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Macro FTIR imaging in transmission under a controlled environment
Vibrational Spectroscopy 42 (2006) 130–134 www.elsevier.com/locate/vibspec
Macro FTIR imaging in transmission under a controlled environment K.L.A. Chan, S.G. Kazarian * Department of Chemical Engineering, Imperial College London, London SW7 2AZ, UK Available online 20 December 2005
Abstract New development in macro FTIR imaging with the use of a specially built accessory has been demonstrated. This accessory expands the possible applications of using macro FTIR imaging in the study of samples under a controlled environment. This new accessory enables the samples (such as films) to be measured in the horizontal position with macro FTIR imaging in transmission. This adds to the flexibility of the sample arrangement and provides the opportunity to use the controlled environment cell for macro FTIR imaging. Using this approach, the crystallisation and the polymorphic changes of amorphous nifedipine film has been monitored by using FTIR imaging over an area of 3.8 mm 3.8 mm under a controlled humidity and temperature environment. The demonstrated imaging approach may impact areas as diverse as polymeric and biomedical materials, pharmaceuticals and high-throughput analysis. # 2005 Elsevier B.V. All rights reserved. Keywords: FTIR imaging; Drugs; Polymorphism; Humidity
1. Introduction The potential of FTIR imaging utilising the focal plane array (FPA) detector has been demonstrated in many areas including polymeric materials [1–7], biomedical [8,9] and pharmaceutical applications [10–12], high-throughput analysis [13] and forensic science [14]. These applications of FTIR imaging included different sampling approaches, such as imaging in transmission [1,3,9,15–18], reflection [14] and with the use of attenuated total reflection (ATR) [10,19,20] infrared spectroscopy. Most of these applications have been demonstrated with the use of an infrared microscope which analysed relatively small area of the sample (typically a fraction of the squared millimetre). The notable exception has been the use of macro ATR accessories pioneered by us [10,20,21] which demonstrated the possibility of studying samples with different sizes of fields of view from 1 mm2 to ca.16 mm2. These macro ATR imaging applications have been achieved without recourse to the infrared microscope and often included imaging without the use of additional optics by collecting radiation directly on FPA
* Corresponding author. Tel.: +44 2075945574; fax: +44 2075945604. E-mail address: [email protected] (S.G. Kazarian). 0924-2031/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2005.10.005
detector. In such case the imaged area was equal to the area of the detector. Macro FTIR imaging in ATR mode has been demonstrated to be a good complementary approach to the micro FTIR imaging when applied to study distribution of excipients in pharmaceutical tablet [10]. A larger field of view provides a better overall picture of a sample and may be particularly useful in studying processes involving relatively large samples. For example, in studies of tablet dissolution and drug release [21–23], a large field of view can provide information on the whole dissolution process of the tablet, namely relaxation of the polymer matrix, diffusion of drug in the polymer gel layer and the distribution and behaviour of the drug in the dissolution medium. Furthermore, sample handling within the millimetre scale is much easier than within the micrometer scale. In high-throughput applications, when combined with a ‘‘dropon-demand’’ system for the preparation of samples array [13], an expanded imaging area allows larger sample drops to be used. This increases the flexibility in the preparation of a broader range of samples with different concentrations. On the other hand, if the sample size remains small, a larger field of view would allow more samples to be measured simultaneously in a single imaging measurement. The benefits of using an enlarged field of view in imaging application has been utilised
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inhigh-throughput analysis using macro ATR imaging with the ATR crystal as an inverted pyramid where samples were deposited on the inverted base of the pyramid [13]. This also provides the opportunity to combine this macro ATR accessory with the controlled environment cell. However, with the current commercially available FTIR instruments, the use of FTIR imaging in transmission under a controlled environment was limited to the use of an infrared microscope because the specialised accessory can only operate in a horizontal position [11,24]. Macro-chambers (large sample compartments) of commercially available FTIR imaging systems, however, are only supplied with vertical sample holders which would not be suitable for the use with the controlled humidity cell. The first application of FTIR imaging and the controlled environment cell involved the use of an infrared microscope to study the sorption of water in a pharmaceutical formulation [11]. In such an arrangement, the field of view was ca. 0.27 mm 0.27 mm. Other relevant applications involved the use of infrared microscope in FTIR transmission imaging mode to study the morphology of semicrystalline polymers [3] and solvent-induced crystallisation of polymers [17]. By contrast, if the macro FTIR imaging mode was employed then the imaged area would be ca. 4 mm 4 mm. In this paper, we demonstrate the first application of macro FTIR imaging in transmission under a controlled environment to study polymorphic transition of a particular drug, nifedipine. Nifedipine exhibits both a glassy phase and a few crystalline states [25]. Conventional infrared spectroscopy had been applied to study different polymorphism of nifedipine under controlled humidity and temperature [25]. However, the location and the size of the crystalline domain were not observed previously because the imaging approach was not used.
