Ultraviolet and Visible Absorption Spectrophotometry note students 2021(2).pptx

ULTRAVIOLET/VISIBLE
ABSORPTION
SPECTROPHOTOMETRY
1

BY
PROF. (MRS.) ADEPOJU-BELLO AA
PHARMACEUTICAL CHEMISTRY
DEPARTMENT
2

MOLECULAR ABSORPTION
SPECTROSCOPY
3

What happens when light is absorbed
by molecules?
• Promotion of electrons
4
http://www.chemguide.co.uk/analysis/uvvisible/analysis.html#top

• When light passes through the
solution of a compound or
substance, energy from the light is
absorbed to promote an electron
from a bonding or non-bonding
orbital into one of the empty anti-
bonding orbitals.
5

THE POSSIBLE ELECTRON JUMPS THAT
LIGHT MIGHT CAUSE ARE:
6

• In each possible case, an electron is
excited from a full orbital into an
empty anti-bonding orbital.
• Each jump takes energy from the
light, and a big jump obviously
needs more energy than a small
one.
7

• Each wavelength of light has a particular
energy associated with it.
• If that particular amount of energy is
just right for making one of these
energy jumps, then that wavelength will
be absorbed – (energy is absorbed in
promoting an electron).
8

• We need to work out what the relationship is
between the energy gap and the wavelength
absorbed.
• Does, for example, a bigger energy gap mean that
light of a lower wavelength will be absorbed - or
what?
9
(http://www.chemguide.co.uk/analysis/uvvisible/analysis.html#top)

• If a high energy jump is required, light of a
higher frequency will be absorbed. The
greater the frequency, the greater the energy.
• UV-visible absorption spectra are always
given using wavelengths of light rather than
frequency.
• That means that we need to know the
relationship between wavelength and
frequency.
10

• You can see from this that the higher the frequency is,
the lower the wavelength is.
• So a bigger energy jump means a higher frequency of of
light is absorbed - which is the same as saying that a
lower wavelegth of light is absorbed.
11

12

• Figure 2 The electromagnetic spectrum
13
The electromagnetic spectrum
The electromagnetic spectrum showing the UV/Visble range clearly

• The human eye is only sensitive to a tiny
proportion of the total electromagnetic
spectrum between approximately 400 and
800 nm
• Within this area, 400 and 800nm, human eye
perceives the colors of the rainbow from
violet through to red (ROYGBIV).
14

• The larger the energy jump, the lower
the wavelength of the light absorbed.
• For absorption spectrometry, some
jumps are more important than others
15

• An absorption spectrometer works in a
range from about 200 nm (in the near
ultra-violet) to about 800 nm (in the
very near infra-red).
• Only a limited number of the possible
electron jumps absorb light in that in
the region.
16

• Let us look at the possible jumps again. The important
jumps are shown in black ink, and a less important one in
grey. The grey dotted arrows indicate jumps which absorb
light outside the region of UV/Visible spectrum we are
working in.
17

The important jumps are:
• from pi bonding orbitals to pi anti-
bonding orbitals; π π*
• from non-bonding orbitals to pi anti-
bonding orbitals; n π*
• from non-bonding orbitals to sigma anti-
bonding orbitals; n σ∗
18

• That means that in order to absorb light
in the region from 200 - 800 nm (which
is where the spectra are measured), the
molecule must contain either π BONDS
or ATOMS WITH NON-BONDING
ORBITALS.
• Remember that a non-bonding orbital,
has a lone pair of electron in it, e.g.
oxygen, nitrogen, sulphur or a halogen.
19

SPECTROPHOTOMETRIC TERMS
CHROMOPHORE
AUXOCHROME
LAMBDA MAX
HYPSOCHROMIC SHIFT OR BLUE SHIFT
BATHOCHROMIC SHIFT OR RED SHIFT
HYPERCHROMIC SHIFT
HYPOCHROMIC SHIFT
20

• CUT OFF WAVELENGTH
• ISOSBESTIC POINT
21

Chromophore
• Is a group responsible for light absorption by
a molecule. Chromophores contain one or
more multiple bonds e.g. dienes, benzene etc
22

