백서

An Introduction to THz-Raman Spectroscopy

Overview

The terahertz (THz) region of the electromagnetic spectrum, from roughly 0.15 THz to 6 THz (5 cm-1 to 200 cm-1), has long been researched as a means of investigating low-energy vibrational modes of materials. This region is particularly attractive to spectroscopists studying intermolecular vibrations within crystals [1], yielding important information about the molecular orientation that governs many key properties of materials. On the most fundamental level, THz spectroscopy is an extension of far-infrared (FIR) absorption spectroscopy. In practice, it is far more challenging to implement because it is at the extreme low-end of the optical frequency domain and extreme high-end of the frequency domain for electronics. That is why this region of the spectrum is often referred to in the published literature as the “terahertz gap” [2]. Currently, commercial THz spectroscopy systems are available on the market, but most are extremely expensive and difficult to use due to the complexity of the ultrafast lasers typically required to generate THz radiation and susceptibility of samples to moisture.

Fortunately, direct absorption of terahertz radiation is not the only way to access THz-domain vibrational modes as many are also Raman active. Raman scattering results from the inelastic scattering of photons, not absorption, therefore decoupling the excitation wavelength from the vibrational modes under investigation. In traditional Raman, the spectral range of 200 cm-1 to 1800 cm-1 represents the “chemical fingerprint,” since most intramolecular vibrations occur in this frequency range. In a complementary way, the THz-Raman™ (also referred to as low-frequency Raman or LFR) region, from 5 cm-1 to 200 cm-1, provides a “structural fingerprint” that corresponds mainly to inter-molecular vibrations, or lattice/phonon modes of the material. Figure 1 shows the relative wavelength locations where THz spectroscopy (50 μm to 2 mm), IR spectroscopy (5 to 50 μm) and THz-Raman spectroscopy occur within the electromagnetic spectrum. Figure 2 correspondingly shows the relative locations of the chemical and structural fingerprints of a typical Raman spectrum.

Raman spectroscopy historically has been challenging to implement in the THz-domain because the inelastic scattering (Raman shift) is orders of magnitude weaker than the elastic scattering (Rayleigh), making it hard to adequately filter out the Rayleigh scatter. As a result, it was difficult to resolve signals with Raman shifts very close to the Rayleigh line (0 cm-1). This situation completely changed with revolutionary developments in volume holographic notch filters that have made it easy to directly measure Raman shifts as small as 5 cm-1 with just a simple monochromator featuring high optical efficiency [3]. This breakthrough has resulted in a paradigm shift for low-frequency Raman: from a relative novelty requiring complex multistage monochromators, to self-contained spectrometers that any scientist can set-up in a matter of minutes, as well as plug and play add-on modules for existing instruments. For example, these THz-Raman modules include immersion probes for reaction monitoring, microscope platforms for mapping the phase of samples, and well plate readers for high-throughput screening applications.

그림 1: 여러 다른 분광 기법의 위치를 보여주는 전자기 스펙트럼 - 라만 분광법은 일반적으로 가시광선(532nm) 또는 NIR(785nm)에서 수행되는 반면 IR 흡수 분광법은 5~50μm에서 THz 분광법은 50μm~2mm에서 수행됩니다.

THz-Raman System Requirements

Every THz-Raman system has four things in common:

  1. A wavelength stabilized laser source
  2. Narrowband (< 5 cm-1) spectral clean-up filters
  3. Narrowband (< 5 cm-1) Rayleigh scatter blocking filters
  4. A spectrometer that can detect the low frequency signals

The Rayleigh filter, a +/- 5 cm1 bandwidth notch filter, should be the limiting factor to how low into the THz-domain the system can detect. However, for the Rayleigh filter to function properly, it is critical that the laser’s output spectrum be extremely stable, have a very narrow linewidth, and exhibit extremely low noise from amplified spontaneous emission (ASE) or side bands within the transmission region of the notch filters. It is also essential that the center wavelengths of both the filters and the laser remain “spectrally synchronized” to one another during operation to avoid leaking of the Rayleigh signal and saturation of the spectrometer’s detector. As an integrated manufacturer of lasers, filters and optical systems, Coherent spectrometers and modules are all designed to perfectly maintain this synchronization, ensuring robust, easy to operate performance at all times.

An additional advantage of these systems is that since Raman spectra are measured as a relative shift from the laser frequency, THz-Raman systems can use lasers at any wavelength in the visible or near infrared (NIR) to probe these low-energy vibrational modes, eliminating the need for a THz laser source. This enables a much simpler and less-costly spectral collection scheme, allowing for the use of glass optics and fibers, compact and inexpensive diode and diode-pumped solid-state (DPSS) laser sources, and silicon detectors and detector arrays. THz-Raman systems are also capable of detecting anti-Stokes Raman shifts, less than -5 cm-1, which provide additional information about the sample, including local effective temperature. Therefore THz-Raman systems can simultaneously measure both the chemical and structural fingerprints of materials with high throughput in a single measurement.

