Spectrofluorophotometer Manual

Document Version: 2.1.9.0

Last modified: 16.04.2008

 Table Of Contents

1 OVERVIEW .............................................................................................1

1.1 Introduction to fluorescence ....................................................................................................... 1

1.2 Installation steps .......................................................................................................................... 1

1.3 Optional Accessories.................................................................................................................... 1

1.4 Preparing the instrument for measurements ............................................................................ 1

1.5 Measurements .............................................................................................................................. 1

1.6 Menus............................................................................................................................................ 2

1.7 Maintenance ................................................................................................................................. 2

1.8 Troubleshooting ........................................................................................................................... 2

1.9 Additional Information ............................................................................................................... 2

2 WHAT IS FLUORESCENCE? .................................................................3

2.1 Principles of Fluorescence........................................................................................................... 3

2.2 Three Basic Laws of Fluorescence.............................................................................................. 4

2.3 Advantages of Fluorometric Analysis ........................................................................................ 5

2.3.1 High selectivity....................................................................................................................... 5
2.3.2 High sensitivity....................................................................................................................... 5

2.4 Important Notes on Fluorometric Analysis ............................................................................... 5

2.4.1 Effect of Sample Temperature................................................................................................ 5
2.4.2 Photochemical reaction of samples......................................................................................... 5
2.4.3 Fluorescence from impurities ................................................................................................. 5
2.4.4 Effects of scattered light ......................................................................................................... 6
2.4.5 High-concentration samples ................................................................................................... 6
2.4.6 Effects of cell contamination .................................................................................................. 7
2.4.7 Effect of dissolved oxygen ..................................................................................................... 7

2.5 Example Scan of Fluorescence Spectra...................................................................................... 7

2.6 Example Scan of Excitation Spectra .......................................................................................... 8

2.7 Working Curve of Fluorescence ................................................................................................. 9

2.8 References................................................................................................................................... 10

3 INSTALLATION OF THE SPECTROFLUOROPHOTOMETER ............11

3.1 Inspection of Parts ..................................................................................................................... 11

3.1.1 RF-5301PC Instrument......................................................................................................... 11

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Spectrofluorophotometer Manual

3.1.2 RF-1501 Instrument.............................................................................................................. 11

3.1.3 Necessary Optional Parts for RF-1501: ................................................................................ 11
3.1.4 panorama fluorescence software package............................................................................. 12

3.2 Safety Precautions ..................................................................................................................... 12

3.3 Part Names and Functions ........................................................................................................ 14

3.3.1 RF-5301PC System .............................................................................................................. 14
3.3.2 RF-1501 System ................................................................................................................... 17

3.4 Hardware Specification............................................................................................................. 21

3.4.1 RF-5301PC System .............................................................................................................. 21
3.4.2 RF-1501 System ................................................................................................................... 22

3.5 Installation of the RF-5301PC or RF-1501 .............................................................................. 24

3.5.1 Location................................................................................................................................ 24
3.5.2 Connection to Personal Computer ........................................................................................ 25
3.5.3 Installation of the Xenon lamp.............................................................................................. 26
3.5.4 IC Cards (Options for the RF-1501) ..................................................................................... 31
3.5.5 Installation of external Accessories ...................................................................................... 32
3.5.6 Powering ON ........................................................................................................................ 33

3.6 Lamp Adjustment ...................................................................................................................... 33

3.6.1 Coarse-adjustment of the lamp position ............................................................................... 33
3.6.2 Fine-adjustment of the lamp position ................................................................................... 37

4 PERFORMANCE TESTS ......................................................................39

4.1 Signal/Noise Test ........................................................................................................................ 39

4.1.1 Prerequisites ......................................................................................................................... 39
4.1.2 Signal to Noise Test Procedure............................................................................................. 41

4.2 Signal to noise report layout ..................................................................................................... 44

4.2.1 Noise spectrum print layout properties ................................................................................. 44

4.3 Wavelength Accuracy Test........................................................................................................ 44

4.3.1 Verifying wavelength accuracy for emission monochromator ............................................. 44
4.3.2 Verifying wavelength accuracy for excitation monochromator............................................ 47

5 MENUS..................................................................................................49

5.1 RF-5301/RF-1501 menu ............................................................................................................ 49

5.1.1 RF-5301/RF-1501 menu contents......................................................................................... 49

6 MEASUREMENTS ................................................................................51

6.1 Measurement Window .............................................................................................................. 51

6.1.1 Online Measurement Monitoring.......................................................................................... 51
6.1.2 Measurement Control Buttons .............................................................................................. 51
6.1.3 Measurement parameters ...................................................................................................... 52

6.2 2D Emission Measurement........................................................................................................55

6.3 2D Excitation Measurement...................................................................................................... 57

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6.4 2D Synchro Measurement......................................................................................................... 58

6.5 3D Emission Increment Measurement..................................................................................... 59

6.6 3D Emission Measurement........................................................................................................61

6.7 3D Excitation Increment Measurement................................................................................... 63

6.8 3D Excitation Measurement...................................................................................................... 64

6.9 Go to Wavelength ...................................................................................................................... 66

6.10 Photometric Measurement .................................................................................................... 67

6.11 Time Measurement ................................................................................................................ 68

7 HARDWARE AND MAINTENANCE .....................................................69

7.1 Maintenance ............................................................................................................................... 69

7.1.1 Cautions for transferring the instrument ............................................................................... 69
7.1.2 Service life of the Xenon Lamp............................................................................................ 69
7.1.3 Safe disposal of the Xenon lamp .......................................................................................... 69
7.1.4 Replacing fuse ...................................................................................................................... 69

7.2 Instrument Parameter............................................................................................................... 69

7.2.1 Instrument Settings ............................................................................................................... 71
7.2.2 Instrument Calibration .......................................................................................................... 72

7.3 Checking Lamp Time ................................................................................................................ 72

7.4 Show Instrument Status ............................................................................................................ 72

7.4.1 General instrument properties............................................................................................... 73
7.4.2 RF-5301 Instrument self test ................................................................................................ 74

7.5 Troubleshooting ......................................................................................................................... 75

7.5.1 Communication problems..................................................................................................... 75
7.5.2 Before suspecting malfunction ............................................................................................. 75

7.6 Instrument Construction........................................................................................................... 77

7.6.1 The optical system of RF-5301PC........................................................................................ 77
7.6.2 The optical system of RF-1501............................................................................................. 77

8 CONTACTING .......................................................................................79

8.1 Contact Shimadzu...................................................................................................................... 79

9 APPENDIX ............................................................................................81

9.1 Working with the Auto-Sampler Accessory ............................................................................ 81

9.1.1 Enabling and disabling the Auto-Sampler ............................................................................ 81
9.1.2 Measurements with Auto-Sampler and Sipper ..................................................................... 81

9.2 Working with the Sipper Accessory ......................................................................................... 82

9.2.1 Installing the Sipper.............................................................................................................. 82

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9.2.2 Enabling and disabling the Sipper Unit ................................................................................ 82

9.2.3 Performing Measurements with the Sipper........................................................................... 82

10 INDEX....................................................................................................85

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1 Overview
Thank you for your purchase a Shimadzu spectrofluorophotometer system. The following instruments
are supported:

• RF-5301PC

• RF-1501 (requires a program card)

For convenience and enhanced operability the system is designed to run on an MS Windows platform
providing an intuitive, graphical user interface. As a result the operator can feel comfortable in a wide
variety of applications ranging from routine analysis to state-of-the-art research work.
The spectrofluorophotometer unit is combined with the panorama Fluorescence Spectroscopy
software package.
For trouble-free and efficient use of the spectrofluorophotometers be sure to study this manual
thoroughly. You are advised to read the following chapters subsequently.

1.1 Introduction to fluorescence

Principles of fluorescence

1.2 Installation steps

Inspection of Parts
Safety Precautions
Part Names and Functions
Hardware Specifications
Installation of the Spectrofluorophotometer
Lamp Adjustment

1.3 Optional Accessories

Sipper Accessory
Auto-Sampler Accessory

1.4 Preparing the instrument for measurements

The following test procedures are applied to test the instrument and the software to produce reliable
results:
Signal/Noise Test
Wavelength Accuracy Test

1.5 Measurements
Any measurements will be carried out from a central measurement window, where instrument
parameters and follow-up actions are configured:
Measurement Window
Before a measurement can be started, the instrument must be initialized:
Instrument Initialization

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Spectrofluorophotometer Manual

The following measurement procedures are available:

2D Emission Measurement
3D Emission Increment Measurement
3D Emission Measurement
2D Excitation Measurement
3D Excitation Increment Measurement
3D Excitation Measurement
2D Synchro Measurement
Time Measurement
Photometric Measurement

1.6 Menus
RF-5301/RF-1501 menu

1.7 Maintenance
Maintenance
Instrument Parameter
Checking Lamp Time
Lamp Adjustment
Show Instrument Status

1.8 Troubleshooting
Troubleshooting

1.9 Additional Information

Instrument Construction

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2 What is Fluorescence?
The phenomenon of certain kinds of substance emitting light on absorbing various energies, without
involving heat generation, is called luminescence. That kind of luminescence that is emitted on exposure
to ultraviolet or visible rays is called photo luminescence.
Fluorescence and phosphorescence, representative of photo luminescence, possess hues different from
the reflected or transparent color a substance, emitting longer wavelengths of light than radiated light.
Familiar examples are the green color observed in a red ink in the daylight (eosin aqueous solution) and
a pale color in shirts.

2.1 Principles of Fluorescence

This section explains the principle of fluorescence with the reference to an organic compound as an
example.
When a molecule in the base state S0 is exposed to light, the kinetic energy of the electrons in the
molecule is altered, moving the molecule into the excited state S1 with higher energy level as shown in
the following figure:

The excited state, however, soon changes back to the base state as the molecule is deactivated by
radiating the energy in the form of heat or light. The molecule then transits, without radiation, to an
excited state having a slightly lower energy level than the excited state S1. The light the molecule emits
as it returns further to the base state S0 is called fluorescence.
Since part of the energy of the light absorbed has been lost as vibration or heat energy, the light covers
a longer wavelength than the light to which the molecule has originally been exposed (Stokes law).
The light the molecule emits as it transits, without radiation, to the triplet state T1 from the excited state
S1, the returns to the base state S0 is called phosphorescence. Phosphorescence has a life longer than
-4
10 seconds because of the need for spin transformation.

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2.2 Three Basic Laws of Fluorescence

• Law 1
In order a substance to emit fluorescence, light absorption must take place.

• Law 2
Generally, fluorescence has a longer wavelength than excitation light.

• Law 3
The quantum yield of fluorescence (Q)is determined by the frequency of non-radiation of the
absorption energy to heat or others.

where
ne: Frequency of light emission
nf: Frequency of non-radiation transition
Law 1 dictates that the task of testing an unknown sample begin with measurement of its absorption
spectrum with a relatively higher concentration. If there is no absorption at all, then it is considered that
fluorescence is not emitted even if the sample is excited at the wavelength. Conversely, fluorescence is
emitted most intensely if the sample is excited with the absorption peak wavelength.
Law 2 indicates that, since part of the energy is reduced, shifting the wavelength of the fluorescence to
longer side. Hence, the task of measuring the fluorescence spectrum can be reduced to a matter of
scanning only the longer wavelength side of the excitation light.
Law 3 uses the quantum yield of fluorescence (Q) to indicate what proportion of energy absorbed is
radiated as fluorescence. The higher the value of Q, the easier the substance produces fluorescence.
The following table lists the quantum yields of typical fluorescent substances:

Compound Solution Quantum yield Q

Fluorescein 0.1N NaOH 0.92

Eosin 0.1N NaOH 0.19

Rhodamine B Ethanol 0.97

Riboflavin Aqueous solution pH 7 0.26

Anthracene Ethanol 0.30

Napthalene Ethanol 0.12

Indole Water 0.45

Chlorophyll a Ether 0.32

Chlorophyll b Ether 0.12

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 Principles of Fluorescence

2.3 Advantages of Fluorometric Analysis

2.3.1 High selectivity

Even if multiple substances are intermixed in a single sample, selective fluorescence measurement of a
particular substance is made possible without having to remove the other substances if these other
substances do not emit fluorescence. Further, even though multiple fluorescence-emitting substances
are intermixed in a sample, measurement can still distinguish them from each other by setting their
wavelength in an appropriate manner if they vary in excitation or emission light wavelength.

