UNIT-I

SPECTRAL METHODS OF ANALYSIS

 SPECTROSCOPY
• Spectroscopy is a technique where we studied the interaction between matter
and electromagnetic radiation.
• Matter may be ions, molecules or atoms.
• The nature of the interactions in spectroscopy techniques between radiation and
matter may includes,absorption, emission or scattering.
• Electromagnetic spectrum is mainly range from shorter wavelengths
to longer wavelengths such as gamma rays.
• Wavelengths of the visible region of the spectrum is ranges between
400-700 nm.
• Blue end of the spectrum has shorter wavelengths, while red end has
longer wavelengths.
• In spectrum, energy associated with the given segment is much
related to the frequency and wavelength.
• Frequency (v) of the electromagnetic radiation is the number of
oscillations made by wave in 1 second.
• Frequency is measured in hertz (Hz), Where, 1Hz = 1 cycle per sec.
• Wavelength (λ): It is the length of on complete wave cycle.
• Wavelength is generally measured in centimeters (cm).
• Wavelength is inversely proportional to the frequency.
• The relation between wavelength and frequency is defined by:
• V= c/ λ, where, c= speed of light.
• E= hv = hc/ λ    where, Planck’s constant (h) = 6.6 X 10-34 joules per second.
• Based on this there is three principles branches of spectroscopy:
• Absorption spectroscopy
• Scattering spectroscopy
• Emission spectroscopy
• Absorption spectroscopy uses the range in which a material absorbs the
electromagnetic spectrum. It involves atomic absorption spectroscopy and different
molecular techniques that area, such as infrared spectroscopy and radio region
nuclear magnetic resonance (NMR) spectroscopy.
• Emission spectroscopy uses the electromagnetic spectrum range in which a material
radiates (emits). The material must consume energy first. This energy may come from
several sources, such as luminescence, which defines the subsequent emission.
Spectro fluorimetry includes molecular luminescence techniques.
• Scattering spectroscopy tests the amount of light at specific wavelengths, incident
angles, and polarization angles that a material scatter. The method of scattering is
much quicker than the process of absorption/emission. Raman spectroscopy is
one of the most beneficial applications of light scattering spectroscopy.
• TRANSMITTANCE AND ABSORBANCE
• Transmittance (T) is the fraction of incident light which is transmitted. In other
words, it’s the amount of light that “successfully” passes through the substance
and comes out the other side. It is defined as T = I/Io, where I = transmitted light
(“output”) and Io = incident light (“input”). %T is merely (I/Io) x 100. For example,
if T = 0.25, then %T = 25%. A %T of 25% would indicate that 25% of the light
passed through the sample and emerged on the other side.
• Absorbance (A) is the flip-side of transmittance and states how much
of the light the sample absorbed. It is also referred to as “optical
density.” Absorbance is calculated as a logarithmic function of T: A =
log10 (1/T) = log10 (Io/I).
• Principles of absorption spectroscopy
• Absorption spectroscopy is based on law known as Beer-Lambert law.
• When electromagnetic radiations pass or fall onto homogeneous
medium, some amount of light is reflected, some amount is absorbed
and remained is transmitted.
• Absorption spectroscopy is governed by two laws i.e., Lambert’s law
and Beer’s law, in the combined form it is known as Beer-Lambert
law.
• Lambert’s law: It states that when monochromatic light passes
through a transparent medium, the intensity of transmitted light
decreases exponentially as the thickness of absorbing material
increases.
• Beer’s law: It states that the intensity of transmitted monochromatic
light decreases exponentially as the concentration of the absorbing
substances increases.
• When a monochromatic light of initial intensity Io passes through a
solution in a transparent vessel, some of the light is absorbed so that
the intensity of the transmitted light I is less than Io.
• There is some loss of light intensity from scattering by particles in the
solution and reflection at the interfaces, but mainly from absorption
by the solution.
• The relationship between I and Io depends on the path length of the
absorbing medium, l, and the concentration of the absorbing solution,
c. These factors are related in the laws of Lambert and Beer.
 Derivation of Beer-Lambert Law

• If material bodies are exposed to radiation, part of the incident radiation is

absorbed, a part is scattered and a part is transmitted.
• As a result of absorption the intensity of light passing through material bodies, i.e.
the intensity of transmitted light, decreases.
• The fraction of incident light absorbed depends on the thickness of the absorbing
medium.
• Lambert derived a quantitative relationship between the decrease in intensity of
a monochromatic light due to the passage through a homogeneous medium of
thickness dx and the intensity of light I. This law is known as Lamberts law, and
may be stated as
• The decrease in intensity of light with thickness of the absorbing medium at any point is
directly proportional to the intensity of light.
• Mathematically it can be expressed as
• – 𝑑𝐼 / 𝑑𝑥 ∝ 𝐼 — — — — — — (1)
• Where dI is a small decrease in intensity of light upon passing through a small
distance dx and I is the intensity of the monochromatic light just before entering the
medium.
• Equation (1) may be written as
• – 𝑑𝐼 / 𝑑𝑥 = 𝑎𝐼 — — — — — — (2)
• Where – 𝑑𝐼/ 𝑑𝑥 is the rate of decrease of intensity with thickness dx , a is called the
absorption co-efficient.
• Integration of equation (2) after rearrangement gives,
• – ln I = ax+C — — — — — — (3)
• Where C is a constant of integration. At x=0, I=Io. So, C = – ln Io. Introducing this in equation (3) we get,
• ln I/ Io = – ax — — — — — — (4)
• Equation (4) can also be written as,
• I = Io 𝑒−𝑎𝑥 — — — — — — (5)
• Equation (5) can also be written as,
• log I/ Io = − a/ 2.303 x — — — — — (6)
• or, log I/ Io = -a` x — — — — — (7)
• Where a` (= a /2.303 ) is called extinction co-efficient and -ln I/ Io is termed absorbance of the medium.
Absorbance is represented by A.
• Lambert’s law was extended by Beer who showed that
• when light passes through a solution of a given thickness the fraction of incident light absorbed is
dependent not only on the intensity I of light but also on the concentration c of the solution.
• This is known as the Beer’s law.
• – 𝑑𝐼 /𝑑𝑥 ∝ 𝑐 — — — — — — (8)
•  The two laws may be combined to write
• – 𝑑𝐼 /𝑑𝑥 ∝ 𝐼 × 𝑐
• Or, – 𝑑𝐼 /𝑑𝑥 = 𝑏 × 𝐼 × 𝑐 — — — — — (9)
• When the concentration, c, is expressed in mol /L, b is called the molar absorption co-efficient.
• As in the case of Lambert’s law equation (9) may be transformed into,
•  log I/ Io = − 𝑏 /2.303 × 𝑐 × 𝑥 — — — — — (10)
•  log I/ Io = – ∈× 𝑐 × 𝑥 — — — — — (11)
• Where ∈ (= 𝑏 / 2.303) is called the molar extinction co-efficient which is expressed in L/mol/cm.
• The molar extinction co-efficient ∈ is dependent on the nature of the absorbing solute as well as
on the wave length of the incident light used. The expression (equation 11) is commonly known
as Beer-Lambert’s law. 
PRINCIPLE OF SPECTROMETER
 TYPES OF SPECTROPHOTOMETER

• There are two types of spectrophotometer as given below:

• 1. Single Beam Spectrophotometer
• 2. Double Beam Spectrophotometer

• SINGLE BEAM SPECTROPHOTOMETER:

• Single beam spectrophotometer is an analytical instrument in which all
the light waves coming from the light source passes through the sample.
• Therefore, the measurements are taken as the intensity of light passes
through the sample.
 Definition
• A single beam spectrophotometer utilizes one beam of light that passes through the
sample and the intensity of light reflected from a reference is measured without the
sample.
• There are following essential parts of a single beam spectrophotometer:
• Light source:
• In spectrophotometer three different sources of light are commonly used to produce
light of different wavelength.
• The most common source of light used in the spectrophotometer for the visible
spectrum is a tungsten lamp.
• For Ultraviolet radiation, commonly used sources of are the hydrogen lamp and the
deuterium lamp. Nernst filament or globar is the most satisfactory sources of IR
(Infrared) radiation.
• Monochromator:
• It consists of entrance slit, dispersive device(maybe prism or diffraction grating).
• To select the particular wavelength, prism or diffraction grating is used to split
the light from the light source.
• Sample holder:
• Test tube or Cuvettes are used to hold the coloured solutions. They are made
up of glass at a visible wavelength.
• Photodetector system:
• When light falls on the detector system, an electric current is generated that
reflects the galvanometer reading.
• Measuring device:
• The current from the detector is fed to the measuring device – the
galvanometer. The meter reading is directly proportional to the
intensity of light.
• WORKING:
• Working of single beam spectrophotometer is explained below:
• Calibration:
• When using a Spectrophotometer, it requires being calibrated first
which is done by using the standard solutions of the known
concentration of the solute that has to be determined in the test
solution.
• For this, the standard solutions are filled in the Cuvettes and placed in
the Cuvette holder in the spectrophotometer that is similar to the
colorimeter.
• Path towards Monochromator:
• There is a ray of light with a certain wavelength that is specific for the
assay is directed towards the solution. Before reaching the solution the
ray of light passes through a series of the diffraction grating, prism, and
mirrors.
• These mirrors are used for navigation of the light in the
spectrophotometer and the prism splits the beam of light into different
wavelength and the diffraction grating allows the required wavelength to
pass through it and reaches the Cuvette containing the standard or Test
solutions. It analyses the reflected light and compares with a
predetermined standard solution.
• Detecting System:
• When the monochromatic light (light of one wavelength) reaches the Cuvette
some of the light is reflected, some part of the light is absorbed by the
solution and the remaining part is transmitted through the solution which
falls on the photodetector system.
• The photodetector system measures the intensity of transmitted light and
converts it into the electrical signals that are sent to the galvanometer.
• Measuring of Current:
• The galvanometer measures the electrical signals and displays it in the digital
form. That digital representation of the electrical signals is the absorbance or
optical density of the solution analysed.
• Concentration of Solution:
• If the absorption of the solution is higher than there will be more light absorbed by
the solution and if the absorption of the solution is low then more lights will be
transmitted through the solution which affects the galvanometer reading and
corresponds to the concentration of the solute in the solution.
• ADVANTAGES:
• Following are the advantages of single beam spectrophotometer:
• • It is simple in construction
• • It is easy to use
• • It is less expensive
• • It is high throughput and hence, high sensitivity.
• DISADVANTAGES:
• Following are the disadvantages of single beam spectrophotometer:
• The primary limitation is, it provides no means to compensate for
instrumental variations during an analysis, such as changes in source
intensity.
• It only measures the absorbance of either sample or reference blank
at a time.
 DOUBLE BEAM
SPECTROPHOTOMETER
• Definition:
• A double beam spectrophotometer splits the beam of light into two different paths, one of which passes
through the sample while the other passes through a reference standard.
• In the Beam splitter:
• In double beam spectrophotometers, the beam splitters are present
which splits the monochromatic light into two beams one for the
standard solution and the other for test solution.
• In this, the absorbance of Standard and the Test solution can be
measured at the same time and any no. of test solutions can be
analyzed against one standard.
• It gives more accurate and precise results, eliminates the errors which
occur due to the fluctuations in the light output and the sensitivity of
the detector.
• ADVANTAGES
• Following are the advantages of double beam spectrophotometer:
▪ It facilitates the rapid scanning over wide wavelength region.
▪ It does not require adjustment of the transmittance at 0% and 100% at each
wavelength.
It gives ratio of intensities of sample and reference beams simultaneously
▪ Fluctuations due to radiation source is minimized.
If absorbance measurement from reference cell changes, spectrophotometer
corrects for that change to find true absorbance of sample cell.
• DISADVANTAGES
• Following are the disadvantages of double beam spectrophotometer:
• It is very expensive
• Working with double beam spectrophotometer is difficult
INFRARED SPECTROPHOTOMETERS
• It is one of the most common and widely used spectroscopic
techniques employed mainly by inorganic and organic chemists due to
its usefulness in determining the structures of compounds and
identifying them.
The method or technique of infrared spectroscopy is conducted with an
instrument called an infrared spectrometer (or spectrophotometer) to
produce an infrared spectrum.
IR instruments is divided in to two classes.They are
1) Dispersive
2) Non dispersive
• Dispersive are similar toUV/Visible spectrometer which uses prism or grating. The difference is that the
source must be infrared.
• Non dispersive uses interference filters,tunable laser sources or an interferometer is very popular for FTIR
spectrometer.
Instrumentation of Infrared (IR)
Spectroscopy
• The main parts of the IR spectrometer are as follows:
• Radiation source
• Sample cells and sampling of substances
• Monochromators
• Detectors
• Recorder
• IR radiation sources
• IR instruments require a source of radiant energy which emits IR radiation which must
be steady, intense enough for detection, and extend over the desired wavelength.
• Various sources of IR radiations are as follows.
• Nernst glower
• Incandescent lamp
• Mercury arc
• Tungsten lamp
• Glober source
• Nichrome wire
• B. Sample cells and sampling of substances
• IR spectroscopy has been used for the characterization of solid, liquid,
or gas samples.
• i. Solid – Various techniques are used for preparing solid samples such
as pressed pellet technique, solid run in solution, solid films, mull
technique, etc.
• ii. Liquid – Samples can be held using a liquid sample cell made of alkali
halides. Aqueous solvents cannot be used as they will dissolve alkali
halides. Only organic solvents like chloroform can be used.
• iii. Gas– Sampling of gas is similar to the sampling of liquids.
• C. Monochromators 
• Various types of monochromators are prism, gratings and filters.
• Prisms are made of Potassium bromide, Sodium chloride or Caesium iodide.
• Filters are made up of Lithium Fluoride and Diffraction gratings are made up of
alkali halides.
• D. Detectors 
• Detectors are used to measure the intensity of unabsorbed infrared radiation.
• Detectors like
• thermocouples,
• Bolometers, thermisters, Golay cell, and pyro-electric detectors are used.
• Thermal Detector (Thermocouple)
• Thermocouples consist of a pair of junctions of different metals; for
example, twopieces 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.
• Bolometer & Thermistor
• • Bolometer
• A bolometer functions by changing resistance when heated. It is constructed ofstrips of metals
such as platinum or nickel or from a semiconductor.Thermistor:Thermistor is a resistance
thermometer, or a resistor whose resistance is dependent on temperature.
• Pyroelectric detectors:
• Pyroelectric detectors are thermal detectors: Temperature fluctuations
produce a charge change on the surface of Pyroelectric crystals, which
produces a corresponding electrical signal. This temperature gradient
can be created by the absorption of light. Unlike other thermal
detectors the Pyroelectric effect depends on the rate of change ofthe
detector temperature rather than on the temperature itself.
• This allows the Pyroelectric detector to operate with a much faster
response time and makes these detectors the choice for Fourier
transform spectrometers where rapid response is essential.
• Photo-conducting Detectors
• Photo-conducting detectors are the most sensitive detectors. They rely
on interactions between photons and a semiconductor. The lead
sulphide detector is used for the near-infrared region of the spectrum.
• For mid- and far-infrared radiationthe mercury cadmium telluride
detector is used. It must be cooled with liquid nitrogen to minimize
disturbance.
• Recorders
• Recorders are used to record the IR spectrum.
 Fourier Transform InfraRed

FT-IR stands for Fourier Transform InfraRed, the preferred method of

infrared spectroscopy.
In infrared spectroscopy, IR radiation is passed through a sample. Some of
the infrared radiation is absorbed by the sample and some of it is passed
through (transmitted).
The resulting spectrum represents the molecular absorption and
transmission, creating a molecular fingerprint of the sample. Like a
fingerprint no two unique molecular structures produce the same infrared
spectrum. This makes infrared spectroscopy useful for several types of
analysis.
• what information can FT-IR provide?
• It can identify unknown materials
• It can determine the quality or consistency of a sample.
• It can determine the amount of components in a mixture.
An infrared spectrum represents a fingerprint of a sample with absorption peaks
which correspond to the frequencies of vibrations between the bonds of the
atoms making up the material.
Because each different material is a unique combination of atoms, no two
compounds produce the exact same infrared spectrum. Therefore, infrared
spectroscopy can result in a positive identification (qualitative analysis) of every
different kind of material.
• Fourier Transform Infrared (FT-IR) spectrometry was developed in order
to overcome the limitations encountered with dispersive instruments.
• The main difficulty was the slow scanning process. A method for
measuring all of the infrared frequencies simultaneously, rather than
individually, was needed.
• A solution was developed which employed a very simple optical device
called an interferometer. The interferometer produces a unique type of
signal which has all of the infrared frequencies “encoded” into it. The
signal can be measured very quickly, usually on the order of one second
or so.
• Most interferometers employ a beam splitter which takes the incoming
infrared beam and divides it into two optical beams.
• One beam reflects off of a flat mirror which is fixed in place. The other beam
reflects off of a flat mirror which is on a mechanism which allows this mirror to
move a very short distance (typically a few millimeters) away from the
beamsplitter.
• The two beams reflect off of their respective mirrors and are recombined when
they meet back at the beamsplitter. Because the path that one beam travels is
a fixed length and the other is constantly changing as its mirror moves, the
signal which exits the interferometer is the result of these two beams
“interfering” with each other. The resulting signal is called an interferogram.
• This means that as the interferogram is measured, all frequencies are
being measured simultaneously. Thus, the use of the interferometer
results in extremely fast measurements.
• The measured interferogram signal can not be interpreted directly. A
means of “decoding” the individual frequencies is required. This can
be accomplished via a well-known mathematical technique called the
Fourier transformation.
• 1. The Source: Infrared energy is emitted from a glowing black-body
source. This beam passes through an aperture which controls the amount
of energy presented to the sample (and, ultimately, to the detector).
• 2. The Interferometer: The beam enters the interferometer where the
“spectral encoding” takes place. The resulting interferogram signal then
exits the interferometer.
• 3. The Sample: The beam enters the sample compartment where it is
transmitted through or reflected off of the surface of the sample,
depending on the type of analysis being accomplished. This is where
specific frequencies of energy, which are uniquely characteristic of the
sample, are absorbed.
• 4. The Detector: The beam finally passes to the detector for final
measurement. The detectors used are specially designed to measure
the special interferogram signal.
• 5. The Computer: The measured signal is digitized and sent to the
computer where the Fourier transformation takes place. The final
infrared spectrum is then presented to the user for interpretation and
any further manipulation.
 ADVANTAGES
• Some of the major advantages of FT-IR over the dispersive technique
include:
• • Speed: Because all of the frequencies are measured simultaneously,
most measurements by FT-IR are made in a matter of seconds rather than
several minutes. This is sometimes referred to as the Felgett Advantage.
• • Sensitivity: Sensitivity is dramatically improved with FT-IR for many
reasons. The detectors employed are much more sensitive, the optical
throughput is much higher (referred to as the Jacquinot Advantage) which
results in much lower noise levels, and the fast scans enable the
coaddition of several scans in order to reduce the random measurement
noise to any desired level (referred to as signal averaging.
• Mechanical Simplicity: The moving mirror in the interferometer is the
only continuously moving part in the instrument. Thus, there is very
little possibility of mechanical breakdown.
• Internally Calibrated: These instruments employ a HeNe laser as an
internal wavelength calibration standard (referred to as the Connes
Advantage). These instruments are self-calibrating and never need to
be calibrated by the user.
 Atomic absorption spectroscopy principle

