Horiba Scientific Quanta-phi F-3029 User manual

Quanta-φrev. C (23 Apr 2010)
i
Quanta-φ
F-3029 Integrating Sphere
Operation Manual rev. C
http://www.HORIBA.com/scientific

Quanta-φrev. C (23 Apr 2010)
ii
Copyright © 2003–2010 by HORIBA Jobin Yvon Inc.
All rights reserved. No part of this work may be reproduced, stored, in a retrieval sys-
tem, or transmitted in any form by any means, including electronic or mechanical, pho-
tocopying and recording, without prior written permission from HORIBA Jobin Yvon
Inc. Requests for permission should be requested in writing.
Information in this manual is subject to change without notice, and does not represent a
commitment on the part of the vendor.
Spectralon®is a registered trademark of Labsphere, Inc. Excel®is a registered trade-
mark of Microsoft Corporation. Freon®is a registered trademark of E.I. du Pont de
Nemours and Company.
April 2010
Part Number J81089

Quanta-φrev. C (23 Apr 2010)
iii
Table of Contents
0: Introduction ................................................................................................. 0-1
About the Quanta-φintegrating sphere..................................................................................................0-1
Disclaimer............................................................................................................................................... 0-2
Safety summary...................................................................................................................................... 0-4
Risks of ultraviolet exposure...................................................................................................................0-5
Additional risks of xenon lamps.............................................................................................................. 0-7
1: Theory of Operation........................................................................................ 1-1
Introduction............................................................................................................................................. 1-1
Spherical enclosures and radiance ........................................................................................................ 1-1
Time decay of signal...............................................................................................................................1-2
Coating of an integrating sphere ............................................................................................................ 1-2
Photoluminescence quantum yield.........................................................................................................1-3
Calculation and evaluation of chromaticity........................................................................................... 1-11
Flowchart of method for data-acquisition..............................................................................................1-14
Additional references............................................................................................................................1-15
2: Requirements & Unpacking .............................................................................. 2-1
Environmental requirements...................................................................................................................2-1
Software requirements............................................................................................................................2-1
Unpacking............................................................................................................................................... 2-2
3: Installation & Use.......................................................................................... 3-1
Method.................................................................................................................................................... 3-1
Continue here if your detector is a CCD.............................................................................................. 3-10
Using a photomultiplier tube as the detector....................................................................................... 3-25
4: Maintenance ................................................................................................ 4-1
Handling..................................................................................................................................................4-1
Cleaning..................................................................................................................................................4-1
5: Generating Correction Files .............................................................................. 5-1
Introduction............................................................................................................................................. 5-1
Flowchart for generating Quanta-φ correction-factor files...................................................................... 5-2
Method for generating Quanta-φ correction-factor files .........................................................................5-3
6: Troubleshooting ............................................................................................ 6-1
Troubleshooting chart.............................................................................................................................6-1
Further assistance... ............................................................................................................................... 6-4
7: Technical Specifications.................................................................................. 7-1
Hardware ................................................................................................................................................ 7-1
Software................................................................................................................................................. 7-2
8: Index ......................................................................................................... 8-1

Quanta-φrev. C (23 Apr 2010)
iv

Quanta-φrev. C (23 Apr 2010) Introduction
0-1
Note: Keep this and the other reference manuals near the system.
0: Introduction
About the Quanta-φintegrating sphere
This manual explains the theoretical and practical issues of operating and maintaining
the Quanta-integrating sphere. The Quanta-is used with the Fluorolog®-3, Fluoro-
Max®-3 and -4, and Fluorolog®-Tau-3 spectrofluorometers to study fluorescence from
solid, powder, thin-film and liquid samples. The main purpose of the Quanta-is the
measurement of photoluminescence quantum yields of such materials.
The Quanta-integrating sphere accessory is external to the spectrofluorometer. Light
from the sample compartment is directed into the sphere via a fiber-optic cable and the
F-3000 Fiber-Optic Adapter, and returned to the sample compartment (and then to the
emission monochromator) via a second fiber-optic cable and the F-3000.