2.3.2 High sensitivity

Fluorescence analysis is 100 to 1,000 times more sensitive than absorptiometry, allowing measurement
of ultra micro-quantity.

2.4 Important Notes on Fluorometric Analysis

2.4.1 Effect of Sample Temperature

In many samples, each rise of 1°C in sample temperature is said to produce a loss of 1-2% in
fluorescence intensity, though this is dependent on the type of the sample. Certain biochemical samples
reportedly produce a change of some 10% in fluorescence intensity in response to a temperature
change of 1°C. Temperature-dependent samples need to be tested in the constant temperature cell
holder.

2.4.2 Photochemical reaction of samples

Exposures to excitation light cause certain samples to produce a photochemical reaction, resulting in a
change in fluorescence intensity. Testing of such samples should benefit from regulating the shutter to
expose the sample to excitation light only for the duration of measurement. Other techniques available
would be increase the spectrum scan speed to the extent possible or narrowing the bandwidth of the
excitation light.

2.4.3 Fluorescence from impurities

Peaks caused by fluorescent components other than the component of interest during fluorescence
spectrum measurement are called fluorescence from impurities. Fluorescence from impurities are
associated with
1. Scattered light and its secondary light
2. RAMAN scattered light of the solvent
3. Fluorescence from the solvent or cell.
(1.) and (2.) are discussed in the following. For (3.), commercially available grades of reagents often
detect fluorescence caused by the presence of impurities solvent.
Remember that high-sensitivity testing in the ultraviolet region is particularly susceptible to the effects of
solvent fluorescence. Fluorescence-free solvents are available for fluorescence analysis use. To remove
concern over the possible effects of solvent fluorescence, rather use these commercial solvents or
refine your solvent by yourself.
General quartz cells will weak fluorescence when they are excited at around 260 nm because of
impurities (aluminum) inherent in the cells. Use of a fluorescence-free cell that contains artificial quartz
(P/N200-34591-03) is recommended for exciting traces of samples at around 260 nm.

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Spectrofluorophotometer Manual

2.4.4 Effects of scattered light

In fluorescence testing, peaks caused by scattered light and RAMAN scattering may be observed in
addition to the fluorescence components of primary interest. Scattered light is associated with the
scattering of excitation light by solvent molecules (Rayleigh scattering) or by particulates or air bubbles,
with the resultant scattered light entering the emission monochromator. Scattered light is manifest
particularly in the testing of solid samples. These peaks are readily distinguished because they appear
at the wavelength of excitation light.
Depending on the characteristics of the grating monochromator, scattered light may also appear in the
wavelength regions two and three times the excitation light wavelength as second order and third order
light, respectively. With an excitation light wavelength of 220 nm, for example, second order light
appears at 440 nm and third order light appears at 660 nm. A short wavelength cutoff is inserted at the
emission side to remove such light.
Further, if the excitation light wavelength is set to visible, light having half the wavelength is also emitted
from the excitation monochromator. With an excitation light wavelength of 450 nm, for example, light of
225 nm is also emitted. A short wavelength cutoff filter is inserted at the excitation side to remove such
light. Use the filter set available as a special accessory if second order or third order light is of concern.
RAMAN scattering appears when the solvent has RAMAN activity. It appears on the longer wavelength
side of excitation light as like fluorescence peak. RAMAN scattering is distinguishable because it
remains essentially unchanged in intensity with changes of the sample concentration and also because
changes of excitation light wavelength vary the position of the peaks caused by RAMAN scattering but
not the peak position of fluorescence. The table below summarizes the relation-ships between the
excitation light wavelength and RAMAN peak.

Solvent and RAMAN peak wavelengths [nm]

Excitation light Water Ethanol Cyclohexane Carbon Chloroform

wavelength [nm] Tetrachloride

248 271 267 267 - -

313 350 344 344 320 346

365 416 409 408 375 410

405 469 459 458 418 461

436 511 500 499 450 502

2.4.5 High-concentration samples

Too high sample concentrations can be a cause of various errors. Because spectrofluorophotometers
are designed to detect fluorescence emitted from the center of the cell, the excitation light would be
absorbed in the vicinity of the inlet of the cell if 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
re-absorbed by the sample in the cell, making the spectrum look as if it were shifted towards the longer
wavelength side. Generally, samples with absorbances up to 0.02 (in a 10 mm cell) in the ultraviolet
region are said to be free from these phenomena.

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 Principles of Fluorescence

2.4.6 Effects of cell contamination

In fluorescence analysis, the slightest smear in the cell could affect measurement accuracy. Especially,
if the cell is left with a sample being loaded inside. the solvent could be evaporated and adhere to the
cell wall as persistent smear. In testing an extremely dilute sample, dirt on the external, as well as
internal, walls of the cell would be of concern. If a sample solution should contact the external walls of
the cell, wipe it off completely with tissue paper (not using a fluorescent dye) before mounting the cell in
the cell holder.

2.4.7 Effect of dissolved oxygen

Generally, oxygen dissolved in a solvent exerts a marked fluorescence extinction effect on certain
samples (quenching). If quenching by dissolved oxygen is not negligible, solvent degassing is required.
Solvent degassing is conveniently accomplished by blowing a nitrogen gas into the solvent or
decompressing the solvent with a vacuum pump.

2.5 Example Scan of Fluorescence Spectra

The figure below shows a fluorescence spectrum of an aqueous solution of sodium salicylate as an
example. Generally, the fluorescence spectrum of a dilute solution not only provides a record of the
fluorescence of the sample but involves a complication of various emission spectra, involving the
scattered light (Rayleigh scattering) that is observed as a result of excitation light being scattered by
dust or molecules in the solution, order light of the scattered light as shown in figure:

A measured emission spectrum of diluted Aspirin tablet looks like this:

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Spectrofluorophotometer Manual

2.6 Example Scan of Excitation Spectra

The figure below shows the excitation spectrum of a sodium salicylate aqueous solution. Excitation.
Excitation at the peak of 302 nm is found to maximize fluorescence with the highest excitation efficiency.
The peak of 405 nm is caused by a scattering of the excitation light. Since the peak of the excitation
spectrum and that of the absorption spectrum correspond essentially, the peak wavelength of the
excitation spectrum of a particular sample can be estimated by analogy if its absorption maximum
wavelength is known. Because, in precise terms, it is a corrected excitation spectrum that is analogous
to the absorption spectrum, the apparent peak of the excitation spectrum does not completely match
that of the absorption spectrum.

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 Principles of Fluorescence

A measured excitation spectrum of diluted Aspirin tablet looks like this:

2.7 Working Curve of Fluorescence

According to Foster, the relationship between the intensity and concentration of fluorescence emitted
from a point in a cell can be stated in an expression as shown in the following:

where
*
db (λ ): Intensity of the fluorescence observed at wavelength
n: Refractive index
p: Reflection coefficient

Eλ: Intensity of the excitation light at wavelength

Fλ (λ') : True fluorescence intensity at wavelength in the spectrum emitted by the excitation light
at wavelength

Kλ: Absorbance at wavelength

dλ' : Bandwidth of wavelength

Since the absorbance is proportional to the concentration C, this equation can be transformed by
integration to:

B(λ') = K C
Hence, the calibration curve that is proportional to the concentration C is straight. As the concentration
of the sample rises, however, the calibration curve would be curved as explained in "High-concentration
samples" section. The following figure shows a calibration curve observed with a diaminostilbene
aqueous solution:

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Spectrofluorophotometer Manual

2.8 References
Applied Engineering Division, Shimadzu Corporation. Principles, Application, Application, and
Equipment Structure of Fluorescence Analysis, Shimadzu Fluorescence Analysis Course, Shimadzu
Corporation.

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3 Installation of the spectrofluorophotometer

3.1 Inspection of Parts

After unpacking the instrument check that all the following parts are included.

3.1.1 RF-5301PC Instrument

Type Part Number Quantity

Optical unit P/N 206-81601-** 1

Power cable (100/120 VAC) P/N 071-60814-01 1
I/F Cable P/N 206-83579-91 1
Phillips-head screwdriver P/N 200-94612 1
Sensitivity adjustment flat-blade screwdriver P/N 200-94613 1
Fuse 5A (100/120 V) P/N 072-01663-14 2
Xenon lamp 150 W P/N 200-81500-01 1

3.1.2 RF-1501 Instrument

Type Part Number Quantity

Optical unit P/N 206-62901 1

Power cable 1
(100/120 VAC)
P/N 206-67 197
(200-240 VAC)
P/N 200-81009-02
1
I/F Cable
P/N 206-83579-91
1
Phillips-head screwdriver
P/N 200-94612
2
Sensitivity adjustment flat-blade screwdriver
P/N 200-94613
1
Fuse
5A(100-120VAC)
3A(200-240VAC) P/N 072-01661-23
1
P/N 072-01661-20
Xenon lamp 150 W
P/N 072-01661-201

3.1.3 Necessary Optional Parts for RF-1501:

Type Part Number Quantity

RS-232C cable P/N 200-86408 1

Communication Program Card P/N 489-04196-24 1

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Spectrofluorophotometer Manual

3.1.4 panorama fluorescence software package

If you ordered only the software package, check the equipment items according to the next table:

Type Part Number Quantity

panorama fluorescence Software P/N 980-00212 1

3.2 Safety Precautions

This chapter lits precautions that are particularly important for ensuring safe operation of the equipment.
Be sure to observe these precautions when operating the instrument. It is dangerous not to comply with
the following points:
1. Do not use the unit for any purpose other than the above-mentioned types of analysis.
2. Follow the procedures described in the user manual.
3. Observe all warnings and cautions.
4. Do not disassemble or modify the unit without the express approval of an authorized Shimadzu
representative. Failing to do may lead to dangerous situations or damage of the unit.
5. Do not use methods which are not indicated in the instrument manual. Failing to do may lead to
dangerous situations or damage of the unit.
6. For internal repair of the product contact your Shimadzu representative.
The precautions are classified into the following three types based on their importance:
Danger: Can lead to death or serious injury.
Warning: Can lead to injury.
Caution: Can cause damage, malfunction or fire to the equipment or loss of data.

Danger: The positive electrode of the Xenon lamp is charged to a voltage as high as
30000 V when the lamp is turned ON. The lamp is energized to about 90 V and it
reaches a temperature of 100 °C or more after it has been lit. Make sure that the
cover of the lamp housing is installed in position before operating the
instrument!
Before removing the cover of the lamp housing be sure to disconnect the power
cable from the outlet!
Remember that even after the power switch is set to the OFF position, the lamp
unit remains electrically charged and can cause electric shock. Disconnect the
power cable from the power outlet, set the power switch to the OFF position
and leave the instrument for five minutes or longer. Only then remove the cover
of the lamp housing!

Danger: The Xenon lamp contains high pressure gas. Impact, vibration or pressure on it
may cause it to burst. Be extremely careful with handling it!

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 Installation of the spectrofluorophotometer

Warning: Be careful not to spill water, organic solvent, etc. on the instrument. Spilling
liquid on the instrument can cause electric shock, fire, damage or malfunction
of the instrument.

Caution: While installing the lamp do not touch the bulb with bar fingers. Finger oil
remaining on the bulb is be baked onto the bulb when the lamp is lit. That can
cause the lamp to burst! Cleaning of the lamp is possible with ethyl alcohol or
the special cleaning agent supplied with the lamp.

Caution: When relocating or shipping the instrument be sure to remove the Xenon lamp!
Store the removed lamp in the special case supplied with the lamp.

Caution: Replace the lamp immediately when ist total service life has exceeded 500
hours (guaranteed service life). Never use a lamp more than 1000 hours! A
lamp used more than 1000 hours is liable to burst. A damage of the lamp unit is
possible.

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Spectrofluorophotometer Manual

3.3 Part Names and Functions

3.3.1 RF-5301PC System

Top View and Front View

(1) Sample compartment. Holds a sample being analyzed.

(2) Front panel Xenon lamp. Can be removed when installing an accessory, etc.
(3) Cover of reagent injection hole. Can be removed to allow reagent to be injected into a cell loaded in
the sample compartment.
(4) Cover of lamp housing. Can be removed for installing or removing the Xenon lamp.
(5) Power indicator lamp. Lights up when the instrument is powered ON.

