chapter 3

Pharmaceutical Analysis

Pharmaceutical Analysis UV-Visible Spectroscopy (Electronic

Electromagnetic Radiation Spectroscopy)
Electromagnetic (EM) radiation is a periodically changing Basic principle Valence shell electronic transition.
or oscillating electric field propagating in a certain direction Graph Plotted between absorbance and wavelength.
with a magnetic field oscillating at the same frequency but
perpendicular to the electric field.
EM radiation may be considered as a travelling wave
or as a stream of massless elementary particles, often called
photons. Antibonding σ∗
n σ* σ σ*
Characteristics of wave Antibonding π∗
Energy

n π* π π*
yy Wavelength λ (the length of one wave) e e Non-bonding η
‡‡ Expressed in nm/ A°/µm n σ*
yy Frequency ν (the number of waves per unit time) e Bonding π
‡‡ Expressed in cycle per second (cps)/Hertz/Fresnel
‡‡ V = c/λ = c * wave number
e Bonding σ
yy Wave number k (the number of waves per unit length)
‡‡ Wave number =1/λ
‡‡ Expressed in cm-1 or Kaiser Energy value order for transition
Max-Plank Equation n → π* < n → σ* < π → π * < σ → σ*
E = h v = h c/λ Transition probablity
Where E is energy of photon 1. Allowed–extinction coefficient value 104 or greater
ν is frequency of EM radiation = c/λ hence v is inversly 2. Forbidden–extinction coefficient value 102 or less
proportional to λ
h is Plank constant (6.6 × 10–27 erg-sec) Absorption band
c is velocity of light 1. K band–due to π → π* transition
λ is wavelength of EM radiation 2. R band–due to n → π* transition

Cut-off wavelength Above which solvent behaves as transparent

Methanol 210 nm Hexane 199 nm Water 190 Diethyl ether 205 nm

nm

Ethanol 207 nm Benzene 280 nm Heptane 200 nm THF 220 nm

Chloroform 247 nm Carbon tetrachloride 257 nm Acetone 331 nm Dichloromethane 233 nm

 Analytical Chemistry  3.87

Instrumentation 2. Provides narrow effective bandwidth with increased

1. Sources (UV and visible) output.
2. Wavelength selector (monochromator)
3. Sample containers Construction of monochromator
4. Detector 1. Entrance slit
5. Signal processor and readout 2. Collimating mirror (Provide parallel beam of radiation
to prism or grating)
Monochromator Reference Detector 3. Diffracting grating
Ratio Output 4. Focussing mirror
Sample Detector
5. Exit slit

yy Most widely used radiation source in UV deuterium Basically, monochromator converts polychromatic
discharge lamp and in visible tungsten halogen lamp. light (EM radiation of more than one wavelength) into
yy Most widely used detector in UV-visible spectroscopy monochromatic light (EM radiation of single wavelength).
is PMT (photo multiplier tube).
Interferometer
Instead of filtering or dispersing the EM radiation, it si-
multaneously allows source radiation of all wavelengths to
reach detector.
Dynode Construction of interferometer
1. Fixed mirror
2. Moving mirror
Grill
3. Beam splitter (Transmit half of the radiation to fixed
Anode Incident mirror and half of the radiation (which are reflected)
radiation to movable mirror). It is a semi reflecting device and
Photoemissive made up of silicon or germanium coated on metal
cathode halide plate.
Isobestic point common point to every absorption curve
Figure 3.1 UV-Visible Spectroscopy
which is obtained in the spectrum of compound taken at
Wavelength selector different pH.
yy It should provide narrow band of radiation and maxi-
mum throughput. IR Spectroscopy
yy Wavelength isolation can be done using filter, mono-
Basic principle Vibrational level changes
chromator and interferometer.
Graph Plotted between % transmittance and wave number.
Filter A wavelength selector that uses either absorption,
or constructive and destructive interference to control range
of selected wavelength. Selection rule in IR
A. Absorption Filter Narrow effective bandwidth 30 to Only those compounds are IR active which show change in
250 nm and Maximum throughput 10%. dipole moment upon interaction with IR radiation.
B. Interference Filter Narrow effective bandwidth 10 Fundamental frequency of Vibration γ = 1/2π * (K/µ)1/2
to 20 nm and Maximum throughput 40%.
Region Wavelength Wavenumber
Monochromator range (Micron) range (cm–1)
A wavelength selector that uses diffraction grating or
Near/Harmonic/Over- 0.78–2.5 12800–4000
prism, and that allows continuous variation of nominal tone
wavelength.
Middle/Fundamental 2.5–50 4000–200
Advantages of monochromator over filter
1. It provide continuous variation of nominal wavelength. Far/Rotational 50–1000 200–10
3.88  Chapter 3

Bending Change in angle between two bonds. There are Sampling techniques
four types of bends: 1. Mull technique
yy Rocking Nujol (mineral oil) is a mixture of paraffin hydrocarbons. To
yy Scissoring avoid Nujols band in spectrum sometimes hexachlorobuta-
yy Wagging diene or chlorofluorocarbon oil is added.
yy Twisting 2. Pressed pellet/KBr
KBr is used 100 times to sample quantity. This technique
Bending vibrations can be used for quantitative analysis.
Near Near Near
Far
NMR Spectroscopy
Basic principle Nuclear spin changes.
In-plane In-plane Out-of-plane Out-of-plane Nuclear-zeeman effect Splitting of nuclei spin state in
rocking scissoring wagging twisting applied external magnetic field.
Stretching vibrations
Energy levels for a nucleus with spin quantum
number 1/2

Applied
No field magnetic field 1
Symmetric Asymmetric m= −
Energy 2
Figure 3.2 Types of Bending and Stretching Vibrations 0

1
Factors affecting vibration frequency m= +
2
1. Nature of bond present
2. Masses of atoms Larmor equation It is fundamental equation of NMR
3. Force constant of bond spectroscopy.
4. Electronic effect
5. Bond angle W (Angular Precessional Frequency) = γ H
6. Hydrogen bonding 2πv = γ H
7. Symmetry of molecule
Precessional frequency (v) = γ/2π
Type of degree of Linear Non-linear where H = Applied magnetic field
freedom γ = 
Magnogyretic ratio or Gyro-
Transitional 3 3 magnetic ratio = 2πµ/hI

Rotational 2 3 where h is Plank constant and I is Spin Quantum Number.

µ is Magnetic moment of spinning nuclei.
Vibrational 3N-5 3N-6 Magnetic moment = γ * Spin angular moment (h/2π *
Total 3N 3N spin quantum number I)
Note only those Nuclei show NMR absorption signal
having spin quantum number (I) greater or equals to ½.
Finger print region
1. 8 µ to 16 µ or 1500 cm–1 to 500 cm–1 1. If the number of neutrons and the number of protons
2. Absorption is unique and complex. are both even, then the nucleus has no spin.
2. If the number of neutrons plus the number of pro-
Fermi resonance tons is odd, then the nucleus has a half-integer spin
Energy of harmonic or overtone region coincides with (i.e., 1/2, 3/2, 5/2)
fundamental mode of vibration. Instead of one band, two 3. If the number of neutrons and the number of protons
bands of almost equal intensity results. It is normally observed are both odd, then the nucleus has an integer spin
in carbonyl compounds. (i.e., 1, 2, 3)
 Analytical Chemistry  3.89

Element H1 H2 C12 C13 N14 O16 O17 P31 Cl35 F19

Spin quantum number (I) 1/2 1 0 1/2 1 0 5/2 1/2 3/2 1/2

No. of spin states (2I + 1) 2 3 1 2 3 1 6 2 4 2

Nucleus Spin (I) Natural Abundance/% Magnetogyric Ratio (γ) /107 kg–1·s·A Relative Frequency (ν) /MHz
1
H 1/2 99.985 26.752196 100.00
2
H 1 0.015 4.106625 15.35
13
C 1/2 1.10 6.72828 25.15
15
N 1/2 0.366 –2.712621 10.14
17
O 5/2 0.037 –3.62808 13.56
19
F 1/2 100.0 25.18147 94.13
29
Si 1/2 4.67 –5.319 19.88
31
P 1/2 100.0 10.8394 40.52
119
Sn 1/2 8.58 –10.0318 37.27

Population Densities of Nuclear Ideally, Relaxation rates to be fast–but not too fast. If
Spin States the relaxation rate is fast, then saturation is reduced. If the
relaxation rate is too fast, line-broadening in the resultant
Saturation of signal If population densities of upper
NMR spectrum is observed.
and lower spin states becomes exactly equals then we
There are two major relaxation processes:
observe no net signal. This is called saturation of signal.
Saturation of signal can be achieved by intense RF signal. yy Spin-lattice (longitudinal) relaxation
Saturation should be avoided in during NMR experiment. yy Spin-spin (transverse) relaxation

Boltzmann distribution Spin-lattice-relaxation

In the presence of an external magnetic field, different Nuclei in an NMR experiment are in a sample. The sample
mI nuclear spin states have different energies. At thermal in which the nuclei are held is called the lattice. Nuclei in
equilibrium, they will also have different populations the lattice are in vibrational and rotational motion, which
according to the Boltzmann equation creates a complex magnetic field. The magnetic field caused
by motion of nuclei within the lattice is called the lattice
N high field. This lattice field has many components. Some of these
= e− ∆ / k⁄
N low components will be equal in frequency and phase to the
1 Larmor frequency of the nuclei of interest. These compo-
=
e − ∆ / k⁄ nents of the lattice field can interact with nuclei in the higher
energy state, and cause them to lose energy (returning to the
where Nhigh and Nlow are the populations of the upper lower state). The energy that a nucleus loses increases the
and lower states respectively, ΔE = Ehigh – Elow is the energy amount of vibration and rotation within the lattice (resulting
difference between the two states, k is the Boltzmann con- in a tiny rise in the temperature of the sample).
stant, and T is the absolute temperature.
Spin-spin-relaxation
Relaxation Processes Spin-spin relaxation describes the interaction between
It is the process by which nuclei undergo (return back) from neighbouring nuclei with identical precessional frequencies
higher energy state to the lower state. but differing magnetic quantum states. In this situation, the
3.90  Chapter 3

nuclei can exchange quantum states; a nucleus in the lower ‡‡ Chemical shift equivalent protons-Nuclei having
energy level will be excited, while the excited nucleus re- identical chemical shift.
laxes to the lower energy state. There is no net change in the ‡‡ Magnetically equivalent protons-Nuclei having
populations of the energy states, but the average lifetime of identical coupling constant.
a nucleus in the excited state will decrease. This can result
in line-broadening. NMR instrumentation
Chemical shift It is a dimensionless quantity and does 1. Magnet-To provide magnetic field
not depends on applied external field. It is expressed in
parts per million (ppm). RF (60-MHz)
oscillator
δ = Frequency shift (In Hz) X 106/Operating frequency (MHz) RF detector Recorder

