BIOCHEMICAL TECHNIQUES “Biochemical analysis techniques aredefined as the methods, assays, and procedures that allow scientists to revealthe substances present in living organisms and the chemical reactionsunderlying life processes.” It is arapidly developing field of science and is a crucial part of modern drugdiscovery and research. This field emphasis on the chemical composition ofliving systems utilizing certain experimental techniques and computationalsimulations.To carry outbiochemical analysis of a biomolecule in a biological system, it is essentialto design an approach to detect the biomolecule and extract it in pure form.For the purification of a biomolecule various techniques such ascentrifugation, electrophoresis, precipitation, chromatography etc. are used. BIOCHEMICALTECHNIQUESØ Centrifugation Ø MicroscopyØ SpectrophotometryØ Chromatography Ø ElectrophoresisØ Flame PhotometryØ FluorometryØ Spectroscopy Ø ELISA CENTRIFUGATIONCentrifugation isa technique employed for the separation of particles from a solution accordingto their size, shape, density, viscosity of the medium and rotor speed.CENTRIFUGEA centrifugeis a device that separates particles from a solution by using a rotor.
Inbiology, the particles are usually cells, subcellular organelles, or large molecules.WORKINGThe particles suspended in a liquid mediumare put in a centrifuge tubeand the tube is then settled in a rotorand spun at a specified speed. The centrifuge grips the top of the tubes andthe bottom is free to angle out. As it spins, a centrifugal force is exerted toevery particle in the sample. The particles will sediment at the speedproportional to the centrifugal force exerted on it. The viscosity of thesample solution and the physical properties of the particles also influence thesedimentation rate of each particle.Thus, bycentrifugation red cells from plasma of blood, nuclei from mitochondria in cellhomogenates, and one protein from another in complex mixtures can be separatedTYPES OF CENTRIFUGESMicrocentrifuges Small Benchtop Centrifuges General Purpose Centrifuges Large Capacity Centrifuges Superspeed CentrifugesUltracentrifuges SPECTROPHOTOMETRYSpectrophotometryis a technique used to measure the amount of light absorbed by a chemicalsubstance by estimating the intensity of light as a beam of lightpasses through sample solution. PRINCIPLEThebasic principle is that each compound absorbs or transmits light over a certainrange of wavelength.
The spectrophotometer measures that amount if lightabsorbed in order to estimate the concentration of an unknown solution.INSTRUMENTATIONAspectrophotometer is an instrument that estimates the intensity of lightabsorbed as it transmits a sample solution. It consists of two devices, aspectrometer and a photometer. · Spectrometer: Aspectrometer is a device that produces, typically scatters and estimateslight. It produces a desired range of wavelength of light.
First acollimator emits a straight beam of light that transmits through amonochromator to disperse it into several component wavelengths referred to asspectrum. Then a wavelength slit passes only the required wavelengths.· Photometer: A photometer is thephotoelectric detector that measures the amount of light. Afterthe desired range of wavelength of light transmits through the samplesolution in cuvette, the photometer detects the intensity of light that isabsorbed and then conveys a signal to a galvanometer or a digital displayprovided.TYPESOF SPECTROPHOTOMETERAspectrophotometer can be classified into two different types depending on therange of wavelength of light sourceUV-visible spectrophotometer: It useslight over the ultraviolet range (185 – 400 nm) and visible range (400 -700 nm) of electromagnetic radiation spectrum.IR spectrophotometer: It uses light over the infrared range (700 – 15000 nm) ofelectromagnetic radiation spectrum. CHROMATOGRAPHYChromatography is a technique that utilizes a groupof methods for separating the componentsof very small quantities present in complex mixtures on the basis of the relativeamounts of each component distributed between a moving fluid streams, calledthe mobile phase, and a contiguous stationary phase.
The mobile phase may beeither a liquid or a gas, while the stationary phase is either a solid or aliquid.HISTORYThe chromatography technique wasintroduced early in the twentieth century when a Russian botanist MikhailTswett used a column packed with calcium carbonate to separate plant pigments.It is one of the most important techniques in environmental analysis.PrincipleThe principle of chromatographyis based on the fact that a substance that is in contact with two immisciblephases, moving and stationary, will equilibrate among them. A reproducible partwill separate into each phase, according to the relative affinity of thesubstance for each phase. A substance which has affinity for the moving or mobile phase will be moved rapidlythrough the system. A material which has a stronger affinity for the stationary phase will spend more timeimmobilized in that phase, and will require longer time to pass through the system.TYPESOF CHROMATOGRAPHYPaper chromatographyThin layer chromatography (TLC)Liquid column chromatographySize exclusion chromatographyIon-exchange chromatographyAffinity chromatographyGas chromatography ElectrophoresisElectrophoresis is a chromatography technique by which a mixture of chargedmolecules is differentiated on the basis of size when placed in an electricfield.
This technique is frequently performed in the lab to differentiatecharged molecules, such as DNA, on the basis of their size.PRINCIPLEDuringelectrophoresis charged molecules move through a gelas an electric current is applied across the gel so that one end of the gel isa positively charged while the other end is a negatively charged. The movementof charged molecules is referred to as migration. Molecules migrate towards theopposite charge.