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 Installation of the spectrofluorophotometer

Right-side View

(1) Screw for horizontal positioning of lamp. For adjusting the horizontal position of the lamp.
(2) Screw for vertical positioning of lamp. For adjusting the vertical position of the lamp.
(3) Lamp fixing screw. Is tightened or loosened for installing or removing the Xenon lamp using the
supplied screwdriver which is inserted through the guide tube.
(4) Power switch. Powers the instrument ON/OFF.
(5) Lamp ON/OFF switch. Usually, keep this switch in the ON position.
(6) Voltage selector 1. Must be set according to the site power supply (100/120/220/240 VAC).
(7) Voltage selector 2. Must be set according to the site power supply (100/200 VAC).
(8) Parallel I/F connector. Not used.
(9) RS-232C I/F connector. Used for communications with the computer.
(10) I/O-1 connector. For connecting the optional auto-sampler.
(11) I/O-2 connector. For connecting the optional sipper.

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Spectrofluorophotometer Manual

Left-side View

(1) Analog OUT terminals. Outputs voltage (1 V/FS) in proportion to the fluorescence intensity. The
Chromatopack or other output devices can be connected here.
(2) Contact IN terminals. Analysis operations can be started by jumpering these terminals.

Rear View

(1) Sensitivity adjustment trimmer VR0. Used to adjust the gain.

(2) Sensitivity adjustment trimmer VR1. Intended for adjustment of detection sensitivity when negative
high-voltage is OFF. This trimmer has been factory-set to a suitable position. Under normal
circumstances this trimmer should not be touched!
(3) Sensitivity adjustment trimmer VR2. Intended for adjustment of detection sensitivity when lamp
compensation is ON. This trimmer has been factory-set to a suitable position. Under normal
circumstances, this trimmer should not be touched!
(4) Grounding terminal Can be connected to a grounding line. If the site power supply does not include
grounding, be sure to connect this terminal to ground to protect the instrument.

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 Installation of the spectrofluorophotometer

3.3.2 RF-1501 System

Front View

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Spectrofluorophotometer Manual

(1) Sample compartment

Set the sample to be measured in the sample compartment. Opening the sample compartment cover
will expose the cell holder forward.
(2) Front panel
Detach the front panel to install accessories or the like.
(3) Top panel
Detach the top panel to install accessories or the like.
(4) Reagent injection hole cover
Detach the reagent injection hole cover to inject a reagent or the like into the sample compartment when
a cell has been set in it.
(5) Light-source compartment cover
Detach the light-source compartment cover to mount or unmount the xenon lamp.
(6) Light-source illumination indicator lamp
This indicator lights when the xenon lamp is ON.
(7) Power switch
Use to turn the power to the unit on and off.
(8) Contrast control
Use to adjust the contrast of the LCD.
(9) IC card slot
Insert optional IC cards (data pack and program pack) into this slot.
(10) Operator panel
Use to operate the unit. (Refer to the description of chapter 4 OPERATING INSTRUCTIONS 4.1
Operator Panel Discription)
(11) Standard cell holder
Cell holder for 10 mm cells.
(12) Side panel
Detach the side panel to install accessories or the like.
(13) LCD (Liquid Crystal Display)
Display of the current mode, parameters, wavelengths and fluorescence intensity. It also displays
messages and warnings or remedies of error. The selection modes are displayed in frames on the
bottom. Those correspond to the function keys F1 to F4. By pressing function key to be selected, the
operating mode is implemented. The selected mode is shown in the frame at upper left side of the
screen.

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 Installation of the spectrofluorophotometer

Right Side View

(1) Light source horizontal dlrection adjusting screw

Use to adjust the horizontal direction of the light source.
(2) Lamp mounting screw
Use to mount or demount the xenon lamp.
(3) Light source vertical direction adjusting screw
Use to adjust the vertical direction of the light source.
(4) Lamp switch
Leave the lamp switch normally at the ON position.
(5) I/0-2 connector
Attach the sipper to this connector.
(6) I/0-1 connector
Attach the auto-sample changer to this connector.
(7) RS-232C connector
Attach a computer In support of an RS-232C interface to the RF-1501.
(8) Printer connector
Attach a printer to the RF-1501.

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Spectrofluorophotometer Manual

Rear View

(1) Sensitivity control VR1

Use to adjust fluorescence intensity when adjusting the light source position.
(2) Sensitivity control VR2
The sensitivity control VR2 is used to set detector sensitivity. It Is factory-adjusted and must not be
touched.
(3) Grounding terminal
Connect a grounding wire to this terminal.
(4) Voltage setting selector
The voltage setting selector offers a choice of four input voltages (100, 120, 220, and 240 VAC).
(5) Power cable Inlet
Plug the accessory power cable into this inlet to power the unit from an AC outlet.
(6) Expansion board slot

20
 Installation of the spectrofluorophotometer

Set an optional interface board or output board into this slot. It is normally covered.

3.4 Hardware Specification

3.4.1 RF-5301PC System

Item Description

Light source 150 W Xenon lamp

Lamp housing Lamp housing with ozone self-decomposition

Emission/Excitation monochromator Blazed holographic concave diffraction grating (F/2.5 for

emission and excitation side)

Density of diffraction grating 1300 lines/mm

Wavelength scan range 220 - 900 nm and zero-order light (EX and EM)

Measuring wavelength range 220 - 750 nm and zero-order light (EX and EM)

Slit width 1.5/3/5/10/15/20 nm (excitation side)

1.5/3/5/10/15/20 nm (emission side)

Wavelength accuracy ±1.5 nm

Wavelength scan speed SURVEY, SUPER, VERY FAST, FAST, MEDIUM, SLOW,
VERY SLOW

Light source compensation system Monochromatic light monitoring diode feedback system

Detector Photomultiplier tube for photometry and monitor sides;

Photometry: R3788-02, Monitor: R212-14

Sample compartment Single non-constant temperature cell holder

Sensitivity selection High/low selectable (difference approx. 50:1)

Response Automatic cumulative smoothing depending upon wavelength;

scan speed and manual response are selectable.

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Spectrofluorophotometer Manual

S/N ratio 150 or greater at the water Raman peak of distilled water,
spectral bandwidth 5 nm for both EX and EM, EX 350 nm

Power supply 100/120/220/240 VAC, 50/60 Hz, 400 VA

Operating temperature range 15 - 35 °C

Operating humidity range 45 - 80 % (70 % or less at room temperature 30 °C or higher)

Dimensions Photometer: 670(W) × 530(D) × 270(H) mm

Weight Photometer: 43 kg

3.4.2 RF-1501 System

Item Description

Light source 150 W Xenon lamp

Light source chamber Ozone self-extinction lamp house

Monochromators Ion-blazed holographic concave grating for both excitation and

emission monochromator F12.4

Number of grating ruled lines 900 lines/mm

Wavelength scan range 220 - 900 nm and zero-order light (EX and EM)

Measuring wavelength range 220 - 750 nm and zero-order light (EX and EM)

Bandwidth 10 and 20 nm for both excitation and emission

Wavelength accuracy ± 5 nm

Wavelength scan speed Selectable from among SUPER (about 3700 nm/min), FAST,
MEDIUM, and SLOW

Slewing speed Approx. 30.000 nm/min

22
 Installation of the spectrofluorophotometer

Light source compensation system Dynode feedback method by monitored excitation light.

Detector Photomultipliers for both fluorescence measurement and

monitoring

Sample compartment Single non-constant temperature cell holder

Sensitivity selection Two selectable settings, HIGH and LOW

(sensitivity difference: about 50 times)

Response 0-98%
0.02, 0.03, 0.1, 0.25, 0.5, 2 and 8 sec

S/N ratio The S/N ratio at the Raman peak of distilled water is 300 or
higher under the measurement conditions:
Excitation wavelength : 350 nm
Emission wavelength : Peak wavelength of the Raman line
Bandwidths : 10 nm for both excitation and emission
Response : 2 sec

Display Backlit LCD (320 x 200 dots)

I/O One RS-232C port

One printer interface (Centronics compatible) port
Sipper interface
Autosampler interface

Measurement functions Wavelength scanning

• Automatic search for optimal combination of excitation

and emission wavelengths

• Excitation and emission spectrum measurement

• Spectrum enlarging and reduction

• Differential spectra

• Spectrum data printout

• Saving of spectra (two spectra stored in the unit's

internal memory and 9 stored per RAM card)
Quantitative measurement

• Calibration curve creation (up to 10 standard sample,

first order approximation)

• Repetitive measurement (up to 5 times)

• Calibration curve storage and modification

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Spectrofluorophotometer Manual

• Saving of quantitative measurement data files (two

files stored in the unit and 9 stored per RAM card)

Storage of measurement files Three files stored in the unit's internal memory and 60 per
RAM card

Input Power 100 V,120 V, 220 V, 240 V AC, 50160 Hz

Power Consumption 400 VA

Operating temperature range 10 - 35 °C

Operating humidity range 45 - 80 % (70 % or less at room temperature 30 °C or higher)

Dimensions 500 (W) x 400 (D) x 255 (H) mm

Weight 23 kg

3.5 Installation of the RF-5301PC or RF-1501

3.5.1 Location
To provide optimal performance of the instrument and to ensure a long trouble-free operating life, be
sure to install the instrument in a location that satisfies the requirements listed below.

Take care of your instrument location!

Note that Shimadzu Corporation shall not be responsible for any damages or costs
arising from deteriorated performance or mechanical damage arising from the use of
the instrument in location that fails to satisfy the following requirements even during
the guarantee period.

• Room temperature within the range of 15 °C to 35 °C.

• A position not exposed to direct sunlight.

• A position not subject to strong vibration or any continuous (even weak) vibration.

• A position free from strong magnetic or electromagnetic fields.

• Relative humidity within the range 45 % to 70 %. (If the room temperature is 30 °C or higher
the relative humidity must be more than 70 %.)

• A location free from exposure to corrosive gas or any organic or inorganic gas that has an
absorption band in the UV region.

• A location substantially free from dirt or dust.

• Place the spectrofluorophotometer on a table or surface which can withstand the load of the
spectrofluorophotometer (weight of RF 5301PC: 43 kg; weight of RF-1501: 23 kg).

24
 Installation of the spectrofluorophotometer

Warning: Be careful not to spill water, organic solvent, etc. on the instrument. Spilling
liquid on the instrument can cause electric shock, fire, damage or malfunction
of the instrument.

3.5.2 Connection to Personal Computer

Using the RF-1501 with panorama Fluorescence.

When using the RF-1501 with the software, please make sure to have the Program Pack
(see "IC Card Options for the RF-1501" below) installed properly. Otherwise the software
cannot control the instrument!

The RF-5301PC or RF-1501

unit must be connected to the
personal computer with the
attached I/F cable.

1. Connect the male

end of the cable to
the connector (serial)
on the right-side face
of the RF-5301PC
unit
2. Adjust the two screws
to secure the
connection.

25
Spectrofluorophotometer Manual

3. Connect the female

end of the cable to
the serial port
4. Tighten the two
screws to secure the
connection. expose
the inside of the lamp
housing.

3.5.3 Installation of the Xenon lamp

Before installing the Xenon lamp make sure that the power switch is in the OFF position and disconnect
the power cable from the power outlet to prevent accidental electrical shock.

1. Loosen the two

mounting screws on
the cover of the lamp
housing and remove
the cover.

26
 Installation of the spectrofluorophotometer

2. Loosen the fixing

screws on the slide
plate.

3. Shift the sliding plate

to expose the inside
of the lamp housing.

27
Spectrofluorophotometer Manual

4. Inset the screw driver

for fixing the lamp
(standard accessory)
into the opening
provided for that
purpose along the
guide tube. Loosen
the lamp fixing screw
by turning it
counterclockwise.

5. Remove the Xenon

lamp from its shipping
case and remove the
knurled screws on the
positive and negative
sides.

6. Line up the lamp so

that the black or red
mark on the cement
part of the negative
side of the lamp is
oriented in the
direction of the lamp
fixing screw. Then
insert the lamp into
the housing.

28
 Installation of the spectrofluorophotometer

Caution: Do not confuse the positive side of the lamp with the negative side when
installing the lamp. If the instrument is powered ON with the lamp installed in
the wrong direction it will be damaged.

7. Insert the screw driver into

the opening again and turn
the lamp fixing screw
clockwise to secure the lamp
in position.

8. Close the sliding plate and

secure it with the fixing
screws.