Auxochromes
• These are functional groups that on their
own do not absorb the UV/Visible light but
when attached to a molecule or moiety it
leads to an absorption near the UV range.
• The functional group must be a non-bonding
electron interaction. e.g. -OH, -NH2, -SO2
• They function by entering into the electronic
interaction with a nearby chromophore.
23

Lambda max, λmax
{uv-visible spectroscopy menu (chemguide.co.uk)}
24

Bathochromic and Hypsochromic
shifts
25
Absorbance
https://www.google.com/search?source=univ&tbm=isch&q=bathochromic+shift+and+hyp
sochromic+shift&client=firefox-b-
d&sa=X&ved=2ahUKEwjjxfvZ1NXuAhVNOBoKHbXwA6UQjJkEegQIIhAB&biw=1366&bih=65
4#imgrc=O6nxb2GEheUvpM&imgdii=WoadkNTx5_X1EM

Cut-off Wavelength
• This is important in the choice of solvent to
dissolve the substance for analysis.
• The cut off wavelength of the solvent should
be below 200 nm or should not be in the
UV/Visible range nor absorb in the range at
which the sample absorbs.
26

Isosbestic point
27
(http://www.chemguide.co.uk/analysis/uvvisible/analysis.html#top

• Isosbestic point in the bromocresol green spectrum. The
spectra of basic, acid and intermediate pH solutions are
shown. The analytical concentration of the dye is the same
in all solutions.
• In spectroscopy, an isosbestic point is a specific wavelength,
wavenumber or frequency at which the total absorbance of
a sample does not change during a chemical reaction or a
physical change of the sample.
• The word derives from two Greek words: "isos", meaning
"equal", and "sbestos", meaning "extinguishable".
{v-visible spectroscopy menu (chemguide.co.uk)}
28

• For the reaction:
• The analytical concentration is the same at any
point in the reaction:
• The absorbance of the reaction mixture
(assuming it depends only on X and Y) is:
29

• But at the isosbestic point both molar absorptivities
are the same:
• Hence, the absorbance
• does not depend on the extent of reaction (i.e., in the
particular concentrations of X and Y)
(http://www.chemguide.co.uk/analysis/uvvisible/analysis.
html#top) 30

T = I/I0,
I0 = intensity of incident
monochromatic radiation,
I = intensity of transmitted
monochromatic radiation.
31

• In the absence of other physico-chemical
factors, the absorbance (A) is proportional to
the path length (b) through which the
radiation passes and to the concentration (c)
of the substance in solution in accordance
with the equation:
A = εcb
ε =molar absorptivity, if b is expressed in
centimetres and c in moles per litre.
•
32

• The expression A
representing the specific absorbance of a
dissolved substance refers to the absorbance of a
10 g/L solution in a 1.0 cm cell and measured at a
defined wavelength so that:
A = 10ε
Mr
where ε is the molar absorptivity
Mr is the molecular mass of the substance
33
1%
1cm
1%
1cm

• Unless otherwise prescribed, the
absorbance is measured at the prescribed
wavelength using a pathlength of 1.0 cm.
• Unless otherwise stated, the measurements
are carried out with reference standard and
with the same solvent or the same mixture
of solvents.
34

• Plot the absorption spectrum with
absorbance or function of absorbance as
ordinate against wavelength or function of
wavelength as abscissa.
35

Apparatus
• Spectrophotometers suitable for measuring
in the ultraviolet and visible range of the
spectrum consist of an optical system capable
of producing monochromatic radiation in the
range of 200-800 nm and a device suitable for
measuring the absorbance.
36

Control of wavelengths
• Verify the wavelength scale using the
absorption maxima of holmium
perchlorate solution R, the line of a
hydrogen or deuterium discharge lamp
or the lines of a mercury vapour arc
shown in Table 2.2.25.-1.(BP, 2014)
37

• The permitted tolerance is ± 1 nm for the
ultraviolet range and ± 3 nm for the visible
range. Suitable certified reference materials
may also be used. (BP, 2014)
38

