Molecular Devices SpectraMax 190, Operator'S Manual

Page: 12 / 54

Chapter 1
1-4 SPECTRAmax
190
Microplate Spectrophotometer Operator’s Manual
PathCheck
™
As predicted by the Beer Lambert law of light absorption, absorbance is pro-
portional to the distance that light travels through the sample—the longer the
pathlength, 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 dif
Þ cult
to perform extinction-based assays and confusing to compare results between
microplate readers and spectrophotometers.
This pathlength correction is accomplished only with the use of SOFTmax
PRO software. SOFTmax
®
PRO software from Molecular Devices provides
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 coef
Þ cients, 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 190
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 2.5% of the
values obtained directly in a 1-cm cuvette.
Horizontal
Horizontal
Light Path
Cuvette
Vertical
Vertical Light Path
Microplate Wells