Transcript Applications of UV

Applications of UV-Vis
Spectroscopy
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The Molar Absorptivity
We have seen earlier that validation of Beer’s
law is dependent on the nature of the molar
absorptivity. It was found that the molar
absorptivity is influenced by:
1. The wavelength of radiation
2. The refractive index and is thus indirectly
related to concentration
3. Electrostatic interactions taking place in
solution; and thus electronic distribution
4. In rare cases, like methylene blue, the molar
absorptivity is directly dependent on
concentration
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The molar absorptivity ,however, is supposed
to be constant for Beer’s law to be valid. The
molar absorptivity is a measure of the ability
of an analyte to absorb light at a specified
wavelength. Therefore, the value of the
molar absorptivity is crucial for the
sensitivity of an analysis. The value of the
molar absorptivity can usually range from
zero (for a nonabsorbing species) to 105 for
highly absorbing species). For quantitative
analysis, a value of at least 103 is necessary
for a reasonable quantitative analytical
determination of an analyte .
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Absorbing Molecular Species
When an incident beam of radiation having a suitable
wavelength hits a molecule, absorption of a photon
takes place and the molecule becomes excited.
Excited molecules will lose excitation energy as heat
or photons (luminescence). Absorption of UV-Vis
radiation is capable of affecting the excitation of
bonding electrons and other valence electrons.
Therefore, excitation of electrons in chemical bonds
(p and s) or nonbonding electrons (n) is the result of
absorption of UV-Vis radiation of a suitable
wavelength. Absorption will thus be dependent on
the availability of p and s bonds or n electrons that
can absorb incident radiation.
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Molecules Containing p, s, and n Electrons
1. Molecules with s Bonds Only
Let us start with a molecule like CH4 and
then expand our discussion to more
complex molecules:
All bonds in methane are s bonds and the
only transition possible is the s-s*
transition. However, the s-s* transition
requires very high energy which occurs in
vacuum UV.
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It is not wise to think of doing UV measurements on
molecular species in the vacuum UV region (125185 nm) for five important reasons:
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The high energy required can cause rupture of the s
bonds and breakdown of the molecule
Air components absorb in vacuum UV which limits
the application of the method
Working in vacuum UV requires special training and
precautions which limit wide application of the
method.
Special sources and detectors, other than those
described earlier, must be used
All solvents contain s bonds
2. Molecules with n Electrons
Electrons in the valence shell that are not used up
in chemical bonds are referred to as nonbonding
electrons (n electrons). Consider a molecule like
ammonia:
The line on nitrogen is a symbol for two nonbonding
electrons. Now, the type of transitions observed
in this molecule can be listed as:
s-s*
n-s*
We have seen earlier that the s-s* transition is not
useful in practical UV-Vis spectroscopy but the
other transition (n-s*) is of lower energy and
should be further discussed.
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The absorption wavelength for a n-s *transition
occurs at about 185 nm where, unfortunately,
most solvents absorb. For example, the most
important solvent is, undoubtedly, water
which has two pairs of nonbonding electrons
that will strongly absorb as a result of the ns *transitions; which precludes the use of
this transition for studies in aqueous and
other solvents with nonbonding electrons. In
summary, it is also impractical to think of
using UV-Vis absorption spectroscopy to
determine analytes based on a n-s *
transition .
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3. Molecules with p Bonds
Absorption of radiation by an alkene, containing a double bond,
can result in s-s* or p-p* transitions. We have seen that a s-s*
transition is not useful but on the other hand, the p-p* turned
out to be very useful since it requires reasonable energy and
has good absorptivity. A molecule having s, p, and n electrons
can show all types of transitions possible in UV-Vis
spectroscopy. For example, an aldehyde molecule shows all
these transitions since it contains s, p, and two pairs of n
electrons.
Two transitions are possible for the n electrons:
n-s*
n-p*
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We have seen that a n-s *
is not very useful due
to absorbance from
solvents and other
frequently used
additives which have n
electrons. The n-p *
transition requires
very little energy and
seem to be potentially
useful. However,
unfortunately, the
absorptivity of this
transition is very small
which precludes its
use for sensitive
quantitative analysis .
