is necessary to compare the ordinate values step-by-step
again (when it is checked) to remove the check mark, and the around the peak wavelength in order to define the precise peak
displayed values are again maxima (peaks). 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
I the X-axis (Wavelength or Time) and the value for the Y-axis
m.
Qptions fdit Outj!ut Beplot (Intensity).
:r.
Batch 1 Sa~ple Hu~ber 1
Created: 15 :33 118/31/94 Mark Graph

Data: Original
When the graph is redrawn on the first selection of Peak Pick, the
Instru~ent: RF-53111 positions of the peaks are automatically marked on the graph by
Spectru~ Type: EM
Scan Range: 41111.lIn~ to 61111.lIn~ number. When the graph is redrawn after changing the limits using
EX Wauelength: 41111. 0n~ Limits on the Graph Pop-up menu or Radar (see Limits, Section
Sa~ple Pitch: 1.11
Slit Width: EX:111.lIn~ EM:5_lIn~ 5.5.2.4 and Radar, Section 5.5.2.7), the peaks (or valleys) are no
Scan Speed: Uery Fast
Sensi tiui ty: High longer numbered. Selecting Mark Graph replaces the numbers on
Response Ti~e: 11.5 Seconds the graph for the selected channel.
Shutter: Manual, Open
Ho. Wauelength (n~) Intensity Edit

1 424.11 -19.288
2 466.11 21.473 This menu allows data to be copied to the Clipboard.
3 514.11 -5.763
) Copy
Figure 5-58 Peak Pick Window
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
Change Threshold
drag to the last line. Release the mouse button and select Copy from
This option allows you to enter a new value for the threshold which the Edit menu. The highlighted text is placed in the Clipboard.
determines peak selection. In general, a larger threshold decreases the
number of peaks found, while smaller threshold values result in more Copy All
peaks. (See the following discussion of Peak Pick algorithm.)
Sends all of the text to the Clipboard.
Peak Pick Algorithm: A four point successive comparison method is Output

used to define a peak as at least four consecuti ve increases in value The Output menu contains items for handling output of Peak Pick data.
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 Print Table
situation is reversed (4 + decreases, bottom, 4 + increases). Select Print Table to automatically print the Peak Pick data in tabular
form with the text printer selected in the PC Configuration dialog box.
The threshold is defined as the distance between a peak point and a
line connecting the two adjacent valley points (or vice versa for
Save Table
valleys). When the threshold value of the peak is less than the
This option creates a tabular text file (with .txt extension) and saves it
threshold value entry (0.001 is default), the peak is rejected. The
to disk in the data directory. Select Save Table and enter a file name
threshold value can be adjusted to filter out unwanted peaks or noise.
(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.

Graphics Plot Point Pick

Select Graphics Plot to automatically plot the Peak Pick graphic data
of a selected channel with the printer selected in the PC Configuration
1"--
,;Qpen ~I >! ~
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.
li.s;ve-~!> ]
~
,.,
Replot
cQ)
:£ 1/ Ii'l\Pb .1
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). 450.0 500.0 5500 600.0
Wavelength (nm)
£loints
5.4.6 Point Pick

1 1-45-8.0-1 I478.0 I 3 I 517.0 I 4 I560.0 I 5 I
2
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 6 I I 7I I 8I I 9I I 10 1====
corresponding Y-axis values are listed in a window. When this command 11 1 I 12 1 I 13 1 I 14 1 I 15 1_ _

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.
Figure 5-59 Point Pick Dialog Box
When only Channel 0 has data, the Point Pick dialog box immediately
appears displaying the graph and a vertical marker line for using the Press the Open push button to load a Point Pick template file previously
mouse to select the points (see Figure 5-59). (In Time Course, the saved to disk using Save as.
marker line may be on the far left at 0.0 and hard to distinguish between
the graph border.) Press the Save as push button to save the points just selected into a
)
template file for later recall.
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 Press Graph to open the Graph Pop-up Menu (see Section 5.6).
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 Press Cancel to close the operation with no changes.
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 Select OK to close the dialog box. The points are marked on the main
using the left mouse button (see Zoom in Graph Cursor, Section 4.6). To graph and the Point Pick window is displayed (Figure 5-60) listing the
return the graph to its original scale, use the Graph Pop-up menu (see channel's acquisition parameters and the selected points. This window
Section 5.6) or use one of the Radar functions (select Radar under the may be moved and resized for convenient viewing of the data.
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.

Copy
Qptions .Edit OutQut Beplot! Copies only the highlighted text to the Clipboard. To highlight text,
Raman Peak 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
Created: 11:21 08/31/94
Data: Original select Copy to place the highlighted text in the Clipboard.
Instrument: RF -5301
Spectrum Type: EM Copy All
Scan Range: 350.0nm to 450.0nm Sends all of the text to the Clipboard.
EX Wauelength: 350.0nm
Sample Pitch: 0.2
Slit Il1dth: EX:5.0nm EM:5.0nm Output
Scan Speed: Medium
Sensitiuity: High The Output menu contains items for handling the output of Point Pick
Response Time: Auto
Shutter: 11anual, Open data.
Ho. Wauelength (nm) Intensity
1 364.2 4.530 Print Table
2 369.0 5.099 Select Print Table to automatically print the Point Pick data in tabular
3 372.8 8.464
4 376.2 15.054 form by the text printer specified in the PC Configuration dialog box.
5 378.8 18.159
6 383.0 13.963
7 385.8 9.788 Save Table
8 390.2 7.470
9 398.2 7.836 This option creates a tabular text file (with .txt extension) and saves it
10 411.4 7.976 to disk in the data directory. After selecting Save Table, enter a file
name (without extension) in the Save .txt File dialog box. The
Figure 5-60 Point Pick Window created file may be used in Plot (see Section 5.5.5) or in a word
The Point Pick window menu bar contains the following commands:
)) J processor or editor.
Graphics Plot
Options Select Graphics Plot to graphically plot the Point Pick data of a
The Options menu contains items for modifying point selection and selected channel by the graphics printer specified in the PC
display. Configuration dialog box. The plot will print with the marked points
and a list of the points as well as the acquisition parameters.
Change Values
This re-displays the Point Pick dialog box shown in Figure 5-59. Replot
Select Replot to redraw the graph and clear it of all marks (including
Mark Graph Peak Pick marks, which may be used in combination with Point Pick; see
When the graph is redrawn after the first selection of Point Pick, the Peak Pick, above).
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.

