2 mark Questions

1. What are the general requirements for doing a separation by column chromatography
Ans General Requirements of doing a separation by column chromatography
1. Stationery Phase
 Particle size and geometry: uniform size and spherical shape (600-200micrometre)
 Increased mechanical stability
 Inert
 Allow free flow of mobile phase
 Inexpensive and freely available
 Insoluble in solvents/ mobile phase
2. Mobile Phase
 Should displace solute from the surface od adsorbant
 Separate mixture into individual component
3. column
4. column preparation
5. development of column
6. Detection technique
2. Enumerate various types of chromatography
1. Column chromatography
2. Thin layer chromatography
3. Paper chromatogtraphy
4. Ion Exchange chromatography
5. High Performance Liquid chromatography (HPLC)
6. High Performance Thin Layer chromatography (HPTLC)
7. Gas Chromatography
8. Electrophoresis
9. Gel Filteration
10. Affinity Chromatography
3. What is migration parameters
Rf value is defined as the ratio of the distance moved by the solute and the distance moved by the
solvent along the paper where both distances are measured from the common origin, that is the
point where the sample is initially spotted on the paper
Rf value= Distance travelled by solute from origin /
Distance travelled by solvent from origin
 Rx value = distance travelled by the sample
Distance travelled by standard
Rx is always closer to 1
Rm value
It is used to quantitative analysis to find out whether the compounds belong to homologous series.
If they belong to a homologous series, the delta Rm values are constant.
The delta Rm values for a pair of adjacent member of homologous series is determined by using
the formula:
Rm = log (1/Rf – 1)

4. What is TLC & Write the principles of separation in TLC

Thin layer chromatography is a technique uses to separate non volatile mixtures on a sheet of glass,
plastic or aluminium foil which is coated by thin layer of adsorbent material usually silica gel,
alumina or cellulose
Principle:
The principle of separation is adsorption. One or more compounds are spotted on a thin layer of
adsorbent coated on a chromatographic plate. The mobile phase solvent flows through because of
capillary action. The components move according to their affinities towards the adsorbent . the
component with more affinity towards the stationery phase travels slower and the component with
lesser affinity towards the stationery phase travels faster

5. What are the general requirements in TLC techniques

1. Stationery phase
2. Mobile phase
3. Glass plate
 Preparation
 Pretreatment
 Layer thickness
 Activation
 Storage
4. development technique
5. development techniques
6. Define TLC & name the stationary phases used in TLC
*refer Q 4 for definition
Stationery phase
 i) silica gel
silica gel G , Silica gel GF254, Silica gel 60 etc
ii) cellulose
iii) alumina
iv) keiselghur G
7. What is the difference between silica gel H,G,GF ?
Silica Gel H: silica gel without binder
High purity grade and without water
Silica Gel G: silica gel with binders
Eg: gypsum (CaSO4)
Silica Gel GF: silica gel with binder and with fluorescent indicator
8. Define Chromatography & Rf values
Chromatography is the method of separation of a mixture that distributes the components into 2
phases
 Stationery phase
 Mobile phase
By chromatography we can separate the mixture into pure form of substance
Rf value *refer Q 3 for definition
Range = 0 to 1
Ideal = 0.3 – 0.8
The Rf value depends upon
 Type of sample
 Medium of separation
 Nature of mixture
 Temperature of developing chamber
9. Name the different grades of alumina
 Smelter grade alumina
 Calcined alumina
 Low soda alumina
 Reactive alumina
 Tabular alumina
 Fused alumina
 High purity alumina
10. Describe the preparation of plates & adsorbents used in TLC
Slurry is the combination of silica gel(stationery phase) and water.
Pretreatment: it involves the cleaning of glass plate with soap water and distilled water and
pouring slurry to it
The prepared slurry is added to the glassplate by 4 methods
 Pouring: the glassplate is taken and the slurry is poured onto it
 Dipping: slurry is taken in a beaker and then 2 plates are dipped into it
 Spraying: slurry is sprayed into the plate
 Spreading: by using TLC spreader the slurry is spread in tlc plate
Adsorbents used in TLC:
 Silica
 Alumina
 cellulose
11. For silica gel G, in what ratio it is mixed with water for making slurry for use in TLC
Silica gel G consists of silica gel + binder like CaSO4
For making slurry to use in TLC the ratio to which it is mixed with water is 1:2
12. What is the thickness of adsorbents layer in a) analytical TLC b) preparative TLC
i) in analytical TLC 0.25 mm
ii) in preparative TLC 2 mm
13. How is activation of TLC plates done?
Activation of TLC plate is nothing but removing water and moisture and other adsorbed
substances from the surface of any adsorbent , by heating at a temperature so that adsorbent
activity is retained.
Procedure:
 Dry at room temperature for 15 minutes
 Heat at 110 degree Celsius for 30 minutes in hot air oven
 Heat at 200 degree for 4 hours for further increase in the activity
14. Why activation of TLC plates necessary. How TLC plates stored
Activation is done to remove water or moisture and also to increase the power to absorb the
sample
Storage:
Stored in a desiccator or thermostatically controlled oven
15. What is edge effect in TLC ? To avoid edge effect what must be done in TLC
 Edge effect happens the solvent front in the middle of the TLC plate moves faster than the
edge.
It is caused when saturation of atmosphere with mobile phase is not done
Remedy;
The development chamber or tank should be lined inside with filter paper moistened with the
mobile phase so as to saturate the atmosphere. When the mobile phase reaches the top of filter
paper, remove the filter paper and insert the glass plate
16. Enumerate four adsorbents and four mobile phases used inTLC
Adsorebents
 Alumina
 Silica gel
 Cellulose
 Kieselghur
Mobile phase
 Petroleum ether
 Carbon tetrachloride
 Benzene
 Methanol
17. Give a specific spray reagent to detect the following compounds by TLC a) sulphanilamide
i. b) amino acids c) alkaloid d) phenols
Ans a) sulphanilamide – florescamine in acetone
b) amino acids – ninhydrine in acetone
c) alkaloid – Dragendrooff’s reagent
d) phenol – Ferric chloride
18. Define paper chromatography ? which type of paper is normally used ; Hydrophilic/hydrophobic
Paper chromatography is an analytical method used to separate coloured chemicals or substances
especially amino acids and dyes
The principle of paper chromatography is paper partition method
Hydrophilic paper especially whatmann filter paper is used in paper chromatography
19. Enumerate the application of paper chromatography
 To detect foreign substances or impurities
 Separation of mixtures of drugs from biological origin or plant extracts
 Separation of carbohydrates, vitamins, proteins, alkaloids, amino acids etc
 Separate metabolites of drug in blood or urine
20. Explain Radial Chromatography
Radial or Circular Paper Chromatography permits separation of sample components in the form of
concentric circular zones through radial movement of liquid phase. A circular shaped filter paper is
employed.

A narrow slit about 2mm wide is cut from the circumference to the centre and the wick formed is bent so
as to dip in the solvent contained in a Petri dish. Sample spotting is done along the circumference of the
smaller inner circle. The petri dish is covered after securing the filter paper to the top and allowed to
develop. The mobile phase reaches the filter paper through capillary action and spreads radially outwards
thereby resolving the sample components radially.

21. What is an ion-exchange resin? Give an example of natural resin a) Cation b) Anion
Ion exchange chromatography is the separation of ions and polar molecules based on their affinity to ion
exchanger
Ion exchange resins are polymetric resin matrix containing exchange sites. The resin is composed of
polystyrene and divinyl benzene . polystyrene contains sites for exchangeable functional groups , divinyl
benzene act as cross linking agent and offers adequate strength.
a) cation – eg: zeolytes, clay
b) anion – eg: dolomite
22. Which portion of resin contains exchangeable sites
Ion exchange resins are polymetric resin matrix containing exchange sites. The resin is composed of
polystyrene and divinyl benzene . polystyrene contains sites for exchangeable functional groups , divinyl
benzene act as cross linking agent and offers adequate strength i.e, mechanical stability
23. Which function group can be present in a) weak Cationic exchange resin b) Strong Cationic exchange
resin c) weak and strong anionic exchange resins
Ans. a) weak Cationic exchange resin – carboxylic methacrylate
b) strong Cationic exchange resin – sulphonated polystyrene
c) weak anionic exchange resin – phenol formaldehyde
d) strong anionic exchange resins – quaternary ammonium polystyrene
24. What is cross linking, rigidity & swelling of ion exchange resin?
 Cross linking offers strength, rigidity and mechanical stability
Eg: divinyl benzene helps in cross linking
Increase in cross linking

Increased rigidity

Decreased swelling

And vise versa

 Rigidity is the inability to be bent or forced
When the rigidity increases swelling decreases and molecules won’t pass through
 Swelling occurs when the pores become wide , no proper separation occurs
25. How is the efficiency of an ion-exchange resin measured?
It is measured by ion exchange capacity
Ion exchange capacity is the total ion exchange capacity in terms of exchangeable functional group
expressed as milliequillants per gram of ion exchange resin
Meq/g = 1000/ eq. wt
26. What are the requirements for a compound to be analysed by Gas- Liquid Chromatography
 Mobile phase i.e, carrier gas like helium, hydrogen or nitrogen
 Flow regulator and flow meter like soap bubble flow meter and rotameter
 Sample injection system: gas, liquid or solid in nature
 Column
 Open tubular column
 Packed column
 Support coated open tubular column
 Detector
 Thermal conductivity detector
 Flame ionisation detector
 Electron capture detector
 Temperature control device
 Pre heater
 Thermostatically controlled oven
  Recorders and integrator
27. Give example of carrier gas used in G.L.C.
 Hydrogen
 Helium
 Argon
 Nitrogen
28. Under what conditions Gas-Solid Chromatography is preferred over Gas- Liquid Chromatography.
Gas solid chromatography (GSC) works on the principle of adsorption
Gas liquid chromatography(GLC) works on the principle of partition
Gas solid chromatography is preferred when
 Low molecular weight gaseous species is used
 Less solubility of solutes in stationery phase
 GSC can be used at high temperature, due to limitations of validity and instability of liquid coating
at high temperature GLC cannot be used
 GSC is used for analysis of gases having no active functional group which interact with adsprbent
surface
29. Write a note on Guard Column & its Significance.
Guard column is seen in between column and sample injector
It has the same composition as that of analytical column
Significance:
 It increases the life span of analytical column
 It act as pre filter to remove particulate matter
 It doesn’t contribute in separation
 It removes impurities of mobile phase
30. What is C18 or ODS? What is its use in Chromatography hplc
It is octadecyl silane. It is used as industry packing material for HPLC application
Uses:
 Surface coating used in reverse phase chromatography
 Column is filled with ODS in HPLC
 It increases theoretical plate number
 It provides rapid equilibration
 It is used as stationery phase in RP-HPLC
31. How will you check the presence of impurities in HPLC & What is internal standard
By comparing the chromatogram of standard and that of sample the presence of additional peaks can be
prevented and impurities can be detected.
From the percentage area of peaks, percentage purity of compounds can be known.
Internal standard:
An internal standard in analytical chemistry is a chemical substance of known concentration that is added
to constant amount of samples in a chemical analysis
It is used to correct loss of analyte
32. What is potentiometry ? How is potential (emf) is measured
It is the measurement of electrical potential (potential difference of a sample) as a method of chemical
analysis in electroanalytical chemistry to find concentration of a solute in a solution
Measurement of emf:
Ɛ = Ɛ˚ + RT / nF Ln Cᵐᵑᵗ
For redox reaction
Ɛ = Ɛ˚ + RT / nF log oxidised state
Reduced state
The potential difference between 2 electrodes are measured using a voltmeter
33. What is Indicator electrode & reference electrode give examples
Indicator electrode:
An electrode that is used to measure potential or pH of a solution whose potential varies depending upon
the concentration of analyte
Eg: glass electrode
Antimony / antimony oxide electrode
Reference electrode:
It is an electrode whose potential remains constant
Eg: standard Hydrogen electrode (SHE)
Calomel electrode
34. What is the relationship between emf & pH.
Ɛ = Ɛ˚ + 0.0592 (-pH outside + pH inside) – (-pH2 + pH1) + (pH1 – pH2)
Where pH 1 is constant
Ɛ = Ɛ˚ - 0.0592 pH outside
35. What are the advantages of glass electrode & saturated Calomel electrode.
Glass electrode
It is a combination of reference and indicator electrode
Advantages:
 It can be used in the presence of oxidation, reduction or complexation
 Most widely used for pH measurement
 It gives rapid response
Saturated Calomel Electrode
Advantages:
  Useful in measurement at high pH range
 Can be employed in various solvents
 Stability of potential
 Ease of construction
36. Name the factors which affect potential of a solution.
 pH of solutions
 temperature
 pressure of gases
 concentration of ions
 ligands presence
37. What are auto titrimeters? What is the principle of operation?
It is an automatic apparatus designed to carryout karl fisher titrations for accurate determination of
moisture content in various samples.
The instrument comprises of 2 units
i. control unit : it controls the complete automatic titration
ii. titration unit : it contains the magnetic stiring mechanism and the solenoid valve assembly.
The cabinet is designed to serve as a titration stand.
Principle
If moisture is present,
I2 + SO2 2 HI + SO3
SO3 + C5H5N C5H5NSO3
Methanol + C5H5NSO3 C5H5NCH3SO3H
Pyridinium methyl sulphate
( straw yellow color)

38. What is dead stop end point techniques? How this techniques is applied in the determination of water
This method is called biamperometry
It is applicable when a redox system is present before and after the end point.
For the determination of water using karl fischer reagent, a small potential is applied between two similar
platinum electrodes. Initially when water is present both electrodes are depolarised . the addition of karl
fischer A & B (solution of iodine and sulphur dioxide in pyridine and methanol )is continued till the end
point, where the diffusion current decreases . At the end point only one electrode is depolarised and the
diffusion current is almost zero or nill
id
end point

vol. of karl fischer reagent

39. What is null point potentiometry

(G)

reference Indicator electrode

unknown solution
in null point potentiometry, the reference electrode and indicator electrode is connected to a galvanometer.
On dipping the electrodes, galvanometer shows deflection
End point is when there is no deflection.
Unknown potential = Ɛ ˚

40. Explain the significance of dead-stop end point potentiometry.

*refer Q 38
41. What is Coductometry, Resistance,
Conductometry is the measurement of conductivity of solution due to the mobility of cations and anions
towards respective electrodes
Resistance:
Conductometry is inversely proportional to the resistance of a solution
C = 1/ R
R=V/I
R also depends upon the length , surface area of the conductor
R=ρl
 a or
ρ = a R/I

42. What is relationship between resistance & conductivity

* refer Q 41 for definitions
C=1 C is inversely proportional to R
R
43. What is relationship between conductivity & Specific conductivity
Specific conductivity ( Kᵥ ) = 1 / ρ
It is the conductivity offered by a substance of 1 cm length and 1 square cm surface area
Unit = mhos /cm
Conductivity ( C ) = 1 / R
We know R = ρ l
a
1 = 1 l
ρ R A
or
K=C l
A
44. Define Specific Conductance & Equivalent conductance
*for specific conductance refer Q43
Equivalent conductance is the conductivity of a solution containing equivalent weight of the solution
between electrodes 1 cm apart and 1 square cm surface area
Unit = mhos/cm
45. What is the principle in Amperometric Titrations
The principle is that the potential applied between polarisable and non polarisable electrode is kept
constant and the diffusion current is measured during the titration
During titration, the concentration of electroreducible ion changes and hence the diffusion current also
changes
At the end point , sharp change in diffusion current is seen.
46. How is the potential selected in Amperometric titrations
The potential selected for the titration is at its limiting value i.e, potential corresponds to the point where
the limiting current is reached
Condition:
The potential applied should correspond to the limiting current
47. What are the advantages of Amperometric titrations over potentiometry/ conductometry?
Advantages
 electro reducible and non electro reducible ions can be analysed
 dilute solution can be analysed
 sensitivity of titration increases
 apparatus is simple and polarography is sufficient
 impurities do not affect result
 reactions can be reversible or irreversible
48. What is the principle in polorographic analysis ?
Gradually increasing negative potential is applied between polarisable and non-polarisable electrode and
the corresponding current is recorded
Sigmoid shaped graph is observed
Instrument -> polarography
Graph -> polarogram
49. What is E1/2 (Half Wave potential)
The point of inflection in the I-V curve is called half wave potential
It is the characteristic for every element or functional group (qualitative aspect)

Ɛ1/2
Voltage
50. What is diffusion current, residual current, migration current ,polarographic maxima.
 Diffusion current is due to the active diffusion of electroreducible ion from the bulk of the sample
to the surface of the mercury droplet due to concentration gradient.
Current carried by such ions under such condition is called as diffusion current.
 Residual current is the sum of charging current and faradaic current
Charging current occurs due to formation of hemholtz double layer and faradiac current is due to the
presence of traces of impurities
 Migration current occurs due to migration of cations from bulk of the solution towards cathode due
to diffusive force, irrespective of concentration gradient
  Polarographic maxima is seen on absence of maxima suppressors
It leads to errors in determining half wave potential and diffusion current
Remedy: use suppresors like gelatin, dyes, surfactants etc
51. Why is DME used? What are the advantages ?
Doping mercury electrode( DME) is the polarisable electrode and provides a gradually increasing negative
potential.
Each drop has a smooth and uncontaminated surface free from impurities
Advantages:
 Surface area is reproducible
 Constant renewal of electrode surface eliminate poisoning effect
 It has a range of +0.4 to -1.8 V
52. What changes in the molecules occur when the following is passed a) UV/Visible radiation b) I.R
radiation
a) UV/Visible radiation- electronic transitions
b) I.R radiation – vibrational transitions

53. Principle involved in a grating & prism monochromators

Principle:
Polychromatic light monochromatic light
Prism:
It disperses the light radiation into individual colors
It is of 2 types:
 Refractive type
 Littrow type
Grating :
It is of 2 types:
 Diffraction grating
Diffraction grating produces reinforcement. The rays are incident upon the grating gets reinforced by
reflection
 Transmission grating
Transmission is similar to diffraction but refraction produces environment
54. How are the primary & secondary filters selected in fluorimetry assay?
Primary filter – absorbs visible radiation and allows uv radiation to pass
Secondary filter – absorbs uv radiation and transmits visible radiation
55. How do you detect the aromaticity of an unknown sample by means of its UV absorption spectrum.

substance Number of rings λmax

benzene 1 261 nm
napthalene 2 312 nm
anthracene 3 375 nm
napthacene 4 480 nm (yellow)
pentacene 5 580 nm (blue)

56. Explain why the intensity of π-π * transition is more than that of n- π * transitions.
Ϭ*
ᴧ*
R band
B n
E
K ᴧ
band ϭ

energy required for ᴧ to ᴧ* trabsition is greater than n to ᴧ* so intensity is more than n to ᴧ*

57. Define transmittance & absorbance in spectrometry

Transmittance is the fraction of incident light which is transmitted i.e, passes through a sample
(OR) amount of monochromatic light absorbed by a sample is determined by comparing the intensities of
incident light and transmitted light
Transmittance = I / I˳
Absorbance is a measure of the capacity of a substance to absorb light of a specified wavelength
It is equal to logarithm reciprocal of transmittence
58. Reasons for Deviation of Beer’s law
i. True deviation
 Only dilute solution [10-50 microgram/ml] follow beer’s law closely
 Increasing concentration of absorbing species leads to collosion and each molecule can’t absorb.
 In increased concentration, index of refraction (n) is changed
ii. instrumental deviation
 stray radiation reaching detector
 changes in sensitivity of detector
  fluctuation of radiation source
 presence of polychromatic light
 wide slit width
iii. chemical deviation
 association
 dissociation
 incomplete reactions
59. Principle involved in Grating & Prism monochromators
*refer Q 53
60. Write various ranges of electromagnetic spectrum
Radiation Wavelength region
Gamma rays 0.2 to 1 A˚
X rays 1 to 10 A˚
UV radiation 200nm to 400 nm
Visible radiation 400nm to 800 nm
Violet 370nm
Indigo 430 nm
Blue 450 nm
Green 490 nm
Yellow 550 nm
Orange 590 nm
Red 650 to 750nm
Infra Red 2.5 to 25 micrometer
Microwaves 0.1 to 100 cm
Radio waves 1 to 1000 cm
61. Define Red & Blue Shift with example
Red shift (bathochromic shift )
Shift to λmax towards longer wavelength
It occurs due to the presence of auxochrome
Eg: cosmological red shift
Blue shift (hypsochromic shift)
Shift of λmax towards shorter wavelength
It occurs due to dealkylation or desaturation

Red shift

λ
62. What are stepwise & Gradient elutions?
Stepwise elution is an elution technique in which the composition of mobile phase is changed in steps
during a single chromatographic run
Gradient elution is the technique in which solvents of gradually increasing polarity or increasing elution
strength are added gradually
63. Importance of Finger prints region in IR Spectroscopy
Range 1500 – 600/cm
It contains a very complicated series of absorption mainly due to all manner of bending bending vibrations
within the molecule

Fingerprint region
Absorption in this region is characteristic of the molecule as a whole
It is used for identification pourpose

64. Define filters and monochromators

Filters absorb unwanted radiations and transmit the rest of radiation which is required for colorimetry
It is of 2 types:
 Absorption filters
 Interference filters
Monochromators are optical devices that transmits a selectable narrow band of wavelength of lights chosen
from wide range of wavelength.
Monochromators are of 2 types:
i. Prisms
 Refraction
  littrow
ii. gratings
 transmission
 diffusion
65. What is natural frequency of vibration & mention different types of vibrations
Free vibrations of elastic body are called natural vibrations ans the frequency at which it occurs is equal to
natural frequency of vibrations.
Each substance has its particular frequency
Types of vibrations

Stretching Bending
(alteration in (change in bond angle)
body length)
In plane out of plane

symmetric asymmetric rocking scissoring wagging twisting

66. What is the effect of conjugation & cross conjugation on λ max

Increased conjugation longer the compound and increased λmax
If it has enough λ max (>400nm ) it becomes coloured
Cross conjugation has no effect on λmax

Progesterone λ max = 241 nm

Prednisolone λ max = 241nm
67. What is Stoke’s & Anti-stoke’s fluorescence
It is based on wavelength of emitted radiation compared to absorbed radiation
Stoke’s law:
Wavelength of emitted radiation is longer than absorbed radiation
Anti-stokes fluroscence :
Wavelength of emitted radiation is shorter than absorbed radiation
68. A solution of P-nitro phenol in water is yellowish but its solution in dilute NaoH Is intense yellow.
Explain why the colors deepens in the latter case.
.
 NO2

NO2

+ NaOH + H2O

OH ONa+ ( yellow colour)

p- nitrophenol is a ph indicator whoch turns bright yellow in alkaline pH while on neutal pH remains
colorless/ yellowish
70. What is the source for UV & Visible radiations ? How is monochromaticity obtained in both case
 visible radiation
 tungsten filament lamp
 tungsten halogen lamp
 carbon arc lamp
 uv radiation
 deuterium discharge lamp
 Hydrogen lamp
 Xenon lamp
Monochromatic light is obtained by
 Filters
 monochromators
71. Which is the common detectors in UV absorption spectrometry & outline its functioning
i) photo multiplier tube
 principle employed in this detector is that, multiplication of photoelectrons by secondary emission
of electrons.
 In a vacuum tube, a primary photo-cathode is fixed which receives radiation from the sample.
 Some eight to ten dynodes are fixed each with increasing potential of 75-100V higher than
preceding one.
 Near the last dynode is fixed an anode or electron collector electrode.
 Photo-multiplier is extremely sensitive to light and is best suited where weaker or low radiation is
received
 ii) photo voltaic cell

 The detector has a thin film metallic layer coated with silver or gold and acts as an collector
electrode.
 It also has a metal base plate which acts as another electrode.
 These two layers are separated by a semiconductor layer of selenium.

iii photo tubes

 Consists of a evacuated glass tube with a photocathode and a collector anode
(metal).
 The surface of photocathode is coated with a layer of elements like cesium,
potassium or silver or oxides of these metals (to increase sensitivity).
 When radiant energy falls on photosensitive cathode, electrons are emitted which
are attracted to anode causing current to flow.
 More sensitive compared to barrier layer cell and therefore widely used.
 The signal from the detector can be amplified using an amplifier circuit.

72. What are the three types of fundamental motions of a molecules?

i. Vibrational
ii. Rotational
iii. translational
73. What are the methods of solvent degassing
 vacuum filtration
 removes air bubbles
 not reliable
 Ultrasonification
 Helium purging
 Pass helium to solvent
 Expensive
 effective
74. Define & explain “Quality Assurance”.?
It is a way of preventing mistakes or defects in manufactured products and avoiding problems when
delivering solution or services to customers.
ISO 9000 is defined as part of quality management focused on providing confidence that quality
requirements will be fulfilled
5 mark questions

1. Explain the term a) HETP b ) Retention time c) Theoretical plate d) Retention volume
a ) HETP is an acronym for Height Equivalent to the Theoretical Plate. It arises from the plate
theory and is numerically equal to the column length divided by the number of theoretical plates in
the column.
A theoretical plates can be of any height, which decided the efficiency of separation. If HETP is
less, the column is more efficient. HETP can be calculated by using the following formula
HETP = Length of the column
Number of theoretical plates
HETP is given by the Van deemeter equation
HETP = A + B + Cu
U
Where A = Eddy effusion term or multiple path diffusion which arises due to packing of the
column.
B = longitudinal diffusion term
C = effect of mass transfer which depends on flow rate
u = flow rate or velocity of mobile phase
b ) retention time
retention time is difference in time between the point of injection and appearance of peak maxima.
Retention time is the time required for 50% of a component to be eluted from a column. Retention
time is measured in minutes or seconds. Retention time is also proportional to the distance moved on
a chart paper which can be measured in cm or mm.
c) Theoretical Plate
A theoretical plate is an imaginary or hypothetical unit of a column where the distribution of
solute between stationery phase and mobile phase has attained equilibrium. A theoretical plate
can also be called as functional unit of the column
d) Retention volume
Retention volume is the volume of carrier gas required to elute 50 % of the component from the
column. It is the product of retention time and flow rate
Retention volume, Vr = retention time x flow rate
2. Define HPLC & write a note on detectors present in HPLC
HPLC is characterized by the use of high pressure to push a mobile phase solution through a
column of stationary phase allowing separation of complex mixtures with high resolution
Advantages
 Sensitivity
 Ready adaptability to accurate quantitative determination
  Suitability for separating nonvolatile species or thermally fragile ones
 Higher resolution and speed of analysis
 HPLC columns can be reused without repacking or regeneration
 Greater reproducibility due to close control of the parameters affecting the efficiency of separation
 Easy automation of instrument operation and data analysis
 Adaptability to large-scale, preparative procedures
Types:
Liquid-liquid: partition between two bulk phases (one immobilized)
Liquid-solid: adsorption on solid which is generally polar (silica gel, alumina, magnesium
silicates) or reverse phase (cellulose, poly amides)
Detectors
Detectors used depend upon the property of the compounds to be separated different detectors
available are
a) UV detector: this detector is based upon the light absorption characteristics of the sample. 2
types of detector available are
 Fixed wavelength detector: works at 254 nm
 Variable wavelength detector: 190 nm to 600 nm
b) Refractive index detector: this is a nonspecific or universal detector. not much used for
analytical application because of low sensitivity and specificity
c) Flourimetric detector: it is based on fluorescent radiation emitted from some class of
compounds. The excitation wavelength and emission wavelength csn be selected for each
compound. It has more specificity and sensitivity.
d) Conductivity detector: based upon electrical conductivity, the response is recorded. This
detector is used when the sample has conducting ions like anions and cations.
e) Amperometric detector: this detector is based on the reduction or oxidation of compounds
whena potential is applied. The diffusion current recorded is proportional to the concentration
of the compound eluted. It is applicable when compounds have functional groups which csn
either be oxidised or reduced.
f) Photodiode array detector : it id similar to uv detector which operates from 190-600nm.
Radiations of all wavelength fall on the detector simultaneously. The resulting spectra is a 3D
plot of response vs time vs wavelength.

