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Semester 1: Biochemical Techniques
pH scale, buffer solution, pH electrode, Clarkes oxygen electrode and applications
Biochemical Techniques
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The pH scale measures the acidity or alkalinity of a solution, ranging from 0 to 14, where 7 is neutral.
Vital for biochemical reactions, as most biological processes occur within a narrow pH range.
Used in various fields including medicine, agriculture, and environmental science.
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A buffer solution is a solution that resists changes in pH upon the addition of small amounts of acid or base.
Typically composed of a weak acid and its conjugate base or a weak base and its conjugate acid.
Buffers are crucial in maintaining consistent pH levels in biological systems, influencing enzyme activity and stability.
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A pH electrode is a sensor used to determine the pH of a solution, commonly consisting of a glass membrane.
The glass membrane generates a voltage in response to the hydrogen ion concentration, which is converted to pH.
Widely used in laboratories, food industry, and environmental monitoring for accurate pH measurement.
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The Clarkes oxygen electrode is an electrochemical device used to measure the partial pressure of oxygen in a sample.
It consists of a cathode and an anode, with a membrane that allows oxygen diffusion.
Utilized in clinical settings for measuring blood oxygen levels and in various research applications related to respiration and metabolic studies.
Microscopy: principles and applications of light, phase contrast, fluorescence, scanning and transmission electron microscopy, specimen preparation for TEM and SEM, organ and tissue slice technique, cell disruption and homogenization, microtomy, staining and fixation
Microscopy: principles and applications
Principles of Light Microscopy
Light microscopy uses visible light to magnify specimens. It involves the use of lenses to focus light and create images of small samples at magnifications up to 1000x. The resolution is limited by the wavelength of light, typically around 200 nanometers.
Phase Contrast Microscopy
Phase contrast microscopy enhances contrast in transparent specimens without the need for staining. It converts phase shifts of light passing through the specimen into brightness changes, making cellular structures more visible.
Fluorescence Microscopy
Fluorescence microscopy uses fluorescent dyes to label specific cellular components. Excitation light causes the dyes to emit light at a longer wavelength, allowing visualization of structures with high specificity and sensitivity.
Scanning Electron Microscopy (SEM)
SEM provides three-dimensional images of surfaces by scanning a focused electron beam across the specimen. It offers high-resolution surface images and can analyze the composition using energy-dispersive X-ray spectroscopy.
Transmission Electron Microscopy (TEM)
TEM involves transmitting electrons through a thin specimen to produce high-resolution images of internal structures. It offers resolutions at the atomic level, making it essential for materials science and biology.
Specimen Preparation for TEM and SEM
Specimen preparation for TEM requires ultrathin sections, while SEM specimens are typically coated with a conductive layer. Proper preparation is crucial for achieving high-resolution images.
Organ and Tissue Slice Technique
This technique involves slicing organs or tissues into thin sections for microscopy analysis. It allows for the observation of cellular architecture and organization.
Cell Disruption and Homogenization
Cell disruption methods (e.g., sonication, mechanical disruption) are used to release intracellular contents. Homogenization further breaks down the cell mixture for analysis.
Microtomy
Microtomy is the process of slicing samples into ultra-thin sections using a microtome. These sections are essential for light and electron microscopy.
Staining and Fixation
Staining enhances the contrast of specimens under a microscope. Fixation preserves cellular structures. Common fixatives include formaldehyde and paraformaldehyde.
Cell sorting and counting, cryopreservation, manometric techniques
Cell sorting and counting, cryopreservation, manometric techniques
Cell Sorting and Counting
Cell sorting is a technique used to separate specific cell populations from a heterogeneous mixture. Methods include fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). Counting cells can be performed using hemocytometers or automated cell counters, which facilitate the determination of cell concentration and viability.
Cryopreservation
Cryopreservation involves the preservation of cells, tissues, or organs at very low temperatures to halt all metabolic and chemical processes. Key factors include the use of cryoprotectants, such as dimethyl sulfoxide (DMSO), to prevent ice crystal formation. The process is crucial for preserving stem cells, sperm, and embryos for medical and research purposes.
Manometric Techniques
Manometric techniques measure the pressure of gases produced during biochemical reactions, providing insight into metabolic rates. Commonly employed in studies of cellular respiration and fermentation, methods such as the Warburg manometer allow for quantitative analysis of the rates of gas production or consumption under varying conditions.
