Cutoff purity

Fig. 6.4. Yield vs. purity in cutoff selection. For the distributions represented in Fig. 6.3, retention probability (solid line) and enrichment factor (dotted line) were calculated as a function of the cutoff fluorescence value. For example, a cutoff value of 1.4 gives 95 % probability of retention of a given mutant cell, but a fairly modest enrichment factor of 11.8-fold. A cutoffof 3.0 increases the enrichment factor two orders of magnitude to 1000 x, at the cost of an increase in probability of clone loss to 50%.

KF as the best basic reagents and towards DMF as the best solvent. An accurate determination of yields and purity of 9.73 in all reaction vessels selected entry 43 (TEA-DMF) as the best compromise for step a, Fig. 9.29. A similar optimization was performed for the f-butyl ester hydrolysis (step b, Fig. 9.29), creating the 39-member reaction library L19 by permutations of acidic reagents and solvents, and addition of adjuvants (Fig. 9.30). The screening outcome (Table 9.3) highlighted the poor performances of ion-exchange resins (no reaction) and TFA (unclean product) to prepare 9.74, and selected entry 7 (HCl/EtOAc) as the best reaction conditions to obtain clean 9.74 (CSA (camphor sulfonic acid) actually performed slightly better, but the reaction work-up was less automation friendly). The whole manual optimization process required three to four days, and the best reaction conditions were used directly to produce a 590-member discrete library, which met the >75% purity cutoff (78). 

Sometimes purity of this reagent is poor and UV cutoff could be greater. 

At the end of the fermentation, protein is separated from cell mass by filtration, typically with a rotary vacuum filter. The crude enzyme concentration is often lower than suitable for commercial applications, so the concentration of enzyme is increased by ultrafiltration. Most cellulase enzymes have a molecular weight of 25,000 to 75,000 and are retained by ultrafiltration membranes of 5000 molecular weight cutoff. The membranes permit the passage of low molecular weight salts, sugars and other impurities, and are sometimes operated in a diafiltration mode to increase the purity of the enzymes. The crude broth at this point is dark brown. 

Dioxane from a variety of manufacturers and of various degrees of purity were all unsatisfactory without extensive purification the measure of quality was a sharp ultraviolet cutoff at 203 n.m. and a low and reproducible rate of DMU photolysis in the neat solvent. This could only be achieved in dioxane fractionated in a nitrogen atmosphere five times from sodium through a ten plate packed column and distilled immediately into flask containing the pyrimidine. Dioxane was not considered a satisfactory solvent. 

The common solvents used in UV/VIS spectroscopy are listed in Table 5.5, along with their low wavelength cutoff. At wavelengths shorter than the cutoff wavelength, the solvent absorbs too strongly to be used in a standard 1 cm sample cell. The cutoff is affected by the purity of the solvent. For spectroscopy, the solvents should be of spectral or spectro-chemical grade, conforming to purity requirements set by the American Chemical Society. 

Table 14-3 lists some common solvents and the approximate wavelength below which they cannot be used because of absorption. These wavelengths, called the cutoff wavelengths, depend strongly on the purity of the solvent. Common solvents for ultraviolet spec-... 

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