KALINGA UNIVERSITY NAYA RAIPUR

DEPARTMENT OF CHEMISTRY

Course: - B.Sc. (BT/PCM/ZBC) B.Sc. VI Sem

Subject: - Instrumental Methods of Analysis

Subject Code:- BSCBT604A/ BZBC603A/BPCM603A

SECTION - A

Very Short Answer type questions.

Unit I

Question 1: What are some key spectroscopic methods covered in the core chemistry syllabus,
and how do they utilize electromagnetic radiation for analytical purposes?

Answer: Several spectroscopic methods are covered in the core chemistry syllabus, including
UV-Visible spectroscopy, infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy
(NMR), and atomic absorption spectroscopy (AAS). These methods utilize electromagnetic
radiation to interact with sample molecules, leading to measurable signals that provide valuable
information about the sample's composition, structure, and concentration.

Question 2: How is analytical data treated in spectroscopy, and why is error analysis important
in the interpretation of spectroscopic results?

Answer: Analytical data in spectroscopy undergoes treatment to extract meaningful information.

This includes calibration processes, baseline corrections, and data normalization. Error analysis
is crucial in spectroscopy to assess the accuracy and precision of measurements. Understanding
and quantifying errors ensure the reliability of spectroscopic results, aiding in the correct
interpretation and validation of analytical findings.

Question 3: Discuss the classification of analytical methods, with a focus on the types of
instrumental methods commonly employed. Provide examples of instrumental techniques within
each classification.
Answer: Analytical methods can be classified into two main categories: classical methods and
instrumental methods. Instrumental methods further divide into electrochemical methods (e.g.,
voltammetry), spectroscopic methods (e.g., UV-Visible spectroscopy), chromatographic methods
(e.g., gas chromatography), and mass spectrometric methods (e.g., mass spectrometry). Each
classification offers distinct advantages, and the choice of method depends on the specific
analytical requirements.

Question 4: How does electromagnetic radiation play a central role in various spectroscopic
methods? Briefly explain its interaction with sample molecules and the information it provides.

Answer: Electromagnetic radiation is essential in spectroscopic methods as it interacts with

sample molecules, leading to measurable signals. UV-Visible radiation induces electronic
transitions in molecules, providing information about their electronic structure and
concentration. Infrared radiation causes molecular vibrations, offering insights into functional
groups in compounds. Nuclear magnetic resonance spectroscopy involves the interaction of
nuclei with magnetic fields and radiofrequency radiation, revealing information about the local
environment of nuclei. Atomic absorption spectroscopy utilizes the absorption of specific
wavelengths of light by atoms, aiding in the determination of elemental concentrations.

Question 5: How does the consideration of electromagnetic radiation contribute to the versatility
and selectivity of spectroscopic methods? Provide an example of a spectroscopic technique and
its application in analytical chemistry.

Answer: The consideration of electromagnetic radiation contributes to the versatility and

selectivity of spectroscopic methods by allowing the targeted interaction with specific molecular
properties. For example, in UV-Visible spectroscopy, the absorption of light corresponds to
electronic transitions, providing information about the concentration of chromophores. This
selectivity allows the determination of the concentration of specific compounds in a mixture.
Infrared spectroscopy is selective for molecular vibrations, aiding in the identification of
functional groups in organic compounds. The ability to tailor the type and wavelength of
electromagnetic radiation used in different spectroscopic methods enhances their applicability
and specificity in analytical chemistry.

Unit II
Q1. Define Infrared Spectroscopy.

Answer: Infrared (IR) Spectroscopy is a form of spectroscopy that deals with the infrared part of
the electromagnetic spectrum. The infrared region’s rays have a longer wavelength than light but
a lower frequency. Absorption spectroscopy is the foundation of infrared spectroscopy.

Infrared spectroscopy (IR Spectroscopy) identifies infrared light frequencies absorbed by a

molecule. Because these specific frequencies of light correspond to the frequency of the
vibration of bonds in the molecule, molecules tend to absorb them.
Q2. Briefly explain the Absorption Spectrum.
Answer: Absorption Spectrum: When a ray of white light strikes a prism, it is refracted twice.
 It does this twice: once when it goes from a rarer medium (air) to a denser medium
(glass), and then again when it travels from a denser medium (glass) to a rarer medium
(air).
 Finally, we see a spectrum, which is a band of colours created by a ray of white light. If
we look carefully at this spectrum, the colour with the shortest wavelength deviates the
most, and vice versa.

 As a result, a spectrum of colours ranging from red to violet can be seen, with red
suffering the least variation due to its longest wavelength.

 As violet merges into blue, blue into green, and so on, this type of spectrum is known as
a continuous spectrum.

Q3. What are the advantages of Mass Spectrometry?

Answer: Mass spectrometry (MS) is an analytical technique that identifies the molecular weight
of compounds and thus allows for the identification of individual elements. When used in
conjunction with other identification techniques like carbon and proton NMR and IR
spectroscopy, mass spectrometry can be extremely helpful in identifying unknown compounds.

MS can also tell you whether a compound contains particular elements like bromine or chlorine.
These halogens can be easily detected by comparing the intensity ratios of ions with atomic mass
units (m/z) that differ by two.

Q4. Briefly explain Proton magnetic resonance spectroscopy.

Answer: Because most organic compounds contain numerous hydrogen atoms, and the
hydrogen atoms absorb the energy of different wavelengths depending on their bonding
environment, proton NMR spectra provide a plethora of data about molecular structure.

Nuclear magnetic resonance with respect to hydrogen-1 nuclei within a substance’s molecules is
known as proton nuclear magnetic resonance (proton NMR, hydrogen-1 NMR, or 1H NMR). In
samples containing natural hydrogen (H), the isotope 1H makes up nearly all of the hydrogen
(hydrogen-1; i.e., having a proton for a nucleus).

Q5. Mention the differences between Spectrometry and Spectroscopy.

Answer: The science of spectroscopy is the study of the interaction between matter and radiated
energy. It’s the study of matter’s absorption properties or absorption behaviour when exposed to
electromagnetic radiation. Spectroscopy is a theoretical method of science that does not produce
any results.

Spectrometry, on the other hand, is a technique for obtaining a quantitative spectrum

measurement. It is the practical application in which results are obtained, assisting in quantifying
absorbance, optical density, and transmittance. In a summary, spectroscopy is a theoretical
science, and spectrometry is a practical measurement in the atomic and molecular balances of
matter.

Unit III

Question 1: What are the primary factors influencing the separation in chromatography, and
how do these techniques differ in terms of separation mechanisms?
Answer: The primary factors influencing separation in chromatography are volatility, solubility,
interactions with the stationary phase, size, and electrical field. Gas chromatography separates
based on volatility, liquid chromatography on solubility, and electrophoresis on size and charge
interactions.
Question 2: Explain the importance of column technology in chromatography, specifically
discussing the role of packing materials and capillaries.
Answer: Column technology is crucial in chromatography for efficient separation. Packing
materials and capillary dimensions influence separation efficiency. Different packing materials
provide diverse stationary phases, allowing for the separation of compounds based on their
interactions with the stationary phase.
Question 3: How is mass spectrometry used to make gaseous molecules into ions, and what are
the different techniques employed for making liquids and solids into ions?
Answer: Gaseous molecules are made into ions in mass spectrometry through techniques like
electron impact and chemical ionization. For liquids and solids, ionization techniques include
electrospray, electrical discharge, laser desorption, and fast atom bombardment.
Question 4: Discuss the detection methods in chromatography and mass spectrometry. How is
detection used as a means of further analysis, including the use of tags and coupling to other
analytical techniques?
Answer: Detection methods in chromatography include simple (e.g., UV-Vis) and specific (e.g.,
MS) techniques. Detection is used for further analysis by incorporating tags and coupling to
infrared (IR) and mass spectrometry (MS) techniques, providing additional information about the
compounds being analyzed.
Question 5: In electrophoresis, how does it contribute to DNA analysis, and what role does it
play in immunological assays (immunoassays)?
Answer: In DNA analysis, electrophoresis is used to separate DNA fragments based on size.
Capillary electrophoresis offers high-resolution DNA sequencing. In immunoassays,
electrophoresis helps separate and analyze proteins, aiding in the detection and quantification of
specific antibodies or antigens.

Unit IV

Q1. What are the different electroanalytical methods and their application?

Answer: The four main categories are potentiometry (the difference in electrode potentials is
measured), amperometry (electric current is the analytical signal), coulometry (charge passed
during a certain time is recorded), and voltammetry (the cell's current is measured while actively
altering the cell's potential).

Q2. What are the important applications of electrochemistry in industry?

Answer: Electrochemistry Applications are as follows:

 Electrosynthesis.
 Electrolysis.
  Electrochemical Corrosion.

 Battery Testing.

 Biological Electrochemistry.

 Photovoltaics.

 Fuel Cells.

 Supercapacitors.

Q3. What is the working principle of pH meter?

Answer: The working of the pH meter is such that the glass electrode and the reference
electrode are dipped in the electrolytic solution which in turn are connected to the test
solution(whose pH is to be found) through a porous ceramic membrane. There is then a
voltmeter which displays the volts in terms of pH.

Q4. What are the types of conductometric titration?

Answer: Types of conductometric titrations:

 Acid-base titration.
 Precipitation titration.

 Replacement titration.

 Redox (oxidation-reduction) titration.

 Complexometric titration.

Q5. Define pH. What is unit for pH?

Answer: pH is defined as the negative log of the hydrogen ion concentration. The range of pH
extends from zero to 14.pH is a logarithm (the negative of the logarithm of H+ activity), and as
such, it has no units.

Unit V

Q1. What are the mobile and stationary phases in chromatography?

Answer: Chromatography relies on two different 'phases': the mobile phase is the solvent that
moves through the paper, carrying different substances with it. the stationary phase is contained
on the paper and does not move through it.
Q2. What do you mean by HPTLC?
Answer: HPTLC is the High-Performance version of Thin Layer Chromatography and a state-
of-the art technique for plant analysis. It features significantly shorter developing times, lower
solvent consumptions and improved resolution. Highly reproducible results and traceable records
are achieved through a standardized methodology and the use of suitable instruments (typically
controlled by software) for all steps of the analysis. A system suitability test is used to qualify
results.
The stationary phase is a HPTLC glass plate or aluminum sheet coated with a uniform thin layer
(typically 200 micron) of porous particles (2 – 10 μm) with an average particle size of 5 μm.
Q3. What is the main principle of solvent extraction?

Answer: The principle behind solvent extraction is extremely basic. The goal is to use a liquid
(solvent) to dissolve (solvate) a target molecule or group of compounds (solute) and to wash
them out of the solid plant material. The solvent is then separated from the solute in order to
concentrate the solute.

Q4. What is adsorption and partition?

Answer: The adsorption chromatography is used for solid-gas chromatography and solid-liquid
chromatography. Whereas, partition chromatography is the liquid–gas, and liquid-liquid. An
example of adsorption chromatography is column chromatography. An example of partition
chromatography in paper chromatography.

Q5. What are the two types of ion exchange chromatography?

Answer: Ion exchange chromatography (IEX) separates molecules by their surface charge, a
property that can vary vastly between different proteins. There are two types of IEX, cation
exchange and anion exchange chromatography.
 SECTION B

Short Answer type questions.

Unit I
Q1. Differentiate between qualitative and quantitative analysis.
Answer: The main distinction between qualitative and quantitative chemistry is that qualitative
chemistry determines the presence or absence of various chemical components in a sample,
whereas quantitative chemistry determines the amount of various chemical components present
in a sample.

Qualitative Analysis Quantitative Analysis

Qualitative vs In chemistry, qualitative analysis is In chemistry, quantitative

Quantitative Analysis a branch of the subject that analysis is a section of the subject
in Chemistry examines the chemical composition that deals with the quantities of
of a material. various components in a sample.

Details The presence or absence of various In chemistry, quantitative

chemical components in a sample is analysis is a section of the subject
determined via qualitative analysis that deals with the quantities of
in chemistry. various components in a sample.

Techniques The amount of different chemical

In chemistry, qualitative analysis
components contained in a given
employs procedures such as
distillation, extraction, colour sample is determined via
change, chromatography, and so on. quantitative analysis in
chemistry.

Q2. Write the main types of Error. Explain.

