Development of Photothermal FTIR Microspectroscopy as a Novel

Article pubs.acs.org/molecularpharmaceutics

Development of Photothermal FTIR Microspectroscopy as a Novel Means of Spatially Identifying Amorphous and Crystalline Salbutamol Sulfate on Composite Surfaces Louise C. Grisedale,† Jonathan G. Moffat,†,§ Matthew J. Jamieson,‡ Peter S. Belton,† Susan A. Barker,†,§ and Duncan Q. M. Craig*,†,§ †

School of Pharmacy, University of East Anglia, Norwich, NR4 7TJ, United Kingdom Particle Generation, Control and Engineering, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom

‡

S Supporting Information *

ABSTRACT: Photothermal Fourier transform infrared (FTIR) microspectroscopy (PTMS), involving the combination of FTIR spectroscopy with atomic force microscopy, has been used to examine compacts of amorphous and crystalline salbutamol sulfate in order to assess the ability of the technique to distinguish between different physical forms in a multicomponent material. Samples of amorphous and crystalline material were assessed using modulated temperature differential scanning calorimetry (DSC), atomic force microscopy, microthermal analysis, and conventional FTIR. Mixed compacts were then prepared such that verification of the location of the forms present was possible via topography and localized thermal analysis. PTMS studies were then performed on selected interrogation points, with spectra obtained which were largely intermediate between those corresponding to the two individual forms. Calculation of the thermal diffusivity indicated a resolution for the technique corresponding to a hemisphere of a major diameter in the region of 40 μm, which is large in relation to the particle sizes involved. However, distinction into amorphous, crystalline, and indeterminate categories was possible using chemometric analysis (hierarchical cluster analysis and principal component analysis). Good agreement was found between the identification methods for the mixed systems. The study has therefore shown the potential, as well as identifying the limitations, of using PTMS as a means of spatially identifying components in complex materials. KEYWORDS: photothermal microspectroscopy, local thermal analysis, salbutamol sulfate, amorphous, chemometrics

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INTRODUCTION Many materials of practical significance such as polymers, sugars, and drugs may exist in amorphous, crystalline, or partially amorphous states, depending on processing and storage conditions. As the physical form may have a marked effect on performance, there is considerable interest in developing novel means of differentiating between these forms, preferably in a quantitative and, ideally, a spatially resolved manner, although the latter has proved to be challenging thus far. In this study we describe the use of photothermal Fourier transform infrared (PT-FTIR) microspectroscopy1−3 (henceforth referred to as PTMS) as a novel means of identifying and potentially mapping amorphous and crystalline forms of a model low molecular weight drug, salbutamol sulfate. PTMS is a recently introduced hyphenated technique which interfaces the thermal probes associated with microthermal analysis,4−6 itself a form of atomic force microscopy (AFM), with FTIR spectroscopy. Microthermal analysis (μTA) is based on the same principles as AFM, but the usual scanning probe is replaced with a thermal probe1,7,8 which has the functionality to © 2013 American Chemical Society

act as both a heating device and a temperature sensor. The thermal probes used in this study are constructed from a Wollaston wire, more specifically a 75 μm diameter silver wire with a 3−5 μm diameter platinum/10% rhodium filament core. The wire is looped to form a sharp bend, where the silver is electrochemically etched away to expose approximately 150 μm of the filament. The exposed filament has a higher resistance than the remaining Wollaston wire and probe, and hence a current passing across the tip will result in Joule heating; hence the tip may be heated in a controlled manner. Similarly, by measuring the voltage across the tip in relation to a remote calibration probe, it is possible to measure the tip temperature; hence the probe may act as both a miniaturized heater and thermocouple. Measurement of the thermomechanical properties of the sample directly beneath the probe is possible by applying a Received: Revised: Accepted: Published: 1815

October 24, 2012 February 1, 2013 March 20, 2013 March 20, 2013 dx.doi.org/10.1021/mp300605s | Mol. Pharmaceutics 2013, 10, 1815−1823