29
Spectrofluorophotometer Manual

Caution: If the positive electrode side is subjected to a pull-force by the power cord
while the lamp is lit, the lamp may be damaged. When securing the terminal of
the positive electrode with the knurled screw make sure that the power cord at
the positive side is a little slack.

9. Connect the power cord

terminal of the positive
electrode with the positive
end of the lamp.
10. Secure the terminal with the
knurled screw.
11. Tighten the knurled screw by
hand! Using a tool such as a
wrench may damage the
lamp.

30
 Installation of the spectrofluorophotometer

12. Install the cover of the lamp

housing. The installation of
the Xenon lamp is complete.

3.5.4 IC Cards (Options for the RF-1501)

The following kinds of IC cards are available to add additional functions to the RF-1501 unit.

Caution: Please pay attention to the following precautions in handling IC cards:

• Do not give rough shocks to the cards.

• Do not fold or bend the cards.

• Do not expose the cards to high temperature or direct sunlight.

• Protect the cards against intense magnetic fields or electrostatic effects. The
stored programs might be destroyed.

• The useful life of the data pack battery is about 7 years.

Program Pack (Gray-labeled IC card)

The program pack adds additional functions to the RF-1501. It supports two modes of measurements in
standard:

• Spectrum mode
in which the excitation or emission spectrum of a sample is measured by wavelength scanning.

• Quantitative measurement mode

in which the concentration of an unknown sample is measured by creating a calibration curve
from a standard sample with a known concentration.

Data Pack (Yellowish green-labeled IC card)

A single data pack provides two memory functions as described below. Battery backup protects the
stored data from erasure when the data pack is removed from the slot in the RF-1501.

• Parameter memory function

The RF-1501 can store three sets of measurement conditions by itself. An additional 60 Sets of
measurement conditions can be stored per data pack.

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Spectrofluorophotometer Manual

• Data memory function

The RF-1501 provides two files to store spectrum, quantitative measurement and other data.
An additional 9 files can be stored per data pack.

Caution: Note on removing data packs

If you remove the data pack while it is being accessed for reading or writing, damage
to the stored data could result. Since reading from or writing to the data pack is
completed in a short time, perform any key operation, then wait for about 3 seconds
before removing the data pack.

Installing the IC Card

Please follow the steps below to install the IC card correctly to your RF-1501 unit:

1. Insert the IC card

firmly into the slot
at the front, lower
right of the RF-
1501.

You can insert or

remove the
program pack
regardless of
whether the
power is on or off.

Never remove the

data pack while
the instrument
reads from or
writes to the data
pack!

3.5.5 Installation of external Accessories

The following external accessories are available with the RF-5301PC or RF-1501 instrument.

• Standard cell holder (default)

• Sipper
Please refer to the section "Working with the Sipper Accessory" for details.

• Auto-Sampler
Please refer to the section "Working with the Auto-Sampler Accessory" for details.

• Constant temperature cell holder (currently not supported by the panorama fluorescence
software!)
Install the external accessory according to the steps described in the particular accessory sub-section.
As long as you run the instrument with external accessories together with the panorama fluorescence
software, there is no need for a special setup of the instrument. The software will take over full control
over all accessories and parameter settings.
In case you like to run the RF-1501 unit as a stand-alone instrument, the external accessories need to
be installed separately via the on-screen display. Please refer to your RF-1501 operation instructions
manual, chapter 4.7 for details.

32
 Installation of the spectrofluorophotometer

3.5.6 Powering ON
After installing the Xenon lamp and optional IC cards or accessories power ON the instrument. Before
setting the power switch to the ON position, make sure of the following aspects.
1. Grounding
The power cable for the instrument is a three-wire type including a ground wire. Connect the
cable to a three-wire type power outlet, and provide reliable grounding. If the power outlets
available on-site are two-wire type, make sure to provide grounding using the grounding
terminal on the power cable, ground the instrument directly.
2. Connecting the power cable
First, be sure that the power switch is in the OFF position. Then insert the attached power
cable into the power inlet located on the right-side face of the instrument. Make sure that the
setting of the AC voltage selector conforms to the voltage of the site power.
3. Xenon lamp ON/OFF switch
Set the Xenon lamp ON/OFF switch to the ON position.
If all items are satisfied please continue as follows:
1. Set the power switch to the ON position. The Xenon lamp will turn ON automatically.

Xenon lamp ignition failure!

If the Xenon lamp fails to light or a continuing ”crackling” sound can be heard,
power OFF the instrument. For troubleshooting take a look at chapter "RF-
5301PC Troubleshooting" or contact Shimadzu.

2. After powering ON the instrument power ON the computer and start MS-Windows.
3. Start the panorama fluorescence software package.

3.6 Lamp Adjustment

The lamp position must be adjusted according to the RAMAN spectrum obtained with distilled water.
This adjustment must be preformed when installing the Xenon lamp for the first time and when replacing
a lamp. Make sure that the instrument has been powered ON for at least 30 minutes before performing
adjustment.

3.6.1 Coarse-adjustment of the lamp position

For coarse-adjustment of the Xenon lamp, please follow the instructions below:
1. Start the panorama Fluorescence software.

33
Spectrofluorophotometer Manual

2. From the RF-5301/RF-1501 menu, select the Go to Wavelength command.

The instrument will be initialized automatically on first time after powering ON showing the
following dialog:

Instrument Initialization is not mandatory!

Instrument initialization is only done, if the spectrofluorophotometer is used
the first time after powering ON or if connection was lost for any reason. If
this dialog is omitted, the instrument has already been initialized successfully.

3. After completion of the initialization process, click the Close button.

What happens on Instrument Initialization Failure?

Please refer to the Troubleshooting section or contact Shimadzu support for
help.

4. In the Go to Wavelength dialog, set the following parameters:

• Excitation Wavelength = 500 nm.

• Emission Wavelength = 350 nm.

34
 Installation of the spectrofluorophotometer

5. Click the Read button to update the actual intensity value (optional).
6. Click the Set button to apply actual values.
7. From the RF-5301/RF-1501 menu, select the Lamp Adjustment command.
The instrument shutter is opened and the following dialog is shown:

The intensity value is updated continuously.

The lamp time reset function is not available for the RF-1501 Instrument!
The current lamp time is only displayed for the RF-1501 instrument.

8. Open the lid of the sample compartment.

35
Spectrofluorophotometer Manual

9. Place a sheet of white paper into the cell holder.

The excitation monochromator should be emitting a green light beam.

10. Adjust the screw for horizontal positioning of the lamp so that the beam spot on the white
paper is of maximum brightness.

Caution: Be sure to adjust the horizontal position of the lamp first. Adjusting the
vertical position first may cause the light beam to deviate and make it
impossible to correct the lamp position.

36
 Installation of the spectrofluorophotometer

11. Similarly to the horizontal position adjust the screw for vertical positioning of the lamp.

12. Remove the white paper.

13. Load a cell containing distilled water into the cell holder.
14. Close the compartment lid.

3.6.2 Fine-adjustment of the lamp position

After coarse-adjustment of the lamp position a fine-adjustment is necessary. Follow the steps below:
1. Activate the Lamp Adjustment dialog.
The display shows an energy value.

2. Adjust the screw for horizontal positioning of the lamp until the energy value indicates a
maximum.
3. Adjust the screw for vertical positioning of the lamp until the energy value indicates a
maximum.
4. If a new lamp was installed, click the Reset button to reset the lamp timer (optional).
5. Click the Close button to finish the procedure.

37
4 Performance Tests

4.1 Signal/Noise Test

After installation of the Xenon lamp and adjustment of the lamp position the performance of the
instrument should be verified to check that it is operating normally.

4.1.1 Prerequisites
A 2D Emission measurement is required in advance to check experimental conditions with the sample.
Please follow the instructions below:
1. Start the panorama fluorescence software and make sure, the instrument has warmed up for
at least 30 minutes.
2. From the RF-5301/RF-1501 menu, select the 2D Emission Measurement command. The
measurement dialog is opened showing the last measured spectrum and last used parameter
settings.
3. In the measurement dialog, set the following parameters:

• Emission start wavelength: 350 nm

• Emission stop wavelength: 450 nm

• Excitation wavelength: 350 nm

• Scanning speed: fast

• Emission slit width:

RF-5301 PC: 5 nm
RF-1501: 10 nm

• Excitation slit width:

RF-5301 PC: 5 nm
RF-1501: 10 nm

• Sensitivity: high

• Sampling interval: 1.0 nm

• Response time: Auto

4. Open the lid of the sample compartment.
5. Load a cell filled with distilled water into the cell holder.
6. Close the lid of the sample compartment.
7. Click the Autozero button.

39
Spectrofluorophotometer Manual

8. Click the Measure button to start scanning.

The resulting spectrum is shown in the following figure:

9. Click the button on top right of the measurement dialog to close it.
The measured 2D emission spectrum is now shown in the main workspace.
10. From the Mathematics menu, select the Find Peaks command.
11. In the Mathematics tab on bottom right, adjust the parameters and click the Calculate
button.
The peaks should be detected automatically and a peak table will be created.

Too less or too many peaks are found!

Please refer to the Find Peaks command section for details on how to setup
parameters for peak finding. Refer to the Find Peaks section in the chapter
Mathematics for an introduction.

40
 Performance Tests

The resulting spectrum looks similar to the figure below:

(Determined wavelength of water band: 400.00 nm)

The obtained peak wavelength should be approximately 397 nm.

12. From the File menu, select the Print command to print a report for the spectrum and
corresponding peak values.
For details on printing and configuring report templates, please refer to the chapter "Printing".

4.1.2 Signal to Noise Test Procedure

The automatic signal to noise test procedure will perform subsequent measurements among a time
interval of 10 minutes. Emission spectra will be recorded automatically and the signal to noise ratio is
calculated from the water peak. Finally a report is generated, which can be printed.
To perform the signal to noise test, please follow the instructions below:

41
Spectrofluorophotometer Manual

1. From the RF-5301 menu, select the Instrument Parameters command.

The instrument parameters dialog is opened:

42
 Performance Tests

2. In the dialog, click the Perform button next to the S/N Ratio Check label.
The Signal Noise Performance Test dialog is opened:

3. In the dialog, click the Start button.

The user is prompted to insert a cell filled with distilled water into the cell holder:

4. Open the lid of the sample compartment.

5. Load a cell filled with distilled water into the cell holder.
6. Close the lid of the sample compartment.
7. Click the OK button to start measurement.

43
Spectrofluorophotometer Manual

8. After the automatic test procedure is finished, the report is completed:

(Determined S/N ratio: 218.861)

The signal to noise value should be ≥ 150 for the RF-5301PC instrument.

4.2 Signal to noise report layout

This report shows the results of the RF-5301 signal to noise test.
Notwithstanding the basic print layout properties and general object properties, some individual
properties can be adjusted for 2D data objects, that need to be printed.

4.2.1 Noise spectrum print layout properties

The same settings are applied as described for the Spectral image in the basic report layout section.

4.3 Wavelength Accuracy Test

Based on the mercury emission line (435.8 nm) of the light from a fluorescent lamp, the wavelength
accuracy can be verified for the monochromators.

4.3.1 Verifying wavelength accuracy for emission monochromator

To verify the emission monochromator a 2D emission measurement must be carried out.
1. Start the panorama Fluorescence software and make sure, the instrument has warmed up for
at least 30 minutes.

44
 Performance Tests

2. From the RF-5301/RF-1501 menu, select the 2D Emission Measurement command. The
measurement dialog is opened showing the last measured spectrum and last used parameter
settings:

3. In the measurement dialog, set the following parameters:

• Emission start wavelength: 400 nm

• Emission stop wavelength: 500 nm

• Excitation wavelength: 350 nm

• Scanning speed: medium

• Emission slit width:

RF-5301 PC: 1.5 nm
RF-1501: 10 nm

• Excitation slit width:

RF-5301 PC: 1.5 nm
RF-1501: 10 nm

• Sensitivity: high

• Sampling interval:
RF-5301 PC: 0.2 nm
RF-1501: 1 nm

• Response time: Auto

3. Open up the lid of the sample compartment to expose the emission monochromator to light
from a fluorescent lamp.
Remove the cell from the sample compartment.
4. Click the Measure button to start scanning.

