Applications

Electrophoresis involves the separation of charged species on the basis of their movement under the influence of an applied electric field. It has found wide applications in the characterization of biological molecules (proteins and nucleic acids). The main applications of electrophoresis have been in the separation of biological molecules, which includes molecules with

Table 1 Methods of detection for quantitative analysis of sample components separated by electrophoresis

Optical methods

UV absorption

Staining

Fluorescence

Raidiochemical methods

Liquid scintillation counting

Autoradiography

Biological assay and

Immunoelectrophoresis

immuno-methods

Rocket electrophoresis

relatively lower relative molecular masses such as amino acids, and also molecules of higher relative molecular masses such as proteins and polynucleot-ides (including RNA and DNA molecules). An example of the use of paper electrophoresis follows.

Paper electrophoresis has been extensively used in almost all laboratories where proteins and other similar macromelecular electrolytes are investigated. The apparatus (Figure 2) consists of two electrode chambers placed 15 cm apart. There is also a device which can support up to six (30 cm) filter paper strips between the electrodes. A d.c. supply source (0-250 V) is used to apply the desired voltage across the electrodes. The two electrode chambers are filled to equal heights with the buffer solution. The buffers commonly used for this purpose are (1) Aronsson and Gronwall buffer, i.e. dimethyl barbiturate buffer, which is a mixture of 20.60 g sodium dimethyl barbiturate and 2.80 g barbituric acid with a pH of 8.6, and (2) Consden and Powell buffer, i.e. borate buffer, which is a mixture of 1.77 g sodium hydroxide and 9.60 g orthoboric acid with a pH of 8.6.

Whatman paper (M540) strips (about 30 cm long) are cut and dipped in a container of buffer until they are thoroughly wet. The excess buffer is then removed by laying them out on a large sheet of filter paper. The strips are then immersed into the electrode chambers so that the ends of the strips dip in the buffer solutions. The sample is applied at the centre of the paper. The paper strips are allowed to stand for about 1 h, to equilibrate the bed with the liquid evenly throughout the paper. The power supply is then switched on and the voltage adjusted to about 75 V. Excellent sharply defined separations of serum proteins into five fractions within a span of 2 cm can be obtained within 1 h. If the run is extended to 16 h, a pattern approximately 12 cm long with five fractions may be obtained. On completing the run the fractions are measured by staining. The dye most commonly employed for this purpose is amido black 10B. The paper strip is dried and developed in a dye bath containing a saturated solution of the dye in a mixture of methanol and glacial acetic acid (9:1

v/v). Staining is allowed for 10 min with constant shaking.

After the electrophoretogram has been stained, the excess of dye is removed (destaining) by dipping the stained paper in baths of methanol-glacial acetic acid (9 : 1 v/v) several times. This destaining procedure is a slow process and can be made more efficient and faster by opting for electrophoretic destaining. After destaining, the paper strip is dried and scanned in a densitometer, i.e. the strip is illuminated and moved along the light source, the transmittance showing the distribution of the separated compounds. On plotting the reciprocal of transmittance against the wavelength one or more maxima are observed, depending upon the number of components. The amount of each component can be estimated by measuring the area under each peak. The estimation of serum proteins can also be done by an elution method. The destained paper strip is cut transversely into small pieces 5 mm wide, one unstained small strip at the end of the paper providing the blank value. The elution is done in 1.0 mol L 1 NaOH in 50% ethanol # 0.25% (0.25 g per 100 mL) ethylenediaminetetraacetic acid (EDTA). After elution is over, the optical density is measured using a colorimeter.

A more rapid separation of serum proteins can be achieved using polyacrylamide gel electrophoresis. However, paper electrophoresis is still of particular interest where small amounts of protein need to be isolated for further analysis or testing.

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Solar Panel Basics

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