SPECTROPHOTOMETRY

Spectroscopy is a powerful tool used for SPECTROPHOTOMETRY

quantitative and qualitative analysis of samples,  an analytical technique used to
and is the most important of all the measure the amount of light of a particular
instrumental methods of analysis. It is a branch wavelength absorbed by a sample in solution 
of science dealing with the study of interaction concentration
of electromagnetic radiation with matter.  a process where we measured
Spectrophotometry is one of the most widely
absorption and transmittance of
applied physicochemical technique, which is
monochromatic light in terms of ratio or a
remarkable for its sensitivity and precision. Two
reasons for the popularity of the function of the ratio, of the radiant power of the
spectrophotometric technique are the two beams as a functional of spectral
instrumentation is fairly easy to operate and wavelength
widely available. The chief advantage of
spectrophotometric method is that they provide
a simple means for determining minute Electromagnetic Spectrum
quantities of substances.  It may be considered as simple
harmonic wave propagated from a
It is possible for a ray of light to be source and travelling in straight lines
absorbed by some material and simply pass except when reflected or refracted.
through others without being affected. When a  This radiation will be associated with
molecule absorbs light, energy is transferred
the properties of the waves.
from the ray of light to the molecule. If the
frequency of the electronic and magnetic fields
of a ray of light match the frequency at which
molecules will vibrate, then light will be
absorbed, if the frequency does not match, then
the light will pass straight through unaltered.
Inert molecules whether solid or liquid appear
colored due to the way they modify light
illuminating the object. Thus different objects
absorb some wavelengths and reflect others.
For example, if a white light passes through a
Regions of the electromagnetic spectrum.
yellow solution, it absorbs all colors except
yellow. Wavelength - the distance between two
successive maxima on an electromagnetic wave
Frequency - number of complete wavelength
a. spectroscopy - the branch of science units passing through a given point in unit time
concerned with the investigation and is called the frequency of radiation (v)
measurement of spectra produced when matter
Wave number - the number of waves per
interacts with or emits electromagnetic
radiation centimeter in vacuum
b. spectrometry – methods of analysis
which deal with the measurement of spectra
c. spectrum - defined as the
characteristic wavelengths of electromagnetic Interaction of Radiation and Matter
radiation (or a portion thereof) that is emitted  If matter is exposed to electromagnetic
or absorbed by an object or substance, atom, or radiation, e.g. infrared light, the
molecule radiation can be absorbed,
d. colorimeter - an instrument that transmitted, reflected, scattered or
compares the amount of light getting through a
undergo photoluminescence.
solution with the amount that can get through a
sample of pure solvent Photoluminescence is a term used to
e. pKa - a number that shows how designate a number of effects,
weak or strong an acid is including fluorescence,
phosphorescence, and Raman
 scattering  measured by a
spectrophotometer.
 The entire range over which
electromagnetic radiation exists is
known as electromagnetic spectrum.
 Spectrophotometry is mainly
concerned with the ultraviolet (200-
400nm) and visible (400-800nm)
regions.
 Main instruments – photometers,
colorimeters and spectrophotometers.
The interaction between light radiation and
matter.
Principles of Spectrophotometry:
Parts of a spectrophotometer.
a. source of light with a continuous spectrum i
b. monochromator (prism or grating)
c. sample holder (glass cuvette)
d. electronic detector systems
Terminology used in Absorption Measurement
1. Radiant Energy  defined as energy
transmitted as electromagnetic radiation. It has
the properties of both particle and wave
motion.
2. Radiant Power (P)  formerly known as
Intensity (I). Energy per unit time is called as
power. Radiant power is the rate at which
energy is transported in a beam of radiant
energy.
3. Transmittance (T)  simply the fraction of
the incident power transmitted by a sample.
4. Absorbance (A)  formerly known as an
optical density (O.D.) or extinction (E). The
absorbance is the logarithm to base 10 of the
reciprocal of the transmittance.
5. Absorptivity (a)  formerly known as the
extinction coefficient or specific extinction. It is
defined as the ratio of the absorbance to the
product of the length of optical path b and the
concentration of the sample C. Absorptivity is
the measure of the ability of sample to absorb
light.
6. Molar absorptivity (€)  formerly known as
molar extinction coefficient or molar absorption
coefficient. Molar absorptivity is the product of
absorptivity and molecular weight of the
material.
7. Path Length (b)  formerly denoted by l or d.
It is the internal thickness (diameter) of the cell
in which the test sample is taken.
Fundamental Laws of photometry
When light is incident upon a
homogeneous medium, a part of the radiant