Reference for measurement of chemical shift

TMS (tetra methyl silane) is used as reference in proton
NMR. Because
1. TMS has 12 equivalent protons N S
~1.41 T ± a
2. Chemically inert and very low B.P. few ppm
3. Miscible with all organic substances
4. Electron negativity of silicon is very low so shield- Variable
magnetic
ing of protons in TMS are most shielded compared to
field
other organic compounds.
Figure 3.3 NMR instrumentation
TMS is not soluble in aqueous solution hence 2,2
dimethyl-2-2-silapentane-5-sulphonate. 2. Sweep generator–To vary the field strength.
There are two methods, one is field sweep another is
Shift reagent These are paramagnetic substances used
frequency sweep method.
to spread the NMR absorption pattern without increasing
3. Rf transmitter–To provide Rf radiation for NMR
the magnetic field strength.
phenomenon
Example-Lanthanide Fluorinated β-di ketones 4. Rf receiver
Splitting of signal
yy The multiplicity of a multiplet is given by the number of Magnet
equivalent protons in neighbouring atoms plus one, Earlier, NMR magnets were iron core permanent or elec-
i.e., the n + 1 rule tromagnets producing magnetic fields of less than 1.5 T.
yy Equivalent nuclei do not interact with each other. Today, most NMR magnets are of the superconducting
type. Superconducting NMR magnets range in field strength
Type of Multiplet Relative Inten- No. of Vicinal from approximately 6 to 23.5 T.
sity Protons A superconducting magnet has an electromagnet made
of superconducting wire. Superconducting wire has a re-
Doublet 1:1 1 sistance approximately equal to zero when it is cooled to
Triplet 1:2:1 2 a temperature close to absolute zero (–273.15° C or 0 K)
by immersing it in liquid helium. Once current is caused to
Quartet 1:3:3:1 3 flow in the coil, it will continue to flow for as long as the coil
is kept at liquid helium temperatures.
Quintet (Pentet) 1:4:6:4:1 4
The superconducting elements of the wire are made of
Sextet 1:5:10:10:5:1 5 (NbTaTi)3Sn. This material is brittle and therefore is embedded
in copper for strength. The Cu has a high resistance compared
Coupling constant (J) to the superconductor which is carrying the current.
This wire is wound into a multi-turn solenoid or coil.
yy Distance of centre of peaks in a given multiplet. It is
The coil of wire is kept at a temperature of 4.2K or less by
expressed in Hz or cycle per second.
immersing it in liquid helium. The coil and liquid helium
yy Normal range 0–20. Ratio of J for trans to cis alkene is
are kept in a large dewar. This dewar is typically surrounded
approximately 2.
 Analytical Chemistry  3.91

by a liquid nitrogen (77.4K) dewar, which acts as a thermal of the molecule OR if they interchange by a rapid process
buffer between the room temperature air (293K) and the (rapid with respect to the NMR timescale).
liquid helium. If a pair of nuclei can be interchanged by rotation about
There is a vacuum region followed by a liquid nitrogen an axis of symmetry of the molecule, then they are chemically
reservoir. The vacuum region is filled with several layers of equivalent and are called homotopic. E.g., the pair of protons
a reflective mylar film. The function of the mylar is to reflect in dichloromethane are chemically equivalent.
thermal photons, and thus diminish heat from entering the If a pair of nuclei can be interchanged by an improper
magnet. rotational symmetry operation of the molecule, then they
are chemically equivalent and are called enantiotopic. E.g.,
Shim Coils pair of protons attached to the alpha-Carbon in glycine amino
The purpose of shim coils on a spectrometer is to correct acid (they are not chemically equivalent if glycine is part of
minor spatial inhomogeneities in the Bo magnetic field. a polypeptide chain).
These inhomogeneities could be caused by the magnet design, If a pair of geminal protons (CH2) cannot be interchanged
materials in the probe, and variations in the thickness of through a symmetry operation of the molecule, then these
the sample tube, sample permeability, and ferromagnetic protons are diastereotopic and are not chemically equivalent.
materials around the magnet. A shim coil is designed to E.g., the b-methylene protons of amino acids where the
create a small magnetic field which will oppose and cancel methylene group is attached to chiral Cα atom.
out an inhomogeneity in the Bo magnetic field. Chemical shift equivalence by rapid interconversion
Shim Coil function: by passing the appropriate amount of structures may occur due to rapid rotation about bonds
of current through each coil, a homogeneous Bo magnetic or due the rapid chemical changes such as keto-enol
field can be achieved. tautomerism.

Sample Probe Magnetic Equivalence

The sample probe is the name given to that part of the (Spin Coupling Equivalence)
spectrometer which accepts the sample, sends RF energy If in a set of chemically equivalent nuclei, each member of
into the sample, and detects the signal emanating from the the set has exactly the same interaction (J-coupling) to ev-
sample. It contains the RF coil, sample spinner, temperature ery other magnetically active nucleus in the molecule, then
controlling circuitry, and gradient coils. the nuclei are also magnetically equivalent. E.g., the pair of
The purpose of the sample spinner is to rotate the NMR protons in dichloromethane are chemically as well as mag-
sample tube about its axis. In doing so, each spin in the netically equivalent.
sample located at a given position along the Z axis and radius A set of nuclei that are magnetically equivalent will also
from the Z axis, will experience the average magnetic field be chemically equivalent; however, chemical equivalence
in the circle defined by this Z and radius. The net effect is a does not guarantee magnetic equivalence. e.g., the two
narrower spectral linewidth. protons ortho to hydroxy group in tyrosine are chemically
equivalent but they are not magnetically equivalent.
RF Coils
RF coils create the B1 field which rotates the net magneti- Nuclear Overhauser Effect (NOE)
zation in a pulse sequence. They also detect the transverse Nuclear Overhauser Effect (NOE) is the transfer of nuclear
magnetization as it precesses in the XY plane. spin polarization from one nuclear spin population to another
Most RF coils on NMR spectrometers are of the saddle via cross-relaxation.
coil design and act as the transmitter of the B1 field and When a proton is saturated or inverted, spatially-close
receiver of RF energy from the sample. You may find one or protons may experience an intensity enhancement, which
more RF coils in a probe. is termed the Nuclear Overhauser Effect (NOE). The NOE
is unique among NMR methods because it does not
Chemical Shift Equivalence depend upon through-bond J couplings but depends only on
If a set of nuclei exists in identical environments, they are the spatial proximity between protons. In other words, the
expected to have the same chemical shift. Such nuclei are strength of the NOE gives information on how close two
called chemical shift equivalent or chemically equivalent. protons are. For small molecules, an NOE may be observed
Pair of nuclei in a molecule is chemically equivalent if between protons that are up to 4Å apart, while the upper
they are interchangeable through any symmetry operation limit for large molecules is about 5Å.
3.92  Chapter 3

The NOE differs from the application of spin-spin Mass Spectroscopy

coupling in that the NOE occurs through space, not through
Basic principle
chemical bonds. Thus, atoms that are in close proximity to
each other can give a NOE, whereas spin coupling is observed Molecules are bombarded with high energetic electron
only when the atoms are connected by 2-3 chemical bonds. beam, positive ion fragments are sorted out depending upon
The inter-atomic distances derived from the observed NOE their m/z ratio. (No EM radiation used)
can often help to confirm a precise molecular conformation, Graph Plotted between Relative Abundance and m/z.
i.e., the three-dimensional structure of a molecule.
Mass Spectrometer Sorting out mechanism
Magic Angle NMR
Conventional MS Energy and momentum
In nuclear magnetic resonance, magic angle spinning
(MAS) is a technique often used to perform experiments in Time of Flight (TOF) MS Energy and Velocity
solid-state NMR spectroscopy.
By spinning the sample (usually at a frequency of 1 to 70 FT-MS Momentum and Velocity
kHz) at the magic angle θm (ca. 54.74°, where cos2θm=1/3)
with respect to the direction of the magnetic field, the normally Ionization mode in MS
broad lines become narrower, increasing the resolution for A. Gas Phase
better identification and analysis of the spectrum.
1. Electron impact ionization (Unimolecular)–Tungsten
In any condensed phase, a nuclear spin experiences a
filament (50–80 ev) used as electron source.
great number of interactions. The main three interactions
2. Chemical mode (Bi molecular)–Methane (mostly
(dipolar, chemical shift anisotropy, quadrupolar) often lead
used gas), Isobutane, Ammonia and inert gases
to very broad and featureless lines. However, these three
(He, N2 Ar2).
interactions in solids are time-dependent and can be averaged
by MAS. B. Desorption/Condensed Phase
1. Field desorption or Laser desorption
Shielding 2. Plasma desorption or californium fission fragments
All atoms in a molecule are surrounded by electrons that 3. FAB (Fast Atom Bombardment)–Solvent used is Glyc-
occupy  core and valence orbitals. The permanent magnetic erol
field β0 induces a current in the surrounding electrons,
 Argon gas ionized Ar+. ions are accelerated
which in turn generates an induced magnetic field βind .                    
According to Lenz’s Law, the induced field is proportional
Accelerated Ar molecule  Again strike to
to the permanent magnetic field but is opposite in direc-
argon gas (Ar)
tion.
In general, the amount of shielding is proportional    
to the local electron density, i.e., higher electron density Strike to sample dissolved in polar solvent E.g.,

causes more shielding and results a lower Larmor frequency. Glycerol
However, it is possible for some chemical groups with cir-    
cular п electron systems, most notably aromatic rings and
Fragmentation of sample result.
triple bonds, to cause induce chemical shifts which are not
the same for all orientations in space, a phenomenon known 4. Electron Spray Ionization (ESI)
as chemical shift anisotropy. Sorting out system or sector analyser
1. Magnetic Sector Analyser
H
m/z =H2 r2/2 V
C
B0 Where, H is magnetic field strength
H H r is ion tranjectory
C V is applied Voltage
Bind H Bind 2. TOF analyser
Ions are allowed to travel in a field free path, each ion
figure 3.4 Shielding
will take different time to travel a particular distance
 Analytical Chemistry  3.93

depending upon their m/z ratio. This time is known as (c) relative low abundance and broader peak
time of flight.
3. Quadruple analyser Maclafferty rearrangement
Both TOF and Quadruple analysers are used in inter- Migration of γ-hydrogen followed by β bond cleavage and
facing with GC. elimination of ethylene or substituted ethylene neutral
Detector system in MS Electron multiplier tube molecule.
Types of peak in MS Instrumentation of Mass spectrometer
1. Molecular ion or Parent peak–Comes at molecular 1. Sample inlet system
weight of compound. Peaks at M+1 and M+2 are due 2. Molecular leak–It is pin-hole restriction (0.01 to 0.05
to isotopic abundance. mm diameter) and made up of gold foil. It is used for
Relative intensity ratio for Br and Cl form and metering the sample to ionization chamber.
M+2 are 1:1 and 1:3 respectively. 3. Ionization Chamber
2. Base Peak 4. Ion separation (Sector analyser)
(a) It is considered as 100% 5. Ion collector (Detector)
(b) Most abundant peak
Recorder 5 separated galvanometers can be used to
3. Metastable peak
record simultaneously, the peaks for fragment ions and
M+ (Origional ion) N+ (daughter ion) parent ions.
+ Z (Neutral molecule)
Meta stable peak M* = (N+)2/M+ Nitrogen Rule It states that organic compound having:

(a) arise due to decomposition of ions in field free 1. an even integral molecular weight must contains either
path none or even number of nitrogen atoms.
(b) appears as weak, diffuse (humped shape) and at 2. odd molecular weight must contain odd number of
non-integral mass nitrogen atoms.

Note
yy Operation of Mass spectrometer requires a collision free path for ions to prevent arching due to high voltage and to
avoid recombination of fragmented ions.
yy For this, Vacuum Systems are used.
yy Vacuum in 1. Ionization Source (10–5 to 10–6 torr) 2. Sector Analyser (10–6 to 10–7 torr)

Luminescence Triplet state One set of electron spins is unpaired.

Luminescence is the emission of light by a substance. It occurs
when an electron returns to the electronic ground state from
an excited state and loses its excess energy as a photon.
Luminescence spectroscopy is a collective name given
to three related spectroscopic techniques. They are:
yy Molecular fluorescence spectroscopy
yy Molecular phosphorescence spectroscopy
yy Chemiluminescence spectroscopy ground
singlet state
excited
singlet state
excited
triplet state
spins is unpaired
Fluorescence and phosphorescence
(photoluminescence) Figure 3.5 Triple Set
The electronic states of most organic molecules can be
divided into singlet states and triplet states. Fluorescence
Singlet state All electrons in the molecule are spin- Absorption of UV radiation by a molecule excites it from a
paired vibrational level in the electronic ground state to one of the
3.94  Chapter 3

many vibrational levels in the electronic excited state. This Jablonski Diagram (Relaxation
excited state is usually the first excited singlet state. Mechanism for Excited State
A molecule in a high vibrational level of the excited
Molecules)
state will quickly fall to the lowest vibrational level of this
state by losing energy to other molecules through collision. Once a molecule has absorbed energy in the form of electro-
magnetic radiation, there are a number of routes by which it
Lowest excited
singlet state can return to ground state (the statistically most common en-
Lowest excited
Ground triplet state ergy state for room temperature chemical species). The follow-
electronic state ing graphic, termed a Jablonski diagram, shows a few of these
processes.
6 1 4 5
Energy

excited vibrational states

Sn (excited rolational states not shown)
4
A = photon absorption
2 3 F = fluorescence (emission)
4 4 S2 P = phosphorescence
IC S = singlet state
Processes involving photons T = triplet state
Radiationless transitions IC = internal conversion
S1
ISC = intersystem crossing
1. Absorption
2. Fluorescence Energy T2
ISC
3. Phosphorescence IC
4. Virbrational relaxation
A F T1
5. Intersystem crossing
6. Internal conversion P
S0
Figure 3.6 Possible physical process following electronic ground state
absorption of a photon by a molecule

