Verification of Multichannel Liquid Dispenser Performance in the 4–30 μL Range by Using Optical Pathlength Measurements in Microplates

Abstract

Pipettors and liquid handling systems should be checked regularly to verify their precision and accuracy. Professional organizations recommend performing 4-replicate tests monthly and 10-replicate tests at least quarterly (1)(2)(3)(4). Traditional gravimetric procedures are adequate to meet these testing requirements for single-channel pipettors but are tedious and impractical for multichannel devices. An alternative is to dispense a solution of colored dye into wells of a microplate and to measure the resulting absorbance values in a microplate reader. Volume calibration is performed with a calibration curve relating absorbance to volume (5). We recently reported a method for verifying multichannel pipettor performance by a spectrophotometric procedure that utilizes the near infrared absorbance of water and does not require addition of a dye (6). Water or other aqueous reagent is dispensed from the pipettor into microplate wells, and the optical pathlength in each well is determined in a microplate spectrophotometer. Water is essentially transparent from 200 to 900 nm but has a distinctive absorbance peak near 977 nm. As predicted by the Lambert law of light absorption, absorbance is proportional to the distance that light travels through the sample; thus the characteristic absorbance of water can be utilized to measure the pathlength of an aqueous sample. The maximum absorbance is affected by temperature; however, temperature dependency can be avoided by making the absorbance measurements at a temperature isosbestic point (near 1000 nm). Baseline absorbance is measured at a wavelength distant from the water absorbance peak, e.g., 900 …