JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY HYDERABAD B.Pharmacy IV ADVANCED INSTRUMENTATION TECHNIQUES Previous Question Paper (July-2021 - R17)

Mass spectrometry separates ions based on their mass-to-charge ratio (m/z). The process begins with the ionization of the sample, where molecules are converted into gas-phase ions. These ions are then accelerated by an electric field into a mass analyzer.

In the analyzer, ions are subjected to electromagnetic fields that exert a force proportional to their mass and charge. According to Newton's second law and Lorentz force principles, the trajectory of an ion is determined by its m/z value. For example, in a magnetic sector analyzer, the magnetic field deflects ions in a circular path with a radius r defined by the equation:

where B is the magnetic field strength, V is the accelerating voltage, m is the mass of the ion, and z is the charge. Ions with different m/z values follow different paths, allowing them to be spatially or temporally separated. By varying the magnetic field or the accelerating voltage, ions of specific m/z values are sequentially focused onto a detector, which records the intensity of the ion current, resulting in a mass spectrum.

Radioimmunoassay (RIA) is a highly sensitive competitive binding technique used to measure the concentration of antigens by using antibodies and radiolabeled antigens. The methodology follows these steps:

1. Preparation of a standard curve using known concentrations of unlabeled antigen.
2. Incubation of a fixed, limited amount of specific antibody with a constant amount of radiolabeled antigen (tracer) and the unknown sample containing unlabeled antigen.
3. Competitive binding occurs where the unlabeled antigen from the sample and the radiolabeled antigen compete for the limited binding sites on the antibody.
4. Separation of the antibody-bound antigen from the free antigen using methods like charcoal adsorption, precipitation, or solid-phase separation.
5. Measurement of the radioactivity in either the bound or free fraction using a scintillation counter.
6. Calculation of the unknown concentration by comparing the radioactivity of the sample to the standard curve, where the amount of bound radiolabeled antigen is inversely proportional to the concentration of unlabeled antigen in the sample.