Molecular Pharmaceutics

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sample suitability for any technique involving Raman spectroscopy;16 therefore there is the desire to develop techniques that involve the more versatile IR spectroscopy. Dazzi et al.17 have developed a technique whereby a conventional AFM probe is used to detect molecular vibrations due to IR excitation, demonstrating resolutions of ∼100 nm. For this technique, however, samples are limited to thicknesses of up to 1 μm. The advantage of photothermal FTIR over conventional FTIR spectroscopy is that it has the ability in principle to directly measure temperature changes caused by the excitation of the molecules within the sample as they absorb infrared radiation, as opposed to measuring the absorbance of light by the sample, thus avoiding the diffraction limit. However, currently the spatial resolution of the technique is not fully utilized due to the influence of probe size,1 although an instrument is now available which appears to have overcome this difficulty.18 Despite the high spatial resolution afforded by the probes, there is a limitation of the technique due to the diffusion of thermal waves through a sample meaning that signals arising from certain distances from the probe can be detected. The length of this thermal diffusion is dependent on thermal properties of the material as well as the modulation frequency of the incident light; for example polymeric materials exposed to light at a modulation frequency of 200 Hz have been shown to have a diffusion length of 11−15 μm.19 The purpose of this study was to develop this method as a means of spatially characterizing the physical properties of a pharmaceutical material, salbutamol sulfate, a β-2 agonist used primarily for the treatment of asthma. Recent studies20 have examined the amorphization of this drug via milling and the water uptake properties of amorphous salbutamol sulfate. In brief, the generation of amorphous material either deliberately or accidentally, is of very considerable importance to the processing of pharmaceutical and related materials due to the accompanying changes in performance that may be manifest as a result. While methods are available for determining the absolute amount of crystalline and amorphous material,20 there is little understanding of how such physical forms may be distributed within a material. In this investigation we develop the use of PTMS as a means of spatially identifying amorphous and crystalline regions in a compressed sample, with a view to providing the foundations for the development of the method as a means of mapping physical components of complex samples.

controlled voltage across the tip, causing a localized heating effect. A predetermined amount of force is applied to the cantilever to maintain the tip in contact with the sample. The tip will penetrate into the sample when the sample undergoes a mechanical response such as thermal expansion or softening on passing through the glass transition or during a melt, the probe position being monitored as a function of temperature; this technique is known as local thermal analysis (LTA). The probe tip can be positioned at precise predetermined locations of interest identified from previously acquired topography images. The small size of the probe tip enables it to heat up extremely fast, in the order of 10−20 K s−1; the use of such high rates minimizes heat dissipation throughout the sample. Photothermal spectroscopy in the broadest sense is an indirect method used to measure the effect of the absorption of electromagnetic energy rather than the transmission of light.9 Initially a source of electromagnetic radiation excites the sample. A temperature change results from the relaxation of the excited molecules, whereby their excess internal energy is dissipated in the form of heat into the sample matrix. This results in the generation of a range of measurement possibilities.10 For example, as the sample molecules become excited at a given absorption frequency, a temperature change proportional to the electromagnetic radiation absorbed will result which may in turn be measured as a frequency spectrum. The photothermal signal can be enhanced and a smaller spatial resolution than the diffraction limit achieved by modulating the frequencies of the excitation radiation.11 Alternatively, thermal gradients result as the sample tries to maintain a thermal equilibrium with both itself and its surroundings. To maintain this equilibrium, thermal diffusion takes place within the material, affecting the density of the material and, therefore, its refractive index which again may be measured, while the rapid changes in temperature cause changes in the pressure of gas at the surface of a solid. This pressure perturbation disperses as an acoustic wave, leading to the possibility of photoacoustic measurement. PT-FTIR microspectroscopy4−6 is a development of the existing photothermal approach and involves interfacing the thermal probe of microthermal analysis (μTA), as outlined above, with a conventional FTIR spectrometer, thereby allowing spectra to be obtained in a highly localized manner. The probe tip is placed at a chosen location on the surface of a sample, and the infrared beam is then focused onto the tip of the probe, which is in contact with the surface of the sample. As the sample absorbs the radiation from the infrared beam, a photothermal signal is generated, which in turn heats the sample. The variations in temperature due to the modulation of the light beam are sensed by the probe, and the signal is then amplified, filtered, and digitized. A spectrum is obtained by Fourier transformation of the resulting interferogram. Due to the diffraction limit, interfacing a conventional FTIR spectrometer with a standard microscope will only achieve a maximum spatial resolution in the order of a few micrometers.12−14 Despite this there are a number of groups looking into improving the spatial resolution of imaging techniques involving molecular spectroscopy. With conventional Raman microscopy resolutions of up to ∼200 nm can be achieved although this technique is limited by issues such as low signal strength and fluorescence. This then lead to the development of higher resolution techniques such as tip-enhanced Raman spectroscopy (TERS), with a resolution of 10 nm having been demonstrated.15 Despite this there still remains the issue of