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
of a scan and output the results (plot and/or table) to the printer or the is located just to the left of the Start edit box. Pressing Alt and the
Clipboard. The Peak Area dialog box appears (see Figure 5-62) when
region number (1, 2, 3, or 4) also selects the Region box.
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
a
a+I3=(.L D[i])*P The Factor is applied to all four regions and is set by pressing the
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
13 baseline). The Divisor is set for each individual region by entering a
13 = ~ * (D s + De) * (A e - As)
value in the Divisor edit box on the appropriate row.
-+1 I+-
Ae P As
The Reset button restores Start, End, Divisor, Factor, and Baseline to
Figure 5-61 Formula for Peak Area Zero to match the currently displayed area(s) and result(s).
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.
1~%it~~*~I~r';1
I) )
@ [~iintTabie:
450.0 500.0 550.0 604.0 o Save table to text file

W.velength (nm)
Region Start End Divisor Area Result

o Graphics Plot
1 11111538.0 1 1586.0 11.000 I 4673.91 B 4673.918 o Copy graph to clipboard
Z I§S!j 1 400.0 I 1 400.0 1 11.000 1 0.000 0.000 o Copy table to clipboard
J 'Btl! 413.0 I I 428.0 I 11.000 I -267.613 -267.613
! lmll !! 1 1 I 11"h~~·<r;p;"·1 I
M ¢i';l~G;J
Figure 5-62 Peak Area Dialog Box
Figure 5-63 Area Output Dialog Box
To calculate the area under a curve for a Region, enter the starting and
ending points in the Start and End edit boxes. Enter the divisor (default The Open push button can be used to recall a template file that was
value is 1.0) in the Divisor edit box and press the Recalc push button. previously saved using the Save as push button. All the values for Start,
The Start and End values can also be graphically selected: move the End, Divisor, Factor, Baseline to Zero, color and brush types for the
mouse over the left marker until the cursor turns into a horizontal double areas, and the graph limits are replaced with those from the template file.
arrow, hold down the left mouse button and drag the marker to the After opening a template file the name of the file is appended to the title
desired starting point. Follow the same procedure to mark the end point of the Peak Area dialog box. The template file blank.pka is provided to
using the right marker. Press Recalc; the area under the curve for that clear all values in the dialog box. When changing the default values
region is displayed and the Area and Result are calculated. contained in this file, set the desired colors, patterns, and parameters and
select the Save as push button.
5 - 60 RF-530 1PC Instruction Manual RF-530 1PC Instruction Manual 5 - 61

The Save as push button saves the current settings (Start, End, graph 5.5 Presentation Menu
limits, etc.) to a template file for later recall using the Open push button.
The Presentation menu consists of items which monitor and control the
5.4.8 Average (Quantitative Unknown only) presentation format of data files on the screen and determine their printout
format (see Figure 5-64).
The Average option is used in conjunction with the Repetitions selection
in the Quantitative parameters.
c::::~( ~... ,:j ...
file Acquire Mode .(;onfigure

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, ~
f:
Section 5.4.4.
50.
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 450.0 500.0
Wavelength (ron)
550.0 600.0
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).
I} )
A minimum number of standards must be present for each order fit. As Figure 5-64 Presentation Menu
enough standards are collected to fit the data to the selected polynomial,
the equation is calculated for review. Additional standards will prompt 5.5.1 Channel Status
for recalculation of the calibration curve. This option is used for monitoring the screen presentation status of the
occupied channels. The Channel Status window is accessed via the
Polynomial Order Minimum Number of Standards Presentation menu or directly from the keyboard by entering Ctrl+C.
No Zero Interception Zero Interception
When Channel Status is selected in Spectrum or Time Course mode, the
window shown in Figure 5-65 appears. In Quantitative mode, the Channel
1st 2
Status dialog box displays whether the Standard and Unknown have been
2nd 3 2 saved to disk.
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.

Line Channel File Hame Displayed Saved File: BATCH1 N1 Batch 1 Sample Number 1
:r.
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
IQfA~
~.,'-'.lW;K~.m"
Slit Width: EX: 10.0 nm EM: 5.0 nm
Response Time: 0.5 sec
Figure 5-65 Channel Status Dialog Box Shutter Mode: Manual Shutter Position: Open
For each occupied channel, the window displays the following information
from left to right:
Figure 5-66 Spectrum Parameters Display Window
Line: Color and type
After reviewing the parameters, select OK to return to the Channel Status
Channel: Channel number (0 - 9)

window.
File Name: Shows the file name (or *No Name*) associated with the
Displayed:
channel
Y / N indicates whether a channel is currently displayed on
I) ) 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
the graph
channels.
Saved: Y / N indicates whether the data has been saved to disk
5.5.2 Graph
The Displayed status may be changed for any channel by selecting the channel
5.5.2.1 Copy
(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 Use this command to automatically copy the main graph to the
channel. 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
The acquisition parameters of the data in any of the channels (see Figure 5-66) that the graph is the appropriate size, and select Copy. Any other
may be viewed by highlighting the channel (using up/down arrow keys or windows (i.e., Data Print, etc.) or parts of other windows that are over
mouse) and selecting the Parameters push button. 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).

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.'
[2QDD.D.[3~
L .:. _]
Channell wj
h:'$'<iI
Channel 2 .~ ~.IiillI.
1*;ffiHChannel 3 .1Iilil• • • • • •
440.0 450.0 Wt;;@'1Channel ..
1:!Phd Channel 5 ••••••••
••••••••
L:¥1wl Channel 6
@'w~l Channel 7 + ••••• ~.D
Line and Annotation llpes: ~ustom CoiOls:
Figure 5-67 Spectrum Graph with a Grid ~MM>II:xl ttFf#iflI#%$?f1 fiI:lfill ffi"Fil DDDDDDDD
~ FFJ r;i'9 If,;'J • !NEI IiZ7iI WFlj
DDDDDDDU
5.5.2.3 Fonts
Allows selection of font, font style, size, effects and color for the
1&~~I2!lli~~ffi1illtili!tJ
Ifil8 ,jif.
4F
'''-~'-'''6jn ~~Bt "
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 type to be associated with the designated channel from the Basic
(see Figure 5-68). Colors or Custom Colors and Line and Annotation Types selection
boxes.
I Graph Limits
'~.~ Note: The color and line type selected for channel 0 is used for the
IIImII 1
Mali:
I -21.345 I
['
}of.ax: 1600.0 1
PopUp Scan. Line color and line type for the Standard and Unknown
Min: Min: 1400.0 I data points and the Standard Curve may be selected in the Colors
dialog box.
Iirt!t~¥?! [!~t&J [lmlfi~j®l
When a black and white printer is used (dot matrix or laser printers),
Figure 5-68 Limits Dialog Box
line patterns can be used to discriminate between scans.
Enter maximum and minimum values for the X- and Y-axes and select
To add a Custom Color, select the Define Custom Colors push
OK to update the graph axes. Select Reset to scale the graph to the
button. The Custom Color Selector dialog box appears as in Figure 5-70.
acquisition parameters currently set in the Parameters dialog box in
the Configure menu. When the minimum value is greater than the
Define the color either by directly entering the numeric value or by
maximum, they are automatically switched.
using the mouse to specify the color coordinates. When the setting is
correct, select Add Color. Multiple custom colors may be added at

