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Crystallization of amorphous lactose at high humidity studied by terahertz time domain spectroscopy
Chemical Physics Letters 558 (2013) 104–108
Contents lists available at SciVerse ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Crystallization of amorphous lactose at high humidity studied by terahertz time domain spectroscopy Alexander I. McIntosh a,b, Bin Yang a, Stephen M. Goldup b, Michael Watkinson b, Robert S. Donnan a,⇑ a b
Queen Mary University of London, School of Electrical Engineering & Computer Science, Mile End Road, London E1 4NS, England, UK Queen Mary University of London, School of Biological & Chemical Sciences, Joseph Priestley Building, Mile End Road, London E1 4NS, England, UK
a r t i c l e
i n f o
Article history: Received 16 November 2012 In final form 21 December 2012 Available online 31 December 2012
a b s t r a c t We report the first use of terahertz time-domain spectroscopy (THz-TDS) to study the hydration and crystallization of an amorphous molecular solid at high humidity. Lactose in its amorphous and monohydrate forms exhibits different terahertz spectra due to the lack of long range order in the amorphous material. This difference allowed the transformation of amorphous lactose to its monohydrate form at high humidity to be studied in real time. Spectral fitting of frequency-domain data allowed kinetic data to be obtained and the crystallization was found to obey Avrami kinetics. Bulk changes during the crystallization could also be observed in the time-domain. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Amorphous materials are of interest to many industries due to their physical properties. In particular the pharmaceutical industry has shown growing interest in using such materials due to their differing physical properties, increased solubility and bioavailability compared to their crystalline forms [1]. With the relative thermodynamic instability of the amorphous phase with respect to the crystalline phase there is a tendency for these materials to crystallize over time, either to anhydrous or hydrate crystalline forms, depending on the environmental conditions. Thermal and gravimetric techniques are well established for the study of such systems, however, the use of spectroscopic techniques is less developed but they have the potential to provide valuable ancillary information such as changes in bonding and the ability to distinguish polymorphs. In particular terahertz time domain spectroscopy (THz-TDS), although still immature, has much potential in this area due to its ability to probe low energy interactions in molecular solids [2]. Terahertz (0.1–10 THz) spectroscopy covers the far-infrared band (33–333 cm1 or 4–40 meV) of the electromagnetic spectrum, commonly referred to as the terahertz region. Due mainly to a lack of powerful sources, this region of the electromagnetic spectrum has traditionally been hard to access until the advent of improved technology in the last 20 years. The frequency of terahertz radiation (relative to mid-infrared, visible and ultraviolet radiation) is low, allowing it to probe low energy molecular motion and interactions such as torsional modes, librations and phonon modes within molecular lattices. It has therefore ⇑ Corresponding author. E-mail address: [email protected] (R.S. Donnan). 0009-2614/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cplett.2012.12.044
been shown that terahertz spectroscopy is particularly adept at characterizing molecular solids [3–6]. With fast spectral resolution it has shown some utility in studying phase changes and dehydration mechanisms in the solid state [7–9]. However, due to the sensitivity of terahertz spectroscopy to atmospheric humidity, this can make measurements at high humidity and the characterization of hydration processes more difficult. Here we demonstrate the use of THz-TDS to investigate the hydration and subsequent crystallization of the molecular solid lactose. Amorphous lactose, compared to its crystalline forms, has quite different physical properties. In particular its improved compaction behavior, in relation to direct compression tableting operations, has led to amorphous lactose being one of the most widely used amorphous materials in the pharmaceutical industry [10]. However, at high humidity it can absorb water leading to the transformation to the more thermodynamically stable a-lactose monohydrate resulting in potential problems during formulation, processing and storage [10]. As a result this phase change has been well studied by X-ray diffraction (XRD), thermal and gravimetric methods, but its study by spectroscopic methods is relatively less well developed [10–14]. In the terahertz region lactose exhibits very different spectra for its various forms [8]. Figure 1 shows spectra we previously recorded using THz-TDS for amorphous lactose and a-lactose monohydrate at low humidity. Due to the inherent sensitivity of terahertz spectroscopy to long range order the difference between the spectra is marked. The disordered amorphous form shows a largely featureless spectrum whereas the crystalline a-lactose monohydrate exhibits distinct spectral features. This clear distinction allowed the hydration and subsequent crystallization of amorphous lactose to be monitored in real time by THz-TDS.
Absorption / cm-1
A.I. McIntosh et al. / Chemical Physics Letters 558 (2013) 104–108
14
Amorphous Lactose
12
α-Lactose Monohydrate
10 8 6 4 2 0 0.2
0.7
1.2
1.7
2.2
Frequency / THz Figure 1. THz-TDS spectra for amorphous lactose (freeze dried) and a-lactose monohydrate. Material was mixed with polyethylene at a concentration of 25% w/w, compressed at 5 tons and spectra were recorded under a nitrogen purge.