The combined benefits of THz spectral information with the ease of use of Raman spectroscopy now make it a powerful solution for applications that are interested in both the chemical and structural properties of materials. THz-Raman has been rapidly adopted by the pharmaceutical industry [4-9] and is also getting traction in polymers [10], semiconductors [11-13], and biomedical diagnostics [14].

 

Low-frequency Vibrational Modes

As described above, low-frequency peaks result from intermolecular vibrations such as phonon modes and lattice vibrations. In crystalline samples, the position (shift) of the peaks are specific to the composition (chemical and structural) details of the crystal. And the bandwidth and intensity of these sharp low-frequency peaks are directly related to the degree of structure (crystallinity) of the sample. In contrast, amorphous solids and liquids have a broad unresolved peak known as the boson peak. Therefore, the THz-Raman spectrum is a useful tool to quantitatively analyze sample crystallinity and to classify different allotropes and polymorphs. Although polymorphic analysis is a more popular application, it is easier to understand by examining the THz-Raman spectra of different allotropes to better illustrate the properties of low-frequency vibrational modes.

그림 2: 0~200cm-1 사이의 분자 간 진동 모드(A) 및 200cm-1~500 cm-1 사이의 분자 내 진동 모드(B)를 보여주는 α황 및 β황의 라만 스펙트럼

30개 이상의 동소체를 가지고 있는 황은 생명 과학 및 산업 응용 분야 모두에서 광범위한 화학 공정에서의 중요성 때문에 폭넓게 연구되어 왔습니다[15]. 동소체 α황은 24가지 다른 분자 간 진동을 가지고 있는 것으로 나타났으며, 그중 다수는 스펙트럼의 30cm-1~100cm-1 영역에서 라만 활성입니다[16]. 2013 SPIE Defense, Security, and Sensing 컨퍼런스에서 발표된 한 논문에서, Heyler 등은 THz-Raman을 사용하여 이러한 진동 모드를 감지할 수 있는 방법을 설명했습니다[3]. 또한 α황 샘플이 95.6°C 이상으로 가열됨에 따라 동소체 β황으로 형태가 변화된 후 115.2°C에서 액화된다는 것을 보여주었습니다. 보손 피크 구조에 더 가까워짐에 따라 증가된 불규칙성이 100cm-1 미만의 라만 스펙트럼에서 모드를 "흐릿하게" 하기 때문에 α 형태(사방정계)에서 덜 규칙적인 β 형태(단사정계)로의 전이를 쉽게 감지할 수 있습니다[그림 2]. 용해되고 y 형태(액체)를 형성한 후, THz-Raman 영역의 구조 모드가 완전히 혼합되어 일반적인 액체의 전형적인 순수 보손 대역만 표시됩니다[17]. 대조적으로, 화학적 지문 영역의 모드는 100cm-1이상의 스펙트럼의 피크 위치를 비교할 때 명백히 형태 변화에 의해 크게 영향을 받지 않았습니다. 그림 2의 모든 스펙트럼은 직립 현미경 및 파이버 결합 분광계에 부착된 Coherent TR-MICRO THz-Raman을 사용하여 수집되었습니다. 그림 3은 개략적으로 나타낸 구성도입니다.

그림 3: α황 및 β황의 THz-Raman 스펙트럼을 수집하는 데 사용된 실험 기구를 개략적으로 나타낸 구성도

The THz-Raman unit contains a single frequency excitation laser, precisely matched to ultra-narrow-band (volume holographic) laser line and notch filters. The notch filters are specifically designed to ensure maximum throughput of the Raman scatter while also attenuating the Rayleigh scatter with an optical density (OD) greater than 9, enabling their use with inhomogeneous samples. Finally, the remaining Raman scatter is coupled to a spectrometer through a fiber optic cable. A more detailed explanation of the optical design is available in the conference proceedings [3].

 

Applications in Pharmaceuticals

Polymorphism is a critical and common characteristic of active pharmaceutical ingredients (APIs). It has a direct impact on drug substance bioavailability, manufacturability, and quality/performance [18]. Since polymorphic compounds have the same base molecular composition but different bulk structural orientations, THz-Raman is far better suited than conventional infrared (IR) and Raman spectroscopies to determine the polymorphic form. For the sake of completeness, it should be noted that in some cases, polymorphic differences can lead to subtle peak shifts in the chemical fingerprint region of the Raman spectra [19,20] due to damping of intramolecular vibrations. That said, the spectral changes in the low-frequency range tend to be far more pronounced (up to 10x stronger) and more easily differentiable without the need for complex chemometric analysis. Additionally, as Larkin et al. demonstrated in a 2014 article published in Applied Spectroscopy, “Low-frequency Raman spectra of large aromatic species typical of APIs provide remarkably intense bands below 200 cm-1 with complex spectral features [9].” They went on to explain that these bands are typically an order of magnitude more intense than those of the surrounding excipients in the same frequency range, which provides the ability for THz-Raman to directly measure “crystalline structure, crystalline disorder, and amorphous states,” with enhanced sensitivity. 