• Atomic absorption spectroscopy (AAS) and 

atomic emission spectroscopy (AES) principle is based upon the
absorption and emission of light by atoms in the gaseous state.
• Atomization, hollow cathode lamp, monochromator, detector, and
recorder are the main components in atomic absorption spectroscopy
instrumentation.
• Elements with low excitation energy can be determined by flame
emission while high excitation energy can be determined by atomic
absorption spectroscopy.
Atomic absorption spectroscopy instrumentation
• For instrumentation, flame, non-flame, and graphite furnace are
available in atomic absorption instruments. Any atomic absorption
spectroscopy instrumentation has the following types of components,
• Atomization
• Hollow cathode lamp
• Monochromator
• Detector
• Recorder
• Atomization
• Atomization can be carried out either by a flame or furnace. Heat
energy is utilized in atomic absorption spectroscopy to convert
metallic elements to atomic dissociated vapor.
• The temperature should be controlled very carefully for the conversion
of atomic vapor. At too high temperatures, atoms can be ionized.
• Fuel and oxidant gases are fed into a mixing chamber which passes
through baffles to the burner. A ribbon flame is produced in the AAS
instrument. The sample is aspirated through the air into the mixing
chamber.
• Hollow cathode lamp
• We need a continuous source of radiation in the AAS instrument. The extreme
narrowness of the absorption line in the sources causes problems. We used a
hollow cathode glow discharge lamp to give sharp emission lines for a specific
element in atomic absorption spectroscopy instrumentation.
• The hollow cathode lamp has two electrodes, one is cup-shaped and made of a
specific element. Radiation from the hollow cathode lamp should not be
continuous due to spurious radiations. Therefore, we used a chopping wheel
between the radiation or pulsed potential.
• The metal which is used in the cathode is the same as that metal that we analyzed.
The lamp is filled with noble gas at low pressure. The lamp forms a glow of
emission from the hollow cathode.
• Monochromator
• A monochromator is an optical device that transmits a narrow band
of wavelengths of light or other radiation from a wider range of
wavelengths. The atoms in the AAS instrument accept the energy of
excitation and emit radiation.
• A desired band of lines can be isolated with a monochromator by
passing a narrow band. The spectra through a monochromator can be
shown by a curve.
• Detector
• A detector can convert light coming from a monochromator to a
simplified electrical signal. Generally, we used a photomultiplier tube as a
detector in the atomic absorption spectroscopy instrument. A detector
can be tuned to respond by a specific wavelength or frequency.
• Recorder
• The recorder can receive electrical signals from the detector to convert
them into a readable response. In atomic absorption spectroscopy
instrumentation, today we used a computer system with suitable 
software for recoding signals coming from the detector.
• APPLICATIONS:
• The atomic absorption spectroscopy technique is the most powerful tool
in analytical chemistry, forensic science, environmental analysis, and food
industries.
• Advantages:
• The most important advantage is the speed of analysis. It can analyze
various samples within a day.
• It is possible to determine all elements at trace concentration.
• It is not always essential to separate the element before analysis because
AAS can be used to determine one element in presence of another.
FLAME PHOTOMETER
 UNIT-2

INDUSTRIAL GAS ANALYZERS

• Types of Gas Analyzers:
• Non Dispersive Infrared Gas Analyzer
• Thermal Conductivity Analyzer
• Analyzer based on Gas Density
• Analyzer based on Ionization of Gases
• Infra red gas analyzer work :-
• An infra red gas analyzer measures trace gases by determining the absorption
of an emitted infrared light source through a certain air sample.
• Trace gases found in the Earth’s atmosphere get excited under specific
wavelengths found in the infrared range. The concept behind the technology
can be understood as testing how much of the light is absorbed by the air.
• IR Gas analyzer can be of two types
• Non-Dispersive Infra Red Analyzer
• Dispersive Infra Red Analyzer
• Non-Dispersive Infra Red Analyzer
• A non dispersive infrared sensor (or NDIR) sensor is a simple spectroscopic device often
used as gas detector.
• It is called non dispersive because wavelength which passes through the sampling chamber
is not pre-filtered instead a filter is used before the detector.
• NDIR analyzer is designed to continuously monitor the concentration of a particular infrared
absorbing component of a gas in a flowing gaseous mixture.
• One of the most commonly measured gases using infrared radiation absorption method is
the carbon monoxide, carbon dioxide or hydrocarbons in a gas.
SCHEMATIC DIAGRAM  OF NDIR
DETECTION SYSTEM
• Infrared radiation is produced from two separate energy sources.
• This radiation is interrupted by a chopper at 5 Hz.
• The two equal beams are then directed through two parallel optical
cell/chamber( sample cell and reference cell).
• A portion of the infrared radiation is absorbed by the sample gas, the quantity
of which is proportional to the sample gas concentration.
• The detector has an optical filter in front of it that eliminates all light except
the wavelength that the selected gas molecules can absorb. Ideally other gas
molecules do not absorb light at this wavelength, and do not affect the
amount of light reaching the detector to compensate for interfering
components.
• DETECTOR PART
• The signal from the two cells after the filter then goes to the two different chambers of the
detector unit.
• The diaphragm is distended depending up to the difference between the two signals which in
turn is proportional to the concentration of the sample gas.
• This value of capacitance is directly proportional to the difference between the reference and
sample cells signals.
• The capacitance is used to modulate the amplitude of a radio frequency voltage, which is then
demodulated into a resulting DC voltage signal.
• The output signal is proportional to the component concentration; it is amplified and sent to
the digital display.
• When chopper blocks the radiation, pressure in both parts of detector is same and diaphragm
remains in neutral position.
• Thermal Conductivity Analyzer
• A universal detector and can detect air, hydrogen, carbon monoxide, nitrogen, sulphur
oxide, inorganic gases and many other compounds.
• 
• Thermal conductivity (TCD) is a commonly used detector in gas
chromatography.
• TCD works by having two parallel tubes both containing gas and
heating coils. The gases are examined by comparing the heat loss rate
from the heating coils into the gas.
• Normally one tube holds a reference gas and the sample to be tested
is passed through the other.
• Using this principle, a TCD senses the changes in the thermal
conductivity of the column effluent and compares it to a reference
flow of carrier gas.
• Most compounds have a thermal conductivity much less than that of
the common carrier gases of hydrogen or helium. Therefore, when an
analyte elutes from the column, the thermal conductivity of the effluent is
reduced and a detectable signal is produced.
• Analyzer Based on Ionization of Gases:
• The spectral regions for maximum radiation absorption for different
gases are of different wavelength. Nitrogen and oxygen analyses are
routinely done by using these absorption bands. However, with
sufficient electrical excitation and at suitable pressure, gases emit
radiation in different ways like spark, arc, and glow discharge in
different part of the radiation spectrum.
• The measurement of the emitted radiation can help in determination
of the unknown concentration of a gas in a mixture. This technique
has been utilized for the measurement of nitrogen gas.
• The presence of nitrogen is detected by the emission of a
characteristic purple colour, when discharge takes place in a low
pressure chamber containing the sample gas.
• Construction: the instrument generally operates in two parts. The
sampling head contains the ionizing chamber, filter and the detector.
The other part contains the power supply, amplifier and the display
system.
• The ionizing chamber or the discharge tube is maintained at absolute
pressure of a few Torr. A rotary oil vacuum pump draws a sample and
feeds it to the discharge. The voltage required for striking the
discharge in the presence of nitrogen is of the order of 1500 V dc. This
voltage is generated by using a dc-dc converter, or by rectifying the
output of a high voltage transformer.
• The light output from the discharger tube is interrupted by means of a
rotating slotted disc, so that a chopped output is obtained. This light is
then passed through optical filters to the wavelength corresponding
to the purple colour.
• The intensity of the light is measured with a photocell and an
amplifier specially tuned to the chopping frequency.The light intensity
is proportional to nitrogen concentration.
• Pollution Monitoring Instruments
• Monitoring methodologies can be divided into three categories according to cost
and the level of accuracy and precision.
• Continuous monitoring methods:
• These are high-resolution methods that provide continuous records of
contaminant levels. They can operate over extended periods (weeks
or months).
• This is the most expensive monitoring methods. A high standard of
maintenance, calibration, and operational and quality control
procedures are required for good data quality.
 Gravimetric particulate methods:
• Monitoring starts when a known volume of air is pumped through a pre-
weighed filter for a known length of time (typically 24 hours). The filter is
reweighed after exposure and a concentration determined. This can be
done on consecutive days.
• Passive monitoring method:
• Diffusion tubes work when a contaminant is diffused into a tube containing
either an adsorbent or reactive material. Analysis of the tubes following a
known exposure time (typically two to four weeks) will provide a time-
averaged contaminant concentration. These methods are simple and cheap.
• Nitrogen oxides:
• NO Monitoring – Chemiluminescence
• An exothermic chemical reaction is one that releases a net sum of energy, as opposed
to an endothermic reaction which requires a greater input of energy than it releases.
Combustion is a common class of exothermic reactions, with the released energy being
very obviously in the forms of heat and light, with heat being the predominant form.
• Some exothermic reactions release energy primarily in the form of light rather than
heat. The general term for this effect is chemiluminescence.
• Certain industrial compounds engage in chemiluminescent reactions, and this
phenomenon may be used to measure the concentration of those compounds. One
such compound is nitric oxide (NO), an atmospheric pollutant formed by high-
temperature combustion with air as the oxidizer.
A spontaneous chemical reaction between nitric oxide and ozone (an unstable molecule
formed of three oxygen atoms: O3) is known to produce chemiluminescence:
• NO + O3 → NO2 + O2 + light
• As with many optical analyzers, a photomultiplier tube serves as the light- detecting
sensor, generating an electrical signal in proportion to the amount of light observed
inside the reaction chamber. The higher the concentration of NO molecules in the
sample gas stream, the more light will be emitted inside the reaction chamber, resulting
in a stronger electrical signal produced by the photomultiplier tube.
• Although this instrument readily measures the concentration of nitric oxide (NO), it is
insensitive to other oxides of nitrogen
• In order to use chemiluminescence to measure all oxides of nitrogen, we must
chemically convert the other oxides into nitric oxide (NO) before the sample enters the
reaction chamber. This is done in a special module of the analyzer called a converter. A
three-way solenoid valve is shown in this diagram, providing a means to bypass the
converter so the analyzer only measures nitric oxide content in the sample gas. With
the solenoid valve passing all the sample
• through the converter, the analyzer responds to all oxides of nitrogen
(NOx) and not just nitric oxide (NO)
• One simple way to achieve the NOx → NO chemical conversion is to
simply heat the sample gas to a high temperature, around 1300 F. At this
o

