18-01-2021

BASICS OF SPECTROSCOPY
Prepared by
Dr. Kashyap N. Thummar
Assistant Professor,
Graduate School of Pharmacy,
Gujarat Technological University,
Mob; 9427979959
Email: ap_kashyap@[Link]

Analytical Methods

■ There are two types of Analytical method

A. Non-Instrumental methods – Physicochemical Properties

B. Instrumental Methods – Physical Properties

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Non-Instrumental/Classical Methods
■ Classical methods/ wet chemical methods/ earliest methods of
analysis; relied mainly on chemical properties of analytes.
■ Analytes are treated with reagents to form products that could be
identified. Gravimetric and titrimetric methods were used for
quantitative analysis.
■ Examples:
– Formation of precipitate and measurement of mass
– Oxidation of analyte and detection of end via the change of color
of the analyte.
– Neutralization of analyte and detection of end point using acid-
base indicator.
– Complexation of analyte and use of metallochromic indicators to
detect end point.
■ Separation of analyte from matrix was achieved using precipitation,
extraction and distillation. 3

Instrumental Methods

■ Instrumental methods largely rely on physical properties of

analyte, thus generally are not destructive.
■ They are not necessarily more sensitive.
■ Methods for the qualitative and quantitative analysis of
analytes

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Instrumental Methods
Characteristic Properties Instrumental Methods

Electric Potential Potentiometry

Electrical resistance Conductometry
Electrical current Amperometry
Absorption of radiation U.V., Visible & IR, NMR, ESR
Emission of radiation Emission spectroscopy (Fluorescence, Phosphorescence
and Luminescence)
Scattering of radiation Raman spectroscopy, Turbidimetry, Nephelometry
Diffraction of radiation X-ray
Rotation of radiation Polarography, Optical rotatory dispersion, Circular dichroism
Mass to charge ratio Mass spectrometry
Refraction of radiation Refractometry
Thermal methods Thermal conductivity, Gravimetric analysis, and enthalpy
Radioactivity Activation and isotope dilution methods
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Spectroscopy
■ It is a instrumental method of analysis that deals with the
study of the interaction of EMR and Chemical analysis.
spectrum – Band of different color
■ Spectroscopy
Skopein – Examination/ To observe

 In the 17th century the word spectrum was introduced into

optics by Isaac Newton, referring to the range of colors
observed when white light was dispersed through a prism.

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Spectrum
■ When a narrow beam of light is allowed to pass through a
prism/grating, it is dispersed or produce into various band
of colors called spectrum

Electromagnetic radiation (EMR)

■ It can be described as a wave occurring simultaneously in
electric and magnetic fields, propagating (radiating) through
space carrying electromagnetic radiant energy.

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Electromagnetic spectrum
■ It is an arrangement obtained by arranging various type of
electromagnetic waves or radiation in order of their increasing or
decreasing frequency called Electromagnetic spectrum.

Region of Electromagnetic spectrum

Region Wavelength, λ Frequency, v, Wavenumber, cm-1
Cosmic rays 5 x 10-5 nm
Gamma rays (ϒ) 10-3 x 0.14 nm
X-rays 0.01 - 15 nm
Far Ultra violet 15 – 200 nm 6,66,667 – 50,000
Near Ultra violet 200 – 400 nm 50,000 – 20,000
Visible 400 – 800 nm 25,000 – 12,500
Near infrared 0.8 – 2.5 µ 12,500 – 400
Vibrational IR 2.5 – 24 µ 4000 – 400
Far IR 0.025 – 0.5 mm 400 – 200
Microwave 0.5 – 300 mm 200 – 0.033
Radio wave 0.3 – 109 m
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Properties of EMR – Wavelength (λ)

■ It is a distance between two successive peaks of wave or crest
is called Wavelength.
■ The wavelength of EMR are expressed in either meters (m),
millimeters (mm), micrometers (µm), or nanometers (nm).

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Frequency (v)
■ The radiation may also be characterized by its frequency (v).
■ It is defined as the number of complete cycle per second or
the number of waves passing through point in one second is called v.
■ Unit : cycle per second, also called Hertz (Hz)

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Wavenumber ( v )
■ Number of waves spread in a unit distance(cm).
■ Wavenumber is reciprocal of wavelength.