Answer: no analysis is free of error or “uncertainty”. The main types of error are as follow:
A. Systematic Error (determinate error)

The error is reproducible and can be discovered and

corrected.
B. Random Error (indeterminate error)
Caused by uncontrollable variables, which can not be
defined/eliminated.
Systematic (determinate) errors:
1. Instrument errors - failure to calibrate, degradation of parts in the
instrument, power fluctuations, variation in temperature, etc.
Can be corrected by calibration or proper instrumentation maintenance.
2. Method errors - errors due to no ideal physical or chemical behavior
- completeness and speed of reaction, interfering side reactions,
sampling problems
Can be corrected with proper method development.
3. Personal errors - occur where measurements require judgment, result
from prejudice, color acuity problems.
Can be minimized or eliminated with proper training and experience.
Random (indeterminate) Error:
• No identifiable cause; Always present, cannot be
eliminated; the ultimate limitation on the
determination of a quantity.
• Ex. reading a scale on an instrument caused by the
finite thickness of the lines on the scale; electrical
noise
• The accumulated effect causes replicate
measurements to fluctuate randomly around the
mean; Give rise to a normal or Gaussian curve; Can
be evaluated using statistics.
Q3. Write a note on Student’s t-test.
Answer: Student’s t-test, in statistics, a method of testing hypotheses about the mean of a small
sample drawn from a normally distributed population when the population standard deviation is
unknown.
In 1908 William Sealy Gosset, an Englishman publishing under the pseudonym Student,
developed the t-test and t distribution. (Gosset worked at the Guinness brewery in Dublin and
found that existing statistical techniques using large samples were not useful for the small
sample sizes that he encountered in his work.) The t distribution is a family of curves in which
the number of degrees of freedom (the number of independent observations in the sample minus
one) specifies a particular curve. As the sample size (and thus the degrees of freedom) increases,
the t distribution approaches the bell shape of the standard normal distribution. In practice, for
tests involving the mean of a sample of size greater than 30, the normal distribution is usually
applied.
It is usual first to formulate a null hypothesis, which states that there is no effective
difference between the observed sample mean and the hypothesized or stated population mean—
i.e., that any measured difference is due only to chance. In an agricultural study, for example, the
null hypothesis could be that an application of fertilizer has had no effect on crop yield, and an
experiment would be performed to test whether it has increased the harvest. In general, a t-test
may be either two-sided (also termed two-tailed), stating simply that the means are not
equivalent, or one-sided, specifying whether the observed mean is larger or smaller than the
hypothesized mean. The test statistic t is then calculated. If the observed t-statistic is more
extreme than the critical value determined by the appropriate reference distribution, the null
hypothesis is rejected. The appropriate reference distribution for the t-statistic is the t
distribution. The critical value depends on the significance level of the test (the probability of
erroneously rejecting the null hypothesis).
For example, suppose a researcher wishes to test the hypothesis that a sample of size n = 25 with
mean x = 79 and standard deviation s = 10 was drawn at random from a population with mean μ

= 75 and unknown standard deviation. Using the formula for the t-statistic, the
calculated t equals 2. For a two-sided test at a common level of significance α = 0.05, the critical
values from the t distribution on 24 degrees of freedom are −2.064 and 2.064. The calculated t
does not exceed these values, hence the null hypothesis cannot be rejected with 95 percent
confidence. (The confidence level is 1 − α.)
A second application of the t distribution tests the hypothesis that two independent random
samples have the same mean. The t distribution can also be used to construct confidence
intervals for the true mean of a population (the first application) or for the difference between
two sample means (the second application).
Q4. How To Reduce Errors In Measurement.

Answer: Keeping an eye on the procedure and following the below listed points can help to
reduce the error.

 Make sure the formulas used for measurement are correct.

 Cross check the measured value of a quantity for improved accuracy.

 Use the instrument that has the highest precision.

 It is suggested to pilot test measuring instruments for better accuracy.

 Use multiple measures for the same construct.

  Note the measurements under controlled conditions.

Q5. Discuss about Normal Distribution.

Answer: A normal distribution implies that if you take a large enough number of measurements
of the same property for the same sample under the same conditions subject only to random
(indeterminate) error, the values will be distributed around the expected value, or mean, and that
the frequency with which a particular result (i.e. value) ocurs will become lower the farther away
the result is from the mean.
Put another way, a normal distribution is a probability curve where there is a high probability of
an event (i.e. a particular value) occurring near the mean value, with a decreasing chance of an
event occurring as we move away from the mean. The normal distribution curve and equation
look like this:

The important thing to know about the Normal Distribution is that the probability of getting a
certain result decreases the farther that result is from the mean. The concept of the normal
distribution will be important when we talk about 1- and 2-tailed tests, confidence levels, and
statistical tests, such as the t-test and F-test.
There are many other types of distributions, but we will only consider the normal distribution
here. This is because we assume that the measurements we perform in this course will be
normally distributed about the mean, and that the random errors will also be normally
distributed. Generally, this is a good assumption, though there are many situations where it does
not apply.
Unit II
Q1: Explain Nuclear Magnetic Resonance (NMR) spectroscopy.
Answer: The study of molecules by recording the interaction of radiofrequency (Rf)
electromagnetic radiations with the nuclei of molecules placed in a strong magnetic field is
known as nuclear magnetic resonance (NMR) spectroscopy.
It’s a study method that takes advantage of the magnetic characteristics of specific atomic nuclei.
The physical and chemical properties of atoms or molecules are determined using NMR
spectroscopy.
It uses the nuclear magnetic resonance phenomena to offer extensive information on a
molecule’s structure, dynamics, reaction state, and chemical environment.

Q2. Give the applications of Mass Spectrometry.

Answer: Mass spectrometry has a wide range of applications. Mass spectrometry is utilised in
practically every branch of science for both qualitative and quantitative study of macromolecules
and low molecular weight substances, whether in pure or applied research. Some of the most
important uses of mass spectrometry are listed below.
 Biomolecules such as proteins, carbohydrates, and nucleic acids have molecular masses
that may be determined.
 Biopolymer sequences, such as nucleic acids, oligosaccharides, and polypeptides, are
determined.

 To ascertain the structure of a protein.

 Elements and their isotopes are identified.

 Toxins and pesticide residues in food are tested.

 Monitoring the environment and climate change by analysing air, water, and soil quality.

 During surgery, monitoring the metabolic gas exchange of patients.

 Carbon dating of samples and determining the composition of rock and soil.

 Quality control in the chemical and petrochemical sectors is examined.

 Particles in aerosols, such as perfumes, are being studied.

  Analyzing drug misuse metabolites in saliva, urine, and blood to determine drug abuse
cases.

Q3. Describe the types of atomic spectroscopy.

Answer: Atomic spectroscopy is classified into three main types, and they are,
a. Atomic emission spectroscopy: Atomic emission spectroscopy involves energy transfer from
the ground state to an excited state. The electronic transition can be explained by atomic
emission.
b. Atomic absorption spectroscopy: For absorption to occur, there should be identical energy
differences between the lower and higher energy levels. According to the atomic absorption
spectroscopy principle, free electrons generated in an atomizer can absorb radiation at a specific
frequency. It measures the absorption of ground-state atoms in gaseous form.
c. Atomic fluorescence spectroscopy: Atomic fluorescence spectroscopy combines atomic
emission and atomic absorption because it involves excitation and de-excitation radiation.
Q4. Write advantages and disadvantages of flame photometer.

Answer: Advantages:

1. Simple quantitative analytical test based on the flame analysis.

2. Inexpensive.

3. The determination of elements such as alkali and alkaline earth metals is performed
easily with most reliable and convenient methods.

4. Quite quick, convenient, and selective and sensitive to even parts per million (ppm) to
parts per billion (ppb) range.

Disadvantages: Moreover the flame photometer has a wide range of applications in the
analytical chemistry, it possess many disadvantages which are explained below:

1. The concentration of the metal ion in the solution cannot be measured accurately..

2. A standard solution with known molarities is required for determining the

concentration of the ions which will corresponds to the emission spectra.

3. It is difficult to obtain the accurate results of ions with higher concentration.

4. The information about the molecular structure of the compound present in the sample
solution cannot be determined.
 5. The elements such as carbon, hydrogen and halides cannot be detected due to its
non-radiating nature.

Q5. Differentiate between UV visible and IR spectroscopy.

Answer:
S.
UV Visible Spectroscopy IR Spectroscopy
No.

UV visible spectroscopy is absorption IR is absorption spectroscopy in the

1. spectroscopy in the ultraviolet-visible spectral infrared spectral region of the
region of the electromagnetic spectrum. electromagnetic spectrum.

It has a shorter wavelength as compared to visible It has a longer wavelength as compared

2.
light. to visible light.

It has low frequency and less energy per

3. It has high frequency and more energy per photon
photon.

It changes the rotational and vibration

4. It changes electronic energy within the molecule.
movements of the molecule.

Unit III
Question 1: Discuss the principles of gas chromatography (GC) and liquid chromatography
(LC), emphasizing the importance of column technology in separation. How do the separation
mechanisms differ in GC and LC, and how does column packing influence efficiency?

Answer: Gas chromatography (GC) and liquid chromatography (LC) are widely used separation
techniques. GC separates volatile compounds based on their vaporization and partitioning in a
column, while LC separates compounds based on solubility and interactions with the stationary
phase. The choice of column technology, including packing materials and capillaries, is pivotal
in achieving efficient separations. The stationary phase's nature, thickness, and column
dimensions impact resolution and selectivity. In GC, compounds with higher volatility elute
faster, whereas in LC, solubility and interactions play a critical role. The variation in column
packing materials allows tailoring separation conditions for specific analytes, making column
technology a key factor in chromatographic performance.

Question 2: Elaborate on the ionization techniques in mass spectrometry, explaining how

gaseous molecules, liquids, and solids are transformed into ions. Discuss the principles of ion
separation using different mass analyzers, including magnetic sector, time-of-flight, and electric
quadrupole.

Answer: Mass spectrometry (MS) is a powerful analytical technique for identifying and
characterizing molecules. Gaseous molecules are ionized through electron impact or chemical
ionization, while liquids and solids undergo ionization methods such as electrospray, electrical
discharge, laser desorption, and fast atom bombardment. The separation of ions is achieved
based on their mass-to-charge ratio (m/z). Different mass analyzers contribute to ion separation.
Magnetic sector analyzers utilize a magnetic field, time-of-flight analyzers rely on ion flight
time, and electric quadrupole analyzers use varying electric fields. Each method offers unique
advantages, and the choice depends on factors such as resolution requirements and the type of
sample being analyzed.

Question 3: Examine the role of detection methods in chromatography and mass spectrometry.
Distinguish between simple and specific detection, and elaborate on how detection is utilized for
further analysis, including the use of tags and coupling to other analytical techniques like
infrared (IR) and mass spectrometry (MS).

Answer: Detection methods play a crucial role in chromatography and mass spectrometry,
providing insights into the identity and concentration of analytes. Simple detection methods,
such as UV-Vis in chromatography, offer general information about the sample. Specific
detection, as seen in mass spectrometry (MS), provides detailed information on individual
compounds. Detection serves as a means for further analysis through the incorporation of tags
and coupling to other techniques. Tags enhance specificity, allowing the detection of specific
functional groups or compounds. Coupling to infrared (IR) and mass spectrometry (MS)
techniques provides additional information about the molecular structure and composition,
contributing to a more comprehensive analysis of complex samples.

Question 4: Explore the applications of electrophoresis in DNA analysis and immunoassays.

Discuss how electrophoresis techniques, including plates and capillaries, contribute to the
separation of DNA fragments and proteins. Highlight the significance of high-resolution
capillary electrophoresis in DNA sequencing.

Answer: Electrophoresis is a versatile technique with applications in DNA analysis and

immunoassays. In DNA analysis, electrophoresis separates DNA fragments based on size,
allowing for the characterization of DNA samples. Capillary electrophoresis, with its high
resolution and efficiency, has become a powerful tool in DNA sequencing. It enables the
separation of DNA fragments with enhanced precision and speed compared to traditional gel
electrophoresis. In immunoassays, electrophoresis is used for the separation of proteins,
facilitating the detection and quantification of specific antibodies or antigens. The technique aids
in profiling complex protein mixtures, enhancing the sensitivity and specificity of immunoassays
for diagnostic purposes.

