Addis Ababa University Department of

Chemistry

Fundamentals of
UV-visible spectroscopy

The Fourth Chemistry Summer School  September 19 – 30, 2005, Addis

Ababa  Ethiopia
 The Electromagnetic Spectrum

E = h =c/

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 The Electromagnetic Spectrum

E = h =c/

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 Primary Colors

red, green, and blue are called the

additive primaries

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 Complimentary Colors
You get blue.

White
Blue

Yellow

Take away yellow and what is left?

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 Complimentary Colors
You get red.

White
Red

Cyan

Take away cyan and what is left?

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 Complimentary Colors
You get green.

White
Green

Magenta

Take away magenta and what is left?

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 Complimentary Colors

•A red object is a good absorber

of cyan and vice versa.

•A blue object is a good absorber

of yellow and vice versa.

•A green object is a good

absorber of magenta and vice
versa.
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 Complementary Colors -
Summarized

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 Electronic Transitions and Spectra

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 Electronic Transitions and UV-
visible Spectra in Molecules

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 Electronic Transitions - Example

*  

*  n

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 The Lambert Law

T I / I 0 e  Const×Pathlength
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 Beer’s Law

Concentration

T I / I 0 e  Const×Concentration
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 The Lambert - Beer Law

Concentration

A  log T  log  I / I 0  log  I 0 / I   cl

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 The Lambert - Beer Law

A  log T  log  I / I 0  log  I 0 / I   cl

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 Total Overlapping of Bands

• Overlay of 2
Gaussian
bands with a NBW of
40 nm separated by
30 nm

• Separated by 4th
derivative

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 Partial Overlapping of Bands

Measure at two
wavelengths
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 No (significant) Absorption

Derivate the
compound
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 Conventional
Spectrophotometer

Schematic of a conventional single-beam

spectrophotometer
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 Intensity Spectrum of the D2 Arc
Lamp

• Good intensity in UV
range
• Useful intensity in visible
range
• Low noise
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• Intensity decreases over
 Intensity Spectrum of the
Tungsten-Halogen Lamp

• Weak intensity in UV
range
• Good intensity in visible
range
• Very low noise
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• Low drift
 Intensity Spectrum of the Xenon
Lamp

• High intensity in UV
range
• High intensity in visible
range
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• Medium noise
 Dispersion Devices

• Non-linear • Linear Dispersion

dispersion • Different orders
• Temperature
sensitive WS-Modern Instrumentation, Dept. Chemistry, AAU
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 Transmission Characteristics
of Cell Materials

Note that all materials exhibit at least

approximately 10% loss in transmittance
at all wavelengths
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 Cell Types I

Open-topped rectangular standard cell (a)

and apertured cell (b) for limited sample volume

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 Cell Types II

Micro cell (a) for very small volumes

and flow-through cell (b) for automated applications

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 Photomultiplier Tube Detector

Anode

• High sensitivity at low light levels

• Cathode material determines spectral
sensitivity
• Good signal/noise
• Shock sensitive
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 The Photodiode Detector (PD)

• Wide dynamic range

• Very good signal/noise at high
light levels
• Solid-state device
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 Schematic Diagram of a PD Array

• Same characteristics as
photodiodes
• Solid-state device
• Fast read-out cycles
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 Conventional
Spectrophotometer

Schematic of a conventional single-beam

spectrophotometer
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 Diode-Array Spectrophotometer

Schematic of a diode-array
spectrophotometer

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 Conventional
Spectrophotometer 1

Optical system of a double-beam

spectrophotometer
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 Definition of Resolution

Spectral resolution is a measure of the ability of an

instrument to differentiate between two adjacent
wavelengths
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 Instrumental Spectral
Bandwidth

The SBW is defined as the width, at half the maximum

intensity, of the band of light leaving the
monochromator
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 Natural Spectral Bandwidth

The NBW is the width of the sample absorption band

at half the absorption maximum

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 Effect of SBW on Band Shape

The SBW/NBW ratio should be 0.1 or better to yield an

absorbance measurement with an accuracy of 99.5%
or better
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 Effect of Digital Sampling

The sampling interval used to

digitize
the spectrum for computer
evaluation
and storage also effects
resolution

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 Wavelength Resettability

Influence of wavelength resettability on

measurements at the maximum and slope of an
absorption band
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 Background Cirrection

Corrects for constant background absorbance

over a range
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 Effect of Stray Light

Effect of various levels of stray light on

measured absorbance compared with
actual absorbance
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 Effect of Integration Time

Averaging of data points reduces

noise by the square root of the
number of points averaged

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 Scattering

Scattering causes an apparent absorbance because less light

reaches the detector

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 Three-Point Correction

• Uses two reference wavelengths

• Corrects for sloped linear background
Feb 3, 2025 absorbance
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 Discrimination of Broad Bands

• Derivatives can eliminate background

absorption
• Derivatives discriminate against broad
absorbance bands
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 Scatter Correction by
Derivative Spectroscopy

Scatter is discriminated like a broad-band

absorbance band
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 Wavelength(s) for Best
Sensitivity

• Calculation of relative standard deviation of the

measured values
at each wavelength
• The wavelength with lowest %RSD likely will yield the
Feb 3,best
2025 sensitivity
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 Wavelength(s) for Best
Selectivity

Selectivity is the ability of a method to quantify

accurately and specifically the analyte or analytes in the
presence of other compounds
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 Ideal Absorbance and
Wavelength Standards

• An ideal absorbance standard would have a constant

absorbance at
all wavelengths
• An ideal wavelength standard would have very narrow,
well-defined peaks
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 Holmium Perchlorate Solution

The most common wavelength accuracy

standard is a holmium perchlorate solution

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 Potassium Dichromate Solution

The photometric accuracy standard required by

several pharmacopoeias is a potassium dichromate
solution

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 Toluene in Hexane (0.02% v/v)

The resolution is estimated by taking the ratio of the

absorbance of the maximum near 269 nm and
minimum near 266 nm

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 Precision of an Analysis

Precision of a method is the degree of agreement

among individual test results when the procedure is
applied repeatedly to multiple samplings
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 Precision and Accuracy

Precision – Precision + Precision – Precision +

Accuracy – Accuracy – Accuracy + Accuracy +

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 Thank You

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 Thank You
[email protected]

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