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EXPERIMENTAL SECTION Materials. Crystalline salbutamol sulfate was used as received (GlaxoSmithKline, Stevenage, UK). The spray dried sample was prepared using a Büchi 290 mini-spray dryer (Büchi Ltd., Oldham, UK). A 10% aqueous solution of salbutamol sulfate was prepared and dried under the following settings: compressed air 5 bar, air flow 357 mL per hour (equivalent to 30 mm height), aspirator operating at 40 m3 per hour (100%), and an inlet temperature of 160 °C. Samples were found to have a water content of ca. 2.20% (w/w). The particle size ranges of the crystalline and amorphous material were ca. 120− 300 μm and 8−12 μm, respectively. The amorphous form of this sample, as well as the crystalline form of the as-received material, was characterized by X-ray powder diffraction in a previous article.20 Sample Preparation. Compressed sample discs were chosen to improve image resolution, reduce noise from topographical dominance, and minimize damage to the probes. 1816

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μm. The adjustable stage enables the focal point of the IR beam to be located onto the probe tip of the SPM. The sample is loaded onto an XY translation and Z adjustment controlled stage and using the SPM the probe is placed in contact with the sample. All subsequent chemometric analysis was carried out using the Pirouette software, Version 4 (Infometrix, Inc.).

Pure amorphous and crystalline samples were prepared at two compression strengths, 1 tonne and 10 tonnes, which was applied to approximately 500 mg of respective samples in a 13 mm diameter evacuable IR press for 10 s. Before any PTMS experiments were conducted, the possible effects of different compression pressures on the spray-dried and crystalline samples were investigated using modulated temperature differential scanning calorimetry (MTDSC) and attenuated total reflectance (ATR)-FTIR spectroscopy. Details of the instruments used can be found in section 1 of the Supporting Information. Preliminary studies indicated evidence for 10 tonne compression resulting in crystallization of the amorphous sample; hence only the lower pressure was used, even though this led to a considerably less smooth surface for subsequent analysis. Mixed sample compacts used in this study were prepared by filling a 13 mm diameter evacuable IR sample press die with approximately 500 mg of spray dried amorphous form, and then the crystalline form was sparingly sprinkled on the top. To compress the sample, a 1 tonne force for 10 s was applied. The presence of the two differing phases was confirmed from the topography images prior to the photothermal spectra and local thermal analysis responses being acquired. These responses were obtained by positioning the probe tip at 16 specific locations on the 100 μm square sampling area forming a 4 × 4 grid. Thermal and (Macroscopic) Spectroscopic Analysis. Bulk samples were analyzed with MTDSC and attenuated total reflectance ATR-FTIR spectroscopy. Details of the experimental procedures for these methods can be found in section 1 of the Supporting Information. Atomic Force Microscopy, Microthermal Analysis, and PT-FTIR Microspectroscopy. Topography, local thermal analysis (LTA), and PTMS were all performed by interfacing an Explorer scanning probe microscope (SPM) equipped with a Wollaston wire thermal probe (both from Veeco instruments, Santa Barbara, CA) to a Bruker Optics IFS66/S spectrometer (Coventry, UK). LTA experiments were conducted with an underlying heating rate of 10 °C per second from 25 °C up to 300 °C. Temperature calibrations were performed according to the recommendations of the manufacture using Anasys Instruments nano-TA calibration samples: polycaprolactone (Tm 60 °C), polyethylene (Tm 130 °C), polyethylene terephthalate (Tm 238 °C) and the thermal feedback at room temperature. A custom built optical interface was used to interface the SPM with the Bruker spectrometer. The photothermal signal was fed through an ultralow noise Wheatstone bridge preamplifier supplied by Specac Ltd. and a Stanford Research Systems Ltd. SR-650 programmable filter amplifier (Sunnyvale, CA). The PTMS spectra were collected in the 4000−500 cm−1 wavenumbers region with 200 scans and a resolution of 8 cm−1 resulting in an acquisition time of approximately 10 min. A background air scan was taken before the probe tip was placed in contact with the sample. A scanner frequency of 1.6 kHz was used which equates to ∼0.05 cms−1. Prior to the acquisition of spectral measurements, an AFM image of the surface was acquired. The samples were imaged over a 100 μm × 100 μm square area with a scan rate of 50 μm sec−1. Details of the instrumental set up can be found in a previous article.21 To summarize, the interface contains a flat mirror, which is used to align the IR beam onto a concave spherical mirror that focuses and condenses the beam to an approximate diameter of 500

