How do we know what the stars or the Sun are made of?

Isaac Newton during his time studied the properties of prisms and its ability of separating a

white light into the visible spectrum. Newton also prepared lenses to use in the telescopes.

Figure 1: Sir Isaac Newton

He observed that the light of celestial objects contains much information hidden in its

detailed colour structure.

Hot, glowing bodies like a light bulb, or the Sun glow. All these colours together appear as

white light. When this white light passes through a prism, or a raindrop, or a diffraction

grating, colours get separated according to their wavelength. Similarly rainbow is formed in

the sky when sunlight hits a raindrop.

Figure 2: Interaction of light with prism

Figure 3: A Telescope

In laboratory we can separate the light from some sources into constituent colours and

observe the chemical constitution of gases. The baseline is a laboratory experiment with

known materials. Results of these experiments can be compared later with the unknown

material’s results. Red, with its wavelength of 600 nm to 700 nm, ends up on one edge of the

spectrum and is deflected the least. Blue, wavelength is around 400 nm, is present at the other

end of the visible spectrum. Between these two edges, an infinite number of elementary

colours are located, each corresponding to its own wavelength. The same procedure is used

for starlight, telling us what its source is composed of.

Almost all that knowledge about planets, stars, and galaxies is achieved from studies of the

light received from them. When we are looking at a matter, we are not actually looking at the

matter but we are observing the interaction light’s with the molecule.

Figure 4: Interaction of Light

This study of interaction between light* and matter is called as Spectroscopy

Spectroscopy is defined as study of properties of matter through its interaction with

various frequency components of the electromagnetic spectrum.

OR

Spectroscopy is set of methods where interaction of electromagnetic radiation with chemical

molecules is measured to obtain its properties, characteristics and quantity.

Few terms are given below related to spectroscopy

Wavelength (λ) - length between two equivalent points on successive waves

Wavenumber (n) – the number of waves in a unit of length or distance per cycle - reciprocal

of the wavelength

Frequency (ν) – is the number of oscillations of the field per second (Hz)

Velocity (c) – independent of wavelength – in vacuum is 3.00 x 1010 cm/s (3.00 x 108 m/s)

Photon (quanta) – quantum mechanics nature of light to explain photoelectric effect

Wavelength (λ)= c/ν- Relations

Where: λ (cm); ν (Hz; s-1); c = light velocity

n is a symbol for wavenumber and is the reciprocal of the wavelength (λ)

therefore n= 1/λ when the wavelength is expressed in centimetres.

As a result

When wavelength λ = 2500 nm

wavenumber (n)= 1/ λ = 4000 cm-1

When wavelength = 50,000 nm

wavenumber (n) = 1/ λ = 200 cm-1.

Multiplication of n by the speed of light, 3×1010cm s-1, gives the frequency that is directly

proportional to the energy.

n×speed of light= frequency (ν)

Units used for wavelength:

Å = angstrom = 10-10 m

nm = 10-9m

μm = 10-6m

Interaction of light with matter causes the light to change its direction

Figure 5: Interaction of light with matter

Important terms

I0 Intensity of incident radiation

Itr Intensity of transmitted radiation

Iabs Intensity of absorbed radiation = (I0- Itr)

Light energy is associated with wavelength:

Long Wavelength is associated with Low Energy,

Short Wavelength is associated with High Energy

In spectroscopy different light frequency gives a different picture for particular molecule. It is

called as spectrum. Such spectra are due to the absorbance of electromagnetic radiation

energy by a sample.

A spectrum is a plot of measure of the absorption of electromagnetic radiation by a

sample versus the wavelength or energy of the electromagnetic radiation. For example, it

is general practice to plot the absorbance versus wavelength for spectra in the ultraviolet and

visible spectral regions as shown below (Fig. 6).

Figure 6: Typical format of absorbance versus wavelength, of ultraviolet and Visible

spectra

Ultraviolet, visible and infrared spectroscopy is the most commonly used spectroscopic

techniques today. Visible region is from 350 to 700 nm of the spectrum whereas ultraviolet

radiation is commonly defined as the wavelengths from 200 to 350 nm. Technically, the

infrared region starts immediately after the visible region at 700 nm. From 700 to 2500 nm is

the near infrared.

