Especificaciones técnicas del microcontrolador

Diferential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), Karl- Fischer analysis. Thin Layer Chromatography (TLC) and hot stage microscopy were used in the characterization of amorphous terfenadine samples.

Saleki-Gerhardt et al. (1994b) showed that DSC could be used to estimate the degree of disorder with an overall sensitivity of +5%. They pointed out however that for highly crystalline samples, this method is not able to differentiate low levels of amorphous content when the level of disorder decreases below 10%. The DSC measures the heat capacity (Cp) as well as the glass transition temperature (Tg) of amorphous solids.

In the case of amorphous solids there is an increase in the heat capacity of the solid at the glass transition temperature, which shows as a shift in the DSC thermogram baseline. At the same time, it should be mentioned that the heat capacity of an amorphous material is always higher than that of its crystalline counterpart (Hancock and Zografi, 1997).

The basic principles of hot stage microscopy were described in Section 2.1.6. A heating stage is incorporated on the optical microscope, which allows viewing of structural transitions on a microscale. Many transitions such as collapse, crystallization, melting and decomposition can be viewed via hot stage microscopy. A built in temperature programmer is used to control the temperature of the sample as well as the heating rate. The general principles of TGA were explained in Section 2.1.5, Karl Fischer titration was discussed in Section 2.1.11, TLC was described in Section 2.1.12.

3.2.2.I. METHODS:

The DSC was performed by placing one bead of slow cooled terfenadine (12 mg + 2mg) in the aluminium pan for testing to be performed. Since the quenched sample shatters while quenching, the only thing that could be done was to take almost the same sample weight as that of the slow cooled terfenadine in the same pan type. Hermetically sealed and open aluminium pans (Perkin-Elmer) were used in the experiments and heating was carried out over a temperature range of 5 - 180 °C while holding isothermally for two minutes at the start temperature. The heating rates used were as shown in Table 3.2 and Table 3.3. The Tg was calculated as the mid point value with half Cp extrapolated. The results reported in Table 3.2 and Table 3.3 are those obtained at a first run on the DSC. TGA experiments were carried out in a ramp mode from ambient temperature to a maximum temperature of 170 °C at a heating rate of 20 °C/min. Open aluminium pans supplied by Perkin-Elmer were employed. For comparison purposes, the TGA experiments were carried out under purged nitrogen gas within the same temperature range and at a heating rate used in the DSC. The Karl-Fischer method and the instrument used in the analysis were discussed in Section 2.1.11.

The method used in Thin Layer Chromatography (TLC) consisted of a mobile phase, a stationary phase and a detection method. Those were described in Section 2.1.12. Terfenadine was dissolved in HPLC grade methanol. The solution was prepared in a concentration of 5 mg/ ml. Samples were prepared by spotting a 5 pi sample by a micropipette on the silica gel plate, 2 cm away from the bottom of the plate (this is considered as point of origin). Closed, glass TLC jars containing the mobile phase were left for a couple of hours to saturate the glass chamber with organic vapours before placing the spotted silica gel plates inside. When the mobile phase travelled up the stationary phase a distance of around 11 cm, the developed plates were taken out, left to dry and later read using UV light at 254 nm.

In carrying out the experiments using hot stage microscopy, a sample was taken and placed on an Ultima plain glass slide having the dimensions of 76 x 26 mm and a thickness of 1 - 1.2 mm. In the case of the slow cooled sample, the bead was lightly crushed with a spatula before being placed on the slide. The sample was then covered by a slide’s cover made from borosilicate glass with the dimensions of 22 x 26 mm (BDH). The slide was then placed on the heating stage and was heated from ambient temperature up to complete melting of the sample at a heating rate of 2 °C/min. The magnification

used was lOx (objective lens) in the case of the slow cooled terfenadine sample and 40x (objective lens) in the case of the quenched sample, multiplied by the magnification of the eyepiece (ocular lens), which was lOx. The net magnification was hence equal to lOOx and 400x respectively.

