In Situ X-ray Diffraction Study of the Crystallization of Spray-Dried Lactose A. Shawqi Barham* and B. Kieran Hodnett Materials and Surface Science Institute (MSSI) and the Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland Received May 28, 2005;
CRYSTAL GROWTH & DESIGN 2005 VOL. 5, NO. 5 1965-1970
Revised Manuscript Received June 29, 2005
ABSTRACT: An in situ technique with X-ray diffraction analysis has been developed to study the crystallization of lactose in humid air. This technique was used in association with ex situ scanning electron microscopy to determine changes in phase composition and morphology during crystallization. Individual spherical particles of spray-dried lactose plasticize and coalesce upon absorption of moisture. Following an induction period, crystallization is rapid with the appearance of the anhydrous 5:3 phase containing both R and β isomers of lactose, R-lactose monohydrate and β-lactose, almost simultaneously. The 5:3 phase decomposed as the other two phases developed. After extended exposure to humid air, only R-lactose monohydrate and β-lactose were observed. The predominant particle habit observed in the fully crystallized lactose is platelet. These observations are rationalized in terms of the restricted molecular diffusion in plasticized lactose preventing movement of the β-lactose isomer toward developing R-lactose monohydrate crystals with the result that normal solution phase inhibition of growth of the (01 h 1) face leading the tomahawk shaped crystal lactose does not occur. The restricted diffusional conditions also favor the formation of the mixed isomer 5:3 phase, resulting in the entrapment of both isomers during crystallization. Introduction The physicochemical properties of amorphous and partially crystalline materials are of increasing importance in many industrial products, such as polymers, metals, foods, ceramics, optical materials (glasses and fibers), and pharmaceuticals.1-5 In particular, the activation energy barrier for crystallization of certain compounds, among them lactose, is lowered on exposure to water vapor and/or heat.1 However, these materials have problems regarding manufacturability, hygroscopicity, shelf life, and stability arising from differences in the thermodynamic stability of the crystalline and amorphous forms.6 Furthermore, material properties, morphology, density, physicochemical properties, hygroscopicity, chemical stability, water solubility, flow properties, and compatibility6-8 are dependent on the solid phase(s) present be they crystalline or amorphous. Lactose is a disaccharide consisting of two moieties of D-glucose and D-galactose joined by a β-1,4-glycosidic linkage. There are two isomeric forms of lactose, R-lactose and β-lactose, differing in the relative positions of H- and OH-groups at the anomeric center of the glucose moiety.9,10 In aqueous solution, the two forms of lactose are balanced through mutarotation.11 The natural crystalline state of lactose is R-lactose monohydrate, but several solid forms exist, such as anhydrous β-lactose, stable and unstable R-lactose, and amorphous lactose.12 Amorphous lactose is a very hygroscopic material without a crystal structure.13 It can be prepared by using several drying techniques such as hot air (fixed or fluidized), freeze-drying, spray drying, and rapid cooling of melt.6,14 Amorphous lactose usually contains both R- and β-lactose isomers.13 Several researchers studied the crystallization of amorphous lactose under storage conditions in humid * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: +353 612324159. Fax: +353 61213529.
air by moisture uptake.15-17 Joupilla et. al reported that the occurrence of crystallization processes of amorphous lactose at a constant temperature depends on relative humidity and water content during storage.18 They concluded that the rate of the crystallization of the amorphous lactose depends on storage RH, water content, and the difference between the storage temperature and the glass transition temperature (Tg).18 As a result of the crystallization processes, water plasticization and depression of the Tg below the storage temperature has been reported.19 When water plasticizes lactose, it will clearly start to change, which results in a collapse, stickiness, and the formation of cakes with densification. When this occurs, the amorphous material changes from its glassy state to a rubbery state, at which point mutarotation and crystallization to R-lactose monohydrate occurs.19 Above the glass transition, the molecular mobility increases as evidenced by a decrease in viscosity and increasing flow. Lost of adsorbed water is an indication of the crystallization processes.18,19 On the basis of the previous discussion, the present study describes a method to characterize the transformation of amorphous lactose to its various crystalline forms in a controlled environment. In situ XRD is used to study the solid-state transformation of spray-dried amorphous lactose to the crystalline phases as a function of time. Quantification of crystallinity is correlated with morphological observations from SEM. Experimental Procedures Materials. Crystalline R-lactose monohydrate (C12H22O11‚ H2O), β-lactose anhydrous (C12H22O11, 73% β-lactose and 27% R-lactose monohydrate), molecular sieves (calcium, sodium alumino-silicate, 1/16 in. pellets, Nominal pore diameter 5 Å), phosphorus pentoxide anhydride (P2O5, 98%), and potassium chloride (KCl, 99%) were purchased from Sigma-Aldrich Ltd. Lactose samples were prepared by spray-drying 15% w/w aqueous R-lactose (Dairy Gold, Mitchlestown, Cork, Ireland)
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Scheme 1.
