ANALYTICAL

CHEMISTRY II
220401
Dr. Aysun DİNÇEL

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 Week-4
Special Techniques in UV/Vis and
Infrared (IR) Spectroscopy

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• Transmittance: denoted by “T" and is the ratio of the intensity of the
light coming out of the sample solution (I) to the intensity of the light
sent on the sample solution (Io):

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 I0 1
• A = log A =-log T or A = log
I T
• Lambert-Beer combines the two releationship of concentration and
absorption
• By these two definitions

A=k.l.C
A=Ɛ.l.C
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Ex.
• A 7.25 x 10-5 M solution of potassium permanganate has a
transmittance of 44.1% when measured in a 2.10-cm cell at a
wavelength of 525 nm. Calculate (a) the absorbance of this solution
and (b) the molar absorptivity of KMnO4.

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Solution
(a) A = -log T = -log 0.441 = -(-0.356) = 0.356

(b) From Equation A = Ɛ . l . C

Ɛ =0.356/(2.10 cm x 7.25 x 10-5 mol/L )
Ɛ = 2.34 x 103 mol-1cm-1

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Absorption Spectra
• An absorption spectrum is a plot of absorbance versus wavelength,
• Absorbance could also be plotted against wavenumber or frequency.
• Modern scanning spectrophotometers produce such an absorption
spectrum directly

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• Typical absorption spectra of
potassium permanganate at
five different concentrations.
(ppm)

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Limits to Beer’s Law
• There are few exceptions to the linear relationship between
absorbance and path length at a fixed concentration
• Beer’s law describes the absorption behavior only of dilute solutions
and in this sense is a limiting law.

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• At concentrations exceeding about 0.01 M, the average distances
between ions or molecules of the absorbing species are diminished to
the point where each particle affects the charge distribution and thus
the extent of absorption of its neighbors.
• Because the extent of interaction depends on concentration, the
occurrence of this phenomenon causes deviations from the linear
relationship between absorbance and concentration.

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• A similar effect sometimes occurs in dilute solutions of absorbers that
contain high concentrations of other species, particularly electrolytes.

• When ions are very close to one another, the molar absorptivity of
the analyte can be altered because of electrostatic interactions which
can lead to departures from Beer’s law.

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• The most important of these is the presence of hydrogen bonds
between the substance and the solvent. Because if there are
hydrogen bonds, the spectrum changes.
• For example, in the spectrum of 0.01M alcohol, while 0.01M is at
λmax 362 nm when it is 1M,
• λmax shifts to 332 nm.
• The reason is that as the concentration increases, by the hydrogen
bonds between them

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Limitations to Beer’s Law
b) If there is scattering due to the particles in the sample,
c) If there is fluorescence or phosphorescence in the sample
d) If changes in the refractive index occur at high concentrations of the
analyte (the analyzed substance),
e) If a shift in chemical equilibrium occurs as a function of concentration,
f) If monochromatic light is not used and
g) Deviations from the Lambert-Beer rule occur if there are interference
lights
h) If analyzed substances change during the study; for example, if there are
complexation reactions
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In some systems, both pH and concentration play
a role;
• the equilibrium 2CrO42− + 2H+ → Cr2O72− + H2O is an example.
• Cr2O72- (red) turns into CrO42- (yellow) in the basic medium, and the
spectrum changes.
• For this reason, it is necessary to perform spectrophotometric studies
in buffer solutions.
• In addition, the oxidation of substances during operation also changes
the spectrum

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The spectra obtained in UV and visible field
spectroscopy are used in the following ways:
1) In qualitative analysis: Because the spectrum of each substance
in a certain solvent is unique.
2) In quantitative analysis: Because absorption is proportional to
the amount of substance (Beer's law)
3) In the determination of the equilibrium constant (Ka and Kb)
4) In the determination of molecular weight
5) In kinetic studies
6) In photometric titrations.

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Quantification operations in UV and
visible field spectrophotometry:
• When working for analytical purposes, UV and visible areas are
deciphered 200 – 800 nm as.
• In UV region studies, the near UV area between the 200 – 400 nm is
used.

