PHARMACEUTICAL ANALYSIS

ACID-BASE TITRATIONS:

Theory ACID BASE

Arrhenius Proton donor Hydroxide donor
Lowry-Bronsted Proton donor Proton acceptor
Lewis Electron pair acceptor Electron pair donor

Law of mass action:

Rate of chemical reaction is proportional to the active masses of the reacting substances.

Indicator pH
Phenolphthalein 8.3-11.0
Thymol blue 1.2-2.8
Bromophenol blue 3.0-4.6
Bromocresol green 3.8-5.4
Methyl orange 3.1-4.4
Methyl red 4.2-6.3
Phenol red 6.8-8.4
Universal indicator: pH meter

Dielectric constant:

Water: 78.5

Sulphuric acid: 100.0

Cyclohexane: 2.02

Solvents:

Type of solvent Nature Examples

Protogenic Acidic Acetic acid, Formic acid, Propionic
acid
Protophilic Basic Pyridine, Dimethyl formamide,
Ethylene diamine
Aprotic Neutral Chloroform, Benzene, Toluene,
Acetone, Dioxane etc.
Amphiprotic Acidic & Neutral Water & Alcohol

REDOX TITRATIONS:

- Oxidising agents: KmNO4, K2Cr2O7

- Reducing agents: FeCl2, H2S
Indicators:

- Internal/Redox indicator: Ferroin, Diphenylamine

- Self indicator: KmNO4, Cerric ammonium sulphate
- External indicator: K2Cr2O7

Precipitation/Argintometric Titrations:

- Mohr’s method: Titration of NaCl with silver nitrate, using potassium

chromate as indicator
- Volhard’s method Formation of soluble coloured compound

- Fajan’s indicator: Adsorption indicator

e.g. Acidic dyes: Eosin, Fluorescein

Basic dyes: Rhodamine, Rose Bengal

Gay Lussac method: Turbidity method

GRAVIMETRIC ANALYSIS:

- Lyophobic colloid (Suspensoid), e.g. Gold

- Lyophilic colloid (Emulsoid), e.g. Gelatin
- Sequestering agents, e.g. Citric acid, Tartrates, EDTA

KARL-FISCHER AQUAMETRY:

- Iodine + Sulphur dioxide + Amine + Methanol

- Primary standard (Std.): Sodium tartrate dehydrate (Std.)

DIAZOTISATION TITRATION (Sodium Nitrite titration):

- NaNO2 + conc. HCl

KJELDAHL’S METHOD: Nitrogen estimation

CONDUCTOMETRY:

- Resistance unit: ohm cm

-
Conductance unit: mho cm-1
-
Current unit: Ampere
-
Voltage unit: Volt

Specific conductance (k) : Reciprocal of resistance of 1cm cube of liquid or solution at a

specified temperature.

Equivalent conductance (٨) : Gram equivalent of solute present in a solution placed

between two large electrodes 1cm apart.
 Titrations Graph Constant Equation Electrode
Potentiometry Potential vs. Potential Nernst -
Volume equation
Amperometry Current vs. Current - Rotating platinum
Volume electrode
Conductometry Conductance Conductance - Platinum electrode
vs. Volume
Polarography Current vs. Half-wave Ilkovic Dropping mercury
Potential potential equation electrode (20-22
(Quality of (id = 607 drops/min)
compd.) nCd1/2m2/3t1/6)
Diffusion
current
(Quantity of
compd.)
Chronopotentiometry: Potential vs. Time (X-axis)

Chronoamperometry: Current vs. Time (X-axis)

Indicators:

Titration Indicator
Complexometric Murexide, Solochrome black, Calcon, Solochrome dark blue, Xylenol
orange
Precipitation Eosin, Fluorescein, Rhodamine, Rose Bengal
Non-aqueous Crystal violet, Methyl red, Oracet blue B
Aqueous Methyl orange, Phenolphthalein, Methyl red, Congo red

Electrodes:

Titration Indicator electrode Reference electrode

Acid-base Glass Calomel or Ag-AgCl
Precipitation Silver Calomel
Redox Platinum Calomel or Ag-AgCl
Chelometric Mercury-mercury Calomel or Ag-AgCl
 PHOTOCHEMISTRY
 Grothur-Draper Law: Only that light which is absorbed by a system can bring about a
photochemical change.
 Stark-Einstein Law: One mole absorbed one quantum.
 Beer’s-Lamberts Law:
Beer’s Law: Intensity of a beam of monochromatic light decreases exponentially with the
increase in concentration of absorbing substance.
Lambert’s Law: When a beam of light is allowed to pass through a transparent medium,
the rate of decrease of intensity with the thickness of medium is directly proportional to
the intensity of the light.
 Pauli’s Exclusion Principle: Maximum 2 electrons in orbital should show opposite spin.
 Hunt’s Principle: Maximum 2 electrons in orbital should show parallel spin.
 Excited triplet more stable than excited singlet.
 Jabolanski diagram:
 Vibrational relaxation
 Intersystem crossing
 Internal conversion
 Photosensitization: Singlet energy transfer and triplet energy transfer
 Stokes’ Law (Stokes’ shift / red shift): λabs > λfluorescence
 Prompt fluorescence (Quick):