Schematic of in Situ XRD under Humid N2
Barham and Hodnett In a typical operation, about 0.17 g of spray-dried lactose was placed in the sample holder, and a flat surface was formed using a clean glass slide. The sample was scanned over a range of 5-23 °2θ using a step size of 0.0167 °2θ and a scan speed of 0.0711 °2θ/s. Scan time was 5 min. During the X-ray diffraction run, the sample was maintained under isothermal conditions. The temperature of the saturator and the in situ XRD chamber was 22 and 25 °C, respectively. The vapor pressure of water at 22 and 25 °C was 2.9 and 3.3 kPa, respectively.29
relative humidity (%) ) saturator water vapor pressure × 100% ) in situ XRD water vapor pressure (2.9 kPa/3.3 kPa) × 100% ) 88 %
using a Niro Atomizer spray-drier (Copenhagen, Denmark). The spray drying settings were as follows: inlet temperature 185-190 °C and outlet temperature 80-85 °C. Materials were collected, immediately packaged into glass containers, and desiccated over phosphorus pentoxide [0% relative humidity (RH) at 25 °C]. Before experiments, the residual moisture was removed by drying the lactose samples in a vacuum oven at 50 °C for 24 h.20-22 Characterization. X-ray Diffraction. The characterization of spray-dried lactose was carried out using an X-ray powder diffractometer model (Philips X’Pert-MPD PRO diffractometer) with nickel filtered copper Cu KR radiation (λ ) 1.542 Å) as the X-ray source. The Cu KR diffractometer anode was run under a tension of 40 kV and a current of 35 mA. The diffraction angle °2θ was varied by rotating the X-ray tube and X’Celerator strip detector to collect the diffracted data. All samples were spun at 10 Hz. In situ X-ray Diffraction under a Controlled Environment. The design principle of the present system is shown in Scheme 1. The in situ XRD chamber (High-Temperature Oven Camera HTK1200, Anton Paar Gmbh, Austria) consists of a housing lid with connectors, a heater (inside housing), and the sample holder flange with a sample holder (rotating). All components of the HTK 1200 housing were made of nickelplated brass. The base plate and wall were water-cooled. The adapter, which was used to mount the HTK 1200 to the goniometer, has to be connected to the housing’s base plate. Vertical alignment of the HTK 1200 versus the goniometer axis was enabled by using the height aligning equipment. The sample holder was inserted through the opening at the bottom. Two guide pins provide for exact centering of the sample holder flange. The housing was slotted along the radiation path (entrance and exit of X-rays). A 125 µm Kapton foil (respectively, Kapton foil + graphite foil double layer) that was pressed against an O-ring by means of two clamps was used to airtighten these window openings. The sample holder was placed in an airtight chamber provided with an inlet and outlet for a gas at a controlled humidity. The chamber was equipped with a wire heater on the inner wall and was thermally insulated. The relative humidity and temperature were controlled as follows: the temperature in the sample chamber was independently controlled by a Temperature Control Unit TCU1000 (Anton Paar Gmbh, Austria), which controls the wire heater of the sample and the inner wall of the chamber. Gas of a specified humidity was formed by passing dry nitrogen (N2) into a saturator filled with Zeolite (Molecular Sieves, SigmaAldrich Ltd.) saturated in water. The temperature of the saturator (wet N2) was controlled using a Clifton (Nickel Electro LTD) water bath (Scheme 1). The gas flow rate of the dry N2 could be varied from 50 to 500 mL min-1 using a mass flow meter (Brooks, Instrument B.V.). A flow rate of 100 mL/min was used in this work. By varying the temperature of the water bath (saturator) and for a set temperature of 25 °C in the in situ XRD chamber (HTK 1200), different relative humidity values were created.