3. Write a note on Guard Column and its significance

Guard column is set between the injector and an analytical column.
It is used to protect analytical columns from chemical impurities in samples.
It is divided into 2 types:
 Cartridge type: it csn be changed easily by hand
  Packed type: it is packed in stainless cartridge like analytical columns.
In order to obtain maximum protection, it is recommended to use a guard column that
contains the same packing material as the analytical column.
Significance:
 It serves to remove the impurities and suspended solids from reaching the
analytical column.
 It has very small quantity of adsorbent and improves the life of the analytical
column.
 It act as a pre filter to remove particulate matter, if any and other materials.
 It has the same material as that of the analytical column.
 Guard column doesnot contribute to any seperation.
4. What are the techniques of separation in HPLC based on a) Principle of separation b) Elution
Techniques c) Types of analysis d) Scale of operation
A) Based on principle of seperation:
i) adsorption chromatography
ii) ion exchange chromatography
iii) size exclusion or gel permeation chromatography
iv) affinity chromatography
v) chiral phase chromatography
B) Based on elution techniques
i) isocratic seperation: in this separation technique, same mobile phase is used throughout
the process of seperation. The same polarity or elution strength is maintained throughout
the process
ii) gradient technique: in this technique a mobile phase combination of lower polarity or
elusion strength is used followed by gradually increasing the polarity or elution strength.
C) based on types of analysis:
i) qualitative analysis: it is used to identify the compound , detect the presence of impurities, to
find out the number of components etc
it is done by using retention time values.
ii ) quantitative analysis: it is done to determine the quantity of the individual or several
components in a mixture. This is done by comparing the peak area of the standard and sample

D) based on scale of operation:

i) Analytical HPLC: where only analysis of the samples are done.
Recovery of the samples for reusing is normally not done,since thr sample us eis very low.
Eg: microgram quantities
 ii )Preparative HPLC: where the individual fractions of pure compounds can be collected using
fraction collector. The collected samples are reused.
Eg: seperation of few grams of mixtures by HPLC

5. What is the principle in a) Normal-Phase Chromatography b) Reverse -Phase Chromatography c)

Ion- Exchange Chromatography d) Ion-pair Chromatography.
a) Normal Phase Chromatography: the principle is adsorption
Stationery phase is polar Eg: silica gel
Mobile phase is non polar
When a mixture of components are introduced into HPLC column, they travel according to
their relative affinities towards the stationery phase.
The component which has more affinity towards the adsorbent, travels slower.
The component which has less affinity towards the stationery phase travels faster. Since no 2
components have the same affinity towards the stationery phase, the components are separated.
b) reverse phase chromatography:
stationery phase is non polar Eg: octadecyl silane
mobile phase is polar
*principle same as normal phase
c) ion exchange chromatography:
the principle of seperation is by reversible exchange of ions between the ions present in the
solution and those present in ion exchange resin.
 Cation Exchange: The cations to be separated are present in solution and exchanges for
similar ions present in cation exchange resin, a solid matrix.
The cations retained by the solid matrix of ion exchange resin can be eluted by using
buffers of different strength and hence separation of cations can be affected
 Anion Exchange: similarly the seperation of anions using anion exchange resin can be
carried out. The anions to be separated are present in anion exchange resin, a solid matrix.
The anions are retained by the solid matrix of ion exchange resin and can be eluted by
using buffers of diiferent strength and hence seperation of anions can be effected.
d ) Ion-pair Chromatography
It is a type of column chromatography in which ions in solution can be paired or neutralised
and separated as an ion pair or on a reverse phase column. Ion pairing agents are usually ionic
compounds that contain a hydrocarbon chain.
By adding pair ions with the opposite charge to the mobile phase, the pair ions form ion pairs
with the ionic compounds. Which neutrilizes the charge, increases hydrophilicity and increases
retention.
 If pair ions with a hydrophobic functional group are added to the mobile phase, the
hydrophobic functional groups are retained by the stationery phase as though a dynamic ion
exchange groups had formed on the surface of the stationery

6. Define chromatography? What are the principles of separations in chromatography

It is a physical process of separation in which components to be separated are distributed in
between two phases, a stationary phase which has large surface area and a mobile phase which is in
constant motion through the stationary phase
Classification Based on the principles of Separation :
 Adsorption Chromatography : Eg Gas solid chromatography, Thin layer chromatography,
Column chromatography.
 Partition Chromatography : Eg Gas liquid chromatography, Paper chromatography.
 Size exclusion chromatography : Eg Gel permeation chromatography
 Ion exchange chromatography :
ADSORPTION CHROMATOGRAPHY
When a mixture of compounds (adsorbate) dissolved in the mobile phase (eluent) moves through a
column of stationery phase (adsorbent), they travel according to the relative affinities towards
stationery phase.
The compound which has more affinity towards stationery phase travels slower and the compound
which has lesser affinity towards stationery phase travels faster. Hence compounds are separated.
No 2 compounds have the same affinity for a combination of stationery phase, mobile phase and
other conditions.
PARTITION CHROMATOGRAPHY
In partition chromatography, the components are separated according to their partition coefficients.
Partition coefficient is the ratio of solubility of a substance distributed between 2 immiscible
liquids at a constant temperature.
The component which is more soluble in stationery phase travels slower and eluted later.
The component which is less soluble in stationery phase, travels faster and eluted out first. No 2
components have the same partition coefficient for a fixed combination of stationery phase, mobile
phase and other conditions.

SIZE EXCLUSION CHROMATOGRAPHY

A gel is used to separate the components of a mixture according to their molecular sizes. Different
gels are used for different molecular weight ranges. The solvent used can be of aqueous or non-
aqueous type. The stationery phase is porous matrix.

ION EXCHANGE CHROMATOGRAPHY

In this type an ion exchange resin is used. Reversible exchange of ions takes place between similar
charged ions and that of ion exchange resin. A cation exchange resin is used for the separation of
cations and an anion exchange resin used for the separation of cations and an anion exchange resin
is used to separate a mixture of anions.
7. Explain the different packing techniques in column chromatography which packing techniques is best
and why?
There are 2 types of preparation of the column, which are called as packing techniques. They are
i. Dry packing:
In this technique the required quantity of adsorbent is packed in the column in dry form
and the solvent allowed to flow through the column till equilibrium is reached. The
demerit with this technique is that air bubbles are entrapped between the solvent and
the stationery phase and the column may not be uniformly packed. Cracks appear in the
adsorbent present in the column. Hence the uniformity in flow characteristics and clear
band of the separated component may not be obtained.
ii. Wet packing technique:
This is the ideal technique.
The required quantity of the adsorbent is mixed with the soluble phase solvent in a
beaker and poured into column. The stationery phase settles uniformly in columnand
there is no entrapment of air buubles. There will not be any crack in the column of
adsorbent. The bands eluted from the column will be uniform and ideal for seperation
8 Classify adsorbents used in column chromatography with example.
An adsorbent used in column chromatography should meet the following criteria:
a) particle size and geometry:
the particles should have uniform size distribution and have spherical shape
particle size: 60-200 micron
b) should have high mechanical stability
c) should be inert and should not react with solute or other components
d) insoluble in solvents or mobile phase used
e) it should be colourless to facilitate absorption of zones and recovery of components
f) it should allow free flow of mobile phase
g) it should be useful for separating wide variety of compounds
h) it should be freely available, inexpensive
Types of adsorbent:
Based upon their adsorbent activity, it can be classified as weak, medium and strong adsorbents.
They are:
weak medium strong
sucrose Calcium carbonate Silica gel
starch Calcium phosphate Activated alumina
inulin Magnesium carbonte Activated charcoal
talc Magnesium oxide Activated magnesia
 Sodium carbonate Calcium hydroxide Fuller’s earth

The most commonly used is silica gel

9. Define partition chromatography and write a note on factors affecting column efficiency
In partition chromatography, the components are separated according to their partition
coefficients. Partition coefficient is the ratio of solubility of a substance distributed between 2
immiscible liquids at a constant temperature.
The component which is more soluble in stationery phase travels slower and eluted later.
The component which is less soluble in stationery phase, travels faster and eluted out first. No
2 components have the same partition coefficient for a fixed combination of stationery phase,
mobile phase and other conditions.
Factors affecting column efficiency:
i) dimensions of the column:
a length: diameter ratio of 20:1 or 30:1 are ideal. But for improving the efficiency , 100:1
may be more satisfactory.
ii) particle size of the adsorbent:
adsorbent activity depends on the surface area of the adsorbent. For increasing the surface
area, particle size can be reduced and hence the adsorbent activity increases.
iii) Nature of solvent:
The flow rate of solvent is affected by its viscosity. The flow rate is inversely
proportional to viscosity. Hence less viscous solvents are better efficient than more
viscous solvents.
iv) Temperature of the column:
Speed of elution is increased at higher temperature. But adsorbent power is decreased at
higher temperature. Hence a compromise is made between speed and elution power.
Normal room temperature is used for all samples.
Difficult samples are separated at higher temperature.
v) High pressure above the column and low pressure below the column increases the
efficiency of seperation. High pressure above the column is achieved by maintaining
reservoir on the top of the column or by using pressure devices.
 Pressure below the column is decreased by applying vaccum.

10. Write a note on Development Techniques in column chromatography

After the introduction of the sample, by elution techniques the individual components are
separated out from the column.
The techniques are:
i) isocratic elution technique:
in this elution technique, the same solvent composition or solvent of same polarity is used
throughout the process of seperation.
Eg: chloroform only , petroleum ether: benzene = 1:1 only
ii) gradient elution technique:
in this elution technique, solvents of gradually increasing polarity or increasing
elution strength are used during the process of separation. Initially low polar
solvents are used followed by gradually increasing the polarity to a more polar
solvent.
Eg: initially benzene ,then chloroform, then ethyl acetate then to methanol etc.
 Other techniques like frontal analysis and displacement analysis where a graph of
concentration of eluate vs volume of eluate will give an idea of how compounds are eluted
from the column.
11. Define Partition chromatography & write a note on Bonded Phase Chromatography
*refer Q9(5 marks) for definition
Bonded Phase Chromatography
A stationary phase chemically bonded to a support that is used for the separation. It is the most
commonly used LC mode. The most popular support used is micro particulate silica gel. An
organosilane, such as octadecyl (for reversed-phase chromatography), is the most accepted
type of bonded phase. Approximately 70 percent of all HPLC is carried out on chemically
bonded phases
In liquid chromatography, liquid-liquid systems are unstable as, however small the solubility of
the stationary phase may be in the mobile phase, the stationary liquid phase will be eventually
stripped from the column. It was therefore found necessary to chemically attach the stationary
phase to the support to ensure a stable system and these materials were called bonded phases.
Different Classes of Bonded Phase
There are basically three classes of bonded phase
1.Brush phase
2.Oligomeric phase
3.Bulk phase
Mono-substituted silanes single layers of organic moieties could be bonded to the silica surface and these
materials were called brush phases


alternately treating silica with di-substituted silanes and water in a heated fluidized-bed system,
oligomeric bonded phases
Employing tri-substituted silanes in the presence of water the organic moieties could be cross linked with
ether groups and form a type of a polymer. These polymeric phases were strongly held to the silica matrix
and were very stable and were given the name bulk phases
Charactersitics of Bonded Phases:
To render the silica dispersive in character, the interactive surface must be chemically
modified. For example, appropriate hydrocarbon moieties could be chemically linked to the
surface hydroxyl groups.
The silicon-oxygen-carbon linkage is, very weak and regenerating the original hydroxyl groups
of the silica gel. An alternative bonding method that involved the use of chlorsilane reagents.
When a chlorsilane reacts with a hydroxyl group of the silica gel surface, the hydrocarbon
chain is attached by the much stronger and stable silicon-carbon link.

12. Write a note on Frontal Analysis & Bonded Phase Chromatography

*refer Q11(5 marks) for bonded phase chromatography
Frontal analysis
Frontal analysis is the process in which the sample is fed continuously onto the column as a
dilute solution in the mobile phase.
Frontal analysis can only separate part of the first component in a relatively pure state, each
subsequent component being mixed with those previously eluted.
For a 3 component mixture, containing solutes (A), (B) and (C) as a diute solution is fed
continuously into the column.
The first component to elute,(A) will have less affinity to the stationery phase.
Then the second solute (B), will elute but it will be mixed with the first solute. Finally the third
solute(C), will elute in conjunction with (A) and (B).
 It is clear that only solute (A) is eluted in a pure form. Thus, frontal analysis is not suitable for
most practical analytical applications.

13. Describe the preparation activation of plates & adsorbents used in TLC
*refer Q 8 for adsorbents (5marks)
*refer Q 13 and Q14 (2 marks) for preparation and activation
14. Define paper chromatography? What are the modes of development in paper chromatography

The mode of separation of paper chromatography is the partition and the basis for the separation is
solubility. As in other chromatographic techniques this also has a stationary phase as well as a mobile
phase. Both phases are liquids. Stationary phase is water that is tightly bound to the paper. Filter papers are
used for this purpose and most common filter paper used is Whatman filter paper- 98-99% Alpha cellulose.
The cellulose paper can well absorb water molecules. Fiber of cellulose acts as the stationary phase.
Mobile phase is a solvent- solvent partially miscible in water. Paper chromatography is usually used for
separating amino acids and anions and also testing histamines and antibiotics.

Procedure of Paper Chromatography

The spot of the test sample is loaded on the filter paper using a capillary tube. This spot should always be a
concentrated but a very minute one. Capillary tubes are used in paper chromatography, because a small
quantity can be taken into the tube without any force. The upper line (solvent front) is drawn on the paper
from 2 cm on the top and the bottom line (base line) is drawn from 2 cm from the bottom of the paper.
Usually 0.01g of the sample is dissolved in the running solvent (1g). Micro liter quantities are used to spot
on the paper by a capillary tube. The diameter of the spot should be only up to few mili meters. The
spotting should be done several times in order to get a concentrated spot. This filter paper is then placed
inside the chamber saturated with the solvent to develop the chromatogram. The chromatogram is then
heated in an oven to high temperatures. This can be done not only by an oven but also using a fan, air etc.

Paper Chromatography Separation Mechanisms

The mobile phase rises up by the capillary action. The testing sample is concentrated as a minute spot at
the bottom of the filter paper. When the mobile phase which is a liquid, rises up in the filter paper the
spotted mixture is gradually rises with the mobile phase. This eventually leads to the separation of the
compounds. Compounds in the mixture will be separated according to their ability of the solubility. In
other words it is again the polarity as in open column chromatography. More polar substances will move
slower and less polar substances will travel faster. Consider “A” substance is more soluble than “B” in the
stationary phase, thus “A” will dissolve in that solvent. This means “A” is more polar than “B” with
respect to the stationary phase solvent. Thus “A” will travel slowly than “B”. And “B” will elute first. This
explains that substances that having more solubility in stationary phase move slower and substances
having less solubility in stationary phase move faster.   

Types of development techniques:

1. Paper Chromatography Ascending Method

In ascending technique the chromatogram is attached in a way that the spot is touched with the solvent
where the solvent is at the bottom. The development of the chromatogram or the separation of the spot is
against the gravity. This is why this is termed as ascending technique. There is a paper support on the top
of this tank. The mobile phase (solvent) is at the bottom of the tank. The filter paper is attached to the tank
by the paper support and filter paper will touch the solvent. But the spot should not touch the solvent. The
mobile phase will gradually rise up wards and carry the spot substances. The most polar substance will be
at the bottom with respect to the tank where as the least polar will be on the top end of the tank. Ascending
technique is relatively a slow process.
2. Paper Chromatography Descending Method
The descending technique is a complex setup. This is built due to its time consuming ability. This develops
along the gravity. Thus there is a force which will make the separation quick and easy. The filter paper is
attached to a paper support. And the developing solvent (mobile phase) is filled into a chamber. The filter
paper is saturated with the stationary phase before it is hung. 

The mobile phase will gradually move downwards carrying the spot of the test sample along the paper.
The term descending is given because the separation or the development of the chromatogram is taking
place towards down. In this technique most polar substance will be on the top with respect to the tank
where as the least polar ones will be at the bottom.

3. Ascending – Descending Chromatography

Ascending – Descending Chromatography

The solvent first travels upwards on the paper which is folded over a rod and it continues with its travel
downwards after crossing the rod. This arrangement permits longer development period for better
resolution of complex mixtures
4. Radial or Circular Paper Chromatography

Radial or Circular Paper Chromatography permits separation of sample components in the form of
concentric circular zones through radial movement of liquid phase. A circular shaped filter paper is
employed.
 Arrangement – 1 : Circular Paper Chromatography
A narrow slit about 2mm wide is cut from the circumference to the centre and the wick formed is bent so
as to dip in the solvent contained in a petridish. Sample spotting is done along the circumference of the
smaller inner circle. The petri dish is covered after securing the filter paper to the top and allowed to
develop. The mobile phase reaches the filter paper through capillary action and spreads radially outwards
thereby resolving the sample components radially.

5.Two Dimensional Techniques in Paper Chromatography

Two dimensional technique is another complex set up which is used to separate complex mixtures. In this
method the development of the chromatogram is done as in the previous methods.

 Solvent is placed at the bottom of the tank and the filter paper saturated with the stationary phase is
then kept inside the tank. The development occurs up wards. But very slowly because it is against
the gravity as well as the compound is a complex one.

 After few hours the filter paper is turned 90 0 clockwise and the tank is filled with a different type
of solvent. If there is no pronounced separation then development is proceeded to the “c” stage.

Again the filter paper is turn 90 0 clockwise and used another solvent. This will probably end up with a
satisfactory separation. If not again it should be turned
15. Explain two dimensional & Reverse-phase Chromatography
*refer Q14 (5marks) for two dimensional technique
Reverse phase
*refer Q5 for reverse phase
16. Explain various development techniques used in paper chromatography
*refer Q14
17. Enumerate the application of paper chromatography
a)alkaloid b) Cardiac glycoside c) Aldehydes or ketones d) proteins.
a) alkaloid

ascending paper chromatography can be used for isolation and identification of various alkaloids like
atropine, morphine, heroin etc.
mobile phase: n butanol saturated with an aqueous solution of acetic acid
Stationery phase: whatman filter paper no; 4
Spray reagent; potassium iodoplatinate
b) Cardiac glycoside
Descending partition chromatography
Mobile phase: chloroform and benzene
Stationery phase: whatman filter paper no;1
Spray reagent: tollen’s reagent
c) Aldehydes or ketones:
Circular chromatography is used for separation of 2,4 dinitrophenyl hydrazones of aldehydes
and ketones
Mobile phase: mixture of isooctane and a light mineral oil
Stationery phase: n,n dimethyl formamide impregnated paper
Visible without spraying
d) proteins:
sample is spotted on a paper, eluted followed by visualisation using ninhydrin which gives
purplr or brown colour

18. How will you perform quantitative analysis in paper chromatography

For qualitative analysis at first preliminary separation is done. Then, the assay can be
performed either after extraction from the paper or in situ on the paper.
I) Estimation after extraction from paper
i) isolation of separated components from paper chromatography
Procedure:
Cut the appropriate part of the filter paper having spot
 Soak it into minimum quantity of solvent

Semi-micro extractor˷Soxhlet apparatus

Eluent is obtained (Elution)

Determination:
The microanalysis of the eluate can be performed by adopting one or more of the
following techniques:
 Gravimetric estimation
 UV spectrophotometry
 Colorimetry
 Polaropgraphy
 Radioactivity
 Flame photometry
II) In situ methods
i) visual assessment:
-not very quantitative
ii) by measurement of areas:
if the outlines of the spots or zones are well defined, the size of the spot may serve for
determining the quantity of the substance.
Then a linear relationship is obtained between the spot area and amount of substance
present.
Disadvantages:
 Random error due to variation of spot shape during separation
 Volume of the applied sample and speed of application should be identical in all
cases
iii) By densitometer:
The intensity of colour of a substance is measured directly on the chromatogram
iv) Potentiometry:
Changes in potential of a metal electrode in contact with filter paper is also utilized
with quadrant electrometer or electronic voltmeters
v) Other methods:
 Conductimetry
 Absorptiometry
 polarography
 19. Compare the principle techniques limitations and application of paper chromatography
with electrophoresis.
Difference Paper chromatography electrophoresis
principle Separation of molecules Separation of charged
based on their partition molecules by attraction
coefficient and adsorption towards opposite charged
properties electrodes
technique Flow of solvents on specially Electrical field id applied to
designed filter paper separate sample molecules
based on their charge
Types  Paper adsorption  Moving boundary
chromatography electrophoresis
 Paper partition  Zone electrophoresis
chromatography
 Capillary
electrophoresis
 Immuno
electrophoresis
 Isoelectric focusing
limitation Expensive instrumentation Inexpensive instrumentation
and even expensive reahents but reagents can be
expensive
application Used for both analytical and Only analytical purposes i.e.
preparatory purposes identification and
quantification
Separation media Liquid mobile phase and Semi solid gel media is used
filter paper impregnated with
silica or alumina or moisture
present in the pores of
cellulose fibres in filter paper
act as stationery phase

20. How does the following factors affect separation efficiency a) cross-linking of Resin b) Ion-Exchange
Capacity
a) Cross linking of Resin
Cross linking and swelling is important factor which depends on the proportion of cross linking agent
divinyl benzene and polystyrene. When more cross linking agents are present, they are more rigid but
swelling is less. When swelling is less separation of ions of different sizes is difficult as they cannot pass
through the pores present and it becomes selective to ions of different sizes.
When less cross linking agent is present, they are less rigid but swell more. When swelling is more,
separation will not be efficient as exchange of functional groups doesnot takes place due to wide pore.
Hence an optimum quantity of crosslinking agent should be added to the polymeric ion exchange resins for
the separation to be effective.
Nature of exchanging ions:
 Valency of ions: at low concentrations and at ordinary temperatures, extent of exchange increases
with increase in valency.
 Size of ions: for similar charged ions, exchange increases with decrease in the size of hydrated
ion.
 Polarizability: exchange is preferred for greater polarizable ion.
 Concentration of solution: in dilute solutions, polyvalent anions are generally absorbed
preferentially
 Concentration and charge of ions: if resin has higher positive charge and solution has lower
positive charge, exchange is favoured at higher concentration. If the resin has lower positive
charge and solution has higher positive charge, the exchange is favoured at low concentration.
B) ion exchange capacity
*refer Q25 ( 2 marks)
21. Write a note on factors affecting the separation efficiency of Ion exchange resin
The factors affecting ion exchange seperations are:
A) nature and properties of ion exchange resin
B) nature of exchanging ions
*refer Q 20 ( 5 marks)

22. What is regeneration of a resin? How will you regenerate cation and anion exchange resin?
Regeneration refers to the replacement of the exchangeable cations or anions present in the original resin.
The ion exchange resin after separation may not be useful for next separation as exchangeable functional
groups are lost. But due to the cost of ion exchange resins, they cannot be disposed off. Hence like
reactivation, regeneration of the resin is important. Regeneration makes the used ion exchange resin to be
as efficient as virgin resin.
Regeneration of cation exchange resin:
Hence regeneration of the cation exchange resin is done by charging the column with strong acid like
hydrochloric acid.
Regeneration of anion exchange resin:
Regeneration of anion exchange resin is done by using strong alkali like sodium hydroxide or potassium
hydroxide.
23. Explain with a neat diagram any two detectors used in G.C.
i) Katharometer or thermal conductivity detector
The principle is based upon thermal conductivity difference between carrier gas and that of component.
Katharometer has 2 platinum wires of uniform dimensions which forms part of wheatstones bridge.
Through one of them pure carrier gas always flows through and through the other, the effluents of the
column passes. The 2 plantinum wires are heated electrically and hence assume equilibrium conditions of
temperature and electrical resistance. when pure carrier gas passes through both of them there is no
difference in temperature or resistance and hence a baseline is recorded. When a component emerges from
the column, it alters the thermal conductivity and resistance of the wire. Hence this produces a difference
in resistance and so conductivity between wires, which is amplified and recorded as signal.
The thermal conductivities of some carrier gases are given as follows:
hydrogen helium nitrogen methane hexane
32.7 33.9 5.2 6.5 3.0
From the tabular column, it can be seen that hydrogen and helium have higher thermal conductivity and
they are the best carrier gases for katharometer. Hydrogen is inflammable and helium is expensive . but
both of them offer good response. If any other carrier gas is used, they give rise to negative peaks beacause
of lower thermal conductivity.
Advantages:
 applicable to most compounds
 linearity is good
 the sample is not destroyed and hence used in preparative scale
 simple, easy to maintain and inexpensive.
ii) Electron Capture Detector
the electron capture detector has 2 electrodes, with the column effluent passing
between them. One of the electrode is treated with a radioactive isotope which emits
electrons as it decays. These emitted electrons produce secondary electrons which are
collected by the anode, when a potential of 20 volt is applied between them.
When carrier gas alone flows through, all the secondary electrons are collected by the
positively polarised electrode. Hence a steady baseline is recorded.
 Effluent molecules which have affinity for electrons, capture these electrons when they
pass through the electrodes. Hence the amount of steady state current is reduced. This
difference is amplified and recorded as output signal.
The carrier gas used in this type of detector depends upon the electron affinity of the
compounds analysed. For compounds with high electron affinity, argon is used as
carrier gas. For compounds with low electron affinity, nitrogen, hydrogen, helium or
carbon dioxide can be used as carrier gas.

Advantages:
 highly sensitive
 even Nano gram quantities can be detected
24. Explain the concept of pre-dervitization & post dervitization techniques in Gas –Chromatography with
relevant examples
Derivatisation is a technique of treatment of the sample to improve the process of separation by column or
detection by detector. They are of 2 types based upon its need
Pre column Derivatisation:
This is done to improve some properties of the sample for separation by column. By this derivatisation
technique, the components are converted to more volatile and thermostable derivatives. Moreover
improved separation and less tailing will be seen after such treatment.
In the following conditions pre-derivatisation is done:
 the component is less volatile
 the compounds are thermolabile (heat sensitive)
 to reduce tailing
 to improve separation factor
Eg: carboxylic acids, sugars , phenols, alcohols etc can be converted to less polar compounds by usinh
reagents like BSA reagent
They can also be converted to acetyl derivative or trifluro acetyl derivative
Post column derivatisation:
It is done to improve the response shown by detector. The components may not be detected by detector
unless derivatisation is done. The components may be converted in such a way that their ionization or
affinity towards electrons is increases . normally this is online detection technique where the flowrate is
neither stopped or altered.

