A simple microscope is an optical instrument that uses a single biconvex lens of short focal length to produce an enlarged, erect, and virtual image of a small object. It is essentially a magnifying glass.
Magnification (M) = 1 + (D/f)
Where D is the least distance of distinct vision (25 cm) and f is the focal length of the lens.
A pH meter is an electronic device used to measure the hydrogen-ion activity (acidity or alkalinity) in a solution. It consists of a voltmeter attached to a pH-responsive electrode (usually glass) and a reference electrode. It provides more precise readings than pH indicator papers.
Electron Microscope: An instrument that uses a beam of accelerated electrons as a source of illumination to visualize objects at a much higher resolution than light microscopes.
Scanning Electron Microscope (SEM) Principle:
The SEM works by scanning a focused high-energy electron beam over the surface of a specimen. When the beam hits the sample, it interacts with the atoms, producing signals (secondary electrons) that contain information about the surface topography and composition.
Instrumentation of SEM:
Difference between Light and Electron Microscopy:
| Feature | Light Microscopy | Electron Microscopy |
|---|---|---|
| Illumination Source | Visible Light | Electron Beam |
| Lenses | Glass Lenses | Electromagnetic Lenses |
| Magnification | Up to 1,500x - 2,000x | Up to 1,000,000x |
| Resolution | ~200 nm | ~0.1 - 0.5 nm |
(i) Fluorescence microscopy:
This technique uses fluorescence instead of, or in addition to, scattering and reflection to study properties of organic or inorganic substances. The specimen is illuminated with light of a specific wavelength (excitation) which is absorbed by the fluorophores, causing them to emit light of longer wavelengths.
(ii) TEM (Transmission Electron Microscope):
In TEM, a beam of electrons is transmitted through an ultra-thin specimen. The electrons interact with the specimen as they pass through, and an image is formed from the interaction.
Atomic Absorption Spectroscopy (AAS) is an analytical technique used to determine the concentration of a specific metal element in a sample. It measures the amount of light absorbed by ground-state atoms in the gaseous state.
The Beer-Lambert Law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species and the path length of the light beam.
A = εcl
Where A = Absorbance, ε = Molar absorptivity, c = Concentration, and l = Path length.
An IR Spectrophotometer measures the absorption of infrared radiation by a sample. It is primarily used to identify functional groups in organic molecules, as different chemical bonds vibrate at specific frequencies within the IR spectrum.
Principle:
The principle is based on the absorption of Ultraviolet light (200-400 nm) by chemical substances, which results in the electronic excitation of electrons from ground state to an excited state. It follows the Beer-Lambert Law.
Working:
Applications:
(i) Atomic emission spectroscopy (AES):
AES uses quantitative measurement of the optical emission from excited atoms to determine analyte concentration. Atoms in the sample are excited (using heat, flame, or plasma) to higher energy levels; when they return to lower states, they emit light at characteristic wavelengths.
(ii) Colorimeter:
A colorimeter is a light-sensitive instrument used to measure the absorbance and transmittance of light passing through a liquid sample. It works in the visible spectrum (400-700 nm) and is used to determine the concentration of colored solutes in a solution.
The sedimentation coefficient (s) is the ratio of the sedimentation velocity of a particle to the applied acceleration due to centrifugal force. It is expressed in Svedberg units (S), where 1 S = 10⁻¹³ seconds.
The isolation of organelles is typically achieved through Differential Centrifugation. The process involves several steps:
(i) Types of rotors:
(ii) Density gradient centrifugation:
A technique where particles are separated based on their buoyant density or sedimentation rate as they move through a medium of increasing density (e.g., Sucrose or CsCl gradient). It includes rate-zonal and isopycnic (equilibrium) centrifugation.
The principle is partition chromatography, where substances are distributed between two phases: a stationary phase (water molecules trapped in cellulose paper) and a mobile phase (solvent). Components move at different rates based on their partition coefficients.
Also known as Size-Exclusion Chromatography, it separates molecules based on their size. Large molecules bypass the pores in the gel beads and elute first, while smaller molecules enter the pores and take longer to elute.
It is based on highly specific biological interactions between an analyte and a ligand (e.g., enzyme-substrate, antigen-antibody, or hormone-receptor) immobilized on a stationary phase.
TLC Principle: Based on adsorption chromatography. The stationary phase (silica gel or alumina) is coated on a glass/plastic plate. The mobile phase travels up the plate by capillary action, carrying the components of the mixture at different speeds based on their affinity for the stationary phase.
TLC Procedure:
Gas Chromatography (GC):
GC is used to separate volatile compounds. The mobile phase is a carrier gas (He or N₂), and the stationary phase is a microscopic layer of liquid or polymer on an inert solid support inside a column. Separation occurs based on the boiling point and vapor pressure of the analytes.
(i) Ion-exchange chromatography:
Separates molecules based on their net charge. It uses a resin (stationary phase) with charged groups. Anion exchangers have positive groups and bind negatively charged molecules, while Cation exchangers have negative groups and bind positively charged molecules.
(ii) HPLC (High-Performance Liquid Chromatography):
An advanced form of column chromatography that uses high pressure to push the solvent through a column packed with very small particles. This results in much faster separation and higher resolution compared to traditional chromatography. It includes a pump, injector, column, and detector.
Electrophoresis is the movement of dispersed charged particles relative to a fluid under the influence of a spatially uniform electric field. It is widely used to separate DNA, RNA, or protein molecules based on size and charge.
Isoelectric Focusing (IEF) is a technique used to separate proteins based on their isoelectric point (pI). Proteins migrate through a pH gradient in an electric field until they reach a position where their net charge is zero (pH = pI).
PFGE is a technique used to separate extremely large DNA molecules (e.g., whole chromosomes) by periodically changing the direction of the electric field. Standard gel electrophoresis cannot resolve DNA larger than ~50 kb, but PFGE can.
SDS-PAGE Principle: Proteins are coated with Sodium Dodecyl Sulfate (SDS), an anionic detergent that denatures them and imparts a uniform negative charge. This ensures that proteins are separated strictly based on their molecular weight rather than their native charge or shape.
Procedure:
Immunoelectrophoresis:
A method that combines electrophoresis with immunodiffusion. Proteins are first separated by electrophoresis, then antibodies are allowed to diffuse toward the separated proteins, forming visible precipitin arcs where they react. It is used to analyze complex protein mixtures like serum.
(i) Agarose gel electrophoresis:
A standard method used to separate nucleic acids (DNA/RNA). Agarose is a polysaccharide that forms a porous matrix. Large DNA molecules move slower through the pores than smaller ones. It is typically run horizontally and visualized using Ethidium Bromide under UV light.
(ii) Native PAGE:
Polyacrylamide gel electrophoresis conducted without denaturing agents (like SDS). Proteins remain in their folded, active state. Separation is based on a combination of the protein's size, shape, and intrinsic net charge. It is useful for studying protein-protein interactions or enzyme activity.