PHA-404, Advanced Pharmaceutical Analysis

Sample Questions for Final

Autumn 2022

Quality

1. Define the following terms:

a) Quality Control b) Quality Assurance C) Regulatory Compliance

d) Validation e) IPQC

a) Quality Control: QC is that part of GMP which is concerned with sampling, specifications, testing
and with in the organization, documentation, and release procedures which ensure that the
necessary and relevant tests are carried out.

b) Quality Assurance: QA is the sum total of organized arrangements made with the object of
ensuring that product will be of the. Quality required by their intended use.

C) Regulatory Compliance: In general, compliance means conforming to a rule, such as a

specification, policy, standard or law.

d) Validation: Validation is establishing documented evidence which provides a high degree of

assurance that a specific process will consistently produce a product meeting its predetermined
specifications and quality attributes.

e) IPQC: It is an important function of the in-process quality assurance program to ensure that
finished dosage forms have uniform purity and quality within a batch and between batches.

2. Define TQM? Write down the major components of TQM.

= Total Quality Management is a managerial approach used by pharmaceutical manufacturers in

ensuring pharmaceutical products meets the required quality with regard to their uses. It is a
potentially beneficial approach to manufacturing pharmaceutical products, as it ensures they exceed
customers' expectations in relation to quality standards, efficiency and also effectiveness.

Major Components of the TQM Approach

• Customer Focus: It is beneficial to recognize product consumers.

• Involvement of Employees: All employees are involved in the quality production process, and so
they should as well take part in the innovation and improvement of quality.

• Constant Improvement: The mission for quality is an endless process in which individuals
are .constantly attempting to improve on the product’s features
3. What are the difference between QC & QA?

QA
QA is the sum total of organized
arrangements made with the object of
ensuring that product will be of the
Quality required by their intended use
QC
QC is that part of GMP which is
concerned with sampling,
specifications, testing and within the
organization, documentation, and
release procedures which ensure that
the necessary and relevant tests are

All those planned or systematic actions Operational laboratory techniques and

necessary to provide adequate activities used to fulfill the requirement
confidence that a product will satisfy the of Quality
requirements for quality
QA is company based QC is lab based

IR:

1. Explain briefly the instrumentation of IR spectroscopy

=Two types of infrared spectrophotometer are in common use in the laboratory.

1. Dispersive infrared spectrophotometer

2. Fourier Transform infrared spectrophotometer

Dispersive infrared instruments are sometimes called grating or scanning spectrometers. ...
This grating is similar to a prism. It separates the wavelengths of light in the spectral range
and directs each wavelength individually through a slit to the detector.

Dispersive IR spectrophotometer consists of the following parts.

 Radiation source

 Sample compartment

 Monochromator

 Detector

 Recorder

Radiation source

• An inert solid is electrically heated to a temperature in the range 1500-2000 0C. The heated
material will then emit infrared radiation.

Sample compartment:

 After preparation of the sample (solid, liquid or gas), itis inserted into a holder and then
placed in the sample compartment in the path of the IR beam. For solutions, a reference cell
is needed that contain solvent only. After passing through the sample and reference, the
beams are chopped by a mirror which focuses each beam alternately into the entrance slit of
the monochromatic.

Glass and plastics absorb strongly throughout the IR region of the spectrum. Thus cells are
constructed by ionic substances like

 Sodium chloride

 Potassium bromide

3. Monochromator: A monochromator selects specific frequency from other extraneous

radiation using either a prism or a diffraction grating.

4. Detector: The detector senses the difference and determines which frequencies have been
absorb by the sample and which frequencies remain unaffected and send a signal which is
amplified by an amplifier.

6. Recorder

The amplified detector signal is recorded as % of transmitted light vs. frequency on a chart.

2. Explain the advantages of FT-IR spectroscopy over dispersible IR spectroscopy

=1. Multiplex Advantage

• An interferometer in an FT-IR instrument does not separate energy into individual

frequencies for measurement of the infrared spectrum. Each point in the interferogram
contains information from each wavelength of light being measured. Every stroke of the
moving mirror in the interferometer equals one scan of the entire infrared spectrum, and
individual scans can be combined to give better representation of the actual absorbance of the
sample.

• In contrast, every wavelength across the spectrum must be measured individually in a

dispersive spectrometer. This is a slow process, and typically only one measurement scan of
the sample is made in a dispersive instrument.

• The FT-IR advantage is that many scans can be completed and combined on an FT-IR in a
shorter time than one scan on a dispersive instrument. The multiplex advantage results in
faster data collection of an FT-IR spectrum.

