eChapters

Abstract

The phosphorus deficiency in soil is a major problem for agricultural production as phosphorus is one of the major constituents in energy metabolism and biosynthesis of nucleic acids and cell membranes. The phosphorous present in soil is an important macronutrient for the growth and development of the plants. The phosphorus occurs in two forms in soil, organic and inorganic. The phytates (salts of phytic acid) constitute the major form of organic phosphorus other than nucleic acids, phospholipids and other compounds in soil. Phytate-phosphorus is not readily available to plants due to its chelation with metal ions forming insoluble complexes or adsorbed to soil particles. Phosphate solubilizing and phytate hydrolyzing microorganisms, bacteria and fungi, are ubiquitous in cultivated, wetland, grassland and forest soils, which play an important role in supplying soluble phosphorus to plants. Microbes hydrolyze organic forms of phosphorus by secreting phytases and acidic/alkaline phosphatases. By genetic engineering, several attempts have been made to develop transgenic plants which are able to utilize phytates as sole source of phosphorus for their growth and development. This review focuses on the applicability of microbial phytases in promoting the growth and development of various crop plants.

Abstract

The greatest challenge facing us in the coming years is to produce adequate amounts of basic necessities of food, feed, fibre, fuel and raw materials for the rapidly shrinking per capita agricultural land. One alternative is to make a sustainable use of existing marginal lands. In many regions of the tropics and sub-tropics, the need to use marginal low-input soils with usually inadequate nutrient(s) supply permits only very low or unreliable yields to be achieved. It is frequently not possible  to supply required amounts of mineral fertilizers or chemical pesticides because of the low purchasing power of the marginal farmers. In particular, it is not possible to compensate for the deficient nutrient supply and this constitutes a yield limiting factor. The role of soil microorganisms in sustainable productivity has been well construed. In recent years, root inhabiting (rhizozosphere) bacteria (rhizobacteria) is gaining importance.

Abstract

With the growing awareness on the benefits of organically cultivated food, biofertilization and biocontrol by plant-associated bacteria are being used as alternatives to chemical fertilizers and conventional control methods. It therefore becomes essential to continue surveying different agroecological niches to identify plant growth promoting bacteria suitable for each microenvironment. Species belonging to the genus Enterobacter are versatile colonizers of numerous environmental niches. Several species and strains from this genus have also been routinely recovered from the rhizosphere of crops such as rice, canola, cucumber, banana, potato, corn, tomato and cotton. These isolates have been studied and shown to have beneficial effects on plant growth and protective action against phytopathogens.

Introduction

Microorganisms are a great source of genetic diversity, still far from completely known and explored. The intimate association of plants with complex microbial communities is of great importance for the growth and productivity of plants in agriculture. There exists a definite difference in microbial community present in the bulk soil and the neighboring micro environments like phyllosphere, endosphere, rhizosphere and endorhizae. Microorganisms that colonize the plants’ rhizosphere are either pathogenic or beneficial according to their effects on plants and the way they interact with roots. The rhizosphere supports a plethora of microbial populations capable of exerting beneficial, neutral or detrimental effects on plant growth and development. Microorganisms such as bacteria, actinomycetes, protozoa, algae, fungi, and viruses coexist in the rhizosphere; bacteria are the most abundant among them.

Abstract

Endophytes are microorganisms  that live inside the living plant tissues for at least part of their life without causing any apparent disease symptoms in the host. In general  they  can be divided into two main types namely, obligate and facultative endophytes. Facultative endophytes are capable to survive in the soil, on the plant surface, inside the plants as well as on artificial nutrients and can be cultured away from their host. Endophytes which inhabit inside plant tissues throughout their lifespan are called as obligate endophytes. Natural products produced by endophytic microbes are found to be antimicrobial, antiviral, anticancer, antioxidants, antidiabetic and immunosuppressant. Therefore, endophytes form an untapped source of novel natural compounds for application in agriculture, medicine and industry.