Chromatography: principles, instrumentation, applications of paper chromatography, exclusion chromatography, column chromatography, chromatofocusing, affinity chromatography, adsorption chromatography, ion exchange chromatography, liquid chromatography including GLC, LC, LPLC and HPLC
Biochemical Techniques
M.Sc. Medical Biochemistry
Biochemical Techniques
I
Tamil Nadu State Council for Higher Education
Core II
Chromatography
Principles of Chromatography
Chromatography is a separation technique based on the differential distribution of compounds between a stationary phase and a mobile phase. The compounds interact with the phases, leading to their separation.
Instrumentation
Instrumentation in chromatography includes various components such as the mobile phase delivery system, injection system, separation column, detector, and data processing system.
Paper Chromatography
Paper chromatography uses a strip of absorbent paper as the stationary phase. This method is commonly used for separating pigments and small molecules in biological samples.
Exclusion Chromatography
Exclusion chromatography, or size-exclusion chromatography, separates molecules based on their size. Larger molecules pass through the column more quickly, while smaller molecules are delayed.
Column Chromatography
Column chromatography involves the use of a column filled with stationary phase materials. It is widely used for the purification of liquids and solids.
Chromatofocusing
Chromatofocusing is a technique that separates proteins based on their isoelectric points. It employs a pH gradient and is used in protein purification.
Affinity Chromatography
Affinity chromatography separates compounds based on specific binding interactions. It is commonly used to purify biomolecules, such as antibodies and proteins.
Adsorption Chromatography
Adsorption chromatography involves the adhesion of molecules to the surface of the stationary phase. This method is used for both qualitative and quantitative analysis.
Ion Exchange Chromatography
Ion exchange chromatography separates ions and polar molecules based on their charge. It is extensively used for protein purification and analysis.
Liquid Chromatography
Liquid chromatography encompasses several techniques, including Gas-Liquid Chromatography (GLC), Liquid Chromatography (LC), Low-Pressure Liquid Chromatography (LPLC), and High-Performance Liquid Chromatography (HPLC). Each variant has specific applications and advantages.
Electrophoresis: principles, instrumentation, applications of paper electrophoresis, agar gel, starch gel, PAGE, capillary electrophoresis, PFGE, high and low voltage electrophoresis, isoelectric focusing, tachophoresis
Electrophoresis
Principles of Electrophoresis
Electrophoresis is a technique used to separate charged particles in a fluid under the influence of an electric field. The movement of particles is dependent on their size and charge. Anionic particles migrate towards the anode while cationic particles move towards the cathode. The process is driven by the electric field strength and the properties of the medium.
Instrumentation of Electrophoresis
Electrophoresis setups generally consist of power supplies, electrophoresis chambers, and supporting media. The power supply provides a constant voltage or current, while the chamber holds the medium and samples. Supporting media can include agarose gel, polyacrylamide gel, or paper, depending on the separation required.
Applications of Electrophoresis
Electrophoresis has various applications including protein analysis, nucleic acid separation, and the purification of biomolecules. Common uses include DNA fingerprinting, protein quantification, and studying mutations in genetic material.
Paper Electrophoresis
Paper electrophoresis involves the use of paper as a medium for separation. Samples are applied to the paper, and an electric field is applied to facilitate migration. This method is often used for the separation of proteins and amino acids.
Agar Gel Electrophoresis
Agar gel electrophoresis is commonly used for the separation of nucleic acids. Agarose, derived from seaweed, creates a porous gel matrix that allows for the resolution of DNA and RNA fragments based on size. It is widely used in molecular biology for analyzing PCR products and restriction digests.
Starch Gel Electrophoresis
Starch gel electrophoresis utilizes starch as a medium for the separation of proteins. This method is particularly useful for biomolecular research involving enzyme activity studies and genetic polymorphism analysis.
Polyacrylamide Gel Electrophoresis (PAGE)
PAGE is a versatile technique for the separation of proteins based on their size. It involves the use of polyacrylamide gels and can be performed under denaturing or non-denaturing conditions. SDS-PAGE, for instance, is used to analyze the molecular weight of proteins.
Capillary Electrophoresis
Capillary electrophoresis is a high-resolution technique that utilizes thin capillary tubes for separation. It offers rapid analysis and requires minimal sample volume. This method is particularly effective for the analysis of small molecules, nucleotides, and peptides.