Figure 7: Visible region

The energies of infrared radiation range from 48 kJ mol_1 at 2500nm to 2.4 kJ mol_1 at

50,000 nm. As infrared radiation have low energies, they are not sufficient to cause electron

transitions but they are sufficient to cause vibrational changes within molecules. Therefore

Infrared spectroscopy is also called as vibrational spectroscopy.

General Types of Spectrum

There are two distinctive classes of spectrum –

Continuous spectrum and Discrete spectrum.

Figure 8: Types of Spectrum.

Continuous Spectrum

The light is composed of a wide, continuous range of colors (energies). Continuous spectra

arise when dense gases or solid objects radiate away its heat through the production of light.

In such case, objects emit light over a broad range of wavelengths, which resulting in

appearance of continuous spectrum.

Stars emit light in a predominantly continuous spectrum. Electric cooking stove burners,

flames, incandescent light bulbs, cooling fire embers and our body are the other examples.

Our body, emits a continuous spectrum, but the light waves we emitted by body lie at infrared

wavelengths. As we don’t have infrared-sensitive eyes, we cannot see people by the

continuous radiation they emit.

Discrete Spectrum

In Discrete spectrum we can observe only bright or dark lines of very distinct and sharply-

defined colors (energies).

There are two types of discrete spectra,

Emission (bright line) spectra

Absorption (dark line) spectra

Discrete spectrum with bright lines are termed as emission spectrum, and those with dark

lines are termed as absorption spectrum.

Emission Line Spectra

Each element on the periodic table has its own set of possible energy levels. These levels are

distinct and identifiable. Unlike continuous spectrum source, the electron clouds surrounding

the nuclei of an atom have very specific energies dictated by quantum mechanics. An atom

will always tend to settle to the ground state (i.e. lowest energy level) by releasing some

energy. An atom releases that energy by emitting a wave of light with that exact energy it

needs to release for reaching ground state.

In the diagram below, a hydrogen atom drops from the 2nd energy level to the 1st, giving out

a wave of light with an energy equal to the difference of energy between levels 2 and 1. Such

energy corresponds to specific wavelength of light or specific colour, thus we can see a bright

line at that exact wavelength.

Figure 9: Emission Line spectrum

An excited Hydrogen atom relaxes from level 2 to level 1, yielding a photon. This results in a

bright emission line.

These minute changes of energy in an atom generate photons having low energies and long

wavelengths, (eg. radio waves). Similarly, large changes of energy in an atom will emit high-

energy, short-wavelength photons (eg. UV, x-ray, gamma-rays).

Emission spectrum can provide the spectrum of each atom.

Absorption Line Spectra

If we fire photon back into a ground state atom, the atom can absorb these ‘specially-

energetic’ photons and would become excited. It will jump from the ground state to a higher

energy level. In this way, a dark-line absorption spectrum is born, as shown in the figure:

Figure 10: Dark-line absorption spectrum

eg. When a hydrogen atom in the ground state is excited by a photon of exactly the `right'

energy needed to send it to level 2, it will absorb the photon in the process resulting in a

dark absorption line

Such absorption spectrum is used in deducing the presence of elements in stars and other

gaseous objects which cannot be measured directly.

By comparing the absorption spectrum with the element’s emission spectrum, people can

build the spectrum of planets.

A spectrophotometer is an instrument employed to measure the amount of light absorbed by a

sample.

Important components of Spectrophotometer

Spectroscopy instrument (Spectrophotometer) has following components

Figure 11: Components of Spectrophotometer

1) Radiation sources

Most common are broad spectrum sources (Tungstun lamp, medium pressure mercury arc,

xenon arc) Particular wavelengths are isolated by means of filters or monochromators

Less common sources are those who emit only one or very few wavelenghts

eg: Low Pressure Mercury arc

Hollow cathode lamp (Atomic spectroscopy only)

Lasers (Some are tunable over narrow wavelength ranges)

2) Wavelength selector (Filter/ Monochromator)

Filters: Commonly used in cheap instruments, In Photometers, eg: HPLC detectors

Monochromators: almost always present in spectrophotometers

Prisms: rarely used now a days

Gratings

3) Detectors of Light Intensity

Detector should be sensitive to radiation of the desired wavelength. Detectors for each region

of the spectrum differ because of the unique properties of either the radiation itself or the

source of the electromagnetic radiation. Light sources produce plentiful amounts of photons

in the visible region and the photon energy is sufficient so that a simple phototube or

phototransistor will generate enough electron flow to measure. In the ultraviolet region of the

spectrum, the available light sources produce relatively few photons when compared to the

visible light sources. Therefore, measurement of ultraviolet photons uses a special

arrangement of a phototube called a photomultiplier to obtain a measurable electrical current.