Abstract

The pressure due to increase in world population necessitates an increase in agricultural production and this warrants the vital importance of research in plant and soil productivity. Applications of chemical fertilizers and pesticides have resulted in soil pollution, desertification and salinisation which disrupted the agriculture production. A suitable microbial technology to improve the soil health and plant productivity has been invented. Increase in the use of bacterial fertilizers substantially reduced the use of chemical fertilizers. Similarly invention of specific antagonistic microbes which control pathogen and stimulate plant growth resulted in lesser use of agrochemicals. Selection, development and application of such microbial strains to the agronomic crops resulted improving the fertility of such affected soil. The studies on plant growth promoting rhizobacteria (PGPR) on the growth and nutrient uptake of various plants have been well studied. Presently, it has been found that the use of beneficial microbes can enhance the resistance ability to adverse stresses. Some mechanisms by which the beneficial microorganisms are able to stimulate plant growth and arrest plant pathogens include mobilization of nutrients, production of phytohormones, siderophores, organic acids, nitrogen fixation, lowering of ethylene levels in plants and antagonism. Here the recent developments that have been taken place on the interaction between plants and PGPR and their mechanism of action resulting in enhanced agriculture production and soil fertility are discussed.

Abstract

Plant-associated microorganisms play a significant role in conferring resistance to abiotic stresses. These organisms include rhizoplane and endophytic bacteria and symbiotic fungi and operate through a variety of mechanisms like triggering osmotic response and induction of novel genes in plants. The development of stress tolerant crop varieties through genetic engineering and plant breeding is essential but a long and strained process, whereas microbial inoculation to alleviate stresses in plants could be a more cost effective environmental friendly option which could be available in a shorter time frame. Considering the current trends available, intensive future research is needed in this area, particularly on field evaluation and application of potential organisms.

Abstract

The medicinal plant, Nithyakalyani, Catharanthusroseus is valued for its alkaloid and the content varied, among root (0.14 to 1.43%), stem (0.48%), fruit (0.4%), seed (0.18%) and pericaup (1.14%). The biofertilizer consortium development includes Catharanthusroseus. Azosppirillumlipoferum CAZS-4, Azotobaterchroococcum CAZB-1, Bacillusmegaterium CPB-18 and Pseudomonasflourescens CPF-14. The relative chemotactic response (RCR) of the strains to anionic fraction is more than that of cationic and neutral fractions. The consortium treated plants possessed 1.120 mg g^-1 of ajmalicine on 180 DAS, and the antioxidant superoxide dismutase (SOD) (13.240 U mg of protein hr-1), peroxide (POX) (11.240 U mg of protein hr-1) and catalase (CAT) (9.000 U mg of protein hr^-1) values were higher on 150 DAS. The consortium increased the biomass production by 50-75 per cent with proportionate increase in the mineral nutrient content. Biofertilizer consortium is the most effective organic input for biomass production.

Abstract

Any environmental variable, can adversely affect the optimal growth conditions of the plants. The stressed environment makes the plants to alter their physiology for survival rather than reproduction. Prolonged drought, intense raining, frequent floods, cold waves high and low temperatures are the predicted abiotic stresses in the future due to climate changes. Water stress is one of the important growth limiting factor which decreases plant growth during vegetative stage. Crops grown under rain-fed conditions are the most affected by seasonal variations in rainfall. Therefore failure of monsoon, high temperature and salinity usually result in water stress. In addition, it causes serious negative impacts on crop growth and yields. A greater stress tolerance capacity could also be largely attributed to the plants by the symbiotically associating microorganisms capable of modulating stress factors. The microorganisms having close association with plants are referred as plant associated bacteria (PAB), which can be grouped into rhizospheric (in vicinity of root) and rhizoplanic (on surface of root), endophytic (inside the plant tissue)  and phyllospheric (on the leaf surface ) bacteria. Among the PAB, pink pigmented facultative methylotrophic (PPFM) bacteria belonging to the  genus Methylobacterium are well known for their intimate ubiquitous occurrence in the plant.

