Supernatant concentration

There are a variety of methods for determining solubility in a relatively high-throughput manner but of these, two methods occur most often. These can be classified as supernatant concentration and precipitate detection.  

The supernatant concentration method uses small volumes of stock solution added to wells containing aqueous buffer in a microtiter filter plate of the type available from Millipore Inc. The solution is incubated for a given amount of time (typically in the range of 1-24 h depending on the requirements of the laboratory) and then filtered or centrifuged. The supernatant is analyzed by UV plate reader or HPLC and the concentration of dissolved species is calculated by reference to a calibration curve. This is often either a three- or a four-point curve prepared from serial dilutions of the stock solution using a solvent such as 80% v/v acetonitrile/water in which the compound is fully soluble. 

According to Beer s law, path length is a fundamental property of the absorbance of the sample as shown in Equation 6.1  

Using a UV plate reader, correction factors for path length are included in the instrument however, it is critical that the path length is the same in the incubation and calibration samples. The actual path length itself is determined by the depth of 

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Figure 11.9 Production of rhGH in . coli (as an intracellular protein). Subsequent to fermentation, the cells are collected by centrifugation or filtration. After homogenization, nucleic acids and some membrane constituents are precipitated by the addition of polyethyleneimine. Ammonium sulfate precipitation of the supernatant concentrates the crude rhGH preparation. Chromatographic purification follows, as illustrated...

Vortexed with methanol and supernatant concentrated (4-NP and its conjugates) HPLC-UV no data >98 (4-NP) >95 (conjugates) Diamond and Quebbemann 1979... 

Bzl-OH (21.6 g, 200 mmol) was treated with NaH (200 mmol) in refluxing THF (200 mL) for 2h. The soln was cooled to rt and CO2 was bubbled into the mixture under efficient stirring and external cooling. After Ih, Z-Q (1 28.5 mL, 200 mmol) was added slowly. After 3h stirring at rt, the precipitate was centrifuged and the supernatant concentrated. The resulting oil crystallized in the cold. The crystalline mass was triturated with cold hexane, filtered, and dried 5deld 45.8 g (79%) mp 28 °C. 

Supernatant Concentration Methods from Solid Material... 

Figure 9-11 Supernatant concentration of RRR and SSR isomers at different solvent compositions.

The fermentation medium (enriched with alanine) is inoculated with B. krzemieniew-ski. After 10 days, the culture is filtered and acetone and ammonium sulfate are added to the filtrate which is acidified to pH 2.0. The precipitated proteins are discarded, and the supernatant concentrated under vacuum. The concentrated extract is shaken with chloroform, and the polypeptin precipitates as a waxy mass, which is dissolved in hot aqueous acetone, and reprecipitated by cooling to —20 C. The polypeptin is then repurified by dissolving in hot aqueous ethanol, followed by its crystallization in the cold. Interesting photographs of the free polypeptin crystals and those of the sulfate are found in the publication of Howell (290). 

The vanadium pentoxide catalyst Is prepared as follows Suspend 5 g. of pure ammonium vanadate in 50 ml. of water and add slowly 7 5 ml. of pure concentrated hydrochloric acid. Allow the reddish-brown, semi-colloidal precipitate to settle (preferably overnight), decant the supernatant solution, and wash the precipitate several times by decantation. Finally, suspend the precipitate in 76 ml. of water and allow it to stand for 3 days. This treatment renders the precipitate granular and easy to 6lter. Filter the precipitate with suction, wash it several times with cold 5 p>er cent, sodium chloride solution to remove hydrochloric acid. Dry the product at 120° for 12 hours, grind it in a mortar to a fine powder, and heat again at 120° for 12 hours. The yield of catalyst is about 3 - 5 g. 

Add 101 g. (55 ml.) of concentrated sulphuric acid cautiously to 75 ml. of water contained in a 1 htre beaker, and introduce 35 g. of finely-powdered wi-nitroaniline (Section IV,44). Add 100-150 g. of finely-crushed ice and stir until the m-nitroaniUne has been converted into the sulphate and a homogeneous paste results. Cool to 0-5° by immersion of the beaker in a freezing mixture, stir mechanically, and add a cold solution of 18 g. of sodium nitrite in 40 ml. of water over a period of 10 minutes until a permanent colour is immediately given to potassium iodide - starch paper do not allow the temperature to rise above 5-7° during the diazotisation. Continue the stirring for 5-10 minutes and allow to stand for 5 minutes some m-nitrophenjddiazonium sulphate may separate. Decant the supernatant Uquid from the solid as far as possible. 