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Fig. 1. Schematic diagram showing the path of the IR beam for horizontal macro transmission accessory in the macro-chamber of the imaging system. The position of the controlled environment cell is indicated by the position of the sample stage.
2.3. The controlled environment cell and sample preparation
2. Experimental
a-Crystalline nifedipine (a-NIF) in powder form was purchased from Sigma and used without further treatment. Glassy nifedipine sample film was prepared by melting a small quantity of a-NIF powder between a non-stick film and a BaF2 window at 190 8C followed by quenching on a cool surface. After quenching, the non-stick film was removed to expose the flat surface of the glassy nifedipine (g-NIF) film. The preparation of the film was performed in the dark to avoid the photochemical degradation of the drug. A controlled environment spectroscopic transmission cell (VGI2000M, Surface Measurement System Ltd.) was used to control the environment (temperature and humidity) of which the glassy nifedipine thin film was exposed to. For further details of this controlled environment cell see our previous publications [11,24]. The crystallisation experiment was performed at 38 8C and at relative humidity levels of 80%.
2.1. FTIR imaging
3. Results and discussion
The FTIR imaging system consisted of an infrared spectrometer (IFS66S, Bruker Optics), a macro-chamber (IMACTM, Bruker Optics) and a 64 64 focal plane array (FPA) detector. The size of the FPA array detector was 3.8 mm 3.8 mm. FTIR images were acquired in step scan mode and the image collection time for 8 cm 1 and 20 coadditions was ca. 300 s.
3.1. HMTA
2.2. Horizontal macro transmission accessory (HMTA) The accessory was built of three plane mirrors and one parabolic mirror (Fig. 1). The parabolic mirror refocuses the IR beam to the sample to increase the energy throughput of the accessory. No magnification or view expansion was expected from this accessory. Samples were placed on the sample stage horizontally during measurement. A copper grid, typically used in SEM, was utilised for validation of the imaged area.
The commercial design of the macro-chamber focuses the IR beam at the centre in the sample compartment. The design of the HMTA built by us directs the IR beam up at ca. 908, then ca. 908 to the right of the sample compartment, then ca. 908 downward towards the sample and then finally another ca. 908 to the right towards the detector, as shown in Fig. 1. In this way, the accessory increase the IR beam path by 2a (see Fig. 1) which reduces the amount of IR radiation that reaches the detector if all four mirrors were plane mirrors. A parabolic mirror is, therefore, introduced just before the beam enters the sample providing the means to refocus light onto the sample hence increases the IR light intensity throughput of the accessory. The design of the accessory allows the distance a to be adjusted to optimise the intensity of the IR radiation falling onto the detector. The focus of image can be adjusted in two ways: either by moving the whole accessory along the original
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Fig. 2. Left image is a photograph of the copper grid taken under a 4 objective. Right image is a FTIR image showing the total IR intensity across the whole imaged area (dark areas indicates a high intensity). The copper grid in the visible image was not in the same orientation as the grid in FTIR imaging measurement.
IR beam path in the macro-chamber or by moving the sample stage up or down. The accessory has been tested for any unpredicted image artefacts due to the optical design. A circular copper grid with regular pattern has been used as a test sample to verify the quality of the FTIR image. The white light image of the copper grid was first captured with an optical microscope and shown in Fig. 2 (left). The scale of the optical image has been calibrated with an objective scale from Nikon and the dimension of the copper has been estimated on the optical image using the video assisted measurement software. The grid has an outer diameter of 3050 mm, the width of the ring is 250 mm and the wire between each hexagon hole is ca. 45 mm wide. Then the macro FTIR image of the whole copper ring was measured with the newly developed accessory whilst the grid was placed in the sample stage of the HMTA. The FTIR image of the grid was generated, and shown in Fig. 2 (right), by plotting the intensities of IR radiation registered by each pixel across the whole FPA detector. The result shows that the size and the shape of the copper grid have been accurately projected on the FPA detector without significant image artefacts. It was not surprising that the wires of the copper grid in the FTIR image shown in Fig. 2 (right) appeared blurred since the width of the wire was smaller than the size of a single pixel. This successful demonstration of the HMTA extends the range of applications for macro FTIR imaging in transmission where sample mounting in vertical position would be difficult, and opens new opportunities to study samples in conjunction with the other accessories which only operate in horizontal orientations such as the controlled environment cell. 3.2. Application of HMTA to drug crystallisation The designed HMTA has been used in conjunction with the controlled environment cell to study the crystallisation of
nifedipine with FTIR imaging in situ. The particular form of nifedipine was analysed by characteristic absorption bands in infrared spectra determined in our previous studies using conventional FTIR microscopy [25]. The absorption bands of a-NIF at 3332 cm 1 (integration range of 3344–3300 cm 1), b-NIF at 3353 cm 1 (integration range of 3380–3344 cm 1) and g-NIF at 1281 cm 1 (integration range of 1292– 1269 cm 1) have been used to characterise the three polymorphs of nifedipine observed under controlled humidity as a function of time. Chemical images representing the distribution of a particular form of nifedipine have been generated by plotting the distribution of the value of the integrated absorbance of characteristic band of that component over the whole measuring area. Fig. 3 confirms that in the beginning of
Fig. 3. FTIR image showing the distribution of amorphous nifedipine before being exposed in high humidity and high temperature. The variation in intensity was due to the non-uniform film thickness. The image size is ca. 3.8 mm 3.8 mm.