T = 10-4 sec (unit)

εmax

 Excimer: Complex of ground state singlet and excited state singlet (gives prompt
fluorescence)
 Efficiency of photo reaction:

Quantamyl = No. of moles yield

No. of quanta absorbed
 Photoisomerisation: Isomerisation by light
Trans-stilbene Cis-stilbene

 Ratio becomes constant: Photostationary state

 Photosensitizer: Benzophenones
  Photoreductive dimerisation: Photolysis of benzophenones.

 Norrish reaction: Cleavage

(1) Type-I (α-cleavage)
 C-C adjacent to carbonyl is removed
 Carbon monoxide (CO) as a byproduct is eliminated.
 e.g. Aldehydes and ketones

(2) Type-II (γ-cleavage)

 γ-hydrogen will be abstracted.
 Intramolecule γ-hydrogen abstraction gives biradical.

Biradical

Cyclobutanol Enol + Olefin (Main product)

 Paterno-Bushir reaction (Photochemical cycloaddition):

O=C + C=C Oxetane (Cyclobutane / Oxacyclobutane)

ANALYTICAL TECHNIQUES
Analytical Light Source Detectors Angle b/w Reference Type of Graph
Techniques source and standard sample
detector
Ultraviolet “XHTDM” Barrier layer 180o Potassium Liquids Absorbance
spectroscopy Xenon arc, cell chromate vs.
(UV) Hydrogen, (Photovoltaic (K2Cr2O7) Concentration
Tungsten cell),
filament, Photomultipli
Deuterium, er tube
Mercury Arc (PMT),
Photocell
Infrared Incandescent Thermal - Polystyrene Solid, %
spectroscopy lamp, Nernst Detector film Liquid, Transmittance
(IR) Glower, (Thermocoupl Gases vs. Wave
Globar e, Bolometer, number
Source, Thermistor,
Mercury Arc, Golay cell),
Tungsten Photon
filament (Semiconduct
 or),
Pyroelectric,
FT-IR
Nuclear Radiofrequen FT-NMR - Tetramethyl Solvents Radiofrequenc
Magnetic cy generator Silane (for y absorption
Resonance organic vs. Field
(NMR) solution) strength
Silapentane
(for aqueous
solution)
Mass Ionisation of Quadrapole - Perfluorokero Liquids, Abundance
Spectrometry electrons mass analyser, sene Gas vs. m/e ratio
(MS) Time of Flight
(TOF)
Electron Spin Klystron Silicon - DPPH (1,1- Liquids Intensity
Resonance Oscillator Crystal diphenyl (signal) vs.
(ESR) picrylhydrazy Magnetic field
l) (Field
strength)
X-ray Collidge tube Photographic, - - Solid Intensity vs.
radiation Counter Wavelength
detectors
(Gieger-
Muller tube
counter,
Semiconducto
r etc.)
Flame Burners Photomultipli - Sodium, Liquid Relative
Photometry (Mecker, er tube (PMT) Potassium, Emission
Total Lithium, Intensity vs.
consumption, Magnesium Wave number
Lundergraph
burner etc.)
Atomic Hollow Photomultipli - Sodium, Solids, -
Absorption Cathode er tubes Potassium, Liquids,
Spectroscopy Lamp, (PMT) Calcium, Gas
(AAS – Electrodeless Magnesium
Kirchoff’s Discharge etc.
law) Lamp
Nephelometr White light, Photomultipli Nephelome - Suspen- Absorbance
y/ Monochroma er tubes ter – 90o sions vs. Volume
Turbidimetry tic radiation, (PMT) Turbidimet (most
Tungsten er – 180o sensitive
lamp for
dilute
suspen-
sions
100
mg/l)
Note: Reference standard is known “RS” as per Indian Pharmacopoeia (I.P)
 Turbidimetry used for determination of growth of bacteria in culture media.

SPECTROSCOPY

Definition: Interaction of electromagnetic radiation with matter (atom or molecule).