Question 5: Explain the factors influencing resolution, time, and multiple separations in mass
spectrometry. Delve into the detection and interpretation processes, emphasizing the linkage to
excitation and its role in analyzing complex samples.

Answer: Resolution, time, and the ability to perform multiple separations are critical aspects of
mass spectrometry (MS). Resolution is influenced by factors such as the type of ionization, mass
analyzer, and magnetic field strength. Time considerations involve the speed of analysis, with
time-of-flight (TOF) analyzers providing rapid results. Multiple separations are achievable by
combining various ionization and separation methods, enhancing the technique's versatility.
Detection in mass spectrometry involves analyzing mass spectra, and interpretation relies on
characteristic peaks corresponding to specific ions. The linkage to excitation refers to the initial
energy input during ionization, influencing the observed mass spectra. Understanding excitation
aids in identifying and characterizing compounds in complex samples, providing valuable
information for detailed molecular analysis and structural elucidation.

Unit IV
Q1. What is Potentiometric Titration? Explain.

Answer: It is the procedure through which the quantity of the given test substance is determined
by the measured addition of titrant until the entire test substance undergoes reaction. After the
titration process, the potential difference between the two electrodes (namely the reference and
indicator electrode) is measured in conditions where a thermodynamic equilibrium is maintained
and the current passing through the electrodes does not disturb this equilibrium.
Potentiometric Titration Principle
Potentiometric titration is a laboratory method to determine the concentration of a given analyte.
It is used in the characterization of acids. In this method, there is no use of a chemical indicator.
Instead, the electric potential across the substance is measured.
Potentiometric Titration Method
Potentiometric Titration is done via the usage of two electrodes – an indicator electrode and a
reference electrode (generally a hydrogen electrode or a silver chloride electrode). One half-cell
is formed with the indicator electrode and the ions of the analyte, which is generally an
electrolyte solution. The other half-cell is formed by the reference electrode.
The overall cell potential can be calculated using the formula given below.
Where the potential drop between the indicator and reference electrodes over the electrolyte
solution is given by Esol.
The overall cell potential, Ecell is calculated in every interval where the titrant is measured and
added. Now, a graph is plotted with the Potential difference on the Y-axis and the volume on the
X-axis as shown below.

It can be observed from the graph that the electric potential of the cell is dependent on the
concentration of ions which are in contact with the indicator electrode. Therefore, the E cell is
measured with each addition of the titrant.
Types of Potentiometric Titration
There are four types of titration that fall under the category of potentiometric titration, namely
acid-base titration, redox titration, complexometric titration, and precipitation titration. A brief
description of each of these types of titration is given below.
Acid-Base Titration: This type of potentiometric titration is used to determine the concentration
of a given acid/base by neutralizing it exactly using a standard solution of base/acid whose
concentration is known.
Redox Titration: This type of potentiometric titration involves an analyte and titrant that
undergo a redox reaction. An example of this type of titration would be the treatment of an
iodine solution with a reducing agent which produces iodide ion (a starch indicator is used to get
the endpoint).
Complexometric Titration: This type of titration can also be referred to as chelatometry. In this
method, a coloured complex is formed, indicating the end point of the titration. This method is
used to determine a mixture of metal ions in a given solution.
Precipitation Titration: This type of titration involves a reaction between the given analyte and
the titrant wherein an insoluble precipitate is formed. The end-point of this titration is noted
when the addition of the titrant no longer forms a precipitate.
Q2. What is pKa? How do we calculate the pKa? Describe the relation between pKa and
pKb.
Answer: The pKa value is the negative base -10 logarithm of the acid dissociation
constant (Ka) of a solution.
The quantitative behavior of acids and bases in solution can be understood only if their pKa
values are known. In particular, the pH of a solution can be predicted when the analytical
concentration and pKa values of all acids and bases are known; conversely, it is possible to
calculate the equilibrium concentration of the acids and bases in solution when the pH is known.
These calculations find application in many different areas of chemistry, biology, medicine, and
geology. For example, many compounds used for medication are weak acids or bases, and a
knowledge of the pKa values.
Definition of pKa: pKa is a number that describes the acidity of a particular molecule. It
measures the strength of an acid by how tightly a proton is held by a Bronsted acid. The lower
the value of pKa, the stronger the acid and the greater its ability to donate its protons. describe
the acidity of a particular molecule. Ka denotes the acid dissociation constant. It measures how
completely an acid dissociates in an aqueous solution. The larger the value of Ka, the stronger
the acid as acid largely dissociates into its ions and has lower pka value. The relationship
between pKa and Ka is described by the following equation:
pKa = -log[Ka]
Acid dissociation constants, or pKa values, are essential for understanding many fundamental
reactions in chemistry. These values reveal the deprotonation state of a molecule in a particular
solvent. There is great interest in using theoretical methods to calculate the pKa values for many
different types of molecules.
Calulation of pKa
 Let us consider a weak acid HA which ionises in the aqueous solution as:
 o HA(acid) + H2O ⇋ H3O+ (aq) + A– (aq)(conjugate base)

 The dissociation constant of acid is defined by

Ka = [H3O+] [A–] / [HA]

pKa = – log Ka = – log { [H3O+] [A–] / [HA]}
 In the case of polyprotic acid which contains more than 1 proton dissociates stepwise and
produces more than one dissociation constant and more than one pKa value.

Examples with Phosphoric acid which contain 3 protons that produced dissociation of the first
proton may be denoted as Ka1 and the constants for the dissociation of successive protons as
Ka2, and Ka3.
H3PO4 ⇋ H2PO4– + H+
Ka1 = [H2PO4– ] [H+] / [H3PO4]
pKa1 = – log Ka1 = – log {[H2PO4– ] [H+] / [H3PO4]}
H2PO4– ⇋ HPO42- + H+
Ka2 = [HPO42- ] [H+] / [H2PO4– ]
pKa2 = – log Ka2 = – log {[HPO42- ] [H+] / [H2PO4– ]}
HPO42- ⇋ PO43- + H+
Ka3 = [PO43- ] [H+] / [HPO42- ]
pKa3 = – log Ka3 = – log {[PO43- ] [H+] / [HPO42- ]}
pKa and pH of buffer solution
From the Henderson equation of acidic buffer, the pH of the solution is defined as the
pH = pKa + log { [salt] / [Acid]}
When [salt] / [Acid] = 10 then,
pH = pKa + 1
When [salt] / [Acid] = 1/10 then,
pH = pKa – 1
Note: So weak acid may be used for preparing buffer solutions having pH values lying within
the ranges pKa + 1 and pKa – 1.
The acetic acid has a pK a of about 4.8. It may therefore be used for making buffer solutions with
pH values lying roughly between the range 3.8 to 5.8.
Relation between pKa and pKb
Let us consider a weak acid HA which ionises in the aqueous solution as:
HA + H2O ⇋ H3O+ (aq) + A– (aq)
(acid) (conjugate base)
The dissociation constant of acid is defined by
Ka = [H3O+] [A–] / [HA] ……….. (1)
The conjugate base A- behaves as a weak base in water
A– + H2O ⇋ HA + OH–
For base Kb = [HA] [ OH–] / [A– ] …………. (2)
Multiply equation (i) and (ii)
Ka x Kb = {[H3O+] [A–] / [HA]} x {[HA] [OH–] / [A– ]} = [H3O+] [OH–] = Kw
Ka x Kb = Kw
On taking negative logarithm on both side
– log Ka – log Kb = – log Kw
pKa + pKb = pKw
Q3. How does optical polarimetry work?

Answer: Polarimetry machines are used in chemistry in a variety of ways. Their primary use is
to measure the angle of rotation of an optically active substance using polarized light. The
polarized light will either rotate clockwise or counter-clockwise and the amount it rotates
indicates the angle of rotation. Polarimetry is important in chemistry due to the fact that it allows
one to distinguish between optically active stereoisomers using optical activity as a measuring
point. There are other key tests that are used in chemistry for identification of substances, such as
melting point. However, these tests would prove non conclusive for identification of some
stereoisomers, such as enantiomers, as they will have identical physical properties, such as
melting points and boiling points. The step by step process of rotation of plane-polarized light in
a polarimeter is depicted by the schematic diagram below:
The specific rotation value of a chiral substance is dependent on numerous variables. The main
variables are concentration of the substance and the length of the tube in the polarimeter
machine.

Q4. Define Equivalence Point.

Answer: The equivalence point is the point in a titration where the amount of titrant added is
enough to completely neutralize the analyte solution. The moles of titrant (standard solution)
equal the moles of the solution with unknown concentration. This is also known as the
stoichiometric point because it is where the moles of acid are equal to the amount needed to
neutralize the equivalent moles of base. Note this does not necessarily mean the acid to base
ratio is 1:1. The ratio is determined by the balanced acid-base chemical equation.
The equivalence point is not the same as the endpoint of a titration. The endpoint refers to the
point at which an indicator changes color. More often than not, the color change occurs after the
equivalence point has already been reached. Using the endpoint to calculate equivalence
naturally introduces error.
Q5. What are the methods of finding the Equivalence Point?
Answer: There are several different ways to identify the equivalence point of a titration:
Color Change - Some reactions naturally change color at the equivalence point. This may be
seen in redox titration, particularly involving transition metals, where the oxidation states have
different colors.
pH Indicator - A colored pH indicator may be used, which changes color according to pH. The
indicator dye is added at the beginning of the titration. The color change at the endpoint is an
approximation of the equivalence point.
Precipitation - If an insoluble precipitate forms as a result of the reaction, it can be used to
determine the equivalence point. For example, the silver cation and chloride anion react to form
silver chloride, which is insoluble in water. However, it can be difficult to determine
precipitation because the particle size, color, and sedimentation rate may make it difficult to see.
Conductance - Ions affect the electrical conductivity of a solution, so when they react with each
other, the conductivity changes. Conductance may be a difficult method to use, especially if
other ions are present in the solution that can contribute to its conductivity. Conductance is used
for some acid-base reactions.
Isothermal Calorimetry - The equivalence point may be determined by measuring the amount
of heat that is produced or absorbed using a device called an isothermal titration calorimeter.
This method is often used in titrations involving biochemical reactions, such as enzyme binding.
Spectroscopy - Spectroscopy can be used to find the equivalence point if the spectrum of the
reactant, product, or titrant is known. This method is used to detect etching of semiconductors.
Thermometric Titrimetry - In thermometric titrimetry, the equivalence point is determined by
measuring the rate of temperature change produced by a chemical reaction. In this case, the
inflection point indicates the equivalence point of an exothermic or endothermic reaction.
Amperometry - In an ampometric titration, the equivalence point is seen as a change in the
measured current. Amperometry is used when the excess titrant is able to be reduced. The
method is useful, for example, when titrating a halide with Ag + because it isn't affected by
precipitate formation.

Unit V
Question 1: Explain the principle of NMR spectroscopy and how it provides information about
the molecular structure of compounds.

Answer: Nuclear Magnetic Resonance (NMR) spectroscopy is based on the principle of nuclear
spin. When a sample is exposed to a strong magnetic field and radiofrequency radiation, certain
nuclei with a nonzero magnetic moment absorb energy and undergo resonance. The energy
absorbed is proportional to the strength of the magnetic field, allowing for the determination of
the chemical environment of the nuclei. NMR provides detailed information about molecular
structure, including the types of atoms present, their connectivity, and the local environment,
making it a powerful tool for structural elucidation in organic and biochemistry.

Question 2: Describe the instrumentation used in NMR spectroscopy and the role of magnets
and radiofrequency pulses in the NMR experiment.

Answer: NMR spectroscopy utilizes specialized instruments consisting of powerful magnets and
radiofrequency (RF) pulse systems. The magnets generate a strong and homogeneous magnetic
field, aligning nuclear spins in the sample. RF pulses are applied perpendicular to the magnetic
field, perturbing the spin alignment. When the RF pulse is turned off, the spins relax back to
their equilibrium positions, emitting signals that are detected and used to generate NMR spectra.
The strength and homogeneity of the magnetic field, as well as the precision of the RF pulses,
are critical for obtaining high-quality NMR data.

Question 3: What are the factors affecting chemical shift in NMR spectroscopy, and how do
they influence the positions of signals in the NMR spectrum?

