Medium-pressure fraction collection

An aqueous extract of P. hysterophorus (collected in Puerto Rico) was partitioned into methylene chloride at pH 7, pH 10 and pH 2. Bioassays of the methylene chloride soluble fractions, using the bean second internode bioassay (13), showed that the highest activity was concentrated in the methylene chloride extract at pH 7. Extensive chromatographic purification (flash chromatography, medium pressure LC, preparative TLC) monitored by bioassay led to the isolation of the four sesquiter-... 

The submitters purified the product by medium pressure liquid chromatography on a 60-cm x 5-cm column packed with 230-400 mesh silica gel 60 purchased from E. Merck. Ethyl acetate was used as eluant at a flow rate of 4 0 mL per min. Fractions (20 mL) were collected and analyzed by thin layer cHromatography. 

The submitters used medium pressure chromatography5 on 25 x 1000 urn of 230-400 mesh silica gel with a 25 x 250 mm scrubber column. The eluant (3 1 1 hexane ethyl acetate methylene chloride) was passed through the column at a rate of 15 mL/min, collecting 15 mL fractions. The checkers were unable to obtain pure product using a gravity column or flash chromatography. 

Sample preparation. For the study of trace compounds we used two different sample preparation procedures SPI and SPII for wine no. 1 and wine no. 2, respectively. The first flavor extract SP I was obtained by liquid-liquid extraction with fluorochloromethane and dichloromethane (9+1) from 45 L Scheurebe wine. For further analysis a portion of 1/3 was used. After separation on silica gel (pentane/diethyl ether) 6 fractions were analyzed. For the second flavor extract SP II we started from 200 L wine stripping off volatile compounds with vapour in a spinning cone column (SCC) system (5). The condensate was sequentially collected in two main portions of 8 and 2 L, respectively. The first condensate was discarded. The second condensate (2 L) was subjected to liquid-liquid extration with fluorochloromethane and dichloromethane (9+1). After separation on silica gel (pentane/diethyl ether) using medium pressure chromatography (MPLC) 4 fractions were analyzed. 

F TSK gel (Tosho Co., Ltd) under medium pressure, and absorptions of the elutes were measured at 280 nm with an UVICON 750 instrument (Advantec Toyo Co., Ltd.). The neutral components was eluted with 20 1 of distilled HjO and 101 of 50% MeOH-HjO, successively. The eluate was collected in every min., and each fraction was concentrated in vacuo and freeze-dried. 

The checkers eluted the columns with a slight positive air pressure on the solvent reservoir to prevent formation of gas bubbles and cracks in the chromatographic medium. Fractions were collected in 25-mL test tubes (Note 1), analyzed by TLC on silica gel, eluting with the column solvent, and visualized with a phosphomolybdic acid solution. The checkers observed a nonvolatile hydrocarbon material (not substrate related) which was eluted in the fractions just prior to the products, which are quite nonpolar themselves and are eluted in the early fractions, ahead of any unreacted ester. Colored, metal-containing components usually remain near the top of the column, although some colored material may accompany the... 

Extrapolation from the Arrhenius plot in Fig. 37 shows that Fe2+—02 has a half-time of 5 hours at the temperature chosen for the chromatography (—35°C). The elution buffer did not contain camphor. At this temperature the high viscosity of the medium (90 cP, Travers et al., 1975) necessitated a stable pressure of 1.5 bar. Fractions of 1.3 ml were collected at —35°C and cooled to 77°K in liquid nitrogen to avoid any further thermal decomposition of the complex. The peak fraction contained 8.3 fiM of the complex. The final recovery of the cytochrome was nearly 100%, and the elution pattern was the same as in the case of camphor-bound oxy-ferrous compound (Fes2+—02). 

In situ rat kidney perfusion experiments were carried out as described in Ref. [5.4]. Briefly, the perfusion medium used was Krebs-Henseleit buffer at pH7.4 containing glucose (5.6 mmol/L), 5.5% bovine serum albumin (fraction V, Sigma), 5-6% washed rat erythrocytes and different amino acids. After cannulation of the ureter and the renal vein and artery, the right kidney was perfused via the renal artery at a constant arterial pressure of 14.5 kPa at 37"C. After an equilibration period of 30-35 min, the agent was added and the perfusion continued for 60 min. Urine samples were collected every 10 min, and midpoint samples of the perfusate were also obtained. Inuhn was used as the standard for the measurement of the glomerular filtration rate. Elimination parameters of labelled peptides in the perfused rat kidney were characterized by the values of total renal clearance (CLr) and free fraction of the peptide in the perfusate (FJ. These values were compared with the glomerular filtration rate (GFR). 

Quantitative IR spectroscopy has turned out to be very usefid for measuring the decay of peroxide concentration or, alternatively, the inaease in the concentration of products from decomposition. Two types of discontinuous procedures have been used at lower reaction temperatures, the peroxide solution is contained in an internal cell (see Figure 3), which is filled and assembled in a glove box under an argon atmosphere. The internal cell is positioned into the optical high-pressure cell and pressurized with n-heptane acting as the pressure-transmitting medium. The assembly is heated to the reaction temperature and the collection of IR spectra is started. In the case that decomposition rate becomes too fast and a major fraction of the peroxide is decomposed before reaaion conditions of constant T and p are reached, the peroxide solution is dirertly fed into a preheated autoclave. The solution is then quickly pressurized and the collection of IR spectra is started. 

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