INSTRUMENTAL METHODS OF ANALYSIS Question Papers (2008, Aug/sep,supple)

SET : 1

1. (a) Define the terms qualitative and quantitative analysis. (b) Differentiate between micro and semi-micro analytical methods. (c) What are advantages and limitations of chemical methods.

2. Compare and contrast the techniques phase contrast microscopy and differential interference contrast microscopy (DIC).

3. Explain the following terms:

• Gravitational force
• Centrifugal force
• Sedimentation coefficient.

4. You have two samples of DNA derived from different organisms, A and B. DNA from A shows an increase in optical density beginning at 70 C and plateau at 80 C. DNA from B shows a corresponding increase in O.D beginning at 65 C and plateau out at 76 C. what is your conclusion. Justify your answer.

5. (a) Define fluorescence. Derive an expression relating intensity of fluorescence and concentration.
(b) Write a brief note on:

• Triplet state
• Doublet state
• Singlet state

6. (a) Discuss the basic principle of infrared spectroscopy? (b) Briefly describe the scanning of infrared spectrum of an organic compoung.

7. Explain the following:

• Free inductive decay
• Hetero nuclear decoupling
• Off resonance coupling
• Chemical shift

8. Give an account on electronic structure and hyperfine splitting in ESR spectroscopy?

SET: 2

1. Give a brief account on methods for reporting analytical data?

2. Compare and contrast the optical magnification obtained by the bright and dark field microscopy along with their specific applications.

3. (a) Give the basic principle involved in analytical centrifugation. (b) What are the important applications of this technique in biochemistry?

4. You have two samples of DNA derived from different organisms, A and B. DNA from A shows an increase in optical density beginning at 70 C and plateau at 80 C. DNA from B shows a corresponding increase in O.D beginning at 65 C and plateau out at 76 C. what is your conclusion. Justify your answer.

5. (a) Define optical activity? What is the principle involved in circular dichroism spectroscopy? (b) Differentiate polarimetry and circular dichroism.

6. Discuss the principle, importance and applications of mass spectrometry?

7. (a) Give the principle underlying in NMR spectrometry? (b) Describe the component of a NMR spectrometer with a neat labeled diagram.

8. (a) What is the principle of ESR. Discuss some important applications of ESR. (b) What are the limitations of ESR.

SET :3

1. Give an account of the instruments involved in electrochemical methods of analysis along with their principle of working.

2. (a) Give the basic principle involved in the viewing an object in dark field. (b) Draw a neat labeled diagram of optical pathway of dark field microscope. (c) Give its specific use.

3. (a) Give the basic principle involved in analytical centrifugation. (b) What are the important applications of this technique in biochemistry?

4. What are the different regions of the electromagnetic spectrum and where are they used for?

5. Define the following terms:
Chiral compounds
Cotton effect
Circular birefringence.
6. How will you distinguish between intermolecular and intramolecular hydrogen bonding by IR spectroscopy?

7. (a) Explain the principle involved in CMR spectra? (b) Why it is not possible to determine relative ratio of carbon atoms in a compound by integration of peak areas in 13C NMR as in PMR?

8. Why ESR spectroscopy is widely used in the study of chemical, photochemical and electrochemical reactions? Justify your answer.

SET: 4

1. Write an account on analytical methods based on the physical property measurement?

2. Narrate the principle of operation of a fluorescence microscope. Give its applications in biology?

3. Calculate the molecular mass of the protein where the sedimentation coefficient of the protein is 7.75 * 10 power -12 s. In subsequent analysis the protein was found to have an average diffusion coefficient of 4.0 * 10 power -11 Sq.mt per sec. and the partial specific volume of 0.734 * 10 power -3 cub.mt per kg.

• Convert the molecular mass to relative molecular mass.
• express the molecular mass in Daltons.

4. Define the terms:
(a) (i) Radiant power (P)
(ii) Path length of radiation
(iii) absorptivity
(iv) molar absorptivity
(b) Describe and derive the Beer's law.
(c) What are its limitations and deviations.

5. (a) What is atomic absorption. What are its advantages and disadvantages. (b) Give some of its limitations.

6. (a) What are the different regions of infrared radiation? (b) Explain various types of streching and bending vibrations with suitable examples.

7. (a) why does a signal for a particular set of protons in split into a multiplet. (b) Explain with suitable examples the term spin-spin coupling.

8. (a) Which of the following two formula is correct for the complex obtained between copper ion and 8 quinolinethanol (C9H7N5)
(i) Cu(I) (C9H7N5) (C9H6N5)
(ii) Cu (II) (C9H6N5)2.
Show that the ESR approach is appropriate for this determination.
(b) Which is one of the best known free radicals used in calibrating ESR spectra?

