Dr/ Sara Atta

Medica biochemistry department

Principle of Spectroscopic techniques
• The term “spectroscopy” refers to a wide range of techniques that employ radiation to
learn about the structure and properties of matter.
• The underlying idea behind all spectroscopic techniques is to radiate a beam of
electromagnetic radiation onto a sample and see how it reacts to it.
• Spectroscopy is most commonly used to identify and explain the elements and compounds
of atoms and molecules.
• They are determined by evaluating the radiant energy absorbed or emitted by the sample
or object.
• In this case, a beam of electromagnetic radiation such as infrared rays, UV rays, and so on is
passed on to the sample, and the response of the sample is measured using the wavelength
of the electromagnetic spectrum applied from an external energy source.
SPECTROPHOTOMETRY

• Photometry is defined as the measurement of light;

• spectrophotometry is defined as the measurement of the intensity of light at selected

wavelengths.

• Spectrophotometric analysis is a widely used method of quantitative and qualitative

analysis in the chemical and biological sciences.

• The method depends on the light-absorbing properties of the substance being analyzed.
The intensity of transmitted light passing through a solution containing an absorbing
substance (chromogen) is decreased by the absorbed fraction.

• This fraction is detected, measured, and used to relate the light transmitted or absorbed to
the concentration of the analyte in question.
Basic Concepts

• Consider an incident light beam with intensity Iо passing through a square cell containing a solution of a compound that
absorbs light of a certain wavelength, λ.

• Because the intensity of the transmitted light beam is Iѕ, then transmittance (T) of light is defined as: ŖŖ

T= Iѕ/Iо

• However, a portion of the incident light may be reflected by the surface of the cell or may be absorbed by the cell wall or
solvent.

• To focus attention on the compound of interest, elimination of these factors is necessary.

• This is done by using a reference cell which is identical to the sample cell, except that the compound of interest is omitted
from the solvent in the reference cell.

• The transmittance (T) through this reference cell is IR divided by Iо. The transmittance for the compound of interest in
solution is thus defined as: Iѕ / IR

• In practice, the reference cell is inserted to adjust the scale reading of the instrument to 100 (corresponding to 100%
transmittance) following which the percent transmittance reading is made on the sample. As the concentration of the
compound in solution increases, transmittance varies inversely and logarithmically
Consequently, it is more convenient to define a new term, absorbance (A), which will be
directly proportional to the concentration. Then, the amount of light absorbed (A) as the
incident light passes through the sample is equivalent to:
A= - log Iѕ = - log T I R
Beer’s law (also known as the Beer-Lambert law) states that the concentration of a
substance is directly proportional to the amount of light absorbed or inversely proportional to
the logarithm of transmitted light. Beer‟s law is expressed as:
A=abc
Where: A = absorbance.
a= constant defined as absorptivity.
b= light path in centimeters.
c = concentration of the absorbing compound, expressed in moles per liter Absorbance and %T relationship.

• This equation forms the basis of quantitative analysis by absorption photometry. When b
is 1 cm and c is expressed in moles per liter, the symbol € (epsilon) is substituted for the
constant a.
• The value for € is a constant for a given compound at a given wavelength under prescribed
conditions of solvent, temperature and pH, and is called molar absorptivity.
• Knowing molar absorptivity and absorbance of a certain compound can be used to
INSTRUMENTATION
• Modern instruments isolate a narrow wavelength range of the spectrum for
measurements. Those that use filters for this purpose are referred to as filter
photometers; those that use prism or gratings are called
spectrophotometers.
• Spectrophotometers are classified as single or double beam.
• The major components of a single and double beam spectrophotometers
are shown in (figure).
• In such an instrument, a beam of light is passed through a monochromator
that isolates the desired region of the spectrum used for measurements.
• The light next passes through an absorption cell (cuvet), where a portion of
the radiant energy is ab sorbed,
• Depending on the nature and concentration of the substance in the
solution.
• Any light not absorzbed is transmitted to a detector, which converts light
energy to electrical energy that is registered on a meter or recorder, or
digitally displayed.
• Some important points when carrying out spectrophotometric measurements:
1. A colored substance (chromophore) exhibits the complementary colour to that which it absorbs, i.e. a yellow compound
absorbs blue light, and therefore must be determined in the blue region of the spectrum.
2. Spectrophotometric assays are most sensitive at the absorbance maximum (λmax) of the chromophore produced, which is
the wavelength of light that is absorbed maximally by the chromophore.
3. The reference cuvet should contain all the reagents (other than the substance being assayed) in the same concentrations as
in the test cuvet.
4. The calibration curve may vary between batches of both reagent and standard, and hence a new calibration curve should be
constructed each time the assay is used, unless ther is evidence (quality control) to suggest that the previously obtained
curve is still valid.
5. Calibration curves should never be extrapolated beyond the highest absorbance value.
6. If the absorbance value lies outside the calibration curve or exceeds the reaction area where Beer‟s law is obeyed
(linearity), the sample should be re-assayed at a concentration that falls within this range.
• This is done by dilution of the sample with a suitable diluent followed at the end of the assay by multiplying the result
(concentration) by the dilution factor.
For example., if 300 µL of the sample is diluted by 300 µL of diluent, the dilution factor is calculated as follows:
• Total Volume = 300 + 300 = 2
• Sample Volume 300
 Types of Spectroscopic techniques
1-Nuclear magnetic resonance (NMR)