Phosphorescence Figure 3.7 Jablonski Diagram

A molecule in the excited triplet state may not always use in-
If the photon emission occurs between states of the
tersystem crossing to return to the ground state. It could lose
same spin state, (e.g., S1 ---> S0) this is termed fluorescence.
energy by emission of a photon. A triplet/ singlet transition
If the spin state of the initial and final energy levels are
is much less probable than a singlet/singlet transition. The
different (e.g., T1 --> S0), the emission (loss of energy) is
lifetime of the excited triplet state can be up to 10 seconds,
called phosphorescence.
in comparison with 10-5 s to 10-8 s average lifetime of an ex-
cited singlet state. Emission from triplet/singlet transitions Since fluorescence is statistically much more likely
can continue after initial irradiation. Internal conversion and than phosphorescence for most molecules, the lifetimes of
other radiationless transfers of energy compete so success- fluorescent states are very short (1 × 10–5 to 10–8 seconds)
fully with phosphorescence that it is usually seen only at low and phosphorescence somewhat longer (1 × 10–4 seconds to
temperatures or in highly viscous media. minutes or even hours).
Three non-radiative deactivation processes are also
Chemiluminescence significant here: internal conversion (IC), intersystem
Chemiluminescence occurs when a chemical reaction pro- crossing (ISC) and vibrational relaxation.
duces an electronically excited species which emits a photon Internal conversion is the radiation less transition be-
in order to reach the ground state. These sort of reactions can tween energy states of the same spin state (compare with
be encountered in biological systems; the effect is then known fluorescence-a radiative process).
as bioluminescence. The number of chemical reactions which Intersystem crossing is a radiationless transition between
produce chemiluminescence is small. A good example of che- different spin states (compare to phosphorescence).
miluminescence is the determination of nitric oxide: Vibrational relaxation, the most common of the three—
NO + O3 → NO2* + O2 for most molecules, occurs very quickly (<1 × 10–12 seconds)
NO2* → NO2 + hv (l = 600–2800 nm) and is enhanced by physical contact of an excited molecule
 Analytical Chemistry  3.95

with other particles with which energy, in the form of vibra- Factor affecting fluorescence and phosphorescence
tions and rotations, can be transferred through collisions. 1. Nature of molecule
This means that most excited state molecules never emit any 2. Nature of substitutents
energy because in liquid samples the solvent or, in gas phase a.  Substituents that delocalize the π electrons
samples, other gas phase molecules that are present “steal” such as –NH2, –OH, –OR etc., enhance the fluo-
the energy before other deactivation processes can occur. rescence.
b. Substituents which withdraw electrons such as
Fluorescence (fluorimetry) and –NO2, –Cl, –Br etc., quench the fluorescence.
phosphorescence (phosphorimetry) 3. Rigidity of molecule
4. Viscosity
Absorption followed by emission i.e., λemitted > λincident 5. Temperature
6. pH
Fluorescence Phosphorescence
Average life time of Average life time for X-ray Diffraction
electron in excited state phosphorescence ranges
Basic principle Inner shell electron transition
is 10–5–10–8 sec. Decay from 10–4 –104 sec. Phos-
rapidly after excitation phorescence may continue Kα line (transition from shell L to Shell K)
source is removed. for sometime after remov- Kβ line (transition from shell M to Shell K)
ing excitation source. Kγ line (transition from shell N to Shell K)
No change in spin state Change in spin state Target material used Co, Ni, Cu, Mn, Mo etc.
Excited singlet state Excited triplet state
(Multiplicity = 1) (Multiplicity = 3) Brag's equation
nλ = 2d sinθ
Where
n = order of diffraction
Ground state (Multiplic- Ground state (Multiplic-
d = lattice spacing or inter planner distance
ity=1) ity=1)
θ = angle between direction of incident beam and that
Fluorescence spectrum is Not a mirror image because of diffracted beam
a mirror image excited triplet energy levels
lies lower than correspond-
ing excited singlet level

Note
Diffraction from crystal is only possible when λ is equal to or less than d.

Detectors
Nephelometry Turbidimetry
1. Photographic film method
2. Counter method 2. Most suitable for di- 2. Most suitable for
(a) Geiger-muller tube lute suspension concentrated suspen-
(b) Proportional counter sion
(c) Scintillation detector 3. Similar to fluorimetry 3. S imilar to colorimetry
(d) Solid state semiconductor because both measure because both
scattered radiations measure transmitted
Nephelometry Turbidimetry but elastic scattering radiations but light
in fluorimetry while intensity decreased
1. Intensity of scattered 1. Intensity of transmit- non-elastic scattering by scattering in
light measured as a ted light measured as a in nephelometry. turbidimetry while
function of concentra- function of concentra- by absorption in
tion of dispersed phase tion of dispersed phase colorimetry.
3.96  Chapter 3

Flame Photometry (Flame Emission 4. Electrode system:

Spectroscopy) a.  Indicator/Working electrode–DME (dropping
mercury electrode)
1. Mainly used for Alkali metal like Li, Na, K and Alkali
b. Reference electrode–Large mercury pool
earth metals like Mg, Ca, Ba, Sr.
2. Principle Supporting Electrolyte
Nebulization (breakdown of liquid into smaller a. It neither reacts with material under investigation
droplets) → Evaporation → Atomization in Flame → nor with DME. It has higher discharge potential
Excitation followed by Emission compared to material under investigation.
b. Quantity of supporting electrolyte taken as 100
Electro Analytical Methods times to material.
c. It carries almost all the current of the solution and
1. Conductometry
raise the conductivity of the solution, thus suppress
Conductivity cell the migration current.
1. Made up of Pyrex glass and Quartz 5. Ilkovic equation Id = 607 n CD1/2 m2/3 t1/6
2. Two platinum electrode system: To avoid polarization, Id =  Diffusion current = Limiting current–residual
Pt electrodes are coated with Pt black (Chloro platinic current
acid and lead acetate mixture). D = diffusion coefficient
3. Cell constant (x=specific conductivity/observed conduc- C = concentration
tivity) is determined using N/50 KCl. m = flow rate of mercury from DME
Specific conductance t = drop time
yy Conductivity offered by 1 ml or cm3 solution. 6. Half wave potential (E1/2)
yy It is the reciprocal of specific resistance (ρ). It has unit Potential corresponding to point of inflexion of polar
ohm-1 cm-1 or mho cm-1 graphic wave. It is a characteristic of nature of reacting
material.
К = 1/ρ = 1/R * L/A 7. Polar graphic Maxima-Developed due to streaming
= Conductance * cell constant (L/A) movement of diffusion layer at interface.
8. Maxima suppressor
πeq = К (Specific conductance) * V (dilution)
Gelatin (0.002–0.01 %) and triton-x-100 (0.02–0.01
K decreases but there is much greater increment in V %) are used as maxima suppressor.
hence overall equivalent conductance increases. Special Note
2. Potentiometry 1. Salt Bridge–It is made up of inert, hot, concentrated,
aqueous solution of KCl, KNO3, NH4NO3 in gelatine
Nernst equation or agar-agar solution. Only those salts can be used in
E = E0-RT/nF loge (P)/(R) preparation of Salt bridge formation which have equal
cations and anions mobility.
Where E = electrode potential of the cell 2. Saturated KCl solution mostly used compared to molar
E0 = standard electrode potential or decimolar solution in the preparation of reference
n = No. of electron consumed in 1 mol oxidation electrodes because it minimize the liquid-liquid junction
or reduction of electro active species potential.
(P), (R) = product and reactant concentration
Chromatography
3. Polargraphy Chromatography terms
1. Polarogram–Graph between current and applied yy Analytical chromatography is used to determine the
voltage existence and possibly also the concentration of analyte
2. Polarograph–Apparatus used for polarography (s) in a sample.
3. Residual/Charging/Capacitance current = Faradic yy Preparative chromatography is used to purify suffi-
current (due to impurity) + Condenser current (due to cient quantities of a substance for further use, rather
supporting electrolyte) than analysis.
 Analytical Chemistry  3.97

yy A bonded phase is a stationary phase that is covalently Distribution of analytes between phases
bonded to the support particles or to the inside wall of The distribution of analytes between phases can often be
the column tubing. described quite simply. An analyte is in equilibrium between
yy A chromatogram is the visual output of the chromato- the two phases;
graph. In the case of an optimal separation, different
peaks or patterns on the chromatogram correspond to Amobile Astationary
different components of the separated mixture. The equilibrium constant, K, is termed the partition
yy A chromatograph is an equipment that enables a coefficient; defined as the molar concentration of analyte in
sophisticated separation e.g., gas chromatographic or the stationary phase divided by the molar concentration of
liquid chromatographic separation. the analyte in the mobile phase.
yy Chromatography is a physical method of separation The time between sample injection and an analyte peak
in which the components to be separated are distrib- reaching a detector at the end of the column is termed the
uted between two phases, one of which is stationary retention time (tR). Each analyte in a sample will have a
(stationary phase) while the other (the mobile phase) different retention time. The time taken for the mobile
moves in a definite direction. phase to pass through the column is called tM.
yy The eluate is the mobile phase leaving the column.
yy The eluent is the solvent that will carry the analyte. tR
yy An eluotropic series is a list of solvents ranked according

Detector singal
to their eluting power.
tM
Chromatogram development technique
1. Frontal analysis–A large volume of sample mixture
is continuously passed through the column. Most weak- Time
ly retained component of the mixture emerges first.
Figure 3.8 Distribution of analyses between phases
2. Displacement analysis–Sample mixture is dissolved
in large volume of solvent and applied to the top of the A term called the retention factor, k ′, is often used to
column. Mobile phase containing displacement agent describe the migration rate of an analyte on a column. You
is passed through the column. may also find it called the capacity factor. The retention fac-
3. Elution Analysis–Most widely used technique. It can tor for analyte A is defined as;
be used for quantitative applications.
A. Isocratic elution (Solvent composition or strength k'A = tR – tM/tM
is not changed during column development)
tR and tM are easily obtained from a chromatogram.
B. Gradient elution (Solvent composition or strength
When an analytes retention factor is less than one, elution
is changed during column development).It is also
is so fast that accurate determination of the retention time is
known as solvent programming.
very difficult. High retention factors (greater than 20) mean
that elution takes a very long time. Ideally, the retention factor
Introduction
for an analyte is between one and five.
Chromatography involves a sample (or sample extract) being We define a quantity called the selectivity factor, α, which
dissolved in a mobile phase (which may be a gas, a liquid describes the separation of two species (A and B) on the column;
or a supercritical fluid). The mobile phase is then forced
through an immobile, immiscible stationary phase. α = k'B/k'A
The phases are chosen such that components of the When calculating the selectivity factor, species A elutes
sample have differing solubilities in each phase. A com- faster than species B. The selectivity factor is always greater
ponent which is quite soluble in the stationary phase will than one.
take longer to travel through it than a component which is
not very soluble in the stationary phase but very soluble in Band broadening and column efficiency
the mobile phase. To obtain optimal separations, sharp, symmetrical chromato-
As a result of these differences in mobilities, sample graphic peaks must be obtained. This means that band
components will become separated from each other as they broadening must be limited. It is also beneficial to measure
travel through the stationary phase. the efficiency of the column.
3.98  Chapter 3

Theoretical plate model of chromatography HETP = A + B/u + C u

The plate model supposes that the chromatographic column Where u is the average velocity of the mobile phase.
is contains a large number of separate layers, called theoret- A, B, and C are factors which contribute to band broadening.
ical plates. Separate equilibrations of the sample between
the stationary and mobile phase occur in these “plates”. The A. Eddy diffusion
analyte moves down the column by transfer of equilibrated
The mobile phase moves through the column which is
mobile phase from one plate to the next.
packed with stationary phase. Solute molecules will take
different paths through the stationary phase at random. This
The column will cause broadening of the solute band, because different
paths are of different lengths.

B. Longitudinal diffusion
Theoretical
plate The concentration of analyte is less at the edges of the band
than at the center. Analyte diffuses out from the center to the
Figure 3.9 Theoretical plate model of chromatography edges. This causes band broadening. If the velocity of the
mobile phase is high then the analyte spends less time on the
It is important to remember that the plates do not column, which decreases the effects of longitudinal diffusion.
really exist; they are a figment of the imagination that help
us understand the processes at work in the column. They also C. Resistance to mass transfer
serve as a way of measuring column efficiency, either by stat-
The analyte takes a certain amount of time to equilibrate
ing the number of theoretical plates in a column, N (the more
between the stationary and mobile phase. If the velocity of
plates the better), or by stating the plate height; the Height
the mobile phase is high, and the analyte has a strong affin-
Equivalent to a Theoretical Plate (the smaller the better).
ity for the stationary phase, then the analyte in the mobile
If the length of the column is L, then the HETP is
phase will move ahead of the analyte in the stationary phase.
HETP = L/N The band of analyte is broadened. The higher the velocity of
mobile phase, the worse the broadening becomes.
The number of theoretical plates that a real column
possesses can be found by examining a chromatographic Van Deemter plots
peak after elution;
A plot of plate height vs average linear velocity of mobile
phase.
5.5t R2
N=
w12/ 2 A typical Van Deemter plot

where w1/2 is the peak width at half-height.

