Critical Aspects of Starch Determination in Plant Tissues and a New Approach Utilizing HPAEC/PAD for the Quantification of Starch-Derived Glucose

This paper investigates error-contributing factors frequently encountered during plant starch content determinations by the widely used methodology based on the enzymatic/colorimetric determination of glucose released from enzymatic hydrolysis of starch. Due to the dynamics and variability of starch levels in plant tissues, inaccurate results were found to be associated with sampling and tissue preparation protocols. Other error-contributing factors included: (1) incomplete removal of interfering soluble sugars before starch hydrolysis, (2) non-specific hydrolysis during gelatinisation of starch granules, (3) incomplete hydrolysis of starch due to insufficient amounts of hydrolyzing enzyme, and (4) improper use of starch standards. A unified procedure that only requires 10-25 mg dry material is presented.

An alternative approach based on high performance anion exchange chromatography and pulsed amperometric detection (HPAEC/PAD) for direct determination of glucose following starch hydrolysis was also implemented to enable quantification of low starch content materials or for micro-scale analysis. The starch values obtained from the HPAEC/PAD approach were directly correlated with the enzymatic/colorimetric method (Slope 1.0113, R² = 0.9993). The advantage of this approach is its greater sensitivity (0.08 mg/L MQL vs 0.6 mg/L in enzymatic/colormetric approach) and the potential to be automated.