• Nuclear magnetic resonance (NMR) spectroscopy is a physicochemical technique for

determining the structural properties of molecules.

• It is described as the study of molecules by recording radiofrequency electromagnetic

radiation interactions with the nuclei of molecules placed within a strong magnetic field.

• NMR Spectroscopy, which is based on the nuclear magnetic resonance phenomenon, offers
comprehensive information on the structure, reaction state, and chemical surroundings of
molecules.
2-Fluorescence Spectroscopy (FS)

It is a set of techniques that deals with the measurement of fluorescence emitted by substances when exposed
to ultraviolet, visible, or other electromagnetic radiation.
3-Vibrational spectroscopy

• It is a type of spectroscopy that allows for the simple interpretation and sensitive structural identification of
trace amounts of substances based on their distinctive vibrational properties

• Because of the inelastic scattering, the resultant light will have a different frequency than the incident light.

• This method is commonly used to investigate vibrational, rotational, and other low-frequency interactions in
molecules.

• This is quite beneficial for identifying molecular structure, identifying functional groups, etc.
4-Infrared spectroscopy

• Infrared spectroscopy is the measurement of the interaction of IR radiation with

compounds.

• It is based on the principle that When IR radiation is passed through the IR active
compounds, they will get excited and show specific vibrational rotational spectra, which are
characteristic of the functional group of compounds.

5-The X-ray fluorescence (XRF)

• The X-ray fluorescence (XRF) spectrometer is an analytical instrument that employs X-ray
technology to perform routine and minimally invasive chemical analyses of various
geological materials such as rocks, minerals, sediments, and fluids.

• The operational principles of this system are based on wavelength-dispersive spectroscopy.

6-UV spectroscopy

• UV spectroscopy is an analytical technique that compares the amount of discrete

wavelengths of UV or visible light absorbed or transmitted by a sample to a reference or
blank sample.

• The sample composition influences this attribute, which may provide information about
the nature and concentration of the sample.

• UV-Vis spectroscopy is a widely used technique in many fields of science, including

bacterial culture, drug identification, and nucleic acid purity checks and quantification, as
well as quality control in the beverage industry and chemical research.
7-Atomic emission spectroscopy

• Atomic emission spectroscopy (AES) is an analytical technique used to quantify metal

atoms by measuring the intensity of light produced by the atoms in excited states.

• When an excited atom returns to its ground state, it emits a specific wavelength of
radiation.

• Excitation (absorption of radiation) and de-excitation (emission of radiation) of electrons

are both involved in atomic emission spectroscopy.
8-Atomic absorption spectrometry

• Atomic absorption spectrometry (AAS) detects elements in liquid or solid samples by using
certain wavelengths of electromagnetic radiation from a light source.

• Individual elements absorb wavelengths differently, and the absorbances of these elements
are evaluated against standards.