Control of absorbance
• Check the absorbance using suitable filters or
a solution of potassium dichromate R at the
wavelengths indicated in Table 2.2.25.-2 (BP,
2014)
39

Woodward's rules
• Woodward's rules, named after Robert Burns
Woodward and also known as Woodward–
Fieser rules (for Louis Fieser) are several sets
of empirically derived rules which attempt to
predict the wavelength of the absorption
maximum (λmax) in an ultraviolet–visible
spectrum of a given compound.
(http://www.chemguide.co.uk/analysis/uvvisibl
e/analysis.html#top)
40

• Inputs used in the calculation are the
type of chromophores present, the
substituents on the chromophores, and
shifts due to the solvent.
• Examples are conjugated carbonyl
compounds, conjugated dienes, and
polyenes.
41

Implementation
• One set of Woodward–Fieser rules for dienes
is outlined in table 1.
• A diene is either homoannular with both
double bonds contained in one ring or
heteroannular with two double bonds
distributed between two rings.
42

Table 1. Rules for wavelength of maximum diene absorption[3][7]
Structural feature λmax
effect
(in
nanometers)
Base value for heteroannular diene 214
Base value for homoannular diene 253
Increments
Double bond extending conjugation + 30
Alkyl substituent or ring residue + 5
Exocyclic double bond + 5
acetate group + 0
Ether group + 6
Thioether group + 30
bromine, chlorine + 5
secondary amine group + 60
43

• With the aid of these rules the UV absorption
maximum can be predicted, for example in
these two compounds:
44

• In the compound on the left, the base value is 214 nm (a heteroannular
diene). This diene group has 4 alkyl substituents (labeled 1,2,3,4) and the
double bond in one ring is exocyclic to the other (adding 5 nm for an
exocyclic double bond).
• In the compound on the right, the diene is homoannular with 4 alkyl
substituents. Both double bonds in the central B ring are exocyclic with
respect to rings A and C.
45

Fieser Kuhn Rules
• Fieser-Kuhn rules is use for polyenes
with more than four (4) conjugated
double bonds.
• Used to calculate wavelength of
maximum Absorption (λmax) of Polyenes
46

Woodward fieser rules work for conjugated
dienes and polyenes with up to four-double
bonds or less.
Some plant pigments e.g. carotenoids have
more than four conjugated double bonds.
For these conjugated polyenes with more
than four double bonds, the Fieser-Kuhn
rules is applied in order to obtain the
wavelength of maximum absorption, (λmax) .
47

• According to the Fieser-Kuhn rule the equation
below can be used to get the wavelength of
maximum absorption λmax and the maximum
absorptivity εmax:
• λmax=114+5M + n(48.0 – 1.7 n) – 16.5 Rendo – 10 Rexo
48

• where,
λmax is the wavelength at maximum absorption
M is the number of alkyl substituents / ring
residues in the conjugated system
n is the number of conjugated double bonds
49

• Rendo is the number of rings with endocyclic
double bonds in the conjugated system
Rexo is the number of rings with exocyclic
double bonds in the conjugated system.
• and
• εmax = (1.74 x 104) n
• where,
εmax is the maximum absorptivity
n is the number of conjugated double bonds.
50

• Thus using the above equations, one can get the
wavelength of maximum absorbance (λmax) and
the maximum absorptivity (εmax)
• Sample Problem 1: β-Carotene
• β-carotene is a precursor of vitamin A which is a
terpenoid derived from several isoprene units.
The observed λmax of β-carotene is 452 nm,
while the observed εmax is 15.2 x 104. Let us
therefore use Fieser-Kuhn rules to calculate the
λmax and the εmax for β-carotene.
51

Name of Compound β-Carotene
Base Value 114 nm
M (number of alkyl substituents) 10
n (number of conjugated double bonds) 11
Rendo (number of endocyclic double bonds) 2
Rexo (number of exocyclic double bonds) 0
Substituting in equation
λmax = 114 + 5M + n (48.0 – 1.7 n) – 16.5 Rendo – 10 Rexo
= 114 + 5(10) + 11 (48.0-1.7(11)) – 16.5 (2) – 10 (0)
= 114 + 50 + 11 (29.3) – 33 – 0
= 114 + 50 + 322.3 – 33
Calc. λmax = 453.30 nm
λmax observed practically 452nm
Calculate εmax using equation:
εmax = (1.74 x 104) n
= (1.74 x 104) 11
Calc. εmax= 19.14 x 104
Practically observed εmax 15.2 x 104
53