Types of Pharmaceutical Analysis:

1. Spectral method, e.g. UV, IR, NMR

2. Chromatography, e.g. GC, HPLC
3. Electroanalytical
4. Physical
5. Biological and microbiological assays of vitamins and antibiotics.
6. Radioimmunoassay

Definitions:

1. Wavelength: Difference between Crest and Trough.

2. Frequency: Number of rotations per second.
3. Wave number: Number of waves per cm.

Parameters Units
Wavelength m, cm, μm, nm, Å o
Frequency Hz, cps (cycles per sec), Fresnel
Wave number m-1, cm-1, Kaiser
Velocity / speed m/sec, cm/sec

Types of Spectroscopy:

I] According to atom or molecule:

1. Atomic Spectroscopy: Change in energy (atom)

e.g. Atomic absorption spectroscopy; Atomic emission spectroscopy

2. Molecular Spectroscopy: Change in energy (molecule)

II] According to absorption or emission:

1. Atomic absorption spectroscopy:

e.g. UV, IR, NMR

2. Atomic emission spectroscopy:

e.g. Flame photometry, Fluorimetry

III] According to electric and magnetic level:

 1. Electric level:
e.g. UV
2. Magnetic level:
e.g. NMR, ESR

V I B G Y O R

Shorter λ Higher λ

Higher energy Shorter energy

ULTRAVIOLET-VISIBLE SPECTROSCOPY

UV Regions:

Regions Values
Near UV 2000-4000 Å
Far / Vacuum UV < 2000 Å
Visible 4000-8000 Å

Conversion of units:

 1 nm = 10-7 cm
 1 Å = 10-8 cm

Origin and Theory of Ultraviolet Spectra:

 Ultraviolet absorption spectra arise from transition of electron or electrons from a lower
to higher electronic energy level.
 Energy absorbed in the UV region produces changes in the electronic energy of the
molecule, resulting from transition of electrons in the molecule.

Beer’s Law: Intensity of a beam of monochromatic light decreases exponentially with

increase in concentration of absorbing substance.

Lambert’s Law: When a beam of light is allowed to pass through a transparent medium, the
rate of decrease of intensity with the thickness of medium is directly proportional to the
intensity of the light.

Deviations of Beer-Lambert’s Law:

1. Only in dilute solutions (not in concentrated solutions).

2. Law does not follow in case of suspensions (due to scattering).
3. Does not obey if any chemical reaction occurs in solution.
4. Does not follow in case of polychromatic light (valid only for monochromatic light).
5. Does not follow in case of broad slit width (valid only for narrow slit width).
 If c is in gm/lit and b is in cm, then A = abc ; where, a is absorptivity; b is path length and
c is concentration.
 If c is in moles/lit and b is in cm, then A = εbc, where, ε is molar absorptivity / molar
extinction coefficient.
 A1% 1cm : Absorbance of solution have concentration 1% and path length 1cm.
 Absorbance (A) = - log T or log10 1/T

Types of electrons Present in Examples

σ- electrons Saturated bonds Paraffin compounds
π- electrons Unsaturated hydrocarbons Trienes, aromatic
compounds
n- electrons Non-bonding electrons Nitrogen, oxygen or
halogens

Types of Transitions:

Transitions Energy levels

σ* Antibonding
π* Antibonding
n* Non-bonding
 Bonding
σ Bonding
Note: Transitions to antibonding π* orbitals
are associated only with unsaturated centres.

Order of transitions:
n π* < π π* < n σ* < σ σ*

Energy of transitions:

Types of transitions Energy

n σ* High energy
π π* High energy
n π* Low energy

Transition Probability:

(1) Allowed transitions:

 Transitions having εmax 104 or more.
 Due to π π*

(2) Forbidden transitions:

 Transitions having εmax less than 104.
 Due to n π*
Bands:

Band obtained from Type of band

π π* R - band
n π* B - band
Aromatic / Heteroaromatic K - band
Electronic oscillation (σ σ* E - band
but not completely)

TERMS:

1. Chromophore: It denotes a functional group which gives a specific colour to a

compound, e.g. nitro group is a chromophore as it gives yellow colour to the
compound. It may be defined as, ‘Any group which exhibits absorption of
electromagnetic radiations in the visible or ultraviolet region’.
2. Bathochromic shift or red shift: It involves the shift of absorption maxima towards
longer wavelength, because of the presence of certain groups, such as OH and NH2,
called auxochromes, or by change of solvent or by extension of conjugation.

3. Hypsochromic shift or blue shift: It involves the shift of absorption maxima towards
shorter wavelength due to removal of conjugation, or by a change of solvent.

4. Hyperchromic shift: This effect involves an increase in the intensity of absorption

caused by introduction of an auxochrome.

5. Hypochromic shift: This effect involves a decrease in intensity of absorption and is

caused by groups that are able to distort the geometry of the molecule.

6. Auxochrome: It is a group that itself does not act as a chromophore, but when
attached to a chromophore, shifts the absorption maximum towards longer
wavelength, along with increase in intensity of absorption, e.g. –OH, NH2, etc.

Choice of solvent:

(i) It should not itself absorb radiations in the region.

(ii) It should be less polar, so that it has minimum interaction with the solute molecules.

Woodward-Fieser rules:

(1) Homoannular diene: It is a cyclic diene having conjugated double bonds in the same
ring.

(2) Heteroannular diene: It is a cyclic diene in which double bonds in conjugation are
present in different rings.
(3) Endocyclic double bond: It is a double bond present in a ring.
(4) Exocyclic double bond: It is a double bond in which one of the double bonded atoms is a
part of a ring system.