one time-each consecutive selection will be placed one box to the Select the Rounded Tick Marks check box to create tick marks at
right. When the last box is reached, the first color will be overwritten. evenly calculated intervals on the graph (i.e., 20.0, 40.0, 60.0, and
Select Close to end the color definition. 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 7,.--_-,---_---,--_---,~___,-_____, 764.27"T'- - - , - - - - - - - , - - - - , - - - - - - - ,
600.00
389.65 400.00
200.00
• HU~:~ ~ 15.03''-----~--'---~-----'--~-'---~---'--~-----' 15.03iL

1 ..LI_ _---"--_ _---'---_ _--'---_-----l
.!led:
37.567 58.456 79.34 37.567 50.000 60.000 70.000 79.34
.2.al:~ ~reen:~
hum: ~ BIQe: ~
ColorlSQ.lid
Fixed Rounded
Figure 5-72 Fixed vs. Rounded Tick Marks

Figure 5-70 Custom Colors Dialog Box ) Select OK to save changes.
5.5.2.6 Options Select Cancel to terminate the changes and close the dialog box.
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 Select Reset to restore the line characteristics to the values last saved.
Minor Tick marks.
5.5.2.7 Radar
1'- Options 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.
Grid bines:
Radar may be applied to the X- or Y-axis independently, or radar may
IIN~on:le I [gJ Bounded Tick Marks
be applied to both axes simultaneously. This option may be selected
Grid Color: D Minor Tick Marks from Graph under the Presentation menu or from the Graph Pop-up
IBJ Teal II
Menu. Or, apply radar to both axes from the keyboard using Ctrl+R.
Line Iype:
5.5.3 Hide All
1-
~ 0 0 11 e d I
~
Select Hide All from the Presentation menu to clear the graph of all
f!.ackground Color: displayed data. (It will not delete any data.) The data is still available in
PH£t:1 Silver ~
~ the respective channels. A check appears next to the Hide All option to
1- - - - - - -
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
Figure 5-71 Options Dialog Box screen is cluttered with previous data that cannot yet be stored or erased.
The screen will be clear for the next acquisition channel.

5.5.4 Show Speed Box ·'W,!WiITl
Select Show SpeedBox from the Presentation menu to display or hide the Graph Params. File Channel/F. Name Quadrant[s)
SpeedBox. The SpeedBox is an accelerator device which uses picture
buttons to represent commonly used menu commands. Figure 5-73 ~ I [8] 0 0
I
Screen
01 0203 04
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. f!1 0 [8] 0 I 0 rzJ1 [8]2 [8]j 04
(See the Microsoft Windows User's Guide for more information on
moving windows.) ~ I 0 0 0
I 01 0203 04
QI 0 0 0 I 0, 0203 04
tffiJ
3 4
Figure 5-73 Configured SpeedBox

Figure 5-74 Plot Layout Dialog Box (Spectrum I Time Course modes)
The SpeedBox control menu has three selections, Move, Close, and
A maximum of four items (Rows A - D in the dialog box) may be chosen for a
About; select Move to move the SpeedBox around the screen via the
single printout, one for each quadrant of the page. Beginning with Row A,
keyboard; Close hides the SpeedBox; About displays the About
select either Graph, Params. (parameters), or File using the check boxes in
SpeedBox dialog box. The SpeedBox is configured using SpeedBox
the selection box adjacent to A.
Configuration under the Configure menu (see Section 5.3.4). The
SpeedBox may also be configured by double-clicking on its caption bar. When Graph is selected, the Plot Data dialog box appears, as shown in Figure
The RF-530IPC software remembers the last position of the SpeedBox 5-75. Select one or more channels of data to be plotted in the conventional X,
when the program is exited or the SpeedBox is hidden. 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
5.5.5 Plot Title box, and the Y-axis range may be modified by entering new upper and/or
This option enables layout of a combination of graph overlays, lower values.
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 ·Jri'flI:roU".
has been installed and is selected in the PC Configuration dialog box. O!lM 05
When Plot is selected while in Spectrum or Time Course modes, the OJ Dr,;
Plot Layout dialog box appears, as shown in Figure 5-74.

O~ O!
OJ Ofl
O~~. O.H
(8) ~creen [ I Spectra Qnly
Graph Iille
~I·----J
fAxis Rimgt
(,1(""''': I <D.WI I
Lm.<fer: I ·1,~11
Figure 5-75 Plot Data Dialog Box (Spectrum I Time Course modes)
5 -70 RF-530 1PC Instruction Manual RF-5301PC Instruction Manual 5 - 71

The Spectra Only option in the dialog box may be checked in the case of @it layout
overlaying data only in a graph which has already been printed. This
option requires a printer which allows multiple printouts on a single page. Std. iinY. Params. File Format Quadrantls)
Select OK to close the Plot Data dialog box and update the ChannellF.
AI[gJ 0001 Graph [gJ1 02 03 04
Name column in Row A in the Plot Layout dialog box.
12 1 000 [gJ1 aa.M 01 [gJ203 04

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
fCO=O 00[ Std. Params. 01 o 2 [gJ[~: 04
Y-axis range given in the Plot Data dialog box is not used when Screen
is selected.
QIO 0001 01 020304
Next, select the appropriate quadrants in the Quadrant(s) column of Row

