Applications of chromatography and spectroscopy

B. Pharm. Vth semester Pharmacognosy and Phytochemistry II

1. Basics of Phytochemistry Dr. Amit Gangwal (Associate Professor) SVKM’s Institute of Pharmacy, Dhule B. Pharm. Vth Semester Pharmacognosy & Phytochemistry II Applications of spectroscopy, chromatography & electrophoresis in isolation, purification & identification of crude drugs

2. Disclaimer Entire content in this video has been taken from textbooks and online resources using Google as search engine. Author has no claim whatsoever on the images or infographics taken from internet. This video is not made for commercial interest, but just to share the educational stuff, purely from academies point of view.

3. Chromatography Chromatography usually consists of mobile phase and stationary phase. The interaction between the mobile phase and the stationary phase results in the separation of the compounds from the mixture depending upon its relative affinity for mobile phase and stationary phase. Differences in a compound's affinity results in differential retention on the stationary phase. 

4. 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.

5. Classification of chromatography On the basis of interaction of solute to the stationary phase Adsorption Chromatography Partition Chromatography Ion Exchange Chromatography Size Exclusion Chromatography On the basis of physical state of mobile phase Liquid Chromatography Gas Chromatography Super Critical Fluid Chromatography

6. Terms used in chromatography Chromatograph - equipment that enables a sophisticated separation eg. 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 particles or on the inner wall of the column tubing. › Examples : Silica layer - Thin Layer Chromatography Mobile phase - Moves in a definite direction. The mobile 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.

7. Column chromatography Column chromatography is a technique in which the substances to be separated are introduced onto the top of a column packed with an adsorbent, passed through the column at different rates that depend on the affinity of each substance for the adsorbent and for the solvent or solvent mixture, and are usually collected in solution as they pass from the column at different times. It is a solid – liquid technique in which the stationary phase is a solid & mobile phase is a liquid or gas.

8. Forms of Column Chromatography There are two forms of column chromatography Liquid chromatography (LC) Gas chromatography (GC) The most widely used forms of column chromatography are Adsorption chromatography Partition chromatography Ion exchange chromatography Size exclusion or gel chromatography

9. Principle of Column Chromatography In column chromatography the stationary phase is packed into a glass or metal column. The mixture of analytes is then applied and the mobile phase, commonly referred to as the eluent, is passed through the column either by use of a pumping system or applied gas pressure. The stationary phase is either coated onto discrete small particles (the matrix) and packed into the column or applied as a thin film to the inside wall of the column. As the eluent flows through the column the analytes separate on the basis of their distribution coefficients and emerge individually in the eluate as it leaves the column.

10. Instrumentation of Column Chromatography A stationary phase : Chosen to be appropriate for the analytes to be separated. A column : In liquid chromatography these are generally 25- 50 cm long and 4mm internal diameter and made of stainless steel whereas in gas chromatography they are 1-3m long and 2- 4mm internal diameter and made of either glass or stainless steel. They may be either of the conventional type filled with the stationary phase, or of the microbore type in which the stationary phase is coated directly on the inside wall of the column. A mobile phase and delivery system : Chosen to complement the stationary phase and hence to discriminate between the sample analytes and to deliver a constant rate of flow into the column. An injector system: To deliver test samples to the top of the column in a reproducible manner. A detector and chart recorder: To give a continuous record of the presence of the analytes in the eluate as it emerges from the column. Detection is usually based on the measurement of a physical parameter such as visible or ultraviolet absorption or fluorescence. A peak on the chart recorder represents each separated analyte. A fraction collector:  For collecting the separated analytes for further biochemical studies.

11. Steps in Column Chromatography Preparation of the Column The column mostly consists of a glass tube packed with a suitable stationary phase. A glass wool/cotton wool or an asbestos pad is placed at the bottom of the column before packing the stationary phase. After packing, a paper disc kept on the top, so that the stationary layer is not disturbed during the introduction of sample or mobile phase. There are two types of preparing the column, they are: 1. Dry packing / dry filling In this the required quantity of adsorbent is poured as fine dry powder in the column and the solvent is allowed to flow through the column till equilibrium is reached. 2. Wet packing / wet filling In this, the slurry of adsorbent with the mobile phase is prepared and is poured into the column. It is considered as the ideal technique for packing. Before using column, it should be washed properly and dried. The column should also be free from impurity and uniformly filled with the stationary phase.