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Fig. 4. Distribution of amorphous nifedipine (top row) and b-NIF (bottom row) of the drug film in the first 4 h of exposure in the controlled environment. The number at the bottom of each column indicates the time at which the image has been measured. Colour scales on right show the integral value of the characteristic absorption band of the corresponding component. The image size is ca. 3.8 mm 3.8 mm.
the experiment (at time = 0), all nifedipine was amorphous. The noises on the top of each image are artefact caused by the aging of the FPA detector. The film thickness was not exactly uniform as shown by the distribution of the absorbance, which is proportional to pathlength (film thickness). The effect of difference in film thickness to the image can be minimised either by ratioing the images against another image generated with an internal absorption band with which absorption does not change with any polymorphic transition or ratioing the images against Fig. 3 where the drug was still pure amorphous nifedipine film and the absorbance only varied with change in film thickness. In this case, the assumption that the film thickness does not change during the polymorphic transition is
made. The latter approach was used because it was more convenient and it has less chance of inducing unexpected artefacts caused by band shift. The resultant images after the ratioing are shown in Figs. 4 and 5. Fig. 4 has demonstrated that the amorphous nifedipine slowly crystallised into b-NIF and all area of the film was fully converted after 4 h of exposure to an environment of 38 8C and relative humidity of 80%. With the imaging capability, the location and the morphology of the initial crystal can been observed. The large imaged area allowed assessing development of crystalline domain. The bNIF crystal grows into large domains progressively starting from a few initial nucleate sites. It should be noted that the area measured under the infrared microscope using the
Fig. 5. Distribution of amorphous b-NIF (top row) and a-NIF (bottom row) of the drug film in the last 18 h of exposure in the controlled environment. The number at the bottom of each column indicates the time at which the image has been measured. Colour scales on right show the integral value of the characteristic absorption band of the corresponding component. The image size is 3.8 mm 3.8 mm.
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controlled humidity cell was ca. 250 times smaller than the area measured in this study. Fig. 5 shows the further polymorphic changes of the nifedipine film after 5 h of exposure to the controlled environment. The b-NIF film begins to crystallise into aNIF after 5 h of exposure time as shown in Fig. 5. The polymorphic change begins at the centre of the film followed by the side of the film, which progresses at a much slower pace than the conversion from g-NIF to b-NIF. There was no clear formation of large a-NIF domains unlike the g-NIF to b-NIF transition (Fig. 4). Perhaps this is due to the b-NIF to a-NIF transition does not require a crystal nucleation step while the gNIF to b-NIF transition requires a nucleation step before b-NIF can be formed under these experimental conditions. Once a bNIF crystal is formed, the crystal can grow relatively fast and forming large domains as shown in Fig. 4. It is important to note that such observation will not be possible with conventional FTIR measurements due to the lack of information about the domain size of the crystal formed. The conversion of nifedipine from b to a form was not completed 5 h after the first appearance of an a-NIF crystal in the film (the first appearance of an a-NIF crystal was 5 h after the beginning of the experiment). The general trend of observation of the nifedipine crystallisation from g-NIF to b-NIF to a-NIF was in good agreement with our previous study. However, the imaging capability allowed us to accurately locate the area where crystallisation or polymorphic transition initiated and how far it propagates through the whole film. This example also underlines the advantage of FTIR imaging over optical microscopy since the characteristic bands of each form of the drug allowed one to assess unequivocally the appearance of the particular form of the drug. This experiment also shows an opportunity to utilise the gradient of film thickness to study the effect of film thickness on crystallisation of amorphous nifedipine; however, this was left for future investigations. 