Open Elective

1. UNIT-I
INTRODUCTION OF SPECTROMETRY
PART-A
1. Discuss instrumental method of analysis?
Instrumental method is a technique based on instrument which converts chemical
information to a form that is more observable. It plays an important role in the
production and evaluation of new products and in the protection of consumers and the
environment.
2. What is a read out device?
It is a transducer that converts information from an electrical domain to a domain that is
understandable by a human observer.
3. What is the advantage of instrumental methods over chemical methods? (Nov/Dec
2015)
 A small amount of a sample is needed for analysis.
 Determination by instrumental method is considerably fast.
 Complex mixture can be analyzed either with or without their separation.
 Sufficient reliability and accuracy of results are obtained by instrumental method.
 When non-instrumental method is not possible, instrumental method is the only
answer to the problem.
4. What are the four basic functions of instrumental analysis?
The four basic function of instrumental
method of analysis are
 Generation of a signal
 Signal transduction
 Amplification of the transformed signal and
 Presentation of signal.
5. What are the basic functions of Instrumentation?
Instrument may be regarded as a communication device which is accomplished by several
steps such as
 Generation of a signal
 Transformation of a signal to one of a different nature
 Amplification of a transformed signal
 Presentation of the signal as a displacement on a scale or on
the chart of the recorder.
6. Classify the instrumental techniques?
Most instrumental techniques are divided into three principal areas.Spectroscopy,
Electrochemistry, andChromatography.
7. Define instrumental method of analysis?

2. This is a newer methods use for separating and deterring chemical species.
Example: chromatography and electrophoretic techniques used to replace
distillation, extraction and precipitation for the separation of components of
complex mixtures prior to their qualitative or quantitative determination.
8. What is the basis of instrumental methods?
Instrumental methods are based on the theory of relations between the content and
the corresponding physico-chemical and physical properties of the chemical system being
analyzed.
9. Classify the sources of noise in instrumental analysis?
 Chemical noise
 Instrumental noise
 Thermal noise or Johnson noise
 Shot noise
 Flicker noise
 Environmental noise
10. How a thermal noise is caused?
It is caused by the thermal agitation of electrons or other charge carriers in
resistors, capacitors, radiation transducers, electrochemical cells, and other resistive
elements in an instrument.
11. When a shot noise is encountered?
It is encountered wherever electrons or other charged particles cross a junction.
12. What is flicker noise?
It is characterized as having a magnitude that is inversely proportional to the frequency of
the signal being observed.
13. List out the read out devices available?
 Oscilloscopes
 Cathode-ray tubes
 Horizontal and vertical control plates
14. Define sensitivity.
It is a measure of its ability to discriminate between small differences in analytic
concentration.
15. What are the factors that limit sensitivity?
 The slope of the calibration curve
 The reproducibility or precision of the measuring device.
16. What is the relation between wavelength and energy of electromagnetic
radiation? (May/June2012)

3. 17. What type of noise can be reduced by hardware techniques? (May/June 2012)
 Environmental Noise
 flicker noise
 noise in transducer
18. Distinguish between sensitivity and detection limit. (May/June 2012,2014)
Sensitivity:
It is a measure of its ability to discriminate between small differences in
analytic concentration.
Detection limit: Detection Limit (Limit of detection, LOD):
The minimum concentration of analytic that can be detected with a specific method at a
known confidence level.
19. Define signal to noise ratio. (May/June 2013)(Nov/Dec 2015)
S/N ratio is defined as the ratio of average amplitude of signal to the average
amplitude of the noise.
S/N = Avg. amplitude of signal / Avg. amplitude of noise
20. Give the sources of IR radiation?
Nernst glower and Globar widely used, Nichrome wire, A tungsten filament lamp for near
IR.UV-radiation -Hydrogen gas lamps and deuterium lamps Visible radiation -Incandescent
tungsten filament lamp.
21. Sort out the ideal requirements of source?
It should provide continuous radiation, It should be stable, It must generate beam with
sufficient powerfor ready detection and measurement.
22. Arrange the different types of electromagnet
They are cosmic rays < r-rays x-rays < UV rays < visible light < infrared rays < microwave
and <radio wave
23. What is electromagnetic radiation?
Electromagnetic radiation is a form of energy that is transmitted through space at a
enormous velocity.
24. Name the materials of which sample containers are made?
Quartz or fused silica -in UV region below 350nm Silicate glasses –350-2000nm. Plastic
containers in visible regions.
25. Name the two filters employed in wavelength selection?
 Interference filters
 Absorption filters.
26. Define monochromator?

4. They are the units which are used to separate a polychromatic radiation into a
monochromatic form.
27. List out the components of monochromators?
An entrance slit-a collimating lens-a prism-a focusing element-an exit slit
28. Explain about crystal monochromator?
A crystal monochromator is made up of a suitable crystalline materials positioned in the x-
rays beam
and satisfies the Bragg’s equation.
29. State the ideal properties of a transducer?
High sensitivity-a high signal-to-noise ratio, and a constant response over
considerable range of wavelengths. In addition it would exhibit a fast response time and a
zero output signal in the absenceof illumination.
30. List out the types of radiation transducers?
Two general types of transducers are: one responds to photons, the other to heat.
31. List out the types of photon transducers?
Photovoltaic cells in which the radiant energy generates a current at the interface
of a semiconductor layer and a metal. Phototubes -in which radiation causes emission of
electrons from a photosensitive solid surface. Photomultiplier tubes -which contain a
photo emissive surface as well as several additional surfaces that emit a cascade of
electrons when struck by electrons from photosensitive area. Photoconductivity
transducers -in which absorption of radiation b a semiconductor produces electrons
and holes. Silicon photodiodes –in which photons increase the conductance across a
reversed biased pn junction.
32. Why is thermal noise called as White noise? (May/June 2014)
Thermal noise called as white noisebecause thermal noise is independent of absolute
frequency.
PART-B
1. What is meant by instrumental noise? What are the types of noise? Explain each with
example. (May/June 2013)
Instrumental Noise:
Noise is associated with each component of an instrument with the source, the input
transducer, signal processing elements and output transducer. Noise is a complex composite
that usually cannot be fully characterized.Certain kinds of instrumental noise are
recognizable.
 Chemical Noise
 Instrumental Noise

5. Chemical Noise:
Arises from an uncontrollable variable that affect the chemistry of the system being
analyzed. Examples are undetected variations in temperature, pressure, chemical equilibria,
humidity, light intensity etc.
Instrumental Noise:
Instrumental noise are classified as into four types,
 Thermal or Johnson noise
 Shot noise
 Flicker or 1/f noise
 Environmental noise
Thermal Noise or Johnson Noise:
Thermal noise is caused by the thermal agitation of electrons or other charge carriers
in resistors, capacitors, radiation transducers, electrochemical cells and other resistive
elements in an instruments. The magnitude of thermal noise is given by
Where, Vrms = root mean square noise, ∆f = frequency band width (Hz), k = Boltzmann
constant (1.38 x 10-23 J/K), T = temperature in Kelvin, R = resistance in ohms of the
resistive element. Thermal noise can be decreased by narrowing the bandwidth, by lowering
the electrical resistance and by lowering the temperature of instrument components.
Shot Noise:
Shot noise is encountered wherever electrons or other charged particles cross a
junction.
Where, irms = root-mean-square current fluctuation,
I = average direct current,
e = charge on the electron (1.60 x 10-19 C),
∆f = band width of frequencies.
Shot noise in a current measurement can be minimized only by reducing bandwidth.
Flicker Noise:
Flicker noise is characterized as having a magnitude that is inversely proportional to
the frequency of the signal being observed. It is sometimes termed 1/f (one-over-f) noise. The
causes of flicker noise are not well understood and are recognizable by its frequency
dependence. Flicker noise becomes significant at frequency lower than about 100 Hz. Flicker
noise can be reduced significantly by using wire-wound or metallic film resistors rather than
the more common carbon composition type.
Environmental Noise:
Environmental noise is a composite of different forms of noise that arise from the
surroundings. Much environmental noise occurs because each conductor in an instrument is
potentially an antenna capable of picking up electromagnetic radiation and converting it to an
electrical signal.

6. 2. Describe the hardwaretechniques for signal to noise enhancement. (May/june 2012)
When the need for sensitivity and accuracy increased, the signal-to-noise ratio often
becomes the limiting factor in the precision of a measurement. Both hardware and software
methods are available for improving the signal-to-noise ratio of an instrumental method.
Hardware method: Hardware noise reduction is accomplished by incorporating into the
instrument design components such as filters, choppers, shields, modulators, and
synchronous detectors. These devices remove or attenuate the noise without affecting the
analytical signal significantly. Hardware devices and techniques are as follows,
 Grounding and Shielding
 AnalogFiltering
 Modulation
 Signalchopping
 Lock-in-Amplifiers
Grounding and Shielding:
Noise that arises from environmentally generated electromagnetic radiation can be
substantially reduced by shielding, grounding and minimizing the length of conductors
within the instrumental system.
Analog Filtering:
By using low-pass and high-pass analog filters S/N ratio can be improved. Thermal,
shot and flicker noise can be reduced by using analog filters.
Modulation:
In this process, low frequency or dc signal from transducers are often converted to a
higher frequency, where 1/f noise is less troublesome. This process is called modulation.
After amplification the modulated signal can be freed from amplifier 1/f noise by filtering
with a high-pass filter, demodulation and filtering with a low-pass filter then produce an
amplified dc signal suitable for output.
Signal chopping:
In this device, the input signal is converted to a square-wave form by an electronic or
mechanical chopper. Chopping can be performed either on the physical quantity to be
measured or on the electrical signal from the transducer.
Lock-in-Amplifiers:
Lock-in-amplifiers permit the recovery of signals even when the S/N is unity or less.
It requires a reference signal that has the same frequency and phase as the signal to be

7. amplified. A lock-in amplifier is generally relatively free of noise because only those signals
that are locked-in to the reference signal are amplified. All other frequencies are rejected by
the system.
3. Describe the software techniques for signal to noise enhancement. (May/June
2014),(Nov/Dec 2015)
Software Method:
Software methods are based upon various computer algorithms that permit extraction
of signals from noisy data. Hardware convert the signal from analog to digital form which is
then collected by computer equipped with a data acquisition module. Software programs are
as follows,
 Ensemble Averaging
 Boxcar Averaging
 Digital filtering
Ensemble Averaging:
In ensemble averaging, successive sets of data stored in memory as arrays are
collected and summed point by point. After the collection and summation are complete, the
data are averaged by dividing the sum for each point by the number of scans performed. The
signal-to-noise ratio is proportional to the square root of the number of data collected.
Boxcar Averaging:
Boxcar averaging is a digital procedure for smoothing irregularities and enhancing the
signal-to-noise ratio. It is assumed that the analog analytical signal varies only slowly with
time and the average of a small number of adjacent points is a better measure of the signal
than any of the individual points. In practice 2 to 50 points are averaged to generate a final
point. This averaging is performed by a computer in real time, i.e., as the data is being
collected. Its utility is limited for complex signals that change rapidly as a function of time.