2. Experimental method 2.1. Materials and methods
a-Lactose monohydrate was purchased from Sigma Aldrich and used as received. Amorphous lactose was prepared by rapidly freezing a 20% (w/v) aqueous solution of a-lactose monohydrate in dry ice followed by freeze drying in a VirTis Advantage freeze dryer at a pressure of 100 mTorr and a condenser temperature of 40 °C. Immediately after freeze drying the amorphous lactose was transferred to a desiccator over SiO2. 2.2. Sample preparation Amorphous lactose was lightly ground and diluted with ultrahigh molecular weight polyethylene powder (Sigma Aldrich) in a pestle and mortar to give a concentration of 65% w/w. This dilution ensured that absorption saturation could be avoided and also offered the possibility of producing thicker pellets so that multiple THz reflections could be prevented, pellets were typically 2.5 mm in thickness. This material was then compressed to form pellets at a low pressure of 0.5 tons. This low pressure ensured moisture could interact with the pellet and its absorption and desorption were not rate-limiting. Pellets for transmission study were then mounted inside a specially designed humidity chamber, which allowed the sample to be exposed to high humidity while allowing as much of the terahertz beam path to remain under as low a humidity condition as possible. This humidity chamber consisted of a pellet holder held between two TPXÒ windows with a path length of 52 mm. Beneath the sample, but within the holder, saturated salt solutions could be placed to keep the atmosphere of the box at a specific relative humidity defined by the salt solution employed. Crystallizations were undertaken using either deionised water or a saturated NaCl solution to give a humidity of 100% and 75% RH respectively at 295 K. 2.3. Terahertz time domain spectroscopy The THz-TDS set up used in our experiments is shown in Figure 2. At the heart of this system is the generation of THz radiation, which is facilitated by use of low temperature GaAs as a photoconductive antenna excited by a pulsed femtosecond laser beam from a Mai Tai Ti:Sapphire laser (Newport). The laser path used for generation is one which has been split just after the source into a pump and probe beam. The pump beam passes through a delay stage prior to excitation of the non-linear crystal. Following generation, the THz radiation is propagated along a beam path to the
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humidity chamber containing the amorphous lactose sample. After passing through the sample the resulting spectra can be detected using electro-optical detection. Here the THz radiation from the sample is directed to a crystal which can become birefringent when exposed to the electric field of the THz radiation. Typically a h1 1 0i ZnTe crystal is used. A portion of the optical beam from the laser is co-propagated through this crystal which results in its polarization rotating in proportion to the magnitude of the THz electric field. By using a Wollaston prism the beam is then split into two orthogonal fields (probe and measurement) which are passed onto the balanced photo-diode detectors. By measuring the signal as a function of time-delay between the THz radiation and the probe beam, the electric field of the THz pulse in the time-domain can be mapped. A Fourier transform of the time-domain data yields a spectrum in the frequency-domain. For further information regarding this technique the reader is directed elsewhere [15]. 3. Results and discussion From Figure 1 it can be seen that the spectrum of amorphous lactose is largely featureless over the 0.25–2.6 THz frequency range while for a-lactose monohydrate clear adsorption peaks at 0.53, 1.20, 1.38, 1.82 and 2.56 THz are observed. This difference in the spectra was used to observe the crystallization of amorphous lactose at high humidity. For our studies the region between 0.3 and 0.8 THz was used to observe and measure the crystallization as it occurred, as this frequency range appeared to be the most stable when the amorphous lactose began to hydrate and above this frequency range poor and irreproducible spectra were recorded. This effect is the result of poor signal to noise at these higher frequencies which is exacerbated by the physical changes in the specimen upon hydration and subsequent crystallization, resulting in large changes in scattering effects. In addition this spectral region contained the peak at 0.53 THz which is well separated from neighboring peaks and also has a well-defined Lorentzian line shape allowing quantitative information to be obtained from the spectra. This absorption peak is believed to be the result of the hindered rotational motion of the lactose molecule along the B axis of the crystal [16]. The overall changes during the hydration and crystallization of amorphous lactose at 100% RH and 295 K can be seen in the surface plot of the frequency-domain data in Figure 3. Qualitatively it can be seen that spectra have increasing absorbance with time, which is especially strong at high frequencies, up to approximately 120 min. Amorphous lactose is hygroscopic and it is well known that when exposed to high humidity, water absorption is the first stage in the crystallization of lactose [10,12]. Since water is strongly attenuating in the terahertz region the steady loss in signal is believed to be a result of this increasing water content of the sample in addition to an increase in scattering effects resulting, possibly, from the reordering and collapse of the amorphous crystallites [11]. After an initial induction-period lactose crystallization occurs and the peak at 0.53 THz can be seen to grow in intensity. At the same time the strong absorption at high frequencies begins to drop. In an attempt to follow the crystallization quantitatively the individual spectra in Figure 3 were fit with Eq. (1) using a Levenberg–Marquardt least squares fitting procedure.
aðv Þ ¼
A
1 þ v cv c
a 2 þ Bv þ C
ð1Þ
This expression for the absorption coefficient a(v) consists of two parts; a contribution from a Lorentzian function to represent the peak at 0.53 THz from the crystalline phase and a baseline fit.