temperature, the molecular structure of NO is favored over more complex

oxides such as NO2, the result being a release of oxygen from the NO2
and NO3 molecules to become NO molecules.
• A disadvantage of this technique is that those same high temperatures
also have a tendency to convert other compounds of nitrogen such as
ammonia (NH3) into nitric oxide, thereby creating an unintended
interference species.
• Sulphur Dioxide:
• Colorimetric method
•  In this method a known volume of air is passed through an aqueous
solution, which contains reagent that absorb sulphur dioxide and
produce a coloured substance. The amount of coloured substance is
proportional to the component of interest (SO2). This determine by
measuring a solution’s optical absorbance spectro-photometrically.
• Gas Chromatography
• A gas chromatograph (GC) is an analytical instrument that measures the content
of various components in a sample.
• Principle of gas chromatography:
• The sample solution injected into the instrument enters a gas stream which
transports the sample into a separation tube known as the "column.“
• (Helium or nitrogen is used as the so-called carrier gas.) The various
components are separated inside the column.
• The detector measures the quantity of the components that exit the column. To
measure a sample with an unknown concentration, a standard sample with
known concentration is injected into the instrument.
• Gas chromatography differs from other forms of chromatography in
that the mobile phase is a gas and the components are separated as
vapours.
• It is thus used to separate and detect small molecular weight
compounds in the gas phase.
• The sample is either a gas or a liquid that is vaporized in the injection
port. The mobilephase for gas chromatography is a carrier gas,
typically helium because of its low molecular weight and being
chemically inert.
• The Principle of separation for gas chromatography is partitioning, and the
components of the sample will partition (i.e. distribute) between the two
phases: the stationary phase and the mobile phase.
• Compounds that have a greater affinity for the stationary phase spend more
time in the column and thus elute later and have a longer retention time (Rt)
than samples that have a higher affinity for the mobile phase.
• Affinity for the stationary phase is driven mainly by intermolecular
interactions and the polarity of the stationary phase can be chosen to
maximize interactions and thus the separation.
• The separation is hence accomplished by partitioning the sample between
the gasand a thin layer of a non-volatile liquid held on a solid support.
• Flame photometric method
• The photoemission from the flame is monitored instead of the electric
conductivity.
•  The sample passes through the hydrogen/ air flame and light emission is
observed due to the excitation of some of the atoms.
• The light in the UV/Visible region of the spectrum is selected by using
suitable filters and is measured by a photomultiplier tube. The light
emitting processes that produce the sulphur and phosphorous sensitivity
occur in the upper portion of the flame that does not emit appreciably in
the absence of sulphur and phosphorous.
• Selective to compounds containing sulphur and phosphorous.
• Fluorescence method
• The advantage of fluorescence method is its high sensitivity for
selective groups of compounds. By using a specific wavelength,
analyte atoms are excited and then emit light signal (fluorescence).
The intensity of this emitted light is monitored to quantify the analyte
concentration. Most pharmaceuticals, natural products, clinical
samples, and petroleum products have fluorescent absorbance
• Hydrocarbons:
• Different methods are available for monitoring hydrocarbons.
• It is emitted in to the atmosphere by natural biological activity as
well as anthropogenic sources such as automobile exhausts,burning
of coal,oil,wood and solvent evaporation.
• Gaseous hydrocarbons may be reactive or non reactive.ethylene and
methane are examples .
• Hydrocarbon pollutants can be determined as total hydrocarbons
• Flame ionization detector:
• FID is the most widely used and generally applicable detector in GC and gas
analyzer.
• Effluent from the column is directed into a small air/ H2 flame and most organic
compounds produce ions and electrons when pyrolyzed at the temperature of an
air /H2 flame.
• Compound is detected by monitoring the current produced by collecting the ions
and electrons.
• A few hundred volts applied between the burner tip and a collector electrode
located above the flame serve to collect the ions and electrons.
• The current is then measure with a sensitive picoammeter.
 Estimation of carbonmonoxide(CO)
• The standard method for the measurement of CO is NDIR spectroscopy
• It is used for the continuous analysis of CO based on the absorption of
IR.
• The NDIR consists of sample cell, reference cell, two infrared sources,
chopper,two filter cells and a detector.
• Reference cell is filled with non absorbing gas such as nitrogen and the
same containing CO which absorbs radiation at 4.6μm.
• The detector consists of two compartments separated by a thin metal
diaphragm and is filled with CO.
• The IR is produced from a hot filament and is passed alternately
through sample and a reference cell with the help of an optical
chopper. After passing through the two cells the radiation reaches a
detector cell which is divided by a pressure sensitive diaphragm.
• The reference cell passes almost all of the infrared energy on to one
part of the detector cell while a varying amount of infrared energy
which is inversely proportional to CO concentration passes through
the sample cell and reaches the other part of detector.
• Since more radiation enters the reference cell side of the detector the
diaphragm is moved towards the sample cell side of the detector.
• The resultant distortion of the diaphragm is converted in to an
electrical signal which can be amplified and recorded.
 UNIT-3
ION CONDUCTIVITY AND DISSOLVED
COMPONENTANALYZER
CONDUCTIVITY METER
 PH METERS
• pH is a scale used to specify the acidity or basicity of an aqueous
solution. Acidic solutions (solutions with higher concentrations of H+
ions) are measured to have lower pH values than basic or alkaline
solutions.
• When the hydrogen ions outnumber the hydroxide ions, the solution is
acidic. If the reverse is true, then the solution is alkaline. So, pH is
defined by the following formula:
pH=-log10[H+]
• In the case of a neutral solution,
• [H+]=10-7 , which we call a pH of 7.
• The methods for measuring pH fall roughly into the following four
categories:
• Indicator methods
• Metal-electrode methods
• Glass-electrode methods
• Semiconductor sensor methods
• Measuring pH using an Indicator
• This category basically includes two methods: One involves comparing the standard color
corresponding to a known pH with the color of an indicator immersed in the test liquid
using buffer solution.
• The other method involves preparing pH test paper which is soaked in the indicator, then
immersing the paper in the test liquid and comparing its color with the standard color.
• This method is simple, but prone to error. A high degree of accuracy cannot be expected.
Various errors include;
• Error due to high salt concentration in the test liquid
• Error due to the temperature of the test liquid
• Error due to organic substances in the test liquid. The indicator method cannot measure
the pH of high-purity water, since the influence of the indicator itself is too large.
• Hydrogen-Electrode Method
• A hydrogen electrode is made by adding platinum black to platinum
wire or a platinum plate. It is immersed in the test solution and an
electric charge is applied to the solution and the solution is saturated
with hydrogen gas.
• The electrode potential is measured between platinum black
electrode and silver chloride electrode. This potential is inversely
proportional to pH of the solution. The hydrogen-electrode method is
a standard among the various methods for measuring pH.
• Quinhydron-Electrode Method
• When quinhydrone is added to a solution, it separates into hydroquinone and quinone.
Because quinone’s solubility varies depending on the pH value of the solution, pH can
be determined from the voltage between a platinum and reference electrode.
Although this method is simple, it does not work when the test solution has a pH above
8 or 9.
• Antimony-Electrode Method
• This method involves immersing the tip of a polished antimony rod into a test solution,
also immersing a reference electrode, and measuring pH from the difference in
potential between them. This method was once widely used because the apparatus is
sturdy and easy to handle. However, its application is now quite limited because results
vary depending on the degree of polish of the electrode, and reproducibility is low.
• Glass-Electrode Method
• The glass electrode method uses two electrodes, a glass electrode and
reference electrode, to determine the pH of a solution by measuring the
voltage (potential) between them.
• This method is the one most commonly used for pH measurement, since
the potential quickly reaches equilibrium and shows good reproducibility,
and because the method can be used on various types of solutions, with
oxidizing or reducing substances having very little impact on the result.
• The glass electrode method is widely used, not only in industry but also
in many other fields.
• Semiconductor sensor methods
• This sensor, known as an ion sensitive field effect transistor (ISFET), is
not only resistant to damage but also easily miniaturized.
Miniaturization allows the use of smaller amounts of sample for
measurement, and makes it possible to perform measurements in
very small spaces and on solid state surfaces. This sensor promises
useful applications in measurement in the fields of biology and
medicine.
 Dissolved oxygen (DO) Analyzer
 