Answer: Chemical shift in NMR spectroscopy is influenced by factors such as electronegativity,

hybridization, and magnetic anisotropy. Electronegative atoms or groups cause deshielding,
leading to higher chemical shifts, while electron-donating groups result in shielding and lower
chemical shifts. Hybridization affects chemical shift through sigma and pi contributions.
Magnetic anisotropy arises from the orientation of neighboring bonds, influencing the local
magnetic field. Understanding these factors is crucial for interpreting NMR spectra and
assigning signals to specific nuclei in a molecule.

Question 4: Discuss the principles of potentiometry and voltammetry in electroanalytical

methods. How do these techniques measure analyte concentrations, and what are their respective
applications?

Answer: Potentiometry involves measuring the potential difference between an indicator

electrode and a reference electrode at zero current. It is used for determining concentrations of
ions in solutions, especially in titrations. Voltammetry, on the other hand, measures the current
flowing through an electrochemical cell under varying applied potentials. It is employed for
studying redox reactions and determining concentration through the measurement of peak
currents or potentials. Both techniques find applications in analytical chemistry, with
potentiometry commonly used in titrations and voltammetry applied in fields such as
environmental analysis, pharmaceuticals, and electroplating.

Question 5: Explain the principles of X-ray analysis and electron spectroscopy in surface
analysis. How are these techniques used to investigate the composition and structure of materials
at the atomic level?

Answer: X-ray analysis and electron spectroscopy are powerful techniques for surface analysis.
X-ray analysis, including X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS),
utilizes X-rays to determine crystal structures and elemental compositions. Electron
spectroscopy, such as Auger electron spectroscopy (AES) and scanning electron microscopy
(SEM), uses electrons for surface analysis. These techniques provide information about the
elemental composition, crystal structure, and morphology of materials at the atomic level. XRD
is particularly useful for crystalline materials, while XPS and AES offer insights into elemental
composition and chemical bonding on surfaces. SEM provides high-resolution images, allowing
for detailed morphological analysis of surfaces.
 SECTION C

Long Answer type questions:

Unit I

Q1. What are the types of errors in analytical chemistry?

Answer: Types of errors: There are three types of errors that are classified based on the source
they arise from; They are:
 Gross Errors
 Random Errors

 Systematic Errors

Gross Errors
This category basically takes into account human oversight and other mistakes while reading,
recording, and readings. The most common human error in measurement falls under this
category of measurement errors. For example, the person taking the reading from the meter of
the instrument may read 23 as 28. Gross errors can be avoided by using two suitable measures,
and they are written below:
 Proper care should be taken in reading, recording the data. Also, the calculation of error
should be done accurately.
 By increasing the number of experimenters, we can reduce the gross errors. If each
experimenter takes different readings at different points, then by taking the average of
more readings, we can reduce the gross errors

Random Errors
The random errors are those errors, which occur irregularly and hence are random. These can
arise due to random and unpredictable fluctuations in experimental conditions (Example:
unpredictable fluctuations in temperature, voltage supply, mechanical vibrations of experimental
set-ups, etc, errors by the observer taking readings, etc. For example, when the same person
repeats the same observation, he may likely get different readings every time.
This article explored the various types of errors in the measurements we make. These errors are
everywhere in every measurement we make.
Systematic Errors:
Systematic errors can be better understood if we divide them into subgroups; They are:
 Environmental Errors
 Observational Errors

 Instrumental Errors
Environmental Errors: This type of error arises in the measurement due to the effect of the
external conditions on the measurement. The external condition includes temperature, pressure,
and humidity and can also include an external magnetic field. If you measure your temperature
under the armpits and during the measurement, if the electricity goes out and the room gets hot,
it will affect your body temperature, affecting the reading.
Observational Errors: These are the errors that arise due to an individual’s bias, lack of proper
setting of the apparatus, or an individual’s carelessness in taking observations. The measurement
errors also include wrong readings due to Parallax errors.
Instrumental Errors: These errors arise due to faulty construction and calibration of the
measuring instruments. Such errors arise due to the hysteresis of the equipment or due to
friction. Lots of the time, the equipment being used is faulty due to misuse or neglect, which
changes the reading of the equipment. The zero error is a very common type of error. This error
is common in devices like Vernier callipers and screw gauges. The zero error can be either
positive or negative. Sometimes the scale readings are worn off, which can also lead to a bad
reading.
Instrumental error takes place due to :
 An inherent constraint of devices
 Misuse of Apparatus

 Effect of Loading

Q2. Explain the Q-Test for Rejecting Data.

Answer: The Q-Test for Rejecting Data: As mentioned previously, outliers are data
measurements occurring from gross errors.

Their value deviates significantly from the mean. The Q-Test can be used to determine

whether an individual measurement should be rejected or retained. The quantity Q is the absolute
difference between the questioned measurement (xq) and the next closest measurement (xn)
divided by the spread (ω), the difference between the largest and smallest measurement, of the
entire set of data.

(xq − xn)ω

Q =Q is compared to a specified confidence levels (the percent probability a measurement

will fall into a range around the mean (x).) If Q is greater than the values listed below for

a particular confidence level, the measurement should be rejected. If Q is less than the

values in the table, the measurement should be retained.

Assume the student measuring the gas volumes collected a ninth measurement equal to

27.58 L. The Q-value for this measurement is calculated as follows:

Q-value: 27.58 − 26.30

Q = 27.58 − 26.05 = 0.82

The limiting value is 0.60 for rejecting data at the 99% confidence level for 9 measurements.

Since 0.82 is greater than 0.60, the student should reject the measurement.

* A shortcut can be used to test a suspect measurement: Calculate the mean and average or

standard deviation without using the suspect measurement and reject the suspect

measurement if its deviation from the mean is greater than four times the average or standard

deviation.

Q3. Comparison between T-test and F-test in analytical chemistry.

Answer: Comparison between T-test and F-test:
Basis for
T-test F-test
Comparison

F-test is statistical test, that

T-test is a univariate hypothesis test, that is
determines the equality of the
Meaning applied when standard deviation is not
variances of the two normal
known and the sample size is small.
populations.

T-statistic follows Student t-distribution, F-statistic follows Snedecor f-

Test statistic
under null hypothesis. distribution, under null hypothesis.

Application Comparing the means of two populations. Comparing two population variances.
 Basis for
T-test F-test
Comparison

Q4. How do we measure the different types of errors? A length was calculated to be 10.1
feet, but the absolute length was 10.5 feet. Calculate the absolute error.

Answer: Errors Calculation- Different measures of errors include:

Absolute Error
The difference between the measured value of a quantity and its actual value gives the absolute
error. It is the variation between the actual values and measured values. It is given by
Absolute error = |VA-VE|
Percent Error
It is another way of expressing the error in measurement. This calculation allows us to gauge
how accurate a measured value is with respect to the true value. Per cent error is given by the
formula
Percentage error (%) = (VA-VE) / VE) x 100
Relative Error
The ratio of the absolute error to the accepted measurement gives the relative error. The relative
error is given by the formula:
Relative Error = Absolute error / Actual value
According to question,
A length was calculated = 10.1 feet and
the absolute length = 10.5 feet
We know that, Absolute error = |VA-VE|
Absolute error = 10.5-10.1 = 0.4 feet
Q5. What do you mean by sampling? Explain the types of sampling and also discuss about
the purpose of sampling.
Answer: Sampling:- Sampling is the process of obtaining a reasonable amount of material that
has all the essential properties of the bulk material. Or sampling is the science of extracting from
a larger quantity of material, a small portion that is truly representative of the bulk material.
Types of Sampling:
For a sampling technique to be reliable and accurate, the method should confirm to following
conditions.
 The sample mean should provide an unbiased estimate of the population mean.
  Sampling procedure should lead us to an accurate estimate of the central tendency and
dispersion of bulk material for given time & money.

 Test of significance should be applicable on the sample to estimate populations variance.

The sampling procedure can be broadly classified into two types

A) Random Sampling, B) Non-random or Systematic sampling.
A) Random Sampling:-
Random sampling is the selection of samples without bias and in a way that gives full freedom to
the operation of chance factors
 In this method every individual item has an equal chance of being selected; if sample size
is large enough then sample produced is most likely represent the bulk.
 This type of sampling requires minimum knowledge of the bulk material in advance of
being selected.

 When bulk is of homogenous nature, random sampling is comparatively easy. However,

when sample is heterogeneous then different procedure followed.

 Heterogeneous material is first divided into relatively homogenous group i.e. material is
first divided into groups possessing similar characteristics. Then from each group
samples are drawn at random it is called as stratified sampling.

E.g.: ores exist as lumps of various sizes; hence lumps of different sizes are grouped on basis of
their sizes then from each group of sizes samples are drawn at random at mixed.
B) Non-random or Systematic Sampling:-
 This type of sampling appears to be more scientific method than random sampling,
though not necessary that it will give better sample.
 In systematic sampling, sample units are drawn in a definite sequence at equal intervals
from one another.

Purpose of Sampling:-
 Judging acceptability :-

A sampling of bulk many times is done to conclude the material from which sample is done to
conclude whether the material from which sample is drawn meets the requirements such as
purchase or sales specifications. If it meets requirements it can be accepted otherwise rejected.
 Detection of contaminations :-

The second purpose of sampling is to assure that the material under consideration is free from
contamination or any unwanted material. E.g.: Urea in drinking milk.
 Identifying material :-
The third purpose of sampling is to identify an unknown material. A carefully drawn sample can
adequately serve to establish the identity of bulk material under study.
 Estimation of material : –

Sampling is sometimes done to make an estimate that a particular element or material is

present or absent in a given material.
Unit II

Q1. Write principle, instrumentation and application of flame photometry.

Answer: The principle of flame photometry is based on the fact that the compounds of some
elements can be thermally dissociated in a flame and that some of the atoms produced in this
process can get excited to a higher energy level. A flame photometer has three essential parts.

Flame photometry (more accurately called Flame Atomic Emission Spectrometry) is a

branch of spectroscopy in which the species examined in the spectrometer are in the form of
atoms • A photoelectric flame photometer is an instrument used in inorganic chemical analysis
to determine the concentration of Parts of a flame photometer.

1. Source of flame:

A burner that provides flame and can be maintained in a constant form and at a constant
temperature.

2. Nebuliser and mixing chamber:

Helps to transport the homogeneous solution of the substance into the flame at a steady
rate.
3. Optical system (optical filter):

The optical system comprises three parts: convex mirror, lens and filter. The convex
mirror helps to transmit light emitted from the atoms and focus the emissions to the
lens. The convex lens help to focus the light on a point called slit. The reflections from
the mirror pass through the slit and reach the filters. This will isolate the wavelength to
be measured from that of any other extraneous emissions. Hence it acts as interference
type color filters.
4. Photo detector:

Detect the emitted light and measure the intensity of radiation emitted by the flame.
That is, the emitted radiation is converted to an electrical signal with the help of photo
detector. The produced electrical signals are directly proportional to the intensity of
light. A schematic representation of flame photometer is shown in figure 1.
Fig 1: A schematic representation of flame photometer
 Mechanism of working:

The working of the flame photometer involves a series of steps which is discussed in the
following sections.
Nebulisation:

The solution of the substance to be analyzed is first aspirated into the burner, which is then
dispersed into the flame as fine spray particles.

A brief overview of the process:

1. The solvent is first evaporated leaving fine divided solid particles.

2. This solid particles move towards the flame, where the gaseous atoms and ions are
produced.

3. The ions absorb the energy from the flame and excited to high energy levels.

4. When the atoms return to the ground state radiation of the characteristic element is
emitted.

5. The intensity of emitted light is related to the concentration of the element.

Fig 2: Brief overview of the process

Events occurring in the flame:

Flame photometry employs a variety of fuels mainly air, oxygen or nitrous oxide (N2O) as
oxidant. The temperature of the flame depends on fuel-oxidant ratio.

The various processes in the flame are discussed below:

1. Desolvation: The metal particles in the flame are dehydrated by the flame and hence
the solvent is evaporated.
2. Vapourisation: The metal particles in the sample are dehydrated. This also led to the
evaporation of the solvent.
3. Atomization: Reduction of metal ions in the solvent to metal atoms by the flame heat.
4. Excitation: The electrostatic force of attraction between the electrons and nucleus of
the atom helps them to absorb a particular amount of energy. The atoms then jump to
the exited energy state.

