Vertical-type chiroptical spectrophotometer (I): Instrumentation and application to diffuse reflectance circular dichroism measurement

Abstract

We have designed and built a novel universal chiroptical spectrophotometer (UCS-2: J-800KCMF), which can carry out in situ chirality measurement of solid samples without any pretreatment, in the UV-vis region and with high relative efficiency. The instrument was designed to carry out transmittance and diffuse reflectance (DR) circular dichroism (CD) measurements simultaneously, thus housing two photomultipliers. It has a unique feature that light impinges on samples vertically so that loose powders can be measured by placing them on a flat sample holder in an integrating sphere. As is our first universal chiroptical spectrophotometer, UCS-1, two lock-in amplifiers are installed to remove artifact signals arising from macroscopic anisotropies which are unique to solid samples. High performance was achieved by theoretically analyzing and experimentally proven the effect of the photoelastic modulator position on the CD base line shifts, and by selecting high-quality optical and electric components. Measurement of microcrystallines of both enantiomers of ammonium camphorsulfonate by the DRCD mode gave reasonable results.

Full text

Vertical-type chiroptical spectrophotometer „I…: Instrumentation and
application to diffuse reflectance circular dichroism measurement
Takunori Harada,1Hiroshi Hayakawa,2and Reiko Kuroda1,3
1Japan Science and Technology Agency, ERATO-SORST Kuroda Chiromorphology Team, 4-7-6 Komaba,
Meguro-ku, Tokyo 153-0041, Japan
2JASCO Corporation, 2967-5, Ishikawa-cho, Hachioji-shi, Tokyo 192-8537, Japan
3Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba,
Meguro-ku, Tokyo 153-8902, Japan
共Received 6 May 2008; accepted 10 June 2008; published online 2 July 2008兲
We have designed and built a novel universal chiroptical spectrophotometer 共UCS-2: J-800KCMF兲,
which can carry out in situ chirality measurement of solid samples without any pretreatment, in the
UV-vis region and with high relative efficiency. The instrument was designed to carry out
transmittance and diffuse reflectance 共DR兲circular dichroism 共CD兲measurements simultaneously,
thus housing two photomultipliers. It has a unique feature that light impinges on samples vertically
so that loose powders can be measured by placing them on a flat sample holder in an integrating
sphere. As is our first universal chiroptical spectrophotometer, UCS-1, two lock-in amplifiers are
installed to remove artifact signals arising from macroscopic anisotropies which are unique to solid
samples. High performance was achieved by theoretically analyzing and experimentally proven the
effect of the photoelastic modulator position on the CD base line shifts, and by selecting
high-quality optical and electric components. Measurement of microcrystallines of both enantiomers
of ammonium camphorsulfonate by the DRCD mode gave reasonable results. © 2008 American
Institute of Physics. 关DOI: 10.1063/1.2952674兴
I. INTRODUCTION
Solid-state chiral spectroscopy offers unique chemistry,
which cannot be obtained by conventional solution spectros-
copy. However, it inevitably suffers from artifact signals aris-
ing from macroscopic anisotropies of the solid samples. To
obtain true circular dichroism 共CD兲and circular birefrin-
gence 共CB兲共optical rotatory dispersion兲in the solid state, we
had already designed and constructed a universal chiroptical
spectrophotometer1共UCS-1 =J-800KCM兲which can mea-
sure CD of all phases including solid state. The instrument
has novel electrical and optical systems though based on a
commercially available CD spectrophotometer, JASCO
J-820. It equipped with two lock-in amplifiers 共50 and
100 kHz兲and an analyzer, and is capable of measuring all
polarization phenomena, that is, linear birefringence 共LB兲,
linear dichroism 共LD兲, CB and CD, simultaneously. Using
this instrument, we have measured several solid samples
such as single crystals 关
␣
-Ni共H2O兲6·SO4,1–3NaClO3,4and
CaF2
4兲, highly stretched films of a polymer 共polyvinyl
alcohol1,5兲, biopolymer 共bovine serum albumin6兲, porphyrin
derivatives,7,8chiral supramolecular fluorophor9and metal
complexes,10–13 and obtained physicochemical information
which was not obtainable on commercially available CD
spectrophotometers.
UCS-1 is highly useful for measuring true CD spectra of
single crystals, however, we cannot always obtain single
crystals big enough for the UCS-1 measurement. Further,
cogrinding of two kinds of crystals produces microcrystal-
lines of a new phase, which is sometime different from crys-
tals obtained from solution crystallization.14 In these cases,
CD measurements must be carried out on the microcrystal-
lines using the KBr disk method or the nujol-mull method.
