Exchanging buffer solutions

Because trls has only meager buffering action at pH 6.1, fluctuations In the pH of Ion exchanger and solutions may occur and may result In abnormal chromatographic behavior. Buffered conditions may be obtained by substituting bls-trls which has a pK of about 6.5, and virtually Identical behavior results If Developer A Is constituted with 0.03 M bls-trls-HCl 0.03 M NaCl 0.01% KCN at pH 6.2. 

Figure 17.7 Electrocatalysis of O2 reduction by Pycnoporus cinnabarinus laccase on a 2-aminoanthracene-modified pyrolytic graphite edge (PGE) electrode and an unmodified PGE electrode at 25 °C in sodium citrate buffer (200 mM, pH 4). Red curves were recorded immediately after spotting laccase solution onto the electrode, while black curves were recorded after exchanging the electrochemical cell solution for enzyme-fiiee buffer solution. Insets show the long-term percentage change in limiting current (at 0.44 V vs. SHE) for electrocatalytic O2 reduction by laccase on an unmodified PGE electrode ( ) or a 2-aminoanthracene modified electrode ( ) after storage at 4 °C, and a cartoon representation of the probable route for electron transfer through the anthracene (shown in blue) to the blue Cu center of laccase. Reproduced by permission of The Royal Society of Chemistry fi om Blanford et al., 2007. (See color insert.)...

To the flasks for the crop and soil samples (Section 6.1), add 2mL of 0.01 M Tris-HCl buffer solution (pH 7.7) and 50 and 100 qL of 1M Tris-HCl buffer solution for wheat grain, bariey grain and rice straw, and for soil, respectively. Adjust the pH to about 7.7 (confirm the pH with a pH test paper using the sample of untreated area). Homogenize the residue with ultrasonication and transfer the homogenate to the top of an ion-exchange column. Wash the flask twice with 2mL of 0.01 M Tris-HCl buffer solution and transfer the washings to the column. Elute the column with 40 mL of the same buffer solution. Discard this eluate. 

Sample preparation techniques vary depending on the analyte and the matrix. An advantage of immunoassays is that less sample preparation is often needed prior to analysis. Because the ELISA is conducted in an aqueous system, aqueous samples such as groundwater may be analyzed directly in the immunoassay or following dilution in a buffer solution. For soil, plant material or complex water samples (e.g., sewage effluent), the analyte must be extracted from the matrix. The extraction method must meet performance criteria such as recovery, reproducibility and ruggedness, and ultimately the analyte must be in a solution that is aqueous or in a water-miscible solvent. For chemical analytes such as pesticides, a simple extraction with methanol may be suitable. At the other extreme, multiple extractions, column cleanup and finally solvent exchange may be necessary to extract the analyte into a solution that is free of matrix interference. 

Before releasing a process column for chromatography, it is advisable to perform some test to measure efficiency, such as calculating height equivalent theoretical plates (HETP), both to forestall any problems in the column bed and to provide a benchmark by which to measure column reproducibility and predict degradation of the bed or material. Examples of compounds that are relatively innocuous for use in pharmaceutical applications are 1% NaCl (for gel filtration), concentrated buffer solutions (for ion exchange), and benzyl alcohol and parabens for reverse phase LC.10... 

Aerosil was converted into the sodium form by treating it with a buffer solution (pH = 8.4) made of sodium hydroxide and sodium hydrogen carbonate solutions, after which it was filtered, washed free of alkali, and dried. This sodium-exchanged aerosil was then suspended in a solution of Ni(en)3(N03)2 prepared by adding the calculated amount of ethylene-diamine to a solution of nickel nitrate. The suspension was agitated for about 30 min and filtered off. The catalyst was then washed and dried at 100°C. 

System C is used when an immobilized enzyme reactor (IMER) is introduced into system B. The analyte(s) separated by HPLC is converted to a suitable species for CL detection with an IMER, and then mixed with the CL reagent. In this system, a buffer solution as a mobile phase and an ion-exchange-type column are preferable for an enzyme reaction. 

Penefsky introduced the centrifuged-column procedure (see diagram below) that can be used for small aliquots of enzymes and other proteins (a) to exchange buffers and/or other solutes, (b) to measure ligand binding, and (c) to concentrate protein by as much as 10 times. His two-step protocol for rapid buffer exchange can be summarized as follows ... 

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