• Sample Problem 2: all-trans-Lycophene
• Lycophene (all-trans-lycophene) is a bright red
carotenoid pigment found in tomatoes and
other red fruits and vegetables. However,
lycophene has no vitamin A like activity.
54

Name of Compound all-trans-lycophene
Base Value 114 nm
M (number of alkyl substituents) 8
n (number of conjugated double bonds) 11
Rendo (number of endocyclic double bonds) 0
Rexo (number of exocyclic double bonds) 0
Substituting in equation
λmax = 114 + 5M + n (48.0 – 1.7 n) – 16.5 Rendo – 10 Rexo
= 114 + 5(8) + 11 (48.0-1.7(11)) – 16.5 (0) – 10 (0)
= 114 + 40 + 11 (29.3) – 0 – 0
= 114 + 40 + 322.3 – 0
Calc. λmax = 476.30 nm
λmax observed practically 474nm
Calculate εmax using equation:
εmax = (1.74 x 104) n
= (1.74 x 104) 11
Calc. εmax= 19.14 x 104
Practically observed εmax 18.6 x 104
56

QUANTITATIVE ASSAY USING
UV/VISIBLE
SPECTROPHOTOMETER
57

A DOUBLE BEAM UV-VISIBLE
ABSORPTION SPECTROMETER
• If you pass white light through a coloured
substance, some of the light gets absorbed. A
solution containing hydrated copper(II) ions, for
example, looks pale blue because the solution
absorbs light from the red end of the spectrum.
• The remaining wavelengths in the light combine
in the eye and brain to give the appearance of
cyan (pale blue).
58

• Some colourless substances also absorb light -
but in the ultra-violet region. Since we can't see
UV light, we don't notice this absorption.
• The amount of absorption is also dependent
on the concentration of the substance if it is
in solution.
• Measurement of the amount of absorption can
be used to find concentrations of very dilute
solutions.
• An absorption spectrometer measures the way
that the light absorbed by a compound varies
across the UV and visible spectrum.
59

A simple double beam spectrometer
60

The light source
• You need a light source which gives the entire visible
spectrum plus the near ultra-violet so that you are
covering the range from about 200 nm to about 800
nm. (This extends slightly into the near infra-red as
well)
• You can't get this range of wavelengths from a single
lamp, and so a combination of two is used - a
deuterium lamp for the UV part of the spectrum,
and a tungsten / halogen lamp for the visible part.
• The combined output of these two bulbs is focused
on to a diffraction grating.
61

The diffraction grating and the slit
• You are probably familiar with the way that a prism
splits light into its component colours. A diffraction
grating does the same job, but more efficiently.
• The blue arrows show the way the various
wavelengths of the light are sent off in different
directions. The slit only allows light of a very narrow
range of wavelengths through into the rest of the
spectrometer.
• By gradually rotating the diffraction grating, you can
allow light from the whole spectrum (a tiny part of
the range at a time) through into the rest of the
instrument.
62

63

The rotating discs
• Each disc is made up of a number of different
segments. Those in the machine we are
describing have three different sections - other
designs may have a different number.
64
(http://www.chemguide.co.uk/analysis/uvvisible/analysis.html#top}

• The light coming from the diffraction grating
and slit will hit the rotating disc and one of
three things can happen.
• If it hits the transparent section, it will go
straight through and pass through the cell
containing the sample. It is then bounced by
a mirror onto a second rotating disc.
• This disc is rotating such that when the light
arrives from the first disc, it meets the
mirrored section of the second disc. That
bounces it onto the detector. 65

• (http://www.chemguide.co.uk/analysis/uvvisible/a
nalysis.html#top) 66

• If the original beam of light from the slit hits the
mirrored section of the first rotating disc, it is
bounced down along the green path. After the
mirror, it passes through a reference cell (more
about the reference cell).
67