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Figure 2. Schematic representation of the terahertz time-domain spectroscopy system used in this study.
Figure 3. Surface plot of terahertz spectra between 0.3 and 0.8 THz for amorphous lactose exposed to 100% RH at 295 K. Spectra were recorded every 270 s over a period of 351 min, pellets were prepared as described in the text.
100
Conversion / %
A Lorentzian function has previously been found to be the best fit for this peak and this was also found to be true for our data [17]. Here A represents the amplitude of the peak, vc the center frequency of the peak and c the half width at half maximum (HWHM). An effective baseline fit for both the amorphous and crystalline phases was modeled by Bva + C and was adapted from the work of Shen et al. [18]. All data fitting using Eq. (1) achieved fits with R2 P 0.99. This fitting allowed us to quantitatively observe changes in the parameters A, vc and c, which can be linked to physical phenomena, as the crystallization progressed with time. Firstly using the Lorenztian contribution, the fraction (/) of a-lactose monohydrate converted could be evaluated using the peak at 0.53 THz. Since an external calibration could not be reliably applied the completion of the crystallization was assumed when there was no further change in the peak at 0.53 THz. / was then calculated by normalizing the peak intensity during the course of the crystallization to this completed value. Using this approach it was apparent that /(t) was sigmoidal in form for both humidity measurements (Figure 4). This sigmoidal form is characteristic of Avrami type kinetics for isothermal solid state
80 75 % RH 60 75 % RH Avrami Fitting
40
100 % RH 20 100 % RH Avrami Fitting
0 50
100
150
200
250
300
350
Time / mins Figure 4. Rate of conversion to a-lactose monohydrate with time for amorphous lactose at 75% RH and 100% RH fitted to Eq. (2).
crystallizations (Eq. (2)) [19–21]. This Avrami type kinetics is consistent with previous observations using gravimetric analysis and XRD [10,22,23].
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0.535
Using the Avrami equation it was possible to fit / with time at 75% RH and 100% RH (Figure 4) with a least squares approach to give the Avrami constants K and n and the induction time tind (Table 1). These best fits (R2 > 0.995%) were found with n equal to 4 suggesting a constant nucleation rate and three dimensional growth of the crystalline domains. Within the Lorentzian term of Eq. (1) the parameters for peak center (vc) and HWHM (c) were found to vary throughout the crystallization (Fig. 5) at both 75% RH and 100% RH. From the induction time onwards peak center frequency blue shifts until it reaches the value of 0.534 THz and the HWHM of peak decreases to a value of 13 GHz. This narrowing and blue shift of the peak as the crystallization proceeds might be understood by considering that as the domains of crystalline a-lactose monohydrate grow the ratio of molecules close to or at the surface of the crystalline domain, to those in the bulk crystalline domain, changes. As the mode at 0.53 THz is believed to be a hindered rotation within the crystal lattice, this mode is expected to be strongly influenced by the intermolecular framework [16]. Therefore such hindered rotations near the surface of the crystalline domain, where little of this intermolecular framework exists and unit cell parameters can be relaxed, will be less hindered resulting in a lower frequency mode. In addition the intermolecular framework is less defined at the surface of the crystalline domain leading to a range of frequencies for this mode and a broadening of the peak. However, within the bulk crystalline phase the intermolecular framework is significantly more ordered leading to a more defined hindered rotational mode with less broadening. As the crystalline domains grow the contribution from these ‘bulk’ modes relative to the less defined ‘surface’ modes increases, resulting in a narrowing of the peak and its shift to higher frequency. Thus the growth of the crystalline phase on the nano-scale is expected to qualitatively lead to changes in the observed terahertz spectra. The growth of these crystalline domains from amorphous lactose can be easily understood by referring to the work of Mahlin et al. where they have recorded and measured their growth using AFM [24]. The changes observed in the terahertz spectra during crystallization are similar to those observed when studying vibrational spectra of nano-particles in aerosols and the crystallization of molecules in these aerosol particles [25–28]. Interestingly the overall bulk changes during the course of the experiment could also be clearly observed by studying the timedomain spectra before a Fourier transform was performed. For example Figure 6 shows the differences observed between the time-domain spectra at the start of the reaction (t = 0) and at the crystallization half-life (t = t1/2). As can be seen the height and position of the maxima in these time-domain spectra varies greatly. Figure 7 shows these changes in the peak maximum and position plotted as a function of time during the hydration and crystallization of amorphous lactose at 100% RH and 295 K. The general decrease in the peak maximum over the first 120 min correlates with the general increasing absorbance observed in Figure 3. A change in the rate of attenuation with time is seen at approximately 90 min and coincides with the onset of crystallization as is observed with the appearance of the peak at 0.53 THz (Fig. 3). After 130 min the peak maximum reaches its minimum value and this is
0.530
Table 1 Avrami constants for amorphous lactose at 75% RH and 100% RH with n = 4. Humidity
K/min4
tind/min
75% RH 100% RH
1.22 107 1.37 107
190.5 76.3
vc / THz
ð2Þ
0.525 0.520
100 % RH 75 % RH
0.515
50
100
150
200
250
300
350
250
300
350
γ (HWHM) / GHz
Time / mins 35 30 25 20 15 10
50
100
150
200
Time / mins Figure 5. Change of peak center frequency (vc) (top) and HWHM (c) (bottom) with time at 75% RH and 100% RH.