■ 𝑉= where v = wavenumber, λ = wavelength (m, cm, mm, µm or nm)

■ Hence the unit is cm-1.

■ It is more frequently used in Infra red spectroscopy.

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Energy (E, ΔE)

■ It is an expression of energy per single Photon or energy per mole of photon.
■ Its unit : Calories, Joules, erg, Ev

■ Equation; 𝐸 =
where, E = energy
h = plank’s constant
c = speed of light
λ = wavelength

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Relation between E and v

■ Since, = v, where v = frequency,
■ Hence, the photon energy can simplified to
E = hv
■ This equation known as planck – Einstein
relation.

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Most important things to remember

■ 𝐹𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝛼

■ 𝐸𝑛𝑒𝑟𝑔𝑦 𝛼

■ As energy increases the wavelength decreases,

■ As wavelength increases the energy decreases.

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Theory
 Reflection is the process by which
electromagnetic radiation is returned either at
the boundary between two media (surface
reflection) or at the interior of a medium
(volume reflection)

 Transmission is the passage of

electromagnetic radiation through a medium.

 Scattering, which is the process of deflecting

a unidirectional beam into many directions.

 Absorption is the transformation of radiant

power to another type of energy, usually heat,
by interaction with matter.
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Absorption and Transmission

■ The spectrum of a compound represent a graph of wavelength versus
Absorbance (A) or percent Transmittance (%T)
■ The %T is the percent of intensity of original radiation which pass
through the sample.
■ %𝑇 = × 100

■ When a compound does not absorb any radiation at a particular

wavelength, % T = 100 at that wavelength.
■ Absorption of radiation at a particular wavelength leads to a decrease
in the %T to appear in the spectrum as dip, called peak or absorption
band.
■ Absorbance is measure of the absorption of radiation by a sample:
■ 𝐴 = log , In this case, Increase in absorption
appears as increase of the signal. 18

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Relation between Absorption and Transmission

■ Iabsorbed = I0 – IT
■ I0 = original intensity
■ IT = reduced intensity transmitted
■ Transmittance (T) =

■ %T = x 100

■ Absorbance (A) = log

■ A = log ( )
■ A= −𝑙𝑜𝑔𝑇
■ A = 2 − log %𝑇

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UV Spectrum/spectra….
 A Spectrum is simply plot of the amount of light which is absorbed versus the wavelength of
light.

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Principle of spectroscopy – Electronic transition

■ Different type of electronic transition…
■ An organic molecule contain three types of electron eg,
– Sigma (σ),
– Pi (π) and
– non bonding electron (n).

■ All saturated compound like - Alkanes

– Ethane
– Methane
Contain only Sigma (σ) bonds and therefore only Sigma (σ) electron

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■ Unsaturated compound like

– Alkynes
– Alkenes
– Aldehydes containing triple or double bond
Contain Sigma (σ) and Pi (π) bonds therefore they have Sigma (σ) as
well as Pi (π) electron

■ Non bonding electron (lone pair of e- , Valence e-)

– The pair of valence electron that are not shared with other atoms.
– Molecular having electronegative element such as Nitrogen,
Oxygen, Sulphur and Halogen etc.

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■ For eg. Acetaldehyde CH3CHO

σ
σ
σ

σ n
π

 On absorption of energy by molecule in the ultraviolet region,

changes are produced in the electronic energy of the molecule due
to transition of valence electron in the molecule.

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Electronic transition
■ These transition consist of the excitation or an electron from occupied molecular
orbital,

Thus, the promotion of an electron, e.g.

Non An anti σ σ*
boding n bonding, π π*
or π* and
bonding σ*, σ*
Energy

π orbital orbital n
π*

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σ σ*
■ A transition of a electron from a bonding sigma orbital to the higher energy
antibonding sigma orbital is designated σ σ*.
■ In alkanes contains only σ bonds, this is only transition available. For eg, Methane,
Ethane.
■ In general, σ bonds are very strong, this is therefore is a high energy process and
these transition require very short wavelength.
■ For eg. High energy UV light 100 nm – 200nm.
■ A study of such transition is done in vacuum UV region since below 200nm Oxygen
present in to the air begin to absorb.
■ So, In ordinary spectrophotometer we never seen σ σ* transition.