3.2.2.1 RESULTS:

Crystallization of the amorphous quenched and slow cooled samples of terfenadine was noticed to take place during the DSC experiments when the DSC experiments were carried out at the lowest heating rate of 10 ° d min. However, no crystallization of either the quenched or the slow cooled terfenadine samples was noted at the higher heating rate of 20°C/ min during the DSC experiments. A typical DSC trace of amorphous terfenadine showing crystallization to occur just prior to melting when the experiments were carried out at a heating rate of 10 °C/ min is shown in Figure 3.3 for slow cooled sample and Figure 3.4 for quench cooled sample. Figures 3.5 and 3.6 show the lack of crystallization of the amorphous samples when the DSC experiments were performed at a higher heating rate of 20°C/ min. It is well known that the heating/cooling rate has an effect on the crystallization of the sample from the amorphous state (Ediger et al., 1996; Hancock et al., 1998; Moynihan et al., 1974). At a higher heating rate, sample crystallization can be avoided through rapid increase in temperature, which does not give sufficient time for crystallization of the amorphous sample to occur since crystallization takes place at a temperature between Tg and Tm. Table 3.2 shows the DSC results of quench cooled terfenadine samples in hermetic and open pans where samples are run from 5 - 180 °C at two heating rates (10, 20 °C min). The samples are basically amorphous with an average enthalpy of fusion of the crystalline portion of 3.1 J/g (Table 3.2). This corresponds to ca. 3.0 % crystallinity of the quenched sample (knowing that the enthalpy of fusion of the completely crystalline sample is 101.5 J/g as seen in Table 3.1). This average, crystallinity value was obtained when the DSC experiments were carried out at a heating rate of 10 °C/min where some crystallization took place during the DSC experiments and hence it reflects the total crystallinity in the sample (the crystalline content present prior to the DSC and that achieved during the DSC experiments). The results in Table 3.2 show that lower average Tg values were achieved when hermetically sealed pans were used as opposed to open pans at the same heating rate. This is probably due to entrapment of water leading to an inability of water to escape when sealed pans were used (the

presence o f w ater is confirmed by Karl-Fischer results below). W ater can be gathered by the sam ple during quenching in liquid nitrogen and, being basically am orphous, water may get absorbed by the sam ple, and cause suppression o f Tg due to its plasticizing effect. The Tg value is affected by heating rate and it can be noted from Table 3.2 that at the low er heating rate o f 10 ”C/ min, a slightly lower average Tg was obtained for the quenched sam ples (ca. 57 °C at 10 °C/ min versus ca. 59 °C at 20 °C/min in open pans and ca. 54 °C at 10 ®C/ min versus ca. 57 °C at 20 °C/ min in hermetically sealed pans). Table 3.3 shows the DSC results o f slow cooled terfenadine samples. The experim ents were carried out at two heating rates (10, 20 ®C/min) in hermetically sealed aluminium pans. As in the case o f the quenched sam ples, it can be seen that the heating rate affected the obtained Tg value with a slightly lower Tg noted at the lower heating rate (slow cooled terfenadine exhibited a Tg value o f ca. 59 °C at 10 °C/min and ca. 61 °C at 20 °C/ min). Ediger et al. (1996) and Moynihan et al. (1974) explained the effect o f heating rate on the Tg value o f am orphous solids. They clarified that a higher Tg value can be obtained at a faster heating rate than that achieved at a lower heating rate. Hancock et al. ( 1998) stated that for all the m aterials they used in their study, the Tg changed by about 7 K for a ten-fold change in scanning rate. The effect o f heating/cooling rate on the Tg value was discussed in Section 1.2. Basically a smaller heating/cooling rate means a low er change in tem perature with time. This can allow for m ore m olecular rearrangem ents resulting in a m ore dense glass and the sam ple will convert from the rubbery to the glassy state at a lower tem perature.

• .0 7 .S - § 6 7 .0 - 8.0 5.6- 5.0-1 Tm Water Crystallization 0.0 6 5 .0 SO.O 7 5 .8 to o .e TwM foten r o las.o 150.0 178.0I

Figure 3.3 Typical DSC thermogram o f the amorphous slow cooled terfenadine at a

10.0- Water fl.ô- Tm 9.0- B . 3 - 8.0- I 7.5- 1 Crystallization 0.5 5.5- 5.0 150.0 100 .0 TMpervtnre (*Q 0 0 .0 0 .0 85.0

Figure 3.4 Typical DSC therm ogram o f the am orphous quench cooled terfenadine at a heating rate o f 10 °C/min showing som e crystallization prior to melting.

Table 3.2 DSC data o f the quenched terfenadine sample. Batches were prepared as described in Section 3.2.1.2.