12. Introduction of the Sample The sample which is usually a mixture of components is dissolved in minimum quantity of the mobile phase. The entire sample is introduced into the column at once and get adsorbed on the top portion of the column. From this zone, individual sample can be separated by a process of elution.

13. Elution By elution technique, the individual components are separated out from the column. It can be achieved by two techniques: Isocratic elution technique:  Same solvent e. g. use of chloroform alone. Gradient elution technique:  Solvents of gradually ↑ polarity or ↑ elution strength are used during the process of separation e.g. initially benzene, then chloroform, then ethyl acetate then chloroform.

14. Detection of Components If the compounds separated in a column chromatography procedure are colored, the progress of the separation can simply be monitored visually. If the compounds to be isolated from column chromatography are colorless then small fractions of the eluent are collected sequentially in labelled tubes and the composition of each fraction is analyzed by TLC.

15. Factors Affecting Column Efficiency Dimensions of the column Particle size of the adsorbent Nature of the solvent Temperature of the column Pressure

16. Applications Separation of mixture of compounds. Removal of impurities or purification process. Isolation of active constituents. Isolation of metabolites from biological fluids. Estimation of drugs in formulation or crude extracts.

17. Ion Exchange Chromatography Ion exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on their affinity to ion exchangers. The principle of separation is thus by reversible exchange of ions between the target ions present in the sample solution to the ions present on ion exchangers. In this process two types of exchangers i.e., cationic and anionic exchangers can be used.

18. Cationic exchangers  possess negatively charged group, and these will attract positively charged cations. These exchangers are also called “Acidic ion exchange” materials, because their negative charges result from the ionization of acidic group. Anionic exchangers  have positively charged groups that will attract negatively charged anions. These are also called “Basic ion exchange” materials.

19. Principle of ion exchange chromatography This form of chromatography relies on the attraction between oppositely charged stationary phase, known as an ion exchanger, and analyte. The ion exchangers basically contain charged groups covalently linked to the surface of an insoluble matrix. The charged groups of the matrix can be positively or negatively charged. When suspended in an aqueous solution, the charged groups of the matrix will be surrounded by ions of the opposite charge. In this “ion cloud”, ions can be reversibly exchanged without changing the nature and the properties of the matrix.

20. Applications of ion exchange chromatography An important use of ion-exchange chromatography is in the routine analysis of  amino acid  mixtures. The 20 principal amino acids from blood serum or from the hydrolysis of proteins are separated and used in clinical diagnosis. This is most effective method for water purification. Complete deionization of water (or) a non-electrolyte solution is performed by exchanging solute cations for hydrogen ions and solute anions for hydroxyl ions. This is usually achieved by method is used for softening of drinking water. In the analysis of products of hydrolysis of nucleic acids. In this way, information is gained about the structure of these molecules and how it relates to their biological function as carriers of hereditary information.  Chelating resins are used to collect trace metals from seawater. To analyze lunar rocks and rare trace elements on Earth

21. Size exclusion chromatography

22. Affinity chromatography

23. Thin Layer Chromatography Chromatography  is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. In this physical method of separation, the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. Depending upon the stationary phase and mobile phase chosen, they can be of different types. Thin Layer Chromatography can be defined as a method of separation or identification of a mixture of components into individual components by using finely divided adsorbent solid / (liquid) spread over a plate and liquid as a mobile phase.

24. Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminum oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved. It is thus based on the principle of adsorption chromatography or partition chromatography or combination of both, depending on adsorbent, its treatment and nature of solvents employed. The components with more affinity towards stationary phase travels slower. Components with less affinity towards stationary phase travels faster. Once separation occurs, the individual components are visualized as spots at a respective level of travel on the plate. Their nature or character is identified by means of suitable detection techniques.

25. HPTLC Fingerprints of Various Secondary Metabolites in the Traditional Medicinal Herb Hypochaeris radicata L.

26. Derivatizing reagents used in TLC & HPTLC

27. Paper Chromatography Paper chromatography (PC) is a type of a planar  chromatography  whereby chromatography procedures are run on a specialized paper. PC is considered to be the simplest and most widely used of the chromatographic techniques because of its applicability to isolation, identification and quantitative determination of organic and inorganic compounds.