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RESULTS Analysis of Pure Amorphous and Crystalline Salbutamol Sulfate Samples. In the first instance it was considered necessary to investigate the effects of compression on the salbutamol sulfate compacts to ascertain whether the compression process itself may be influencing structure, a phenomenon that is well-recognized.22−24 On this basis MTDSC studies were conducted on scrapings from the sample surfaces of compressed amorphous and crystalline materials, while ATR-FTIR spectroscopy, AFM, and microthermal analysis were performed on the intact compacts. The results of the macroscopic techniques, MTDSC and ATR-FTIR spectroscopy, can be found in section 1 of the Supporting Information, while the results from the localized techniques are summarized below. Atomic Force Microscopy. Figure 1a and b shows the contact mode topography images of the surfaces of the crystalline and amorphous compacts, respectively. From Figure 1a, the clear spherical shape of the spray dried amorphous particles can be seen. The morphology of the crystalline particles obtained from the AFM image was less clear. It is acknowledged that the images are, in AFM terms, of poor quality and higher compression would be expected to result in a flatter surface and hence a greater surface resolution. However the intention in the present study is to generate surfaces whereby we minimize the risk of altering sample structure and also are able, at a very basic level, to identify the constituent components so as to validate the PTMS approach. Microthermal Analysis. Microthermal analysis was performed whereby the thermal probe was positioned at the coordinates where the photothermal spectra were also obtained. A calibrated and controlled voltage was passed through the probe tip causing localized heating. The local thermal analysis (LTA) responses for the crystalline and amorphous compacts at representative points across the surface are shown in Figure 2. Both samples showed expansion beneath the probe as temperature increased, leading to steady increase in deflection of the tip. A discontinuation of the signal was seen as the probe tip penetrated the sample at approximately 200 °C for the crystalline sample and approximately 120 °C for the amorphous material. These values coincided reasonably with the onset of decomposition in the crystalline sample and the glass transition in the amorphous sample. Any discrepancies between the thermal onsets of events determined by LTA and MTDSC could be attributed to the differences in thermal interrogation by the two techniques, particularly in terms of the differences in speed of temperature ramping.5 Photothermal-FTIR Microspectroscopy Studies. The photothermal spectra for both the crystalline and amorphous samples were obtained by positioning the thermal probe at nine specific locations forming a 3 × 3 grid on the image area. Figure 3 shows the photothermal spectra of the crystalline and amorphous samples; reproducibility within the two sets of nine locations was excellent. Spectral differences were particularly apparent in the 3600 cm−1 to 2250 cm−1 region where the amorphous sample 1817



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Figure 3. Typical photothermal FTIR spectra obtained for amorphous (black line) and crystalline (red line) compacts of salbutamol sulfate.