5. Emission process: Since the higher energy state is unstable the atoms jump back to
the stable low energy state with the emission of energy in the form of radiation of
characteristic wavelength, which is measured by the photo detector.
The energy level diagram of the sodium atom is shown in figure 3.

Fig 3: Energy level diagram for atomic sodium

The intensity of the light emitted could be described by the Scheibe-Lomakin equation:

Where: I = Intensity of emitted light; C = the concentration of the element; k= constant of

proportionality; n ~ 1 (at the linear part of the calibration curve)

Then,
That is the intensity of emitted light is directly related to the concentration of the sample.

The comparison of emission intensities of unknown samples to either that of standard

solutions (plotting calibration curve), or to those of an internal standard (standard addition
method), helps in the quantitative analysis of the analyte metal in the sample solution.

The flame emissions of the alkali and alkaline earth metals in terms of the emission
wavelength and the characteristic color produced by each element is shown in table 1
 Name of the element Emitted wavelength
Observed colour of the flame
range (nm)

Potassium (K) 766

Violet

Lithium (Li) 670

Red

Calcium (Ca) 622

Orange

Sodium (Na) 589

Yellow

Barium (Ba) 554 Lime green

Applications:

Flame photometer has both quantitative and qualitative applications. Flame photometer with
monochromators emits radiations of characteristic wavelengths which help to detect the
presence of a particular metal in the sample. This help to determine the availability of alkali
and alkaline earth metals which are critical for soil cultivation. In agriculture, the fertilizer
requirement of the soil is analyzed by flame test analysis of the soil. In clinical field, Na+ and
K+ ions in body fluids, muscles and heart can be determined by diluting the blood serum and
aspiration into the flame. Analysis of soft drinks, fruit juices and alcoholic beverages can also
be analyzed by using flame photometry.

Q2. What is FTIR ? Explain the instrumentation.

Answer: Fourier-transform infrared spectroscopy (FTIR) is a technique used to obtain an

infrared spectrum of absorption or emission of a solid, liquid, or gas. An FTIR spectrometer
simultaneously collects high-resolution spectral data over a wide spectral range. This confers a
significant advantage over a dispersive spectrometer, which measures intensity over a narrow
range of wavelengths at a time.
The term Fourier-transform infrared spectroscopy originates from the fact that a Fourier
transform (a mathematical process) is required to convert the raw data into the actual spectrum.
Instrumentation of a FTIR Spectrometer
In FTIR spectroscopy, a Michelson interferometer splits a collimated beam of polychromatic
infrared light into two different optical paths that cause constructive and destructive interference
based on the position of a stationary and moving mirror. This interference waveform
(interferogram) is converted from the time domain to the frequency domain via a Fast Fourier
Transform (FFT). The final spectrum is obtained by separating a broad spectrum of IR radiation
into individual frequencies using the precisely known wavelength of a timing laser.
A FTIR spectrometer consists of four major parts: a light source, an interferometer, a sample
chamber, and a detector (Figure 1).

Fig.1. Optical system diagram a Fourier transform infrared spectroscope.

FTIR Spectrometer Light Sources
The light source for a FTIR spectrometer is typically a broadband emitter, such as a mid-IR
ceramic source (50 – 7,800 cm-1), a near-IR halogen lamp (2,200 – 25,000 cm -1), or a far-IR
mercury lamp (10 – 700 cm -1). JASCO FTIR spectrometers come standard with a high-intensity
ceramic source.
FTIR Spectrometer Michelson Interferometer
The interferometer is the heart of a FTIR spectrometer and consists of a beamsplitter, a
stationary mirror, and a moving mirror. The beamsplitter is a semi-transparent mirror that splits a
collimated beam of light into two different optical paths. Half of the light is transmitted to the
moving mirror while the other half of the light is reflected to the stationary mirror. The two light
beams are reflected from the moving and stationary mirror where they are recombined back at
the beamsplitter before passing through the sample chamber and onto the detector. The
difference in the path of the mirrors causes constructive and destructive interference over the
course of time it takes for the moving mirror to make a pass. The power of the beam (Pin) is
attenuated by the sample due to absorbance by the sample (Pout). The relationship between
power, transmittance, and absorbance can be seen in Figure 5.
FTIR Spectrometer Beamsplitters
In many FTIR systems, the beamsplitter is placed at 45° to the incident beam, but for high
throughput applications, a low angle interferometer (such as the 28° design used by JASCO) is
preferred as the S and P polarizations converge close to Brewster’s Angle. Common beamsplitter
materials are KBr (375 – 12,000 cm-1) for mid-IR, Quartz (4,000 – 25,000 cm-1) for near-IR, and
Mylar (30 – 680 cm-1) for far-IR. JASCO FTIR spectrometers come standard with a Germanium
coated KBr beamsplitter.
FTIR Spectrometer Detectors
FTIR detectors are used to measure the transmitted or reflected light from a sample and convert
it into an electrical signal. The type and material of the detector will determine the sensitivity
and wavelength range of the data that can be acquired. Common FTIR spectrometer detectors
include room temperature DLaTGS (220 – 15,000 cm -1) for routine analysis, liquid nitrogen
cooled MCT (450 – 12,000 cm-1) for high sensitivity applications, silicon photodiodes (10,000 –
25,000 cm-1) for visible and near-IR measurements, and silicon bolometers (10 – 650 cm -1) for
far-IR measurements. JASCO FTIR spectrometers come standard with a DLaTGS detector and
have the option to install a second detector, such as a liquid nitrogen cooled MCT detector.
Q3. Explain the term single beam and double beam spectrophotometer.
Answer: Different types of Spectrophotometers:
The Design of spectroscopic systems is based on the fundamental principle of light
absorption by absorbing species-the Beer Lambert law. Over the years basic design has been
based on single beam or double beam optics with the latter gaining prominence due to its
distinct advantages.
Advances in electronics and detection systems have contributed further to the
popularity of double beam systems. In the present article the discussion will be
limited to Atomic Absorption Spectroscopy systems.

It is important to understand the optical layout of both single beam and double beam
systems before you begin to appreciate the advantages of one over the other. So let’s
get into the details and know these systems better.
Understanding Single Beam Spectrophotometer

Single
Beam Schematic Diagram
This spectrophotometer is used to measure the relative intensity of light before and
after inserting a test sample. It functions by either blanking the instrument or
standardizing it with respect to the reference. There are four basic single beam
spectrophotometer components:
 Monochromator
 Detector
 Light source (should be stable over time, low cost, wide wavelength range
brightness, and long service life)
 Sample (object)
Working: The light source comprising of a hollow cathode lamp emits sharp atomic
line of the element whose determination is required. The light is modulated (switched
on and off) rapidly by means of a rotating chopper located between the light source
and the flame.
Modulation can also be achieved by pulsing the power (switched on and off rapidly)
to the light source. Modulation serves to differentiate the light coming from the
source lamp from the emission from the flame.
The modulated light is led to the flame where ground state atoms of the element of
interest are present and after absorption is led to the monochromator which isolates
the wavelength of interest which is then led to the detector.
Now that you know what a single beam spectrophotometer is and how it works, let’s
see some of the advantages and disadvantages of this instrument.
Advantages of a Single Beam Spectrophotometer
 Cost-Effective: Single beam instruments are less expensive as compared to
the other alternative.
  Better Performance: High energy throughput due to the non-splitting of the
source beam results in high sensitivity of detection.
Disadvantages of a Single Beam Spectrophotometer
 Instability: This happens due to lack of compensation for disturbances like
electronic circuit fluctuations, voltage fluctuations, mechanical component’s
instability, or drift in the energy of light sources. Such drifts cause abnormal
fluctuations in the results.
Understanding Double Beam Spectrophotometer

Double Beam AAS Schematic Diagram

Unlike the single beam variant, this one does not standardize or blank the instrument
before use. Instead, it splits the beam into two parts for the same purpose. One of the
beam parts is passed through the given object while the other passes through a
reference standard. The number of components also get increased in this
spectrophotometer.
 Sample Holder
 Interpreter
 Light Source
 Detector
 Monochromator
Working of spectrophotometer: The light beam from the source is split into sample
beam and reference beam by the mechanical chopper. The reference beam monitors
the lamp energy whereas the sample beam reflects sample absorption.
The observed absorbance measurement is the ratio of the sample and reference beams
which are recombined before moving to the monochromator. This arrangement
compensates the effects due to drift in lamp intensity, electronic and mechanical
fluctuations which affect both the sample and reference beams equally.
Advantages of using a double beam spectrophotometer are:
 More Reliable Detection: Modern improvements in optics permit a high
level of automation and offer the same or even better level of detection as
 compared to earlier single beam systems. Instability factors due to lamp drift,
stray light, voltage fluctuations do not affect the measurement in real-time.
 No Warm-Up Time: Little or no lamp warm-up time is required. This not
only improves the throughput of results but also conserves lamp life.

Q4. What is IR Spectroscopy? Give a note.

Answer: An IR spectrum is essentially a graph plotted with the infrared light absorbed
on the Y-axis against. frequency or wavelength on the X-axis. An illustration
highlighting the different regions that light can be classified into is given below.
IR Spectroscopy detects frequencies of infrared light that are absorbed by a molecule.
Molecules tend to absorb these specific frequencies of light since they correspond to the
frequency of the vibration of bonds in the molecule.

The energy required to excite the bonds belonging to a molecule, and to make them
vibrate with more amplitude, occurs in the Infrared region. A bond will only interact
with the electromagnetic infrared radiation, however, if it is polar.
The presence of separate areas of partial positive and negative charge in a molecule
allows the electric field component of the electromagnetic wave to excite the vibrational
energy of the molecule.
The change in the vibrational energy leads to another corresponding change in the dipole
moment of the given molecule. The intensity of the absorption depends on the polarity of
the bond. Symmetrical non-polar bonds in N≡N and O=O do not absorb radiation, as
they cannot interact with an electric field.
Regions of the Infrared spectrum
Most of the bands that indicate what functional group is present are found in the region
from 4000 cm-1 to 1300 cm-1. Their bands can be identified and used to determine the
functional group of an unknown compound.

Bands that are unique to each molecule, similar to a fingerprint, are found in the
fingerprint region, from 1300 cm-1 to 400 cm-1. These bands are only used to compare the
spectra of one compound to another.
Samples in Infrared Spectroscopy
The samples used in IR spectroscopy can be either in the solid, liquid, or gaseous state.
 Solid samples can be prepared by crushing the sample with a mulling agent
which has an oily texture. A thin layer of this mull can now be applied on a salt
plate to be measured.
 Liquid samples are generally kept between two salt plates and measured since the
plates are transparent to IR light. Salt plates can be made up of sodium chloride,
calcium fluoride, or even potassium bromide.

 Since the concentration of gaseous samples can be in parts per million, the
sample cell must have a relatively long pathlength, i.e. light must travel for a
relatively long distance in the sample cell.

Thus, samples of multiple physical states can be used in Infrared Spectroscopy.

Q5. What is beer lambert law?
Answer: The beer lambert law states that there is a linear connection between the
absorbance and the concentration of the solution. The intensity of the beam of
monochromatic radiation decreases exponentially with an increase in the thickness x and
the concentration c of the absorbing medium.
A = log (I° / I) = εLc
Here,
A = Absorbance
I° = Intensity of the incident beam,
I = Intensity absorbed by the sample
ε = Molar Extinction Coefficient
L = Distance covered by the light through the solution
c = Concentration of the absorbing species
Unit III

Question 1: Discuss the principles of gas chromatography (GC) and liquid

chromatography (LC), emphasizing the importance of column technology in separation.
How do the separation mechanisms differ in GC and LC, and how does column packing
influence efficiency?

Answer: Gas chromatography (GC) and liquid chromatography (LC) are sophisticated
separation techniques widely employed in analytical chemistry. GC relies on the
vaporization and partitioning of volatile compounds in a column, while LC separates
compounds based on their solubility and interactions with the stationary phase. The
choice of column technology, including packing materials and capillaries, significantly
impacts the efficiency and effectiveness of chromatographic separations. The stationary
phase's nature and thickness, along with column dimensions, play crucial roles in
achieving optimal resolution and selectivity.
In GC, the separation mechanism is primarily based on the volatility of compounds.
Compounds with higher volatility elute faster, resulting in a chromatogram that reflects
their relative boiling points. In contrast, LC separation involves factors such as
solubility, interactions with the stationary phase, and molecular size. Column packing
materials, which can vary in polarity and selectivity, allow tailoring separation
conditions for specific analytes.
The significance of column technology is evident in the chromatographic performance it
affords. Packed columns, consisting of particles coated with a stationary phase, provide
efficient separations for a wide range of compounds. Capillary columns, with their
smaller diameter and increased surface area, offer improved resolution and sensitivity.
The careful selection of column packing materials ensures optimal interactions between
the stationary phase and analytes, contributing to the success of chromatographic
analyses.
Q2. Discuss the basic p rinciple and instrumentation of Mass
Spectrometry.

