M aterials and Methods

2.2.1 Preparation o f samples

The pure and untreated G50/13 sample was received from the manufacturers in 500g containers. As the molten gelucire was filled into the containers, their large size could have resulted in different cooling environments within the solidifying mass. This could have led to a segregation of components depending on the position of the gelucire in the container, as Sutananta et al (1994a) postulated that various cooling rates caused the emergence of such segregations. Preliminary studies were conducted on the G50/13 obtained from the top, middle and bottom positions of three different batches prior to any heat treatments in order to examine the homogeneity of the sample within the container.

In the other studies, the DSC scans were performed on the previously prepared melt-fused tablets (see Section 2, Chapter 4) as it is better to perform the thermal analysis on the final dispersion rather than on physical mixes, in order to elucidate the real nature o f the dispersion. The tablets were equilibrated for 12-24 hours over silica gel after preparation and wholly comminuted in order to get a uniformed sample. This was a reasonable technique to use in order to fit solid fat into the pans. De Muynck and Remon (1992) obtained samples for the DSC from scraping it off from the fracture in the middle of a glyceride suppository. 9-10.5 mg of the sample was then placed in non-hermetic aluminium pans (Perkin-Elmer, Beaconsfield) and their lids were crimped into place. This amount was needed to cover the base of the pan without being too excessive so as to cause a great thermal lag. Preliminary studies of different sample masses o f 7.5 to 12.5mg had shown that the smaller masses gave non-reproducible thermal profiles probably due to gaps on the base of the pan whilst the larger masses caused a leakage during melting. This type of pan was chosen as it gave a satisfactory contact with the sample once crimped. A hermetic pan was not ideal as some of the samples were sticky and could not be distributed evenly at the

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base. The reference was an empty, crimped pan weighing within 0.01 mg of the sample pan.

2.2.2 Scanning conditions

The DSC scans were performed using a TA instruments DSC 2920 (Leatherhead) equipped with a Refrigerated Cooling Accessory (RCS) which would allow the temperature to go below ambient and additionally, stabilise the heating process. This instrument uses the heat- flux principle of DSC (see Section 1.1). The heating rate used for the scan was 2°C/min and the purge gas employed was nitrogen at 40cmVmin. A similar sample size (6-lOmg) and the heating rate of 2°C/min were chosen so that a maximum number of transitions could be detected in the melting profile (Liversidge et al, 1981; De Muynck and Remon, 1992). The relatively slow rate was chosen so that the resolution o f the peaks was optimal (see Section 2.1). This would also allow each thermal process to go to completion and the different stages of melting could possibly be correlated to the findings of HSM. A much lower scanning speed however, could anneal the fatty matrix and permit structural reorganisation to occur over the time scale of the experiment. In a previous study using G50/13, the scanning speed of 2°C/min was utilised as no evidence was found to indicate that lower speeds detected recrystallisation into higher melting point forms (Sutananta et al, 1994a). As no exothermic peak was seen between two overlapping endotherms of PEG 4000, it was regarded that the rate was fast enough to only cause the melting of the structures already present at the beginning o f the scanning process but not the melting of the folded form, followed by exothermic recrystallisation to the extended form and subsequently the melting of this form, as would happen in the structural reorganisation of the PEG (Lloyd, 1997).

2.2.3 Data collection

The melting temperature was often taken to be the temperature o f the endotherm at its most minimum heat flow value (peak), as gelucires do not have a sharp onset but instead melt over a broad range (Serajuddin et al, 1988). During preliminary studies, G50/13 was also found to give a broad melting range and so, the peak o f the endotherm as computed by Universal Analysis’*^ (TA instruments, Leatherhead) rather than onset was taken to be the

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melting temperature as it was not always clear where the onset melting temperature was. Moreover, the peak of the endotherm was the temperature at which the rate of melting was at its maximum. This would correspond better to the melting temperature found under Hot Stage Microscopy (see Chapter 3) as the exact temperature at which the gelucire started to melt is difficult to ascertain accurately under microscopy. In addition, the onset that was given by the analysis programme was calculated from the tangent to the first peak and not the point where the deflection from the basehne first occurred. The significance of this onset temperature was incorporated into the calculation of Solid Fat Content (SFC) instead (see below). The onset temperature measured from the tangent would depend on the slope of the leading edge o f the endotherm and this slope would also affect the SFC profile.

In other studies where several peaks were detected in the melting profile (Liversidge et al, 1981; De Muynck and Remon, 1992), the temperature of the last peak maximum was taken to be the melting point. In this current study, the melting point was not assigned to any one particular peak as the sizes of the peaks can be altered by certain conditions and so, the choice of the maximum peak could change. Moreover, preliminary studies showed that the peaks towards the end of the melting range were significant enough to not be overlooked even though they were much smaller than the other peaks. Therefore, the melting points of all the peaks are reported here.

The enthalpy or heat o f fusion was calculated as the area under the curve from the baseline, using the TA instruments software (Universal Analysis'*^). The heat flow is given as power (mW or W/g) and the heat o f fusion is given in J/g, noting the relationship between power and heat is Watt = Joules/second. The area under the curve was taken between the temperature where the first inflection o f the baseline occurred, to the temperature where the endothermie curve returned to the baseline. Projections from the baseline to the point of curve transitions are taken to be the borders o f the area under the curve o f the mid-range peaks (refer to Appendix 3 for an example of this step). This is analogous to the suggestion that a DSC curve may be integrated in parts when there is an overlapping o f peaks, the total enthalpy change thus being the sum o f these areas (Wendlandt, 1986). Other suggestions in analysing the enthalpies o f complex peaks include the sum o f Gaussian peaks or first- order peaks (Haines et al, 1998). The fitting of the Gaussian equation to complex peaks can

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be performed by using specialised calorimetry software such as the Origin™ software (Microcal™ Software Inc, USA). These methods are beyond the scope of the current investigation and could be explored in future studies.

Temperature calibrations were performed using n-octadecane (Riedel de Haén, Sigma- Aldrich GmbH, Germany), indium and tin (both are GPR grade supplied with TA instruments DSC cell accessory unit, Leatherhead). Their melting temperatures of 28.24°C, 156.60°C and 231.93°C respectively ensured that the temperature calibrations encompassed the range that is encountered in the current study for accuracy. Heat of fusion calibration was performed using indium (as above) with its standard heat given as 28.71 J/g. All the data generated were analysed using Universal Analysis, converted to ASCII format by the same program and imported into the Excel program (Version 7, Microsoft®) which plotted the profiles shown in Section 2.3.

The Solid Fat Content (SFC) of the samples at a certain temperature was calculated by taking the area under the curve up to that temperature and dividing it with the total area of the temperature range of interest. The SFC is then the percentage of the remaining area to the total area, which therefore gives the indication of the amount of solid matrix left at a certain point when the SFC is plotted against temperature. The integration of the partial areas were performed by using the Origin™ software (Microcal™ Software Inc., USA). A diagrammatic example of this procedure is given in Appendix 4. Some of the details have been discussed in Chapter 1 and more will be covered in Chapter 6. All measurements were performed five times.