Study On Sequential Injection Coupled With Spectrophotometry For The Determination Ofα-amylase Activity And Phosphorus Species

In this dissertation, the sequential injection analysis coupled with spectrophotometric technique was proposed and applied to the determination of α-amylase activity and phosphorus contents.The first section of the dissertation introduced the history of enzyme, the classification of enzyme, α-amylase, influence factors of the enzyme reaction, the applications of α-amylase and the review on the progress of analytical methods for the determination of α-amylase activity in recent years. The methods including spectrophotometry, fluorescence analysis, biosensor, capillary electrophoresis and the flow injection analysis were introduced and their analytical performance was evaluated briefly.The second section of the dissertation developed a sequential injection analysis coupled with spectrophotometric method for the determination of of α-amylase activity. The proposed method was based on the iodine-starch colorimetry, in which starch was hydrolyzed by α-amylase under the appropriate condition, and then α-amylase activity can be quantified by monitoring the decolourization of the blue iodine-starch complex. In the optimized experiment condition, the linear response range was74-555U/L. The relative standard deviation (RSD) for11repeated measurements was1.2%for370U/L α-amylase. The detection limit (3σ) was16.7U/L. A throughput of50samples/h can be achieved. The proposed method was applied to determination of α-amylase activity in the samples of urine and saliva with a recovery between98.5%and110%. The method was simple, rapid, sensitive and was successfully applied to monitoring the process of the stress response.The third section of the dissertation proposed an automated flow-through microcolumn fractionation system for monitoring phosphorus in soil samples. This system includes dynamic extraction and on-line determination of phosphorus.0.25mol/L H2SO4and0.5mol/L NaHCO3were used as extractant for extraction of phosphorus in soil samples. The determination was based on Molybdenum Blue reaction. The proposed flow assembly implies several advantages as compared to extraction analysis in the batch mode in terms of saving of extractant and other reaction reagents, shortening the operational times from hours to minutes. In the optimized experiment condition, the linear range was0.1-6mg/L and the detection limit (3σ) was0.03mg/L. The relative standard deviation for thirteen repeated measurements was0.8%for3.5mg/L phosphorus. The proposed method was successfully applied to extraction and determination of phosphorus in different soil samples. The results for the determination of phosphorus in soil samples were in good agreement with results obtained with conventional manual methods.In the last section, characteristics of the experiments were summarized, and the prospect of application of SI coupled with spectrophotometry was described.