Instrumental Methods of Analysis

2 methods of analysis

1. Classical/Wet method
2. Instrumental

Classical/Wet method

- more accurate and precise
- high amount of analyte

Instrumental

- faster
- analyze trace amounts of analyte
- complenteray w/ classical

qualitative analysis

for identification and characterization (specific quality/attribute)

quantitative analysis

- for measurement and quantification (amount of substance/analyte)

signal generator

• Produces a signal that reflects presence and usually the
concentration of the analyte
- generates analytical signal
- 2 components: light/energy source and sample/chemical system

input transducer/detector

device that converts from analytical signal to electrical signal

signal processor

• Modifies the transduced signal to make it more convenient
for the operation of the readout device

o Amplification → signal is magnified/ increased
o Attenuation → signal is reduced/decreased
o Filtration → unwanted noise is removed/reduced from the signal

readout device

• Converts a processed signal to a signal that is
understandable by a human operator
• Examples: analog meter, digital meter, computer monitor

electromagnetic spectrum

- Complete system of energy propagated in a wave form
- 2 waves: Electric and magnetic wave
- ↓ Lower frequency, ↑ longer wavelength

Electric wave

vertical electromagnetic wave

Magnetic wave

horizontal electromagnetic wave

spectrum

RMIVUXG

spectroscopic methods

o Absorption of radiation → UV-Visible, AAS, NMR, IR

o Emission of radiation → Fluorometry, MES, AES

o Scattering of radiation → Turbidimetry, Nephelometry

o Refraction of radiation → Refractometry

o Diffraction of radiation → X-ray and electrondiffraction

o Rotation of radiation → Polarimetry

chromatographic methods

o Gas Chromatography
o High Performance Liquid Chromatography

electrochemical methods

o Electric charge → Coulometry
o Electric current → Polarography1
o Electric potential → Potentiometry

Miscellaneous Methods

o Mass-to-charge → Mass spectrometry
o Radioactivity → Radioactive emissions

spectroscopy

• Is a general term for the science that deals with the interactions of various types of radiation with matter
• Interaction of light with matter

spectrometry/spectrometric methods

refer to the measurement of the intensity of radiation with a photoelectric transducer or other type of electronic device.

spectrophotometry

branch of spectrometry which embraces the
measurement of the absorption, by chemical species, of
radiant energy of definite and narrow wavelength,
approximating monochromatic radiation

colorimetry

branch of spectrometry in which absorption measurement is made in the visible region of the spectrum

colorimeter/flame photometer

Instruments designed to measure radiant power with the aid
of filter, instead of prism or diffraction grating, for the
purpose of increasing the sensitivity of the measurement

absorption

transition from a lower level to a higher level
with transfer of energy from radiation field to an absorber

chromophore

functional group which absorbs radiant
energy in the UV or visible regions of the spectrum

laws governing absorption

Beer's Law
Lambert's Law

Beer's Law

- the power of transmitted radiant
beam decreases exponentially as the concentration of the
solution containing the absorbing chemical species increases
arithmetically
- A = acl, A = ɛcl
(absorptivity/molar coefficient, concentration, length)

Lambert's law

absorbance will increase as the thickness of the absorbing material increases
- A = log (l0/l)

Theory of Absorptivity

A = log (I0/I) = -log T = ɛcl

• When an EMR with an intensity of Io impinges a solution of
concentration c and pathlength l, its intensity is diminished in an exponential fashion (I).

absorption of radiation

• UV-Vis Spectroscopy
• Infrared Spectrophotometry
• Atomic Absorption Spectrophotometry
• Nuclear Magnetic Resonance

uv-vis spectroscopy principle

- 200-780 nm
• The electrons in the bonds within the molecule become
excited so that they occupy a higher quantum state and in
the process absorb some of the energy passing through a
solution.
• The more loosely held the electrons are within the bonds of
the molecule, the longer the wavelength of the radiation
absorbed.

