Visible and Ultraviolet (UV)

Spectroscopy

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Learning Objectives
By the end of this lecture, the students should be
able to:
1.Describe the visible and ultraviolet (UV) region in
the electromagnetic spectrum.
2.Explain the absorption of radiation.
3.State the reason why compounds appear
colored.
4.Explain electronic transitions and the different
types.
5.State the relationship between Absorbance,
Transmittance and Intensity.
6.Determine unknown concentration using Beers
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Law Plot.

Visible Spectroscopy
A study of interaction of the visible part ( = 380
720 nm) with a chemical species
Main uses:
1. Allows determination of concentration of an
unknown sample.
2. Monitoring of reaction progress as a function of
time.
3. Other quantitative uses.; identification to a
certain extent.

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Visible Light
Each photon has a specific energy; related to certain frequency
or wavelength
380 (blue violet) 700 nm (red)
When all of these wavelength is present, the light appears
white to our eyes
If any wavelength is removed (absorbed), we perceive the
remaining wavelengths of light as the complimentary color

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Visible Light Spectrum

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Coloured Compounds and

The Colour Wheel
Colored because of the
absorption of visible
radiation
Absorption of a certain
color, leads to the
perception of the
compound being the
complimentary color
Example:

Colour Wheel

Why radiation is absorbed

When a photon is absorbed, an electron transitions from
lower energy orbital to a higher energy orbital
Energy absorbed is equal to the energy difference between
the highest energy electronic occupied orbital (OO) and the
closest unoccupied orbital UO
Many transition metals are brightly colored because the
energy is within the visible region of the electromagnetic
spectrum

Energy levels and

transitions

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Chromophore
The nuclei that the electrons hold together in
bonds play an important role in determining which
wavelength of radiation are absorbed
This property of transition and absorption is a
characteristic of a group of atoms not the
electrons themselves
These group of atoms are called chromophores

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Substituents

Attachment of substituent groups on a basic

chromophore structure changes position and
intensity of absorption
Substituents that increase the intensity of the
absorption, and possibly the wavelength, are called
auxochromes
Typically auxochromes include methyl, hydroxyl,
Other substituents may have 4 different kinds of
alkoxyl, halogens and amino groups
effect on absorption
1.Bathochromic Shift (Red Shift) - Shifts to lower
energy or longer wavelength
2.Hypsochromic Shift (Blue Shift)- Shifts to higher
energy or shorter wavelength
3.Hyperchromic effect - An increase in intensity
4.Hypochromic effect- A decrease in intensity
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How is it measured ?
Usually measures absorbance in liquid solutions
The amount of a particular energy passing
(transmitted) through that solution is quantified
as transmittance

Solvent Cutoffs
Acetonitrile

190 nm

n-Hexane

201 nm

Chloroform

240

Methanol

205

Cyclohexane

195

Isooctane

195

1,4-Dioxane

215

Water

190

95% Ethanol

205

Trimethyl
phosphate

210

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Spectrometer or
Spectrophotometer
1. A light source
2. A diffraction grating
or prism to split light
into different
wavelengths
3. A sample holder or
cell
4. A photosensitive
detector which
measures the light
passing through the
sample
5. An amplifier and an

Absorbance Spectrum
A plot of the
absorbance of a
sample as a function
of wavelength
Can be used to identify
an unknown sample,
since compounds have
characteristic
absorption spectra

Maximum absorption = ~
560 nm

Absorbance and Beers Law

Beers Law Plot

Is a calibration curve
First: determine the wavelength of maximum
absorbance; max
All absorbances are acquired at this wavelength
setting
Create the plot, using at least three solutions of
known concentration
Construct a graph of absorbance versus
concentration and draw a best fit straight line
through the data points
Then the unknown concentration is determined by
comparison to the Beers Law Plot

Example

Absorbance readings are

taken for seven standard
cobalt (II) chloride solutions
and a Beers Law Plot is
created with the equation: y =
4.8571x + 0.0038.

A cobalt (II) chloride solution

of unknown concentration is
found to have an absorbance
of 0.28.

The unknown concentration

can be calculated by setting y
= 0.28 and solving for x.

Also, by a visual comparison

to the plot, the concentration
of the unknown is 0.057 mol/L.

Interferences
Path length
Other absorbing species in the solution
Impurities

Please Watch
By RSC
http://www.youtube.com/watch?v=O
39avevqndU&playnext=1&list=PL44C
6193128D03E4D
By khanacademy.org
http://www.youtube.com/watch?v=qbC
ZbP6_j48&feature=related

Application Question 1
Assay Example 1: Furosemide (frusemide) in tablet form
A typical example of a straightforward tablet assay is the analysis of furosemide (frusemide)
tablets:
1.Tablet powder containing ca 0.25 g of furosemide (frusemide) is shaken with 300 ml of 0.1 M
NaOH to extract the acidic furosemide (frusemide).
2.The extract is then made up to 500 ml with 0.1 NaOH.
3.A portion of the extract is filtered and 5 ml of the filtrate is made up to 250 ml with 0.1 M NaOH.
4.A portion of the extract is filtered and 5 ml of the filtrate is made up to 250 ml with 0.1 M NaOH.
5.The absorbance of the diluted extract is measured at 271 nm.
6.The A (1%, 1 cm) value at 271 nm is 580 in basic solution.
From the data below, calculate the % of stated content in a sample of furosemide (frusemide)
tablets:
Stated content per tablet: 40 mg of furosemide (frusemide)
Weight of 20 tablets : 1.656 g
Weight of tablet powder taken for assay : 0.5195 g
Absorbance reading : 0.596

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Application Question 2
Calculate the percentage of stated content of promazine hydrochloride
in promazine tablets from the following information:
(i)Tablet powder containing ca 80 mg of promazine hydrochloride is
ground to a paste with 10ml of 2 M HCl.
(ii)The paste is then diluted with 200 ml of water, shaken for 15 min
and finally made up to 500 ml.
(iii)A portion of the extract is filtered.
(iv)5 ml of the filtrate is taken and diluted to 100 ml with 0.1 m HCl.
(v)The absorbance is read at a wavelength of 251 nm.

A (1%, 1cm) value of promazine.HCl at 251 nm = 935

Stated content of promazine.HCl per tablet = 50 mg
Weight of 20 tablets = 1.667g
Weight of tablet powder taken for assay = 0.1356 g
Absorbance reading = 0.755
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