8. Digital filtering:
Digital filtering can be accomplished by number of different well-characterized
numerical procedure such as
(a) Fourier transformation and
(b) Least squares polynomial smoothing.
(a) Fourier transformation:
In this transformation, a signal which is acquired in the time domain is converted to a
frequency domain signal in which the independent variable is frequency rather than time.
This transformation is accomplished mathematically on a computer by a very fast and
efficient algorithm. The frequency domain signal is then multiplied by the frequency
response of a digital low pass filter which removes frequency components. The inverse
Fourier transform then recovers the filtered time domain spectrum.
(b) Least squares polynomial data smoothing:
This is very similar to the boxcar averaging. In this process first 5 data points are
averaged and plotted. Then moved one point to the right and averaged. This process is
repeated until all of the points except the last two are averaged to produce a new set of data
points. The new curve should be somewhat less noisy than the original data. The signal-to-
noise ratio of the data may be enhanced by increasing the width of the smoothing function or
by smoothing the data multiple times.

9. 4. Explain about wavelength selectors/Explain the various components of optical
instruments (May/June 2012 & 13),(Nov/Dec 2015)
A. Filters are used to pass a band of wavelengths
Interference Filters:
They rely on optical interference to provide narrow bands of radiation. It consists of a
transparent dielectric that occupies the space between two semitransparent metallic films.
They are available with transmitter peaks throughout the ultraviolet region and visible regions
and up to about 14µm in the infrared.
Interference Wedges:
An interference wedge consists of a pair of mirrored, partially transparent plates separated by
a wedge-shaped layer of a dielectric material. They are available for the visible region, the
near-infrared region, and for several parts of the infrared region. They can serve in place of
prisms or gratings in monochromators.
Absorption Filters:
They are generally less expensive than interference filters and are widely used for
band selection in the visible region. They function by absorbing certain portions of the
spectrum. The most common type consists of colored glass or of a dye suspended in gelatin
and sandwiched between glass plates. The former have the advantage of greater thermal
stability.
B.Monochromators:
One color - pass a narrow band of wavelengths. For many spectroscopic methods, it is
necessary or desirable to be able to vary the wavelength of radiation continuously over a
considerable ran-c. This process is called scanning- a spectrum. Monochromators are
designed for spectral scanning. Monochromators for ultraviolet, visible, and infrared
radiation are similar in mechanical construction in the sense that they employ slits, lenses,
mirrors, windows, and -ratings or prisms.
Components of monochromators:
The optical elements found in all monochromators, which include
(1) An entrance slitthat provides a rectangular optical image,
(2) A collimating- lens or mirror that produces aparallel beam of radiation,
(3) A prism or a grating that disperses the radiation into itscomponent wavelengths,
(4) A focusing element that reforms the image of the slit and focusesit on a planar surface
called a focal plane
(5) An exit slit in the focal plane that isolates the desired spectral band.
C. Prism
Two types of dispersing elements are found in monochromators: reflection gratings
and prisms. For the grating monochromator, angular dispersion of the wavelengths results
from diffraction, which occurs at the reflective surface; for the prism, refraction at the two
faces results in angular dispersal of the radiation.
1) Dispersing prisms:
Separation of wavelengths due to differences in index of refraction of the glass in the
prism with each different wavelength. This leads to constructive and destructive
interference. Dispersion is angular (nonlinear). Single order is obtained. The larger the
focal length, the better the dispersion.
2) Reflecting prisms:
Designed to change direction of propagation of beam, orientation, or both
3) Polarizing prisms:
Made of birefringent materials.

10. D.The Echellette Grating
It is grooved or blazed such that it has relatively broad faces from which
reflectionoccurs and narrow unused faces. Each of the broad faces can be considered to be
apoint source of radiation.
E.Radiation Transducers
 High sensitivity
 High S/N
 Constant response over range of wavelengths
 Fast response
 Zero output in absence of illumination
 Electrical signal directly proportional to radiant power
F. Slits
Slits are used to limit the amount of light impinging on the dispersing element as well as to
limit the light reaching the detector. There is a dichotomy between intensity and resolution.
Atomic lines are not infinitely narrow due to types of broadening
1) Natural
2) Doppler:
3) Stark
4) Collisional broadening
The use of entrance and exit slits convolutes this broadening as a triangular function -the slit
function.
G. Sample Containers
Sample containers are required for all spectroscopic studies except emission
spectroscopy. In common with the optical elements of monochromators, the cells or cuvettes
that hold the samples must be made of material that passes radiation in the spectral region of
interest. Quartz or fused silica is required for work in the ultraviolet re-ion (below '150 nm)
both of these substances are transparent in the visible region and up to about 3um in the
infrared region as well. Silicate glasses can be employed in the region between 350 and
2000nm. Plastic containers have also found application in the visible re-ion. Crystalline
sodium chloride is the most common substance employed for cell windows in the infrared
region. Must be made of material that is transparent to the spectral region of interest

11. H. Photomultiplier Tubes
1. Sensitivity: Significantly more sensitive than simple phototube
2. Process of Multiplication: Electrons emitted from cathode surface and accelerated
towards dynode (each successive dynode is 90 V more positive than preceding dynode)
3. Construction
 Photocathode: made of alkali metals with low work functions
 Focusing electrodes
 Electron multiplier (dynodes) amplification by factor of 106 to 107 for each
 photon
 Electron collector (anode)
 Window: borosilicate, quartz, sapphire, or MgF2
4. Features fast response time and low noise
5. Spectral response
 Depends on photocathodic material
 Conversion efficiency varies
 Lower cutoff determined by window composition
I. Array Detectors
 An "electrical photographic plate"
 Detect differences in light intensity at different points on their photosensitivesurfaces
 Fabricated from silicon using semiconductor technology
 Originally conceived as television camera sensing elements
 Placed at focal plane of polychromator in place of the exit slit
 Sensitive for detection of light in 200-1000 nm range
 Major advantage is simultaneous detection of all wavelengths within range
 Types
 SIT : silicon intensifier target
 PDA : photodiode array
 CCD : charge-coupled device
 CID : charge injection device
Photodiode Arrays (PDA)
 Usually 1-3 cm long; contains a few hundred photodiodes (256 - 2048) in a
lineararray
 Partitions spectrum into x number of wavelength increments
 Each photodiode captures photons simultaneously
 Measures total light energy over the time of exposure (whereas PMT
measuresinstantaneous light intensity)
Process
 Each diode in the array is reverse-biased and thus can store charge like a capacitor
 Before being exposed to light to be detected, diodes are fully charged via a
 transistor switch
 Light falling on the PDA will generate charge carriers in the silicon which
 combine with stored charges of opposite polarity and neutralize them
 The amount of charge lost is proportional to the intensity of light
 Amount of current needed to recharge each diode is the measurement made
whichis proportional to light intensity
 Recharging signal is sent to sample-and-hold amplifier and then digitized
 Array is however read sequentially over a common output line
 Use minicomputer to handle data

12. Disadvantages
 Must have fast data storage system
 High dark noise
 Must cool PDA to well below room temperature
 Diode saturates within a few seconds integration time
 Resolution not good, limited by diodes/linear distance
 Stray radiant energy (SRE) is a killer
 Used as detectors in Raman, fluorescence, and absorption
5. Discuss in detail the different types of and properties of electromagnetic radiations
and interaction with matters (Nov/Dec2016)
Electromagnetic radiation (EMR) is a form of energy that is produced by oscillating
electric and magnetic disturbance, or by the movement of electrically charged particles
traveling through a vacuum or matter. The electric and magnetic fields come at right angles to
each other and combined wave moves perpendicular to both magnetic and electric oscillating
fields thus the disturbance.
General Properties of all electromagnetic radiation:
 Electromagnetic radiation can travel through empty space. Most other types of waves
must travel through some sort of substance. For example, sound waves need either a
gas,solid, or liquid to pass through in order to be heard.
 The speed of light is always a constant. (Speed of light: 2.99792458 x 108 m s-1)
 Wavelengths are measured between the distances of either crests or troughs. It is
usually characterized by the Greek symbol λ.
In general, as a wave’s wavelength increases, the frequency decreases, and as wave’s
wavelength decreases, the frequency increases. When electromagnetic energy is released as
the energy level increases, the wavelength decreases and frequency decreases. Thus,
electromagnetic radiation is then grouped into categories based on its wavelength or
frequency into the electromagnetic spectrum. The different types of electromagnetic
radiations how in the electromagnetic spectrum consists of radio waves, microwaves, infrared
waves, visible light, ultraviolet radiation, X-rays, and gamma rays. The part of the
electromagnetic spectrum that we are able to see is the visible light spectrum.
Radiation Types
Radio Waves are approximately 103 m in wavelength. As the name implies, radio
waves are transmitted by radio broadcasts, TV broadcasts, and even cell phones. Radio waves
have the lowest energy levels. Radio waves are used in remote sensing, where hydrogen gas
in space releases radio energy with a low frequency and is collected as radio waves. They are
also used in radar systems, where they release radio energy and collect the bounced energy
back. Especially useful in weather, radar systems are used to can illustrate maps of the
surface of the Earth and predict weather patterns since radio energy easily breaks through the
atmosphere.
Microwaves can be used to broadcast information through space, as well as warm food. They

13. are also used in remote sensing in which microwaves are released and bounced back to
collect information on their reflections. Microwaves can be measured in centimeters. They
are good for transmitting information because the energy can go through substances such as
clouds and light rain. Short microwaves are sometimes used in doppler radars to predict
weather forcasts.
Infrared radiation can be released as heat or thermal energy. It can also be bounced
back,which is called near infrared because of its similarities with visible light energy.
Infrared Radiation is most commonly used in remote sensing as infrared sensors collect
thermal energy, providing us with weather conditions.
Visible Light is the only part of the electromagnetic spectrum that humans can see with an
unaided eye. This part of the spectrum includes a range of different colors that all represent a
particular wavelength. Rainbows are formed in this way; light passes through matter in which
it is absorbed or reflected based on its wavelength. Thus, some colors are reflected more than
other, leading to the creation of a rainbow.
Interference
An important property of waves is the ability to combine with other waves. There are
two type of interference: constructive and destructive. Constructive interference occurs when
two or more waves are in phase and their displacements add to produce a higher amplitude.
On the contrary, destructive interference occurs when two or more waves are out of phase
and their displacements negate each other to produce lower amplitude.
Wave-Particle Duality
Electromagnetic radiation can either acts as a wave or a particle, a photon. As a wave,
it isrepresented by velocity, wavelength, and frequency. Light is an EM wave since the speed
of EM waves is the same as the speed of light. As a particle, EM is represented as a photon,
which transports energy. When a photon is absorbed, the electron can be moved up or down
an energy level. When it moves up, it absorbs energy, when it moves down, energy is
released. Thus, since each atom has its own distinct set of energy levels, each element emits
and absorbs different frequencies. Photons with higher energies produce shorter wavelengths
and photons with lower energies produce longer wavelengths.