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Effect of Solvent Polarity on Absorption Wavelength
The molar absorptivity for a n-p* transition is
rather small (10-100 L mol-1 cm-1) and the
energy required for transition is affected by
solvent polarity. In presence of a polar
solvent, nonbonding electrons will interact
with protic solvents to form hydrogen bonds.
The solvation of n electrons is the result;
which lowers the energy of the orbitals
holding the n electrons. Partial stabilization
of the polar p* is also observed but to a much
lower extent than the n electrons.
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A net increase in energy required for a n-p* transition
is thus observed in protic solvents; like water or
alcohols. Therefore, an increase in energy will reflect
a decrease in transition wavelength, or what is called
hypsochromic shift or blue shift.
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On the other hand, a p-p* transition is affected in an opposite
manner with solvent polarity. In presence of a polar solvent, the
more polar p* orbital will be more stabilized than the p orbital
leading to a net decrease in the transition energy. This results
in an increase in transition wavelength or what is called a
bathochromic shift (red shift).
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Spectral nomenclature of shifts
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Conclusions on Electronic Transitions
A s-s* and a n-s* are not useful for reasons discussed earlier.
The n-p* transition requires low energy but the molar
absorptivity is also low and transition energy will increase in
presence of polar solvents. The n-p* transition is seldom used
in quantitative UV-Vis spectroscopy.
The most frequently used transition is the p-p* transition for the
following reasons:
1. The molar absorptivity for the p-p* transition is high allowing
sensitive determinations.
2. The energy required is moderate, far less than dissociation
energy.
3. In presence of the most convenient solvent (water), the energy
required for a p-p* transition is usually smaller.
It is therefore primitive that an analyte to be determined by UVVis absorption spectroscopy be of unsaturated nature (or!!).
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Applications of UV-Vis
Spectroscopy
Lecture 26
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Organic Chromophores
Molecules having unsaturated bonds or free
nonbonding electrons that can absorb
radiation of relatively low energy are called
chromophores. Examples include alkenes,
alkynes, ketones, aldehydes, phenyl and
other aromatic species, etc.
Auxochromes
An auxochrome is a functional group that does
not absorb by itself (like -NH2 and -OH) , but
its presence in a molecule can increase
absorption and usually results in a red-shift.
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The human eye sees the complementary color to that
which is absorbed
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Chromophore
Example
Excitation
λmax ,nm
ε
Solvent
C=C
Ethene
π g π*
171
15,000
hexane
C≡C
1-Hexyne
π g π*
180
10,000
hexane
C=O
Ethanal
n g π*
π g π*
290
180
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10,000
hexane
hexane
C-X X=Br
X=I
Methyl
bromide
Methyl
Iodide
n g σ*
n g σ*
205
255
200
360
hexane
hexane
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Effect of Conjugation of Chromophores
As conjugation is increased in a molecule, more
delocalization (stability) of the p electrons results.
The effect of this delocalization is to decrease the p*
molecular orbital. The result is a decrease in
transition energy from p-p* and thus a red or
bathochromic shift. The molar absorptivity will
increase in this case and better quantitative analysis
will be achieved. In cases of introduction of more
unconjugated double bonds, the molar absorptivity
will increase as well depending on the number of the
double bonds. For example, at 185 nm,1-hexene has
a molar absorptivity of about 10,000 L mol-1 cm-1 but
hexa-1,4-diene has a molar absorptivity of twice as
much as 1-hexene. However, when the double bonds
are conjugated as in hexa-1,3-diene the molar
absorptivity is about 21,000 L mol-1 cm-1.
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Rule of thumb for conjugation
If greater then one single bond apart
- e are relatively additive (hyperchromic shift)
- l constant
CH3CH2CH2CH=CH2
lmax= 184 emax = ~10,000
CH2=CHCH2CH2CH=CH2
lmax=185
emax = ~20,000
If conjugated
- shifts to higher l’s (red shift)
H2C=CHCH=CH2
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lmax=217 emax = ~21,000
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Effect of Aromaticity of Chromophores
On the other hand, aromaticity results in
extraordinarily high degree of delocalization
of electrons and thus stabilization of the p*. If
we assume a molar absorptivity of about
10,000 L mol-1 cm-1 for each double bond, we
expect the sum of the three double bonds in
benzene to be just above 30,000 L mol-1 cm-1
(at 185 nm) but actually the value is about
60,000 L mol-1 cm-1 due to increased
delocalization as a result of aromaticity. It is
therefore advantageous to use UV-Vis
absorption spectroscopy for determination
of compounds having aromatic character.
