Molecular Devices SPECTRAmax 340PC, Operator'S Manual

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Chapter 1
1-4 SPECTRAmax 340PC Microplate Spectrophotometer Operator’s Manual
PathCheck
™
Sensor
As predicted by the Beer Lambert law of light absorption, absorbance is propor-
tional to the distance that light travels through the sample—the longer the path-
length, the higher the absorbance.
Microplate readers use a vertical light path so the distance of the light through the
sample depends on the volume. This variable pathlength makes it difficult to per-
form extinction-based assays and confusing to compare results between micro-
plate readers and spectrophotometers.
Factory-stored values derived from deionized water are used to normalize the OD
data for microplate wells. This pathlength correction is accomplished only with
the use of SOFTmax
®
PRO software from Molecular Devices which also pro-
vides full instrument control and statistical data analysis.
The standard pathlength of a cuvette is the
conventional basis for quantifying the unique
absorptivity properties of compounds in solution.
Quantitative analyses can be performed on the
basis of extinction coefficients, without standard
curves (e.g., NADH-based enzyme assays). When
using a cuvette, the pathlength is known and is
independent of sample volume, so absorbance is
proportional to concentration.
In the microplate, pathlength is dependent on the
liquid volume, so absorbance is proportional to both
the concentration and the pathlength of the sample.
Standard curves are often used to determine analyte
concentrations in vertical-beam photometry of
unknowns, yet errors can still arise from pipetting the
samples and standards. The PathCheck feature of the
SPECTRAmax 340PC automatically determines the
pathlength of aqueous samples in the microplate and
normalizes the absorbance in each well to a
pathlength of 1 cm. This novel approach to
correcting the microwell absorbance values is accurate
to within 5% of the values obtained directly in a
1-cm cuvette.
Horizontal
Light Path
Cuvette
Vertical Light Path
Microplate Wells
Horizontal
Vertical