Mr. T. P. Nimbekar Bajiraoji Karanjekar College of Pharmacy, SakoliTn methods of extraction » Application of latest techniques like Spectroscopy, chromatography and electrophoresis in the isolation, purification and identification of crude drugsExtraction ¥ It is the method of removing active constituents from a solid or liquid by means of liquid solvent. ¥ It can be defined as the process of isolation of soluble material from am insoluble residue by treatment with solvent. Yt is done for separation of medicinally active rtions of plant or animal tissues from the inactive or _ inert components by using selective solvents. vIn this method the wanted components are dis: solved by the use of selective solvents known as menstrum & undissolved part is a mare. ¥ After the extraction unwanted matter is removed.Extract Mare Preparations Solid Solvent Dichter (IT ote Cg UM mM eT : ; Tay obtain after extraction constituents extraction Auli ees ULC ima ity hota¥ Extraction is the process of ‘efficiently dissolving and separating the desired constituents from the crude drug with the use of solvent/s. It is controlled by mass transfer. ¥ Plant constituents are usually contained inside the ‘ore, The solvent used for extraction must » into the cell to dissolve the desired compounds the solution must pass the cell wall in the opposite direction and mix with the surrounding liquid. An equilibrium is established between the solute inside the cells and the solvent surrounding the fragmented plant tissues. ¥ The choice of solvent depends upon the char: of secondary metabolites like polarity and thermal stability. FIdeal properties of solvent ¥ Be highly selective for the compound to be extracted. Y Not react with the extracted compound or with other compounds in the plant material. ¥ Have a low price. ¥ Be harmless to man and to the environment. ¥ Be completely volatile. ¥ Should not mix up with water. ¥ Should have the big capacity in relation to extractive. ¥ The density of solvent should be difference from water density.Successful determination of biologically active compounds depends on the type of solvent used in the extraction procedure. Choice of solvents Property of a good solvent in plant extraction. Low toxicity Ease of evaporation at low heat Promotion of rapid physiologic absorption of the extract Preservative action Choice of Extraction Methods Sample size |) Quantity of the exctract required Extraction time {| Choice of solvent (|) CostThe factors affecting the choice of solvent «Quantity of phytochemicals to be extracted «Rate of extraction Diversity of different inhibitory compounds extracted «Ease of subsequent handling of the extracts Toxicity of the solvent in the bioassay process «Potential health hazard of the extractants‘} Solvents used for active component extraction Ethanol Tannins Polyphenols Polyacetylenes Flavonols Terpenoids Sterols Alkaloids Methanol ‘Chloroform — Ether Acetone Anthocyanins Terpenoids Alkaloids Phenol Terpencids Flavonoids TerPeneids Fig yonols ‘Coumarins ‘Tannins Fatty acids ‘Xanthoxyllines Totarol Quassinoids Lactones Flavones Phenones Polyphenolscome extraction Supercritical fluid extraction \ ‘hydrofluotocarbon sExtraction techniques For aromatic _ plants Hydrodistillation techniques Headspace trapping (water distillation) Steam distillation Solid phase micro- extraction Water and steam distillation Protoplast extraction Hydrolytic maceration Microdistillation followed by distillation Expression and enfleurage Thermo-microdistillation (cold fat extraction)Steps Involved in the Extraction of Medicinal Plants 1, Size reduction- Maximises surface area, increase mass transfer, 30-40 mesh size is optimum 2. Extraction- Maceration, perccolation, soxhlation, etc 3. Filtration- Through musciline cloth, filter paper, filter press - 4, Concentration- Evaporation of sol oven, vacuum camber drying, rota evaporator § DPDrvine- Snrav drvine. etcMaceration Maceration= soaking ¥ The whole / coarsely powdered crude drug is placed in a stoppered container with the solvent. ¥ Allow to stand at room temperature for a period of at least 3 days with frequent agitation until the soluble matter gets dissolved. ¥The mixture then is strained, the mare (the damp solid material) is pressed. “The combined liquids are clarified by filtration or decantation after standing. “This method is best suitable for use in case of the thermolabile drugs.Modified maceration Is used for unorganised drug like gums and resins » Pressing is avoided » Fresh menstrum is used to wash mare and make the volume. Multiple maceration Used t obatain concentrated extract, total menstrum divided into 