I':"~~:p'~ 'I tffiJ
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) Figure 5-76 Plot Layout Dialog Box (Quantitative mode)
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 As above, a maximum of four items (Rows A - D in the dialog box) may
appear when printed. Click on the Preview screen or press any key to be chosen for a single printout, one for each quadrant of the page.
return to the Plot Layout dialog box. Beginning with Row A, select either Std. (Standard), Unk. (Unknown),
Params. (parameters) or File, using the check boxes in the selection box
To include data acquisition parameters in the printout, assuming that at adjacent to A.
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
) When Std or Unk is selected, the Plot Data dialog box appears as shown
parameters may be selected per item. Select OK to close the dialog box in Figure 5-77. Select Table to display the data in a tabular format and
and update the ChannellF. Name column in Row B of the Plot Layout enter a title when desired. Note that any portion of the table that does not
dialog box. 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
Select an available quadrant from the Quadrant(s) column as described desired limits for the X- and Y-axes.
above.
Any text file (.txt) may also be included as another item in the page
Format------------,
format when at least one quadrant remains available. These include Data
Print, Peak Pick, Point Pick, Activity Calculation, or any file created in a o Iable @[~i~p~l
word processing or editor program (such as Microsoft Notepad).
Select File in the next available row, and then select a file name (only one Tit!e:
per item) from the Open dialog box which appears. The default directory Max Min
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
~Axis:
I 100.00 II 0.00 I
row of the Plot Layout dialog box. 't Axis: 11000.000 II 0.000 I
When Plot is selected while in Quantitative mode, a Plot Layout dialog kO~¥41 Il'B6;e't:"1 I'cancel I
box similar to the one described above appears (see Figure 5-76).
Figure 5-77 Plot Data Dialog Box (Quantitative mode)
5 - 72 RF-5301 PC Instruction Manual RF-5301 PC Instruction Manual 5 -73

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
After ascertaining the acceptability of the overall page format using the
Chi =
I f(C)
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
continues in the background until the assigned plot is finished. ,1·'11"; :'! .~)
[ile Acquire Mode l;onfigure Manipuia!e Eresenta!ian tlelp
Standard
When Cancel is selected, the printout is cancelled and the dialog box is
Standard
Humber Cone. Int.
closed.
1 56.000 2.251
3 '"
0.0000 3.443 >
4 0.0000 3.154
Note: In some cases the use of the Print Manager may not be desired
5
~
~
1:
(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) 2O.00J
mgll
<O.OOJ 61.600
Select Int/Conc Toggle from the Presentation menu to toggle the units
Cone - klint' kO
k1 = -11 ~.5
used for the Y-axis of the Unknown display between the intensity and
kO = 378.2
concentration units. Each time this is selected, the Unknown graph
Chi-Square: O.2~208
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.
Figure 5-78 Quantitative Window Showing Equation under the Graph

5.5.7 Display Equation (Quantitative mode only)
Select Display Equation from the Presentation menu to display or hide
the equation below the graph.
5 -74 RF-5301 PC Instruction Manual RF-530 1PC Instruction Manual 5 - 75

5.6 Graph Pop-up Menu 5.7 Help

The Graph Pop-up Menu is accessed by pressing on the right mouse button The Help selections offer a summary of the RF-530l PC software. Help is
when the cursor is inside the graph area. The menu appears on the screen especially useful when you require information quickly or when the instruction
wherever the mouse cursor is placed. Select any Graph Pop-up menu function manual is not available.
to display a dialog box for that function. Clicking on the main screen with the
left mouse button removes the Graph Pop-up menu. 5.7.1 Help for RF-5301 PC
Select this menu item to open the Windows Help program and load the
5.6.1 Copy RF-5301 PC Help file. The initial Help window displays the acquisition
Use this command to automatically copy the main graph to the Clipboard. modes for which help is available. When selecting one of these, help on
(See Section 5.5.2.1) any menu item is available. The user may also select Search to locate
information on a specific topic. Please refer to the Microsoft Windows
5.6.2 Cross Hair User's Guide for more information on the Windows Help System.
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 5.7.2 How to Use Help
intersection of the cross hair (see Figure 3-9, Tutorial). To remove the Select this item to access information on the Windows Help system. This
cross hair, reselect the Graph Pop-up menu and click on Display. may be used as a quick reference in addition to the Microsoft Windows
User's Guide.
Select Lock to lock the cross hair in a selected place on the graph.
5.7.3 About RF-5301 PC
5.6.3 Fonts Select About RF-5301PC from the Help menu to see the program
Allows selection of font, font style, size, effect and color for the
coordinate values, axes labels and headings. II )
version, the software licensee, and the software serial number. To exit
this dialog box, select OK.
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.)
5 - 76 RF-5301 PC 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 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
RS-232C serial cable

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
Computer RS-232C port
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.

Troubleshooting Troubleshooting
6.2 Software Operational Error Messages
Table 6-2a Error Messages and Remedial Actions Table 6-2b Error Messages and Remedial Actions (continued)
IVIessage Remedial Action Message Remedial Action
The instrument attempted to perfonm an AutoZero and the Windows was unable to create a Device Control Block for i
· signal level was too high for this operation. Verify that DCB Initialization Failure! the specified Asynchronous Communications Port. This is
there is no sample in the cell holder and repeat the last usually caused by a corrupted configuration (.CFG) file.
AutoZero Level Over
operation. Separate the excitation and emission Default configuration file Default configuration file cannot be deleted as it is
wavelengths when they are too close; lower the sensitMty cannot be deleted! required for the software to load.
setting; or decrease the bandwidth.

The directory entered must exist on the disk. See MKDIR
The configuration file is conrupted or is not in the proper Directory Does Not Exist!
in the MS-DOS manual.
Bad or Missing directory. The default configuration file is used at
Configuration File! initialization. This may be copied into the correct directory · When the time units are changed in Parameters under the
• from the original master disk. · Configure menu, all current channels are changed to
Error Converting Channel
I those units for consistency. This message will appear
Time Units! Delete
i PC has ost connection with fluorometer unit. Can be when there is not enough memory available. Delete any
Unnecessary Channels!
Cannot Establish channels not needed at this time to make room for new
caused by loose or broken cable. Verify proper cable
Communcations! i data.
oonnections and continuity.
: A temporary file is written to disk for Peak Pick and Point
Cannot Set -HV mode to Xenon lamp is OFF. Negatwe high voltage cannot be Error Creating Temporary
0\1 tumed on when the lamp is not lit.
) File!
· Pick operations. If the file -TEMP_PK.TXT exists in the
: data directory, delete it. Also see Rle Creation Error.
There was a problem sending or receiving data to or from Error Opening Rle! I same as Rle Creation Error!
Co d Checksu E r1 the instrument. Please try your last operation again. If
mman m rro. this problem persists, please contact your Service · Generally, this is a hardware problem. The disk file has
Error Reading Rle! been corrupted, the door of the disk drive is open, etc.
1 Representa~.~~~~_
Consult the computer and MS-DOS documentation.
! Windows was unable to initialize the specified
Communcations Asynchronous Communcations Port for some unknown The emission monochromator of the fluorometer unit has
slipped while moving. Discontinue communications with
Initialization Failure! reason. Try rebooting PC. If the port still fails, check for
Emission Monochromator the instrument by turning off the Fluorometer Window,
hardware problems.
Slip and power the instrument OFF and ON. If the problem
Windows was unable to open the specified Asynchronous persists, consult the hardware section of the manual or
contact your Service Representative.
Communications Port. This can be caused by another
program (i.e., Windows Tenminal) having oontrol of the The excitation monochromator of the fluorometer unit has
COM Port. Another cause oould be a Fatal Windows slipped while moving. Discontinue communications with
Error (one which returns the PC to MS-DOS). This will Excitation Monochromator the instrument by turning off the Fluorometer Window,
Communications Open
force any programs attached to the COM Port to exit Slip and power the instrument OFF and ON. If the problem
Failure!
without detaching from the port. If Windows receives a persists, consult the hardware section of the manual or
· Fatal Error, always reboot the PC. If you select a contact your Service Representative.
I Fluorometer Serial Port in the PC Configuration Parameter
! Entry Fonm that does not exist, this error message will be
· issued.
Communications Time Out! Same as Cannot Establish Communications!

