Applied Photophysics Chirascan Integrating Sphere User manual

Applied Photophysics Limited
21 Mole Business Park
Leatherhead
Surrey
KT22 7BA
United Kingdom
Tel: [phone]
Fax: [phone]
Email: [e-mail]
Applied Photophysics, Inc.
100 Cummings Center
Suite 440-C
Beverly
MA 01915
United States
Tel: [phone]
Email: [e-mail]
www.photophysics.com
User Manual
Chirascan Integrating Sphere

Document 4207Q266 V3.00 January 2018 Page 2 of 23
Chirascan Integrating Sphere User Manual
This document contains important safety information. Read this document and the
Chirascan V100 Spectrometer User Manual before attempting to install or use the
Chirascan Integrating Sphere. Failure to do so could result in deathor serious injury.

Document 4207Q266 V3.00 January 2018 Page 3 of 23
Chirascan Integrating Sphere User Manual
CONTENTS
USE OF THIS DOCUMENT ...............................................................................................................................4
HAZARD AND OTHER INDICATORS ...............................................................................................................5
ESSENTIAL SAFETY INFORMATION...............................................................................................................6
CHIRASCAN INTEGRATING SPHERE INSTALLATION AND OPERATIONAL REQUIREMENTS.................7
GLOSSARY........................................................................................................................................................8
1 INTRODUCTION.............................................................................................................................................9
2 INSTALLATION .............................................................................................................................................11
2.1 Preparing the Chirascan sample chamber .........................................................................................11
2.2 Installing the Integrating Sphere.........................................................................................................13
3 OPERATION..................................................................................................................................................16
3.1 Diffuse reflectance mode....................................................................................................................16
3.1.1 Typical use......................................................................................................................................... 16
3.1.2 Sample preparation........................................................................................................................... 16
3.1.3 Mounting the sample ........................................................................................................................ 16
3.1.4 Acquiring a spectrum ........................................................................................................................ 18
3.1.5 Example: D- and L-alanine................................................................................................................. 18
3.2 Transmission mode.............................................................................................................................20
3.2.1 Typical use......................................................................................................................................... 20
3.2.2 Sample preparation........................................................................................................................... 20
3.2.3 Mounting the sample ........................................................................................................................ 20
3.2.4 Acquiring a spectrum ........................................................................................................................ 22
4 MAINTENANCE AND STORAGE .................................................................................................................23
4.1 Maintenance .......................................................................................................................................23
4.2 Storage................................................................................................................................................23

Document 4207Q266 V3.00 January 2018 Page 4 of 23
Chirascan Integrating Sphere User Manual
USE OF THIS DOCUMENT
This document is intended to inform the operator of Applied Photophysics Integrating Sphere on its design,
installation and operation with the Chirascan V100 Spectrometer. It is assumed that the user of this document
is familiar with the operation of the Chirascan spectrometer, and with Applied Photophysics Pro-Data software.
In particular it is assumed that the user is familiar with the hazards associated with the operation of the
spectrometer, and has read the safety information contained in its User Manual.
Sample preparation for the Integrating Sphere may require the use of a hydraulic press generating a pressure
of up to 10 tons and operating under high vacuum. It is assumed that the user has read the instruction manuals
supplied with the press and vacuum pump and is aware of the hazards associated with their use.
The information in this document is subject to change without notice and should not be construed as a
commitment by Applied Photophysics, who accept no responsibility for errors that may appear herein. This
document is believed to be complete and accurate at the time of publication, and in no event shall Applied
Photophysics be held responsible for incidental or consequential damages with or arising from the use of this
document.
COPYRIGHT 2018 APPLIED PHOTOPHYSICS LIMITED. ALL RIGHTS RESERVED. THIS DOCUMENT
OR PARTS THEREOF SHALL NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN
PERMISSION OF THE PUBLISHER.
THE SOFTWARE PROVIDED WITH THE CHIRASCAN AND ITS ACCESSORIES (PRO-DATA CHIRASCAN,
PRO-DATA VIEWER, ETC.) IS THE PROPERTY OF APPLIED PHOTOPHYSICS LIMITED AND IS
SUPPLIED UNDER LICENSE. APPLIED PHOTOPHYSICS IS WILLING TO LICENSE THE SOFTWARE
ONLY UPON THE CONDITION THAT THE LICENSEE ACCEPTS ALL THE TERMS CONTAINED IN THE
LICENSE AGREEMENT. THESE INCLUDE THAT THE LICENSEE MAY NOT SELL, RENT, LOAN OR
OTHERWISE ENCUMBER OR TRANSFER LICENSED SOFTWARE IN WHOLE OR IN PART TO ANY
THIRD PARTY. FOR A FULL COPY OF THE LICENSE PLEASE CONTACT APPLIED PHOTOPHYSICS OR
SEE THE SOFTWARE INSTALLATION DISK.
Chirascan is a trademark of Applied Photophysics Limited.
All other trademarks or registered trademarks are the sole property of their respective owners