Quanta-φrev. C (23 Apr 2010) Introduction
0-2
Disclaimer
By setting up or starting to use any HORIBA Jobin Yvon product, you are accepting
the following terms:
You are responsible for understanding the information contained in this document. You
should not rely on this information as absolute or all-encompassing; there may be local
issues (in your environment) not addressed in this document that you may need to ad-
dress, and there may be issues or procedures discussed that may not apply to your situa-
tion.
If you do not follow the instructions or procedures contained in this document, you are
responsible for yourself and your actions and all resulting consequences. If you rely on
the information contained in this document, you are responsible for:
Adhering to safety procedures
Following all precautions
Referring to additional safety documentation, such as Material Safety Data Sheets
(MSDS), when advised
As a condition of purchase, you agree to use safe operating procedures in the use of all
products supplied by HORIBA Jobin Yvon, including those specified in the MSDS
provided with any chemicals and all warning and cautionary notices, and to use all
safety devices and guards when operating equipment. You agree to indemnify and hold
HORIBA Jobin Yvon harmless from any liability or obligation arising from your use or
misuse of any such products, including, without limitation, to persons injured directly
or indirectly in connection with your use or operation of the products. The foregoing
indemnification shall in no event be deemed to have expanded HORIBA Jobin Yvon’s
liability for the products.
HORIBA Jobin Yvon products are not intended for any general cosmetic, drug, food, or
household application, but may be used for analytical measurements or research in
these fields. A condition of HORIBA Jobin Yvon’s acceptance of a purchase order is
that only qualified individuals, trained and familiar with procedures suitable for the
products ordered, will handle them. Training and maintenance procedures may be pur-
chased from HORIBA Jobin Yvon at an additional cost. HORIBA Jobin Yvon cannot
be held responsible for actions your employer or contractor may take without proper
training.
Due to HORIBA Jobin Yvon’s efforts to continuously improve our products, all speci-
fications, dimensions, internal workings, and operating procedures are subject to
change without notice. All specifications and measurements are approximate, based on
a standard configuration; results may vary with the application and environment. Any
software manufactured by HORIBA Jobin Yvon is also under constant development
and subject to change without notice.
Any warranties and remedies with respect to our products are limited to those provided
in writing as to a particular product. In no event shall HORIBA Jobin Yvon be held lia-

Quanta-φrev. C (23 Apr 2010) Introduction
0-3
ble for any special, incidental, indirect or consequential damages of any kind, or any
damages whatsoever resulting from loss of use, loss of data, or loss of profits, arising
out of or in connection with our products or the use or possession thereof. HORIBA Jo-
bin Yvon is also in no event liable for damages on any theory of liability arising out of,
or in connection with, the use or performance of our hardware or software, regardless
of whether you have been advised of the possibility of damage.

Quanta-φrev. C (23 Apr 2010) Introduction
0-4
Safety summary
The following general safety precautions must be observed during all phases of opera-
tion of this instrument. Failure to comply with these precautions or with specific warn-
ings elsewhere in this manual violates safety standards of design, manufacture and in-
tended use of instrument. HORIBA Jobin Yvon assumes no liability for the customer’s
failure to comply with these requirements. Certain symbols are used throughout the text
for special conditions when operating the instruments:
A WARNING notice denotes a hazard. It calls
attention to an operating procedure, practice, or
similar that, if incorrectly performed or adhered
to, could result in personal injury or death. Do
not proceed beyond a WARNING notice until
the indicated conditions are fully understood and
met. HORIBA Jobin Yvon Inc. is not responsi-
ble for damage arising out of improper use of the
equipment.
A CAUTION notice denotes a hazard. It calls
attention to an operating procedure, practice, or
similar that, if incorrectly performed or adhered
to, could result in damage to the product. Do not
proceed beyond a CAUTION notice until the
indicated conditions are fully understood and
met. HORIBA Jobin Yvon Inc. is not responsi-
ble for damage arising out of improper use of the
equipment.
Intense ultraviolet, visible, or infrared light!
Wear light-protective goggles, full-face shield,
skin-protection clothing, and light-blocking
gloves. Do not stare into light.
Wear protective gloves.
General information is given concerning opera-
tion of the equipment.
Note:
Caution:
Caution:
Warning:

Quanta-φrev. C (23 Apr 2010) Introduction
0-5
Caution: This instrument is used in conjunction with ultra-
violet light. Exposure to these radiations, even reflected
or diffused, can result in serious, and sometimes irre-
versible, eye and skin injuries.
Overexposure to ultraviolet rays threatens human health
by causing:
Immediate painful sunburn
Skin cancer
Eye damage
Immune-system suppression
Premature aging
Risks of ultraviolet exposure
Do not aim the UV light at anyone.
Do not look directly into the light.
Always wear protective goggles, full-face shield and skin protection clothing and
gloves when using the light source.
Light is subdivided into visible light, ranging from 400 nm (violet) to 700 nm (red);
longer infrared, ―above red‖ or > 700 nm, also called heat; and shorter ultraviolet
radiation (UVR), ―below violet‖ or < 400 nm. UVR is further subdivided into UV-
A or near-UV (320–400 nm), also called black (invisible) light; UV-B or mid-UV
(290–320 nm), which is more skin penetrating; and UV-C or far-UV (< 290 nm).
Health effects of exposure to UV light are familiar to anyone who has had sunburn.
However, the UV light level around some UV equipment greatly exceeds the level
found in nature. Acute (short-term) effects include redness or ulceration of the skin.
At high levels of exposure, these burns can be serious. For chronic exposures, there
is also a cumulative risk of harm. This risk depends upon the amount of exposure
during your lifetime. The long-term risks for large cumulative exposure include
premature aging of the skin, wrinkles and, most seriously, skin cancer and cataract.
Damage to vision is likely following exposure to high-intensity UV radiation. In
adults, more than 99% of UV radiation is absorbed by the anterior structures of the
eye. UVR can contribute to the development of age-related cataract, pterygium,
photodermatitis, and cancer of the skin around the eye. It may also contribute to
age-related macular degeneration. Like the skin, the covering of the eye or the cor-
nea, is epithelial tissue. The danger to the eye is enhanced by the fact that light can
enter from all angles around the eye and not only in the direction of vision. This is
especially true while working in a dark environment, as the pupil is wide open. The
lens can also be damaged, but because the cornea acts as a filter, the chances are re-

Quanta-φrev. C (23 Apr 2010) Introduction
0-6
duced. This should not lessen the concern over lens damage however, because cata-
racts are the direct result of lens damage.
Burns to the eyes are usually more painful and serious than a burn to the skin. Make
sure your eye protection is appropriate for this work. NORMAL EYEGLASSES OR
CONTACTS OFFER VERY LIMITED PROTECTION!
Training
For the use of UV sources, new users must be trained by another member of the labora-
tory who, in the opinion of the member of staff in charge of the department, is suffi-
ciently competent to give instruction on the correct procedure. Newly trained users
should be overseen for some time by a competent person.
Caution: UV exposures are not immediately felt. The us-
er may not realize the hazard until it is too late and the
damage is done.