2 parts (double maceration) or 3 part ple maceration) and each part is used for maceration separately. Vacuum maceration When pressure is reduced to desired extent menstrum is allowed to enter, increases permeal walls, reduced extraction time. ssKinetic maceration: The vessel is agitated in constant motion for first 24 hours > 20-50% more yield usually Re-maceration: First maceration is carried with small amount of menstrum, followed by remaining amount of solvent. Circulation maceration: Solvent is continuously circulated by pumpPercolate= I pass through It is continuous flow of the solvent through the bed of crude drug material to get the extract. It implies a slow passage of menstrum under the influence of gravity through column of drug powder and during this movement it goes on extracting the drug molecules layerwise. In percolation the drug is exhaustively extracted by fresh menstrum Used most frequently to extract active ingredients in the preparation of tinctures and fluid extracts. The solid ingredients are moistened with an appropriate amount of the specified menstrum. Allowed to stand for approximately 4 hours in a well closed container, After stand time, the mass is packed & the top of the percolator is closed. The mixture is allowed to macerate in the closed percolator for 24 h.Modifications Intermittent percolation: 24 hr maceration and 12 hr maceration are alternated with percolation to effect extraction. Re-pereolation: drug is divided into 4/5 lots, percolation followed, ext used as menstrum for next lot. Same menstrum for every new lot. olation: percoaltion at elevated temperature the efiiciency Reserved percolation: first part of percolate is reserved and ‘susequent percolate is collected separately. Most of active constituents are in first percolate. Second percoate contains less active constituents. Circulatory percolation: menstrum is continuously circulated. Diffusion percolation: menstrum is x form bottom to top under hydrostatic pressur Diacolation: same as diffusion ju: Jae of hydrostatic pressure, positive pressure of com d air is utilizedHot Continuous Extraction (Soxhlet) crude drug is placed in a porous of strong filter paper, which is ‘the Soxhlet eppaTavie) vapors condense in rdeaser ¥The condensed extractant drips into the thimble containing the crude drug & extracts it by contact. ~ When the level of liquid in chamber rises to the top of siphon tube, the liquid contents of chamber siphon into flask. ¥ This process is continuous and is car ut until a drop of solvent from the siphon tube does not leave residue when evaporated,Aqueous Alcoholic Extraction by Fermentation Some-medicinal preparations of Ayurveda (asava & arista ) adopt the technique of fermentation for extracting the active principles. The extractio: sdure involves soaking the crude drug, [powder / a ction (kasaya )], for a specified period of time. Under; fermentation & generates alcohol in situ. cilitates the extraction of the active constituents ined in the plant material. The alcohol thus generated also serves as a preservative. Some examples of Ayurvedic preparations: karpurasaya, , kanakasava, dasmularista . If the fermentation is to be carried out in an earthen ve should not be new: water should first be boiled in the vessel. In large-scale manufacture, wooden vats, porcelain jars or metal vessels are used in place of eartheCounter-current Extraction v Wet raw material is pulyerized using toothed disc disintegrators to produce a fine slurry. ¥ Material to be extracted is moved in one direction generally in the form of a fine slurry within a cylindrical extractor where it comes in contact with extraction solvent. ¥The further the starting material moves, the more concentrated the extract becomes. ¥Complete extraction is thus possible when the quantities of solvent & material. Their flow rates should be optimized. ¥ Sufficiently concentrated extract comes out at one end of the extractor while the marc, practically free of visible solvent falls out from the other end.Ultrasound Extraction (Sonication) ¥ The procedure involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz. ¥This increases the permeability of cell walls & produces cavitation. Eg: extraction of rauwolfia root. ¥ Deleterious effect: Ultrasound energy (+20 kHz) on the active constituents of medicinal plants through formation of free radicals and consequently undesirable changes in the drug molecules.Supercritical Fluid Extraction ¥ Cylindrical extraction vessels are used. ¥ The collection of the extracted analyte following SFE is another important step: significant analyte loss can occur during this step. ¥ CO? as the extracting fluid. Y Organic solvents are frequently added to the CO2 extracting fluid to alleviate the polarity limitations ¥ The component recovery rates generally increase with increasing pressure/temperature. ¥ The highest recovery rates in case of argon: @ 500 atm & 150°C.Microwave Assisted Extraction Microwaves are (frequency 300 MHz- 300 GHz) nonioinizing electromagnetic waves. High temperature produced by microwaves evaporate the moisure in cell--dehydrates cellulose-- ruptures cellwall-- extraction with solvent Rapid extraction within 5-10 min Types: » Solvent extractor » Solvent free extractor > Microwave reflux » Microwave assisted extractor » Sub- 500 W microwave extractor » Drydist model of milestone » Monolithic equipment for MAE » Closed vessel mono model of CEM Co.Analytical techniques in th aration and purification Due to the variability the mratirs of phenolic compounds (Polarity, chemical structure, glycosidic linkages and _ spectral characteristics) there is no single method that may be universally appropriate for separation of all extracts, which should be carefully sel lecte tification and isolation of bioactive compounds from extracts is the starting point for drug development for ntially new mechanisms against human diseases. To ples are subjected to a range of solvents of varied polarii and then separated using chromatographic techniques. There two broad types of chromatography; liquid and gas chromatography, Liquid chromatographic techniques are techniques where the mobile phase is a liquid whilst gas chromatography has gas phase as the mobile phase. The mechanisms of interaction vary d stationery phase and type of equilibriur ng on the states of theChromatography It.is a laboratory technique for the separation of a mixture of phytoconstituents. Chromatography=from Greek chroma "color and graphein "to write“. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The separation is based on differential partitioning between the mobile and stationary phases.Chromatography usually consists of mobile phase and stationary phase. i phase = a porous solid matrix through which le phase along with sample percolates. action between the mobile phase and the stationary phase results in the separation of the compounds from the ‘mixture depending upon its partition coefficient between ‘mobile phase and stationary phase. Subtle differences in a compound's partition coefficient result in differential retention on the stationary. Due to this various constituents of th ixture travel at different speeds along with mobile phase, causi separate.Purpose of Chromatography Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for later use, and is thus a form of purification. Analytical chromatography is done normally with | smaller amounts of material and is for establishing the presence or measuring the relative proportions of analytes in a mixture.Chromatograph - equipment that enables a sophisticated separation » EX. Gas chromatography or Liquid chromatography. Eluent - Fluid entering column/ solvent that carries the analyte. Eluate - Mobile phase leaving the column. Stationary phase - Immobilized phase » Immobilized on the support particl r on the imner wall of the column tubing. > : Silica layer - Thin Layer Chromatography - Moves in a definite direction. Liquid (LC), Gas The mabe phase moves through the chromatography column (the stationary phase) where the sample interacts with the ‘stationary phase and is separated. Retention time: Time takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. Sample (Analyte) :Substance analyzed in chromatography. Solvent: Any substance capable of solubilizing « another substance.Liquid chromatographic techniques In.this chromatographic technique, molecules in test samples are separated based on their shapes, sizes and charges. A solvent containing analyte which is the mobile phase is passed through a molecular sieve, called the stationary phase that categorically separates components of the extracts. Chromatographic techniques can be categorized based on phase states and mechanisms, phase polarities, separation region geometries, Gradient of experimental parameters and duration and column dimensions.Adsorption chromatography This is also called liquid/solid or displacement chromatography, based on solute interactions with active sites on the solid stationery phase. The stationery phase particularly interacts with specific functior al groups in the mobile phase by non-polar interactions, non-covalent bonds, hydrophobic interactions and Van-Der-Vaals forces. The compounds in the mobile phase get separated on the basis of