Peak Max t = 0
1.00
Relative Intensity
t=0 t=0 0.75
t = t1/2 t=t1/2
Peak Max t = t1/2
0.50 0.25 0.00 -0.25 -0.50 -5
0
5
10
15
20
25
Relative Peak Position / ps
Peak Maximum Relative Intensity
Figure 6. Expanded time-domain spectra for amorphous lactose exposed to 100% RH at 295 K showing the change in peak maximum and position at the beginning of the reaction (t = 0) and at the crystallization half-life (t = t1/2).
1.00
1.00
0.90 0.90
0.80 0.70
0.80
0.60 0.70
0.50 0.40
0.60
0.30 0.20
0.50
Relative Peak Position / ps
/ ¼ 1 eKðttind Þ
0.10 99 %
1%
0.40 0
50
t1/2 150 Time / mins
100
0.00 200
250
Figure 7. Change in peak maximum (solid line) and relative peak position (dashed line) with time for the time-domain spectra of amorphous lactose exposed to 100% RH at 295 K. Crystallization half-life (t1/2), 1% and 99% conversion to a-lactose monohydrate are marked.
the time at which the rate of crystallization is at its maximum (i.e. crystallization half-life, t1/2). After this point an increase in peak maximum is observed which may be as a result of the loss of the water of crystallization as is observed in dynamic vapor sorption experiments of the same phenomenon [10]. This change in the
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peak maximum with time is largely mirrored with the relative change in the peak position with time, however this is observed at much reduced resolution due to the resolution of the delay stage. This use of the main time-domain peak in THz-TDS spectra for observing bulk changes in the sample has also been demonstrated previously and could provide a valuable method for detecting bulk changes in molecular solids, since it can be done with greater time resolution and speed compared to frequency-domain observations [9]. 4. Conclusions Due to the high sensitivity of terahertz spectroscopy to water, measurements at high humidity can be challenging. Through careful experimental design it was shown that qualitative changes in amorphous lactose exposed to high humidity could be observed with THz-TDS both in the frequency and time-domains. Using an equation to describe the spectral data, quantitative data regarding the rate of crystallization and its kinetics could be obtained. This allowed changes in spectral peak position and width to be observed during the preliminary phase of crystallization, implying that THz-TDS is sensitive to size-effects in nano-crystallites. Further investigations may enable these effects to be modeled, potentially allowing terahertz spectroscopy to make estimations of crystallite size and growth-rate during crystallization. Acknowledgments The authors thank the Engineering and Physical Sciences Research Council (EPSRC) of UK for financial support under Grant EP/1014845/1.
References [1] A. Newman, G. Knipp, G. Zografi, J. Pharm. Sci. 101 (2012). [2] A.I. McIntosh, B. Yang, S.M. Goldup, M. Watkinson, R.S. Donnan, Chem. Soc. Rev. 41 (2012) 2072. [3] M.D. King, W.D. Buchanan, T.M. Korter, Anal. Chem. 83 (2011) 3786. [4] D.G. Allis, P.M. Hakey, T.M. Korter, Chem. Phys. Lett. 463 (2008) 353. [5] C.J. Strachan, T. Rades, D.A. Newnham, K.C. Gordon, M. Pepper, P.F. Taday, Chem. Phys. Lett. 390 (2004) 20. [6] P.F. Taday, I.V. Bradley, D.D. Arnone, M. Pepper, J. Pharm. Sci. 92 (2003) 831. [7] H.B. Liu, X.C. Zhang, Chem. Phys. Lett. 429 (2006) 229. [8] J.A. Zeitler et al., Int. J. Pharm. 334 (2007) 78. [9] J.A. Zeitler, P.F. Taday, M. Pepper, T. Rades, J. Pharm. Sci. 96 (2007) 2703. [10] E.A. Schmitt, D. Law, G.G.Z. Zhang, J. Pharm. Sci. 88 (1999) 291. [11] G. Buckton, P. Darcy, Int. J. Pharm. 136 (1996) 141. [12] R.A. Lane, G. Buckton, Int. J. Pharm. 207 (2000) 57. [13] T.A.G. Langrish, Food Res. Int. 41 (2008) 630. [14] R. Price, P.M. Young, J. Pharm. Sci. 93 (2004) 155. [15] C.A. Schmuttenmaer, Chem. Rev. 104 (2004) 1759. [16] D.G. Allis, A.M. Fedor, T.M. Korter, J.E. Bjarnason, E.R. Brown, Chem. Phys. Lett. 440 (2007) 203. [17] E.R. Brown, J.E. Bjarnason, A.M. Fedor, T.M. Korter, Appl. Phys. Lett. 90 (2007) 061908. [18] Y.C. Shen, P.F. Taday, M. Pepper, Appl. Phys. Lett. 92 (2008) 051103. [19] A. Khawam, D.R. Flanagan, J. Phys. Chem. B 110 (2006) 17315. [20] M. Avrami, J. Chem. Phys. 7 (1939) 1103. [21] M. Avrami, J. Chem. Phys. 8 (1940) 212. [22] M.K. Haque, Y.H. Roos, Carbohydr. Res. 340 (2005) 293. [23] K. Jouppila, J. Kansikas, Y.H. Roos, J. Dairy Sci. 80 (1997) 3152. [24] D. Mahlin, J. Berggren, G. Alderborn, S. Engstrom, Pharm. Sci. 93 (2004). [25] O.F. Sigurbjoernsson, G. Firanescu, R. Signorell, Annu. Rev. Phys. Chem. 60 (2009) 127. [26] G. Firanescu, D. Hermsdorf, R. Ueberschaer, R. Signorell, Phys. Chem. Chem. Phys. 8 (2006) 4149. [27] O.F. Sigurbjornsson, R. Signorell, Phys. Chem. Chem. Phys. 10 (2008) 6211. [28] A. Bonnamy, R. Georges, A. Benidar, J. Boissoles, A. Canosa, B.R. Rowe, J. Chem. Phys. 118 (2003) 3612.
Contents lists available at SciVerse ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Crystallization of amorphous lactose at high humidity studied by terahertz time domain spectroscopy Alexander I. McIntosh a,b, Bin Yang a, Stephen M. Goldup b, Michael Watkinson b, Robert S. Donnan a,⇑ a b
