eChapters

Camphor plant is valued for presence of camphor in its wood and leaves. Camphor is chiefly a pharmaceutical product though it is used in preparation of artificial essential oils like lavender and lavendin.  It is used as incense and in balms of various kinds.  It is employed as a masking agent in perfumery and sometimes used to achieve lift in perfume blends. Redistilled brown oil is used directly in soap perfumes for a masking effect. Oil is a source of safrole which is a starting material for production of various perfumery chemicals. In 13th century, Marco Polo noted that camphor oil was highly valued by Chinese as a medicine, scent, and embalming fluid.

Ajowan is a profusely branched winter annual herb, seed oil of which is a major source of thymol, being present to extent of 35-60%. Ajowan oil is aromatic, stimulant and carminative.  It possesses antimicrobial activity. Ajowan seeds are employed either alone or in combination with spices and condiments. It is used in pickles, confectionery and beverages.  It is a good remedy for indigestion.  A paste of crushed fruit is applied externally for relieving colic pains.  It is also used in lotions and ointments.

Commercial crops include a variety of high value plantation crops, which play a vital role in the agricultural economy and export trade of many developing and developed countries (Table 1). Tea, coffee, cocoa, rubber, coconut, arecanut, oil palm, cashew, palmyra and cardamom are the major crops grown on a plantation scale. These crops are perennial in nature and the cultivation is labour intensive, generating considerable employment opportunity during on farm operations and off farm processing activities. The most obvious characteristic is that plantations are monocultures; the same species occupying large contiguous area usually in even aged stands. Exceptions are coconut, arecanut, cocoa and coffee, which are grown either as mono crop or as multiple crops. These crops are grown in humid tropics, mainly between 20°N and 20°S of equator in an area of 35.7 million ha yielding about 74.4 million tonnes of produce and contributing US $ 11 billion to global trade during 2002.

Arecanut palm (Areca catechu L.) is the source of common masticatory nut, popularly known as arecanut, betelnut, or supari. It is used extensively in India by all sections of people as masticatory, and is an essential requisite for several religious and social ceremonies.

Cashew (Anacardium occidentale L.) is an export-oriented crop grown for its nuts, a true drupe. Often referred as ‘wonder nut’, it is highly delicious and nutritive. Its nutritional composition compares well with almond, hazel nut and walnut. It is a zero-cholesterol nut with high content of unsaturated fatty acids, proteins and carbohydrates with a right combination of amino acids, vitamins and minerals. Kernels extracted from nuts are often consumed as such or used in confectionery items and various kinds of dishes. Kernel oil of pale yellow colour is used in pharmaceutical and cosmetic industries. Cashew nut shell liquid (CNSL) extracted from its shell has many industrial applications - for making paints, resins, flame-retardants, etc. Testa covering the kernel, rich in tannin, can be an acceptable substitute for wattle bark tannin in leather industry. Cashew apple, the pseudo-fruit, is rich in vitamin C. It is used to cure scurvy, diarrhea, uterine complaints and dropsy.

Cocoa (Theobroma cacao L.) is the third important beverage crop grown for its seeds known as beans, used mainly in chocolate industry. It is the source of cocoa butter and cocoa mass.

Coconut is the most beautiful and useful of palms known to mankind. Coconut is unique, producing a host of products and by products with distinctive applications at home and in industry. It is essentially an oil seed crop providing all amenities for human life, which include food, energetic beverage, medicine, fibre, wood, shell and a variety of raw materials for production of array of products of commercial importance. In view of its versatile uses and affinity to Indian culture, it is rightly called the “tree of heaven” or “Kalpavriksha”.

Coffee (Coffea species) occupies a pride of place, in the international trade of commodities, next to petroleum in trade volume and money value. Coffee is also the second important popular beverage for its aroma and mild stimulant action. Its dried beans are roasted, ground and brewed to make a stimulating & refreshing beverage. Its production forms the backbone of more than 60 developing nations. Use of coffee has evolved from original chewing of leaves and beans of the plant to relieve pain, hunger and fatigue to the present more sophisticated uses, like espresso and decaffeinated coffees. Its use was first discovered in Arabia around mid-15th century.