4. Conclusions A novel approach has been developed which allows macro FTIR imaging in transmission of samples under controlled environment. This approach required construction of an optical accessory which enables the measurement of samples placed in a horizontal position in the macro-chamber of the imaging system. A combination of this new accessory with a controlled humidity cell provided an opportunity to obtain chemical images of the large areas of the samples under controlled humidity. In this study, this approach was applied to study the effect of humidity on polymorphic transitions of thin film of amorphous nifedipine. It has been shown that exposure of the drug to the conditions set in the experiment for 4 h resulted in a transition of the drug from amorphous to b-crystalline form while further exposure for several hours resulted in the
conversion of the b-form to the a-form of nifedipine. The FTIR imaging demonstrated in this experiment shows advantages of the chemical imaging approach compared to optical microscopy due to the chemical specificity of FTIR spectroscopy. Imaging capability allows one to visualise the distribution and evolution of domains of different forms of drug which can be useful in studies of crystallisation processes. Developed macro FTIR imaging approach in transmission can find broad range of applications from studying crystallisation processes in polymers and pharmaceuticals to high-throughput analysis of materials under controlled environments. Acknowledgements We thank Drs. G.D. Chryssikos and V. Gionis and for providing nifedipine, Mr. Richard Wallace in the workshop, EPSRC for support (GR/T08746/01). References [1] J.L. Koenig, Microscopic Imaging of Polymers, ACS, Washington, DC, 1998. [2] B.A. Miller-Chou, J.L. Koenig, Macromolecules 35 (2002) 440. [3] C.M. Snively, J.L. Koenig, J. Polym. Sci. Polym. Phys. 37 (1999) 2353. [4] K. Artyushkova, B. Wall, J.L. Koenig, J.E. Fulghum, J. Vac. Sci. Technol. A 19 (2001) 2791. [5] R. Bhargava, S.-Q. Wang, J.L. Koenig, Adv. Polym. Sci. 163 (2003) 13. [6] A. Gupper, S.G. Kazarian, Macromolecules 38 (2005) 232. [7] A. Gupper, P. Wilhelm, M. Schmied, S.G. Kazarian, K.L.A. Chan, J. Reussner, Appl. Spectrosc. 56 (2002) 1515. [8] R. Salzer, G. Steiner, H.H. Mantsch, J. Mansfield, E.N. Lewis, Fresenius J. Anal. Chem. 366 (2000) 712. [9] D.C. Fernandez, R. Bhargava, S.M. Hewitt, I.W. Levin, Nat. Biotechnol. 23 (2005) 469. [10] K.L.A. Chan, S.V. Hammond, S.G. Kazarian, Anal. Chem. 75 (2003) 2140. [11] K.L.A. Chan, S.G. Kazarian, Vib. Spectrosc. 35 (2004) 45–49. [12] Y. Roggo, A. Edmond, P. Chalus, M. Ulmschneider, Anal. Chim. Acta 535 (2005) 79. [13] K.L.A. Chan, S.G. Kazarian, J. Comb. Chem. 7 (2005) 185. [14] M. Tahtouh, J.R. Kalman, C. Roux, C.I. Lennard, B.J. Reedy, J. Forensic Sci. 50 (2005) 64. [15] C.M. Snively, J.L. Koenig, J. Polym. Sci. B: Polym. Phys. 37 (1999) 2261. [16] T. Ribar, J.L. Koenig, R. Bhargava, Macromolecules 43 (2001) 8340. [17] A. Gupper, K.L.A. Chan, S.G. Kazarian, Macromolecules 37 (2004) 6498. [18] J. Koenig, Adv. Mater. 14 (2002) 457. [19] A.J. Sommer, L.G. Tisinger, C. Marcott, G.M. Story, Appl. Spectrosc. 55 (2001) 252. [20] K.L.A. Chan, S.G. Kazarian, Appl. Spectrosc. 57 (2003) 381. [21] S.G. Kazarian, K.L.A. Chan, Macromolecules 36 (2003) 9866. [22] S.G. Kazarian, K.W.T. Kond, M. Bajomo, J. Van der Weerd, K.L.A. Chan, Food Bioproducts Process. 83 (C2) (2005) 127. [23] J. Van der Weerd, S.G. Kazarian, J. Pharm. Sci. 94 (2005) 2096. [24] H. Jervis, S.G. Kazarian, K.L.A. Chan, D. Bruce, N. King, Vib. Spectrosc. 35 (2004) 225. [25] K.L.A. Chan, O.S. Fleming, S.G. Kazarian, D. Vassou, G.D. Chryssikos, V. Gionis, J. Raman Spectrosc. 35 (2004) 353.