45
Spectrofluorophotometer Manual

5. After scanning has stopped, check that the resultant peak in the spectrum is within the range
of 435.8 ± 1.5 nm.

(Determined wavelength: 436.4 nm)

6. Click the button on top right of the measurement dialog to close it.
The measured 2D Emission spectrum is shown in the main workspace now.
7. From the Mathematics menu, select the Find Peaks command.
8. In the Mathematics tab on bottom right, adjust the parameters and click the Calculate
button.
The peak should be detected automatically and a peak table will be created.

Too less or too many peaks are found!

Please refer to the Find Peaks command section for details on how to setup
parameters for peak finding. Refer to the Find Peaks section in the chapter
Mathematics for an introduction.

The resulting spectrum looks similar to the figure below:

46
 Performance Tests

(Emission spectrum of a Neon Lamp)

9. From the File menu, select the Print command to print a report for the spectrum and
corresponding peak values.
For details on printing and configuring report templates, please refer to the chapter "Printing".

Check the resultant peak position in the spectrum!

If the emission monochromator fails to satisfy the range mentioned above, contact Shimadzu
or its nearest representative. You will find contact data in the "Contact Shimadzu" chapter.

4.3.2 Verifying wavelength accuracy for excitation monochromator

To verify the excitation monochromator a 2D Excitation measurement must be carried out.
1. From the RF-5301 menu, select the 2D Excitation Measurement command. The
measurement dialog is opened showing the last measured spectrum and last used parameter
settings (see figures above).
2. In the measurement dialog, set the following parameters:

• Excitation start wavelength: 400 nm

• Excitation stop wavelength: 500 nm

• Scanning speed: medium

• Emission slit width: 3.0 nm

• Excitation slit width: 1.5 nm

• Sensitivity: high

• Sampling interval: 0.2 nm

• Response time: Auto

3. Set the Emission wavelength parameter exactly to the peak maximum value determined
from the Emission wavelength accuracy test above. The value should be within the limits of 435.8 ± 1.5
nm.
4. Open the lid of the sample compartment.

47
Spectrofluorophotometer Manual

5. Load a cell filled with distilled water into the cell holder.
6. Close the lid of the sample compartment.
7. Click the Measure button to start scanning.
8. After scanning has stopped, check that the resultant peak in the spectrum is within the range
of 435.8 ± 1.5 nm.

(Determined wavelength: 436.3 nm)

9. To produce a printed report, please follow the instructions from step 7 on in chapter
"Verifying wavelength accuracy for emission monochromator".
The wavelength of the resultant peak is around the emission wavelength that was recorded in chapter
"Verifying wavelength accuracy for emission monochromator".
However, this is an easier method for obtaining the wavelength accuracy of the instrument. To obtain
the exact wavelength accuracy you need a Mercury lamp. Please contact Shimadzu or its nearest
representative for further information. You will find contact data in the "Contact Shimadzu" chapter.

48
5 Menus

5.1 RF-5301/RF-1501 menu

The RF-5301/RF-1501 menu holds various commands to control and drive a Shimadzu RF-5301PC or a
RF-1501 spectrofluorophotometer. It provides a collection of measurement commands being short
hands to pre-defined instrument configurations. Some instrument maintenance commands are also
available.

Why is the menu missing sometimes?

The RF-5301/RF-1501 menu is only available when running a panorama
fluorescence software.

5.1.1 RF-5301/RF-1501 menu contents

The RF-5301/RF-1501 menu provides the following commands:

Commands are dynamically updated according to the connected instrument type.

• 2D Emission Measurement

• 3D Emission Increment Measurement

• 3D Emission Measurement

• 2D Excitation Measurement

• 3D Excitation Increment Measurement

• 3D Excitation Measurement

• 2D Synchro Measurement

49
Spectrofluorophotometer Manual

Why is the menu entry missing?

The 2D Synchro Measurement is only available for the RF-5301PC system! It is not
supported with RF-1501 systems.

• Time Measurement

• Photometric Measurement

• Show Instrument Status

• Go to Wavelength

• Instrument Parameter

• Lamp Adjustment

• Accessories

• Sipper

50
6 Measurements

6.1 Measurement Window

The measurement window is the main dialog where all measurement parameters for the RF-5301PC
and the RF-1501 instrument can be adjusted. The dialog always shows the last used settings and
spectra. From this dialog, measurements can be started and stopped.

During measurement no other operations can be performed!

The measurement window is always shown on top of the application, because during
measurement no other actions can be performed in the software.

The contents of the measurement window are described in more detail in the following:
To show the measurement window, select one of the measurement procedures from the RF-5301/RF-
1501 menu. It looks like this:

6.1.1 Online Measurement Monitoring

In order to follow the measurement progress, on the left side of the dialog either a 2D spectrum view or
a 2D and 3D spectrum view are displayed. The data views show the current measured spectra and will
be updated subsequently during measurement.

6.1.2 Measurement Control Buttons

On top right of the dialog, several buttons are available to control the instrument and measurements.
The following buttons are available:

51
Spectrofluorophotometer Manual

Measure Button / Sip and Measure Button

To start a measurement with actual parameter settings, click this button. For details on measurement
parameter settings, please see below.
In case a sipper accessory is connected to the instrument and activated,the button text shows Sip and
Measure. For details on how to work with the sipper accessory, please refer to the section "Working
with the sipper accessory".

Abort Button
To abort the current measurement, click this button.

Why is abort delayed sometimes?

Depending on the measurement, the response time of the instrument delays abort of
a measurement sometimes.

Autozero Button
While working with the instrument adjustment of the photomultiplier must be re-calibrated from time to
time to obtain a constant baseline for spectra.
Click this button to reset the baseline.

Shutter Button
The shutter protects the photomultiplier from permanent exposure to light in order to prevent damage.
Usually, the shutter is controlled by the measurement procedures automatically. For maintenance
purposes it might be useful to control the shutter manually.
Click this button to open or close the shutter manually. The image inside the button shows the actual
shutter condition.

Automatic shutter function can be switched off!

The Auto-shutter function can be permanently switched off in the Instrument
Parameter dialog. Please refer to this section for details.

Go to WL Button
Please refer to the Go to Wavelength section for details.

Close Button
Closes the measurement window and returns to the main software.

Reset Button
This button is only available with the photometric measurement. By default, results of photometric
measurements will be appended to the current report on the left pane of the measurement window.
Click Reset to clear the current report and recent measurements and restart with a new report.

Recent data is stored automatically!

When performing a reset, all previous measurement results are stored in a report
within the main software automatically.

6.1.3 Measurement parameters

Some general parameters are available in all measurement procedures. They are described in more
detail in the following:

52
 Measurements

Instrument Parameters
The following parameters are available:

• Emission Slit Width

This parameter specifies the slit width of the slit in front of the emission monochromator in [nm].
Allowed settings are
RF-5301 PC: 1.5 nm, 3 nm, 5 nm, 10 nm, 15 nm and 20 nm.
RF-1501: 10 nm and 20 nm.

• Excitation Slit Width

This parameter specifies the slit width of the slit in front of the emission monochromator in [nm].
Allowed settings are
RF-5301 PC: 1.5 nm, 3 nm, 5 nm, 10 nm, 15 nm and 20 nm.
RF-1501: 10 nm and 20 nm.

• Sensitivity
This parameter controls the receiver gain. It can be adjusted to High or Low sensitivity. The
high setting is about 50 times more sensitive than the low setting.

Online Display
The following parameters are available:

• Autoscale Display
This parameter controls the display of the 2D and 3D online monitors on the left of the dialog.
On Yes, spectra will be automatically scaled after completion of measurement.

• Displayed Intensity Maximum

This parameter specifies the upper limit of the intensity axis.

• Displayed Intensity Minimum

This parameter specifies the lower limit of the intensity axis.

Sample Information
The following parameters are available:

• Folder
This parameter specifies the destination folder in the panorama project, where measured data
are stored. If the folder does not exist, it will be created automatically.

• Project
This parameter specifies the destination panorama project, where measured data are stored. If
the project does not exist, it will be created automatically. For details on how to work with
projects please refer to the Projects and Project Explorer section of the panorama help
manual.

• Sample Name
In the text field the name of the measured spectrum must be entered. In order to allow
automatic naming, some wildcards can be used as described in the following:

• %c
This wildcard specifies a counter starting with 0. It will be increased automatically.

• %d
This wildcard specifies the actual date. The date format of the regional settings of the
operating system is applied.

• %t
This wildcard specifies the actual time. The time format of the regional settings of the
operating system is applied.

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Spectrofluorophotometer Manual

• Show Sample Name Dialog

After completion of a measurement the user can be forced to enter a spectrum name. A default
name is proposed automatically. The following sample name dialog is shown:

Scan Parameters
The following parameters are available:

• Response Time
This corresponds to the response speed of the RF-5301 unit relative to the variation in the
fluorescence intensity on a particular sample. The lower the setting (in seconds) the more
swiftly the instrument can follow variations in the fluorescence intensity with time, although the
resulting noise level will be greater. In contrast, the higher the setting, the less rapidly the
instrument can follow variations, but the lower the noise. The allowed settings are
RF-5301 PC: 0.02, 0.03, 0.1, 0.25, 0.5, 2.0, 4.0, 8.0 and Auto.
RF-1501: 0.02, 0.03, 0.1, 0.25, 0.5, 2.0 and 8.0.

• Sampling Interval
Sets up the wavelength intervals for scanning. The allowed settings are
RF-5301 PC: 0.2 nm, 1.0 nm and 2.0 nm.
RF-1501: 1.0 nm and 2.0 nm.

Limitation of settings!
However, that the scanning interval of 2.0 nm is automatically selected only
when the scan speed is set to "Survey" and cannot be used for other speed
settings. Also, the setting of 0.2 nm cannot be selected for speed setting
"Survey", "Super" and "Very Fast".

• Scanning Speed
This parameter specifies the scanning speed for data acquisition. The seven settings are
RF-5301 PC: Survey, Super, Very Fast, Fast, Medium, Slow and Very Slow.
RF-1501: Super, Fast, Medium and Slow.

Sipper Parameters
For a general introduction into functionality of the sipper unit and how to use it with the instrument,
please refer to the section "Working with the Sipper Accessory". The following parameters are available,
which control the sipper accessory functions:

• Use Sipper
Specifies, whether the sipper accessory is enabled and used during measurement or not.
Parameters will be expanded or collapsed in the measurement window according to the
settings.
The parameter can be also adjusted from the Accessories sub menu in the RF-5301/RF-1501
menu.

• Yes
Make sure the sipper accessory is connected to the instrument before you change the
flag status to Yes.

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 Measurements

• No
The sipper accessory is not used.

• Pump Speed
This parameter controls the pump speed of the built-in sipper pump. The following pump speed
levels are applicable:

• Slow

• Medium

• Fast

• Sipping Time
Specifies the pumping duration in seconds.

• Dwell Time
Specifies the rest period after pumping and before performing the measurement. This interval
is applied to let the sample rest in the cuvette before measurement.

• Purging Time
Specifies the purging duration after completion of the measurement in seconds. The sample is
removed from the cuvette by pumping the solvent through the cuvette.

• Number of Rinses
Specifies the number of purge intervals required to clean the cuvette.

Sipper Sampling Parameters (Auto-Sampler Control)

For a general introduction into functionality of the auto-sampler unit, please refer to the "Working with
the Auto-Sampler Accessory" section. The following parameters are available to setup the auto-sampler
unit:

• Use Auto-Sampler

• Yes
The auto-sampler is enabled

• No
The auto-sampler is disabled

• Number of Samples
Denotes the number of samples to be measured and provided in the auto-sampler.

6.2 2D Emission Measurement

The 2D emission measurement is performed to obtain a selective fluorescence spectrum of a chemical
substance by excitation of a particular wavelength. Even within compound mixtures individual
substances can be analyzed without prior separation.
A 2D emission measurement is carried out as described in the following:

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Spectrofluorophotometer Manual

1. From the RF-5301/RF-1501 menu, select the 2D Emission Measurement command. The
measurement dialog is opened showing the last measured spectrum and last used parameter
settings:

If the measurement window is already open...

... the measurement type can be directly changed in the Measurement Mode
drop down box.