Document 4207Q266 V3.00 January 2018 Page 5 of 23
Chirascan Integrating Sphere User Manual
HAZARD AND OTHER INDICATORS
HAZARD INDICATORS USED IN THIS DOCUMENT
The sign to the left is used to indicate a hazardous situation, which, if not avoided, could result
in death or serious injury.
The sign to the left is used to indicate a hazardous situation, which, if not avoided, could result
in minor or moderate injury.
OTHER INFORMATORY INDICATORS USED IN THIS DOCUMENT
The sign to the left is used to indicate a situation which, if not avoided, could result in damage
to the instrument.
HAZARD INDICATORS USED ON THE SPECTROMETER OR ITS ACCESSORIES
Note that these hazard indicators may be either colored as below or as black and white.
The sign to the left is a general hazard indicator, indicating the presence of a hazard that is either
described by text accompanying the sign or in this User Manual.
The sign to the left is a high voltage hazard indicator.
The sign to the left is a flammable hazard indicator

Document 4207Q266 V3.00 January 2018 Page 6 of 23
Chirascan Integrating Sphere User Manual
ESSENTIAL SAFETY INFORMATION
MAKE SURE THAT YOU HAVE READ AND UNDERSTOODALL THE SAFETY INFORMATION CONTAINED
IN THIS DOCUMENT BEFORE ATTEMPTING TO INSTALL OR OPERATE THE INTEGRATNG SPHERE.
IF YOU HAVE ANY QUESTIONS REGARDING THE OPERATION OF THE ACCESSORY, PLEASE
CONTACT APPLIED PHOTOPHYSICS TECHNICAL SUPPORT AT THE ADDRESS SHOWN ON THE FIRST
PAGE OF THIS USER MANUAL
OBSERVE ALL SAFETY LABELS AND NEVER ERASE OR REMOVE SAFETY LABELS.
PERFORMANCE OF INSTALLATION, OPERATION OR MAINTENANCE PROCEDURES OTHER THAN
THOSE DESCRIBED IN THIS USER MANUAL MAY RESULT IN A HAZARDOUS SITUATION AND WILL
VOID THE MANUFACTURERS WARRANTY.
The photomultiplier tube (PMT) and Large Area Photodiode (LAAPD) detectors used with the
Chirascan Spectrometer operate at high voltages and can produce a shock leading to serious
injury or death. Do not connect or disconnect the detector from the spectrometer unless the
spectrometer is powered off.
Sample preparation for the Integrating Sphere may require the use of a hydraulic press
generating a pressure of up to 10 tons and operating under high vacuum. It is assumed that
the user has read the instruction manuals supplied with the press and vacuum pump and is
aware of the hazards associated with their use.
Disconnecting electrical components without powering off the spectrometer can cause
damage to the components. Power off the spectrometer before removing any accessories or
installing the Integrating Sphere.
The internal surface of the Integrating Sphere is coated with a highly reflective material and
the performance of the sphere will deteriorate if the surface becomes contaminated. Do not
touch the surface or the white parts of the reflective plug used when the sphere is operating in
transmission mode. Do not allow them to come into contact with other materials, and do not
attempt to clean them except by blowing gently with clean, dry air or nitrogen.
Avoid subjecting the Integrating Sphere to humid environments, or to extremes of temperature.
When not in use, the sphere should be stored in a clean, dry place.