14. UNIT II
MOLECULAR SPECTROSCOPY
PART-A
1. Explain Beers Law.(May/June 2012).
Beer's law states that the absorbance is directly proportional to the concentration of a solution. If
you plot absorbance versus concentration, the resulting graph yields a straight line.
2. Explain the term chromophore and give two examples.(May/June 2012)
A chromophore is the part of a molecule responsible for its color. The color arises
when a molecule absorbs certain wavelengths of visible light and transmits or reflects
others. The chromophore is a region in the molecule where the energy difference between
two different molecular orbitals falls within the range of the visible spectrum. Examples:
Lycopene, Beta carotene, azo dyes.
3. What is Lambert’s (May/June2014) law?
When a beam of light is allowed to pass through a transparent medium, the rate of decrease of
intensity(I) with the thickness(t) of medium is detect proportional to the intensity
-dI/dt =KI Or It =Ioe-kt
4. What is absorbance?
The absorbance(A) is the logarithm to the base of the reciprocal of the transmittance.
A= log(1/ T) = -log T or log I0 / It
5. What is absorptivity, Explain?
Absorptivity (a) is the ratio of the absorbance to the product of the concentration and length of
optical path. It is a constant characteristic of ( a= A / bc) substance and wavelength. The
alternate of this term is extinction coefficient or absorbance index.
6. What are the reasons for deviation from Beer Law?
Deviation from the Beer’s law are there reported the as resultant curve is concave upwards or
concave downwards. The factors involved in deviation from Beer’s law may be chemical &
instrumental.
7. Define colorimeter?
Any instrument used for measuring absorption in the visible region is generally called colorimeter.
8. Define spectrophotometer.
The instrument which measures the ratio or a function of the two, of the radiant power of
two electromagnetic beam over a large wavelength region.
9. Define monochromators.
A monochromators used to isolates band of interest of wavelengths. It allows the light of the
required wavelength to pass through but absorb the light of other wavelengths. It contains
entrance slit, dispensing elements and exit slit.
10. What is Detector and what are the detectors used in visible spectroscopy?
Detector is used for measuring the radiant energy transmitted through the sample. There are
three types of photo devices used 1) photovoltaic cell 2) phototube and Photomultiplier tubes.
11. What is meant of single and double beam spectrophotometer?
Single beam have only one light path. Involve three controls: wavelength, zero adjustment and 100
per cent adjustment. The double-beam design provides two equivalent paths for radiation, both
originating with the same source. One of these beams passes through the sample and other through
reference. The two beams are measured separately, ether by duplicate detector or rapidly alternating
use of the same detector.
12. What are the application of IR spectroscopy?
To estimation of organic compounds, inorganic compounds, geometrical isomerism, presence of
water in the samples, shape of symmetry of a molecules, determination of purity etc.
13. Discuss about the sources of AA spectroscopy.
The most successful line spectra source for AA is the hollow-cathode
lamp.
14. What are the applications AA?

15. AA is useful in the determination of a large number of metals, specially at trace levels. 2) It is
widely used in such field as water and pharmaceutical analysis and in metallurgy.
15. Mention the basic components of instruments that measure transmittance or absorbance.
A stable light source, Monochromator, Sample containers for sample and solvent, A radiation
detector, A signal indicator.
16. State the advantages of spectroscopy?
 More rapid and less time consuming
 Gives more information.
 Requires small amount of the compound to be anlysed
 Precise and reliable
 More selective and sensitive
 Continuous operation is often possible.
17. Explain molecular spectroscopy.
This is deals with the interaction of electromagnetic radiation with molecules. The results in
transition between rotational and vibrational energy levels in addition to electronic transition.
Molecular spectra extend from the visible through infrared into the microwave region.
18. Define transmittance.
It is the ratio of the radiant power transmitted by the sample (It) to the radiant power incident on the
sample (I0), both being measured at the same spectral position and with the same slit
width. This transmittance T is defined by It/ I0 .
19. Discuss atomic absorption.
This is most powerful technique for the quantitative determination of trace metals in liquids. e.g.
total sodium content of a water. The sample should be gaseous state and volatilization of liquids or
solid followed by the dissociation of molecules to give free atoms.
20. What are amphiprotic compounds? Give examples.
Can act as both acid or base. example: amino acids, water, proteins.
21. Proportionality: how it is used in the determination of unknown concentration?
(May/June 2013)
Absorbance Varies linearly with the change i law.
22. The absorptivity of a compound is 1.5M
-1
cm
-1
. What is the concentration of solution
of this compound if 2cm sample has an absorbance of 1.20? (May/June 2013)(Nov/Dec 2015)
A = abc
Where, a= 1.5M
-1
cm
-1
; B=1.2cm; A=1.2 ; C=? Answer’s=0.4
23. What is interference?
Interference are confined mainly to phenomena that affect the number of atoms in the flame which
are given as –spectral interference-caused by overlapping of any radiation of the test
elements to be estimated, chemical interference-due to presence of chemicals, it may be cationic or
anionic etc.
24. What is an Interferogram? (Nov/Dec 2015)
To make an interferogram, we combine light from two different sources. In practice, we use the
same light source (a laser ) and split the light into two beams. One is the reference beam, which will
provide a comparison wavefront. The other is the test beam , which is passed through the optical
system to be tested. The two beams are combined together to make an interferogram.

16. PART B
1. Discuss about the Jablonski’s Diagram. (Nov/Dec 2015)
PARTIAL ENERGY DIAGRAM FOR A PHOTOLUMINESCENT SYSTEM
Singlet: all electron spins are paired; no energy level splitting occurs when the molecule is exposed to a
magnetic field;
Triplet: the electron spins are unpaired and are parallel; excited triplet state is less energetic than the
corresponding singlet state.
Diamagnetic: no net magnetic field due to spin paring. The electrons are repelled by permanent magnetic
fields.
Paramagnetic: magnetic moment and attracted to a magnetic field (due to unpaired electrons).
Deactivation processes for an excited state:
Vibrational relaxation: Fluorescence always involves a transition from the lowest vibrational states of an
excited electronic state; electron can return to any one of the vibrational levels of the ground state; 10 -12
s.
Internal conversion: Intra molecular processes by which a molecule passes to a lower-energy electronic
state without emission of radiation.
External conversion: Interaction and energy transfer between the excited molecule and the solvent or
other molecules.
Intersystem crossing: The spin of an excited electron is reversed and a change in multiplicity of the
molecule results.
Phosphorescence: an excited triplet state to give radioactive emission.
Emission: A photon is emitted.

17. Resonance fluorescence: Absorbed radiation is re-emitted without a change in frequency.
Stokes shift: Molecular fluorescence bands are shifted to wavelengths that are longer than the resonance
line.
2.Explain the important components of Infrared spectroscopy with diagram. (May/June 2013)
Spectroscopy is an instrumentally aided study of the interactions between matter (sample being
analyzed) and energy (any portion of the electromagnetic spectrum)IR spectroscopy is concerned with the
study of absorption of infrared radiation, which causes vibrational transition in the molecule. Hence, IR
spectroscopy also known as vibrational spectroscopy. IR spectra mainly used in structure elucidation to
determine the functional groups. It is absorption (4000 - 200cm-1
) in this region which gives structural
information about a compound.
Instrumental components
Sources
An inert solid is electrically heated to a temperature in the range 1500-2000 K. The heated material will
then emit infra red radiation.
The Nernst glower is a cylinder (1-2 mm diameter, approximately 20 mm long) of rare earth oxides.
Platinum wires are sealed to the ends, and a current passed through the cylinder. The Nernst glower can
reach temperatures of 2200 K.
The Globar source is a silicon carbide rod (5mm diameter, 50mm long) which is electrically heated to
about 1500 K. Water cooling of the electrical contacts is needed to prevent arcing. The spectral output is
comparable with the Nernst glower, execept at short wavelengths (less than 5 m) where it's output
becomes larger.
The incandescent wire source is a tightly wound coil of nichrome wire, electrically heated to 1100 K. It
produces a lower intensity of radiation than the Nernst or Globar sources, but has a longer working life.
Detectors
There are three catagories of detector
 Thermal
 Pyroelectric
 Photoconducting
Thermocouples consist of a pair of junctions of different metals; for example, two pieces of bismuth
fused to either end of a piece of antimony. The potential difference (voltage) between the junctions
changes according to the difference in temperature between the junctions
Pyroelectric detectors are made from a single crystalline wafer of a pyroelectric material, such as
triglycerine sulphate. The properties of a pyroelectric material are such that when an electric field is
applied across it, electric polarisation occurs (this happens in any dielectric material). In a pyroelectric
material, when the field is removed, the polarisation persists. The degree of polarisation is temperature
dependant. So, by sandwiching the pyroelectric material between two electrodes, a temperature dependant
capacitor is made. The heating effect of incident IR radiation causes a change in the capacitance of the
material. Pyroelectric detectors have a fast response time. They are used in most Fourier transform IR
instruments.