2. Set the Excitation Wavelength to an appropriate value in [nm].

3. Set the Emission Start Wavelength to an appropriate start value of the detection range in
[nm].
4. Set the Emission Stop Wavelength to an appropriate end value of the detection range in
[nm].
5. Set the other parameters in the dialog optionally. For details on general parameter settings,
please refer to the Measurement Window section.
6. Open up the lid of the sample compartment
7. Load a cell filled with a sample into the cell holder.
8. Close the lid of the sample compartment.
9. Click the Autozero button.

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 Measurements

10. Click the Measure button to start scanning.

During measurement the user can see evolving the spectrum. A sample spectrum of a neon
lamp is shown below:

11. Click the button on top right of the measurement dialog to close it.
The measured spectrum is now shown in the main workspace and a new spectrum node has
been added to the destination project.

6.3 2D Excitation Measurement

The 2D excitation measurement is performed to observe emission signals at a discrete emission
wavelength as a response to excitation. This way the fluorescence with a defined energy transition can
be studied in different molecules.
A 2D excitation measurement is carried out as described in the following:
1. From the RF-5301/RF-1501 menu, select the 2D Excitation Measurement command. The
measurement dialog is opened showing the last measured spectrum and last used parameter
settings.

If the measurement window is already open...

... the measurement type can be directly changed in the Measurement Mode
drop down box.

2. Set the Emission Wavelength to an appropriate value in [nm].

3. Set the Excitation Start Wavelength to an appropriate start value of the detection range in
[nm].
4. Set the Excitation Stop Wavelength to an appropriate end value of the detection range in
[nm].
5. Set the other parameters in the dialog optionally. For details on general parameter settings,
please refer to the Measurement Window section.
6. Open up the lid of the sample compartment

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Spectrofluorophotometer Manual

3. Load a cell filled with a sample into the cell holder.

4. Close the lid of the sample compartment.
5. Click the Autozero button.
6. Click the Measure button to start scanning.
During measurement the user can see evolving the spectrum. A sample excitation spectrum of
anthracene-9-carboxylic acid is shown below:

7. Click the button on top right of the measurement dialog to close it.
The measured spectrum is now shown in the main workspace and a new spectrum node has
been added to the destination project.

6.4 2D Synchro Measurement

The synchro measurement excites a sample at a particular wavelength and measures corresponding
emission at the same wavelength as a direct response.

The 2D synchro measurement is not supported by the RF-1501 instrument!

A 2D excitation measurement is carried out as described in the following:

58
 Measurements

1. From the RF-5301/RF1501 menu, select the 2D Excitation Measurement command. The
measurement dialog is opened showing the last measured spectrum and last used parameter
settings:

If the measurement window is already open...

... the measurement type can be directly changed in the Measurement Mode
drop down box.

2. Set the Start Wavelength to an appropriate start value of the detection range in [nm].
3. Set the Stop Wavelength to an appropriate end value of the detection range in [nm].
4. Set the other parameters in the dialog optionally. For details on general parameter settings,
please refer to the Measurement Window section.
5. Open up the lid of the sample compartment
6. Load a cell filled with a sample into the cell holder.
7. Close the lid of the sample compartment.
8. Click the Autozero button.
9. Click the Measure button to start scanning.
10. During measurement the user can see evolving the spectrum.

11. Click the button on top right of the measurement dialog to close it.
The measured spectrum is now shown in the main workspace and a new spectrum node has
been added to the destination project.

6.5 3D Emission Increment Measurement

The 3D emission increment measurement is performed to identify and separate fluorescent wavelengths
of a sample caused by particular excitation. Significant emission wavelengths can be determined by
scanning along the emission dimension.

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Spectrofluorophotometer Manual

This measurement is an automated sequence of 2D emission measurements collected in a 3D data

object. The emission wavelength is increased subsequently by a user defined amount during
measurements.
A 3D emission increment measurement is carried out as described in the following:
1. From the RF-5301/RF-1501 menu, select the 3D Emission Increment Measurement
command. The measurement dialog is opened showing the last measured spectrum and last
used parameter settings.

If the measurement window is already open...

... the measurement type can be directly changed in the Measurement Mode
drop down box.

2. Set the Excitation Wavelength to an appropriate value in [nm].

3. Set the Emission Start Wavelength to an appropriate start value of the detection range in
[nm].
4. Set the Emission Stop Wavelength to an appropriate end value of the detection range in
[nm].
5. Set the Wavelength Increment to an appropriate value in [nm].
6. Set the other parameters in the dialog optionally. For details on general parameter settings,
please refer to the Measurement Window section.
7. Open up the lid of the sample compartment
8. Load a cell filled with a liquid sample into the cell holder.
9. Close the lid of the sample compartment.
10. Click the Autozero button.

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 Measurements

11. Click the Measure button to start scanning.

During measurement the user can see the 2D spectrum of the current cycle and the 3D
spectrum evolving. As an example, the top view of a 3D emission increment measurement of
distilled water is shown below:

The 3D spectrum can be displayed in several ways!

For 3D data several display modes are available. They can be selected from
the 3D View menu:

• 3D View

• Top View

• 2D Overlay

12. Click the button on top right of the measurement dialog to close it.
The measured spectrum is now shown in the main workspace and a new spectrum node has
been added to the destination project.

6.6 3D Emission Measurement

The 3D emission measurement is performed to collect several 2D emission spectra in fixed time
intervals. This is useful to study reactions and see evolving of reactant signals.
All recorded 2D emission spectra will be collected in a 3D data object. This measurement is similar to
the time measurement.
A 3D emission measurement is carried out as described in the following:

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Spectrofluorophotometer Manual

1. From the RF-5301/RF1501 menu, select the 3D Emission Measurement command. The
measurement dialog is opened showing the last measured spectrum and last used parameter
settings:

If the measurement window is already open...

... the measurement type can be directly changed in the Measurement Mode
drop down box.

2. Set the Excitation Wavelength to an appropriate value in [nm].

3. Set the Emission Start Wavelength to an appropriate start value of the detection range in
[nm].
4. Set the Emission Stop Wavelength to an appropriate end value of the detection range in
[nm].
5. Set the Timing Conditions for the measurement. Changing one of the following parameters
will cause automatic recalculation of dependent parameters:

• Acquisition Rate

• Total number of Acquisitions

• Total time.
6. Set the other parameters in the dialog optionally. For details on general parameter settings,
please refer to the Measurement Window section.
7. Open up the lid of the sample compartment
8. Load a cell filled with a sample into the cell holder.
9. Close the lid of the sample compartment.
10. Click the Autozero button.
11. Click the Measure button to start scanning.
During measurement the user can see evolving the 2D spectra and the 3D spectrum.

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 Measurements

The 3D spectrum can be displayed in several ways!

For 3D data several display modes are available. They can be selected from
the 3D View menu:

• 3D View

• Top View

• 2D Overlay

12. Click the button on top right of the measurement dialog to close it.
The measured spectrum is now shown in the main workspace and a new spectrum node has
been added to the destination project.

6.7 3D Excitation Increment Measurement

The 3D excitation increment measurement is performed to identify fluorescent wavelengths of a sample.
Particular excitation will return corresponding response signals in the emission dimension of the 3D
excitation spectrum. Significant wavelengths can be determined and used in further analyses of the
sample.
This measurement is an automated sequence of 2D excitation measurements collected in a 3D data
object. The excitation wavelength is increased by a user defined amount during measurements.
A 3D excitation increment measurement is carried out as described in the following:
1. From the RF-5301/RF-1501 menu, select the 3D Excitation Increment Measurement
command. The measurement dialog is opened showing the last measured spectrum and last
used parameter settings.

If the measurement window is already open...

... the measurement type can be directly changed in the Measurement Mode
drop down box.

2. Set the Emission Wavelength to an appropriate value in [nm].

3. Set the Excitation Start Wavelength to an appropriate start value of the detection range in
[nm].
4. Set the Excitation Stop Wavelength to an appropriate end value of the detection range in
[nm].
5. Set the Wavelength Increment to an appropriate value in [nm].
6. Set the other parameters in the dialog optionally. For details on general parameter settings,
please refer to the Measurement Window section.
3. Open up the lid of the sample compartment
4. Load a cell filled with a liquid sample into the cell holder.
5. Close the lid of the sample compartment.
6. Click the Autozero button.

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Spectrofluorophotometer Manual

7. Click the Measure button to start scanning.

During measurement the user can see the 2D spectrum of the current cycle and the 3D
spectrum evolving. As an example, the 3D excitation increment measurement of a mixture of
anthracene and naphthaline is shown below:

The 3D spectrum can be displayed in several ways!

For 3D data several display modes are available. They can be selected from
the 3D View menu:

• 3D View

• Top View

• 2D Overlay

8. Click the button on top right of the measurement dialog to close it.
The measured spectrum is now shown in the main workspace and a new spectrum node has
been added to the destination project.

6.8 3D Excitation Measurement

The 3D excitation measurement is performed to collect several 2D excitation spectra in fixed time
intervals. This is useful to study reactions and see evolving of reactant signals.
All recorded 2D excitation spectra will be collected in a 3D data object. This measurement is similar to
the time measurement.
A 3D excitation measurement is carried out as described in the following:

64
 Measurements

1. From the RF-5301/RF-1501 menu, select the 3D Excitation Measurement command. The
measurement dialog is opened showing the last measured spectrum and last used parameter
settings:

If the measurement window is already open...

... the measurement type can be directly changed in the Measurement Mode
drop down box.

2. Set the Emission Wavelength to an appropriate value in [nm].

3. Set the Excitation Start Wavelength to an appropriate start value of the detection range in
[nm].
4. Set the Excitation Stop Wavelength to an appropriate end value of the detection range in
[nm].
5. Set the Timing Conditions for the measurement. Changing one of the following parameters
will cause automatic recalculation of dependent parameters:

• Acquisition Rate

• Total number of Acquisitions

• Total time.
6. Set the other parameters in the dialog optionally. For details on general parameter settings,
please refer to the Measurement Window section.
7. Open up the lid of the sample compartment
8. Load a cell filled with a sample into the cell holder.
9. Close the lid of the sample compartment.
10. Click the Autozero button.
11. Click the Measure button to start scanning.
During measurement the user can see evolving the 2D spectra and the 3D spectrum.

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Spectrofluorophotometer Manual

The 3D spectrum can be displayed in several ways!

For 3D data several display modes are available. They can be selected from
the 3D View menu:

• 3D View

• Top View

• 2D Overlay

12. Click the button on top right of the measurement dialog to close it.
The measured spectrum is now shown in the main workspace and a new spectrum node has
been added to the destination project.

6.9 Go to Wavelength
The Go to Wavelength function is used to adjust a particular excitation and emission wavelength
directly. By adjusting the pair of parameters, the response signal can be directly detected. This
procedure is useful for single value detection or instrument maintenance.
To set particular excitation and emission wavelengths, please follow the instructions below:
1. From the RF-5301/RF-1501 menu, select the Go to Wavelength command. The Go to
Wavelength dialog is opened as shown in the following:

Go to Wavelength button is also available in measurement window!

The go to wavelength function is also accessible from the Measurement
Window. Please refer to this section for details.

2. Set the Excitation Wavelength to an appropriate value in [nm] or move the slider to adjust.
3. Set the Emission Wavelength to an appropriate value in [nm] or move the slider to adjust.
4. Click the Read button to update the actual intensity value (optional).
5. Click the Set button to apply actual values and close the dialog.

Setting a wavelength...
... will be applied instantly! The instrument will move to the selected excitation
and emission wavelength and will keep this settings until new positions are
applied by any other measurement procedure.

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 Measurements

6. Click the Cancel button to discard changes and close the dialog.

6.10 Photometric Measurement

The photometric experiment allows single point measurements of up to 10 different excitation/emission
pairs. results are displayed in a comprehensive report and trend plot. Follow the steps below to setup a
photometric measurement:
1. From the RF-5301/RF-1501 menu, select the Photometric Measurement command. The
measurement dialog is opened with last used results:

2. Click the Reset Button to clear current report contents.

If you like to append the current measurements to the most recent ones, skip this step
3. Set the Show Trend Plot flag, if you like to perform multiple measurements of the same
wavelengths (see below).
4. Set the Number of Wavelengths to the total number of wavelengths being measured
simultaneously .
Up to 10 different wavelengths can be measured at a time.
5. For each wavelength, a new "Wavelength n" category is appended to the properties with the
following parameters:

• Set the Excitation Wavelength to an appropriate value of the detection range in

[nm].

