The word electrophoresis, as Gray pointed out, means "borne by electricity." In a broad sense the movement of charged pith balls in an electrostatic field is an electrophoretic effect. So is the Cottrell process for eliminating smoke particles from flue gases by passing them between electrodes of high potential difference. After picking up a charge from one electrode, the particles are attracted to the other, where they clump and fall into a collecting bin. The electrodeposition of colloidal rubber suspensions on electrodes of special shape-a process widely used in the manufacture of rubber gloves and other common articles-is another example.

But electrophoresis is the special name given to the technique of separating molecular mixtures into fractions. Most suspensions of molecules in water are charged and hence can conduct an electric current. Even molecules which normally do not carry a charge tend to adsorb ions from the water. Some molecules pick up more charge than others, depending upon their chemical nature and the concentration of ions in the solution. If the ionic concentration (pH) is properly adjusted, all closely related molecules, such as those of the proteins, appear to adsorb charges of the same sign. Consequently when they are subjected to an electric field they migrate in the same direction, although at rates which vary with the amount of charge on each member of the family. Many amateur microscopists have observed such migration on a gross scale with objects such as blood cells or protozoa. If a voltage is applied across a drop containing cells in suspension, the cells will migrate. Alexander Reuss first described the experiment 148 years ago, and it was a favorite of Michael Faraday.

To analyze molecular mixtures Tiselius hit upon a radically different scheme. He poured the material to be studied into the bottom of a U-shaped tube and carefully laid a buffered solution on top in each arm of the U so that sharp boundaries formed between the mixture and buffer. When a current was passed through the three-part solution, the material under analysis migrated down one arm of the U and up the other. Each of its fractions moved at a characteristic rate. The boundaries of each fraction were made visible by an elaboration of the schlieren optical technique devised by Leon Foucault for testing the figure of parabolic mirrors and lenses. Like the ruling engine for making diffraction gratings, the Tiselius technique of "free" electrophoresis is simple in principle. Like the ruling engine, too, the method 3 appears easy until you set up the apparatus and try to make it work! In this domain the gifted professional appears safe from amateur challenge.

The less precise yet powerful method 3 of zone electrophoresis has found wide application during the past five years. In the zone method, particles move in liquid that fills the spaces of a finely divided solid instead of a U-tube. Molecules of like kind migrate as distinct zones which can easily be identified and recovered as purified products. Porous solids of many kinds can serve as the medium. One medium frequently used is filter paper. Zone electrophoresis thus bears a superficial resemblance to partition chromatography. The electrophoretic separation, however, depends not upon the properties of solubility and adsorption, as in the case of chromatography, but upon the electrical charge carried by the molecules of the substance that is being analyzed.

The amount and sign of the charge picked up by compounds in solution depend both upon the chemical nature of the compound and upon the pH of the solvent. Molecules which normally carry a weak charge, such as the slightly alkaline proteins, are highly sensitive to changes in pH. A small shift in acidity or alkalinity can cause a substantial change in the rate at which such particles migrate and may even reverse the direction of their movement. One therefore selects for the solution electrolytes (sources of charge) which have a "buffering" action: that is, which tend to supply positive and negative ions to the solution at a rate precisely offsetting that at which ions are removed or dissipated. Many common salts have a strong buffering action, although table salt (sodium chloride) is not one of them.

Unfortunately there are no textbooks on electrophoresis, and most of the literature is confined to biological and medical journals not readily available. Hence an amateur who takes up electrophoresis will have to find his way through woods where few trees are blazed. Except for protein chemistry, he must develop his own electrolytes, buffers and solid media, and must find out by experiment just what voltages and current densities work best for the substance under analysis. The field of electrophoresis has barely been scratched. If you enjoy original work, you can dig in almost anywhere, certain that you are breaking fresh ground.

To give you a start, we picture here the essentials of an apparatus which uses filter paper as the solid medium [Figure 1]. You can set it up and put it into operation in a single evening.

The ends of the paper dip into two vessels containing an electrolytic solution connected through carbon electrodes to a source of direct current. To retard evaporation of the solution from the paper, we sandwich it within a pair of glass plates. The plates, about two inches wide and eight inches long, are cut from window glass. As a safety precaution it is a good idea to round the edges and corners of the glass on either a whetstone (using water as a lubricant) or on a sheet of glass smeared with a slurry of carborundum.

It is desirable to maintain an even pressure of the glass on the filter paper, so that migration proceeds in a symmetrical and reproducible pattern. Pressure improves the sharpness of the zones, because it reduces the amount of fluid in the paper. However, if the pressure is too high, it will bend the glass and distort the zones. Some workers have attempted to solve the problem by using plates an inch or more thick. The bottom plate is supported by a flat base and the top one rests on the paper as a weight. Others suspend the paper from glass rods laid across the buffer vessels. The apparatus is then covered by a bell jar and operated in a buffer-saturated atmosphere. The latter method has the disadvantage that the buffer tends to gravitate toward the low point of the strip with consequent distortion of the pattern of separation.

Capillary effects between the glass plates and the paper also introduce some distortion. This is minimized by coating the plates with a film of grease. Vaseline will work, but not so well as silicone grease of the type used for lubricating the stopcocks of chemical glassware.

Glass containers of any convenient shape can be used as buffer vessels. Heavy Pyrex icebox dishes, available from hardware dealers, work as well as specially made glassware. The principal considerations in the selection of containers are chemical inertness and enough weight so the empty vessels will support the plates, paper strip and clamps without upsetting.

