Div 1#agawanesir

Instrumental Techniques of
Chemical Analysis
Department of Applied Sciences

COEP
College of Engineering Pune (COEP)

Forerunners in Technical Education
Contents
1 Introduction
2 Qualitative and Quantitative Analysis: an overview
3 Electromagnetic spectrum and Mol. Interactions
4 UV-Vis Spectro., Beer-Lambert’s Law, Deviation
5 UV-Vis Spectro: Instru, Working and Appl.
6 IR Spectro: Instru, Working and Appl.

Instrumental Analysis
What is it?
Instrumental Analysis: It is the study and use of instruments and methods to

separate, identify and quantify the matter.

Types of Instrumental Analysis
College of Engineering
College of Engineering PunePune
(COEP) (COEP)
Brief info on Instrumental Analysis
Sr. No. Instrument Principle/Law Applications
1 UV-Vis spectrophotometer 1) Lambert-Beer’s law 1) To identify the molecule
2) With UV –Vis light the 2) To quantify the sample
molecule undergoes 3) To identify the functional group of organic
electronic transitions sample
2 IR spectrophotometer With IR radiation the 1) Identification of the structure and
molecules undergo functional group present in the sample
stretching and bending 2) To study the progress of reaction.
vibrations 3) To determine the type of hydrogen
bonding of the sample.
3 X-ray Diffraction Bragg’s law: nλ = 2dsinθ 1) Identification of the material.

2) Identification of phase of the single
material.
3) Determination of the composites
4) Determination of crystallite size
NaCl
4 Turbidimetry Intensity of transmitted 1) Determination of turbidity in natural
light from the turbid waters and in processing streams.
sample is measure of the 2) Protein content in urine
Concentration A= e.c
5 Nephelometry Intensity of back scattered 1) Pollution monitoring
light from the turbid 2) Climate monitoring
sample is measure of the
concentration
6 Scanning Electron Microscopy Electron beam interacts 1) To obtain the image of the material
(SEM) with the surface of the 2) To determine the morphology of the
sample and back scattered sample till 50-100 nm
electrons are collected to 3) To get Elemental mapping – distribution
create image of the sample and percentage of the different elements in
under study. the sample

7 Transmission Electron Electron beam penetrates the sample 1) To obtain the image of the material
Microscopy (TEM) surface throughout the depth of 2) To determine the structure/morphology of
sample and transmitted to create the the sample till 2 nm
image of the sample 3) To obtain the interplanar d-spacing in the
materials
8 Voltammetry Constant and/or varying potential at 1) To determine the oxidation /reduction

an electrode and measures the potential.
resulting current with a three 2) To determine the CB and VB of the material
electrode system. Based on ohm’s
law
9 Coulometry The total current or charge 1) To determine the no of electrons utilized to
(Current/time) passed is directly or complete the reaction
indirectly proportional to the number 2) To determine columbic efficiency of the
of electrons passed in the system.
electrochemical cell. Based on
Faraday’s law
10 Chromatography Separation of the components 1) Separation a mixture of chemical
based differential partitioning substances into its individual components.
(liquid) between the mobile and 2) Identification of the components.
stationary phases.
11 Gravimetry Weight gain/loss after 1) To determine the composition of major

transformation of the sample is component of the sample
measured
12 Thermogravimetric Measures the weight change 1) Composition of the material
versus temperature or time
analysis (TGA) 2) Water of crystallization or melting point
3) Thermal stability of the material
13 Differential scanning Quantitatively measures the heat 1) To determine the phase transition of the
calorimetry (DSC) flow changes (heat loss/gain) material
versus temperature or time 2) To determine the melting point and glass
during physical and chemical transition temperature of polymers
transformation
College ofof sample Pune (COEP)
Engineering
Electromagnetic spectrum
Radiations that can reach the surface of earth

Electromagnetic spectrum
Valence elec. Inner shell

elec.
Spin orientation Mol. Vibra. Ionization of atoms Nuclear changes
Energy
strec. and
College bend
of Engineering Pune (COEP) and molecules
changes Forerunners in Technical Education
Ultraviolet, Visible and Infrared region

Ultraviolet-Visible Spectrometer
Principle of UV-Vis spectroscopy

UV-Vis spectroscopy obeys the Beer-Lambert law, which states that: when a beam of
monochromatic light is passed through an absorbing medium, the intensity of the absorbed
light is directly proportional to the thickness and the concentration of the absorbing
medium.
Mathematical expression of Beer-Lambert’s law is:
A = log (I0/It)
= ε.c.l
Useful links to understand the working of UV-Vis. Spectroscopy.