Parent values and increments for different substituents/groups

Substituents / Groups λmax (nm)

Parent values
Acyclic conjugated diene and heteroannular 215
conjugated diene
Homoannular conjugated diene 253
Acyclic triene 245
Increments
Alkyl substituent or ring residue 5
Exocyclic double bond 5
Double bond extending conjugation 30
For base line separation, value of R (resolution) > 0.5

INFRARED SPECTROSCOPY

 Gives information about structure of compound

 Determines functional group.
 IR spectroscopy produces changes in vibrational / rotational energy in the molecule.

Ranges λ (μ) ν ()

Near IR 0.78 – 2.5 12800 – 4000
Middle IR 2.5 – 50 4000 – 200
Far IR 16 – 200 625 – 10
IR 2.5 – 16 4000 – 625
Fingerprint 400 - 1500 cm-1
Functional group 1500 - 4000 cm-1

Origin of IR: Change in dipole moment (partial +ve and partial –ve)

Theory of IR:

1. Correct wavelength
2. Electric Dipole

Transmittance (%T): The ratio of radiant power transmitted by a sample to radiant power
incident on a sample.

%T = It / Io

Absorbance: Logarithm to the base 10 of the reciprocal of transmittance.

A = log10 1/T

SOURCE:
1. Incandescent Lamp:
 Used in near IR region due to its low emissivity.

2. Nernst Glower:
 Consists of hollow rod.
 Composed of earth oxides such as zirconia, yttria, thoria.
 Heated at temperature 1000-1800 oC
 Maximum radiation 7100 cm-1

3. Globar Source:
 Consists of sintered silicon carbide rod.
 Self-starting
 Heated at a temperature 1300–1700 oC
 Maximum emission at 5200 cm-1

4. Mercury Arc:
 Only source used in far IR region.
 Similar to black body sources (emits 99% radiation).

5. Tungsten filament lamp:

 Closed nichrome wire
 Temperature – 1100oC

SAMPLING TECHNIQUES:

Mull Technique:

Solid sample + Heavy mineral oil (Nujol) Paste Sandwiched between two salt
plates Spectral measurement

 Nujol is highly purified liquid paraffin.

 Alternative to Nujol is Hexachlorobutadiene.
 Absorption maxima at 2915, 1462, 1376, 719 cm-1.
 Disadvantage is, Absorption maxima of drug and Nujol show same peaks.

Pressed Pellet Technique:

Sample finely grinded mix it with 100 times of wt. of KBr 25,000 psig (pressure)
IR tablet press (pellet)

DETECTORS:

1. Thermal Detector
(a) Thermocouple
(b) Bolometer
(c) Thermistor
 (d) Golay cell
2. Photon / Semiconductor Detector
3. Pyroelectric Detector

Bolometer

 Consists of a thin blackened metal strip in an evacuated vessel.

 Uses Wheatstone Bridge.

Golay cell:

 Spectrophotometer
 Consists of a small metal cylinder, one end of which is closed by a blackened metal plate,
and at the other end is a metallic diaphragm.
 Cylinder is filled with xenon (inert gas).

Photon / Semiconductor detector:

 It consists of semiconductor:

(i) Lead telluride

(ii) Lead sulphide
(iii) Germanium
(iv) Indium antimonide

Pyroelectric detector:

 Converts electromagnetic radiation into electrical signal.

 Uses Triglycine (Deuteriated) sulphate, which deteriorates at 45oC; hence, is cooled in
liquid N2).

FT-IR (FOURIER TRANSFORM INFRARED):

A monochromator is not used in FT-IR. In FTIR the following system is used:

a) Drive mechanism
b) Beam splitter
c) Transducer

INFRARED FREQUENCIES:

Compounds Frequencies (cm-1)

Amide 1690
Carboxylic acid 1710
Ketone 1715
Aldehyde 1725
Ester 1735
 MASS SPECTROMETRY
 Qualitative technique
 Sample should be in vaporized form or ionized form.
 Ionization Potential: Energy required to remove, or add, one electron.
 Pico molar concentrations are used.

Ionization Techniques:

 Hard Ionisation techniques:

1) Electron bombard on sample
2) Chemical Ionization: Methane Gas used
3) Field ionization: Contain metal Plates placed at high voltage.
4) Thermal ionization: Heat sample and convert to ions.

 Soft ionization techniques:

1) FAB (Fast atom bombardment): Argon, Xenon Used.
2) MALDI (Matrix assisted laser desorbed ionization)
- They are used to ionize enzymes, vitamins, proteins.
- Detector used in MS is Faraday Cup.

 Types of ions in MS:

1) Molecular ion/ Parent ion: Ion formed by removing one electron.

2) Multiple charge ion: Double/ triple charge ion.
3) Fragmentation ion: Different ions are formed leads to different peaks.
4) Metastable ions: It is not stable throughout mass analyzer.
5) Base peak: Most intense peak on graph.
6) Rearrangement ion.