• The basis of atomic absorption spectroscopy (AAS) is that free atoms in their ground state
can absorb light of a specific wavelength.
Atomic absorption spectrophotometry
• Atomic absorption (AA) spectrophotometry is used widely in clinical laboratories to
measure elements such as aluminum, calcium, copper, lead, lithium, magnesium, zinc
and other metals.
• AA spectrophotometry is the inverse of flame emission photometry in many
respects.
• In AA photometry, the element is not excited in the flame, but dissociated from its
chemical bonds, and placed in an unexcited or ground state (neutral atom).
• The neutral atom is at a low energy level in which it is capable of absorbing
• radiation at a very narrow bandwidth corresponding to its own spectrum.
• When light from the hollow-cathode lamp enters the flame, some of it is absorbed
by the ground-state atoms in the flame, resulting in a net decrease in the intensity of
the beam from the lamp. This process is called “atomic absorption”.
• Atomic absorption methods are 100 times more sensitive than flame emission
methods.
• Owing to the unique specificity of the wavelength from the hollow-cathode lamp,
these methods are highly specific for the element being measured.
• AA is most frequently used for calcium analysis
9-Mass spectroscopy

• It is the accurate method for determining of molecular mass of the compound and its
elemental composition.

• It can be used to determine the mass-to-charge ratio (m / z) of one or more molecules in a

sample.

• These measurements are frequently used to determine the exact molecular weight of the
sample components.

• It is used in determining the molecular mass of a molecule that has indirectly contributed
to the identification of isotopes.
Applications of Spectroscopic techniques

• Spectroscopic techniques are mostly used to investigate the structure of molecules and
atoms. Spectroscopy will explore the structure and electron configurations of atoms and
molecules using a long wavelength.

• Spectroscopic techniques can also be used to determine the chemical makeup of unknown
materials. The emission spectrum of spectroscopy will aid in identifying a few parts per
million of a trace element in a substance.

• Spectroscopic techniques are used to examine the atmospheres of cool stars for molecular
species, molecular clouds where new stars emerge, and the Doppler shifts of galaxies to
trace their movements.
• Spectroscopic techniques aid in biochemical studies such as DNA melting, proteins,
enzymes, nucleic acids, and so on, as well as surface characterization, chemical kinetic
studies on reactants and products of reactions, qualitative and quantitative analysis of
transition metals, and studies of chemical and biochemical processes, and isotopic studies.

• The analysis of air pollutants, organic and inorganic contamination in wastewaters, and
natural water bodies is made easier by spectroscopic techniques. The analysis of air
pollutants, organic and inorganic contamination in wastewaters, and natural water bodies is
made easier by spectroscopic techniques.

• Spectroscopic techniques aid in the improvement of different qualities such as heating,

irradiation, and impregnating gems with oils, waxes, and so on.
FLUOROMETRY
• Fluorescence refers to the condition when a molecule absorbs light at one wavelength and reemits light at a longer
wavelength.
• The interaction of radiant energy with molecules or particles in solution can result in either luminescence or light
scattering.
• Luminescence is the emission of light or radiant energy when an electron returns from an excited or higher energy level to
a lower energy level.
• Light scattering occurs when radiant energy passing through a solution strikes a particle and is scattered in all directions.
• Fluorescence: light is absorbed by a molecule and subsequently emitted as light at a longer wavelength.
• Phosphorescence: light is absorbed by a molecule, but in contrast to fluorescence, the subsequently emitted light is from
an excited triplet state; compared with fluorescence, the emission time and wavelength of light are both much longer.
• Chemiluminescence: light is emitted as a result of chemical energy; no light is absorbed.
• Fluorometry is defined as the measurement of emitted fluorescent light.
• Fluorometric analysis is a widely used method of quantitative analysis in the chemical and biological chemistry.
TURBIDIMETRY AND NEPHELOMETRY
• Light scattering is a physical phenomenon resulting from the interaction of light with particles in solution. Unlike
fluorescence emission, the scattered light is of the same frequency as the incident light.
• Turbidimetry and nephelometry are techniques used to measure scattered light. Light scattering measurements are applied
best to immunoassays of specific proteins.
Turbidimetry
• Turbidimetry causes the intensity of the incident beam of light to decrease as it passes through a solution of particles.
• The measurement of the decrease in intensity of the incident light beam that is caused by scattering of light is called
turbidimetry.
Nephelometry
• Nephelometry is the detection of light energy that is
scattered at a variety of angles that is not in the direct path
of the transmitted light.
• Common nephelometers measure scattered light at right angles to the incident light.
FLAME EMISSION SPECTROPHOTOMETRY

• Flame emission spectrophotometry is based on the

emission of light by atoms of many metallic elements
when given sufficient energy, such as that supplied by a
hot flame.

• Under constant and controlled conditions, the light

intensity of the characteristic wavelength produced by
each atom is directly proportional to the concentration of
the substance of interest in the sample.

• Flame emission photometry is used for quantitative

determination of sodium and potassium in body fluids.