ELECTRON SPIN RESONANCE (ESR/EPR)

Electron spin resonance is also called as Electron Paramagnetic Resonance.
ESR is a spectroscopic technique used to detect “species that have unpaired electrons”. In case of an organic molecule the species are the free radicals and in case of inorganic complexes they are the transition metal ions.
Because most stable molecules have a closed shell configuration with out a suitable unpaired spin, the ESR spectroscopy is less widely used than the NMR(Nuclear Magnetic Resonance) spectroscopy.
ESR was first discovered by a Soviet physicist Yevgeniy Zavoyskiy in 1944.
The basic physical concepts of ESR Spectroscopy are analogous to those of NMR spectroscopy.
Spins of the electron are excited in ESR instead of the spins of the atom’s nuclei as in NMR.
For electrons in a magnetic field of 0.3 tesla, spin resonance occurs at around 10GHz.
ESR is used in solid-state physics, for the identification and quantification of radicals (in chemistry) to identify reaction pathways, as well as in biology and medicine for tagging biological spin probes.
Since radicals are very reactive, they do not normally occur in high concentrations in biological environments. With the help of specifically designed nonreactive radical molecules that attach to specific sites in a biological cell, its possible to obtain information on the environment of these so called spin label or spin probe molecules.
To detect some subtle details of some systems, ‘high field high frequency’ electron spin resonance spectroscopy is required.

UNITS & CONSTANTS :

A magnetic field is described by some constants and units :
· Magnetic induction in teslas (T)
· Magnetic flux density in amperes per meter (A/m)
· The CGS unit for magnetic induction is the gauss (G) which is equivalent to 10-4 T.
Further more, in describing ESR, the following are very important :
Planck’s constant h = 6.63 × 10-34 Js
Boltzmann constant K = 1.38 × 10-23 J/K
Bohr magneton µB = 9.27 × 10-24 J /T

BASICS
An electron has a magnetic moment. When placed in an external magnetic field of strength Bo, this magnetic moment can align itself parallel or antiparallel to the external field. The former is a lower energy state than the latter and the energy separation between the two is ∆E = geµBo, where ge is the gyromagnetic ratio/g factor of the electron .
Gyromagnetic ratio is the raito of electrons magnetic dipole moment to its angular momentum.
To move between the two energy levels, the electron can absorb electromagnetic radiation of the correct energy :

∆E = hν = geµBBo

and this is the fundamental equation of EPR spectroscopy. The paramagnetic centre is placed in a magnetic field and the electron caused to resonate between the two states; the energy absorbed as it does so is monitored, and converted into the EPR spectrum.

A free electron on its own has a value of 2.002319304386. This is ge, the electronic g factor. This means that for radiation at the commonly used frequency of 9.5 GHz (known as X – band microwave radiation, and thus giving rise to X – band spectra), resonance occurs at a magnetic field of about 0.34 tesla / 3400 gauss.
EPR signals can be generated by resonant energy absorption measurements made at different electromagnetic radiation frequencies in a constant external magnetic field (i.e we can scan with a range of different frequency radiation whilst holding the field constant, like in an NMR experiment). Conversely, measurements can be provided by changing the magnetic field B and using a constant frequency radiation; due to technical considerations, this second way is more common. This means that an EPR spectrum is normally plotted with the magnetic field along the X – axis, with peaks at the field that cause resonance (whereas an NMR has peaks at the frequencies that cause resonance).
In practice a single, isolated, paramagnetic centre never occurs, but only a population of a large number centres. If this population of centres is in thermodynamic equilibrium, its statistical distribution is described by the Boltzmann distribution

K is Boltzmann constant
T is temperature in Kelvin
For X – band radiation (ν = 9.75 GHz) at room temperature,

Because the lower level has more electrons than the higher one, transitions from the lower to the higher level are more probable, which is why there is net absorbtion of radiation.

EPR SPECTRAL PARAMETERS

The g Factor :

Knowledge of the g factor gives us information about the paramagnetic center’s electronic structure. When an unpaired electron is in an atom, it is an atom, it feels not only the external magnetic field Bo applied by the spectrometer, but also the effects of any local magnetic fields. Therefore the effective B(eff) felt by the electron is
B(eff) = Bo(1 - σ)
Where σ allows for the effects of the local fields (it can be positive or negative), and therefore the resonance condition is ∆E = hν = geµBB(eff) = geµBBo(1 - σ)
The quantity ge(1 - σ) is called the g factor, given by the symbol g, so