Abstract

Current trend of global agricultural systems entails preference towards sustainable crop production through organic agriculture (ecology based) as compared to conventional (synthetic input based). In order to claim as organic, it needs to be certified as per requirements organic standard. The soil fertility management organic standard requirements of major countries are outlined. This review article compares the agricultural management practices of organic and conventional systems so as to assess its impacts on microbial biodiversity, microbial activities on soil functioning and long term fertility based on long term field studies conducted in different parts of the globe.

Abstract

Microorganisms execute many key functions that regulate the processes related to the fertility status, nutrient transformation, nutrient conservation, and soil health management that are crucial for agronomic sustainability. Rice soils offer peculiar conditions for microorganisms that support diametrically opposite biochemical reactions under flooded conditions. It is pertinent to note that both reduction and oxidation reactions of several essential nutrients mediated by microbes occur simultaneously in the flooded soils at various depths, profiles and stages of rice crop growth in concurrence. This is peculiar and specific to rice soils as the cultivation process demands changes in water regimes, operative and management practices.

Abstract

Of the various rhizosphere associated bacteria, Azospirillum species are probably the most studied and appear to have significant potential for commercial application. During the last 35 years of studies of Azospirillum–plant interaction, over 20 proposals were suggested for the mechanism of action by which Azospirillum  enhances plant growth, yield and nutrient uptake. Besides, fixing the nitrogen with associative symbiosis, they impart a number of beneficial effects on plants such as phytohormone production, siderophore production, enzyme production, proliferation of root system, enhanced water and mineral uptake, mobilization of minerals, mitigation of environmental stressors of plants, and moderate biocontrol potential. The following plant responses to Azospirillum inoculation in agriculture crops were often reported: increase the germination percentage and height of the seedlings, increase the total number and length of adventitious roots and root hairs, increase the hydraulic conductivity of the root system, increase in the height of the shoot and dry matter production and yield of the crop plants. The effect of Azospirillum inoculation on the total yield increases of field grown plants generally ranged from 10 to 30 per cent. This article reviews the recent researches on role of Azospirillum in plant growth promotion.

Abstract
 
This work aimed to evaluate agromicrobes preponderance and activity in maize ecosystem of same soil types (diara land/fragile land) under different agricultural practices. We conducted a five year field survey of grower’s field to investigate the effect of tillage system, maize plant genotypes and soil organic carbon level on preponderance of diazotroph (Azotobacter), plant growth promoting rhizobacteria (Pseudomonas) and arbuscular mycorrhiza (AM) colonization of maize ecosystem. No tillage, traditional maize cultivar and soil organic carbon (>1%) appear to support the preponderance of targeted diazotrophs, plant growth promoting rhizobacteria (PGPR) in maize rhizosphere and AM colonization of maize root. Elevation in the preponderance of bacteria, Azospirillum population in maize root, AM colonization, microbial biomass, nitrogen mineralization, phosphate solubilising microbes, cellulose hydrolyzing microbes and acetylene reducing assay (ARA)  of maize root appeared due to organic fertilization of maize crop.

Abstract

The nutrition of tobacco plants relies upon the plant genotypic variation and soil characteristics which also influence the various microflora that inhabit the rhizosphere. The investigations were aimed to analyze the role of native and inoculated plant growth promoting bacteria on the uptake of major plant nutrients viz., nitrogen, phosphorus and potassium and also their supportive nature in reducing the infection caused by  Pythium aphanidermatum, the damping-off fungus of tobacco in nursery. Rhizosphere microorganisms include PGPR viz., Azotobacter, Azospirillum, Pseudomonas and Bacillus, AM fungi and Streptomyces. The addition of these microbes as bioinoculants in tobacco integrated nutrient and disease management resulted in better quality tobacco worth premium price.