• Dissolved oxygen is a key measure of water quality relied upon in

various applications.
• In industrial water treatment, dissolved oxygen levels can be an
indicator of water quality issues that lead to corrosion of
equipment.
• In aquaculture, fish transport, and aquarium applications, dissolved
oxygen is monitored to ensure that aquatic species have enough
oxygen in their habitat to survive, grow, and reproduce.
• In municipal water treatment facilities, dissolved oxygen in
wastewater is monitored during aeration water treatment processes.
• Measuring dissolved oxygen concentration
•  The concentration of dissolved oxygen in water can be sampled or
monitored continuously using a dissolved oxygen sensor. Commercially
available dissolved oxygen sensors typically fall into 3 categories:
• Galvanic cell dissolved oxygen sensors
• Polarographic dissolved oxygen sensors
• Optical dissolved oxygen sensors
•  Each type of dissolved oxygen sensor has a slightly different working
principle. Therefore, each dissolved oxygen sensor type has advantages
and disadvantages depending on the water measurement application.
• Galvanic cell method
•  The membrane has high permeability to oxygen and is constructed so that
the electrodes and electrolyte are isolated from the water being measured.
• The counter electrode (anode) is a base metal and the working electrode is
a noble metal (cathode) and potassium hydroxide is used as the electrolyte.
• Oxygen passes through themembrane and is reduced on the working
electrode, and so the method measures the reduction current flowing
between both electrodes, which is proportional to the concentration of
dissolved oxygen.
• Advantages
• No warm up time, it can be set immediately after turn-off
• The electrolyte is never used up; in theory it can be used forever.
• Fast response time.
• Limitations
• The sensor continuously consumes the anode, even when turned off. Therefore
the lifetime of the sensor is much shorter than of the Polarographic sensor.
• Since the electrode consumes oxygen, readings are affected by flow across the
sensor tip. Thus enough flow rate at the membrane (or sample renewal rate)
must be ensured for accurate results.
• Polarographic method
•  The sensor construction is almost the same as that of the galvanic
cell method. The counter electrode is silver-silver chloride and the
working electrode is gold or platinum.
• When a voltage of 0.5–0.8 V is applied between both electrodes,
oxygen that has permeated through the membrane initiates a
reduction reaction on the working electrode, causing a Polarographic
limiting current to flow which is proportional to the oxygen
concentration. This method measures the concentration of dissolved
oxygen based on this current value.
• Optical method
• An optical dissolved oxygen sensor does not have an anode or
cathode, and oxygen is not reduced during measurement. Instead, the
sensor cap contains a luminescent dye, which glows red when
exposed to blue light. Oxygen interferes with the luminescent
properties of the dye, an effect called “quenching.” A photodiode
compares the “quenched” luminescence to a reference reading,
allowing the calculation of dissolved oxygen concentration in water.
• Advantages:
• Reliable measurements: Optical sensor technology delivers accurate DO
monitoring with no drift and no minimum flow
• Long operating life with minimal maintenance: No membranes, electrolyte
solutions, or anodes – just replace the cap once in every 1 or 2 years.
• Disadvantages:
• Higher initial Cost.
• Slower measurement response time.
• Higher power consumption than other sensors.
SODIUM ANALYZER
SILICA ANALYZER
 UNIT-4
CHROMOTOGRAPHY
• ‘Chromatography’ is an analytical technique commonly used for
separating a mixture of chemical substances into its individual
components, so that the individual components can be thoroughly
analyzed.
• Principle of separation
• Principle of separation of different components: Differential affinities
(strength of adhesion) of the various components of the analyte
towards the stationary and mobile phase results in the differential
separation of the components. Affinity, in turn, is dictated by two
properties of the molecule: ‘Adsorption’ and ‘Solubility’.
• We can define adsorption as the property of how well a component of the
mixture sticks to the stationary phase, while solubility is the property of how
well a component of the mixture dissolves in the mobile phase.
• Higher the adsorption to the stationary phase, the slower the molecule will
move through the column.
• Higher the solubility in the mobile phase, the faster the molecule will move
through the column.
• So, the interplay between the above two factors determines the differential
rates at which the different components of the analyte will move through the
column. Adsorption and solubility of a molecule can be manipulated by
choosing the appropriate stationary phase and mobile phase.
 Gas chromatography

• A gas chromatograph (GC) is an analytical instrument that measures the content of

various components in a sample.
• Principle of gas chromatography: The sample solution injected into the
instrument enters a gas stream which transports the sample into a separation tube
known as the "column." (Helium or nitrogen is used as the so-called carrier gas.)
• The various components are separated inside the column. The detector measures
the quantity of the components that exit the column. To measure a sample with an
unknown concentration, a standard sample with known concentration is injected
into the instrument.
• The standard sample peak retention time (appearance time) and area are
compared to the test sample to calculate the concentration.
• Gas chromatography differs from other forms of chromatography in
that the mobile phase is a gas and the components are separated as
vapours.
• It is thus used to separate and detect small molecular weight
compounds in the gas phase.
• The sample is either a gas or a liquid that is vaporized in the injection
port. The mobile phase for gas chromatography is a carrier gas,
typically helium because of its low molecular weight and being
chemically inert.
• The pressure is applied and the mobile phase moves the analyte
through the column. The separation is accomplished using a column
coated with a stationary phase.
• The Principle of separation for gas chromatography is partitioning,
and the components of the sample will partition (i.e. distribute)
between the two phases: the stationary phase and the mobile phase.
• Compounds that have a greater affinity for the stationary phase spend
more time in the column and thus elute later and have a longer
retention time (Rt) than samples that have a higher affinity for the
mobile phase.
• Affinity for the stationary phase is driven mainly by intermolecular
interactions and the polarity of the stationary phase can be chosen to
maximize interactions and thus the separation.
• Ideal peaks are Gaussian distributions and symmetrical, because of the
random nature of the analyte interactions with the column.
• The separation is hence accomplished by partitioning the sample
between the gas and a thin layer of a non-volatile liquid held on a solid
support.
• Procedure:
• A sample containing the solutes is injected into a heated block where it is
immediately vaporized and swept as a plug of vapour by the carrier gas stream
into the column inlet.
• The solutes are absorbed by the stationary phase and then desorbed by a fresh
carrier gas.
• The process is repeated in each plate as the sample is moved toward the
outlet.
• Each solute will travel at its own rate through the column.
• Their bands will separate into distinct zones depending on the partition
coefficients, and band spreading.
• The solutes are eluted one after another in the increasing order of their Rt,
and enter into a detector attached to the exit end of the column.
• Here they register a series of signals resulting from concentration changes and
rates of elution on the recorder as a plot of time versus the composition of
carrier gas stream.
• The appearance time, height, width, and area of these peaks can be measured
to yield quantitative data.
 Liquid Chromatography
• Liquid chromatography (LC) is a separation process used to isolate the
individual components of a mixture.
• This process involves mass transfer of a sample through a polar
mobile phase and non-polar stationary phase.
• The device is a column packed with the porous medium made of a
granular solid material (i.e., stationary phase), such as polymers and
silica, where the sample is injected and the solvent (i.e., mobile
phase) passes to transport the sample.
• When a sample is injected, it is adsorbed on the stationary phase, and
the solvent passes through the column to separate the compounds
one by one, based on their relative affinity to the packing materials
and the solvent.
• The component with the most affinity to the stationary phase is the
last to separate. This is because high affinity corresponds to more
time to travel to the end of the column.
 Differences between LC and HPLC
• High-performance liquid chromatography (HPLC), also known as high-
pressure liquid chromatography, is an advanced type of LC. HPLC is
amenable to a wide range of applications, such as pharmaceuticals and
food analysis.
• It is especially useful for low or non-volatile organic compounds, which
cannot be handled with gas chromatography.
• The difference between traditional LC and HPLC is that the solvent in LC
travels by the force of gravity. In the application of HPLC, the solvent
travels under high pressure obtained by means of a pump to overcome the
pressure drop in the packed column, which reduces the time of
separation. A continuous flow syringe pump is very useful in HPLC.
 HPLC
• High performance liquid chromatography or commonly known as HPLC is an
analytical technique used to separate, identify or quantify each component in a
mixture.
• The mixture is separated using the basic principle of column chromatography
and then identified and quantified by spectroscopy.
• In the 1960s the column chromatography LC with its low-pressure suitable glass
columns was further developed to the HPLC with its high-pressure adapted
metal columns.
• HPLC is thus basically a highly improved form of column liquid chromatography.
Instead of a solvent being allowed to drip through a column under gravity, it is
forced through under high pressures of up to 400 atmospheres.
 Principle of HPLC

