The CD-Spectrometer

The CD-spectrometer utilized in this work is an Aviv Model 42 Spectrometer manufactured by Aviv® biomedical, inc. It allows time- and wavelength dependent CD-measurements in a wide wavelength range (200 – 2000 nm) with an excellent resolution of up to 0.1 nm.

The light source is a commercially available tungsten halogen low-voltage lamp (OSRAM HLX 64663) with a nominal voltage of 36 V and a nominal wattage of 400 W. The luminous flux is 16200 lm and the color temperature is 3250 K. According to Planck’s law for black body radiation^122,123, this color temperature corresponds to a maximum in spectral energy density u (,T) at approximately 900 nm, as shown in Figure 17. At higher wavelengths, the spectral energy density slowly decreases while at lower wavelengths this decrease is much steeper. The light source is placed in a ventilated box at the backside of the spectrometer and a screw allows the adjustment of the height in order to optimize the light intensity reaching the sample (Figure 17).

The polychromatic light provided by the light source is guided to a Cary 14 double monochromator, which disperses the light into its individual wavelengths. It consists of a Czerny-Turner fused silica prism monochromator in series with a 600 lines/mm echelette grating. Figure 18 illustrates the arrangement of the optical elements: The radiation from the lamp enters the monochromator through the entrance slit, gets dispersed by the prism and the grating and the resulting monochromatic radiation leaves through the variable exit slit.¹²⁴ The prism-grating design not only improves wavelength resolution compared to single monochromators but also reduces stray light.

Figure 17: Left: Black body radiation spectrum at 3250 K. Around 200 nm, the energy density gets close to zero and determines the high energy limit of the spectrometer. Right: Photograph of the halogen low-voltage lamp used in the CD-spectrometer.

Design and Setup of the MCD-Spectrometer 53

Figure 18: Arrangement of the optical elements in the prism-grating double monochromator.¹²⁴

While prisms suffer from light absorption of the glass leading to poor dispersion curves in the NIR, gratings exhibit more uniform dispersion curves. However, gratings show different orders of reflection besides the used first order. Due to second-order stray light at a given angle, multiples of the prime wavelengths may be reflected. One way to solve these issues is the combination of prisms and gratings: At long wavelengths, a prism does not separate the wavelengths efficiently anymore, but it eliminates second order stray light. The grating then provides the necessary spectral resolution.¹²¹

The dispersive elements are connected to a wavelength cam, which converts the non-linear dispersion into the non-linear motion of an external gear drive mechanism moved by a stepper motor. Each motor step corresponds to a wavelength change of 0.01 nm and thus sets the limit for wavelength specification. The wavelength repeatability is better than 0.05 nm in the entire wavelength range (typically 0.02 nm) and the spectral resolution is ca. 0.1 nm in most of the UV-Vis range. Due to the linear wavelength scale, only one point needs to be specified for wavelength calibration. The calibration wavelength corresponds to the position of an optical beam switch linked to the motion of the wavelength cam. More details about wavelength calibration will be given below. The monochromator slit widths contributing to the spectral bandwidth are controlled by a stepper motor, and a potentiometer on the drive senses the slit positions. The bandwidth can be chosen between 0.005 nm and 10 nm and in the constant bandwidth mode the spectrometer software converts the desired bandwidth to the corresponding slit width using a stored version of the monochromator dispersion function.

The slit height can be manually regulated using a knob adjusting a mask. This option allows optimizing the light intensity and the beam position on the sample.

After the monochromator, an achromatic lens and a Rochon polarizer generating linearly polarized light are placed. In the Aviv Model 42 CD Spectrometer, the Rochon polarizer consists of two optically connected prisms of single crystal magnesium fluoride.

Magnesium fluoride was chosen because of its high and rather uniform transparency in the spectral range from 200 to 6000 nm,¹²⁵ making it an ideal material for the application in the desired MCD-spectrometer. It exhibits a tetragonal, i.e. uniaxial crystal system resulting in optical anisotropy. Due to different refractive indices along different axes, birefringence is observed whenever the light path is not parallel to the principal axis.^121,126 A schematic illustration of the utilized Rochon polarizer is given in Figure 19.¹²⁷ The optical axes of the MgF2 prisms are oriented perpendicular to each other, with one of them being parallel to the direction of light propagation. At the interface, double refraction occurs and the incident ray splits up into two separate beams with vertical and horizontal polarizations, i.e. the ordinary and extraordinary ray. While the ordinary beam passes straight through the polarizer, the extraordinary ray is refracted. The angular separation between the ordinary and the extraordinary ray is 5.1 degrees at 200 nm and 4.6 degrees at 546 nm.¹²⁷ For the CD- or MCD-measurements in this work, the extraordinary ray is discarded by a mask after the photoelastic modulator and only the ordinary ray is forwarded to the sample (single-beam setup).