The oil palm (Elaeis guineensis Jacq.) is recognised as the cheapest edible oil-yielding crop and has become one of the major oil crops in the world. The most productive oil-bearing crop, oil palm yields about three times the oil yield of coconut, seven times that of rapeseed, and ten times that of soybean. In terms of world production, palm oil maintains second place after soybean as a source of vegetable oil and has overtaken other crops such as rapeseed, sunflower, groundnut, and cottonseed. Under good agricultural management, a hectare of oil palm yields 5 to 7 tonnes of oil / year (Henson, 1991). The crop is unique in producing 2 distinct types of oil- palm oil and palm kernel oil- used for culinary and industrial purposes. Being rich in b-carotene, palm oil contributes substantially to nutritional and energy requirements of masses.

Palmyra (Borassus flabellifer L.) is a tropical dioecious palm adapted to arid zone tracts. Though it is a versatile plant which yields many edible and non-edible products it is a less exploited species. It is used for sap, fruit, seed, leaf and fiber and timber products. From nutritional point of view, fruit pulp and seed shoot flour are the most important.Successful promotion of these as foods for general consumption depends on subjecting the fruit pulp to a debittering process, and the seed shoot flour, to initial detoxification processes (Thabrew and Jansz, 2004). Palmyra palms are the most numerous palms in the world after coconut palm (Fox, 1977). Despite this, they are the least studied of all the palms.

Natural rubber, cis-l,4 polyisoprene, produced in the milky cytoplasm (latex) of specialized cells called laticifers in certain plants is one of most important biological macromolecules, which is used as an industrial raw material for the manufacture of a variety of products. Natural rubber is found in the latex of over 2000 species of plants belonging to 311 genera under 79 families. However, Hevea brasiliensis, also known as Para rubber tree after the Brazilian port of Para, is the only commercial source of natural rubber mainly because of its abundance in latex, quality and convenience of harvesting. Hevea was first used as a source of rubber by the French.

Tea is the most important non-alcoholic beverage worldwide gaining further popularity as an important ‘health drink’. It is consumed as a morning drink by 2/3^rd of world population. Tea was used initially as a medicine and subsequently as beverage and now has proven to be a potential raw material for pharmaceutical industry. Tea is mainly consumed in the form of ‘fermented tea’ or ‘black tea’, ‘non-fermented’ or ‘green tea’ and semi-fermented or ‘oolong tea’ are also popular in Japan and China. Green leaves are also used as vegetables such as ‘leppet tea’ in Burma and ‘meing tea’ in Thailand.

Plants have been a major source of therapeutic agents since time immemorial. The increasing acceptance of traditional herbal systems of medicine, like Ayurveda, within India and outside has resulted in the revival of ancient traditions of medicine. Medicinal plants and their derivatives are thus looked upon not only as a source of affordable healthcare but also as an important commodity item of international trade and commerce. As per World Health Organization estimates, traditional medicines, mostly plant drugs, cater to the health needs of nearly 80% of the world population. The medicinal plant related trade is growing rapidly every year due to the growing recognition of natural products being non-narcotic, with relatively less side effects and being easily available at affordable costs.

In India, medicinal plant sector has traditionally occupied an important position in the socio-economic, cultural and spiritual arena of rural and tribal lives. About 8000 flowering plants, 650 lichens, 650 algae, 200 pteridophytes and 150 bryophytes are attributed with medicinal properties. The Indian coded systems encompasses a large number of treaties on recognized systems of medicine viz. Ayurveda, Unani and Siddha. The Ayurvedic system of medicine, which cater to the health needs of a major segment of population currently utilizes as many as 1000 single drugs and over 8000 compound formulations of recognized merit. Other systems of medicine viz. Siddha, Unani and Amchi (Tibetan) systems of medicine together utilize about 1800-1900 medicinal species. Many medicinal plants are the source of clinically useful prescription drugs being used in modern systems of medicine. Also incredible knowledge on phyto medicine is acquired in non-coded form by tribals and rural community, as is clear from evidences related to folklore medicine. Recognizing their importance, medicinal plant sector has been identified as one of the key areas by the Government of India and the Department of Indian System of Medicine and Homeopathy.