However, the methods often suffer from interactions of
samples with the matrix15 or dissolution of samples in nujol.
We ourselves have noticed the collapse of crystal lattices by
simple grinding of microcrystallines.16 Thus, it is ideal to
measure CD spectra of microcrystallines in situ, and for this
purpose DR spectroscopy is most suited. It is applicable to
all crystallines irrespective of the size as well as noncrystal-
line materials.
The diffuse reflectance 共DR兲CD spectrophotometer mea-
sures light diffused or reflected from samples through pref-
erential absorption of either left or right-handed incident cir-
cularly polarized light. It was first developed by Bilotti et al.
in 2002 and used for microcrystalline samples.17 However,
due to the arrangement of the optical trains in the instrument
and the low grade of the optical elements, the CD measure-
ment was limited to the visible wavelength range and the
sensitivity was low.
To achieve high-quality in situ chirality measurements,
i.e., measurements over a wide wavelength range with
higher sensitivity, we have designed and built UCS-2
共J-800KCMF兲. The novel instrument is equipped with an in-
tegrating sphere and a right angle prism which makes it pos-
sible to set a sample on a horizontal stage. By investigating
the photoelastic modulator 共PEM兲position and selecting
high-quality optical elements, we could overcome the defect
of the prototype DRCD spectrophotometer and succeeded in
developing a new DRCD spectrophotometer which can mea-
sure signals over UV-vis regions. In fact, this new instrument
REVIEW OF SCIENTIFIC INSTRUMENTS 79, 073103 共2008兲
0034-6748/2008/79共7兲/073103/6/$23.00 © 2008 American Institute of Physics79, 073103-1
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was designed to measure not only DRCD but transmittance
CD, simultaneously. In this paper, we shall report instrumen-
tation of the novel vertical-type chiroptical spectrophotom-
eter UCS-2 and application of its DRCD mode to powdered
samples. Application of transmittance CD mode to real-time
phase-transition measurements of biological samples will be
reported elsewhere.
II. EXPERIMENTAL SECTION
A. Outline of the new instrument
A block diagram of the vertical-type universal chiropti-
cal spectrophotometer 共UCS-2兲is shown in Fig. 1.Itwas
designed to measure DRCD and transmittance CD, simulta-
neously, and hence possesses two photomultiplier tubes
共PMTs兲. One of the characteristics of UCS-2 is that the inci-
dent light beam proceeds to samples vertically. Thus, the
new instrument is ideal for measuring DRCD of loose pow-
ders without any pretreatment, and transmittance CD of soft
materials such as gels that are affected by the gravity.
The light from a Xe lamp passes through a double prism
monochrometor. It then passes through linear polarizer, pile
of plates polarizer 共P兲tilted at Brewster angle with respect to
the horizontal plane, and strikes a right angle prism. The
right angle prism deviates a beam normal to the incident face
by 90°. Notice that the top and bottom of the coming out
light image have been exchanged while the right and left
sides have not. Upon emerging from the right angle prism,
the light traverses the PEM which consists of a transparent
quartz block, driven to oscillate at a resonant frequency of
50 kHz by a piezoelectric transducer. It is fixed at 45° with
respect to the horizontal plane, which imparts a relative
phase to the orthogonal components of the transmitted light.
The emitted light from PEM strikes a sample. In the case of
transmission mode, the light from sample impinges on a
PMT directly or after passing through the analyzer 共A兲,
angled in the optical axis at 45° with respect to the horizontal
plane. The analyzer can be inserted or taken out from the
light path to measure CD and LD, and this is to remove
artifact signals arising from macroscopic anisotropies of the
sample, as UCS-1.
In the case of DRCD mode, the diffused and reflected
light from samples is integrated by the integral sphere and
finally falls in a detector. The signal from the photomultiplier
is a photocurrent, which is converted into a voltage by a
transimpedance preamplifier. The resultant voltage is read
out by a lock-in amplifier. UCS-2 houses two lock-in ampli-
fiers and their reference frequency is set to the frequency of
the PEM 关
␻
共=50 kHz兲and 2
␻
kHz兲. The PEM setting was
␦
m
0=2.4 rad, that is, J0共
␦
m
0兲=0, J1共
␦
m
0兲=0.52 and J2共
␦
m
0兲
=0.43, in order to measure the
␻
and 2
␻
kHz signals
simultaneously.1Here
␦
is the periodic phase difference in-
duced between the xand yaxes of PEM operating at fre-
quency
␻
/2
␲
Hz, and
␦
m
0is the peak modulator retardation.
