Q1.

objective and types of derivatization with example

Objectives for derivatization

*Improvement of resolution and reduce tailing of polar compounds which may contain - OH, -COOH,
NH, -NH. -SH, and other functional groups,

*Analysis of relatively nonvolatile compounds

*Reduction of volatility of compounds prior to GC analysis.

*Improvement of analytical efficiency and hence increase detectability.

Types of Derivatization

1.Silylation : The most prevalent method, readily volatilizes the sample .

Mechanism: This process produces silyl derivatives which are more volatile, and more thermally
stable. Replaces active hydrogens with TMS (trimethyl silyl groups). It occurs by the nucleophilic
substitution attack. e.g. N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA).

2. Alkylation: Alkylation reduces molecular polarity by replacing active hydrogens with an alkyl group.
These reagents are used to modify compounds with acidic hydrogens, such as carboxylic acids and
phenols. These reagents make esters, ethers, alkyl amines, and alkyl amides. e.g. Boron trichloride in
chloroethanol or methanol.

3. Acylation: Alkylation reduces molecular polarity by replacing active hydrogens with an alkyl group.
These reagents are used to modify compounds with acidic hydrogens, such as carboxylic acids and
phenols. These reagents make esters, ethers, alkyl amines, and alkyl amides.eg . Acetic anhydride

4. Chiral derivatization: these reagents target one specific functional group and produce individual
diastereomers of each of the enantiomers.eg. tartaric acid and the amine brucine.
Q2. Evaluate the instrumentation of HPLC

Principle :(Hplc) high-pressure liquid chromatography, is a chromatographic technique that can

separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify,
quantify and purify the individual components of the mixture.

types of pump

1. Gas-driven discontinuous displacement system

2. Discontinuous displacement pumps

3. Membrane piston pumps

4. Short-stroken piston pump

Method

1. Internal diameter and Length

The internal diameter (ID) of an HPLC column is a critical aspect that determines quantity of sample
that can be loaded onto the column and also influences sensitivity.

2. Particle size

Most traditional HPLC is performed with the stationary phase of small spherical silica particles (very
small beads). These particles come in a variety of sizes with Sum beads being the most common.

3. Pore size

Many stationary phases are porous (3, 5 or 10 um in size) to provide greater surface area. Small
pores provide greater surface area while larger pore size has better kinetics especially for larger
sample.

4.Pump pressure

Pumps vary in pressure capacity, but their performance is measured on their ability to yield a
consistent and reproducible flow rate. Pressures may reach as high 400 atmospheres (IN HPLC) .

5.Injection Volume -For a fixed volume auto-sampler or manual valve, this is set by changing the
loop. For a variable volume auto-sampler, it is set in the auto-sampler method. 10 micro liter

6.Flow rate - Usually around I ml/min for an analytical column, slower if using polymer based
columns

7.Run time -Usually from 2-60 min.

Q3. Evaluate the instrumentation of Gas chromatography

Gas chromatography differs from other forms of chromatography in that the mobile phase is a gas
and the components are separated as vapors.

Instrumentation:

Carrier gas: Helium, N2, H, Argon are used as carrier gases.

Sample injection system:Liquid samples are injected by a microsyringe with a needle inserted
through a self-scaling, silicon-rubber septum into a heated metal block by a resistance heater.

The separation column:The heart of the gas chromatography is the column which is made of metals
bent in U shape or coiled into an open spiral or a flat pancake shape

Liquid phases:An infinite variety of liquid phases are available limited only by their volatility, thermal
stability and ability to wet the support.

Support-The support should be inert but capable of immobilizing a large volume of liquid phase as a
thin film over its surface.

Detector:Detectors sense the arrival of the separated components and provide a signal. These are
either concentration-dependent or mass dependant
Q4. . Explain the principles and types and application of HPLC

the principle of HPLC is to force the sample through the column of the stationary phase by pumping
the mobile phase at high pressure. The sample to be analyzed is introduced in small volume to the
stream of mobile phase and the sample molecules are retained by specific chemical and physical
interactions with the materials of the stationary phase as it travels the length of the column. The
amount of retention depends on-

(1) the nature of the sample.

