VOLUME 1 -methyl-, 5-phosphate

Suspend a 20 g sample of the fortified feed in 100 ml of acid-acetone mixture, shake for one hour and separate in a centrifuge. Neutralise a 25-ml portion of the supernatant solution with N sodium hydroxide using methyl orange as indicator and make up to 100 ml in a graduated flask with phosphate buffer solution, pH 4-5. Measure 25 ml of this solution into a 50-ml beaker, add 2-5 ml of N sodium hydroxide and boil gently for fifteen minutes. Cool and add 2-5 ml of N hydrochloric acid. Transfer quantitatively to a 50-ml graduated flask and add a known amount of a solution of the chlortetracycline standard equal to the amount estimated to be present in the test sample. Make up to volume with phosphate buffer solution and from this prepare further final dilutions as required in the assay. 

Of interest is the manner in which cavities of the appropriate size are introduced into ion-selective membranes. These membranes typically consist of highly plasticized poly(vinyl chloride) (see Membrane technology). Plasticizers (qv) are organic solvents such as phthalates, sebacates, trimelLitates, and organic phosphates of various kinds, and cavities may simply be the excluded volumes maintained by these solvent molecules themselves. More often, however, neutral carrier molecules (20) are added to the membrane. These molecules are shaped like donuts and have holes that have the same sizes as the ions of interest, eg, valinomycin [2001-95-8] C H QN O g, and nonactin [6833-84-7] have wrap around stmctures like methyl monensin... 

A solution of bis-triethylamine phosphate was prepared by slowly adding 2.36 ml of B5% phosphoric acid to 20 ml of acetonitrile containing 9.9 ml of triethylamine at 20°C. This solution was added to a stirred solution of 4.70 g of 9a-fluoro-11(3,170,21 -trihydroxy-160-methyl-1,4-pregnadiene-3,20-dione 21 -methanesulfonate and 20 ml of acetonitrile. The mixture was heated under reflux for four hours and then evaporated under reduced pressure to a volume of 12 ml. This mixture was a concentrated solution of 9a-fluoro-11(3,170,21 -tri-hydroxy-160-methyl-1,4-pregnadiene-3,20-dione 21 -phosphate triethylamine salt with some inorganic phosphate. 

A solution of 5 grams of 6,7-dimethoxy-3-methyl-1-(4 -ethoxy-3 -methoxybenzyl)-lsoquin-oline in 100 cc of ethanol is treated with a solution of 1.5 grams of phosphoric acid in 10 cc of ethanol. 10 cc of water are added to effect complete solution, and the reaction mixture is then cooled and ether is added until precipitation of the salt is complete. The precipitate of 6,7-dimethoxy-3-methyl-T(3 -methoxy-4 -ethoxybenzyl)-isoquinoline phosphate is filtered off and recrystallized from 85% ethanol by the addition of 2 volumes of ether. 

Fig. 1. — Effect47 of Tomato Pectinesterase on the Activity of Pectate Lyase Acting on Highly Esterified Pectin. [Reaction mixtures contained 0.5% of D-galacturonan (broken line) or poly(D-galacturonic acid methyl ester) methyl glycoside (solid lines), 5 mM CaCl2, 33 mM phosphate buflFer (pH 7.0), and 0.25 unit of lyase in a final volume of 3.0 ml. In addition, the reaction mixture represented by the middle curve contained 0.031 unit of tomato pectinesterase in a final volume of 3.3 ml.] Reprinted, with permission, from M. Lee and J. D. Macmillan, Biochemistry, 9, 1930-1934 (1970). Copyright by the American Chemical Society.

To [Co(en)2((S)-GluOBzl)]I2 (5.0 g, 7.3 X 10 3 mol) in dry trimethyl-phosphate (18 ml, 4A sieves) contained in a conical flask equipped with a drying tube was added methyl trifluoromethane sulfonate (8.0 g, 4.9 x 10 2 mol) and the mixture was stirred at room temperature for 30 min (Caution The alkylating agent is believed to be extremely toxic. Use a hood and avoid skin and vapor contact). The deep orange solution was then slowly poured into rapidly stirred dry ether (600 ml) and the precipitated semisolid recovered by decantation. The residue was dissolved in the minimum volume of dry methanol (10-20 ml), the product reprecipitated using further dry ether (400 ml), and the solid recovered as before. A further precipitation using methanol (10-20 ml) and dry ether (800 ml) produced the complex as a finely divided solid. This was recovered by filtration (porosity 4 sin-... 