J0共
␦
m
0兲,J1共
␦
m
0兲, and J2共
␦
m
0兲are the Bessel function of zeroth,
first, and second order, respectively.
B. Materials
1. Optical and electronic components
The optical and electronic components used in UCS-2
共J-800KCMF兲are as follows. The light source: a 450 W Xe
lamp for JASCO J-820 CD spectrophotometer; the mono-
chromator and the polarizer: a double prism monochromator
and pile of plate for JASCO J-820 CD spectrophotometer;
the right angle prism: strain-free fused quartz glass RPSQ-
25-10H 共Sigma Koki Co., Ltd.兲the PEM 共
␣
=0.75° at
250 nm兲: a PEM for JASCO J-820 CD spectrometer; inte-
grating sphere: integrating sphere 共
␸
120 mm兲for JASCO
共coated with a white standard, barium sulfate, showing a
high DR兲; the detectors: a Hamamatsu R-376 head-on type
PMT; the lamp and photomultiplier power supply: JASCO;
the lock-in amplifiers 共50 and 100 kHz兲: a SRS SR830 and a
JASCO’s lock-in amplifiers; analyzer 共Glan–Taylor prism兲;
and stage controller 共Mark-102兲. J-800KCMF and a personal
computer 共PC兲are interfaced with RS-232C interface. PC
controls all the measurement conditions. Analog signals de-
tected by the lock-in amplifiers and the PMT voltage are
transformed into digital signals and are transmitted to PC by
RS-232C interface.
Spectralon 共Labsphere Co. Ltd.兲was used as an ideal
diffuser. Spectralon is a material with very high diffuse re-
flection, with reflectance higher than 95% for UV-vis wave-
length region. Filter, lustrous, and normal white papers were
FIG. 1. Block diagram of the vertical-type universal chiroptical spectropho-
tometer 共UCS-2兲: Mo; monochrometor, P; polarizer, PM; pulse motor, St;
stage controller, LA; lock-in amplifier, PMT1 and 2; photomultiplier for
transmission and DR, P.P; photomultiplier power supply, and PC; personal
computer.
073103-2 Harada, Hayakawa, and Kuroda Rev. Sci. Instrum. 79, 073103 共2008兲
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purchased from Whatman Co. Ltd., Epson Co. Ltd., and
ASKUL Co. Ltd. and used without any further treatment.
2. Chemicals
Ammonium 共d兲- and 共l兲-10-camphorsulfonate 共ACS兲
were purchased from Katayama Chemical Co. Ltd., and used
without any further treatment.
III. RESULTS AND DISCUSSION
A. PEM position
As a right angle quartz prism used in UCS-2 inevitably
has intrinsic birefringence, a PEM position is highly impor-
tant for the instruments performance. We have investigated
two possible positions of PEM 关A and B in Fig. 2共a兲兴.
We can formulate intensities, Id, of the light at the detec-
tor for the two arrangements by the Mueller matrix calcula-
tion as follows:
Id关A兴=D
ˆ·P
ˆris ·M
ˆ共45兲·P
ˆ共90兲·I
ˆ0,
Id关B兴=D
ˆ·M
ˆ共45兲·P
ˆris ·P
ˆ共90兲·I
ˆ0.共1兲
The Stokes vector of the light emitted from a light source, I
ˆ0,
can be expressed as
I
ˆ0=
冢
1
0
0
0
冣
.共2兲
The Mueller matrix for the polarizer whose optical axis
parallel to the yaxis can be expressed as
P
ˆ共90兲=1/2
冢
1 00−1
0000
0000
−100 1
冣
.共3兲
When the PEM is set at 45° with respect to the xaxis, the
Mueller matrix for the PEM, M
ˆ共45兲, can be expressed as
M
ˆ共45兲=
冢
10 0 0
01 0 0
00cos共
␦
+
␣
兲− sin共
␦
+
␣
兲
00sin共
␦
+
␣
兲cos共
␦
+
␣
兲
冣
.共4兲
The crystal quartz right angle prism has inherently large re-
sidual static LB, LBp. Thus the imperfect property of the
right angle prism is given as
P
ˆris =
冢
10 0 0
01−LB
p0
0LBp1−LB
p
⬘
00 LBp
⬘1
冣
.共5兲
The Mueller matrix of the photomultiplier acting as a
partial polarizer, D
ˆ, is formulated as
D
ˆ=
冢
共Px
2+Py
2兲共Px
2−Py
2兲sin 2a0共Px
2−Py
2兲cos 2a
共Px
2−Py
2兲sin 2a共Px+Py兲2cos22a+2PxPysin22a0共Px−Py兲2cos 2asin 2a
002PxPy0
共Px
2−Py
2兲cos 2a共Px−Py兲2cos 2asin 2a0共Px+Py兲2cos22a+2PxPysin22a
冣
.共6兲
FIG. 2. 共A兲Optical arrangement showing two alternative PEM positions 共A and B兲in UCS-2; Mo; monochromator, P; polarizer, R; right angle prism, PMT;
photomultiplier tube, I; integrating sphere. 共B兲Transmission 共upper兲and DR 共bottom兲CD base lines of UCS-2 depending on the alternative PEM positions
关A共dotted line兲and B 共solid line兲兴.