uv-vis spectroscopy instrumentation

- tungsten (visible); H2/D2 (UV)
- monochromator
- sample
- detector
- amp
-r ecorder

monochromator

- prism/graduating
o Separates the components of light based on wavelength range
o As a result, only the wavelength chosen will interact with the sample

quartz cuvette

UV and vis cuvette

optical glass, plastic cuvette

vis only cuvette

uv-vis spectroscopy applications

I- identity
P - pKa, partition coefficient
Q - quantity
D - dissolution testing, degradation kinetics

spectral absorbance curve

• Plot of absorbance readings of the analyte versus the wavelength
o When wavelength changes, so does absorbance
• Used to determine the wavelength at which maximum
absorption occurs (Amax)
o Usually specified in USP monographs

Beer's plot

• Plot of absorbance values against a series of known solute
concentrations
o High absorbance = high concentration
• Should yield a straight line
• Used to determine the unknown solute concentration

Infrared spectrophotometry principle

- between 2500 and
20000 nm
- Sample is contained within discs or cells made of alkali
metal halides (KCl or NaCl)
• Sample may be in solid or liquid form

dispersive IR

makes use of monochromator to select each
wavenumber in turn in order to monitor its intensity after the
radiation has passed through the sample

fourier transform IR

makes use of an interferometer that
generates a radiation source in which individual wave
numbers can be monitored within 1s pulse radiation without
dispersion being required

solid sample preparation

mull technique
pellet technique
film technique
solution technique

mull technique

the sample is ground with a mulling agent
(mineral oil like Nujol [brand]) in a mortar or pestle to a
fine paste. The paste is placed on a plate and covered with
another.

pellet technique

the solid is suspended in KBr pellet and
prepared under high pressure

film technique

the sample is cut into sheets of suitable
thickness with a microtome or melted at low heat and
allowed to dry as a film.

solutino technique

sample is dissolved in a suitable solvent and used as a liquid sample

Infrared spectrophotometry applications

P - polymorphs
F - fingerprinting
C - characterization

3-15μm

region for identification

Group Frequency Region (fx groups)

3-8 μm (4000 to 1500 cm-1)

fingerprint region

8-15 μm (1500 to 500 cm-1)

atomic absorption spectrum (AAS) principle

• metal being determined is measured
• hollow cathode lamp
• HCl + metal

atomic absorption spectrum (AAS) instrumentation

- hollow cathode lamp
- flame
- nebulizer (test soln)
- monochromator
- detector
- data processor

atomic absorption spectrum (AAS) applications

- determine metal residudes
- analyze trace minerals in multivitamins

nuclear magnetic resonance (NMR) principle

• Radiation in the radiofrequency region is used to excite
atoms, usually protons or carbon-13 atoms, so that their spins switch from being aligned with to being aligned
against an applied magnetic field.

NMR

N - nuclei 13C and 1H
M - magnetic field (aligned with or against)
R -
radiofrequency region

nuclear magnetic resonance (NMR) applications

N - eNantiomeric impurities
M - molecular structure

Emission of radiation

MES
AES

emission

transition from a higher level to a lower level,
and the radiation is transferred to the radiation field;
nonradiative decay if no radiation is emitted

Molecular Emission Spectroscopy (MES) principle

C - chromophore
R - rigid
S -structure

aka fluorescence spectrophotometry or
fluorometry.

Molecular Emission Spectroscopy (MES) instrumentation

- D2/H2 lamp
- excitation monochromator
- exciting light
- sample holder
- emission monochromator
- detector

Molecular Emission Spectroscopy (MES) applications

- fluorescent drugs in low-dose formulations
- limit test
- binding of drugs
- Analysis of vitamins, particularly thiamine (Vitamin B1) and riboflavin (Vitamin B2)

atomic emission spectrophotometry (AES) principle

• Analyte atoms in solution are aspirated into the excitation
region (higher energy levels) where they are desolvated,
vaporized and atomized by a flame, discharge or plasma.
• atoms decay back to lower levels by emitting light.
• aka optical emission spectrophotometry
(OES).