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Absorption by Inorganic Groups
Inorganic groups containing double bonds absorb in
the UV-Vis region. The most transitions are a result
of n-p* transitions as in nitrate (313 nm), carbonate
(217 nm), nitrite (280 and 360 nm) and azide (230 nm)
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Absorption Involving d and f Orbitals
Many transition metals have colored solutions and are
also colored in the solid state. The transition metals
have some of their d orbitals empty where a d-d
transition can occur. The d-d transitions require
excitation energy in the UV-Vis region. The direct
interaction of the d electrons with ligands around the
transition metal results in a spectrum of broad band
nature. On the other hand, inner transition elements
show transitions by absorption of UV-Vis radiation (ff transitions). Since the electrons in the f orbitals are
far inside the metal orbitals and are screened by
electrons in orbitals of higher principal quantum
numbers, f-f electronic transitions will not be
affected by the nature of ligands or solvent around
the inner transition metals. Therefore, the spectra of
inner transition metals have narrow bands.
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The crystal field theory is usually used to explain
splitting of the d orbital energy so that a transition
from a lower energy d orbital electron can be excited
to a higher energy d orbital. The theory will be
described for a transition metal with six ligands or
molecules of water around it. An octahedral (only
this case will be discussed) arrangement of these
ligands is most appropriate where ligands will be
located at the z axis and at the x and y axis (will repel
electronic cloud in the dz 2and dx-2y 2orbitals and thus
will make these two orbitals to have higher
energies). The other four ligands will be arranged in
between axis( dxy ,dxz ,and dyz )which will increase
their energies but to a lower extent. The net result is
the splitting of the degenerate d orbital into two
groups of d orbitals of different energies (the energy
difference is referred to as D).
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Transition metal ions
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Charge Transfer Absorption
When a ligand permanently donates an electron to a
metal ion, a charge transfer is said to take place. The
net outcome of the process is an oxidation reduction
phenomenon occurring within the complex. An
example is the reaction of Fe3+ with thiocyanate
where the product is an excited species with neutral
thiocyanate and Fe3+. In less common situations, the
transfer of electrons can take place from the electron
deficient metal ion to the ligand. An example is the
Fe2+ or Cu+ complexes with 1,10-phenanthroline
where Fe2+ and Cu+ metal ions donate electrons to
1,10-phenanthroline. The complex will then have Fe3+
and Cu2+ ions. Charge transfer complexes are of
special interest their molar absorptivities are
unusually high; allowing very sensitive
determinations.
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Applications
of Absorbance
Qualitative Analysis
Measurement
to
As seen earlier, the broad band absorption spectra
obtained in UV-Vis absorption spectroscopy is
usually featureless and lacks details that can be
used in qualitative analysis. Therefore, this
technique is mainly a quantitative technique.
Plotting Spectral Data
A plot of either the absorbance or %transmittance
against wavelength can be made. However, the most
common practice is to plot the absorbance versus
wavelength.
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Applications of UV-Vis
Spectroscopy
Lecture 27
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Solvents
We have seen earlier that solvent polarity affects the
absorptivity of the analyte molecules; due to change
in transition energies. Usually, polar solvents are
used when possible. However, polar solvents like
water or alcohol tend to remove the fine spectral
details. Therefore, in cases where the fine spectral
details are really needed (as in qualitative analysis) a
non polar solvent like hexane should be used. In
addition, the solvent must be optically clear (does
not absorb incident radiation), well dissolve the
sample, and chemically pure.
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Quantitative Analysis
The basis for quantitative analysis in the UV-Vis relies on
Beer’s law. Several characteristics of quantitative
measurements using UV-Vis absorption spectroscopy can
be rationalized:
1. Applicability to all types of analytes as far as they absorb
in the UV-Vis region.
2. Moderate sensitivities in the range from 10-4 to 10-6 with
possibility to extend this range under certain conditions.
3. The relative standard deviation occurs within 1-3% which
reflects good precision.
4. Easy to perform and convenient.
5. Can be used for quantitative analysis in liquid
chromatographic separations.
6. Non absorbing species can also be determined if they are
derivatized with an absorbing species as the case of
metal ions when complexed to ligands.