(2) the nature of the composition of the stationary phase and

(3) the nature of the composition of the mobile phase

types of HPLC

[ 1). Normal phase HPLC (NP-HPLC) separates samples based on polarity.

This method uses (i)a polar stationary phase (ii)a non-polar mobile phase, and (iii)is used when the
samples is polar in nature.

2).Reversed phase HPLC chromatography In reversed phase HPLC (RP-HPLC) following applies:

(a)The stationary phase is very non-polar (b)The mobile phase is relatively polar

(c)A polar solvent such as water elutes more slowly than a less polar solvent such as acetonitrile.

Applications

• Water purification.

• Detection of impurities in pharmaceutical industries.

• Pre-concentration of trace components.

• Ligand-exchange chromatography.

• Ion-exchange chromatography of proteins.

• High-pH anion-exchange chromatography of carbohydrates and oligosaccharides.

Q5. Explain the factors affecting ion exchange and it's application.

FACTORS AFFECTING ION EXCHANGE

1. NATURE AND PROPERTIES OF ION EXCHANGE RESINS

• Cross linking and swelling are important factors, when more cross linking agent is present,
the resin becomes more rigid and swells less (has small pore size). This makes separations of
ions of different sizes more difficult as they can not pass through the pores present and it
becomes selective to ions of different (smaller) sizes.
• The nature of resin whether cationic or anionie exchanger, which determines strongly its
selectivity. Cationic resin is selective for cations and vice versa.
• Also, the resin capacity (number of m° equivalents of replaceable ions per gram of dry resin)
is important.

2.NATURE OF EXCHANGING IONS IN THE SAMPLE

a. Valence of ions: At low concentrations, the extent of exchanges increases with increase in valence;
Ions with higher charge is more selective;

b. Size of ions: For similarly charged ions; the exchange selectivity increases with decrease in the size
of hydrated ions

C.Polarizability: Highly polarizable ions are more selective.

d.Concentration of solutions: In dilute solutions poly-valent ions are generally absorbed

preferentially.

3.NATURE OF MOBILE PHASE AND PH

•The presence of other ions that compete with the sample for binding to the ion exchanger (using of
electrolyte).

The pH of the solution which influences the net charge of the sample (as in case of amino acids)

APPLICATIONS

1.Clinical applications: As in separation of amino acids and proteins. Ion exchange chromatography is
a principle technique for analyses of the amino acids and proteins.

2.Separation of similar ions: By passing much water over a resin and then elute with a high
concentration of acid. Cation exchangers trap cations. It is important for trace analysis, where
solubility

[s] is extremely low . It is important for environmental problem.

5.Water deionization and softening: Removal of cations by cation exchangers and anion exchangers
for amino removal.
Q6. Explain the principles and methodology of IEC (ion exchange chromatography)

PRINCIPLE

•The principle involved is the reversible exchange of ions between the ions present in the solution
and those present in the resin.

METHOD

1. Column: Column used in the laboratories are made up of glass but those used in industries are
made up of either high quality stainless steel or polymer, which are resistant to strong acids and
alkalis.

• A Dimension of column is 20:1 to 100:1 for the higher efficiency can be used.

2. Mobile phase: The organic solvents are less useful so they are not used these days. Only different
strength of acids, alkalis and buffer are used as cluting solvent.

• E.g.: 0.1 N HCI, IN NaOH, phosphate buffers, acetate buffers, borate buffers, phthalate buffers, etc.