As shown in Figure 20, the photoelastic modulator (PEM)¹²¹ is positioned after the Rochon polarizer and converts the linearly polarized light into circularly polarized light. The PEM consists of a metal plated crystalline quartz block, which acts as a large piezoelectric oscillator with a resonance frequency of 50 kHz.

Figure 19: Schematic illustration of the MgF2 Rochon polarizer utilized in the Aviv Model 42 CD Spectrometer.

OA denotes the optical axes of the MgF2 prisms and D denotes the angular separation between the ordinary and the extraordinary ray. Reprinted with permission from Karl Lambrecht Corporation, Chicago.¹²⁷

Design and Setup of the MCD-Spectrometer 55

Figure 20: Left: Polarizer compartment containing an achromat, the Rochon polarizer and the photoelastic modulator. A cuvette holder behind the PEM allows conventional CD-measurements at room temperature. Right:

View through the fused silica block of the PEM towards the polarizer.

At one end it is attached to a clear fused silica block where the light passes through. By applying a varying voltage to the metal plated part (1 V per 400 nm), a mechanical strain is induced, which is transferred as pressure waves to the fused silica block. The pressure waves cause birefringence in the usually optically isotropic fused silica, resulting in different refractive indices for light with vertical and horizontal polarizations. The PEM is mounted at a 45 degree angle relative to the linear polarization of the incident rays, i.e. relative to the PEM the incident light exhibits equal portions of vertical and horizontal polarization in phase with each other. Due to the strain-induced birefringence, the vertical and horizontal components traverse the glass at different rates, leading to a phase shift when the light emerges the PEM.

Since the pressure waves are continuously passing back and forth through the fused silica block, the phase shifts periodically between +90 and –90 degrees resulting in alternatingly left and right circularly polarized light. Different conventions regarding the definition of lcp and rcp exist. The convention used throughout this work is demonstrated in Figure 21: Lcp corresponds to the case where the electric field vector rotates counter-clockwise when propagating towards the observer, while rcp corresponds to a clockwise rotation.¹²⁸ The periodically alternatingly left and right circularly polarized radiation passes the sample, which is either placed inside a cuvette behind the PEM (see Figure 20) for conventional CD-measurements at room temperature or inside a magnet for MCD-CD-measurements.

Detection is carried out using a photomultiplier (PMT) for the UV and visible range and an indium gallium arsenide (InGaAs) photodiode for the NIR range. As shown in Figure 22, both detectors are placed in the detector compartment, which is detachable from the polarizer compartment in order to allow MCD measurements. A software controlled detector motor allows automatic detector crossover at a user specified wavelength.

Figure 21: Definition of left and right circularly polarized light used throughout this work.

The PMT is a custom-built end window photomultiplier tube manufactured by Hamamatsu Photonics K.K. with an S20 response range from 190 to 870 nm. S20 is the spectral number and refers to multialkali photocathodes. During the CD-measurements an adjustable voltage is applied to the dynodes while the DC current induced by the photoelectric effect is held constant. This leads to increased sensitivity. The applied voltage is thus a measure for the light intensity reaching the detector, e.g. low light intensity due to absorption by a sample leads to a positive peak in the dynode voltage. The typical dynode voltage profile between 200 and 900 nm for the model 42 MCD spectrometer is shown on the right hand side in Figure 22. It was recorded without any sample and with the detector compartment being directly attached to the polarizer compartment. The DC level was fixed to the default value of 1 V and the bandwidth was set to 1 nm.

Figure 22: Left: Detector compartment containing the InGaAs NIR detector and the photomultiplier tube. Right:

Characteristic dynode voltage profile of the photomultiplier recorded at a fixed bandwidth of 1 nm and a DC level of 1 V.

Design and Setup of the MCD-Spectrometer 57 The increase of the dynode voltage at low wavelengths ( < 300 nm) is attributed to the emission limit of the light source (Figure 17) and the limited transmission of the optics. At high wavelengths ( > 800 nm), a second increase is observed because the lower energy of the incoming photons leads to less emission of electrons from the metal. Very high dynode voltages result in increased noise in the CD-spectrum and if the dynode voltage reaches a plateau above ca. 800 V, the CD-measurement is not reliable at all anymore. When measuring an absorbing sample, the bandwidth should be chosen in a way that finds a compromise between the spectral resolution and the light intensity reaching the detector.