Queen Mary University of London, School of Electrical Engineering & Computer Science, Mile End Road, London E1 4NS, England, UK Queen Mary University of London, School of Biological & Chemical Sciences, Joseph Priestley Building, Mile End Road, London E1 4NS, England, UK
a r t i c l e
i n f o
Article history: Received 16 November 2012 In final form 21 December 2012 Available online 31 December 2012
a b s t r a c t We report the first use of terahertz time-domain spectroscopy (THz-TDS) to study the hydration and crystallization of an amorphous molecular solid at high humidity. Lactose in its amorphous and monohydrate forms exhibits different terahertz spectra due to the lack of long range order in the amorphous material. This difference allowed the transformation of amorphous lactose to its monohydrate form at high humidity to be studied in real time. Spectral fitting of frequency-domain data allowed kinetic data to be obtained and the crystallization was found to obey Avrami kinetics. Bulk changes during the crystallization could also be observed in the time-domain. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Amorphous materials are of interest to many industries due to their physical properties. In particular the pharmaceutical industry has shown growing interest in using such materials due to their differing physical properties, increased solubility and bioavailability compared to their crystalline forms [1]. With the relative thermodynamic instability of the amorphous phase with respect to the crystalline phase there is a tendency for these materials to crystallize over time, either to anhydrous or hydrate crystalline forms, depending on the environmental conditions. Thermal and gravimetric techniques are well established for the study of such systems, however, the use of spectroscopic techniques is less developed but they have the potential to provide valuable ancillary information such as changes in bonding and the ability to distinguish polymorphs. In particular terahertz time domain spectroscopy (THz-TDS), although still immature, has much potential in this area due to its ability to probe low energy interactions in molecular solids [2]. Terahertz (0.1–10 THz) spectroscopy covers the far-infrared band (33–333 cm1 or 4–40 meV) of the electromagnetic spectrum, commonly referred to as the terahertz region. Due mainly to a lack of powerful sources, this region of the electromagnetic spectrum has traditionally been hard to access until the advent of improved technology in the last 20 years. The frequency of terahertz radiation (relative to mid-infrared, visible and ultraviolet radiation) is low, allowing it to probe low energy molecular motion and interactions such as torsional modes, librations and phonon modes within molecular lattices. It has therefore ⇑ Corresponding author. E-mail address: [email protected] (R.S. Donnan). 0009-2614/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cplett.2012.12.044
been shown that terahertz spectroscopy is particularly adept at characterizing molecular solids [3–6]. With fast spectral resolution it has shown some utility in studying phase changes and dehydration mechanisms in the solid state [7–9]. However, due to the sensitivity of terahertz spectroscopy to atmospheric humidity, this can make measurements at high humidity and the characterization of hydration processes more difficult. Here we demonstrate the use of THz-TDS to investigate the hydration and subsequent crystallization of the molecular solid lactose. Amorphous lactose, compared to its crystalline forms, has quite different physical properties. In particular its improved compaction behavior, in relation to direct compression tableting operations, has led to amorphous lactose being one of the most widely used amorphous materials in the pharmaceutical industry [10]. However, at high humidity it can absorb water leading to the transformation to the more thermodynamically stable a-lactose monohydrate resulting in potential problems during formulation, processing and storage [10]. As a result this phase change has been well studied by X-ray diffraction (XRD), thermal and gravimetric methods, but its study by spectroscopic methods is relatively less well developed [10–14]. In the terahertz region lactose exhibits very different spectra for its various forms [8]. Figure 1 shows spectra we previously recorded using THz-TDS for amorphous lactose and a-lactose monohydrate at low humidity. Due to the inherent sensitivity of terahertz spectroscopy to long range order the difference between the spectra is marked. The disordered amorphous form shows a largely featureless spectrum whereas the crystalline a-lactose monohydrate exhibits distinct spectral features. This clear distinction allowed the hydration and subsequent crystallization of amorphous lactose to be monitored in real time by THz-TDS.