A point of some importance is the delivery of moisture achieved using the in situ method. At a N2 flow rate 100 mL min-1 containing a partial pressure of water of 3.3 kPa, the system delivers 1.33 × 10-4 mol of water (2.40 × 10-3) per minute, corresponding to 1.4 wt % of the mass of spray-dried lactose in the in situ cell. The stoichiometric requirement for R-lactose monohydrate is 5 wt%, corresponding to the amount of water delivered to the in situ cell in 4 min. Typical water sorption data show maximum water uptake of 10 wt% at high relative humidity, corresponding to a delivery time of 8 min in our in situ cell.20-22 In this work, there was no variation in crystallization time when the N2 flow rate was varied from 50 to 500 mL min-1. These data indicate that the slow step in the crystallization process is not the rate of delivery of water to the in situ cell. Scanning Electron Microscopy (SEM). In this study, the in situ crystallization procedure was interrupted at regular intervals, and small amounts of sample were taken for ex situ SEM analysis. The electron micrographs were obtained using a JEOL JSM-5600 electron microscope. A small amount of unground material was scattered evenly onto the surface of an aluminum stub covered with a 12 mm diameter carbon tab. Excess material was removed using dry compressed air, and the sample was sputter coated with a thin conductive film of gold for approximately 2 min in an Edwards S150B sputter coater. Before coating, a vacuum was established in the chamber after which Ar gas was introduced. The stub containing the coated sample was then placed in the specimen chamber under high vacuum, the accelerated electron beam was directed onto the sample, and the image was produced. The accelerating voltage used was 20 keV.
Results and Discussion Figure 1 presents the in situ X-ray diffraction XRD patterns of spray-dried lactose exposed to water vapor for up to 320 min at 25 °C (RH ) 88%). The starting material contained small amounts of β-lactose (note the small peak at 2θ ) 10.5°), but these seemed to disappear, and the sample was X-ray amorphous after 70 min. At 100 min, the features of β-lactose started to appear and were very strong at 110-125 min (note peaks at 2θ ) 10.5° (110) and 21° (111)). At 110 min, peaks of other phases of lactose also started to appear including R-lactose monohydrate. A point of interest is that the XRD technique is about 3 times more sensitive in the detection of the 2θ ) 10.5° (110) peak of β-lactose than the 2θ ) 12.5° (110) peak of R-lactose monohydrate, so that simple visual inspection of the intensities of the peaks in Figure 1 gives an erroneous impression of the true percentages of each present. At least six phases have been associated with crystalline lactose. The structures of two of these, namely, R-lactose monohydrate and β-lactose, are well-established. The structures of the four remaining phases are
Crystallization of Spray-Dried Lactose
Figure 1. Typical in situ X-ray diffraction patterns of spraydried lactose exposed to water vapor for the times indicated at T ) 25 °C, RH ) 88%, and pH2O ) 3.3 kPa.