Answer: Mass Spectrometry: Mass spectrometry is a powerful analytical

technique used to quantify known materials, to identify unknown compounds
within a sample, and to elucidate the structure and chemical properties of
different molecules. The complete process involves the conversion of the
sample into gaseous ions, with or without fragmentation, which are then
characterized by their mass to charge ratios (m/z) and relative abundances.

This technique basically studies the effect of ionizing energy on molecules. It

depends upon chemical reactions in the gas phase in which sample molecules
are consumed during the formation of ionic and neutral species.

Basic Principle
A mass spectrometer generates multiple ions from the sample under
investigation, it then separates them according to the ir specific mass-to-
charge ratio (m/z), and then records the relative abundance of each ion
type.

The first step in the mass spectrometric analysis of compounds is the

production of gas phase ions of the compound, basically by electron
ionization. This molecular ion undergoes fragmentation. Each primary
product ion derived from the molecular ion, in turn, undergoes
fragmentation, and so on. The ions are separated in the mass spectrometer
according to their mass -to-charge ratio, and are detected in proportion to
their abundance. A mass spectrum of the molecule is thus produced. It
displays the result in the form of a plot of ion abundance versus mass-to-
charge ratio. Ions provide information concerning the nature and the
structure of their precursor molecule. In the spectrum of a pure
compound, the molecular ion, if present, appears at the highest value of m/z
(followed by ions containing heavier isotopes) and gives the molecular
mass of the compound.

Component/instrumentation

The instrument consists of three major component s:

1. Ion Source: For producing gaseous ions from the substance

being studied.

2. Analyzer: For resolving the ions into their

characteristics mass components according to their mass-to-
charge ratio.

3. Detector System: For detecting the ions and recording

the relative

abundance of each of the resolved ionic species.

In addition, a sample introduction system is necessary to admit the samples to

be studied to the ion source while maintaining the high vacuum
requirements (~10 -6 to 10-8 mm of mercury) of the technique; and a
computer is required to control the instrument, acquire and manipulate data,
and compare spectra to reference libraries.
 Figure:Components of a Mass Spectrometer

With all the above components, a mass spectrometer should always perform
the following processes:

1. Produce ions from the sample in the ionization source.

2. Separate these ions according to their mass -to-charge ratio in

the mass analyzer.

3. Eventually, fragment the selected ions and analyze the fragments

in a second analyzer.

4. Detect the ions emerging from the last analyzer and

measure their abundance with the detector that converts the ions
into electrical signals.

Process the signals from the detector that are transmitted to the computer
and control the instrument using feedback.
Question 3: Examine the role of detection methods in chromatography and mass
spectrometry. Distinguish between simple and specific detection, and elaborate on how
detection is utilized for further analysis, including the use of tags and coupling to other
analytical techniques like infrared (IR) and mass spectrometry (MS).
Answer: Detection methods in chromatography and mass spectrometry are integral
components that contribute to the identification and quantification of analytes. Simple
detection methods, such as UV-Vis spectroscopy in chromatography, provide general
information about the sample, often based on the absorption of light by compounds.
Specific detection, as seen in mass spectrometry (MS), offers detailed information on
individual compounds through their unique mass spectra.
Detection serves as a means for further analysis by incorporating tags and coupling to
other analytical techniques. Tags, or chemical labels, are introduced to the analytes to
enhance specificity. For instance, in liquid chromatography-mass spectrometry (LC-
MS), specific tags can be attached to compounds, allowing for enhanced detection and
identification. Coupling chromatography or electrophoresis with mass spectrometry
provides additional information about the molecular structure and composition of the
compounds being analyzed.
Infrared (IR) spectroscopy is another powerful technique often coupled with
chromatography or mass spectrometry. It provides information about molecular
vibrations, aiding in the identification of functional groups within compounds. The
combination of chromatography, mass spectrometry, and infrared spectroscopy allows
for a more comprehensive analysis of complex samples, offering insights into both the
identity and structure of analytes.

Question 4: Explore the applications of electrophoresis in DNA analysis and

immunoassays. Discuss how electrophoresis techniques, including plates and capillaries,
contribute to the separation of DNA fragments and proteins. Highlight the significance
of high-resolution capillary electrophoresis in DNA sequencing.
Answer: Electrophoresis is a powerful technique with diverse applications in molecular
biology and immunoassays. In DNA analysis, electrophoresis plays a crucial role in
separating DNA fragments based on their size. Gel electrophoresis, utilizing plates, is
commonly employed for this purpose. The technique allows for the visualization of
DNA fragments, facilitating tasks such as DNA profiling and restriction fragment length
polymorphism (RFLP) analysis.
Capillary electrophoresis (CE) has emerged as a high-resolution technique with profound
implications in DNA sequencing. Capillaries, with their small diameter and high surface-
to-volume ratio, offer advantages over traditional gel electrophoresis. CE allows for the
separation of DNA fragments with enhanced precision and speed, making it a preferred
method for DNA sequencing projects. The technique is particularly valuable in the
Human Genome Project and other large-scale genomics initiatives.
In immunoassays, electrophoresis is employed for the separation and analysis of
proteins. The technique contributes to the detection and quantification of specific
antibodies or antigens, enhancing the sensitivity and specificity of immunoassays. By
separating proteins based on their charge and size, electrophoresis aids in profiling
complex mixtures, providing valuable information for diagnostic purposes.

Question 5: Explain the factors influencing resolution, time, and multiple separations in
mass spectrometry. Delve into the detection and interpretation processes, emphasizing
the linkage to excitation and its role in analyzing complex samples.
Answer: Resolution, time, and the ability to perform multiple separations are critical
considerations in mass spectrometry (MS), influencing the technique's analytical
performance. Resolution is influenced by various factors, including the type of ionization
technique, mass analyzer, and the strength of the magnetic field. Higher resolution
allows for the distinction of closely spaced peaks in a mass spectrum, providing more
detailed information about the components of a sample.
Time considerations in MS involve the speed of analysis. Time-of-flight (TOF)
analyzers, for example, provide rapid results by measuring the time ions take to travel a
specific distance. Faster analysis times are advantageous in high-throughput applications
where efficiency is paramount.
The ability to perform multiple separations is a characteristic feature of advanced MS
setups. By combining different ionization and separation methods, researchers can
achieve comprehensive separations of complex samples. For example, liquid
chromatography-mass spectrometry (LC-MS) combines the high separation efficiency of
liquid chromatography with the sensitivity and specificity of mass spectrometry.
Detection and interpretation processes in MS are essential for extracting meaningful
information from mass spectra. Detection involves the identification of ions based on
their mass-to-charge ratio (m/z). Interpretation includes the analysis of characteristic
peaks and fragmentation patterns in mass spectra. The linkage to excitation refers to the
initial energy input during ionization. Understanding excitation processes is crucial as it
influences the observed mass spectra and aids in the identification and structural
elucidation of compounds in complex samples.
In summary, MS is a powerful analytical technique that continues to evolve, driven by
advancements in resolution, speed, and the ability to perform multiple separations,
making it an indispensable tool in modern analytical chemistry.

Unit IV

Q1. Write a notes on the following:

(a) Square wave Voltammetry Purpose
(b) Cyclic Voltametry
Answer: Square-wave Voltammetry Purpose

Square-wave Voltammetry (SWV) is used for both quantitative chemical analysis and
study of the mechanism, kinetics, and thermodynamics of chemical reactions.
SWV used as an analytical tool offers three major advantages when compared to other
electrochemical techniques.
 SWV is very sensitive, often allowing direct analyses at the ppb (parts per
billion) level and even the low ppt (parts per trillion) level when used in a
stripping mode.
 SWV requires less time per sweep than older techniques such as differential
pulse polarography. A SWV sweep can often be recorded in less than ten
seconds, in contrast with a differential pulse polarogram that typically requires
more than two minutes for data acquisition.

 The square-wave frequency can be used to differentiate between processes with

fast and slow kinetics. In some cases, kinetically fast processes can be measured
without interference from slower processes that occur in the same potential
range.
Other techniques, such as cyclic voltammetry, are generally preferred over SWV for
mechanistic and kinetic studies. However, square-wave voltammetry’s sensitivity allows
mechanistic and kinetic measurements in solutions that are too dilute for more
conventional study.
SWV is generally performed on a stationary solid electrode or a hanging mercury drop
electrode. The SWV script in the Pulse Voltammetry software provides for mercury-drop
generation, solution de-aeration, and experiment sequencing suitable for the most
common applications for square-wave voltammetry.
Choose the type of electrode in the Electrode Setup Panel.
Additional sequencing steps suitable for square-wave anodic (or cathodic) stripping are
implemented in the Pulse Voltammetry’s SWS (square-wave stripping) technique.

(ii) Cyclic Voltametry: Cyclic Voltammetry (CV) is an electrochemical technique

which measures the current that develops in an electrochemical cell under conditions
where voltage is in excess of that predicted by the Nernst equation. CV is performed by
cycling the potential of a working electrode, and measuring the resulting current.

A cyclic voltammogram is obtained by measuring the current at the working electrode

during the potential scans.² Figure 2 shows a cyclic voltammogram resulting from a
single electron reduction and oxidation. Consider the following reversible reaction:
M++e−⇌M(1)
 Figure: Voltammogram of a Single electron oxidation-reduction

In Figure, the reduction process occurs from (a) the initial potential to (d) the switching
potential. In this region the potential is scanned negatively to cause a reduction. The
resulting current is called cathodic current (i pc). The corresponding peak potential occurs
at (c), and is called the cathodic peak potential (E pc). The Epc is reached when all of the
substrate at the surface of the electrode has been reduced. After the switching potential
has been reached (d), the potential scans positively from (d) to (g). This results in anodic
current (Ipa) and oxidation to occur. The peak potential at (f) is called the anodic peak
potential (Epa), and is reached when all of the substrate at the surface of the electrode has
been oxidized.
Instrumentation:
A CV system consists of an electrolysis cell, a potentiostat, a current-to-voltage
converter, and a data acquisition system. The electrolysis cell consists of a working
electrode, counter electrode, reference electrode, and electrolytic solution. The working
electrode’s potential is varied linearly with time, while the reference electrode maintains
a constant potential. The counter electrode conducts electricity from the signal source to
the working electrode. The purpose of the electrolytic solution is to provide ions to the
electrodes during oxidation and reduction. A potentiostat is an electronic device which
uses a dc power source to produce a potential which can be maintained and accurately
determined, while allowing small currents to be drawn into the system without changing
the voltage. The current-to-voltage converter measures the resulting current, and the data
acquisition system produces the resulting voltammogram.
Applications: Cyclic Voltammetry can be used to study qualitative information about
electrochemical processes under various conditions, such as the presence of
intermediates in oxidation-reduction reactions, the reversibility of a reaction. CV can
also be used to determine the electron stoichiometry of a system, the diffusion
coefficient of an analyte, and the formal reduction potential, which can be used as an
identification tool. In addition, because concentration is proportional to current in a
reversible, Nernstian system, concentration of an unknown solution can be determined
by generating a calibration curve of current vs. concentration.

Q2. What is Titration? Explain different types of titrations.

Answer: A quantitative and volumetric technique, to determine the unknown

concentration of a solution by the known concentration of a solution in the presence of
indicator is called Titration
Titration is a common laboratory method of using quantitative chemical analysis. This
method is used to determine the unidentified concentration of a known analyte. The
volume measurement is known as volumetric analysis, and it is important in the titration.

Types of Titration
There are many types of titration when considering goals and procedures. However, the
most common types of titration in quantitative chemical analysis are redox titration and
acid-base titration.

