JP2012202812A - Circular dichroism measuring apparatus and method for measuring circular dichroism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a CD measuring apparatus having enhanced measurement sensitivity and improved reliability, and a CD measuring method.SOLUTION: A circular dichroism measuring apparatus comprises elliptical polarization modulation means which modulates the phase difference (δ) between the two polarization components orthogonal to each other of a linear polarization so as to alternately form the right and left elliptical polarizations having a large flatness factor with identical long axis orientation, polarization intensity measuring means which sorts the polarization component in short axis orientation from the right and left elliptical polarizations transmitting through a measurement sample so as to measure the change in intensity of the polarization component, and calculation means which samples each of the direct current (DC) component of the change in intensity and the alternate current (AC) component of the change in intensity synchronized with the modulation of the phase difference so as to obtain the circular dichroism of the measurement sample through the calculation of the product of the ratio (AC/DC) of the alternate current component to the direct current component and the modulation amplitude (δ) of the phase difference.

Description

  The present invention relates to an improvement in a measurement method and an apparatus for improving sensitivity and accuracy of circular dichroism (CD for short) measurement. The circular dichroism measuring device is a device that measures circular dichroism (called ECD) mainly related to electronic transition in the ultraviolet, visible, and near infrared regions, and a circular dichroism measuring device mainly related to vibrational transition in the infrared region. There is a device for measuring chromaticity (vibration circular dichroism: called VCD), and the present invention targets both devices.

  CD measurement is widely used as almost the only spectroscopic method that can directly analyze the three-dimensional structure of a molecule. Initially, it was used to determine the absolute structure of biologically active natural organic compounds and to study stereochemistry of complex compounds. Later, in the biochemical field, analysis of higher-order structures of proteins and other biopolymers Has been applied to. It is an extremely useful tool for measuring the thermal stability of biopolymers and analyzing the reaction process of enzyme reactions. In the field of pharmacy / pharmaceuticals, it is also heavily used to reduce side effects by grasping molecular asymmetry and drug efficacy, and to manage the activity of enzymes and the like incorporated in drugs. In addition, since the VCD measurement has good comparability between the spectrum obtained by the measurement and the spectrum predicted from the calculation based on the molecular structure, its application is expanding to the structural analysis of drugs and physiologically active substances.

  Increasing the sensitivity of devices, not limited to CDs, is an eternal issue for all analytical and measurement devices. Efforts have been made to improve and improve the results so that more reliable measurement results can be obtained with a smaller amount of sample. There is. In particular, in the biological research field, it is rare that a sufficient amount of samples of biological components to be analyzed can be secured, and only a very small amount of samples can be prepared. Moreover, in recent years, research has become more sophisticated, and the amount of target components has become very small. Efforts are being made to improve and improve devices and systems in order to meet this demand, but as a matter of fact, orthodox improvement means such as increasing the amount of light, increasing the light throughput, and increasing the signal utilization efficiency Is almost exhausted, and it is no longer possible to meet the demands for soaring sensitivity on its extension.

(Common issues for CD measuring devices)
Returning to the basics, considering the factor that has become a bottleneck in increasing the sensitivity in CD measurement, it can be mentioned that CD has a minute value of about 1/100 to 1/1000 of the absorbance. CD is defined as the difference (ΔA = A ₁ −A _r ) between the absorbance (A ₁ ) for left circularly polarized light and the absorbance (A _r ) for right circularly polarized light. However, the difference is small, and even in the case of a large molecule, the absorbance is usually 1/100, usually 1/1000. This fact means that, with a simple estimate, the CD measurement can only be expected to have a sensitivity on the order of 1/100 to 1/1000 of the spectrophotometric method. Still, in the current CD measuring apparatus, by combining the polarization modulation method and the lock-in amplification method, a sensitivity improvement of about 10 times the simple expected value is achieved, but the absorbance is 1/100 to 1/1000. The difference is still detected.

  This consideration suggests a breakthrough means for increasing the sensitivity of CD measurements. Detecting a difference of 1/1000 in absorbance means measuring the difference between 1000 and 999. If the measurement method can be improved to measure this as a difference between 100 and 99, or 10 and 9, then detection is possible. Sensitivity can be expected to improve by a factor of 10 or 100. And in the high-speed CD time change measurement which cannot be achieved by the conventional polarization modulation CD measuring apparatus, the detection sensitivity is improved by using the above measurement principle.

  The inventor has devised this principle to apply to a conventional polarization modulation CD measuring apparatus, not for high-speed CD time change measurement, and has arrived at the present invention. First, common problems of the above-described CD measuring apparatus will be described in the order of ECD, high-speed CD, and VCD measuring apparatus. JP-A-2001-133399 (Patent Document 1) can be cited as the prior art of the ECD measuring apparatus, and JP-A-2005-43100 (Patent Document 2) can be cited as the prior art of the VCD measuring apparatus. . The technique of the high-speed CD measuring device is described in detail in Japanese Patent Application No. 2010-190514.

