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ATTO 3 Manual 1.6 12
Typically, Beer’s law is used for the calculation of absorbance for liquid/solution
samples. According to Beer’s Law (also known as Beer-Lambert law) that the
absorbance of a solution will depend directly on the concentration ( c ) of the
absorbing molecules and the pathlength ( l ) travelled by light through the solution.
𝐴(𝜆) = 𝜀(𝜆)𝑐𝑙
where 𝜀(𝜆) is the molar absorptivity of the sample. By measuring the transmittance
also given as
𝑇(𝜆) =
𝐼(𝜆)
𝐼
0
(𝜆)
= 𝑒
−𝜀𝑐𝑙 , we can estimate the absorbance as follows:
𝐴(𝜆) = − log 𝑇 = 𝜀(𝜆)𝑐𝑙
where A(λ) is dimensionless.
Fluorescence
Fluorescence is a very important technique to resolve single molecules and
commonly used to understand the electronic structure of a molecule or an atom.
Fluorescence is the light emitted by an electron that is excited from ground state to
excited state by a photon. The emitted light typically has longer wavelength than the
wavelength of excitation.
Most of the dyes generally have an absorption peak in the UV (360-400 nm) region
of the electromagnetic spectrum. Attonics spectrometers can be customized to UV
LED or any desired illumination for measuring fluorescence. For example, if a
sample is excited at UV, the UV light is filtered using a long pass filter above the
excitation wavelength to avoid saturation of the detector caused by the UV light
source.
Colorimetry
As for every spectroscopy system, the Atto 3 sample measurement consists of two
independent measurements, a reference and a sample spectrum measurement:
First, a bright spectrum must be taken. The bright spectrum measures the
emission of the spectrometers light source alone. To take the bright spectrum the
device is placed onto, e.g. a white reference surface meant for colorimetry and the
bright or reference spectrum is recorded. This measurement is commonly referred
to as the source spectrum I
0 (
λ
)
. The user confirms the measurement of the white
standard by pressing “collect bright spectrum”.
Second, the sample measurement is taken I
S (
λ
)
by placing the device onto the
sample surface.
The device then computes the sample reflectance spectrum R
(

)
by:
𝑅
(𝜆)
=
𝐼
0 (𝜆)
𝐼
𝑆 (𝜆)
In the Atto
3
software, once the bright spectrum is taken, the computation of
reflectance spectrum R
(
λ
)
is automatically implemented, subsequent spectra are
compared against the bright spectrum. To be able to see the reflectance spectrum,
please enable the Reflectance Tab on the plot. This disables the Scope tab.
Reflectance is plotted as a normalized spectrum ranging from 0 to 1. Since the