Pharma Applications of THz-Raman Quantitative Analysis


THz-Raman™ spectroscopy (also called low-frequency Raman) is based on the detection of vibrations in the 0-200 cm-1 range in addition to the chemical fingerprint region from 200-1800 cm-1 collected by conventional Raman. It has recently emerged as a powerful quantitative analysis modality that provides real-time sample information about structure and phase, with all the advantages – ease of use, remote sensing, and direct measurement – typically associated with optical spectroscopy techniques such as NIR or FTIR. This structural and phase information can be particularly important in pharma applications, but it previously required cumbersome sample prep and the use of more complex off-line methods such as powder x-ray diffraction (PXRD). THz-Raman systems now can be used as standalone instruments or integrated with existing conventional Raman instruments to increase their sensitivity in the low wavenumber region. They are available for use with immersion probes, as benchtop instruments for sample vials, as microscope accessories, and integrated in well plate readers for high throughput screening applications. There are numerous applications in the pharmaceutical industry, and this white paper examines some representative applications selected to highlight the breadth of THz-Raman utility for pharma.

Polymorph Identification

Many active pharmaceutical ingredients (APIs) can be crystallized in multiple forms or polymorphs. This is particularly important in poorly soluble drugs, where various polymorphs can have quite different dissolution characteristics in the gastrointestinal tract, impacting their bioavailability and hence efficacy. In the past, the polymorphism of many drugs was detected fortuitously or through manual time-consuming methods. Today, drug crystal engineering via combinatorial chemistry and high-throughput screening, makes it possible to easily and exhaustively identify stable polymorphic and/or hydrate/anhydrous forms. This means that rapid and reliable identification of polymorphs during the development, manufacturing, and quality assurance process is critical in pharmaceutical manufacturing.

Polymorphs have the same molecular composition but only differ in the way the molecules are packed in a crystal lattice. So traditional optical analysis methods struggle to identify different polymorphs. For example, FTIR, NIR, and conventional Raman analysis are all based on measuring spectral peaks due to intramolecular vibrations that indicate molecular composition. Consequently, polymorph analysis applications have often resorted to other methods such as powder x-ray diffraction (PXRD) or solid-state NMR (ss-NMR), requiring prep of captured samples and complex instrumentation. THz-Raman has been shown to be ideal for this application because it measures low-energy vibrations in samples and these vibrational spectral peaks arise mainly from intermolecular vibrations such as phonon modes and lattice vibrations. The THz-Raman spectrum is thus highly sensitive to even subtle differences in crystal forms. Also, since THz-Raman does not require any special sample preparation, it is compatible with Green Chemistry initiatives to reduce solvent use.


図 1:明確に区別できるピークを示すさまざまなAPIの多形のTHz-Ramanスペクトル。

たとえば、Applied Spectroscopy誌の論文[1]では、Coherent(旧Ondax)とBristol-Myers Squibbの科学者が、カルバマゼピン(CBZ)、カフェイン、テオフィリン、アピキサバンなど、多くの共通するAPIの多形と非晶質を定量的に区別する目的で、THz-Raman分析を使用することに成功したことを実証しています。研究チームは、THz-Ramanがいわゆる光学フォノンを検出し、芳香族分子構造では約130 cm-1(4THz)までの周波数範囲が期待できると説明し、各多形体が格子モードといくつかの低周波(ねじれなど)分子内振動からなる特徴的なTHz Ramanシグネチャーを持っていることを示しました。 

関連する研究で、Coherent [2]の科学者は、インドメタシン、プロブコール、アセトアミノフェンなど、他のいくつかのAPIの多形間の明確なスペクトルの違いを示しました。図1を参照してください。同科学者たちは、THz-Ramanシステムが多形の高速で明確な区別を提供し、化学的同定に使用される高周波ラマン「化学的指紋」領域を完全に補完するとまとめました。


Co-Crystal Formation

Optimizing polymorphs alone is not always sufficient to enable formulation of an API with the requisite thermal stability, dissolution rates and so on. So the pharma industry has developed a range of other approaches to modify these properties. An example is the use of cocrystals to regulate the API’s solubility, stability, and bioavailability. Cocrystals are molecular crystals formed by weak intermolecular interactions such as hydrogen bonding between two different molecular or ionic compounds. They are also of interest in explosives, agrochemicals, and pigments. In pharma, their main use is with low-solubility APIs.