Phytochemistry

Pytochemistry is concerned with enormous variety of organic substances that are elaborated and accumulated by plants and deals with chemical structures of these substances and biosynthesis, and their natural distribution and biological function. Chemical constituents of plants can be classified in different ways. Based on biosynthetic origin, solubility properties and presence of certain key functional groups, it is classified as phenolic compounds, substances that are readily recognized by their hydrophilic nature and by their common origin from aromatic precursor shikimic acid. Terpenoids share lipid properties and a biosynthetic origin from isopentenyl pyrophosphate (Fig. 1). Organic acids, lipids, alkenes and related hydrocarbons, polyacetylenes and sulphur compounds are derived biosynthetically from acetate. Nitrogen compounds of plants like amino acids, amines, alkaloids, cynogenic glycosides, indoles, purines, pyramidines and cytokinins and chlorophyll are basic substances recognized by their positive responses to either ninhydrin or Dragendorff reagent. Monosaccharides, oligosaccharides, sugar alcohols and cyclitols are water-soluble carbohydrates and their derivatives. Macromolecules of plants such as nucleic acids, proteins and polysaccharides are easily separated from other constituents by their high molecular weights (Harborne, 1984). Some of the therapeutically active compounds are discussed below:

Medicinal plants and their various products are extensively utilized not only in traditional systems of medicine but also in modern medicine throughout the world. In recent times, they have also attracted attention of scientists encompassing various disciplines of medicine and biotechnology for search of new drug molecules and modern drug development. Although several research strategies are followed for drug development only the traditional, empirical and molecular methods/strategies are mostly applied. There is also need of scientific validation for safety, efficacy and rational use of traditional form of medicine along with development of standards for identity, purity and strength (Singh and Bagchi, 2002).

Approaches of Drug Developement

Following approaches are generally employed to discover and invent new therapeutic agents from plants viz. screening of plant materials, identification of active constituents, pharmacological and clinical evaluation and drug design.

Good Agricultural Practices

Guidelines for Good Agricultural Practices of Medicinal and Aromatic Plants is intended to apply to the growing and primary processing of all such plants traded and used in food, feed, pharmaceutical, flavouring and perfume industries. The main aim is to ensure that the raw material meets the demands of the consumer and maintains standards of the highest purity. Important aspects are that, these are produced hygienically, in order to reduce microbial load to a minimum, and are produced with care, so that the negative impacts affecting plant during cultivation, processing and storage can be minimised. The guidelines provide guidance for producers to reduce raw material contamination to the lowest level. This document can be used in bilateral relationship between the supplier and the customer and the products documentation is regarded confidentiall (EUROPAM).

Aloe, known for laxative property is one of the old natural remedies that have emerged as modern miracle drug. Popularly known as ‘nature’s sun screen’ has growing use as skin tonic in cosmetic industry and as a nutraceutical. Aloes are also quite popular among succulent ornamental plants because of their stiff, harsh and rugged habit and are commonly grown in deserts and other dry situations. Genus Aloe is indigenous to African continent and Mediterranean countries such as Greece and Southern Italy, and got naturalized in arid tracts of India. In India, Aloe barbadensis is cultivated on a small scale in Rajastan, Andhra Pradesh and Gujarat (CSIR, 1998).

Botany

Genus Aloe belonging to Liliaceae comprises 275 species distributed in Africa, Asia and America. Forty-two species belong to Madagascar region, 12-15 to Arabian Peninsula and rest are distributed over tropical South Africa. Aloe barbadensis, A. ferox and A. arborescens are used as herbal drugs. In India, only four species occur and of these, Aloe barbadensis is the most widely naturalized species. These taxa comprise several varieties viz., officinalis, chinensis, littoralis and they cross freely and several intermediary forms occur in semi wild state.