2200 cm−1 the sharper bands arise from aliphatic CH vibrational modes; in the amorphous material these show some broadening due to generation of a range of bonding environments and overlap of NH modes.20,25 A comparison of the photothermal and ATR-FTIR spectra (Figure S-1b) of the crystalline and the amorphous samples shows good agreement overall. This provided confidence in the technique, particularly when considering that the photothermal signal was generated from the thermal signal measured at a localized point rather than an average taken across the whole surface, as is the case in conventional measurements. The ATR spectra did, however, show greater signal intensity in the fingerprint region (1500 cm−1 to 500 cm−1) of the spectra. In general the photothermal spectrum will not be equivalent to that of an absorption spectrum as the phase and amplitude of the signal are a function of the thermal diffusion depth and absorption coefficient of the material.26 For the amorphous sample the photothermal spectrum in the 3600 cm−1 to 2250 cm−1 region showed fewer features and greater broadening compared to the ATR spectrum. Some variations between the spectra may also be expected due to the different penetration depths of the two techniques. The ATR signal can only penetrate the sample to approximately 1 μm, whereas the photothermal signal can penetrate to depths of approximately 10−20 μm depending on the mirror speed used. Analysis of Mixes of Amorphous and Crystalline Salbutamol Sulfate. The ability of PTMS to distinguish between compacts of crystalline and amorphous salbutamol sulfate in isolation was demonstrated in the previous section. The objective of this section was to apply knowledge gained in the previous section in the study of mixed samples, containing both the amorphous and crystalline phase of salbutamol sulfate. Figure 4 shows the AFM topography image of a representative compact containing the combined amorphous and crystalline salbutamol sulfate, prepared by sprinkling crystalline material on the amorphous powder prior to compacting. Based on previous observations (Figure 1) the spherical particles in the resulting image can be assumed to be the spray-dried amorphous phase and the more block-like structures, approximately outlined in Figure 4, the crystalline phase. Therefore, from its topography alone the 100 μm square sample was expected to be predominantly amorphous. Due to the size of the crystalline particles used (120−300 μm) it was impossible to obtain an image that was predominantly crystalline.

Figure 1. AFM contact mode topography image of the surface of an (a) amorphous and a (b) crystalline salbutamol sulfate compact.

Figure 2. Local thermal analytical responses on the surface of an amorphous (black lines) and a crystalline (red lines) compact of salbutamol sulfate.

demonstrated characteristic band broadening. Within this area of the spectrum the 3500 to 3150 cm−1 region represents alterations in the hydrogen bond distribution, particularly reflected in changes in the OH stretching frequency (3475 cm−1) and NH stretching (3272 to 3162 cm−1). From 3130 to 1818



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to the probe landing on areas of interface between the two phases, with the softening of one material influencing the penetration into the second.27 The penetration of the probe into the surface is typically 1−2 μm but can be up to 4 μm depending on the material.27 Potentially the probe could penetrate further, but the measurement is stopped once a transition is observed to minimize damage to the probe. The LTA for column 2 (Figure 5b) indicates that the material at the tested points was amorphous, as indicated by the temperature of penetration. However, the photothermal responses (Figure 5a) showed characteristics of both the amorphous and crystalline regions. More specifically, the higher wavenumber region (3000−3400 cm−1) showed little evidence for the absorption bands associated with the crystalline material, yet in the region 2800−2500 cm−1 there was indeed evidence of such characteristic bands. Similarly for Figure 5d, one region was amorphous and the others crystalline, as detected by the LTA, but the spectra (Figure 5c) associated with the amorphous region again showed some evidence of crystalline bands. It therefore appears to be the case that there may be issues with resolution associated with the PTMS technique in that the technique is interrogating a region which is larger than that scrutinized by the LTA studies, thereby not allowing such unequivocal assignment of the physical state of the sample. We address this issue in two ways below; first we calculate the theoretical resolution of the technique, and second we employ chemometric analysis to further delineate the spectra.

Figure 4. AFM contact mode topography image of the surface of a mixed amorphous and crystalline salbutamol sulfate compact.

