Studies of the separation of amylase isoenzymes by high performance liquid chromatography using post-column detection

Abstract

This thesis deals with the separation and detection of alpha-amylase isoenzymes by High Performance Liquid Chromatography (HPLC) using a post-column reaction system. Although there is no literature concerning a post-column reaction method for the detection of amylase isoenzymes, there is literature for other isoenzymes. The assay was performed using a coupled enzyme system containing a new substrate, a blocked p-nitrophenyl maltoheptaoside. After a very short lag phase, the enzyme-substrate reaction is linear, which is ideal for post-column work. A 50μL injection of filtered control serum or diluted saliva was eluted from the cation exchange column using a mobile phase of sodium cacodylate containing sodium chloride. This was accomplished by using a 25 min step gradient at the flow rate of 0.3mL/min. The substrate was then added by another pump at 0.5mL/min. Mixing occurred in a tee connector before the eluated went to a temperature controlled reaction coil, which was maintained at 43°C. The detection of the PNP released in the reaction was accomplished with a spectrophotometer at 405nm. Much trial and error testing was done to find the optimum conditions for the assay system. These tests included: changing the pH and composition of the substrate solution; changing the ionic strength of the mobile phase buffer; varying the temperature of the reaction coil; evaluating the mixing of the streams at the tee connector; and limiting the diffusion in the reaction coil. As a result of these tests, a partial separation of the isoenzymes was achieved. Further research must be done to make this method clinically useful. Resolution may be improved by fine tuning the existing step gradient, using lithium chloride in the mobile phase instead of sodium chloride, using a more efficient post-column reactor, such as a packed bed column, or changing to a longer column. The temperature of the reaction should also be decreased to 37°C before clinical studies can be done.