Absorption / cm-1
A.I. McIntosh et al. / Chemical Physics Letters 558 (2013) 104–108
14
Amorphous Lactose
12
α-Lactose Monohydrate
10 8 6 4 2 0 0.2
0.7
1.2
1.7
2.2
Frequency / THz Figure 1. THz-TDS spectra for amorphous lactose (freeze dried) and a-lactose monohydrate. Material was mixed with polyethylene at a concentration of 25% w/w, compressed at 5 tons and spectra were recorded under a nitrogen purge.
2. Experimental method 2.1. Materials and methods
a-Lactose monohydrate was purchased from Sigma Aldrich and used as received. Amorphous lactose was prepared by rapidly freezing a 20% (w/v) aqueous solution of a-lactose monohydrate in dry ice followed by freeze drying in a VirTis Advantage freeze dryer at a pressure of 100 mTorr and a condenser temperature of 40 °C. Immediately after freeze drying the amorphous lactose was transferred to a desiccator over SiO2. 2.2. Sample preparation Amorphous lactose was lightly ground and diluted with ultrahigh molecular weight polyethylene powder (Sigma Aldrich) in a pestle and mortar to give a concentration of 65% w/w. This dilution ensured that absorption saturation could be avoided and also offered the possibility of producing thicker pellets so that multiple THz reflections could be prevented, pellets were typically 2.5 mm in thickness. This material was then compressed to form pellets at a low pressure of 0.5 tons. This low pressure ensured moisture could interact with the pellet and its absorption and desorption were not rate-limiting. Pellets for transmission study were then mounted inside a specially designed humidity chamber, which allowed the sample to be exposed to high humidity while allowing as much of the terahertz beam path to remain under as low a humidity condition as possible. This humidity chamber consisted of a pellet holder held between two TPXÒ windows with a path length of 52 mm. Beneath the sample, but within the holder, saturated salt solutions could be placed to keep the atmosphere of the box at a specific relative humidity defined by the salt solution employed. Crystallizations were undertaken using either deionised water or a saturated NaCl solution to give a humidity of 100% and 75% RH respectively at 295 K. 2.3. Terahertz time domain spectroscopy The THz-TDS set up used in our experiments is shown in Figure 2. At the heart of this system is the generation of THz radiation, which is facilitated by use of low temperature GaAs as a photoconductive antenna excited by a pulsed femtosecond laser beam from a Mai Tai Ti:Sapphire laser (Newport). The laser path used for generation is one which has been split just after the source into a pump and probe beam. The pump beam passes through a delay stage prior to excitation of the non-linear crystal. Following generation, the THz radiation is propagated along a beam path to the
105
humidity chamber containing the amorphous lactose sample. After passing through the sample the resulting spectra can be detected using electro-optical detection. Here the THz radiation from the sample is directed to a crystal which can become birefringent when exposed to the electric field of the THz radiation. Typically a h1 1 0i ZnTe crystal is used. A portion of the optical beam from the laser is co-propagated through this crystal which results in its polarization rotating in proportion to the magnitude of the THz electric field. By using a Wollaston prism the beam is then split into two orthogonal fields (probe and measurement) which are passed onto the balanced photo-diode detectors. By measuring the signal as a function of time-delay between the THz radiation and the probe beam, the electric field of the THz pulse in the time-domain can be mapped. A Fourier transform of the time-domain data yields a spectrum in the frequency-domain. For further information regarding this technique the reader is directed elsewhere [15]. 