A molecule with a negative charge will move towards the positiveend and the molecule with positive charge will move towards the negative end.Smaller molecules move through the gel more rapidly and therefore move fartherthan larger fragments that move more slowly and therefore travel a shorterdistance. Eventually the molecules are differentiated according to size. WORKING To perform electrophoresis DNA samples are loaded into wellsat one end of a gel and an electric current is applied to pull them through thegel. As DNA fragments are negatively charged they move towards the positive electrode.When a gel is stained with a DNA-binding dye, the DNA fragments can be visualizedas bands, each presenting agroup of same-sized DNA fragments. SPECTROSCOPYSpectroscopy is the study of the interaction ofelectromagnetic radiation in all its forms with matter. It can be morespecifically defined as the study of the interactions between particles such aselectrons, protons, and ions, as well as their interaction with other particles asa function of their collision energy.
PRINCIPLEWhen a beam of white light strikes a triangular prism it isdifferentiated into its various components (ROYGBIV) which is called aspectrum. The optical system which enables production and visualizing of thespectrum is called a spectroscope. There are many other forms of light whichare invisible to the human eye and spectroscopy is extended to cover all these.
APPLICATIONSSpectroscopic analysis has been necessary in the developmentof the most fundamental theories in physics, including quantum mechanics, the specialand general theories of relativity, and quantum electrodynamics. It has been avital tool in developing scientific understanding of the electromagnetic forceand strong and weak nuclear forces. FLAMEPHOTOMETRY FlamePhotometry is a branch of spectroscopy in which the species studied in thespectrometer are in the form of atoms.
FlamePhotometer is used in inorganic chemical analysis to analyze the concentrationof certain metal ions such as sodium, potassium, calcium and lithium. It is basedon measurement of amount of the light emitted when a metal is introduced intoflame. The wavelength of color determines the element qualitatively. The colorintensity determines the element quantitatively. PRINCIPLEThe generalprinciple of Spectroscopy is based on the fact that “Matter absorbs lightat the same wavelength at which it emits light.” Atoms of elements when subjectedto hot flame its orbital electrons absorbed Specific quantum of thermal energy andbecome unstable at high energy level. As the excited electron move back toground state they release energy as photons which is of particular wavelength.
INSTRUMENTATIONA burnerthat supplies flame and can be kept in a constant form and at a constanttemperature. There is a Nebulizer and Mixing Chamber which helps to take thehomogeneous solution of the substance into the flame at a steady rate. Theoptical system consists of three parts: convex mirror, lens and filter. The PhotoDetector detects the emitted light and measure the intensity of radiationemitted by the flame.
Therefore the emitted radiation is converted to an electricalsignal with the aid of photo detector. The given electrical signals aredirectly proportional to the intensity of light. FLUORIMETRYFluorimetry is a spectroscopic analytical techniquefor the detection and measurement of fluorescence in compounds such as cells,proteins, or nucleotides that are labeled with fluorescence agents.PRINCIPLEFluorescenceis a phenomenon of emission of radiation when the molecules are excited byradiation at certain wavelength.
Molecule contains electrons that may bepresent in bonding molecular orbital which is called as highest occupiedmolecular orbital (HOMO).It has least energy and is more stable. When themolecules absorbs radiant energy from a light source, the bonding electrons maybe excited to anti bonding molecular orbital that has more energy and hence isless stable.
As the molecule falls back from the excited state to the groundstate it emits radiation that can the measured by fluorimeter as fluorescence.INSTRUMENTATIONA fluorimeter is a spectrometer that is very similar to thespectrophotometer, and contains a light source and a filter or monochromator toset a specific group of excitation wavelengths, which are then targeted into asample. The light produced from the sample is then transmitted through anotherfilter or monochromator which determines the emission wavelength of interest aswell as eliminating most of the excitation light, prior to the measurement by adetector.Types of FluorimeterThere are two basic types of fluorimeters:· Filter fluorimeter· Spectrofluorimeter ELISAThe Enzyme-Linked Immunosorbent Assay (ELISA) is a techniqueused to observe antibodies or infectious agents in a sample. ELISA testdiagnoses infections such as HIV (human immunodeficiency virus) and some allergicdiseases like food allergies.
TYPES OF ELISAThere are four types or kinds ofELISA tests:Direct ELISADirect ELIZA is the attachment of anantigen to a polystyrene plate proceeded by an enzyme-labeled antibody that canreact with the antigen and a substrate that can be measured.Indirect ELISAIndirect ELIZA is the attachment ofan antigen to a polystyrene plate proceeded by an unlabeled or primary antibodyproceeded by an enzyme-labeled antibody that can react with both the primaryantibody and substrate.· Sandwich ELISA During a sandwich ELIZA a capturedantibody is bound to the polystyrene plate and an antigen is introduced thatspecifically attaches or captures the antigen. A second antibody that is alsospecific for the antigen but not the same as the capture antibody is introducedand “sandwiches” the antigen. This second antibody is then proceeded byan enzyme-labeled antibody specific for the second antibody that can react witha substrate that can be measured.
· Competitive ELISAThis test involves the addition of competing antibodies orproteins as the second antibody is added. This results in a decrease in the substratesignal that is produced. This test gives highly specific results.