Procedure for Practical Method Development

The selection of the column is of crucial importance and, therefore, has the utmost priority in any method development. In this chapter, a guide is given to the strategy by which it should be possible to rapidly find the most appropriate column. But, before we come to the list, first some important rules - these are often ignored but, when complied with, lead sooner to success. 

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Variational methods - theoretically the variational approach offers the most powerful procedure for the generation of a computational grid subject to a multiplicity of constraints such as smoothness, uniformity, adaptivity, etc. which cannot be achieved using the simpler algebraic or differential techniques. However, the development of practical variational mesh generation techniques is complicated and a universally applicable procedure is not yet available. 

The first practical method for asymmetric epoxidation of primary and secondary allylic alcohols was developed by K.B. Sharpless in 1980 (T. Katsuki, 1980 K.B. Sharpless, 1983 A, B, 1986 see also D. Hoppe, 1982). Tartaric esters, e.g., DET and DIPT" ( = diethyl and diisopropyl ( + )- or (— )-tartrates), are applied as chiral auxiliaries, titanium tetrakis(2-pro-panolate) as a catalyst and tert-butyl hydroperoxide (= TBHP, Bu OOH) as the oxidant. If the reaction mixture is kept absolutely dry, catalytic amounts of the dialkyl tartrate-titanium(IV) complex are suflicient, which largely facilitates work-up procedures (Y. Gao, 1987). Depending on the tartrate enantiomer used, either one of the 2,3-epoxy alcohols may be obtained with high enantioselectivity. The titanium probably binds to the diol grouping of one tartrate molecule and to the hydroxy groups of the bulky hydroperoxide and of the allylic alcohol... 

Rapid Approximate Design Procedure. Several simplified approximations to the rigorous solutions have been developed over the years (57—60), but they aU. remain too compHcated for practical use. A simple method proposed in 1989 (61,62) uses a correction factor accounting for the effect of axial dispersion, which is defined as (57)... 

The procedure is technically feasible, but high recovery of unconverted raw materials is required for the route to be practical. Its development depends on the improvement of catalysts and separation methods and on the avaHabiUty of low cost acetic acid and formaldehyde. Both raw materials are dependent on ample supply of low cost methanol. 

The only practical method of preparing 1,4-aminonaphthol is from a-naphthol through an azo dye, the nitroso compound not being readily available. The majority of investigators have reduced technical Orange I with stannous chloride Mi.is.is.ir.is by the procedures discussed above, and benzeneazo-a-naphthol has been reduced by the same reagent. In order to make possible the use of crude, technical a-naphthol a method has been developed for the preparation of the benzeneazo compound, its separation from the isomeric dye coming from the d-naphthol present as well as from any disazo compound by extraction with alkali, and the reduction of the azo compound in alkaline solution with sodium hydrosulfite. The process, however, is tedious and yields an impure product. 

It will be noted that at lOO C that the color comes up rapidly, but then rapidly fades, and many have noticed that in the o-toluidine method unless one waits a substantial period of time,, one is dealing with a fading color. However, it should be noted that when heated at 37 C that the color will not develop for at least 2 hours, but will yield a higher absorb-ence, because the color is more stable. A good compromise is to heat at temperatures of the order 55 C or 65 C in which case one can then conveniently read at the end of 10 or 15 minutes with adequate reading time for practical purposes. This is not done in most laboratories. Most laboratories continue to heat at 100 C, which is not the correct way to use this procedure. ... 

One of the primary requirements for methods is that it be practicable [Section 512(b)(1)(G)]. A method that cannot be used in Federal laboratories has no value in the protection of the food supply. Method developers should avoid the use of rare or custom-made equipment, prohibitively expensive equipment, untested technologies, or reagents that are not commercially available. For a determinative procedure, an analysis should not exceed two working days, and methods should have a minimum sample throughput of at least six samples per analyst-day. 

Although most of the methods for the arylation of enolizable compounds so far developed rely on special phosphine ligands, there is a report of an unusually mild and efficient phosphine-free procedure for the arylation of diethyl malonate, the key to success of which is announced to be the use of a heterogeneous base. In this procedure all three halobenzenes, including PhCl, showed practically identical reactivity (49).198... 

The palladium-catalyzed Heck carbonylation reaction is a powerful means of generating amides, esters, and carboxylic acids from aryl halides or pseudohalides [28]. The development of rapid, reliable, and convenient procedures for the introduction of carbonyl groups is important for the development of high throughput chemistry in general and high-speed microwave-mediated chemistry in particular. Unfortunately, the traditional method of introducing carbon monoxide into a reaction mixture via a balloon or gas tube is not practical because of the special requirements of microwave synthesis. 

Methods development starts with a relatively high number of techniques to characterize and test samples. The number of protocols is often reduced once the critical parameters and the methods that identify them have been defined. The analyst must evaluate the initial techniques with respect to their purposes. If the goal is to generate research data, the practicality of the method and its limitations are not of primary concern if the goal is to use the technique as part of a test procedure, it has to be evaluated in terms of its potential to meet full validation. Critical procedures (e.g., release testing) that cannot be validated will bring a project to an expensive halt. For these reasons, this chapter provides basic principles as well as limitations of capillary electrophoresis (CE) as applied to the analysis of real biopharmaceutical molecules. 

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