2. Throughput Advantage

• An FT-IR instrument does not use a slit to limit the individual frequency reaching the
sample and detector as a dispersive instrument does.
• There are also fewer mirror surfaces in an FT-IRspectrometer, so there are less reflection
losses than in a dispersive spectrometer.

• Overall, more energy reaches the sample and hence the detector in an FT-IR spectrometer
than in a dispersive spectrometer. This means that the signal-to-noise ratio of an infrared
spectrum measured on an FT-IR is higher than the signal-to-noise ratio attained on a
dispersive instrument which increase the sensitivity of small peaks that result in sharp and
more distinguishable peak in FTIR.

3. Precision Advantage

• An FT-IR spectrometer requires the use of a laser to control the velocity of the moving
mirror and to time the collection of data points throughout the mirror stroke length for each
scan.

• This laser is also available as a source of wavelength calibration within the instrument. The
laser wavelength is a constant value, and the x-axis data points of the FT-IR spectrum are
automatically referenced to this known value to maintain internal precision and accuracy of
the wavelength positions.

• Spectra collected with an FT-IR spectrometer can be compared with confidence whether
they were collected five minutes or five years apart. This capability is not available on a
dispersive infrared system. External calibration standards are required to control the accuracy
of dispersive instrument, making spectra less comparable due to instrumental unknowns
during and between scans.

• Accuracy and precision in infrared spectra are much higher when collected on an FT-IR.

3. What is finger print region? What is its significance in IR spectroscopy

=The region between 600 cm-1 and 1200 cm-1 in an IR spectrum is known as the fingerprint
region

Infrared spectroscopy (IR) provides a sensitive probe for specific functional groups in

polymers. Especially when dealing with hydrocarbon polymers such as polystyrenes and
polydienes, functional groups such as hydroxy, amine, and carbonyl can be easily detected
by IR.

AAS:

1. Breifly describe the radition source that is commonly used in atomic absorption

=    The most common line source used for atomic absorption spectroscopy is the HCL.
Structurally, this is an air-tight lamp filled with argon or neon and kept at around 1 to 5 torr.
The inert gas is ionized as a high voltage potential difference is created between the tungsten
anode and use-specific cylindrical cathode. Cations of the ionized argon or neon gas dislodge
metal ions from the cathode. These produce an atomic cloud; where some of the atoms in the
cloud are in an excited state and emit an element specific radiation upon returning to ground
state. There are many commercially available models of HCLs, some can possess multiple
metal cathodes for analysis of several metals.

2. Explain the applications of atomic absorption spectroscopy

= 1. Clinical analysis: Analyzing metals in biological fluids like blood and urine etc.

2. Environmental analysis: Finding out the levels of various elements

in rivers, sea water, drinking water, air, petrol etc.
3. Pharmaceuticals: In pharmaceutical manufacturing process minute quantities of a
catalyst is often required which may be present in the
final product. Besides, there are pharmaceuticals containing metals
(e.g.: antacids, TPN etc.)
4. Industry: Raw materials are examined by atomic absorption spectroscopy to check
the presence of major elements and often toxic impurities (e.g.: concrete)
5. Mining: By using atomic absorption spectroscopy, the amount of gold in rocks for
example can be determined

3. Explain the principle of atomic absorption spectroscopy with application.

 4. Show a schematic diagram of an atomic absorption spectrophotometer. Mention
the role of monochromator

Mention the role of monochromators:

 Also it is called wavelength selector

 Select the specific wavelength
 Polychromatic light →monochromatic light
 Simple one is enough for AAS

Atomizer in atomic absorption:

The function of atomizer is to convert the atoms of the liquid sample into free gaseous atoms.

 The most common atomizer is Flame.

 The flame breaks the liquid sample into droplets which are then evaporated and leave a
solid residue.
 The residue is decomposed to give free atoms. These free atoms then absorb radiation
characteristic for that particular metal liberated from the radiation source

5. Write down the steps of emission of radiation:

= Emission: The excited metal atoms then emit radiation characteristics of the metal as they
fall back to ground state.

• The shape of the cathode is such that it concentrates the radiation into a beam which then
passes through a quartz window. The shape of cathode also facilitates the redisposition of sputtered
atoms.

Steps of emitting radiation:

 These characteristic radiation is absorbed by neutralatoms of the same element in ground

state , which occur in the flame, when sample solution is sprayed.

 It is not possible to use a source of light with a monochromator because this arrangement
gives a radiation with a band width of 1nm, where as the hollow cathode
lamp gives a band width of 0.001 to 0.01nm, which is highly desirable to achieve specificity.
 Moreover, light source should provide a line width less than the absorption line width of the
element to be determined
6. Write down the difference between molecular and atomic absorption
spectroscopy and how it is minimized?