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Procedural Details
1. Selection of Wavelength
The first step in a successful determination is to find the suitable
wavelength for the analysis. This is accomplished by plotting
the absorbance/wavelength curve. However, the following
points should also be considered:
1. If more than an absorption maximum is available, the
wavelength far from the instrument extremes should be
preferred
2. A wavelength at the maximum of a broad peak should be
preferred to another of a sharp peak
3. The peak with a maximum peak height is preferred
4. If interferences are present, the wavelength that is far away
from interferences should be selected
5. Working in the visible region should be preferred
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2. Cleaning and Handling the Cell
First, one should appreciate the use of good quality
matched cells that are free from wearing, etching,
and scratches. In addition, cleaning procedures of
external and internal cell surfaces are also
important. A suggested cleaning procedure
involves moistening a lens paper with methanol
and wiping the external surface, then leaving the
cell to evaporate. The interior of the cell is first
washed with water followed by methanol and the
solvent is also allowed to evaporate. Disposable
polypropylene cuvettes are incompatible with non
polar solvents and formulations having these
solvents should be avoided; or large errors will be
encountered.
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3. Calibration Curves
Usually, a plot of the absorbance of a series of standards is
plotted versus the concentration. The absorbance of
the unknown is then determined and the prepared
calibration plot is used for the determination of the
analyte concentration. If the absorbance of the analyte
was located outside the calibration plot, more standars
should be made or the analyte concentration must be
adjusted to occur on the calibration plot. We have seen
earlier that it is not allowed to theoretically extrapolate
or interpolate a calibration plot. It should also be
appreciated that the composition of standard solutions
must approximate that of the sample solution. In cases
where the sample composition is not clear, the method
of standard addition should be used. The slope of the
linear calibration plot is the molar absorptivity when
the path length is 1.00 cm. Larger slopes mean higher
sensitivities.
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4. Standard Addition method
The use of this method provides better correlations between
absorbances of standards and sample due to constant
matrix effects. The method involves addition of the same
sample volume to a set of tubes or containers. Variable
volumes of a standard are added to the tube set followed
by completion to a specific volume. Now, all tubes contain
the same amount of sample but different concentrations
of analyte. A plot is then made for the volume of standard
and absorbance. This plot will have an intercept (b) with
the y axis and a slope equals m. The concentration of the
analyte can be determined by the relation:
Cx = bCs/mVx
Where, Cx and Vx are concentration and volume of analyte
and Cs is the concentration of standard.
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One can only use two points to get the analyte
concentration using the relation:
Cx = AxCsVs/(At –Ax)Vx
Where, Ax is the absorbance of the analyte
solution brought to a final volume Vt and At is
the total absorbance of the solution (final
volume is Vt) containing same amount of
analyte and a volume of the standard (Vs).
However, this procedure assumes that both
points occur at the linear portion of the
calibration plot. In case where this is not
true, an error will be encountered.
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5. Analysis of Mixtures of Absorbing Substances
When the sample solution contains more than one absorbing
species, the absorbance of the solution will be the sum of all
absorbances:
At = A1 + A2 + A3 + ….
The different constituents can be determined if we build equations
equal to the number of unknowns. However, this procedure,
if manually performed, is impractical due to lengthy and
difficult math involved especially when more than two
components are present. Remember that we need to
calculate n2 values for e where n is the number of
components.
Al’ = ex’bcx + ey’bcy
(1)
Al” = ex”bcx + ey”bcy
(2)
ex’, ex”, ey’, ey” can be determined from standards of analytes x and
y at l’, l” and values obtained are inserted in equations 1
and 2 where two equations in two unknowns can be easily
solved.
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6. Derivative UV-Vis Absorption Spectroscopy
In derivative spectroscopy, DA/Dl is plotted against the
wavelength. The derivative spectrum contains
sharper peaks where better location of peaks and
wavelengths maxima can be achieved. Not only the
first derivative of the absorbance spectrum can be
obtained but up to the fourth derivative is possible.
However, as the differentiation order increases, the
noise increases as well and if a lower derivative is
fine, going to higher derivatives is a waste of time
and effort. Derivative spectroscopy is excellent for
determination of multi components in a sample, if
they can be resolved.