25. write a note on paper electrophoresis

Electrophoresis is a method whereby charged molecules in a solution, chiefly proteins and nucleic acids,
migrate in response to an electric field.
PAPER ELECTROPHORESIS
One of the simplest process in electrophoresis involved spotting of a mixture of solute in middle of paper
moistening the paper with some electrolyte and placing it between 2 sheets of glass.
The ends of paper strip extending beyond glass plate are immersed in beaker of electrolyte.
A potential of 5V/cm of paper length is placed from a DC source.
It is allowed to continue for a period of several hours.
Advantages:
 it is economical
 easy to use
Disadvantages:
 Certain compounds such as proteins, hydrophilic molecules cannot be resolved due to adsorptive
and ionogenic properties of paper which results in tailing and distortion of component bands.
 Electro osmosis
There are 3 types of paper electrophoresis:
1. Horizontal
2. Vertical
3. Continuous
26. What is electrophoresis? Mention their types
 Electrophoresis is a method whereby charged molecules in a solution, chiefly proteins and nucleic acids,
migrate in response to an electric field.
The rate of migration through the electric field depends on:
 The strength of the field
 The net charge
 Size and shape of the molecules
 Ionic strength
 Viscosity and temperature of the medium in which the molecules are moving
As an analytical tool, electrophoresis is simple, rapid and highly sensitive.
It can be used analytically to study the properties of a single charged species or mixture of
molecules. It can also be used preparatively as a separating technique.

Types of electrophoresis:
A. Moving boundary electrophoresis
B. Zone electrophoresis
C. Isotachophoresis
D. Isoelectric focusing
E. Capillary electrophoresis
F. Immuno electrophoresis
A. Moving boundary electrophoresis:
It allows charged species to migrate in a free moving solution, without the supporting
medium
The main features of this method are:
 Formation of sharp boundaries
 Large electrode vessels containing reversible electrodes
 An optical system for following movement of boundaries
B. Zone electrophoresis:
It involves migration of charged particles. Which are supported on relatively inert and
homogenous solid or gel framework.
In this method the separated components are distributed into discrete zones on stabilizing
media.
It is classified based on supporting material used:
 Paper electrophoresis
 Cellulose acetate electrophoresis
  Thin layer electrophoresis
 Gel electrophoresis
C. Isotachophoresis
Isotachophoresis (ITP) is a technique in analytical chemistry used for selective separation and
concentration of ionic analytes. It is a form of electrophoresis: charged analytes are separated based on
ionic mobility, a factor which tells how fast an ion migrates through an electric field.
D. Isoelectric focusing:
It is mainly used for separation of electrolytes as proteins.
When electrophoresis is run in solution buffered at constant pH, proteins have net charge
will migrate towards opposite electrode.
E Capillary electrophoresis
Capillaries are typically of 50 micro meter inner diameter and 0.5 to 1 m in length
Due to electro osmotic flow, all sample components migrate towards the negative
electrode.
The capillary can also be filled with a gel, which eliminates the electro osmotic flow.
F immuno electrophoresis
Each antibody binds specifically to one feature (epitope) on one macromolecule (antigen). This allows the
use of antibodies for detection and quantitation of specific proteins in a complex mixture.
When electrical potential is applied to study of antigen-antibody reactions, it is called immuno
electrophoresis.
27. Describe the principles and application of electrophoresis
*for definition refer Q26 (5marks)
Principle:
Biological molecules exist in a solution as electrically charged particles at given pH.
Anionic (positively charged / basic) zwitterions
Cationic (negatively charged/ acidic) or amphoteric molecules
pH greatly influences the total charge of molecules
when electricity is applied to the medium containing biological molecules, depending on their net charge
and molecular size, they migrate differentially, thus different protiens/DNA can be separated.
The velocity (v) of charged molecule in an electric field:
V = Eq/F
Where F= Frictional coefficient, which depends upon the mass and shape of the molecule
E = electric field(V/cm)
Q = the net charge on molecule
V = velocity of the molecule
Isoelectric point:
There is a pH at which there is no net charge on a protein, this is the isoelectric point
Above the isoelectric point, a protein has a net negative charge and migrated towards the anode in an
electric field
Below its isoelectric point, the protein is positive and migrated towards the cathode.
Application:
 DNA sequencing
 Medical research
 Protein research/ purification
 Agricultural testing
 Separation of organic acid, alkaloid, carbohydrates, amino acids, phenols, insulin
 In food industry
 It is employed in biochemical and clinical fields i.e., in the study of protein
mixtures such as blood serum, haemoglobins and in the study of antigen-antibody
interactions.
 Electrophoresis is combination with autoradiograph is used to study the binding of
iron to serum proteins.
 Used for analysis of terpinoids, steroids and antibiotics.
 Used for diagnosis of various diseases of kidney, liver and CVS.
28. Write a note on HPTLC.
 High Performance Thin Layer Chromatography.
 It is a sophisticated & automated form of TLC.
 It is also known as planar chromatography or Flat-bed chromatography.
PRINCIPLE
 The principle of separation is adsorption
 One or more compounds are spotted on a thin layer of adsorbent coated on chromatographic plate
 The mobile phase solvent flows through because of capillary action
 The components move according to their affinities towards the adsorbent.
 The component with more affinity towards the stationary phase travel slower and lesser affinity
towards stationary phase travel faster.
 Thus the components are separated
 Separation tracks are scanned in densitometer with light beam in visible or uv region
Steps involved in HPTLC:
 Selection of chromatography layer
 Depends on nature of material to be separated.
 Commonly used
(silica gel, alumina)
Support for chromatographic layer

Materials Advantages Disadvantages

Glass Resistant to heat and chemicals Fragility

Easy to handle and offers Relatively High weight
superior flat surface for work
Polyester  More economical as produced even in It react if temperature
sheets  roll forms exceeds 120 °C as the
(0.2 mm Unbreakable, plates are dimensionally
thick) Less packing material unstable beyond this
Spots can be cut and eluted temperature
Aluminum  Increased temperature resistance Eluents containing high
sheets concentration of mineral
(0.1mm acids or ammonia can
thick) attack chemically on
aluminum

Adsorbents used in HPTLC:

S.NO. EXAMPLE APPLICATIONS

1 Silica gel F60 80% of analysis is done on this layer.
2 Aluminium oxides Basic substances ,alkaloids and steroids

3 Cellulose (microcrystalline ) Amino acids ,peptides ,sugars and other liable compoun
cannot be analyzed on the active layers of silica gel.
 4 Silica gel chemically modified -COOH ,Phenols,  Nucleotides Pharmaceutical preservat
-NH2 and -CN
Binders and other agents used
 Gypsum (G)
 Starch (S)
 Layer containing fluorescent indicator (F)
Plate size
 20X20 cm, 10X20 cm, 5X10 cm, 5X7.5 cm
 Good cut edges of sheets is Important to obtain constant R values.
f
precoated plates
 The plates with different support materials and adsorbent layers with different format and
thickness are used.
 Plates with adsorbent thickness of 100 – 250 µm are used.
Pre washing of precoated plates
 The main purpose of the pre-washing is to remove impurities which include water vapour and
other volatile substances from the atmosphere when plates get exposed in the lab environment.
 Silica gel 60 F is most widely used adsorbent.
 The major disadvantage of this adsorbent is that it contain iron as impurity.
 This iron is removed by using Methanol : water in the ratio of 9:1.
 This is the major advantage of this step of pre-washing.
 Prewashing methods include ascending running, dipping and continuous running
Solvents used for pre-washing
 Methanol
 Chloroform: methanol (1:1)
 Chloroform: Methanol: Ammonia (90:10:1)
 Methylene chloride: Methanol (1:1)
 Ammonia solution (1%)
 After washing, dry for sufficient time for removal washing liquids. Avoid hot/cold air dryer.
 Use desiccators for storing
Activation of plates
 Freshly opened box of  plates doesn’t need activation.
 Plates exposed to high humidity or kept in hand for long time require activation.
 Plates are placed in oven at 110 ° - 120 °c for 30 min prior to the sample application.
 Activation at higher temperature for longer period is avoided as it may lead to very active layers
and risk of the samples being decomposed.
Sample Preparation and application
Proper sample preparation is require for proper separation.
For normal chromatography: Solvent should be non-polar.
For reversed chromatography: Polar solvent is used for dissolving the sample
Sample and reference substances should be dissolved in the same solvent to ensure comparable
distribution at starting zones.
The selection of sample application technique and device to be used depends on
 Sample volume
 No. of samples to be applied
 Precision required
Some applicators used for spotting are
Capillary tubes, Micro bulb pipettes, Micro syringes, Automatic sample applicator.
Mobile phase
 Mobile phase should be of high graded.
 Mobile phase selection is depends upon the chemical property of analytes and adsorbent layer.
 Use of mobile phase containing more than three or four components should normally be avoided
as it is often difficult to get reproducible results for different components.
 Mobile phase optimization is necessary in HPTLC
 Various components of MP should be measured separately and then placed in mixing vessel.
This prevents contamination of solvents and also error arising from volumes expansion or
contraction on mixing.
Trough chambers are used in which smaller volumes of MP usually 10 -15 ml is required.
Different components of MP are mixed first in mixing vessel and then transferred to developing chambers
Chambers containing multi component MP are not generally used for re-use for any future development,
due to differential evaporation and adsorption by layer and also once the chamber is opened , solvents
evaporate disproportionally depending on their volatilities.
Development 
The different methods used for development are
Ascending, descending, two dimensional, horizontal, multiple overrun, gradient, radial, anti-radial,
multimodal, forced flow planar chromatography
Drying
Drying of chromatogram should be done in vacuum desiccators with protection from heat and light.
29. Write a note on instrumentation and application of HPTLC
*refer Q28 (5mark) for definition
Instrumentation
Post Chromatography Steps
 Detection & Visualization
 Densitometry measurements
 Photo documentation
Detection & Visualization
Detection is of two types:
Qualitative & Quantitative
Qualitative detection is carried by comparing Rf by observing spots under UV light-nondestructive
Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm(long wave
length)
Spots of non fluorescent compounds can be seen -fluorescent stationary phase is used -silica gel GF
Non UV absorbing compounds like ethambutol, dicylomine etc., is done by dipping the plates in 0.1%
iodine solution
Quantitative detection can be performed by
 Spectrophotometry
 Fluorimetry
 Colorimetry
 Densitometry
Densitometry measurements:
It involves resolving of a compound on thin layer plate, visualizing the spots and measuring the optical
density of each spot / band directly on the plate
The amount of material / compound in the unknown is measured by comparing them to a standard curve
from reference standard used under the same condition
Chromatographic zones remit a lower light intensity than the environment around it. Absorption spectra
can be directly determined on the plate by comparison with substance free area of sorbent layer.
Measurements are usually made by reflection from the plate using single beam, double beam or single-
beam dual wavelength operation of scanning instrument.
The scanner present converts the spot / band on the layer into chromatogram consisting peaks
Position of scanned peaks on recorder chart is related to Rf values of the spots on the layer and peak height
or area is related to the concentration of the substance on spot.
Signals which are measured represent the adsorption of transmitted or reflected light passes through the
spot compared to blank portion of sorbent layer.
Advantages  of densitometer / Scanner
 The purpose of scanner is to convert the spot /band on the layer in to densitogram consisting of
peaks similar in appearance to HPLC.
 The position of the scanned peaks on recorder chart is related to R f values.
 Peak height/area is related to the concentration of the substance in the spot.
 Quantitation is faster, reliable, accurate and reproducible.
 Photo-documentation can be done by a digital camera
Applications:
  Pharmaceutical industry
 Quality control, content uniformity, uniformity test, identity/purity check.
 Food Analysis
 Quality control , additives ,pesticides, stability testing
 Clinical Applications
 Metabolism studies, drug screening, stability testing, therapeutic drug monitoring to determine its
concentration and metabolites in blood urine etc.
 Quantitative determination of prostaglandins and thromboxanes in plasma. Etc
 Industrial Applications
 Process development and optimization
 In-process check ,validation etc.,
 Forensic
 Poisoning investigations
 Environmental Application
 Analysis of environment pollution level.

30. Define Validation? Classify and explain each type in briefs

Validation means rectification or confirmation or to be certified. It is a concept which establishes
documented program of specified and reliable performance.
Therefore, process of validation is a concept that is fundamental to GMP and is a part of quality assurance
and thus validation studies leads to:
> process optimisation
> better productivity
>Lowering manufacturing cost
Importance of validation process:
i) it ensured conformance to CGMP requirements
ii) it helps to identify root cause of problems
iii) it defines a baseline of parameters against which changes can be evaluated before
implementation
iv) it documents that the product is made by reliable manufacturing process
v) it documents that the process or activity is in control
Types of validation
a) process validation;
 process validation is a documented program which provides a high margin of
assurance that a specific process will consistently produce a product that meets its
predetermined specifications and quality attributes.
It is essential to instill confidence in personnel
b) Computer (Process Controller) Validation:
It is a documented program which provides a high degree of assurance that the
computer system (or process controller) consistently performs as expected.
It is concerned with performance, security, integrity and accountability
c) Cleaning validation:
It is a documented programme which provides a high degree of assurance that a
specific cleaning procedures, when performed appropriately, will consistently clean
a particular piece of equipment to a predetermined level of cleanliness
d) Analytical methods of validation:
It is a documented programme which provides a high degree of assurance that an
an analytical assay method will consistently determine the presence, absence or
quality of one or more attributes with accuracy and precision.

31. Define & Explain (1) Accuracy (2) Precision (3) Significant figure
1) Accuracy:
The accuracy of an analytical procedure expresses the closeness of agreement between the value which is
accepted either as a conventional true value or an accepted reference value and the value found
Should be established across specified range of analytical procedure.
Should be assessed using a minimum of 3 concentration levels, each in triplicate (total of 9 determinations)
Should be reported as:
Percent recovery of known amount added or
The difference between the mean assay result and the accepted value
 Accuracy: drug substance:
Application of procedure to an analyte of known concentration (reference material).
Comparison of results between proposed method and second well characterized procedure (of known
accuracy).
Inferred from precision, linearity, and specificity studies.
 Accuracy: Drug product
Application of procedure to synthetic mixture of drug products components (placebo) to which known
amounts of drug substance have been added.
Adding known amounts of drug substance to drug product and assaying by method.
Comparison of results between proposed method and second well characterized procedure (of known
accuracy).
Inferred from precision, linearity, and specificity studies.
 Accuracy: Impurity assay
Application of procedure of drug substance or drug product samples spiked with known amounts of
impurities.
Comparison of results between proposed method and second well characterized procedure (of known
accuracy).

For 1 or 2, may be assayed as weight/weight or area percent.

2) precision:
The precision of an analytical procedure expresses the closeness of agreement between a series of
measurements obtained from multiple sampling of the same homogenous sample under the prescribed
conditions. Precision may be considered at 3 levels:
(i) Repeatability:
This expresses the precision under same operating conditions over a short interval of time.
Repeatability is also called as intra-assay precision.
(ii) Intermediate precision:
This expresses within laboratoris variations; different days, different analysis,
different equipments etc.
(iii) Reproducibility:
This expresses the precision between laboratories (collaborative studies, usually
applied to the standardisation of methodology)
Thus precision should be investigated using homogenous, authentic samples and
the precision of an analytical procedure is usually expressed as variance; Standard
deviation and coefficient of variation of series of measurements.
3) significant figure
The significant figures of a number are digits that carry meaning contributing to its
measurement resolution.
This includes all digits except:
 All leading zeros
 Trailing zeros when they are merely placeholders to indicate the scale of the number
 Spurious digits introduced, for example, by calculations carried out to greater precision than that of
the original data, or measurements reported to a greater precision than the equipment supports.

Concise rules:
 All non-zero digits are significant
Eg: 1, 2, 3 etc
  Zeros between non-zero digits are significant
Eg: 102, 50009 etc
 Leading zeros are never significant
Eg: 0.02, 001.887 etc
 In a number with a decimal point, trailing zeros (those to the right of the last non-zero
digit) are significant Eg:2.02000, 5.400 etc
 In a number without a decimal point, trailing zeros may or may not be significant.

32. Describe different steps involved in validation master plan

*refer Q30 (5 marks) for definition and importance
Validation is a systemic approach to indentifying, measuring, evaluating, documenting and re-evaluating
the critical steps in manufacturing process that requires in control to ensure reproducible final product.
The following 3 basic steps involved in validation are as follows:
(1) Establishment of specifications and performance characteristics
(2) Selection of methodology, process and equipment to ensure that the product meets specifications
(3) Final product testing by using validated analytical methods in order to meet specifications
The following steps also have been added in validation
1) Calibration or verification and maintenance of process and equipment
2) Qualification or validation of both process and equipment
3) challenge audit, monitor or sample the recognise critical steps in the process
4) Requalification or re-validation
due to the addition of these steps, the validation concept can be made dynamic and self
improving
Validation of analytical equipments
Analytical equipment is one of the basic components of pharma processing hence, need a
critical validation. To validate a pharmaceutical process, the equipment used must be validated
and caliberated well.
The equipment validation process generally covers following steps:
a) Customer requirements
Customer or the user of equipment has certain expectations about the equipment generally
put in the form of his requirements
The general requirements may be stated as:
(i) size of the equipment
 (ii) speed of the equipment
(iii) effectiveness of equipment
(iv) availability of spares, and prompt services at reasonable cost
(v) ease of operation, cleaning and maintenance
(vi) low dust and sound generation
(vii) materials of plant construction
(viii) auto-control systems
(ix) easy changeovers
(x) overall good construction and workmanship
the equipment manufacturer, after discussion on the selection of equipment with
customer, prepares the design qualification report and sends it to the customer/user for
approval
b) preparation of design qualification and its certification:
to purchase standard equipment as per user’s requirements, the detailed design qualification
document is very important. The detailed equipment specifications must be worked out by
the manufacturer and purchaser
the various stages during the fabrication of equipment must be identified where visual ,
instrumental or physico-chemical testing are performed.
Generally, Factor Acceptance Test (FAT) is performed at the manufacturer’s premises
before dispatching the equipment to the purchaser.
c) Installation Qualification (IQ) : IQ may be defined as “documented verification of
installation adhere to manufacturers recommendation, appropriate codes and approved
design qualification.
The equipment can be installed when it passes IQ test
d) Operational Qualification (OQ): operational qualification may be defined as:” Documented
verification that the system performs throughout all specified operating ranges”. The
equipment should be operated only when in passes OQ test,
e) Performance Qualification (PQ): PQ is the documented verification of performance of the
system or subsystem with load. Here, the equipment should be performed well.
33. Explain the procedure to calibrate wavelength of UV Instrument
Selection of absorption filter is done according to the following procedure:
Draw a filter wheel
  Write the color VIBGYOR in clockwise or anticlockwise manner, omitting Indigo.
 If solution to be analyzed is BLUE in color a
filter having a complimentary color
ORANGE is used in the analysis.
 Similarly, we can select the required filter in
colorimeter, based upon the color of the
solution.

34. Write a note on ICH Guidelines

ICH Guidelines
Validation of Compendial Methods
ICH Validation requirements
 Specificity or selectivity
 Linearity
 Range
 Accuracy
 Repeatability
 Intermediate Precision
 Limit of Detection
 Limit of Quantitation
 Robustness
1) Specificity
 Ability of an analytical method to measure the analyte free from interference due to
other components
 An investigation of specificity should be conducted during the validation of an
identification test, an impurities assay, and a potency assay.
 Procedures used will depend on the intended objective of the analytical procedure.
  If a method can not completely discriminate, two of more procedures are
recommended.
Specificity: Identification
 Should be able to discriminate between compounds closely related in structure.
 Confirmed by obtaining negative results for samples with spiked related compounds
and positive results for samples with analyte.
 Choice of potential interfering substances should be based on sensible scientific
judgment considering substances that could likely occur.
Specificity: Impurites/assay
 Chromatographic Methods
 Demonstrate Resolution
 Impurities/Degradants Available
 Spike with impurities/degradants
 Show resolution and a lack of interference
 Impurities/Degradants Not Available
 Stress Samples
 For assay, Stressed and Unstressed Samples should be compared.
 For impurity test, impurity profiles should be compared.

Peak purity tests can also be evaluated with

 The spectra of Photodiode array detectors
 Mass spectrometry
2) Linearity
Ability of an assay to elicit a direct and proportional response to changes in analyte
concentration.
Linearity should be evaluated by
 By Visual Inspection of plot of signals vs. analyte concentration
 By Appropriate statistical methods
 Linear Regression (y = mx + b)
 Correlation Coefficient, y-intercept (b), slope (m), residual sum of squares

Requires a minimum of 5 concentration levels

3) Range:
 The interval between the upper and lower concentrations of analyte in the sample that have
been demonstrate to have a suitable level of precision, accuracy, and linearity.
Normally derived from Linearity studies.
Established by confirming that the method provides acceptable degree of linearity, accuracy, and
precision.
Specific range dependent upon intended application of the procedure.
Minimum specified range;
 For Drug Substance & Drug product Assay
 80 to 120% of test Concentration
 For Content Uniformity Assay
 70 to 130% of test Concentration
 For Dissolution Test Method
 +/- 20% over entire Specification Range
 For Impurity Assays
 From Reporting Level to 120% of Impurity Specification for Impurity Assays
 From Reporting Level to 120% of Assay Specification for Impurity/Assay Methods
4) Accuracy *refer Q31(5marks)
5) Precision *refer Q31( 5 marks)
6) Repeatability
 Express the precision under the same operating conditions over a short interval of time.
 Should be assessed using minimum of 9 determinations
 (3 concentrations/ 3 replicates) or
Minimum of 6 determinations at the 100% level.
7) Intermediate precision
Express within-laboratory variations.
Expressed in terms of standard deviation, relative standard deviation (coefficient of variation)
and confidence interval
8) Detection limit (DL)
Lowest amount of analyte in a sample that can be detected but not necessarily quantitated.
Estimated by Signal to Noise Ratio of 3:1.
9) Quantitation limit (QL)
Lowest amount of analyte in a sample that can be quantified with suitable accuracy and
precision.
Estimated by Signal to Noise Ratio of 10:1.
 10) Robustness
 Definition: Capacity to remain unaffected by small but deliberate variations in method
parameters
 Determination: Comparison results under differing conditions with precision under
normal conditions
 Variations may include: stability of analytical solution, variation of pH in a mobile
phase, different column (lot/supplier), temperature, flow rate.

35. Describe the pharmaceutical water system Validation.

To prove the performance of processes or systems under all
conditions expected to be encountered during future operations.
 To prove the performance, one must demonstrate (document) that
the processes or systems consistently produce the specified quantity
and quality of water when operated and maintained according to
specific written operating and maintenance procedures.
 validation involves proving-
1. Engineering design
2. Operating procedures and acceptable ranges for control
parameters
3. Maintenance procedures to accomplish it
 the system must be carefully,
-designed
-installed
-tested during processing, after construction, and under all operating
conditions.
 Variations in daily, weekly and annual system usage patterns must
be validated.
Importance of validation
 To ensure reliable, consistent production of water of required quality
 To operate system within design capacity
 To prevent unacceptable microbial, chemical and physical
contamination during production, storage and distribution
 To monitor system performance, storage and distribution systems
 Water System Qualification

Validation Master Plan

User Requirement Specification

Design Qualification

Operation Qualification

Performance Qualification

Re- Qualification

Sign-off Report
DESIGN QUALIFICATION (DQ)
DQ is documented the design of the system & will include :
 Functional Specification.(Storage, purification, etc)
 Technical/Performance specification for equipment.(requirements of
 water volume and flow, define pumps and pipe sizes )
 Detailed layout of the system.
 Quality attribute of input water
 Required plant output capacity
 Selection of membrane
 Designing of holding tanks pumps, heat exchangers and piping.
Design must be in compliance with GMPs and other regulatory
Requirements
Operational Qualification
The purpose of OQ is to establish, through documented testing, that all
critical components are capable of operating within established
limits and tolerances.
Operation Qualification Checks-
  Identification of dead legs
 Slope verification
 Weld inspection
 Pressure test
 Passivation
The purpose of OQ is also to verify and document that the water
supply system provides acceptable operational control under “at-rest”
conditions
Installation Qualification
IQ is in the form of checklist and it should include-
 Water velocity test
 Turbulence flow test (Reynolds number)
 Quality attribute of output water
 Prepare operational SOP
 Prepare cleaning and sanitation SOP
 Design action and alert limit
 Collection and collation of supplier operating and working
instructions and maintenance requirements
Performance Qualification
The purpose of PQ is to verify and document that water supply system
provides acceptable control under ‘ Full Operational ‘ conditions.
Qualification phases
 Phase I (investigational step): sampling and testing (4-6 weeks)
 Phase II (verification step): sampling and testing (2-4 weeks)
 Phase III (satisfactory completion step): sampling and testing
(Yearly activity, seasonal monitoring)
 Establish action and alert limit
PQ should follow successful completion of IQ and OQ.
36. Define Validation? Explain types of “process Validation”?
*refer Q30 (5 marks)
37. What is the principle in potentiometric titration and How is the end point determined in Potentiometric
titrations
 • It is a method of analysis in which we determine the concentration of an ion or substance by
measuring the potential developed when a sensitive electrode is immersed in the solution of the
species to be determined.
Determination of the substances by potentiometric
technique can be carried out by two ways:
1. Direct potentiometry and
2. potentiometric titrations
• The potential of the indicator electrode cannot be measured alone;

• For any potentiometric measurement we must have:

1. Reference electrode
2. Indicator electrode.
3. Potentiometer
4. Salt bridge to connect the two electrode solutions and complete the circuit.