• Finally the light gets to the second disc which is
rotating in such a way that it meets the
transparent section. It goes straight through to
the detector.
• If the light meets the first disc at the black
section, it is blocked - and for a very short while
no light passes through the spectrometer. This
just allows the computer to make allowance for
any current generated by the detector in the
absence of any light.
68

The sample and reference cells
• These are small rectangular glass or quartz
containers. They are often designed so that
the light beam travels a distance of 1.0 cm
(pathlength) through the contents.
• The sample cell contains a solution of the
substance you are testing - usually very
dilute. The solvent is chosen so that it
doesn't absorb any significant amount of
light (remember the cut off wavelength) in
the wavelength range we are interested in
(200 - 800 nm).
70

• The reference cell just contains the
pure solvent used to dissolve the
sample and reference standard (the
blank).
71

THE DETECTOR AND COMPUTER
The detector converts the
incoming light into a current. The
higher the current, the greater the
intensity of the light.
72

• For each wavelength of light passing through
the spectrometer, the intensity of the light
passing through the reference cell is measured.
This is usually referred to as Io
Io = Intensity of incident light
• The intensity of the light passing through the
sample cell is also measured for that
wavelength - given the symbol, I.
I = intensity of light coming out of the sample cell
• If I is less than Io, then obviously the sample
has absorbed some of the light. A simple bit of
maths is then done in the computer to convert
this into something called the absorbance of the
sample - given the symbol, A.
73

• The relationship between A and the two
intensities is given by:
• Transmittance, T = I
IO
74

• For quantitative analysis, the absorbance ranges
from 0 to 1, but it can go higher than that for
qualitative measurements.
• An absorbance of 0 at some wavelength means
that no light of that particular wavelength has
been absorbed. The intensities of the sample and
reference beam are both the same, so the ratio
Io/I is 1. Log10 of 1 is zero.
75

• An absorbance of 1 happens when 90%
of the light at that wavelength has been
absorbed - which means that the
transmitted light intensity, I, is 10% of
what it would otherwise be.
• In that case, Io/I is 100/I0 (=10) and log10
of 10 is 1.
76

The chart recorder
• Chart recorders usually plot absorbance against
wavelength. The output might look like this:
 This particular substance has what are known as
absorbance peaks at 255 and 395 nm. 77

THE BEER-LAMBERT LAW
• THE IMPORTANCE OF CONCENTRATION
• The proportion of the light absorbed will depend
on how many molecules it interacts with.
Suppose you have got a strongly coloured
organic dye.
• If it is in a reasonably concentrated solution, it
will have a very high absorbance because there
are lots of molecules to interact with the light.
• Beer’s law states that the absorbance, A, is
directly proportional to concentration, c. 78

THE IMPORTANCE OF THE CONTAINER
SHAPE
• Suppose this time that you had a very dilute solution of the dye in a cube-shaped container so that the
light travelled 1 cm through it. The absorbance isn't likely to be very high.
• On the other hand, suppose you passed the light through a tube 100 cm long containing the same
solution. More light would be absorbed because it interacts with more molecules.
• This is Lambert’s law which states that the absorbance, A, is directly proportional to the pathlength, l.
• The pathlength (l) is the inner width of the couvette.
•
The cuvette
79

• Again, if you want to draw sensible
comparisons between solutions, you have to
allow for the pathlength of the solution the
light is passing through.
• Both concentration and solution length are
allowed for in the Beer-Lambert Law.
80

• You will find that various different symbols
are given for some of the terms in the
equation - particularly for the concentration
and the solution length. The concentration
of the solution is "c" and the pathlength is
"l".
81
A =

• You should recognise the expression on
the left of this equation as what we
have just defined as the absorbance, A.
You might also find the equation written
in terms of A:
82

• That's obviously easier to remember than
the first one, but you would still have to
learn the equation for absorbance. It
might be useful to learn it in the form:
83

• The Greek letter epsilon, ε, in these
equations is called the molar
absorptivity - or sometimes the molar
absorption coefficient.
84

