BIOCHEMISTRY:The branchof science concerned with the chemical and physico-chemical processes andsubstances which take place within living organisms.BIOCHEMICAL TECHNIQUES:Biochemical analysis techniques referto a set of methods, assay,and procedures that enable scientists to study the substances found in livingorganisms and the chemical reactions essential life processes. The most complicated ofthese techniques are reserved for specialty research and diagnostic laboratories,while simplified sets of these techniques are used in such common procedures astesting for banned drug abuse in competitive athletic events and monitor ofblood sugar by diabetic patients.
LIST OF BIOCHEMICAL TECHNIQUES:Biochemicallab methods, systematic and analysis methods.· Spectroscopicmethods· Electrophoretictechniques· Chromatography· Calorimeter· Photometery· Nuclearmagnetic resonance· Centrifugation· ELISA· DNAcloning and sequencing· useof radioisotopes· immunoassaymethods 1.CHROMATOGRAPHY:Chromatography is one of the mosthelpful and accepted tools of biochemistry.
It is an analytical techniquedealing with the separation of closely associated compounds from a mixture.These consist of proteins, peptides, amino acids, lipids, carbohydrates,vitamins and drugs.Principles and classification:Chromatography usually consists of a mobile phase and a stationary phase. · MOBILE PHASE:The mobile phase refers to the combinationof substances (to be separated), dissolve in a liquid or a gas. · STATIONARY PHASE:The stationary stage is a poroussolid matrix through which the sample contained in the mobile phase percolates.
The contact between the mobileand stationary phases results in the separation of the compounds from themixture. These interactions consist of the physicochemical principles forexample adsorption, partition, ion-exchange, molecular sieving and affinity.The interaction betweenstationary phase and mobile phase is frequently employed in the classificationchromatography e.g. partition, adsorption, ion- exchange.
More, the categorizationof chromatography is also base either on the nature of the stationary phase(paper, thin layer, column), or on the nature of both mobile and stationaryphases (gas-liquid chromatography).2.ELECTROPHORESIS:The movement of charged particles(ions) in an electric field resulting in their migration towards the oppositelycharged electrode is called as electrophoresis. Molecules with a net positivecharge (cations) move towards the negative cathode whereas those with netnegative charge (anions) transfer towards positive anode. Electrophoresis is awidely used analytical method for the separation of biological molecules forexample plasma proteins, lipoproteins and immunoglobulin’s.
TYPES OF ELECTROPHORESIS:· Zone of electrophoresi· Immunoelectrophoresis:· Isoelectric focusing:3.PHOTOMETERY:Photometry broadly deals with the learn of the phenomenon oflight absorption by particles in solution. The specificity of a compound to takeup light at a particular wavelength (monochromatic light) is exploited in thelaboratory for quantitative measurements.COLORIMETER:Colorimeter (or photoelectriccolorimeter) is the tool used for the measurement of coloured substances. This apparatusis operative in the observable range (400-800 nm) of the electromagneticspectrum of light.
The functioning of colorimeter is based on the principle ofBeer-Lambert law.The colorimeter, in generalcontain light source, filter sample holder and detector with display (meter ordigital). A string lamp usually serves as a light source. The filters allow thepassage of a minute range of wave length as incident light.4.SPECTROPHOTOMETERY:The spectrophotometer primarily differfrom colorimeter by covering the ultraviolet region (200- 400 nm) of theelectromagnetic spectrum. additional the spectrophotometer is more complicatedwith numerous additional devices that eventually raise the sensitivity of itsoperation several fold when compare to a colorimeter.
A precisely selected wavelength ( 234 nm or 610 nm) in bothultra violet and visible range can be use for measurements. In place of glasscuvettes (in colorimeter), quartz cells are used in a spectrophotometer. Thespectrophotometer has similar basic parts describe for a colorimeter 5.ULTRACENTRIFUGATION:Ultracentrifugation is anindispensable instrument for the isolation of subcellular organelles, proteinsand nucleic acids. as well, this technique is also in use for the purpose ofmolecular weights of macromolecules.
The rate at which the sedimentation occurin ultracentrifugation primarily based on the mass and shape of the particlesor macromolecules (i.e. on the molecular weight). It is expressed in terms ofsedimentation coefficients).6.
CENTRIFUGATION:Centrifugation is the use of the centrifugal forces generatedin a spinning rotor to divide biological particles,it includes cells, viruses,sub?cellular organelles, macromolecules (principally proteins and nucleicacids) and macromolecular complexes (such as ribonucleoproteins and lipoproteins).The three mainprocedures of separation are differential pelleting, rate?zonalcentrifugation and isopycnic centrifugation. The first two methods separateparticles primarily on the basis of volume while isopycnic centrifugationseparates particle on the basis of their density. The choice of centrifugation techniquebased on the nature of the particles and often above one separation techniqueis compulsory for example, membrane fractionation often involves first makingan enriched fraction from a cell homogenate by differentialpelleting followed by isopycnic 7.NUCLEAR MAGNETIC RESONANCE:Nuclear Magnetic Resonance(NMR) spectroscopy is an analytical chemistry method used in quality control and research for determining thecontent and purity of a test withits molecular structure. centrifugation to attain purified fractions.8.
MASS SPECTROMETRY:Massspectrometry is a useful analytical technique used to quantify known materials,to identify unidentified compounds in a sample, and to elucidate the structureand chemical properties of dissimilar molecules. The whole process includes thechange of the sample into gaseous ions, with or not including fragmentation,which are then characterize by their mass to charge ratios (m/z) and relativeabundances.This method mainlystudies the effect of ionizing energy on molecules. It based upon chemicalreactions in the gas phase wherein sample molecules are consumed during theformation of ionic and neutral species.
PRINCIPLE:The initial step in the mass spectrometric analysis of compounds is the manufacturingof gas phase ions of the compound, mostly by electron ionization. Thismolecular ion undergoes fragmentation. Each primary product ion derived fromthe molecular ion, in turn, undergoes fragmentation, and so on. The ions areseparated in the mass spectrometer in accordance with their mass-to-chargeratio, and are detected in proportion to their great quantity A mass spectrumof the molecule is thus formed. It displays the result in the form of a plot ofion abundance versus mass-to-charge ratio. Ions give information concerning thenature and the structure of their precursor molecule. In the spectrum of a purecompound, the molecular ion, if present, appears at the highest value of m/z(followed by ions containing heavier isotopes) and gives the molecular mass ofthe compound.
9.ELISA:ELISA is dependon the immunochemical principles of antigen-antibody effect 1.Theantibody in opposition to the protein to be determined is set on an inert solidfor example polystyrene.2. The biological sample containthe protein to be estimated is useful on the antibody coated surface.3. The protein antibody complexis then reacted with a second protein specific antibody to which an enzyme iscovalently related.
These enzymes must be easily assayable and make preferablycoloured products. Peroxidase, amylase and alkaline phosphatase are normallyused.4. Later than washing the unboundantibody linked enzyme, the enzyme bound to the second antibody complex isassayed.5. The enzyme activity isidentifies by its action on a substrate to form a product (usually coloured).This is related to the concentration of theprotein being estimated.APPLICATIONS:ELISA is widely used for thedetermination of small quantities of proteins (hormones, antigens, antibodies)and other biological substances.
The most commonly used pregnancy test for thedetection of human chorionic gonadotropin (hCG) in urine is based on ELISA. Bythis test, pregnancy can be detected within few days after conception. ELISA isalso useful for the diagnosis of AIDS.