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n σ*
■ This transition involves saturated compound with one hetero atom
with unshared pair of electron (n electron)
■ For example; Saturated halides, Alcohol, ethers, aldehydes, ketones,
amines, etc.
■ This transition required comparatively less energy than σ σ*
transitions.
– H20 = 167 nm
– MeOH = 174 nm
– CH3Cl = 169 nm Due to greater electronegativity of Cl than I, the n electron on Cl
atom are difficult to excite hence more energy is required to
– CH3I = 258 nm excite therefor…
Moreover n electron on Iodine atom are loosely bound.

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■ In presence of polar solvent such as Water of Ethanol the non bonding pair of an
analyte interact with electropositive atom of solvent, it is formation of H band.
■ Due to formation of H bond, some energy will be released hence, system becomes
more stable.
■ Therefore, the more polar solvent will become more stable system, so to excite this
higher energy is required, so shorter wavelength is used.

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π π* (K – Band)
■ A transition of a electron from a bonding π orbital to the
higher energy antibonding π orbital is designated π π*.
■ This transition is available in compounds with unsaturated
centres.
■ For example; simple alkene, aromatics, carbonyl
compounds, etc.
■ This transition required lesser energy then n σ*
transition.
■ Simple alkene
Simple alkene Structure λmax
Ethene CH2 = CH2 171 nm
1,3 – Butadiene CH2 = CH – CH = CH2 217 nm
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n π* (R – Band)
■ In this transition, an electron of unshared electron pair on a
hetero atom is excited to π* anti bonding orbital.
■ This transition involves least amount of energy than all the
transitions and therefore, this transition gives rise to an
absorption band at longer wavelength.
■ For example, Saturated aliphatic ketones,
– n π* transition around 280 nm

■ This transition is forbidden transition, thus the intensity of

the band due to this transition is low, although the
wavelength is long.

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Terminology in UV Spectroscopy
Chromophore Auxochrome
• It is a Covalently unsaturated • It is a saturated group containing
group responsible for electronic unshared electrons which when
absorption attached to chromophore changes
• Groups which gives rise to both the intensity as well as
electronic absorption are termed wavelength of the absorption
chromophore maximum.
For eg.-OH, -COOH, -RNH2 , -CONH2
For eg. C = C, C = O and NO2
i.e. Ethene, benzene, and acetone

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■ Red shift: when absorption maximum moves

to longer wavelengths (lower energy) is called
Hyperchromic Shift as Red shift or Bathochromic shift.
■ Blue Shift: when absorption maximum moves
Blue Shift to shorter wavelength (higher energy) is called
Red Shift as Blue shift of Hypsochomic Shift.
■ Hyperchromic shift: Increase in conjugation
usually results in increased intensity, termed
as Hyperchromic shift.
■ Hypochromic shift: A decrease in intensity of
Hypochromic Shift an absorption band is termed hypochromic
shift.

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Absorption law

= 0

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Lambert’s law – depend on path length (b)

When monochromatic radiation is pass through transparent
medium, the decreasing in the intensity of radiation with
increasing pathlength is directly proportional to the intensity of
incident radiation.

A= 0

A=
Where,
a = absorbtivity or Specific absorbance, or absorbance index or
extinction coefficient
b = Pathlength 33

Beer’s law – depend on Concentration(c)

When monochromatic radiation is pass through transparent
medium, the decreasing in the intensity of radiation with
increasing Concentration is directly proportional to the intensity of
incident radiation.

A= 0

A=
Where,
a = absorbtivity or Specific absorbance, or absorbance index or
extinction coefficient
c = Concentration 34

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Lambert - Beer’s law – depend on both (b) & (c)

When monochromatic radiation is pass through transparent
medium, the decreasing in the intensity of radiation with
increasing pathlength and Concentration is directly proportional
to the intensity of incident radiation.