1) https://www.youtube.com/watch?v=tbUx-RaZS7M
2) https://www.youtube.com/watch?v=wxrAELeXlek
3) https://www.youtube.com/watch?v=s5uIVQGFDE4
4) https://www.youtube.com/watch?v=mNnjhoeutow

Ultraviolet-Visible Spectrometer
Introduction
UV-Vis spectroscopy is an important tool in analytical
chemistry. The other name of UV-Vis spectroscopy is
Electronic spectroscopy as it involves the promotion
of the electrons from the ground state to the higher
energy or excited state. In this article I will explain the
basic principle, working and all the applications of
UV-Vis spectroscopy.
Principle of UV-Vis spectroscopy
UV-Vis spectroscopy obeys the Beer-Lambert law, which states that: when a beam of
monochromatic light is passed through an absorbing medium, the intensity of the absorbed
light is directly proportional to the thickness and the concentration of the absorbing
medium.
Mathematical expression of Beer-Lambert’s law is:
A = log (I0/I)
= ε.c.l
A = absorbance, I0 = intensity of the incident light, I = intensity of the transmitted light,
c = concentration of solution, l = length of sample cell (cm), ε = molar absorptivity

Absorption of visible and ultraviolet light
hν hν
TiO2+ZnO TiO2+ZnO
A = log (I0/I) A = log (I0/I)

= log (1/0.95) = log (1/0.05)
∴A = 0.022 ∴A = 1.30
Visible light Plot of A vs. λ Ultraviolet light

Absorption of UV-Vis photons
• Electronic transitions
• Π, σ, and n electrons
• d and f electrons
• Charge transfer reactions
• Π, σ, and n (non-bonding) electrons
hν
Formaldehyde

Electronic transitions
σ*
(anti-bonding) These are normally empty
Π*
n (non-bonding) These contain lone pair e-
σ
(bonding) These contain normal
Π bonding e-
Energy hν
Molecular orbitals

Electronic transitions
• σ → σ*
• UV photon required, high energy Alkanes -CH3
σ σ*
• Methane at 125 nm
• Ethane at 135 nm σ Π* Carbonyls
• n → σ*
• Saturated compounds with unshared e- Π Π* Unsaturated cmpds.
• Absorption between 150 nm to 250 nm
• e between 100 and 3000 cm-1 mol-1 n σ* O, N, S, halogens
• Shifts to shorter wavelengths with polar
n Π* Carbonyls
solvents
• Minimum accessibility

UV-Visible Spectroscopy: Instrumentation

Video of UV-Visible Spectro. working

Ultraviolet-Visible Spectroscopy
Instrumentation and working
1. Source- Two sources are required to scan the entire UV-Vis band:
• Deuterium lamp – covers the UV – 200-330 nm
• Tungsten lamp – covers the Vis 330-780 nm
2. Monochromator- As with the dispersive IR, the lamps illuminate the entire band of UV or
visible light; the Monochromator (grating or prism) gradually changes the small bands of
radiation sent to the beam splitter
3. Cuvette- The beam splitter sends a separate band to a cell containing the sample solution
and a reference solution
4. Detector- The detector measures the difference between the transmitted light through the
sample (I) vs. the incident light (I0) and sends this information to the recorder
3. Amplifier- Alternating current generated in the photocells is transferred to the amplifier.
4. The amplifier is coupled to a small servometer. Generally current generated in the photocell
is of very low intensity, the main purpose of amplifier is to amplify the signals many times
5. so we can get clear and recordable signals
6. Recording device- Most of the time amplifier is coupled to a pen recorder which is
7. connected to the computer. Computer stores all the data generated and produces the
8. spectrum of the desired compound