Table 6-2c Error Messages and Remedial Actions (continued) Table 6-2d Error Messages and Remedial Actions (continued)
Message Remedial Action Message Remedial Action
An attempt has been made to write a file to the disk which The configuration file in question (those with a .CFG
violates DOS protocol (e.g., a directory that is nonexistent extension) cannot be loaded because it is not the right
or a full disk) or computer has encountered a bad sector Invalid Configuration File! type for the instrument being used. This may occur when
File Creation Error! or improperly formatted disk. Check the PC Configuration a completely foreign file type was stored with the .CFG
and consult the computer and DOS manuals. Check that extension.
the disk drive door is properly closed when using floppy
disks. Fluorometer unit was unable to interpret the instruction
Invalid Fluorometer
sent by the PC. This may be caused by a corrupted .CFG
When printing a text file on a graphics printer, the file may Commard
file or by a loose or broken communications cable.
, be too large to fit within the defined graphics area. When
Continue is selected, the text will be trimmed and not fUlly
File will not be displayed printed. When cancelled, the text file may be edited (e.g., A value was input in a dialog box that was outside the
entirely. Do you want to editing the file with Microsoft Notepad) to make it smaller Invalid Entry! acceptable limits or of an invalid fonmat. Enter the correct
continue? to fit into the graphics space. The printer resolution can value and proceed.
also be redefined in the Microsoft Control program to
produce small fonts and allow the file to fit into the Invalid Numerical The lower limit of the recording range must be lower than
allowable graphics range. Relationship! the higher limit.
I ' --------
File Write Error! , Same as File Creation Error. Xenon lamp illumination time is abnonmal. Please reset
Lamp Time is Invalid ! the lamp time to zero (0). If it occurs again, call your
General Communications
Error!
- - ---,_.~
Same as Transmit or Receive Error!
--------
II ) 1- - -
: Service Representative.
--I- ~--- -,-- - -
: Other programs are conflicting with Microsoft Windows
Graphics Initialization Error! See Main Initialization Error.
Main Initialization Error! ! and are not compatible. Discontinue the operation of
Windows has run out of graphics memory. Windows must ; other loaded software running under Windows.
Graphics Memory Error! ;...-----_._-
be restarted.
Main Window Creation i
Error!
i See Main Initialization Error.
Graphics Printer Error! Same as Text Printer Error.
i
, The proper printer must be selected in the software as Memory Error!

1Sam~ ~~ Not ~~gh Me~ory. When this error appea~,
Graphics Printer has NOT even when memory is freed up, reboot the system.
I well as in the Microsoft Windows Control Panel program.
been Selected!
Save to disk under Save Parameters.
, No More Channels , All RAM channels for scans are occupied. Select Erase
Available! Channel under the File menu.
The file in question cannot be loaded because it is not
Incorrect File Type!

valid for the mode of operation or analysis.
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 Instnument~ j Use ~configuratio~fi~e f()r_t~e_fluorome~erunit's software.
6-4 RF-5301PC Instruction Manual RF-530 1PC Instruction Manual 6-5

Table 6-2e Error Messages and Remedial Actions (continued) Table 6-2/ Error Messages and Remedial Actions (continued)
Message Remedial Action Message Remedial Action
Not Enough Memory for Not enough computer RAM space free to perform the 1) The printer driver is not installed. Install the proper
Text Printer Error!

Calculations! designated operation. Restart Windows. printer drivers in Windows Control Panel.
Not Enough Memory for 2) The printer is not online. See that the printer is online
Same as Not Enough Memory for Calculations! and ready to accept commands from the computer.
Operation!
3) The printer was not initialized by the computer when it

Not Enough Memory for was turned on. Turn the computer OFF and ON and
Same as Not Enough Memory for Calculations!
Peak Pick! reinitialize.
. The Optimal Wavelength Search could not locate the EX 4) An improper connection was made. Verify that the
Optimal Wavelengths Not and EM wavelengths. The fluorescence peak was not correct interface port (COM1, LPT1, etc.) is selected to
Found detected during a search. Please adjust the parameters correspond to the hardware configuration.
and try again.

Text Printer has NOT been
Same as Text Printer Error!
The instrument option is not responding. Please check Selected!
Options Are Not Attached the attachments for proper cable connection to the
instrument. This is NOT a Valid The selected configuration file is not compatible with this
Configuration File! software.
In Time Course mode, the total time (Acquisition Rate x
Reaction time may not Number of Readings) may not exceed 100,000. When ) Too Many Timers for Opt.
Bench!
Other programs may be running that use the clock.
Discontinue other applications using the timer.
exceed 100,000 using manual timing mode, reduce either Acquisition Rate
or Number of Readings.
Too Many Timers,
The instrument interrupted a scan that was being Time/Date Update Same as Too Many Timers for Opt. Bench!
performed on the computer. Turn the Fluorometer Disabled!
Scan Interrupted by EOT Window OFF and turn the instrument OFFand ON. When
PC has lost connection with fluorometer unit. Can be
the problem persists, refer to the instruction manual or
caused by loose or broken cable. May also be caused by
contact your Service Representative. Transmit or Receive Error
running other programs while communicating with the
optical bench.
A motor in the instrument is not working properly. Please
turn the instrument OFF and ON and try again. If the Unknown Communication
Slit Origin Not Found Same as Transmit or Receive Error.
problem persists, please contact your Service Error Status!
Representative.
Refer to the section in this manual which lists the
Value Out of Range