Heating rate rc/min)

Pan type Sample no. Tg CC) Tm (*C) A H f J/g 10 Hermetic pans 1 55J 147.2 2.4 2 54.4 146.8 3.7 3 53J 147.2 3.1 Average 54.3 147.1 3.1 S.D. 1 . 0 0.3 0.7 20 1 55^ 146.9 0.4 2 57.0 146.4 1 . 2 3 5&2 148.7 0 . 8 Average 57.0 147.3 0 . 8 S.D. 1 . 2 1 . 2 0.4 10 Open pans 1 57.3 148.8 1.5 2 56.6 148.5 2 . 1 Average 57.0 148.7 1 . 8 S.D. 0.5 0 . 2 0.4 20 1 58.1 148.3 0.3 2 59J 147.6 0.4 Average 58.7 147.9 0.3 S.D. 0.9 0.5 0 . 1

Table 3.3 DSC data for slow cooled terfenadine using hermetically sealed aluminum pans. Heating rate (T/min) Sample no. Tg

CO

Figure 3.5 Typical DSC thermogram o f quenched terfenadine at a heating rate o f 20

Water

Tm

I

SS

Figure 3.6 Typical DSC thermogram o f slow cooled terfenadine at a heating rate o f 20

^C/min showing the lack o f crystallization during the DSC experiment.

Table 3.4 C om parison o f average DSC values and average crystallinity for quenched and slow cooled sam ples o f terfenadine using hermetically sealed aluminium pans.

Heating Sample Average Average Average %

rate treatment Tg, (S.D.) Tm, (S.D.) AHf, (S.D.) Crystallinity

("C/min) °C J/g 1 0 Quenched 54.3 147.1 3.1 3.1 (1.0) (0.3) (0.7) Slow cooled 58 j 149.2 7.4 7.3 (1.3) (1.0) (0.2) 2 0 Quenched 57.0 147.3 0 . 8 0 . 8 (1 2) (1.2) (0.4) Slow cooled 60.5 148.9 1.3 1.3 (1.3) (1.0) (0.6)

C om parison o f the DSC results for quenched versus slow cooled terfenadine sam ples is dem onstrated in Table 3.4. It can be noticed that quenching produced the less therm odynam ically stable polym orph (polym orph II which shows a melting range o f 146 - 148 °C as reported by Badwan et al., 1990), whereas slow cooling produced the m ost therm odynamically stable polym orph with a melting range o f 149 - 152 °C. This was

noticed from the DSC results of experiments carried out at a heating rate of 20 ®C/min (where no crystallization of the amorphous sample was seen during the DSC experiments). Comparing the DSC results of quenched and slow cooled samples at a heating rate of 10 °C/ min (where crystallization was seen during the DSC experiments) showed that the slow cooled samples crystallized to the most stable polymorph (polymorph I), whereas the quenched samples crystallized into a metastable polymorph (polymorph II) during the DSC experiments. Yoshioka et al. (1994) reported that rapidly cooled indomethacin crystallized under nonisothermal conditions predominantly to the less thermodynamically stable a form, whereas the slowly cooled sample predominantly gave the more thermodynamically stable y crystal.

Comparison of the percentage crystallinity between quench cooled and slow cooled terfenadine samples and of the same sample type (quenched or slow cooled) at the two heating rates of 10 and 20 °C/ min is shown in Table 3.4. It can be observed from Table 3.4 that the quenched terfenadine samples exhibited lower crystallinity than the slow cooled samples (3.1% crystallinity in quench cooled sample versus 7.3% crystallinity in the slow cooled sample at a heating rate of 10 °C/ min in the DSC and 0.8% crystallinity in the quench cooled sample versus 1.3% crystallinity in the slow cooled sample at a heating rate of 20 °C/ min in the DSC). Seymour and Carraher (1996) pointed out that quench cooling might result in lower percentage of crystallinity in an essentially amorphous sample whereas slow cooling favours a comparatively higher level of crystallinity. The quenched and slow cooled samples have crystallinity of 0.8 and 1.3 % respectively, which reflects the inherent crystallinity in the samples prior to the DSC run. This is obtained from the enthalpy of fusion seen in the DSC experiments when a heating rate of 20 °C/ min was applied (Table 3.4), since at this heating rate no crystallization of the samples during the DSC experiments was noted (Figures 3.5 and 3.6 for quench cooled and slow cooled terfenadine samples respectively). Since both quench cooled and slow cooled samples showed some crystallization taking place during the DSC experiments when a heating rate of 10 °C/ min was applied, while the same samples failed to crystallize at the higher heating rate of 20 °C/ min during the DSC experiments, the difference in crystallinity for the same sample type (quenched or slow cooled) at the two heating rates corresponds to that amount of amorphous solid that crystallized during the DSC experiments. This amounts to around 2.3% crystallinity of the quenched sample and 6% crystallinity of the slow cooled sample obtained during the DSC experiments.