Aconites, the tuberous roots of which contain highly toxic alkaloids, are deadly poisons. In small doses, aconites are beneficial in nasal catarrh, sore throat, gibbous, paralysis and chronic fevers. Aconitum is a genus of perennial, erect or rarely twining herbs distributed over North temperate regions of world. The mountains of Eastern Asia form the primary centre, presenting the greatest diversity of genus. About 28 species are reported from India, mostly confined to alpine and subalpine belts of Himalayas. Of the Aconitum spp. occurring in India, roots of nine species are obtained in Indian market. In fact, aconite sold in Indian market is a mixture of 304 species. Still, A. ferox, known as Indian Aconite, is considered the most important.

A. ferox is a perennial herb with tuberous roots. Roots are paired, daughter tuber, ovoid oblong to ellipsoid, with filiform root fibres. Leaves are scattered, orbicular-cordate to reniform, palmately 5 lobed, resembling melon leaves. Petioles are pubescent. Flowers are pale dirty blue, borne in a dense terminal raceme, 10-25 cm long. Follicles are oblong with long, obovoid winged seeds.

Introduction

Fruits are undoubtedly God’s gift to mankind. Fruits are protective foods as they contain vitamins, minerals and enzymes, necessary for better functioning of human body and for fighting diseases. History of fruits is as old as that of Adam and Eve who ate the forbidden apple. Date palm seems to be the earliest fruit cultivated by man as early as 7000 B.C. Pomegranate is also one of the oldest fruits which was under cultivation as early as 3500 B.C. During ancient times, monks survived solely on fruits and milk giving an indication about importance of fruits and antiquity of fruits in India. India is the place of origin of mango, banana, phalsa, jack fruit, bael, aonla and a few citrus fruits whereas all other fruits are introduced from foreign countries. Litchi, loquat, persimmon, peach and sweet orange are from China; papaya, guava, sapota, avocado, custard apple and pineapple are from tropical America; and apple, plum, pear, date palm, mangosteen, strawberry, cherry, almond, walnut, apricot, grape and pomegranate are from the Central Asiatic Regions.

Introduction

India has a long history in the cultivation of vegetable crops and it sustains the livelihood of a large section of population directly or indirectly. Vegetable crops offer high returns,  have a potential to alleviate the poverty of people, provide nutritional security and essential raw materials for agro industries. These crops also offer greater rural employment opportunity and have high potential for foreign exchange earning. India bestowed with varied soil and high climatic conditions has excellent potential for growing both tropical and temperate vegetables. India is the second largest producer of vegetables in the world after China. The total area under vegetables is estimated 5.9 million ha which contributes more than 2.5% of total cropped area. The production is estimated  90.83 milion tonnes. China having a production of about 278.59 million tonnes  is the largest producer of vegetable crops in the world. The annual growth rate in vegetables is of the order of 2.6%. Potato, brinjal, tomato, chilli, cabbage, onion, cauliflower,okra and peas are grown on commercial scale. West Bengal, Uttar Predesh, Bihar, Orissa and Karnataka are the leading vegetable producing states. Maharashtra, Gujarat, Karnataka, Uttar Pradesh and Andra Pradesh are the leading states in onion production where as West Bengal, Uttar Predesh and Bihar are the leading potato producing states. Bengal, Orissa, Bihar and Assam are the leading brinjal producing states.

Introduction

Flowers are the most fascinating objects in the world and many poems are written glorifying them. They are associated with mankind from the dawn of civilization. Flowers are an integral part of our day to day life and they find a place in worshiping, religious and social functions, wedding, interior decoration and self adornment. Flowers are used to convey the human feelings. To say it with flowers is very common in western culture. Emergence of a strong middle class and changing life style has created a great demand for cut flowers in India.

Introduction

Plantation crops are high value industrial crops with great economic importance and play vital role in trade. It contributes to environment protection and sustains a large number of agrobased industries. It has great potential for utilization of waste lands in varied agro-ecosystems like rainfed, dryland, hilly, arid and coastal, providing higher employment opportunities, nutritional security, eco-friendly, high potential for foreign exchange earnings and above all providing livelihood security to people. They contribute 25% of AGDP. Major plantation crops grown in India are coconut,cardamom,cinchona, arecanut, oilpalm, cashew, palmyra, rubber, cocoa, tea and coffee. These crops grown between 20° N and 20° S of equator occupy nearly 3.2 million ha with a production of 9.8 million tonnes. All these crops are perennial in nature and demand  labour round the year. All plantation crops enter international trade and all of them have a long juvenile period. Coconut, arecanut and cashew are cultivated by individual land  owners who are not organized to the same extent as  planters of tea, coffee and rubber.