similarities with the nature of the stationery phase, eluting loosely bound molecules first.Partition chromatography Also known as liquid/liquid chromatography, the method involves this method is based on the interaction of the molecules to be separated, with two immiscible liquids phases relative to their solubility, where the stationery liquid phase is adsorbed on a solid. ‘The components that are soluble in one are held strongly by it, if mobile phase, then they are the first to be eluted, but if they are held more strongly by stationery phase, they will be delayed within the system.Affinity chromatography and ion chromatography Extracts are introduced into the columns and they interact with the stationery phase ligands. If they have a high affinity to the ligands, they are drawn towards the stationery p If they have low or no affinity, they get washed away easily through the system using buffers of different pH or higher ionic strength, resulting in early elution. Usually, the desired components are bound to the ligands. On the other hand, closely related io chromatography separates ionic compo : molecules in extracts based on properties. The stationery phase is n eir electrical e of ion resins.Size exclusion chromatography This technique is also known as gel permeation, molecular sieve and gel filtration chromatography and is based on molecular size, relative to the sizes of the permeation spaces on the stationery phases. It enables measurements of molecular weights and ‘their distribution for compounds, particularly ‘polymeric compounds. Liquid stationary phase forms interstices on a polymeric solid. PI Equilibrium is achieved through s partitioning. There is no chemical interaction in this technique.Paper chromatography Paperchromatography is a technique that involves placing a small dot or line of sample solution onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent and sealed. As the soly rises through the paper, it meets the sample mixture, which starts to travel up the paper with the solvent. } paper is made of cellulose, a polar substance, and the compounds within the mixture travel farther if they are non- polar. Mote polar substances bond with the cellulose paper more quickly, and therefore do not travel as far. ’ Retention factor : Rf = zero, - Solute remains in the stationary phase and thus it is immobile. - Rf = | - Solute has no affinity for the stationary phase and travels with the solvent front.Sometimes, it is rather difficult to separate a complex mixture of substances by a single run with one solvent system. In such a case, a second run is carried out by a different solvent system, in a direction perpendicular to the first run. This is referred to as two dimensional chromatography. The paper need to have high porosity for high rate of capillary action and thick to accommodate a reasonably higher amount of samples better. The advantage of paper chromatography is that it is relatively cheaper and has a _ considerable reproducibility of retention factor (RF) values right on the paper.Bewus Solvent front Blue Purple OriginThin layer chromatography (TLC) Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, folves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose. ‘Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. The mobile phase runs upward along the plate due to capillary action and the components get separated depending upon their polarity towards stationary phaseThis isan adsorption chromatography, where the separation is based on the interaction of samples with a thin adsorbent layer attached to a plate. This works best low molecular weight molecular compounds. Thi advantage of this technique over paper chromatography is that it versatile, more sensitive and quick to tell the researcher possible number of compounds i in a sample. orbents are carefully selected depending on the ponents they are good at separating. The m commonly used adsorbents with components separate are Silica gel (Alkaloids, amino aci D sugars and fatty acids among others), Aluminum (Phenols, alkaloids, carotenes, steroids and vitamins), Celite (Inorganic cations and steroids), Starch (amino acids) and Sephadex (proteins and ; amin acids)RF = distance travelled by the component distance travelled by the solvent Steps of Thin Layer ChromatographyGas chromatography (GC) Gas chromatography (GC), also sometimes known as Gas- Liquid chromatograph (LC), is a separation technique in which the mobile phase is a gas. It is the method of choice for the separation of volatile substances or the volatile derivatives of certain non-volatile substances. Stationary phase is an inert