7. Explain the principle and interferences in atomic spectroscopy

= Spectral: in spectroscopy, interference occurs when another emission line (e.g. from other
elements in the sample) is close to the emitted line of the test element and is not resolved
by the monochromators
 Chemical: formation of undesired species during
Atomization
 Physical: variation of instrument parameters such as uptake in the burner and atomization
efficiency (gas flow rate, sample viscosity etc.)
8. What do you mean by ionic interferences in atomic absorption spectroscopy and
how is it minimized?
= Ionization interference is a phenomenon which shows a change in emission
intensity, causing the ioniza- tion equilibrium to shift, when coexisting elements
include easily ionizable elements such as Na, K, Rb, and Cs.
9. Point out the merits and limitations of atomic absorption spectroscopy?

= Advantages of AAS:
1. Simplicity: The principle of measurement is simple
2. Wide spread application: Atomic absorption spectroscopy is used widely for
quantitative determination of most of the metals
3. Sensitivity: Metals can be analyzed at a level of ppm and even ppb
4. Interference: It is possible to determine a metal in the presence of other metals
without any interference
5. The technique is relatively inexpensive
6. The equipment is easy to use
Disadvantages of AAS

1. Atomic absorption spectroscopy is sequential (i.e. one element at a time). A separate lamp
for each element is needed. So it is suitable for small scale measurement.

2. It is applicable for liquid sample not for solids or gases. So it is greatly relies on sample
dissolution in solvent

3. It is not applicable for determination of non-metals

Mass Spectroscopy:

1. Breifly outline the instrumentation of mass spectrometer

= Mass Spectrometry Instrumentation:

The four main parts of mass spectrometry are discussed below:
1. Ionizer – The bombarding of the sample is done by the electrons. These electrons move
between cathode and anode.  When the sample passes through the electron stream
between the cathode and anode, electrons with high energy knock electrons out of the
sample and form ions.
2. Accelerator – The ions placed between a set of charged parallel plates get attracted to
one plate and repel from the other plate. The acceleration speed can be controlled by
adjusting the charge on the plates.
3. Deflector – Magnetic field deflects ions based on its charge and mass. If an ion is heavy
or has two or more positive charges, then it is least deflected. If an ion is light or has one
positive charge, then it is deflected the most.
4. Detector – The ions with correct charge and mass move to the detector. the ratio of
mass to charge is analyzed through the ion that hits the detector.
2. Define the following terms:
i) Mass spectrum ii) Base peak iii) Molecular ion peak iv) Detector v)
Ionization

i) Mass spectrum
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-
to-charge ratio of ions. The results are presented as a mass spectrum, a plot of
intensity as a function of the mass-to-charge ratio.

ii) Base peak: The most intense (tallest) peak in a mass spectrum, due to the ion with
the greatest relative abundance (relative intensity; height of peak along the spectrum's
y-axis).

iii) Molecular ion peak: The molecular ion peak is the peak in a mass spectrum that
represents the molecular ion (

iv) Detector : a device for detecting the presence of electromagnetic waves or of

radioactivity
 v) Ionization: ionization, in chemistry and physics, any process by which
electrically neutral atoms or molecules are converted to electrically charged
atoms or molecules (ions) through gaining or losing electrons.

10. Describe the theory of Mass spectrometry

11. Discuss in detail various ionization techniques employed in the Mass
Spectroscopy.

12. Explain the principle involved in the Chemical ionization. Write the merit and
demerits. Explain the working of Quadra pole mass analyzer.
6. Which is the simplest and most common method for converting the sample to ions?
Explain with diagram.

7. Define fragmentation and explain the fragmentation process. What are the factors
influencing fragmentation process?

= When a molecule is bombarded with high energy electrons in the ionization chamber of a
mass spectrometer, it not only loses an electron to form the molecular ion but also absorbs
some energy.

Fragmentation Processes:

A fragmentation process requires the energy transfer to the nucleus, and can be induced by
electromagnetic forces — this is called electromagnetic dissociation, or by strong forces —
this is called nuclear fragmentation. We will discuss these two processes separately, as they
differ in many aspects, in particular in the cross-section behaviour with increasing incident
energy. We will also address fragmentation in its extended meaning which is the notion used
at very high energies.

Fragmentation also involves-

• cleavage of bonds,

• rearrangements,

• secondary fragmentation of already fragmented ions etc.

8. Write a short note on Quasi- Equilibrium theory.

= The initial ionization is “vertical”, i.e., without change in position andkinetic energy of the
nuclei while it takes place. With the usual electron energy any valence shell electron may be
removed.