As can be seen from this equation, columns behave as Optimum velocity
Plate height

if they have different numbers of plates for different solutes

in a mixture.
Minimum plate
Rate theory of chromatography height

A more realistic description of the processes at work inside

Mobile phase velocity
a column takes account of the time taken for the solute to
equilibrate between the stationary and mobile phase (unlike Figure 3.10 Van Deemter plots
the plate model, which assumes that equilibration is infinitely
fast). The resulting band shape of a chromatographic peak is Such plots are of considerable use in determining the
therefore affected by the rate of elution. It is also affected by optimum mobile phase flow rate.
the different paths available to solute molecules as they travel
Resolution
between particles of stationary phase. If we consider the vari-
ous mechanisms which contribute to band broadening, we ar- Although the selectivity factor, R, describes the separation of
rive at the Van Deemter equation for plate height; band centres, it does not take into account peak widths.
 Analytical Chemistry  3.99

Another measure of how well species have been separated Instead, to increase the number of plates, the height
is provided by measurement of the resolution. The resolution equivalent to a theoretical plate can be reduced by reducing
of two species, A and B, is defined as the size of the stationary phase particles.
2 [ (t R ) B − (t R ) A ] It is often found that by controlling the capacity
R= factor, k′, separations can be greatly improved. This can be
W A + WB achieved by changing the temperature (in Gas Chromatog-
Baseline resolution is achieved when R = 1.5 raphy) or the composition of the mobile phase (in Liquid
It is useful to relate the resolution to the number of Chromatography).
plates in the column, the selectivity factor and the retention The selectivity factor, α, can also be manipulated to
factors of the two solutes; improve separations. When α is close to unity, optimising
k′ and increasing N is not sufficient to give good separa-
N  – − 1  1 + K B′  tion in a reasonable time. In these cases, k′ is optimised
R=    K ′ 
4  –  B
first, and then R is increased by one of the following
procedures:
To obtain high resolution, the three terms must be
maximized. 1. Changing mobile phase composition
An increase in N, the number of theoretical plates, 2. Changing column temperature
By lengthening the column leads to an increase in 3. Changing composition of stationary phase
retention time 4. Using special chemical effects (such as incorporating
By increasing band broadening—which may not be a species which complexes with one of the solutes into
desirable. the stationary phase)

Note
Column chromatography is used to separate and purify components of a mixture.

TLC and GC are usually used only to analyse mixtures: Instrumentation

to determine the number of components and to see if a
Gas chromatography Specifically, gas-liquid chroma-
desired component is present.
tography–involves a sample being vaporized and injected
TLC is often used to determine the “ideal solvent system”
onto the head of the chromatographic column. The sam-
for a column chromatography or flash chromatography.
ple is transported through the column by the flow of inert,
The ideal system is the one that moves the desired com-
gaseous mobile phase. The column itself contains a liquid
ponent of the mixture to a TLC Rf of 0.25–0.35 and will
stationary phase which is adsorbed onto the surface of an
separate this component from its nearest neighbour by dif-
inert solid.
ference in TLC Rf values of at least 0.20. Therefore, a mix-
ture is analysed by TLC to determine the ideal solvent (s)
for a flash chromatography procedure. Injector
Flow port
Controller
Gas Chromatography
Principle Adsorption (GSC) or partition (GLC) Recorder
Main requirement Thermal stability and volatile na-
ture of compound.
Detector
Column
Derivatization in GC
1. To improve thermal stability of compound (polar com-
pound to non-polar compound). Column oven
2. To introduce a detector oriented tag in molecule.
3. For purposeful adjustment of volatility. Figure 3.11 Gas Chromatography
3.100  Chapter 3

Most common stationary phases The injector can be used in one of the two modes; split
1. Separation of mixture of polar compounds or splitless. The injector contains a heated chamber containing
a glass liner into which the sample is injected through the
Carbowax 20M (polyethylene glycol) septum. The carrier gas enters the chamber and can leave by
2. Separation of mixtures of non-polar compounds three routes (when the injector is in split mode). The sample
vapourises to form a mixture of carrier gas, vapourized
OV101 or SE-30 (polymer of methylsilicone) solvent and vapourised solutes. A proportion of this mix-
3. Methylester of fatty acids ture passes onto the column, but most exits through the
split outlet. The septum purge outlet prevents septum bleed
DEGS (diethylene glycol succinate) components from entering the column.

Columns
Instrumental Components
There are two general types of column, packed and capillary
Carrier gas (also known as open tubular).
The carrier gas must be chemically inert. Commonly used Packed columns contain a finely divided, inert, solid
gases include nitrogen, helium, argon, and carbon dioxide. support material (commonly based on diatomaceous earth)
The choice of carrier gas often depends upon the type of coated with liquid stationary phase. Most packed columns are
detector used. 1.5–10m in length and have an internal diameter of 2–4mm.
Capillary columns have an internal diameter of a few
Sample injection port tenths of a millimeter. They can be of one of the two types:
The most common injection method is where a micro sy- wall-coated open tubular (WCOT) or support-coated open
ringe is used to inject sample through a rubber septum into a tubular (SCOT).
flash vaporizer port at the head of the column. The tempera- Wall-coated columns consist of a capillary tube whose
ture of the sample port is usually about 50°C higher than the walls are coated with liquid stationary phase.
boiling point of the least volatile component of the sample. In support-coated columns, the inner wall of the capillary
For packed columns, sample size ranges from tenths of is lined with a thin layer of support material such as diato-
a microliter up to 20 microliter. maceous earth, onto which the stationary phase has been
Capillary columns, on the other hand, need much less adsorbed. SCOT columns are generally less efficient than
sample, typically around 10–3 microliter. For capillary GC, WCOT columns. Both types of capillary column are more
split/splitless injection is used. efficient than packed columns.
A new type of WCOT column was devised–the Fused
Silica Open Tubular (FSOT) column;
The split/splitless injector
Cross section of a Fused Silica Open Tubular Column
Rubber septum
Septum purge Polyimide coating
outlet Fused silica tube
Carrier gas Chemically bonded
inlet stationary phase

Split cutlet
Heated Figure 3.13 Cross section of a Fused Silica Open
metal block
Tubular Column
Vapourisation
Glass liner chamber These have much thinner walls than the glass capillary col-
umns, and are given strength by the polyimide coating. These
Column columns are flexible and can be wound into coils. They have the
advantages of physical strength, flexibility and low reactivity.
Column temperature
For precise work, column temperature must be controlled
to within tenths of a degree. The optimum column tempera-
ture is depends upon the boiling point of the sample. As
Figure 3.12 Capillary columns a rule of thumb, a temperature slightly above the average
 Analytical Chemistry  3.101

boiling point of the sample results in an elution time of a specific detector responds to a single chemical compound.
2–30 minutes. Minimal temperatures give good resolution, Detectors can also be grouped into concentration
but increase elution times. If a sample has a wide boiling dependant detectors and mass flow dependant detectors.
range, then temperature programming can be useful. The The signal from a concentration dependant detector is related
column temperature is increased (either continuously or in to the concentration of solute in the detector, and does not
steps) as separation proceeds. usually destroy the sampled dilution of with make-up gas
will lower the detectors response. Mass flow dependant
Detectors detectors usually destroy the sample, and the signal is related
A non-selective detector responds to all compounds except to the rate at which solute molecules enter the detector. The
the carrier gas, a selective detector responds to a range of response of a mass flow dependant detector is unaffected by
compounds with a common physical or chemical property and make-up gas.

Detector Type Support gases Selectivity Detectability Dynamic

range

Flame ionization Mass flow Hydrogen and Most organic com- 100 pg 107
(FID) air pounds

Thermal Concentration Reference Universal 1 ng 107

conductivity
(TCD)

Electron capture Concentration Make-up Halides, nitrates, nitriles, 50 fg 105

(ECD) peroxides, anhydrides,
organometallics

Nitrogen- Mass flow Hydrogen and Nitrogen, phosphorus 10 pg 106

phosphorus air

Flame photomet- Mass flow Hydrogen and Sulphur, phosphorus, tin, 100 pg 103
ric (FPD) air possibly boron, arsenic, germani-
oxygen um, selenium, chromium

Photoionization Concentration Make-up Aliphatics, aromatics, ke- 2 pg 107

(PID) tones, esters, aldehydes,
amines, heterocyclics,
organosulphurs, some
organometallics

Hall electrolytic Mass flow Hydrogen, Halide, nitrogen,

conductivity oxygen nitrosamine, sulphur

Flame Ionization Detector

The effluent from the column is mixed with hydro-
gen and air, and ignited. Organic compounds burning in
the flame produce ions and electrons which can conduct
Collector electricity through the flame. A large electrical potential is
Flame electrode applied at the burner tip, and a collector electrode is located
+300V
ignition above the flame. The current resulting from the pyrolysis of
coil Polarizing
voltage any organic compounds is measured. FIDs are mass sensi-
tive rather than concentration sensitive; this gives the advan-
tage that changes in mobile phase flow rate do not affect the
Air Hydrogen detector’s response. The FID is a useful general detector for
Column the analysis of organic compounds; it has high sensitivity, a
large linear response range, and low noise. It is also robust
Figure 3.14 Flame Ionization Detector and easy to use, but unfortunately, it destroys the sample.
3.102  Chapter 3

Thermal conductivity detector 4. Analytical Column

Principal When a compound elutes, the thermal conduc- yy Made up of stainless steel or fused silica
tivity of the gaseous mixture of carrier gas and compound yy Particle size below 5 µm
gas is lowered, and the filament in the sample column yy Mostly used silica gel as stationary phase.
becomes hotter than the other control column. (NP-HPLC)
Its resistance increases, and this imbalance between yy Mostly used bonded phase silica gel as stationary
control and sample filament resistances is measured by a phase. (RP-HPLC)
simple gadget and a signal is recorded. E.g., RP 18 (ODS), RP 8, RP 2
yy Type of material used for column packing
(a) Totally porous
Electron capture detector
(b) Superficially porous/pellicular type
For pesticide analysis (picogram).3H or 63Ni which emits b 5. Detector
particles. Accept electrons of carrier gas. A. UV-Visible spectrometer–(Most widely used)
Ionization: N2 (Nitrogen carrier gas) + b (e) = N2+ + 2e (a) Fixed wavelength (254 nm)
(b) Diode array detector
These N2+ establish a “base line” B. Refractive Index Detector
X (F, Cl and Br) containing sample + b (e) → X
– Temperature sensitive detector and cannot be
–
used in case of gradient elution method.
Ion recombination: X + N2+ = X + N2 C. Fluorescent Detector
The “base line” will decrease and this decrease consti- D. Conductrometric Detector
tutes the signal. Insecticides, pesticides, vinyl chloride, and
fluorocarbons. Ion Exchange Chromatography
Principle
HPLC Ionic compounds of solute are selectively separated by
Derivatization in HPLC forming temporary electrostatic chemical bond with counter
1. To improve sensitivity of the method such as formation ion of stationary phase.
of fluorescent derivative of amino acids. Resin......SO3H + Na+ Resin.....SO3Na + H+
2. To improve resolution by adding functional group
Resin......N (CH3)3OH + Cl–  Resin..... N (CH3)3
that enhances interaction of solute with stationary
Cl + OH–
phase.
Instrumentation Cation exchanger
1. Pump Strong – ..... SO3H Weak – ..... COOH
a. Pneumatic pump
b. Reciprocating pump Anion exchanger
Pressure up to 6000 psi but most of the analytical work
done in 400 to 1500 psi. Strong – ..... NR3Cl Weak – ..... NR2H
2. Sample Injector
a. Micro litre syringe Stationary phase
b. Rotary valve Polymeric Matrix e.g., Styrene (Vinyl benzene)-Divinyl
c. Loop Injector Benzene (DVB) Copolymer
3. Precolumn/Guard/Support column Divinyl Benzene is added to cross-link the chains
yy It is similar to analytical column but differs in formed from Styrene polymerization and gives a three-
particle size (30–50 µ) from analytical column dimensional bead structure.
(Size below 5µ).
yy Function Size Exclusion Chromatography
(a) Prevents dissolution of silica gel (Column Bleed-
ing) by previously saturating the mobile phase. Principle Molecular sieve basis i.e., larger molecule
(b) Removes irreversibly, adsorption of particulate unable to fit into pores are eluted first while small molecules
matter. enters into pores and are eluted later.
 Analytical Chemistry  3.103