Figure 5 shows representative photothermal spectra and LTA responses for the tip positions analyzed. The LTA results for the mixed compact fell into two thermal categories: those with a softening onset at approximately 120 °C, corresponding to the glass transition of the amorphous sample, and those with a softening onset at approximately 200 °C, corresponding to the decomposition of the crystalline sample. On occasion, an intermediate penetration temperature (pink line, Figure 5d) was observed which we have previously noted and may be due

Figure 5. (a) Photothermal FTIR spectra and (b) μTA responses at locations 2.1 (pink), 2.2 (red), 2.3 (green), and 2.4 (blue) in Figure 4. (c) Photothermal FTIR spectra and (d) μTA responses at locations 3.1 (pink), 3.2 (red), 3.3 (green), and 3.4 (blue) in Figure 4. 1819



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DISCUSSION Calculation of the Spatial Resolution of the PTMS Technique. Based on the results and evidence of the first two samples studied above, it can be concluded that LTA can clearly distinguish between amorphous and crystalline salbutamol sulfate; this principle has been previously demonstrated in a number of studies.5 Differences in the photothermal spectra were, however, less clear-cut, as the same bands are present in both spectra with the biggest difference being in the broadening of bands of the amorphous form. Had the study been focused on different chemical entities, one would reasonably expect a greater level of discrimination for the PTMS as bands at different locations would be observed for each component. Nevertheless, the question arises as to the spatial resolution of the PTMS technique. This may be anticipated to be dependent on a number of factors including probe size as well as the density, heat capacity, and thermal conductivity of the sample directly in contact with the probe. The frequency range of the interferometer modulation will also influence the response of the sample.8 These effects can to some extent be accounted for by calculating the thermal diffusion length of the material. Thermal diffusivity (α) can be calculated by eq 1:19 α=

k Cpρ

Figure 6. Schematic diagram illustrating the thermal diffusion length from the tip through the sample.

accurate but they both illustrate the issue of spectral interference from neighboring material. In either case these models are intuitively compatible with observations, that the detected signal is from a domain larger than that of the tip. A hemisphere of dimensions significantly larger than this would be expected to show characteristics of both types of material (crystalline and amorphous); hence the appearance of characteristics of both in virtually all the samples examined. This effect will be exacerbated by the needle-shape of the crystalline particles, rendering it even less likely that the probe will land on a region whereby a “clean” response will be observed. Moreover, the LTA response is fundamentally determined by the nature of the upper surface of the sample as that provides the barrier to penetration, hence it is a reasonably surface specific technique even though it involves probe transition into the material. The PTMS, however, may be indicating subsurface structure, as indicated in Figure 6, thus rendering the detection of a single material even more unlikely. At present the PTMS technique represents a significantly poorer spatial resolution compared to other chemical imaging techniques. Raman imaging techniques such as TERS can provide resolutions of up to ∼10 nm, but the PTMS technique does not have the same issues of fluorescence and signal strength. Other IR based imaging techniques exist such as micro-ATR imaging12−14 and have been shown to provide fast (significantly faster than the current PTMS acquisition time) and reliable chemical images of pharmaceutical samples. A similar issue of wavelength-dependent penetration exists with micro-ATR and also relies on firm contact between the ATR crystal and sample which can influence the relative intensity of each measurement. There are, however, approaches that could address some of the current shortfalls of the PTMS technique which could include using more intense sources to increase signal and hence reduce both acquisition time and thermal diffusion length. The PTMS technique also has the possibility to adjust the thermal diffusion length by utilizing a spectrometer in step-scan mode as has been demonstrated recently.29 Moreover, this approach allows a unique range of sample manipulation and measurement modes, both in terms of the additional verification afforded by localized thermomechanical studies and the possibility of studying extremely small quantities of sample such as individual particles. Indeed, it has been demonstrated that the technique is capable of detecting femtograms27 of material. It is, however, also reasonable to suggest that chemometric analysis may provide a means of classifying each measurement by providing a cluster based analysis which may in turn indicate predominance of one or other physical form. The intensity of the thermal wave traveling through the sample decays exponentially with distance traveled, 19 so if a spectrum is classified to contain predominantly one particular form over another, then it can

(1) −1

−1

where k is the thermal conductivity (W m K ), Cp the heat capacity (J K−1 kg−1), and ρ the density (kg m−3) of the sample. The thermal diffusion length can then be calculated from eq 2: ⎡ α ⎤0.5 μ=⎢ ⎥ ⎣ πf ⎦

(2)

where μ is the sample depth (m) and f the modulation frequency (Hz) determined by eq 3: f = 2wv