(Outline of conventional ECD measuring device)
First, a conventional ECD measurement method will be described. The optical system is as shown in FIGS. The light emitted from the light source lamp 10 is split into monochromatic light by the spectroscope 11, converted into linearly polarized light having a polarization plane in the YZ plane by the polarizer 12, and passed through a phase modulator (PEM) 13. This PEM is an element that uses the piezoelectric effect, and is installed so that its axis intersects the plane of polarization of linearly polarized light that is incident at an angle of 45 °. The incident linearly polarized light can be expressed as the sum of two linearly polarized lights in the direction of ± 45 °, but when they pass through this PEM, they are given a phase difference and are determined by the modulation frequency f (50 kHz in a commercially available device). Circularly polarized light that alternates left / right in the cycle. However, this expression is intended to aid understanding and is not accurate. To be exact, the polarization state is periodically changed by the applied phase difference δ = δ ₀ · sin 2πft. The intensity of the modulation, that is, the modulation amplitude δ ₀ is set to δ ₀ = 1.84 where the first-order Bessel function J ₁ (δ ₀ ) becomes the maximum value. This is an optimum value that maximizes the AC component reflecting the CD in the conventional apparatus. Since the phase difference δ becomes circularly polarized when δ = π / 2 = 1.57, the ellipse in which the linearly polarized light passes through the circularly polarized light and the length is changed at δ = ± δ ₀ where the phase difference is maximum. The polarization will be modulated.

  The left and right circularly polarized light is incident on the sample unit 14. The sample part 14 contains a sample having a CD. When left and right circularly polarized light passes through the sample, the sample part 14 receives absorption of different magnitudes on the left and right, and the light thereafter varies in intensity depending on this CD. Will be included. In the CD device, the light intensity detected by a detector (a photomultiplier tube, PMT for short in a commercially available device) 15 is changed to an electric signal, which is expressed by Expression (1).

Here, I _l and I _r are transmitted light intensities of left and right circularly polarized light. This electric signal is sent to a DC amplifier and an AC amplifier. In the DC amplifier, an electric signal is amplified, and a direct current component (DC), that is, (I ₁ + I _r ) / 2 is obtained therefrom. The electric signal amplified by the AC amplifier is further sent to the lock-in amplifier. The AC amplifier and the lock-in amplifier obtain an AC component (AC) having a frequency synchronized with the modulation frequency f, that is, (I ₁ −I _r ) · J ₁ (δ ₀ ). Then, the circular dichroism ΔA is calculated by the equation (2) from AC divided by DC.

(Problem of improving S / N ratio of conventional ECD measuring device)
As mentioned at the outset, generally the circular dichroism is very small, and even a large molecule usually has a ΔA / A of at most 10 ⁻² , usually 10 ⁻³ to 10 ⁻⁴ . Then, in the measurement of the conventional ECD measuring apparatus described above, an AC component that is superposed slightly on a very large DC component is extracted, and a relatively large signal fluctuation derived from the large DC component is dominant. It was. This is a major factor that makes it difficult to extract the AC component with a higher S / N and increase the CD measurement sensitivity.

(Outline of high-speed CD time change measuring device)
The high-speed CD time change measuring apparatus is an apparatus improved from the conventional polarization modulation CD measuring apparatus in order to enable high-speed CD time change measurement, and its basic configuration is shown in FIG. In order from the light source side of white light, a polarizer 31, a phase shifter 32, a sample unit 34, an analyzer 35, a spectroscope 36, and a detector 37 are arranged on the optical axis. The white light is converted into linearly polarized light having a polarization plane in the XZ plane by the polarizer 31. The linearly polarized light passes through the phaser 32. The phase advance axis of the phase shifter 32 is inclined by a minute angle θ from the X axis. In addition, a phase shifter 32 having an appropriately small phase difference δ is selected. As a result, the light after passing through the phase shifter 32 becomes not only the polarization component in the X direction but also the polarization component having a small amount of the polarization component in the Y direction, that is, elliptical polarization that is close to linear polarization and has an extremely large flatness. Yes. When this elliptically polarized light is transmitted through the sample, the flatness changes depending on the CD of the sample. The changed elliptically polarized light is transmitted through the analyzer 35 installed at the position of the crossed Nicols with the previous polarizer 31. The analyzer 35 extracts only the polarization component in the Y-axis direction from the elliptically polarized light. The extracted polarization component is passed through the spectroscope 36 to be monochromatic, and the polarization intensity is detected by the detector 37. The measurement result at this time is defined as I ₊ (λ). In addition to acquiring data of only a single wavelength, a system that efficiently obtains a CD spectrum may be obtained by simultaneously acquiring a plurality of pieces of data for each wavelength with an array detector after the spectrum is split by the spectroscope 36.

Next, the phase shifter 32 is rotated reversely by the phase shifter driving mechanism 33, and the same measurement is performed with the angle set to -θ. The measurement result at this time is defined as I ₋ (λ). The circular dichroism ΔA is calculated from I ₊ (λ), I ₋ (λ) and δ according to the equation (3).

(Problem of high-speed CD time change measuring device)
In a conventional polarization modulation CD measurement apparatus, the limit of time resolution in CD measurement is determined by the speed of polarization modulation. On the other hand, the above-mentioned high-speed CD time change measuring apparatus does not modulate the phase difference, and uses the phase shifter 32 having a constant phase difference to adjust the fast axis to an angle of ± θ with respect to the polarization plane. . In this way, the states that are biased to the left and right circularly polarized light are individually made, high-speed time change measurement is performed in each state, and the difference in absorbance with respect to the left and right circularly polarized light along with the time change (circle) by the calculation of Equation (3). Dichroism ΔA) was obtained. As an effect, an improvement in sensitivity has been obtained.