Titrations can be classified as:

1. Acid-base Titrations
2. Redox Titrations.
3. Precipitation Titrations.
4. Complexometric Titrations.

Four Types of Titration

1. Acid-Base Titration

The strength of an acid can be determined using a standard solution of a base. This
process is called acidimetry. In the same way, the strength of a base can be found with
the help of a standard solution of an acid, which is known as alkalimetry. Both titrations
involve in the neutralization reaction of alkali.

What is Acid-Base Titration?

It is a quantitative analysis method to determine an acid’s or bases’ concentration by

precisely neutralizing them with a standard solution of either acid or base of known
concentration. It is monitored with the help of a pH indicator to know the development
of the acid-base reaction.

HA+BOH→BA+H2O

Acid + Alkali→Salt + Water

Or H+ + A– + B+ + OH– → B+ + A– + H2O

Or H+ + OH– → H2O

The acid-base titration is based on the reaction that neutralization is between a base or an
acidic and analyte. In this type, a reagent is mixed with the sample solution until it
reaches the required pH level. This type of titration majorly depends on the track change
in pH or a pH meter.

2. Redox Titrations

The redox titration is also known as an oxidation-reduction reaction. In this type of

titration, the chemical reaction takes place with a transfer of electrons in the reacting ions
of aqueous solutions. The titrations are named after the reagent that is used in are as
follows;

 Permanganate Titrations
 Dichromate Titrations
 Iodimetric and Iodometric Titrations

Permanganate Titrations

In this titration, the potassium permanganate is used as an oxidizing agent. It is

maintained with the use of dilute sulphuric acid. Here is the equation.

2KMnO4 + 3H2SO4 → K2SO4 + 2MnSO4 + 3H2 + 5O

Or MnO4– + 8H + 5e → Mn2++ 4H2O

Further, the solution remains colourless before the endpoint. The potassium
permanganate is used to estimate oxalic acid, ferrous salts, hydrogen peroxide, oxalates
and more. While the solution of potassium permanganate is always standardized before it
is used.

Dichromate Titrations

These are titrations in which, potassium dichromate is used as an oxidising agent in

acidic medium. The medium is maintained acidic by the use of dilute sulphuric acid. The
potential equation is:

K2Cr2O7 + 4H2SO4 → K2Cr2(SO4) + 4H2O + 3[O]

Or Cr2O27- + 14H + 6e → 2 Cr3+ + 7H2O

The solution of potassium dichromate can be directly used for titrations. It is mainly used
for the estimation of ferrous salts and iodides.

Iodimetric and Iodometric Titrations

The reduction of free iodine to iodide ions and

oxidation of iodide ions to free occurs in these titrations.

l2 + 2e → 2l–……………. (reduction)
2l– + 2e → 2e ……………. (oxidation)

The solution is used as an indicator. Free iodine is used in the iodometric titration, while
in the iodometric titration an oxidation agent is used to react to liberate free iodine.

3. Precipitation Titrations

The titration is based on the insoluble precipitate formation when the two reacting
substances are brought into contact are called precipitation titration. For instance, when
the solution of silver nitrate is used to a solution of ammonium thiocyanate or sodium
chloride, it reacts and forms a white precipitate of silver thiocyanate or silver chloride.

AgNO3 + NaCl → AgCl + NaNO3

AgNO3 + NH4CNS → AgCNS + NH4NO3

4. Complexometric Titrations

The complexometric titration is where an undissociated complex is formed at an

equivalence point. It is greater than the precipitation titrations, and there will be no error
due to co-precipitations.

Hg2+ + 2SCN– → Hg(SCN)2

Ag+ + 2CN– → [Ag(CN)2]–

Ethylenediaminetetraacetic acid (EDTA) is an important reagent that forms complexes

with metals.

Q3. What is Redox Titration? Explain with example.

Answer: Redox Titration is a laboratory method of determining the concentration of a

given analyte by causing a redox reaction between the titrant and the analyte.
These types of titrations sometimes require the use of a potentiometer or a redox
indicator.

Redox titration is based on an oxidation-reduction reaction between the titrant and the
analyte. It is one of the most common laboratory methods to identify the concentration of
unknown analytes.

In order to evaluate redox titrations, the shape of the corresponding titration curve must
be obtained. In these types of titration, it proves convenient to monitor the reaction
potential instead of monitoring the concentration of a reacting species.

As discussed earlier, redox reactions involve both oxidation and reduction. The key
features of reduction and oxidation are discussed below.
Reduction

A substance can undergo reduction can occur via:

 The addition of hydrogen.

 The removal of oxygen.
 The acceptance of electrons.
 A reduction in the overall oxidation state.

Oxidation

The following points describe a substance that has undergone oxidation.

 The addition of oxygen.

 Removal of hydrogen which was attached to the species.
 The donation/loss of electrons.
 An increase in the oxidation state exhibited by the substance.

Thus, it can be understood that redox titrations involve a transfer of electrons between
the given analyte and the titrant. An example of a redox titration is the treatment of an
iodine solution with a reducing agent. The endpoint of this titration is detected with the
help of a starch indicator.

In the example described above, the diatomic iodine is reduced to iodide ions (I –), and
the iodine solution loses its blue colour. This titration is commonly referred to as
iodometric titration.

Redox Titration Example

An example of a redox titration is the titration of potassium permanganate (KMnO 4)

against oxalic acid (C2H2O4). The procedure and details of this titration are discussed
below.

Titration of Potassium Permanganate against Oxalic Acid

 Prepare a standard Oxalic acid solution of about 250 ml.

 The molecular mass of oxalic acid is calculated by adding the atomic mass of
each constituent atom
 The molecular mass of H2C2O4.2H2O = 126
 Since the weight of oxalic acid that is required to make 1000 ml of 1M solution is
126 g. Hence, the weight of oxalic acid needed to prepare 250 ml of 0.1 M
solution = 126/1000 x 250 x 0.1 = 3.15 g
Determining the Strength of KMnO4 using Standard Oxalic Acid Soln

In this titration, the analyte is oxalic acid and the titrant is potassium permanganate. The
oxalic acid acts as a reducing agent, and the KMnO 4 acts as an oxidizing agent. Since the
reaction takes place in an acidic medium, the oxidizing power of the permanganate ion is
increased. This acidic medium is created by the addition of dilute sulfuric acid.

MnO−4+8H++5e−→Mn2++4H2O
KMnO4 acts as an indicator of where the permanganate ions are a deep purple colour. In
this redox titration, MnO4– is reduced to colourless manganous ions (Mn2+) in the acidic
medium. The last drop of permanganate gives a light pink colour on reaching the
endpoint. The following chemical equation can represent the reaction that occurs.

Molecular equation

2KMnO4+3H2SO4→K2SO4+2MnSO4+3H2O+5[O]
H2C2O4.2H2O+[O]→2CO2+3[H2O]×5
Complete Reaction

2KMnO4+3H2SO4+5H2C2O4.2H2O→K2SO4+2MnSO4+18H2O+10CO2
Ionic equation

MnO−4+8H++5e−→Mn2++4H2O]×2
C2O2−4→2CO2+2e−]×5
Complete Reaction

2MnO−4+16H++5C2O2−4→2Mn2++8H2O+10CO2

From the above-balanced chemical reaction, it can be observed that 2 moles of

KMnO4 reacts with 5 moles of oxalic acid.

Q4. Discuss about Complexometric titration in brief.

Answer: Complexometric titration (sometimes chelatometry) is a form of volumetric

analysis in which the formation of a colored complex is used to indicate the end point of
a titration. Complexometric titrations are particularly useful for the determination of a
mixture of different metal ions in solution. An indicator capable of producing an
unambiguous color change is usually used to detect the end-point of the
titration .Complexometric titration are those reactions where a simple ion is transformed
into a complex ion and the equivalence point is determined by using metal indicators or
electrometrically.

Reactions for Complexometric titration

In theory, any complexation reaction can be used as a volumetric technique provided
that:
1. The reaction reaches equilibrium rapidly after each portion of titrant is added.
 2. Interfering situations do not arise. For instance, the stepwise formation of several
different complexes of the metal ion with the titrant, resulting in the presence of
more than one complex in solution during the titration process.
3. A complexometric indicator capable of locating equivalence point with fair
accuracy is available.

In practice, the use of EDTA as a titrant is well established.

Complex titration with EDTA

EDTA, ethylenediaminetetraacetic acid, has four carboxyl groups and two amine groups
that can act as electron pair donors, or Lewis bases. The ability of EDTA to potentially
donate its six lone pairs of electrons for the formation of coordinate covalent bonds to
metal cations makes EDTA a hexadentate ligand. However, in practice EDTA is usually
only partially ionized, and thus forms fewer than six coordinate covalent bonds with
metal cations.
Disodium EDTA is commonly used to standardize aqueous solutions of transition metal
cations. Disodium EDTA (often written as Na2H2Y) only forms four coordinate covalent
bonds to metal cations at pH values ≤ 12. In this pH range, the amine groups remain
protonated and thus unable to donate electrons to the formation of coordinate covalent
bonds. Note that the shorthand form Na4−xHxY can be used to represent any species of
EDTA, with x designating the number of acidic protons bonded to the EDTA molecule.
EDTA forms an octahedral complex with most 2+ metal cations, M 2+, in aqueous
solution. The main reason that EDTA is used so extensively in the standardization of
metal cation solutions is that the formation constant for most metal cation-EDTA
complexes is very high, meaning that the equilibrium for the reaction:
M2+ + H4Y → MH2Y + 2H+

lies far to the right. Carrying out the reaction in a basic buffer solution removes H + as it
is formed, which also favors the formation of the EDTA-metal cation complex reaction
product. For most purposes it can be considered that the formation of the metal cation-
EDTA complex goes to completion, and this is chiefly why EDTA is used in titrations
and standardizations of this type.

Unit V

Question 1: NMR Spectroscopy

Principle:

Nuclear Magnetic Resonance (NMR) spectroscopy is based on the principles of nuclear

spin and magnetic resonance. When placed in a strong magnetic field, certain nuclei
absorb and re-emit radiofrequency energy, providing information about their chemical
environment and molecular structure.

Instrumentation:
NMR spectrometers consist of a powerful magnet, radiofrequency transmitter, and a
detector. The magnet aligns nuclear spins, RF pulses perturb the spin alignment, and the
resulting signals, detected by the instrument, generate the NMR spectrum.

Factors Affecting Chemical Shift:

Chemical shift in NMR is influenced by factors such as electronegativity, hybridization,

and magnetic anisotropy. Electronegative groups cause deshielding, leading to higher
chemical shifts, while electron-donating groups result in shielding and lower chemical
shifts.

Spin Coupling:

Spin coupling in NMR occurs when adjacent nuclei influence each other's magnetic
environment. This phenomenon, known as coupling or splitting, provides additional
information about the connectivity and structure of a molecule.

Applications:

NMR spectroscopy finds diverse applications in chemistry, biology, and medicine. It is

widely used for structural elucidation of organic compounds, determination of molecular
dynamics, and in studies of biomolecular interactions.

Question 2: Electroanalytical Methods

Potentiometry:

Potentiometry involves measuring the potential difference between an indicator electrode

and a reference electrode at zero current. It is commonly used in pH measurements,
titrations, and the determination of ion concentrations.

Voltammetry:

Voltammetry measures the current flowing through an electrochemical cell under

varying applied potentials. It is extensively used in redox reactions, metal ion analysis,
and environmental monitoring.

Applications:

Both potentiometry and voltammetry are crucial in analytical chemistry. Potentiometry is

employed in the pharmaceutical industry for drug formulation, while voltammetry is
applied in the detection of heavy metals in environmental samples.

Question 3: Radiochemical Methods

Principle:

Radiochemical methods involve the use of radioactive isotopes to study chemical

processes. Radioactive tracers are incorporated into compounds, enabling the tracking of
reactions and transformations.

Applications:

Radiochemical methods find applications in medicine, biology, and industry. In nuclear

medicine, radioisotopes are used for diagnostic imaging, and in industry, they are
employed for process monitoring and quality control.

Question 4: X-ray Analysis and Electron Spectroscopy

X-ray Analysis:

X-ray analysis includes techniques such as X-ray diffraction (XRD) and X-ray
photoelectron spectroscopy (XPS). XRD is used for determining crystal structures, while
XPS provides information on elemental composition and chemical bonding.

Electron Spectroscopy:

Electron spectroscopy methods, including Auger electron spectroscopy (AES) and

scanning electron microscopy (SEM), use electrons for surface analysis. AES reveals
elemental composition, while SEM provides high-resolution images for morphological
analysis.