solid material ( kieselgurh/ firebrick) impregnated with a non-volatile liquid. (silicon/PEG). / This is packed in narrow column and maintained at high temperature around 2000C. A sample is rapidly heated and vaporized at the The sample is transported through the column phase consisting of an inert gas (Ar/He/N). ‘i Sample components are separated based partion coefficeint between mobile phase and staionary phase — ction port. a mobileThe higher a component's affinity for the stationary phasey the slower it comes off the column. The components are then detected and represented as peaks on a chromatogram. Tt is commonly used for quantitative estimation of lipids, drugs and vitamins chromatography has three most common categories ) on phase states and mechanism. The gas-bonded phase has already been described in bonded phases section. The second one are gas-liquid, where a liquid adsorbed onto a solid acts as the stationery phase and e rium is reached through partition between gas and liquid. The last one is gas-solid technique, where a solid is the stationery phase and equilibrium is reached by adsorption.Gas flow regulator % Detector Computer’ Carrier gasColumn chromatography Column chromatography involves the following: 1. Adsorption/retention of substance on stationary phase 2. Separation of adsorbed substance using mobile phase. 3. Recovery of individual components by continuous - flow of mobile phase. 4. Stationary phase: silica gel, alumina, 5. These are chromatographic techniques that involve use solid phase packed columns as a separation housing. Sometime, they use regulat d pressure to pump mobile phase through a the « chamber laid with an adsorbent stationery phase.omatography COLUMN CHROMATOGRAPHY Bewus ® Mobile phase Somple separation Stron ‘interactions Stationary phase Weaker interactions ti e a Fluted __» Hee S ‘molecilesHigh Performance Liquid Chromatography Also called as high pressure liquid chromatography pass a pressurized (5000-1000 psi) mobile phase containing the sample a column filled with a solid adsorbent ry phase-immobilized thin layer liquid on micro glass or plastic beads tightly packed on narrow column. Mobile phase- solvent system passed under high pressure through column. The eluents can be detected by detectors. It can be applied inn the form of adsorption, ion exchange, partition or molecular sieve chromatography. Due to rapidity in detection its is used for detection of amino acids, peptides, Ce proteins, lipi is, nucleic acids, vitamins, hormones, drugs, etcThis techniques is used for separation, quantification and identification of inorganic and organic solutes in industrial, environmental, biological or pharmaceutical samples It is based on solute interactions in the mobile phase (usually polar combinations of water and other solvents) with tightly packed solid particles of the stationery phase (usually non-polar particles like C18).. Diode Array Detector (DAD) is one of % 3 to gas chromatographic technique in} Ee analysesChromatograr Peat + Vein. Hd, ee Solvent Manager Satvent Delrvary SystemHigh Performance Thin Layer Chromatography This technique is an advanced technology to TLC, with better efficiency esolution, an auto sampler and ization of spots and capability to allow for quantitative analysis. This technique does not use columns but rather, chambers: which contain the separation plates. The preparation of HPTLC has plate adsorbent sizes of between 5 and 7 microns and coating layer of between 150 and 200 microns, which is thicker the C Mobile phase is pumped over the plates using s pressure. ; HPTLC is also used in combination with spectroscopic techniques to maximize analytical potential of these techniques. dyImmerse plate in the Stationary phaselon Exchange Chromatography It retains analyte molecules based on it’s ionic interaction. Tt can be further divided into: » Cation exchange chromatography- retains cations n negatively charged stationary phase with negatively charged functional groups and is used when the molecule of interest is positively cha ed. > Inion exchange- retains anions on charged stationary phase with positively charged functional groups is used when the molecule of interest is negatively charged.When amino acid mixture is passed through the cation exchange chromatgraph, individual amino acid can be eluted using buffers of different pH. ‘A change in pH affects the charge on the particular molecules and, therefore, alters binding. Bound proteins are cluted out by utilizing a gradient of linearly increasing salt concentration, usually NaCl. With increasing ionic strength of the buffer, the salt ions will e with