3. Results and discussion From Figure 1 it can be seen that the spectrum of amorphous lactose is largely featureless over the 0.25–2.6 THz frequency range while for a-lactose monohydrate clear adsorption peaks at 0.53, 1.20, 1.38, 1.82 and 2.56 THz are observed. This difference in the spectra was used to observe the crystallization of amorphous lactose at high humidity. For our studies the region between 0.3 and 0.8 THz was used to observe and measure the crystallization as it occurred, as this frequency range appeared to be the most stable when the amorphous lactose began to hydrate and above this frequency range poor and irreproducible spectra were recorded. This effect is the result of poor signal to noise at these higher frequencies which is exacerbated by the physical changes in the specimen upon hydration and subsequent crystallization, resulting in large changes in scattering effects. In addition this spectral region contained the peak at 0.53 THz which is well separated from neighboring peaks and also has a well-defined Lorentzian line shape allowing quantitative information to be obtained from the spectra. This absorption peak is believed to be the result of the hindered rotational motion of the lactose molecule along the B axis of the crystal [16]. The overall changes during the hydration and crystallization of amorphous lactose at 100% RH and 295 K can be seen in the surface plot of the frequency-domain data in Figure 3. Qualitatively it can be seen that spectra have increasing absorbance with time, which is especially strong at high frequencies, up to approximately 120 min. Amorphous lactose is hygroscopic and it is well known that when exposed to high humidity, water absorption is the first stage in the crystallization of lactose [10,12]. Since water is strongly attenuating in the terahertz region the steady loss in signal is believed to be a result of this increasing water content of the sample in addition to an increase in scattering effects resulting, possibly, from the reordering and collapse of the amorphous crystallites [11]. After an initial induction-period lactose crystallization occurs and the peak at 0.53 THz can be seen to grow in intensity. At the same time the strong absorption at high frequencies begins to drop. In an attempt to follow the crystallization quantitatively the individual spectra in Figure 3 were fit with Eq. (1) using a Levenberg–Marquardt least squares fitting procedure.
aðv Þ ¼
A
1 þ v cv c
a 2 þ Bv þ C
ð1Þ
This expression for the absorption coefficient a(v) consists of two parts; a contribution from a Lorentzian function to represent the peak at 0.53 THz from the crystalline phase and a baseline fit.
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Figure 2. Schematic representation of the terahertz time-domain spectroscopy system used in this study.
Figure 3. Surface plot of terahertz spectra between 0.3 and 0.8 THz for amorphous lactose exposed to 100% RH at 295 K. Spectra were recorded every 270 s over a period of 351 min, pellets were prepared as described in the text.
100
Conversion / %
A Lorentzian function has previously been found to be the best fit for this peak and this was also found to be true for our data [17]. Here A represents the amplitude of the peak, vc the center frequency of the peak and c the half width at half maximum (HWHM). An effective baseline fit for both the amorphous and crystalline phases was modeled by Bva + C and was adapted from the work of Shen et al. [18]. All data fitting using Eq. (1) achieved fits with R2 P 0.99. This fitting allowed us to quantitatively observe changes in the parameters A, vc and c, which can be linked to physical phenomena, as the crystallization progressed with time. Firstly using the Lorenztian contribution, the fraction (/) of a-lactose monohydrate converted could be evaluated using the peak at 0.53 THz. Since an external calibration could not be reliably applied the completion of the crystallization was assumed when there was no further change in the peak at 0.53 THz. / was then calculated by normalizing the peak intensity during the course of the crystallization to this completed value. Using this approach it was apparent that /(t) was sigmoidal in form for both humidity measurements (Figure 4). This sigmoidal form is characteristic of Avrami type kinetics for isothermal solid state
80 75 % RH 60 75 % RH Avrami Fitting
40
100 % RH 20 100 % RH Avrami Fitting
0 50
100
150
200
250
300
350
Time / mins Figure 4. Rate of conversion to a-lactose monohydrate with time for amorphous lactose at 75% RH and 100% RH fitted to Eq. (2).
crystallizations (Eq. (2)) [19–21]. This Avrami type kinetics is consistent with previous observations using gravimetric analysis and XRD [10,22,23].