Functional groups and its max values
Compounds and its λmax values
Solvent λnm
Acetonitrile 190
Chloroform 240
Cyclohexane 195
1,4-dioxane 215
95% ethanol 205
n-hexane 201
Methanol 205
Isooctane 195
Water College of Engineering Pune (COEP) 190
Functional groups and its max values
1. Chromophores: functional groups that give electronic transitions.
Group Structure nm
Carbonyl >C=O 280

Azo -N = N- 262
Nitro -N=O 270
Thioketone -C =S 330
Nitrite -NO2 230
Conjugated Diene -C=C-C=C- 233
Conjugated Triene -C=C-C=C-C=C- 268
Conjugated Tetraene -C=C-C=C-C=C-C=C- 315

Red and Blue Shift
2. Auxochromes: substituents with unshared paire's like OH, NH,
SH, when attached to π chromophore they generally move the
absorption max. to longer λ.
3. Bathochromic shift: shift to longer λ, also called red shift.

4. Hypsochromic shift: shift to shorter λ, also called blue shift.
Hyperchromic
e Hypsochromic Bathochromic
Hypochromic
200 nm 700 nm

Applications
1. Detection of functional groups
2. Detection of extent of conjugation With the increase in double

bonds the absorption shifts towards the longer wavelength.
3. Identification of an unknown compound The spectrum of unknown

compound is compared with the spectrum of a reference compound.
100 1.0
(a)
80 0.8
4. Determination of Film Thickness
Transmittance (%)
Transmittance
Absorbance (t)
45 min.
60 60 min. 0.6
75 min.
40 0.4
Absorbance
0.2
20
0.0
5. Determination of the purity of a substance 300 400 500 600
Wavelength (nm)
700 800
The intensity of the absorption can be used for the relative calculation of the purity of the sample
substance. College of Engineering Pune (COEP)
UV-Vis. Spectra of ZnO, KMnO4 & Eg
αhν = A×(hν - Eg)n

Where,
α = linear absorption coefficient; h = Planck's
constant; ν = frequency of light; A = the
proportionality constant; Eg = gap energy;
n = 1/2 for direct band gap material.

Infrared Spectroscopy


Introduction
1. Electromagnetic radiation displays the properties of both particles and waves
2. The particle component is called a photon
3. The energy (E) component of a photon is proportional to the frequency. Where h

is Planck’s constant and n is the frequency in Hertz (cycles per second)
E = h
4. The term “photon” is implied to mean a small, massless particle that contains a
small wave-packet of EM radiation/light – we will use this terminology further

Introduction
5. Because the speed of light, c, is constant, the frequency, n, (number of cycles

of the wave per second) can complete at the same time, must be inversely
proportional to how long the oscillation is, or wavelength:
E = h where
h= 6.62607004×10-34 m2kg/s
c = 2.99 m×108 m/s
6. Amplitude, A, describes the wave height, or strength of the oscillation
7. Because the atomic particles in matter also exhibit wave and particle properties
(though opposite in how much) EM radiation can interact with matter in two
ways:
• Collision – particle-to-particle – energy is lost as heat and movement
• Coupling – the wave property of the radiation matches the wave property
of the particle and “couple” to the next higher quantum mechanical
energy level
The IR Spectroscopic Process

1. The quantum mechanical energy levels observed in IR spectroscopy are those of
molecular vibration
1. We perceive this vibration as heat
2. When we say a covalent bond between two atoms is of a certain length, we are
citing an average because the bond behaves as if it were a vibrating spring
connecting the two atoms
1. For a simple diatomic molecule, this model is easy to visualize

The IR Spectroscopic Process
There are two types of bond vibration:

• Stretch – Vibration or oscillation along the line of the bond
H H
C C
H H
symmetric asymmetric
• Bend – Vibration or oscillation not along the line of the bond
H H H H
C C C C
H H H
H
scissoring rocking twisting wagging
in plane College of Engineering Pune (COEP) out of plane
Video showing Instrumentation and working