Introduction

Spices hold a special place in world trade and were a catalyst in many historical events, discoveries, wars and inventions. It has even been cited as an important element in the spread of knowledge and culture. Spices are non-leafy parts of plants used as a flavoring or seasoning. Asia is known as ‘Land of Spices’ as it is the place of origin, production, consumption and export of most spices. Out of the world’s 70 plant species grown as spices, 13 are considered major spices produced in Asia. Spices had many uses, from adding flavour to food to medicinal properties, but only grew in a few places. In the last decade, the annual average growth in spice production was 4.3% and trade in spices grew at an average of 5.8% /year. More sophisticated consumers, health-conscious eating habits (but also the rise in the amount of processed foods that use herbs and spices as inputs), rising incomes, increased world trade and globalisation are driving this new era of profitability. Spices are ideal commodities for international trade. They are normally exported in raw or dried form and are usually easy to store and transport. Consequently they do not need expensive infrastructure such as cold chain management or advanced storage facilities and they are not particularly time sensitive. In addition, they are recognised as high value products that can be produced relatively easily on small farms and do not require investments in advanced technology and machinery.

Analysis of materials or samples is essential to understand their nature and quality. In recent years it has become mandatory to display the composition of food as well as non-food items. This is because of the need for their safety from the point of view of their day to day use. Environmental awareness has further imposed many restrictions on items of wide ranging nature. Analytical information gained from a laboratory is highly useful in knowing the qualitative and quantitative composition of the material.

Chromatography is a general technique available for the separation of closely related compounds in a sample. The term chromatography is derived from Greek where chroma means colour and graphein means to write. It is a very useful technique for both qualitative (preparative ) and quantitative (analytical) analyses. The separation is effected by the differential distribution of the components between two immiscible phases, namely, stationary and mobile phases. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning (partition coefficient) between the mobile and stationary phases. The stationary phase is a porous medium through which the sample mixture percolates under the influence of a moving solvent (mobile phase).

Paper chromatography (P.C.) is the easiest to perform and requires simple and ordinary apparatus. It easily provides qualitative information since components of a mixture can be separated based on their mobilities or molecular weights. It also can provide quantitative information if some special attention and calculation procedures are followed. Paper chromatography is a technique that involves placing a small dot or line of sample solution on to a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent and sealed. As the solvent rises through the paper, it meets the sample mixture, which starts to travel up the paper with the solvent. This paper is made of cellulose, a polar substance, and the compounds within the mixture travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far.

Column chromatography is a separation technique in which the stationary bed is in a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase through the tube (open tubular column). Differences in rates of movement through the medium are calculated as different retention times of the sample.
 
The separation of compounds in the column chromatography takes place based on the phenomenon of adsorption. Here the substance gets attracted by electrostatic forces to the surface of a unit particle. If the substance is in solution and the particle insoluble in the solvent, then part of the substance is adsorbed and part remains in solution. The ratio between the amount adsorbed and the amount in solution is a constant called adsorption coefficient. The separation takes pace based on the differences in the adsorption coefficients.

Kirchner was the first in 1950 to think of thin layers. But it was Egon Stahl in 1958 who was responsible for bringing out standard equipment and related procedures to popularize thin layer chromatography (TLC). TLC is a modified form of column chromatography. Hence it is also called open column chromatography. The adsorbent is spread over a supporting material (glass or plastic sheets) to form a thin layer of adsorbent. As a binder, plaster of Paris or gypsum is added to the adsorbent material like silica gel (hence silica gel TLC). The adsorbent is spread over the plate as a thin layer of 0.25 or 0.5 mm. The adsorbent to water ratio is 1:2. It is initially allowed to air dry, followed by drying in an oven at 110oC for 1-2hr or overnight (activation of TLC plates) after which the plate is ready for thin layer chromatography.