Document 4207Q266 V3.00 January 2018 Page 7 of 23
Chirascan Integrating Sphere User Manual
CHIRASCAN INTEGRATING SPHERE INSTALLATION AND OPERATIONAL
REQUIREMENTS
Environmental and electrical requirements
The Integrating Sphere has no environmental requirements additional to those of the Chirascan spectrometer.
Bench space
The Integrating Sphere has no bench space requirements additional to those of the Chirascan spectrometer.
Nitrogen purge gas
The recommended Integrating Sphere purge gas rate is 3 liters per minute; this gives a total recommended
purge gas rate to the spectrometer of 7 liters per minute.
Circulating water
The Integrating Sphere has no temperature control, and no requirement for circulating water
Servicing
Servicing of the Integrating Sphere should only be undertaken by qualified personnel. If you are in any doubt
at all please contact the Applied Photophysics Technical Support at the address given on the first page of this
User Manual.

Document 4207Q266 V3.00 January 2018 Page 8 of 23
Chirascan Integrating Sphere User Manual
GLOSSARY
The following abbreviations may be found in this User Manual
CD circular dichroism
IR infrared
IS Integrating Sphere
UV ultraviolet
HYPERLINKS
This document contains hyperlinks between references (for example the Contents tables, or references to
Sections or Figures in the text), and sources. To follow a link, place the cursor over the reference and use
CTRL+click. Hyperlinks in the text are indicated by underlined blue font.

Document 4207Q266 V3.00 January 2018 Page 9 of 23
Chirascan Integrating Sphere User Manual
1 INTRODUCTION
The Chirascan Integrating Sphere, shown in Figure 1.1, is designed to be used with the Chirascan V100
spectrometer.
Figure 1.1: the Integrating Sphere
Integrating spheres were developed early in the twentieth century, and were originally used to measure the
total output of a light source, without reference to the original direction of the light. They are now commonly
used when measuring the UV/visible or IR absorption spectra of solid samples, and their use can be extended
to the UV/visible CD spectroscopy of solids.
Light enters the sphere through an inlet port, and the white internal surfaces of the sphere are reflective and
diffuse, so that the light becomes equally distributed within the sphere through multiple scattering reflections.
A detector is placed at an outlet port and the intensity of the light is measured. For CD spectroscopy, the
sample can be placed either at the inlet port, so that the light passes through it before entering the sphere, or
at a point on the sphere opposite the inlet port, so that light is diffusely reflected from the sample after entering
the sphere.
These two operating modes are called respectively transmission and diffuse reflectance. In both cases the
sample is prepared in powder form. For transmission mode it must then be dispersed in either potassium
bromide or potassium chloride and compressed into a disc. For diffuse reflectance mode, it remains as a free
powder, if necessary dispersed in a diluent such as barium sulphate or polytetrafluoroethylene (PTFE).
The ideal integrating sphere should have as high an inner surface area as possible, with the ratio of the port
area to the total inner surface as low as possible. The Applied Photophysics Integrating Sphere has a diameter
of 100 mm, with the port sizes the minimum for light inlet and the detector active surface, giving a total reflective
surface of over 300 cm2. The inner surfaces of the sphere are coated with proprietary diffuse reflectance
material, although small areas around the ports are coated with barium sulphate.
A schematic of the Integrating Sphere is shown in Figure 1.2.