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Quantity determination operations are usually
performed in 2 ways :
• First of all, 1. a calibration curve is prepared with a standard
substance to be determinate substance.
• For this purpose, the absorbance values of standard solutions varying
and known amounts of analyte are measured and the absorbance
values are plotted against the concentration

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Calibration curve
• a linear line is obtained.
• has an equation in the form of y = mx + n
• m = slope and n = intercept
• R: correlation constant  1
• R2: Regression constant  1

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• 2. The absorbance (A1) of the standard substance at a known
concentration (C1) is read.
• In addition, the absorbance (As) of the solution of unknown
concentration (Cx) is read.
•Then, according to the Lambert-Beer law if two equations are
divided into
A1 = Ɛ . l . C1
As = Ɛ . l . Cx and
A1 / As = C1 / Cx
Cx and it can be easily calculated.
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• Method 2 is used for if the medium contains only for one substance

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to be considered during the quantity
determination procedures:
1. In qualitative analysis, it is preferred to measured the absorbances at
wavelengths where it is maximum absorption.
the measuring errors are minimized
2. It is desirable that the absorbance values be between 0.200 – 0.800 in
order to measure with the least error during the studies.
This is due to the effect of the absolute error on the transmittance
measurement.

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•A=Ɛ.l.C
log T = Ɛ . l . C if the left side is converted to a logarithm of course, if
the differential is taken:

dT = Ɛ . l . dC
T
If this equality is divided by – log T = Ɛ . l . C and arranged:
dC 0.434
= dT happes :
C T log T
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can be written in
the form

ΔC 0.434
= ΔT.
C T log T

• ΔC/C indicates the relative error

• The transmittance value at which this
error is minimal is 0.368, which
corresponds to an absorbance value of
0.434.
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• It is possible to make measurements with a relative error of less than
2% at concentration values between approximately 0.200 – 0.800
absorbance values. The result is the prediction that the minimum
relative error in the concentration theoretically occurs when T = 0.368
or A = 0.434.

Relative
concentration error as
function of
transmittance for
1% uncertainty
in % T.

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• If both species of a chemical equilibrium absorb, and if there is some
overlap of the same at the isosbestic point.
• their absorption curves, the wavelength at which this occurs is called
the isosbestic point, and the molar absorptivity of both species is the
same.

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• The spectra are plotted at different pH values since the pH generally
causes the shift in the equilibrium. Obviously, the effect of pH could
be eliminated by making measurements at the isosbestic point, but
the sensitivity is decreased
• By making the solution either very acid or very alkaline, one species
predominates and the sensitivity is increased by measuring at this
condition.

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• For a two-component system in which the two
absorbing species are in equilibrium, all curves
intersect at the isosbestic point where they
have the same Ɛ value.
• The existence of an isosbestic point is a
necessary (although not sufficient) condition
to prove that there are only two absorbing
substances in equilibrium with overlapping
absorption bands..
• Illustration of isosbestic point of bromothymol
blue (501 nm): (A) pH 5.45, (B) pH 6.95, (C) pH
7.50, (D) pH 11.60. A represents the spectrum
of the acid form, and D of the base form, of
bromothymol blue. B and C represent the
sums of the spectra of the two species in
equilibrium as the pH shifts. They both have
the same absorbance at 500 nm.

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Analysis of Mixtures
• The total absorbance of a solution at any given
wavelength is equal to the sum of the
absorbances of the individual components in
the solution.
• This relationship makes it possible in principle
to determine the concentrations of the
individual components of a mixture even if
their spectra overlap completely.
• Absorption spectrum of a two-component
mixture (M + N), with spectra of the individual
components M and N.

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Solvent effect:
• Hypsochromic shift occurs as the solvent polarity increases in the
bands belonging to n → π * transition. This is because the orbitals
that have not made bonds in polar solvents cause a decrease in their
energy as much as hydrogen bonding, and more energy is required for
n → π * transition.
• By taking advantage of this property, the strength of hydrogen bonds
can also be calculated

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The use of UV-Visible
region spectrophotometry in
mixture analysis:

1) use in the analysis of a mixture of two substances

• In a situation where there is a spectrum of 2 components,
• such as a and b,
• which have spectra as shown in Figure
• in their pure state that is, such as λ1 and λ2, where one
substance has a maximum, while the other has a minimum
• on the other hand, if there are 2 wavelengths in which the
other substance has a maximum, while it has a minimum
• for example, a mixture of paracetamol + methocarbamol

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• The Lambert-Beer equation is used for determination of two substance
concentration.
1. first of all, Ɛ values at λ1 and λ2 wavelengths are calculated using the
measured absorbances of solutions of known concentrations of two
substances such as a and b (for pure solutions)
A=Ɛ.l.C

in λ1 A1 = Ɛaλ1 . l . Ca + Ɛbλ1 . l . Cb

in λ2 A2 = Ɛaλ2 . l . Ca + Ɛbλ2 . l . Cb can be written.