A cocrystal consists of an active pharmaceutical ingredient (APIs) and a so-called coformer. Different cocrystals are often possible with the same API/coformer pairing, based on different stoichiometries. For example, the carbamazepine:4-aminobenzoic acid cocrystal system can exist in 1:1, 2:1, and 4:1 stoichiometric combinations, each with specific dissolution rates and other important physiochemical properties. Cocrystals can be formed by a variety of solid state, slurry, and solution-based methods, and cocrystal preparation remains an active area of research.

Thus, there is a common need from research through QA, screening, and in-process monitoring for a reliable and fast method to quantitatively identify specific cocrystals. Traditional optical methods are focused on spectral peaks based on covalent bonds and can at best show only subtle data differences between cocrystals and mixtures and then between different cocrystals. Powder x-ray diffraction (PXRD) and nuclear magnetic resonance (NMR) spectroscopy can provide the data but need captured and prepared samples and costly instrumentation for off-line measurements. However, since THz-Raman is an optical method that mainly detects intermolecular vibrations and lattice modes it is very well-suited to this analysis challenge as shown by the example in figure 2.



Industrial & Industrial Chemical Research [3]に掲載された論文で、Coherent(旧Ondax)、明治薬科大学(東京都清瀬市)、国立医薬品食品衛生研究所(東京都世田谷区)の研究者は、THz-Raman分光器によって共結晶形成を定量的に監視できることを示しました。具体的には、エタノールを溶媒とする反応晶析法(RCM)によって生成するカルバマゼピンと4-アミノ安息香酸の1:1および2:1共結晶を研究しました。THz-Ramanデータは、液浸プローブにファイバー結合した分光器によりリアルタイムで取得され、多変量曲線分割(MCR)に基づくデータフィッティングが行われました。また、試料の定量分析だけでなく、化学量論が異なる共結晶間の変化を追跡し、反応速度や終点までの時間を決定できることも示して、共結晶化プロセスにおけるリアルタイム有用なプロセス解析ツールとしてのTHz-Ramanの有用性が確認されました。


図 3: カルバマゼピンと4-アミノ安息香酸の1:1共結晶から2:1共結晶への変換をTHz-RamanスペクトルデータのMCRフィッティングに基づきリアルタイムでモニタリングしたもの。[3]より許可を得て転載。

Crystallinity vs. Amorphous Monitoring

The low frequency Raman spectrum is dominated by sharp peaks due to crystal lattice modes and optical phonons, which means that a THz-Raman analyzer can measure the concentration of a particular crystal in a mixture of phases, i.e., act as a “crystallinity monitor” [4] The gold standard in crystal identification is powder x-ray diffraction (PXRD) which produces data in the form of a pattern of bright spots at specific diffraction angles based on the lattice properties of the material. However, amorphous materials produce a broad halo of diffracted x-rays due to their semi-random, disordered orientation of molecules, which acts as a noise floor and sets the limit of detection (LOD). This means that it is usually not possible to reliably measure crystallinity content with PXRD, when that content falls below about 5%.







Coherent THz-Raman分光アプリケーションラボの他のシステムからのデータは、プロセスモニタリングのケースでは、より単純な分析でも結晶化度を定量化するのに十分であることを示しています。具体的には、非晶質背景の幅広いボソン特徴に比べ、結晶性ピークは普遍的にシャープであるため、低周波領域の平均スペクトル微分(傾き)の分析だけで結晶性の程度を近似的に知ることができる場合がよくあります。


Multiple Hydrates (Pseudo-Polymorphism)

Pseudopolymorphism refers to crystal phases of the same compound that vary by the amount of solvent material incorporated in the crystal lattice. For example, in the case of aqueous mixtures these take the form of hemi-, mono-, di-, and trihydrate phases, as well as mixtures of these. In another published study [5], scientists at Coherent (formerly Ondax), Meiji Pharmaceutical University (Kiyose, Tokyo) and the Japan National Institute of Health Sciences (Setagaya, Tokyo) demonstrated the ability of THz-Raman spectroscopy to determine the amount of various hydrates in solid samples of magnesium stearate (Mg-St). They noted that previous studies had showed that powder lubrication, densification, and flowability were all different for the monohydrate and dihydrate forms of Mg-St. They also noted that the identification of the magnesium salt, the relative content of stearic acid by gas chromatography, and the water content are collectively not sufficient to uniquely determine the pseudopolymorphism of Mg-St. This uncertainty can be increasingly problematic where global corporations source raw materials from different locations with varying levels of good manufacturing practices.