less well-established. Buma and Wiegers presented the XRD patterns of stable R-anhydride and unstable R-anhydride.23 In addition, Simpson et al. have described anhydrous compounds crystallized from solutions containing molar ratios of anhydrous R/β of 5:3 and 4:1.24 The principal XRD peaks of each of the six compounds in question are presented in Figure 2, either as recorded XRD patterns for R-lactose monohydrate and β-lactose (traces E and F) or as bar charts taken from the literature (traces G-J) for the other phases.23,24 Its clear that many intense peaks associated with these phases occur in the 19-20 °2θ region, making phase identification based on this region alone very difficult. All the peaks observed by in situ XRD in spraydried lactose after 320 min exposure to humid air can be associated for mixtures of R-lactose monohydrate and β-lactose. The same result was recorded when the sample crystallized in situ for 320 min was ground into a fine powder and the XRD pattern again was recorded. In addition, all the peaks observed in the sample of spray-dried lactose exposed to humid air at RH ) 76% for 144 h in ex situ conditions only exhibited the peaks of R-lactose monohydrate and β-lactose.22 The situation is different for XRD patterns recorded in situ after 120 min (see Figure 2, trace D). In this case, the peaks of β-lactose are clearly present (2θ ) 10.5°). There are also indications of R-lactose monohydrate (2θ ) 12.5 and 16.4°), but the three main characteristic peaks of this phase (2θ ) 19.1, 19.6, and 19.9°) have not developed fully, and the principal peak present in this pattern is at 20.1°. There are additional small peaks
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Figure 2. X-ray diffraction patterns of (A) spray-dried lactose exposed to humid air at 76% RH for 144 h. (B) Sample taken from the in situ cell after 320 min at 88% RH. (C) In situ pattern for spray-dried lactose exposed to RH ) 88% for 320 min. (D) In situ pattern for spray-dried lactose exposed to RH ) 88% for 120 min. (E) R-Lactose hydrate monohydrate (Sigma). (F) β-Lactose anhydrous (Sigma). (G) Anhydrous R/β) 5:3.24 (H) Anhydrous R/β) 4:1.24 (I) Stable R-lactose.23 (J) Unstable anhydrous R-lactose.23
at 2θ ) 18.3 and 22.1°. These features are characteristic of the so-called anhydrous 5:3 phase. One additional feature remains in the XRD patterns recorded at between 110 and 185 min, namely, a peak at 2θ ) 19.4°. Assignment of this peak is difficult as no other unassigned peaks were observed. The closest match is with the stable R-lactose anhydrous.23 However, this assignment is very tentative (see Figure 2). On the basis of this analysis, phase evolution during the crystallization of spray-dried lactose (Figure 3) was determined using the XRD peaks summarized in Table 1. The (040) of R-lactose monohydrate at 2θ ) 16.4° was selected as the signature peak for this phase over the (110) peak at 2θ ) 12.5° because the anhydrous 5:3 phase also exhibits an interfering peak at 2θ ) 12.4°. β-Lactose does exhibit a small peak at 2θ ) 16.4° also, but it is very weak. The (110) peak of β-lactose at 2θ ) 10.5° was selected as the signature peak for this phase, and the 2θ ) 18.3 and 22.1° peaks were both selected for the anhydrous 5:3 phase. Figure 3 summarizes the temporal evolution of phases during crystallization of spray-dried lactose. The characteristic peaks extracted from Figure 1 to create Figure 3 are identified in the former with broken lines. Quantification of phase content has been addressed for R-lactose monohydrate and for β-lactose. Unfortunately, standards are not available for any of the other
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Figure 3. Crystallization profile for spray-dried lactose exposed to water vapor for the times indicated at T ) 25 °C, RH ) 88%, and pH2O ) 3.3 kPa. The letters in this figure indicate the times at which samples were captured for SEM analysis, presented in Figure 6.
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Figure 4. XRD calibration plot for R-lactose monohydrate: (9 (040); 16.4° 2θ). Standards were prepared by mixing pure R-lactose monohydrate, KCl, and glass. Each sample contained 10 wt% KCl as an internal standard, and all the peaks for R-lactose monohydrate were normalized against (200) KCl peak at 28.3° 2θ (R2 ) 0.9721).
Table 1. Summary of the Peaks Used to Construct Figure 3 and Other Peaks Used to Analyze Phase Identification peaks used to construct Figure 3
phase β-lactose R-lactose monohydrate R/β) 5:3
other characteristic peaks of this phase
10.5° (110) 16.4° (040)
19.1 and 21.0° 12.5, 19.1, 19.6, and 19.9° 19.1 and 20.1°
18.3 and 22.1°
Table 2. Calibration Constants Determined by the Linear Fit between Normalized Peaks of β-Lactose Anhydrous and r-Lactose Monohydrate at Selected Miller Planes Using 10% w/w KCl as Internal Standarda
β-lactose R-lactose
(hkl)
d-spacing Å
°2θ
1/slope
R2
(110) (040)
8.42 5.41
10.5 16.4
166.7 322.6
0.9753 0.9721
a wt % of β- and R-lactose hydrate was determined as follows: wt % ) [I(hkl)(1/slope)].