(3)

where w is the wavenumber (cm−1) and v is the scanner velocity (cm s−1). Unfortunately there is little published data on the thermal conductivity of pharmaceuticals, and therefore, the precise thermal diffusivity depth of salbutamol sulfate could not be calculated. However, an approximation was made based on the calculated thermal diffusion lengths of pure sucrose,28 a similar low molecular weight organic compound to salbutamol sulfate. Using eqs 1, 2, and 3 the thermal diffusion length for sucrose was found to be approximately 15 μm at 4000 cm−1 and 44 μm at 500 cm−1. Therefore, it can be assumed that the probe tip of the AFM will detect the thermal response of a salbutamol sulfate sample within 15−44 μm diameter a hemisphere from where it makes contact with the surface of the sample. This principle is illustrated in Figure 6. Thus, it can be assumed that the intermediate photothermal spectra results recorded were the thermal responses of neighboring amorphous and crystalline material within the hemisphere. Despite this possible model it has been shown in previous work21 that the probe is assumed to have zero heat capacity so heat would not be drawn to the tip along the surface. As such, if this model was appropriate then there would only be vertical travel of thermal waves and no horizontal movement. This would mean that the model would effectively be a cylinder with the diameter of that of the tip and length that of the diffusion length as represented in Figure 6. As yet, it is not clear as to which model is the most 1820



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be assumed that the material directly under the probe is likely to be that form. This approach is outlined below. Chemometric Analysis. Chemometric analysis was then used to further explore the ability of PTMS to distinguish between amorphous and crystalline salbutamol sulfate, particularly in the light of the difficulties identified in utilizing unequivocal fingerprint regions for this purpose for the reasons outlined above. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were both utilized. HCA is based on the principle of identifying the distances in hyperspaces between pairs of samples or variables and presents the data in a format where samples with similar attributes are grouped together in clusters. For photothermal data, the dimensions of the hyperspace equate to the number of wavelengths or wavenumber points used to construct a spectrum. The output of the analysis is a dendrogram, whereby the length of a branch connecting two clusters relates to the similarity of the leaves within that cluster. Similarity is plotted on the top of the dendrogram with 1.0 corresponding to an exact duplicate and 0.0 indicating maximum distance and, therefore, dissimilarity. The dashed vertical line shown in the plot (Figure 7a) corresponds to the similarity value and partitions the dendrogram into four distinguishable clusters. Principal component analysis is a means of identifying pattern or relationships in multidimensional data sets. PCA identifies the linear combinations of the original independent variables that are responsible for maximal amounts of variation, thereby reducing the number of dimensions but without much loss of information. The first principal component (PC1) describes the direction of the largest variance (trend) of the data in multidimensional space. By drawing a line in this direction through the middle of the data swarm, the first (PC1) can be assigned. Once PC1 has been assigned, the next largest variance in the data (PC2) is then determined orthogonal to the direction of PC1. This creates a two-dimensional “window” in this space with axes of PC1 and PC2, which will show where the data points lie in relation to the two largest variances in the data. Scores plots can, of course, be made from any choice of particular components of the PCA; it is simply the case that PC1 and PC2 are the most common choice when the aim is simply to preserve the maximum amount of variation possible in a two-dimensional (and hence printable) plot. In fact, it is possible to explore score plots in three (or even more) dimensions, but specialized software is needed to view projections from these manipulations. The results of the HCA and PCA performed on the photothermal spectra generated from the compact are shown in Figure 7. The HCA dendrogram (Figure 7a) identified three clusters, indicating that the photothermal spectra had been able to differentiate between three distinguishable chemical properties on the surface of the compact. This agreed with the LTA results, in that some data points showed two transitions which were ascribed to interrogation sites whereby mixes of the two components are present. Nevertheless, this method showed a greatly enhanced ability to distinguish between the two physical forms within the sample compared to simple inspection of the spectra. The similarity index here was relatively low, approximately 0.25, which indicated that the spectra within each cluster were relatively dissimilar. This can also be seen from the PCA score plot (Figure 7b) where the clustering is relatively dispersed. The principal component analysis scores also showed the photothermal spectra being divided into three groups. If each measurement on the plot is compared to the

Figure 7. (a) Hierarchical cluster analysis (HCA) dendrogram for photothermal spectra taken from the surface of mixed compact containing amorphous and crystalline salbutamol sulfate. (b) Principal component analysis scores for photothermal spectra taken from the surface of mixed compact containing amorphous and crystalline salbutamol sulfate. The nature of each measurement within each was classified using LTA profiles.