• The purification takes place in a separation column between a

stationary and a mobile phase.
• The stationary phase is a granular material with very small porous
particles in a separation column.
• The mobile phase, on the other hand, is a solvent or solvent mixture
which is forced at high pressure through the separation column.
• Via a valve with a connected sample loop, i.e. a small tube or a
capillary made of stainless steel, the sample is injected into the
mobile phase flow from the pump to the separation column using a
syringe.
• Subsequently, the individual components of the sample migrate
through the column at different rates because they are retained to a
varying degree by interactions with the stationary phase.
• After leaving the column, the individual substances are detected by a
suitable detector and passed on as a signal to the HPLC software on
the computer.
• At the end of this operation/run, a chromatogram in the HPLC
software on the computer is obtained.
• The chromatogram allows the identification and quantification of the
different substances.
Instrumentation of HPLC
• The Pump
• The development of HPLC led to the development of the pump system.
• The pump is positioned in the most upper stream of the liquid chromatography
system and generates a flow of eluent from the solvent reservoir into the
system.
• High-pressure generation is a “standard” requirement of pumps besides
which,it should also to be able to provide a consistent pressure at any
condition and a controllable and reproducible flow rate.
• Most pumps used in current LC systems generate the flow by back-and-forth
motion of a motor-driven piston (reciprocating pumps). Because of this piston
motion, it produces “pulse”.
• Injector
• An injector is placed next to the pump.
• The simplest method is to use a syringe, and the sample is introduced
to the flow of eluent.
• The most widely used injection method is based on sampling loops.
• The use of the auto sampler (auto-injector) system is also widely used
that allows repeated injections in a set scheduled-timing.
• Column
• The separation is performed inside the column.
• The recent columns are often prepared in stainless steel housing,
instead of glass columns.
• The packing material generally used is silica or polymer gels compared
to calcium carbonate. The eluent used for LC varies from acidic to
basic solvents.
• Most column housing is made of stainless steel since stainless is
tolerant towards a large variety of solvents.
• Detector
• Separation of analyte is performed inside the column, whereas a
detector is used to observe the obtained separation.
• The composition of the eluent is consistent when no analyte is
present. While the presence of analyte changes the composition of
the eluent. What detector does is to measure these differences.
• This difference is monitored as a form of an electronic signal. There
are different types of detectors available.
• Recorder
• The change in eluent detected by a detector is in the form of an electronic
signal, and thus it is still not visible to our eyes.
• In older days, the pen (paper)-chart recorder was popularly used. Nowadays,
a computer-based data processor (integrator) is more common.
• There are various types of data processors; from a simple system consisting
of the in-built printer and word processor while those with software that are
specifically designed for an LC system which not only data acquisition but
features like peak-fitting, baseline correction, automatic concentration
calculation, molecular weight determination, etc.
• Degasser
• The eluent used for LC analysis may contain gases such as oxygen that
are non visible to our eyes.
• When gas is present in the eluent, this is detected as noise and causes
an unstable baseline.
• Degasser uses special polymer membrane tubing to remove gases.
• The numerous very small pores on the surface of the polymer tube
allow the air to go through while preventing any liquid to go through
the pore.
• Column Heater
• The LC separation is often largely influenced by the column
temperature.
• In order to obtain repeatable results, it is important to keep
consistent temperature conditions.
• Also for some analysis, such as sugar and organic acid, better
resolutions can be obtained at elevated temperatures (50 to 80°C).
• Thus columns are generally kept inside the column oven (column
heater).
 Types of HPLC
• Normal phase:
• Column packing is polar (e.g silica) and the mobile phase is non-polar.
It is used for water-sensitive compounds, geometric isomers, cis-trans
isomers, and chiral compounds.
• Reverse phase:
• The column packing is non-polar (e.g C18), the mobile phase is water+
miscible solvent (e.g methanol). It can be used for polar, non-polar,
ionizable and ionic samples.
• Ion exchange:
• Column packing contains ionic groups and the mobile phase is buffer.
It is used to separate anions and cations.
• Size exclusion:
• Molecules diffuse into pores of a porous medium and are separated
according to their relative size to the pore size. Large molecules elute
first and smaller molecules elute later.
• Applications of HPLC
• The HPLC has developed into a universally applicable method so that
it finds its use in almost all areas of chemistry, biochemistry, and
pharmacy.
• Analysis of drugs
• Analysis of synthetic polymers
• Analysis of pollutants in environmental analytics
• Determination of drugs in biological matrices
• Isolation of valuable products
• Advantages of HPLC
• Speed
• Efficiency
• Accuracy
• Versatile and extremely precise when it comes to identifying and quantifying
• chemical components.
• Limitations
• Cost: Despite its advantages, HPLC can be costly, requiring large quantities of
expensive organics.
• Detectors in HPLC
• 1. Refractive Index Detector
• 2. Evaporative Light Scattering Detector(ELSD)
• 3. UV/VIS Absorption Detector
• 4. Fluorescence Detector
• 5. Electrochemical Detector
• 6. Conductivity Detector
• Refractive Index Detector
• Measures the overall ability of the mobile phase and its solutes to
refract or bend light.
• When a solute is in a sample compartment, refractive index changes
will shift the light beam from detector.
• Refractive index detector measures the molecule's ability to deflect
light in a flowing mobile phase in a flow cell relative to a static mobile
phase contained in a reference cell.
• The amount of deflection is proportional to the concentration of the
solute in the mobile phase.
Evaporative Light Scattering Detector
(ELSD)
• Detection is based on the scattering of beam of light by particles of compound
remaining after evaporation of the mobile phase. It is universal and
destructive detector.
There are three steps in detection:
• 1. Nebulization: The flow from the column is nebulized with a stream of inert
gas (liquid into a fine spray or mist).
• 2. Mobile Phase Evaporation: The mobile phase, which must be volatile, is
evaporated, leaving tiny particles of the analyte.
• 3. Detection: The Particles are passed through a laser beam and they scatter
the laser light. The scattered light is measured at right angles to the laser
beam by a photodiode detector.
 Electrochemical Detector
• It is based on the measurement of the current resulting from an
oxidation/reduction reaction of the analyte at a suitable electrode.
• The level of current is directly proportional to the analyte
concentration.
• Three electrodes are employed which are:
• ➢ Working electrode: The elute is oxidized or reduced at the working
electrode.
• ➢ Auxiliary electrode
• ➢ Reference electrode
Thermal Conductivity Detector
• The thermal conductivity detector (TCD), also known as a
katharometer, is a bulk property detector and a chemical specific
detector commonly used in gas chromatography.
• This detector senses changes in the thermal conductivity of the
column eluent and compares it to a reference flow of carrier gas.
Since most compounds have a thermal conductivity much less than
that of the common carrier gases of helium or hydrogen, when an
analyte elutes from the column the effluent thermal conductivity is
reduced, and a detectable signal is produced.
 UV/VIS Absorption Detector
• The UV, VIS, and PDA (Photodiode-Array Detection) detectors are categorized as
absorbance detectors.
• They provide good sensitivity for light-absorbing compounds at ~pg level. They are
easy to operate and provide good stability.
• UV detector is a very commonly used detector for HPLC analysis. During the analysis,
sample goes through a clear color-less glass cell, called flow cell.
• When UV light is irradiated on the flow cell, sample absorbs a part of UV light. Thus,
the intensity of UV light observed for the mobile phase (without sample) and the
eluent containing sample will differ.
• By measuring this difference, the amount of sample can be determined. Since the UV
absorbance also differs depend on what wavelength is used, it is important to choose
an appropriate wavelength based on the type of analyte.
 Fluorescence Detector
• The advantage of fluorescence method is its high sensitivity for
selective groups of compounds. By using a specific wavelength,
analyte atoms are excited and then emit light signal (fluorescence).
• The intensity of this emitted light is monitored to quantify the analyte
concentration. Most pharmaceuticals, natural products, clinical
samples, and petroleum products have fluorescent absorbance.
• For some compounds which do not have fluorescence absorbance or
low absorbance, they can be treated with fluorescence derivatives
such as dansylchloride. The system is easy to operate and relatively
stable.
 Gas chromatography Detectors