Macro FTIR imaging in transmission under a controlled environment K.L.A. Chan, S.G. Kazarian * Department of Chemical Engineering, Imperial College London, London SW7 2AZ, UK Available online 20 December 2005
Abstract New development in macro FTIR imaging with the use of a specially built accessory has been demonstrated. This accessory expands the possible applications of using macro FTIR imaging in the study of samples under a controlled environment. This new accessory enables the samples (such as films) to be measured in the horizontal position with macro FTIR imaging in transmission. This adds to the flexibility of the sample arrangement and provides the opportunity to use the controlled environment cell for macro FTIR imaging. Using this approach, the crystallisation and the polymorphic changes of amorphous nifedipine film has been monitored by using FTIR imaging over an area of 3.8 mm 3.8 mm under a controlled humidity and temperature environment. The demonstrated imaging approach may impact areas as diverse as polymeric and biomedical materials, pharmaceuticals and high-throughput analysis. # 2005 Elsevier B.V. All rights reserved. Keywords: FTIR imaging; Drugs; Polymorphism; Humidity
1. Introduction The potential of FTIR imaging utilising the focal plane array (FPA) detector has been demonstrated in many areas including polymeric materials [1–7], biomedical [8,9] and pharmaceutical applications [10–12], high-throughput analysis [13] and forensic science [14]. These applications of FTIR imaging included different sampling approaches, such as imaging in transmission [1,3,9,15–18], reflection [14] and with the use of attenuated total reflection (ATR) [10,19,20] infrared spectroscopy. Most of these applications have been demonstrated with the use of an infrared microscope which analysed relatively small area of the sample (typically a fraction of the squared millimetre). The notable exception has been the use of macro ATR accessories pioneered by us [10,20,21] which demonstrated the possibility of studying samples with different sizes of fields of view from 1 mm2 to ca.16 mm2. These macro ATR imaging applications have been achieved without recourse to the infrared microscope and often included imaging without the use of additional optics by collecting radiation directly on FPA
* Corresponding author. Tel.: +44 2075945574; fax: +44 2075945604. E-mail address: [email protected] (S.G. Kazarian). 0924-2031/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2005.10.005
detector. In such case the imaged area was equal to the area of the detector. Macro FTIR imaging in ATR mode has been demonstrated to be a good complementary approach to the micro FTIR imaging when applied to study distribution of excipients in pharmaceutical tablet [10]. A larger field of view provides a better overall picture of a sample and may be particularly useful in studying processes involving relatively large samples. For example, in studies of tablet dissolution and drug release [21–23], a large field of view can provide information on the whole dissolution process of the tablet, namely relaxation of the polymer matrix, diffusion of drug in the polymer gel layer and the distribution and behaviour of the drug in the dissolution medium. Furthermore, sample handling within the millimetre scale is much easier than within the micrometer scale. In high-throughput applications, when combined with a ‘‘dropon-demand’’ system for the preparation of samples array [13], an expanded imaging area allows larger sample drops to be used. This increases the flexibility in the preparation of a broader range of samples with different concentrations. On the other hand, if the sample size remains small, a larger field of view would allow more samples to be measured simultaneously in a single imaging measurement. The benefits of using an enlarged field of view in imaging application has been utilised
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inhigh-throughput analysis using macro ATR imaging with the ATR crystal as an inverted pyramid where samples were deposited on the inverted base of the pyramid [13]. This also provides the opportunity to combine this macro ATR accessory with the controlled environment cell. However, with the current commercially available FTIR instruments, the use of FTIR imaging in transmission under a controlled environment was limited to the use of an infrared microscope because the specialised accessory can only operate in a horizontal position [11,24]. Macro-chambers (large sample compartments) of commercially available FTIR imaging systems, however, are only supplied with vertical sample holders which would not be suitable for the use with the controlled humidity cell. The first application of FTIR imaging and the controlled environment cell involved the use of an infrared microscope to study the sorption of water in a pharmaceutical formulation [11]. In such an arrangement, the field of view was ca. 0.27 mm 0.27 mm. Other relevant applications involved the use of infrared microscope in FTIR transmission imaging mode to study the morphology of semicrystalline polymers [3] and solvent-induced crystallisation of polymers [17]. By contrast, if the macro FTIR imaging mode was employed then the imaged area would be ca. 4 mm 4 mm. In this paper, we demonstrate the first application of macro FTIR imaging in transmission under a controlled environment to study polymorphic transition of a particular drug, nifedipine. Nifedipine exhibits both a glassy phase and a few crystalline states [25]. Conventional infrared spectroscopy had been applied to study different polymorphism of nifedipine under controlled humidity and temperature [25]. However, the location and the size of the crystalline domain were not observed previously because the imaging approach was not used.
