Chromatographic separations can be carried out using a variety of different experimental arrangements. In paper chromatography the sample is spotted on to the bottom of a sheet of paper. The paper is usually impregnated with a liquid, that acts as the stationary phase. The bottom of the paper is placed in a vessel that contains a solvent. The solvent acts as the mobile phase because it carries the sample with it as it moves up the paper due to capillary forces. The molecules are separated according to the strength of their interaction with the stationary phase: the stronger the interaction with the stationary phase, the slower they move. More-sophisticated separations can be carried out using two-dimensional paper chromatography.

Thin-layer chromatography (TLC) has largely replaced paper chromatography because it is quicker, more sensitive, has greater resolution, and has better reproducibility. The general principles of TLC are similar to paper chromatography, except that the paper is replaced by a glass plate coated with a thin layer of porous material that acts as the stationary phase.

Column Chromatography. Originally, column chromatographic techniques used glass columns with either gravity or a slight vacuum to move the mobile phase through them. Steel columns and high pressures are now widely used to speed up this process. In high-pressure (or performance) liquid chromatography (HPLC) the sample to be analyzed is dissolved in a liquid mobile phase and passed through a column containing the stationary phase (which may be either solid or liquid). In gas chromatography the sample is usually volatilized and then carried by a gaseous mobile phase through a thin tube that is coated with the stationary phase. The efficiency of the separation can be manipulated by carefully selecting the most appropriate combination of stationary and mobile phases.

Electrical Techniques. A number of analytical instruments utilize measurements of the electrical properties of foods to obtain information about their composition. Po-tentiometric methods rely on measurements of the potential difference between an indicator electrode and a reference electrode. The magnitude of the potential difference is related to the concentration of a specific component within a solution. Coulometric methods are based on the measurement of the electrical charge required to completely electrolyze the substance being analyzed: the greater the electrical charge, the higher the concentration of the substance. The composition of some foods can be determined by measuring their electrical conductivity. The current passing between a pair of electrodes is measured and then related to the composition of a specific component within the food.

Electrophoresis relies on differences in the migration of charged macromolecules or colloidal particles in a solution when an electrical field is applied across it. It is used to separate molecules and particles on the basis of their size, shape, or charge. A buffered solution containing the substance to be analyzed is poured onto a porous matrix (usually a strip of paper or a gel) and a voltage is applied across it. The charged substance moves through the gel in a direction that depends on the sign of their electrical charge, and at a rate that depends on the magnitude of the charge and the friction to their movement. The friction of a substance is a measure of its resistance to movement through the matrix and is largely determined by the relationship between the effective size of the substance and the size of the pores in the matrix. The smaller the size of the substance, or the larger the size of the pores in the matrix, the lower the resistance and therefore the faster the substance moves through the matrix. Matrices with different porosities can be purchased from chemical suppliers, or made up in the laboratory, so that substances with different sizes can be analyzed. Electrophoresis techniques can be carried out in two dimensions to improve resolution. Substances are separated in one direction on the basis of their size, and then in a perpendicular direction on the basis of their charge.

Biochemical Analysis. A number of biochemical techniques are available that can be used to determine the concentration or identity of specific food components. Enzymic techniques utilize specific enzyme reactions, whereas immunological techniques utilize specific interactions between an antigen and an antibody. Biochemical assays can be purchased commercially for many important food components. These kits contain the chemicals and instructions required to carry out the analysis and are usually easy to use, rapid, extremely sensitive, and specific.

Because some of the nutritional requirements of microorganisms and experimental animals are similar, it is possible to employ analytical microbiology to determine some substances that are essential constituents of living cells. Microorganisms, as reagents, have been used to determine amino acids, vitamins, nucleic acids, heavy metals, growth factors, nutritional value of proteins, and antibodies. The basic principle is that in the presence of limiting amounts of certain compounds, the amount of microbial growth is a function of the amounts of the compounds. The microorganisms used for assay are primarily bacteria, but yeasts, fungi, and protozoans also have been used. The assay methods include diffusion in a gel, turbidimetric and dilution methods, gravimetric methods, and metabolic response methods.

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