Basic concept of Instrument analysis

What is UV Visible Spectroscopy?

• UV-visible spectroscopy is a technique that measures the amount of light absorbed by a
chemical substance.
• It is absorption spectroscopy or reflectance spectroscopy technique within the ultraviolet and
visible regions of the electromagnetic spectrum.
• When continuous radiation is passed through a compound a portion of that compound is
absorbed by the compound.
• The residual radiation after passing through the compound yields a spectrum with gaps in it
due to absorption by the compound, this spectrum is called the absorption spectrum.
• Absorption of UV-Visible radiation results in the electronic transition of the compound, i.e.,
an electron in the ground state (occupied orbital) is promoted to the excited state (unoccupied
orbital), and the amount of radiation absorbed corresponds to the energy difference between
the ground state and the excited state.

What is Atomic Absorption Spectroscopy (AAS)?

• Atomic absorption spectroscopy, or AAS, is a technique for measuring the concentrations of
metallic elements in different materials.
• As an analytical technique, it uses electromagnetic wavelengths, coming from a light source.
• Distinct elements will absorb these wavelengths differently. It gives a picture of what
concentrations of a specific element there is in whatever material, or liquid, is being tested.
• Spectroscopy is the study of how radiated energy and materials interact.
• Matter absorbs energy, which will create some sort of change in its state.
• The atomic part refers to the atoms in a material, which will absorb radiated energy from a
light source.
• These atoms will each have their own characteristics when it comes to absorbing energy
because each element has a unique electronic structure.
• Therefore, using AAS, you can measure for a specific element in a material, based on the
amount of light absorbed at a defined wavelength, which corresponds to the known
characteristics of the element you are testing for.

What is flame photometry?

• Flame photometry is defined as the measurement of intensity of the light emitted when any
alkali or earth metal is introduced into the flame.

• Because of the emission of radiation, it is also known as flame emission spectroscopy.

• Based on the element present in the sample, it produces emission spectra and different
colours to the flame.

What is mass spectroscopy?

• Mass spectrometry is an analytical tool useful for measuring the mass-to-charge ratio (m/z) of

one or more molecules present in a sample.

 • These measurements can often be used to calculate the exact molecular weight of the sample

components as well.

• Typically, mass spectrometers can be used to identify unknown compounds via molecular weight

determination, to quantify known compounds, and to determine structure and chemical

properties of molecules.

• How does a mass spectrometer perform such a feat? Every mass spectrometer consists of at least

these three components:

• Ionization Source

• Mass Analyzer

• Ion Detection System

What is gas chromatography?

• Gas chromatography differs from other forms of chromatography in that the mobile phase
is a gas and the components are separated as vapours.
• It is thus used to separate and detect small molecular weight compounds in the gas phase.
• The sample is either a gas or a liquid that is vaporized in the injection port. The mobile phase
for gas chromatography is a carrier gas, typically helium because of its low molecular weight
and being chemically inert.
• The pressure is applied and the mobile phase moves the analyte through the column. The
separation is accomplished using a column coated with a stationary phase.

What is chromatography?
• Chromatography is an important biophysical technique that enables the separation,
identification, and purification of the components of a mixture for qualitative and
quantitative analysis.
• The Russian botanist Mikhail Tswett coined the term chromatography in 1906.
• The first analytical use of chromatography was described by James and Martin in 1952, for
the use of gas chromatography for the analysis of fatty acid mixtures.
• A wide range of chromatographic procedures makes use of differences in size, binding
affinities, charge, and other properties to separate materials.
• It is a powerful separation tool that is used in all branches of science and is often the only
means of separating components from complex mixtures.

Types of Chromatography
• Substances can be separated on the basis of a variety of methods and the presence of
characteristics such as size and shape, total charge, hydrophobic groups present on the
surface, and binding capacity with the stationary phase.
• This leads to different types of chromatography techniques, each with their own
instrumentation and working principle.
 • For instance, four separation techniques based on molecular characteristics and interaction
type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion.
• Other chromatography techniques are based on the stationary bed, including column, thin
layer, and paper chromatography.

What is the Beer-Lambert Law?

Beer’s law states the following:
For a given material, the sample path length and concentration of the sample are directly
proportional to the absorbance of the light.

The Beer-Lambert law is expressed as:

A = εLc
where,

• A is the amount of light absorbed for a particular wavelength by the sample

• ε is the molar extinction coefficient
• L is the distance covered by the light through the solution
• c is the concentration of the absorbing species

What is photoelectric colourimeter?

• A colorimeter is a device that is used in Colorimetry. It refers to a device which helps specific
solutions to absorb a particular wavelength of light.
• The colorimeter is usually used to measure the concentration of a known solute in a given
solution with the help of the Beer-Lambert law.
• The colorimeter was invented in the year 1870 by Louis J Duboscq.

Working of Colorimeter
• Step 1: Before starting the experiment, it is important to calibrate the colorimeter. It is done
by using the standard solutions of the known solute concentration that has to be
determined. Fill the standard solutions in the cuvettes and place it in the cuvette holder of
colorimeter.
• Step 2: A light ray of a certain wavelength, which is specific for the assay is in the direction of
the solution. The light passes through a series of different lenses and filters. The coloured
light navigates with the help of lenses, and the filter helps to split a beam of light into
different wavelengths allowing only the required wavelength to pass through it and reach
the cuvette of the standard test solution.
• Step 3: When the beam of light reaches’ cuvette, it is transmitted, reflected, and absorbed
by the solution. The transmitted ray falls on the photodetector system where it measures
the intensity of transmitted light. It converts it into the electrical signals and sends it to the
galvanometer.
• Step 4: The electrical signals measured by the galvanometer are displayed in the digital form.
• Step 5: Formula to determine substance concentration in test solution.
What is spectrometer?

• A spectrometer is a device for measuring wavelengths of light over a wide range of the
electromagnetic spectrum.

• It is widely used for spectroscopic analysis of sample materials.

• The incident light from the light source can be transmitted, absorbed or reflected through
the sample.

• The changes occurred during the interaction of incident light with the sample reveals the
sample characteristics.

• Two types of radiation sources are generally employed in spectrometer – continuous and
line sources. Continuous sources are heated solid substances or lamps that emit light over a
wide wavelength range, and line sources are specialized lamps and lasers.

• Incident light can be adjusted to the wavelength of interest with the help of dispersive or
non-dispersive elements.