phases. The XRD patterns of R-lactose monohydrate and β-lactose anhydrous used as standards in this work are presented in Figure 2, traces E and F. Pure β-lactose is not commercially available. β-Lactose (Sigma-Aldrich) contains 73% as the β-phase and 27% as the R-phase. Known amounts of crystalline R-lactose monohydrate or β-lactose, glass, and 10 wt% KCl were mixed in an agate mortar and pestle with grinding. Each sample was mixed further using a Vortex Mixer (Fisherbrand) for 10 min. The mixed samples are compressed manually into an aluminum sample holder. The samples are scanned over a range of 5-40 °2θ using a step size of 0.0167 °2θ and a scan speed of 0.0356 °2θ/s. X-ray diffraction calibration plots for R-lactose monohydrate and β-lactose were generated by dividing the areas under selected peaks in the diffraction patterns by the area of 2θ ) 28.3° (200) of KCl. Accordingly, the results using the (040) peak at 2θ ) 16.4° for R-lactose monohydrate and the (110) peak of β-lactose at 2θ ) 10.5° are presented in Figures 4 and 5 (see Table 2). These calibration plots were used to determine the % crystalline R-lactose monohydrate and % β-lactose present in samples exposed to different relative humidities and
Figure 5. XRD calibration plot for β-lactose: (9 (110); 10.5° 2θ). Standards were prepared by mixing β-lactose (73% β-lactose and 27% R-lactose hydrate), KCl, and glass. Each sample contained 10 wt% KCl as an internal standard, and all the peaks for β-lactose were normalized against (200) KCl peak at 28.3 °2θ (R2 ) 0.9753).
mixed with 10 wt% KCl prior to X-ray analysis. On this basis, the % R-lactose monohydrate and β-lactose for the sample exposed to 76% RH for 144 h (see Figure 2, trace A) was 62 and 35%, respectively. Quantification of the samples crystallized using the in situ method is difficult because it is not possible to add KCl standard to the in situ cell because it might affect the rate of uptake of water. However, the XRD patterns of our samples after 320 min using the in situ technique (see Figure 2, traces B and C) are very similar to the sample exposed to water vapor at RH ) 76%. Figure 6 presents a selection of SEM images from samples taken at the indicated times from the in situ cell. Care was taken during the procedure to avoid exposure of the captured samples to the ambient conditions, and the SEM image was recorded within minutes of its removal from the XRD cell. Images of samples withdrawn at short exposure times (10 min) show the typical appearance of individual spherical particles of spray-dried lactose (Figure 6A) (bulk density ) 0.81 g/cm3 and BET surface area ) 8 m2/g). The first change observed at 75 min is the coalescence of individual particles driven by the absorption of water (see Figure 6B). At 85 min, still prior to the onset of crystallization, there was a striking change
Crystallization of Spray-Dried Lactose
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Figure 6. SEM micrographs of the in situ X-ray diffraction patterns of spray-dried lactose exposed to water vapor for the times indicated at T ) 25 °C, RH ) 88%, and pH2O ) 3.3 kPa. (A) At 10 min, (B) at 70 min, (C and D) at 85 min, (E) at 105 min, (F) at 115 min, (G) at 125 min, (H) at 185 min, and (I) at 320 min.