• Set the Emission Wavelength to an appropriate value of the detection range in [nm].
6. Set the other parameters in the dialog optionally. For details on general parameter settings,
please refer to the Measurement Window section.
7. Open up the lid of the sample compartment.
8. Load a cell filled with a sample into the cell holder.
9. Close the lid of the sample compartment.
10. Click the Autozero button.
11. Click the Measure button to start scanning.

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Spectrofluorophotometer Manual

After completion of a measurement, the trend plot and report are updated accordingly.

6.11 Time Measurement

The time measurement is useful for studies of chemical reactions or life time of excited states. A
substance is excited at a particular wavelength and its response is detected at the emission wavelength.
A time measurement is carried out as described in the following:
1. From the RF-5301/RF-1501 menu, select the Time Measurement command. The
measurement dialog is opened showing the last measured spectrum and last used parameter
settings.

2. Set the Excitation Wavelength to the appropriate value in [nm].

3. Set the Emission Wavelength for detection of the response signal to the appropriate value in
[nm].
4. Set the timing conditions for the measurement. Changing one of the following parameters will
cause automatic recalculation of dependent parameters:

• Acquisition Rate

• Total number of Acquisitions

• Total time.
5. Set the other parameters in the dialog optionally. For details on general parameter settings,
please refer to the Measurement Window section.
6. Open up the lid of the sample compartment
7. Load a cell filled with a liquid sample into the cell holder.
8. Close the lid of the sample compartment.
9. Click the Measure button to start scanning.
During measurement the user can see the emission evolving.

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7 Hardware and Maintenance

7.1 Maintenance

7.1.1 Cautions for transferring the instrument

When transferring or shipping the RF-5301PC/RF-1501 instrument be sure to remove the Xenon lamp.
Store the removed lamp in a special case. The Xenon lamp contains high pressure gas. Impact,
vibration or pressure on it may cause it to burst, posing a serious danger. Be extremely careful when
handling it. If the lamp is touched with naked hand, clean the surface before lighting it. Cleaning is
possible with ethyl alcohol or the special cleaning agent included with the lamp. Finger oil remaining on
the bulb can be baked onto the bulb when the lamp is lit, possibly causing the lamp to burst.

7.1.2 Service life of the Xenon Lamp

The Xenon lamp is a consumable part. When the lamp is used for a long time, the emission point moves
around or flickers. If such a problem occurs the noise level will become greater and an accurate data
acquisition will be impossible. The time during which the lamp remains operative before the first
occurrence of flickering is called the flicker life.
The guaranteed flicker life of a 150 W Xenon lamp is 500 hours. When the lamp time used reaches 500
hours, replace the lamp immediately.

Caution: Never use a lamp in excess of 1000 hours. A lamp used for more than 1000
hours may burst, possibly damaging the lamp unit.

7.1.3 Safe disposal of the Xenon lamp

Special care must be taken to dispose the used Xenon lamp. The lamp will pose a potential danger in
ordinary waste disposable processes, because it contains high pressure gas. Before disposing it, wrap it
up carefully in thick cloth (to prevent glass fragments from flying) and crash the bulb portion with a
hammer, etc. Be careful not to injure yourself with glass fragments.

7.1.4 Replacing fuse

When a fuse is blown replace it. The fuses are right beside the ON/OFF power switch of the instrument
RF-5301PC/RF-1501.

7.2 Instrument Parameter

Some general instrument settings can be adjusted here. These parameters are most of all used for
maintenance purposes and should not be changed.
To open the instrument parameter dialog, please follow the instructions below:
1. From the RF-5301/RF-1501 menu, select the Instrument Parameter command.
For the RF-5301PC system the instrument parameter dialog is opened as follows:

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Spectrofluorophotometer Manual

For the RF-1501 system the dialog looks like this:

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 Hardware and Maintenance

2. Enter the serial number of the RF-1501 instrument, which is located on a label on the right
side of the instrument. (RF-1501 System only!)

Caution: Entering the serial number is only required for the RF-1501
instrument!
With the RF-5301PC system, the serial number is available
automatically.

3. Configure the parameters in the Instrument Settings section.

Caution: These options should only be used by service persons or

experienced users. Wrong settings might cause permanent
damage to any instrument components!
With the RF-1501 system, the PMT control is not available!

4. Perform any instrument calibration from the Instrument Calibration section.

5. Click the Close button to finish.

7.2.1 Instrument Settings

HV Control
This parameter controls the high voltage gain (HV) of the Xenon lamp which is regulated automatically
by default. Regulation is done with regard to the sensitivity of the photomultiplier. For instrument
maintenance, automatic regulation can be switched of. E.g. for measurement of specific instrument
backgrounds or stray light measurements, which can be used for background correction of spectra.

• On (default)
Automatic regulation of Lamp voltage is applied.

• Off
No regulation of Lamp voltage is applied.

PMT Control
This parameter controls the sensitivity of the photomultiplier tube (PMT) with regard to the amount of
light exposure. By default the sensitivity of the PMT will be controlled automatically. For service and
maintenance purposes automatic regulation can be deactivated.

• On (default)
PMT sensitivity is regulated automatically.

• Off
The PMT sensitivity is not regulated.

Auto Shutter
This parameter controls the shutter which protects the photomultiplier from permanent irradiation with
light. By default, the auto shutter is on to avoid damages to the photomultiplier. For some dark or
background measurements carried out by service persons the shutter must be controlled manually.

• On (default)
The shutter opens and closes automatically during measurements.

• Off
The shutter must be opened and closed manually before starting measurements.

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Spectrofluorophotometer Manual

7.2.2 Instrument Calibration

This section provides the following operations:

• Dark Level Correction

The dark level correction measures the background noise of the instrument. The characteristic
noise level will be used to adjust the photomultiplier.

• S/N Ratio Check

Please review the Signal to Noise Test section in the chapter "Performance Tests" for details.

• Lamp Alignment
Please review the Lamp Adjustment section in the chapter "Hardware and Maintenance" for
details.
Click the Perform button next to the desired operation to start it.

7.3 Checking Lamp Time

The spectrofluorophotometer keeps a record of the total amount of time that the Xenon lamp has been
lit. The time is displayed in units of hours in the Lamp Adjustment dialog.
After installing the lamp for the first time or after replacing it, zero the lamp time as followed:
1. From the RF-5301/RF-1501 menu, select the Lamp Adjustment command.
The instrument shutter is opened and the following dialog is shown:

The actually measured intensity value is updated continuously.

In the upper part of the dialog the amount of time the lamp has been lid is shown.
2. To zero the lamp time click the Reset button.

The lamp time reset function is not available for the RF-1501 Instrument!
The current lamp time is only displayed for the RF-1501 instrument.

3. Click the Close button to finish.

7.4 Show Instrument Status

The instrument status shows the actual status of the spectrofluorophotometer or is used for initialization.
Initialization is required to ensure correct communication between the instrument and the panorama
fluorescence software. The initialization procedure includes various internal self test procedures, which
must be passed to ensure reliable measurement results.

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 Hardware and Maintenance

When is the instrument initialized?

Initialization is only required, when the instrument is used the first time after powering
ON and after a certain standby period. In this case, initialization is done automatically
before the next measurement is started.

The steps of the initialization process are displayed on screen:

7.4.1 General instrument properties

In the upper part of the dialog, some general instrument information are displayed. During initialization
process all COM ports will be scanned for attached instruments. After successful auto-detection of the
RF-5301 spectrofluorophotometer the following data is read from the instrument:

• Instrument name

• Serial number

RF-1501 Instrument Serial Number!

For the RF-1501 system the serial number needs to be entered manually. It is
imprinted on a label on the right side of the device.
With RF-5301PC instruments, the serial number is stored on the internal EPROM and
will be displayed automatically.

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Spectrofluorophotometer Manual

If no valid serial number is entered, you will be prompted by the following message when you try
to close the dialog:

• ROM version

• Serial port

7.4.2 RF-5301 Instrument self test

Various internal self tests are performed to check the basic instrument functions. The test status is
shown as colored square in front of the particular test item.
All tests need to be passed before working with the instrument will be possible.

What shall I do, if any self test fails?

If any internal self test fails, this might be due to any damage on one of the system
components. Please contact Shimadzu for assistance.

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 Hardware and Maintenance

After a successful initialization the instrument is ready for measurement.

In case of an unsuccessful initialization an error message appears as shown below:

For details on remedial actions please follow the instructions in the error message or refer to the RF-
5301 troubleshooting section of this manual.
Click the Close button to finish.

7.5 Troubleshooting

7.5.1 Communication problems

The following items should be checked:

Items to be Remedial action

checked

Cable connection Check the cable connections between the instrument and the computer.
Next, try again to establish a communication.

COM port setting Study the documentation for your computer to check that the COM port in
question has been addressed correctly.

If a normal communication cannot be established in spite of the above remedial actions contact
Shimadzu or its nearest representative.

7.5.2 Before suspecting malfunction

The following items should be checked:

Problem Cause Remedial action

Instrument is Power cable is Securely insert both end of the cable into the inlet on the
not powered not securely instrument and the outlet at the site.
although the connected.
power switch is
in the ON
position.

Power fuse is Install a new fuse.

blown.

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Spectrofluorophotometer Manual

Other causes. Contact Shimadzu or its nearest representative.

Xenon lamp Lamp ON/OFF Move the switch in ON position.

does not light. switch is in OFF
position.

Wiring is Before opening the cover of the lamp housing be sure to

disconnected. disconnect the power cable from the outlet!

Lamp is still Allow the lamp to cool down for about 10 minutes.
hot.

Other causes. Contact Shimadzu or its nearest representative.

Signal does not Lamp is unlit. See problem above.

come out.

Xenon lamp is Correct the lamp position.

misaligned.

The Shutter or Open the shutter or the slit.

the slit on the
emission side is
closed.

Wrong Correct the parameters.

acquisition
parameters.

Other causes. Contact Shimadzu or its nearest representative.

S/N ratio does Xenon lamp is After powering ON the instrument wait 30 minutes until the
not satisfy the not stably lit. Xenon lamp is stably lit.
guaranteed
value.

Xenon lamp is Use a new Xenon lamp.

aged.

Distilled water Use only clean distilled water.

is
contaminated.

Other causes. Contact Shimadzu or its nearest representative.

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 Hardware and Maintenance

7.6 Instrument Construction

7.6.1 The optical system of RF-5301PC

The optical system of the RF-5301PC instrument is illustrated in the figure below. A 150 W Xenon lamp
(1) serves as the light source. The lamp housing contains generated ozone. The ozone is decomposed
by means of the heat produced by the lamp. The bright spot of the Xenon lamp is expanded and
collected by the ellipsoidal mirror (2). Then, the beam is collected again onto the entrance slit of the
excitation-side slit assembly (3) by the concave mirror (4). A part of beam scattered by the concave
grating (5) passes through the exit slit to irradiate the sample cell thought the condenser lens (11). To
achieve light source compensation a part of the excitation light is reflected by the beam splitter quartz
plate (6). Next it is directed to the teflon reactor plate 1 (7). The diffusely reflected light from the reflector
plate 1 (7) then passes through the aperture for light balancing (21) and illuminates the teflon reflector
plate 2 (8). Reflected by the reflector plate 2 (8) the diffuse light is attenuated to a specific ratio by the
optical attenuator (9). Then it reaches the photomultiplier for monitoring (10).
The fluorescence occuring from the cell passes the condenser lens (13) and then it is introduced into the
emission monochromator which comprises the slit assembly (14) and the concave grating (15). Then the
isolated light is introduced through the concave mirror (16) into the photomultiplier for photometry (17).
The resultant electrical signal is conducted to the preamplifier.

7.6.2 The optical system of RF-1501

The Optical system of the RF-1501 is explained below with reference to an Optical system diagram (see
Figure below).

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Spectrofluorophotometer Manual

Light emitted from the xenon lamp is concentrated in the entrance slit S1 at the excitation
monochromator by the light-concentrating ellipsoidal mirror M1. Light passing the slit S1 is reflected by
the plane mirror M2 and enters the excitation grating G1 before it is dispersed to enter the exit slit S2.
Light emitted from S2 (excitation light) is concentrated in the sample cells through the lenses L1 and L2.
Part of the excitation light is dispersed by the beam splitter 82 and enters the monitor photomultiplier
PM1 for light source compensation.
Fluorescence passing the slit S3 is dispersed by the emission grating and, passing through the exit slit
S4, enters the fluorescence photomultiplier PM2 for fluorescence intensity measurement.

