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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

The hybrid breeding program in pigeonpea is unique and the first among the food legume. The concept of hybrid in this crop was conceived in 1974; and it is based on a small window of natural out-crossing present in the crop. This natural out-crossing in pigeonpea is mediated by a range of flying insects, which forage on large bright colored flowers of the crop in search of nectar and in the process affect the cross-fertilization. In the last 40 years a lot of basic and applied research has been successfully completed and over 50 research papers have been published in national and international journals of repute. A coordinated effort of scientists of different institutions has led to the release of two hybrids, one for central and another for peninsular zone of India.

With the advent of hybrid technology, it is natural to assess its potential benefits at the national level. To meet the domestic requirement of pigeonpea, about 50.000–60,000 metric tons of pigeonpea is imported annually from Myanmar and Africa. In this communication, an attempt has been made to look into the possibility if we can meet the entire domestic needs of pigeonpea through the adoption of hybrid technology. It is a big question but the goal is not impossible. The estimates shown in Table 18 show that if only 2% of total pigeonpea area can be brought under hybrids, the target can be met with ease. This will require seed production in only 700-800 hectares.

To overcome the challenge of popularizing pigeonpea hybrids in the country, it is considered fair enough to compare the hybrid rice with respect to their seed production technology, yield advantages and profitability to the farmers and seed producers. Since both pigeonpea and rice are grouped under self-pollinated crops, this comparison, in the views of authors, may help in encouraging hybrid pigeonpea seed producers and those involved in marketing of hybrid seed.
  
Rice is a highly self-pollinated crop and earlier hybrids were not possible because it was essential that large quantities of hybrid seeds are produced easily and economically. In China, the birth place of hybrid rice, this impossible looking task was accomplished. To launch this program, the Chinese scientists decided to alter the pollination system of rice.

Fig. 25: ICPH 2671 in field

5.1 Seed back up programs

In hybrid breeding program, testing of new hybrid combinations is a continuous process and in most cases, the availability of crossed (hybrid) seed for their annual evaluation of selected hybrids is an important issue. Since, the selection of hybrids for advancement is based on yield data, one year will be required to produce seed for testing in the subsequent season. This means for each cycle of advancement, one valuable season will be consumed in their seed production. To avoid this situation and enhance the testing program at a fast pace, an aggressive hybrid seed production program should be followed. This can be achieved by visually selecting relatively more number of hybrids based on their flowering time, plant vigor, disease resistance, and early pod load about 4-5 weeks before harvesting. During this period, the new seed of the selected hybrids can be produced manually. This operation is facilitated by the perennial nature of pigeonpea plant, which can flower for extended periods of 40-50 days; that allows completion of desired number of pollinations of the target crosses in the same cropping season. One trained person can pollinate about 300 buds in 6-7 hours; and with 20% success, about 150 seeds can be produced. Experience has shown that, with good planning and crop management, this program can be achieved with ease.

4.1. Flower and flowering in pigeonpea

Growth habit: Primarily, there are two major growth types in pigeonpea. These are determinate and non-determinate. The determinate growth in the pigeonpea plants is characterized by flowers in bunches at the top of the canopy (Fig 17a). In this case the epical growing points are transformed in to reproductive buds and bunches of flowers appear on the top of the canopy. Such cultivars are not popular as the management of insects such as Helicoverpa and Maruca becomes very difficult. Most of the cultivated types belong to another growth category called as non-determinate. In such genotypes the apical growing points remain vegetative and the plants keep growing under favorable conditions and it results in tall canopy with many spreading or compact branches. The inflorescence in this case emerges from the axil of secondary and tertiary branches (Fig 17b); and hang sideways. These types show less susceptibility to insects due to non-clustering nature of flowers.