073103-3 Diffuse reflectance circular dichroism Rev. Sci. Instrum. 79, 073103 共2008兲
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Thus, from Mueller matrix calculations of
D
ˆ·P
ˆris ·M
ˆ共45兲·P
ˆ共90兲·I
ˆ0and D
ˆ·M
ˆ共45兲·P
ˆris ·P
ˆ共90兲·I
ˆ0, the
photocurrent detected by the lock-in amplifier can be ex-
pressed as
Id=G1共Px
2−Py
2兲sin 2a关−LB
psin共
␦
+
␣
兲兴 共case A兲,
共7兲
Id=G1共Px
2−Py
2兲cos 2a关−LB
p
⬘sin共
␦
+
␣
兲− cos共
␦
+
␣
兲兴 共case B兲.共8兲
Here, G1is the apparatus constantly related to the sensi-
tivity of the spectrometer at 50 kHz, Px
2and py
2are the prin-
cipal transmittance of the detector in the xand ydirections,
respectively, and ais the azimuth angle of the partial polar-
izer with respect to the xaxis.
␣
is the residual static bire-
fringence of PEM. Making use of Fourier series expansion,
we can express cos共
␦
+
␣
兲and sin共
␦
+
␣
兲as
cos共
␦
+
␣
兲=2J2共
␦
m
0兲cos 2
␻
mtcos
␣
−2J1共
␦
m
0兲sin
␻
mtsin
␣
+J0共
␦
m
0兲cos
␣
,共9兲
sin共
␦
+
␣
兲=2J1共
␦
m
0兲sin
␻
mtcos
␣
−2J2共
␦
m
0兲cos 2
␻
mtsin
␣
+J0共
␦
m
0兲sin
␣
.共10兲
In order to minimize the CD and LD base line shifts that
influence the performance of the instrument, the optical axis
of the photomultiplier was set at 45° with respect to the xand
yaxes. Thus, cos 2abecomes nearly zero.1Therefore, Eqs.
共7兲and 共8兲can be approximated as
CDbase关A兴=G1共Px
2−Py
2兲sin 2a关−LB
p兴,共11兲
CDbase关B兴=0. 共12兲
Thus, in case B, CD base line shift should become flat
over a wide wavelength range.
Experimental results are shown in Fig. 2共b兲. As ex-
pected, setting of a PEM at position “A” exhibits nonflat
transmittance and DRCD base lines with particularly high
signals in the wavelength region shorter than ⬃350 nm. This
is due to the intrinsic static strain birefringence of a right
angle prism. On the other hand, by setting a PEM at position
“B,” we could obtain flat base line shifts on both transmit-
tance and DR CD modes, as shown in Fig. 2共b兲. Base line
shifts differs substantially depending on the PEM position
and thus, it is clear from our theoretical analyses and experi-
mental results that we have to choose position B.
It is well known that the intrinsic birefringence of optical
elements governs a base line shift.18 We suppose that one of
the reasons that the first DRCD spectrophotometer was lim-
ited in visible wavelength range lies in the PEM position in
the optical train, i.e., in front of the right angle prism.17
B. Relative efficiency
To compare the efficiency of the DRCD with the trans-
mittance CD, we measured dc voltages of the photocurrent
detected by the photomultipliers for the two modes while
keeping the high tension voltage on the PMT constant
共250 V兲. Bandwidth of the monochromator, response time,
and scanning speed were also set constant at 1 nm, 1 s, and
100 nm/min, respectively. For the transmittance CD mode,
the dc voltage of the air blank was measured. For the DRCD
mode, a standard white plate 共spectralon兲was placed in the
sample holder of the integrating sphere and used as an ideal
diffuser. Figure 3shows an efficiency curve, which is what
we call the relative efficiency, calculated from the transmit-
tances on both modes at constant high tension voltage
applied to the PMTs. The results show that the efficiency of
the instrument is about 4%–10% throughout the long-
wavelength UV-vis range, but significantly lower in the
short-wavelength UV region due to the reflectance property
of barium sulfate used for the integrating sphere. Still, the
relative efficiency of our instrument is ten times higher than
that of the prototype17 over the UV-vis wavelength range as
compared in Fig. 3共inset兲. Thus, we have succeeded in ob-
taining high relative efficiency on DRCD mode by selecting
good optical elements.