Quanta-φrev. C (23 Apr 2010) Introduction
0-7
Warning: Xenon lamps
are dangerous. Please
read the following pre-
cautions.
Additional risks of xenon lamps
Among the dangers associated with xenon lamps
are:
Burns caused by contact with a hot xenon lamp.
Fire ignited by hot xenon lamp.
Interaction of other nearby chemicals with intense ultraviolet, visible, or infrared
radiation.
Damage caused to apparatus placed close to the xenon lamp.
Explosion or mechanical failure of the xenon lamp.
Visible radiation
Any very bright visible light source will cause a human aversion response: we either
blink or turn our head away. Although we may see a retinal afterimage (which can last
for several minutes), the aversion response time (about 0.25 seconds) normally protects
our vision. This aversion response should be trusted and obeyed. NEVER STARE AT
ANY BRIGHT LIGHT-SOURCE FOR AN EXTENDED PERIOD. Overriding the
aversion response by forcing yourself to look at a bright light-source may result in per-
manent injury to the retina. This type of injury can occur during a single prolonged ex-
posure. Excessive exposure to visible light can result in skin and eye damage.
Visible light sources that are not bright enough to cause retinal burns are not necessari-
ly safe to view for an extended period. In fact, any sufficiently bright visible light
source viewed for an extended period will eventually cause degradation of both night
and color vision. Appropriate protective filters are needed for any light source that
causes viewing discomfort when viewed for an extended period of time. For these rea-
sons, prolonged viewing of bright light sources should be limited by the use of appro-
priate filters.
The blue-light wavelengths (400–500 nm) present a unique hazard to the retina by
causing photochemical effects similar to those found in UV-radiation exposure.
Infrared radiation
Infrared (or heat) radiation is defined as having a wavelength between 780 nm and 1
mm. Specific biological effectiveness ―bands‖ have been defined by the CIE (Commis-
sion Internationale de l’Eclairage or International Commission on Illumination) as fol-
lows:
IR-A (near IR) (780–1400 nm)
IR-B (mid IR) (1400–3000 nm)
IR-C (far IR) (3000 nm–1 mm)

Quanta-φrev. C (23 Apr 2010) Introduction
0-8
The skin and eyes absorb infrared radiation (IR) as heat. Workers normally notice ex-
cessive exposure through heat sensation and pain. Infrared radiation in the IR-A that
enters the human eye will reach (and can be focused upon) the sensitive cells of the re-
tina. For high irradiance sources in the IR-A, the retina is the part of the eye that is at
risk. For sources in the IR-B and IR-C, both the skin and the cornea may be at risk from
―flash burns.‖ In addition, the heat deposited in the cornea may be conducted to the lens
of the eye. This heating of the lens is believed to be the cause of so called ―glassblow-
ers’ ‖ cataracts because the heat transfer may cause clouding of the lens.
Retinal IR Hazards (780 to 1400 nm): possible retinal lesions from acute high irra-
diance exposures to small dimension sources.
Lens IR Hazards (1400 to 1900 nm): possible cataract induction from chronic lower
irradiance exposures.
Corneal IR Hazards (1900 nm to 1 mm): possible flashburns from acute high irra-
diance exposures.
Who is likely to be injured? The user and anyone exposed to the radiation or xenon
lamp shards as a result of faulty procedures. Injuries may be slight to severe.

Quanta-φrev. C (23 Apr 2010) Theory of Operation
1-1
1: Theory of Operation
Introduction
An ideal integrating sphere is designed to integrate light for collection over all emission
angles from the sample. No integrating sphere is ideal, and so there are various approx-
imations required.
Spherical enclosures and radiance
When light hits a diffuse surface, such as the interior of an integrating sphere, radiation
exchange occurs. Imagine an area dA1that reflects light to another area dA2. We can
write the exchange factor dF, the fraction of energy leaving dA1, traveling a distance S,
and going to dA2.
2
221 coscos dA
Sπ
θθ
dF 
(1)
For a spherical enclosure, the area dA1actually exchanges light with an area A2of defi-
nite size. With geometrical considerations, we can integrate the differentials to get
sphere
AA
rπ
A
F2
2
2
4
(2)
Equation (2) tells us that the fraction of light Fthat A2receives is just A2’s fraction of
the sphere’s total surface area. The parameter Fis independent of the viewing angle, is
the same when measured from anywhere within the integrating sphere, and is propor-
tional to the area of the sphere.
Radiance, L, is the flux density of light emanating per unit solid angle. For a diffuse
surface receiving an incident flux Φi,
Aπ
ρ
Li
Φ

