Phosphate buffer, preparation

Ammonium persulfate solution, 1.0 g/ 10 mL of phosphate buffer (prepare fresh just before use)... 

Immediately add the 250 ml of enzyme solution (potato filtrate) to 500 ml of the solution of starch plus phosphate buffer prepared in step 3. Record the total volume, and store the solution in a stoppered Erlenmeyer flask at room... 

Sample preparation 200 xL Serum + 5 x.L 50 p,g/mL 2-ethyl-4-oxoquinazoline in EtOH + 100 p,L reagent, let stand at room temperature for 1 h, add 1.8 mL ethyl acetate, mix, centrifuge at 1800 g for 5 min, remove 1.5 mL of the supernatant, repeat the extraction. Combine the organic layers and evaporate them to diyness under reduced pressure below 30°, reconstitute the residue in 100 p.L initial mobile phase, iqject a 90 p.L aliquot. (Reagent was 30 mg N-ethylmaleimide in 2 mL 50 mM pH 7.0 phosphate buffer, prepare fresh daily.)... 

Mobile phase MeCN buffer 18 82, after run was over flush at 35 65 for 5 min then reequilibrate with 18 82 for 9 min. (Buffer was 10 mM pH 7 phosphate buffer prepared from 1.36 KH2PO4 and 2.84 g Na2HP04 in 3 L water.)... 

ARX-500 MHz NMR Spectrometer at ambient temperature. The polymerization reaction was carried out in the 5 mm NMR tube. The polymerization of phenol and p-cresol was carried out in an aqueous-organic medium (0.4 mL of acetone-dfi and 0.1 mL of pH 7.0, O.OIM phosphate buffer (prepared in D2O)) to avoid the inhibition of polymerization due to insolubility of the monomers and the resulting dimers and oligomers in aqueous medium. However, polymerization of... 

Hydrolysis rates of dissolved yperite were studied in a phosphate buffer prepared with deionised water (R = 10" Mom x cm) at the temperature of 17 1°C. Yperite was detected by highly effective liquid chromatography using an LKB liquid chromatograph (Sweden) with a UV absorption detector at a wavelength of 210 nm. 

All reagents for phosphate buffer preparation in Milli-Q water were of analytical grade. 

Kieselguhr G (20 g) is mixed with 40 ml of pH 5 phosphate buffer (prepared by mixing equal volumes of 0.1 M phosphoric acid and 0.1 M disodium hydrogen phosphate). The plates are spread to a thickness of 250 fx and dried overnight in the air. From 5 to 25 [xg of carbohydrate is applied to the starting point. 

A variety of cyclic ortho esters,including cyclic orthoformates, have been developed to protect czs-1,2-diols. Cyclic ortho esters are more readily cleaved by acidic hydrolysis (e.g., by a phosphate buffer, pH 4.5-7.5, or by 0.005-0.05 M HCl) than are acetonides. Careful hydrolysis or reduction can be used to prepare selectively monoprotected diol derivatives. 

FIGURE 7.10 Dependence of the resolution on the sample volume. A preparative Superformance column 1000-200 (bed volume 20 liters) packed with Fractogel END BioSEC (S) (bed height 63 cm) was loaded with 60 ml (top) and 300 ml of a mixture of bovine serum albumin (5 mg/ml), ovalbumin (5 mg/ml), and cytochrome c (3 mg/ml) (bottom) (20 m/VI sodium phosphate buffer, 0.3 M NaCI, pH 7.2 flow rate 100 ml/min corresponding to 19 cm/hr). When the sample volume is 300 ml the separation efficiency for BSA and ovalbumin is similar. Thus the column can be loaded with larger sample volumes, resulting in reasonable separations. 

FIGURE 7.13 Preparative separation of various proteins on Fractogel EMD BioSEC (S). The length of the column was 1000 mm and the inner diameter 100 mm. The flow rate was 6.2 ml/min with 20 sodium phosphate buffer (pH 7.2) containing 0.3 M NaCI as the eluent. The injected standard proteins can be used to create a calibration curve. 

Given 0.1 M solutions of Na3P04 and H3PO4, describe the preparation of 1 L of a phosphate buffer at a pH of 7.5. What are the molar concentrations of the ions in the final buffer solution, including Na and H ... 

The choline ester is prepared by treating the 2-bromoethyl ester with trimethyl-amine. The ester is cleaved with butyrylcholine esterase (pH 6, 0.05 M phosphate buffer, rt, 50-95% yield). As with the morpholinoethyl ester, the choline ester imparts greater solubility to the C-terminal end of very hydrophobic peptides, thus improving the ability to cleave enzymatically the C-terminal ester. ... 