In DRCD measurement, it is necessary to mount a
sample on a sample holder which has as high as possible
reflectance. Although a standard white plate 共spectralon,
ideal Teflon diffuser兲with high reflectance is used for cali-
bration and for the evaluation of the relative efficiency, it is
not desirable to use this as a sample holder for the ordinary
measurements. The material is porous so that contamination
of samples cannot be avoided even with repeated washing.
This may result in incorrect spectra. Thus, we surveyed al-
ternative materials, which are disposable and have high re-
flectance equivalent to the standard white plate. Figure 4
shows DRCD and DRLD base line shifts of several reflective
materials. In UV wavelength region, the DRCD and DRLD
base line shifts of normal white paper is too large for the use
of a sample holder. Flat DRCD base line shifts of the filter
and the lustrous papers are obtained throughout whole range
from visible to UV. Especially, DRCD base line shift of lus-
trous paper was almost equivalent to the standard white plate
although DRLD base line shift is slightly larger. Therefore,
FIG. 3. Efficiency curve calculated from transmission CD and DRCD spec-
tra at constant high tension voltage 共250 V兲applied to temperature. 共Inset兲
Figure is adopted and modified from Ref. 16.
073103-4 Harada, Hayakawa, and Kuroda Rev. Sci. Instrum. 79, 073103 共2008兲
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we adopted lustrous paper as a disposable sample holder.
In order to evaluate the performance of DRCD mode of
UCS-2, we measured powdered ACS which is usually used
as a standard sample for CD spectrophotometer calibration in
UV wavelength region.19,20 As shown in Fig. 5, DRCD spec-
tra of the microcrystalline ACS enantiomers are mirror im-
ages of each other, indicating that all the equipments, both
optical and electric, work well as predicted. The spectra are
compared to solution spectra in Fig. 5. The agreement in
general is quite good, but slight redshifts of the peak maxi-
mum, 9 and 6 nm, were observed compared to transmission
CD in solution and in the solid state 共KBr matrix method兲,
respectively. It has been reported that the redshifts in DR
spectra depend on specular reflectance21,22 which is defined
as the reflected radiation which reaches the detector but
never penetrates the sample particles. In contrast, DR is de-
fined as the reflected radiation which is transmitted and/or
refracted through one or more sample particles and finally
reflected onto the detector. Thus, it might be suggested that
the observed DRCD signal contains both specular and DR
lights. DRCD measurement mode of UCS-2 can remove ar-
tifact signals arising from LD which is not coupled with LB,
as LB contribution cannot be measured at the moment. We
are currently investigating the reason of redshift in the
DRCD measurements which may be reported later.
IV. CONCLUSION
A new type of CD spectrophotometer, UCS-2, with high
performance was constructed. By theoretically analyzing and
experimentally proven the effect of PEM position on CD
base line shifts, we could obtain flat CD base lines despite
the fact a right angle quartz prism having substantial intrinsic
birefringence is installed. We could achieve ten times higher
reflective efficiency throughout whole wavelength ranges as
compared with the prototype.
In situ chirality measurement of the powdered material is
of great importance in many research fields such as chemis-
try and material science as transmittance spectroscopy is of-
ten not applicable to these materials. It may also give in-
sights into the origin of chirality, i.e., chirality of molecular
and intermolecular origins. Thus, the new spectrophotometer
UCS-2 certainly provides various physicochemical proper-
ties, which are not obtainable with commercially available
CD spectrophotometers.
ACKNOWLEDGMENTS
The authors thank Dr. Y. Shindo, an emeritus Professor
of Fukui University and Dr. H. Masago, Dr. T. Fukazawa,
and Mr. Y. Miyoshi of Jasco Corp. Ltd. for discussion and
technical assistance.
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Teflon diffuser, b 共dotted line兲: lustrous paper, c 共long dashed and short
dashed line兲: filter paper, d 共broken line兲: normal white paper.
FIG. 5. DRCD spectra of 共d兲-共solid line兲and 共l兲-ACS 共dotted line兲micro-
crystallines pulverized to small particles. 共Inset兲Their transmittance CD
spectra in solution state.
073103-5 Diffuse reflectance circular dichroism Rev. Sci. Instrum. 79, 073103 共2008兲
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