Two types Super critical fluid chromatography

1. Gel Filtration-S.P. used are cross-linked carbohydrates Supercritical point is a point at which a gas cannot be
(Soft gel) Liquified no matter how high is the pressure. The resulting
E.g., Sephadex (Cross linked dextran), Agarose liquid has density, viscosity and diffusivity characteristics
(Sepharose), Polyacrylamide (Bio-gel) midway between gaseous and liquid states.
2. Gel Permeation-S.P. used are semi-rigid or rigid gel The most commonly used mobile phase, carbon dioxide
E.g., Cross-linked polystyrene, Alkylated Dextran, has a critical temperature of 31 degree C at 73 atm pressure.
Controlled porosity Glass beads
Thin layer chromatography (TLC)
Principle may be adsorption, partition, ion exchange or
Chiral Chromatography
molecular sieve depending upon the stationary phase used.
Principle
Separation of particular isomer from enantionmeric mixture Paper Chromatography
involves formation of Diastereomers.
Principle Partition
Methods Stationary phase Bound water in pores of cellulose fil-
1. Chiral Stationary Phase ter paper act as S.P.
e.g., Naphthyl Alanine, Naphthyl Leucine, Dinitro
Rf Value = distance travelled by solute/distance
benzoyl phenyl glycine, β-Cyclodextrin
travelled by Solvent front
2. Chiral mobile phase
Rf Value cannot be greater than 1.
Affinity chromatography
Derivatization in TLC
1. Affinity ligand is immobilized by covalent attachment
with inert support E.g., Silica or polysaccharide matrix. Compound class Derivatizing Agent
Affinity ligands selectively adsorb a single molecular
General Iodine Vapor
species which is complementary to it, from a mixture of
solute. General Sulphuric acid (50%)
2. Adsorption is reversible and non-covalent.
Acids Bromo cresol green
3. It exploit lock and key binding.
4. It is specially used for purification and separation of Aldehyde and Ketones 2, 4-dinitro phenyl hydrazine
biological macromolecules.
Amines and amino acids Ninhydrin
Analyte Affinity ligand Alkaloids Mercuric nitrate
Enzyme Coenzyme or inhibitor Barbiturates Diphenylcarbazone
Antigen Antibody Lipids Bromo thymol blue
Lectin Carbohydrate Steroids Antimony trichloride
Hormone Carrier Carbohydrate Aniline Phthalate

Important Table: Pharmaceutical Analysis

Spectral Region Frequency (Hertz) Wavelength Wave number (cm–1) Special Phenomenon

Gamma Rays 3 × 1018 – 3 ×1020 – – Nuclear reaction and Mossbau-

er spectroscopy

X Rays 3 × 1016 – 3 × 1018 0.01–2 nm – Inner shell electron transition

Vacuum or Far UV 1.5 × 1015 – 3 × 1016 2nm–200 nm – Ionization of atoms or molecule

3.104  Chapter 3

Spectral Region Frequency (Hertz) Wavelength Wave number (cm–1) Special Phenomenon

UV 8 × 1014 – 1.5 ×1015 200–400 nm 50, 000–25, 000 Outer or valence shell electron
transition

Visible 4 × 1014 – 4 × 1014 400–800 nm 25, 000–12, 500 Outer or valence shell electron
transition

IR 1012 – 4 × 1014 0.8 µm–1 mm 12, 500–20 Molecular vibration

Micro wave 1010 – 1012 1 mm–30 cm – Molecular rotation

Radio wave 106 – 1010 10 m–2000 m – Nuclear spin change

Spectroscopy Sample Window or Sample cell Spectroscopy Radiation Source Detector

UV Quartz or Fused silica UV 1. H ydrogen 1. B arrier layer

discharge cell (Photo
Visible Glass or plastic lamp Voltaic Cell)
2. Deuterium 2. Photo Emissive
IR Metal halide salts e.g., NaCl, KBr, discharge Cell
CsBr (for Non-Aqueous samples) lamp 3. Photo Multipli-
LiF, CaF2 (For Aqueous samples) 3. Xenon arc er Tube (PMT)
lamp 4. Silicon Diode
ATR-IR (Attenuated Silicon, Germanium, Sapphire 5. Charge coupled
total Reflectance) (single crystal of Al2O3) device (CCD)
6. Photo Diode
NMR Cylindrical Glass Tube
Array

Visible Tungsten Same as UV

halogen lamp
Detector Composition Principle
IR 1. N ernst glower 1. B olometer
Bolometer Pt strip in Evacu- Resistance operates at 2. Themocouple
ated vessel change 1500 degree C or Thermopile
(Rod of fused 3. Thermister
Thermocouple Two dissimilar Voltage or EMF or sintered 4. Golay cell
and metals e.g., Change at mixture of 5. Pyroelectric
Thermopile Bismuth and junction rare earth 6. Semiconductor
Antimony oxide e.g., 7. Photo
Zirconium, conductivity
Thermister Oxide of Mn, Co, Resistance Ytterbium, detector
Ni change Erbium or
Thorium)
Goley cell Xenon gas Membrane 2. Globar source
(Pneumatic) displacement operates at
or Expansion 1300°C (Rod
of gas of Silicon
carbide)
Pyroelectric Non-centrosym- Electric 3. Nichrome
metric crystal polarization wire or coil
below its Curie producing
point. TGS (Tri current NMR Radio Frequency Radio Frequency
glycine sulphate) Oscillator or Receiver or Phase
used as medium Generator Sensitive Detector
 Analytical Chemistry  3.105

Spectroscopy Radiation Source Detector Bathochromic shift Shift towards longer wave-
(Red Shift) length or lower energy
Fluorescence 1. Mercury PMT (Photo
and Phos- vapour Lamp Multiplier Tube) λmax The wavelength at maximum
phorescence 2. Xenon Arc absorption
lamp

Type of Potentio- Indicator Reference Elec- Electrode System Construction

meric Titration Electrode trode
Standard Hydro- It is a primary reference electrode.
Acid-base Glass Calomel (Hg/Hg- gen electrode It consists of Pt electrode immersed
(Neutralization) electrode Cl2) or Ag/AgCl (SHE) in a solution whose hydrogen ion
2
activity is 1.0 and in which H2 gas is
Complexometric Mercury- Calomel (Hg/Hg- bubbled at 1 atm Pressure.
or Chelometric Mercury (II) 2
Cl2) or Ag/AgCl
electrode Calomel ( (Hg/ Solid mercury surrounded with
Hg2Cl2) electrode Hg, Hg2Cl2 Paste and kept in satu-
Precipitation Silver Calomel (Hg/Hg- rated solution of KCl.
Electrode 2
Cl2) or Ag/AgCl
Ag/AgCl elec- Silver wire is coated with thin
Redox Pt elec- Calomel (Hg/Hg- trode film of silver chloride and kept in
trode 2
Cl2) or Ag/AgCl saturated solution of KCl.

Glass electrode Most widely used H+ ion sensitive

Electro-Analytical Basic principle electrode used in pH metre.
Method Calibration of pH metre carried
Conductometry Conductance V/S volume of out in following buffers 7.0, 4.0,
Titrant added 10.0 (sequence order). It is made
up of 22%Na2O, 6%CaO, and
Potentiometry Potential V/S volume of Titrant 72% SiO2.
added (No current flow i.e., I = 0)

Amperometry Current V/S volume of Titrant

added (V=Constant) Woodward Fieser Rule for conjugated Diene, triene
systems
Polargraphy Current V/S Applied Potential
Parent Values Homoannular conjugated 253 nm
diene
Bathochromic shift Shift towards longer wave-
Heteroannular conjugated 214 nm
(Red Shift) length or lower energy
diene
Hypsochromic shift Shift towards shorter wave- Acyclic conjugated diene 217 nm
(Blue Shift) length or higher energy Acyclic triene 245 nm

Hyper chromic shift Increase in intensity Increment Each alkyl substituent or + 5 nm

Ring residue
Hypo chromic shift Decrease in intensity Exocyclic double bond + 5 nm
Double bond extending + 30 nm
Auxochrome A group which extend the conju- conjugation
gation of a chromophore by shar-
ing of non-bonding electrons. Auxo- -Cl, -Br + 5 nm
chromes -OH/-OR/-SH + 6 nm
Chromophore Structural unit responsible for -SR + 30 nm
absorption -NR2 + 60 nm
The molar absorption at λmax -OCOCH3 + 0 nm
max
 SRM Parameter checked

Potassiurm iodide Stray Light in UVNisible

Didym ium o r Holomium Wavelength in UVNis-

Oxide ible

Polystyrene Fi lm Wavelength in IR

Beer-Lambert Law

Karl Fisher Titration (Coulometric end point

detection)
• It determines water content (moister content) in
10 c:===:> c==:>r,
pharmaceuticals.
• Reagent consists of mixture of anhydrous metha-
nol, anhydrous pyridine, and iodine and sulphur
dioxide.
• End point detection-presence of water causes con- It states that the proportion of the light absorbed
version of iodine to iodide through its reduction by the solute in a homogenous, transparent med i-
by sulphur dioxide. um is independent of the intensity of the incident
• Sodium tartarate d ihydrate is used in standardiza- light and proportional to the number of absorbing
tion of Karl-Fisher reagent. molecules and path length.

Standard reference materials used in calibration of A = ECI = log (1 0 ) 1) = log 1/T = - log T = 2 - log % T
spectroscopic instrument
A d irect ly proportional to path length (Lambert law)
SRM Parameter checked A directly proportional to concentration (Beer's law)
Potassium dichromate Absorbance in UVNisible W here A = Absorbance
Quartz cuvett Path length in UVNisible € =molar absorptivity/molar extinction coef-
f icient
Toluene in Hexane Resolution in UVNisible
C =concentration (mol per litre)
I =path length
T =Transmittance= 1{1 0

If concentration is taken in g per litre, then the for-

mula becomes
A=aCI
a= absorptivity or extinction coefficient

When molecular weight of absorbing molecule is not

known, the A 1,m1% is used to compare absorption
intensity instead of €.

Where C =concentration (gm per 100 ml)

Thus € = (A1crn1% x [Link])/10
 Analytical Chemistry  3.109

Proton (H1) NMR V/S Carbon (C13) NMR Flame Temperature in Kelvin (K)

1. Gyro magnetic (Magnogyretic) Ratio for proton Fuel Oxidant

NMR is 4 times than carbon-13 NMR.
Air Nitrous Oxide (N2O) Oxygen (O2)
2. Proton NMR provide information of periphery while
C-NMR about Backbone. Acetylene 2400 K 3200K 3400K

3. C
 hemical shift normal range for Proton NMR Hydrogen 2300K 2900K 2900 K
(0–10) while for C-NMR (0–200).
Propane 2200K 3000K 3100K
4. P
 roton NMR spectrum is more complex than
C-NMR because homo (H1-H1) as well as hetero Normal Phase Chromatography (NPC)
(H1-H2) nuclear coupling are possible in PMR but in      Stationary Phase (S.P.) Polar
C-NMR hetero nuclear coupling is not possible due
     Mobile Phase (M.P.) Non-Polar
to spin quantum number of C12 is zero and prob-
ability of homo nuclear coupling is very low due Elution Order-Alkane → Olefins → Aromatics →
to natural isotopic abundance of C13 is only 1.1%. Organic halides → Sulfide → Ether → nitro compounds
5. PMR is more sensitive than C13-NMR. → Ester/Aldehyde/Ketone → Alcohol/Amines → Sulfone
→ Sulfoxide → Amides → Carboxylic Acids
Information from PMR Non-polar compound will elute first and most polar will
elute last in the NPC.
Number of Signals Different sets of equivalent
protons in molecule Reverse Phase Chromatography (RPC)
     Stationary Phase (S.P.) Non-Polar
Intensity of Signal Relative number of protons of      Mobile Phase (M.P.) Polar
different kinds
Eluotropic Series–Increasing order of Solvent Polarity
Splitting or Multi- Environment of proton with
plicity of Signal respect to neighbouring proton Hexane/Pentane < Petroleum ether < Cyclo Hexane
< Xylene <Toluene < Diethyl ether < Chloroform
Area of Peak Number of absorbing protons
< Dichloromethane < THF < Acetone <Dioxane <
giving rise to a signal
Acetonitrile < Methanol <Water

List of important Chemical Shift Values (δ)

Protons on Protons on unsaturated Protons on Saturated CH3, CH2, Saturated CH3, CH2,
unsaturated carbons carbons e.g., Benzene, unsaturated carbons and CH protons next CH protons not next
next to oxygen e.g., Aromatic Hydrocarbons e.g., Alkenes to oxygen e.g., CH3O, to oxygen
Aldehyde CH2O

10.5 8.5 6.5 4.5 3.0 0.0

Large Chemical Shift Sm a ll

L ow (Down F ield ) Field H igh (Up F ield )

H igh Frequency L ow

De Shielded Shielding Shielded

Figure 3.16
3.110  Chapter 3

1. System Suitability Parameters for HPLC Method development

Parameters Acceptance criteria

Capacity factor The peak should be well resolved from other peaks.