Molar absorptivity
• The equation could be rearranged to
give an expression for epsilon (the
molar absorptivity):
ε = A
lc
85

• Note: The absorbance of a solution will vary as
the concentration or the size of the container
varies.
• Molar absorptivity compensates for this by
dividing by both the concentration and the length
of the solution that the light passes through.
• ε, works out a value for what the absorbance
would be under a standard set of conditions - the
light travelling 1.0 cm through a solution of
1.0 mol dm-3. 86

• That means that you can then make comparisons
between one compound and another without having
to worry about the concentration or solution length.
• Values for molar absorptivity can vary hugely. For
example, ethanal has two absorption peaks in its UV-
visible spectrum - both in the ultra-violet.
• One of these corresponds to an electron being
promoted from a lone pair on the oxygen into a pi
anti-bonding orbital;
• the other from a pi bonding orbital into a pi anti-
bonding orbital.
87

• The table below gives values for the molar
absorptivity of a solution of ethanal in hexane.
• The units of molar absorptivity are mol-1 dm3
cm-1 assuming the length is in cm and the
concentration is mol dm-3.
88

The ethanal obviously absorbs much more strongly at 180
nm than it does at 290 nm.
(Although, in fact, the 180 nm absorption peak is outside
the range of most spectrometers).
electron jump
wavelength of
maximum absorption
(nm)
molar absorptivity
lone pair to pi anti-
bonding orbital
290 15
pi bonding to pi anti-
bonding orbital
180 10000
89

• The ε, is molar absorptivity If the concentration is in
mol/L. The constant in Beer-Lambert,s law is A if
the unit of concentration, c, is g/100ml.
• A is also known as Known as specific Absorbance
• ε = A X molecular weight
10
1%
1.0cm
1%
1.0cm
1%
_______________________
1.0cm
90

Methods of Assay of single
component Samples
• One point assay or 1x1 assay
• Calibration plot or graph method
• Use of molar absorptivity or A
(ALL POINTS WELL EXPLAINED IN CLASS)
1.0cm
1%
91

One point assay
% Purity = Absorbance of test sample x 100
Absorbance of standard 1
AT = CT
AS CS
92
CT = AT X CS
AS
% Purity = CT x 100
CS 1
% Purity = AT X 100
A 1

Calibration Plot Method
• A reference standard of the sample to be analysed is
obtained from a reliable source.
• Suitable stock solution should be made with suitable
solvent as explained in class.
• Various concentrations are prepared from the stock and
the absorbance obtained from the spectrophotometer.
• A graph of Absorbance against concentration of reference
standard should be plotted and the regression equation
should be obtained from the graph. 93

Test Sample
• After suitable extraction of the sample from the
matrix, suitable concentration should be prepared as
carried out in the practical class.
• The absorbance of the test sample should be
obtained from the spectrophotometer.
• The concentration of the sample can be obtained
from the regression equation from the calibration
plot.
94

Calibration curve for ciprofloxacin reference
standard in 0.1N HCl
y = 0.1102x + 0.0038
R² = 0.9998
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Absorbance.
Concentration (µg/ml)

Finding concentration using
molar absorptivity
• Using a solution in a cell of length 1.0 cm. If the
absorbance of the solution at a particular
wavelength using a spectrophometer 1.92. If the
molar absorptivity is 19400 for that wavelength.
A = ε.l.c
1.92 = 19400 x 1 x c
c = 1.92
19400
= 9.90 x 10-5 mol dm-3
96

A very low concentration can be measured if the
molar absorptivity of the sample is very high.
• An accurate value of molar absorptivity must be
known. This method assumes that the Beer-
Lambert Law works over the whole range of
concentration which is not correct.
• It is much better to measure the concentration
by plotting a calibration curve.
97

Using UV-absorption spectra to help
identify organic compounds
• The wavelength of maximum absorption
(lambda-max) depends on the presence of
particular chromophores (light-absorbing
groups) in a molecule.
• A simple carbon-carbon double bond (for
example in ethene) has a maximum absorption
at 171 nm. The two conjugated double bonds in
buta-1,3-diene have a maximum absorption at a
longer wavelength of 217 nm. 98