It is used for all types of volumetric analysis: acid base, precipitimetry, complexometry and redox
It is used when it is not easy or impossible to detect the end point by ordinary visual methods i.e:
1. For highly coloured or turbid solutions.
2. For very dilute solutions 10-3, 10-6 M.
3. When there is no available indicator
Types of potentiometric titrations:
1) Acid-Base or neutralisation titrations
2) Redox titrations
3) Complex titrations
4) Precipitation titrations
5) Diazotisation titrations
6) Non-aqueous titrations
1) Acid-Base or neutralisation Titrations
Acid-base titrations are based on neutralisation reactions
H+ + OH ̄ H20
It involves reaction between the analyte and an acidic or basic titrant to give a salt along with
neutral water.
Acid (titrant) + Base (analyte) salt + H2O
Water is formed by the interaction of H+ ions of the acid and OH- ions of the base
Acid base titrations involve changes in the concentration of H+ and OH ions. In this titration,
Glass electrode is used as an indicator electrode and Saturated Calomel Rlrctrode (SCE) is thr
reference electrode.
The acid to be titrated is taken in the titration vessel.
Indicator and reference electrodes are dipped into titrate and the circuit is completed by
connecting these electrodes to a potentiometer which records the changes in e.m.f of the titrate
As soon as the acid(titrate) is titrated with a base (titrant), the acidic solution shows changes in
the e.m.f which are recorded by the potentiometer.
Changes in the e.m.f of an acid is measured after each successive addition of the base. These
values of e.m.f are plotted against volume of base to give a titration curve as follows:

Change in emf
End point
Volume of base (in ml)

2) Redox Titrations
Redox titrations are based on oxidation-reduction reactions between the titrant and the analyte.
In these titrations, both oxidations and reduction occurs simultaneously. One substance
becomes reduced in the process of oxidising the other.
 Redox titrations are based on the oxidation-reduction reactions between the analyte and the
titrant. It involves the transfer of electrons from the substance being oxidised to the substance
being reduced.
Redox titrations involve 2 half reactions. Each half reaction involves a redox conjugate pair
whose standard potentials are used to calculate the net standard potential of the reaction.
Fe+2 Fe+3 + e ̄{oxidation half cell reaction Ɛ˚= -0.76 V}
Ce+4 + e ̄ Ce+3 {Reduction half cell reaction Ɛ˚= +1.61 V}
The net standard potential of the reaction is given as:
Ce+4 + Fe+2 Fe+3 + Ce+3
At the beginning of the titration, when Ce+4 ions are added to Fe+2 ions, Ce+4 ions are
converted to Fe+3 ions(oxidation). The number of Fe+3 ions created during the reaction will
remain equal to the number of Ce+3 ions because, for each mole of Fe+3 created, a mole of
Ce+3 is created. Therefore, throughout the titration.
[Fe +3 ] = [Ce +3]
Before the equivalence point, the concentration of Fe+2 ions is more.
Potentiometric redox titrations are applied in agricultural and biochemical studies, monitoring
water pollution, sewage treatment, cyanide esters from metal plating industries, chlorine
compounds in bleaching agents and paper manufacturing.
3) Complexometric titrations
It is based on the formation of a complex between the analyte and the titrant.
During complexometric titrations, addition of titrant to the solution of metal ions (analyte)
produces a metal complex which is chemically stable, stotiometric and undissociated.
Ethylenediamine Tetraacetic Acid (EDTA) is the most commonly used titrant for the titration
of metal ions.
It forms covalent bonds with the metal ions to give a stable metal complex, the characteristics
of which are different from that of the free metal ion.
Complexometric titrations are carried out potentiometrically using an indicator electrode made
up of the same metal, the ion of which is involved in the complex formation.
Eg: titration of mercuric cyanide with silver chloride in the presence of silver electrode.
Titration of cyanide ions with silver ions results in the formation of silver cyanide complex
which is seen as follows:
Ag+ + 2 CN [Ag(CN)2]
4) Precipitation titrations:
Precipitation titration involve reaction between the titrant and the analyte to form sparingly
soluble salts.
Precipitation titrations are carried out for metallic ions like Ag, Au, Cu, Hg, Pb which form
sparingly soluble salts with the titrants.
The titrants used in such type of titrations are called as precipitating agents or precipitants as
they lead to precipitation of the salt by reacting with the analyte. The end point of
 potentiometric precipitation titration depends on the solubility of the precipitate and also on
concentration of the analyte.
The indicator electrode used in this type of titration should be reversible with one of the
precipitating ions i.e, it should be in equilibrium with one of the ions.
Reference electrode can be any electrode of stable potential which may be saturated calomel
electrode or standard hydrogen electrode.
In the titration of AgNO3 with KCl, KCl is added in small volumes to the titrate.
As the titration proceeds, Ag+ ions get precipitated as AgCl.
55AgNO3 + KCl AgCl + KNO3
With each increment of KCl, concentration of Ag+ ions decreases and potential of the
electrode increases. Near the end point, the electrode shows a sharp change in the potential due
to precipitation of all the Ag+ ions as AgCl. The values of electrodes potential are plotted
against the volume of KCl added.
End point is depicted at the point of maximum inflection in the titration curves.
5) Diazotisation Titrations:
Analytes containing primary aromatic amino groups are titrated against sodium nitrite in acidic
medium to give diazonium salts. The end point of titration is determined by potentiometry.
Examples of drugs primary amino groups which are potentiometrically titrated are dapsone,
sulphacetamide, procainamide, amino alkaloids etc.
The indicator electrode used in glass electrode and the reference electrode used in saturated
calomel electrode.

38. Enumerate the different reference electrodes & Indicators electrodes in potentiometric titrations
Reference electrode
Reference electrode must:
1. Have a constant potential
2. Its potential must be definite
To express any electrode we have to mention:
1. Redox reaction at the electrode surface.
2. Half cell and Nernst equation.
3. Sketch of its design.
4. Any necessary conditions for its preparation.
5. Any necessary precautions for its use.
1.Standard Hydrogen Electrode
It’s a primary reference electrode. Its potential is considered to be zero.
Electrode reaction:
half cell: pt/ H2 , H+ (1N) 
Eo = zero
d-Limitation
1. It is difficult to be used and to keep H 2 gas at one atmosphere during all determinations.
2. It needs periodical replating of Pt. Sheet with Pt. Black

2.Saturated calomel electrode (S.C.E.)

Hg | Hg2Cl2 (sat’d), KCl (sat’d) | |
electrode reaction in calomel hal-cell
Hg2Cl2 + 2e = 2Hg + 2Cl–
Eo = + 0.268V

E = Eo – (0.05916/2) log[Cl–]2 = 0.244 V

Potential of the electrode depends on the chloride ion

Ksp = 1.8 ×10–18

Hg2Cl2 ⇌ 2 Hg22+ + 2Cl-

Sp Hg2Cl2 = [Hg22+]2 [Cl-]2

Ksp = 1.8 ×10–18

o –2
E = E – (0.0591/2) log[Cl ] = 0.244 V

The crystal structure of calomel(Hg2Cl2), which has limited solubility in water (Ksp = 1.8 ×10 –18).

KCl E volt

Saturated 0.241

1M 0.280

0.1 M 0.334

Hg2Cl2  Hg22+ + 2Cl– Ksp = 1.8 ×10–18

Saturated KCl = 4.6 M KCl
3.Silver-silver chloride electrode
Ag(s) | AgCl (sat’d), KCl (xM) | |
AgCl(s) + e = Ag(s) + Cl–
Eo = +0.244V
E = Eo – (0.05916/1) log [Cl–]
E (saturated KCl) = + 0.199V (25oC)
Indicator electrode
its potential is sensitive to the concentration of analyte
Ecell=Eindicator-Ereference
It must be:
(a) give a rapid response and
(b) its response must be reproducible.

Metallic electrodes: where the redox reaction takes place at the electrode surface.
1.Membrane (specific or ion selective) electrodes: where charge exchange takes place at a specific
surfaces and as a result a potential is developed

2.standard hydrogen electrode

electrode reaction:
1
H+ + 2e ⃗
Pt . balck
2
H2
 Nernst equation
1
E = zero - 0.059 log
[ H+ ]
E = -0.059 pH
When it is connected with NHE as reference electrode the e.m.f. of the cell :
Ecell = zero –(–0.059 pH)
= 0.059 pH
pH = E / 0.059

Demerits:
1. It cannot be used in solution containing oxidising agent which will oxidiose [ ½ H 2 = H+ + e ]
or reducing substances which will reduce [ H + + e = ½ H2 ] especially in presence of platinum
black
2. It cannot be used in reactions involving volatile constituent’s e.g. CO 2, as it will be bubbled out by
the H2 gas.
3. It cannot be used in presence of catalytic poisons which will affect Pt black which catalyses the
electrode reaction.
4. It needs repletion with Pt black.
5. It is not easy to keep H2 gas at one atmospheric pressure during all measurements.
3 Antimony electrode Sb/Sb2O3
Advantages
Easy to use, cheep and durable.
Disadvantages
1. can only be used within pH range 2 – 8 at lower pH Sb 2O3 dissolves and at higher pH Sbo
dissolves.
2. It cannot be used in presence of oxidizing agents, reducing agents, complexing agents and noble
metals
39. Explain the principle underlying “Dead- Stop end point technique” and Null point potentiometry.
Dead stop end point technique
This method is called biamperometry.It is applicable when a redox system is present before and after the
end point.
Principle:The principal is that, the potential applied between polarisable and non-polarisable electrode is
kept constant and the diffusion current is measured during the titration. During the titration, the
concentration of the electroreducible ion changes and hence the diffusion current also changes. At the end
point, there is a sharp change in the diffusion current as shown by the curve of diffusion current Vs volume
of titrant. The titration is performed between a reducible or non-reducible ion and a counter ion of which
atleast the titrate or the titrant or both can give rise to diffusion current.
The potential selected for the titration is at its limiting value, i.e. the potential coreesponds to the point
where limiting current is reached.
The principle is explained from the following curve:

Id1id2id3id4id5

0 E1/2 applied emf(V)

E ½ = Half wave potential

I1 = limiting current
Id 1,2,3,4,5 = diffusion current of solutions of increasing concentrations

id
end point
vol. of karl fischer reagent
This method is applicable when a redox system is present before and after the end point. An example is the
titration of water using Karl fischer reagent. A small potential is applied between 2 similar platinum
electrodes. Initially when water is present both electrodes are depolarised. The addition of Karl Fisher A &
B (solution of iodine and sulphur dioxide in pyridine and methanol) is continued till the end point, where
the diffusion current decreases. At the end point, only one electrode is depolarised and the diffusion
current is almost zero or nil.
null point potentiometry

(G)

reference Indicator electrode

 unknown solution
in null point potentiometry, the reference electrode and indicator electrode is connected to a galvanometer.
On dipping the electrodes, galvanometer shows deflection
End point is when there is no deflection.
Unknown potential = Ɛ ˚

40. Explain with graphs the methods of potentiometric end point determination
Types of potentiometric titrations:
1. Acid-Base or neutralisation titrations
2. Redox titrations
3. Complex titrations
4. Precipitation titrations
5. Diazotisation titrations
1) Acid-Base or neutralisation Titrations
Acid-base titrations are based on neutralisation reactions
H+ + OH ̄ H20
It involves reaction between the analyte and an acidic or basic titrant to give a salt along with
neutral water.
Acid (titrant) + Base (analyte) salt + H2O
Water is formed by the interaction of H+ ions of the acid and OH- ions of the base
Acid base titrations involve changes in the concentration of H+ and OH ions. In this titration,
Glass electrode is used as an indicator electrode and Saturated Calomel Rlrctrode (SCE) is thr
reference electrode.
The acid to be titrated is taken in the titration vessel.
Indicator and reference electrodes are dipped into titrate and the circuit is completed by
connecting these electrodes to a potentiometer which records the changes in e.m.f of the titrate
As soon as the acid(titrate) is titrated with a base (titrant), the acidic solution shows changes in
the e.m.f which are recorded by the potentiometer.
Changes in the e.m.f of an acid is measured after each successive addition of the base. These
values of e.m.f are plotted against volume of base to give a titration curve as follows:
Change in emf
End point
Volume of base (in ml)
2) Redox titrations
Oxidation reduction titration can be followed by an indicator electrode. The electrode assumes
a potential proportional to the logarithm of the concentration of the two oxidation state of the
reactant or the titrant, whichever is capable of properly poising the electrode

emf

Equivalence point

Volume of titrant
Such type of reactions consists essentially of two half reactions whose standard potential may be used to
calculate the standard potential of the reaction.
Fe2+ Fe3+ + e E˚ = +1.61 V
Ce4+ + e Ce3+ E˚ = -0.77V
Fe2+ + Ce4+ Fe3+ +Ce3+ E˚=+0.84 V
3) Complexometric titrations
It is based on the formation of a complex between the analyte and the titrant.
During complexometric titrations, addition of titrant to the solution of metal ions (analyte)
produces a metal complex which is chemically stable, stotiometric and undissociated.
Ethylenediamine Tetraacetic Acid (EDTA) is the most commonly used titrant for the titration
of metal ions.
It forms covalent bonds with the metal ions to give a stable metal complex, the characteristics
of which are different from that of the free metal ion.
Complexometric titrations are carried out potentiometrically using an indicator electrode made
up of the same metal, the ion of which is involved in the complex formation.
Eg: titration of mercuric cyanide with silver chloride in the presence of silver electrode.
Titration of cyanide ions with silver ions results in the formation of silver cyanide complex
which is seen as follows:
 Ag+ + 2 CN [Ag(CN)2]

emf

Volume of titrant

4) precipitation titrations
Precipitation titration involve reaction between the titrant and the analyte to form sparingly
soluble salts.
Precipitation titrations are carried out for metallic ions like Ag, Au, Cu, Hg, Pb which form
sparingly soluble salts with the titrants.
The titrants used in such type of titrations are called as precipitating agents or precipitants as
they lead to precipitation of the salt by reacting with the analyte. The end point of
potentiometric precipitation titration depends on the solubility of the precipitate and also on
concentration of the analyte.
The indicator electrode used in this type of titration should be reversible with one of the
precipitating ions i.e, it should be in equilibrium with one of the ions.
Reference electrode can be any electrode of stable potential which may be saturated calomel
electrode or standard hydrogen electrode.
In the titration of AgNO3 with KCl, KCl is added in small volumes to the titrate.
As the titration proceeds, Ag+ ions get precipitated as AgCl.
55AgNO3 + KCl AgCl + KNO3
With each increment of KCl, concentration of Ag+ ions decreases and potential of the
electrode increases. Near the end point, the electrode shows a sharp change in the potential due
to precipitation of all the Ag+ ions as AgCl. The values of electrodes potential are plotted
against the volume of KCl added.
End point is depicted at the point of maximum inflection in the titration curves.

emf
 Volume of KCl

5) Diazotisation Titrations:
Analytes containing primary aromatic amino groups are titrated against sodium nitrite in acidic
medium to give diazonium salts. The end point of titration is determined by potentiometry.
Examples of drugs primary amino groups which are potentiometrically titrated are dapsone,
sulphacetamide, procainamide, amino alkaloids etc.
The indicator electrode used in glass electrode and the reference electrode used in saturated
calomel electrode.

pH of
analyte

Volume of titrant added

41. With titration curves, describe the principles of Conductometric titrations?
Conductivity is the measurement od conductivity of a solution due to the mobility of cations and anions
towards respective electrodes.
A. ACID-BASE TITRATIONS
a. Strong acid Vs Strong base
Eg: Hydrochloric acid Vs Sodium hydroxide
When HCl is taken in beaker as titrate, the initial conductivity is high, because strong acid
completely dissociates into H+ ions and the ionic conductivity of H+ is 250.
When NaOH is added as titrant, the OH- and K+ reacts to produce water and the no; of H+
decreases and the conductivity gradually decreases after every addition. After the end point,
when all the H+ has reacted, the addition of NaOH causes increase in the no; of OH- and hence
the conductivity starts to increase.
A plot of conductivity Vs volume of NaOH added shows a V shaped curve

H+ OH

conductivity
 End point
Vol. of NaOH added
b. Strong acid Weak base
Eg: Hydrochloric acid Vs Ammonium hydroxide
When HCl is taken in a beaker as titrate, the initial conductivity is high, because strong acid
completely dissociates into H+ ions and the ionic conductivity of H+ is 350. When NH4OH is
added as titrant the OH- and H+ decreases and the conductivity gradually decreases after every
addition. After the end point, when all the H+ has reacted, the addition of NH4OH doesnot
cause increase in the no; of OH- since it poorly dissociates into OH- and hence the conductivity
remains constant.

conductivity Addition of NH4OH

(mhos) end point
Vol. of NH4OH
c. Weak acid Vs Strong base
Eg: Acetic acid Vs Sodium Hydroxide
When CH3COOH is taken in a beaker as titrate, initial conductivity is low, beacause weak acid
does not dissociate into H+ ions. When NaOH is added as titrant, formation of CH3COONa
takes place and there is only slight increase in conductivity till the end point, the addition of
NaOH causes increase in the no; of OH- and hence the conductivity starts to increase steeply

Conductivity OH-
salt formation
end point

Vol. of NaOH
d. Weak acid Vs weak base
Eg: Acetic acid Vs Ammonium hydroxide
Addition of NH4OH
Salt
Conductivity formation
End point
vol of NH4OH
 B. PRECIPITATION TITRATIONS
In this titration, any one of the product of the titration can be a precipitate and the other soluble or
both products can be in the form of precipitate
1. only one product is a precipitate
Eg: KCl + AgNO3  AgCl + KNO3
KCl is taken in a beaker and silver nitrate is the titrant. When silver nitrate is added, the first
part of the curve shows no increase in the conductivity as there is only replacement of chloride
ions with nitrate ions. As the silver chloride is precipitated, it doesnot contribute to
conductivity. The second part of the curve after the end point increases because of increase in
the concentration of silver as well as nitrate ions.

Add. of
Replacement of Cl AgNO3

Conductivity end point

Vol. of AgNO3
2. when both precipitates are sparingly soluble
Eg: Magnesium sulphate and Barium hydroxide
Precipitation

conductivity Add. Of Ba(OH)2

End point
Vol of Ba(OH)2
When magnesium sulphate is thr titrate and barium hydroxide is the titrant, both the products,
i.e. magnesium hydroxide and barium sulphate are sparingly soluble and are precipitated.
C. DISPLACEMENT TITRATIONS
These are titrations in which there is displacement of one ion by the other
a. Salt of strong acid and weak base Vs Strong base
NH4Cl + NaOH  NH4OH + NaCl
The first part of the titration is a plateau because there is only displacement of Ammonium and
Chloride ions with Sodium ions with sodium and chloride till the end point. After the end
point, the addition of sodium hydroxide causes a steep increase in the conductivity.
conductivity
end point
Vol. of NaOH
b. Salt of strong base and weak acid Vs strong acid
CH3COONa + HCl  Ch3COOH + NaCl
In this titration, sodium acetate is used as titrate and hydrochloric acid is used as titrant. The
first part of the titration is gradual increase in the conductivity because there is displacement od
acetate ions by chloride ions till the end point. After the endpoint, the addition of HCl causes a
steep increase in the conductivity.

conductivity
End point
Vol of HCl
D. REDOX TITRATIONS
It is usually conducted in acid medium. The end point is determined because of decrease in
hydrogen ion concentration and decrease in conductivity at the end point

Conductivity
End point
Vol of Cr2O7
E. Complexometric titrations
The conductivity changes that could be observed or measured near the end point is small.
The following graph is obtained in the titration of potassium chloride with mercuric chlorate. The 2
inflections indicate the formation of HgCl4 2- and the completion of the reaction.

conductivity
end point end point
 vol. of Hg(ClO4)2
42. Explain with graph, the conductometric titration of a mixture of weak & strong acids with alkali
*refer Q 41(5marks)
43. Explain the conductometric titration curve for strong acids against weak base?
*refer Q41(5marks)
44. What is quenching? Explain various types of quenching with suitable examples?
 Decrease in fluorescence intensity due to specific effects of constituents of the solution.
Due to concentration, ph, pressure of chemical substances, temperature, viscosity, etc.
Types of quenching
o Self quenching
o Chemical quenching
o Static quenching
o Collision quenching
1. Self quenching:
 At low concentration fluorescence increases with concentration ,but at high concentration
corresponding increase does not occur.

Caliberation curve(low conc.)

fluorescence

concentration of
fluorescing species

Caliberation curve(High conc.)

fluoresence

concentration of
 fluorescing species
Deviations at higher concentrations can be attributed to self-quenching or self-absorption.

2. Chemical quenching
o Here decrease in fluorescence intensity due to the factors like change in ph,presence of
oxygen, halides &heavy metals.
o ph- aniline at ph 5-13 gives fluorescence but at ph <5 &>13 it does not exhibit
fluorescence.
 halides like chloride,bromide,iodide & electron withdrawing groups like no2,cooH
etc. leads to quenching.
o Heavy metals leads to quenching, because of collisions of triplet ground state.
o Oxygen: due to paramagnetism
3. Static quenching
This occurs due to complex formation:
(eg) caffeine reduces fluorescent intensity of riboflavine due to complexation.
4. Collosion Quenching
It reduces fluorescence by collision. where no. of collisions increased hence quenching takes place.
a. Reduce fluorescence by dissipating absorbed energy as heat.
b. Due to various factors like halide , heavy metals, temp , viscosity, where collisions are
increased.
45. What is the number of Fundamental Vibration modes for linear and non-linear molecules containing
‘n’ atoms? Explain how these numbers are obtained
Vibrational modes
N atoms in a molecule have 3N degrees of freedom which constitute translations, rotations, and vibrations.
For non-linear molecules, there are 3 degrees of freedom for translational (motion along the x, y, and z
directions) and 3 degrees of freedom for rotational motion (rotations in Rx, Ry, and Rz directions) for each
atom. Linear molecules are defined as possessing bond angles of 180°, so there are 3 degrees of freedom
for translational motion but only 2 degrees of freedom for rotational motion because the rotation about its
molecular axis leaves the molecule unchanged. [2] When subtracting the translational and rotational
degrees of freedom, the degrees of vibrational modes is determined.
Example of a linear molecule:
Number of degrees of vibrational freedom for nonlinear molecules: 3N-6

Number of degrees of vibrational freedom for linear molecules: 3N-5

Degrees of freedom
The location of a molecule in a 3-dimensional space can be described by the total number of coordinates.
Each atom is assigned a set of x, y, and z coordinates and can move in all three directions. Degrees of
freedom is the total number of variables used to define the motion of a molecule completely. For N atoms
in a molecule moving in 3-D space, there are 3N total motions because each atom has 3N degrees of
freedom.
46. What is nebulization? Write a note on types of Burner’s used in Atomic emission spectrometer
Nebulization is the technique of converting liquids into a fine mist by applying pressure through small
holes with an apparatus called nebulizer. Nebulizers generally use gas flows to deliver the mist.
Its main purpose is to deliver a fine mist to spectrometric instruments for elemental analysis.
There are 2 types of burners in common use:
i) Total consumption burner
ii) Premixed burner
TOTAL CONSUMPTION BURNER
In the total consumption burner, the sample solution, the fuel and oxidising gases are passed through
separate passages to meet at the opening of the base of the flame. As the sample containing metallic
elements to be estimated by atomic spectroscopy is a liquid, the flame breaks up the liquid sample into
droplets which are then evaporated or burnt, leaving the residue which is reduced to atoms.
Total consumption burners do use oxygen, with hydrogen or acetylene, and give very hot flames.
Demerits:
 Noisy and hard to use
 Efficiency is not very good
 PREMIXED BURNER
In the premixed burner, a mixture of the sample (liquid) and premixed gases (C2H2 +O2) is
allowed to enter the base M. From the base M, the gases enter the region A. From the region A the
unburnt hydrocarbon gaseous mixture and liquid droplets are allowed to enter the region B which
is a region of hydrocarbon gaseous mixture and liquid droplets are allowed to enter region B which
is a region of heating and about 1mm in thickness. In the region B, the liquid is evaporated leaving
a residue.
The heating in this region is done by the heat obtained from the region C by conduction and
convection and by diffusion of radicals into it which initiate the combustion. After this the sample
residue is burnt into regions C and D to produce atoms. The production of atoms is initiated in the
region C and is complete in the region D.
The premixed burner is very suitable for the atomic absorption studies of metals of groups IA, IB
and IIB together with Ga, Ti, Te, Mn, Ni and Pd.

47. Depict the different modes of fundamental vibrations in a tri-atomic group (stretching & deformation)
by means of a neat sketch for each mode
Linear triatomic molecules such as CO2 and CS2 have four vibrational normal modes but just three
fundamental vibration frequencies because two modes are degenerate.1 The symmetric stretching
mode is totally symmetric so it is inactive in infrared spectra and active in Raman spectra.
The asymmetric stretching vibration and the degenerate bending vibrations are infrared active and
Raman inactive. Infrared activities follow from the gross selection rule that “displacements of a normal
mode must cause a change in dipole moment in order to be spectroscopically active in the infrared.”1 The
CO2 and CS2 molecules have a centre of symmetry located at the carbon atom

Stretching vibrations are of 2 types:

 Symmetric
 Asymmetric
These are denoted by ‘v’.
(a) illustrates the symmetric vibration
(b) the asymmetric stretching vibration of AB2
(c) & (d) illustrated the symmetric and asymmetric vibrations of AB3 molecule respectively.

(a) (b) (c) (d)

Deformation vibrations are more compared to stretching vibrations.