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Fig. 1. Schematic diagram showing the path of the IR beam for horizontal macro transmission accessory in the macro-chamber of the imaging system. The position of the controlled environment cell is indicated by the position of the sample stage.
2.3. The controlled environment cell and sample preparation
2. Experimental
a-Crystalline nifedipine (a-NIF) in powder form was purchased from Sigma and used without further treatment. Glassy nifedipine sample film was prepared by melting a small quantity of a-NIF powder between a non-stick film and a BaF2 window at 190 8C followed by quenching on a cool surface. After quenching, the non-stick film was removed to expose the flat surface of the glassy nifedipine (g-NIF) film. The preparation of the film was performed in the dark to avoid the photochemical degradation of the drug. A controlled environment spectroscopic transmission cell (VGI2000M, Surface Measurement System Ltd.) was used to control the environment (temperature and humidity) of which the glassy nifedipine thin film was exposed to. For further details of this controlled environment cell see our previous publications [11,24]. The crystallisation experiment was performed at 38 8C and at relative humidity levels of 80%.
2.1. FTIR imaging
3. Results and discussion
The FTIR imaging system consisted of an infrared spectrometer (IFS66S, Bruker Optics), a macro-chamber (IMACTM, Bruker Optics) and a 64 64 focal plane array (FPA) detector. The size of the FPA array detector was 3.8 mm 3.8 mm. FTIR images were acquired in step scan mode and the image collection time for 8 cm 1 and 20 coadditions was ca. 300 s.
3.1. HMTA
2.2. Horizontal macro transmission accessory (HMTA) The accessory was built of three plane mirrors and one parabolic mirror (Fig. 1). The parabolic mirror refocuses the IR beam to the sample to increase the energy throughput of the accessory. No magnification or view expansion was expected from this accessory. Samples were placed on the sample stage horizontally during measurement. A copper grid, typically used in SEM, was utilised for validation of the imaged area.
The commercial design of the macro-chamber focuses the IR beam at the centre in the sample compartment. The design of the HMTA built by us directs the IR beam up at ca. 908, then ca. 908 to the right of the sample compartment, then ca. 908 downward towards the sample and then finally another ca. 908 to the right towards the detector, as shown in Fig. 1. In this way, the accessory increase the IR beam path by 2a (see Fig. 1) which reduces the amount of IR radiation that reaches the detector if all four mirrors were plane mirrors. A parabolic mirror is, therefore, introduced just before the beam enters the sample providing the means to refocus light onto the sample hence increases the IR light intensity throughput of the accessory. The design of the accessory allows the distance a to be adjusted to optimise the intensity of the IR radiation falling onto the detector. The focus of image can be adjusted in two ways: either by moving the whole accessory along the original
132
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Fig. 2. Left image is a photograph of the copper grid taken under a 4 objective. Right image is a FTIR image showing the total IR intensity across the whole imaged area (dark areas indicates a high intensity). The copper grid in the visible image was not in the same orientation as the grid in FTIR imaging measurement.
IR beam path in the macro-chamber or by moving the sample stage up or down. The accessory has been tested for any unpredicted image artefacts due to the optical design. A circular copper grid with regular pattern has been used as a test sample to verify the quality of the FTIR image. The white light image of the copper grid was first captured with an optical microscope and shown in Fig. 2 (left). The scale of the optical image has been calibrated with an objective scale from Nikon and the dimension of the copper has been estimated on the optical image using the video assisted measurement software. The grid has an outer diameter of 3050 mm, the width of the ring is 250 mm and the wire between each hexagon hole is ca. 45 mm wide. Then the macro FTIR image of the whole copper ring was measured with the newly developed accessory whilst the grid was placed in the sample stage of the HMTA. The FTIR image of the grid was generated, and shown in Fig. 2 (right), by plotting the intensities of IR radiation registered by each pixel across the whole FPA detector. The result shows that the size and the shape of the copper grid have been accurately projected on the FPA detector without significant image artefacts. It was not surprising that the wires of the copper grid in the FTIR image shown in Fig. 2 (right) appeared blurred since the width of the wire was smaller than the size of a single pixel. This successful demonstration of the HMTA extends the range of applications for macro FTIR imaging in transmission where sample mounting in vertical position would be difficult, and opens new opportunities to study samples in conjunction with the other accessories which only operate in horizontal orientations such as the controlled environment cell. 3.2. Application of HMTA to drug crystallisation The designed HMTA has been used in conjunction with the controlled environment cell to study the crystallisation of
nifedipine with FTIR imaging in situ. The particular form of nifedipine was analysed by characteristic absorption bands in infrared spectra determined in our previous studies using conventional FTIR microscopy [25]. The absorption bands of a-NIF at 3332 cm 1 (integration range of 3344–3300 cm 1), b-NIF at 3353 cm 1 (integration range of 3380–3344 cm 1) and g-NIF at 1281 cm 1 (integration range of 1292– 1269 cm 1) have been used to characterise the three polymorphs of nifedipine observed under controlled humidity as a function of time. Chemical images representing the distribution of a particular form of nifedipine have been generated by plotting the distribution of the value of the integrated absorbance of characteristic band of that component over the whole measuring area. Fig. 3 confirms that in the beginning of
Fig. 3. FTIR image showing the distribution of amorphous nifedipine before being exposed in high humidity and high temperature. The variation in intensity was due to the non-uniform film thickness. The image size is ca. 3.8 mm 3.8 mm.