• FLAME IONIZATION DETECTOR (FID)

• THERMAL CONDUCTIVITY DETECTOR (TCD)
• ELECTRON CAPTURE DETECTOR (ECD)
• FLAME PHOTOMETRIC DETECTOR (FPD)
• PHOTOIONIZATION DETECTOR (PID)
• ELECTROLYTIC CONDUCTIVITY DETECTOR (ELCD)
• NITROGEN PHOSPHORUS DETECTOR (NPD)
• Flame Ionization Detector (FID)
• FID is the most widely used and generally applicable detector in GC.
• Effluent from the column is directed into a small air/ H2 flame and most
organic compounds produce ions and electrons when pyrolyzed at the
temperature of an air /H2 flame.
• Compound is detected by monitoring the current produced by collecting the
ions and electrons.
• A few hundred volts applied between the burner tip and a collector electrode
located above the flame serve to collect the ions and electrons.
• The current is then measure with a sensitive picoammeter.
• Thermal Conductivity Detector
• It works on the principle of Wheatstone bridge.
• Out of four resistances in the circuit, the magnitude of three resistances
remains constant.
• But that of fourth resistance varies as per changes in the temperature.
• This change is because of the difference in the capacity of solute and the
carrier gas to absorb heat (thermal conductivity differences).
• The change in the temperature changes the resistance and hence the current
in circuit.
• Electron Capture Detector (ECD)
• The electron capture detector has become one of the most widely used
detector for environmental samples
• In ECD, the sample elute from a column is passed over a radioactive beta
emitter.
• An electron from the emitter causes ionization of the carrier gas (often N2)
and the Production of a burst of electrons.
• In the absence of organic species, a constant standing current between a pair
of electrodes results from this ionization process.
• The current decreases in the presence of organic molecules containing
electronegative functional groups that tend to capture electrons.
• Flame Photometric Detector (FPD)
• The photoemission from the flame is monitored instead of the electric
conductivity.
• The sample passes through the hydrogen/ air flame and light emission is
observed due to the excitation of some of the atoms.
• The light in the UV/Visible region of the spectrum is selected by using suitable
filters and is measured by a photomultiplier tube. The light emitting processes
that produce the sulphur and phosphorous sensitivity occur in the upper
portion of the flame that does not emit appreciably in the absence of sulphur
and phosphorous.
• Selective to compounds containing sulphur and phosphorous.
Photo Ionization Detector (PID)

• Photo Ionization Detector or GC-PID is a technique used to analyse a wide range of aromatic
hydrocarbons and other organic compounds. A typical application is the analysis of hydrocarbon
pollution of water. The PID uses ultraviolet light to ionize the components exiting the column.
ELECTROLYTIC CONDUCTIVITY DETECTOR (ELCD)

• The electrolytic conductivity detector is for compounds containing halogens,

sulphur, or nitrogen.
• Compounds are mixed with a reaction gas in a small high temperature
reaction tube.
• Specific reaction products are created which mix with a solvent liquid and
pass through an electrolytic conductivity cell.
• The change in conductivity as a result of the presence of the active compound
is then measured.
• Reaction tube temperature and solvent determine which types of compounds
are detected.
NITROGEN PHOSPHORUS DETECTOR
(NPD)
• The Nitrogen – Phosphorous Detector is also known as Thermionic Specified
Detector (TSD) or Alkali flame ionization detector & is commonly used in gas
chromatography.
• NPD uses a Hydrogen/ Air flame through which the sample is passed.
• NPD uses a rubidium/ cesium bead which is heated by a coil, over which the
carrier gas mixed with Hydrogen passes.
• The hot bead emits electrons by which are collected at the anode and
provides the background current.
• When a component that contains N/P exits the column, the partially
combusted N/P materials are absorbed on the surface of the bead.
• This then increases the emission of electrons.
 Applications

• GC analysis is used to calculate the content of a chemical product, for example in

assuring the quality of products in the chemical industry; or measuring toxic
substances in soil, air or water.
• Gas chromatography is used in the analysis of:
• air-borne pollutants
• performance-enhancing drugs in athlete’s urine samples
• oil spills
• essential oils in perfume preparation
• GC is very accurate if used properly and can measure Pico-moles of a substance in a 1
ml liquid sample, or parts-per-billion concentrations in gaseous samples.
 UNIT-5
NMR, X-RAY AND MASS SPECTROMETRIC
TECHNIQUES
• Nuclear Magnetic Resonance (NMR) Spectroscopy
• Nuclear magnetic resonance spectroscopy, most commonly known as NMR
spectroscopy or magnetic resonance spectroscopy (MRS),is a spectroscopic
technique to observe local magnetic fields around atomic nuclei.
• It is a spectroscopy technique which is based on the absorption of
electromagnetic radiation in the radio frequency region 4 to 900 MHz by nuclei
of the atoms.
• Over the past fifty years, NMR has become the most powerful technique for
determining the structure of organic compounds.
• Of all the spectroscopic methods, it is the only one for which a complete
analysis and interpretation of the entire spectrum is normally expected.
Principle of Nuclear Magnetic Resonance (NMR) Spectroscopy
 

• The principle behind NMR is that many nuclei have spin and all nuclei
are electrically charged. If an external magnetic field is applied, an
energy transfer is possible between the base energy to a higher
energy level (generally a single energy gap).
• The energy transfer takes place at a wavelength that corresponds to
radio frequencies and when the spin returns to its base level, energy
is emitted at the same frequency.
• The signal that matches this transfer is measured in many ways and
processed in order to yield an NMR spectrum for the nucleus
concerned.
 Types of NMR

• Continuous Wave Nuclear Magnetic Resonance (NMR) Spectroscopy(CW- NMR)

• Pulsed Fourier Transform Nuclear Magnetic Resonance (NMR) Spectroscopy (FT-
NMR)
• Working of CW-NMR Spectroscopy
• The sample is placed in a magnetic field and the NMR signal is produced by excitation
of the nuclei sample with radio waves into nuclear magnetic resonance, which is
detected with sensitive radio receivers.
• The intramolecular magnetic field around an atom in a molecule changes the
resonance frequency, thus giving access to details of the electronic structure of a
molecule and its individual functional groups.
• As the fields are unique or highly characteristic to individual compounds, NMR
spectroscopy is the definitive method to identify monomolecular organic compounds.
• Besides identification, NMR spectroscopy provides detailed information about the
structure, dynamics, reaction state, and chemical environment of molecules.
• The most common types of NMR are proton and carbon-13 NMR spectroscopy, but
it is applicable to any kind of sample that contains nuclei possessing spin.
• Spectrum Scan in CW-NMR
• 
• The spectrum can be scanned by the two methods.
• Frequency Sweep method
• In the frequency sweep method the magnetic field is held constant, which keeps
the nuclear spin energy level constant then the RF signal is swept(varied
continuously) to determine the frequencies at which energy is absorbed.
• Field sweep method
• In the field sweep method , the RF signal is held constant then the magnetic field is wept which
varies the energy level to determine the magnetic field strength that produce resonance at a fixed
resonance frequency.
• Sample holder
• Glass tube with 8.5 cm long, 0.3 cm in diameter.
• Permanent magnet
• It provides homogeneous magnetic field at 60-100 MHZ
• Magnetic coils
• These coils induce magnetic field when current flows through them
• Sweep generator
• To produce the equal amount of magnetic field pass through the
sample
• Radio frequency transmitter
• A radio transmitter coil transmitter that produces a short powerful
pulse of radio waves
• Radio frequency receiver
• A radio receiver coil that detects radio frequencies emitted as nuclei
relax to a lower energy level
• Read out systems
• A computer that analyses and record the data.
 Mass Spectrometry
• Mass Spectrometry (MS) is an analytical chemistry technique that helps
identify the amount and type of chemicals present in a sample by measuring
the mass-to- charge ratio and abundance of gas-phase ions.
• In this instrumental technique, sample is converted to rapidly moving ions by
electron bombardment and charged particles are separated according to their
masses.
• Mass spectrum is a plot of relative abundance against the ratio of
mass/charge (m/e).
• These spectra are used to determine the elemental or isotopic signature of a
sample, the masses of particles and of molecules, and to elucidate the
chemical structures of molecules and other chemical compounds.
• Principle of Mass Spectrometry (MS)
• There are four stages in a mass spectrometer which we need to consider, these
are – ionisation, acceleration, deflection, and detection.
• 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 ion 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.
• The ions pass through magnetic and electric fields to reach detector where they
are detected and signals are recorded to give a mass spectra.
Working of Mass Spectrometry (MS)