Changing the mode of data acquisition or exiting the acceptable range for the parameter entered.
Some data has NOT been program may leave data in channels which have not been
saved to disk! Do you wish stored to disk. When permanent storage to disk is A motor in the instrument is not working properly. Please
to continue? necessary, answer NO to cancel the request to change Wavelength Origin Not turn the instrument OFF and ON and try again. If the
the acquisition mode or exit the program. Found problem persists, please contact your Service
Representative.
When printing data tables via Plot, data which does not fit
into the allotted quadrant(s) is not printed. To fit the table . A window is requested to appear but the command cannot
Table will not fit in area on one page, change the graphics printer to Portrait Window Creation Error! I be carried out. In almost all cases this is due to a
selected. orientation (see PC Configuration) or select more : shortage of memory. See memory-related items above.
quadrants. Otherwise, use Data Print under the
Manipulate menu for large data tables.
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.
6-8 RF-530 1PC Instruction Manual

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, ~
~ 'Ion-radiation transition
T.
Fluorescence
Light abso 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 Law 2 indicates that, since part of the energy of the light absorbed is lost as
molecule is deactivated by radiating the energy in the form of heat or light. heat or vibration, the residual radiation energy is reduced, shifting the
The light which the molecule emits as it returns to the ground state So is called wavelength of the fluorescence to the red. Hence, the task of measuring the
fluorescence. fluorescence spectrum is simplified by scanning only the wavelengths longer
than the excitation light.
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 Law 3 uses the quantum yield of fluorescence (Q) to indicate what proportion
the molecule was originally exposed (Stokes law). 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
The light which the molecule emits as it returns from the triplet state T[ to the yields of typical fluorescent substances.
ground state So is called phosphorescence. Phosphorescence has a lifetime
longer than 10- 4 seconds because of the need for spin transformation. Table A-l Compounds and Quantum Yield of Fluorescence
Compound Solution Quantum Yield

1.3 Three Basic Laws of Fluorescence
Fluorescein 0.1 N-NaOH 0.92
Law 1: In order for a substance to fluoresce, light absorption must first take
place. Eosin 0.1N-NaOH 0.19
Law 2: Generally, fluorescence has a longer wavelength than excitation Rhodamine B Ethanol 0.97
light. Riboflavin Aqueous solution, pH7 0.26
Law 3: The quantum yield of fluorescence (Q) is determined by the Anthracene Ethanol 0.3
frequency of non-radiative transfer of the absorbed energy to heat or
others.
] ') ) Naphthalene Ethanol 0.12
Indole Water 0,45
Q
ne Chlorophyll a Ether 0.32
ne + nf Chlorophyll b Ether 0.12
where ne Frequency of light emission
and nf Frequency of non-radiative transition

1.4 Advantages of Fluorescence Analysis
Law 1 dictates that the task of testing an unknown sample begins with
1.4.1 High Selectivity
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 When multiple substances are intermixed in a single sample, selective
emitted even when the sample is excited at the wavelength. Conversely, fluorescence measurement of a particular substance is possible without
fluorescence is emitted most intensely when the sample is excited with the removing substances which do not fluoresce.
absorption peak wavelength.
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

1.4.2 High Sensitivity Generally, quartz cells will produce weak fluorescence when they are
Fluorescence analysis may be 100 to 1,000 times more sensitive than excited at around 260nm because of impurities (aluminum) inherent in
absorptiometry, allowing measurement of ultra micro-quantity. 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 Considerations for Fluorescence Measurement
1.5.4 Effects of Scattered Light
1.5.1 Sample Temperature Effect In fluorescence testing, peaks caused by scattered light and Raman
scattering may be observed in addition to the fluorescence components of
In many samples, each rise of 1°C in sample temperature is said to
primary interest. Scattered light is associated with the scattering of
produce a loss of I - 2% in fluorescence intensity, though this is
excitation light by solvent entering the emission monochromator.
dependent on the type of sample. Certain biochemical samples reportedly
Scattered light is manifest particularly in the testing of solid samples.
produce a change of some 10% in fluorescence intensity in response to a
These peaks are readily distinguished because they appear at the
temperature change of 1°C. Temperature-dependent samples need to be
tested in the constant temperature cell holder. wavelength of the excitation light.
Depending on the characteristics of the grating monochromator, scattered

1.5.2 Photochemical Reaction of Samples light may also appear in the wavelength regions two and three times the
Exposure to excitation light causes certain samples to produce a excitation light wavelength as second order and third order light,
photochemical reaction, resulting in a change of fluorescence intensity. respectively. With an excitation light wavelength of 220nm, for example,
Testing of such samples may benefit from regulating the shutter to second order light appears at 440nm and third order light appears at
expose the sample to excitation light only for the duration of 660nm. A short wavelength cutoff filter is inserted at the emission side
measurement. Other techniques available would be to increase the to remove such light.
spectrum scan speed to the maximum or to narrow the bandwidth of the
excitation light.
) Further, when the excitation light wavelength is set to visible, light
having half the wavelength is also emitted from the excitation
1.5.3 Fluorescence from Impurities monochromator. With an excitation light wavelength of 450nm, for
example, light of 225nm may also be observed. A short wavelength cut
Peaks caused by fluorescent compounds other than the component of
off filter is inserted at the excitation side to remove such light. An
interest during fluorescence spectrum measurement are called
optional the filter set is available when second order or third order light is
fluorescence from impurities. Fluorescence from impurities are
of concern.
associated with CD scattered light and its secondary light, ClJ Raman
scattered light of the solvent, and (J) fluorescence from the solvent or cell.
Raman scattering appears at wavelengths longerthan the excitation
wavelength and may be confused with sample fluorescence. Raman
CD and ClJ are discussed later in Section 1.5.4. For (J), commercially
scattering is distinguishable because it remains essentially unchanged in
available grades of reagents often detect fluorescence caused by the
intensity with changes of the sample concentration, and also because
presence of impurities in the solvent. Remember that high-sensitivity
changes of excitation light wavelength vary the position of the peaks
testing in the ultraviolet region is particularly susceptible to the effects of
caused by Raman scattering but not the peak position of fluorescence.
solvent fluorescence. Fluorescence-free solvents are available for
Table A-2 summarizes the relationships between the excitation light
fluorescence analysis use. To remove concern over the possible effects
wavelength and Raman peak.
of solvent fluorescence, either use these commercial solvents or refine
your own solvent.
A-6 RF-5301PC Instruction Manual RF-5301 PC Instruction Manual A-7