  However, when measuring the circular dichroism in the steady state without following the time change, or even following the time change, the time change within the limit of the speed of the polarization modulation in the conventional polarization modulation CD measuring device is sufficient. In the case where only an improvement in sensitivity is expected, a satisfactory measurement result cannot be obtained by simply using this system as it is. In this system, the phase shifter 32 is mechanically moved to create a polarization state related to the left and right circularly polarized light. Therefore, if the accuracy of the mechanical unit for moving the phase shifter 32 is not strict, the deviation is measured value. Will give an error. Further, in the procedure of measuring with the phase shifter 32 set to one side and then moving to the other side, the time difference between the two measured values can be made shorter than the sub-seconds no matter how fast. It was difficult, and this difference in time could cause an error when taking the difference in absorbance between the left and right.

(Outline of conventional VCD measuring device)
A conventional VCD measuring device is also an object to be improved by the present invention. The basic configuration of a conventional VCD measuring apparatus is shown in FIG.

  White light emitted from the infrared light source 40 is converted into interference light by the interferometer 41. The polarizer 42 extracts linearly polarized light from the interference light, and is then phase-modulated by the PEM 43. The light is incident on the sample portion 44 and the intensity of light transmitted through the sample is detected by the detector 45. The detected signal is amplified by the preamplifier 47, and the DC component extracted therefrom is sent to the data processing device 49 as it is. The signal amplified by the preamplifier 47 is also sent to the lock-in amplifier 48, and the lock-in amplifier 48 extracts the AC component using the signal synchronized with the modulation frequency of the PEM 43 and sends it to the data processing device 49. The DC component and the AC component are taken in as a time-series signal synchronized with the moving mirror position of the interferometer 41 by the data processor 49. Further, an AC / DC value is calculated. Although this value is a signal sequence related to a CD, it is not a CD itself but an interferogram including CD information. This is sent to a data processing PC, where it is subjected to Fourier transform to obtain a CD spectrum.

(Problems with conventional VCD measuring devices)
In the above VCD measuring apparatus, an interferogram through an interferometer is used for measurement. Since light of all wavelengths λ (wave number) is included therein, it is impossible to adjust the phase modulation amplitude δ ₀ to an appropriate size for each wavelength λ. Therefore, the center wavelength is appropriately selected, and the modulation amplitude δ ₀ is determined so that the sensitivity is optimal. However, the measurement wave number range (measurement wavelength range) of VCD measurement ranges from 600 cm ⁻¹ (16 μm) to 5000 cm ⁻¹ (2 μm). As a result, there is a dead zone where the sensitivity is lost due to an excessive phase difference for light of a certain wavelength, and further, the signal is reversed after that. Although the reversal can be dealt with by applying a negative coefficient, it is impossible to deal with the dead zone in which the sensitivity is lost, and the measurement has to be repeated by changing the modulation amplitude δ ₀ of the phase difference.

This situation, defined as the center wavenumber ^{1250 cm} -1 to (8 [mu] m), where determined such modulation amplitude [delta] ₀ is optimal to give the sensitivity (efficiency) of the modulation amplitude [delta] ₀ and CD in other wave number J ₁ Try to estimate (δ ₀ ). The optimum modulation amplitude δ ₀ is δ ₀ = 1.84 giving the extreme value of the first-order Bessel function. The modulation amplitude at each wave number is δ ₀ = 2π · Δn · d / λ, and is determined by the thickness d of the PEM, the maximum refractive index difference Δn induced, and the wavelength λ. Δn depends on the wavelength (wave number), but when Δn is regarded as constant and sensitivity J ₁ (δ ₀ ) is calculated, the following table is obtained.

Even in the vicinity of the center wave number (1250 cm ⁻¹ ), the sensitivity changes depending on the wave number, and this must be corrected as a coefficient. However, 2604 cm ^-1 is a dead band where the sensitivity is zero, and the signal is reversed at a wave number band higher than that, and 4762 cm ^-1 is also a dead band. Further, in the higher wave number band, the sign of the signal is rotated in the normal direction, but the sensitivity is as low as about 1/6. In practice, it is possible to measure normally up to about 2000 cm ^{−1 under} this condition, and in order to measure a higher wave number region than that, an appropriate wave number in that region is reset to the center wave number, and the modulation amplitude δ ₀ optimization will be redone. If only one central wave number is set in this way, a dead band or a wave number region where the sign of a signal is inverted is generated in the measured wave number region, and the wave number region that can be measured at one time is limited.

  Furthermore, in the case of the VCD measuring device, as in the case of the electronic transition CD measuring device, a slight difference in absorbance with respect to the left and right circularly polarized light included in the large absorption is detected, and the ratio is only about 1/10 compared with the electronic transition. However, it is a larger wall for improving sensitivity.

JP 2001-133399 A JP-A-2005-43100

  The present invention is based on the technology achieved in a high-speed CD time variation measuring apparatus. In other words, by applying a small phase difference δ to linearly polarized light to make elliptically polarized light with an extremely large flatness, an analyzer is placed in a direction perpendicular to the major axis direction, and only the minor axis direction component is detected. The major axis component corresponding to is removed. As a result, only the signal that directly reflects the CD is preferentially extracted, and the sensitivity is improved. An object of the present invention is to improve the measurement sensitivity by applying the above technique to the conventional ECD measurement apparatus and VCD measurement apparatus, and in particular, to the conventional VCD measurement apparatus. In addition to improving sensitivity, it also solves the problem of having to limit the measurement wavelength range, and provides an apparatus and measurement method that can measure all wavelength regions involving vibration spectroscopy with high sensitivity at once. is there.