18. Photoelectric detectors such as the mercury cadmium telluride detector comprise a film of
semiconducting material deposited on a glass surface, sealed in an evacuated envelope. Absorption of IR
promotes nonconducting valence electrons to a higher, conducting, state. The electrical resistance of the
semiconductor decreases. These detectors have better response characteristics than pyroelectric detectors
and are used in FT-IR instruments - particularly in GC - FT-IR.
Types of instrument
Dispersive infra red spectrophotometers
These are often double-beam recording instruments, employing diffraction gratings for dispersion
of radiation. Radiation from the source is flicked between the reference and sample paths. Often,
an optical null system is used. This is when the detector only responds if the intensity of the two beams is
unequal. If the intensities are unequal, a light attenuator restores equality by moving in or out of the
reference beam. The recording pen is attached to this attenuator.
Fourier-transform spectrometers
Any waveform can be shown in one of two ways; either in frequency domain or time domain.
Dispersive IR instruments operate in the frequency domain. There are, however, advantages to be gained
from measurement in the time domain followed by computer transformation into the frequency domain. If
we wished to record a trace in the time domain, it could be possible to do so by allowing radiation to fall
on a detector and recording its response over time. In practice, no detector can respond quickly enough
(the radiation has a frequency greater than 1014
Hz). This problem can be solved by using interference to
modulate the IR signal at a detectable frequency. The Michelson interferometer is used to produce a new
signal of a much lower frequency which contains the same information as the original IR signal. The
output from the interferometer is an inte rferogram.

19. Radiation leaves the source and is split. Half is reflected to a stationary mirror and then back to the
splitter. This radiation has travelled a fixed distance. The other half of the radiation from the source
passes through the splitter and is reflected back by a movable mirror. Therefore, the path length of this
beam is variable. The two reflected beams recombine at the splitter, and they interfere (e.g. for any one
wavelength, interference will be constructive if the difference in path lengths is an exact multiple of the
wavelength. If the difference in path lengths is half the wavelength then destructive interference will
result). If the movable mirror moves away from the beam splitter at a constant speed, radiation reaching
the detector goes through a steady sequence of maxima and minima as the interference alternates
between constructive and destructive phases.
If monochromatic IR radiation of frequency, f ( ir ) enters the interferometer, then the output
frequency, fm can be found by;
Where v is the speed of mirror travel in mm/s,because all wavelengths emitted by the source are present,
the interferogram is extremely complicated. The moving mirror must travel smoothly; a frictionless
bearing is used with electromagnetic drive. The position of the mirror is measured by a laser shining on
a corner of the mirror. A simple sine wave interference pattern is produced. Each peak indicates mirror
travel of one half the wavelength of the laser. The accuracy of this measurement system means that the
IR frequency scale is accurate and precise.
In the FT-IR instrument, the sample is placed between the output of the interferometer and the detector.
The sample absorbs radiation of particular wavelengths. Therefore, the interferogram contains the
spectrum of the source minus the spectrum of the sample. An interferogram of a reference (sample
cell and solvent) is needed to obtain the spectrum of the sample.
After an interferogram has been collected, a computer performs a Fast Fourier Transform, which results
in a frequency domain trace (i.e intensity vs. wavenumber) that we all know and love.
The detector used in an FT-IR instrument must respond quickly because intensity changes are rapid (the
moving mirror moves quickly). Pyroelectric detectors or liquid nitrogen cooled photon detectors must be
used. Thermal detectors are too slow. To achieve a good signal to noise ratio, many interferograms are
obtained and then averaged. This can be done in less time than it would take a dispersive instrument to
record one scan.
Advantages of Fourier transform IR over dispersive IR
 Improved frequency resolution
 Improved frequency reproducibility (older dispersive instruments must be recalibrated for each
session of use)
 Higher energy throughput

20.  Faster operation
 Computer based (allowing storage of spectra and facilities for processing spectra)
 Easily adapted for remote use (such as diverting the beam to pass through an external cell and
detector, as in GC - FT-IR)
3. What are the applications of IR spectroscopy (Nov/Dec 2015)
Infrared spectroscopy is widely used in industry as well as in research. It is a simple and reliable
technique for measurement, quality control and dynamic measurement. It is also employed in forensic
analysis in civil and criminal analysis.
Some of the major applications of IR spectroscopy are as follows:
1. Identification of functional group and structure elucidation
Entire IR region is divided into group frequency region and fingerprint region. Range of group
frequency is 4000-1500 cm-1
while that of finger print region is 1500-400 cm-1
.In group frequency
region, the peaks corresponding to different functional groups can be observed. According to
corresponding peaks, functional group can be determined.
Each atom of the molecule is connected by bond and each bond requires different IR region so
characteristic peaks are observed. This region of IR spectrum is called as finger print region of the
molecule. It can be determined by characteristic peaks.
2. Identification of substances
IR spectroscopy is used to establish whether a given sample of an organic substance is identical
with another or not. This is because large number of absorption bands is observed in the IR spectra of
organic molecules and the probability that any two compounds will produce identical spectra is almost
zero. So if two compounds have identical IR spectra then both of them must be samples of the same
substances.
IR spectra of two enatiomeric compound are identical. So IR spectroscopy fails to distinguish between
enantiomers. For example, an IR spectrum of benzaldehyde is observed as follows.
C-H stretching of aromatic rings 3080 cm-1
C-H stretching of aldehyde 2860 cm-1
and 2775 cm-1
C=O stretching of an aromatic aldehyde 1700 cm-1
C=C stretching of an aromatic ring 1595 cm-1
C-H bending 745 cm-1
and 685 cm-1
No other compound then benzaldehyde produces same IR spectra as shown above.
3. Studying the progress of the reaction
Progress of chemical reaction can be determined by examining the small portion of the reaction
mixture withdrawn from time to time. The rate of disappearance of a characteristic absorption band of
the reactant group and/or the rate of appearance of the characteristic absorption band of the product
group due to formation of product is observed.
4. Detection of impurities
IR spectrum of the test sample to be determined is compared with the standard compound. If any
additional peaks are observed in the IR spectrum, then it is due to impurities present in the compound.

21. 5. Quantitative analysis
The quantity of the substance can be determined either in pure form or as a mixure of two or
more compounds. In this, characteristic peak corresponding to the drug substance is chosen and log I0/It
of peaks for standard and test sample is compared. This is called base line technique to determine the
quantity of the substance.
6. OTHER APPLICATIONS
 Determination of unknown contaminates in industry using FTIR
 Determination of cell wall of mutant & wild type plant verities using FTIR
 Biomedical studies of human hair to identify disease state
 Identify color &taste component of the system
 Determine atmospheric pollutant s from atmosphere itself
 It is also used in forensic analysis in both criminal and civil case, example in identifying the
polymer degradation and determining the blood alcohol content.
4. Explain the theory, instrumentation & applications of Raman spectroscopy. (May/June 2014)
THEORY OF RAMAN SPECTROSCOPY
Raman spectra are acquired by irradiating a sample with a powerful laser source of visible or
near-infrared monochromatic radiation. During irradiation, the spectrum of the scattered radiation is
measured at some angle (often 90 deg) with a suitable spectrometer. At the very most, the intensities of
Raman lines are 0.001 % of the intensity of the source; as a consequence, their detection and
measurement are somewhat more difficult than are infrared spectra.
Excitation of Raman Spectra
A Raman spectrum can be obtained by irradiating a sample of carbon tetrachloride with an
intense beam of an argon ion laser having a wavelength of 488.0 nm (20492 cm-1
). The emitted radiation
is of three types
1. Stokes scattering
2. Anti-stokes scattering
3. Rayleigh scattering

22. The raman spectrum is the wave number shift ∆v which is defined as the difference in wave
numbers (cm-1
) between the observed radiation and that of the source. For CCl4 three peaks are found on
both sides of the Rayleigh peak and that the pattern of shifts on each side is identical. Anti-Stokes lines
are appreciably less intense that the corresponding Stokes lines. For this reason, only the Stokes part of a
spectrum is generally used. The magnitude of Raman shifts is independent of the wavelength of
excitation.
Mechanism of Raman
Rayleigh scattering
The heavy arrow on the far left depicts the energy change in the molecule when it interacts with
a photon. The increase in energy is equal to the energy of the photon hν.The second and narrower arrow
shows the type of change that would occur if the molecule is in the first vibrational level of the
electronic ground state.
The middle set of arrows depicts the changes that produce Rayleigh scattering. The energy
changes that produce stokes and anti-Stokes emission are depicted on the right. The two differ from the
Rayleigh radiation by frequencies corresponding to ±∆E, the energy of the first vibrational level of the
ground state. If the bond were infrared active, the energy of its absorption would also be ∆E. Thus, the
Raman frequency shift and the infrared absorption peak frequency are identical. The relative populations
of the two upper energy states are such that Stokes emission is much favored over anti-Stokes.
Rayleigh scattering has a considerably higher probability of occurring than Raman because the
most probable event is the energy transfer to molecules in the ground state and reemission by the return
of these molecules to the ground state. The ratio of anti-Stokes to Stokes intensities will increase with
temperature because a larger fraction of the molecules will be in the first vibrationally excited state
under these circumstances.
Raman Depolarization Ratios
Polarization is a property of a beam of radiation and describes the plane in which the radiation
vibrates. Raman spectra are excited by plane-polarized radiation. The scattered radiation is found to be
polarized to various degrees depending upon the type of vibration responsible for the scattering.
Experimentally, the depolarization ratio may be obtained by inserting a polarizer between the sample
and the monochromator.