28. Types of Paper chromatography Paper Adsorption Chromatography Paper impregnated with silica or alumina acts as adsorbent (stationary phase) and solvent as mobile phase. Paper Partition Chromatography Moisture / Water present in the pores of cellulose fibers present in filter paper acts as stationary phase & another mobile phase is used as solvent. In general paper chromatography mostly refers to paper partition chromatography. Pores of cellulose fibers present in filter paper act acts as stationary phase. Organic solvents/buffers are used as mobile phase. The developing solution travels up the stationary phase carrying the sample with it. Components of the sample will separate readily according to how strongly they adsorb onto the stationary phase versus how readily they dissolve in the mobile phase.

29. Instrumentation of Paper chromatography Stationary phase & papers used Mobile phase Developing Chamber Detecting or Visualizing agents

30. STATIONARY PHASE AND PAPERS Whatman filter papers of different grades like No.1, No.2, No.3, No.4, No.20, No.40, No.42 etc In general the paper contains 98-99% of α- cellulose, 0.3 – 1% β - cellulose. Other modified papers Acid or base washed filter paper Glass fiber type paper. Hydrophilic Papers – Papers modified with methanol, formamide, glycol, glycerol etc. Hydrophobic papers – acetylation of OH groups leads to hydrophobic nature, hence can be used for reverse phase chromatography. Impregnation of silica, alumna, or ion exchange resins can also be made.

31. PAPER CHROMATOGRAPHY MOBILE PHASE Pure solvents, buffer solutions or mixture of solvents can be used. Examples- Hydrophilic mobile phase Isopropanol:ammonia:water 9:1:2 Methanol:water 4:1 n-butanol : glacial acetic acid : water 4:1:5 Hydrophobic mobile phases dimethyl ether: cyclohexane kerosene : 70% isopropanol The commonly employed solvents are the polar solvents, but the choice depends on the nature of the substance to be separated. If pure solvents do not give satisfactory separation, a mixture of solvents of suitable polarity may be applied.

32. CHROMATOGRAPHIC CHAMBER The chromatographic chambers are made up of many materials like glass, plastic or stainless steel. Glass tanks are preferred most. They are available in various dimensional size depending upon paper length and development type. The chamber atmosphere should be saturated with solvent vapor.

33. Steps in Paper Chromatography In paper chromatography, the sample mixture is applied to a piece of filter paper, the edge of the paper is immersed in a solvent, and the solvent moves up the paper by capillary action. The basic steps include: Selection of Solid Support Fine quality cellulose paper with defined porosity, high resolution, negligible diffusion of sample and favouring good rate of movement of solvent. Selection of Mobile Phase Different combinations of organic and inorganic solvents may be used depending on the analyte. Example.   Butanol : Acetic acid: Water (12:3:5) is suitable solvent for separating amino-acids. Saturation of Tank The inner wall of the tank is wrapped with the filter paper before solvent is placed in the tank to achieve better resolution. Sample Preparation and Loading If solid sample is used, it is dissolved in a suitable solvent. Sample (2-20ul) is added on the base line as a spot using a micropipette and air dried to prevent the diffusion.

34. Development of the Chromatogram Sample loaded filter paper is dipped carefully into the solvent not more than a height of 1 cm and waited until the solvent front reaches near the edge of the paper. Different types of development techniques can be used: ASCENDING DEVELOPMENT Like conventional type, the solvent flows against gravity. The spots are kept at the bottom portion of paper and kept in a chamber with mobile phase solvent at the bottom. DESCENDING TYPE This is carried out in a special chamber where the solvent holder is at the top. The spot is kept at the top and the solvent flows down the paper. In this method solvent moves from top to bottom so it is called descending chromatography. ASCENDING – DESCENDING DEVELOPMENT A hybrid of above two techniques is called ascending-descending chromatography. Only length of separation increased, first ascending takes place followed by descending. CIRCULAR / RADIAL DEVELOPMENT Spot is kept at the centre of a circular paper. The solvent flows through a wick at the centre & spreads in all directions uniformly. Drying   of Chromatogram After the development, the solvent front is marked and the left to dry in a dry cabinet or oven. Detection Colourless analytes detected by staining with reagents such as iodine vapour , ninhydrin etc. Radiolabeled and fluorescently labeled analytes detected by measuring radioactivity and florescence respectively.

