Basic steps:
(1) Make a series of dilutions of known concentration for the analyte.
(2) Analyze the known samples and record the results.
(3) Determine if the data is linear if expressed as the direct data vs semilogarithmic.
(4) Draw a line through the data and determine the line's slope and intercept.
(5) Test the unknown sample in duplicate or triplicate. Use the line equation to determine the concentration of the analyte.
concentration of analyte =
= ((test reading) – (intercept)) / (slope)
Issues:
(1) line statistics: The line statistics should indicate how good a line fits the data.
(2) closeness of the results: The replicate readings should be sufficiently close to indicate a valid analysis. The limits depend on the concentration of the analyte in the sample. If the readings are too far apart, then the sample should be retested.
(3) linearity range of assay: The unknown result should lie within the testing interval known to be linear.