Resolution (Rs) Rs > 2 between the peak of interest

Tailing factor (T) T is less than or equal to Two.

Theoretical plate (N) In general should be greater than 2000

2. Analytical method validation parameters

Specificity Specificity is the ability to assess unequivocally the analyte in the presence of components
which may be expected to be present. Typically these might include impurities, degradants,
matrix, etc.

The specificity of the method is determined by comparing the spectra (for UV) and chro-
matogram (for RP-HPLC) of the standard and sample solutions of analyte and both are
spectra/chromatogram are overlap.

Precision (n=6) Repeatability(n=6): Repeatability expresses the precision under the same operating
conditions over a short interval of time.

Reproducibility: Reproducibility expresses the precision between laboratories

Intermediate precision (n=3): Intermediate precision expresses within-laboratories variations:

different days, different analysts, different equipment, etc.
The intermediate precision of the method was confirmed by intraday (variation of results
within the same day) and interday (variation of results between days) analysis. The
intraday and interday precision of the proposed methods were performed by analyzing the
corresponding responses three times on the same day for intraday precision and over a
period of three days for inter day with three different concentrations of standard tertiary
mixture solutions.

LOD (Limit of The detection limit of an individual analytical procedure is the lowest amount of
Detection) analyte in a sample which can be detected but not necessarily quantitated as an exact
Value.

LOQ (Limit of The quantitation limit of an individual analytical procedure is the lowest amount of
Quantification) analyte in a sample which can be quantitatively determined with suitable precision and
accuracy. The quantitation limit is a parameter of quantitative assays for low levels of
compounds in sample matrices, and is used particularly for the determination of impurities
and/or degradation products.

The LOD and LOQ can be determine as per following

LOD = 3.3 σ / S and LOQ = 10 σ / S
Where, σ = standard deviation of y intercept of calibration curve (n = 6)
S = slope of a regression equation.

Linearity (n=6) The linearity of an analytical procedure is its ability (within a given range) to obtain test
results which are directly proportional to the concentration (amount) of analyte in the
sample.
Linearity is checked by diluting standard stock solution at six different concentrations and
correlation coefficients (r2) is greater than 0.995.
 Analytical Chemistry  3.111

Range The range of an analytical procedure is the interval between the upper and lower
concentration (amounts) of analyte in the sample (including these concentrations) for
which it has been demonstrated that the analytical procedure has a suitable level of
precision, accuracy and linearity.

Accuracy (n=3) 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. This is sometimes termed trueness.
The accuracy of the method will be carried out at three levels 80, 100 and 120 % of the
working concentration of sample. This procedure was repeated for three times for each
concentration.

Robustness(n=3) The robustness of an analytical procedure is a measure of its capacity to remain unaffected
by small, but deliberate variations in method parameters and provides an indication of its
reliability during normal usage.
Robustness of the method will be determined by changes in following parameters.

· pH ± 0.2
· Flow rate ± 10 %
· Wavelength ± 2 nm
· Change in mobile phase ratio ± 2 %
*For all validation parameters, % RSD value should not be more than 2.

3. List of Indicator used in different titration methods

Titration Indicators

Complexometric Murexide, solochrome black, Patton and Reedder’s indicator, Calcon or solochrome
dark blue, Xylenol orange, bromopyrogallol, thymolphthalexone, methylthymol
blue, zincon, variamine blue
Precipitation Eosin , fluorescin, Rose Bengal, tartrazine, alizarin red S, rhodamine 6G,
Phenosafranine
Nonaqueous Crystal violet, methyl red, 1- napthol benzein, oracet blue
Aqueous Methyl orange, phenolphthalein, methyl red, thymol phthalein, methyl yellow,
neutral red, congo red etc.

4. List of ICH Guidelines

Quality Guidelines

Q1A - Q1F: Stability

· Q1A(R2)Stability Testing of New Drug Substances and Products

· Q1BStability Testing : Photostability Testing of New Drug Substances and Products

· Q1A(R2)Stability Testing of New Drug Substances and Products

· Q1BStability Testing : Photostability Testing of New Drug Substances and Products

· Q1A(R2)Stability Testing of New Drug Substances and Products

· Q1BStability Testing : Photostability Testing of New Drug Substances and Products

3.112  Chapter 3

Q2(R1): Validation of Analytical Procedures: Text and Methodology

Q3A - Q3D: Impurities

· Q3A(R2)Impurities in New Drug Substances

· Q3B(R2)Impurities in New Drug Products

· Q3C(R5)Impurities: Guideline for Residual Solvents

· Q3DGuideline for Elemental Impurities

· Q3d Training Implementation of Guideline for Elemental Impurities

Q4 - Q4B: Pharmacopoeias

· Q4Pharmacopoeias

· Q4APharmacopoeial Harmonisation

· Q4BEvaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions

Q5A - Q5E: Quality of Biotechnological Products

· Q5A(R1)Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal
Origin

· Q5BAnalysis of the Expression Construct in Cells Used for Production of r-DNA Derived Protein Products

· Q5CStability Testing of Biotechnological/Biological Products

· Q5DDerivation and Characterisation of Cell Substrates Used for Production of Biotechnological/

Biological Products

· Q5EComparability of Biotechnological/Biological Products Subject to Changes in their Manufacturing

Process

Q6A- Q6B: Specifications

· Q6ASpecifications : Test Procedures and Acceptance Criteria for New Drug Substances and New Drug
Products: Chemical Substances

· Q6BSpecifications : Test Procedures and Acceptance Criteria for Biotechnological/Biological Products

Q7 : Good Manufacturing Practice

Q8 : Pharmaceutical Development

Q9 : Quality Risk Management

Q10: Pharmaceutical Quality System

Q11: Development and Manufacture of Drug Substances

Q12: Lifecycle Management

 Analytical Chemistry  3.113

Safety Guidelines

S1A - S1C: Carcinogenicity Studies

· S1Rodent Carcinogenicity Studies for Human Pharmaceuticals

· S1ANeed for Carcinogenicity Studies of Pharmaceuticals

· S1BTesting for Carcinogenicity of Pharmaceuticals

· S1C(R2)Dose Selection for Carcinogenicity Studies of Pharmaceuticals

S2: Genotoxicity Studies

S3A - S3B: Toxicokinetics and Pharmacokinetics

· S3ANote for Guidance on Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies

· S3A Q&AsQuestions and Answers: Note for Guidance on Toxicokinetics: The Assessment of Systemic
Exposure - Focus on Microsampling

· S3BPharmacokinetics: Guidance for Repeated Dose Tissue Distribution Studies

S4: Toxicity Testing

S5: Reproductive Toxicology

S6: Biotechnological Products

S7A - S7B: Pharmacology Studies

· S7ASafety Pharmacology Studies for Human Pharmaceuticals

· S7BThe Non-Clinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval
Prolongation) by Human Pharmaceuticals

S8: Immunotoxicology Studies

S9: Nonclinical Evaluation for Anticancer Pharmaceuticals

S10: Photosafety Evaluation

S11: Nonclinical Safety Testing

Efficacy Guidelines

E1: Clinical Safety for Drugs used in Long-Term Treatment

E2A - E2F: Pharmacovigilance

· E2A:Clinical Safety Data Management: Definitions and Standards for Expedited Reporting

· E2B(R3):Clinical Safety Data Management: Data Elements for Transmission of Individual Case Safety
Reports
3.114  Chapter 3

· E2B(R3): Implementation: Electronic Transmission of Individual Case Safety Reports

· E2C(R2):Periodic Benefit-Risk Evaluation Report

· E2C(R2:) Questions & Answers: Periodic Benefit-Risk Evaluation Report

· E2D:Post-Approval Safety Data Management: Definitions and Standards for Expedited Reporting

· E2E:Pharmacovigilance Planning

· E2F:Development Safety Update Report

E3: Clinical Study Reports

E4: Dose-Response Studies

E5: Ethnic Factors

E6: Good Clinical Practice

E7: Clinical Trials in Geriatric Population

E8: General Considerations for Clinical Trials

E9: Statistical Principles for Clinical Trials

E10: Choice of Control Group in Clinical Trials

E11: Clinical Trials in Pediatric Population

E12: Clinical Evaluation by Therapeutic Category

E14: Clinical Evaluation of QT

E15: Definitions in Pharmacogenetics / Pharmacogenomics

E16: Qualification of Genomic Biomarkers

E17: Multi-Regional Clinical Trials

E18: Genomic Sampling

Multidisciplinary Guidelines

M1: MedDRA Terminology

M2: Electronic Standards

M3: Nonclinical Safety Studies

M4: Common Technical Document

M5: Data Elements and Standards for Drug Dictionaries

M6: Gene Therapy

M7: Genotoxic Impurities

M8: Electronic Common Technical Document (eCTD)

 Analytical Chemistry  3.115

Fourier transform (F.T.) concept

It is a mathematical operation which converts Time Domain Spectra into Frequency Domain Spectra.

Cosine Interferogram FT IR Frequency Domain Spectra

Free Induction DecaySignal FT NMR Frequency Domain Spectra

Time domain trace Frequency domain trace

Stationary
mirror

Time

Beam splitter

Source Movable
mirror

Sample
position

Detector

Advantage of FT of high frequency signals to measurable ones.

1. Fellgett or Multiplex–Increase in S/N ratio Radiation source is He-Ne laser lamp.
2. Jacquinot–Increased energy throughput Laser beam positioning It tells about Sampling Inter-
3. High resolution val time.
4. High sensitivity
Zero position referencing (White light system) It
Michelson interferometer indicates starting point of data sampling for each scan.
It acts as modulating device. Modulation means conversion

Stretching Vibrational Frequencies (cm–1)

C-X
O-H C-C, C-N (Triple bond)
Transparent C=N C-O
C-H X=C=Y C=O
Region C=C C-N
N-H X, Y=C, O, N, S
C-C

4000 2500 2000 1800 1650 1550 500

Alkane – -C-Hstretch (3000 cm–1) -C-Hbend (1440, 1375 cm–1)

(-C-C-) Isopropyl (1375 cm–1) t-Butyl (1375 cm–1)
Alkene – C-Hbend in Cis alkene (700 cm–1) Trans alkene (970cm–1)
(-C=C-)
Alkynes – C-H in triple bonded carbon atom (3300 cm–1)
C triple bond C ( 2150 cm–1)
3.116  Chapter 3

Aromatics – C-Hbend in O-disubstituted 750, 690 cm–1

m-disubstituted 690, 780 cm–1
p-disubstituted 800–850 cm–1
Alcohols – O-H stretching at 3500–3700 cm–1 (without hydrogen bonding)
3200–3500 cm–1 (hydrogen bonding)
C-O stretching at 1100–1300 cm–1
Carbonyl and
Acid derivatives – C=Ostretch order
 Anhydride I band (1810 cm–1) > Acid Halide (1800 cm–1) > Anhydride II band (1760 cm–1) > Esters (1735 cm–1) >
Aldehydes (1725 cm–1) > Ketones (1715 cm–1) > Carboxylic Acid (1710 cm–1) > Amides (1690 cm–1)
Aldehyde can be differentiated from Ketone due to presence of doublet of C-Hstretch at 2750 cm-1 and 2850 cm-1

C-N stretching in Nitriles at 2250 cm–1

N=O stretching at 1550 and 1650 cm–1
S-H stretching at 2550 cm–1
S=O stretching at 1350 and 1150 cm–1

Analytical method based on different properties

Measured Analytical Methods
Measured Analytical Methods Property
Property
Mass to charge Mass Spectroscopy
Mass Gravimetric ratio