The circular dichroism measuring apparatus of the present invention is characterized by comprising the following elliptically polarized light modulating means, polarized light intensity measuring means, and computing means. The elliptically polarized light modulating means modulates the phase difference (δ) between two orthogonally polarized light components of linearly polarized light, and alternately forms left and right elliptically polarized light having a large flatness that matches in the major axis direction. The polarization intensity measuring means takes out a polarization component in the minor axis direction from the left and right elliptically polarized light transmitted through the sample to be measured, and measures an intensity change of the polarization component. The computing means extracts the DC component (DC) of the intensity change and the AC component (AC) of the intensity change synchronized with the modulation of the phase difference, respectively, and the ratio of the AC component and the DC component (AC / DC) is multiplied by the modulation amplitude (δ ₀ ) of the phase difference to obtain the circular dichroism of the sample to be measured.

According to this configuration, the elliptically polarized light modulating means does not modulate the linearly polarized light until it becomes circularly polarized light, but modulates the linearly polarized light to an elliptically polarized state having a large flatness. Therefore, left-handed and right-handed elliptically polarized light having a large aspect ratio are alternately formed from linearly polarized light. Furthermore, the polarization intensity measuring means does not detect all the left and right elliptically polarized light beams that have passed through the sample, but extracts only the short-axis polarized light component of the left and right elliptically polarized light, and the light beam of the polarized component. And the intensity change of the polarization component according to the modulation is measured. In this way, only the signal that directly reflects the CD can be preferentially extracted. Therefore, it is possible to improve the sensitivity and accuracy of circular dichroism measurement. In order to obtain the desired circular dichroism, a DC component and an AC component are extracted from the measured intensity change, and an operation of multiplying the ratio (AC / DC) by the modulation amplitude (δ ₀ ) may be performed. .

The circular dichroism measuring device of the present invention is characterized by comprising the following interference means, elliptically polarized light modulating means, polarization intensity measuring means, and computing means. The interference means divides the infrared light from the light source into two so as to go to the fixed mirror and the movable mirror, and combines the reflected light from the fixed mirror and the movable mirror to change the optical path difference (D). Corresponding interference light is formed. The elliptically polarized light modulation means extracts linearly polarized light from the interference light, and has a phase difference between two polarization components orthogonal to each other at a modulation frequency (f) greater than the change in the optical path difference (D) ( δ) is modulated to alternately form left and right elliptically polarized light having a large flatness that matches in the major axis direction. The polarization intensity measuring means takes out a polarization component in the minor axis direction from the left and right elliptically polarized light transmitted through the sample to be measured, and measures an intensity change of the polarization component. The computing means extracts the DC component (DC) of the intensity change and the AC component (AC) of the intensity change synchronized with the modulation of the phase difference, respectively, and the ratio of the AC component and the DC component (AC / DC) is the vertical axis, and the optical path difference (D) is the horizontal axis. The interferogram is subjected to Fourier transform, and a value obtained by multiplying the value after Fourier transform by the modulation amplitude (δ ₀ ) of the phase difference is calculated. The circular dichroism of the sample to be measured is acquired.

In this configuration, the measurement light incident on the elliptically polarized light modulating means is interference light. When monochromatic light is passed through the interferometer, the interference light draws a sine curve corresponding to the optical path difference. However, in the measurement of infrared absorption, the measurement light is continuous light in a predetermined wavelength region, and the interference light is a superposition of sine curves drawn by monochromatic light. Such a waveform is called an interferogram. According to the configuration of the present invention, the elliptically polarized light modulation means takes out linearly polarized light from the interference light that draws the interferogram, and does not modulate the linearly polarized light until it becomes circularly polarized light as in the above-described invention. Modulate to a state of large elliptical polarization. Therefore, left and right elliptically polarized light in an interferogram state are alternately formed. Similarly to the above-described invention, the polarization intensity measuring means does not detect all the left and right elliptically polarized light beams that have passed through the sample, but extracts only the short axis polarization component of the left and right elliptically polarized light. Then, the light flux of the polarization component is detected, and the intensity change of the polarization component corresponding to the modulation is measured. Since the polarization component measured here is also in an interferogram state, in order to obtain a desired circular dichroism, a DC component and an AC component are extracted from the measured intensity change, and the ratio (AC / DC) is obtained. ) May be subjected to Fourier transform. Then, an operation of multiplying the converted value by the modulation amplitude (δ ₀ ) may be performed. Therefore, similar to the above-described invention, an improvement in sensitivity can be obtained. Furthermore, insensitivity or inversion of the sign does not occur in the measurement wavelength region, and the measurement wavelength region is not limited to the region limited from the center wavelength that sets the modulation amplitude of the elliptical modulation means. Therefore, the entire wavelength region can be measured with high sensitivity at a time.

The elliptically polarized light modulation means includes a polarizer that extracts linearly polarized light from incident light, and a phase modulator that modulates the linearly polarized light to form left and right elliptically polarized light. The polarization intensity measuring means includes an analyzer that transmits linearly polarized light in the short axis direction of the left and right elliptically polarized light that has passed through the sample to be measured, and a detector that detects the intensity of the transmitted linearly polarized light. . And in this invention, it is preferable that the analyzer is installed in crossed Nicols with respect to the polarizer.
According to this configuration, since the polarizer and the analyzer have a crossed Nicols relationship, the analyzer can accurately transmit the polarization component in the short axis direction of the left and right elliptically polarized light.

The present invention is also a circular dichroism measuring device that acquires a circular dichroism spectrum in a predetermined measurement wavelength region, wherein the modulation amplitude (δ ₀ ) of the phase difference (δ) of the elliptically polarized light modulation means is measured. It is preferable that it is 1/10 radians or less in the entire wavelength region.
According to this configuration, the modulation amplitude is extremely small as compared with the modulation amplitude in the conventional ECD measurement method being δ ₀ = 1.84 radians. Therefore, a phase modulator having a small phase difference and high modulation accuracy can be employed.