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Fig. 4. Distribution of amorphous nifedipine (top row) and b-NIF (bottom row) of the drug film in the first 4 h of exposure in the controlled environment. The number at the bottom of each column indicates the time at which the image has been measured. Colour scales on right show the integral value of the characteristic absorption band of the corresponding component. The image size is ca. 3.8 mm 3.8 mm.
the experiment (at time = 0), all nifedipine was amorphous. The noises on the top of each image are artefact caused by the aging of the FPA detector. The film thickness was not exactly uniform as shown by the distribution of the absorbance, which is proportional to pathlength (film thickness). The effect of difference in film thickness to the image can be minimised either by ratioing the images against another image generated with an internal absorption band with which absorption does not change with any polymorphic transition or ratioing the images against Fig. 3 where the drug was still pure amorphous nifedipine film and the absorbance only varied with change in film thickness. In this case, the assumption that the film thickness does not change during the polymorphic transition is
made. The latter approach was used because it was more convenient and it has less chance of inducing unexpected artefacts caused by band shift. The resultant images after the ratioing are shown in Figs. 4 and 5. Fig. 4 has demonstrated that the amorphous nifedipine slowly crystallised into b-NIF and all area of the film was fully converted after 4 h of exposure to an environment of 38 8C and relative humidity of 80%. With the imaging capability, the location and the morphology of the initial crystal can been observed. The large imaged area allowed assessing development of crystalline domain. The bNIF crystal grows into large domains progressively starting from a few initial nucleate sites. It should be noted that the area measured under the infrared microscope using the
Fig. 5. Distribution of amorphous b-NIF (top row) and a-NIF (bottom row) of the drug film in the last 18 h of exposure in the controlled environment. The number at the bottom of each column indicates the time at which the image has been measured. Colour scales on right show the integral value of the characteristic absorption band of the corresponding component. The image size is 3.8 mm 3.8 mm.
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controlled humidity cell was ca. 250 times smaller than the area measured in this study. Fig. 5 shows the further polymorphic changes of the nifedipine film after 5 h of exposure to the controlled environment. The b-NIF film begins to crystallise into aNIF after 5 h of exposure time as shown in Fig. 5. The polymorphic change begins at the centre of the film followed by the side of the film, which progresses at a much slower pace than the conversion from g-NIF to b-NIF. There was no clear formation of large a-NIF domains unlike the g-NIF to b-NIF transition (Fig. 4). Perhaps this is due to the b-NIF to a-NIF transition does not require a crystal nucleation step while the gNIF to b-NIF transition requires a nucleation step before b-NIF can be formed under these experimental conditions. Once a bNIF crystal is formed, the crystal can grow relatively fast and forming large domains as shown in Fig. 4. It is important to note that such observation will not be possible with conventional FTIR measurements due to the lack of information about the domain size of the crystal formed. The conversion of nifedipine from b to a form was not completed 5 h after the first appearance of an a-NIF crystal in the film (the first appearance of an a-NIF crystal was 5 h after the beginning of the experiment). The general trend of observation of the nifedipine crystallisation from g-NIF to b-NIF to a-NIF was in good agreement with our previous study. However, the imaging capability allowed us to accurately locate the area where crystallisation or polymorphic transition initiated and how far it propagates through the whole film. This example also underlines the advantage of FTIR imaging over optical microscopy since the characteristic bands of each form of the drug allowed one to assess unequivocally the appearance of the particular form of the drug. This experiment also shows an opportunity to utilise the gradient of film thickness to study the effect of film thickness on crystallisation of amorphous nifedipine; however, this was left for future investigations. 