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Obtaining a Derivative Spectrum
Derivative spectra cab either be recorded on time or by
manipulation of data obtained in an
absorbance/wavelength spectrum. The later is easy
and can be made by simple electronic or mathematic
operations; by taking DA for a fixed Dl (few nm) and
plotting the data versus wavelength. However, the
most common on line instrumental recording of
derivative spectra involves the use of a dual
wavelength instrument with two monochromator
operated at the same speed but with a lag of few nm
from each other. A chopper will sequentially pass the
beams from both monochromators and thus their
difference divided by the constant nm lag value is
recorded versus the average wavelength. The
following schematic is helpful to visualize how this
is accomplished:
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Applications of Derivative Spectra
1. Better qualitative analysis and identification of the
number of absorbing species in a sample
2. Accurate determination of lmax
3. Obtaining spectra in solutions with high
scattering was possible using dual wavelength
instruments
4. Spectral resolution of multi component systems
by measurement at two wavelengths; where the
interferent has identical molar absorptivity while
the analyte does not, can result in good exclusion
of interferences.
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Photometric Titrations
In cases where an analyte reacts with a reagent
so that the analyte, the reagent or the
product absorbs UV-Vis radiation, the
technique can be used for determination of
the analyte by a photometric titration
reaction. Photometric titrations are similar to
conventional visual titrations but following
the course of a photometric titration occurs
with the aid of a UV-Vis detector, rather than
the naked eye.
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Photometric Titration Curves
A plot of absorbance versus titrant volume is
called a photometric titration curve. The
titration curve is supposed to consist of two
linear lines intersecting in a point
corresponding to the end point of the
reaction. The absorbance reading should be
corrected for volume where as a titrant is
added to the reaction mixture the
absorbance will change. Several shapes of
photometric titration curves can be identified
depending on the reaction.
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Instrumentation
A conventional photometer or
spectrophotometer can be adapted to
performing photometric titrations where the
analyte is placed in the sample cell which
contains a small magnet and is located on
the top of a magnetic stirrer. The wavelength
is selected and the titrant is added, from a
dark burette, gradually and the absorbance is
recorded.
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Advantages and Applications of Photometric Titrations
1. Usually, photometric titrations are more accurate than visual
titrations.
2. Photometric titrations are faster than visual titrations as only
few points at the beginning and end of the titration is
necessary. Extrapolation of the straight lines will intersect at
the end point.
3. Titration reactions that are slow at the end point can not be
performed by visual titrations but are well suited for
photometric titrations. Only few points at the beginning and
end of the titration, well away from the equivalence point where
the reaction is slow, are necessary. Extrapolation of the straight
lines will intersect at the end point. Therefore, dilute solutions
or weak asids and bases can be also titrated photometrically.
4. Mixtures can be easily titrated using photometric titrations if
absorbance is measured at the absorption wavelength of one of
the two analytes. In addition, if both analytes give colored
products but with different molar absorptivities, they can be
determined photometrically.
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For example, figure a below shows the EDTA titration of Bi +3 and
Cu +2 at the wavelength of the Cu-EDTA where the Bi-EDTA
does not absorb. Figure b shows a hypothetical reaction where
two products absorb incident radiation. The first substance (x)
reacts first and followed by the second one which has a lower
molar absorptivity(.
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Photoacoustic Spectroscopy (PAS)
UV-Vis spectra of solid, semisolid, and turbid samples
can be obtained by photoacoustic spectroscopy,
although PAS is more important in infrared
absorption spectroscopy. The technique of
photoacoustic spectroscopy involves subjecting the
sample in a photoacoustic cell (containing a gas) to
a chopped beam of radiation. If the sample absorbs
the incident wavelength, as the chopper rotates, the
sample emits the absorbed energy as heat. Periodic
absorption and reemission causes periodic
fluctuations in the gas temperature and thus
pressure. If the chopper rotates in a rate that occurs
in the acoustical range, the fluctuations in pressure
can be detected by a sensitive microphone.
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Applications of Photoacoustic Spectroscopy
1. Recording UV-Vis spectra of solids and
turbid solutions. Samples as complex as
whole blood were tested by PAS.
2. Study of the absorption characteristics of
polymers, paints archeological samples.
3. Spectra of samples on surfaces, like silica
thin layer chromatographic plates, can be
recorded.
3. In the IR region, qualitative analysis is
possible to a variety of samples.
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