Electrophoresis is the migration of particles under the influence of a direct electric current. There are two requirements for this process to be carried out. First the particles to be separated should be charged or at least should accept charge. Proteins, amino acids, nucleotides etc. conform to this requirement. Second, the medium used for separating the compounds also should carry charge. The mobilities of the ions depend on the nature of the electrolyte solution, the concentration and temperature. The cations and anions move in opposite directions with the cations moving towards the anode and the anions towards the cathode.

This technique is used in the separation of ionised and ionisable compounds.It consists of an insoluble matrix to which charged groups are covalently bound. The charged groups are associated with mobile counter ions. These counter ions can be reversibly exchanged with other ions of the same charge without altering the structure of the matrix.

The ion exchanger is complex and polymeric. The polymer carries an electric charge that is exactly neutralized by the charges on the counter ions. These active ions are cations in a cation exchanger and anions in an anion exchanger. A cation exchanger consists of polymeric anions and active cations whereas anion exchanger consists of polymeric cations and active anions. Amberlite IR 120 and Dowex 50 WX are cation exchangers while Amberlite IRA 400 and Dowex 1X are anion exchangers. Ion exchangers should be insoluble in water and hydrophilic to allow the ions to pass through. It should be chemically stable and should be denser than water when swollen.

Gel-filtration chromatography or molecular sieve chromatography is a method based on molecular dimension. The gel consists of beads which are composed of dextran polymer (sephadex, agarose or polyacrylamide gels). The separation depends upon the different abilities of the various sample molecules to enter pores which contain the stationary phase. Very large molecules, which never enter the stationary phase, move through the chromatographic bed fastest. Smaller molecules, which can enter the gel pores, move very slowly through the column, because they spend a proportion of their mobility, time in the stationary phase. Molecules are, therefore eluted in the order of their decreasing molecular size.

Solvent extraction is mainly used to remove dissolved substances from solutions or soluble material from solids /powders/ solid mixtures. Solvent extraction is explained using distribution law or partition law. The law states that if to a system of two liquid layers made up of two immiscible liquids/ slightly miscible liquids is added a third substance soluble in both layers , then the substance distributes itself between the two layers so that the ratio of concentration in one solvent to the concentration in the second solvent remains constant at a constant temperature.

Dialysis
 
The main purpose of dialysis in biological research is the removal of small molecular weight (M.W.) constituents from biological fluids. This is achieved by dialysing the material in a cellulose tubing with a pore size of 40-80 A0 (4-8mm) diameter which allows the passage of compounds with molecular weight less than 10,000. The following is the procedure followed: cut the length of the dialysis tubing depending upon the volume to be processed. Open the sides of the tubing with a flow of distilled water and transfer it to a boiling water bath. Do not allow the tubing to dry as it will distort the pore size. Make a bag by tying a knot at one end of the tubing. Transfer the solution to be dialysed into the bag. Tie another knot at the top of the bag. The bag is then put in a beaker containing the solution against which dialysis is to be carried out. In general, one volume of the desired solution is dialysed against 50-100 volumes of buffer. The total period of dialysis could range from 8-48hr. The efficiency of dialysis could be increased by stirring and changing the dialysate solution at frequent intervals.

When a particle revolves around an axis, a force is developed and acts away from the axis of rotation. This force is called centrifugal force. A particle suspended in a medium takes some time to sediment, which depends upon the size, molecular weight, and viscosity or density of the medium under normal “g” (gravity). To speed up the process of sedimentation, the external field of force is allowed to act on the particle which depends also on the distance of the particle from the axis of rotation in addition to the other factors.

Gas chromatography is one of the most valuable techniques available for organic analysis. It has been applied for the separation, identification and analysis of virtually all volatile compounds. In gas chromatography, the mobile phase is gas. When the stationary phase is liquid , it is called gas liquid chromatography (GLC). If the stationary phase is solid, it is called gas solid chromatography (GSC). The sample is transported through a stationary phase with help of an inert carrier gas, namely the mobile phase. The fractionation of the components in the sample mixture takes place based on the partitioning between the stationary and mobile phases.