Document 4207Q266 V3.00 January 2018 Page 10 of 23
Chirascan Integrating Sphere User Manual
Figure 1.2: schematic of the Integrating Sphere (top view).
The light enters at the light inlet port (Port A). In transmission mode it passes through the sample situated at
the light inlet port, while the diffuse reflectance port (Port B) is occupied by a reflective plug. In diffuse
reflectance mode the light inlet port is occupied by an aperture, and the sample is situated at the diffuse
reflectance port. In both cases the detector port (Port C) is occupied by the detector. The baffles are positioned
to prevent light entering the detector directly from the light inlet port or the sample, without diffuse reflectance.
Light, 
Port C
Port B
Port A
Detector
Baffle

Document 4207Q266 V3.00 January 2018 Page 11 of 23
Chirascan Integrating Sphere User Manual
2 INSTALLATION
2.1 Preparing the Chirascan sample chamber
Disconnecting electrical components without powering off the spectrometer can cause
damage to the components. Power off the spectrometer before removing any accessories or
installing the Integrating Sphere.
The photomultiplier tube (PMT) and Large Area Photodiode (LAAPD) detectors used with the
Chirascan Spectrometer operate at high voltages can produce a shock leading to serious
injury or death. Do not connect or disconnect the detector from the spectrometer unless the
spectrometer is powered off.
The Chirascan sample chamber is the lidded chamber that mounts to the right of the monochromator unit,
where it is held in place by four thumbscrews. Normally, the sample chamber is not removed from the
spectrometer, although this can easily be done for transportation or to install the Chirascan Stopped-Flow
Accessory.
There are two detector ports on the sample chamber, to the right and rear. Normally the right port is used for
CD and the rear port for fluorescence. The purge gas inlet is to the left rear upper. Before installing the
Integrating Sphere, remove any detectors from the sample chamber. The detectors are held in place by knurled
locking rings, and are released by turning the ring anticlockwise (counterclockwise); the detectors can then be
slid gently from their ports. Remove the locking ring from the CD detector port and insert the port blanking plug
provided into the port from the outside.
Remove the purge gas line that runs from the ‘Y’ connector close to the inlet to the spacer between the CD
detector and the sample holder. The line is secured by push fit connectors, and is released by pressing the
outer flange of the connector towards the connector body. Remove any installed accessories from the sample
chamber (for instructions on how to do this, see the relevant accessory User Manual). The interior of the
sample chamber should appear as in Figure 2.1. The exterior of the sample chamber should appear as in
Figure 2.2.

Document 4207Q266 V3.00 January 2018 Page 12 of 23
Chirascan Integrating Sphere User Manual
Figure 2.1: the interior of the Chirascan sample chamber prepared for the Integrating Sphere
Figure 2.2: the exterior of the Chirascan sample chamber prepared for the Integrating Sphere
Fluorescence
detector port
CD detector port
Purge gas inlet
Support
plate
Locking ring
Locating peg
Locating peg
Blanking plate
‘Y’ Connector

Document 4207Q266 V3.00 January 2018 Page 13 of 23
Chirascan Integrating Sphere User Manual
2.2 Installing the Integrating Sphere
The temperature control unit stands on a mounting plate which locates on a support plate within the sample
chamber. The unit on its mounting plate is shown in Figure 2.3 (the diffuse reflectance port is to the rear of the
Integrating Sphere when viewed from this angle).
Figure 2.3: Integrating Sphere on its mounting plate
When the sample chamber has been prepared as described in Section 2.1, the Integrating Sphere can be
installed. Remove the blue storage plugs from the sphere. Lift the sphere by the handle and place the mounting
plate over the support plate in the sample chamber, with the purge gas inlet to the rear. The plate seats down
with the holes at the front left and rear right locating over the corresponding pegs on the support plate. Make
sure that the ‘V’ ring seal is properly inserted into the light inlet port on the spectrometer. The Integrating
Sphere may need to be manipulated into position slightly to achieve this.
When the Integrating Sphere is fully in place and seated down, secure it by tightening the two captive
thumbscrews positioned at the right front and left rear of the mounting plate.
Purge gas inlet port
Mounting plate
Handle
Light inlet port
Detector port
Captive thumb screw
‘V’ ring seal
Diffuse reflectance port