in appearance of the sample (Figure 6C,D). Complete coalescence appears to have occurred with a large increase in particle size and almost total disappearance of the original spherically shaped particles. This change is associated with the plasticization of lactose generated by the absorption of water. Some granular features started to appear just at the onset of crystallization, namely, at 105 min (Figure 6E), and these became more pronounced as the XRD intensity developed (Figure 6F,G). The principal crystallization feature that developed at this time was poorly defined and sometimes particles with platelet habit were found. At 125 min (Figure 6G), nearly the full intensity of the XRD patterns had developed. However, the morphology of the sample continued to develop. Some 3-D granular particles had developed after 185 min (Figure 6H), and at 320 min, well-developed platelets were the predominant feature in the SEM images. A major outcome of this study is the elaboration of an in situ X-ray diffraction technique that allows individual lactose phases to be monitored during crystallization in humid air. Quantification of phase compositions within the in situ cell was not possible, but the results recorded are entirely consistent with quantitative measures made with samples generated in ex situ conditions to which a KCl internal standard was
added prior to XRD analysis. An important outcome of this work, summarized in Figure 3, is the observation of the so-called anhydrous 5:3 phase as a transient phase in the crystallization of amorphous spray-dried lactose. This phase appeared at the start of the crystallization process and was a dominant feature for the first 30 min. Thereafter, its intensity declined, and at longer crystallization times, only R-lactose monohydrate and β-lactose appeared. A point to note from Figure 3 is that the lower intensity peaks were used to generate the temporal plots. Consequently, perception of the percentage of each phase present should not be taken from the relative intensities of the peaks used to generate this figure. Unfortunately, the structure of the anhydrous 5:3 phase is not known. The original report by Simpson et al. describes it as a crystalline phase generated from a solution made up of a 5:3 molar ratio of R/β isomers.24 This phase is likely to contain both the R and β isomers of lactose. Interestingly, as the crystallization progresses, this phase transforms into R-lactose monohydrate and β-lactose. Recent work by Raghavan et al. on the composition of R-lactose monohydrate formed from aqueous solutions of both isomers indicates that the R-lactose monohydrate contains very little of β-lactose.25
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At this point, it is also useful to comment on the SEM analysis. There is ample evidence in the literature to indicate that water uptake is immediate on contact of spray-dried lactose with humid air but that there is a considerable induction period associated with crystallization. However, this work shows that the initial crystallization event itself is very rapid. The time from the onset of crystallization to the generation of 75% of the final intensities is of the order of 30 min. The rapid crystallization is facilitated by the plasticization of the lactose material by absorbed water. This, in turn, allows a limited amount of molecular movement within the lactose particles. A second factor is the tendency for lactose particles to coalesce in the plasticized state, so that a single nucleation point is sufficient to induce crystallization throughout the coagulated particles. The speed of the crystallization process together with limited molecular movements in the plasticized lactose can be presented as a reason the anhydrous 5:3 phase forms initially, namely, both R-lactose monohydrate and β-lactose isomers become trapped within the crystallization event. By contrast, crystallization from aqueous solution with R and β isomers both present but free to move through the liquid phase favors the formations of pure R-lactose monohydrate on β-lactose. Indeed, the anhydrous 5:3 phase observed in this work appears to be unstable, and a driving force may be the preference for these systems to form mixtures of pure R-lactose monohydrate and pure β-lactose crystals. A noteworthy feature of the SEM data is that the familiar tomahawk shaped particles often associated with R-lactose monohydrate crystallized from aqueous solutions were not observed. Instead, our materials tended to show platelet type morphology. This result is consistent with the