Abstract

Cyanobacteria comprise a group of photosynthetic prokaryotes, which exhibit versatility in their morphological, biochemical and molecular characteristics and represent an untapped source of novel metabolites and genes. Their structural-functional flexibility makes them successful in diverse and extreme environments including high temperatures or levels of UV light and salinity, and co-exist in symbiotic/parasitic/mutualistic associations with various members of Plant and Animal kingdom. The intracellular colonization of cyanobacteria in wheat and rice roots and their potential as plant growth promoting agents for several crops, besides rice, has led to the deployment of novel formulations of cyanobacteria and agriculturally useful bacteria as biofilms and disease suppressive options in field studies.

Abstract

Agricultural production is often limited by low phosphorus (P) availability. In developing countries, which have limited access to P fertiliser, there is a need to develop plants that are more efficient at low soil P. Arbuscular mycorrhizal (AM) symbioses are widespread in the plant kingdom and contribute significantly to plant P nutrition and growth in natural ecosystems. Production and application of these beneficial fungi for agricultural purposes has grown over the last decades worldwide. However, the necessity of AM inoculum production via a host plant is still an obstacle to ample utilization of AM fungi in agricultural crops. The present article deals with the basic and applied aspects of AM research and its role in sustainable agriculture.

Abstract

Soil is the store house of 1/4^th of the global biodiversity. Arbuscular mycorrhizal (AM) fungi is one of the important group of organism occurring in soil. The beneficial effect of AM fungi in improving plant growth is well documented. Many of the functions performed by AM fungi can provide essential services to human society. These services include nutrient cycling, soil structure, plant growth promotion, resistance against biotic and abiotic stresses and bioregulation of plant development. These services are not only essential to ecosystem function but also a critical resource for the sustainable management of agricultural systems. Recent studies have brought out that both plant biodiversity and ecosystem productivity increases with the increasing number of AM fungi. Thus, it appears that the diversity of AM fungi in soil is a major factor contributing to the maintenance of plant biodiversity and the ecosystem functioning. The role of AM fungi in ecosystem services, plant diversity and land productivity are discussed in this review.

Abstract

Soil microorganisms play a major role in the biodissolution of nutrients bound in the soil silicate minerals. Plant nutrients like K, P, Ca, Mg, Fe, Zn, and Mn are bound in them. Silicon is considered as agronomically important as it confers rigidity and strength to crop plants and enhances yield. Biodissolution of these nutrients result in their availability to crops influencing the growth, biomass and yield. The microbial metabolites like organic acids, amino acids, inorganic acids, amines, ammonia, phenolic compounds, extracellular polysaccharides, siderophores, ligands of cations and cation exchange are involved in the biodissolution of K, Zn and Si. Frateuria aurantia, Bacillus mucilaginosus and B. edaphicus and Bacillus sp are recognized as potash mobilizers, silica and zinc solubilizers.

Abstract

Discovery of the agricultural utility of microbes during the later part of the eighteenth century, led to the formulation and utilization of superior microbial strains as biofertilizers, for a wide variety of crops and growing conditions across the world. Though microbial inoculants have been in usage for almost two centuries, they are yet to capture the collective imagination of farmers across the globe, which is essential to meet the larger objective of reducing chemical input usage in cropping systems. One probable reason for this is the existence of large growing tracts that are subject to various abiotic stresses of atmospheric and edaphic origin, which limit the proliferation and efficiency of the microbial strains that are commonly used in inoculant production.  This is obvious, since unlike chemical fertilizers, microbial cells being living systems need to grow and thrive in the introduced eco-systems in order to achieve the desired effects of inoculation. But unfortunately microbial inoculant technology has largely adopted the one size-fit all mindset, which had led to failures of microbial inoculants especially in abiotically stressed growing environments. In such a scenario it would be wise to adopt a location or region specific approach for the selection of superior and locally adapted microbial strains for inoculant production.