3. Developments of the chromatogram and elution: The choice of the mobile phase depends on the
selectivity of the resin for the solute ions. Two types of elution techniques are used:

a.Isocratic elution

b.Gradient elutio

4. Analysis of the lute or Detection: Different fractions are collected with respect to the volume or
time is analyzed for their contents. Several methods of analysis can be used which depends upon the
nature and quantity of the ionic species are;

a..Conductometric method

b.Ampherometric methods

c.Flame photometric method.

d.Polarography

e.UV. Spectroscopy

f.Radiochemical methods using Geiger Muller counter, ionization chamber method

Conductivity detector is the most common and useful detector in ion exchange chromatography.
Conductivity detection gives excellent sensitivity when the conductance of the luted solute ion is
measured in an eluent of low background.
Q7. separation procedure of gel chromatography.

Gel chromatography, also known as size exclusion chromatography, separates molecules based on
their size.

1.Column Preparation :Pack a column with a gel matrix, typically composed of porous beads. The
beads have different pore sizes, allowing molecules of various sizes to enter the gel.

2.Sample Application: Inject the sample mixture onto the top of the gel column. Larger molecules
can't enter the pores and move through the gel matrix more quickly, while smaller molecules enter
the pores and take longer paths.

3.Elution: As the sample moves through the gel, the larger molecules pass through the column more
quickly, eluting first. Smaller molecules take longer paths through the pores and elute later.

4.Detection: Monitor the elution process, often using UV absorbance or other detectors to identify
and quantify the separated molecules.

5.Calibration: Calibrate the column using standards of known molecular weights to create a standard
curve, relating elution volume to molecular size.

6.Fraction Collection: Collect fractions as different molecules elute from the column, allowing for
further analysis or use.
Q8. principle , disadvantage and application of Gel chromatography.

Principle:

*The gel chromatography is based on the molecular size and the hydrodynamic volume of the
components.

*It is a technique in which the separation of components is based on the difference in molecular
weight or size.

•The stationary phase used is a porous polymer matrix whose pores are completely filled with the
solvent to be used as the mobile phase.

• The molecules in the sample are pumped through specialized columns containing such
microporous packing material (gel).

•The basis of the separation is that molecules above a certain size are totally excluded from the
pores.

Disadvantages

The disadvantages of gel electrophoresis are:

• The gel can be altered and provide inaccurate

results.

• User error can be catastrophic, depending on

the mistake

*gels can melt during electrophoresis, the buffer can become exhausted, and different forms of
genetic material may run in unpredictable forms.

Application

1. Proteins fractionation

2. Purification

3. Molecular weight determination.

4. Separation of sugar, proteins, peptides, rubbers, and others on the basis of their size.

5. Can be used to determine the quaternary structure of purified proteins.

6. Group separations and desalting.

7.separation of DNA fragments

Q9. instrumentation and application of TEM (Transistion electron microscopic)

application

The main application of a transmission electron microscope is to provide high marnification images
of the internal Structure of a sample.

• Being able to obtain an internal image of a sample opens new possibilities or wha sor or
informacion can be gathered from it.

• A TEM operator can investigate the crystalline structure of an obiect, see the stress or internal
fractures of a sample, or even view contamination within a sample chrough the use or dittraction
patterns. to name juss a row kinds or Studios

Instrumentation

The main components of the TEM include a vacuum system, specimen stage, electron gun, electron
lens, and apertures.

Vacuum system: The vacuum system in the TEM requires several stages to obtain the operating
stage. Initially, low vacuum is achieved using the vane pumps setting up low pressure which allows
the operation of diffusion pumps to establish high vacuum levels for operations.

Specimen stage: The specimen stage design is an airlock system allowing the insertion of the
specimen with minimal loss of vacuum. There are various designs of stages and holders that exist
which depend on the type of application.

Electron gun: The electron gun is used to produce the beam of electrons that is focused on the
specimen. It consists of a filament, biasing circuit, wehnelt cap, and an extraction anode,

Electron lens: These are the electromagnetic lenses that are used to focus the beam of electrons
onto the specimen. These lenses are designed in a way that emulates the optical lens by focusing
parallel electrons at a constant focal distance.

