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Learning Center
Micro-Volume Sampling—How it Works
Thermo Scientific NanoDrop One User Guide 177
Related Topics
• Instrument Models and Features
• Measure a Micro-Volume Sample
Absorbance Spectrum
The light passes through the liquid column to the detector, which
generates a spectrum of absorbance versus wavelength. The
spectrum shows the amount of light absorbed by the molecules of
the sample at each measured wavelength.
Note: To prevent evaporation, which affects measurement accuracy,
close the arm quickly after you finish loading a sample or blank.
The example at the left shows a typical absorbance spectrum taken
of a nucleic acid sample. The spectrum is measured from 190 nm to
850 nm. The displayed range may vary for each application.
Sample Absorbance
When the instrument is blanked, a reference spectrum is taken of
the blanking solution and stored in memory. For each sample
measurement, the sample intensities along with the blank intensities
are used to calculate the total absorbance of the sample according to
the equation at the left.
Beer-Lambert equation
where:
A = absorbance in absorbance units (A)
= wavelength-dependent molar absorptivity coefficient (or
extinction coefficient) in liter/mol-cm
b = pathlength in cm
c = analyte concentration in moles/liter or molarity (M)
Sample Concentration
The Beer-Lambert equation (Beer’s law) shown at the left is used to
correlate sample absorbance with concentration.
The pathlength is the distance between the two pedestals, which
varies in real time during each measurement. This auto-ranging
pathlength technique produces accurate concentration results over a
wide dynamic range.
Baseline Correction
For some applications, the instrument can be set up to apply a
baseline correction to each measurement to minimize any offset
caused by light scattering particulates in the sample spectra. The
correction subtracts the absorbance value at a reference wavelength
that is close to zero from the absorbance value at each wavelength
across the spectrum, essentially “anchoring” the spectrum to zero
absorbance units at the reference wavelength.
Absorbance
intensity
sample
intensityblank
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