*refer Q51(5marks) for wagging, twisting, rocking

48. Explain what is meant by allowed transition and forbidden transition of valence electron in absorption
spectroscopy
Selection rules
– Electronic transitions that occur without change in number of unpaired electrons (spin multiplicity) are
allowed
– Electronic transitions that involve a change in the number of unpaired spins are “forbidden” and are
therefore of low intensity.
>e.g., solutions of high-spin d5, e.g., Mn(II), complexes are lightly colored
Forbidden transitions
In spectroscopy, a forbidden transition is a spectral line associated with absorption or emission of light by
atomic nuclei, atoms, or molecules which undergo a transition that is not allowed by a particular selection
rule but is allowed if the approximation associated with that rule is not made.[1] For example, in a
situation where, according to usual approximations (such as the electric-dipole approximation for the
interaction with light), the process cannot happen, but at a higher level of approximation (e.g. magnetic
dipole, or, electric quadrupole) the process is allowed but at a much lower rate
allowed transitions
A transition between two energy levels in an atom or a molecule that does not violate any selection rules.
These transitions are characterized by large absorption cross sections
Allowed transitions are those that have high probability of occurring, as in the case of short-lived
radioactive decay of atomic nuclei. In three-millionths of a second, for instance, half of any sample of
unstable polonium-212 becomes stable lead-208 by ejecting alpha particles (helium-4 nuclei) from
individual atomic nuclei
49. Describe the terms fluorescence & phosphorescence Depict both the phenomena by energy diagram
Fluorescence
When a beam of light is incident on certain substances they emit visible light or radiations. This is known
as fluorescence.
Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut
off.
The substances showing this phenomenon are known as flourescent substances.
 Transition from excited singlet state to ground singlet state.
 Emission of uv/visible radiation.
 The process is instantaneous and ceases immediately as the light source is removed.
Life time of the excited state is 10 -6-10-4 sec
Types of Fluorescence:
Based upon wavelength:
 (a) stokes fluorescence: wavelength of emitted radiation is longer than absorbed radiation.
 (b) Anti stokes: wavelength of emitted radiation is shorter than absorbed radiation.
 (c) Resonance fluorescence: wavelength of emitted radiation is equal to absorbed radiation.
Stokes Shift
Stokes shift is the difference (in wavelength or frequency units) between positions of the band
maxima of the absorption and emission spectra of the same electronic transition.
 Based upon Phenomenon:
 Sensitized fluorescence-
When elements like thalium,zn,cadmium or an alkali metal are added to mercury vapour these
elements are sensitised and thus gives fluorescence.
 Direct line fluorescence
Even after the emission of radiation, the molecules retain in metastable state and finally comes
to the ground state after loss of energy by vibrational transmit.
 Step wise fluorescence
This is conventional type of fluorescence where a part of energy is lost by vibrational transmision
before the emission of fluorescent radiation.
 Thermally assisted fluorescense
The excitation is partly by electromagnetic radiation and by partly thermal energy.
PHOSPHORESCENCE
 When light radiation is incident on certain substances they emit light continuously even after the
incident light is cut off.
 This type of delayed fluorescence is called phosphorescence.
 Substances showing phosphorescence are phosphorescent substances.
 Transition from singlet excited to triplet state via intersystem crossing.
 From triplet state to ground state.
 Delayed fluorescence-emit light continuously even when light is cut off.
 At favorable conditions-low temp and absence of oxygen.
 Life time of excited triplet state is
10-4 - several seconds.
Energy Diagram
50. What is flame emission & atomic Absorption spectrometry? How do you estimate the amount of
sodium by the above techniques
Flame emission spectroscopy
A sample of a material (analyte) is brought into the flame as either a gas, sprayed solution, or directly
inserted into the flame by use of a small loop of wire, usually platinum. The heat from the flame
evaporates the solvent and breaks chemical bonds to create free atoms. The thermal energy also excites the
atoms into excited electronic states that subsequently emit light when they return to the ground electronic
state. Each element emits light at a characteristic wavelength, which is dispersed by a grating or prism and
detected in the spectrometer.
A frequent application of the emission measurement with the flame is the regulation of alkali metals for
pharmaceutical analytics
Atomic Absorption Spectroscopy
Atomic absorption spectrometry (AAS) is an analytical technique that measures the concentrations of
elements. Atomic absorption is so sensitive that it can measure down to parts per billion of a gram (μg dm–
3) in a sample. The technique makes use of the wavelengths of light specifically absorbed by an element.
They correspond to the energies needed to promote electrons from one energy level to another, higher,
energy level.
Sodium estimation by flame emission spectroscopy:
Quantitative analysis of these species is performed by measuring the flame emission of solutions
containing the metal salts. Solutions are aspirated into the flame. The hot flame evaporates the solvent,
atomizes the metal, and excites a valence electron to an upper state. Light is emitted at characteristic
wavelengths for each metal as the electron returns to the ground state. Optical filters are used to select the
emission wavelength monitored for the analyte species. Comparison of emission intensities of unknowns
to either that of standard solutions, or to those of an internal standard, allows quantitative analysis of the
analyte metal in the sample solution.
Sodium estimation by atomic absorption spectroscopy:
When a solution containing sodium is introduced into the flame, the vapour of sodium will be obtained.
Some of the sodium atoms may be raised to an energy level sufficiently high to emit the characteristic
radiation of sodium.
But a large percentage of sodium atoms will remain in the non-emitting ground state. These ground states
atoms of a particular element are respective of light radiation of their own specific resonance wavelength.
Thus , when a light of this wavelength is allowed to pass through a flame having atoms of the metallic
species, part of that light will be absorbed and the absorption will be proportional to the density of the
atoms in the flame. Thus we can determine the amount of light absorbed.
Once this value of absorption is known,the concentration of sodium can be known.
51. Explain the different modes of fundamental vibrations occurring in IR Spectroscopy
Fundamental vibration can divided in to two principle groups.
1. STRETCHING VIBRATIONS
These are vibrations in which the bond length is altered i.e. increased or decreased
TYPES OF STRETCHING VIBRATIONS
a. Symmetrical stretching:- in this type of vibration the atoms of a molecule vibrate in the same
direction.
Here the two bonds increase or decrease in length, symmetrically

b. Asymmetric Stretching:- in this type of vibration one atom approach towards the central atom
while other departs from it like one bond length increases, the other one decreases.

2. Bending vibrations:
In this type of vibration the position of atoms changes with respect to original bond axis.
More energy is require to stretch a spring than that required to bend it.
like we can say that stretching absorption of bond appears in high frequency as compared to
bending absorption of same bond
Types of bending vibration
1.In plane vibration : in these vibrations, there is change in bond angle. Bending of bond takes
place within the same plane.
a. Scissoring : in which bond angle decreases
 b. Rocking: In which bond angle is maintained, but both bonds moves within the plane
2. Out plane vibration
a. Wagging: in which both atoms move to one side of plane
b. Twisting: in which one atom is above the plane and the other is below the plane

1.In plane vibration

 Scissoring: in this type of vibration two atoms approach each other.
 Rocking: in this type of the movement of atom take place in the same direction.

2. Out plane vibration:

a. Wagging:- two atoms moved up and below the plane with respect to the central atom.
b. Twisting:- in this type one of atom moved up the plane while other down the plane with respect to
central atom.

52. What are the factors affecting the fluorescence

The factors are as follows:
 Nature of molecule
 Nature of substituent
 Effect of concentration
 Adsorption, Light
  Oxygen, pH
 Photodecomposition
 Temp . &viscosity
 Quantum yield
 Intensity of incident light
 Path length
NATURE OF MOLECULE
 All the molecules cannot show the phenomenon of fluorescence.
 Only the molecules absorbs uv/visible radiation can show this phenomenon.
 Greater the absorbency of the molecule the more intense its fluorescence.
NATURE OF SUBSTITUEINT

 Electron donating group enhances fluorescence – e.g.:NH 2,OH etc.

 Electron withdrawing groups decrease or destroy fluorescence. e.g.:COOH,NO 2, N=N etc.
 High atomic no: atom introduced into  electron system decreases fluorescence.
EFFECT OF CONCENTRATION
 Fluorescence is directly proportional to concentration.
 FI = Q X Ia
 i.e, F = QIOact
 Q = Constant for a particular substance
 IO = Constant for an instrument
 a = Molecular extinction coefficient
 t = Path length
 C = Concentration of the substance
 F = KC Where K represents all constants
 FI α Concentration.
ADSORPTION
 Extreme sensitiveness of the method requires very dilute solution.
 Adsorption of the fluorescent substances on the container wall create serious problems.
 Hence strong solutions must be diluted.
LIGHT
 Monochromatic light is essential for the excitation of fluorescence because the intensity will vary
with wavelength.
OXYGEN
 The presence of oxygen may interfere in 2 ways.
 1] by direct oxidation of the fluorescent substances to non fluorescent.
 2] by quenching of fluorescence.
pH
 Alteration of the ph of the solution will have significant effect on fluorescence.
 Fluorescent spectrum is different for ionized and un-ionized species.
TEMPARATURE & VISCOSITY
 Increase in temperature/decrease in viscosity will decrease fluorescence.
INTENSITY OF INCIDENT LIGHT
 Increase in intensity of light incident on sample increases fluorescence intensity.
 The intensity of light depends upon
1)light emitted from the lamp.
2)Excitation monochromaters
3)Excitation slit width
PATH LENGTH
 The effective path length depends on both the excitation and emission slit width.
 Use of microcuvette does not reduce the fluorescence.
 Use of microcell may reduce interferences and increases the measured fluorescence

53. Write a note on spectrophotometric titration

Spectrophotometric Titrations
In usual titrimetric methods, the equivalence point in a reaction id detected visually, either by the color
produced by an indicator (acid base titrations etc.) or by colour of the reactants (permanganate etc.) in such
titrations , one can only obtain precision within a few tenths of one percent. further good results are not
available in such titrations where the colour change is gradual or the colour of 2 forms do not contrast
sharply.
The above mentioned difficulties, can be overcome if we employ spectrometric titrations.
In a spectrometric titrations, the equivalence point is determined with a spectrophotometer. In this
technique, the titration vessel is kept kirectly in the light path of the instrument. Then the absorbance can
be measured.
1.) Use Absorbance of Light to Follow Progress of Titration
Example:
- Titrate a protein with Fe3+ where product (complex) has red color
- Product has an absorbance maximum at 465 nm
- Absorbance is proportional to the concentration of iron bound to protein

(analyte) colourless titrant (colourless) Red

1.) Use Absorbance of Light to Follow Progress of Titration
Example:
- As more Fe3+ is added, red color and absorbance increases,
- When the protein is saturated with iron, no further color can form
- End point – intersection of two lines (titrant has some absorbance at 465nm)

1.) Use Absorbance of Light to Follow Progress of Titration

Example:
- As more Fe3+ is added, concentration changes due to dilution
- Need to correct absorbance for dilution.

 total volume 
Corrected absorbance    observed absorbance 
 initial volume 
Precipitation Titration Curve

1.) Graph showing how the concentration of one of the reactants varies as titrant is added

Understand the chemistry that occurs during titration

Learn how experimental control can be exerted to influence the quality of an analytical titration
- No end point at wrong pH
- Concentration of analyte and titrant and size of Ksp influence end point
- Help choose indicator for acid/base and oxidation/reduction titrations

54. Explain deformation vibrations in IR Spectroscopy

*refer Q51 (5marks)
55. What are the effect of solvent & conjugation in UV Spectroscopy
Effect of solvent
Solvents play an important role in UV spectra since compound peak could be obscured by solvent peak
hence the solvent for a sample is selected in such a way that the solvent neither absorbs in the region of
measurement nor affects the absorption of the sample.
Some common solvents used and their absorption regions are:
Water 191nm
Cyclohexane 195nm
Methanol 203 nm
A potential source of interference is the solvent.
In general, metals in aqueous solutions yield lower absorbance reading than the same concentration of such
metals when present in an organic solvent.
The main reason for this is that metal is more difficult to atomize from an aqueous solution than from an
organic solution
Effect of conjugation
Cross conjugation has no effect on λmax at 241nm and has structure where a prednisone and prednisolone
have structure but the λmax is at 241 nm and remains unaffected hence cross conjugation has no effect in
uv spectroscopy.
56. Write the structure and chemical name of BMR Reagent. Write the principle involved in the reaction of
BMR.with Sulphanilamide along with chemical reactions.
BMR is Bratton Marshall Reagent
Chemical name: N-1-naphthyl ethylene diamine dihydrochloride

Reaction of BMR with sulphanilamide

The diazonium salt formed is treated with naphyl ethylene diamine dihydrohydrochloride gives reddish
pink colour as the final product of the reaction and hydrochloric acid is liberated. The reddish pink colour
is estimated colorimetrically to measure absorbents absorbance maxima at 540nm and optical density is
noted thus, absorbance is read for different concentration is find out.
 (red colour)

57. State & explain the mathematical expression for Beer’s & Lamberts Law
Beer’s Law
Beer’s law states that “The intensity of a beam of monochromatic light decreases exponentially with
increase in concentration of absorbing species arithmetically”
−dI
Accordingly, cx I [ the intensity of incident light (I) with concentration (c ) is proportional to
dc
intensity of incident light (I)
(removing and introducing the constant of proportionality “k”)
−dI −dI
=k =kII
dc dc

-lnI = kc + b ------------------------(1)
On integration, b is constant of integration
When concentration=0, there is no absorbance, hence I=0
Therefore, substituting in equation 1,
-lnIo = k x 0 + b
-lnIo = b
Substituting the value of b in equation 1,
-lnI = kc - lnIo
lnIo – lnI = kc
ln Io/ I = kc (since log A- log B = LogA/b)
Io/I =e kc (removing natural logarithm0
Io/I = ē kc (making inverse on both sides)

I = Io ē kc ------------------( 2)[equation for Beer’s law)

Lambert’s law
“The rate of decrease of intensity (monochromatic light) with the thickness of the medium is directly
proportional to the intensity of incident light.”
−dI
i.e, , cx I
dt

this equation can be simplified, similar to equation (2) to get the following equation
I = Io ē kt ----------------------(3)
Equation (2) and (3) can be combined to get
I = Io ē kct

I = Io 10−kct (converting natural logarithm to base 10 & K= k x 0.4343)

I/Io = 10−kct

Io/I = 10kct (inverse on both sides)

Log Io/I = kct (taking log on both sides)----------------------(4)
It can be learnt that transmittance (T) = I/Io and absorbance (A) = log 1/T
Hence A = log 1
I/Io
A= log Io/I ------------------------------(5)
Using equation 4 & %, since A=log Io/I and log Io/I = kct we can infer that
A=kct (instead of k we can use Ɛ)
A= Ɛct (mathematical equation for Beer lambert’s law)
Where A=Absorbance or optical density or extinction co-efficient
Ɛ= molecular extinction coefficient
c= concentration of drug
t= path length

58. Explain the term- Red Shift, Blue Shift, hypochromic shift, hyperchromic shift giving suitable
examples for each along with λmax and Σ values.
(i) Red shift: it involves shift of absorption maximum towards longer wavelength because of presence of
certain groups such as OH and NH3 called auxochromes
Eg.: decreasing the polarity of solvents causes a red shift in n-ᴧ* absorption of carbonyl componds
Eg.: I,3 butadiene shows λmax at217 nm
(ii) hypsochromic shift or blue shift;
It involves the shift of absorption maxima towards shorter wavelength and may be caused by removal of
conjugation in a system or by change of solvent.
Eg; in the case of aniline absorption maxima takes place at 280nm because the pair of electrons on
hydrogen atom is on conjugation with the ᴧbond system of the benzene ring.
(iii) hyperchromic effect:
This effect involves an increase in the intensity of absorption and is usually bought about by introduction
of an auxochrome for example, introduction of methyl group in position 2 of pyridine increases Ɛmax
(λmax to 262nm) from 2750 to 3560 (λmax 262nm) for ᴧ-ᴧ* transition
(iv) hypochromic effect
it involves a decrease in intensity of absorption and is bought by groups which are
able to distort the geometry of the molecule, for example, when a methyl group is
introduced in position 2 of biphenyl group hypochromic effect occurs because of
distortion causes by methyl group

59. What is chromophore & auxochrome? Give two examples of each term
Chromophore:
Chromophore or chromophoric group is a group or part of a molecule, responsible for characteristic
absorption at a wavelength.
Eg: -N=N-Cl,
>C=C<.
Chromophores are covalently unsaturated.
Auxochrome:
Auxochromes are co-ordinately saturated/ unsaturated. They do not have any characteristic absorption on
their own but can modify the absorption of chromophore.
Eg: -OH
-NH2
Halogens
They have nonbonding valence electrons which do not absorb above 200nm.
60. What is the effect of polar& Non-polar solvent on π-π* transition of alkenes ? Give one example of
each case with λmax and Σ values..
The absorption band moves to a longer wavelength by increasing the polarity of the solvent. The dipole-
dipole interactions with the solvent molecules lower the energy of the excited state more than that of the
ground state. Hence the value of absorption maximum in ethanol will be greater than that observed in
hexane.
 ᴧ* B
D

CD>AB
ᴧ A
Non polar
Polar
c solvent
The most commonly employed solvent is 95% ethanol. It is cheap has good dissolving power and doesnot
absorb radiation above 210nm. In other words it is transparent above 210nm. Commercial ethanol should
not be used as its contains some benzene which undergoes absorption in the uv range at about 280nm.
Some other solvents which are transparent above 210nm are n-hexane, cyclohexane ,methanol, water and
ether. Benzene, choloroform and carbon tetrachloride cannot be used because they absorb in the range of
about 240-280 nm.

61. Depict their energy diagram with respect to sigma bond , π-bond & non-bonding electrons on
absorption of UV energy.

The energy required for excitation for different transitions are: nλ* < λλ*<nϭ*<ϭϭ*.
Of these transitions nλ* required lowest energy and ϭϭ* requires the highest energy for excitation in
the UV region.
After absorption of UV radiations, these electronic structures have greater or lesser polar character than in
ground state. Some of them exists as biradicals or as activated structures. In general, re-distribution of
electrons within the molecule may take place.
Eg: >C=O  >C+ --O-  Cδ+ - Oδ-
Polar solvents shift nλ* and nϭ* to shorter wavelengths and λλ* to longer wavelengths.

62. What are K bands, R-bands,B-bands & E-bands. Give their significance individually
Λ-λ* transitions give rise to B, E and K bands
Type Due to
B-bands (benzenoid bands) Aromatic and hetero aromatic systems
E-bands (ethylene bands0 Aromatic systems
 K-bands (λ-λ*) Conjugated systems

The energy requirement of this transition is between nϭ* and nλ*. But extended conjugation(addition
of more double/triple bonds) and alkyl substituents shifts the λmax towards lower wavelength
(Bathochromic shift). Also trans isomer of olefin absorbs at longer wavelength with more intensity than cis
isomer (bathocromic shift and hyperchromic shift)
Extended conjugation shifts λmax to such an extend that it falls in the colorimetric region.
Eg; plant pigments like beta carotene , lycophene etc

63. Explain why UV/Visible Spectroscopy is widely applicable in pharmacy

1. Qualitative Analysis
a) Detection of impurities:
To limit the presence of impurities, we can use UV spectrophotometric measurements. Additional peaks
can be due to impurities in the sample and can be compared with that of standard raw material.
Also by absorbance measurements at specific wavelengths, the impurities can be detected.
b) Structural elucidation of organic compounds:
The presence or absence of unsaturation, the presence of hetero atoms like S, O, N or halogens can be
determined.
e) Structural analysis of organic compounds:
Some of the aspects of structural analysis are already discussed under electronic transition, like
effect of conjugation, alkyl substitution etc.
(i) Effect of conjugation
(ii) Effect of geometric isomerism
(iii) Effect of cross conjugation
(v) Effect of alkyl conjugation
(vi) No; of rings
2. Quantitative Analysis
(i) simultaneous multicomponent analysis
(ii) derivative spectrophotometric method
(iii) difference spectrophotometric method
3. Determination of molecular weight
4. Determination of dissociation constant of acids and bases
5. Chemical kinetics:
 Kinetics of a reaction can also be studied using UV spectroscopy. The UV radiation is passed
through the reaction cell and the absorbance changes the follows.
6. Keto-enol tautomerism:
Keto and enol forms of a substance have different UV absorption pattern. Using this principle,
the percentage of keto and enol form in a mixture, the presence of enol in ether or alcohols can
be calculated.
7. Quantitative analysis of pharmaceutical substances:
Many drugs either in the form of raw material or in the form of formulation can be conveniently
assayed by making a suitable solution of the drug in a solvent and measuring the absorbance at
specific wavelength.
8. Assay of medicinal substances

64. Give any four Important application of UV & Visible absorption spectroscopy
1.QUANTITATIVE ANALYSIS
determination is based on Beer Lambert's law
A=log I0/It =ABC

DIFFERENT METHODS FOR QUANTITATIVE DETERMINATION

use of calibration graph

use of standard absorptivity value
single or double point standardization
USE OF CALIBRATION GRAPH
 wavelength of maximum absorption is selected
 absorbance is measured for different concentration of the sample solution
USE OF STANDARD ABSORPTIVITY VALUE
A(1%1cm) or E value avoids the need to prepare a standard solution of the reference substance
A=A1%1cm bC

C=A / A1%1cm b
SINGLE -POINT STANDARDISATION
it involves the measurement of the absorbance of a sample solution and a standard solution
 CTEST= (ATEST X CSTD ) / ASTD
DOUBLE-POINT STANDARDISATOIN
Absorbance of the two standard solutions and sample are measured
Ctest = [Atest-Astd1][Cstd1-Cstd2]+Cstd1 [Astd1-Astd2]
Astd1 - Astd2
2.CHARGE TRANSFER TRANSITIONS
 Iodine + Benzene = Charge transfer complex
 The molar absorptivity is very high .
 Absorption band is due to the electronic transitions occurred between donor and the acceptor.
3.MOLECULAR WEIGHT DETERMINATION
Amine Amine picrate
c = A A = absorbance
a X t c = conc: in gm/l
a = absorptivity in l/gm cm

Mol:wt m = E E = 13,400
a a = absorptivity in litre/gm cm
From c & w (wt: of amine picrate) molecular weight can be calculated
5. KINETIC ASSAY
 UV spectroscopy can be used to study the kinetics of a reaction
 One of the reactant or product should exhibit a suitable absorption in the uv region
 Reactions with half lives down to milliseconds can be studied

65. How do you determine the amount of paracetamol in a given tablet according to IP by means of UV
using 1cm cell (a=0.715 at 257nm)
66. Write a short note on ORD & give its applications.
Optical rotatory dispersion is the variation in the optical rotation of a substance with a change in the
wavelength of light.
Optical rotatory dispersion can be used to find the absolute configuration of metal complexes.
Measuring optical rotation as a function of wavelength is termed Optical rotatory dispersion (ORD)
spectroscopy
FUNDAMENTAL PRINCIPLES OF ORD:
• Plane/Linearly polarized light.
• Optical activity.
• Specific rotation.
• Circular Birefringence/Optical Rotation
APPLICATIONS OF ORD
• Determination of optically active substance such as amino
acids, polypeptides, proteins, steroids, antibiotics, terpenes.
• Stereochemistry of Aliphatic amino acids: Aliphatic amino
acids show a unique cotton effect. α -amino acids of levo
configuration show positive effect around 215nm while dextro
enantiomers show negative effect.
• Stereochemistry of Steroids: In one form the specific rotation
increases with decreasing wave length(positive curve) and in
the other form the specific rotation decreases with increasing wave length( negative curve) .
 Quantitative Analysis: specific rotation is a good measure of concentration.
 Determination of Absolute configuration.
 Conformational studies example :(+) 3 methyl cyclohexanone.
 Equilibrium studies: If an optically active chromophore takes
 part in a reaction the extent of reaction can be observed by means of cotton effect.
67. Describe why UV/Visible spectrometry is widely used for assay of a drug sample than other methods.
The determination of an analyte’s concentration based on its absorption of ultraviolet or visible radiation is
one of the most
frequently quantitative analytical methods. One reason for its popularity is that many organic and inorganic
compounds
have strong absorption bands in the UV/Vis region of the electromagnetic spectrum.
Environmental Applications
Methods for the analysis of waters and wastewaters relying on the absorption of UV/Vis radiation are
among some of the most frequently employed analytical methods.
Clinical Applications
UV/Vis molecular absorption is one of the most commonly employed techniques for the analysis of
clinical samples, several examples of which are listed in Table below. The analysis of clinical samples is
often complicated by the complexity of the sample matrix, which may contribute a significant background
absorption at the desired wavelength
Industrial Analysis
 UV/Vis molecular absorption is used for the analysis of a diverse array of industrial samples, including
pharmaceuticals, food, paint, glass, and metals.
• In many cases the methods are Products that have been analyzed in this fashion include antibiotics,
hormones, vitamins, and analgesics.
• One example of the use of UV absorption is in determining the purity of aspirin tablets
Forensic Applications
UV/Vis molecular absorption is routinely used in the analysis of narcotics and for drug testing.
• One interesting forensic application is the determination of blood alcohol using the Breathalyzer test. In
this test a 52.5-mL breath sample is bubbled through an acidified solution of K2Cr2O7. Any ethanol
present in the breath sample is oxidized by the dichromate, producing acetic acid and Cr3+ as products.
Qualitative Applications
The energy at which the absorption occurs, as well as the intensity of the absorption, is determined by the
chemical environment of the absorbing moiety.
For example, benzene has several ultraviolet absorption bands due to p - p* transitions. The position and
intensity of two of these bands, 203.5 nm (e = 7400) and 254 nm (e = 204), are very sensitive to
substitution. For benzoic acid, in which a carboxylic acid group replaces one of the aromatic hydrogens,
the two bands shift to 230 nm (e = 11,600) and 273 nm (e = 970). Several rules have been developed to aid
in correlating UV/Vis absorption bands to chemical structure.
68. What is the minimum requirements for a molecules to show I.R bands. State selection rule for
exhibiting IR Vibrations
REQUIREMENT FOR IR RADIATION ABSORPTION
1. correct wave length of radiation:-
EX: HCI has natural frequency of vibration that is about 2890cm-1.
2. ELECTRIC DIPOLE:
A molecule is said to have electric dipole when there is a slightly positive and negative charges on it’s
component system( rate of vibration more)
Diatomic mole. Like O2 & N2 don’t posses electric dipole hence cannot be excited by radiation and hence
no IR absorption spectra.

Matter Mode

State Type Translation Rotation Vibration

Gas Atomic Yes No No
Molecular Yes Yes Yes
Liquid Atomic Yes No No
Molecular Yes Yes Yes
Solid Atomic No No Yes
 Molecular No No Yes

Types of Vibrations
Vibrations Min no; of atoms How are atoms What changes?
connected
Stretching 2 Bond length
Bending 3 Bond angle

Internal rotation 4 Torsion angle( the angle

between 2 bonds)
v

NUMBER OF VIBRATIONAL MODES

• A molecule can vibrate in many ways, and each way is
called a vibrational mode.

•If a molecule contains ‘N’ atoms, total number of vibrational modes

• For linear molecule it is (3N-5)
• For non linear molecule it is (3N-6)
•Eg: H2O, a non-linear molecule, will have 3 × 3 – 6 = 3 degrees of vibrational freedom, or modes.