Rotation of Polarimetry, Optical rotatory dis-

Volume Volumetric
radiation persion (ORD) and Circular Dichro-
Electrical Potentiometry, Chronopotentiom- ism (CD)
potential etry
Thermal Thermal conductivity and enthalpy
Electrical Conductometry, Properties methods
Conductance
Titrimetic Analysis
Electrical Polarography, Amperometry
current, Titration A measured amount of a solution of unknown
concentration is added to a known volume of a second solu-
Quantity of Coulometer tion until the reaction between them is just complete; the
electricity concentration of the unknown solution (the titer) can then
be calculated.
Absorption of Spectrophotometry (UV, Visible, IR,
radiation X-ray), Calorimetry, Atomic absorp- Analyte (Titrant) An analyte is a weak base or acid. Its
tion spectroscopy, NMR and ESR structure is made from any compound that can be converted
to a strong acid or base.
Emission of Emission spectroscopy (UV, Visible,
Titrant The titrant is a strong acid or base that is slowly
radiation X-ray), Flame photometry, Fluores-
added to the analyte until it reaches any visible change in
cence, Radiochemical methods
the colour of the solution under observation.
Refraction of Refrectometry, Interferometry Indicator It is a pH marker added to the analyte that trig-
radiation gers a change in colour when equilibrium is reached. It should
have a weaker acid/base concentration than the analyte.
Scattering of Turbidimetry, Nephelometry, Ra-
radiation man Spectroscopy Acidimetry Volumetric analysis using standard solutions
of acids to measure the amount of a base present.
Diffraction of X-ray electron diffraction methods
Alkalimetry Volumetric analysis using standard solu-
radiation
tions of alkali to measure the amount of acid present.
 Analytical Chemistry  3.117

Standards endpoint of the normal titration. They are also useful if the
Certain chemicals which are used in defined concentrations reaction between the analyte and the titrant is very slow.
as reference materials.
Types of titrations
yy Primary standards
yy Secondary standards 1. Acid-base titration
Indicators for Acid-Base titration
Primary standards
Available in pure form, stable and easily dried to a constant Indicator Colour Range of Colour on
known composition. on Acidic Colour Basic Side
yy Stable in air. Side Change
yy High molecular weight.
Methyl Violet Yellow 0.0–1.6 Violet
yy Readily soluble.
yy Undergoes stoichiometric and rapid reactions. Bromophenol Blue Yellow 3.0–4.6 Blue

Methyl Orange Red 3.1–4.4 Yellow

Titration Method Primary Standards
Methyl Red Red 4.4–6.2 Yellow
Acid-base reactions Na2CO3, Na2B4O7, KH (C8H4O4), HCl

Complex forma- AgNO3, NaCl Litmus Red 5.0–8.0 Blue

tion reactions
Bromothymol Blue Yellow 6.0–7.6 Blue
Precipitation reac- AgNO3, KCl
tions Phenolphthalein Colorless 8.3–10.0 Pink

Redox reactions K2Cr2O7, Na2C2O4, I2 Alizarin Yellow Yellow 10.1–12.0 Red

Secondary standards pH meter and Conductivity metre can be used for end
A substance that can be used for standardisations, and point detection.
whose concentration of active substance has been deter-
2. Redox titration
mined by comparison to a primary standard.
Most commonly, a potentiometer or a redox indicator is
Standard solution used to determine the end point of the titration. For exam-
It is a solution of accurately known concentration prepared ple, when one of constituents of the titration is the oxidiz-
from a primary standard (a compound which is stable, of ing agent potassium dichromate, the colour change of the
high purity, highly soluble in water and of a high molar solution from orange to green is not definite and thus an indi-
mass to allow for accurate weighing) that is weighed accu- cator such as sodium diphenylamine is used. The analysis of
rately and made up to a fixed volume. wines for their sulfur dioxide content requires the use of iodine
as an oxidizing agent. In this case, starch is used as an indica-
Types of volumetric titrations tor; a blue starch-iodine complex is formed once an excess of
There are three types of volumetric titration, which are iodine is present, thus signalling the endpoint of the titration.
classified based on the rate of their reaction. Direct titra- Some redox titrations do not require an indicator, due
tion method (DTM) is a one-step titration process. Indirect to the intense colour of some of the constituents. For instance,
method (ITM) involves a two-step titration process. Back in a titration where the oxidizing agent potassium perman-
titration method (BTM) uses a three-step titration process. ganate (permanganometry) is present, a slightly faint
persisting pink colour signals the endpoint of the titration,
Back titration and no particular indicator is therefore required.
The term back titration is used when a titration is done Standardization of Potassium Permanganate or Cerium
“backwards”; instead of titrating the original analyte, one IV sulphate done by Sodium Oxalate or Arsenic III oxide.
adds a known excess of a standard reagent to the solution, Standardization of Potassium Dichromate is done by metallic
then titrates the excess. A back titration is useful if the end- iron. Standardization of Iodine is done by Sodium Thiosul-
point of the reverse titration is easier to identify than the phate or Arsenic III oxide.
3.118  Chapter 3

Application–Determination of Copper, Dissolved salt to ammonia. The amount of ammonia present (hence
oxygen, Chlorine, Arsenic IV, Sulphides. the amount of nitrogen present in the sample) is determined
by back titration. The end of the condenser is dipped into
3. Complexometric titration
a solution of boric acid. The ammonia reacts with the acid
These titrations are based on the formation of a complex and the remainder of the acid is then titrated with a sodium
between the analyte and the titrant. The chelating agent carbonate solution with a methyl orange pH indicator.
EDTA is very commonly used to titrate metal ions in solution.
These titrations generally require specialized indicators that Degradation Protein + H2SO4 → (NH4)2 SO4 (aq) + CO2 (g)
+ SO2 (g) + H2O (g)
form weaker complexes with the analyte. A common exam-
ple is Eriochrome Black T for the titration of calcium and Liberation of ammonia (NH4)2 SO4 (aq) + 2NaOH →
magnesium ions. Indicators-Murexide, Solochrome Black, Na2SO4 (aq) + 2H2O (l) + 2NH3 (g)
Xylenol orange, Eriochrome Black etc. Capture of ammonia B (OH)3 + H2O + NH3 → NH4+
Application Determination of cations and hardness of + B (OH)4–
water. Back-titration B (OH)3 + H2O + Na2CO3 → NaHCO3
(aq) + NaB (OH)4 (aq) + CO2 (g) + H2O.
4. Zeta potential titration
These titrations characterize heterogeneous systems, such
as colloids. Zeta potential plays role of indicator. One of the Precipitation Titration
purposes is determination of iso-electric point when surface Titrations with precipitating agents are useful for deter-
charge becomes 0. mining certain analytes. E.g., Cl– can be determined when
titrated with AgNO3.
5. Iodometry
Usual reagents are sodium thiosulfate as titrant, starch as an Detection of end point
indicator (it forms blue complex with iodine molecules—
though polyvinyl alcohol has started to be used recently as yy Chemical
well), and an iodine compound (iodide or iodate, depending ‡‡ Precipitation Type–Mohr’s method
on the desired reaction with the sample). ‡‡ Adsorption–Fajan’s method
The principal reaction is the reduction of iodine to ‡‡ For silver analysis–Volhard method
iodide by thiosulfate: yy Sensors–Potentiometric or amperometric
I2 + 2S2O32− → S4O62− + 2I−
6. Precipitation reactions The chemical types are also classified into:
1. Mohr method-Using Ag+ as a titrant in chlorides (or 1. Indicators reacting with titrant forming specific colour.
bromides) determination. 2. Adsorption indicators.
End point detection-small amount of sodium or potas-
sium chromate Indicators reacting with the titrant
2. Volhard method-Titration with thiocyanates, can be Two methods will be discussed where this type of indicators
used for Ag+ determination, or for indirect determina- are applied; namely: Mohr and Volhard.
tion (thorough back titration) of chlorides.
End point detection-Iron (III) thiocyanate complex I) Mohr method for determining chloride
3. Kjeldahl method or Kjeldahl digestion Chloride is titrated with AgNO3 solution. A soluble chro-
Quantitative determination of nitrogen in chemical mate salt is added as the indicator. This produces a yellow
substances. colour solution. When the precipitation of the chloride is
The method consists of heating a substance with sulphuric complete, the first excess of Ag+ reacts with the indicator to
acid, which decomposes the organic substance by oxidation precipitate red silver chromate:
to liberate the reduced nitrogen as ammonium sulphate. In
this step, potassium sulphate is added in order to increase 2 Ag+(aq) + CrO42–(aq) → Ag2CrO4(s)
the boiling point of the medium. Chemical decomposition
Yellow red ppt
of the sample is complete when the medium has become
clear and colourless (initially very dark). The Mohr method must be performed at a pH about
The solution is then distilled with sodium hydroxide 8. This method is useful for determining Cl– in neutral or
(added in small quantities) which converts the ammonium unbuffered solutions such as drinking water.
 Analytical Chemistry  3.119

II) Volhard titration rofluorescein (DCF) on the surface of the positively charged
This is an indirect titration procedure for the determination silver chloride particles formed in the precipitation titration
of anions that precipitate with silver like Cl–, Br–, I–, SCN-, when Ag+ ion is in excess.
and it is preferred in acid (HNO3) solution. A measured Application of PPtion Titration Determination of an-
excess of AgNO3 is added to precipitate the anion, and the ions such as halides, divalent anions, mercaptans.
excess of Ag+ is determined by back titration with standard
potassium thiocyanate solution: Non-aqueous titration
Ag+(aq) + Cl–(aq) → AgCl(s) + excess Ag+ Non-aqueous titration is the titration of substances
dissolved in non-aqueous solvents. It is the most com-
excess Ag+(aq) + SCN–(aq) → AgSCN(s) mon titrimetric procedure used in pharmacopoeial assays
and serves a double purpose: it is suitable for the titration
The end point is detected by adding iron III (Fe3+) as
of very weak acids and very weak bases, and it provides a
ferric ammonium sulfate which forms a soluble red com-
solvent in which organic compounds are soluble.
plex with the first excess of titrant.
The most commonly used procedure is the titration of
Fe3+ (aq) + SCN–(aq) → [FeSCN]2+(aq) organic bases with perchloric acid in anhydrous acetic acid.
These indicators must not form a compound with
Non-aqueous solvents
the titrant that is more stable than the precipitate or the
colour reaction would occur on addition of the first drop of Aprotic solvents
titrant. These are neutral, chemically inert substances such as benzene
and chloroform. They have a low dielectric constant, do not
Adsorption indicators react with either acids or bases and therefore do not favour
The indicator reaction takes place on the surface of the ionization. Since dissociation is not an essential preliminary
precipitate. The indicator, which is a dye, exists in solution to neutralization, aprotic solvents are often added to ‘ionizing’
as the ionized form, usually an anion. solvents to depress solvolysis (which is comparable to hydroly-
sis) of the neutralization product and so sharpen the endpoint.
Principle of adsorption Protophilic solvents
Consider the titration of Cl– with Ag+. Before the equivalent These are basic in character and react with acids to form
point, Cl– is in excess and the primary layer is Cl– (go back solvated protons.
to precipitation process in gravimetry). This repulses the
indicator anions; and the more loosely held the secondary HB + Sol. ⇌ Sol.H+ + B–
(counter) layer of adsorbed ions is cations, such as Acid + Basic solvent ⇌ Solvated proton + Conjugate
Na +
: AgCl : Cl : : Na
– + base of acid
A weakly basic solvent has less tendency than a strong-
Beyond the equivalent point (end point as well), Ag+ ly basic one to accept a proton. Similarly, a weak acid has
is in excess and the surface of the precipitate becomes less tendency to donate protons than a strong acid. As a
positively charged, with the 1° layer being Ag+. This will result a strong acid such as perchloric acid exhibits more
now attract the indicator anion and adsorb it in the 2° strongly acidic properties than a weak acid such as acetic
(counter) layer: acid when dissolved in a weakly basic solvent.
On the other hand, all acids tend to become indistin-
AgCl : Ag+ : : indicator –
guishable in strength when dissolved in strongly basic
The colour of the adsorbed indicator is different from solvents owing to the greater affinity of strong bases for
that of the unadsorbed indicator, and this difference signals protons. This is called the leveling effect. Strong bases are
the completion of the titration. The degree of adsorption of leveling solvents for acids, weak bases are differentiating
the indicator can be decreased by increasing the acidity. solvents for acids.
The titration of chloride using this kind of indicator is
called Fajan’s Method. Protogenic solvents
Fajan’s method is the most recent and most accurate These are acidic substances, e.g., sulphuric acid. They exert
silverhalide method. It is based on the adsorption of dichlo- a levelling effect on bases.
3.120  Chapter 3