4. Conclusions A novel approach has been developed which allows macro FTIR imaging in transmission of samples under controlled environment. This approach required construction of an optical accessory which enables the measurement of samples placed in a horizontal position in the macro-chamber of the imaging system. A combination of this new accessory with a controlled humidity cell provided an opportunity to obtain chemical images of the large areas of the samples under controlled humidity. In this study, this approach was applied to study the effect of humidity on polymorphic transitions of thin film of amorphous nifedipine. It has been shown that exposure of the drug to the conditions set in the experiment for 4 h resulted in a transition of the drug from amorphous to b-crystalline form while further exposure for several hours resulted in the
conversion of the b-form to the a-form of nifedipine. The FTIR imaging demonstrated in this experiment shows advantages of the chemical imaging approach compared to optical microscopy due to the chemical specificity of FTIR spectroscopy. Imaging capability allows one to visualise the distribution and evolution of domains of different forms of drug which can be useful in studies of crystallisation processes. Developed macro FTIR imaging approach in transmission can find broad range of applications from studying crystallisation processes in polymers and pharmaceuticals to high-throughput analysis of materials under controlled environments. Acknowledgements We thank Drs. G.D. Chryssikos and V. Gionis and for providing nifedipine, Mr. Richard Wallace in the workshop, EPSRC for support (GR/T08746/01). References [1] J.L. Koenig, Microscopic Imaging of Polymers, ACS, Washington, DC, 1998. [2] B.A. Miller-Chou, J.L. Koenig, Macromolecules 35 (2002) 440. [3] C.M. Snively, J.L. Koenig, J. Polym. Sci. Polym. Phys. 37 (1999) 2353. [4] K. Artyushkova, B. Wall, J.L. Koenig, J.E. Fulghum, J. Vac. Sci. Technol. A 19 (2001) 2791. [5] R. Bhargava, S.-Q. Wang, J.L. Koenig, Adv. Polym. Sci. 163 (2003) 13. [6] A. Gupper, S.G. Kazarian, Macromolecules 38 (2005) 232. [7] A. Gupper, P. Wilhelm, M. Schmied, S.G. Kazarian, K.L.A. Chan, J. Reussner, Appl. Spectrosc. 56 (2002) 1515. [8] R. Salzer, G. Steiner, H.H. Mantsch, J. Mansfield, E.N. Lewis, Fresenius J. Anal. Chem. 366 (2000) 712. [9] D.C. Fernandez, R. Bhargava, S.M. Hewitt, I.W. Levin, Nat. Biotechnol. 23 (2005) 469. [10] K.L.A. Chan, S.V. Hammond, S.G. Kazarian, Anal. Chem. 75 (2003) 2140. [11] K.L.A. Chan, S.G. Kazarian, Vib. Spectrosc. 35 (2004) 45–49. [12] Y. Roggo, A. Edmond, P. Chalus, M. Ulmschneider, Anal. Chim. Acta 535 (2005) 79. [13] K.L.A. Chan, S.G. Kazarian, J. Comb. Chem. 7 (2005) 185. [14] M. Tahtouh, J.R. Kalman, C. Roux, C.I. Lennard, B.J. Reedy, J. Forensic Sci. 50 (2005) 64. [15] C.M. Snively, J.L. Koenig, J. Polym. Sci. B: Polym. Phys. 37 (1999) 2261. [16] T. Ribar, J.L. Koenig, R. Bhargava, Macromolecules 43 (2001) 8340. [17] A. Gupper, K.L.A. Chan, S.G. Kazarian, Macromolecules 37 (2004) 6498. [18] J. Koenig, Adv. Mater. 14 (2002) 457. [19] A.J. Sommer, L.G. Tisinger, C. Marcott, G.M. Story, Appl. Spectrosc. 55 (2001) 252. [20] K.L.A. Chan, S.G. Kazarian, Appl. Spectrosc. 57 (2003) 381. [21] S.G. Kazarian, K.L.A. Chan, Macromolecules 36 (2003) 9866. [22] S.G. Kazarian, K.W.T. Kond, M. Bajomo, J. Van der Weerd, K.L.A. Chan, Food Bioproducts Process. 83 (C2) (2005) 127. [23] J. Van der Weerd, S.G. Kazarian, J. Pharm. Sci. 94 (2005) 2096. [24] H. Jervis, S.G. Kazarian, K.L.A. Chan, D. Bruce, N. King, Vib. Spectrosc. 35 (2004) 225. [25] K.L.A. Chan, O.S. Fleming, S.G. Kazarian, D. Vassou, G.D. Chryssikos, V. Gionis, J. Raman Spectrosc. 35 (2004) 353.