distance above the specimen illuminating both sides of the surface contour. The detector which is placed at an angle, collects electrons originating from one side of the contour, forming a bright image. The opposite side of the contour appears in shadow, because electrons leaving this area fail to reach the detector.
4.1.4 Preparation of Powder Samples for SEM
At times, photographs of samples may show portions which are too bright or too dark due to the samples being partially charged. To prevent this it is important to prepare surfaces with uniform conductivity. This can be achieved by applying conductive paint i.e. carbon, to the specimen portions which are hard to coat. In the case of powder samples, if the particles are piled on each other, charge-up easily takes place causing them to move during observation. To prevent this, once the adhesive used to fix the powder has dried, the piled particles are blown off using a hand blower. When a non-conductive specimen is directly illuminated with an electron beam, its electrons with a negative charge collect locally (specimen charge-up), thus preventing normal emission of secondary electrons. It is therefore usual to coat the surface of an unconductive material with a conductive metal prior to observation. One of the methods by which this can be achieved is known as sputter coating. It allows easy and rapid deposition of a uniform coat of metal to be applied. A noble gas such as argon is ionised and the ions allowed to impinge on an electrode made of gold. The dislodged material is physically sputtered onto the specimen (Bell and Revel, 1980).
4.1.5 Applications of Scanning Electron Microscopy
The main area of SEM applications has been in the semi-conductor industry, however its use in biological applications has increased over the last decade. Its advantage in the latter application is that whole cells can be observed as a single image at a much higher resolution than with a light microscope. Images of biological objects can be visualised and recorded at magnifications ranging from lOOOx (the maximum used in light microscopy) up to about 20 GOGx (Slayter and Slayter, 1992).
Chapter 4 ....Scanning Electron M icroscopy / 9 7
4.1.6 Aim of Scanning Electron Microscopy Studies
The aim of the SEM studies was to visually assess the surface morphology of SACA during ongoing dissolution in a range of buffers. The media used were the same as for the dissolution studies allowing for a correlation to be made between factors affecting drug release i.e. buffer pH and composition, and possible changes to the structure of the spheres.
4.2
Materials
Single batches of cefuroxime axetil and SACA were used, information regarding these materials may be found in Sections 2.1 and 2.2. The following dissolution media were used, Sorensens pH 8.0, 7.0 and 5.9, citrate-phosphate, borate and phosphate mixed. Sodium chloride was used to alter the molarity of selected buffers. Information regarding all buffer components can be found in Section 2.4.
4.3
Methodology
In order to visualise SACA during the dissolution process, a method by which the material could be removed from the media at set time intervals and be prepared in such a way that SEM could be carried out was required. This was achieved by removing some of the dissolution media containing SACA using a syringe, then washing the material with water in order to stop any reaction which may be occurring, the material was then dried. Details of this process are described in the following section.
4.3.1 Isolation, Washing and Drying of SACA
Dissolution media were prepared as described in Section 3.3.1. Approximately 3g of SACA were weighed into a sample pot and 10ml of pre-warmed dissolution media
Chapter 4 ....Scanning Electron M icroscopy / 98
were added. The pot was shaken, then its contents emptied into a single dissolution vessel containing 900ml of the chosen dissolution media. A Ihr dissolution run was performed using a Pharma Test PTWS dissolution bath which was held at 37±0.5°C and used a paddle speed of lOOrpm.
At ten minute intervals a 10ml aliquot of the dissolution media containing SACA was removed using a syringe. The liquid was transferred onto a Whatman filter paper placed on top of a bruchner funnel. A continuous vacuum was applied in order to remove excess buffer and the material was washed using distilled water. The solid material was transferred onto a petri dish, placed into a Heraeus vacuum oven, and dried at 31°C under vacuum until constant weight was obtained. Dry SACA which had not been in contact with the dissolution media were subjected to the same drying conditions in order to provide an initial sample, while material which had been kept at room temperature was used as a control.
4.3.2 Examination of SACA by SEM
The dry powder samples were adhered to a SEM stub using carbon impregnated disks. The disk was divided into several segments, one per sample, and excess powder was removed using a can of compressed air. It was then transferred to a sputter coater and coated with gold for two minutes at 20 mAmps. Microscopic examination was carried out using a Phillips XL20 SEM (Phillips Electron Optics, Eindhoven, Netherlands). The image was transmitted onto a computer monitor where is was analysed, and a hard copy was produced using a black and white video print.
4.4
Results and Discussion
4.4.1 SEM of Cefuroxime Axetil and SACA
A scanning electron micrograph of cefuroxime axetil is shown in Plate 4.1. The drug substance consists of discreet spherical particles, which are hollow in nature. The
Chapter 4 ....Scanning Electron M icroscopy / 99
outer and inner surfaces are smooth and are relatively free o f visible imperfections. The size o f the particles range from 2pm to 30pm. The hollow, spherical nature o f the
drug substance is a result o f its processing conditions, see S ection 2.1.
Acc.V SpotM agn Det WD
ie.00kV 4.0 386x SE 5.4 CEFUROXIME AXETIL
P la te 4.1 C efuroxim e Axetil, BN: U O P W 2565.
P la te 4.2 shows SACA to be spherical in nature with a mean diameter o f 40-60pm. This particular shape and constant particle size is also a result o f processing
conditions, {S ection 2.2). The outer surface o f SACA, though not as smooth as
cefuroxime axetil, is still relative free o f visible imperfections. P la te 4.3 shows a
single sphere which has been fractured by grmding the material between two glass microscope slides. Due to the small diameter o f SACA, splitting a sphere proved difficult and earlier attempts using a razor blade were unsuccessful. Grinding the material produced a very small amount o f good cross-sections, however the single
sphere in P la te 4.3 is adequate to illustrate the internal structure. A more suitable way
in which particles o f SACA can be halved is by using a freeze-fracture technique. This work has been carried out by Glaxo Wellcome and corroborates the information
provided here. As can be seen from P la te 4.3 SACA is composed o f solid spheres.
Small indentations in which the discrete drug particles were embedded can also be obsei"ved.