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• A1 and A2 are the absorbance values measured in the
spectrophotometer for the mixture sample of a and b at wavelengths λ1
and λ2.
• l, is the length of the sample container and is constant, as it will not
change with measurements in λ1 and λ2, and l = 1
• Ɛaλ1, Ɛbλ1, Ɛaλ2 and Ɛbλ2 were previously calculated at λ1 and λ2
wavelengths for a and b.
• λ1 and λ2 are read from the spectrophotometer.
• In these equations, the unknown is only the concentrations of a and b
in the mixture sample, so Ca and Cb. Ca and Cb can be calculated with
the help of 2 unknown equations

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2) Derivative spectrophotometry:
• A spectrum in the UV-visible region is obtained by plotting the
absorbance values against the wavelengths
• In other words, A= f(λ) is a function.
• The derivative of this function at each point can be calculated (dA/dλ).
• If these derivative values are to be graphed against the wavelength, a
“derivative spectrum” occurs and can be from 1 to n degrees.
dA d2A ……….. dnA
dλ dλ 2 dλ n

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•Derivative spectrophotometry, on the other hand, is an
analysis method based on the use of derivative spectra.
• In fact, the derivative is the slope of the tangent drawn on the
curve,

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• Nowadays, the derivative curve from the 1st to the 4th is being drawn
• On the curve in the original spectrum, it shows a maximum and a
minimum corresponding to the points where the slope is the largest,
that is, the half-heights of the curve,
• the half-heights of the curve, and at the point where the slope is the
lowest, it becomes zero.
• Accordingly, the maximum point is equal to the Gaussian curve
corresponds to in a “intercept (zero-crossing)”

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• In the 2nd derivative curve,
as a result of the same
applications, a minimum
corresponding to the
maximum point of the
original curve is observed

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• In the 3rd derivative curve, the maximum point of the original curve
corresponds to a intercept point
• In the 4th derivative, the maximum in the original spectrum
corresponds to a maximum again.
• For this reason, the 2nd and 4th derivative curves are used in the
quantitative determination of a single substance

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 By taking derivatives

• An important point is that the width of the peaks decreases due to the fact that the derivative curves are
taken within the same wavelength zone.
• peaks that cannot be separated in the original spectrum appear in more detail, and quantity
determinations of the components in the mixture can be made without applying any separation process
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Method
•Advantages:
a. The study of the derivative gives information about the slopes of the
original spectrum and causes the shoulder points and turning points
to become more prominent.
b. With derivatization, absorption curves take on a more detailed shape
and thus it is possible to see the peaks separately.
c. when working with turbid solutions in spectrophotometry, a large
percentage of errors can be made. This can be eliminated by
preparing derivative curves.

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d. if there is more than one substance in a mixture, any separation
process such as extraction and chromatography are not required.
e. It is ensured that the noise peaks caused by the reaction environments
are eliminated.

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Disadvantages:
a. Although it is very easy to use, there is a need for expensive
spectrophotometers and a rather complex electronic structure.
b. A large number of satellite peaks are also observed in the derivative
spectra.

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Other uses fields of UV - visible field
spectrophotometry
a) Calculation of the equilibrium constant
b) Determination of molecular weight
c) Chemical kinetics

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COLORIMETRY:
• It is a photometric method that is studied with colored solutions.
• It is studied in the visible region, that is, in the region between 400 –
800 nm.
• the Lambert-Beer’s law applied
• If the substances are colorless, they can be colored with various
reagents and analyzed in the visible area.

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• It is also possible to work roughly by eye in colorimetry.
• For this, a number of standard solutions are prepared.
• standard substance solutions of varying and increasing concentrations
are placed in a large number of absolutely identical sample containers
• The volume is completed with a solvent to a certain value.
• By comparing the resulting color with the colors of the solutions in the
series by eye, a rough determination of the amount can be made based
on the concentration

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UV-VISIBLE and IR SPECTROPHOTOMETRY
DEVICE INFORMATION
•Spectrophotometers:
1) Light source
2) Monochromator
3) Sample container
4) Detector
5) Recorder

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• For the ultraviolet region, a low-pressure deuterium discharge lamp is
generally used as the source. The deuterium continuum emission
extends from 185 to to 370 nm but the lamp has useful spectral
output out to 600 nm.
• Ultraviolet sources must have a quartz window, because glass is not
transparent to ultraviolet radiation.
• For simple absorptiometers a tungsten lamp is the usual source of
illumination.