A stricter control was applied over sample preparation through leaving the sample for a longer time over the hot plate once melting was achieved before slow cooling (fixed at ca. 5 minutes once complete melting was accomplished) and to check the effect that this had on percentage crystallinity of the resulting samples. The temperature of the samples did not exceed 165 °C and no change in colour or odour was observed. The data showed substantial reduction in crystalline content of the resulting slow cooled samples with two out of the three samples tested showing the absence of crystalline material with no melting peaks seen in the DSC thermograms and one of the tested samples showing a negligible enthalpy of fusion of a crystalline solid of 0.5 J/g which corresponds to ca. 0.5% crystallinity. The DSC experiments were carried out at a heating rate of 10 °C/min in a temperature range of 5-180 °C in hermetically sealed aluminium pans in a nitrogen atmosphere. This lower level of crystallinity may be due to reducing the possibility of leaving any crystalline seeds in the melted sample, which was achieved through leaving the sample for a longer time over the hot plate once melting was achieved. The lack of crystalline seeds might be the reason behind the failure of crystallization of this slow cooled terfenadine sample during the DSC experiments at a heating rate of 10 °C/ min. One may thus deduce that the percentage crystallinity is sensitive to the history of sample preparation as indicated by different crystallinity values for quenched versus slow cooled samples and for samples left for a longer time over the hot plate after melting was achieved verses the ones quenched or slow cooled immediately once complete melting was reached. Table 3.5 shows the Tg values of the amorphous slow cooled terfenadine left for 5 minutes over the hot plate (after melting was accomplished) prior to slow cooling.

Table 3.5 DSC data for slow cooled terfenadine left for 5 minutes over the hot plate after melting. DSC carried out at a heating rate of 10 °C/ min from 5 - 1 8 0 °C.

Sample no. Tg (*C) 1. 53.9 2. 55.7 3. 56.4 Average 55.3 S.D 1.3

Therm ogravim etric analysis was carried out in order to explain the appearance o f a peak at a tem perature o f ca. 89 °C in the DSC therm ogram o f quench cooled terfenadine (Figures 3.4 and 3.5) and at ca. 92 °C for slow cooled terfenadine (Figures 3.3 and 3.6). The TGA results are shown in Table 3.6 and Figure 3.7 for quench cooled terfenadine and in Table 3.7 and Figure 3.8 for slow cooled terfenadine.

Table 3.6 TGA results for quench cooled terfenadine at a heating rate o f 20 °C. Run was carried out from start until 170 °C under purged nitrogen in open pans.

Sample no. Approximate

temperature range of weight loss (”C) % Weight loss 1. S t a r t - 95 °C 0.14 2. S ta rt- 9 5 °C 0.16 Average S t a r t - 95 ”C 0.15 0.5 9 9 ,4 - - -0.5 20 43 Tcnpora tu re (*C)

Figure 3,7 Typical TGA thermogram o f quench cooled terfenadine at a heating rate o f 20

Table 3.7 TGA results for slow cooled terfenadine at a heating rate o f 20 °C. Run was carried out from start until 170 under purged nitrogen in open pans.

Sample no. Approximate temperature range of weight loss (”C) % Weight loss 1 S ta r t- 9 0 "C 0.07 2 S ta r t- 9 0 "C 0.05 Average S ta r t- 9 0 "C 0.06 S3.5H 140 Temperature ( " O

Figure 3.8 Typical TGA therm ogram o f slow cooled terfenadine at a heating rate o f 20 "C/m in.

It can thus be concluded from the TGA results above that a loss in weight within the tem perature range o f the peak seen in the DSC was noticed in the TGA (Table 3.6 and T able 3.7 for quench cooled and slow cooled terfenadine respectively). A K arl-Fischer test was then carried out in order to check if this loss in weight corresponds to the presence o f water in the samples. The results from the Karl-Fischer test (Table 3.8) gave a percentage o f water, which is very close to that o f the percentage weight loss in the TGA (Tables 3.6 and 3.7 for quench cooled and slow cooled terfenadine respectively). Karl- Fischer results thus confirm ed that the peaks seen in the DSC therm ogram s at ea. 92 "C and ca. 89 "C for slow cooled and quench cooled terfenadine sam ples respectively and the loss in weight in the TGA within the same tem perature range corresponded to w ater loss from the samples.

Table 3.8 Karl Fischer titration results for both quenched and slow cooled terfenadine.

Type of sample Water %

(!"* trial) Water % (2“* trial) Average water content (%) Quenched 0.21 0.22 0.22 Slow cooled 0.06 0.06 0.06

An initial weight gain can be seen in the TGA thermograms (Figures 3.7 and 3.8 for quench cooled and slow cooled samples respectively). This can be due to an initial water uptake by the amorphous sample at the beginning of the TGA experiments where nitrogen gas was still incompletely filling the TGA compartment and thus was still unable to get rid of all the air in the atmosphere surrounding the furnace in the TGA, which