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Electrophoresis
EXPERIMENT
75
Electrophoresis Discussion If a background electrolyte is allowed to flow down a sheet of filter paper across which an electric field is applied, then an ion applied at an intermediate position on the paper will deviate laterally in accord ance with its inherent mobility. This principle is known as electro phoresis and can be applied to the separation of a mixture of ions. An apparatus which may be used to demonstrate this principle is represented diagrammatically in Fig. 1. The filter paper sheet A is suspended from a trough B in which a constant level of electrolyte is maintained. The test substance is in a small container C from which it is fed to the filter paper sheet by means of a paper wick D. Electrically neutral species will follow a straight path in the direc tion of the electrolyte flow whilst those which are positively charged will deviate towards F and those negatively charged towards E. The extent of deviation will be governed by the size and charge of the various species. Apparatus and Chemicals Shandon Continuous Electrophoresis Apparatus, Power Pack and Dipping Tray, electric fan, drying oven, Whatman 3 M.M. paper,* 101. of 1 M acetic acid, pyridine, bromine, 0-2 per cent solution of ninhydrin in acetone, 0-5 M sodium hydroxide, 0-2 per cent solution of isatin in acetone, 0*1 per cent solution of 8-hydroxy quinoline in ace tone and 50 ml of an aqueous mixture of 0-04 M arginine, 0-04 M glycine and 0-04 M aspartic acid. Method The continuous electrophoresis apparatus is set up as described in the instruction pamphlet. The reservoirs, trough B and electrode vessels G and H are filled with 1 M acetic acid. The paper sheet is folded 1 -5 cm from the non-serrated end and then folded again in the opposite direction 8 cm from the same end. The filter paper sheet is placed so that the first fold dips into trough B where it is held in position by a * This paper is supplied by Reeve Angel and Co. Ltd. 202
Electrophoresis
203
heavy glass rod. The second fold rests on the glass rod at the edge of the trough. The cabinet is completely closed and the paper allowed to become saturated with acetic acid solution. During this period of saturation the working potential difference of 1000 V is applied across the paper. This will produce a current of about 5-6 mA once the filter paper is saturated with buffer.
F I G . 1. Schematic diagram of continuous electrophoresis apparatus.
The specimen container is prepared for use by cutting a piece of filter paper as shown in Fig. 2, wrapping it around a short length of wick and inserting this into the spout. The container is then filled with the solution of amino acids. The power pack is switched off, the cabinet is opened and the container placed in the holding bracket so that the wick touches the paper about 3 cm from the anode side. The cabinet is closed and electrophoresis is allowed to proceed for 18 hr. At the end of this time, the power is switched off, the paper removed from the cabinet and the acetic acid solution evaporated off. The evaporation is carried out in a fume cupboard using an electric fan.
Experiments in Physical Chemistry
204
The dried paper is dipped in a 0-2 per cent solution of ninhydrin in acetone to which a few drops of pyridine have been added and then hung in an oven at about 100°C for 5 min. Colour will develop in curved bands indicating the paths along which the individual amino acids have migrated. In order to determine the extent of separation and to identify the amino acid corresponding to each path, the following tests are applied to the solutions in the appropriate test tubes.
This part rolled round wick
ZÛ mm
5 mm 20 mm FIG. 2. The paper wick.
A spot of solution is placed on a filter paper strip and dipped into a 0-1 per cent solution of 8-hydroxy quinoline in acetone. The acetone is allowed to evaporate. The paper is then dipped into a solution of 0-3 ml of bromine in 100 ml of 0·5 M sodium hydroxide and allowed to dry. An orange-red colour indicates the presence of arginine. This is an application of the Sakaguchi reaction which is exclusive for monosubstituted guanidines. Again a spot of solution is placed on a strip of filter paper and the paper dipped in a 0 -2 per cent solution of isatin in acetone to which a few drops of pyridine have been added. The paper is heated at 105°C for 2-3 min. A blue colour indicates the presence of aspartic acid. This reaction is given by a number of other amino acids but not by glycine or arginine. The coloured band leading to the samples which give neither of the above reactions corresponds to the path followed by the glycine molecules.