35. Applications of Paper Chromatography To check the control of purity of pharmaceuticals, For detection of adulterants, Detect the contaminants in foods and drinks, In the study of ripening and fermentation, For the detection of drugs and dopes in animals & humans In analysis of cosmetics

36. HPLC

37. HPLC process flow

38. Schematic diagram of HPLC

39. HPLC is the most popular technique among all the analytical techniques used today. It is therefore understandable that most happenings are taking place in the modernization of this technique. There are two applications of HPLC: one to generate the profile, for which TLC is preferred, and one to estimate the quantity of markers, where HPLC is preferred. The initial steps of sample preparation are similar to those for TLC with the exception that the samples for HPLC are filtered through a filter of 0.45 μm or less.

40. Furthermore, it is assured that the test sample does not contain substances which are permanently retained on the HPLC column, which means in most cases purification procedures are applied to extracts before injecting them onto the column. After the sample has been prepared, it is injected onto the column and the response is recorded preferably using a variable wavelength ultraviolet detector.

41. As the nature of all the compounds of the extract cannot be known beforehand, the photodiode array detector is useful, especially when constructing profiles of plant extracts. The fingerprint profile of plant extracts can be used for identification purposes and also for obtaining semi-quantitative information if the sample preparation was not done for quantitative analysis. Similarly, the profile can be generated for the finished product and used to record batch to batch variations. The fingerprint profile can be used to study changes in the composition of the finished product or, in other terms, to indicate the stability of the product.

42. HPLC is an analytical technique for the separation and determination of organic and inorganic solutes in any samples especially biological, pharmaceutical, food, environmental, industrial etc. The another name for HPLC is high – pressure liquid chromatography, separates compounds on the basis of their interactions with solid particles of tightly packed column and the solvent of the mobile phase. Modern HPLC uses a non-polar solid phase, like C18 and a polar liquid phase, generally a mixture of water and another solvent. High pressure up to 400 bars is required to elute the analyte through column before they pass through a diode array detector (DAD). A DAD measures the absorption spectra of the analytes to aid in their identification. HPLC is useful for compounds that cannot be vaporized or that decompose under high temperature, and it provides a good complement to gas chromatography for detection of compounds.

43. Applications of HPLC

44. The most important use of HPLC is in estimation of markers in plant drugs. The steps in HPLC analysis are fundamentally the same as used for any other analytical technique. The response of the test sample is compared to that of a known quantity of the marker to quantify the marker in the test substance. The HPLC method is developed from knowledge of the technique and chemistry of the marker. In chemical analysis, HPLC has no parallel and can be customized to produce the most precise and accurate results. The HPLC analysis is vital in the analysis of a finished product and the expected results are superior to those from TLC, as the separations in HPLC are better. However, run time of HPLC analyses usually varies from 15 to 30 min, which restricts its use if large numbers of samples are to be analyzed. HPLC in Quality Control of Plant Products

45. TLC vs HPTLC

46. HPLC vs HPTLC

47. HPLC vs GC

48. Applications of GC: It is used for the identification of non-volatile materials (plastics, natural and synthetic polymers, and some microbiological materials. It is based on the fingerprint chromatogram for the sample, which results from its thermal dissociation and fragmentation.  Food Analysis: Analysis of foods is concerned with confirming the presence and determination the quantities of the analytes (lipids, proteins, carbohydrates, preservatives, flavours , colorants, and also vitamins, steroids, and pesticide residues).  Drug Analysis: GC is widely applied to identification of the active components, possible impurities as well as the metabolites.  Environmental Analysis: The environmental contaminants; e.g. dichlorodiphenyltrichloro - ethane (DDT) and the polychlorinated biphenyls (PCBs) are present in the environment at very low concentrations and are found among many of other compounds. GC, with its high sensitivity and high separating power, is mostly used in the analysis of environmental samples.