In this study, 10 samples of Mg-St acquired from different suppliers were analyzed by PXRD as well as thermogravimetry and differential thermal analysis (TGDTA), allowing them to be described by hydrate type(s). Conventional (chemical fingerprint) Raman at high frequencies showed very minor differences between the hydrates. But as shown in figure 6, when each pseudopolymorph of Mg-St was measured using the low-frequency region, there were differences in peaks between 30 and 60 cm−1 and between 100 and 180 cm−1. In particular, a peak in the spectrum of each hydrate was clearly separated in the region between 100 and 180 cm−1, and this enabled discrimination between the monohydrate and the other hydrates even where the Mg-St was a mixture of pseudopolymorphs.


図6:独立した複数のMg-Stサンプルの規格化されたTHz-Ramanスペクトルの拡大図。(118 cm-1の緑色のピークは三水和物、129 cm-1の黒いピークは二水和物に関連しています。)[5]より許可を得て転載。

High-Throughput Screening

The above applications highlight how THz-Raman spectroscopy is a convenient and simpler alternative to PXRD and ss-NMR for rapidly determining the average phase of a sample. Neither of those somewhat cumbersome older techniques can perform spatial mapping. In contrast, the ability to make Raman and THz-Raman measurements through an objective lens supports both adjustment (zooming) and scanning of the sampling area, uniquely enabling mapping (imaging) of a sample under a microscope for both composition and structure/phase. This imaging capability also enables mapping of well plates and mapping within wells for high throughput screening (HTS) applications, and Coherent offers a fully automated well plate reader – the TR-WPS – to provide turnkey analysis for most common well plate formats from 6 to 1536 wells [6].



図7は、Coherent応用研究所のTR-WPSの簡単なデモによるフォールスカラー(偽色)データです。具体的には、Excedrin®の錠剤1錠をウェルに入れ、錠剤の直径3.5 mmの領域で27,000点以上のスペクトルデータを9分間で収集しました。主成分分析(PCA)とクラスタリングを使用して、3つの構成化合物(アセトアミノフェン、アスピリン、カフェイン)と境界領域の混合物を特定しました。偏光顕微鏡(PLM)画像は、その領域で同時に撮影され、視覚的に比較するためにラマンマップでオーバーレイされました。対応するスペクトルは、高品質で強力な光度データを明確に示し、TR-WPSによって素材または多形を迅速に特定し識別して、視覚データとスペクトルデータの両方をキャプチャできることを示しています。




THz-Raman spectroscopy enables real-time, non-contact analysis of samples, delivering structural information which would otherwise require sample prep and off-line measurements via unwieldy techniques such as PXRD and ss-NMR. It is particularly useful when seamlessly integrated with conventional Raman enabling samples to be simultaneously interrogated for both composition and structure. This survey of several pharma applications illustrates the breadth of potential uses, with benefits that also directly translate into numerous other industries, including polymer sciences, semiconductor materials, and industrial chemicals.




    1. P.J. Larkin et al, Polymorph Characterization of Active Pharmaceutical Ingredients (APIs) Using Low-Frequency Raman Spectroscopy, Applied Spectroscopy, Vol 68, Number 7, 2014

    2. Coherent, Inc., Polymorph Identification in Pharmaceuticals, Application Highlights, 2019

    3. M. Inoue et al, Real-Time Formation Monitoring of Cocrystals with Different Stoichiometries Using Probe-Type Low-Frequency Raman Spectroscopy, Ind. Eng. Chem. Res., Vol, 56, #44, 12693–12697, 2017

    4. J. Wallace, Terahertz-Raman Instrument Becomes a Crystallinity Phase Monitor, Laser Focus World, 6, 2020

    5. T. Koide et al, Identification of Pseudopolymorphism of Magnesium Stearate by Using Low-Frequency Raman Spectroscopy, Org. Process Res. Dev., Vol 20, 1906−1910, 2016

    6. Coherent, Inc., High Throughput Screening Using THz-Raman, Application Highlights, 2020