23. The depolarization ratio is dependent upon the symmetry of the vibrations responsible for
scattering. Polarized band: p = < 0.76 for totally symmetric modes (A1g)
INSTRUMENTATION
Instrumentation for modern Raman spectroscopy consists of three components,
 A laser source,
 A sample illumination system and
 A suitable spectrometer.
Source
The sources used in modern Raman spectrometry are nearly always lasers because their high
intensity is necessary to produce Raman scattering of sufficient intensity to be measured with a
reasonable signal-to-noise ratio. Because the intensity of Raman scattering varies as the fourth power of
the frequency, argon and krypton ion sources that emit in the blue and green region of the spectrum have
an advantage over the other sources.
Sample Illumination System
Liquid Samples:
A major advantage of sample handling in Raman spectroscopy compared with infrared arises
because water is a weak Raman scattere but a strong absorber of infrared radiation. Thus, aqueous
solutions can be studied by Raman spectroscopy but not by infrared. This advantage is particularly
important for biological and inorganic systems and in studies dealing with water pollution problems.
Solid Samples:
Raman spectra of solid samples are often acquired by filling a small cavity with the sample after
it has been ground to a fine powder. Polymers can usually be examined directly with no sample
pretreatment.
Gas samples:
Gas are normally contain in glass tubes, 1-2 cm in diameter and about 1mm thick. Gases can also
be sealed in small capillary tubes.
Raman Spectrometers
 Raman spectrometers were similar in design and used the same type of components as the
classical ultraviolet/visible dispersing instruments.
 Most employed double grating systems to minimize the spurious radiation reaching the
transducer. Photomultipliers served as transducers.
 Now Raman spectrometers being marketed are either Fourier transform instruments equipped
with cooled germanium transducers or multichannel instruments based upon charge coupled
devices.
APPLICATIONS OF RAMAN SPECTROSCOPY
Raman Spectra of Inorganic Species

24. The Raman technique is often superior to infrared for spectroscopy investigating inorganic
systems because aqueous solutions can be employed. In addition, the vibrational energies of metal-
ligand bonds are generally in the range of 100 to 700 cm-1, a region of the infrared that is
experimentally difficult to study. These vibrations are frequently Raman active, however, and peaks with
∆ν values in this range are readily observed. Raman studies are potentially useful sources of information
concerning the composition
Raman Spectra of Organic Species
Raman spectra are similar to infrared spectra in that they have regions that are useful for
functional group detection and fingerprint regions that permit the identification of specific compounds.
Raman spectra yield more information about certain types of organic compounds than do their infrared
counterparts.
Biological Applications of Raman Spectroscopy
Raman spectroscopy has been applied widely for the study of biological systems. The advantages
of his technique include the small sample requirement, the minimal sensitivity toward interference by
water, the spectral detail, and the conformational and environmental sensitivity.
Quantitative applications
Raman spectra tend to be less cluttered with peaks than infrared spectra. As a consequence, peak
overlap in mixtures is less likely, and quantitative measurements are simpler. In addition, Raman
sampling devices are not subject to attack by moisture, and small amounts of water in a sample do not
interfere. Despite these advantages, Raman spectroscopy has not yet been exploited widely for
quantitative analysis. This lack of use has been due largely to the rather high cost of Raman
spectrometers relative to that of absorption instrumentation.
5. Explain the deviations in detail on Beer’s law. (May/June 2012)
This relationship is a linear for the most part. However, under certain circumstances the Beer
relationship gives a non-linear relationship. These deviations from the Beer Lambert law can be
classified into three categories:
Real Deviations :
These are fundamental deviations due to the limitations of the law itself.
Chemical Deviations :
These are deviations observed due to specific chemical species of the sample which is being
analyzed.
Instrument Deviations :
These are deviations which occur due to how the absorbance measurements
are made.
1- Real Deviation
Beer law and Lambert law is capable of describing absorption behavior of solutions containing
relatively low amounts of solutes dissolved in it (<10-3M).When the concentration of the analyte in the
solution is high (>10-3M), the analyte begins to behave differently due to interactions with the solvent
and other solute molecules and at times even due to hydrogen bonding interactions. It is also possible
that the concentration is so high, that the molecules create a screen for other molecules thereby
shadowing them from the incident light.
2- Chemical Deviations
Chemical deviations occur due to chemical phenomenon involving the analyte molecules due to
association, dissociation and interaction with the solvent to produce a product with different absorption
characteristics. For example, phenol red undergoes a resonance transformation when moving from the
acidic form (yellow) to the basic form (red). Due to this resonance, the electron distribution of the bonds
of molecule changes with the pH of the solvent in which it is dissolved.

25. 3- Instrumental Deviations
A. Due to Polychromatic Radiation
Beer-Lambert law is strictly followed when a monochromatic source of radiation exists. In
practice, however, it is common to use a polychromatic source of radiation with continuous distribution
of wavelengths along with a monochromators to create a monochromatic beam from this source.
B. Due to Presence of Stray Radiation
Stray radiation or scattered radiation is defined as radiation from the instrument that is outside
the selected wavelength band selected. Usually, this radiation is due to reflection and scattering by the
surfaces of lenses, mirrors, gratings, filters and windows. If the analyte absorbs at the wavelength of the
stray radiation, a deviation from Beer-Lambert law is observed similar to the deviation due to
polychromatic radiation.
C. Due to Mismatched Cells or Cuvettes
If the cells holding the analyte and the blank solutions are having different path-lengths, or
unequal optical characteristics, it is obvious that there would be a deviation observed in Beer-Lambert
law.

26. UNIT-III
MAGNETIC RESONANCE SPECTROSCOPY AND MASS SPECTROMETRY
PART-A
1. What is NMR spectroscopy?
Is one of the most powerful tool, based on the measurement of absorption of electromagnetic
radiation in the radio-frequency region of roughly 4 to 900 MHz.
2. Compare NMR with UV, Visible and IR absorption spectroscopy.
In contrast to UV, Vis and IR absorption, nuclei of atoms rather than outer electrons are involved in the
absorption process.
3. What are the uses of NMR spectroscopy?
A powerful tool available to chemists and biochemists for elucidating the structure of chemical species. The
technique is also useful for the quantitative determination of absorbing species.
4. List the types of NMR spectroscopy.
Two general types of spectrometers are currently in use, continuous-wave(CW) and pulsed or
Fourier-Transform(FT-NMR).
5. What are the various types of NMR spectra?
Wide line spectra, high resolution spectra.
6. List the factors that decide the type of NMR spectra?
Kind of instrument used –type of nucleus involved –the physical state of the sample –the
environment of the analyte nucleus and the purpose of the data collection.
7. Define wide line spectra of NMR.
Wide line spectra are those in which the bandwidth of the source of the lines is large enough that the fine
structure due to chemical environment is obscured.
8. List the uses of wide line NMR spectra.
Are useful for the quantitative determination of isotopes and for studies of the physical environment
of the absorbing species.
9. Define high-resolution spectra.
Most NMR spectra are high resolution and are collected by instruments capable of differentiating
between very small frequency differences of 0.01 ppm or less.
10. What are the two types of relaxation processes important in NMR spectroscopy? (Nov/Dec 2015)
Spin-lattice or longitudinal relaxation and spin-spin or transverse relaxation.
11. Define relaxation time.
Is the measure of the average life time of the nuclei in the higher-energy state.
12. What is meant by free induction decay?
In Fourier Transform NMR, free induction decay (FID) is the observable NMR signal generated by non-
equilibrium nuclear spin magnetization precessing about the magnetic field(conventionally along z).
13. What is a NMR spectrum?
The NMR spectrum is a plot of the intensity of NMR signals Vs Magnetic Field (Frequency) in
reference to TMS.
14. List the components of NMR instrument.
Sample holder, Permanent magnet, magnetic coils, sweep generator, radio frequency transmitter and radio
frequency receiver and read out systems.
15. Name some solvents used in NMR spectroscopy.
The following solvents are normally used in NMR in which hydrogen is replaced with deuterium.

27.  CCl4- carbon tetrachloride,
 CS2- carbon disulfide, D2O- deuterium oxide,
 CDCl3 –Deuteriochlorofor& C6D6 - HexaDeutriobenzene.
16. Define Chemical shift.
A chemical shift is defined as the difference in parts per million (ppm) between the resonance frequency of
the observed proton and tetramethylsilane (TMS) hydrogens.
17. Name the reference compound mostly used in TMS.
TMS (tetramethylsilane) is the most reference compound in NMR, it is set at
18. List the factors affecting chemical shift.
Electronegative groups –magnetic anisotropy–hydrogenbondingof. π electrons
19. What is n+1 rule?
The multiplicity of signal is calculated by using n+1 rule. This is one of the rule to predict the splitting of
proton signals. This is considered by the nearby hydrogen nuclei. Therefore, n = number of protons in the
nearby nuclei.
20. Define spin-spin coupling (splitting).
The interaction between the spins of neighboring nuclei in a molecule may cause the splitting of NMr
spectrum. This is known as spin-spin coupling or splitting. The splitting pattern is relted to the number of
equivalent H-atom at the nearby nuclei.
21. List the rules for spin-spin coupling.
 Chemically equivalent protons do not show spin-spin coupling.
 Only non equivalent protons couple.
 Protons on adjacent carbons normally will couple.
 Protons separated by four or more bonds will not couple.
22. Define coupling constant.
The distance between the peaks in a given multiplet is a measure of the splitting effect known as the
coupling constant. It is denoted by the symbol J, Expressed in Hz.Coupling constants are the measure of the
effectiveness of spin-spin coupling and very useful in 1H NMR of complex structures.
23. Define NOE.
NOE: Nuclear Over hauser Effect, caused by dipolar coupling between nuclei. The local field at one
nucleus is affected by the presence of another nucleus. The result is a mutual modulation of resonance
frequencies. The intensity of the interaction is a function of the distance between the nuclei according to the
following equation
24. Give the general applications of NMR spectroscopy.
 NMR is used in biology to study the biofluids, cells, per fused organs and biomacromolecules such as
Nucleic acids (DNA, RNA), carbohydrates, proteins and peptides. And also labeling studies in
biochemistry.

28.  NMR is used in physics and physical chemistry to study high pressure diffusion, liquid
crystals, liquid crystal solutions, membranes and rigid solids.
 NMR is used in food science.
 NMR is used in pharmaceutical science to study pharmaceuticals and drug metabolism.
 NMR is used in chemistry to determine the enantiomeric purity, elucidate chemical structure of
organic and inorganic compounds and macromolecules –ligand interaction
25. List the applications of 1H NMR spectroscopy.
1H NMR mainly used for structure elucidation. To examine hydrogen bonding and acidity in
polymers and rubbers. To study about proteins and peptides.
26. Give the applications of NMR in medicine.
MRI is the specialist application of multi-dimensional fourier transformation NMR. Anatomical
imaging-measuring physiological gunctions-flow measurements and angiography-tissue perfusion
studies-tumors.
27. What is shielding in NMR?
When the magnetic moment of an atom blocks the full induced magnetic field from surrounding
nuclei.
28. Define mass spectroscopy.
Is one of the primary spectroscopic methods for molecular analysis available to organic chemist. It is a
microanalytical technique requiring only a few nanomoles of the sample to obtain characteristic
information pertaining to the structure and molecular weight of the analyte.
29. Give the basic principle involved in mass spectroscopy.
In this technique, molecules are bombarded with a beam of energetic electrons. The molecules
are ionized and broken up into many fragments some of which are positive ions. Each kind of ions has a
particular ratio of mass to charge. i.e. m/e ratio (value). For most ions, the charge is one and thus, m/e ratio is
simply the molecular mass of the ion.
30. State Stevensons rule.
When an ion fragments, the positive charge will remain on the fragment of lowest ionization potential.
31. List the factors influencing fragmentation process.
 Bombardment energies
 Functional groups
 Thermal decomposition.
32. Define EPR spectroscopy.
Is a technique for studying materials with unpaired electrons.