Document 4207Q266 V3.00 January 2018 Page 14 of 23
Chirascan Integrating Sphere User Manual
Now reinstall the CD detector in the fluorescence port. Before installation the detector should be fitted with the
spacer and ‘V’ ring (Figure 2.4). Slide the detector into the fluorescence port until the ‘V’ ring seals against the
outer face of the Integrating Sphere detector port, and secure by tightening the detector port locking ring.
Figure 2.4: detector fitted with spacer and ‘V’ ring
Finally connect the purge gas supply to the Integrating Sphere using the tubing provided. The interior of the
sample chamber should now appear from the front as in Figure 2.5, and from the right as in Figure 2.6.
Figure 2.5: interior of the sample chamber with all connections made (front view)

Document 4207Q266 V3.00 January 2018 Page 15 of 23
Chirascan Integrating Sphere User Manual
Figure 2.6: interior of the sample chamber with all connections made (right view)

Document 4207Q266 V3.00 January 2018 Page 16 of 23
Chirascan Integrating Sphere User Manual
3 OPERATION
3.1 Diffuse reflectance mode
3.1.1 Typical use
In diffuse reflectance mode, the sample is in powder form, and is placed in the sample port directly opposite
the light inlet port. The method is easier to use and requires less sample preparation than transmission mode,
but normally uses more sample.
3.1.2 Sample preparation
The sample can be used either undiluted or with an inert reflective diluent that does not absorb strongly in the
UV, such as barium sulphate or powdered PTFE. The Integrating Sphere usually gives best results when the
sample is in powder rather than granular form, and if necessary the sample and diluent should be ground to a
fine powder with a mortar and pestle; the sample and diluent should be thoroughly mixed before use.
The diameter of the sample platen is 13 mm, giving an area of about 135 mm2that needs to be covered. A
typical sample volume, allowing for a depth of 0.5 mm, is therefore about 70 l, including the diluent. It is
important that the sample and diluent are dry, and it is good practice to dry the diluent in an oven at 110°C
before use. High quality spectroscopic grade diluent should be used whenever possible.
As usual with CD spectroscopy, the optimal absorbance is about 0.9 AU, and the sample concentration should
be adjusted with diluent to be below about 2 AU, and centered on about 0.9 AU.
3.1.3 Mounting the sample
The sample holder for diffuse reflectance is shown in Figure 3.1. There are two sections to the holder. The
inner section consists of a ring with pillar and sprung platen and two slotted holes. The outer section consists
of a knurled breechlock ring mounted on a degree marked piece containing a well for the sample. A 13 mm
diameter quartz plate is also provided; this locates at the bottom of the well in the outer section.
Figure 3.1: the diffuse reflectance sample holder outer section (left) and inner section (right)
To assemble the holder, insert the pillar of the inner section into the well on the outer with the caphead screws
passing through the slotted holes, and gripping at the finger holes, press and rotate the inner anticlockwise
(counterclockwise) so that the flanges on the slotted holes lock under the caphead screws. To disassemble
the holder, perform the procedure in reverse.
Sprung platen
Well
Pillar
Breechlock ring

Document 4207Q266 V3.00 January 2018 Page 17 of 23
Chirascan Integrating Sphere User Manual
To load the sample, disassemble the holder and insert a 13 mm quartz plate into the well in the outer section,
ensuring that it lies flat at the bottom of the well. Add the sample / diluent mix to a depth of about 0.5 mm (i.e.
about 70 l volume), and ensure that it is evenly spread on the plate. Assemble the holder as described above.
It should now appear as in Figure 3.2.
Figure 3.2: the assembled diffuse reflectance sample holder with sample in place
The holder can now be mounted on the Integrating Sphere. Fit the male breechlock connector on the sample
holder to the female breechlock connector on the Integrating Sphere diffuse reflectance port, and lock in place
by rotating anticlockwise (counterclockwise). Mount the aperture shown in Figure 3.3 in the position at the light
inlet port on the Integrating Sphere. The aperture mounts with the ‘O’ ring seal towards the sphere; ensure that
it is seated down fully in position. 
Figure 3.3: the inlet port aperture
The Integrating Sphere should now appear as in Figure 3.4 (for clarity the sphere is shown dismounted from
the spectrometer).