Apertures: Apertures are used to exclude the electrons which are scattered at high angles and are
unwanted. It is made up of metallic plates which are sufficiently thick to prevent from passing
through it.
Q10. instrumentation and application of SEM ( Scanning Electron microscope)

A Scanning Electron Microscope (SEM) is a powerful imaging tool that uses electrons to visualize the
surface details of specimens at a high resolution.

Instrumentation:

1.Electron Source: SEMs use an electron gun to generate a focused beam of electrons.

2.Electron Lenses: Magnetic lenses focus and control the electron beam.

3.Specimen Chamber: The specimen is placed in a vacuum chamber to prevent electron scattering.

4.Detectors: Various detectors capture signals emitted or scattered by the specimen.

5.Control and Imaging System: Computer systems control the microscope and process the signals to
generate images.

Applications:

1.Biology: SEM is used to examine biological specimens like cells, tissues, and microorganisms,
offering detailed surface information.

2.Geology: Geologists use SEM to investigate the structure and composition of rocks, minerals, and
geological samples.

3.Nanotechnology: SEM plays a vital role in nanoscale research, enabling the visualization and
characterization of nanomaterials.

4.Forensics: SEM aids forensic investigations by examining trace evidence like fibers, hairs, and
particles.

5.Archaeology: SEM helps in the analysis of archaeological artifacts, providing information about
their composition and surface features.
Q11. instrumentation of AAS ( atomic absorption spectroscopy)

Atomic Absorption Spectroscopy (AAS) is a technique used to analyze the concentration of elements
in a sample by measuring the absorption of light. The instrumentation for AAS typically includes the
following components:

1.Light Source: A hollow cathode lamp is commonly used as the light source. It emits light at specific
wavelengths corresponding to the element of interest.

2.Monochromator: This component is responsible for selecting the desired wavelength of light. A
diffraction grating or prism is often employed to disperse the light into its individual wavelengths,
and a slit allows only the desired wavelength to pass through.

3.Sample Introduction System: The sample is introduced into the instrument through various
methods, such as a nebulizer for liquid samples or a graphite furnace for solid samples. The goal is to
convert the sample into a vaporized form for analysis.

4.Atomizer: The atomization process converts the sample into free atoms, which is crucial for
accurate absorption 5.measurements. Common atomization techniques include flame atomization
for liquid samples and electrothermal atomization (furnace) for solid samples.

6.Detector: A photodetector, typically a photomultiplier tube (PMT), is used to measure the intensity
of the transmitted light after it passes through the sample. The detector generates a signal
proportional to the absorption of light by the atoms.

7.Signal Processor: The signal from the detector is processed to quantify the amount of absorbed
light. This involves comparing the intensity of the transmitted light with that of a reference or blank
solution.

8.Display: The final result, usually in terms of concentration or absorbance, is displayed for analysis.
Q12. Discuss the instrumentation of Flame photometry

Flame photometry is a technique used for elemental analysis based on the emission of light by
excited atoms in a flame.

1.Flame Source: A burner generates a stable and reproducible flame. Commonly used burners
include the premixed air-acetylene flame for general use and the nitrous oxide-acetylene flame for
higher temperatures.

2.Sample Introduction System: The sample is introduced into the flame in a controlled manner. This
can be done through nebulization, aspiration, or other techniques depending on the sample type.

3.Monochromator: A monochromator is used to isolate specific wavelengths of light emitted by the

excited atoms in the flame. This allows for the selective analysis of elements based on their
characteristic emission lines.

4.Photodetector: A photodetector, such as a photomultiplier tube (PMT), detects the intensity of the
emitted light at the selected wavelength. The signal is then converted into an electrical signal.

5.Amplification and Signal Processing: The electrical signal is often amplified and processed to
enhance sensitivity and reduce noise. Signal processing may involve filtering and other techniques to
improve the signal-to-noise ratio.