LTA profiles, then the cluster at the bottom right contains profiles that were crystalline. The large cluster at the top contains entirely amorphous profiles while the remaining cluster contains two crystalline, one amorphous, and the one identified two-component profile (pink line, Figure 5d). The identification of mixed spectra with the photothermal technique highlights the issue of sampling area illustrated in Figure 6. The LTA technique heats the area directly beneath the probe and typically penetrates 1−2 μm from the surface while PTMS can detect signals from depths of up to 44 μm, subsequently increasing the possibility of detection of both components. The comparisons between the three methods are shown in Table 1; the techniques showed very good agreement, despite the LTA and PTMS measurements being based on very different principles. Overall, therefore, the use of chemometric analysis appears to show considerable promise in assisting the PTMS method to distinguish between components in mixed sample surfaces, particularly bearing in mind that the spectroscopic differences between the two forms are relatively 1821



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the ability to manipulate the tip temperature allows the operator to study the spectra at a range of temperatures, even for extremely small samples. The present study has focused on distinguishing between components of complex materials, but the point being made is that this is part of a much wider range of studies that are ongoing in which the strengths and weaknesses of the method are being critically evaluated.

Table 1. Summary of Localized Thermal Analysis (LTA), Hierarchical Cluster Analysis (HCA), and Principal Component Analysis (PCA) of a Mixed Salbutamol Sulfate Compact. A = Amorphous, C = Crystalline, and I = Indeterminate point location

LTA

HCA

PCA

1.1 1.2 1.3 1.4 2.1 2.2 2.3 2.4 3.1 3.2 3.3 3.4 4.1 4.2 4.3 4.4

A A A A A A A A I C C C A C C C

A A A A I A I A I I C C I I C C

A A A A A A A A I I C C I I C C

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ASSOCIATED CONTENT

S Supporting Information *

Analysis of pure amorphous and crystalline salbutamol sulfate samples with macroscopic techniques. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +44 207 753 5819. Fax: +44 207 753 5560. Present Address §

School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom

Notes

The authors declare no competing financial interest.

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subtle; hence when attempting to distinguish between different materials that are different chemical entities entirely one may reasonably expect a far greater level of discrimination.

ACKNOWLEDGMENTS We would like to thank GlaxoSmithKline for financial assistance for LG. We also acknowledge the assistance of Mike Reading in providing access to and discussion of the necessary equipment.

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CONCLUSIONS The study has examined the use of PTMS as a novel means of distinguishing between two forms of a drug, salbutamol sulfate, within compacts. Such distinction is extremely important for a number of applications, not only involving pharmaceuticals whereby spatial characterization has proved to be challenging but also in the vast majority of branches of material science where composite materials are almost invariably used. As may be expected for the development of a nascent technique, the study has revealed both the strengths and weaknesses of the approach. The ability of PTMS to derive spectra which are in good agreement with conventional FTIR analysis has been demonstrated, while the supplementary use of thermal and imaging techniques has allowed verification of the conclusions drawn from the PTMS, with good agreement shown between techniques. It should be noted that lightly compacted materials were used precisely to allow independent verification via topographic features; a “real life” sample would be unlikely to have any such distinguishing features, and hence the operator would be much more dependent on the spectral data. The weakness of the technique appears to lie in the resolution, which necessitated the (successful) use of chemometric analysis to distinguish between the components. Overall, therefore, the technique has been shown to demonstrate considerable promise in the analysis of complex multicomponent samples. A question that naturally arises is whether this method shows advantages over conventional IR and Raman microscopy. We would argue that the PTMS method is potentially highly useful as a complementary technique for several reasons. It is not diffraction length limited, so with further development (now achieved and commercialized30) it could operate at much greater, submicrometer resolution. Second, the method is highly versatile in that instead of landing the tip on a sample, the tip can pick up or be coated with that sample and interactions studied. Finally,

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REFERENCES

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