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) Figure A-2 shows a fluorescence spectrum of an aqueous solution of sodium
Water Ethanol Cyclohexane Carbon Tetrachloride Chloroform
salicylate. Generally, the fluorescence spectrum of a dilute solution provides a
record of the fluorescence of the sample as well as contributions from
248 271 267 267 Rayleigh and Raman scatter, second-order light, and solvent impurities.
313 350 344 344 320 346 Raman scattered light of water; fluorescence of solvent impurities, sodium
Excitation salicy late; and second-order scattered light are shown in the spectrum
light 365 416 409 408 375 410
wavelength 405 displayed in Figure A-2.
469 459 458 418 461
(nm)
436 511 500 499 450 502 Excitation scattered light (282nm)
100
Raman spectrum of solvent

1.5.5 High Concentration Samples
Relative Fluorescence of
Samples with high concentrations can be a source of errors. Because intensity sodium salicylate
spectrofluorometers are designed to detect fluorescence emitted from the
50
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 Fluorescence
reabsorbed by the sample in the cell, making the spectrum look as if it of impurities
were shifted toward the longer wavelength side. ) contained in solvent ~,
300
=:':(-L / / / / ~
400 500
" !
600
Generally, samples with absorbances up to 0.02 (in a 10 mm cell) in the Figure A-2 Spectrum of a Sodium Salicylate Aqueous Solution
ultraviolet region are said to be free from these phenomena.
1.5.6 Effects of Cell Contamination 1.7 Excitation Spectrum

In fluorescence analysis, the slightest smear on the cell could affect Figure A-3 shows the excitation spectrum of a sodium salicylate aqueous
measurement accuracy. Especially, when the sample is left in the cell, solution. Excitation at the peak of 302nm is found to maximize fluorescence
the solvent could evaporate and adhere to the cell wall as a persistent with the highest excitation efficiency. The peak of 405nm is caused by a
smear. In testing an extremely dilute sample, dirt on the external, as well scattering of the excitation light.
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 Since the peak of the excitation spectrum and that of the absorption spectrum
with tissue paper (free of bleaching agents) before inserting the cell in the essentially correspond, the peak wavelength of the excitation spectrum of a
cell holder. particular sample can be estimated by analogy when its absorption maximum
wavelength is known. In precise terms, because it is a corrected excitation
1.5.7 Effect of Dissolved Oxygen spectrum that is analogous to the absorption spectrum, the apparent peak of the
Generally, oxygen dissolved in a solvent exerts a marked fluorescence excitation spectrum does not completely match that of the absorption
extinction effect on certain samples (quenching). When quenching by spectrum.
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.
A-8 RF-5301 PC Instruction Manual RF-530 1PC Instruction Manual A-9

100 302nm (peak wavelength) Since the absorbance is proportional to the concentration C, this equation can
be transformed by integration to
B (A. ') = KC
Relative
intensity
Hence, the calibration curve that is proportional to the concentration C is a
50 straight line. As the concentration of the sample rises, however, the
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

The relationship between the intensity and concentration of fluorescence
o 2 4 6 8 10 o 20 40 60 80 100
emitted from a point in a cell can be stated as:

1) ) (x 1O-'fl g/ml) (flg/ml)
Concentration Concentration
db (A ') =~EAFA(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 A in the
spectrum emitted by the excitation light at wavelength A'
K Absorbance at wavelength
dA' Bandwidth at wavelength A'
A - 10 RF-530 1PC Instruction Manual RF-530 1PC 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 Symbols Configure Menu 4-3, 5-22
sample for qualitative and quantitative analysis. Figure A-5 illustrates the -HV Control 5-38
Copy 5-59, 5-62
arrangement of a spectrofluorometer using the RF-5301PC as an example. Copy All 5-59
A
Correlation Coefficient 5-52
Acquire Mode Menu 4-3,5-12

Cross Hair 4-9, 5-76
Acquisition Rate 5-32

Custom Colors 5-67
Activity Calculation 5-52
Area under a curve 5-61

D
rnO;~~I\~-g~l~torlll( ( Dark Level Correction 5-38
Arithmetic 4-4, 5-44
ASCII Export 4-2, 5-6

Data Acquisition Modes 4-1
Attachments 4-4, 5-40

Data Directory 5-2, 5-34
Auto File 5-26

Data Interchange Format 5-7
Emission Auto Scan Mode 5-30

Data Print 4-4, 5-49
monochromator
Auto Shutter 5-38
Data Translation 5-6
Auto Timing Mode 5-32

Default Parameters 4-1
Auto Zero 5-15

Delete 5-5
Average 4-5, 5-62

Derivative 4-4, 5-45
Figure A-5 Arrangement afthe RF-5301PC DIP Export 5-7
B
Directory
Default 4-3
The excitation monochromator CD disperses light from a light source to

isolate the excitation light. A large aperture grating, measuring 55mm by 1) )
Baseline to Zero 5-61
c
Export 5-34
55mm, is used in the excitation monochromator to collect as much light as Display Equation 5-74
possible, because the brighter the excitation light is, the more sensitive the Cable Connections 2-2
Display Grid 4-5, 5-65
unit becomes. The grating can be rotated by a motor so that simple key Calibration Curve A-1O
Dwell Time 5-40
operations can set the excitation light at the desired wavelength. Cell Blank 5-20
Cell Contamination A-8

E
A cell loaded with a sample is set into the cell holder ell. The emission Change Threshold 5-54
Edit 5-51, 5-59
monochromator (j) disperses the fluorescence emitted from the sample for Change Values 5-59
Standard 5-17
measurement by a photomultiplier. The fluorescence monochromator also Channel 3-6, 5-7

Unknown 5-18
uses a grating as large as that of the excitation monochromator to collect as Calculations 5-48
Emission Wavelength 5-29
much light as possible. Erase 5-9