6.Control System: Automated control systems ensure the stability of the flame and other operational
parameters, enhancing precision and reproducibility.
Q1). Analyse the absorption laws with their example

The Absorption Laws

•There are two laws which govem the absorptio

1.) Lambert's law and

2.) Beer's law

1 Lambert's Law:

When a beam of monochromatic radiation passes through a homogeneous absorbing medium, the
rate of decrease of intensity of radiation with thickness of absorbino miedian as proportional to the
intensity of the scient radiation

Mathematically ,the law expressed as

-di*/dx=ki

Where, I=intensity of radiation after passing through a thickness x,of the medium

Di*= rate of decrease of intensity of radation

K=proportionality constant

2. ) Beer's Law: This law states that: When a beam of monochromatic radiation is passed through a
solution of an

absorbind sabytance, the rate a decrase of menatyo radiation win the thickness of the dosortane
solution is proportional to the intensity of incident radiation as well as the concentration of the
solution.

Mathematically, this law is stated as

- dl/dh = kI

where i = intensity of radiation after passing throuch a concentration h of the medium

- dl/dh=rate of decrese of intensity of radiation

Q2. type of UV visible spectrophotometer with graphical representation

Type of UV-visible spectrophotometers

1. Single beam spectrophotometer: As the name suggests, these instruments contain a single beam
of light. The sample and detector are placed in series in the single-beam configuration of a single-
beam spectroscopie monochromator. Here, low-intensity monochromator light is transmitted
through the sample to excite electrons from a lower to a higher energy one.

2. Double-beam spectroscopy: In double-beam spectroscopy, the splitter or chopper divides

monochromatic light into two beams, one of which goes through the sample and the other through
the standard/reference.

Instrumentation of UV Visible Spectrophotometer (Parts of UV- Spectroscopy)

'The basic instrumentation of the UV-vis spectrometer comprises of

1.Light source

2.Wavelength selector

3.Computer

4.Sample container or cuvette

5.Detector

6.recorder

Digram
Q3. Different parts of UV visible spectrophotometer

Ans:- Instrumentation of UV Visible Spectrophotometer (Parts of UV-v Spectroscopy)

1. Light Source: Light sources that lie in the ultraviolet and visible region are used as UV-visible
spectrometer sources.

a) Hydrogen & deuterium lamps range 160-380nm

b) Xenon arc lamps range 250-600nm
c) Tungsten halogen lamps range 240-2500nm

2. Wavelength selector: Although a single wavelength is not possible, a band of radiation could be
used. So an instrument with narrow bandwidth would be better.

Types of wavelength selectors :- A. Filters: Filters are used to permit a certain band of wavelength.
The simplest type of filter is the absorption filter. B. Monochromators: A monochromator is an
optical device that is used to select a narrow band of a wavelength of light. It may be a quartz prism
or grating.

• Components of a monochromator: All monochromators are similar in mechanical construction. The

essential components of a monochromator are: I. Slit II. Mirror III. Lense IV. Grating/prism

3. Sample container/cells or cuvettes: Sample containers or cuvettes may be made up of 1. Quartz

2. Borosilicate 3. Plastic

• Only quartz is transparent in both UV & visible regions (200-700nm range). • Glass & plastic are
suitable for the visible region only. • Glass is not suitable for the UV region because it absorbs UV
radiation i.e. it is not transparent in the UV region. • Plastic cells are not used for organic solvents.

4. Detectors: Detectors are devices that indicate the existence of some physical phenomenon. Some
examples of simple detectors are

a. Transducers: A transducer is a special type of detector that converts signals such as light intensity,
pH, mass, and temperature, etc into electrical signals.

b. Photodetectors:- ❖ Photo tubes ❖ Photomultiplier tubes ❖ Silicon photodiodes ❖ Photovoltaic

cells

c. Photographic films (photoemissive, semiconductor)

d. Mercury level in thermometers (temperature detector) e. Human eye

Q4. the wavelength selector and detector of UV visible spectrophotometer

Wavelength selector: Although a single wavelength is not possible, a band of radiation could be
used. So an instrument with narrow bandwidth would be better.

