Precipitation buffer

Place solvent line sinkers (lO-pm filters) into intermediary solvent reservoirs when switching solvents to prevent cross-contamination or buffer precipitation. Cover the flask with parafilm to minimize atmospheric contamination and evaporation. 

Borate buffer treatment (pH 8.3), vortexmixing, centrifugation, pellet dissolved in 6M urea and dithiodu eitol Tryptic digestion of borate buffer precipitate Centrifugation at 5000rpm (for skimming), filtration, dilution... 

Figure 3.2 Growing crystals by the hanging-drop method. The droplet hanging under the cover slip contains buffer, precipitant, protein, and, if all goes well, growing protein crystals.

Do not switch from organic solvents to buffer solution or vice versa. Always do an intermediate wash with water. Buffer precipitation is a major cause of system pressure problems. You may be able to go from less than 25% buffer to organic and get away with it, but you are forming a very bad habit and that will get you into trouble later on. I usually keep a bottle of my mobile phase minus buffer on the shelf for column washout at the end of the day. This also can be used for buffer washout, but a water bridge is still the best. 

Be sure not to jump from buffer to pure organic or from organic to buffer. This can lead to buffer precipitation, plugging, and pressure problems. Always use a wash out, intermediate solvent or wash out with water. Allow six column volumes for reequilibration true equilibration takes as much as 24 hours, but this six-volume equilibration is reproducible and sufficient. 

Pacification after removing the column is an excellent cleaning technique for the whole system. It tends to remove buffer precipitation from check valves, organics from the injector rotor, and deposits on detector flow cell windows. Such a treatment is recommended to protect the system from halide attack on stainless steel. Experienced protein people have told me that pacification will protect a system against 200 mM NaCl for a month. This assumes you wash out the NaCl before you shut the system down. 

Check Valve—Mechanism in the pump head inlet and outlet to ensure oneway solvent flow usually a sapphire ball in a stainless steel cone. Major point of buffer precipitation and pump pressure loss. 

Working at subzero temperatures also introduces the important practical problem of the solubility of phosphate and Tris buffers as a function of temperature. At 0.01 M, phosphate buffer precipitates at —60° and —40°C when the solutions contain 70% and 50% methanol, respectively. In the same conditions, Tris buffer precipitates at —70° and — 50°C in solutions containing 70% methanol or dimethylforma-mide and 50% methanol, respectively. In the presence of a high concentration of proteins or other solutes, the precipitation can occur even at higher temperatures. The same buffers do not precipitate at... 

Prepare a concentration range of the precipitant in the same buffer. Precipitant ammonium sulphate,... 

Some of these criteria are self-explanatory, but a discussion of others is needed. A buffer with a high ratio of water solubility to solubility in relatively nonpolar solvents can affect how much organic modifier can be used. A buffer with a low ratio can result in buffer precipitation when an organic solvent is used. Most MEKC experiments are performed at a high pH (>9), but one needs to ensure that the buffer works well in this range. Many buffers are supplied as crystalline acid or bases, and the pH of these buffers is usually not near the pKa when the buffer is placed into solution, so the pH must to be adjusted.The buffer should be selected with a pKa near the desired working pH, and if the pH needs to be adjusted, then the appropriate acid or base should be used. 

Direct synthesis is perhaps the single most widely used method of preparation. This method involves nucleating and growing the metal hydroxide layer by mixing an aqueous solution containing the salts of two metal ions, in the presence of the desired anion, and a base 50% sodium hydroxide is particularly useful for this purpose, since the common ion effect keeps it relatively free of carbonate (see later). It has been demonstrated that LDH materials form in preference to a mixture of the individual metal hydroxides (68) and that, in the case of aluminum as the trivalent cation, they generally do so through an aluminum hydroxide intermediate. Variations of this method include titration at constant or varied pH and buffered precipitation. 

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