High pressure (or performance) liquid chromatography (HPLC) is more versatile than GC since it is not limited to volatile compounds. The choice of mobile and stationary phases is wider. HPLC has high resolving power, separations are speedier, quantifications are more precise and it is in general, more reproducible. Liquid chromatography basically involves separation of components due to differences in the equilibrium distribution of sample components between the mobile (liquid) and stationary phases.

The mass spectrometry (MS) is not very well suited to the analysis of mixtures of similar substances because of the multiplicity of fragment ions. The gas chromatography (GC), of course,  has no ability to identify compounds on its own. The GC/MS can easily overcome these defects and can identify the components (fractionated by GC). The combined instrument of GC/MS is an unexcelled instrument that can identify the compounds after their  fractionation.

The GC-MS is composed of two major building blocks: the gas chromatograph and the mass spectrometer. The gas chromatograph utilizes a capillary column which depends on the column’s dimensions as well as the stationary phase properties. The difference in the chemical properties between different molecules in a mixture and their relative affinity for the stationary phase of the column will promote separation of the molecules as the sample travels the length of the column. The molecules are retained by the column and then elute (come off) from the column at different times and this allows the mass spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized molecules separately.

Spectrophotometry measures how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through the sample solution. Chemical compounds normally absorb or transmit light over a certain range of wavelength. Hence spectrophotometry is used to measure the amount of a known chemical substance. It is one of the most useful methods of quantitative analysis in the laboratory and is widely used.

A  colorimeter or spectrophotometer is an instrument that measures the amount of photons (the intensity of light) absorbed after it passes through the sample solution. With the spectrophotometer, the amount of a known chemical substance (concentrations) can also be determined by measuring the intensity of light detected. Depending on the range of wavelength of light source, it can be classified into two different types, namely,1) UV-visible spectrophotometer which uses light over the ultraviolet range (185-400nm) and visible range (400-700nm) of electromagnetic radiation spectrum and 2) IR spectrophotometer which  uses light over the infrared range (700-15000nm) of electromagnetic radiation spectrum.

Infrared light (800-1500nm) interacts with molecules at the level of atomic vibrations. An infrared spectrum gives valuable information about the molecular structure. It is rarely used for quantitative purposes and hence concentration measurements are not possible. Infrared light detects vibrations characteristic of functional groups. For example the C=O (carbonyl) group absorbs at 1700 cm-1 (wave number). The main region of interest for analytical purposes is from 2.5- 25µm or 4000- 400 cm-1. At wavelengths below 25µm the radiation has sufficient energy to cause changes in the vibrational energy levels of the molecule, and these are accompanied by changes in the rotational energy levels. The pure rotational spectra of molecules occur in the far infra red region (50-1000 µm) and are used for determining molecular properties.

When an element burns in a flame it emits radiation characteristic of its identity. Flame photometry is the determination of an element in solution by measurement of emission of light by atoms of the element in flame. Initial tests for identification of a few elements by flame tests are well known. Na burns with yellow flame, K with lilac flame, Ca, with red flame, Cu, with green flame etc. Emission of characteristic radiation by an element and correlation of emission intensity with the concentration of that element form the basis for flame photometry.

The sample to be analyzed is prepared in solution and sprayed under controlled conditions into a flame. The light from the flame enters a monochromator to isolate the desired region of the spectrum. A photocell and an amplifier measure the intensity of the isolated radiation. After proper calibration of the photometer with solutions of known composition and concentration, it is possible to correlate the intensity of a given spectral line with the unknown concentration of that element. A calibration curve prepared using known concentrations is useful. Internal standards and addition of known concentration of sample to be quantified are more useful.

Atomic-absorption (AA) spectroscopy uses the absorption of light to measure the concentration of gas-phase atoms. Since samples are usually liquids or solids, the analyte atoms or ions must be vaporized in a flame or graphite furnace. The atoms absorb ultraviolet or visible light and make transitions to higher electronic energy levels. The analyte concentration is determined from the amount of absorption. Applying the Beer-Lambert law directly in AA spectroscopy is difficult due to variations in the atomization efficiency from the sample matrix, and non-uniformity of concentration and path length of analyte atoms (in graphite furnace of AA). Concentration measurements are usually determined from a working curve after calibrating the instrument with standards of known concentration.