Instrumentation and working
Filters: -Here filters are used as wavelength selectors, commercially available

for dedicated applications. For example, an instrument for determination of the quality
parameters of gasoline (Zeltex Inc.) employs 14 interference (Fabri-Perrot) filters.
LED as an IR light source :Using Light Emitting Diodes (LED) in the field, price and size of the
instrument can be reduced, produce NIR radiation with a band width of about 30-50 nm. LEDs
function as both the light source and the wavelength selection system, typically cover the range
400–1700 nm. They have the advantages that the measurement is very fast (e.g. one spectrum
per second) and noninvasive. These features are particularly useful where a high sample
throughput or ultra-rapid on-line measurements are required.

EXAMPLES OF APLICATIONS OF IR SPECTROSCOPY
❖ Thermal sensors – IR spectrophotometers are used in thermal detector for remote
body tem. sensing
❖ Soils – high-throughputs soil analysis, mapping and monitoring of soil properties,
remote sensing, digital soil mapping, precision agriculture, monitoring schemes for
good agricultural practice and environmental services payments schemes, sediment
analysis, soil pollution, mobile rural IR spectroscopy oil testing services.
❖ Crop agronomy/breeding/plant sciences –Tissue testing and crop response,
Germplasm
screening /breeding, seed viability/treatment, metabolomics –relating plant tissue
biochemical fingerprints to ecological factors
❖ Crop and livestock products quality and processing – grain quality and storage, cash
crops- tea and coffee etc, fruits and vegetables, Beverages and juice, Dairy products –
butter, cheese, Meat, Wood and paper, Biofuels.
❖ Water quality – Long term monitoring of aquatic systems and Heavy metals pollution
of freshwater sediments e.g. Cd, Cu, Zn, Pb , Mn, Fe in sediments.


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Instrumental Techniques of

Department of Applied Sciences

College of Engineering Pune (COEP)

College of Engineering Pune (COEP)

Instrumental Analysis: It is the study and use of instruments and methods to

College of Engineering Pune (COEP)

3 X-ray Diffraction Bragg’s law: nλ = 2dsinθ 1) Identification of the material.

College of Engineering Pune (COEP)

8 Voltammetry Constant and/or varying potential at 1) To determine the oxidation /reduction

11 Gravimetry Weight gain/loss after 1) To determine the composition of major

College of Engineering Pune (COEP)

Valence elec. Inner shell

College of Engineering Pune (COEP)

Principle of UV-Vis spectroscopy

Useful links to understand the working of UV-Vis. Spectroscopy.

College of Engineering Pune (COEP)

College of Engineering Pune (COEP)

A = log (I0/I) A = log (I0/I)

College of Engineering Pune (COEP)

College of Engineering Pune (COEP)

n (non-bonding) These contain lone pair e-

College of Engineering Pune (COEP)

College of Engineering Pune (COEP)

College of Engineering Pune (COEP)

College of Engineering Pune (COEP)

College of Engineering Pune (COEP)

Carbonyl >C=O 280

College of Engineering Pune (COEP)

3. Bathochromic shift: shift to longer λ, also called red shift.

College of Engineering Pune (COEP)

1. Detection of functional groups

2. Detection of extent of conjugation With the increase in double

3. Identification of an unknown compound The spectrum of unknown

αhν = A×(hν - Eg)n

College of Engineering Pune (COEP)

College of Engineering Pune (COEP)

College of Engineering Pune (COEP)

1. Electromagnetic radiation displays the properties of both particles and waves

2. The particle component is called a photon

3. The energy (E) component of a photon is proportional to the frequency. Where h

College of Engineering Pune (COEP)

5. Because the speed of light, c, is constant, the frequency, n, (number of cycles

The IR Spectroscopic Process

College of Engineering Pune (COEP)

There are two types of bond vibration:

• Bend – Vibration or oscillation not along the line of the bond

College of Engineering Pune (COEP)

Filters: -Here filters are used as wavelength selectors, commercially available

College of Engineering Pune (COEP)

College of Engineering Pune (COEP)

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