75
Electrophoresis Discussion If a background electrolyte is allowed to flow down a sheet of filter paper across which an electric field is applied, then an ion applied at an intermediate position on the paper will deviate laterally in accord ance with its inherent mobility. This principle is known as electro phoresis and can be applied to the separation of a mixture of ions. An apparatus which may be used to demonstrate this principle is represented diagrammatically in Fig. 1. The filter paper sheet A is suspended from a trough B in which a constant level of electrolyte is maintained. The test substance is in a small container C from which it is fed to the filter paper sheet by means of a paper wick D. Electrically neutral species will follow a straight path in the direc tion of the electrolyte flow whilst those which are positively charged will deviate towards F and those negatively charged towards E. The extent of deviation will be governed by the size and charge of the various species. Apparatus and Chemicals Shandon Continuous Electrophoresis Apparatus, Power Pack and Dipping Tray, electric fan, drying oven, Whatman 3 M.M. paper,* 101. of 1 M acetic acid, pyridine, bromine, 0-2 per cent solution of ninhydrin in acetone, 0-5 M sodium hydroxide, 0-2 per cent solution of isatin in acetone, 0*1 per cent solution of 8-hydroxy quinoline in ace tone and 50 ml of an aqueous mixture of 0-04 M arginine, 0-04 M glycine and 0-04 M aspartic acid. Method The continuous electrophoresis apparatus is set up as described in the instruction pamphlet. The reservoirs, trough B and electrode vessels G and H are filled with 1 M acetic acid. The paper sheet is folded 1 -5 cm from the non-serrated end and then folded again in the opposite direction 8 cm from the same end. The filter paper sheet is placed so that the first fold dips into trough B where it is held in position by a * This paper is supplied by Reeve Angel and Co. Ltd. 202
Electrophoresis
203
heavy glass rod. The second fold rests on the glass rod at the edge of the trough. The cabinet is completely closed and the paper allowed to become saturated with acetic acid solution. During this period of saturation the working potential difference of 1000 V is applied across the paper. This will produce a current of about 5-6 mA once the filter paper is saturated with buffer.
F I G . 1. Schematic diagram of continuous electrophoresis apparatus.
The specimen container is prepared for use by cutting a piece of filter paper as shown in Fig. 2, wrapping it around a short length of wick and inserting this into the spout. The container is then filled with the solution of amino acids. The power pack is switched off, the cabinet is opened and the container placed in the holding bracket so that the wick touches the paper about 3 cm from the anode side. The cabinet is closed and electrophoresis is allowed to proceed for 18 hr. At the end of this time, the power is switched off, the paper removed from the cabinet and the acetic acid solution evaporated off. The evaporation is carried out in a fume cupboard using an electric fan.
Experiments in Physical Chemistry
204
The dried paper is dipped in a 0-2 per cent solution of ninhydrin in acetone to which a few drops of pyridine have been added and then hung in an oven at about 100°C for 5 min. Colour will develop in curved bands indicating the paths along which the individual amino acids have migrated. In order to determine the extent of separation and to identify the amino acid corresponding to each path, the following tests are applied to the solutions in the appropriate test tubes.
This part rolled round wick
ZÛ mm
5 mm 20 mm FIG. 2. The paper wick.
A spot of solution is placed on a filter paper strip and dipped into a 0-1 per cent solution of 8-hydroxy quinoline in acetone. The acetone is allowed to evaporate. The paper is then dipped into a solution of 0-3 ml of bromine in 100 ml of 0·5 M sodium hydroxide and allowed to dry. An orange-red colour indicates the presence of arginine. This is an application of the Sakaguchi reaction which is exclusive for monosubstituted guanidines. Again a spot of solution is placed on a strip of filter paper and the paper dipped in a 0 -2 per cent solution of isatin in acetone to which a few drops of pyridine have been added. The paper is heated at 105°C for 2-3 min. A blue colour indicates the presence of aspartic acid. This reaction is given by a number of other amino acids but not by glycine or arginine. The coloured band leading to the samples which give neither of the above reactions corresponds to the path followed by the glycine molecules.