78
8 Contacting

8.1 Contact Shimadzu

Shimadzu Deutschland GmbH is located in Germany:

Shimadzu Deutschland GmbH

Albert-Hahn-Str. 6-10

D-47269 Duisburg
Address:
Telephone:

Telefax:

e-mail:

Telephone: +49 (0)203-7687-0

Fax: +49 (0)203-766625

World Wide Web: www.shimadzu.de

Further headquarters are located around the world. Please find the nearest headquarter in the internet
at
http://eu.shimadzu.de/company/international

79
9 Appendix

9.1 Working with the Auto-Sampler Accessory

The auto-sampler unit is an optional add-on accessory for measurement automation. It provides an
option to prepare a set of samples being measured subsequently in a predefined sequence. For details
on how to install, setup and use the auto-sampler, please refer to the AUTO-SAMPLER OPERATION
MANUAL.

The auto-sampler can only be used together with the sipper!

For technical reasons, the auto-sampler can only be used in conjunction with the sipper
accessory. It allows to provide the samples one after the other to the instrument fully
automated.

9.1.1 Enabling and disabling the Auto-Sampler

The sipper needs to be enabled before the auto-sampler can be enabled or disabled, because it can
only be used in combination with the sipper unit. For details on how to enable the sipper, please refer to
the section "Working with the Sipper Accessory".
In the Measurement Window, set the Use Auto-Sampler flag

• Yes
The auto-sampler is enabled

• No
The auto-sampler is disabled

9.1.2 Measurements with Auto-Sampler and Sipper

Performing measurements with the auto-sampler, please follow the steps below to setup experiments:
1. Prepare a number of samples in suitable reservoirs for the auto-sampler.
2. Put the samples in correct order into the auto-sampler.
3. Setup the auto-sampler itself to support the desired number of samples.
Please refer to the AUTO-SAMPLER OPERATION MANUAL for details.
4. Now setup the measurement in panorama Fluorescence by selection of the desired
measurement in the Measurement Window.
5. In the Sipper Sampling category of the measurement properties on bottom right of the
Measurement Window set the Use Auto-Sampler flag to Yes.
6. Set the Number of Samples value to the number of prepared samples available in the auto-
sampler.
7. Setup the sipper parameters as described in the section "Working with the Sipper Accessory".
8. Click the Sip and Measure button to start the measurement sequence.
Alternatively, press the Start button on the auto-sampler.

All samples will be processed and measured one after the other automatically. On any error the system
will stop and an error message is displayed.

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Spectrofluorophotometer Manual

Choose the automatic sample naming to avoid intermediate breaks and messages!
In the measurement Window setup the following parameters correctly to avoid interruptions:
1. In the Sample Information category set the flag Show Sampler Name Dialog to
No.
2. Setup Sample Name parameter.
For details, please refer to the section Sample Information in the chapter Measurement
Window.

9.2 Working with the Sipper Accessory

The sipper accessory is an optional accessory to the sample compartment for subsequent measurement
of various liquids and dissolved samples. It has a built-in cuvette chamber, which is filled and emptied
with a pump via a pipe system. Liquid samples can be easily measured without extra sample
preparation. The liquid is directly pumped from your sample reservoir into the cell for measurement.

9.2.1 Installing the Sipper

The following steps are required to prepare the instrument for measurements with the sipper accessory:
1. Power OFF the instrument.
2. Open the sample compartment lid and demount the standard sample holder.
3. Mount the sipper accessory into the sample compartment.
4. Connect the sipper cable to the I/O-2 connector (Please refer to section "Part Names and
Functions" to locate it).
5. Power ON the instrument again.

9.2.2 Enabling and disabling the Sipper Unit

Before the sipper can be used in panorama Fluorescence, it needs to be enabled in the software.
There are two ways to do this as described in the following:
1. From the RF-5301/RF-1501 menu, expand the Accessories sub-menu.
2. Check or uncheck the Sipper option

• Checked
The sipper is enabled

• Unchecked
The sipper is disabled
Alternatively, the sipper can be enabled or disabled directly in the Measurement Window. The sipper
unit has a few parameters that can be adjusted in the software as described in the Measurement
Window section.

9.2.3 Performing Measurements with the Sipper

The work flow for routine measurement using the sipper accessory is described in the following:
1. Prepare the sample and provide the liquid in a reservoir.
2. Setup the desired measurement and corresponding parameters.
For details on available measurements, please refer to the particular "Measurements" section.
The Measurement Window is shown after selection of the measurement.

82
 Appendix

3. In the Measurement Window, setup the Sipper parameters.

For detailed parameter explanations, please refer to the "Sipper Parameters" section.
Example:

• Pump Speed = Fast

• Sipping time = 4.0 seconds

• Dwell time = 1.0 seconds

• Purge time = 4.0 seconds

• Number of Rinses = 1
4. Click the Sip and Measure button to start the measurement.

Starting a measurement with the external sipper button.

The whole front panel of the sipper accessory is a button. If you press the
front panel, the measurement will be started with current settings.

5. Put the tube to the ground of the reservoir to avoid bubbles being pumped into the cuvette
chamber and confirm the message box with OK.

The measurement progress and steps are shown in the status dialog:
1. The sample is pumped into the cell.
The following status dialog is shown:

Aborting measurements.
Whenever you like to abort the current measurement, click the Abort
button in the status dialog.
Abort takes some time to complete already started operations. This may
take up to 30 seconds.
The sample remains in the cuvette chamber on abort. It will not be removed
automatically!

2. Pumping stops and the instrument is idle for the specified dwell time in order to let the sample
rest for a while.

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Spectrofluorophotometer Manual

3. The measurement is carried out.

If not aborted, the sample is removed from the cell by purging and rinsing after completion of
the measurement.

84
10 Index
Coarse-adjustment 33
2 COM 72
Contact IN 14
2D
Cyclohexane 3
monochromator 44
Cylce 63
2D 39, 44
2D 44
2D 55 D
2D 57
Damages 69
2D 58
Dark Level Correction 69
2D 59
Determined S/N 39
2D 61
Diaminostilbene 3
2D 63
2D 64
2D Emission 39, 44 E
2D Emission Measurement 39, 44, 55
EM 21
2D Excitation
Emission 44
monochromator 44
Emission Start Wavelength 55, 59, 61
2D Excitation 44
Emission Stop Wavelength 55, 59, 61
2D Excitation Measurement 44, 57, 58
Emission Wavelength 33, 57, 63, 64, 66, 68
2D Synchro Measurement 58
Emission/Excitation monochromator 21
EX 21
3 Excitation Start Wavelength 57, 63, 64
Excitation Stop Wavelength 57, 63, 64
3D 59, 61, 63, 64
Excitation Wavelength 33, 55, 59, 61, 66, 68
3D Emission Increment Measurement 59
Excitation-side 77
3D Emission Measurement 61
3D Excitation Increment Measurement 63
3D Excitation Measurement 64 F
Face
A RF-5301PC 24
File menu 39, 44
Absorptiometry 3
Find Peaks 39, 44
Acquisition Rate 61, 64, 68
Fine-adjustment 33
Acquisitions
Fluorescein 3
number 61
Fluorescence 3
Acquisitions 61
Fluorescence Analysis 3
Acquisitions 64
Acquisitions 68
Adjust G
photomultiplier 69
Germany 79
Adjust 69
Grating 3
After coarse-adjustment 33
Analog OUT 14
Anthracene 3, 63 H
Anthracene-9-carboxylic 57
Hardware 69
Aspirin 3
Hardware Specification 21
Autozero button 39, 55, 57, 58, 59, 61, 63, 64
High/low 21

C
I
Calculate button 39, 44
Illuminates 77
Cancel button 66
Indole 3
Carbon Tetrachloride 3
Inspection 11
Chromatopack 14
Installation
Close button 33, 69, 72
RF-5301PC 24
Coarse-adjustment
Installation 24
Xenon 33

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Spectrofluorophotometer Manual

Installing set 12
Xenon 24, 33 switch 12
Installing 33 OFF 12
Instrument Calibration 69 OFF 14
Instrument Construction 77 OFF 75
Instrument Initialization 33 OK button 39
Instrument Initialization Failure 33 ON
Instrument Parameters 39, 69 powering 24, 33, 75
Instrument Settings 69 ON 12, 14
ON 24
ON 33
J
ON 75
Jumpering 14 ON/OFF 14, 24, 69

L P
Lamp 69 P/N 071-60814-01 11
Lamp Adjustment 33, 72 P/N 072-01663-14 11
Lamp Adjustment dialog 33, 72 P/N 200-81500-01 11
Lamp Alignment 69 P/N 200-94612 11
Lamp ON/OFF 14, 75 P/N 200-94613 11
Lamp Time 72 P/N 206-81601 11
Lits 12 P/N 206-83579-91 11
P/N 980-00071 11
Parallel I/F 14
M Part Names 14
Maintenance 69 Part Number 11
Mathematics 39, 44 Perform button 39, 69
Mathematics menu 39, 44 Performance Tests 69
Measure button 39, 44, 55, 57, 58, 59, 61, 63, 64, 68 Phosphorescence 3
Measurement Mode 55, 57, 58, 59, 61, 63, 64 Photometry 21
Misaligned 75 Photomultiplier
Monitor 21 adjust 69
Monochromator damages 69
2D 44 Photomultiplier 21
2D Excitation 44 Photomultiplier 69
Monochromator 3, 33 Photomultiplier 77
Monochromator 44 Place
Monochromator 77 spectrofluorophotometer 24
Monochromators 44 PMT 69
Position
fine-adjustment 33
N Position 33
Naphthaline 63 Powering
Napthalene 3 ON 24, 33, 75
Neon Lamp 44 Powering 24
Nm 33, 61, 64, 66 Powering 33
Noise report layout Powering 75
Signal 44 Principles, Application 3
Noise report layout 44 Print 39, 44
Noise Test Procedure Purchase
Signal 39 Shimadzu spectrofluorophotometer 1
Noise Test Procedure 39
Number R
Acquisitions 61
Number 61 R212-14 21
R3788-02 21
RAMAN 3, 21, 33
O Rayleigh 3
Occuring 77 Read button 33, 66
OFF Remove

86
 Index

Xenon 12, 14, 24 Start Wavelength 58

Remove 12 Stop Wavelength 58
Remove 14 Survey, Super 21
Reset button 33, 72 Switch
RF 5301PC 24 OFF 12
RF-5301 menu 33, 39, 44, 49, 55, 57, 58, 59, 61, 63, Switch 12
64, 66, 68, 69, 72 Synchro 58
RF-5301 spectrofluorophotometer 72
RF-5301PC
T
Installation 24
RF-5301PC 1 Temperature-dependent 3
RF-5301PC 24 Time Measurement 68
RF-5301PC 39 Timing Conditions 61, 64
RF-5301PC 69
RF-5301PC 72
U
RF-5301PC 77
RF-5301PC Instrument 11 UV 24
Rhodamine 3
ROM 72
RS-232C I/F 14
V
V/FS 14
S VAC 14
Views
S/N 75 3D 59
S/N Ratio Check 39, 69 Views 59
Safety Precautions 12
Salicylate 3
Self-decomposition 21
W
Set Wavelength 33, 66
OFF 12 Wavelength Accuracy Test 44
Set 12 Wavelength button 66
Set button 33, 66 Wavelength dialog 33, 66
Shimadzu 12, 33 Wavelength Increment 59, 63
Shimadzu Croporation 3
Shimadzu Deutschland GmbH 79
Shimadzu Fluorescence Analysis Course 3
X
Shimadzu RF-5301 spectrofluorophotometer 49 Xenon
Shimadzu spectrofluorophotometer 1 coarse-adjustment 33
Show Instrument Status 72 installing 24, 33
Shutter 75 remove 12
Signal removing 14
noise report layout 44 spot 77
Noise Test Procedure 39 Xenon 12
Signal 39 Xenon 14
Signal 44 Xenon 21
Signal Noise Performance Test dialog 39 Xenon 24
Signal/Noise Test 39 Xenon 33
Spectrofluorophotometer 1, 33, 72 Xenon 69
Spectrofluorophotometers 1, 3 Xenon 69
Spot Xenon 72
Xenon 77 Xenon 75
Spot 77 Xenon 77
Start button 39 Xenon Lamp 69

87