Amphiprotic solvents The quantitative determination of a substance by the

They have both protophilic and protogenic properties. precipitation method of gravimetric analysis involves
Examples are water, acetic acid and the alcohols. They are isolation of an ion in solution by a precipitation reaction,
dissociated to a slight extent. The dissociation of acetic acid, filtering, washing the precipitate free of contaminants,
which is frequently used as a solvent for titration of basic conversion of the precipitate to a product of known composi-
substances, is shown in the equation below: tion, and finally weighing the precipitate and determining
its mass by difference. From the mass and known composi-
CH3COOH ⇌ H+ + CH3COO– tion of the precipitate, the amount of the original ion can
Here, the acetic acid is functioning as an acid. If a very be determined.
strong acid such as perchloric acid is dissolved in acetic
acid, the latter can function as a base and combine with Steps involved in gravemetric analysis
protons donated by the perchloric acid to form protonated 1. Coprecipitation
acetic acid, an onium ion:
This is anything unwanted which precipitates with the
HClO4 ⇌ H+ + ClO4– thing you do want. Coprecipitation occurs to some degree
CH3COOH + H+ ⇌ CH3COOH2+ (onium ion) in every gravimetric analysis (especially barium sulfate
and those involving hydrous oxides). You cannot avoid
Since the CH3COOH2+ ion readily donates its proton it—all you can do is minimize it by careful precipitation and
to a base, a solution of perchloric acid in glacial acetic acid thorough washing.
functions as a strongly acidic solution.
When a weak base, such as pyridine, is dissolved in 2. Surface adsorption
acetic acid, the acetic acid exerts its levelling effect and en- Here unwanted material is adsorbed onto the surface of the
hances the basic properties of the pyridine. It is possible, precipitate. Digestion of a precipitate reduces the amount
therefore, to titrate a solution of a weak base in acetic acid of surface area and hence the area available for surface
with perchloric acid in acetic acid, and obtain a sharp end- adsorption. Washing can also remove surface material.
point when attempts to carry out the titration in aqueous
solution are unsuccessful. 3. Occlusion
Visual indicators for non-aqueous titration This is a type of coprecipitation in which impurities are
trapped within the growing crystal.
Indicator Colour Colour Colour
change change change 4. Postprecipitation
basic neutral acidic Sometimes a precipitate standing in contact with the mother
Crystal violet violet blue- yellow- liquor becomes contaminated by the precipitation of an im-
(0.5 per cent in green ish-green purity on top of the desired precipitate.
glacial acetic acid)
5. Washing and filtering
α-Naphtholbenzein blue or orange dark- Problems with coprecipitation and surface adsorption may
(0.2 per cent in blue- green
be reduced by careful washing of the precipitate. With many
glacial acetic acid) green
precipitates, peptization occurs during washing. Here part
Oracet Blue B blue purple pink of the precipitate reverts to the colloidal form e.g.,
(0.5 per cent in
glacial acetic acid) AgCl (colloidal) AgCl (s)

Quinaldine Red magenta almost This results in the loss of part of the precipitate because
(0.1 per cent in colo the colloidal form may pass through on filtration. By washing
methanol) with ice cold water, this can be minimized.

6. Drying of solid
Gravemetric Analysis Generally, the solids are dried at about 120oC but condi-
Gravimetric analysis, which by definition is based upon the tions for drying can vary considerably. To determine the
measurement of mass, can be generalized into two types: correct drying regime, a thermogravimetric balance may
precipitation and volatilization. be used.
 Analytical Chemistry  3.121

Commercial Acids and Bases

Solution Formula Weight Molarity Normality Weight (%) Specific Gravity

Acetic acid 60.05 17.4 17.4 99.8 1.05

[CH3COOH]

Ammonia 35.05 14.8 14.8 57 0.90

[NH4OH]

Hydrochloric acid 36.46 12.1 12.1 37 1.19

[HCl]

Nitric acid 63.01 15.8 15.8 70 1.42

[HNO3]

Sulfuric acid 98.08 18.0 36.0 96 1.84

[H2SO4]

Phosphoric acid 97.1 14.8 44.6 85 1.70

[H3PO4]

Pharmaceutical Impurities x. The amount of drug substance administered per day.

Impurities in pharmaceuticals are the unwanted chemicals that y. Higher reporting thresholds should be scientifically
even in small amounts may influence the efficacy and safety of justified.
the pharmaceutical products. Impurity profiling is the identity z. Lower thresholds can be appropriate if the impurity is
as well as the quantity of impurity in the pharmaceuticals. unusually toxic.

Sources of Impurities Thresholds for degradation products in Drug Products

Associated Related to Upon Aging Maximum daily dose (a) Reporting threshold (b,c)
with API Formulation ≤1 g 0.1%
1. Organic 1. Process /Method 1. Ingredient >1 g 0.05%
Impurities Related interaction Maximum daily dose (a) Reporting threshold (b,c)
2. Inorganic 2. Dosgae form 2. Functional <1 mg 1.0% or 5 μg TDI,
Impurities related group whichever is lower
3. Residual 3. Environment degradation
1 mg–10 mg 0.5% or 20 μg TDI,
Solvents related whichever is lower

ICH Guideline on Impurities >10 mg–2 g 0.2% or 2 mg TDI,

whichever is lower
Q3A Impurities in New Drug Substances
Q3B(R2) Impurities in New Drug Products x>2 g 0.10%
Q3C Guidelines for Residual solvents Maximum daily dose (a) Reporting threshold (b,c)
Q3D Guidelines for Elemental impurities <10 mg 1.0% or 50 μg TDI,
whichever is lower
Drug Substances Impurities thresholds
10 mg–100 mg 0.5% or 200 μg TDI,
Maximum Reporting Identification Qualification whichever is lower
daily threshold Threshold (z) threshold
Dose (x) (y, z) >100 mg–2 g 0.2% or 3 mg TDI,
whichever is lower
< 2g/day 0.05% 0.1% or 1 mg 0.15% or 1 mg
>2 g 0.15%
per day intake per day intake
(whichever is (whichever is a The amount of drug substance administered per day.
lower) lower) b Thresholds for degradation products are expressed ei-
> 2g/day 0.03% 0.05% 0.05% ther as a percentage of the drug substance or as total
3.122  Chapter 3

daily intake (TDI) of the degradation product. Lower N-methyl pyrrol- 48.4 4840
thresholds can be appropriate if the degradation product idone
is unusually toxic.
c Higher thresholds should be scientifically justified Pyridine 2 200
Toluene 8.9 890
Residual Solvents Xylenes 21.7 2170
Class I solvents: Solvents to be Avoided Known human
Methyl cyclo hex- 11.8 1180
carcinogens strongly suspected human carcinogens Envi-
ane
ronmental hazards.
Methyl butyl 0.5 50
Residual solvent Concentration limit (ppm) ketone
Benzene 2 ( Carcinogenic) Nitromethane 0.5 50
Carbon tetrachloride 4 (Toxic) Sulfolane 1.6 160
1,1 Dichloro ethene 8 (Toxic) Tetralin 1 100
1,2 Dichloro ethene 5 (Toxic)
1,1,2-Trichloro 0.8 80
1,1,1 trichloro ethane 1500 (Environmental hazard) ethane

Class II solvents: Solvents to be Limited Nongenotoxic ani- Class III Solvents: These are less toxic and possess lower
mal carcinogens or possible causative agents of other irre- risk to human health than class I or class II solvents. Long-
versible toxicity, such as neurotoxicity or teratogenicity. Sol- term toxicity or carcinogenicity not reported, which is
vents suspected of other significant but reversible toxicities. evident from the available data for the solvents under this
category. The use of class III solvents in pharmaceuticals
Solvent Permissible Concentration
daily exposure limit (ppm)
does not have any serious health hazard.
(mg/day) Solvents with Low Toxic Potential Solvents with low toxic
Acetonitrile 4.1 410 potential to humans; no health-based exposure limit is need-
ed. [NOTE—Class 3 residual solvents may have PDEs of up
Chlorobenzene 3.6 360 to 50 mg or more per day.]
Chloroform 0.6 60
Acetic acid Dimethyl Isobutyl acetate
Cyclohexane 38.8 3880 sulfoxide
1,2-Dichloroethene 18.7 1870 Acetone Ethanol Isopropyl acetate
Dichloromethane 6 600 Anisole Ethyl acetate Methyl acetate
1,1-Dimethoxy- 1 100 1-butanol Ethyl ether Propyl acetate
ethane
2-butanol Ethyl formate Pentane
N,N-Dimehtyl 10.9 1090
acetamide Butyl acetate Formic acid Methyl ethyl ketone

N,N-Dimethyl for- 8.8 880 Cumene Heptane Methyl isobutyl ketone

mamide 1- pentanol 1-propanol 2-propanol
1,2-Dioxane 3.8 380
Class IV Solvents: Class IV solvents, adequate toxicologi-
2-Ethoxyethanol 1.6 160
cal data is not available. The manufacturers should justify
Ethylene glycol 6.2 620 the residual levels for these solvents in pharmaceutical prod-
Formamide 2.2 220 ucts. The solvents under class IV are1, 1-diethoxy propane,
1-1-dimethoxy propane, 2-2-dimethoxy propane, methyl
Hexane 2.9 290
isopropyl ketone, isooctane, isopropyl ether, methyl tetrahy-
Methanol 30 3000 drofuran, petroleum ether, trichloro acetic acid.
 Analytical Chemistry  3.123

Guideline for Elemental Impurities ICH Q3D the risk assessment. For parenteral and inhalation products,
Three Class based on their Toxicity (PDE) and Occurrence the potential for inclusion of these elemental impurities
Type of Class Elemental Impurities should be evaluated during the risk assessment, unless the
route specific PDE is above 500 μg/day. The elements in this
Class 1 As, Cd, Hg & Pb
class include: Ba, Cr, Cu, Li, Mo, Sb, and Sn.
Class 2A Co, Ni & V
Other elements: Some elemental impurities for which
Class 2B Ag, Au, Ir, Os, Pd, Pt, Rh, Ru, Se & Tl PDEs have not been established due to their low inherent
Class 3 Ba, Cr, Cu, Li, Mo, Sb & Sn toxicity and/or differences in regional regulations are not
addressed in this guideline. If these elemental impurities are
* PDE-Permitted daily exposure present or included in the drug product they are addressed
Class 1: The elements, As, Cd, Hg, and Pb, are human by other guidelines and/or regional regulations and practices
toxicants that have limited or no use in the manufacture that may be applicable for particular elements (e.g., Al for
of pharmaceuticals. Their presence in drug products compromised renal function; Mn and Zn for patients with
typically comes from commonly used materials (e.g., compromised hepatic function), or quality considerations
mined excipients). Because of their unique nature, these (e.g., presence of W impurities in therapeutic proteins) for
four elements require evaluation during the risk assessment, the final drug product. Some of the elements considered
across all potential sources of elemental impurities and include: Al, B, Ca, Fe, K, Mg, Mn, Na, W and Zn.
routes of administration. The outcome of the risk assessment Permitted Daily Exposures(PDEs) for Elemental Impurities
will determine those components that may require additional
controls which may in some cases include testing for Class 1 Oral PDE Parenteral Inhalation
Element Class
elements. It is not expected that all components will require μg/day PDE, μg/day PDE, μg/day
testing for Class 1 elemental impurities; testing should Cd 1 5 2 2
only be applied when the risk assessment identifies it as the Pb 1 5 5 5
appropriate control to ensure that the PDE (permitted daily
As 1 15 15 2
exposure) will be met.
Hg 1 30 3 1
Class 2: Elements in this class are generally considered
Co 2A 50 5 3
as route-dependent human toxicants. Class 2 elements are
further divided in sub-classes 2A and 2B based on their V 2A 100 10 1
relative likelihood of occurrence in the drug product. Ni 2A 200 20 5
• Class 2A elements have relatively high probability of Tl 2B 8 8 8
occurrence in the drug product and thus require risk Au 2B 100 100 1
assessment across all potential sources of elemental Pd 2B 100 10 1
impurities and routes of administration (as indicated).
Ir 2B 100 10 1
The class 2A elements are: Co, Ni and V.
Os 2B 100 10 1
• Class 2B elements have a reduced probability of
Rh 2B 100 10 1
occurrence in the drug product related to their low
abundance and low potential to be co-isolated with Ru 2B 100 10 1
other materials. As a result, they may be excluded Se 2B 150 80 130
from the risk assessment unless they are intentionally Ag 2B 150 10 7
added during the manufacture of drug substances, Pt 2B 100 10 1
excipients or other components of the drug product.
Li 3 550 250 25
The elemental impurities in class 2B include: Ag, Au,
Ir, Os, Pd, Pt, Rh, Ru, Se and Tl. Sb 3 1200 90 20
Ba 3 1400 700 300
Class 3: The elements in this class have relatively low
toxicities by the oral route of administration (high PDEs, Mo 3 3000 1500 10
generally > 500 μg/day) but may require consideration in Cu 3 3000 300 30
the risk assessment for inhalation and parenteral routes. Sn 3 6000 600 60
For oral routes of administration, unless these elements are
Cr 3 11000 1100 3
intentionally added, they do not need to be considered during