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Verifying Multichannel Pipettor Performance with Standard Dispense Solutions in the SpectraMax Plus (MaxLine Application Note #28)

en calculated from the absorbance, the light path, the extinction coefficient and the diluent volume. Various chromogens have been used, including Ponceau S dye, methyl red, Evans Blue dye and nicotinamide.1 ,2,10,11 The College of American Pathologists and the NCCLS recommend potassium dichromate because it can be obtained as a primary standard.1,2

The NCCLS and the ASTM recommend that for every pipettor, 10-replicate tests for calibration verification, measuring both inaccuracy and imprecision, be performed at least quarterly and after any maintenance.7,9 For the monthly quick check they recommend 4-replicate tests. If a laboratory has only a few single-channel pipettors, these guidelines can be reasonably accommodated using the gravimetric method. However, in laboratories having numerous pipettors, including multi-channel devices and automated pipetting stations, the testing requirement becomes extremely tedious at best, and impractical at worst. A much more practical approach for multichannel pipettors would be a spectrophotometric procedure in a 96-well format such as that suggested by Kaufman and Wobig.10 The delivery volume of one channel is checked gravimetrically, and the calibration adjusted if necessary. The remaining channels are then compared to the calibrated channel by dispensing a dye solution into the wells of a 96-well plate and measuring the absorbance in a microplate reader. Accuracy is calculated relative to the first channel with a separately-determined calibration factor of absorbance change per microliter.

An important (though often ignored) consideration in pipettor calibration is the fact that variations in surface tension, viscosity, density and temperature of the dispense reagent will lead to differences in dispensed volumes.1,4,7,8,12,13 Addition of a solute (whether buffer, preservativ
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