Continuous flow method for monitoring protein precipitation by ammonium sulfate

I have proposed "Centrifugal Precipitation Chromatography"(1) as a new method for the precipitation of proteins using a linear gradient of ammonium sulfate (AS). However data on the solubility of proteins under these specific conditions are not available. Therefore, the following method is proposed to obtain these data. It uses an HPLC pump to form a linear concentration gradient between a protein solution and a concentrated ammonium sulfate (AS) solution (90% saturation) which is continuously monitored with a uv detector at 280 nm. At a low flow rate of 0.1 mL/min the protein is exposed to a gradually increasing AS concentration until it reaches the critical point where precipitation takes place. The resulting light scattering increases the absorbance reading to form a distinct peak on the uv gradient curve where the valley and summit of the peak correspond to the starting and ending points of the protein precipitation, respectively. The method has been tested with a set of stable proteins at various pHs and ionic strengths of potassium phosphate buffer. Ten protein samples tested may be divided into two groups: The first group represented by albumin precipitates at 60-70% AS saturation and decreases in solubility at an acidic pH. The second group includes globulins which precipitate at 35-40% AS saturation and tend to increase their solubility at an acidic pH. The present method may be improved by the use of a nephelometric detection system with a small capacity flow cell.