 McLafferty Rearrangement:

Molecule should contain:

- One double bond

- One hetero atom (N, S, O)
- One beta-bond
- One gamma hydrogen
- One carbon atom

 N-Rule:
- To find out presence or absence of nitrogen in molecule.
 - If molecular weight is odd then N is present and if molecular weight is
even then N may be present or absent in even number.

 Ring Rule: (HID: Hydrogen Ion Deficiency)

“To find out presence of unsaturation character in structure”.

HID= No. of double bond + No. of Ring + 2(twice) No. of triple bond

e.g. C6H6 (Benzene)

HID = 3+1+0= 4 Double Bonds, i.e., 8 Hydrogen less.

Modifications of MS:

LC-MS is used to detect all organic compounds, except Phosphoric Acid.

NUCLEAR MAGNETIC RESONANCE (NMR)

 Qualitative Technique
 Absorption Phenomenon
 Involves Radiowaves (60-600 mHz)
 NMR is used to confirm the drug structure.
1
H, 13C (odd no.) assymetrical charge distribution (shows “Nuclear magnetic moment”)
2
H, 14C (even no.) symmetrical charge distribution (does not shows NMR)

Identification of Protons (H) present in molecule:

- Determine number of types of chemical environment.

- Number of protons in each environment
- CH3-CH2-OH 3 Types of Hydrogen

(1) (2) (3)

1st contain 3 Hydrogen
2nd contain 2 Hydrogen
3rd contain 1 Hydrogen
- No. of hydrogen atoms present on neighbouring carbons.

- Spin + Earth gravity = Gyroscopic motion

- Gyromagnetic ratio = 26750 (for proton)

 - Principle: Any charged body develops its own magnetic field around
itself.

- In NMR, maximum frequency is observed when radiofrequency (Rf) is

equal to precessional frequency (Pf).

Types of NMR instrument:

1. Frequency sweep: Change frequency of source and field constant.

2. Field sweep: Change field and frequency constant.

Coupling (Relaxation): Excited to ground (Emission)

Spin-Spin Coupling Spin-Lattice Coupling

(Transverse Relaxation ) (longitudinal Relaxation )

Any proton transfers its energy to Any proton transfers its energy
neighbouring proton placed on to surrounding medium (solvent
Neighbouring carbon through molecule) not through bonding
bonding.

Occurs only upto 3- bond distance

Shielding and Deshielding:

1. Shielding (High energy): Effective magnetic field is less than the applied magnetic
field (opposing magnetic field).

2. Deshielding (Low energy): Effective magnetic field is more than the applied
magnetic field (with magnetic field).

Unit of applied magnetic field: Gauss / Weber / Tesla

Chemical shift:

 Shift in the absorption of NMR signals.

 Proportional to field strength
 Expressed in ppm (parts per million) / dimensionless

Shielding / Upfield
0 (σ) 10 (τ)

Deshielding / Downfield

τ = 10 - δ
Features of TMS:

(i) TMS has 12 equivalent protons and gives an intense single signal.
(ii) The electronegativity of Silicon is very low (1.8 as compared to Carbon’s 2.5)
(iii) TMS is chemically inert and has a very low boiling point (300 K).

Factors affecting Chemical shift:

1) Electronegativity: Electronegative group shows Deshielding.

2) Hydrogen Bond: Shows Deshielding.
 Chances of inter-hydrogen bond can be reduced by dilution, but not in case of intra-
hydrogen bond.
 Very very dilute solutions are used.
3) Anisotropic Effect: i.e., Molecule shows different behaviour. Arrangement of atom in
space is also referred as Space Effect.

Compound Delta value Indicates

Ethane 0.95 Shielding
Ethene 4-5 Deshielding
Acetylene 1.5-2 Shielding

4) Van der Waals deshielding: In overcrowded molecules, electron clouds of bulky groups
(hindering group), tend to repel electron clouds surrounding protons. Such protons will
deshield and resonate at slightly higher values.

Rules for Splitting of Proton Signals:

(i) Splitting of a proton signal is caused only by neighbouring or vicinal protons, i.e.,
protons on adjacent carbon atoms.
(ii) Splitting of one proton by another on the same carbon very rarely occurs, because
such protons are equivalent to each other.
(iii) The number of peaks (N) into which a proton signal is split, is equal to one more
than the number of vicinal protons (n).
N=n+1

Thus, the NMR signal due to a proton is split into:

A doublet (with peak intensities 1:1) by one vicinal proton.

A triplet (with peak intensities 1:2:1) by two vicinal protons.

A quartet (with peak intensities 1:4:4:1) by three vicinal protons.

A quintet (with peak intensities 1:4:6:4:1) by four vicinal protons and so on.
 (iv) All the peaks of a given multiplet are not of exactly the same intensity.