While the diazotisation is in progress, cautiously add 165 ml. of concentrated sulphuric acid to 150 ml. of water in a 1-litre round-bottomed flask. Heat the mixture just to boiling. Add the supernatant Uquid (diazonium solution) from a separatory funnel supported over the flask at such a rate that the mixture boils very vigorously (about 30 minutes). Then add the residual damp soUd (or suspension) in small portions avoid excessive frothing. When aU the diazonium salt has been introduced, boil for a further 5 minutes and pour the mixture into a 1-Utre beaker... 

Prepare a solution of 41 g. of anhydrous palladium chloride (1) in 10 ml. of concentrated hydrochloric acid and 25 ml. of water (as in A). Add all at once 60 ml. of 6iV-sulphuric acid to a rapidly stirred, hot (80°) solution of 63 1 g. of A.R. crystallised barium hydroxide in 600 ml. of water contained in a 2-htre beaker. Add more 6iV-sulphuric acid to render the suspension just acid to htmus (5). Introduce the palladium chloride solution and 4 ml. of 37 per cent, formaldehyde solution into the hot mechanically stirred suspension of barium sulphate. Render the suspension slightly alkaline with 30 per cent, sodium hydroxide solution, continue the stirring for 5 minutes longer, and allow the catalyst to settle. Decant the clear supernatant hquid, replace it by water and resuspend the catalyst. Wash the catalyst by decantation 8-10 times and then collect it on a medium - porosity sintered glass funnel, wash it with five 25 ml. portions of water and suck as dry as possible. Dry the funnel and contents at 80°, powder the catalyst (48 g.), and store it in a tightly stoppered bottle. 

Description of Method. Copper and zinc are isolated by digesting tissue samples after extracting any fatty tissue. The concentration of copper and zinc in the supernatant are determined by atomic absorption using an air-acetylene flame. 

M HNO3. The concentration of Cu and Zn in the diluted supernatant is determined by atomic absorption spectroscopy using an air-acetylene flame and external standards. Copper is analyzed at a wavelength of 324.8 nm with a slit width of 0.5 nm, and zinc is analyzed at 213.9 nm with a slit width of 1.0 nm. Background correction is used for zinc. Results are reported as micrograms of Cu or Zn per gram of FFDT. 

Zinc. The 2—3 g of zinc in the human body are widely distributed in every tissue and tissue duid (90—92). About 90 wt % is in muscle and bone unusually high concentrations are in the choroid of the eye and in the prostate gland (93). Almost all of the zinc in the blood is associated with carbonic anhydrase in the erythrocytes (94). Zinc is concentrated in nucleic acids (90), and found in the nuclear, mitochondrial, and supernatant fractions of all cells. 

In the United States, Hquid HLW from the reprocessing of defense program fuels was concentrated, neutralized with NaOH, and stored in underground, mild steel tanks pending soHdification and geologic disposal (see Tanks AND PRESSURE VESSELS). These wastes are a complex and chemically active slurry. Suspended in the supernatant Hquid are dissolver soHds which never went into solution, insoluble reaction products which formed in the tank, and salts which have exceeded their solubiHty limit. The kinetics of many of the reactions taking place are slow (years) so that the results of characterization... 

The clear supernatant solution is decanted and sold in Hquid form or concentrated to approximately 61.5 ° Bh and then allowed to soHdify to form blocks that are cmshed, ground, and graded. A typical analysis for the dry product is total A117.0—17.5% Fe202 <0.5% water of composition 42—43% insoluble <1.0%. Liquid alum contains 7.5—8.5% Al O. At concentrations >8.5% AI2O2, crystallisation of the solution may occur. 

When the overflow clarity is independent of overflow rate and depends only on detention time, as in the case for high soHds removal from a flocculating suspension, the required time is deterrnined by simple laboratory testing of residual soHd concentrations in the supernatant versus detention time under the conditions of mild shear. This deterrnination is sometimes called the second-order test procedure because the flocculation process foUows a second-order reaction rate. 

In hydroseparator tests, it is necessaiy to measure solids concentrations and size distributions of both the supernatant sample withdrawn and the frac tion remaining in the cylinder. The volume of the latter sample should be such as to produce a solids concentration that would be typical of a readily pumped underflow shiny. 

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