2. The molecular ion will be of low symmetry and have an odd electron.It will have as many
low-lying excited electronic states as necessary to form essentially a continuum.
Radiationless transitions then will result in transfer of electronic energy into vibrational
energy at times comparable to the periods of nuclear vibrations.

3. These low-lying excited electronic states will in general not be repulsive; hence, the
molecular ions will not dissociate immediately, but rather remain together for a time
sufficient for the excess electronic energy to become randomly distributed as vibrational
energy over the ion.

4. The rates of dissociation of the molecular ion are determined by the probabilities of the
energy randomly distributed over the ion becoming concentrated in the particular fashions
required to give several activated complex configurations yielding the dissociations.

5. Rearrangements of the ions can occur in a similar fashion.

6. If the initial molecular ion has sufficient energy, the fragment ion wilin turn have enough
energy to undergo further decomposition.

9. What is MS? Write down some uses of MS.

It is the most accurate method for determining the molecular mass of the compound and its
elemental composition. Mass spectrometers are used in industry and academia for both
routine and research purposes. The following list is just a brief summary of the major mass
spectrometric applications:

 Biotechnology: the analysis of proteins, peptides, oligonucleotides

 Pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics, drug

metabolism

 Clinical: neonatal screening, hemoglobin analysis, drug testing

 Environmental: Polycyclic aromatic hydrocarbons (PAH)s, Polychlorinated biphenyls

(PCBs), water quality, food contamination

 Geological: oil composition

10. What are the advantages of Mass spectroscopy over other spectroscopic methods?
Mention its disadvantages.

Advantages:
1. It is used for both qualitative and quantitative analysis of chemical substances.
2. Rather than only molecular masses it also classifies sample isotopes and helps us about
finding the chemical structure of the unknown compound.
3. This can calculate the purity of the sample.
4. The main advantage is it is very much sensitive from other techniques.

Disadvantages:
1. The main disadvantage of mass spectroscopy is that those hydrocarbons which produce
similar ions are not identified.
2. It is also not able to separate optical and geometrical isomers.
3. Mass spectroscopy also fails to distinguish between hydrocarbons having similarly
fragmented ions.

13. What do you mean by McLafferty rearrangement? Give an example.

In the McLafferty rearrangement, a hydrogen atom on a carbon 3 atom away from the radical
cation of an alkene, arene, carbonyl, or imine (a so-called γ-hydrogen) is transferred to the
charge site via a six-membered transition state, with concurrent cleavage of the sigma bond
between the α and β positions of the tether.

An example of McLafferty rearrangement is the fragmentation of 2-hexanone, producing

acetone enol radical cation and a neutral molecule of propene. Other examples include
carboxylic acid and ester.

NMR

1. Explain the fundamental principles and instrumental arrangements of NMR.

= NMR Spectroscopy Principle

Many nuclei have spin, and all nuclei are electrically charged, according to the NMR principle. An
energy transfer from the base energy to a higher energy level is achievable when an external
magnetic field is supplied.
 All nuclei are electrically charged and many have spin.
 Transfer of energy is possible from base energy to higher energy levels when an external
magnetic field is applied.
 The transfer of energy occurs at a wavelength that coincides with the radio frequency.
 Also, energy is emitted at the same frequency when the spin comes back to its base
level.
 Therefore, by measuring the signal which matches this transfer the processing of the
NMR spectrum for the concerned nucleus is yield.

NMR Spectroscopy Instrumentation:

This instrument consists of the following major parts:

1. Sample holder: It is a glass tube which is 8.5 cm long and 0.3 cm diameter.
2. Magnetic coils: it generates magnetic field whenever current flows through it

3. Permanent magnet: It helps in providing homogenous magnetic field at 60 – 100 MHZ

4. Sweep generator: Modifies the strength of magnetic field which is already applied.

5. Radio frequency transmitter: It produces a powerful but short pulse of radio wave.

6. RF detector: It helps in determining unabsorbed radio frequencies.

7. Recorder: It records the NMR signals which are received by the RF detector.

8. Readout system: A computer that records the data.

2. Discuss the principle of NMR Spectroscopy.

Many nuclei have spin, and all nuclei are electrically charged, according to the NMR principle. An
energy transfer from the base energy to a higher energy level is achievable when an external
magnetic field is supplied.
 All nuclei are electrically charged and many have spin.
 Transfer of energy is possible from base energy to higher energy levels when an external
magnetic field is applied.
 The transfer of energy occurs at a wavelength that coincides with the radio frequency.
 Also, energy is emitted at the same frequency when the spin comes back to its base
level.
 Therefore, by measuring the signal which matches this transfer the processing of the
NMR spectrum for the concerned nucleus is yield.