Atomic-absorption (AA) spectroscopy uses the absorption of light to measure the concentration of gas-phase atoms. Since samples are usually liquids or solids, the analyte atoms or ions must be vaporized in a flame or graphite furnace. The atoms absorb ultraviolet or visible light and make transitions to higher electronic energy levels. The analyte concentration is determined from the amount of absorption. Applying the Beer-Lambert law directly in AA spectroscopy is difficult due to variations in the atomization efficiency from the sample matrix, and non-uniformity of concentration and path length of analyte atoms (in graphite furnace of AA). Concentration measurements are usually determined from a working curve after calibrating the instrument with standards of known concentration.

Inductively coupled plasma atomic emission spectroscopy (ICP-AES) uses an inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. A plasma is a neutral gas containing a good number of positive and negative ions or free electrons. A plasma can only be created. A combustion flame is one example of a plasma. Advantages of ICP-AES are excellent limit of detection and linear dynamic range, multi-element capability, low chemical interference and a stable and reproducible signal. Disadvantages are spectral interferences (many emission lines), high cost and operating expense and the fact that samples typically must be in solution.

Nuclear Magnetic Resonance spectroscopy is a powerful and theoretically complex analytical tool. The nuclei of certain isotopes are continuously spinning with an angular momentum, which can give rise to an associated magnetic field.

In 1896, French physicist Becquerel observed that uranium compounds affected a photographic plate that was wrapped inside a black paper. He found that all uranium compounds gave off penetrating rays. He called the production of radiation by uranium compounds “radioactivity”. Madame and Pierre Curie later identified thorium, polonium, and radium as the other radioactive elements.

Mass spectrometry (MS) is a  useful  technique which produces spectra of the masses of the atoms or molecules in a sample.  The spectra are used to determine the elemental or isotopic   details  of a sample. The masses of particles and of molecules, are used to elucidate the chemical structures of molecules, such as peptides and other chemical compounds. Mass spectrometry works by ionizing chemical compounds to generate charged molecules and measuring their mass-to-charge ratios.

A sample, which may be solid, liquid, or gas, is ionized, by bombarding with electrons. This may cause some of the sample’s molecules to break into charged fragments. These ions are then separated according to their mass-to-charge ratio, typically by accelerating them and subjecting them to an electric or magnetic field. Ions of the same mass-to-charge ratio will undergo the same amount of deflection. The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier. Results are displayed as spectra of the relative abundance of detected ions as a function of the mass-to-charge ratio. The atoms or molecules in the sample can be identified by correlating known masses to the identified masses or through a characteristic fragmentation pattern.

Compounds having one or more asymmetric carbon atoms can rotate the plane of polarized light. Such compounds are called optically active. Measurement   of this property is called polarimetry.
 
The magnitude and direction in which the plane of polarized light is rotated depend on 1.The nature  of the compound, 2. The  concentration of the solution, 3. The path length of light, 4. The solvent, 5. The wave length of light and 
6. The temperature. These variables are related and can be presented in the formula

When a fixed voltage is applied to two electrodes dipping into a solution,a current will flow, depending on the conductivity of the solution. Conductivity measurements do not give information about the nature of the ions in solution. However, they are useful in the quantitative measurement of the ions.

When a metal M is immersed in a solution containing its own ion Mⁿ⁺, then  an electrode potential  develops. The value of  the  electrode potential  is given by the Nernst equation.

Immunoassays rely on the ability of an antibody to recognize and bind a specific macromolecule in what might be a complex mixture of macromolecules. In immunology the particular macromolecule bound by an antibody is referred to as an antigen and the area on an antigen to which the antibody binds is called an epitope.

In some cases an immunoassay may use an antigen to detect for the presence of antibodies, which recognize that antigen, in a solution. In other words, in some immunoassays, the analyte may be an antibody rather than an antigen.