49. Application of TLC and HPTLC in separation and identification of Curcumin from Curcuma longa rhizome extract  Sample: Curcuma longa Rhizome  Common name: Haldi powder  Mobile phase: Chloroform: Ethanol: Glacial acetic acid (95:5:1)  Sample Preparation: The marc obtained after successive petroleum ether and chloroform extraction of the powdered sample, further extracted with methanol. The methanol extract concentrated and residue dissolved in methanol.  Detection: Normal light (Figure 01)  Densitometer scan: 254 nm (Figure 02)

50. TLC offers many advantages but also has some disadvantages. It fails to match the sensitivity of HPLC and has not kept with the pace of developments and advancements happening in the area of HPLC. TLC is an open system and hastens the degradation of compounds sensitive to light and air, which in the case of HPLC pass through an enclosed environment. Detection of the analyte in HPLC occurs in solution, permitting high sensitivity, whereas in TLC the solid phase interaction makes detection less sensitive. Finally, recent advances and efficient flow kinetics of HPLC allow more complex separations than TLC.

51. GC

52. Gas chromatography is a method for the separation of volatile compounds. In this method, species distribute between gas and a liquid phase. The gas phase is flowing and the liquid phase is stationary. The rate of migration for the chemical species is determined through it’s distribution in the gas phase. For example, a species that distributes itself 100% into gas phase will migrate at the same rate as the flowing gas, whereas, a species that distributes itself 100% into stationary phase will not migrate at all. Species that distribute themselves partly in both phases will migrate at an intermediate rate . Gas chromatography involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is then transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.

53. Summary of Chromatographic Techniques

54. Paper chromatography In paper chromatography a sheet of paper is used for the inert phase. One of the advantages of paper chromatography is that separations are carried out simply on sheets of filter paper, which acts as both support as well as medium for separation . Another advantage is the considerable reproducibility of Rf (retention factor) values determine on paper. In paper chromatography, filter paper used as solid phase, which is inert phase. A sample is placed near the bottom of the filter paper. Then this filter paper is placed in chromatographic chamber with solvent. The solvent move forwards by capillary action carrying soluble molecules along with it. Low porosity paper will produce a slow rate of movement of the solvent and thick papers have increased sample capacity .

55. Thin layer chromatography (TLC) The first practical application of thin layer chromatography was given by Stahl. Compared to paper chromatography, the special advantage of TLC is the versatility, speedy and sensitive. TLC is an adsorption chromatography where samples are separated based on the interaction between a thin layers of adsorbent attached on the plate. The technique mostly employed for the separation of low molecular weight compounds.

56. Size exclusion chromatography It also termed as gel filtration, gel permeation chromatography and molecular sieve chromatography. In this chromatography, no chemical attraction or interaction occurs between the solutes and stationary phase and the molecules are separated according to their size.

57. Identification of phytochemicals Plant extracts contains various type of bioactive compounds having different polarities their separation still remains a big challenge for the process of identification and characterization of bioactive compounds. It is a common practice in isolation of these bioactive compounds using different separation techniques such as TLC, HPTLC, paper chromatography, column chromatography, Gas chromatography, OPLC and HPLC, should be used to obtain pure compounds. The pure compounds are then used for the determination of structure and biological activity.

58. Adsorption chromatography Adsorption chromatography also termed as displacement or liquid/solid chromatography and is based on interactions between the solute and fixed active sites on the stationary phase. The active sites of the stationary phase interact with the functional groups of compounds to be separated by noncovalent bonds, non-polar interactions, van der Waals forces and hydrophobic interactions. The compounds which are loosely bound will be eluted out firstly by the mobile phase at and classes of compounds can be separated.

59. Partition chromatography In partition chromatography the molecules to be separated will interact between two immiscible liquid phases according to their relative solubility. This process is also referred as liquid/liquid chromatography. Ion-exchange chromatography Ion-exchange chromatography allows the separation of ions and polar molecules on the basis of electrical properties of the molecules.

60. Affinity chromatography In affinity chromatography, separations are based on the specific interactions between interacting pairs of substances such as macromolecules and it’s substrates, cofactor, allosteric effector or inhibitor. During this chromatography, a mixture of substances applied to the columns. Substances that have no affinity with the ligand are washed through with the buffer and desired compound is bind to ligand. Buffer having different pH or an increased ionic strength is used to elutes the analyte out.

61. Chromatography is a technique where the molecules are separated based on their size, shape and charge. During chromatography analyte in solvent and move through solid phase that acts as a sieving material. As molecule proceeds further through molecular sieve it gets separated. Paper and thin layer chromatography are the chromatographic techniques which readily provides qualitative information and through which it become possible to obtain quantitative data.