3 Explain the principle and instrumentation of NMR Spectroscopy

= Answer 1

4. Definition: NMR, NMR spectrum, Chemical shift.

= Nuclear magnetic resonance (NMR)

Nuclear magnetic resonance (NMR) spectroscopy is the study of molecules by recording the
interaction of radiofrequency (Rf) electromagnetic radiations with the nuclei of molecules
placed in a strong magnetic field.

1. Chemical shift: The difference in the absorption of the proton with respect to TMS
signal , is called chemical shift

5. Briefly describe the source of energy in NMR.

= The source of energy in NMR is radio waves which have long wavelengths, and thus low
energy and frequency.
 2. All protons of a molecule may not absorb same radiofrequency. Why?
= The radiofrequency pulses and the gradients have completely different purposes
in NMR. Radiofrequency pulses are the basic building block of NMR experiments,
you use them to manipulate your spin populations and coherences. While each
proton has a different frequency depending on its environment, you generally
can't (or don't) target individual protons with your pulses. In most experiments
you hit either all protons with your pulses, or a specific group of protons.

Gradients have different purposes in NMR and MRI, what they allow you to do is
to change the magnetic field depending on the position inside your sample. In
NMR this allows you to remove unwanted signals and select specific coherences.

3. Write down the instrumentation of NMR spectroscopy with schematic diagram.

4. Write down the aspects of NMR spectrum.

= Aspects of NMR spectrum

• 1. The number of peaks: Tells us how many different kinds of protons are there in molecule.
The frequency of energy neede to flip the magnet is different for hydrogen atoms that are
indifferent positions.

• 2. The intensities of peaks: Tells us how many protons of each kind are there. If one
hydrogen releases a set amount of energy when it falls back in line (e.g. 2 units). Then 3
hydrogen atoms will release 3 times that amount when they fall in line (6 units). This affects
the size of the peak in the NMR spectrum. Larger the number of hydrogen atoms, bigger the
peak.

3. The positions of the peaks: Tells us something about the electronic environment of each
kind of protons. So the type of energy given off tells us the position of the hydrogen in the
molecule.
4. The splitting of a peak into several peaks: Tells us about the environment of a proton with
respect to other nearby protons.

9. Write down the factors that influence the chemical shift.

= Factors affecting chemical shift

•Following are the factors which influence the chemical shift:

1. Intramolecular factor

a) Inductive effect

b) Anisotropic effect

c) Van der waal’s effect

2. Hydrogen bonding

3. Effects of solvent, temperature and concentration

10. Why does TMS (Tetra methyl silane) choose as an internal standard? Write down
the meritsof TMS as internal standard in NMR?

= Tetra methyl silane (TMS) was chosen initially because the proton of its methylgroups are
more shielded than any other known compounds. TMS affords asharp signal for the methyl
protons at a high value of the magnetic field and therefore used as an internal standard and
the chemical shift positions of various protons in a molecule are reported as the distance from
the TMS signal.

1. TMS is-

• miscible with almost all organic compounds

• highly volatile liquid (bp 27° C) and can be removed easily from the system

• inert and non-toxic

• 2. It has least molecular interaction with sample or solvent. So, sample can be recovered
easily.

• 3. It has twelve equivalent protons and gives only one peak and is more shielded than any
other proton in organic compounds. So, absorbance value of TMS can be taken arbitrarily
zero.

11. What is the procedure should be followed for preparing sample in liquid NMR
= Preparation of sample in liquid NMR

• In liquid NMR, a NMR tube is used. Samples for NMR analysis should be prepared in 8
inch × 5 mm NMR tubes. The sample must be sufficiently soluble to yield an NMR
spectrum. It is recommended to dissolve between 2 and 10 mg in between 0.6 and 1 mL of
solvent so that the sample depth is at least 4.5 cm in the tube. The suggested sample volume
for NMR instrument is 700 μL. If necessary, sample is filtered using glass wool. Finally
internal standard is added to the tube.

12. Write down the application of NMR

= NMR Spectroscopy Applications

 NMR spectroscopy is a Spectroscopy technique used by chemists and biochemists to

investigate the properties of organic molecules, although it is applicable to any kind
of sample that contains nuclei possessing spin.
 For example, the NMR can quantitatively analyze mixtures containing known
compounds. NMR can either be used to match against spectral libraries or to infer the
basic structure directly for unknown compounds.
 Once the basic structure is known, NMR can be used to determine molecular
conformation in solutions as well as in studying physical properties at the molecular
level such as conformational exchange, phase changes, solubility, and diffusion.