VIBRATIONAL FREQUENCY
occurs when atoms in a molecule are in periodic motion while the molecule as a whole has constant
translational and rotational motion.
• The frequency of the periodic motion is known as a vibration frequency.
• The value of stretching vibrational frequency of a bond can be calculated by the application
of hooke’s law.
ν/c = ν¯ = 1/2пc[k/m1m2/m1+m2]1/2
= 1/2пc√k/µ

Where, µ→reduced mass

m1&m2 →masses of the atoms k →force constant

c →velocity of radiation
69. What are the different sampling techniques for mounting a sample in the form of a solid, thin film,
liquid or gas in the beam of IR spectrometer.
SAMPLING OF SOLIDS:
Generally 4 techniques are employed for preparing solid samples:
1. Solids run in solution.
2. Solid Films.
3. Mull technique.
4. Pressed pellet technique
SOLIDS RUN IN SOLUTION
 Solids may be dissolved in non-aqueous inert solvent and a drop of this solution is placed on
an alkali metal disc and solvent is allowed to evaporate, leaving a thin film of solute (or the entire
solution is placed in a liquid sample cell) which is then mounted in spectrometer.
 If the solution of solid can be prepared in a suitable solvent then the solution is run in
concentration of cells for liquids.
Some solvents used are chloroform, carbon tetrachloride, acetone, Cyclohexane etc.
Demerit:
 This method can’t be used for all solids because suitable solvents are limited in number & there is
no single solvent which is transparent throughout IR region.
SOLID FILMS
• If a solid is polymer resins & amorphous solids, the sample is dissolved in any reasonable volatile
solvent & this solution is poured on a rock salt plate (Nacl or KBr) & solvent is evaporated
by gentle heating.
If solid is non-crystalline, a thin homogenous film is deposited on the plate which can be mounted and
scanned directly.
• Sometimes polymers can be “hot pressed” onto plates.
Merit and Demerit:
• This method is useful for rapid qualitative analysis but becomes useless for carrying out quantitative
analysis.
MULL TECHNIQUE:
• In this technique a small quantity of sample is thoroughly ground in a clean mortar until the powder is
very fine.
After grinding, the mulling agent (mineral oil or Nujol - heavy paraffin oil ) is introduced in small
quantities just sufficient to take up the powder (mixture approximates the consistency of a
toothpaste).
•The mixture is then transferred to the mull plates & the plates are squeezed together to adjust the
thickness of the sample between IR transmitting windows.
• This is then mounted in a path of IR beam and the spectrum is run.
Demerit:
•Although Nujol is transparent throughout IR region, yet it has a disadvantage that it has absorption
maxima at 2915,1462, 1376 & 719 cm-1.
•So when IR spectrum of solid sample is taken in Nujol mull, absorption bands of solid sample that happen
to coincide with the absorption bands of the Nujol mull will be hidden (but others will be clearly seen in IR
spectrum) and then interferes with the absorption of the sample.
This interference can be avoided by using Hexachlorobutadiene in combination with
nujol which absorbs in regions 1630-1510 cm -1, 1200-1140 cm-1, 1010-760 cm-1 and thus permits the
recording of IR spectra of only the sample.
• This method is good for qualitative analysis but not for quantitative analysis.
Pressed pellet technique:
• In this technique a small amount of finely ground solid sample is intimately mixed with about 100
times its weight of powdered Potassium bromide, in a vibrating ball mill.
•This finely ground mixture is then pressed under very high pressure (25000 p sig) in evacuable die or
minipress to form a small pellet (about 1-2 mm thick and 1cm in diameter).
 The resulting pellet is transparent to IR radiation and is run as such
GASES
Gas sample cell –
 Cylindrical glass body
 Surfaces in the light path are made of KBr, NaCl.
 Cell length-10cm
 To end of the cell are attached disk of appropriate window material with wax epoxy cement or
pressure plate.
 The beam is directed to 90degree angle from its normal direction
and thus longer path length is provide.
 The use of longer cell even with effective path length as long as
1km.
 For some special application-
Example: Corrosive gases or vapors metals such as stainless
 steel, nickel are used for cell bodies.
 The gas sample is carried from the vessel to the detector site, where
it flows through a tube only to return, unchanged, to the sterilizer.
 The IR source is placed at one end of the tube and the IR detector at the other.
 The IR beam passes through the gas sample, and the detector collects the spectral reading and
transmits the data to a microprocessor .
 Gas can be conducted through the beam path, or the beam can be reflected through a window in the
sterilizer so that it crosses the headspace gas and returns to the detector.
LIQUIDS
 Liquid samples are used at room temperature.
There IR spectra are obtained directly
 To obtain the transmittance in the range of 15%-70% ,select-
 Sample concentration
 Path length should be selected
Parameters:
 Rectangular cell(NaCl, KBr)
 Cell thickness-0.02mm in the case of most neat liquids
 Concentration-10%
 Cell length-0.1 mm
 The solvent selected must be transparent
Solvents used:
Acetone Methane
Hexane Ethane
Benzene Dioxene
CCl2 Tetrachloro ethylene

Method
• Put it into rectangular cells of KBr, NaCl etc.
• I R spectra obtained.
• Sample thickness … such that transmittance lies
between 15 – 20 % i.e., 0.015 – 0.05 mm in thickness.
• For double beam, matched cells are generally employed
• One cell contains sample while other has solvent used in sample.
 • Matched cells should be of same thickness, protect
from moisture.
Neat liquids can be analyzed between salt plates made of NaCl or KBr.
Non- or low volatility liquids can be analyzed by placing a drop of the sample onto
specially prepared thin polyethylene polymer substrates.
Use a fixed pathlength cell.
Determine pathlength when empty by counting interference fringes

70. Why carbon-di-oxide shows some IR bands though the molecule as a whole does not possess any
dipole moment?

Fundamental Vibrational modes of CO2 (3 atoms –Linear)

• Fundamental Vibrational modes (degrees of freedom) = 3 x 3 – 5 = 4

• These normal modes of vibration:
–1
• The asymmetrical stretch of CO gives a strong band in the IR at 2350 cm (may noticed
2
in samples due to presence of CO2in the atmosphere).

• The two scissoring or bending vibrations are equivalent and therefore, have the same
-1.
frequency and are said to be degenerate , appearing in an IR spectrum at 666 cm
71. Give approximate stretching wave number values for the following groups C=O, C=N, C=C, C ≡N.
group Range (cm-1)
C=O Stretching (ketone) 1705-1725
C=O Stretching (aldehyde) 1720-1740
C=O stretching (ester) 1735-1750
C=O stretching (acid) 1700-1725
C=C Stretching (aromatic) 1450-1600
C=C stretching (alkene) 1680-1620
C=C stretching (alkyne) 2100-2200
C=N Stretching 1630-1690
, C ≡N Stretching 2240-2260

72. What is the basic requirement for a nucleus to exhibit NMR phenomenon?
• Nuclear magnetic resonance (NMR) spectroscopy is based on the measurement of absorption of
electromagnetic radiation in the radio-frequency region of roughly 4 to 900 MHz.
• Subatomic particles like electrons, protons and neutrons are associated with ‘spin’- a fundamental
property like charge or mass.
• In the case of nuclei with even number of protons and neutrons, individual spins are paired and the
overall spin becomes zero. However, there are many cases such as 1H and 13C, where the nuclei
possess a net spin, which is important in Nuclear Magnetic Resonance (NMR) Spectroscopy. Spin
of nuclei could be correlated with the number of protons and neutrons as:
• When there are even number of protons and even number of neutrons in the nucleus, the net spin
is equal to zero.
• When there are odd number of neutrons and odd number of protons in the nucleus, it will have an
integer spin (i.e. 1, 2, 3)
• If the sum of the number of neutrons and the number of protons is odd number, the nucleus will
have a half-integer spin (i.e. 1/2, 3/2, 5/2).

=
>
 • It is used to study a wide variety of nuclei:
• 1H
• 13C
• 15N
• 19F
• 31P

73. What are the main advantages of mass spectrometry.

 In mass spectrometry, molecule is broken down into the fragments, which are separated on the
basis of their mass to charge ratio m/z; these fragments on reaching the detector give a response
peak which is recorded in the form of mass spectrum.

 Advantages

 Mass Spectrometry- It is an analytical spectroscopic tool primarily concerned with the separation
of molecular or atomic species according to their mass.
 To measure relative mol masses with very high accuracy.
 To detect with in a molecule the places at which it perform to fragment.
 Only Pico molar concentrations required.
 Within an accuracy of 0.01% of total weight of sample and within 5 ppm for small organic
molecule
• Gives molecular mass and also the fragmentation pattern of the sample.
• Extensive fragmentation and consequent large number of peaks gives structural information.
• Gives reproducible mass spectra.

74. Calculate (a) frequency (b) wave number) for the radiation of wavelength 530nm.(c=38x108 m/s)
Frequency =c/ Wavelength = (3 x108)/530 = 5.66037 x 105
Wave number =Frequency/c = (5.66037 x 105)/(3 x108)=1.88679 x 10-3
75. Calculate the wavelength corresponding to a radiation in which the energy of photon 5x10-22 J
Solution:
1) Determine the frequency:
E = hν
5 x 10-22 J = (6.626 x 10¯34 J s) (x)
x = 0.754 x 10 12
2) Determine the wavelength:
λν = c
(x) (0.754 x 10 12) = 3.00 x 108 m/s
X, wavelength =592 x 10 -4 m

76. Calculate the frequency of a radiation of wavelength 700nm

Convert nm to m:

700.0 nm = 700.0 x 10¯9 m = 7.000 x 10¯7 m

2) Determine the frequency:

λν = c

(7.000 x 10¯7 m) (x) = 3.00 x 108 m/s

X, frequency = 4.2 x 1014 s¯1

77. Give reason why you will get absorption curve rather than peak in Ultraviolet region

Every molecule has some absorption for UV rays. And at a particular wavelength of UV rays, there is a
maximum absorption. This is called as λmax. This λmax is a characteristic (specific) for every molecule.
When we scan the sample using the UV rays (usually between 200 nm-400 nm), the sample absorbs the
UV rays accordingly and gives a broad spectrum. This means that the sample absorbs all the different
wavelengths of the UV rays and gives an absorption value for each wavelength absorbed, along with the
wavelength at which maximum absorption took place i.e., λmax. So we get an intense peak for the λmax
and that peak denotes the λmax of the sample.
The Broad Peak in UV-VIS spectra:

The Gap between rotational and vibrational levels continuously keep changing. And UV-VIS spectroscopy
is generated by ELECTRONIC TRANSITION which is associated with simultaneous rotational and
vibrational transitions as well. The result is that absorption takes place over a range of wavelengths rather
than at one fixed one. Hence we get a Broad Peak which looks like a typical Gaussian Distribution in a
Absorbance vs Wavelength plot

78. What are the factors affecting.fluorescence and phosphorescence?

*refer Q52 (5 marks)
79. What is Quenching? Explain various types of quenching with suitable examples?
*refer Q44 (2marks)
80. What are self quenching and true quenching
*refer Q44 for self quenching
True quenching
*explain chemical static and collision quenching from Q44
81. Show the relationship between fluorescence intensity and concentration. Describe any four factors that
influence fluorescence intensity?
Fluorescence intensity is directly proportional to concentration of the substance.
But this is true in low concentrations (micro gram or nano gram/ml). But in high concentration (mg /ml) it
does not obey linearity.
*refer diagram of self quenching in Q44 (5marks)
At high concentrations, often we can see deviations from linearity i.e. when concentration increases,
fluorescence intensity does not increase proportionally. This is because of a phenomenon called as self
quenching or conventional quenching. This is because the emitterd radiation before falling on detector, is
being absorbed and re-emitted by adjacent molecules, which leads to internal circulation of radiation (or
energy). Hence this leads to wrong interpretation of results.
*refer Q52(5marks) for other factors

10 mark questions

1. Discuss the principle & development techniques used in column chromatography Add a note on the
adsorbents mobile phase & detection systems in column chromatography.
Principle;
A solid stationery phase and a liquid mobile phase is used and the principal of separation is
adsorption. When a mixture of components dissolved in mobile phase is introduced into the
column, the individual components move with different rates depending upon their relative
affinities. The compouns with lesser affinity towards the stationery phase(adsorbent) moves faster
and hence it is eluted out of the column first.
The one with greater affinity towards the stationery phase(adsorbent) moves slower down the
column and hence it is eluted out later. Thus the components are separated.
Development techniques:
*refer Q10(5marks)
Adsorebents *refer Q8 (5marks)
Mobile phase
 Mobile phase : (Solvent) Different mobile phases are used in increasing order of polarity. Mobile
phase used during the development of chromatogram. Apart from the use of solvent as mobile
phase, a solvent is useful to transfer the mixture to be separated in to the column.
 
 Functions of a Mobile Phase are :
 To introduce the mixture to the column – As solvent.
 To develop the zones of separation – As developing agents.
 To remove pure component out of the column – As eluent.
 A solvent system arranged in an increasing order of polarilty is called as Eluotropic series.
 Increasing order of Polarity : Petroleum ether > CCl4 > Cyclohexane > Diethyl ether > Benzene
> Ethyl acetate > Ethyl alcohol > Methyl alcohol > Water > Formamide.
 Detection of Components :
The detection of coloured components can be done visually. Different coloured bands move down
the column which can be collected separately. But for colourless compound, the techniques
depends upon the properties of components.
Different properties which can be used are:
i) absorption of light (uv/vis) using UV/Vis detector
ii) flourscence or light emission characteristics using fluorescence detector
iii) by using flame ionisation detector
iv) refractive index detector- based on the refractive index difference between the mobile phase
and mobile phase+component
v) Evaporation of the solvent and weighing the residue
vi) by monitoring the fractions by thin layer chromatography
any one of the above techniques can be used for the detection of compounds so that it can be used
for qualitative analysis and for isolation of compounds.
2. Write a note on development techniques in column chromatography.
*refer Q10 (5 marks)
Other development techniques
(1) Frontal.
(2) Elution.
(3) Displacement.

Frontal analysis : Components to be separated is distributed in large excess of mobile phase. No

more fresh mobile phase is used after commencement of development. The entire portion of the
liquid which contains dissolved components of mixture is added.
Draw back : (i) Incomplete separation, (ii) Components may be retained within the column
without being eluted.
 
Elution Analysis : Mixture to be separated is dissolved in the solvent and transffered into the
column. Elution analysis can complete the separation and eluate containing components can be
recovered within a short duration of time.
Advantage : (i) Complete separation of component, (ii) This is widely used development.
 
Displacement : If a component can not be eluted on account of strong adsorption behaviour, then a
displacing substance to be added in to the column to elute strongly adsorbing substance as it has
higher affinity towards S.P. than substances to be eluted.
 3. Describe the preparation, activation of plates &adsorbents used in the TLC & write its application.
*refer Q13(5marks) for preparation, activation of plates

1) Stationary phase:
 A large number of coating materials can be used are silica gel, alumina, calcium phosphate,
magnesium trisilicate, polyamide, acetylated cellulose, ferric oxide hydrate, etc.
 Some adsorbents do not adhere directly to the glass plates satisfactorily. So, binders like gypsum or
starch are added to the adsorbent.
 In some cases, fluorescent indicators like zinc silicate are added to the adsorbent to facilitate easy
detection of components.
 In selecting, the adsorbent as a stationary phase, various properties of the adsorbent such as
solubility, acidic, basic, or amphoteric nature is to be identified.
 A chemical reaction should not occur between the adsorbents or binders.

NAME COMPOSITION
Silica gel H Silica gel without binder
Silica gel G Silica gel + CaSO4
Silica gel GF Silica gel + Binder + fluorescent indicator
Alumina Al203 Without Binder
Al203 G Al203 + Binder
Cellulose powder Cellulose with/without Binder
Kieselguhr G Diatomaceous earth + binder
Polyamide powder Polyamide
Fuller’s earth Hydrous magnesium alumina
Magnesium Silicate magnesol

Coating Materials Used in TLC:

Adsorben Acidic Activity Separatory Components
t or Basic Mechanis to be
m separated
Silica gel Acidic Active Adsorption Acidic and
partition neutral
substances
 Alumina Basic Active Adsorption Basic and
partition Neutral
Kiesleguhr Neutral Inactive Partition Strongly
hydrophilic
substances
Cellulose Neutral None Partition Water
powder soluble
compounds

Application:

 Separation of mixture of drug of chemical, biological, plant origin.

 Separation of carbohydrates, vitamin, antibiotics, proteins, etc.
 Identification of drug. Ex : Amoxicillin, Levodopa.
 Detection of foreign substances.
 To detect the decomposition products of drug.

4. Define paper chromatography? What are the modes of development of paper chromatography &
Enumerate the application of paper chromatography
Definition:-
 Paper chromatography is the technique in which the separation of an unknown substance is mainly
carried out by the flow of solvents on the specially designed chromatographic paper.
 In this case the solvent goes up by capillary action.
 The separation is effected by differential migration of the substance due to difference in
distribution coefficients.
*refer Q14 (5marks) for development techniques
APPLICATIONS
•Separation of mixtures of drugs

•Separation of carbohydrates, vitamins, antibiotics, proteins, etc.

•Identification of drugs

•Identification of impurities

•Analysis of metabolites of drugs in blood , urine

 To check the purity of the sample obtained by other methods.
 To identify the pure substances.
 To identify the nature of the by products.
 To isolate purify, identify the organic compounds.
 To test the stability of the decomposition products of the medicinal substances.
 To separate complex mixture of drugs, metal ions, amino acids.
ADVANTAGES OF P.C
Simple ,rapid ,inexpensive ,excellent resolving
5. Give a detailed account of principle, classification of Ion-Exchange process in pharmaceutical
analysis
ION EXCHANGE CHROMATOGRAPHY
» The process by which a mixture of similar charged ions can be separated by using an ion exchange resin

 Ion exchange resin exchanges ions according to their relative affinities.

 There is a reversible exchange or similar charged ions
 Mostly similar charged ions like cations or anions can be separated by this technique

PRINCIPLE
Reversible exchange of ions b/w ions present in the solution & ion exchange resin
Cation Exchange:

Solid – H+ + M+ Solid - M+ + H+ (Solution)

(Solution)

Cation Exchange:

Solid – OH- + M- Solid – M- + OH- (Solution)

(Solution)

Ion Exchange Mechanism

• Surface exchange of ions – in homogenous solution

•Diffusion of ion through the matrix structure of the exchanger to the exchanger site

• Exchange of ions at the exchange site – cation and anion exchange

• Diffusion of the exchanged ion through the exchanger to surface

• Selective desorption – by eluant and diffusion of molecule to solution

The exchange depends on charge and size

Na+ < Ca2+ < Al3+

Li+ < Na+ < K + < Cs+ < Mg2+ < Cu2+

Ion Exchange Resins

• Resins
• Organic or inorganic polymer used to exchange cations or anions from a solution phase
General Structure
• Polymer backbone not involved in bonding
• Functional group for complexing anion or cation
CLASSIFICATION OF RESINS
1. Synthetic – Organic and inorganic exchangers
2. Natural organic ion exchangers

 Synthetic
 Inorganic – Alumino-silicate, TiO2, ThO2….. & Trivalent and tetravalent metal
hydroxides.
 Organic - cross linked polymers with functional groups – cation and anion
Polystyrene (sites for exchangeable functional groups), Divinyl benzene (Cross linking agent)-
offers stability
 Natural – sulphonated -coal, paper , cotton & clay, dolomite, etc.,
According to the chemical nature they classified as-
1. Strong cation exchange resin  SO3H
2. Weak cation exchange resin  COOH, OH, SH, PO3H2
3. Strong anion exchange resin  N+R3, NR2
4. Weak anion exchange resin  NHR, NH2
Common ion exchange resins
Class of Nature pH applications
resin range

Cation- Sulfonated 1-14 Fractionation of cations, inorganic separations, peptides,

strong polystyrene aminoacids, B vitamins

Cation Carboxylic 5-14 Fractionation of cations, biochemical separations, org bases,

weak methacrylate antibiotics

Organic resin groups

linkage group

SO3H CH2Cl
Common ion exchange resins
 Class or resin Nature pH Applications
range
Anion Quaternary ammonium 0-12 Fractionation of anions
polystyrene
–
strong Alkaloids, vitamins,
fattyacids

Anion- Polyamine polystyrene 0-9 Fractionation of anionic

or phenol
weak complexes, anions of diff valency vitamins,
aminoacids
H-CHO

Inorganic Resins

• More formalized structures

 Silicates (SiO4)
 Alumina (AlO4)
 Both tetrahedral
 Can be combined
* (Ca,Na)(Si4Al2O12).6H2O
 Aluminosilicates
* zeolite, montmorillonites
* Cation exchangers
* Can be synthesized
 Zirconium, Tin- phosphate
Inorganic ion exchange resin
Easy to synthesis

Metal salt with phosphate

Precipitate forms
 Grind and sieve

• Zr – Zirconium can be replaced by other tetravalent metalsSn, Th, U

Required properties
» It must be chemically stable
» It should be insoluble in common solvents
» It should have a sufficient degree of cross linking
» The swollen resin must be denser than water
» Should be sufficiently hydrophilic for diffusion of ions
» It must contain sufficient no. of ion exchange groups
» It must contain suitable size and shape
» Should have ability for regeneration and reuse

Structural types of ion exchange resins

a) Pellicular type with ion exchange resins: Pellicular – membrane or thin film
» 30 - 40µ with 1-2µ film thickness
» Very low exchange capacity
» Ion exchange efficiency: 0.01 – 0.1 meq/g of ion exchange resin. b) Porous resin coated with
exchanger beads
» Size 5 - 10µ
» Totally Porous & highly efficient
c) Macroreticular resin bead
» A reticular network of the resin is seen superficially on the resin beads
» They are not highly efficient & have very low exchange capacities
d) Surface sulfonated & bonded electrostatically with anion exchanger
» They are less efficient & have low exchange capacity.
» Exchange capacity is 0.02meq/g of exchange resin.

Physical properties of ion exchange resins

1. Cross linking:
It affects swelling & strength & solubility
2. Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte conc.
3. Particle size & Porosity
↑surface area & ↓particle size will ↑rate of ion exchange
Particle size 50-100 mesh / 100-200 mesh
Regeneration

 Cation exchange resin are regenerated by treatment with acid, then washing with water

 Anion exchange resin are regenerated by treatment with NaOH, then washing with water until
neutral

6. What are the Ion-Exchange resins? Explain Mechanism of Ion-Exchange process and application of
Ion-Exchange chromatography
ION EXCHANGE CHROMATOGRAPHY

» The process by which a mixture of similar charged ions can be

separated by using an ion exchange resin
 Ion exchange resin exchanges ions according to their relative affinities.
 There is a reversible exchange or similar charged ions
 Mostly similar charged ions like cations or anions can be separated by this technique

PRINCIPLE AND MECHANISM

*refer Q5 (10 marks)

APPLICATIONS
 Softening of water
 Demineralization or deionization of water
 purification of solutions free from ionic impurities
 separation of inorganic ions
 separation of sugars, amino acids
  ion exchange column in HPLC
 Organic separations: mixture of pharmaceutical compounds can be separated
 Biochemical separations like isolation of drugs or metabolites from blood, urine etc.
 Conc of ionic solutions

7. Explain with a neat diagram any three detectors used in Gas Chromatography
*refer Q 23 for 2 detectors
3) Flame ionisation detector

The ionisation detectors are based upon the electrical conductivity of carrier gases. At normal temperature
and pressure, gases act as insulators, but become conductive if ions are present.
The carrier gas used with this type of detector can be hydrogen. If the carrier gas is either nitrogen or
argon, it can be mixed with hydrogen and reach the burner tip made up of platinum capillary, which act as
one electrode (Cathode).
The anode is silver gauze placed little above the burner tip. When pure carrier gas alone passes, there is no
ionisation and no current flows. When a component emerges from the column, number of ions are
produced because of ionisation by the thermal energy of the flame.
This causes a potential difference and causes a flow of current which is amplified and recorded as signal.
Advantages:
1. this detector is extremely sensitive and background noise is low. Hence microgram quantities of
the solute can be detected
2. stable and insensitive to small changes in the flow rate of carrier gas and water vapour.
3. Responds to most of the organic compounds
4. Linearity is excellant
8. Describe the construction and working of a Gas Chromatography? Emphasize on the ideal
characteristics of stationary phases and mobile phases used in Gas Liquid Chromatography.
DEFINITION
Gas chromatography is basically a separation technique in which the compounds of a vaporized sample
are separated and fractionated as a consequence of partition between a mobile gaseous phase and
stationary phase held in column. The partition takes place between a gas and a liquid or gas and solid.
Construction

The apparatus consists of the following main components:

1. A tank of carrier gas
2. An injection port of the sample
3. The column
4. Detector with appropriate read-out

Carrier Gas
The carrier gas is allowed to flow through the system (carrying the sample in a vapour state). The
mobile phase (carrier gas) in GLC is usually helium or nitrogen, although carbon dioxide and
hydrogen from tank sources have also been tried.
The most important requirements of a carrier gas are:
1. It should be inert.
2. It should be available at low cost, because large quantities are used.
3. It should allow the detector to respond in an adequate manner.
4. Purifiers (traps) to remove O2, H2O and organic traces
In spite of its cost, helium is far the most common carrier gas. There are 2 main reasons for this
choice:
1. One of the most useful detectors depends on thermal conductivity of the gas, a property
that is much greater for hydrogen and helium than for other gases.
2. The other advantage, also shared by hydrogen, is that because of its low density, greater
flow rates can be employed. This reduces the time required for separation.

Thermal Conductivities of some gases (cal. s-1 .cm-1 .deg-1)

Gas Thermal Gas Thermal

Conductivity Conductivity
H2 44.5 O2 6.35
He 36.0 N2 6.24
Ne 11.6 CO2 3.96
CH4 8.18 CH3OH 3.68

The carrier gas flow can be quantified by either linear velocity, expressed in cm/sec, or volumetric
flow rate, expressed in mL/min. The linear velocity is independent of the column diameter while the
flow rate is dependent on the column diameter.

There are 2 main disadvantages of hydrogen.

1. Its fire and explosion hazards &
2. Its reactivity towards reducible or unsaturated sample compounds.

Hydrogen has a specific advantage, however, in that it can be generated electrolytically.

Other gases, such as argon or nitrogen, are also required by certain detectors.

 A high pressure gas cylinder (in which carrier gas is filled in compressed form) is used as a
carrier gas reservoir.
 The cylinder is also attached with a pressure regulator to reduce and control the gas flow to
the separation column.
 A soap bubble meter is also an accurate device for reproducing the rate of the carrier gas.
 In soap bubble meter, a soap film is formed in the path of the gas, when a rubber bulb
containing an aqueous solution of soap or detergent is squeezed.
  The time required for the soap film to move between 2 graduations on the burette is then
measured and converted to flow rate.
 The carrier gas system also contains a molecular sieve to remove water and other impurities.

 The Injection Port

 For optimum column efficiency, the sample should not be too large, and should be
introduced onto the column as a "plug" of vapor - slow injection of large samples causes
band broadening and loss of resolution.
 The most common injection method is where a microsyringe is used to inject sample
through a rubber septum into a flash vapouriser port at the head of the column.
 The temperature of the sample port is usually about 50°C higher than the boiling point of
the least volatile component of the sample.
 For packed columns, sample size ranges from tenths of a microliter up to 20 microliters.
 Capillary columns, on the other hand, need much less sample, typically around 10-3 mL.
For capillary GC, split/splitless injection is used.

Types of injection systems

In gas chromatography two basic types of sampling system are used, those suitable for packed
columns and those designed for open tubular columns.

Packed column injections

In general, the sample injected onto a packed GC column ranges in volume from 0.5ml to 5ml and
usually contains the materials of interest at concentrations ranging from 5%v/v to 10%w/v.
The sample is injected by a hypodermic syringe, through a silicone rubber septum directly into the
column packing or into a flash heater. Although the latter tends to produce broader peaks it also
disperses the sample radially across the column.
The silicone septum is compressed between metal surfaces in such a manner that a hypodermic
needle can pierce it, but when it is withdrawn the hole is closed as a result of the septum
compression and there is no gas leak.
The glass liner prevents the sample coming in contact with the heated metal wall and thus, reduces
the chance of thermal decomposition.

Open Tubular (capillary) Column Injection Systems

Due to the very small sample size that must be placed on narrow bore capillary columns, a split
injection system is necessary, a diagram of which is shown below.
 The basic difference between the two types of injection systems is that the capillary column now
projects into the glass liner and a portion of the carrier gas sweeps past the column inlet to waste.
As the sample passes the column opening, a small fraction is split off and flows directly into the
capillary column, ipso facto this device is called a split injector.
The split ratio is changed by regulating the portion of the carrier gas that flows to waste which is
achieved by an adjustable flow resistance in the waste flow line.
This device is only used for small diameter capillary columns where the charge size is critical.