power of the incident light is reflected, a part is
absorbed and the remainder is transmitted.
1. Lambert’s Law  states the relationship
between the radiant power of absorbed light
with the thickness of the medium. When a
beam of monochromatic light is allowed to pass
through a transparent medium, the rate of
decrease of radiant power with the thickness of
the medium is directly proportional to the
radiant power of the incident light.
2. Beer’s Law  states the relationship between
the radiant power of absorbed light with the
concentration of the medium. When a beam of
monochromatic light is allowed to pass through
a transparent medium, the rate of decrease of
radiant power with the concentration of the
medium is directly proportional to the radiant
power of the incident light.
3. Beer-Lambert’s Law  deals with the
relationship between the radiant power of the
incident light and transmitted light as a function
of both the thickness of the medium and the
concentration of the absorbing species. It states
that, for the given system and the thickness of
the medium, the absorption of the medium is
directly proportional to the concentration of an
absorbing species.
A=abc
Where:
a = absorbity constant of the
specimen
b = light path length
 c = concentration
For a given sample, absorbance depends on six
factors:
(1) the identity of the absorbing substance,
(2) its concentration,
(3) the pathlength l,
(4) and wavelength of light,
(5) the identity of the solvent, and
(6) the temperature.
ULTRAVIOLET AND VISIBLE SPECTROSCOPY
 used to analyze liquids, gases and solids by
using radiative energy corresponding to far and
near ultraviolet (UV), visible (Vis) and near
infrared (NIR) regions of electromagnetic
spectrum.
An example of a UV-Vis spectrophotometer.
Applications:
1. A robust, workhorse method for the
quantification of drugs in formulations where
there is no interference from excipients.
2. Determination of the pKa values of some
drugs.
3. Determination of partition coefficients and
solubilities of drugs.
4. Used to determine the release of drugs from
formulations with time (dissolution).
5. Can be used to monitor the reaction kinetics
of drug degradation.
6. The UV spectrum of a drug is often used as
one of a number of pharmacopoeial identity
check
Strengths:
1. An easy-to-use, cheap and robust method
offering good precision for making quantitative
measurements of drugs in formulations.
2. Routine method for determining some of the
physicochemical properties of drugs, which
need to be known for the purposes of
formulation.
3. Some of the problems of the basic method
can be solved by the use of derivative spectra.
Limitations:
1. Only moderately selective. The selectivity of
the method depends on the chromophore
(functional group which absorbs radiant energy)
of the individual drugs (colored drug with an
extended chromophore is more distinctive than
a drug with a simple benzene ring
chromophore).
2. Not readily applicable to the analysis of
mixtures.
Instrumentation:
Parts of a UV-Vis Spectrophotometer.
a. The light source – a deuterium lamp for the
UV region from 190-350nm and a quartz
halogen or tungsten lamp for the visible region
from 350-900nm
b. The monochromator – used to disperse the
light into its constituent wavelengths, further
selected by the slit
c. The optics – designed to split the light beam
Factors governing absorption of radiation in
the UV-Vis region:
1. Absorption compound - increases absorption
of UV (chromophore and auxochrome)
2. Solvent effect – can absorbed UV radiations,
thus, must be avoided (benzene , carbon
tetrachloride, chloroform)
3. Temperature
- Low temperature is suitable for uv
visible spectroscopy.
- In high temperature, absorption of UV
radiation is increase.
4. Inorganic material – can increase absorption
(Ag, Au)
Diode Array Instruments
 photomultiplier tube – used for
detection in older types of UV-Vis
instrument
 photodiode – used as detectors in
spectrophotometers
 diode array – series of photodiode
detectors positioned side by side on a
silicon crystal
- useful in dissolution testing
Practical Aspects
1. Care should be taken to avoid touching the
optical surface of sample cells with the finger
since fingerprints can cause significant
absorbance. The optical surfaces of the cell can
be wiped carefully with tissue.
2. The precision of the path length cell is
important. Tolerances for cells of good quality
are +/- 0.1 mm for path length. For maximum
quantitative accuracy, the same cell should be
used for measurement of both the standard and
the sample.
The glass cuvette.
3. Distilled water is the ideal solvent but is not
suitable for many organic compounds.
Methanol and ethanol are next best but they
cannot be used below a wavelength of 210nm.
4. The solvent used to dissolve the sample,
concentration, pH and temperature can affect
the position and intensity of absorption bands
of molecules.
5. Ideally absorbances measured should be in
the range of 0.4-1.0.
6. Scattering gives an apparent increase in
absorbance and is caused by particles
suspended in solution.
Instrument Calibration
1. Calibration of absorbance scale:
 0.006% w/v solution of potassium
dichromate solution in 0.005 M H2SO4 (BP)