It is a figure which shows the basic composition of CD measurement apparatus of this invention. It is an optical path figure of the ECD measuring device of a 1st embodiment of the present invention. It is a whole block diagram of the said ECD measuring apparatus. It is a whole block diagram of the VCD measuring apparatus of 2nd Embodiment of this invention. It is a figure which shows the basic composition of the conventional CD measuring apparatus. It is the schematic of the optical system which concerns on the detection principle of the conventional CD. It is the schematic of the optical system of a high-speed CD time change measuring apparatus. It is the schematic of the conventional VCD measuring apparatus.

  A basic configuration of the circular dichroism measuring apparatus of the present invention will be described with reference to FIG. The measurement optical system includes a polarizer 501, a phase modulator (PEM) 502, a sample unit 503, an analyzer (analyzer) 504, and a detector 505, which are arranged on the optical axis of the measurement light 500 in this order.

The polarizer 501 is arranged so as to extract linearly polarized light having a polarization plane in the YZ plane from the measurement light 500. The PEM 502 is arranged with its principal axis inclined 45 ° from the X direction. In a conventional ECD measurement apparatus, the PEM is driven by a program that gives a modulation amplitude of δ ₀ = 1.84 (radians) at which the first-order Bessel function is maximum at each measurement wavelength. However, in the present invention, the driving power of the PEM is controlled so that δ ₀ = 0.1 (radian), which is much smaller than that in the entire wavelength region, and optimally 0.01 (radian). Note that δ ₀ indicates a modulation amplitude when phase modulation is expressed as δ = δ ₀ sin 2π ft, and is obtained and stored in advance by measurement using a standard sample.

The phase difference of the linearly polarized light is modulated by the PEM 502. Specifically, the phase difference (δ) between two polarization components orthogonal to each other of linearly polarized light is modulated by the PEM 502. Then, left and right elliptically polarized light having a large flatness that coincides in the major axis direction (Y direction) are alternately formed.
The analyzer 504 is provided downstream of the sample portion 503 so as to be crossed with the polarizer 501. By this analyzer 504, the polarization component in the minor axis direction (X direction) is extracted from the left and right elliptically polarized light transmitted through the sample of the sample portion 503. The detector 505 measures the intensity change of the polarization component in the minor axis direction.

  The data processing system includes a preamplifier 506, a lock-in amplifier 508, a DC amplifier 507, a PEM driver 509, an A / D converter 510, and a data processing device 511.

The light intensity signal detected by the detector 505 is amplified by the preamplifier 506, and then its DC component and AC component are separately amplified. The lock-in amplifier 508 receives a signal synchronized with the drive frequency of the PEM 502 from the PEM driver 509, and extracts an AC component signal (AC) that is the same frequency component as the drive frequency from the light intensity signal. The DC amplifier 507 extracts a direct current component signal (DC) from the light intensity signal. The DC component signal and the AC component signal are digitized by an appropriate A / D converter 510 and taken into the data processing device 511. In the data processing device 511, the CD value (ΔA) is calculated according to the equation (4). That is, a value obtained by multiplying the ratio of the AC component and the DC component (AC / DC) by the modulation amplitude δ ₀ of the phase difference is calculated to obtain the circular dichroism of the sample to be measured.

[First Embodiment]
FIG. 2 is an optical path diagram showing the configuration of a specific measurement optical system of the ECD measurement apparatus of this embodiment. The measurement optical system is roughly classified into a light source chamber, a spectroscope chamber, a phase modulation chamber, a sample chamber, and a detection chamber. A light source lamp 600 and a condensing mirror 601 are arranged in the light source chamber, and supply measurement light to the spectroscope chamber.

  A spectroscope 602 including optical elements of the first prism system and the second prism system is formed in the spectroscope chamber, and the measurement light from the light source is monochromatized using the two crystal quartz prisms 604 and 606 as the spectroscopic elements. . The first prism system includes a slit 603, a collimator mirror 605, and a first prism 604, and splits the measurement light from the light source chamber into light of a predetermined wavelength region. Similarly, the second prism system includes a slit 603, a collimator mirror 605, and a second prism 606. In the conventional system, the second prism 606 is also used as a polarizer, but it is preferable to follow this. That is, the measurement light from the first prism system is split into predetermined monochromatic light and linearly polarized light. The linearly polarized light is collected by the lens 607 and sent to the phase modulation chamber.

  A PEM 608 and a shutter 609 are arranged in the phase modulation chamber. The linearly polarized light from the spectroscope 602 is subjected to small phase modulation of about 0.01 radians by the PEM 608, and becomes left and right elliptically polarized light having a large flatness and close to linearly polarized light. The left and right elliptically polarized lights are alternately formed according to the modulation frequency of the phase difference, and sent to the sample chamber through the shutter 609.

  The left and right elliptically polarized light passes through the sample portion 610 and is then sent to the detection chamber. In the detection chamber, an analyzer 611 and a detector 612 characteristic of the present invention are arranged. The left and right elliptically polarized light is passed through the analyzer 611 to extract the linearly polarized light component which is the short axis component of the ellipse, and the intensity is detected by the detector 612.

  Note that the second prism 606 and the exit slit 603 functioning as a polarizer are arranged so that the polarized light extracted from the measurement light is linearly polarized light parallel to the paper surface. On the other hand, the analyzer 611 is arranged so as to extract only the polarization component perpendicular to the paper surface from the elliptically polarized light.