Split/splitless injection
The injector can be used in one of two modes; split or splitless

When complete injection may be too much for capillary column:

Split injection:

 It uses valves so only 0.1-10% of injected sample reaches column

 It passes through silanized glass wool for complete vaporization and good mixing

For quantitative and trace analysis:

Splitless injection:
valves closed so ~80% of injected sample reaches column

Auto Sampler Injection System

 The injector is a hollow, heated, glass-lined cylinder where the sample is introduced into the GC.
 The temperature of the injector is controlled so that all components in the sample will be
vaporized.
 The glass liner is about 4 inches long and 4 mm internal diameter

GC Oven
 GC ovens incorporate a fan, which ensures uniform heat distribution throughout the oven.
 They can be programmed to produce a constant temperature, isothermal condition or a gradual increase
in temperature.
Columns
The column is the heart of the chromatography. In the column, the different solutes in the vaporized
samples are separated from each other by virtue of their different interaction with the column packing. As
the solutes emerge individually from the end of the column, they enter the detector.
Gas chromatography columns are basically with two designs.

1. Packed columns
2. Open tubular or capillary columns

Packed columns

 Usually 2 to 4 mm I.D. and 1 to 4 meters long and, packed with a suitable adsorbent, are mostly used
for gas analysis
 Constructed from stainless steel or Pyrex glass
 Pyrex glass is favored when thermally labile materials are being separated such as essential oils and
flavor components.
 However, glass has pressure limitations and for long packed columns, stainless steel columns are used
as they can easily tolerate the necessary elevated pressures.
 Pyrex glass columns are formed to the desired shape by coiling at about 700˚C and metal columns by
bending at room temperature
 Glass columns are sometimes treated with an appropriate silanizing reagent to eliminate the surface
hydroxyl groups which can be catalytically active or produce asymmetric peaks.
 Stainless steel columns are usually washed with dilute hydrochloric acid, then extensively with water
followed by methanol, acetone, methylene dichloride and n-hexane. This washing procedure removes
any corrosion products and traces of lubricating agents used in the tube drawing process.

Capillary column

 Made of fused silica (SiO2) coated with polyimide (plastic, 350oC) or Al support
 Typically available in 10 – 100 m in length with a 250 μm inner diameter that has he stationary phase
coated on the internal surface.

They are classified into three types. Namely:

1. Wall-coated open tubular (WCOT)

2. Porous layer open tubular (PLOT)
3. Support-coated open tubular (SCOT).

Wall coated open tubular (WCOT) columns

 Wall-coated columns consist of a capillary tube whose walls are coated with thin uniform film of
liquid stationary phase.

Fused Silica Open Tubular (FSOT) columns

 These have much thinner walls than the glass capillary columns, and are given strength by the
polyimide coating.
 These columns are flexible and can be wound into coils.
 They have the advantages of physical strength, flexibility and low reactivity.

Porous layer open tubular (PLOT) columns

 The PLOT columns are largely used for gas analysis and the separation of low molecular weight
hydrocarbons.
 The external diameter of PLOT columns range from 320 to 530 m with a porous layer that can
be 5 to 50 m thick.
 The inner surface of the column has an embedded layer that contains the stationary phase. Solid
particles are the active stationary phase

Support coated open tubular (SCOT) columns

 The inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth,
onto which the stationary phase has been adsorbed i.e., solid particles coated with stationary liquid
phase
SCOT columns are generally less efficient than WCOT columns
Based on stationary phase used in column, G.C is of 2 types :
a. Gas solid chromatography (GSC)
b. Gas liquid chromatography (GLC).
a. GSC : Mobile phase – gas
Stationary phase – solid
In GSC, when a carrier gas containing analytes is passed through a column containing solid
Stationary phase, the analytes get adsorbed on to the solid Stationary phase & the separation is due
to differences in their adsorptive behavior.

b. GLC : Mobile phase – gas

Stationary phase – liquid
.
In GLC, Stationary phase is liquid that is retained/coated on the surface of an inert solid by
adsorption or chemical bonding.
The main purpose of carrier gas is to transport sample components through the column.
It determines the efficiency of the column, the time of analysis and the sensitivity of a given
detector.
Factors are considered while selecting a carrier gas :
Chemically Inert
Available in pure and stable form
Free from moisture
Should give best column performance consistent with desired speed of analysis
Suitable for detector used
Inexpensive, readily available
Safe in handling
Mobile phases or carrier gases used in Gas liquid chromatography

GAS APPLICATION COMMENTS

Helium General carrier gas or make Expensive

up gas

Nitrogen General carrier gas or make Very cheap but not good for
up gas
capillary columns as it gives a long run
time

Oxygen Combustion gas for FID Not normally used

Argon Carrier gas for TCD ------------

Hydrogen Carrier gas for capillary Cheap & explosive

column & combustion gas
for FID

Air Combustion gas for FID, Cheap & readily available

NPD, FPD

Argon + Make up gas & carrier gas Better linearity & selectivity than N2 but
methane for packed columns with
ECD.
 Gas-Liquid chromatography
In gas liquid chromatography, the fixed phase is a non volatile liquid held as a thin layer on an
inert solid support. The most common support is diatomaceous earth or kieselguhr.
Stationery phase
Stationery phase Nature Temperature maximum in use
Polydimethyl siloxane Non- polar -60 to 320˚
Poly(diphenyl) dimethyl Non-polar bonded phase -60 to 320˚
siloxane
Polycyano propyl phenyl Intermediate polarity Upto 280˚
dimethyl siloxane
Polyalkylene glycol polar 30 to 220˚
Polyethylene glycol polar 50 to 280˚
PEG modified with Polar bonded phase 60 to 200˚
nitroterephthalic acid
Poly bis cyano propyl siloxane Very polar non-bonded phase Upto 250˚

9. Describe instrumentation and application of HPLC

HPLC is a type of liquid chromatography in which we use a pressure of up to 6000 psi to push the
mobile phase through the column and allows the separation of the complex mixture with high
resolution.
INSTRUMENTATION
Mobile Phase Reservoir
Pumping Systems
Sample Injection Systems
Columns
Detectors & recorders.
MOBILE PHASE RESERVOIR
œ Glass/stainless steel vessel
œ 200-1000mL capacity.
œ Filtering unit having porosity 0.45-1u.
œ Degasing unit.
 DEGASING METHODS
 Sparging
 Vaccum pumping
 Heating & stirring
 Sonication
Criteria for solvent selection
 Able to dissolve sample components with no chemical reaction.
 Low viscosity
 Compatible with detection methods
 Readily available in pure form.
 Elution methods
Isocratic elution
 separation with single solvent or solvents having same polarity.
gradient elution
Seperation with two or more solvents of differing polarity used; seperating efficiency is
enhanced.
Pumping systems
Pumps mobile phase through the column at high pressure & at a controlled flow rate
• Generate High pressures upto 6000 psi.
• Constant & reproducible flow
• Pulse free output(without fluctuation)
• Flow rate(0.1-10mL)
• Adaptable to gradient analysis
• Corrosion resistant .
• Easy to dismantle & repair
• Low maintenance cost

 Constant pressure pumps

Direct gas pressure system
Pneumatic intensifier pump
 Constant flow pumps
Syringe/displacement pumps
Reciprocating pumps
Direct gas pressure system
 Pneumatic amplifier system
 Pressure applied to liquid
 = Gas pressure . Area of gas piston
Area of hydraulic piston
 Cheap
 Noisy and pulsatile
 Used for slurry packing of columns as high pressures can be delivered
SYRINGE PUMP.
 Advantage;
 The flow is pulseless
 Flow can be varied by changing motor speed.
 Disadvantage;
 Limited solvent capacity
 Reciprocating pumps
 ~90% of HPLCs
 Advantages:
small internal volume (35- 400 L)
high output pressures (up to 10,000 psi)
 ready adaptability to gradient elution
constant flow rates
 Problem: pulsed flow
Pulse damping
 Multiple piston heads
 Mechanical/physical methods
Eg; diaphragm pump
 Electronic pulse damping
Sample Injection Systems:
◦ Syringe injection.
◦ Stop flow injection.
◦ Valve type injection
Syringe injection
 Easiest & simplest method
 Use micro syringes able to withstand pressures upto 1500 psi
Advantage;
 Simple and cheap
Disadvantage;
 Short septum life
 Blockage
 Low reproducibility
Stop flow injection
 Advantage;
 Simple and cheap
 Disadvantage;
 Low reproducibility
Valve type injectors
Advantages
 Excellent precision
 Compatibility with high pressure
 Easy to use
 Facilitates automatic operation
Chromatographic Column
X Heart of HPLC system
X Usually made up of stainless steel tubing to withstand high pressures upto 6000psi
X Hundreds of packed columns differing in
size and packing are available from
manufacturers
 Column thermostats
◦ maintaining column temperatures constant to a few tenths degree centigrade
◦ column heaters control column temperatures (from ambient to 150 oC)
◦ columns fitted with water jackets fed from a constant temperature bath
Types of column
 Analytical columns
 Microbore columns
 Preparative columns
 Pre column
 Guard column
Analytical column
 Sample used is very low. So recovery of sample for reusing is not done.
 straight column having dimensions of
4.0-8.0 mm(id) X 15cm or 25cm length
 Particle size - 5 or 10 micron
 Contain 40000-60000 plates/m.
Preparative column
 25-50mm (id) X 25 cm (length)
 For preparative works
 Transfer of analytical data to preparative data
Guard column
 Between sample injector and analytical column
 Protects analytical column
 Removes particulates from samples
 Larger sized packing material
 Minimize pressure drop
 Prolongs the life of the more expensive analytical column
 Column packing
 Pellicular packing - Alumina or silica coated
spherical non porous polymer/glass beads
(30-40 µm in diameter) for guard columns
 Porous particle packing- 3-10 micrometer
– silica, alumina, synthetic resins,
polystyrene divinyl benzene.
 Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support
-C18,C8
Extremely stable
Reproducible
TYPES OF DETECTORS
1.Bulk Property Detectors: These respond to mobile phase bulk property with respect to solute
RI, Dielectric constant, density etc.
2. Solute Property Detectors: These respond to some property of solutes
UV absorbance, fluorescence, diffusion current
 UV/Visible detector
 Fluorescence detector
 Refractive index detector
 Evaporative Light Scattering Detector
 Electrochemical detector
 Conductivity detector
 Transport detector
 Radioactive detector
 Optical activity detector
 Hyphenated system
Selection of detectors

Detectors Type of compounds can be detected

Compounds with chromophores, such as aromatic rings or multiple alternating do

bonds.
UV-Vis & PDA
 Fluorescent compounds, usually with fused rings or highly conjugated planar syste
RF

Charged compounds, such as inorganic ions and organic acid.

CDD

ECD For easily oxidized compounds like quinones or amines.

For compounds that do not show characteristics usable by the other detectors, eg. polym
sccharides.
RID & ELSD

Commonly used detectors in HPLC

Ultraviolet (UV)
This type of detector responds to substances that absorb light.
•The UV detector is mainly to separate and identify the principal active components of a mixture.
•UV detectors are the most versatile, having the best sensitivity and linearity.
•UV detectors cannot be used for testing substances that are low in chromophores (colorless or virtually
colorless) as they cannot absorb light at low range.
•They are cost-effective and popular and are widely used in industry
Advantage:
• Sensitivity is high
• Relative robust to temperature and flow rate change
• Compatible with gradient elution
Disadvantage:
• Only compounds with UV or visible absorption could be
detected.
Fluorescence detector
This is a specific detector that senses only those substances that emit light. This detector is popular for
trace analysis in environmental science.
•As it is very sensitive, its response is only linear over a relatively limited concentration range. As there are
not many elements that fluoresce , samples must be synthesized to make them detectable

Advantage
• Sensitivity is higher than UV-Vis detector
• Selectivity is high because relatively few compounds fluorescence
• Compatible with gradient elution
Disadvantage
• Difficult to predict fluorescence
• Greatly affected by environment
– Solvent
– pH
– Temperature
– Viscosity
– Ionic strength
– Dissolved gas
Refractive Index (RI) Detection
The refractive index (RI) detector uses a monochromator and is one of the least sensitive LC detectors.
•This detector is extremely useful for detecting those compounds that are non-ionic, do not absorb
ultraviolet light and do not fluorescence
•e.g. sugar, alcohol, fatty acid and polymers.

Advantage
 Responds to nearly all solutes
 Unaffected by flow rate
Disadvantage
 Not as sensitive as most other types of detectors
 Could not be used with gradient elution

10. Describe the principles and application of Electrophoresis

*refer Q26 and Q27 (5marks)
11. Explain various methods of preparing TLC plates and its application.
*refer Q 3 (10 marks)
12. How development is carried out in column chromatography,TLC & Paper chromatography
*for development techniques in column. Refer Q1 (10 marks)
Development technique in TLC
. Linear development is the most common and familiar of these techniques
a. This technique involves applying solute to a support on a rectangular plate and eluting
them with mobile phase flowing in only one direction.
Linear development may be performed with the sample and mobile phase applied at the bottom of the
support (ascending development), at the top of the support (descending development), or to the edge of a
support lying flat (horizontal development).
Ascending and descending development

Ascending development:
The sample is spotted at the one end of the plate .
Then it is placed in the chamber containing the mobile face. The spot must not be dipped into the mobile
phase
The mobile phase rises through the capillary action against the gravitational forces
As the distance of height of traveling is increased the rate of movement of the solvent is decreased.
This slowness in the rate provides enough time for partition equilibrium between the two phase to take
place
Descending development:
In case of paper chromatography it is faster than the ascending chromatography.
The sample is spotted at the upper end .
The solvent is developed from the upper portion to the lower portion.
It traveled the capillary action and by the pull of the gravitational force.

In case of thin layer chromatography it is not so faster than the ascending chromatography than that of
paper chromatography
Radial Development is an elution technique also used in planar chromatography. This can be performed in
one of two ways: circular development and anti-circular development.
a. In circular development, a flat circular support is used with mobile phase applied to the center.
b. Samples are applied near the center. As mobile phase enters the center of the circle and moves
towards the edges, it carries solutes with it, separating them as they travel through the system.
c. . One advantage of using circular development instead of linear development is that resolution is
increased (specifically for solutes with high retention factors. Rf(linear) = Rf(circular)
d. Circular development also avoids the “edge effects” seen in linear development
e. This system can be used either with capillary flow or forced flow of the mobile. Forced flow can
be produced by placing the support disk on a centrifuge and spinning it while mobile phase is
applied. This results in a constant flow-rate for the mobile phase, which can be controlled by the
rate of rotation of the disk. This technique, also know as centrifugal development, is not only more
reproducible and more easily controlled than capillary flow, but also create faster flow-rates and
decreases analysis times. The centrifugal development is very useful for preparative separation.
Multiple development:

The solvent must be evaporated from the layer between each development in order that it must
migrate through again by capillary action.

nRf values are introduced in order to be able to quote the position of the separated substances
Multi-dimensional development is a technique that separates solute by combining two or more
different elution methods.
In the simplest form of this technique, solutes are applied to one corner of a planar support and
eluted with a give mobile phase. The support is then rotated 90o and solute are eluted in the
second direction by a different mobile phase system. By choosing mobile phase with different
strengths and selectivities, the result is a separation of solutes in two directions instead of one.
Besides using mobile phase, a separation technique may also be used in the second step of a multi-
dimensional separation. A common example of this is the use of electrophoresis along with paper
chromatography in the separation of amino acids.

*refer Q16 (5 marks) for development terchniqoes in paper chromatography

13. Describe the principle of a Potentiometric titrations? Write the construction and working of a calomel
electrode & glass electrode?
Potentiometry is the field of electro analytical chemistry in which potential is measured under the
conditions of no current flow. The measured potential may then be used to determine the analytical
quantity of interest, generally the concentration of some component of the analytic solution. The potential
that develops in the electrochemical cell is the result of the free energy change that would occur if the
chemical phenomena were to proceed until the equilibrium condition has been satisfied.
These methods rely on the measurement of Ecell for quantification. A number of common reference and
ion selective electrodes are reviewed along with general calculations and analytical approaches.
*refer Q37(5 marks) for principle
CALOMEL ELECTRODE

Calomel is mercury(I) chloride, Hg2Cl2. The reference half cell onvolving it may be represented :

KCl (satd) HgCl2(satd) Hg

And the electron reaction is:
Hg2Cl2(s) + 2 e 2Hg (s) + 2Cl-
Construction:
It consists of a central tube which contains the components of the electrode reaction, i.e., mercury,
mercury(I) chloride and free chloride ions supplied by the KCl. The bridge solution fulfils 2 functions.
Firstly it acts as a “top up” to prevent the cotton wool plug from drying out. Secondly it act as the salt
bridge linking reference and indicator half cells.
The porous divisions the boundaries at the liquid junctions are usually made from a ceramic material. This
provides good electrical contact between the solution, but prevents a major interchangeof the 2 liquids.

Calomel electrode: Calomel electrode consists of mercury, solid mercurous chloride and a solution of
potassium chloride. The electrode is represented as
Hg, Hg2Cl2 (s) ; KCl (aq)
Two types of cell reactions are possible in calomel electrode:
1. If the electrode reaction involves reduction the Hg22+ ion furnished by the sparingly soluble
mercurous chloride would be discharged at the electrode. Hence, more and more of calomel would
pass into solution. The result is increase in concentration of Cl- ions. The reaction is

Hg2 Cl2 (s)Hg22+ (aq) + 2Cl- (aq)Hg22+ (aq) + 2e-2Hg (l)

2. On the other hand, the electrode reaction involves oxidation it would liberate electrons and send
Hg22+ ions into solution. The Hg22+ ions will combine with Cl- ions/ furnished by KCl forming
sparingly soluble Hg2Cl2. The reaction is

2HgHg22+ (aq) + 2e-Hg22+ + 2Cl-Hg2Cl2 (s)

Thus in case of calomel electrode the electrode reaction may be represented as
Hg2Cl2 (s) + 2e-2Hg (l) +2Cl- (aq)
The electrode therefore is reversible with respect to chloride ions.
The potential of calomel electrode on the H2 scale has been measured carefully by connecting it to a
standard H2 electrode. The potential of the electrode depends upon the concentration of KCl solution. The
values for 0.1M KCl, 1.0M KCl and saturated solution of KCl are 0.3335V, 0.2810V and 0.2422V
respectively at 25 °C. The calomel electrode with saturated KCl is most commonly used and is referred as
saturated calomel electrode (SCE).
GLASS ELECTRODE
Construction
Scheme of typical pH glass electrode
1. a sensing part of electrode, a bulb made from a specific glass
2. sometimes electrode contain small amount of AgCl precipitate inside the glass electrode
3. internal solution, usually 0.1M HCl for pH electrodes or 0.1M MeCl for pMe electrodes
4. internal electrode, usually silver chloride electrode or calomel electrode
5. body of electrode, made from non-conductive glass or plastics.
6. reference electrode, usually the same type as 4
7. junction with studied solution, usually made from ceramics or capillary with asbestos or quartz fiber.

A typical modern pH probe is a combination electrode, which combines both the glass and reference
electrodes into one body. The bottom of a pH electrode balloons out into a round thin glass bulb. The pH
meter is best thought of as a tube within a tube. The inside most tube (the inner tube) contains an
unchanging saturated KCl and a 0.1M HCl solution. Also inside the inner tube is the cathode terminus of
the reference probe. The anodic terminus wraps itself around the outside of the inner tube and ends with
the same sort of reference probe as was on the inside of the inner tube. Both the inner tube and the outer
tube contain a reference solution but only the outer tube has contact with the solution on the outside of the
pH probe by way of a porous plug that serves as a salt bridge.
This device is essentially a galvanic cell. The reference end is essentially the inner tube of the pH meter,
which for obvious reasons cannot lose ions to the surrounding environment (as a reference is good only so
long as it stays static through the duration of the measurement). The outer tube contains the medium,
which is allowed to mix with the outside environment (and as a consequence this tube must be replenished
with a solution of KCl due to ion loss and evaporation).
The measuring part of the electrode, the glass bulb on the bottom, is coated both inside and out with a ~10
nm layer of a hydrated gel. These two layers are separated by a layer of dry glass. The silica glass structure
(that is, the conformation of its atomic structure) is shaped in such a way that it allows Na+ ions some
mobility. The metal cations (Na+) in the hydrated gel diffuse out of the glass and into solution while H+
from solution can diffuse into the hydrated gel. It is the hydrated gel, which makes the pH electrode an ion
selective electrode.
H+ does not cross through the glass membrane of the pH electrode, it is the Na+ which crosses and allows
for a change in free energy. When an ion diffuses from a region of activity to another region of activity,
there is a free energy change and this is what the pH meter actually measures. The hydrated gel membrane
is connected by Na+ transport and thus the concentration of H+ on the outside of the membrane is 'relayed'
to the inside of the membrane by Na+.
All glass pH electrodes have extremely high electric resistance from 50 till 500 MOhm. Therefore glass
electrode could be used only with high impedance measuring device like pH meter or more universal
measuring device - ionometer.
Between measurements any glass and membrane electrodes should be kept in the solution of its own ion
(Ex. pH glass electrode should be kept in 0.1M HCl or 0.1M H2SO4). It is necessary to prevent insiccation
of the glass membrane.

14. Give an account of the construction and working of glass electrode? Write the application of
potentiometric titrations
*refer Q13(10 marls) for glass electrode
APPLICATIONS
a)Neutralization reactions: glass / calomel electrode for determination of Ph
b) Precipitation reactions: Membrane electrodes for the determination of the halogens using silver nitrate
reagent
c) Complex formation titration: metal and membrane electrodes for determination of many cations
(mixture of Bi3+, Cd2+ and Ca2+ using EDTA)
d) Redox titration: platinum electrode For example for reaction of Fe3+/ Fe2+ with Ce4+/Ce3+
e) potentionmetric titrations are used in the assay of drugs like amoxicillin, disulphuram, phenobarbitone
etc.
f) It is used for all types of volumetric analysis: acid base, precipitimetry, complexometry and redox
It is used when it is not easy or impossible to detect the end point by ordinary visual methods i.e:
1. For highly coloured or turbid solutions.
2. For very dilute solutions 10-3, 10-6 M.
3. When there is no available indicator

15. Describe the construction and working, advantages, disadvantages & application of a Standard
Hydrogen Electrode.
STANDARD HYDROGEN ELECTRODE

It’s a primary reference electrode. Its potential is considered to be zero.

Construction
Platinum
electrode

Platinum foil(B)
This consists of a glass tube (A) having holes at its bottom. Inside this tube is another glass tube (D)
having a platinum or copper wire with a platinum foil(B). the surface of platinum foil is coated electrically
with Platinum black. Pure hydrogen gas at 1 atmosphere pressure is passed through the opening C of glass
tube A and it escapes through small holes at the bottom of electrode. The electrode is dipped in a solution
of standard acid like hydrochloric acid at unit activity (1.8 M of HCl at 25˚C). some hydrogen gas is
absorbed by pt. black of electrode and it permits the exchange from gaseous to ionic of hydrogen and
reverse process to occur without any obstacle.
The way it acts as hydrogen electrode. Under fixed conditions of pressure of hydrogen gas passed and
hydrogen ions in solution with contact of electrode.
Working:
Electrode reaction:
half cell: pt/ H2 , H+ (1N) 
Eo = zero
d-Limitation
1. It is difficult to be used and to keep H 2 gas at one atmosphere during all determinations.
2. It needs periodical replating of Pt. Sheet with Pt. Black
Nernst equation

1
E  zero - 0.059 log
[H  ]
E = -0.059 pH
When it is connected with NHE as reference electrode the e.m.f. of the cell :
Ecell = zero –(–0.059 pH)
= 0.059 pH
pH = E / 0.059
Advantage:
1. It is a fundamental electrode and is used as standard in pH measurements.
2. It can be used over wide pH range
3. It exhibits no salt error.
4. It establishes equilibrium rapidly and gives accurate results.
Disadvantage
1. It cannot be used in solution containing oxidising agent which will oxidiose [ ½ H 2 = H+ + e ]
or reducing substances which will reduce [ H + + e = ½ H2 ] especially in presence of platinum
black
2. It cannot be used in reactions involving volatile constituent’s e.g. CO 2, as it will be bubbled out by
the H2 gas.
3. It cannot be used in presence of catalytic poisons which will affect Pt black which catalyses the
electrode reaction.
4. It needs repletion with Pt black.
5. It is not easy to keep H2 gas at one atmospheric pressure during all measurements.
Application:
 used as standard reference electrode
 Used in standard pH measurements
 To measure emf
 Used as redox electrode
16. Explain the theory & the types of Conductometric Titrations.
*refer Q41(5 marks)
17. Write the basic principle of a potentiometry .Describe in detail Dead Stop End point techniques.
For principle refer Q40 (5marks)
Dead stop end point technique
This method is called biamperometry.It is applicable when a redox system is present before and after the
end point.
Principle:The principal is that, the potential applied between polarisable and non-polarisable electrode is
kept constant and the diffusion current is measured during the titration. During the titration, the
concentration of the electroreducible ion changes and hence the diffusion current also changes. At the end
point, there is a sharp change in the diffusion current as shown by the curve of diffusion current Vs volume
of titrant. The titration is performed between a reducible or non-reducible ion and a counter ion of which
atleast the titrate or the titrant or both can give rise to diffusion current.
The potential selected for the titration is at its limiting value, i.e. the potential corresponds to the point
where limiting current is reached.
In a uniformly stirred solution of an analyte when two small but similar platinum electrodes are dipped and
a small potential of 1-100 mV is applied, current flows as long as electrodes remain depolarised. When one
component gets consumed or removed by the addition of titrant current cases to flow. For the method to be
applicable, only requirement is that a reversible oxi-red system be present either before oe after the end
point.
In the titration of iodine against thiosulphate, when 2 platinum electrodes are immersed in the iodine
solution and connected to the battery appreciable current flows through cell. The amount of oxidised form
reduced at cathode is equal to that formed by oxidation of the reduced form at anode.
Both electrodes remain depolarised (seen by current flowing through galvanometer) until the oxidised
component or the reduced component of the system is consumed by the titrant.
Current thus, flows until end point. At or after the end point the current becomes zero. In the iodine against
thiosulphate reaction a rapid decrease in current is observed near the vicinity of end point. Such titartions
are given the name as “dead stop end point”.