the desired proteins in order to bind to charged groups on the surface of the medium. This will cause desired proteins to be eluted out of the column. Proteins that have a low net charge will be eluted the salt concentration increases causing the ionic strength to increase. Proteins with high net charge will need a higher ionic strength for them to be eluted out of the coSpectroscopy Spectroscopy is the study of electromagnetic radiation ¥ Traditionally, spectroscopy involved the visible spectrum of light, but X- tay, gamma, and UY spectroscopy also are valuable analytical techniques. ‘the interaction between matter and tion, emission, scattering, etc. im of electromagnetic radiation passes through a sample, the They may be absorbed, reflected, refracted, ete. ¥ Absorbed radiation affects the electrons and chemical bonds | energy photons. ¥ Spectroscopy looks at how the incident radiatio ¥Emitted and absorbed spectra can be used to. material. ‘ormation about theStructural elucidation basically involves the use of spectroscopic techniques to characterize chemical constituents in samples Structure elucidation as the full de novo identification of structures, completely resulting into molecular connections with the data provided by the other. ‘The structures are identified from the interpretation of spectra or by searching and comparing with already discovered, identified and known data from spectral libraries. This presents techniques in thi fication of structures and subsequently names of ‘compounds isolated from plant extracts.UY-Visible spectroscopy This is absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet (10-400nm) and the full, adjacent visible spectral regions (400- 800 nm). Absorption of the ultra-violet radiations results in the excitation of the electrons from the ground state to higher energy stateUV spectroscopy obeys the Beer-Lambert law, which states that: when a beam of monochromatic light is passed through a solution of an absorbing substance, the rate of decrease of intensity of radiation with ; e absorbing solution is proportional to radiation as well as the concentration of the solution. The expression of Beer-Lambert law is- A= log (Io/l) = EcL Where, A = absorbance Io = intensity of light incident upon sample cell I = intensity of light leaving sample cell C = molar concentration of solute L = length of sample cell (cm.) E = molar absorptivityIR spectroscopy IR spectroscopy (which is short for infrared spectroscopy) deals with the infrared region of the electromagnetic spectrum, i.e. light having a longer slength and a lower frequency than visible light. The IR spectroscopy concept can generally be analyzed in three ways: by measuring reflection, emission, and absorption. The major use of infrared spectroscopy is to determine the functional groups of molecules, relevant to both organic and inorganic «Theory- IR radiation does not have enough energy to induce electronic ameiiox Absorption of IR is energy differenc rotational ee. within a Petectle must cause a net chung in the dipole moment of the molecule. The alternating electrical field of the radiation (remember that electromagnetic radiation consists of an oscillating electrical field and an oscillating mag) field, perpendicular to each other) interact fluctuations in the dipole moment of the mole: If the frequency of the radiation matches frequency of the molecule then ra absorbed, causing a change in the amplitude of molecular vibration* What is a vibration in a molecule? » “Athy change in shape of the molecule- stretching of bonds, bending of bonds, or internal rotation around single bonds”. * Why we study the molecular vibration? >» Because whenever the interaction b/w electromagnetic waves & matter occur so change appears in these vibrations. * FUNDAMENTAL VIBRATIONS > Vibrations which appear as band in the spectra. * NON- FUNDAMENTAL VIBRATIONS » Vibrations which appears as a result of fundamental vib. Mol.In IR spectroscopy the changes in the vibrationa energy depends upon i. Mass of the atoms present in a molecule ii. Strength of the bonds ili. Arrangement of atoms within the molecule No two compounds except the enantiomers can have the similar IR spectra.FATE OF ABSORBED RADIATION There are 3 main process by which a molecule can absorb radiation, each of these route involve an increase of energy which is proportional to the light absorbed. i. First route occurs when absorption of radiation leads to a higher rotational energy level in a rotational transition. ii. Second occurs when absorption of radiation leads to a higher vibrational energy level in a vibrational transition. Third occurs