29. UNIT-III
MAGNETIC RESONANCE SPECTROSCOPY AND MASS SPECTROMETRY
Theory of NMR
The nuclear magnetic resonance phenomenon can be described in a nutshell as follows. If a
sample is placed in a magnetic field and is subjected to radiofrequency (RF) radiation (energy) at the
appropriate frequency, nuclei in the sample can absorb the energy. The frequency of the radiation
necessary for absorption of energy depends on three things. First, it is characteristic of the type of
nucleus (e.g., 1H or 13C). Second, the frequency depends on chemical environment of the nucleus. For
example, the methyl and hydroxyl protons of methanol absorb at different frequencies, and amide
protons of two different tryptophan residues in a native protein absorb at different frequencies since they
are in different chemical environments. The NMR frequency also depends on spatial location in the
magnetic field if that field is not everywhere uniform.
The principle of NMR usually involves two sequential steps:
 The alignment (polarization) of the magnetic nuclear spins in an applied, constant magnetic field B0.
 The perturbation of this alignment of the nuclear spins by employing an electro-magnetic, usually
radio frequency (RF) pulse. The required perturbing frequency is dependent upon the static magnetic
field (H0) and the nuclei of observation.
The two fields are usually chosen to be perpendicular to each other as this maximizes the NMR
signal strength. The resulting response by the total magnetization (M) of the nuclear spins is the
phenomenon that is exploited in NMR spectroscopy and magnetic resonance imaging. Both use intense
applied magnetic fields (H0) in order to achieve dispersion and very high stability to deliver spectral
resolution, the details of which are described by chemical shifts, the Zeeman effect, and Knight
shifts (in metals).
Nuclear spins
Nuclei have positive charges. Many nuclei behave as though they were spinning. Anything that
is charged and moves has a magnetic moment and produces a magnetic field. Therefore, a spinning
nucleus acts as a tiny bar magnet oriented along the spin rotation axis (Figure 1.1). This tiny magnet is
often called a nuclear spin. If we put this small magnet in the field of a much larger magnet, its
orientation will no longer be random. There will be one most probable orientation. However, if the tiny
magnet is oriented precisely 180° in the opposite direction, that position could also be maintained (play
with a couple magnets to test this). In scientific jargon the most favorable orientation would be the low-
energy state and the less favorable orientation the high-energy state.

30. This two-state description is appropriate for most nuclei of biologic interest including 1H,
13C,15N, 19F, and 31P; i.e., all those which have nuclear spin quantum number I = l/2. It is a quantum
mechanical requirement that any individual nuclear spins of a nucleus with I = l/2 be in one of the two
states (and nothing in between) whenever the nuclei are in a magnetic field. It is important to note that
the most common isotopes of carbon, nitrogen and oxygen (12C, 14N and 16O) do not have a nuclear
spin.
Values of spin angular momentum
The angular momentum associated with nuclear spin is quantized. This means both that the
magnitude of angular momentum is quantized (i.e. S can only take on a restricted range of values), and
also that the orientation of the associated angular momentum is quantized. The associated quantum
number is known as the magnetic quantum number, m, and can take values from +S to −S, in integer
steps. Hence for any given nucleus, there are a total of 2S + 1 angular momentum states.
The z-component of the angular momentum vector (S) is therefore Sz = mħ, where ħ is the
reduced Planck constant. The z-component of the magnetic moment is simply:
The Resonance Phenomenon
The small nuclear magnet may spontaneously "flip'' from one orientation (energy state) to the
other as the nucleus sits in the large magnetic field. This relatively infrequent event is illustrated at the
left of Figure 1.2. However, if energy equal to the difference in energies ( E) of the two nuclear spin
orientations is applied to the nucleus (or more realistically, group of nuclei), much more flipping
between energy levels is induced (Figure 1.2). The irradiation energy is in the RF range (just like on
your FM radio station) and is typically applied as a short (e.g., many microseconds)
pulse. The absorption of energy by the nuclear spins causes transitions from higher to lower energy as
well as from lower to higher energy. This two-way flipping is a hallmark of the resonance process. The
energy absorbed by the nuclear spins induces a voltage that can be detected by a suitably tuned coil of
wire, amplified, and the signal displayed as free induction decay (FID). Relaxation processes (vide infra)
eventually return the spin system to thermal equilibrium, which occurs in the absence of any further
perturbing RF pulses. The energy required to induce flipping
and obtain an NMR signal is just the energy difference between the two nuclear orientations to depend
on the strength of the magnetic field Bo in which the nucleus is placed

31. Where h is Planck's constant (6.63 x 10-27 erg sec). The Bohr condition (∆E = hν) enables the frequency
νo of the nuclear transition to be written as
above equation is often referred to as the Larmor equation, and ωo = 2πνo is the angular
Larmor resonance frequency. The gyromagnetic ratio γ is a constant for any particular
type of nucleus and is directly proportional to the strength of the tiny nuclear magnet.
Lists the gyromagnetic ratios for several nuclei of biologic interest. At magnetic field
strengths used in NMR experiments the frequencies.
For nuclei (I=1/2) in a magnetic field of strength Bo at thermal equilibrium, i.e.,un perturbed,
there will be infrequent flips of individual nuclear spins between the two different energy levels. When a
radiofrequency (RF) pulse with appropriate energy is applied (i.e., equal to the difference n energies of
the two levels), transitions between the two energy levels will be induced, i.e., the nuclear spin system
will "resonate" the spin system absorbs the energy. Following the RF pulse, a signal termed free
induction decay or FID can be detected as a result of the voltage induced in the sample by the energy
absorption. Eventually the nuclear spin system relaxes to the thermal equilibrium situation.
Environmental effects on NMR spectra
Types of environmental effect:
Chemical shift:
Overall position of peak changes due to shielding of magnetic field by other nearby nuclei.
Spin-Spin Coupling:
Fine structure or splitting within a peak due to other nuclei that are 1, 2 or 3 chemical bonds
away.
Other Nuclear Magnetic Resonance Parameters
The various NMR spectral parameters to be discussed subsequently are illustrated in Figure.
Clearly, a one-dimensional spectrum is represented. However, as we encounter two-, three or four
dimensional spectra, it should be apparent how the features mentioned here may be manifest in those
multidimensional spectra.
1
1
2
2

32. Nuclear magnetic resonance spectral parameters
Chemical Shift
The shift in the positions of NMR signals (compared with a standard reference) resulting from
the shielding and deshielding by electrons are referred to as Chemical shift.
It is obvious from Equation 2 that nuclei of different elements, having different gyromagnetic
ratios, will yield signals at different frequencies in a particular magnetic field. However, it also turns out
that nuclei of the same type can achieve the resonance condition at different frequencies. This can occur
if the local magnetic field experienced by a nucleus is slightly different from that of another similar
nucleus; for example, the two 13C NMR signals of ethanol occur at different frequencies because the
local field that each carbon experiences is different.
The reason for the variation in local magnetic fields can be understood from the below Figure. If
a molecule containing the nucleus of interest is put in a magnetic field Bo, simple electromagnetic
theory indicates that the Bo field will induce electron currents in the molecule in the plane perpendicular
to the applied magnetic field. These induced currents will then produce a small magnetic field opposed
to the applied field that acts to partially cancel the applied field, thus shielding the nucleus. In general,
the induced opposing field is about a million times smaller than the applied field. Consequently, the
magnetic field perceived by the nucleus will be very slightly altered from the applied field, so the
resonance condition of Equation 2 will need to be modified.
Where Blocal is the local field experienced by the nucleus and σ is a non dimensional screening or
shielding constant. The frequency ν at which a particular nucleus achieves resonance clearly depends on
the shielding which reflects the electronic environment of the nucleus.
3

33. Electron currents around a nucleus are induced by placing the molecule in a magnetic field Bo.
These electron currents, in turn, induce a much smaller magnetic field opposed to the applied magnetic
field Bo.
There will be more electronic currents induced in the molecule than just those directly around the
nucleus. In fact, some of those currents may increase Blocal (below the Figure 1.15). Therefore, the
shielding and the resulting resonance frequency will depend on the exact characteristics of the electronic
environment around the nucleus. The induced magnetic fields are typically a million times smaller than
the applied magnetic field. So if the Larmor resonance frequency νo is on the order of several megahertz,
differences in resonance frequencies for two different hydrogen nuclei, for example, will be on the order
of several hertz. Although we cannot easily determine absolute radiofrequencies to an accuracy of ±1
Hz, we can determine the relative positions of two signals in the NMR spectrum with even greater
accuracy. Consequently, a reference signal is chosen, and the difference between the position of the
signal of interest and that of the reference is termed the chemical shift.
Although a chemical shift could be expressed as the frequency difference in hertz, it is clear from
either Equation 2 or 3that the chemical shift in Hz would depend on the magnetic field in which the
sample was placed. To remove the dependence of the chemical shift on magnetic field.
Effects at nucleus X caused by the secondary magnetic field arising from induce electronic currents at
nucleus Y