• In a typical procedure, a sample, which may be solid, liquid, or gas, is ionized, for example by
bombarding it with electrons.
• This may cause some of the sample’s molecules to break into charged fragments. These ions
are then separated according to their mass-to-charge ratio, typically by accelerating them
and subjecting them to an electric or magnetic field.
• Ions of the same mass-to-charge ratio will undergo the same amount of deflection.
• The ions are detected by a mechanism capable of detecting charged particles, such as an
electron multiplier. Results are displayed as spectra of the relative abundance of detected
ions as a function of the mass-to-charge ratio.
• The atoms or molecules in the sample can be identified by correlating known masses (e.g. an
entire molecule) to the identified masses or through a characteristic fragmentation pattern.
Instrumentation and Steps of Mass Spectrometry (MS)
 GM COUNTER(Geiger Muller Counter)

• Geiger Muller Counter is named after its developers: Geiger and

Muller.
• A Geiger counter (Geiger-Muller tube) is a device used for the
detection and measurement of all types of radiation: alpha, beta and
gamma radiation.
• It is a metal cylinder filled with low-pressure gas sealed with a plastic
or ceramic window at one end. Basically it consists of a pair of
electrodes surrounded by a gas. The electrodes have a high voltage
across them. The gas used is usually Helium or Argon.
• This counter works in Geiger region with two specialities.
• The gas multiplication factor is so large that an avalanche dies in at
one point but spreads all over the entire length of the central wire.
• Large output pulses are obtained as the output pulse is independent
both of the energy and nature of the particles detected.
• The Principle of GM Counter
• The voltage of detector is adjusted so that the conditions
correspond to the Geiger-Mueller.
• In this region, the voltage is high enough to provide the primary
electrons with sufficient acceleration and energy so that they can
ionize additional atoms of the medium.
• These secondary ions (gas amplification) formed are also accelerated
causing an effect known as Townsend avalanches. These avalanches
can be triggered and propagated by photons emitted by atoms
excited in the original avalanche.
• Since these photons are not affected by the electric field, they may
interact far (e.g. laterally to the axis) from the primary avalanche, the
entire Geiger tube is participating in the process.
The Working of GM Counter
 

• The Geiger counter has a cathode and an anode that are held at high
voltage, and the device is characterized by a capacitance that is
determined by the geometry of the electrodes.
• In a Geiger counter the fill gas of the chamber is an inert gas which is
ionized by incident radiation, and a quench gas of 5–10% of an organic
vapor or a halogen gas to prevent spurious pulsing by quenching the
electron avalanches.
• As ionizing radiation enters the gas between the electrodes, a finite
number of ion-pairs are formed. In air, the average energy needed to
produce an ion is about 34 eV, therefore a 1 MeV radiation completely
absorbed in the detector produces about 3 x 104 pair of ions.
• The behavior of the resultant ion-pairs is affected by the potential gradient of the
electric field within the gas and the type and pressure of the fill gas. Under the
influence of the electric field, the positive ions will move toward the negatively
charged electrode (outer cylinder), and the negative ions (electrons) will migrate
toward the positive electrode (central wire).
• The electric field in this region keeps the ions from recombining with the
electrons. In the immediate vicinity of the anode wire, the field strength
becomes large enough to produce Townsend avalanches.
• These avalanches can be triggered and propagated by photons emitted by atoms
excited in the original avalanche. Since these photons are not affected by the
electric field, they may interact far (e.g. laterally to the axis) from the primary
avalanche, the entire Geiger tube is participating in the process.
• A strong signal (the amplification factor can reach about 1010) is
produced by these avalanches with shape and height independently of
the primary ionization and the energy of the detected photon. The high
amplification factor of the Geiger counter is the major advantage over
the ionization chamber. Geiger counter is therefore a much more
sensitive device than other chambers. It is often used in the detection of
low-level gamma rays and beta particles for this reason.
• Since the positive ions do not move far from the avalanche region, a
positively charged ion cloud disturbs the electric field and terminates
the avalanche process. In practice the termination of the avalanche is
improved by the use of “quenching” techniques.
• The collection of all these electrons will produce a charge on the
electrodes and an electrical pulse across the detection circuit. Each
pulse corresponds to one gamma ray or neutron interaction. The
pulse height is not proportional to the number of original electrons
produced.
• Therefore, Geiger counters are not capable of particle identification
and energy measurement (spectroscopy). Since the process of charge
amplification greatly improves the signal-to-noise ratio of the
detector, the subsequent electronic amplification is usually not
required.
• It is very useful for general measurement of nuclear radiation, but it
has two important disadvantages.
• Since the pulse height is independent of the type and energy of
radiation, discrimination is not possible. There is no information
whatsoever on the nature of the ionization that caused the pulse.
• Because of the large avalanche induced by any ionization, a Geiger
counter takes a long time (about 1 ms) to recover between successive
pulses. Therefore, Geiger counters are not able to measure high
radiation rates due to the “dead time” of the tube.
Resolving Time or Dead Time and Quenching
• After a count has been recorded, it takes the G-M tube a certain amount of time to
reset itself to be ready to record the next count. The resolving time or ”dead time”,
T, of a detector is the time it takes for the detector to ”reset” itself.
• Since the detector is ”not operating” while it is being reset, the measured activity
is not the true activity of the sample. If the counting rate is high, then the effect of
dead time is very important.
• For Geiger counters, external quenching, sometimes called “active quenching” or
“electronic quenching“, is also a possibility. Electronic quenching uses simplistic
high speed control electronics to rapidly remove and re-apply the high voltage
between the electrodes for a fixed time after each discharge peak in order to
increase the maximum count rate and lifetime of the tube.
• Advantages of GM Counter
• It can count alpha, beta, gamma particles as well as cosmic rays.
• It has high sensitivity.
• Power supply need not be precisely regulated as the pulse height is constant over a large range.
• Because of the fact that output pulse is very high, so the Amplification needed is also very subtle.
• They are relatively inexpensive
• They are durable and easily portable
• Disadvantages of GM Counter
• They cannot differentiate which type of radiation is being detected.
• They cannot be used to determine the exact energy of the detected radiation
• They have a very low efficiency.
• It cannot detect uncharged particles like Neutrons.
 Proportional Counter

• The proportional counter, also known as the proportional detector, is

electrical device that detects various types of ionizing radiation. The
voltage of detector is adjusted so that the conditions correspond to the
proportional region.
• In this region, the voltage is high enough to provide the primary electrons
with sufficient acceleration and energy so that they can ionize additional
atoms of the medium. These secondary ions (gas amplification) formed
are also accelerated causing an effect known as Townsend avalanches,
which creates a single large electrical pulse. Gaseous proportional
counters usually operate in high electric fields of the order of 10
kV/cm and achieve typical amplification factors of about 105.
• An ionization chamber will produce a current that is proportional to the number
of electrons collected each second.
• This current is averaged and is used to drive a display reading in Bq, or μSv/h.
Proportional counters do not work in this way. Instead, they amplify each of the
individual bursts of ionisation so that each ionising event is detected separately.
They the number therefore measure of ionising events.
• The process of charge amplification greatly improves the signal-to-noise ratio of
the detector and reduces the subsequent electronic amplification required.
When instruments are operated in the proportional region, the voltage must be
kept constant. If a voltage remains constant the gas amplification factor also
does not change. Proportional counter detection instruments are very sensitive
to low levels of radiation.
• By proper functional arrangements, modifications, and biasing, the proportional
counter can be used to detect alpha, beta, gamma, or neutron radiation in mixed
radiation fields.
• Moreover, proportional counters are capable of particle identification and energy
measurement (spectroscopy). The pulse height reflects the energy deposited by the
incident radiation in the detector gas. As such, it is possible to distinguish the larger
pulses produced by alpha particles from the smaller pulses produced by beta particles
or gamma rays.
• While ionization chambers can be operated in current or pulse mode, proportional
counters or Geiger counters are almost always used in pulse mode. Detectors of
ionizing radiation can be used both for activity measurements as well as for dose
measurement. With knowledge about the energy needed to form an pair of ions – the
dose can be obtained.
• Basic Principle of Proportional Counters
• The proportional counter has a cathode and an anode that are held at
some voltage (above 1000 V), and the device is characterized by a
capacitance that is determined by the geometry of the electrodes. In
a proportional counter the fill gas of the chamber is an inert gas which
is ionized by incident radiation, and a quench gas to ensure each pulse
discharge terminates; a common mixture is 90% argon, 10% methane,
known as P-10.
• As ionizing radiation enters the gas between the electrodes, a finite
number of ion- pairs are formed. In air, the average energy needed to
produce an ion is about 34 eV, therefore a 1 MeV radiation completely
absorbed in the detector produces about 3 x 104 pair of ions.
• The behavior of the resultant ion-pairs is affected by the potential
gradient of the electric field within the gas and the type and pressure
of the fill gas. Under the influence of the electric field, the positive ions
will move toward the negatively charged electrode (outer cylinder),
and the negative ions (electrons) will migrate toward the positive
electrode (central wire).
• The electric field in this region keeps the ions from recombining with
the electrons. In the immediate vicinity of the anode wire, the field
strength becomes large enough to produce Townsend avalanches.
This avalanche region occurs only fractions of a millimeter from the
anode wire, which itself is of a very small diameter. The purpose of
this is to use the multiplication effect of the avalanche produced by
each ion pair. This is the “avalanche” region. A key design goal is that
each original ionizing event due to incident radiation produces only
one avalanche. Gas amplification factors can range from unity in the
ionization region