The enzymatic reaction was performed at 30 °C for 2 hours in a volume of 1 ml of 250 mM phosphate buffer (pH 6.5) containing 50 mM of KOH, 32 U/ml of the enzyme, and [1- C]-substrate. The product was isolated as the methyl ester. When the (S)-enantiomer was employed as the substrate, C remained completely in the product, as confirmed by C NMR and HRMS. In addition, spin-spin coupling between and was observed in the product, and the frequency of the C-O bond-stretching vibration was down-shifted to 1690 cm" (cf. 1740 cm for C-O). On the contrary, reaction of the (R)-enantiomer resulted in the formation of (R)-monoacid containing C only within natural abundance. These results clearly indicate that the pro-R carboxyl group of malonic acid is ehminated to form (R)-phenylpropionate with inversion of configuration [28]. This is in sharp contrast to the known decarboxylation reaction by malonyl CoA decarboxylase [1] and serine hydroxymethyl transferase [2], which proceeds with retention of configuration. 

The reaction mixture is heated at 80°C for 5 min to cease the reaction, followed by centrifugation. The supernatant is run onto a column of 100 parts by volume of cation-exchange resin [Amberlite IRC-50, NH4+-form]. The column is washed with water, and then eluted with 1 N-aqueous ammonia to give fractions which contain kanamycin B-3 -phosphate. The fractions are collected and concentrated under reduced pressure, and then the concentrate is run onto a column of 100 parts by volume of cation-exchange resin [carboxy-methyl Sephadex C-25, NH4+-form]. The column is washed with water, and eluted with 0.2 N-aqueous ammonia to give fractions which contain kanamycin B-3 -phosphate. The fractions are collected, concentrated and lyophilized, whereby 4.5 parts of kanamycin B-3 -phosphate. 

For abbreviation of analyte names see Sect. Abbreviations . ACN acetonitrile, f -glucu solution of (3-glucuronidase (E. coli), EE ethyl acetate, hep-SA heptane-sulfonic acid,, soprop iso-propanol, IS internal standard, MeOH methanol, metab. metabolites, Me-O- Bu, methyl- butyl ether n.s. not specified, OAc acetate, PB phosphate buffer Numbers give volume ratio v/v... 

Phosphorus may be determined gravimetrically as magnesium pyrophosphate after oxidation of the phosphite to phosphate by bromine and nitric acid. A sample is hydrolyzed in sodium hydroxide solution and diluted to 500 ml. in a volumetric flask. The analyses are run with 50-ml. aliquots. Each aliquot is acidified with 5 ml. of concentrated, 16 M nitric acid, and M nil. (5 drops) of bromine is added (a slight excess over the amount required for complete oxidation of an aliquot containing 0.2 g. of methyl ehlorofluorophosphite). The solution is swirled for a few seconds and allowed to stand for 15 minutes, with swirling at intervals. It is then heated on a steam cone until the color of the bromine disappears (the volume is usually reduced considerably during this process). The solution is... 

A typical method was described by Kim et al. (1999). for the determination of a dopamine D receptor antagonist in rat plasma. 0.2 mL volume of plasma was diluted with 1.5 mL of 0.25 M potassium phosphate buffer, pH 8, followed by the addition of 20 xL of the internal standard (1000 ng/mL in methanol), which was a methyl analog of the analyte. The diluted sample was slowly applied on a 1 mL disposable ethyl cartridge which had been successively prewashed... 

Assay Accurately weigh a portion of sample equivalent to about 475 mg of Calcium Phosphate, Monobasic, Anhydrous [Ca(H2P04)2], dissolve it in 10 mL of 2.7 N hydrochloric acid, add a few drops of methyl orange TS, and boil for 5 min, keeping the volume and pH of the solution constant during the boiling period by adding hydrochloric acid or water... 

An example is shown in Figure 8-71, where the column stability was evaluated with two different buffers, phosphate buffer ("pFI 11) and 1-methyl piperidine (wpH 11) [43]. Plots of H (plate height) or N versus number of column volumes can be generated. Remember that H is inversely proportional to the efficiency ... 

As catalysts for this reaction, inorganic acids having low volatility and high stability at the temperatures necessary may be used. Phosphoric acid or phosphates, boric acid, boric anhydride or borates may be used either in the molten state, supported on inert carriers, or as the solid salts.108 The use of excess amounts of carbon monoxide and high pressure are specified. Thus, when one volume of methyl chloride and 8 volumes of carbon monoxide are passed, as a mixture, over sodium metaphosphate on pumice at 700° to 800° C. a conversion to 10 or 15 per cent of acetyl chloride is obtained. The use of pressure enables much higher yields to be obtained. 

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