62. Different adsorbent used to separate various compounds 1 Silica gel for Amino acids, alkaloid, sugars, fatty acids, lipid etc. 2 Aluminium for Alkaloids, phenols, steroids, vitamins and carotenes. 3 Celite for Steroids and inorganic cations 4 Cellulose powder for Amino acids, food dyes, alkaloids 5 Starch for Amino acids 6 Sephadex for Amino acids, proteins

63. High performance thin layer chromatography (HPTLC) is a planar chromatography where separation of sample components is achieved on high performance layers with detection and data acquisition. These high performance layers are pre-coated plates coated with a sorbent of particle size 5-7 microns and a layer thickness of 150-200 microns. The reduction in thickness of layer and particle size results in increasing the plate efficiency as well as nature of separation. HPTLC gives chromatogram i.e. separated samples after chromatography can be inspected by the eyes only in case of HPTLC. The main difference between TLC and HPTLC are the particle and pore size of sorbents.

64. Column chromatography involves ion exchange, molecular sieves, and adsorption phenomenon. The flushing in conventional chromatography greatly dilutes the material, and the fractions usually require another step for concentration. A newer method called displacement chromatography elute with some compounds that has great affinity for the adsorbent. Fractions of elute materials can be more concentrated than the original solution applied to column.

65. HPTLC applications It facilitates ease of separation and identification of Lipids, Metabolism studies, Drug screening and Doping control. HPTLC provides high degree of precise, accurate and reproducible measurable data of presence of Phytoconstituents in complex nature of herbal extracts. This technique can be employed in food and feed stuff quality control studies. Standardization of herbal formulations/ Ayurvedic polyherbal formulations Identification of unknown compounds can be done using standard compounds.

66. Spectroscopy UV Visible NMR Mass IR Electrophoresis

67. Fourier-transform infrared spectroscopy ( FTIR ) Fourier- transform infrared spectroscopy is a valuable tool for the identification of functional groups present in the plant extract. It helps for identification and structure determination of the molecule . Samples for FTIR can be prepared in a number of ways. For liquid samples, the easiest is to place one drop of sample between two plates of sodium chloride. The drop forms a thin film between the plates. Solid samples can be milled with potassium bromide ( KBr ) to and then compressed into a thin pellet which can be analyzed. Otherwise, solid samples can be dissolved in a solvent such as methylene chloride, and the few drop of solution is then placed onto a single High Attenuated Total Reflectance (HATR) plates and spectra was recorded in terms of percentage transmittance. The peaks at specific wave number were assigned by bonding and functional group as per the reference given in Varian FTIR instrument manual.

68. IR Instrumentation

69. Nuclear Magnetic Resonance Spectroscopy (NMR) Nuclear Magnetic Resonance Spectroscopy gives physical, chemical and biological properties of matter. One dimensional technique is routinely used but the complicated structure of the molecules could be achieved through two dimensional NMR techniques. Solid state NMR spectroscopy is used for the determination of molecular structure of solids. Radiolabelled C NMR is used to identify the types of carbon are present in the compound. H-NMR is used to find out types of hydrogen are present in the compound and to find out how the hydrogen atoms are connected.

70. Mass spectrometry (MS) Mass spectrometry is a powerful analytical technique for the identification of unknown compounds, quantification of known compounds and to elucidate the structure and chemical properties of molecules. Through MS spectrum the molecular weight of sample can be determined. This method mostly employed for the structural elucidation of organic compounds, for peptide or oligonucleotide sequencing and for monitoring the existence of previously characterizes compounds in complex mixtures with a high specificity by defining both the molecular weight and a diagnostic fragment of the molecule simultaneously.

71. UV Instrumentation

72. Application of UV spectroscopy A crude plant extract may contain up to hundreds of different secondary metabolites of different chemical nature and spectroscopic study. Here, it is used for detection, identification and quantification of unknown phytoconstituents in crude drug. Used for the standardization of phenolics for its antioxidant activities. To determine the nature of contaminants in a sample. U.V spectroscopy is one of the best methods for determination of impurities in crude drug. Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material. UV Spectroscopy is beneficial in qualitative analysis as we get spectra with specific solvent extraction. Spectra got can be used as fingerprint of the sample extract. Adulterant can be found out by spectral analysis.

73. Following are the images I have taken from well known book on phytochemistry by JB Harborne