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• A tungsten or deuterium lamp is used if working only in the visible
region, and a deuterium or mercury lamp is used if working in the UV
and visible region.

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• Infrared radiation is essentially heat, and so hot wires, light bulbs, or
glowing ceramics are used as sources. The energy distribution from
the black-body sources tends to peak at about 100 to 2000 nm (near-
IR) and then tails off in the mid-IR.
• Infrared spectrometers usually operate from about 2 to 15 μm, and
because of the relatively low-intensity radiation in this region,
• typical infrared source
• is the Nernst glower. This is a rod consisting of a mixture of rare-earth
oxides.

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• It has a negative temperature coefficient of resistance and is
nonconducting at room temperature.
• Therefore, it must be heated to excite the element to emit radiation,
but once in operation it becomes conducting and furnishes maximum
radiation at about 1.4 μm, or 7100 cm−1 (1500 to 2000◦C). Another
infrared source is the Globar.
• This is a rod of sintered silicon carbide heated to about 1300 to
1700◦C.
• Its maximum radiation occurs at about 1.9 μm (5200 cm−1), and it
must be water-cooled.
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• The Globar is a less intense source than the Nernst glower,
• but it is more satisfactory for wavelengths longer than 15 μm because
its intensity decreases less rapidly.

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• Single-beam instruments of the type just described are well suited for
quantitative absorption measurements at a single wavelength. With
these instruments,
• simplicity of instrumentation, low cost, and ease of maintenance offer
distinct advantages.

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a singlebeam instrument

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• Many modern photometers and spectrophotometers are based on a
double-beam design. Figure shows two double-beam
• two beams are formed by a V-shaped mirror called a beam-splitter.
• One beam passes through the reference solution to a photodetector,
and the second simultaneously passes through the sample to a
second, matched photodetector.
• The two outputs are amplified, and their ratio, or the logarithm of
their ratio, is obtained electronically or computed and displayed on
the output device.

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a doublebeam-in-space instrument

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Monochromators
•Monochromator A device for resolving polychromatic
radiation into its component wavelengths.
•Monochromatic light: It is the light of a single wavelength.
• used in spectrophotometers are used to obtain it

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• Types of monochromators:
• (a) grating monochromator;
• (b) prism monochromator. The
monochromator design in
• (a) is a Czerny-Turner design, while the
prism monochromator in
• (b) is a Bunsen design. In both cases,
• λ1 > λ 2.

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 Sample container:
• If working in the UV area: quartz cuvette (cell) (because glass absorbs
UV light, so it cannot be used)

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IR spectroscopy:
• When working with solid samples:
• the powdered substance is thoroughly mixed with powdered KBr or
NaCl in an agate mortar in a ratio of about 2% and compressed under a
pressure of about 10 tons
• If the concentration is high, it is diluted by adding KBr or NaCl. There
is no absorption of KBr or NaCl between 4000 – 650 cm–1.

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• When working with solutions: IR spectra of the solutions prepared in
solvents in which the solid is dissolved can be taken.
• But in this case, there should be no absorption of the solvent in the
studied area. Salts such as NaCl or KBr in the sample solution made of
compressed, cylindrical or rectangular prism-shaped between two
blocks (called Window) is placed on,

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iii. When working with gases: Gases are filled with pressure in special
containers with tightly closed and light-permeable windows (made of
KBr, NaCl or CaF2) and their spectrum is taken

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Detector:
• A detector is a device that identifies, records, or indicates a change in
one of the variables in its environment such as pressure,
temperature, or electromagnetic radiation.
• Familiar examples of detectors include photographic film for
indicating the presence of electromagnetic or radioactive radiation,
the pointer of a balance for indicating mass differences, and the
mercury level in a thermometer for indicating temperature.

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• For this purpose, phototubes (or photocells) are used in UV-visible
field studies.
• Photomultiplicators are used more precisely than phototubes
• This consists of an electron-emitting cathode and a series of electrodes
(diodes), each with a positive voltage of 60 - 90 V more than the
previous one.
• When an electron hits this cathode surface, an electron is emitted. The
emitted electron goes to the first diode and hits it. As a result, it causes
the emission of a large number of secondary electrons.

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• These electrons go to the second diode and cause more electrons to be
released from there, and thus the number of electrons is increased
gradually
• After all, the electrons are collected by the anode. Thus, the output is
electronically amplified and an electric current of measurable intensity
occurs.

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• DAD detector (diode array detector) is used for
• As a detector in IR; deuterium triglycinsulfate (DTGS) is used in the
middle IR region, which is the most used region.

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