(3)
where Ais the area under illumination, ρis the reflectance, and πis the solid angle from
the surface. The main issue here is the reflectance for an integrating sphere. The reflec-
tance can be rewritten as a simplification of a power series from multiple reflections
within the sphere. Thus we rewrite the radiance as
M
Aπ
Li Φ
(4)

Quanta-φrev. C (23 Apr 2010) Theory of Operation
1-2
where Mis a factor called the sphere multiplier. The sphere multiplier takes into ac-
count the total fractional area fthat the entrance and exit ports occupy (and thus reduce
the reflectance), plus multiple reflections:
 
fρ
ρ
M 11
(5)
For a typical integrating sphere whose ρ~ 0.95 and f~ 0.03, Mis between 10 and 30.
Time decay of signal
An incoming signal (such as a rapid fluorescence-decay) can be stretched temporally
because of the multiple diffuse reflections inside an integrating sphere. This can be im-
portant for fluorescence lifetime determinations. The impulse response of an integrating
sphere takes the form
τ
t
e
where τis the time constant of the integrating sphere, and is
ρc
d
τsphere
ln
1
3
2
(6)
Equation (6) considers also the diameter of the integrating sphere, dsphere, the average
reflectance,
ρ
, and the speed of light, c. A typical τmight range from several ns to sev-
eral dozen ns.
Coating of an integrating sphere
The interior reflective coating of an integrating sphere affects its overall performance.
The interior of the HORIBA Scientific integrating sphere is made from a proprietary
material known as Spectralon®, which has a very wide, flat reflectance of over 95%
from 250 nm to 2.5 μm (see graph below). Thus this integrating sphere is useful
throughout the
spectrofluorome-
ter’s scanning
range, from the
UV through the
near-IR.

Quanta-φrev. C (23 Apr 2010) Theory of Operation
1-3
Photoluminescence quantum yield
An important use of the integrating sphere in conjunction with your spectrofluorometer
is the determination of a sample’s photoluminescence quantum yield. The photolumi-
nescence quantum yield for a particular molecular species is determined by the first-
order rate processes that compete for the excitation energy. The three main processes
can be defined with first-order rate constants, such as kF, the rate constant of fluores-
cence decay (in units of s–1), kNR, the rate constant of nonradiative decay, and kT, the
rate constant of photochemical energy transfer. Using these three rate constants, the
fluorescence quantum yield φis defined as
  
 
The fluorescence quantum yield is directly related to the fluorescence lifetime τ:
  
 
Any increase in the nonradiative decay processes (kNR) related to events such as
quenching and thermal dissipation or energy-transfer (kT) processes—including Förster
Resonance Energy Transfer (FRET)—will decrease both the quantum yield φand fluo-
rescence lifetime τ. Likewise any decrease in kNR or kTwill increase φand τ.
The multiple randomized, diffused reflections in the integrating sphere eliminate
any isotropic (directional) features of the sample emission from the excited molecular
species. Hence, measurement of polarization is not possible within an integrating
sphere.
Following is a brief description of the theory and recommended procedure for measur-
ing quantum yield using the Quanta-φintegrating sphere.
General approach
For a general approach, not requiring an integrating sphere, see Joseph R. Lakowicz’s
book, Principles of Fluorescence Spectroscopy.1
Theory
The sample is placed in the integrating sphere, and excited with a monochromatic
source of wavelength λ. The film absorbance, A, is
b
cb LLL
A 
(7)
1Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed., New York, Springer, 2006, pp.
8–10, 54–55.