Pulsed Field Gel Electrophoresis (PFGE)
PFGE is a specialized form of gel electrophoresis that allows for the separation of large DNA fragments. It employs alternating electric fields to reorient DNA, making it possible to analyze larger sizes than standard agarose gel electrophoresis.
High and Low Voltage Electrophoresis
High voltage electrophoresis can significantly increase the speed of separation but may cause heating and should be closely monitored. Low voltage electrophoresis allows for greater resolution of molecules but takes more time.
Isoelectric Focusing
Isoelectric focusing is a technique used to separate proteins based on their isoelectric point. Proteins migrate in a pH gradient and stop at their isoelectric point where the net charge is zero.
Tachophoresis
Tachophoresis involves rapid movement of particles in a medium due to applied electric fields, used for the separation of charged species at high speeds. This method can enhance the efficiency of separative techniques.
Centrifugation: principles, laws of sedimentation, preparative and analytical centrifugation, differential and density gradient centrifugation, analytical ultracentrifuges and sedimentation equilibrium methods, purity criteria of macromolecules
Centrifugation
Principles of Centrifugation
Centrifugation relies on the principle of sedimentation, where particles in a liquid are separated based on their density when subjected to centrifugal force. The force is generated by the rotation of a centrifuge, resulting in the sedimentation of heavier particles toward the bottom of the tube. Smaller and lighter particles remain in the supernatant.
Laws of Sedimentation
The laws governing sedimentation include Stokes' law, which describes the settling velocity of spherical particles in a viscous medium. The equation involves parameters such as particle size, density, and the viscosity of the medium. The sedimentation rate is crucial for determining the separation efficiency in centrifugation.
Preparative Centrifugation
Preparative centrifugation is utilized for the isolation and purification of biological macromolecules such as proteins, nucleic acids, and organelles. The process involves choosing appropriate rotor types and centrifugal speeds based on the sample's characteristics. It aims to provide high yield and purity of the target biomolecule.
Analytical Centrifugation
Analytical centrifugation focuses on measuring the physical properties of macromolecules in solution. It aids in determining molecular weight, size distribution, and sedimentation coefficients. This technique provides valuable data for characterizing biological substances and is vital for understanding their behavior in diverse conditions.
Differential Centrifugation
Differential centrifugation involves successive spins at increasing speeds to separate cellular components based on size and density. Initially, large organelles and debris are pelleted, followed by smaller organelles at higher speeds. This method is extensively used for cell fractionation.
Density Gradient Centrifugation
Density gradient centrifugation separates particles based on their buoyancy in a gradient medium. A density gradient is created using sucrose or cesium chloride, and particles migrate until they reach their buoyant density. This technique is valuable for purifying viruses, organelles, and DNA.
Analytical Ultracentrifugation
Analytical ultracentrifugation involves high-speed centrifugation to analyze macromolecules. It provides sedimentation velocity and sedimentation equilibrium data, allowing for the determination of molecular weights and interactions among molecules. This highly precise technique assists in understanding intricate biomolecular behaviors.
Sedimentation Equilibrium Methods
Sedimentation equilibrium is determined when the centrifugal and buoyant forces are balanced. This method allows for the study of macromolecular interactions and provides important information on molecular size and shape through the analysis of concentration profiles.
Purity Criteria of Macromolecules
Purity criteria for macromolecules in the context of centrifugation involve assessing yield, structural integrity, and functional activity post-isolation. Methods such as gel electrophoresis, mass spectrometry, and enzymatic assays are employed to ensure desired purity levels are achieved during the separation processes.
Spectroscopy: basic laws of light absorption, optical rotatory dispersion, instrumentation and applications including UV and visible spectrophotometry, spectrofluorimetry, atomic flame photometry, plasma emission spectroscopy, infrared spectrophotometry, mass spectrometry, tandem mass spectrometry, ESR, NMR
Spectroscopy
Basic Laws of Light Absorption
Light absorption follows Beer-Lambert Law, which states that absorbance is directly proportional to the concentration of the absorbing species and the path length of the light through the sample. Key parameters include molar absorptivity, concentration, and path length. This law is fundamental in quantitative analysis.
Optical Rotatory Dispersion
Optical rotatory dispersion (ORD) refers to the rotation of polarized light as it passes through an optically active substance. The amount of rotation is related to the compound's concentration and the wavelength of light. ORD is useful in determining the specific rotation of chiral molecules.
