In this application note, aqueous dispense volumes are obtained by measuring pathlength in microplate wells. The volume is calculated from the pathlength-vs.volume calibration curve previously obtained with a calibrated pipettor. Thus an important determinant for accuracy and precision is the reproducibility of the pathlength measurements. Molecular Devices provides a specification for pathlength-corrected absorbance, but not a specification for pathlength itself. During preparation of this application note, several in-house instruments were surveyed for pathlength precision using a 1 cm aqueous pathlength fixture. Within-row and between-row standard deviations were <0.01 mm and <0.04 mm respectively. The standard deviation values for the instrument used to generate the data for this application note were 0.005 mm and 0.025 mm, respectively. Pathlength measurements on microplates filled with 75 to 300 L water with high-quality pipettors typically have standard deviations of 0.03 to 0.04 mm. A pathlength of 0.03 mm corresponds to 1.0 L in a standard microplate and 0.5 L in a half-area microplate.
Well volumes in a microplate can be determined quickly and accurately by using the PathCheck feature of the SPECTRAmax PLUS microplate spectrophotometer. No special colored solutions are needed; pipettor performance can be determined with the same buffers or reagents that are normally dispensed by the pipettor - thus eliminating the uncertainty that its performance differs with a calibrator solution compared to the reagent(s) typically dispensed. Using flexible custom formulas and SOFTmax PROs spreadsheet capabilities, the data are calculated automatically and results are displayed as Pass/Fail for each pipettor. SOFTmax PRO also allows the user great flexibility in creating customized summary sections.
1. Curtis, R.H. Performance verification of manual action pipets. Pa