A= 0

A=
Where,
a = absorbtivity or Specific absorbance, or absorbance index or extinction
coefficient
b = Pathlength
c = Concentration 35

Absorptivity (a) and molar absorptivity (∈)

■ A = 𝑎bc
■ a= ,
Molar absorptivity(∈ ):
the absorbance for a molar concentration of a substance with a
path length of 1 cm determined at a specific wavelength. Its
value obtained by…
Am = ∈ bc
∈=

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The fundamental of instrumentation

Five main component of UV instrumentation

1. A source of EMR
2. A monochromator (to isolate particular W.L.
3. A sample compartment
4. A detector (to measure the intensity of EMR)
5. A Recorder 37

1a. Source of UV radiation

■ It is important that power of radiation does not change over its wavelength range
■ The electronic excitation at deuterium or hydrogen at low pressure produce
continuous UV spectrum
■ The mechanism involves formation of excited molecular species, which breaks upto
give two atomic & UV photon
D2 + Electrical energy  D2*  D’ + D”+ hv
■ Both D2 and hydrogen lamp emit radiation in the range of 160 – 380 nm
■ Quartz windows must be used in these lamp because glass absorbs radiation of
W.L. less then 350nm.

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1b. Source of visible radiation

■ Tungesten filament lamp is used for Visible radiation
■ Lamp used in this having a range of 350 – 2500nm
■ Glass cuvette is used because the glass material is transparent to visible light.

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2. Monochromator
■ All monochromator contain following component
1. Entrance slit
2. A collimating lense
3. A dispersing device (Prism or grating) a. Grating monochromator
4. A focusing lense
5. An exit slit

Purpose:
To resolved radiation into its
component W.L. to permit the
isolation of any desired b. Prism monochromator
portion of spectrum
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Monochromator -
■ Polychromatic light to monochromatic light
■ Increase the resolution of band width
■ Slit width: Wavelength range of light pass through slit is called slit width.
■ Two types slit
1. Entrance slit
2. Exit slit
Both have a same slit width

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Slit width
1. Spectral slit width (SSW)
It is the total range of wavelength transmitted to pass through slit.
2. Effective slit width (ESW) or nominal slit width
It is the wavelength range pass through slit over which the transmission of
radiation is 50 % of its maximum value.
3. Theoretically SSW is twice of ESW.

Purpose:
To control the spectral resolution of
the monochromator, thus ability to
separate closed wavelength
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A dispersing device
1. Prism
■ When beam of monochromatic light passed through prism ,
it is bent or refracted.
■ The amount of deviation is dependent on the wavelength
■ If polychromatic light leads to separation of different
wavelength to form a spectrum, from which the required
wavelength may be selected for spectrometric
measurement.
■ Prism is made of Quartz for use in UV region, Glass Prism
used for visible region
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A dispersing device
2. Grating
■ The dispersing element in the monochromator of most
modern UV/Visible/IR – Diffraction grating
■ It consist of very large no. of equispaced lines (grooves)
■ Increase the grooves – increase the resolution

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Detector
■ Generally there are four detectors are used
1. Barrier layer cells or photovoltatic cells
2. Photoemmisive tubes
3. Photomultiplier tubes
4. Photodiode array detector

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1. Barrier layer cell or photovoltaic cells

■ One of the simplest detector, which has produce current under suitable condition, is
directly proportional to the light intensity.
■ Sensitivity is similar to human eye.
■ Useful for working in the range of 380 – 780nm hence lies in visible region.
■ Lack of sensitivity, compared to other detector so it is used in cheapest colorimeter and
flame photometer.

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2. Photoemmisive tube

■ It consist of semicylindrical cathode and wire anode sealed inside in evacuated

transparent envelop.
■ Cathode contains photoemmisive material –
– alkali metals ; caesium, Potassium
– alkali metal oxides; caesium oxide, Silver oxide

 Photoemmisive surface – emit electron when irradiate light

 This electron have potential to reaches to anode hence, photocurrent is produced.
 The number of electron ejected from the PET surface is directly proportional to
radiant power of light.

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3. Photomultiplier tube (PMT)

■ Cathod surface is similar to phototube
■ It also contain additional electrodes known as dynodes
■ So greater sensitivity achieves.
■ Electron from photo cathode are attracted to anode 1 and liberate more electron
which will travel to anode 2 because of higher potential relative to anode 1.
■ Process continues to last anodes results in a final photocurrent 106 – 108 greater
than primary current.
■ It is ideal for weak light intensity.

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[Link] array detector

■ The diode is charged to preset potential by continuously scanning of electron beam

■ PMT and PET – are single channel detector
■ PDA – Multiple channel detector
■ It’s a very sensitive detector
■ Generally used in HPLC.
■ Expensive detector

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General Application of UV spectroscopy

■ The UV Spectroscopy is largely limited to conjugated system.