Erase Channel 5-9
Rename 4-2, 5-10

Errors 6-1
The xenon lamp generally used in spectrofluorometers provides the Save 5-7
Software 6-2
advantages of exceptionally high light intensity and a continuous emission Status 4-5, 5-2, 5-4, 5-9, 5-63
Excitation Spectrum A-9
spectrum. But its fluctuated illumination would magnify the signal noise Clear after Save 5-9
Excitation Wavelength 5-29
unless it is compensated. Moreover, because the emission spectrum of the Clipboard 4-10, 5-59
lamp, spectral sensitivity of the photomultiplier, etc.-collectively known as Colors 4-6, 5-67, 5-76
F
instrument functions-are not uniform, the spectrum tends to be distorted. Communication Problems 6-1
File Menu 4-2, 5-1
These problems are alleviated by monitoring part of the excitation light with Concentration 5-17, 5-28, A-8
File/Chnl Calc 4-4, 5-47
the monitor photomultiplier @ and feeding it back to the fluorescence Range 5-30
Find Valleys 5-54
photomultiplier (3). (This scheme of measurement is called light source Units 5-30
compensation. ) Configuration Files 4-1
A - 12 RF-5301 PC Instruction Manual RF-5301PC Instruction Manual 1-1
Index Index
Fluorescence A-3
Mark Graph 5-55
PopUp Scan
S
Measurement A-6
Menu
Parameters 4-3, 5-32
SIN Ratio Check 5-38
Principles A-3
Configure 5-22
PushButton 5-15
Sampling Interval 5-24
Spectrum A-9
File 5-1
Presentation Menu 4-5
Save 4-8
Fluorescence intensity. See Intensity Help 5-77

Print Table 5-55
Parameters 4-4, 5-41
Fluorometer Manipulate 5-43

Printer
Save As 5-4
Serial Port 5-34

Presentation 5-63
Text and Graphics 5-34
Save Channel 5-7
Fluorometer Communication 5-38

Method of Quantification 5-28
Printer Setup 2-3
Save Table 5-55
Fonts 5-66
Minor Tick Marks 5-68
Purge Time 5-40
Scan Speed 5-24
Mode
Push Button
Scattered Excitation Light 5-14
G Quantitative 5-15
AutoZero 5-15
Scattered Light A-7
Go To WL 5-13
Spectrum 4-3, 5-12
Go to WL 5-13
Search Optimal WL 5-14
Graph 5-65
Time Course 4-3, 5-19
PopUp Scan 5-15
Selectivity A-5
Graph Cursor 4-9

Multi-Point WorlGng Curve 5-28
Search Optimal WL 5-14
Sensitivity 5-24, A-6
Graph Pop-up Menu 5-39, 5-76

Shutter 5-14
Set Limits 3-6, 4-8
Graphics Plot 5-56

N Standard 5-16
Show SpeedBox 5-70
No Name 5-3, 5-8

Start 5-15
H
Shutter
Number of Readings 5-32

Stop 5-15
Auto Shutter 5-38
Hide All 4-6, 5-69

Number of Rinses 5-40
Unknown 5-16
Push button 5-14
I
o Q
Single Point WorlGng Curve 5-28
ill numbers 5-62

Sip Button 5-41
Open 5-1
Quadrants 5-72
Impurities A-6
Sip Speed 5-40
Optimal Wavelength 5-14

Quantitative Mode 4-1, 4-3, 5-2, 5-15
Sip Time 5-40
Instrument 4-4
Instrument Parameters 5-37
Options 5-59
1) Quantitative Parameters 5-27
Slit Width 5-24
Output 5-55
Quick save 5-3
Int/Conc Toggle 4-6, 5-74

Smoothing 5-45
Intensity
p R
Software Errors 6-2
fluorescence 5-13
Parameters 4-3
Software Installation 2-2
Radar 4-6, 5-69
International Support 4-10

Attachment 5-40
Spectrofluorometer A-12
Raman Light 5-14
Default 4-1
Spectrum, Excitation A-9
Raman Scattering A-7
K Instrument 5-37
Raw Data Measurement 5-28
Spectrum Mode 4-1, 4-3, 5-12
K-Factor 5-28
Load 4-4, 5-41
Spectrum Parameters 5-22
Raw Data Mode 5-2
Peak Area 5-61

Spectrum Type 5-23
L Reaction A-6
PopUp Scan 5-32

SpeedBox Configuration 4-3, 5-35
Reaction Time 5-32
Lamp Align 5-39

Quantitative 5-27
SpeedBox® 4-9
Read Push Button 5-17
Lamp Time 5-40

Save 4-4, 5-41
Standard Push Button 5-16
Reciprocal 5-45
Line Colors 5-67, 5-76

Spectrum 5-22
Start Push Button 5-15
Recording Range 5-24
List Files of Type 5-4

Time Course 5-30
Status Bar 5-25
Rename Channel 4-2, 5-10
Load Parameters 4-4, 5-41

PC Configuration 4-3, 5-33
Stop Push Button 5-15
Repeat Scan 5-24
Logarithm 5-45
Peak Area 4-5, 5-60
Survey Scan 5-21
Repetitions 5-30
Parameters 5-62
)
M Replot 5-56
T
Peak Pick 4-5, 5-53

Response Time 5-24
Manipulate Menu 4-4, 5-43

Temperature A-6
Algorithm 5-54
Rounded Tick Marks 5-69
Manual Timing Mode 5-32

Text and Graphics Printer 5-34
Plot 4-6, 5-70
Threshold 4-5, 5-54
PMT Protect 5-38
Tick Marks 5-68
Point Pick 4-5, 5-56

Index
Time Course 5-7, 5-10, 5-19

W
Parameters 5-30
Warning 5-26, 5-43
Time Course Mode 4-1, 4-3

Wavelength
Timing Mode
Emission 5-29
Manual 5-32
Excitation 5-29
Transforms 4-4, 5-45

Range 5-23
Translate
Wavelength Selection
DIP 4-2
Read Push Button 5-13
Set Push Button 5-13
U Working Curve 4-5
Unknown Push Button 5-16
Multi-Point 5-28
v
Single Point 5-28
Version
Z
Instrument ROM 5-39
Zoom 4-9
1-4 RF-5301PC Instruction Manual

Manual Spectroflourometer softwareRF5301PC

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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

3.3 Push Buttons " "" " "" .. 3-2

this publication at any time.

services in your country. 4.3.3 Configure Menu " 4-3

4.3.4 Manipulate Menu " " """ 4-4

in accordance with the terms of such license.

The following registered trademarks are used throughout this document:

4.8 Clipboard .. " "". "."." ""."."."" " "."."",," 4-10

Microsoft and MS-DOS are registered trademarks and Windows is a trademark of

5.1.3 Save as " " 5-4

5.1.5 Data Translation " 5-6

5.1.6 Channel 5-7

© Copyright 1995 Shimadzu Scientific Instruments, Inc., U.S.A.

5.2 Acquire Mode Menu 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 Fluore~cence? A-3

5.3 Configure Menu 5-22

5.3.1 Parameters 5-22

1.2 Principles of Fluorescence A-3

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

5.4.6 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

5.5 Presentation Menu 5-63

5.5.4 Show Speed Box 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

6.1 Communication Problems 6-1

6.2 Software Operational Error Messages 6-2