Ultraviolet/Visible Molecular

Absorption Spectrometry I

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Outline

• Measurement of Transmittance and

Absorbance
• Beer’s Law
– Beer’s Law and its derivation
– Limitations to Beer’s Law

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Measurement of transmittance and absorbance

Light attenuation:
• Absorbing loss
• scattering loss
• Reflection losses

Psolution P
T= ≈
Psolvent Po
Experimental True
transmittance transmittance

Psolvent P
A = log ≈ log o
Psolution P

T = 10 − A
A = − log T
About 8.5% of a beam of yellow light is lost by reflection in
passing through a glass cell containing water.
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Derivation of Beer’s law
dn: absorbing particles within a block of cross section
area S and thickness dx.
dS: the surface area associated with the cross section
of dn particles within the block, i.e., photon capture
surface within the cross section area S.
dS/S: probablity for the capture of photons within S.
dPx: the quantity of radiation absorbed within the
section S.
dPx/Px: the average probability for photon capture

n 1000cm3 / L
dPx dS c= mol ×
− = 6.02 ×10 23 V
dPx a dn P
Px S − = − lg = εbc = A Po a nb
Px S Po lg =
dS = a dn P 2.303 V

P an
− lg =
− dPx P an
P n
a dn − ln =
∫P Px ∫0 S= Po 2.303 S
Po S
o V
S=
b
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Example: Beer’s law

Calibration curve
showing the validity of
Beer’s law for the
(ferrozine)3Fe(II)
complex.
(Source: Harris, Quantitative
Chemical Analysis, pp 417.) 6
Application of Beer’s law to mixtures

Beer’s law applies to a medium containing more than one kind of

absorbing substance, if the species do not interact.

Atotal = A1 + A2 + ... + An
= ε 1bc1 + ε 2bc2 + ... + ε nbcn

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Deviations
Deviations of
of Beer’s
Beer’s Law
Law

A = εb c
Conditions for its applicability :

(1) No molecular interactions ( Dilute solution; usually < 0.01 M )

(2) Analyte does not undergo association, dissociation, or

reaction with the solvent to give products with absorbing
characteristics that differ from those of the analyte

(3) Light beam is single wavelength ( monochromatic light )

Reasons for Deviations:
(1) Molecular interactions (When C>0.01M, the average distance
between two neighboring molecules is small enough that the extent of solute-solute
interactions or H-bonding can affect the analyte environment and its absorptivity.)
(2) Chemical deviations
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(3) Instrumental deviations with polychromatic beam
Chemical Deviation example: unbuffered solution of acid-base indicators

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Instrumental Deviation: Deviation due to polychromatic radiation

At λ’

At λ’’

The measured absorbance Am is:

Only when the molar absorptivities are

the same at the two wavelength λ’ and
λ”, the above equation simplifies to:

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Instrumental deviation due to polychromatic radiation

Band A: the absorptivity of the analyte is nearly

constant over Band A.
Band B: The absorptivity shows substantial
changes over Band B.

Greater departure from linearity was seen with

increasing difference between ε and ε’.

Implication: For quantitative absorption

measurement, one should select a wavelength
band near the wavelength of maximum absorption
where the analyte absorptivity changes little with
wavelength.

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Instrumental deviation due to stray radiation

Stray lights: radiation from the

instrument that is outside the
nominal wavelength band chosen
for the determination.
Stray lights are a result of scattering
and reflection off the surfaces of
grating, lenses ore mirrors, filters,
and windows.
Stray lights often have different
wavelength than the principal
radiation for absorbance. At higher conc and longer path
lengths, stray radiation has more
Stray lights may not have passed significant impact because
through the sample. smaller P.
Po + Ps
A' = log
P + Ps

Instrumental deviations always lead to negative absorbance errors.. 12

Deviation due to mismatched cells

If the cells holding the analyte and the blank solutions are mismatched in path
length or in optical characteristics, Æ A = ε b c + k

Solution:
1. Use matched cells
2. Use linear regression procedure to get both slope and intercept
3. Use only one cell for both the sample and the blank (in the case of single-
beam instrument).

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