The assembly consists of a beaker of suitable size mounted on magnetic stirrer containing a solution to be
titrated. Immersed in this solution are two bright platinum electrodes which are connected to a suitable
potentiometer which in turn is connected to 1.5 volts battery. A microammeter galvanometer is
incorporated in the circuit. Small volume of titrant is added through microburette and current flowing
through galavanometer is noted.
When the current stops, the volume corresponding to it is recorded.
18. Describe the construction and working of a Double- Beam Recording Dispersive IR Spectrophotometer
with its advantages and disadvantages.
DOUBLE BEAM SPECTROPHOTOMETER
SOURCE
IR source consist of an inert solid that is heated electrically to a temperature between 1500 and 2200 K
The material is chosen so that its emission approximates
As closely as possible to that of black body radiator.
(in candescent solid-the glow due to the great heat)
We get continuum radiation approximating that of black body results
1)THE NERNST GLOWER
 Nernst Glower is Fabricated from rare earth oxides
 (e.g. ZrO2+Y2O3)
 Diameter 1-2mm length 20mm
 Pt. leads are sealed to the end of cylinder to permit electric connection.
 When electric current pass it glows ,at temp 1200-2200 k
2) THE GLOBAR SOURCE
 Silicon carbide rod, 50 mm length ,5 mm D
 Electrically heated at 1300-1500 k
  Advantage of +ve temp. Coefficient of resistance
 Water cooling required to prevent arcing
 Spectral energy is comparable except in the region 5 μm where provide greater output.
3) INCANDENSCENT WIRE SOURCE(NICHROM WIRE)
Tightly wound spiral of NICHROM WIRE heated to about 1100 k by an electric current
 Lower intensity but larger life than previous 2
 A rhodium wire heater sealed in ceramic cylinder has similar property as source
4) MERCURY ARC
 Used for far IR region (above 50 μm)
 High pressure Hg arc is used (1 atm pressure)
 Passage of electricity through the vapour, forms an internal plasma source
that provides comtinuum radiation in far IR
5) THE CO2 LASER
 A tunable co2 laser is used as an IR source for monitoring the concentration of certain atmospheric
pollutants and for determine absorbing species in aq. Solution
 Range 900-1100 cm-1 which consist of about 100 of closely spaced discreet line
 Imp. For quantitative determination of no. of species like ammonia, butadiene, benzene, ethanol,
nitrogen dioxide, trichloroethylene
 An important property of the laser source is the radiant power available in each line which is
several order of magnitude greater than that of black body source
*for sampling technique refer Q69 (5 mark)
DETECTORS or TRANSDUCERS
 There are 3 types of detector (transducer)
 1) THERMAL DETECTOR
 2) PYROELECRIC DETECTOR
 3) PHOTON (QUANTUM) DETECTOR
THERMAL DETECTOR
 Thermal detector whose response depend on heating effect.
 Which in terns alters the physical properties of transducers such as resistance
 It is a transducer that changes thermal energy in to an electric signal. The electric signal is
amplified and routed to the read out device
 But the problem of measuring IR radiation by thermal means is compounded by thermal noise
from the surrounding for this reason thermal transducers are housing in a vacuum and are carefully
shielded from thermal radiation emitted by other near by objects.
 Therefore temp.of room is mainted
 1)THERMOCOUPLE

 Most widely used IR detector

 Consist of 2 pieces of metal such as “Bi” which are joined with dissimilar metal such as “sb”
(antimony) and form a pair of junction (2 jn)
 Junction. Is usually blackened (to improve its heat absorb capacity) With black metallic oxides
One junction between 2 dissimilar metal is heated with IR radiation other junction. Kept at const.
temperature.
PRINCIPLE
 “A CHANGE IN THE TEMP. AT THE BOTH DIFF.JN. BETWEEN 2 UNLIKE METAL
CAUSES AN ELECTRIC POTENTIAL TO DEVELOPE BETWEEN SPECIES”
Or
 “DUE TO DIFRENCE IN WORK FUNCTION OF METALS WITH TEMP. A SMALL
VOLTAGE DEVELOP ACROSS THE THERMOCOUPLE”
 This potential diff. is depend (proportional) to the amt of IR radiation falling on hot jn.
 Whole assembly is evacuated in still housing with IR transparent KBr window to minimize
conductivity heat loss.
 Capable to responding temp. diff. of 10-6 k corresponding to potential diff. of 6-8 μV/ μW
 Advantage that independent of response with change in wave length
 2) THERMISTOR or BOLOMETER



 When irradiation by IR beam produced an increase in resistance of the metal strip which measured
with a wheatstone bridge
 A potential diff. between the 2 elements produced a proportional voltage difference
3)PNEUMATIC DETECTOR or GOLAY DETECTOR
 Pneumatic detectors respond to change in vol. of non absorbing gas or liq with temp. change
 In pneumatic device if gas is used as medium called golay detector
 Here the absorbing radiation heats an inert gas (usually xenon) in a pneumatic chamber behind the
plate and cause the gas to expand
 As the gas expands the flexible diaphragm at the opposite end of the chamber from the metallic
plate is pushed outward
PYROELECTRIC DETECTOR

 The most recently developed IR detector is pyroelectric detector

 It is constructed from single crystalline wafers of pyroelectric material which are insulator
(dielectric material ) with very thermal and electric properties
 certain crystal such as
 TGS (tri glycine sulphate ((NH2,CH2,COOH)3.H2SO4)
 DTGS (deuterated triglycine sulfate
 Lithium tantalat(LITAO3)
 Lithium niobate (LINbO3)
Is the imp. Pyroelectric material used in ir detector
This crystal possesses internal electric polarizations. This internal property is taken in account.
Crystal is placed between 2 electrodes one of which is IR transparent
 When IR radiation fall on crystal its temp. is change, which alters the charge distribution across the
crystal, which can be detected as the current in the external electric circuit connecting the 2 side of
the capacitor.
 The magnitude of this current is proportional to the surface area of crystal and the rate of change of
polarization with temp.
 This is known as pyroelectric effect.
  Pyroelectric crystals lose their residual polarization when they are heated to a temp. Called the
Curie point
 For TGC curie point 47 0C

Advantages:
 Rapid
 More sensitive
 Accurate
 Has more computational capabilities

Diadvantage:
 High cost
 more labour required
 not widely available

19. Outline the working of a double beam recording of UV/Visible spectrophotometer .Name each part of
the system & its functioning.
Double beam instrument is the one in which two beams are formed in the space by a U shaped mirror
called as beam splitter or beam chopper .its called “Double beam in space instrument”.

SOURCE:
REQUIREMENTS OF AN IDEAL SOURCE

 It should be stable and should not allow fluctuations.

 It should emit light of continuous spectrum of high and uniform intensity over the entire
wavelength region in which it’s used.
  It should provide incident light of sufficient intensity for the transmitted energy to be detected at
the end of optic path.
 It should not show fatigue on continued use.
For Visible radiation:
TUNGSTEN HALOGEN LAMP
 Its construction is similar to a house hold lamp.
 The bulb contains a filament of Tungsten fixed in evacuated condition and then filled with inert
gas.
 The filament can be heated up to 3000 k, beyond this Tungsten starts sublimating .
 To prevent this along with inert gas some amount of halogen is introduced (usually Iodine).
 Sublimated form of tungsten reacts with Iodine to form Tungsten –Iodine complex.
 Which migrates back to the hot filament where it decomposes and Tungsten get deposited.
DEMERIT:
It emits the major portion of its radiant energy in near IR region of the spectrum.
Source for UV Radiation
I.HYDROGEN DISCHARGE LAMP:
 In Hydrogen discharge lamp pair of electrodes is enclosed in a glass tube (provided with silica or
quartz window for UV radiation to pass trough) filled with hydrogen gas.
 When current is passed trough these electrodes maintained at high voltage, discharge of electrons
occurs which excites hydrogen molecules which in turn cause emission of UV radiation.
II.DEUTERIUM LAMP:
 It’s similar to Hydrogen discharge lamp but instead of Hydrogen gas, Deuterium gas is used.
MERIT:
 Intensity of radiation is more as compare to Hydrogen discharge lamp.

DEMERIT:
 Expensive.
III. XENON DISCHARGE LAMP:
 It possesses two tungsten electrodes separated by some distance.
 These are enclosed in a glass tube (for visible) with quartz or fused silica and xenon gas is filled
under pressure.
 An intense arc is formed between electrodes by applying high voltage. This is a good source of
continuous plus additional intense radiation.
DEMERIT:
 The lamp since operates at high voltage becomes very hot during operation and hence needs
thermal insulation.
MERCURY ARC LAMP:
 In mercury arc lamp, mercury vapor is stored under high pressure and excitation of mercury atoms
is done by electric discharge.
DEMERIT:
Not suitable for continuous spectral studies, because it doesn’t give continuous radiations.

COLLIMATING SYSTEM
The radiation emitted by the source is collimated (made parallel) by lenses, mirrors and slits.
LENSES:
 Materials used for the lenses must be transparent to the radiation being used.
 Ordinary silicate glass transmits between 350 to 3000 nm and is suitable for visible and near IR
region.
 Quartz or fused silica is used as a material for lenses to work below 300nm.
MIRRORS
 These are used to reflect, focus or collimate light beams in spectrophotometer.
 To minimize the light loss, mirrors are aluminized on their front surfaces.
SLITS:
 Slit is an important device in resolving polychromatic radiation into monochromatic radiation.

 To achieve this, entrance slit and exit slit are used.

 The width of slit plays an important role in resolution of polychromatic radiation.
Monochromators:
It’s a device used to isolate the radiation of the desired wavelength from wavelength of the continuous
spectra. Following types of monochromatic devices are used:

Filters:
 Selection of filters is usually done on a compromise between peak transmittance and band pass
width; the former should be as high as possible and latter as narrow as possible.
1. Absorption filters
2. Interference filter
 Absorption filters:
Absorption filters works by selective absorption of unwanted radiation and transmits the radiation
which is required.
Selection of absorption filter is done according to the following procedure:
 Draw a filter wheel.
 Write the color VIBGYOR in clockwise or anticlockwise manner, omitting Indigo
 If solution to be analyzed is blue in color a filter having a complimentary color
orange is used in the analysis.
 Similarly, we can select the required filter in colorimeter, based upon the color of the solution.
 An Absorption glass filter is made of solid sheet of glass that has been colored by pigments which
is dissolved or dispersed in the glass.

 The color in the glass filters are produced by incorporating metal oxides like (V, Cr, Mn, Fe,
Ni, Co, Cu etc.).
Merits:-
 Simple in construction
 Cheaper
 Selection of the filter is easy
Demerits:-
 Less accurate
 Band pass (bandwidth) is more (±20-30nm) i.e. if we have to measure at 400nm; we get radiation
from 370-430nm. Hence less accurate results are obtained.
Interference filter
 Works on the interference phenomenon, causes rejection of unwanted wavelength by selective
reflection.
It’s constructed by using two parallel glass plates, which are silvered internally and separated by thin
film of dielectric material of different (CaF2, Sio, MgF2) refractive index. These filters have a band
pass of 10-15nm with peak transmittance of 40-60%.
MERITS:-
 Provide greater transmittance and narrower band pass (10-15nm) as compare to absorption filter.
 Inexpensive
 Additional filters can be used to cut off undesired wavelength.

b) PRISM:- (Dispersing devices)

 Prism is made from glass, Quartz or fused silica.
 Quartz or fused silica is the choice of material of UV spectrum.
 For visible region, glass prisms are preferable as the dispersion is much greater than that obtained
with quartz.
 When white light is passed through glass prism, dispersion of polychromatic light in rainbow
occurs. Now by rotation of the prism different wavelengths of the spectrum can be made to pass
through exit slit which fall on the sample.
 The effective separation of wavelength depends on the dispersive power of prism material and the
apical angle of the prism.
There are two types of mounting of the prisms in an instrument one is called ‘Cornu type’ (refractive
type) which has an apical angle of 600 and it is adjusted such that on rotation the emerging light is
allowed to fall on exit slit
 The other type is called “Littrow type” (reflective type), which has optical angle 30° and its one
surface is aluminized which reflect light back to pass through prism and to emerge on the same
side of the light source i.e. light doesn’t pass through the prism on other side.

c) DIFFRACTION GRATINGS:
 More refined dispersion of light is obtained by means of diffraction gratings.
 These consist of large number of parallel lines ( grooves) about 15000-30000/ inch is ruled on
highly polished surface of aluminum.
 These acts as scattering centers for light beam impinging on it. Because of constructive
interference, the separation of desired wavelength is accomplished.
 Resulting radiation λ = b (sin i ± sin r) / m
 The resolved power of grating depends on the number of lines. Generally resolving power of
grating is better than that of prism and hence grating is used and is preferred.

 Band pass ± 0.1nm

SAMPLE HOLDERS/ CUVETTES
 The cells or cuvettes are used for handling liquid samples.
 The cell may either be rectangular or cylindrical in nature.
 The cells that may hold the sample must be made of substances which are transparent in the
spectral region of interest.
 For study in UV region; the cells are prepared from quartz or fused silica whereas color corrected
fused glass is used for visible region.
 The internal diameter of the cells is 0.5cm, 1cm, 2cm, or 4cm
 The cuvettes with lid are used for handling volatile type solvents and solutions.
 The surfaces of absorption cells must be kept scrupulously clean. No fingerprints or blotches
should be present on cells. Cleaning of the cells is carried out washing with distilled water or with
dilute alcohol, acetone.
DETECTORS
 The light or the intensity of transmitted radiation by a sample is collected on a detector device.
 The generated currents are often amplified and pass on to a meter (a galvanometer or recorder).
 The following types of detectors are employed in instrumentation of absorption
spectrophotometer:-
1. Barrier layer cell/Photovoltaic cell
2. Phototubes/ Photoemessive tube
3. Photomultiplier tubes

1) Barrier layer cell/ photovoltaic cell

The detector has a thin film metallic layer coated with silver or gold and acts as an collector
electrode.
It also has a metal base plate which acts as another electrode.
These two layers are separated by a semiconductor layer of selenium.

 When light radiation falls on selenium layer, electrons become mobile and are taken up by
transparent metal layer.
 This creates a potential difference between two electrodes & causes the flow of current.
 When it is connected to galvanometer, a flow of current observed which is proportional to the
intensity and wavelength of light falling on it.

2) Photomultiplier tube
The principle employed in this detector is that, multiplication of photoelectrons by secondary
emission of electrons.
In a vacuum tube, a primary photo-cathode is fixed which receives radiation from the sample.
Some eight to ten dynodes are fixed each with increasing potential of 75-100V higher than
preceding one.
Near the last dynode is fixed an anode or electron collector electrode.
Photo-multiplier is extremely sensitive to light and is best suited where weaker or low radiation is
received
 3) Photo tubes

 Consists of a evacuated glass tube with a photocathode and a collector anode (metal).
 The surface of photocathode is coated with a layer of elements like cesium, potassium or silver or
oxides of these metals (to increase sensitivity).
 When radiant energy falls on photosensitive cathode, electrons are emitted which are attracted to
anode causing current to flow.
 More sensitive compared to barrier layer cell and therefore widely used.
 The signal from the detector can be amplified using an amplifier circuit.

 Depending upon the monochromators (filters or dispersing device) used to isolate and transmit a
narrow beam of radiant energy from the incident light determines whether the instrument is
classified as Photometer or a Spectrophotometer.
Spectrophotometers used here detects the percentage transmittance of light radiation, when light of
certain intensity & frequency range is passed through the sample
DOUBLE BEAM UV-VIS SPECTROPHOTOMETER
 Double beam instrument is the one in which two beams are formed in the space by a U shaped
mirror called as beam splitter or beam chopper .
 Chopper is a device consisting of a circular disc. One third of the disc is opaque and one third is
transparent, remaining one third is mirrored. It splits the monochromatic beam of light into two
beams of equal intensities.
Advantages of double beam spectrophotometer
 It facilitates rapid scanning over wide λ region.
 Fluctuations due to radiation source are minimised.
 It doesn’t require adjustment of the transmittance at 0% and 100% at each wavelength.
 It gives ratio of intensities of sample & reference beams simultaneously.
Disadvantages:
 Construction is complicated.
 Instrument is expensive.

20.Describe the construction and working of Double-Beam UV/Visible spectrophotometer.

Mention the advantages of double beam over single beam spectrophotometer
*refer Q19 for construction and working
Advantages of double beam over single beam
 Caliberation is done only in the beginning for double beam whereas caliberation should
be done with blank every time before measuring the absorbance or transmittance of
sample in single beam
 Double beam permits a large degree of inherent compensation for fluctuations in the
intensity of the radiant energy while in single beam radiant energy intensity changes
with fluctuation of voltage.
 Double beam measures the percentage of light absorbed by the sample while measure
the total amount of transmitted light reaching the detector
 In double beam it’s possible to do direct one step comparison of sample in one path
with a standard in the other path. But it’s not possible in single beam.
 In double beam scanning can be done over a wide wavelength region while in single
beam radiant energy wavelength has to be adjusted every time
 Working on double beam is fast and non tedious while working on single beam is
tedious and time consuming.
21. a) Derive Beer’s & Lamberts Law .b) what are the applications advantages and limitations of Beer’s
law.
a) *refer Q57 (5 marks)
b) applications:
* Quality control of purity
* Quantitative analysis
> using E 1%cm values for the estimations from formulations or raw material when reference standard
is not available
* structural elucidation of organic componds
* determination of ligand/ metal ratio in metallic complexes
*determination of pKa value of indicators
Advanatages
 As radiation passes through the cell, some radiation is reflected at each surface where there is a
change in refractive index.
 Can be almost entirely compensated by taking P0 as the radiant power transmitted through a cell
that contains only pure solvent.
 It provides a direct correlationship between the absorbance of a molecule to the concnetration
Limitations
*only applicable to monochromatic radiations
*refer Q58 (2 marks)
22. Describe the principle and Application of IR Spectroscopy for the following A) Detection of functional
group(two example) B) Study of Hydrogen Bonding.
A ) identification of functional group
The entire IR region is divided into:
Group frequency region – 4000/cm to 1500/cm
Finger print region – 1500/cm to 400/cm
In the group frequency region, the peaks corresponding to different functional groups can be observed
Eg: amino group, alcoholic group etc
ABSORPTION OF COMMON FUNCTIONAL GROUPS
 group Range (cm-1)
C-H Stretching (alkane) 2960-2850
C-H stretching (alkene) 3040-3010
C-H stretching (aromatic) 3030
C-H bending (alkane) 1340
C-H bending (aromatic) 700-850
C=C stretching (alkene) 1680-1620
C=C stretching (alkyne) 2100-2200

Every part of the molecule has different atoms and are connected by bonds. Each bond requires different
IR region for absorption and so characteristic peaks are observed. Hence this region of IR spectrum is
called as finger print region of a molecule.
*refer Q63 (5 marks) for importance of fingerprint region
)Hydrogen bonding:

•Hydrogen bonding brings about remarkable downward frequency shifts.

• Stronger the hydrogen bonding, greater is the absorption
shift towards lower wave length than the normal value.
•There is 2 types of hydrogen bonding a) inter molecular→broad bands
b) intra molecular → sharp bands
 INTERMOLECULAR H - BONDING

O HO
H C C H

 OH O Formic acid dimer

INTAMOLECULAR H- BONDING

OH -
O
N
O

o-nitro phenol
 The inter and intramolecular h-bonding can be distinguished by dilution.
  The intramolecular H-bonding is independent of the conc. Because it is an internal effects and
hence intramolecular h-bonds remains unaffected on dilution and so absorption band also remains
unaffected giving bonded O-H absorption
 But intermolecular h-bonding is dependent on dilution the H-bonds are broken on dilution (or at
low conc.) and hence there is decrease in the bonded O-H absorption
 on successive dilution the intensities of the bands due to intermolecular h-bonding gradually
decrease and finally disappear
•hydrogen bonding in O-H and N-H compounds deserve special attention.
• Eg: alcohols&phenols enols & chelates
23. Explain the principal instrumentation and factors affecting fluorescence intensity.
*refer Q49 (5marks) for principle
INSTRUMENTATION
 SOURCE OF LIGHT
 FILTERS AND MONOCHROMATORS
 SAMPLE CELLS
 DETECTORS
SOURCE OF LIGHT
 XENON ARC LAMP
 MERCURY ARC LAMP
 TUNGSTEN LAMP
 TUNABLE DYE LASERS
 MERCURY ARC LAMP
 Produce intense line spectrum.
 High pressure lamps give lines at 366,405, 436, 546,577,691,733nm.
 Low pressure lamps give additional radiation at 254nm.
XENON ARC LAMP
 Intense radiation by passage of current through an atmosphere of xenon.
 Spectrum is continuous over the range between over 250-600nm,peak intensity about 470nm.
TUNGSTEN LAMP
 Intensity of the lamp is low.
 If excitation is done in the visible region this lamp is used.
 It does not offer UV radiation.
TUNABLE DYE LASERS
 Pulsed nitrogen laser as the primary source.
  Radiation in the range between 360 and 650 nm is produced.
 FILTERS AND MONOCHROMATORS
 FILTER
 Primary filter : absorbs visible & transmits UV radiation.
 Secondary filter : absorbs UV & transmits visible radiation.
 MONOCHROMATOR
 Excitation monochromator : isolates
 radiation for excitation.
 Emission monochromator : isolates only
 radiation emitted by fluorescent molecule.
 Interference and absorption filters are used in fluorometers.
 Grating monochromaters are used in spectrofluorometers.
SAMPLE AND SAMPLE HOLDER
 The majority of fluorescence assays are carried out in solution.
 Cylindrical or rectangular cells fabricated of silica or glass used.
 Path length is usually 10mm or 1cm.
 All the surfaces of the sample holder are polished in fluorimetry.
DETECTORS
 PHOTOVOLTAIC CELL
 PHOTO TUBE
 PHOTOMULTIPLIER TUBES – Best and accurate.
*refer Q19 (10 marks) for explanation of detectors
INSTRUMENT DESIGNS
 SINGLE BEAM(FILTER) FLUOROMETER
 DOUBLE BEAM(FILTER) FLUOROMETER
 SPECTROFLUOROMETER(DOUBLE BEAM)
SINGLE BEAM FLUOR0METER
 Tungsten lamp as source of light.
 The primary filter absorbs visible radiation and transmits uv radiation.
 Emitted radiation measured at 90o by secondary filter.
 Secondary filter absorbs uv radiation and transmits visible radiation.
ADVANTAGES
 SIMPLE IN CONSTRUCTION
  CHEAP
 EASY TO OPERATE
DISADVANTAGES
 IT IS NOT POSSIBLE TO USE SAMPLE AND REFERENCE SOLUTION AT A TIME
 RAPID SCANNING TO OBTAIN EXCITATION AND EMISSION SPECTRUM OF THE
COMPOUND IS NOT POSSIBLE
DOUBLE BEAM FLUOROMETER
 Similar to single beam instrument.
 Two incident beams from light source pass through primary filters separately and fall on
either sample or reference solution.
 The emitted radiation from sample or reference pass separately through secondary filter.
ADVANTAGE
 SAMPLE AND REFERENCE SOLUTION CAN BE ANALYSED SIMULTANEOUSLY
DISADVANTAGE
 RAPID SCANNING IS NOT POSSIBLE DUE TO USE OF FILTERS
SPECTROFLUOROMETER
 The primary filter in double beam fluorometer is replaced by excitation monochromator.
 The secondary filter is replaced by emission monochromator.
 The incident beam is split into sample and reference beam using a beam splitter.
 The detector is photomultiplier tube.
ADVANTAGES
 RAPID SCANNING TO GET EXCITATION AND EMISSION SPECTRUM.
 MORE SENSITIVITY AND ACCURACY WHEN COMPARED TO FILTER
FLUOROMETER.

*for factors refer Q52(5marks)

24. Describe the Instrumentation and application of HPLC.

*refer q9 (10 marks)
25. Explain with the help of a neat diagram, the construction and working of UV/Visible
spectrophotometer with special emphasis on the monochromators and detectors present in them.
*refer Q19 (10 marks)
26. Describe the instrumentation of I.R. Spectrometry
*refer Q18 (10 marks)
27. How are different samples handled (solid,liquid,and gaseous) in I.R. Spectroscopy
*refer Q 69 (5 marks)
28. List out the sources of UV,Visible spectrophotometers and I.R.Spectrometers
*refer Q19 (10 marks) for UV , visible
*refer Q18 (10 marks) for IR
29. What are pharmaceutical application of fluorimetry? How is fluorimetry more sensitive and specific
than spectrophotometry.

APPLICATIONS OF FLUOROMETRY
Determination of inorganic substances.
 Determination of ruthenium ions in presence of other platinum metals.
 Determination of aluminum (III) in alloys.
 Determination of boron in steel by complex formed with benzoin.
 Estimation of cadmium with
2-(2 hydroxyphenyl) benzoxazole in presence of tartarate.
Nuclear research
 Determination of uranium salts by fluorimetry
 Determination of vitamin B1 and B2 in food samples
 Fluorescent indicators
 Used in acid/base titrations
 Eosin-(3.0-4.0)-colourless to green
 Fluorescein-(4.0-6.0)-colourless to green
 Quinine sulphate-(3.0-5.0)-blue to violet
 Acridine-(5.2-6.6)-green to violet-blue
organic analysis
 Qualitative and quantitative analysis of organic aromatic compounds present in cigarette smoke, air
pollutants, automobile exhausts etc.
pharmaceutical analysis
compound reagent excitation wavelength fluorescence

hydrocortisone 75%v/v H2SO4 in ethanol 460 520

nicotinamide cyanogen chloride 250 430

Liquid chromatography
 Fluorescence is an imp method of determining compounds as they appear at the end of
chromatogram or capillary electrophoresis column.
FLURIMETRY
Ultraviolet and visible (UV/Vis) spectrometers have been in general use for the past 35 years and
are important analytical instruments in the laboratory. Here are some features to consider when
selecting a UV/Vis spectrophotometer
The main problems with spectrophotometer is;
Selectivity: Keep in mind that a UV/Vis spectrophotometer does not discriminate between the
sample of interest and contaminants that absorb at the same wavelength. For example, all nucleic
acids exhibit a peak at or around 260 nm; therefore, intact RNA and double-stranded DNA, as well
as the degraded species of single-stranded DNA in a sample solution, contribute to total
absorbance at 260 nm.
• Stray light: The detectors used in spectrophotometers are broadband, meaning they respond to all
the light that reaches them. If there are impurities in the sample that reflect light, an erroneous
reading may be recorded. Stray light also causes a decrease in absorbance and reduces the linearity
range of the instrument.
• Sample conditions: Absorption results can be influenced by temperature, pH, impurities and
contaminants. All of these factors can change the absorption properties of the sample, leading to
inaccurate readings.
• Low sensitivity: The sensitivity of a spectrophotometer is often inadequate at low sample
concentrations. Researchers may need to concentrate their sample, adding additional steps and time
to their workflow
Although measuring fluorescence may not be as common as measuring absorbance, it may even be a more
powerful method for sample characterization. It is important to understand the strengths and weaknesses of
this method as it applies to different applications.

Advantages

• Sensitivity: The sensitivity of fluorescence detection is approximately 1,000 times greater than absorption
spectrophotometric methods. This leads to greater limits of detection, while potentially using less sample
material. This is important especially when working with precious or limited-quantity materials.
• Specificity: Only molecules that fluoresce are detected by this method, resulting in greater specificity
compared with UV/Vis absorption.
• Wide concentration range: Fluorimetry generally can detect more than three to six log orders of
concentration without sample dilution or modification of the sample.
• Accurate results: The sensitivity and specificity of fluorescence measurement leads to potentially more
precise and accurate readings.
30. Discuss the phenomenon of fluorescence. Explain the working of fluorimeter with suitable diagram?
*refer Q23(10 marks)