Basics and Principles : The term "Flow Cytometer" refers to an instrument which is capable of measuring various properties of cells which are in motion. Flow = Motion, Cyto = Cell, Meter = Measurement. Flow Cytometry is a biomedical technique which is used to analyze, count and sort different populations of cells from a heterogeneous population of cells. This instrument is used to measure the biophysical and biochemical properties of cells or particles under study by understanding their light scattering especially fluorescence scattering properties. Instrument allows the measurement of thousands of cells per second, which makes it a quantitative instrument capable of quantifying the fluorescence intensity. It has been extensively used in immunological studies, disease monitoring, biomedical studies, identification and characterization of microorganisms, identification of variety of cellular markers, bio-marker detection, the list g
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Affinity chromatography was originally developed for the enzyme purification. Now, it has been extended to immunoglobulins, membrane receptors, nucleic acids, nucleotides, whole cells and cell fragments. It is theoretically capable of giving absolute purification from complex mixtures in a single process. Affinity precipitation , in which the ligand is attached to a soluble carrier that can be subsequently precipitated by for example pH change. Affinity Partition , in which the ligand is attached to a water-soluble polymer such as polyethylene glycol and which, with the ligand bound, partitions into an aqueous polymer phase that is in equilibrium with a pure aqueous phase. In this chromatography, the most commonly used matrix are, cellulose, agarose, polyacrylamide, polystyrene, cross-linked dextrans and porous glass antisilica. Applications of Affinity Chromatography : 1. used to purify a large variety of macromolecule such as enzymes, membrane recept
This type of chromatography is also called as "Molecular Sieve Chromatography" or "Molecular Exclusion Chromatography". When a gel having pores on its matrix is packed in the column and a heterogeneous mixture is pass through it, smaller ions enter the gel and come out of the column phase. The bigger molecules can not enter the gel, they move only with the mobile phase through the inner phases of the gel. In this process, a type of sieving of molecules occur. Widely used gels are Agarose, Porous Glass, Sephadex, Polyacrylamide, Polystyrene and Silica Granules. Gel Permeation chromatography can be carried out using the technique of column chromatography. (By determining the distribution coefficient, molecular weight of macro molecules can be determined).
Thin Layer Chromatography principle is similar to paper chromatography. The sample is spotted at one end of a coated glass plate, with the slurry of the adsorbents. Usually, Silica gel or Sodium Acetate. Using solvent systems develops the spotted plate and the spots are deducted as in paper chromatography.
In this chromatography technique, the substance is analyzed by allowing the flow of solvents on a a specially designed filter paper. Due to capillary action, the solvent rises up on the paper and due to differential migration of the substances of a mixture, the separation is achieved. In this type of chromatographic technique, Whatman Paper I is extensively used and this paper is available in different grades. The sample is prepared by using proper solvent and is applied on the paper as bands.
An analytical technique which is used for the isolation, identification and separation of compounds based on differences in affinity for a stationary phase and a mobile phase. In chromatography, the stationary phase may be a solid, liquid, gel or a solid/liquid mixture.. and mobile phase may be a liquid or a gas.
Electron Microscopy Image Source: sv.rkriz.net In electron microscope, the source of illumination is electron beam. The construction and principles of electron microscope are easily related to those of light microscope. The range of wavelength of visible light used in light microscope is 4000Å-7000Å, while with an electron microscope employing 60-80KV electron, the wavelength is only 0.05Å. In the instrument, the electron gun generates electron beam. These electrons are concentrated by other components of electron gun producing a fast moving narrow beam of electron. Electrons are focused by electromagnetic lenses. Electromagnetic lens consists of wire encased in soft iron casing. When electric current is passed through the coil, it generates an electromagnetic field through which electrons are focused. There are three types of electromagnetic lenses. The one is placed between the source of illumination and the specimen. This fo