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FluoroMax

-4 & FluoroMax

-4P with USB v. B (23 Oct 2009)

Troubleshooting

7-9

Further assistance...

Read all software and accessory manuals before contacting the Fluorescence Service

Department. Often the manuals show the problem’s cause and a method of solution.

Technical support is available for both hardware and software troubleshooting.

Before contacting the service department, however, complete the following steps.

If this is the first time the problem has occurred,

try turning off the system and accessories.

After a cool-down period, turn everything back on.

Make sure all accessories are properly confi-

gured, and turned on as needed.

Following the instructions in Chapter 3, System

Operation, run a lamp scan and a water Raman

scan to make sure the system is properly cali-

brated.

Print the spectrum for each and note the peak intensities.

Check this chapter to see if the problem is dis-

cussed.

Try to duplicate the problem and write down the

steps required to do so.

The service engineers will try to do the same with a test system. Depending on

the problem, a service visit may not be required.

If an error dialog box appears in FluorEs-

sence™, write down the exact error displayed.

In FluorEssence™, in the FluorEssence main

window’s toolbar, choose Help:

A drop-down menu appears.

Under Help, choose About FluorEssence....

This opens the About FluorEssence window:

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FluoroMax

-4 & FluoroMax

-4P with USB v. B (23 Oct 2009)

Troubleshooting

7-10

The version of the software (both

FluorEssence™ and Origin

) is

listed here.

Click the View Sys-

tem Info button.

The Installed Components window

appears, displaying all the software

required for FluorEssence™.

Record the informa-

tion by clicking the:

• Save To File... button, which

saves the information to a file;

• Zip Info button, which com-

presses the information while sav-

ing it;

• Print Info button, which prints

out the software information.

Click the OK button

to close the Installed Components window.

Click the OK button to close the About FluorEs-

sence window.

Write down the software’s version numbers,

along with the purchase dates, model numbers,

system configuration, and serial numbers of the

instrument and its accessories.

Determine the SpectrAcq firmware version:

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FluoroMax

-4 & FluoroMax

-4P with USB v. B (23 Oct 2009)

Troubleshooting

7-11

Open the Experiment Setup window:

Click the Detectors icon

Move the mouse over the detectors’ table in the Select area.

The SpectrAcq firmware version appears in a small pop-up window:

If the problem persists or is unlisted, call the Fluorescence Service De-

partment at (732) 494-8660 × 160.

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FluoroMax

-4 & FluoroMax

-4P with USB v. B (23 Oct 2009)

Troubleshooting

7-12

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FluoroMax

-4 & FluoroMax

-4P with USB v. B (23 Oct 2009)

Producing Correction Factors

8-1

8: Producing Correction Factors

Introduction

Gratings, detectors and other spectrometer components have response characteristics

that are functions of wavelength. These characteristics are superimposed on spectra,

and may yield a potentially misleading trace. For accurate intensity comparisons, such

as those required for quantum-yield determinations, spectrometer-response characteris-

tics must be eliminated. Corrections are made for each of these potential problems by

using radiometric correction factors.

Supplied with the FluoroMax

-4 are sets of excitation and emission correction factors

designed to eliminate response characteristics. These files, xcorrect.spc and

mcorrect.spc, are included with the software and should be copied to the hard

disk. The excitation correction range is from 240–600 nm, and the correction range for

emission spectra is from 290–850 nm.

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FluoroMax

-4 & FluoroMax

-4P with USB v. B (23 Oct 2009)

Producing Correction Factors

8-2

Generating emission correction factors

Generate a new correction-factor file only when the gratings or detectors have been re-

placed with those of different specifications than the original hardware.

Required kits

Emission correction factors should be updated periodically or whenever different grat-

ings or signal detectors are installed. The correction factors can be updated either at the

user’s location, or by a representative from the Fluorescence Service Department. To

arrange for a visit and a fee estimate, call the Fluorescence Service Department. To up-

date the correction factors without a service visit, call the Fluorescence Service De-

partment for instructions and further information.

One way to generate correction factors for the instrument is to scan the spectrum of a

standard lamp. Because the actual irradiance values of the standard lamp as a function

of wavelength are known, dividing the irradiance values by the lamp spectrum results

in a set of relative correction values. These values can then be applied to the raw fluo-

rescence data. The emission correction factor file mcorrect.spc was acquired in

this manner.