Applications:

X-ray and electron spectroscopy techniques are essential in material science, providing
insights into crystallography, surface composition, and morphology. They are widely
used in fields such as nanotechnology, catalysis, and semiconductor research.

In summary, these questions cover the principles, instrumentation, influencing factors,

applications, and significance of each analytical method. The comprehensive answers
offer a detailed understanding of NMR spectroscopy, electroanalytical methods,
radiochemical methods, and X-ray analysis with electron spectroscopy.

Extra Questions

Q1. Write a short note about the following

(i) Paper chromatography with applications
(ii) Retardation factor
Answer: (i) paper chromatography, in analytical chemistry, technique for separating
dissolved chemical substances by taking advantage of their different rates of migration
across sheets of paper. It is an inexpensive but powerful analytical tool that requires very
small quantities of material.
The method consists of applying the test solution or sample as a spot near one corner of a
sheet of filter paper. The paper is initially impregnated with some suitable solvent to
create a stationary liquid phase. An edge of the paper close to the test spot is then
immersed in another solvent in which the components of the mixture are soluble in
varying degrees. The solvent penetrates the paper by capillary action and, in passing over
the sample spot, carries along with it the various components of the sample. The
components move with the flowing solvent at velocities that are dependent on their
solubilities in the stationary and flowing solvents. Separation of the components is
brought about if there are differences in their relative solubilities in the two solvents.
Before the flowing solvent reaches the farther edge of the paper, both solvents are
evaporated, and the location of the separated components is identified, usually by
application of reagents that form coloured compounds with the separated substances.
The separated components appear as individual spots on the path of the solvent. If the
solvent flowing in one direction is not able to separate all the components satisfactorily,
the paper may be turned 90° and the process repeated using another solvent.
Applications of paper chromatography

 To study the process of fermentation and ripening.

 To check the purity of pharmaceuticals.

 To inspect cosmetics.

 To detect the adulterants.

 To detect the contaminants in drinks and foods.

 To examine the reaction mixtures in biochemical laboratories.

(ii)Retardation factor: The Rf (retardation factor) value is the ratio of the solute’s
distance travelled to the solvent’s distance travelled.
RF Value Explanation
The stationary phase in paper chromatography is water molecules found in the pores of
the filter paper, whereas the moving phase is a solvent such as hexane, toluene, acetone,
or a mixture of solvents such as methanol-water mixture. As the moving phase passes
through the area where the sample has been adsorbed, it dissolves the components more
or less quickly, depending on their solubility, and carries them with it as it moves across
the spot.
It is possible to determine the characteristic rate of movement of each substance on the
chromatography paper as the moving phase moves at a certain temperature and for a
specific solvent. This is represented by the Rf value, which stands for relative front or
retardation factor. Even if the mobile phase (solvent) is the same, various compounds
have varying Rf values. Furthermore, the Rf value of a chemical may vary depending on
the solvent. The following expression can be used to calculate Rf values:
Rf = Distance travelled by the substance from reference line (cm)/Distance travelled by
the solvent front from reference line (cm)
need of the RF value: In chromatography, Rf values are the most basic prerequisite of
the experiment. These numbers indicate whether the analyte (solute) prefers the
stationary or mobile phase. With stationary and mobile phases, R f values are used to
determine polarity, relative masses, and relative solubilities, among other things.
When comparing the results of one chromatogram to those of another, retention factors
come in handy. The retention factor for a given substance should stay constant if the
chromatogram is conducted under the same conditions (same mobile and stationary
phases). This enables the comparison of unknown materials to known materials. They
are not the same compound if the retention factor of an unknown substance differs from
that of a known substance.
Calculation of RF Value
Depending on the nature of the analytes and the stationary phases, a chromatogram must
first be generated with an appropriate solvent (mobile phase). After drying the
chromatogram, the locations (migration values) of the analytes and the solvent front are
measured.

Q3. What is Partition Chromatography? Explain Partition Chromatography

Principle, Procedure and Applications.

Answer: Partition Chromatography technique is defined as the separation of components

between two liquid phases viz original solvent and the film of solvent used in the
column.

This separation theory was introduced in the year 1940s which was published by
Richard Laurence Millington Synge and Archer Martin. It is also known as
Liquid-liquid chromatography (LLC). Or if gas is the mobile phase it is called Gas-
liquid chromatography (GLC).

Partition Chromatography Principle

The separation of the components from the sample mixture is carried out by the
process of partition of the components between 2 phases. Both phases are in liquid
form. In this process, the immiscible solid surface coated with the liquid surface on the
stationary phase is in the mobile phase. The liquid surface is immobilized by a
stationary phase which results in making its a stationary phase. The mobile phase
moves from the stationary phase and components get separated. The separation
depends on different partition coefficient.
Partition Chromatography Procedure

Below we have explained the procedure to conduct Paper Chromatography

Experiment for easy understanding.
Apparatus required – chromatography jar, liquid impregnated paper (stationary
phase), capillary tube (to apply sample mixture), mobile phase (example chloroform,
methanol, acetone, ethanol).
1. Take a clean and dry chromatography jar.
2. To make sure that the environment of the jar is saturated with solvent vapors, a paper
impregnated in mobile phase is set to the walls.
3. Add mobile phase to the jar. Around 0.5 cm to 1 cm from the bottom of the jar.
4. Close the jar.
5. Allow attaining equilibrium.
6. Mark the baseline on adsorbent.
7. Apply sample to the paper with the help of capillary tube.
8. Air-dry the sample spot.
9. Place the paper in the jar and close it.
10. Allow the system to stand till the solvent moves to some distance from the baseline.
11. Take out the paper and dry it.
12. If the sample components are separated showing colors then dry it in ordinary light. If it is
a colorless component then dry it in UV lamp.
13. Store the chromatogram
14. Calculate Rf value.
Partition Chromatography Applications

There are various applications of Paper Chromatography. Some of the

applications are mentioned below:

 To separate and identify amino acids.

 To separate and identify tannins.

 To separate and identify alkaloids.

 To separate and identify carbohydrates.

 To separate and identify glycosides.

Q4. Explain the term Distribution coefficient and Distribution Ratio.

Answer: Distribution coefficient: A partition coefficient (P) or distribution
coefficient (D) is the ratio of concentrations of a compound in a mixture of two
immiscible solvents at equilibrium. This ratio is therefore a comparison of the
solubilities of the solute in these two liquids. The partition coefficient generally refers
to the concentration ratio of un-ionized species of compound, whereas the distribution
coefficient refers to the concentration ratio of all species of the compound (ionized plus
un-ionized).

In the chemical and pharmaceutical sciences, both phases usually are solvents.[2]
Most commonly, one of the solvents is water, while the second is hydrophobic,
such as 1-octanol. Hence the partition coefficient measures how
hydrophilic ("water-loving") or hydrophobic ("water-fearing") a chemical substance
is. Partition coefficients are useful in estimating the distribution of drugs within the
body. Hydrophobic drugs with high octanol-water partition coefficients are mainly
distributed to hydrophobic areas such as lipid bilayers of cells. Conversely,
hydrophilic drugs (low octanol/water partition coefficients) are found primarily in
aqueous regions such as blood serum.

If one of the solvents is a gas and the other a liquid, a gas/liquid partition coefficient
can be determined. For example, the blood/gas partition coefficient of a general
anesthetic measures how easily the anesthetic passes from gas to blood. Partition
coefficients can also be defined when one of the phases is solid, for instance, when
one phase is a molten metal and the second is a solid metal, or when both phases are
solids. The partitioning of a substance into a solid results in a solid solution.

Partition coefficients can be measured experimentally in various ways (by shake-

flask, HPLC, etc.) or estimated by calculation based on a variety of methods
(fragment-based, atom-based, etc.).

Distribution Ratio

In solvent extraction, a distribution ratio is often quoted as a measure of how well-

extracted a species is. The distribution ratio (Kd) is equal to the concentration of
a solute in the organic phase divided by its concentration in the aqueous phase.
Depending on the system, the distribution ratio can be a function of temperature, the
 concentration of chemical species in the system, and a large number of other
parameters. Note that D is related to the ΔG of the extraction process.

Sometimes, the distribution ratio is referred to as the partition coefficient, which is

often expressed as the logarithm. Note that a distribution ratio for uranium and
neptunium between two inorganic solids (zirconolite and perovskite) has been
reported.[2] In solvent extraction, two immiscible liquids are shaken together. The
more polar solutes dissolve preferentially in the more polar solvent, and the less polar
solutes in the less polar solvent. In this experiment, the nonpolar halogens
preferentially dissolve in the non-polar mineral oil.

Although the distribution ratio and partition coefficient are often used synonymously,
they are not necessarily so. Solutes may exist in more than one form in any particular
phase, which would mean that the partition coefficient (Kd) and distribution ratio (D)
will have different values. This is an important distinction to make as whilst the
partition coefficient has a fixed value for the partitioning of a solute between two
phases, the distribution ratio changes with differing conditions in the solvent.

Q5. Explain Solid-phase extraction (SPE) and Solid-phase microextraction (SPME).

Answer: Solid-phase extraction (SPE)In general, SPE involves four steps:
1. Column preparation or prewash step.
2. Sample loading or the retention of the analytes of interest on the cartridge and/or
sorbent.
3. Column postwash to remove undesirable contaminants. In reality, the compounds of
interest are retained on the sorbent, while interferences are washed away.
4. Analyte desorption from the cartridge. The adsorbed analytes are recovered by an
appropriate eluting solvent.
SPE sorbents are commercially available in three formats:

1. Cartridge.
2. Columns fashioned like syringe barrels.
3. Disks.
Also, sorbent phases can be purchased, and typical column housings are manufactured of
polypropylene or glass. In order to contain the column with the sorbent phase, porous
frits made of polyethylene, stainless steel, or Teflon can be used [2].

There are some examples for applying SPE as sample preparation step before GC
detection of different analytes in a variety of samples. Some of them are pointed below.

Lee et al. reported the determination of endocrine-disrupting phenols, acidic

pharmaceuticals, and personal care products in sewage by solid-phase extraction and gas
chromatography- mass spectrometry [8]. In this work, an anion exchanger was used as a
solid-phase extractor, and a multiresidue method was developed and optimized for the
 extraction of 21 phenols and acids in sewage. The phenols and acids were then
selectively eluted in separate fractions and were converted into volatile derivatives, by
suitable reagents, for GC-MS determination.

Solid-phase microextraction (SPME)

Miniaturization of analytical processes into microchip platforms designed for micro total
analytical systems is a new and rapidly developing field. Solid-phase microextraction
(SPME) is a modern technique that consists in direct extraction of the analytes with the
use of a small-diameter fused silica fiber coated with an adequate polymeric stationary
phase. On the other hand, in two designs of SPME, a thin layer of sorbent is coated
on the outer surface of fibers (fiber design), and the inner surface of a capillary tube (in-
tube design) is covered. The fiber design can be used as an interface in both GC and
HPLC, but in-tube design has been just applied as an easier approaching interface with
HPLC. In fiber design, a thin film of liquid polymer or mixture of a solid sorbent with a
liquid polymer has covered on the surface of a fused silica core fiber.

The properties of extraction process, by SPME, are as follows:

1. By SPME, samples are analyzed after equilibrium is reached or at a specified time

prior to achieving equilibrium.
2. Exhaustive extraction of analyte from the sample matrix is not achieved by SPME.
3. So, SPME operationally encompasses non-exhaustive, equilibrium and preequilibrium,
batch, and flow-through microextraction techniques.
4. SPME is directly applicable for field applications in air and water sampling.
5. It does not require continuation of extraction by SPME until equilibrium is reached.
6. A quantitative extraction may be obtained by careful control of time and temperature.
7. SPME is a solventless sample preparation procedure.
8. SPME is compatible with chromatographic analytical systems, and the process is easily
automated.
SPME can be conducted in three modes:

1. Direct extraction, in which the coated fiber is immersed in the aqueous sample.
2. Headspace configuration, for sampling air or the volatiles from the headspace above an
aqueous sample. However, headspace techniques are more applicable to volatile
organics than to the semivolatile organic compounds.
3. Membrane protection configuration, in which the coated fiber is protected with a
membrane, for analyzing the analytes in too much dirty samples.