∆E = hν = gµBBo

Give this last equation, you can measure g from the ESR experiments by measuring the field Bo and the frequency ν at which resonance occurs. If g differs from ge (2.0023), this implies that the ratio of the electron’s magnetic momentum to its angular momentum has changed from the free electron value. Since the electrons magnetic moment is constant (its Bohr magneton), then the electron must have gained or lost momentum. It does this through spin – orbit coupling, and because the mechanisms of spin – orbit coupling are well understood, the magnitude of the change can be used to give information about the nature of the atomic or molecular orbital containing the electron. In real life the electrons are associated with atoms. There are three important consequences of this. Firstly the electron may gain or loose angular momentum (through spin – orbit coupling) which will affect the value of the g – factor. Secondly, this change in angular momentum is not the same for all orientations of the atom or molecule in an external magnetic field. It means that the g – factor changes according to the orientation of the paramagnetic atom in the magnetic field – it is anisotropic. This anisotropy depends upon the electronic structure of the atom in question, and so can yield information about the atomic/molecular orbitals containing the unpaired electron. Thirdly, if the atom(s) which the electron is associated with has /have a non – zero nuclear spin, the magnetic field associated with this atom will affect electron too. This leads to the phenomenon of hyperfine coupling, which is analogous to coupling in NMR in splitting the resonance signal into doublets, triplets and so forth.

RESONANCE LINE WIDTH DEFINITION
Resonance line widths are defined in magnetic induction units B and are measured along the axis, from line center to y value crossing chosen point of energy spectrum. These defined widths are called half widths and posses some advantages : for asymmetric lines values of left and right half width can be given. Half width ∆Bh is distance measured from center of line to point in which absorption value has half of maximal absorption curve inclination. In a practical approach, full definition of line width is used. In the case of symmetric lines, half width, and full inclination width ∆Bmax = 2∆B1s .

INSTRUMENTAL METHODS OF ANALYSIS Question Papers (Regular, 2007)

SET : 1

1. Give an account of the instruments involved in the electrochemical methods of analysis along wuth their principle of working.

2. Narrate the principle of operation of a fluorescence microscope. Give its applications in biology?

3. A protein has a sedimentation coefficient value of 3.12 × 10^-13 sec in water. Its diffusion coefficient in water is found to be 8.2 × 10^-7 /cm. both the above values have been corrected for 20C in water. The partial specific volume of the protein is 0.735, & the density of water at 20C is 0.9982. Determine the molecular weight of the protein?

Explain the principle involved in the above method.

4. Discuss the effect of hydrogen bonding in UV absorption. Explain by taking example.

What is the effect of electronegativity in UV absorption. Explain by taking examples of methyl chloride & methyl iodide.

5. What are fluorometric reagents? Mention some important fluorometric reagents.

How is fluorometric is used in the determination of thiamine in a meat sample?

6. Write short notes on :

a) Brainbridge mass spectrograph

b) Dempsters mass spectrometer

7. Write a brief account on :

a) Pulse – acquired method

b) Fourier transform method

c) Multidimensional NMR

8. Why electron spin resonance is also called electron paramagnetic resonance?

Calculate the ESR frequency of an unpaired electron in a magnetic field 0.33τ. Given, for electron g = 2 and β = 9.273 × 10^-24 J/T

INSTRUMENTAL METHODS OF ANALYSIS Question Papers (supple,2006)

SET:1

1. What are the instrumental methods? How are they classified? Name two or three instrumental methods for each of the physical property.

2. (a) Write briefly about origin and theory of UV spectroscopy

(b) What are the various components of UV spectrophotometer and discuss each of them in detail and briefly the scanning of UV spectrophotometer?

3. Write short notes on the following UV applications:

(a) Quantitative analysis

(b) Molecular weight determination.

(c) Impurities in organic compounds.

4. (a) What are the advantages in atomic absorption of a heated graphite atomiser over a flame atomiser.

(b) Why are spectral interferences less severe in atomic amsorption and atomic flourescence spectroscopy than in flame emission spectroscopy?

(c) What is the purpose of densitometer and how is it used in qiantitative determination.

5. Explain the analytical applications of emission spectroscopy with examples.

6. Comment on:

(a) Relaxation time

(b) Magic number

(c) Tetramethyl silane and its significance

(d) Multiplicity in NMR

7. Discuss ESR instrumentation with a block diagram.

8. Out of syllabus(so not mentioned!)

SET: 2

1. (a) Distinguish between accuracy and precision.

(b) What are determinate and indeterminate errors

(c) Write briefly about determinate and indeterminate errors.

(d) Distinguish between relative and absolute error.

2. (a) What are different regions of infra red radiation? Explain various types of stretching and bending vibrations with suitable examples.

(b) What is force constant? How is it determined? To what use is it put?

3. (a) What is the difference between molar absorptivity and absorbance?

(b) An absorbance of 0.436 was obtained after 11.5 ml of titrating agibt was added to 68ml of an initial solution. What was the corrected absorbance of the solution? What would be the % error have been if the correction was not made?

(c) A water solution of a coloured compound has a molar absorptivity of 3200 at 525nm. Calculate the absorcance and % transmittance of a3.40 * 10^-4 solution if a 1.0 cm

4. (a) Write the advantages of atomic absorption spectroscopy over flame emission spectroscopy?