To generate emission correction factors, several items are needed: a standard lamp, ap-

propriate holders, and a light-scattering assembly. HORIBA Scientific offers the F-

3026 Standard Lamp Accessory.

The F-3026 Standard Lamp Assembly is a complete correction-factor kit, which in-

cludes the following items:

• Calibrated lamp

• Irradiance values for the calibrated lamp

• Power supply

• Neutral-density filter

• Sample-compartment cover

FluoroMax

-4 & FluoroMax

-4P with USB v. B (23 Oct 2009)

Producing Correction Factors

8-3

Calculating emission correction factors

Introduction

For more information about the theory and application of radiometric correction, con-

sult Accuracy in Spectrophotometry and Luminescence Measurements, Mavrodineau,

Schultz, and Menis, NBS Spec. Publ. 378 (1973), especially p. 137, “Absolute Spec-

trofluorometry,” by W.H. Melhuish.

Irradiance values for a standard lamp, packaged with the lamp, usually are expressed in

–6

W·cm

–2

·nm. With photon-counting systems like the FluoroMax

-4 spectrofluoro-

meter, however, data usually are collected in units of photons·s

–1

·cm

–2

·nm. To convert

the units, multiply each irradiance value by the wavelength at which it is valid. (The da-

ta will still be off by a factor of c, but normalizing the correction factors compensates

for this.) Such a mathematical procedure can be done in a spreadsheet program.

Load the irradiance values.

Enter the irradiance values into a spreadsheet.

Load the irradiance file supplied by HORIBA Scientific by opening the

FluorEssence™ software, choose File in the toolbar, choose Import, and

then choose SPC file.

Save the file as IRR.

Now you have the two files: IRR and stdlamp2. These files are re-

quired to calculate the emission correction factors for the FluoroMax

-4.

FluoroMax

-4 & FluoroMax

-4P with USB v. B (23 Oct 2009)

Producing Correction Factors

8-4

Note:

Naming the file

mcorrect overwrites

the mcorrect file sup-

plied with the software.

Calculate the correction factors.

Using the Simple Math Tool window

under the Analysis/HJY Tools menu,

divide IRR by stdlamp2, and name

the resulting file mcorrect:

stdlamp2

IRR

mcorrect =

Normalize the new mcorrect file.

Using the HJY_normalize window under the Analysis/HJY Tools

menu, divide the mcorrect file by this minimum signal intensity, a

constant.

Save this new file as mcorrect.

(That is, overwrite the existing mcorrect file.) This normalizes the

correction factor file so that the minimum intensity of mcorrect will

be 1 count s

–1

. mcorrect contains the emission correction factors for

the system. The correction-factor file should look similar to this:

Once the emission correction factors are found, determination of the excitation correc-

tion factors may be necessary. The following procedures describe how to obtain excita-

tion correction factors using the photomultiplier and the photodiode. Follow the proce-

dure that applies to your configuration.

FluoroMax

-4 & FluoroMax

-4P with USB v. B (23 Oct 2009)

Producing Correction Factors

8-5

Note:

The source/integrating-sphere module may arrive pre-

mounted atop the special gap-bed.

bed.

Calculating emission correction factors

Set up the F-3026 accessory.

Attach the correction-

factor light-source to its

gap-bed with the brass

thumbscrews.

Use this orientation. Be sure the power

cable doesn’t block optical apertures.

Plug the power cable

into the front of the gap-

bed.

Remove the standard

sample-compartment

gap-bed, and insert the

correction-factor kit

gap-bed into the sam-

ple compartment.

Fix the gap-bed firmly to the sample compartment with screws through the front

panel.

Run the scan.

Start FluorEssence™

and initialize the instru-

ment.

Turn the xenon lamp OFF, and the lamp

for the correction-factor kit ON.

FluoroMax

-4 & FluoroMax

-4P with USB v. B (23 Oct 2009)

Producing Correction Factors

8-6

Note:

We recommend keeping a log of the amount of time the

source is on. Calibration is necessary after every 50 h of use.

Put the raised lid on the

sample compartment.

Make sure it fits securely, and there is no light

leak into the sample compartment.

Collect an emission scan using the following

scan parameters:

Range: 290–850 nm; increment: 5 nm; integration time: 1 s

Warning:

Wear appropriate eye-

protection against UV, visible,

and IR when the tungsten-

halogen lamp is on and the sam-

ple-compartment is uncovered.

Caution:

Do not op-

erate the accessory

without the lid com-

pletely covering the

sample compart-

ment.

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