그림 4: Coherent의 TR-BENCH(탁상형 THz-Raman 모듈)

 

Between the study mentioned above [9] and a follow-up study published in 2015 [10], researchers at Bristol-Myers Squibb have provided a detailed analysis of the low-frequency Raman bands for polymorphs of some common APIs. In those two publications, they analyzed indomethacin, carbamazepine, caffeine, theophylline, and apixaban. Larkin et al. used a benchtop THz-Raman sampling system, similar to the TR-BENCH currently available from Coherent shown in figure 4. The TR-BENCH has the same internal optical architecture as the TR-MICRO discussed in the previous section. As an example of this research, figure 5 shows the THz-Raman spectra of three different forms of carbamazepine. 

그림 5: (A) 형태 III, (B) 의사 다형 이수화물 형태 및 (C) 형태 II의 카르바마제핀에 대한 THz 라만 스펙트럼 (모두 실온에서 측정됨[8])

 

In recent years, THz-Raman spectroscopy has transitioned out of research laboratories and into pharmaceutical process monitoring applications, often referred to as process analytical technology (PAT) [21-24]. In process applications, fiber-coupled THz-Raman probes enable in-line, on-line, and at-line measurement, where the analyzer can be remote from the sample and does not need the sample to be captured and brought to the analyzer. Depending on the demands, the probe tip can be designed with a short working distance for direct immersion into a reaction chamber through an access port, or with a longer working distance to collect spectra through a viewing window. Figure 6 shows an example of the TR-PROBE with an immersion probe tip attached.

thz-raman-intro-figure-6.jpg

그림 6: 11인치(27.94cm) 길이 스테인리스 스틸 소재의 침지 팁이 부착된 Coherent의 TR-PROBE(THz-Raman 프로브 모듈)

 

Inoue 등은 다양한 농도의 에탄올과 물에서 카르바마제핀 III형에서 카르바마제핀 이수화물로의 전이를 모니터링하기 위해 이 접근 방식을 사용했습니다[22]. 그림 7에 표시된 결과는 카르바마제핀 이수화물(111 cm-1) 및 카르바마제핀 III(39cm-1)의 지배적인 THz-Raman 대역을 가진 다변량 곡선 분해능(MCR) 알고리즘을 사용하여 계산되었습니다. 이러한 데이터를 기반으로 연구원들은 이 반응의 경우 62.5% 에탄올과 37.5% 물의 용액 사용 시 가장 빨리 변환했음을 알아낼 수 있었습니다. 

thz-raman-intro-figure-7.jpg

그림 7: 에탄올과 물의 다양한 용제 비율에서 카르바마제핀 III형이 카르바마제핀 이수화물로의 변환에 대한 농도 동역학[21]

The Future of THz-Raman

While the pharmaceutical industry was the first to widely adopt THz-Raman, other sectors are using it to analyze crystallinity and polymorphism as well. A recent example is the use of THz-Raman to explore the relationship between mobility and strain [25], as well as charge transport and low-frequency vibrations [12] in organic semiconductors. It is also being used to analyze phonon modes in quantum dots [11], and layered semiconductor alloys [13]. Recently studies have been also published on polymer crystallization [10] and the formation of lamella during the cooling process [26], providing critical information about structural properties of products for the polymer industry. Perhaps the most exciting up and coming applications of THz-Raman are in biology and biomedical diagnostics. At the 2019 SPIE BiOS conference in San Francisco, Marble et al. presented the first talk on using THz-Raman for biological molecules [14], and a year later, THz-Raman was already being explored as a potential diagnostics tool for COVID-19 [27].

 

Summary

THz-Raman has repeatedly been shown to provide users with robust and simultaneous structural and chemical compositional information. There is no doubt that the applications will continue to grow and expand as THz-Raman analyzers transition out of the lab and into industrial settings. It is inevitable at this point that researchers will continue to find new uses for the highly differentiating information revealed by THz-Raman that have not yet been conceived. For additional information on THz-Raman instrumentation, applications, or capabilities, please visit www.thz-raman.com or request a consultation with an applications scientist via our website www.coherent.com

 

References:

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[2]  Dexheimer, S.L. ed., 2017. Terahertz spectroscopy: principles and applications. CRC press.
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[21] Inoue, M., Hisada, H., Koide, T., Carriere, J., Heyler, R. and Fukami, T., 2017. In situ monitoring of crystalline transformation of carbamazepine using probe-type low-frequency Raman spectroscopy. Organic Process Research & Development, 21(2), pp.262-265. 
[22] Nomura, K., Titapiwatanakun, V., Hisada, H., Koide, T. and Fukami, T., 2020. In situ monitoring of the crystalline state of active pharmaceutical ingredients during high-shear wet granulation using a low-frequency Raman probe. European Journal of Pharmaceutics and Biopharmaceutics, 147, pp.1-9.
[23] Walker, G., Römann, P., Poller, B., Löbmann, K., Grohganz, H., Rooney, J.S., Huff, G.S., Smith, G.P., Rades, T., Gordon, K.C. and Strachan, C.J., 2017. Probing pharmaceutical mixtures during milling: The potency of low-frequency Raman spectroscopy in identifying disorder. Molecular Pharmaceutics, 14(12), pp.4675-4684. 
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