Instrumentation
Modern spectroscopy employs various instrumentation techniques including spectrophotometers, monochromators, and detectors. Key components include light sources, optical components like lenses and mirrors, and detectors such as photomultiplier tubes and CCDs, which translate light into measurable signals.
UV and Visible Spectrophotometry
UV and visible spectrophotometry utilize the absorption of ultraviolet or visible light by a sample to determine concentration. Applications include analyzing nucleic acids, proteins, and other biomolecules. The concentration is calculated based on absorbance measurements at specific wavelengths.
Spectrofluorimetry
Spectrofluorimetry measures the fluorescence emitted by a sample after it absorbs light. It is highly sensitive and able to detect low concentrations of analytes. Applications include biochemical assays and environmental monitoring.
Atomic Flame Photometry
Atomic flame photometry measures the intensity of light emitted by atoms in the gas phase. It is used for detecting and quantifying metal ions in samples. The technique involves nebulizing the sample and introducing it into a flame.
Plasma Emission Spectroscopy
Plasma emission spectroscopy, including inductively coupled plasma (ICP) techniques, detects light emitted from excited atoms in a plasma state. It is widely used for multi-element analysis of metals in various samples.
Infrared Spectrophotometry
Infrared spectrophotometry analyzes molecular vibrations through absorption of infrared light. It is employed in qualitative and quantitative analysis of organic compounds, providing information about functional groups.
Mass Spectrometry
Mass spectrometry identifies compounds based on their mass-to-charge ratio. It involves ionization, fragmentation, and detection of ions. Applications span pharmaceuticals, proteomics, and metabolomics.
Tandem Mass Spectrometry
Tandem mass spectrometry (MS/MS) combines two mass spectrometry steps to enhance sensitivity and specificity. It is particularly valuable for complex mixtures, enabling detailed structural analysis of molecules.
Electron Spin Resonance (ESR)
Electron spin resonance (ESR) detects unpaired electrons in a magnetic field, providing information on radical species. It has applications in chemistry, biology, and materials science.
Nuclear Magnetic Resonance (NMR)
Nuclear magnetic resonance (NMR) exploits the magnetic properties of atomic nuclei. It provides detailed structural information on organic compounds and is used in metabolomics and drug development.
Tracer techniques: radioactive isotopes, half-life of isotopes, principles, applications in biology and medical sciences, measurement of alpha, beta, gamma radiations, radiation dosimeter, autoradiography, Geiger Muller counter, liquid scintillation counter
Tracer techniques
Radioactive isotopes
Radioactive isotopes are unstable atoms that release energy in the form of radiation as they decay. They are used as tracers in various scientific fields due to their identifiable signatures and ability to interact with biological systems.
Half-life of isotopes
Half-life is the time required for half of the radioactive isotopes in a sample to decay. This property is crucial for determining the timing and dosage of tracers in experiments and medical treatments.
Principles of tracer techniques
Tracer techniques utilize radioactive isotopes to track biological processes. The principles involve the detection of radiation emitted from the isotope, allowing scientists to visualize and quantitate biological pathways.
Applications in biology and medical sciences
Tracer techniques are widely used in biology and medical sciences. They help in studying metabolic pathways, diagnosing diseases, monitoring treatment responses, and conducting research in genetics and microbiology.
Measurement of alpha, beta, gamma radiations
Measurement is done through different detectors. Alpha particles are detected using specialized detectors, beta particles can be measured with Geiger counters, and gamma radiation requires more sophisticated systems like scintillation counters.
Radiation dosimeter
A radiation dosimeter is a device used to measure exposure to ionizing radiation. It provides information regarding the dose received by an individual or entity, ensuring safety in environments where radioactive isotopes are used.
Autoradiography
Autoradiography is a technique that uses photographic films or imaging plates to visualize the distribution of radioactive isotopes in a sample. It is valuable for studying biological tissues and cellular processes.
Geiger Muller counter
The Geiger Muller counter is a device used for detecting and measuring ionizing radiation. It provides real-time monitoring of radiation levels and is commonly used in both research and clinical settings.
Liquid scintillation counter
A liquid scintillation counter measures the intensity of light emitted from a scintillation solution as radioactive isotopes decay. This method is sensitive and allows for quantification of low levels of radioactivity.