■ For practical
– Visual range – 750 nm (red) to 400 nm (violet)
– Ultraviolet (near UV) – 200 – 400 nm (near UV)
– Far UV – 100 – 200 nm (vacuum UV)
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a. Extent of conjugation
■ The longer conjugation – longer maximum wavelength
■ Sufficient conjugation shifts the absorbance to wavelength
which will reach the visible region.
■ A compound with sufficient conjugation becomes colored.

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b. Distinction between conjugated and non conjugated compounds

■ Electronic spectroscopy can differentiate – conjugated dienes to non-
conjugated dienes/ triene and α-β- unsaturated analogues (carbonyl group
- ketone, aldehyde, etc..)
■ Compounds can have more than one double bond or triple bond
■ If they are separated by only one single bond they are conjugated and
their orbitals interact
■ The conjugated diene 1,3 – butadiene has properties very different from
those of the non conjugated diene, 1,5 - pentadiene

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c. Detection of chromophore from unknown compound

■ If it is unknown compound, Comparison of its spectrum with that of
known compound Similar spectrum.
■ Absorbance near UV at above 200nm indicate the presence of
saturated group or atoms with unshared pair of electrons.
■ The saturated hydrocarbon which donot have this structural elements
absorbs below 200nm. Hence, it is not significant for organic
compounds.
■ If UV spectrum of small known molecule is comparable with an
unknown large molecule, the two may have same chromophore.

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d. Identification of Chromophore (Functional group)

■ This function is more used in IR for functional group identification.
■ In UV, to know how intense the absorbance is  єmax is reported.
■ The values varies in the range of (very weak to very strong intensity)
10 - 10000 єmax

Functional group єmax

Low transition probability (forbidden < 1000
transition)
Simple conjugated chromophore, i.e., 10000 – 20000
dienes, α-β- unsaturated analogues
Aromatic compound (always) 1000 – 10,000
Aromatic conjugation Above 10,000

■ Hence, The molar absorptivity is high, intensity is also high, vice versa.

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Forbidden vs Allowed transition

Forbidden transition Allowed transiton
єmax 10 – 100 єmax 104
Excitation of one e- from the lone Π  π*
pair present in hetero atom to an
antibonding orbital
n  π*
Near 300nm near 200nm

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e. Study of geometric isomerism

■ The trans isomer absorb at a longer wavelength and with a larger molar extinction
coefficient than Cis isomer.

■ In cis structure due to repulsion (because of both group are present in same plane)
there will be increase energy as well as stability.
■ So, It will required high energy to excite.
■ In trans, both group are in different plane so, it have less energy and required low
energy to excite.
■ So, λmax of cis is less than trans isomers.

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Other applications are…

f. Study of structural features in different solvent
g. As an analytical tool
- reaction rates, pKa values etc.
h. Study of strains
i. Qualitative analysis
j. Quantitative analysis
k. Multicomponent analysis

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Woodward rule

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Calculation of UV absorption Wavelength in conjugated Dienes

and Trienes
Structure π π* Transition (K – band)
λmax
Acyclic and Heteroannular dienes 214 nm

Homoannular dienes 253 nm

Addition for each substituent

- R alkyl (including part of a carbocyclic 5 nm

ring)

- OR alkoxy 6 nm

- Cl, -Br 5 nm

-CH=CH (extended conjugation) 30 nm

If one double bond is exocyclic to one ring 5 nm

If exocyclic to two rings simultaneously 10nm

Rules for α, β- Unsaturated Aldehydes and Ketones (carbonyl

componds)
Structure π π* Transition (K – band) λmax

Ketones 215 nm
β α
l l
---C =C –CO--- , Acyclic or 6 ring cycle, 5 ring cycle 202 nm
Aldehydes 210 nm
β α
l l
---C =C –CHO---
Acids and Esters 197 nm
β α
l l
---C =C –CO2H(R)---
Extended conjugation Add 30 nm
δ γ β α
l l l l
---C =C ---C =C –CO---

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Rules for α, β- Unsaturated Aldehydes and Ketones (carbonyl componds)

Structure π π* Transition (K –
band) λmax
Homodiene component Add 39 nm
If one double bond is exocyclic to one ring 5 nm
If exocyclic to two rings simultaneously 10nm

Thank you

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