when absorption of radiation leads to a higher electronic energy level its electronic transitions. iii.Two criteria must be satisfied by a molecule for the absorption of IR radiation: i. The molecule should possess vibrational and rotational frequency. ii. The molecule must give rise to asymmetrical charge distribution. Three main type of absorption bands occur in IR spectra: 1. Fundamental ii. Overtone . CombinationalAPPEICATIONS OF IR SPECTROSCOPY . Identification of an organic compound - The identity of an organic compound can be established from its fingerprint region by comparing the sample spectrum with the known spectrum of the compound. ¢ E.g. spectrum of n- hexanal 2. Qualitative determination of functional groups - The presence or absence of absorption bands help in predicting the presence of certain functional group in the compound.3. Distinction between 2 types of H-bonding - If a compound having intra-1 lecular H-bonding it will show broad bands in IR spectrum and if a compound having intermolecular H-bonding then it will show sharp well defined bands. itrophenol shows broad bands due to intra- it H-bond whereas p-nitrophenol shows sharp bands due to intermolecular H-bonding. 4, Quantitative analysis - It can be done by measuring the intensity of the absorption bands. E.g. xylene exists as mixture of 3 compounds which shows absorption bands at ortho-740cm-1 meta- 880cm-1 para-830cm-15. Study of chemical reactions ° It is useful for studying the chemical reactions. ° E.g.: reduction of butan-2-one to form butan-2-ol Butan-2-one Butan-2-ol (1710 cm-l) (3300 cm-1) 6. Study of keto-enol tautomerism ° Diketones and ketoesters exhibit keto-enol tautomerism and this can be studied using IR spectrum of the compound.Mass spectrometry Mass spectrometry is a powerful analytical technique used to quantify known materials, to identify unknown compounds within a sample, and to elucidate the structure and chemical properties of different molecules. The complete process involves the conversion of ‘the sample into gaseous ions, with or withou fragmentation, which are then characterized by their mass to charge ratios (m/z) and relative abundances. A mass spectrum is the plot of relative abundance of ions against their mass/charge ratio.The basic aspect of organic mass spectrometry consist of bombarding the vapour of an organic compound with a beam of energetic electron accelerated from a filament to an energy of 70 eV to form positively charged ions (molecular ions). The additional energy of the elecrons is dissipated in breaking the bonds in the molecular ion, which undergoes fragmentation to yield several neutral or positively charged species. The ions are then separated according to their mass to charge ration by virtue of magnet and are detected by detector aaInstrumentation Sample introduction and vacuum. Ton source. Electrostatic accelerator. Magnetic field. Mass separator or analyzer. Detector or Collector . Recording or Read out.Basic. principle Mass spectroscopy is the most accurate method for determining the molecular mass of the compound and its elemental composition. In this technique, molecules are bombarded with a beam of energetic electrons. ‘The molecules are ionised and broken up into many fragments, some of which are positive ions. Each kind of ion has a particular ratio of mass to charge, ie. m/e ratio(value). For most ions, the charge is one and thus, m/e ratio is simply the molecular mass of the ion. PMass spectra is used in two general ways: 1) To prove the identity of two compounds. 2) To establish the structure of a new a compound. The mass spectrum of a compound helps to establish the structure of a new compound in several different ways: 1) It can give the exact molecular mass. 2) It can give a molecular formula or it can reveal the presence of certain structural units in a molecule.Applications of MS Proteomics Determine protein structure, function, folding and interactions Identify a protein from the mass of its peptide fragments Detect specific post-transiational modifications throughout complex biological mixtures Quantitate (relative or absolute) proteins in a given sample Monitor enzyme reactions, chemical modifications and protein digestion Drug Discovery Determine structures of drugs and metabolites Screen for metabolites in biological systems Clinical Testing Perform forensic analyses such as confirmation of drug abuse Detect disease biomarkers (e.g., newborns screened for metabolic diseases) Genomics Sequence oligonucleotides Environment Test water quality or food contamination Geology Measure petroleum composition and Perform carbon dating