• Two peaks are present in the spectrum of
ethanal (containing a simple carbon-oxygen
double bond) at 180 and 290 nm.
• In carefully chosen simple cases, if you
compare the peaks on a given UV-visible
absorption spectrum with a list of known
peaks, it would be fairly easy to pick out
some structural features of an unknown
molecule.
99

• Lists of known peaks often include molar
absorptivity values as well. That might help you
to be even more sure of the unknown
compound.
• For example using ethanal (again using the
simple carbon-oxygen double bond), data shows
that the peak at 290 has a molar absorptivity of
only 15, compared with the one at 180 of 10000.
100

Finding concentration using the molar
absorptivity
• If you know the molar absorptivity of a solution
at a particular wavelength, and you measure the
absorbance of the solution at that wavelength,
it is easy to calculate the concentration.
• The only other variable in the expression above
is the length of the solution. That's easy to
measure and, in fact, the cell containing the
solution may well have been manufactured with
a known length of 1 cm.
101

• For example, let's suppose you have a
solution in a cell of length 1 cm. You measure
the absorbance of the solution at a particular
wavelength using a spectrometer. The value
is 1.92. You find a value for molar
absorptivity in a table of 19400 for that
wavelength.
102

Substituting those values:
• Notice what a very low concentration can be measured provided you
are working with a substance with a very high molar absorptivity.
• This method, of course, depends on you having access to an accurate
value of molar absorptivity. It also assumes that the Beer-Lambert
Law works over the whole concentration range (not true!).
• It is much better to measure the concentration by plotting a
calibration curve.
• (http://www.chemguide.co.uk/analysis/uvvisible/analysis.html#top)
103

Finding concentration by plotting a
calibration curve
• Doing it this way you don't have to rely on a value of
molar absorptivity, the reliability of the Beer-Lambert
Law, or even know the dimensions of the cell
containing the solution.
• What you do is make up a number of solutions of the
compound you are investigating from a Reference
standard - each of accurately known concentration.
• Those concentrations should bracket the
concentration you are trying to find - some less
concentrated; some more concentrated.
104

• For each solution, you measure the
absorbance at the wavelength of strongest
absorption (λmax)- using the same container
for each one.
• Then you plot a graph of that absorbance
against concentration. This is a calibration
curve.
105

• According to the Beer-Lambert Law,
absorbance is proportional to
concentration, and so you would expect
a straight line.
• That is true as long as the solutions are
dilute, but the Law breaks down for
solutions of higher concentration, and
so you might get a curve under these
circumstances. 106

• As long as you are working from values either
side of the one you are trying to find, that
isn't a problem.
• Having drawn a best fit line, the calibration
curve is a straight line passing through the
origin as shown below:
107

• Notice that no attempt has been made to force the line back
through the origin. If the Beer-Lambert Law worked perfectly, it
would pass through the origin, but you can't guarantee that it is
working properly at the concentrations you are using.
• Now all you have to do is to measure the absorbance of the
sample solution with the unknown concentration at the same
wavelength. If, for example, it had an absorbance of 0.600, you
can just read the corresponding concentration from the graph
as below:
108

• Ultraviolet/visible spectrophotometer was used to
measure the spectra of two solutions, A and B.
• The analyst recorded the absorbance of each solution
over a range of wavelengths on the same axes. The
resultant absorbance spectrum is shown below:
i. If 15.00 mL of Solution A was mixed with 15.00 mL of
Solution B, which wavelength should be used to measure
the absorbance of Solution B in this mixture? Justify your
answer.
ii. Mention a method that could be used to quantify
solution A in the mixture. (4 Marks)
109

• QUESTION
• Fifty (50) Carbamazepine tablets (Tegretol(R))
weighing 25.000g were powdered. A 0.3000g
of the powdered sample was boiled in 50 ml
ethanol for a few minutes. The hot mixture
was stirred for 10 minutes and filtered. The
cooled mixture was made up to 100ml with
ethanol. An aliquot 5ml of the extract was
diluted to 250ml with ethanol and the
absorbance of the resulting solution was
found to be 0.588 absorbance units at a
wavelength of 285nm.
•
110