(b) What is a single beam and a double beam atomic absorption spectrophotometer and explain the instrumentation involved?

(c) Define sensitivity and detection limits in atomic absorption spectroscopy.

5. (a) Explain the function of a monochromater.

(b) How plasma emission is used for analytical purpose?

6. Write short notes on

(a) Longitudinal relaxation

(b) Transverse relaxation

(c) Spin-Spin coupling.

7. Out of syllabus

8. Out of syllabus

SET: 3

1.

(a) Define sensitivity and detection limits

(b) Give the detection limits of

1. IR Spectrometry

2. UV Spectrometry

3. NMR

4. Mass Spectrometry

(c) What are the advantages and disadvantages of chemical and instrumental methods.

2. Write short notes on:

(a) Molar extinction coefficient

(b) Laws of absorption

(c) Photometric titrations

(d) Photometric accuracy

3. Write short notes on the following UV applications:

(a) Chemicals kinetics

(b) Charge transfer transitions

(c) Dissociations constants of acids and bases

4. Write short notes on:

(a) Total consumption burner

(b) Premix burner

(c) Nebulizers

5. (a) Explain how the output is detected.

(b) Explain how two lines are separated.

6. (a) What is spin-spin splitting?

(b) A methylene group (CH₂) is adjacent to a CH group. Into how many peaks is the CH₂

peak split by the single adjacent hydrogen?

7, 8 out of syllabus.

SET:4

1. Repeat (set 2 - Q1)

2. (a) why far U.V region is called vacuum ultra region

(b) discuss the advantages and disadvantages of spectrophotometry in vacuum-ultra violet region of the spectrum.

(c) why are the absorption bands appear instead of sharp lines in UV spectra

3. Repeat (set1 – Q3)

4. (a) in atomic absorption, the elements such as Al, Ti, Mo, V, Si can not be detected when a flame is used to produce the atomic state. Why?

5. Explain the analytical applications of emission spectroscopy with examples

6. Draw a schematic of an NMR instrument and discuss the parts.

7. Out of syllabus

8. Out of syllabus

INSTRUMENTAL METHODS OF ANALYSIS syllabus, JNTU (2007-2008)

UNIT I: INTRODUCTION

Types of Analytical Methods – Instruments for Analysis – Uncertainties in Instrumental measurements – Sensitivity and detection limit for instruments.

UNIT II: MICROSCOPY

Bright field, Dark field, Fluorescent, Phase contrast, confocal microscopy, SEM & TEM Microscopy, Flow Cytometry.

UNIT III: CENTRIFUGATION

General Principals, Ultra Centrifugation, velocity Sedimentation & measurements, Equilibrium Ultracentrifugation – Density Gradient centrifugation

UNIT IV : SPECTROSCOPY - I

General principles – Radiation, energy and atomic structure- types of spectra and their biochemical usefulness – basic laws of light absorption. Electromagnetic radiation & Spectrum, Beer – Lambert’s Law and apparent deviations; UV - VIS Spectrophotometer,Spectrofluorimetry, Atomic absorption & Atomic emission spectroscopy, Cirular Dichroism (CD)- principles, instrumentation and applications.

UNIT V: SPECTROSCOPY-II

Infra Red Spectroscopy. Mass spectroscopy-Introduction, analysis, applications in

biology ESR principles - instrumentation-applications

UNIT VI: ONLINE MONITORING AND CONTROL DEVICES

pH, temperature, dissolved oxygen, agitation, sensors and their operation.

UNIT VI: X RAY DIFFRACTION AND CRYSTALLOGRAPHY

Principle, Mode of Operation and Applications

UNIT VII: SEPARATION EQUIPMENTS – PRINCIPLES AND OPERATION:
HPLC, Gas chromatography, Ion – exchange Chromatography, Gel – filtration Chromatography, Affinity Chromatography, Membrane separations, Ultrafiltration , Reverse Osmosis

UNIT VIII: NMR

High resolution NMR –Chemical shift-Spin-spin coupling Frequency lock- double resonance-applications of proton NMR-quantitative analysis-qualitative analysis, application of NMR in biology and study of macromolecules

TEXT BOOKS:

1. A Biologist Guide to principles and techniques of practical Biochemistry. By

Keith Wilson, Kenneth H. Goulding 3rd ed. ELBS Series.

2. Skoog & West, Fundamentals of Analytical Chemistry, 1982

REFERENCES:

1. Vogel, Text Book of Quantitative Inorganic Analysis, 1990

2. Ewing, Instrumental Methods of Analysis, 1992

3. Hobert H Willard D. L. Merritt & J. R. J. A. Dean, Instrumental Methods of

Analysis, CBS Publishers & Distributors, 1992

4. F. Settle. Hand book of Instrumental techniques for Analytical Chemistry, Prentice Hall, 1997.