Specimen preparation method replication methods

Cellulose nitrate and cellulose acetate (CA) were among the first asymmetric, reverse osmosis membranes to be produced [121]. Plummer et al. [122] described 13 specimen preparation methods for observation of CA membrane structures. They pointed out the lack of contrast in epoxy embedded sections and that one of the best stains, osmium tetroxide, reacts with the polymer. Freeze fractured membranes were found by these authors to be of questionable value. In our experience, if care is taken, SEM study of fractured membranes can provide an informative view of the structure even though some structures collapse, and their sizes cannot be accurately determined. A method found acceptable was ultrathin sectioning of gelatin embedded wet membranes (TEM). The structure of CA membranes was shown by replication [123] and SEM [124]. 

This subject is too broad to permit detailed description of any kind of microscope or of the theory by which it is employed. Since many excellent books have been written on the microscope itself (Klemperer, 1953 Thomas, 1962 Haine eta/., 1961 Heidenreich, 1964 Grivet, 1965 Hirsh e/a/. 1965 Amelinckx, 1964, 1970 Hall, 1966 Wyckofi 1949) on methods of preparing specimens (Wyckofi 1949 Kay, 1961 Thomas, 1971), and on the theory of contrast (Heidenreich, 1964 Hirsh eta/., 1965 Amelinckx, 1964, 1970), only a very brief description is provided of contrast principles, specimen preparation methods and applications where replication and sectioning techniques have been successfidly employed to study surfaces. 

A convenient size for a circular coupon is 3 8 cm dia., a thickness of 0 - 32 cm and a central hole of 1 1 cm. Although inherent in the philosophy of corrosion testing, the use of coupons with surfaces that simulate those in service has been found to be unsatisfactory owing to irreproducibility, and the standard procedure normally adopted is to abrade down to 120-grit. ASTM Method G4 1984 gives details of preparation of specimens, evaluation of replicate exposures and the application of statistical methods. 

Specimen preparation for TEM generally in-voles the formation of a thin film of the material less than 100 nm thick. The methods used for this preparation depend upon the nature of the polymer and its physical form. In the case of thick or bulk specimens, microtomy is generally used. In the case of solutions, powders or particulates, simpler methods can provide a thin, dispersed form of the material. Three types of simple preparations will be described later in this section dispersion, disintegration and film casting. The more complex methods such as microtomy, replication, etching and staining will be described in other sections of this chapter. 

Replication is one of the oldest methods used for the production of thin TEM specimens. The procedure was first introduced by Bradley [287, 288], and it is well docximented in texts on specimen preparation [289, 290]. Replicas have the surface characteristics or topography of the... 

Direct or single stage replicas have the best possible resolution, are the fastest and, unfortunately, are the most difficult to prepare. The method involves the deposition of the replicating media and its removal or dissolution of the polymer. Materials used for direct replication include polymers, evaporated carbon films or metal oxides. Carbon is widely used for replication, especially if the polymer specimen to be replicated can be readily removed or dissolved. 

Direct isolation of sufficient quantities of each metabolite for structural characterization, assay validation and pharmacological or toxicological testing from in vivo studies using biological specimens is, therefore, often impossible, particularly from dmgs with a low therapeutic index. Furthermore, many metabolites have structural modifications which are difficult to replicate by traditional chemical methods. A number of synthetic steps may be required to prepare such metabolites from the API, or, in the worst case, a completely new synthetic route may need to be developed. 

In literature the following methods of preparation of polymer material specimen and studying their morphology are described methods of ultrathin section and films with contrasting of osmium tetroxide (10.11). method of replication of the brittle fractur-ed surface (11.12) oxygen and chemical etch of the polished surface or the fractured surface with the following replication for electron microscopy (11. 

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