Quanta-φrev. C (23 Apr 2010) Theory of Operation
1-4
Caution: Always attenuate or adjust the signal intensities to prevent satu-
ration of the detector. Saturated signals can seriously deteriorate the
measurement precision and accuracy, and possibly damage the detec-
tion electronics.
where Lbis the integrated excitation profile when the sample is diffusely illuminated by
the integrating sphere’s surface; and Lcis the integrated excitation profile when the
sample is directly excited by the incident beam.
The quantum yield, υ, is, by definition, photons emitted to photons absorbed:
 
1
cb
ca
a a c
E A E EE
L A L L

   


(8)
where Ecis the integrated luminescence of the film caused by direct excitation, and Eb
is the integrated luminescence of the film caused by indirect illumination from the
sphere. The term Lais the integrated excitation profile from an empty integrating sphere
(without the sample, only a blank). Here Eais the integrated luminescence from an
empty integrating sphere (only a blank).
For integration of function Lover the wavelength, λ, the integration limits can be from
10 nm below the excitation wavelength to 10 nm above the excitation wavelength.
The spectra recorded must be background corrected, using a blank sample holder, and
corrected for wavelength dependence of the spectrofluorometer, sampling optics, and
integrating sphere.
Example
An example is given for rhodamine 101 (see right) in anhydrous ethanol (with a peak
absorbance value of OD = 0.063 at 563 nm) excited
using 545-nm light (OD = 0.035) using 1 nm bandpass
for both excitation and emission.
Using a CCD detector, the scattered 545 nm excitation spectra were recorded for both
the blank cuvette containing only ethanol (red, La), and the sample cuvette (black, Lc).
Integration of the instrument-corrected incident beam’s signal intensities Iwas per-
formed from 535 to 553 nm for 0.5 s (20 summed accumulations).

Quanta-φrev. C (23 Apr 2010) Theory of Operation
1-5
 
553 6
535
2.27954 10
a
L I d    

(9)
 
553 6
535
1.9856 10
c
L I d    

(10)
Next, the sample and blank’s fluorescence spectral region intensities were measured us-
ing a 4-second integration time under otherwise the same instrument conditions as the
Laand Lc. Integration of the emission-signal region Ea(blank) and Ec(sample) was
from 553 to 800 nm.
 
800 5
553
3.29376 10
a
E I d    

(11)
 
800 6
553
2.51648 10
c
E I d    

(12)

Quanta-φrev. C (23 Apr 2010) Theory of Operation
1-6
Because the integration time constant used to collect Eaand Ecwas 8 times longer than
for Laand Lc, we need to divide the difference between Eaand Ecby 8 to calculate the
quantum yield, υ, below using the four parameters Ec, Ea, La, and Lc,.
65
66
[( )/8]
100%
[(2.51648 10 3.29376 10 )/8]
100% 2.27954 10 1.9856 10
93.01%
ca
ac
EE
LL



  
   

This υis slightly lower compared to some literature reports for rhodamine 101 in aci-
difed ethanol (~ 96–99%) primarily due to the anhydrous ethanol used as the solvent.
Error-propagation analysis
In addition to the calculation of υ, the Quanta-φsoftware performs an error-propagation
analysis to help evaluate when signal levels are not properly balanced. This may occur
with dilute concentrations of the sample, or when samples exhibit very low quantum ef-
ficiency. The error propagation is based on the Poissonian statistics of photon-counting,
where the standard deviation σis equal to the square-root of the photon-count value,
i.e.,
 
The error propagation is performed in a stepwise manner.
First, find the standard deviations for Ea, Ec, La, and Lc, respectively as ,
,  and .
Second, evaluate the standard deviation of the numerator num = [(Ec–Ea)/8] of the υ
equation (considering the constant factor of 8 in the integration time) as
 󰇡
󰇢󰇡 󰇢
Then evaluate the standard deviation of the denominator den = (La–Lc), as
 
Propagation of the respective relative (σrel) and absolute errors (σabs) of υis as follows:
 󰇡
󰇢󰇡 󰇢
 