Coupling constant (J):

 Distance between two split lines

 Expressed in Hertz (HZ) or cycles per second (cps)
 Independent on magnetic field.
 Ortho, meta, para- isomers are differentiated on the basis of Coupling constant.

Compounds Coupling Constant values (Hz)

Anti 5 – 12
Gauche 2–4
Cis 6 – 14
Trans 11 – 18
Geminal protons 0 – 20
Vicinal protons 2 – 18

Solvents in NMR:

 Deprotonated
 DMSO (Dimethyl sulfoxide): Highly polar solvent
 Chemically inert
 Magnetically isotropic
 Particularly devoid of H atoms
 e.g. Pyridine, Dioxane, Dimethylformamide (DMF), Acetonitrile, Benzene, Carbon
tetrachloride (CCl4), Carbon disulphide (CS2), Hexachloroacetone, CDCl3

Type of solvent Examples

Universal solvent DMSO
Aqueous solvent 2, 2-dimethyl, 2-silapentane-5-sulphonate
Organic solvent Tetramethyl silane (0.01-1 %)
19
F solvent CFCl3
31
P solvent H3PO4

Interpretation of NMR Spectra:

(acronym: “PINS”)

(1) Number of signals – how many kinds of protons are there in different chemical
environments.
(2) Positions of signals – electronic environment of each kind of proton.
(3) Intensities of signals – relative number of protons of different kinds.
(4) Splitting of signals – environment of the neighbouring protons.
OTHER IMPORTANT POINTS TO REMEMBER:

Paramagnetism: Proton present at peripheral part of molecule, i.e., direction of proton

magnetic field and external magnetic field is same and it causes Deshielding.

Diamagnetism: Proton present at central part of molecule, i.e., direction of proton magnetic
field and external magnetic field is opposite and it causes Shielding.

Shift Reagent: Used to convert complex spectrum (overlapped) into simple spectrum, e.g.
Europium (Eu), Praeseodymium (Pr) from Lanthanides and Actanides series of the periodic
table.

Spin-Spin Decoupling: Used to convert complex spectrum to simple spectrum. Also called
as Double Resonance, or Double Irradiation.

Magic Angle NMR: 54.7

ELECTRON SPIN RESONANCE / ELECTRON PARAMAGNETIC RESONANCE

Substances showing Magnetic moment of

Paramagnetism Unpaired electrons
Dimagnetism Paired electrons

The techniques used to simplify the spectra are:

 ENDOR (Electron Nuclear Double Resonance): Sample irradiated with Microwave

Frequency and Radiofrequency.

 ELDOR (Electron Double Resonance): Sample irradiated with 2 Microwave

Frequencies.

Difference between NMR and ESR:

Sr. NMR ESR/EPR

No.
1. Related with Proton Related with Electron
2. Radiation: Radiowaves Radiation: Microwaves
3. Rf source and Rf coil Klystron Source
4. Rf detector Crystal detector
5. Constant: Gyromagnetic Constant: Bohr’s
Ratio (γ) Magneton (β)
6. Spin-Spin splitting & Spin-Spin splitting &
coupling between TWO coupling between
Protons proton and electron
7. Hydrogen containing Electron containing
species show NMR species show ESR
 Magic –T(Circulating-T),

X-RAY DIFFRACTION (XRD)

X-rays was discovered by William K. Roentgen

XRD relationship : Henry Moseley’s Theory

Bragg’s equation:

nλ = 2d sin θ

Where,

n = integer

d = distance between crystals

θ = the angle of diffraction.

If, n = 1, first order

n = 2, second order

Instrumentation Name
Source Coolidge tube (simple x-ray tungsten
tube)
Filter Zirconium filter (k line)
Detector Scintillator, Photon counter
Target material Copper

X-ray Crystallography:

 Provides configuration of compound.

 Structure of enzyme is determined.

CHROMATOGRAPHY

Chromatography is an analytical technique employed for purification and separation of

organic and inorganic substances.

Chromatographic techniques:

1. High performance (pressure) liquid chromatography (HPLC).

2. Adsorption column chromatography.
3. Partition paper chromatography.
 4. Thin layer chromatography (TLC).
5. Ion exchange chromatography (IEC).
6. Gel permeation (Size exclusion) chromatography (SEC).
7. Gas chromatography (GC).

Non-Hazardous substitute for Radioimmunoassay – HPLC

Polar compound: A compound which is held by the stationary phase is called as Polar
compound.

Non-polar compound: A compound which tends to move forward in the mobile phase is
called as Non-polar compound.

ADSORPTION CHROMATOGRAPHY

Principle: Adsorption chromatography is a technique in which stationary phase is a solid

(e.g. alumina or silica gel) and the mobile phase is either a liquid or gas.

The most strongly adsorbed component forms the topmost band, while the least strongly
adsorbed component forms the lowermost band.