Preparation of PhAcOZ amino acids proceeds from the chloroformate, and cleavage is accomplished enzymatically with penicillin G acylase (pH 7 phosphate buffer, 25°, NaHS03, 40-88% yield). In a related approach, the 4-ace-toxy derivative is used, but in this case deprotection is achieved using the lipase, acetyl esterase, from oranges (pH 7, NaCl buffer, 45°, 57-70% yield). 

An amount of enzyme preparation equivalent to 900 mg of wet cells was made up to 25 ml with the above potassium phosphate buffer solution. 150 mg (1.15 mmol) of 5-fluorouracil and 1.0 gram of thymidine (4.12 mmol) were dissolved in 15 ml of the above potassium phosphate buffer solution. The mixture was incubated at 37°C for 18 hours. After this time, enzyme action was stopped by the addition of four volumes of acetone and one volume of peroxide-free diethyl ether. The precipitated solids were removed by filtration, and the filtrate was evaporated under nitrogen at reduced pressure until substantially all volatile organic solvent had been removed. About 20 ml of aqueous solution, essentially free of organic solvent, remained. This solution was diluted to 100 ml with distilled water. 

This is a crystalline product of insulin and an alkaline protein where the protein/insulin ratio is called the isophane ratio. This product gives a delayed and uniform insulin action with a reduction in the number of insulin doses necessary per day. Such a preparation may be made as follows 1.6 g of zinc-insulin crystals containing 0.4% of zinc are dissolved in 400 ml of water, with the aid of 25 ml of 0.1 N hydrochloric acid. To this are added aqueous solutions of 3 ml of tricresol, 7.6 g of sodium chloride, and sufficient sodium phosphate buffer that the final concentration is As molar and the pH is 6.9. 

Bj Pivaloyloxymethyl D(—)-Ot-aminobenzylpenicillinate. hydrochloride To a solution of pivaloyloxymethyl D(—)-a-azidobenzylpenicillinate (prepared as described above) in ethyl acetate (75 ml) a 0.2 M phosphate buffer (pH 2.2) (75 ml) and 10% palladium on carbon catalyst (4 g) were added, and the mixture was shaken in a hydrogen atmosphere for 2 hours at room temperature. The catalyst was filtered off, washed with ethyl acetate (25 ml) and phosphate buffer (25 ml), and the phases of the filtrate were separated. The aqueous phase was washed with ether, neutralized (pH 6.5 to 7.0) with aqueoussodium bicarbonate, and extracted with ethyl acetate (2 X 75 ml). To the combined extracts, water (75 ml) was added, and the pH adjusted to 25 with 1 N hydrochloric acid. The aqueous layer was separated, the organic phase extracted with water (25 ml), and the combined extracts were washed with ether, and freeze-dried. The desired compound was obtained as a colorless, amorphous powder. 

Reagents. Supporting electrolyte. Prepare a 0.1M phosphate buffer of pH = 8 containing 0.1M potassium iodide and 0.025 M potassium tartrate (0.17 g Na2HP04,12H20, 3.38 g NaH2P04,2H20, 4.15 g KI and 1.5 g potassium tartrate, in 250 mL of water). 

To circumvent these difficulties, a preparation of water-soluble coelenterazine has been developed (Teranishi and Shimomura, 1997a). The preparation contains coelenterazine and 50-times (by weight) of hydroxypropyl-P-cyclodextrin. To prepare this material, 0.1 ml of 3.0 mM coelenterazine in methanol and 0.2 ml of 45 mM solution of the cyclodextrin are mixed and dried under reduced pressure. The dried residue is extracted with 1.0 ml of lOmM phosphate buffer, pH 7.0, containing 2 mM EDTA (if needed), and the extract (after centrifugation) is again dried under reduced pressure. With this preparation, an aqueous solution containing up to 3 mM coelenterazine can be made. 

Preparation of luciferase. Organisms were freeze-dried, powdered, and washed with ethyl acetate to destroy the majority of catalase activity. The washed residue was extracted with 50 mM potassium phosphate buffer, pH 6. The extract was fractionated by ammonium... 

Preparation of Balanoglossus luciferin. The residue of the first pH 6 extraction above was re-extracted with 50 mM potassium phosphate buffer, pH 8. After centrifugation, the supernatant was used as the standard luciferin preparation. Luciferin was highly labile and easily inactivated at an extreme pH, by heat, and also by freezing and thawing. The instability resembled that of certain proteins. 

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