Types of wavelength selectors :- A.) Filters: Filters are used to permit a certain band of wavelength.
The simplest type of filter is the absorption filter. B.) Monochromators: A monochromator is an
optical device that is used to select a narrow band of a wavelength of light. It may be a quartz prism
or grating.

• Components of a monochromator: All monochromators are similar in mechanical construction.

The essential components of a monochromator are

I. Slit II. Mirror III. Lense IV. Grating/prism

.Detectors: Detectors are devices that indicate the existence of some physical phenomenon. Some
examples of simple detectors are

a.) Transducers: A transducer is a special type of detector that converts signals such as light
intensity, pH, mass, and temperature, etc into electrical signals. This electrical signal is amplified and
manipulated. Properties of transducers-Transducers produce a fast response to low levels of radiant
energy, Suitable for a wide range of wavelengths, Electrical signals produced by transducers should
have low noise, The signal produced by the transducer is directly proportional to the beam of power.

b.) Photodetectors:- ❖ Photo tubes, ❖ Photomultiplier tubes, ❖ Silicon photodiodes, ❖

Photovoltaic cells

c.) Photographic films (photoemissive, semiconductor)

d.) Mercury level in thermometers (temperature detector) e. Human eye.

Q5. factors influenc vibration frequency

1. Coupled Vibrations and Fermi Resonance: We expect one stretching absorption frequency for an
isolated C—H bond but in the case of methylene (—CH2—) group, two absorptions occur which
correspond to symmetric and asymmetric vibrations as follows:

•Insuchcases,asymmetric vibrations always occur at higher wave number compared with the
symmetric vibrations. These are called coupled vibrations since these vibrations occur at different
frequencies than that required for an isolated C—H stretching.

• In Infra-red spectrum, absorption bands are spread over a wide range of frequencies. It may
happen that the energy of an overtone level chances to coincide with the fundamental mode of
different vibration. A type of resonance occurs as in the case of coupled pendulums. This type of
resonance is called Fermi Resonance. This can be explained by saying that a molecule transfers its
energy from fundamental to overtone and back again.

2. Electronic Effects. Changes in the absorption frequencies for a particular group take place when
the substituents in the neighbourhood of that particular group are changed. The frequency shifts are
due to the electronic effects which include Inductive effect, Mesomeric effects, Field effects etc.
These effects cannot be isolated from one another and the contribution of one of them can only be
estimated approximately.

Example- (a). +I effect

(i) Formaldehyde (HCHO) 1750 cm–1 (ii) Acetaldehyde (CH3CHO) 1745 cm–1 (iii) Acetone
(CH3COCH3) 1715 cm–1

(b). –I effect

(i) Acetone (CH3COCH3) 1715 cm–1

(ii) Chloroacetone (CH3COCH2Cl) 1725 cm–1

(iii) Dichloroacetone (CH3COCHCl2) 1740 cm–1

(iv) Tetrachloroacetone (Cl2CH—CO—CHCl2) 1750, 1778 cm–1

3. Hydrogen bonding. Hydrogen bonding brings about remarkable downward frequency shifts.
Stronger the hydrogen bonding, greater is the absorption shift towards lower wave number than the
normal value. Two types of hydrogen bonds can be readily distinguished in Infra-red technique.
Generally, intermolecular hydrogen bonds give rise to broad bands whereas bands arising from
intramolecular hydrogen bonds are sharp and well defined. Intermolecular hydrogen bonds are
concentration dependent.

Example: In aliphatic alcohols, a sharp band* appears at 3650 cm–1 in dilute solutions due to free
O—H group.
Q6. Fundamental model of vibration in molecule.

A) Stretching. In this type of vibrations, the distance between the two atoms increases or decreases
but the atoms remain in the same bond axis.

(b) Bending. In this type of vibrations, the positions of the atoms change with respect to the original
bond axis. We know that more energy is required to stretch a spring than that required to bend it.
Thus, we can safely say that stretching absorptions of a bond appear at high frequencies (higher
energy) as compared to the bending absorptions of the same bond.