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0.535
Using the Avrami equation it was possible to fit / with time at 75% RH and 100% RH (Figure 4) with a least squares approach to give the Avrami constants K and n and the induction time tind (Table 1). These best fits (R2 > 0.995%) were found with n equal to 4 suggesting a constant nucleation rate and three dimensional growth of the crystalline domains. Within the Lorentzian term of Eq. (1) the parameters for peak center (vc) and HWHM (c) were found to vary throughout the crystallization (Fig. 5) at both 75% RH and 100% RH. From the induction time onwards peak center frequency blue shifts until it reaches the value of 0.534 THz and the HWHM of peak decreases to a value of 13 GHz. This narrowing and blue shift of the peak as the crystallization proceeds might be understood by considering that as the domains of crystalline a-lactose monohydrate grow the ratio of molecules close to or at the surface of the crystalline domain, to those in the bulk crystalline domain, changes. As the mode at 0.53 THz is believed to be a hindered rotation within the crystal lattice, this mode is expected to be strongly influenced by the intermolecular framework [16]. Therefore such hindered rotations near the surface of the crystalline domain, where little of this intermolecular framework exists and unit cell parameters can be relaxed, will be less hindered resulting in a lower frequency mode. In addition the intermolecular framework is less defined at the surface of the crystalline domain leading to a range of frequencies for this mode and a broadening of the peak. However, within the bulk crystalline phase the intermolecular framework is significantly more ordered leading to a more defined hindered rotational mode with less broadening. As the crystalline domains grow the contribution from these ‘bulk’ modes relative to the less defined ‘surface’ modes increases, resulting in a narrowing of the peak and its shift to higher frequency. Thus the growth of the crystalline phase on the nano-scale is expected to qualitatively lead to changes in the observed terahertz spectra. The growth of these crystalline domains from amorphous lactose can be easily understood by referring to the work of Mahlin et al. where they have recorded and measured their growth using AFM [24]. The changes observed in the terahertz spectra during crystallization are similar to those observed when studying vibrational spectra of nano-particles in aerosols and the crystallization of molecules in these aerosol particles [25–28]. Interestingly the overall bulk changes during the course of the experiment could also be clearly observed by studying the timedomain spectra before a Fourier transform was performed. For example Figure 6 shows the differences observed between the time-domain spectra at the start of the reaction (t = 0) and at the crystallization half-life (t = t1/2). As can be seen the height and position of the maxima in these time-domain spectra varies greatly. Figure 7 shows these changes in the peak maximum and position plotted as a function of time during the hydration and crystallization of amorphous lactose at 100% RH and 295 K. The general decrease in the peak maximum over the first 120 min correlates with the general increasing absorbance observed in Figure 3. A change in the rate of attenuation with time is seen at approximately 90 min and coincides with the onset of crystallization as is observed with the appearance of the peak at 0.53 THz (Fig. 3). After 130 min the peak maximum reaches its minimum value and this is
0.530
Table 1 Avrami constants for amorphous lactose at 75% RH and 100% RH with n = 4. Humidity
K/min4
tind/min
75% RH 100% RH
1.22 107 1.37 107
190.5 76.3
vc / THz
ð2Þ
0.525 0.520
100 % RH 75 % RH
0.515
50
100
150
200
250
300
350
250
300
350
γ (HWHM) / GHz
Time / mins 35 30 25 20 15 10
50
100
150
200
Time / mins Figure 5. Change of peak center frequency (vc) (top) and HWHM (c) (bottom) with time at 75% RH and 100% RH.
Peak Max t = 0
1.00
Relative Intensity
t=0 t=0 0.75
t = t1/2 t=t1/2
Peak Max t = t1/2
0.50 0.25 0.00 -0.25 -0.50 -5
0
5
10
15
20
25
Relative Peak Position / ps
Peak Maximum Relative Intensity
Figure 6. Expanded time-domain spectra for amorphous lactose exposed to 100% RH at 295 K showing the change in peak maximum and position at the beginning of the reaction (t = 0) and at the crystallization half-life (t = t1/2).
1.00
1.00
0.90 0.90
0.80 0.70
0.80
0.60 0.70
0.50 0.40
0.60
0.30 0.20
0.50
Relative Peak Position / ps
/ ¼ 1 eKðttind Þ
0.10 99 %
1%
0.40 0
50
t1/2 150 Time / mins
100
0.00 200
250
Figure 7. Change in peak maximum (solid line) and relative peak position (dashed line) with time for the time-domain spectra of amorphous lactose exposed to 100% RH at 295 K. Crystallization half-life (t1/2), 1% and 99% conversion to a-lactose monohydrate are marked.
the time at which the rate of crystallization is at its maximum (i.e. crystallization half-life, t1/2). After this point an increase in peak maximum is observed which may be as a result of the loss of the water of crystallization as is observed in dynamic vapor sorption experiments of the same phenomenon [10]. This change in the
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peak maximum with time is largely mirrored with the relative change in the peak position with time, however this is observed at much reduced resolution due to the resolution of the delay stage. This use of the main time-domain peak in THz-TDS spectra for observing bulk changes in the sample has also been demonstrated previously and could provide a valuable method for detecting bulk changes in molecular solids, since it can be done with greater time resolution and speed compared to frequency-domain observations [9]. 4. Conclusions Due to the high sensitivity of terahertz spectroscopy to water, measurements at high humidity can be challenging. Through careful experimental design it was shown that qualitative changes in amorphous lactose exposed to high humidity could be observed with THz-TDS both in the frequency and time-domains. Using an equation to describe the spectral data, quantitative data regarding the rate of crystallization and its kinetics could be obtained. This allowed changes in spectral peak position and width to be observed during the preliminary phase of crystallization, implying that THz-TDS is sensitive to size-effects in nano-crystallites. Further investigations may enable these effects to be modeled, potentially allowing terahertz spectroscopy to make estimations of crystallite size and growth-rate during crystallization. Acknowledgments The authors thank the Engineering and Physical Sciences Research Council (EPSRC) of UK for financial support under Grant EP/1014845/1.
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