It is not difficult to generate sufficient photons in the infrared region but the photons

produced are of such low energy that devices to rapidly measure infrared radiation have just

been recently developed.

Phototube: It is used for Photoionization of the cathode, provided that radiation has

sufficient energy. Emitted electrons migrate to the anode and current is measured. Each type

has its own long cut off depending on ionization energy of the cathode.

Photomultiplier tube

Diode array Detector

Photovoltaic detector

4) Amplifier / readout

Spectroscopy can be divided into 2 types based on the analysis

Figure 12: Types of Spectroscopy

Quantitative Analysis: Quantitative analysis refers to the determination of how much of a

given component is present in a sample. The quantity may be expressed in terms of mass,

concentration, or relative abundance of one or all components of a sample.

Methods

Ultraviolet Spectroscopy

Figure 13: Components of UV Spectrophotometer

Figure 14: UV spectrophotometer

All atoms absorb in the Ultraviolet (UV) region because these photons are energetic enough

to excite outer electrons. If the frequency is high enough, photoionization takes place. UV

Spectroscopy is also used in quantifying protein and DNA concentration as well as the ratio

of protein to DNA concentration in a solution. Several amino acids usually found in protein,

such as tryptophan, absorb light in the 280 nm range and DNA absorbs light in the 260 nm

range. Due to which, the ratio of 260/280 nm absorbance is considered as a good indicator of

relative purity of a solution in terms of these two macromolecules. Reasonable estimates of

protein or DNA concentration can be made with the help of Beer's law.

Visible spectroscopy

Many atoms emit or absorb visible light. In order to obtain a fine line spectrum, the atoms

must be in a gas phase. This means that the substance has to be vaporised. The spectrum is

studied in absorption or emission. Visible absorption spectroscopy is often combined with

UV absorption spectroscopy in UV/Vis spectroscopy. Although this form may be uncommon

as the human eye is a similar indicator, it still proves useful when distinguishing colours.

(UV-Visible) Spectroscopy: -

UV-visible spectroscopy is used primarily to measure liquids or solutions. This mode is

simpler and allows more accurate quantitative analysis. 95% of all quantitative analysis in

health care field is done by UV-Vis Spectroscopy. This technique is widely used in organic

and inorganic analysis. The data acquisition is easy and accurate.

Tools and techniques in biochemistry (Ref)

The ground state (or ground electron state) is refers to the lowest energy configuration of the

atom or molecule (electron filling AOs or MOs from the lowest energy in order)

An Excited State refers to any electron configuration other than the ground state

In Absorption Spectroscopy, a valence electron is promoted to higher energy atomic or

molecular orbiltal. The “amount” of light absorbed from an incident beam is monitored.

In Emission Spectroscopy, a pre-excited valance electron drops into a lower enegy atomic or

molecular orbital. The intensity of emitted light is monitored.

Virtually all the atoms and molecules exist in their ground electronic state at room

temperature. Therefore absorption spectroscopy almost always involves transition from

ground state to an excited state to the ground state.

Qualitative Analysis : in this the nature of the chemical species in a sample is determined.

Qualitative analysis can tell us whether a perticular atom, ion, or compound is present or

absent in a sample, but it does not provide information about its quantity of that species.

Methods

Infrared Spectroscopy

Infrared Spectroscopy is based on absorption of infrared light. Absorption excites molecular

vibration and rotation, which have frequencies within the infrared range. Infrared

spectroscopy (IR spectroscopy or Vibrational Spectroscopy) deals with the infrared region

of the electromagnetic spectrum. IR is the light with a longer wavelength and lower

frequency than visible light. This technique is mostly based on absorption spectroscopy.

Beer-Lambert law relates the absorption of IR light to the properties of the material through

which the light is traveling

Figure 15: Components of IR Spectrophotometer

Infrared spectrometer (or spectrophotometer) is used to produce an infrared spectrum. It is

used to indicate the ionic character in an molecule.