• The various stretching and bending vibrations of a bond occur at certain quantised frequencies.
When Infra-red radiation is passed through the substance, energy is absorbed and the amplitude of
that vibration is increased.

Types of stretching vibrations. There are two types of stretching vibrations:

(i) Symmetric stretching. In this type, the movement of the atoms with respect to a particular atom in
a molecule is in the same direction.

(ii) Asymmetric stretching. In these vibrations, one atom approaches the central atom while the
other departs from it.

Types of bending vibrations. (Deformations). Bending vibrations are of four types:

(i) Scissoring. In this type, two-atoms approach each other.

(ii) Rocking. In this type, the movement of the atoms takes place in the same direction.

(iii) Wagging. Two atoms move ‘up and down’ the plane with respect to the central atom.

(iv) Twisting. In this type, one of the atoms moves up the plane while the other moves down the
plane with respect to the central atoms.
Q7. the wavelength selector and detector of FTIR spectrometer

In a Fourier Transform Infrared (FTIR) spectrometer, the wavelength selector is typically a Michelson
interferometer. It modulates the incoming infrared beam by splitting it into two paths, introducing a
path difference that varies with time. The interferogram produced is then Fourier-transformed to
obtain the spectrum.

The detector, commonly a single-element or array detector, measures the intensity of the modulated
infrared beam after it passes through the sample. It converts the varying intensity at different
wavelengths into an electrical signal, which is then processed to generate the FTIR spectrum.
Common detectors include photovoltaic, photoconductive, or thermal detectors, depending on the
specific instrument design.

Q8. elaborate the instrumentation of FTIR spectrometer

A Fourier Transform Infrared (FTIR) spectrometer is an analytical instrument used to measure the
absorption, emission, and reflection of infrared light in a sample. Its key components include:

1.Source: Typically, a broadband infrared light source emits radiation across a range of wavelengths.

2.Interferometer: The heart of FTIR, it modulates the incoming infrared light, creating an
interferogram—a pattern of interference. Common types include Michelson and Fabry-Perot
interferometers.

3.Sample Compartment: This is where the sample is placed. The infrared light interacts with the
sample, and the transmitted or reflected light carries information about the sample's composition.

4.Detector: Detects the interferogram produced by the interferometer. Common detectors include
mercury cadmium telluride (MCT) or deuterated triglycine sulfate (DTGS).

5.Beam Splitter: Splits the incoming infrared beam into two paths—one directed to the sample and
the other to a reference mirror. The interferogram is generated by the recombination of these
beams.

6.Data System: Collects and processes the interferogram, converting it into a spectrum. The
transformation is done using a mathematical process called Fourier Transform.

7.Computer: Controls the instrument, manages data acquisition, and displays the resulting infrared
spectrum.
Q9. principal and methodology of column chromatography

PRINCIPLE • Adsorption • Mixture of components dissolved in the M.P is introduced in to the

column. Components moves depending upon their relative affinities. • Adsorbents: The usual
adsorbents employed in column chromatography are silica, alumina, calcium carbonate, calcium
phosphate, magnesia, starch, etc.

Adsorbent in C.C should meet following criteria

1. 1.Particles should be spherical in shape & uniform in size

2. Mechanical stability must be high a They shouldn't react chemically
3. It should be useful for separating for wide variety of compounds
4. It should be freely available & inexpensive

Selection of Stationary Phase:- • Success of chromatography depends upon proper selection of S.P,
it depends on the following:

1. Removal of impurities, 2. No. of components to be separated 3. Length of the column used

Mobile Phase • They act as solvent, developer & eluent.

The function of a mobile phase are: • As developing agent, • To introduce the mixture into the
column - as solvent, • To developing agent

The length of the column depends upon: • Number of compounds to be separated, • Type of
adsorbent used, • Quantity of the sample

PREPARATION OF THE COLUMN:- It consists of a glass tube with bottom portion of the column -
packed with glass wool/cotton wool or may contain asbestos pad, Above which adsorbent is packed,
After packing a paper disc kept on the top, so that the adsorbent layer is not disturbed during the
introduction of sample or mobile phase.

