James V. Sullivan

Spiral disk assembly for HSCCC: Column design and basic studies on chromatographic resolution and stationary phase retention

Abstract A set of four separation disks equipped with spiral channel(s) was designed for our type‐J high speed countercurrent chromatography (J‐HSCCC) centrifuge to improve retention of the stationary phase of polar solvent systems. Four different spiral disks were tested: two had a single spiral channel with different depths and the other two had four spiral channels connected in series to provide a greater spiral pitch. Performance of each disk was tested in terms of chromatographic resolution and/or stationary phase retention using three different two‐phase solvent systems, including 1‐butanol/acetic acid/water (4:1:5, v/v/v) for dipeptide separation; 12.5% (w/w) polyethylene glycol (PEG) 1000–12.5% (w/w) dibasic potassium phosphate for protein separation; and 4% (w/w) PEG8000–5% (w/w) dextran T500 in 10 mM dibasic sodium phosphate for determination of stationary phase retention. The results show that the spiral column retains a satisfactory amount of stationary phase for all solvent systems, even at a relatively high mobile phase flow rate where the spiral pitch plays a significant role. Separation of dipeptides with the butanol solvent system was possible using 10 mL/min flow rate of the mobile phase and 4‐spiral‐disks with their greater spiral pitch. In protein separations with the PEG–phosphate system, the single‐spiral disks yielded the best separation using the upper phase in tail‐to‐head elution mode at 1 mL/min, while the 4‐spiral disks show higher retention of the stationary phase. The retention of the PEG–dextran system is improved in the 4‐spiral disks, which exceed 60% at a flow rate of 0.5 mL/min. Various parameters, which affect the performance of the spiral disk separation column are discussed.

A chemical quenching apparatus for studying rapid reactions

Abstract A chemical quench-flow apparatus is described for studying enzymatic reactions with half-lives of 0.005 sec or longer. The syringe pistons are driven by a stepping motor which provides precise control over the volume and rate of flow of reactants. The drive mechanism also ensures a rapid approach to a steady flow velocity and thus minimizes the amount of material used per stroke. Improved mixing efficiency is accomplished by means of ball mixers which utilize the zone of turbulence in the wake of a sphere as the mixing mechanism. The instrument was used to follow the presteady state time course of phosphorylation and dephosphorylation of a microsomal preparation of (Na + + K + )-stimulated ATPase.

A thin-layer multistrip agarose gel electrophoresis apparatus for Ferguson plot analysis at the sub-microgram load level☆

A method for the simultaneous horizontal agarose gel electrophoresis on thin-layer strips of different gel concentrations was developed for the purpose of generating Ferguson plots at the sub-microgram load level. Seven independent gel strips on a common GelBond support were formed by filling channels created by a comb-shaped spacer (polycarbonate) in a vertical multistrip cassette. Electrophoresis on a horizontal Peltier-cooled surface employed commercial apparatus (E-C Apparatus Corp.) with a modified cover which is airtight and holds anodic and cathodic voltage measurement probes for each strip. The application of the apparatus to Ferguson plot analysis in a single experiment was exemplified on the RNA-containing turnip crinkle virus (TCV) at a load of 50 ng/gel strip, using an optimized silver staining method (a modification of a procedure of FMC Corp. BioProducts) for detection. Within the range of 3.5 to 12.5 V/cm, the plot was found to be independent of field strength. Mobility is also independent of the concentration of detergent (CHAPS) up to 10 mM.

Method of making small‐diameter tubing from porous film

A method is described for preparing membrane or porous film in tubing form from sheet material by a heat sealing technique. This should be generally useful with a variety of materials which are not available in tubing form. Tubing sizes both smaller and larger than 1‐mm diameter, of indefinite length, can be easily formed.