Units of IR frequency = reciprocal centimetres, symbol cm−1 (sometimes called wave

numbers),

Units of IR wavelength = micrometers, symbol μm

Instrument= A common laboratory instrument using this technique is Fourier transform

infrared (FTIR) spectrometer

Fourier transform infrared (FTIR) spectroscopy is a measurement technique for recording

infrared spectra.

We can see working of IR spectrophotometer from Figure.15.

A data-processing technique, Fourier transform converts this raw data into the sample's

spectrum. The sample's spectrum is always compared with a reference.

Two interferograms are produced:

1) With the sample in the light beam

2) Without a sample in the light beam (Reference)

The final transmission spectrum is obtained by dividing the sample spectrum by the reference

spectrum

Results:-

Figure 16: Examples of FTIR Microspectroscopy Interferograms

Advances in IR Sectroscopy:

FTIR microspectroscopy is IR spectrometer in combination with a microscope facility. It

facilitates study of very minute samples (5-10 μm).

• FTIR microspectroscopy is useful in obtaining 2D or 3D “chemical image” of a sample

At a time thousands of interferograms can be collected and transformed into infrared spectra.

It can work in transmission and reflection (ATR) modes

A basic IR spectrum is essentially a graph of infrared light absorbance (or transmitted) on the

vertical axis vs. frequency or wavelength on the horizontal axis.

Figure: 17: Example of IR Results

Figure 18: IR spectra interpretation

The mid-infrared spectrum (4000-400 cm-1) can be approximately divided into four regions:

1. X-H stretching region (4000-2500 cm-1)

2. Triple-bond region (2500-2000 cm-1)

3. Double-bond region (2000-1500 cm-1)

4. Fingerprint region (1500-600 cm-1)

1. X-H stretching region (4000-2500 cm-1)

Ø O-H stretching à 3700-3600 cm-1

Ø N-H stretching à 3400-3300 cm-1

Ø C-H stretching à 3100-2850 cm-1

2. Triple-bond region (2500-2000 cm-1)

Ø C≡C bonds à 2300-2050 cm-1

Ø C≡N bonds à 2300-2200 cm-1

3. Double-bond region (2000-1500 cm-1)

Ø C=O bond à 1830– 1650 cm-1

Ø C=C stretching à ≈1650 cm-1

Infrared spectroscopy measure different types of inter atomic bond vibrations at different

frequencies. With the help of IR absorption spectra type of bonds are present in an organic

sample can be determined. Can analyse polymers and constituents like fillers, pigments and

plasticizers.

Near Infrared (NIR)- IR spectroscopy

The near infrared NIR range, Much greater penetration of NIR radiation into the sample than

IR spectroscopy range. Currently employed for practical applications such eg- imaging of

intact organisms, medical diagnosis pharmaceuticals/medicines, proteomic analysis,

biotechnology, food analysis genomics analysis, interactomic research, and chemical

imaging, plastics, textiles, insect detection, forensic lab application, crime detection, various

military applications, and so on.

RAMAN Spectroscopy

In RAMAN Spectroscopy, the sample is irradiated by intense laser beams of UV-visible

region resulting in scattering of light. In Raman spectroscopy, the vibrational frequency (νm)

is measured as a shift from the incident beam frequency (ν0 )

Such scattering light consists of

Rayleigh scattering: Rayleigh scattering is Strong

It’s frequency is same as the incident light

Rayleigh is filtered out from the signal

Raman scattering: Raman scattering is very weak (~10-5 of the incident light)

It has frequencies v0 ± vm

v0 - vm is called the Stokes line

v0 + vm is called the anti-Stokes line

Figure 19: RAMAN Spectrophotometer Instrumentation

Figure 20: RAMAN Spectrophotometer

Significance of Raman Spectroscopy

• Easy identification of chemical structure, Indicating covalent character in an

molecule.

• It is flexible, simple, sensitive and fast

No sample preparation, non invasive, non destructive method

• Scattering in general is dependent on the frequency of the excitation radiation to the

fourth power

• Stokes and Anti-Stokes scattering are related to the population in the ground state and

the first excited vibrational level

• Applicable for analysis of wide range of material solid or liquid

• Applicable in Pharmaceuticals, biomedicals, material science, nanotechnology,

forensic/ anti crime/ anti terrorism field, gemology, mineralogy, Archeology, art,

heritage

Scattering in general is dependent on the frequency of the excitation radiation to the fourth

power

Stokes and Anti-Stokes scattering are related to the population in the ground state and the

first excited vibrational level

Rayleigh scattering is about 105 times stronger than Raman scattering

Nuclear Magnetic Resonance

Figure 21: NMR Instrumentation

NMR is the most powerful tool to determine organic structure

Nuclei of atoms have magnetic properties. This phenomenon can be used to revile the

chemical information. Subatomic particles like protons, neutrons and electrons have spin. The

spinning charged nucleus generates a magnetic field. To determine the spin of a given

nucleus one can use the following rules:

If the number of neutrons and the number of protons are both even, the nucleus has no spin. If

the number of neutrons plus the number of protons is odd, then the nucleus has a half-integer

spin (i.e. 1/2, 3/2, 5/2). If the number of neutrons and the number of protons are both odd,

then the nucleus has an integer spin (i.e. 1, 2, 3).

When such nucleus is placed in an external field, spinning protons act like bar magnets.

A photon with the right amount of energy can be absorbed and cause the spinning proton to

flip.

If all protons absorbed the same amount of energy in a given magnetic field, information

could be obtained.

But protons are surrounded with electrons which shield them from an external field.

Circulating electrons create an induced magnetic field that opposes the external magnetic

field

If we irradiate the sample with radio waves (in the MHz frequency range) the proton will

absorb the energy and be promoted to the less favorable higher energy state. This energy

absorption is called resonance because the frequency of the applied radiation and the

precession coincide or resonate.

Application of NMR

Solution structure The only method for atomic-resolution structure determination of

biomacromolecules in aqueous solutions under near physiological conditions or membrane

mimeric environments.

Molecular dynamics The most powerful technique for quantifying motional properties of

biomacromolecules.

Protein folding The most powerful tool for determining the residual structures of unfolded

proteins and the structures of folding intermediates.

Ionization state The most powerful tool for determining the chemical properties of

functional groups in biomacromolecules, such as the ionization states of ionizable groups at

the active sites of enzymes.

Weak intermolecular interactions Allowing weak functional interactions between

macrobiomolecules (e.g., those with dissociation constants in the micromolar to millimolar

range) to be studied, which is not possible with other technologies.

Protein hydration A power tool for the detection of interior water and its interaction with

biomacromolecules.

Hydrogen bonding A unique technique for the DIRECT detection of hydrogen bonding

interactions.

Drug screening and design Particularly useful for identifying drug leads and determining

the conformations of the compounds bound to enzymes, receptors, and other proteins.

Native membrane protein Solid state NMR has the potential for determining atomic-

resolution structures of domains of membrane proteins in their native membrane

environments, including those with bound ligands.

Metabolite analysis A very powerful technology for metabolite analysis.

Chemical analysis A matured technique for chemical identification and conformational

analysis of chemicals whether synthetic or natural.

Material science A powerful tool in the research of polymer chemistry and physics.

Mass Spectroscopy

Applications

• Can be used for qualitative and quantitative analysis.

• Used to identifying unknown compounds from various samples

• To determine isotopic composition of elements in a molecule,

• To determining the structure of a compound.

• Use to identify and quantify the amount of a compound in a sample

• To study the chemistry of ions and neutrals in a vacuum.

• Use to study physical, chemical, or biological properties of a great variety of

compounds

Figure 22: Mass Spectroscopy instrumentation and model

Uses of spectroscopy

To understand how light interaction of light with the matter,

For quantitative estimation of various compounds in an known or unknown sample.

Generalization for the rage 200-800nm absorption by various groups of compounds:

Simple alkanes, monoalkenes, alcohols, ethers, amines, halides etc. absorb very

Poorly (in fact they are good solvents for UV-visible Spectrometry)

Polyunsaturated compounds such as dienes, trienes, unsaturated ketones and

aromatics absorb strongly

Simple carbonyl compounds such as aldehydes and ketones absorb weakly

Transition metal ions absorb weakly in the visible range

Metal complexes often absorb strongly in the UV range

Photometer:- can operate at one or more fixed wavelengths and are used exclusively

for quantitative analysis eg: HPLC detector

Spectrophotometers:- Are capable of scanning trough wavelength to record spectrum.

They are more versatile as a) can obtain qualitative and quantitative information

b) can quantify any desire wavelength

To monitor the changes in energy states of a molecule.

Refs-

Osterberg EC, Laudano MA, Li PS. Clinical and investigative applications of Raman

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