  FIG. 3 is an overall configuration diagram of the ECD measuring apparatus according to the present embodiment. Here, the processing system of the intensity signal measured by the detector 612 will be described. The light intensity signal detected by the detector 612 is amplified by a preamplifier 613 and sent to an AC amplifier and a DC amplifier. The AC amplifier 618 amplifies the AC component in the intensity signal. Furthermore, the lock-in amplifier 619 extracts the same frequency component as the modulation frequency from the amplified AC component signal. The extracted AC component signal is digitized by the A / D converter 621 and taken into the data processing device 622.

  On the other hand, the DC amplifier 620 amplifies a direct current component in the intensity signal. The amplified direct current component signal is digitized by the A / D converter and taken into the data processing device 622. Further, a part of the direct current component from the DC amplifier 620 is taken into the differential amplifier 617 and compared with a reference voltage of 1V. Then, the PMT HTV 616 controls the applied voltage of the PMT that is the detector 612 so that the output of the direct current component is 1 V, which is the same as the reference voltage. As a result, AC / DC can be automatically calculated at a desired signal level.

The signal captured by the data processing device 622 is basically AC / DC, and is multiplied by the coefficient (−δ ₀ / ln10) shown in the equation (4) in the data processing device 622 to become CD data.

In the present embodiment, it is convenient for simplifying the system that the driving power of the PEM is constant in the entire wavelength region. As a result, the modulation amplitude δ ₀ changes depending on the wavelength, and is corrected as a coefficient. Instead, it is also possible to program the driving power of the PEM so that the modulation amplitude δ ₀ is constant over the entire wavelength region.

(Method for obtaining δ ₀ by actual measurement)
As described above, it is necessary to multiply the coefficients (modulation amplitude [delta] ₀₎ related to the intensity of the modulation AC / DC obtained from the detection signal of the light intensity to calculate the CD, advance the modulation amplitude [delta] ₀ is You must know. In the preceding high-speed CD time change measuring apparatus that triggered the present invention, the phase shifter 32 is mechanically moved to create two polarization states as shown in FIG. It was necessary that the phase difference was known. However, the phase shifter 32 is static and can be measured by known means such as ellipsometry. However, in the present invention, the modulation amplitude δ ₀ gives vibration to the crystal constituting the PEM 502 as shown in FIG. 1 and causes distortion from the vibration, and the distortion causes anisotropy of the refractive index in the crystal, It gives a phase difference to two orthogonal linearly polarized lights, which are dynamic in intensity. Therefore, it cannot be actually measured by ordinary means such as ellipsometry for measuring a static phase difference. If there is no means for actually measuring and determining the dynamic phase modulation intensity (modulation amplitude δ ₀ ), the present invention cannot be realized. This is also a crucial difference from the prior art.

For the purpose of the present invention, a method for obtaining the modulation amplitude δ ₀ of the dynamic PEM 502 by actual measurement will be described. As a reference sample for this purpose, an element having static birefringence is used. As such an element, a transparent uniaxial crystal plate such as crystal quartz or sapphire is suitable. A phase difference of about 0.1 radians is appropriate. Then, the phase difference is measured by ellipsometry as a spectrum with respect to the wavelength.

  The reference sample whose phase difference is obtained in this way is placed on the sample portion 503 with its axis inclined by 45 ° with respect to the X axis, that is, in a direction matching the axis of the PEM. When the measurement according to the present invention is performed in this state, the direct current component is DC = 1/2 (the light intensity when the light is linearly polarized through the polarizer is 1), and the alternating current component is expressed by equation (5). .

Δ is the phase difference of the reference sample. Since this is already known, the modulation amplitude δ ₀ of the target PEM can be obtained by Expression (6).

[Second Embodiment]
Next, a VCD measuring apparatus according to the second embodiment of the present invention will be described.
FIG. 4 is an overall configuration diagram of the VCD measuring apparatus. The basic configuration is the same as the configuration of the optical system in FIG. 1, but in the first embodiment, the monochromatic light from the spectrometer is used as the measurement light, whereas in this embodiment, the measurement is performed. The difference is that interference light from an interferometer is used as light.

  That is, light emitted from the infrared light source 700 becomes interference light that draws an interferogram through the interferometer 702. Infrared light reflected by the condenser mirror 701 is supplied to the interferometer 702. The interferometer 702 includes a beam splitter 706 that divides the measurement light into two light beams. One of the divided light beams (reflected light from the beam splitter) is reflected by the fixed mirror 705 and returns to the beam splitter 706, and the other light flux (transmitted light from the beam splitter) is reflected by the movable mirror 707 and similarly the beam splitter 706. Return to. The two light beams are combined by a beam splitter 706 and emitted as interference light. The interference light is emitted as interference light having an intensity corresponding to the moving position of the movable mirror 707, that is, an intensity corresponding to the optical path difference (D) between the two light beams.

  Note that the interference light is converted into a linearly polarized interferogram by the polarizer 708, phase-modulated by the PEM 709, and incident on the sample unit 710, which is basically the same as that of a conventional vibration CD measuring apparatus. However, the PEM is operated in a state where the modulation intensity is 0.1 radians or less, and optimally 0.01 radians over the entire wave number region in accordance with the basis of the present invention. Further, in the present invention, an analyzer 711 is newly attached after the sample portion 710. The light that has passed through the analyzer 711 is condensed on the detector 713 by the condenser lens 712. The analyzer 711 is installed in a crossed Nicols state with the polarizer 708 provided in the preceding stage.