Packing techniques in C.C :- • There are two types of preparing the column, they are:, • i. Dry
packing / dry filling, • li. Wet packing / wet filling

Development technique (Elution):- 1.By elution technique, 2.Isocratic elution technique

DETECTION OF COMPONENTS

• If the compounds separated in a column chromatography procedure are colored, the progress of
the separation can simply be monitored visually.

• If the compounds to be isolated from column chromatography are colorless. In this case, small
fractions of the eluent are collected sequentially in labelled tubes and the composition of each
fraction is analyzed by TLC.
Q10. explain the principal and methodology of TLC

It is based on the principle of adsorption chromatography or partition chromatography or

combination of both, depending on adsorbent, its treatment and nature of solvents employed The
components with more affinity towards stationary phase travels slower. Components with less
affinity towards stationary phase travels faster.

the stationary phase that is applied to the plate is made to dry and stabilize.

To apply sample spots, thin marks are made at the bottom of the plate with the help of a pencil.

Apply sample solutions to the marked spots.

Pour the mobile phase into the TLC chamber and to maintain equal humidity, place a moistened filter
paper in the mobile phase.

Place the plate in the TLC chamber and close it with a lid. It is kept in such a way that the sample
faces the mobile phase.

Immerse the plate for development. Remember to keep the sample spots well above the level of the
mobile phase. Do not immerse it in the solvent.

Wait till the development of spots. Once the spots are developed, take out the plates and dry them.
The sample spots can be observed under a UV light chamber.
Q11. instrumentation of fluorimetry

* SOURCE OF LIGHT

• FILTERS AND MONOCHROMATORS

• SAMPLE CELLS

• DETECTORS

1) SOURCE OF LIGHT:-

Mercury vapor lamp:

Mercury vapour at high pressure give intense lines on continuous background above 350m.low
pressure mercury vapour gives an additional line at 254mm.it is used in filter fluorimeter.

xenon arc lamp:

It give more intense radiation than mercury vapour lamp. it is used in spectrofluorimeter.

tungsten lamp:

If excitation has to be done in visible region this can be used. It is used in low cost instruments.

2) FILTERS AND MONOCHROMATORS:

Primary filter:-absorbs visible radiation and transmit UV radiation.

Secondarv filter: -absorbs UV radiation and transmit visible radiation.

3) Sample cells:

These are meant for holding liquid samples. These are made up of quartz and can have various
shapes ex: cylindrical or rectangular etc.

4. Detectors: Photometric detectors are used they are

*Barrier layer cell/Photo voltaic cells

*Photomultiplier cells

INSTRUMENTS

The most common types are:

• Single beam (filter) fluorimeter

• Double beam (filter )fluorimeter

•Spectrofluorimeter (double beam)

Q12. principal and development techniques of paper chromatography

PRINCIPLE OF SEPERATION

The principle of separation is mainly partition rather than adsorption.

Cellulose layers in filter paper contains moisture which acts as stationary phase & organic
solvents/buffers are used as mobile phase.

Different types of development tech. are

1) ASCENDING DEVELOPMENT

• Like conventional type, the solvent flows against gravity. The spots are kept at the bottom portion
of paper and kept in a chamber with mobile phase solvents at the bottom.

2) DESCENDING TYPE (a downward slope)

•This is carried out in a special chamber where the solvent holder is at the top. The spot is kept at
the top and the solvent flows down the paper.

•In this method solvent moves from top to bottom so it is called descending chromatography.

3)ASCENDING - DESCENDING DEVELOPMENT

A hybrid of above two technique is called ascending-descending chromatography.

Only length of separation increased, first ascending takes place followed by descending

4)CIRCULAR / RADIAL DEVELOPMENT

Spot is kept at the centre of a circular paper. The solvent flows through a wick at the centre &
spreads in all directions uniformly.