Document 4207Q266 V3.00 January 2018 Page 18 of 23
Chirascan Integrating Sphere User Manual
Figure 3.4: the Integrating Sphere configured for diffuse reflectance
3.1.4 Acquiring a spectrum
It is difficult to distribute the powder entirely symmetrically in the holder. To minimize the effect of uneven
sample distribution, the spectrum can be recorded at several positions and the results averaged. The sample
holder is graduated with degree markings to help with this. For example, set the holder to zero degrees (i.e.
with the unmarked point between 45° and 315° uppermost). Take a spectrum, then rotate the holder by 45° or
90° and take another spectrum, and so on to give a total of eight or four spectra, which can be averaged. This
should be done for both the baseline spectrum and the sample spectrum.
The baseline for a diffuse reflectance circular dichroism spectrum should be taken with the diluent without
sample in pace. Use the same sample size and particle size as used for the sample spectrum.
3.1.5 Example: D- and L-alanine
The CD spectra of D- and L-alanine were measured using the conditions shown in Table 3.1. Barium sulfate
(BaSO4) was used for the baseline.
Table 3.1: conditions used for the measurement of the CD spectra of D- and L-alanine
Wavelength range
280 to 200 nm
Wavelength step
1 nm
Bandwidth
4 nm
Time-per-point
1 second
Number of spectra
8 at 45rotation intervals
Sample holder
Aperture

Document 4207Q266 V3.00 January 2018 Page 19 of 23
Chirascan Integrating Sphere User Manual
The results before data manipulation are shown in Figure 3.5 and after averaging and baseline subtraction in
Figure 3.6.
Figure 3.5: CD spectra of D- and L-alanine and barium sulfate before data manipulation
Figure 3.6: CD spectra of D- and L-alanine after averaging and baseline subtraction
L-alanine
D-alanine
BaSO4
L-alanine
D-alanine

Document 4207Q266 V3.00 January 2018 Page 20 of 23
Chirascan Integrating Sphere User Manual
3.2 Transmission mode
3.2.1 Typical use
In transmission mode the sample is ground to a fine powder with a dispersant such as potassium bromide
(KBr), or potassium chloride (KCl), and pressed to a transparent disc. This method requires less sample than
for diffuse reflectance mode, but sample preparation is less straightforward and the method is not suitable for
some samples.
3.2.2 Sample preparation
Sample preparation for the Integrating Sphere in transmission mode requires the use of a
hydraulic press generating a pressure of up to 10 tons and operating under high vacuum. It is
assumed that the user has read the instruction manuals supplied with the press and vacuum
pump and is aware of the hazards associated with their use.
The use of KBr discs for solid samples is an established technique in infrared spectroscopy. Usually the details
of their preparation will be given in the user manuals supplied with the press and its accessories, and will not
be described in detail here. However, there are several points to note:
•KBr is almost always used as the dispersant for making the discs for IR spectroscopy, but KCl is often
a better option for UV spectroscopy because of its lower absorbance in the UV region. The density of
KBr is 2.74 g cm-3, and KCl is 1.98, so where the quantities for making KBr discs are specified by
weight, these should be multiplied by about 1.4 for KCl discs to give the same volume.
•Use only spectroscopic or equivalent grade dispersant.
•The dispersant must be free from water. It is good practice to dry the dispersant overnight in an open
vessel at 110°C and then store in a desiccator.
3.2.3 Mounting the sample
The sample holder for transmission is shown in Figure 3.7. There are two sections to the holder. The inner
section consists of a ring with two caphead screws. The outer section consists of a degree marked ring with
two slotted holes, containing a 13 mm recess for the sample disc.
Figure 3.7: The transmission sample holder outer section (left) and inner section (right)
To assemble the holder, insert the caphead screws on the inner through the holes on the outer, and press and
rotate the inner anticlockwise (counterclockwise) so that the flanges on the slotted holes lock under the
caphead screws. To disassemble the holder, perform the procedure in reverse.
To load the sample, disassemble the holder and place the sample disc in the 13 mm recess in the sample
holder outer section. Assemble the holder as described above. It should now appear as in Figure 3.8.
13 mm recess for the
sample disc