The detection signal of the detector 713 is amplified by the preamplifier 714 as in the conventional VCD measurement device, and then the direct current component is taken in the data processing device 718 as it is. The AC component of the detection signal is extracted by a lock-in amplifier 717 synchronized with the modulation frequency of the PEM, and is taken into the data processing device 718 as a time-series signal synchronized with the position of the interferometer moving mirror. The data processing device 718 calculates an AC / DC value from each component. Although this calculation result is a signal sequence related to the CD, it is not a CD itself but an interferogram. Therefore, this is sent to the control PC 720 for data processing, where it is subjected to Fourier transform to obtain a spectrum for the wavelength (wave number ν). By multiplying this spectrum by a modulation amplitude δ ₀ having a parameter of the wave number ν, a CD spectrum can be obtained by Expression (7). F [] in the formula represents a Fourier transform.

[Proof of the measurement principle of the present invention]
Next, the appropriateness of the CD measurement principle of the present invention will be described using the Jones vector and Jones matrix methods. The linearly polarized light in the Y-axis direction after passing through the polarizer 501 in FIG. 1 is represented by the Jones vector, and J ₁ is expressed as in Expression (8).

Further, the PEM 502 that gives a modulation phase difference δ (= δ ₀ sin 2πft), a sample that exhibits circular dichroism with amplitude transmittances t ₁ and t _r for the left and right circularly polarized light, and an analyzer in the X-axis direction Jones matrix _{M PEM} shown, _M S, _{M A} is expressed by equation (9).

The Jones vector J ₂ after passing through the PEM 502, the sample portion 503, and the analyzer 504 is obtained by multiplying J ₁ by these matrices in order from the left, as shown in Equation (10).

The detected signal strength is the square of this absolute value, which is expressed by equation (11).

Since [delta] is less sin [delta = [delta], can be approximated with sin2δ = 2δ, the difference between t _l and t _r is small, it can be approximated as ^{_{(t l -t r) 2 =}} 0. Equation (11) becomes the following equation (12).

From the definition of the phase difference δ and the approximate expression of (t ₁ −t _r ) ² = 0, the expression (13) indicating δ ² and t _l · t _r is derived.

When the relationship of the above equation (13) is substituted, the intensity I finally becomes the equation (14).

Then, the alternating current component AC having the same frequency as the direct current component DC and the modulation frequency f is expressed by Expression (15).

Therefore, the relationship between CD (ΔA) and light intensity I is expressed by equation (16).

Here, I and ΔI are expressed by Expression (17).

Therefore, Expression (16) becomes Expression (18), and the validity of Expression (4) described above is illuminated.

[Proof of the measurement method of modulation amplitude]
Next, the basis of a method for actually measuring the modulation amplitude δ _{0 of the} PEM using a static phase shifter having a phase difference Δ as a reference sample will be described using the Jones vector and Jones matrix methods.

In FIG. 1, a Jones matrix M _std when a phase shifter having a static phase difference Δ is tilted in the direction of 45 ° with respect to the X-axis and used as a reference sample is expressed by Expression (19).

When this is calculated in place of M _S in equation (10), equations (20) and (21) are obtained.

Since δ is small sin2δ = 2δ, the equation (22) can be approximated with cos2δ = 1-2δ ^2.

Similar to the previous equation (13), when the equation (23) is substituted, the equation (24) is obtained.

Therefore, equation (25) is obtained, and the validity of equation (6) is proved.

(Detection sensitivity of CD of the present invention)
Next, it will be explained that the method according to the present invention is superior to the conventional method in terms of sensitivity. For this purpose, a direct current signal and an alternating current signal detected by the conventional polarization modulation CD measurement apparatus and the polarization modulation CD measurement apparatus according to the present invention are compared. For the sake of simplicity, the signal intensity is compared with the intensity of the light that is linearly polarized light passing through the polarizer as 1.0. For the sake of simplicity, the comparison is performed with the average absorbance of the sample being 1.0 and ΔA / A being 1/1000.

The signal intensity according to the conventional method is expressed by the equation (1). The DC signal DC is DC = (I ₁ + I _r ) / 2, and the AC signal AC is AC = (I ₁ −I _r ) · J _1. (δ ₀ ) = 0.5819x (I ₁ −I _r ). On the other hand, the method according to the present invention is expressed by the equation (15), and the modulation amplitude δ ₀ here is set to 0.01 which is optimum.

  Table 2 shows the calculation based on these conditions.

From this calculation result, it is necessary to detect (AC / DC) of about 1.3 × 10 ^{−3 in} the conventional method, whereas in the present invention, if (AC / DC) of about 3.7 × 10 ⁻² is detected. Well, it turned out to be about 27 times more advantageous.

  This relationship is exactly the same in the vibration circular dichroism measuring device, and the detection sensitivity of the present invention is about 27 times more advantageous for the vibration circular dichroism measuring device than in the conventional method.

(Expansion of VCD wave number range of the present invention)
In the method according to the present invention, it is optimal to set the modulation amplitude δ _{0 of the} PEM to 0.01 radians. In the wave number 1250 cm ^-1 (8 [mu] m in wavelength), when this modulation amplitude [delta] _0, the modulation amplitude [delta] ₀ as shown in Table 3 in other wavenumber. In all of these wave number regions covered by the vibration circular dichroism measurement, the measurement can be performed under conditions where a suitable sensitivity can be obtained because the modulation amplitude δ ₀ where insensitivity or sign inversion occurs is sufficiently small.