34. Strength and therefore operating frequency, the chemical shift is usually expressed in terms of parts per
million (ppm), actually a dimensionless number, by
Where the difference between the resonance frequency of the reference and the sample (ν ref –ν sample)
measured in hertz (e.g., 75 Hz) divided by the spectrometer’s operating frequency (e.g., 500 MHz) gives
the chemical shift (e.g., 0.15 ppm). Typical ranges in chemical shifts for signals emanating from
biochemically important samples are 1H, 15 ppm; 13C, 250 ppm; 15N, 400 ppm; and 31P, 35 ppm.
Spin-Spin Coupling (Splitting)
A nucleus with a magnetic moment may interact with other nuclear spins resulting in mutual
splitting of the NMR signal from each nucleus into multiplets. The number of components into which a
signal is split is 2nI+1, where I is the spin quantum number and n is the number of other nuclei
interacting with the nucleus. For example, a nucleus (e.g., 13C or 1H) interacting with three methyl
protons will give rise to a quartet. To a first approximation, the relative intensities of the multiplets are
given by binomial coefficients: 1:1 for a doublet, 1:2:1 for a triplet, and 1:3:3:1 for a quartet. The
difference between any two adjacent components of a multiplet is the same and yields the value of the
spin-spin coupling constant J (in hertz). One important feature of spin-spin splitting is that it is
independent of magnetic field strength. So increasing the magnetic field strength will increase the
chemical shift difference between two peaks in hertz (not parts per million), but the coupling constant J
will not change. To simplify a spectrum and to improve the S/N ratio, decoupling (usually of protons) is
often employed, especially with 13C and 15N NMR. Strong irradiation of the protons at their resonance
frequency will cause a collapse of the multiplet in the 13C or 15N resonance into a singlet.
NMR spectroscopy
NMR spectroscopy is one of the principal techniques used to obtain physical, chemical,
electronic and structural information about molecules due to either the chemical shift, Zeeman effect, or
the Knight shift effect, or a combination of both, on the resonant frequencies of the nuclei present in the
sample.
Types of NMR spectroscopy
 Continuous wave spectroscopy
 Fourier transforms spectroscopy
Continuous-wave (CW) spectroscopy
In its first few decades, nuclear magnetic resonance spectrometers used a technique known
as continuous-wave spectroscopy (CW spectroscopy). Although NMR spectra could be, and have been,
obtained using a fixed magnetic field and sweeping the frequency of the electromagnetic radiation, this
more typically involved using a fixed frequency source and varying the current (and hence magnetic
field) in an electromagnet to observe the resonant absorption signals. This is the origin of the
counterintuitive, but still common, "high field" and "low field" terminology for low frequency and high
frequency regions respectively of the NMR spectrum.
4
4
Type equation here.

35. CW spectroscopy is inefficient in comparison with Fourier analysis techniques, since it probes
the NMR response at individual frequencies in succession. Since the NMR signal is intrinsically weak,
the observed spectrum suffers from a poor signal-to-noise ratio. This can be mitigated by signal
averaging i.e. adding the spectra from repeated measurements. While the NMR signal is constant
between scans and so adds linearly, the random noise adds more slowly – proportional to the square
root of the number of spectra (see random walk). Hence the overall signal-to-noise ratio increases as the
square-root of the number of spectra measured.
Fourier-transform spectroscopy
Most applications of NMR involve full NMR spectra, that is, the intensity of the NMR signal as
a function of frequency. Early attempts to acquire the NMR spectrum more efficiently than simple CW
methods involved illuminating the target simultaneously with more than one frequency. A revolution in
NMR occurred when short pulses of radio-frequency radiation began to be used—centered at the middle
of the NMR spectrum. In simple terms, a short pulse of a given "carrier" frequency "contains" a range of
frequencies centered about the carrier frequency, with the range of excitation (bandwidth) being
inversely proportional to the pulse duration, i.e. the Fourier transform of a short pulse contains
contributions from all the frequencies in the neighborhood of the principal frequency. The restricted
range of the NMR frequencies made it relatively easy to use short (millisecond to microsecond) radio
frequency pulses to excite the entire NMR spectrum.

36. Applying such a pulse to a set of nuclear spins simultaneously excites all the single-quantum
NMR transitions. In terms of the net magnetization vector, this corresponds to tilting the magnetization
vector away from its equilibrium position (aligned along the external magnetic field). The out-of-
equilibrium magnetization vector precesses about the external magnetic field vector at the NMR
frequency of the spins. This oscillating magnetization vector induces a current in a nearby pickup coil,
creating an electrical signal oscillating at the NMR frequency. This signal is known as the free induction
decay (FID), and it contains the vector sum of the NMR responses from all the excited spins. In order to
obtain the frequency-domain NMR spectrum (NMR absorption intensity vs. NMR frequency) this time-
domain signal (intensity vs. time) must be Fourier transformed. Fortunately the development of Fourier
Transform NMR coincided with the development of digital computers and the digital Fast Fourier
Transform. Fourier methods can be applied to many types of spectroscopy
Applications of 1H and 13C NMR
13C NMR
We will first concentrate on Carbon. The most abundant isotope 12C has no overall nuclear spin,
having an equal number of protons and neutrons. The 13C isotope however does have spin 1/2, but is
only 1% abundant. Carbon NMR spectra are characterized by the following;
* A chemical shift range of about 220 ppm, normally expressed relative to the 13C resonance of TMS.
* A natural line width of ca 1Hz, related to the values of the relaxation times T1 and T2.
* A Larmor frequency in the range of 20-100 MHz, for typical spectrometers.
* Typically about 5-20 mg of sample dissolved in 0.4 - 2 ml of solvent (normally CDCl3) are required,
and a good spectrum would be obtained in 64 - 6400 scans.

37. Let’s start by looking at a 13C spectrum of diethyl phthalate obtained by the FT technique,
First we note the wide chemical shift range of the signals. Note also that the signal we attribute
to the methyl group is approximately a 1:3:3:1 quartet, the methylene approximately a 1:2:1 triplet, and
the aromatic CHs approximately 1:1 doublets. The intensity ratios suggest this is due to coupling and the
multiplicities that it is due specifically to coupling of the spin 1/2 13
C with the spin 1/2 protons and
nothing else (ie the 2nI+1 rule, I being the spin number). Before we move on to discuss how this
coupling may be useful to us, let us remind ourselves of how the coupling arises. Remember the energy
level diagram of one spin 1/2 nucleus in a magnetic field.
The processing magnetization vector either reinforces or opposes Bo, so that locally at least,
other nuclei will perceive two slightly different values of Bo. Since the populations of each energy level
are practically identical), another nucleus close-by will resonate with equal probability at two slightly
different Larmor frequencies. The difference between these two frequencies is what we know at the
coupling constant J. Its value depends on how the perturbation in Bo is transmitted between the two
nuclei, and this is normally achieved via the intervening electrons (hence the term through bond
coupling). When two identical nuclei are involved, three slightly different and equally spaced values
of Bo, with the middle one being twice as probable as the highest or lowest, hence the 1:2:1 triplet

38. coupling pattern we are familiar with. In spin terms, we say that four configurations are possible; +1/2,
+1/2; +1/2, -1/2; -1/2, +1/2; -1/2, -1/2. As the middle two are of equal energy, this manifest as a double
height peak, i.e. a 1:2:1 triplet. If we remember that J(coupling) = x ω o/106
, these visible in the carbon
spectrum above look in the range JC-H ~ 6 x 22 ~ 130 Hz. Notice that carbon appears not to couple with
other carbons, only with protons in the same molecule. This is because the probability that two 13
C-13
C
nuclei will be close enough to couple is 100 times less than the probability of finding 13
C-12
C as adjacent
nuclei.
These spectra give the following information;
 The proton decoupled spectrum tells the number of unique types of carbon atoms in the molecule
(i.e. a mono-substituted phenyl group has four unique carbon atoms)
 The off-resonance spectrum tells how many hydrogen atoms are attached to each unique carbon
(quartet=3, triplet=2, doublet=1, singlet=none).
 From the chemical shift of each carbon, much information about the environment of the carbon
can be gleaned.
The typical chemical shift ranges of carbon nuclei are as follows;
1H NMR
The application of 1H NMR to living cells is used to determine metabolites in complex mixtures
and has been widely used for identification and quantification of the bacterial species. This technique
has also been applied for antimicrobial drug susceptibility studies on different species of yeast, and in
the last few years, it has also been developed for bacterial studies. Furthermore, other determinations
directly in body fluids have emerged to help in the diagnosis of different diseases and conditions.
1. Bacterial identification and metabolic studies
1H NMR spectroscopy has been used for bacterial identification and quantification and for
metabolic pathways studies. Several studies have been conducted for the diagnosis of the bacteria that
cause urinary tract infections (UTI). These focus on the use of 1H NMR spectroscopy for the
identification and quantification of common uropathogens such as Pseudomonas aeruginosa, Klebsiella
pneumoniae, Escherichia coli, and Proteus mirabilis in urine samples. These studies are based on
specific properties of the metabolism of the studied bacteria, and the results showed that 1H NMR is a
simple and fast tool compared with the traditional methods.
The qualitative and quantitative determination of P. aeruginosa using NMR spectroscopy is
based on the specific property of the bacteria to metabolize nicotinic acid (NA) to 6-hydroxynicotinic
acid (6-OHNA). Only this bacterium can produce this reaction. The addition of NA to urine samples

39. after incubation and the subsequent analysis by 1H NMR spectroscopy showed that NA signals
disappeared from the medium after some time, while the appearance of new signals of the metabolite 6-
OHNA indicated the presence of P. aeruginosa. The increase in the intensity of the metabolite signals,
together with the decrease in the NA signals, involved a proportional increase in the number of bacteria.
This shows the potential offered by this technique for quantitative and qualitative identification,
simultaneously, on the bacteria.
2. Antimicrobial susceptibility assays
Application of 1H NMR spectroscopy to antimicrobial susceptibility studies was first carried out
on different species of yeast. The standardized methods currently available for fungal susceptibility
studies are unreliable and relatively slow, so, 1H NMR spectroscopy can be a simple indicator, an
objective and fast method (metabolic changes detected by this method are more easily observed than
growth inhibition in broth). 1H NMR spectroscopy is potentially valuable in determining the metabolic
composition of yeast suspensions incubated with a drug. In addition, it is a high performance automated
method with low operating costs, so that both operator time and reagent cost are greatly reduced.
Therefore, it has great potential to emerge as an alternative method for the antifungal drug susceptibility
determination of different yeast species.
3. Biofluids
1H NMR has been used to directly analyse biofluids and to diagnose different diseases directly
from body fluids. In this sense, it has been applied to analyse human microbiota from faeces and urine
samples, to study the metabolic implications that take place in sepsis, or even to diagnose hepatitis C
virus infection, distinguish HIV-1 positive patients from negative individuals or to diagnose pneumonia
from urine.
4. Other types of analyses
The combination of NMR spectroscopy, with the use of isotopically substituted molecules as
tracers is a well‐established protocol in microbiology. These NMR analyses appear to be the most
appropriate for such studies because of their analytical power (provided that the labeling of products can
be easily monitored non‐invasively), their non‐destructive features, and the large number of compounds
that can be analysed simultaneously. However, despite the great potential of this combination in clinical
practice.

