Hi There, I am optimising an enzyme assay that measures the appearance of NADPH. I began by creating a standard curve ([NADPH] (mM) vs. ABS@340nm) for NADPH in the buffer that I intend to use to identify my lower and upper detection limits. I performed it in triplicate and got nice tight data (all STDEV were less than 10% of each point - in most cases less than 1%). The linear portion of the graph has an R2 value of 0.9981 (5 data points). The only problem is that the slope is only 4.98, but it should by around 6.022, as this would be the correct millimolar extinction coefficient for NADPH. Can variables like temperature or buffer composition change the extinction coefficient of a compound? Should I be worried if the slope is wrong, considering everything else looks well on the graph? Shane.