• i). What was the content of a single tablet if the
(A1%,1.0cm) was 490? (M.wt. of
carbamazepine=236.269 g/mol) (8 marks)
• ii). Calculate the molar absorptivity, ε, of
carbamazepine in 1b above. (2 marks)
111

QUESTION
• Ten brands of Ciprofloxacin Tablets were assayed
using the Ultraviolet/Visible spectrophotometry.
• The calibration data obtained from the reference
standard are shown below:
Table 1: Calibration data for ciprofloxacin reference
standard.
112
Absor
Bance
0.0823 0.167 0.268 0.358 0.456 0.5723 0.6605 0.7505 0.8368 0.9398
Conc.
(µg/ml)
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

• Ten (10) milligram equivalent of the test samples
were weighed and diluted with deionized water to
100ml (100µg/ml stock). The stock, 0.5ml, was
diluted to 10ml with deionized water.
• The absorbances of the various brands are shown
in Table 2 below:
Table 2: Absorbances of the Ten Brands of
Ciprofloxacin hydrochloride Tablets
113
Brand
Code
CF SP AP CB CM CJ CL CY CG VP
Mean
Abso
rbance
0.5020 0.4915 0.5022 0.5090 0.4940 0.5233 0.4513 0.5032 0.4999 0.5001

• Calculate the % Purity of the ciprofloxacin
hydrochloride tablet brands using the
calibration plot method. (The BP 2005
Specification is 95-105%). (15 Marks).
114

QUALITATIVE USE OF UV/VISIBLE
SPECTROSCOPY
1. Detection of Impurities
• UV absorption spectroscopy is one of the best
methods for determination of impurities in
organic molecules. Additional peaks can be
observed due to impurities in the sample and it
can be compared with that of standard raw
material. By also measuring the absorbance at
specific wavelength, the impurities can be
detected.
Benzene appears as a common impurity in
cyclohexane. Its presence can be easily detected
by its absorption at 255 nm. 115

2. Structure elucidation of organic compounds.
UV spectroscopy is useful in the structure
elucidation of organic molecules, the presence
or absence of unsaturation, the presence of
hetero atoms.
From the location of peaks and combination of
peaks, it can be concluded that whether the
compound is saturated or unsaturated, hetero
atoms are present or not etc. 116

3. UV absorption spectroscopy can characterize
those types of compounds which absorbs UV
radiation. Identification is done by comparing
the absorption spectrum with the spectra of
known compounds.
4. UV absorption spectroscopy is generally used
for characterizing aromatic compounds and
aromatic olefins.
117

REFERENCES
• http://www.chemguide.co.uk/analysis/uvvisible/analysis.html#top
• https://www.google.com/search?source=univ&tbm=isch&q=bathoc
hromic+shift+and+hypsochromic+shift&client=firefox-b-
d&sa=X&ved=2ahUKEwjjxfvZ1NXuAhVNOBoKHbXwA6UQjJkEegQIIh
AB&biw=1366&bih=654#imgrc=O6nxb2GEheUvpM&imgdii=Woadk
NTx5_X1EM
• https://www.learnpick.in/prime/documents/ppts/details/4118/instr
umental-techniques
118
https://www.google.com/search?source=univ&tbm=isch&q=bathochromic+shift+and+hyp
sochromic+shift&client=firefox-b-
d&sa=X&ved=2ahUKEwjjxfvZ1NXuAhVNOBoKHbXwA6UQjJkEegQIIhAB&biw=1366&bih=65
4#imgrc=O6nxb2GEheUvpM&imgdii=WoadkNTx5_X1EM

Ultraviolet and Visible Absorption Spectrophotometry note students 2021(2).pptx

Recommended

Recommended

More Related Content

Similar to Ultraviolet and Visible Absorption Spectrophotometry note students 2021(2).pptx

Similar to Ultraviolet and Visible Absorption Spectrophotometry note students 2021(2).pptx (20)

Recently uploaded

Recently uploaded (20)

Ultraviolet and Visible Absorption Spectrophotometry note students 2021(2).pptx