  That is, in the conventional vibration circular dichroism measuring apparatus, the measurement wave number region is limited to a region limited from the center wave number for setting the modulation intensity of the PEM, but the limitation is removed by the present invention. I understand that.

501 and 708 Polarizer (elliptic polarization modulation means)
502, 608, 709 Phase modulator (elliptical polarization modulation means)
504, 611, 711 Analyzer (polarization intensity measuring means)
505, 612, 713 detector (polarization intensity measuring means)
507, 620 DC amplifier (calculation means)
508, 619, 717 Lock-in amplifier (calculation means)
511, 622, 718 Data processing device (calculation means)
720 Control PC (calculation means)
602 Spectrometer 606 Second prism (polarizer)
702 Interferometer

Claims (6)

An elliptical polarization modulator that modulates a phase difference (δ) between two polarization components orthogonal to each other of linearly polarized light to alternately form left and right elliptically polarized light having a large flatness that matches in the major axis direction;
A polarization intensity measuring means for taking out the polarization component in the minor axis direction from the left and right elliptically polarized light transmitted through the sample to be measured, and measuring the intensity change of the polarization component;
The DC component (DC) of the intensity change and the AC component (AC) of the intensity change synchronized with the modulation of the phase difference are extracted, respectively, to obtain the ratio of the AC component and the DC component (AC / DC). A circular dichroism measuring apparatus comprising: an arithmetic unit that calculates a value obtained by multiplying the modulation amplitude (δ ₀ ) of the phase difference and acquires circular dichroism of the sample to be measured. The infrared light from the light source is divided into two so as to go to the fixed mirror and the movable mirror, and the reflected light from the fixed mirror and the movable mirror is synthesized, and the interference light according to the change in the optical path difference (D) Interfering means to form,
The linearly polarized light is extracted from the interference light, and the phase difference (δ) between the two polarization components orthogonal to each other is modulated at a modulation frequency (f) greater than the change in the optical path difference (D). , Elliptical polarization modulation means for alternately forming left and right elliptically polarized light with a large flatness matching the major axis direction;
A polarization intensity measuring means for taking out the polarization component in the minor axis direction from the left and right elliptically polarized light transmitted through the sample to be measured, and measuring the intensity change of the polarization component;
The DC component (DC) of the intensity change and the AC component (AC) of the intensity change synchronized with the modulation of the phase difference are extracted, respectively, and the ratio of the AC component and the DC component (AC / DC) is calculated vertically. A sample to be measured is calculated by performing Fourier transform on the interferogram with the axis and the optical path difference (D) as the horizontal axis, and multiplying the value after Fourier transform by the modulation amplitude (δ ₀ ) of the phase difference. And a circular dichroism measuring device. The measuring apparatus according to claim 1 or 2,
The elliptically polarized light modulation means includes a polarizer that extracts linearly polarized light from incident light, and a phase modulator that modulates the linearly polarized light to form left and right elliptically polarized light,
The polarization intensity measuring means includes an analyzer that transmits linearly polarized light in the short axis direction of the left and right elliptically polarized light transmitted through the sample to be measured, and a detector that detects the intensity of the transmitted linearly polarized light. ,
The circular dichroism measuring apparatus, wherein the analyzer is installed in a crossed Nicol direction with respect to the polarizer. The measurement apparatus according to any one of claims 1 to 3, wherein the measurement apparatus acquires a circular dichroism spectrum in a predetermined measurement wavelength region,
The circular dichroism measuring device characterized in that the modulation amplitude (δ ₀ ) of the phase difference (δ) of the elliptically polarized light modulating means is 1/10 radians or less in the entire region of the measurement wavelength region. An elliptical polarization modulation step of alternately forming left and right elliptically polarized light having a large flatness that coincides in the major axis direction by modulating a phase difference (δ) between two polarization components orthogonal to each other of linearly polarized light;
A polarization intensity measuring means for taking out the polarization component in the minor axis direction from the left and right elliptically polarized light transmitted through the sample to be measured, and measuring the intensity change of the polarization component;
The DC component (DC) of the intensity change and the AC component (AC) of the intensity change synchronized with the modulation of the phase difference are extracted, respectively, to obtain the ratio of the AC component and the DC component (AC / DC). A circular dichroism measurement method comprising: a calculation step of calculating a value obtained by multiplying the modulation amplitude (δ ₀ ) of the phase difference to obtain circular dichroism of the sample to be measured. The measurement method according to claim 5, wherein
Further, the infrared light from the light source is divided into two so as to go to the fixed mirror and the movable mirror, and the reflected light from the fixed mirror and the movable mirror is synthesized to respond to the change in the optical path difference (D). An interference light forming step for forming interference light;
In the elliptical polarization modulation step, linearly polarized light is extracted from the interference light, and a phase difference between two polarization components orthogonal to each other at a modulation frequency (f) larger than a change in the optical path difference (D). Modulate (δ),
In the calculation step, the direct current component (DC) of the intensity change and the alternating current component (AC) of the intensity change synchronized with the modulation of the phase difference are respectively extracted, and the ratio of the alternating current component and the direct current component ( AC / DC) is the vertical axis, and the optical path difference (D) is the horizontal axis. The interferogram is subjected to Fourier transform, and the value obtained by multiplying the value after Fourier transform by the modulation amplitude (δ ₀ ) of the phase difference is calculated. Then, the circular dichroism measuring method characterized by acquiring circular dichroism of a sample to be measured.

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