atomic emission spectrophotometry (AES) applications

• Quantification of alkali metals like sodium, potassium and
lithium in alkali metal salts, infusion and dialysis solutions (MgBaLiK Ca Na plAES)
• Determination of metallic impurities in some of the other
inorganic salts used in preparing solutions

scattering of radiation

nephelometry
turbidimetry

scattering

- Redirection of light due to its interaction with matter, and may or may not involve transfer of energy
• Crucial: position of detector

nepheLometry

- 90°
• Reflected light is measured after radiant energy passes
through a turbid solution or suspension

turbidimetry principle

- 0°
• Transmitted light is measured after radiant energy passes
through a turbid solution or suspension
• Light transmittance is a measure of turbidity

turbidimetry applications

- official assay for antibiotics
- Testing of official chemicals to ensure absence of excessive
amounts of chloride and sulfate

diffraction of radiation

X-ray and electron diffraction methods

x-ray diffraction principle

- bending of light
• scattered radiation characteristic is manifested as a diffraction pattern.
o Crystals or materials provide you with a diffraction pattern that allows you to identify them
• Source of light: X-ray tube or cathode ray tube

diffraction

A scattering phenomenon that either enhances
or reduces the amplitude of the vibration
• Light is bent when it passes through a narrow opening
- CONSTRUCTIVE WAVES

x-ray diffraction applications

• Characterization of pharmaceutical solids
• Study/identification of crystals and polymorphs
o Polymorphs were also encountered in IR

chromatography

• A separation process in which components of a mixture are
repetitively equilibrated between two phases, a stationary phase and a mobile phase.
• The main purpose is to separate and quantify the
components of the sample.

Stationary phase

a porous solid used alone or coated with a liquid

mobile phase

a liquid or a gas that carries through
the stationary phase, referred to as eluent or carrier

chomatographic method according to physical configuration

- column: packed or tubular
- planar

column

the stationary phase is held in a column
through which the mobile phase is pushed under pressure or drawn by gravity. The two types are
packed column and open tubular

packed column

packed, filled with stationary phase

tubular column

- hollow, usually used for gas chromatography

planar

the stationary phase is a flat strip of paper
or a solid coated onto a glass plate. The liquid mobile
phase moves through the stationary phase by capillary
wetting or a combination of wetting and gravity

chomatographic method according to sample development

frontal
displacement
elution

partition

is a bulk-phase distribution process in which the solute forms homogeneous solution in each phase

adsorption

involves interaction at a surface or fixed
sites on a normally solid stationary phase

exclusion

relies on the ability of a porous solid
stationary phase to discriminate on the basis of size by
admitting small molecules to its pores but excluding
larger ones.

chomatographic method according to mechanism of retention

partition
adsorption
exclusion

frontal

the sample is fed onto the column continuously and acts as the mobile phase.

displacement

characterized by a mobile phase that
is strongly attracted to the stationary phase, causing
the sample components to be "pushed" through the
column by the advancing solvent.

elution

sample components are carried along the
column by a mobile phase, and separation is the result
of their spending different fractions of time in that
phase. May be isocratic mode (1 mobile phase with a
constant composition) or gradient mode (multi-mobile
phases with changing composition).

chromatogram

• Is a plot of some function of solute concentration versus
elution time or elution volume.
o Identification - based on comparing with standard sample
o Quantification - based on calculating the peak area of the sample
• Time in minutes vs intensity

retention time (TR)

Is the time required by an average molecule of component
to pass from the injection point through the column to the
detector.

dead time (TM)

Is the time after the sample injection for the small peak of
the species that is not retained by the stationary phase.

retention volume (VR)

Is the volume of carrier gas necessary to carry an average
molecule of component from the point of injection to the detector.

high-pressure liquid chromatography (HPLC)

• It is a liquid chromatography which uses high efficiency
columns and relatively high mobile phase inlet pressure.

normal phase

o Column: polar property
o Solvent: non-polar property

reversed phase mode (RP)

o Column: non-polar property
o Solvent: polar property

ion-exchange mode (IC)

o Cationic exchanger: with sulfonic acid/carboxylic acid;
alkaloid bases
o Anionic exchanger: with quaternary ammonium/amino;
alkaloidal salts

size exclusion chromatography

o Gel Permeation Chromatography (GPC) - molecular
weight (MW) measurement of polymers
o Gel Filtration Chromatography (GFC) - separation of
proteins

HPLC applications

D - drugs
D - degradation products
M - metabolties in bio fluids
P - partition coefficients and pKa

gas chromatography principle

A gaseous mobile phase flows under pressure through a
heated tube either coated with a liquid stationary phase or
packed with liquid stationary phase coated onto a solid
support.

gas chromatography instrumentation

- gas cylinder
- injector
- sample
- column
- oven
- detector
- recorder

gas chromatography applications

v - volatile oils
i - impurities
s - solvent residues