The peaks arising in the PMT voltage due to light absorption by a standard sample were employed for the wavelength calibration of the spectrometer. The wavelength calibration was performed in factory by using a solution of 40 g L^-1 holmium oxide in 10 % (volume fraction) perchloric acid and repeated during the installation of the instrument. The solvated Ho³⁺ cation has a very stable coordination and shows characteristic narrow f-f-transitions. Most of the observed bands in the visible range are thus NIST-certified (NIST:

National Institute of Standards and Technology) as intrinsic traceable wavelength standards.¹²⁹ Especially the most intense peak attributed to the transition to the ⁵F4 free ion state and located at 536.4 nm for a spectral bandwidth of 0.1 nm¹²⁹ was employed for assigning the calibration wavelength (the so-called “home wavelength”). The calibration wavelength corresponds to the starting position of the optical beam switch linked to the motion of the wavelength cam.

The InGaAs NIR detector was manufactured by Teledyne Judson Technologies (model J23D-M204-R02M-60-2.6-CSW) and operates over the spectral range from about 800 nm to 2200 nm. In order to reduce the dark current, the detector is surrounded by a metal dewar (model M204) enabling cooling with liquid nitrogen. Suprasil quartz glass was chosen for the windows. Since the detector’s active area is only 2 mm, special care has to be taken when focusing and adjusting the incoming light beam. Details about the utilized optics and optomechanics will be given in section 4.1.4.

If a CD active sample is placed in the light path, lcp and rcp will be absorbed to a different extent and the light intensity after the sample will oscillate with the resonance frequency of the PEM. Compared with the overall light intensity, this oscillation is very small and as a result a signal with a small AC component superimposed on the DC component is obtained. The signal path is schematically shown in Figure 23: The components of the detected mixed signal are separated with the help of a band-pass filter and after amplification the AC signal reaches a sample-and-hold circuit, which also receives square wave time

signals from the PEM. The sample-and-hold circuit samples the signal at a given time and stores the information for a given time interval. The time signals arriving at the sample-and-hold circuit correspond to the phase shifts of +90 and –90 degrees and are thus used to trigger the measurement of the lcp and rcp amplitudes. The difference is the amplitude of the AC signal and is sent to the computer, where an A/D card samples the AC amplitude as well as the amplified DC signal. The software builds the AC/DC ratio, which is scaled to become the CD signal. CD signal intensity calibration is performed by adjusting the gain on the amplifier in the AC signal circuit.

Also the CD signal intensity calibration was performed in factory and repeated during the installation of the instrument. As a reference sample, a solution of 1.0 mg mL^-1 (1S)-(+)-10-camphor sulfonic acid (CSA) in water was used that shows an intense positive CD signal at 290.5 nm. However, after adding the additional optics for the MCD-setup, some slight recalibration was required and the corresponding data will be discussed in section 4.1.5.

Both detectors use the same circuit but for the InGaAs detector the data need to be corrected for the dark current. The dark signal is measured with the help of a (closed) chopper, which is located between the light source and the monochromator entrance slit (not shown in Figure 18). The CD signal is then calculated as

𝐶𝐷_𝑁𝐼𝑅 = 100 ∙ ∆𝐺𝑎𝑖𝑛 ∙ ∆𝐼 − ∆𝐼_{𝑂𝑓𝑓𝑠𝑒𝑡}

𝐼 − 𝐼_{𝐷𝑎𝑟𝑘}− 𝐼_{𝑂𝑓𝑓𝑠𝑒𝑡} (64) where I and I are the measured total intensity and intensity difference and IDark accounts for the dark signal. IOffset is a correction for electrical offsets and IOffset is an optional additional correction besides the dark correction. The factor 100 is a scaling factor for converting voltage to millidegrees and Gain is included to correct for differences between the PMT and the InGaAs detector outputs. Signal calibration in the NIR range thus includes the appropriate adjustment of the offset parameters.

Figure 23: Schematic illustration of the electronic circuit used to extract the CD signal (according to a draft by Dr. Glen Ramsay, Chief Scientist at Aviv Biomedical, Inc.).

Design and Setup of the MCD-Spectrometer 59