40. UNIT –IV
SEPARATION METHODS
TYPES OF CHROMATOGRAPHY
Chromatography can be classified by various ways
 On the basis of interaction of solute to the stationary phase
 On the basis of chromatographic bed shape
 Techniques by physical state of mobile phase
ON THE BASIS OF INTERACTION OF SOLUTE TO STATIONARY PHASE:
(i) ADSORPTION CHROMATOGRAPHY
Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes
a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The
equilibration between the mobile and stationary phase accounts for the separation of different solutes
(ii) PARTITION CHROMATOGRAPHY
This form of chromatography is based on a thin film formed on the surface of a solid support by a
liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid.

41. ION EXCHANGE CHROMATOGRAPHY
In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently
attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to
the resin by electrostatic forces
MOLECULAR EXCLUSION CHROMATOGRAPHY
Also known as gel permeation or gel filtration, this type of chromatography lacks an attractive interaction
between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which
separates the molecules according to its size. The pores are normally small and exclude the larger solute
molecules, but allow smaller molecules to enter the gel, causing them to flow through a larger volume. This
causes the larger molecules to pass through the column at a faster rate than the smaller ones.

42. ON THE BASIS OF CHROMATOGRAPHIC BED SHAPE
COLUMN CHROMATOGRAPHY
Column chromatography is a separation technique in which the stationary bed is within a tube. The
particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the
whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall
leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular
column). Differences in rates of movement through the medium are calculated to different retention times
of the sample.
The technique is very similar to the traditional column chromatography, except for that the solvent is
driven through the column by applying positive pressure. This allowed most separations to be performed in
less than 20 minutes, with improved separations compared to the old method. Modern flash chromatography
systems are sold as pre-packed plastic cartridges, and the solvent is pumped through the cartridge. Systems
may also be linked with detectors and fraction collectors providing automation. The introduction of gradient
pumps resulted in quicker separations and less solvent usage.
In expanded bed adsorption, a fluidized bed is used, rather than a solid phase made by a packed bed.
This allows omission of initial clearing steps such as centrifugation and filtration, for culture broths or
slurries of broken cells.
PLANAR CHROMATOGRAPHY
Planar chromatography is a separation technique in which the stationary phase is present as or on a
plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper
chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer
chromatography). Different compounds in the sample mixture travel different distances according to how
strongly they interact with the stationary phase as compared to the mobile phase. The specific Retention
factor (Rf) of each chemical can be used to aid in the identification of an unknown substance.
PAPER CHROMATOGRAPHY
Paper chromatography is a technique that involves placing a small dot or line of sample solution
onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent and
sealed. As the solvent rises through the paper, it meets the sample mixture which starts to travel up the paper
with the solvent. This paper is made of cellulose, a polar substance, and the compounds within the mixture
travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and
therefore do not travel as far.
THIN LAYER CHROMATOGRAPHY
Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to paper
chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a
thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it
has the advantage of faster runs, better separations, and the choice between different adsorbents. For even
better resolution and to allow for quantification, high-performance TLC can be used.
TECHNIQUES BY PHYSICAL STATE OF MOBILE PHASE
GAS CHROMATOGRAPHY
Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a
separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a
column, which is typically "packed" or "capillary". Gas chromatography (GC) is based on a partition
equilibrium of analyte between a solid stationary phase (often a liquid silicone-

43. based material) and a mobile gas (most often Helium).The stationary phase is adhered to the inside of a
small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed
column). It is widely used in analytical chemistry; though the high temperatures used in GC make it
unsuitable for high molecular weight biopolymers or proteins (heat will denature them), frequently
encountered in biochemistry, it is well suited for use in the petrochemical, environmental monitoring, and
industrial chemical fields. It is also used extensively in chemistry research.
LIQUID CHROMATOGRAPHY
Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid
chromatography can be carried out either in a column or a plane. Present day liquid chromatography that
generally utilizes very small packing particles and a relatively high pressure is referred as high performance
liquid chromatography (HPLC). In the HPLC technique, the sample is forced through a column that is
packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a
liquid (mobile phase) at high pressure. HPLC is historically divided into two different sub-classes based on
the polarity of the mobile and stationary phases. Technique in which the stationary phase is more polar than
the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) is called normal phase
liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the mobile phase and C18
= octadecylsilyl as the stationary phase) is called reversed phase liquid chromatography (RPLC). Ironically
the "normal phase" has fewer applications and RPLC is therefore used considerably more.
Specific techniques which come under this broad heading are listed below. It should also be noted
that the following techniques can also be considered fast protein liquid chromatography if no pressure is
used to drive the mobile phase through the stationary phase.
AFFINITY CHROMATOGRAPHY
Affinity chromatography is based on selective non-covalent interaction between an analyte and
specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the purification
of proteins bound to tags. These fusion proteins are labeled with compounds such as His-tags, biotin or
antigens, which bind to the stationary phase specifically. After purification, some of these tags are usually
removed and the pure protein is obtained. Affinity chromatography often utilizes a biomolecule's affinity for
a metal (Zn, Cu, Fe, etc.). Columns are often manually prepared. Traditional affinity columns are used as a
preparative step to flush out unwanted biomolecules. However, HPLC techniques exist that do utilize
affinity chromatography properties. Immobilized Metal Affinity Chromatography (IMAC) is useful to
separate aforementioned molecules based on the relative affinity for the metal (I.e. Dionex IMAC). Often
these columns can be loaded with different metals to create a column with a targeted affinity.
HIGH PERFORMANCE LC

44. High performance liquid chromatography is now one of the most powerful tools in analytical
chemistry. It has the ability to separate, identify, and quantitate the compounds that are present in any
sample that can be dissolved in a liquid. Today, compounds in trace concentrations as low as parts per
trillion (ppt) may easily be identified. HPLC can be, and has been, applied to just about any sample, such as
pharmaceuticals, food, nutraceuticals, cosmetics, environmental matrices, forensic samples, and industrial
chemicals. The components of a basic high-performance liquid chromatography (HPLC) system are shown
in the simple diagram below.
A reservoir (Solvent Delivery) holds the solvent (called the mobile phase, because it moves). A high-
pressure pump solvent manager is used to generate and meter a specified flow rate of mobile phase, typically
milliliters per minute.An injector (sample manager or auto sampler) is able to introduce (inject) the sample
into the continuously flowing mobile phase stream that carries the sample into the HPLC column. The
column contains the chromatographic packing material needed to effect the separation. This packing
material is called the
stationary phase because it is held in place by the column hardware. A detector is needed to see the
separated compound bands as they elute from the HPLC column (most compounds have no color, so we
cannot see them with our eyes).
Separation of two peaks with resolution values of (a) 0.75, (b) 1.0 and (c) 1.5.
The mobile phase exits the detector and can be sent to waste, or collected, as desired. When the
mobile phase contains a separated compound band, HPLC provides the ability to collect this fraction of the

45. eluate containing that purified compound for further study. This is called preparative chromatography. The
high-pressure tubing and fittings are used to interconnect the pump, injector, column, and detector
components to form the conduit for the mobile phase, sample, and separated compound bands.
The detector is wired to the computer data station, the HPLC system component that records the
electrical signal needed to generate the chromatogram on its display and to identify and quantitate the
concentration of the sample constituents. Since sample compound characteristics can be very different,
several types of detectors have been developed. For example, if a compound can absorb ultraviolet light, a
UV-absorbance detector is used. If the compound fluoresces, a fluorescence detector is used. If the
compound does not have either of these characteristics, a more universal type of detector is used, such as an
evaporative-light-scattering detector (ELSD). The most powerful approach is the use multiple detectors in
series. For example, a UV and/or ELSD detector may be used in combination with a mass spectrometer
(MS) to analyze the results of the chromatographic separation. This provides, from a single injection, more
comprehensive information about an analyte. The practice of coupling a mass spectrometer to an HPLC
system is called LC/MS.
Ion Exchange Liquid Chromatography
Elution order in ion exchange chromatography is determined by the charge density (charge/radius) of the
hydrated ion. In organic acids and bases the elution order is determined by their pKa or pKb (strength of
acid or base).
Different Types of Ion Exchange Resins:

46. Gel Permeation Chromatography --Molecular Sieve Chromatography
The separation is based on the molecule size and shape by the molecular sieve properties of a variety of
porous material

48. Band Broadening and Column Efficiency
• Band broadening affects the efficiency of the chromatographic column
• Why do bands become broader as they move down the column?
Rate theory of Chromatography
Random-walk mechanism
 Although the general direction of migration is towards the bottom of the column, random walk is
superimposed on the general movement forward – Random motion during migration explains the
shape and the breath of chromatographic peaks.
 Gaussian distribution around mean retention time.
 Residence time in either phase is irregular a few particles travel faster because they are
accidentally included in the mobile phase most of the time. Some particles lag behind because
they are incorporated in the stationary phase for a time longer than the average.
 Width of band/zone is directly related to the residence time and inversely related to the velocity
of the mobile phase flow.

49. Capillary Electrophoresis
Capillary Electrophoresis (CE) is one of the possible methods to analyse complex samples. In High
Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) the separating force is the
difference in affinity of the sample components to a stationary phase, and or difference in boiling point. With
both techniques the most important factor is the polarity of a sample component. In CE the separating force
is the difference in charge to size ratio. Not a flow through the column, but the electric field will do the
separation.
In Capillary Electrophoresis a capillary is filled with a conductive fluid at a certain pH value. This is
the buffer solution in which the sample will be separated. A sample is introduced in the capillary, either by
pressure injection or by electro kinetic injection. A high voltage is generated over the capillary and due to
this electric field (up to more than 300 V/cm) the sample components move (migrate) through the capillary
at different speeds. Positive components migrate to the negative electrode, negative components migrate to
the positive electrode. When you look at the capillary at a certain place with a detector you will first see the
fast components pass, and later on the slower components.