2. Calibration of wavelength scale:
 5% w/v solution of holmium
perchlorate
 deuterium or mercury discharge
lamps
3. Determination of instrumental resolution:
 controlled by its slit width settings
 0.02% w/v solution of toluene in
hexane
4. Determination of stray light:
Stray light – light which falls on the detector
within a UV instrument without having passed
through the sample (light scattering, entry of
light)
 gives a false-low
absorbance reading
 1.2% solution of KCl in
water against a water blank at a wavelength of
200nm
ATOMIC SPECTROSCOPY
a. mass spectrum - a distribution of
ions shown by the use of a mass spectrograph
or mass spectrometer
 b. alkali metals - any of the elements
lithium, sodium, potassium, rubidium, cesium,
and francium, occupying Group IA (1) of the
periodic table
c. alkaline earth metals - any of the
elements beryllium, magnesium, calcium,
strontium, barium, and radium, occupying
Group IIA (2) of the periodic table
ATOMIC SPECTROSCOPY
 is the determination of elemental
composition by its electromagnetic or mass
spectrum
Three techniques:
(1) atomic emission spectrophotometry (AES),
(2) atomic absorption spectrophotometry (AAS)
(3) atomic fluorescence spectrophotometry
(AFS)
How the three techniques are implemented.
Atomic Emission Spectrophotometry
Principles:
 Atoms are thermally excited so that
they emit light and the radiation
emitted is measured.
Applications:
 Quantification of alkali metals in: alkali
metal salts, infusion and dialysis
solutions.
 Determination of metallic impurities in
some of the inorganic salts used in
preparing these solutions.
Strengths:
 Flame photometry provides a robust,
cheap and selective method based in
relatively simple instrumentation for
quantitative analysis of some metals.
Limitation:
 Only applicable to the determination of
alkali and some alkaline earth metals.
Introduction
 plays an important role in the control
of sodium, potassium, barium, calcium
and lithium
 Atoms contain various energy states
and the normal unexcited state is the
ground state. If energy is gained by the
atom, this electron may be excited to a
higher state and then subsequently
lose its excess energy by falling back to
a lower energy orbital.
Instrumentation
1. Flame – the metal is volatilized in a natural
gas/compressed air flame at 2000ºC
- a higher temp.
(2500ºC) may be obtained using air/acetylene
and is required for analysis of Mg by AES.
2. Monochromator/Filter – where radiation
emitted by the excited atoms is passed
3. Detector – measures the intensity of the
selected radiation using a photosensitive cell
Interferences
1. Ionisation
 an equilibrium and may be shifted
to the left by addition of another readily ionized
element to the sample (produces electrons)
Ex: strontium chloride is added in the assay of
effervescent KCl tablets
2. Viscosity
 can either increase or decrease the
rate of emission
Ex: sucrose decreases the rate, ethanol
increases the rate
3. Anionic interference
 reduces the reading of the sample
 may be removed by the addition of
lanthanum chloride
Atomic Absorption Spectrophotometry
Principles:
 Atoms of a metal are volatilized in a
flame and their absorption of a narrow
band of radiation produced by a hollow
 cathode lamp, coated with the
particular metal being determined, is
measured.
 All atoms can absorb light.
 The wavelength at which light is
absorbed is specific for each element.
If a sample containing nickel, for
example, together with elements such
as lead and copper is exposed to light Parts of Atomic Absorption
at the characteristic wavelength for Spectrophotometry.
nickel, then only the nickel atoms will
absorb this light. 1. Light source – hollow cathode lamp
 The amount of light absorbed at this 2. Flame – air/acetylene (2500ºC); nitrous
wavelength will increase as the number oxide/acetylene (3000ºC) for Al or Ca
of atoms of the selected element in the 3. Monochromator – narrows down the width
light path increases, and is proportional of the band of radiation being examined
to the concentration of absorbing 4. Detector – a photosensitive cell
atoms.
 The relationship between the amount
of light absorbed and the Examples
concentration of the analyte present in 1. Assay of Calcium and Magnesium in
known standards can be used to Haemodialysis Fluid
determine unknown concentrations by 2. Assay of Lead in Sugars
measuring the amount of light they 3. Assay of Mg and Sr in Calcium acetate
absorb. An atomic absorption 4. Assay of Palladium in Carbenicillin Sodium
spectrometer is simply an instrument 5. Assay of Copper and Iron in Ascorbic acid
in which these basic principles are
applied to practical quantitative
analysis.

Applications:
 Determination of metal residues
remaining from the manufacturing
process in drugs.
Strengths:
 More sensitive than AES. A highly
specific method of analysis useful in
some aspects of quality control.

Limitations:
 Only applicable to metallic elements.
 Each element requires a different
hollow cathode lamp for its
determination.

Introduction:
 The energy difference between their
ground state orbital and the excited
state is too great for thermal
excitation. If the energy is too great,
AAS may be used.

Instrumentation