Abstract

Microbial inoculants are low cost inputs with high benefits in agriculture. Solid carrier based (peat or lignite) inoculants are used in most tropical countries. The carrier based inoculants have short shelf life, are liable to have high contaminants and have shown inconsistent field performance. In recent years, special liquid formulations containing not only the desired microorganisms and their nutrients but also added cell protectants or chemicals that promote formation of resting spores or cysts have been developed. The liquid formulations have longer shelf-life (12 months at room temperature) and are also more tolerant to adverse conditions encountered in storage. Liquid Rhizobium biofertilizers enhanced the soybean crop yields in the United States of America, Brazil, Europe and Asia over carrier based inoculants.

Abstract

Understanding the impacts of organic or inorganic nutrient management on soil biology and biochemistry may help to develop more sustainable fertilization strategies. The results from comprehensive study made on a field experiment comparing the long-term (more than 100 years) effects of organic manures and inorganic chemical fertilizers on soil biological and biochemical properties are reviewed. The soils adopted with three nutrient managements viz., no fertilization (control), inorganic chemical fertilizers (IC) and organic amendments (OM) obtained from long-term fertilizer experiment were assessed for physical, chemical, microbiological and biochemical properties in three successive years. The response of most of the assessed soil variables to long-term organic nutrient management was significantly higher than those from inorganic fertilization and control (OM > IC = control).

Abstract

The potent greenhouse gases N₂O and CO₂ are relased by aerobic/anaerobic respiring bacteria, archaea, fungi and protists in increasing amounts from converting virgin rain forest and permafrost soils, over fertilized agricultural fields and grasslands including animal husbandry, compost/waste water treatment plants, oceans, savannahs, forests of the temperate climate zones in annual amounts of 0.5 to 4.2 terragrams (Tg) N₂O-N into the atmosphere after autotrophic and hetreotrophic NH₄⁺ to NO₃- oxidation. With estimated 0.25% industries contribute to the annual atmospheric N₂O-N concentration increase.

Abstract

Complex industrial wastewater by using conventional treatment method is a significant challenge for its safe disposal. Recently, two step treatments by aerobic bacteria using activated sludge process followed by rhizofiltration has been recommended as a promising technology for detoxification of complex industrial waste. In activated sludge process recalcitrant compounds are biotransformed along with biodegradation of organic waste. Aerobic treatment may be activated sludge process, trickling filter or aerobic lagoon process, which one is used for treatment  it is based upon the characteristics of wastewater. Carbon and nitrogen ratio along with other organic compound is also play important role for microbial growth and degradation of pollutants. During degradation of various pollutants, metal microbes interaction play significant role for its bioremediation either through adsorption or bioaccumulation.

Soil is the most precious basic natural resource, on which depends the security of food, nutrition, income, biodiversity, livelihood and environment. But, its indiscriminate and unscientific management is a major threat to sustainability of agriculture. Over the years, there is a loss in the productive potential of soils, which is attributed to imbalance in nutrient use, mining of nutrients from soil, poor replenishment, loss of carbon, pollution, decrease in biological activities, waterlogging, soil salinity etc. Thus, land use is not commensurate with land capability.

Plant nutrient disorders result from nutrient imbalances in soils. Nutrients are considered essential for plants, when it satisfies the following criteria (Arnon and Stout,1939):
    1.    The deficiency of the nutrient element makes it impossible for the plant to complete its life cycle
    2.    The deficiency of the element is specific and can be corrected only by supply of that element
    3.    The element is directly involved in the nutrition of the plant

Soils of India vary widely among different regions. The first soil map of India was prepared and classified on the basis of existing knowledge of Russian pedological principles. Govind Rajan (1971) and Govind Rajan and Rao Gopal (1978) have discussed in detail the later developments on the preparation of soil map of India and giving the equivalents of different soil groups in US Comprehensive System of Soil Classification. Soil resource mapping was initiated in 1986 and in a span of 10 years (1986-1996), a detailed database in terms of soils, their area and extent, characteristics and grouping following soil taxonomy (Soil Survey Staff, 1999) was created. The present chapter on “soils of India-nutrient status and management concerns” takes into account the following aspects:

A. Soil Sampling

Soil is a store house of several nutrients. These are present in available and reserve forms and a dynamic equilibrium always exists between these forms of nutrients in soil. If the available form of a given nutrient element is depleted by crop uptake or by any other means, there will be immediate replenishment from the reserve source. In this context, it is also appropriate to note that soil is not an eternal source of plant nutrients. In a bid to produce more, soils are exploited very badly and they are gradually becoming deficient in several nutrients. There are several soil related constraints coming in the way of achieving sustainability in crop production.

Plant analysis includes tests on fresh tissue in the field and analysis of total nutrients in plants in the laboratory. Plant analysis is based on the relationship between nutrients in a plant and nutrient availability in a soil. Plant analysis is not a direct method of soil fertility evaluation, but it can serve as a valuable supplement to soil testing in the task of soil fertility evaluation. Plant analysis is useful in confirming nutrient deficiencies, toxicities and imbalance and identifying hidden hunger. Plant analysis generally provides more current plant-based information than soil testing, entails more effort in terms of sampling, sample handling and analysis and, perhaps, expensive.

In agricultural systems, water quality is usually determined to  assess its suitability for irrigation and/or its use for aquaculture. Analysis of irrigation water is carried out, to know their nutrient load, suitability for irrigation purposes in general and to salt sensitive crops in particular, and their significance as nutrient suppliers as in practical crop production (Tandon,1993). For aquaculture, on the other hand, maintenance of pond environment for survival and growth of aquatic lives, forms the major purpose of such exercises. While both the assessments are mostly governed by some common variables, a few additional attributes are also used for identifying the water quality for these two different purposes. The most important characteristics, that determine the quality of irrigation water are:

Earth provides enough to satisfy every man’s need but not every man’s greed.
—Mahatma Gandhi
The term ‘environment’ is derived from French word ‘environner’, which literary means ‘to surround’ or ‘to encircle’ i.e. everything either living (plants, animals, microbes) or nonliving (soil, water, air, temperature, humidity etc.) surrounding us makes our environment. The universe is full of uncountable number of stars, planet and satellites, which has specific type of environment but earth is the only known planet in the universe that contains life supporting environment. The earth environment is a collective term for the nature or ecosystems, that we use and benefit from on a local, national and global scale. The earth ecosystems provide number of benefits called ecosystem services, free of cost.

Spatial variability in soil parameters including nutrients has been attributed to the parent material, topography, landforms, cropping pattern, fertilization and manuring practices.. Blanket nutrient recommendations further widen the variability and enhance the risk of soil degradation in terms of soil organic carbon and nutrient mining, acidity, green house gas emission, water and environmental pollution. Unfavorable economics on account of blanket recommendation, adversely influence farmers to increase investments in new crop management technologies. This results in low farm productivity and poor soil health, that jeopardize food security and agricultural sustainability.

1. Why Plant Nutrient Disorders?

Plant Nutrient Disorders result from nutrient imbalances in soils and plants. In-adequate use of one or more nutrients can result in nutrient imbalances. Presence or use of a nutrient in excess amount can hinder the absorption or uptake of another nutrient and thus,cause nutrient disorders. Lime induced iron chlorosis or use of excess Potassium can hinder the absorption of Ca²⁺/and, or Mg²⁺ by plants. Enhanced level of Mg can decrease the uptake of Zn. Excessive use of NPK fertilisers can result in micronutrient deficiencies.

Soils are being regularly depleted of their native reserves, due to intensive cropping and non-application of essential nutrients (primary, secondary and micronutrients). Lack or restricted use of quality organic manures, such as compost, green manures, crop residues etc. and cultivation of HYVs in most crops have accentuated the problem.

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.