Adsorbent: Adsorbents may be classified according to the force with which they hold the
ions:

(a) Weak force: Sucrose and starch

(b) Intermediate force: Calcium carbonate, calcium phosphate, calcium hydroxide etc.
(c) Strong force: Alumina, charcoal etc.

Factors affecting column efficiency:

(i) Dimension of the column: As the length/width ratio of the column is increased,
column efficiency increases.
(ii) Particle size of the packing: As the particle size of the packing is decreased,
separation is improved.
(iii) Packing of the column: In dry packing, the material is added in small amounts so as
to give maximum packing intensity. In wet packing, the material is made into slurry
with the solvent and added to the column in portions.

Application: Used to determine amino acid contents of protein hydrolysate.

PAPER CHROMATOGRAPHY

Paper chromatography may be defined as the technique in which the analysis of an unknown
substance is carried out mainly by the flow of solvent on specifically designed filter paper.

Types of Paper Chromatography:

1. Paper partition chromatography: This is a technique in which paper is used as an inert

support with one solvent as mobile phase and other as stationary or immobile phase.
2. Paper adsorption chromatography: In this technique, a modified paper (first
impregnated with an adsorbent like silica or alumina) is used as an adsorbent and a single
solvent is allowed to flow over the unknown components.

Principle

Developer: A drop of solution containing the sample is introduced at the starting point on
the paper. Migration occurs as a result of flow of the mobile phase across the paper.

Ascending development: When the movement of the mobile phase is in upward direction.

Descending development: When the movement of the mobile phase is in downward

direction.

Radial or disc development: When the flow of mobile phase is outward from a central spot.

Technique

- The liquid rises up the strip of paper (15-30 cm in length) by capillary

action.
- The dried paper is called paper chromatogram.

The individual components can be characterised by the Rf values (rate of flow), where

Rf = distance moved by the component

distance moved by the solvent

-Whatmann filter paper is widely used in PC.

Materials

Materials Examples
Paper α-cellulose (98.99%), β-cellulose, ammonia, nitrogen
compounds etc.
Mobile phases Isopropanol: Ammonia: Water (9:1:2)
n-butanol: Acetic acid: Water (4:1:5)
Water-phenol etc.
Solvents Methanol, formamide, glycol, aromatic and aliphatic
hydrocarbons etc.
Applications: Quantitative analysis (Separation of amino acids)

ISOTHERMS:

Type of isotherm Type of chromatographic peak

Adsorption isotherm Tailing
Partition isotherm Fronting
 THIN LAYER CHROMATOGRAPHY (TLC)

Principle: In TLC partition, occurs on a layer of finely divided adsorbent, which is supported
on a glass plate.

TLC Plate Thickness: 0.1-2 mm

Basic operations involved in TLC:

1. Methods for the production of thin layers on plates (0.25-2 mm)

2. Application of sample on the chromoplates (0.1-1 %)
3. Choice of adsorbent
4. Choice of solvent
5. Detecting reagent
6. Developing chamber
7. Development and detection

Materials:

Materials Examples
Adsorbent Silica gel (acidic), alumina, kiesulghur and powdered cellulose
Binding agent Calcium sulphate
Solvents Petroleum ether, CCl4, benzene, chloroform, diethyl ether, ethyl
alcohol, water etc.
Detecting agents Iodine vapour and sulphuric acid (mixed with aromatic
aldehydes or oxidising agents like KmNO4, HNO3, chromic acid
etc.)

Derivatizing agents:

TLC of Derivatizing agents

Steroids Antimony trichloride
Lipids Bromothymol blue

GAS CHROMATOGRAPHY

Gas chromatography is based on distribution of gaseous solutes between a gas and liquid or
solid phase.

GC is the most sensitive chromatography.

Type Stationary phase Mobile phase

Gas solid chromatography Granular silica, alumina or Gas
carbon
Gas liquid chromatography Kiesulghur or diatomaceous Gas
earth
THEORY:

Performance – Resolution

Rs = tb – ta = 2 (tb – ta)

(Wa + Wb)/2 (Wa + Wb)

Where,

W= band width

t = time taken for the band to appear

 Band broadening  1/column efficiency (inversely proportional to column effeciency)

 Band separation  solvent efficiency (directly proportional to solvent efficiency)

Plate theory

N=L/H

Where,

N = no. of plates

L = length

H = height of each plate

- As N increases, resolution increases.

- As H decreases, resolution increases.

N = 16 (t/w)2

OR,

N = 5.14 (t/w1/2)2

Van Deemter Equation (control performance):

H = A + B/u + Cu

Where,

A = Eddy diffusion (multiple path)

B = longitudinal diffusion (important in GC)

C = mass transfer (mass transfer to another solvent must be low)

u = velocity of mobile phase

If increase Increases Decreases

Eddy diffusion Height of plates Efficiency
Longitudinal diffusion Height of plates Efficiency
Mass transfer Height of plates Efficiency
Velocity of mobile phase Efficiency -

Instrumentation:

1. Carrier gas
2. Injection port
3. Columns
4. Detectors

1. Carrier gas: The mobile phase in GLC is usually helium or nitrogen.

2. Injection port: Liquid and gas samples are injected by syringe through a silicon rubber
diaphragm in the injection port.
3. Columns:
(i) Capillary columns: It is fabricated from capillary tubing.
(ii) Packed columns: It is usually a stainless steel or copper tube packed with either a
solid substrate (GSC) or a liquid coating on an inert solid (GLC).