work of Roetman, who also observed this type of particle shape for lactose crystallized in humid air.26 Several researchers have extensively studied the origin of R-lactose monohydrate particle shapes in the principal phase present in spray-dried lactose crystallized in humid air.17,26,27 It is now clearly established that when R-lactose monohydrate crystallizes from an aqueous solution containing R and β isomers, tomahawk shaped particles result because the β isomer inhibits growth of the (01h 1) facet of R-lactose monohydrate. When the β isomer is not present, then needle or platelet shaped particles result. Dincer et al. reported that the β-lactose significantly influences the morphology of R-lactose monohydrate crystals grown from DMSO solution.28 They concluded that at low concentrations of β-lactose, the fastest growing face is the (01h 1) face resulting in long thin prismatic crystals. At higher β-lactose concentrations, the main growth occurs in the b direction, and the (020) face becomes the fastest growing face since the (01h 1) face is blocked by β-lactose, producing pyramid and tomahawk shaped crystals. Our observation of a predominance of platelet shaped particles in this work is entirely consistent with those findings. First, in the plasticized state prior to the onset of crystallization, molecular motion is restricted by comparison with the solution state, so preventing the necessary movements of the β isomer toward specific
Barham and Hodnett
developing facets of the crystallizing R-lactose monohydrate. The rapidity of the crystallization event is also a factor as the rate maybe significant by comparison with diffusion in the plasticized lactose. Finally, the formation of the anhydrous 5:3 phase and indeed β-lactose at the onset of crystallization ties up most of the β isomer, leaving less available to attach to developing facets of R-lactose monohydrate. Acknowledgment. The authors acknowledge financial support from PRTL1 Cycle3 of the Higher Education Authority of Ireland, and Prof. Yrjo¨ Roos and Kamrul Haque, University College Cork for the provision of spray dried lactose and for useful discussions. References (1) Price, R.; Young, P. M. J. Pharm. Sci. 2004, 93, 155-164. (2) Lian, Yu. Adv. Drug Delivery Rev. 2001, 48, 27-42. (3) Mazzobre, M. F.; Soto, G.; Aguilera, J. M.; Buera, M. P. Food Res. Int. 2001, 34, 903-911. (4) Sebhatu, T.; Angberg, M.; Ahlneck, C. Int. J. Pharm. 1994, 104, 135-144. (5) Vivienne, L. H.; Craig, Q. M.; Feely, L. C. Int. J. Pharm. 1998, 161, 95-107. (6) Gombas, A.; Antal, I.; Szabo-Revesz, P.; Marton, S.; Eros, I. Int. J. Pharm. 2003, 256, 25-32. (7) Gombas, A.; Szabo-Revesz, P.; Kata, M.; Regdon, G.; Eros, I. J. Therm. Anal. Calorim. 2002, 68, 503-510. (8) Figura, L. O. Thermochim. Acta 1993, 222, 187-194. (9) Michaels, A. S.; Van Kreveld, A. Neth. Milk Dairy J. 1966, 20, 163-181. (10) Wong, N. P.; Jenness, R.; Keeney, M.; Marth, E. H. Fundamentals of Dairy Chemistry; Van Nostrand Reihold Co.: New York, 1988. (11) Roelfsema, W. A.; Coberco; F.; Kuster, F. M. Lactose and Derivatives. In Ullmann’s Encyclopedia of Industrial Chemistry; Wiley-VCH: Weinheim, 2002. (12) Fox, P. F.; McSweeney, P. L. H. Dairy Chemistry and Biochemistry; Blackie Academic and Professional: London, 1998. (13) Roetman, K.; Van Schaik, M. Neth. Milk Dairy J. 1975, 29, 225-237. (14) Bhandari, B. R.; Howes, T. J. Food Eng. 1999, 40, 71-79. (15) Nakai, Y.; Fukuoka, E.; Nakajima, S.; Morita, M. Chem. Pharm. Bull. 1982, 30 (5), 1811-1818. (16) Olano, A.; Corzo, N.; Castro, I. Milchwissenschaft 1983, 38 (8), 471-474. (17) Faldt, P.; Bergenstahl, B. Lebensm.-Wiss. Technol. 1996, 29, 438-446. (18) Jouppila, K.; Kansikas, J.; Roos, Y. H. J. Dairy Sci. 1997, 80, 3152-3160. (19) Buckton, G.; Darcy, P. Int. J. Pharm. 1996, 136, 141-146. (20) Haque, M. K.; Roos, Y. H. J. Food Sci. 2004, 69, 23-29. (21) Haque, M. K.; Roos, Y. H. J. Food Sci. 2004, 69, 384-391. (22) Haque, M. K.; Roos, Y. H. Carbohydr. Res. 2005, 340, 293301. (23) Buma, T. J.; Wiegers, G. A. Neth. Milk Dairy J. 1967, 21, 208-213. (24) Simpson, T. D.; Parrish, F. W.; Nelson, M. L. J. Food Sci. 1982, 47, 1948-1954. (25) Raghavan, S. L.; Ristic, R. I.; Sheen, D. B.; Sherwood, J. N. J. Pharm. Sci. 2001, 90 (7), 823-832. (26) Roetman, K. Neth. Milk Dairy J. 1979, 33, 1-11. (27) Buma, T. J.; Henstra, S. Neth. Milk Dairy J. 1971, 25, 7580. (28) Dincer, T. D.; Parkinson, G. M.; Rohl, M. I.; Ogden, M. I. J. Cryst. Growth 1999, 205, 368-374. (29) CRC Handbook of Chemistry and Physics, 73rd ed.; Lide, D. R., Ed.; CRC Press: Boca Raton, FL, 1992.
CG050237C