Note: Coiled column (50 metre) is used to improve sensitivity.

Solid inert support: The main purpose of this is to provide support to the thin, uniform film
of liquid phase, e.g. kiesulghur (diatomaceous earth).

Tailing of peaks can be minimised by using a polar liquid phase, which is adsorbed strongly
on the surface of the solid and which can be reduced by treating a solid with a silanizing
agent, such as Hexamethyl disilazane.

Stationary liquid phase:

(i) Non-polar liquids: Squalene

(ii) Polar liquids: Polyethylene glycol

4. Detectors:
(i) Thermal conductivity detector (Katharometer)
(ii) Flame ionisation detector (Destructive type)
(iii) Electron capture detector

- Flame ionisation detector is the most commonly used GC detector.

- Flame ionisation detector is used for detecting all organic compounds
except Formic acid.
- Flame ionisation detector shows greatest response to Hydrocarbons.
- Electron capture detector is the only detector which is sensitive to
halogenated compounds and pesticides etc.
 - Novel detector in GC: Chlorine selective pulse discharged emission
detector (CL-PDED).
- Head space analysis is done in GC.

Sample derivatisation: is done to

- Increase vapour pressure (to get volatile sample)

- Increase detectability
- Modify solubility

Applications:

1. Qualitative analysis: These are based on the time required for the peaks to appear at the
end of the column.
2. Quantitative analysis: These are based on peak heights, or on peak areas.
- Gel Permeation Chromatography
- Size Exclusion Chromatography

Only chromatography in which sample does not react with solvent, i.e., there is no interaction
between solute and solvent.

Stationary phase: Styrene divinylbenzene co-polymer / Cross-linked dextran

Applications:

(i) Separation of polymers (macromolecules).

(ii) Determination of molecular weight of polymer.

CHIRAL CHROMATOGRAPHY

- Either column or mobile phase is chiral

- Used to separate enantiomers.

Column derivatisation:

Type of chromatography Stationary phase Mobile phase

Normal phase Polar Non-polar
Reverse phase Non-polar Polar

Octadecyl silane (ODS) C-18

-Si – OH -Si – CH3

-Si – OH -Si – CH3

-Si – OH -Si – CH3

(Hydrophilic) (Hydrophobic)
Silanol effect: Interference in separation by unreplaced –OH groups.

End capping: Derivatisation done at last (To overcome silanol effect, column is reacted at
end of derivatisation with small size alkyl (CH3) group).

Acetonitrile column (ANC): Hydrophilic as compared to alkylated column.

AFFINITY CHROMATOGRAPHY

Only chromatography based on lock and key mechanism, in which enzyme reacts with the
substrate, e.g. Dihydrofolate reductase (DHFR) has affinity towards methotrexate.

Application: Used in biotechnology for separation of enzyme.

ION EXCHANGE CHROMATOGRAPHY

In ion exchange chromatography, a reversible exchange of ions is possible between ions in a

liquid phase (mobile phase) and a solid, insoluble substance containing ionic sites (ion
exchange resin).

Ion exchange resins:

Type Examples Used in fractionation of

Strongly acidic cation-
exchange resins
Weakly acidic cation-
exchange resins
Strongly basic anion-
exchange resins
Weakly basic anion-
exchange resins
Sulphonated polystyrene resins
Carboxylic polymethacrylate
resins
Quarternary ammonium
polystyrene resins
Phenol formaldehyde and
polyamine polystyrene resins
Cations, inorganic separations,
vitamins, amino acids etc.
Cations, transition elements,
amino acids, antibiotics etc.
Anions, halogens, alkaloids,
vitamin B complex etc.
Anionic complexes of metals,
vitamins and amino acids

Crosslinking:

- As the crosslinking in the resin decreases, the resin swelling increases.

- Divinylbenzene is most commonly used crosslinking agent.

Particle size range (50-100 mesh or 100-200 mesh)

IMPORTANT CHARTS TO REMEMBER

Type of structure Techniques used

Structural isomer Nuclear magnetic resonance (NMR), Adsorption chromatography
Crystal structure X-ray diffraction (XRD), Electron spin resonance (ESR)
Cis and trans isomer Mass spectrometry (MS), X-ray diffraction (XRD)
 IMPORTANT POINTS TO REMEMBER

 In Limit of Detection, signal to noise ratio = 3:1

 In Limit of Quantitation, signal to noise ratio = 10:1