Chemical inertness is a major consideration in the choice of electrodes. Most professionals use platinum, but carbon rods work almost as well. Avoid the cored carbons used in sun lamps. These cores are charged with finely divided metal (to enrich the emission of ultraviolet rays ) and will contaminate the solution. Solid carbons designed for low-intensity motion-picture projectors are good and can be procured from theater supply dealers.

The amount of electric current needed varies with the substance under analysis. A rectifier capable of operating between 50 and 300 volts at an output of 20 milliamperes will be ample for most work. You may get a good rectifier from a junked radio receiver. Just connect a 40,000-ohm wire-wound resistor (of the type fitted with an adjustable tap) across the filter condenser. The resistor should be of at least the 20-watt size. Take the output from across the ground side of the resistor and the tap. If no old radio set is at hand, you can get the parts specified in the drawing [opposite page] from radio supply dealers.

It is frequently desirable, particularly during the experimental phase of analyzing unknown substances, to maintain either a constant voltage across the paper strip or a constant current through it. Power supplies with automatic regulating features can be constructed, but they are costly and complex. Good results can be achieved with a manual control. Substituting a continuously variable potentiometer for the tapped resistor makes adjustment easy, and the knob will protect your fingers from the hot resistance element.

Almost any soft paper will demonstrate zone electrophoresis. You can use strips cut from white blotters, paper towels, cleansing tissues, even the unprinted parts of old newspapers. Clear, reproducible patterns, however, require a specially made paper of uniform texture and free of contamination. A good paper is Whatman 3MM, supplied in 600-foot rolls by the Fisher Scientific Company of New York City, which also has most of the chemicals used in electrophoresis experiments. You can order the Fisher materials at drug stores.

The separation of the artificial coloring used in a cheap wine is a nice electrophoretic project for a beginner. You can make your own mixture for analysis by adding a few drops of food coloring to grape juice. For an electrolyte you can use a weak solution of common salt buffered with a small amount of baking soda (sodium bicarbonate). Later you can investigate electrolytes made with other salts, many of which provide their own buffering action.

Food coloring migrates nicely in an electric field of 25 volts per inch at a current of 10 milliamperes. This means that the buffer-moistened filter paper should have a resistance of about 2,500 ohms. To obtain this value of resistance you will have to experiment with various dilutions of the electrolyte. Begin by drawing enough tap water to fill the icebox dishes to within half an inch of the top. Put all this water in one container and add a level teaspoon of salt. After it 11 dissolves, immerse the paper strip in the solution. Remove the strip, blot it thoroughly and clamp it between the glass plates. Then pour the solution into the icebox dishes, suspend the ends of the paper in it [drawing on page 92], connect the power supply to the solution through the carbon electrodes and adjust the potentiometer or tapped resistor to the prescribed potential of 200 volts.

If the resulting current is less than 10 milliamperes, turn off the power, remove the strip, return the solution to the common container, add more salt and try again. Usually a level teaspoon of salt for each 12 ounces of water produces the desired conductivity, but the amount needed varies with the purity o f the tap water. Finally add a quarter teaspoon of baking soda for each 12 ounces of solution. (It will affect the resistance only slightly.)

After you have an electrolyte with the proper resistance, draw a light pencil line across the middle of a fresh strip of filter paper, dip the strip into the buffered electrolyte, blot it and then apply a drop of wine to the pencil line with the blunt end of a toothpick. The wine should first be concentrated by letting it evaporate at room temperature to half or less of its normal volume. Now spread a film of grease on the inner faces of the glass plates. Clamp the paper between them, seal the edges of the plates with grease, immerse the protruding ends of the paper into the buffer and switch on the power.

If the wine sample contains artificial coloring, in about five minutes the edge of the wine spot nearest the anode should become sharper and the edge toward the cathode should grow fuzzier. Within an hour a blotch of dye, probably comet-shaped, will have migrated a substantial distance from the point of origin. As the process continues, comets of other colors, each a constituent of the dye, will trail the first one down the length of the paper [see top pattern above]. The dye fractions in the wine should be fully resolved in about six hours. (The blotch made by the wine itself will move little, if at all.) By spacing drops along the pencil line you can analyze several samples of fluid simultaneously on the same strip of paper.

The tendency of zones to smear, trail, assume comet shapes and otherwise depart from sharpness is one of the undesirable features of zone electrophoresis on filter paper. It represents a challenge to the experimenter. In general the drier you can run the filter paper- (or other solid medium), the sharper the zones will be. Within limits dryness can be achieved by applying heavy pressure on the glass plates: in effect you try to squeeze out the buffer. The spots should be dry enough so that you can rub your hand across the paper as it comes from the apparatus at the end of a run without smearing the pattern. The amateur who resolves the dilemma of applying enough pressure without bending the glass will make a contribution to science.

Substances which migrate more rapidly than others along the electrophoretic paper are said to have "high mobility." Mobility is determined in large part by the strength of ionization of the particles. Measuring the mobility of substances is an interesting project for beginners. You simply time the rate of migration of each substance along a scale ruled on the strip of paper, using a control buffer of a certain pH and concentration. Stains used for coloring organisms to show them under the microscope make nice test specimens. A particularly good series is eosin Y, methylene blue, basic fuchsin, malachite green, Bismarck brown, safranine and gentian violet. The chemical properties of these stains are listed in reference texts. Each migrates in a saline solution at a characteristic rate. The third pattern from the top on page 96 shows the relative migration rates of positively ionized methylene blue (top) and basic fuchsin (bottom), and negatively ionized eosin Y (middle). These were resolved on filter paper with a saline solution buffered with sodium bicarbonate. The same test showed that the malachite green stain migrates an inch per hour at 70 degrees Fahrenheit under 200 volts and 10 milliamperes.

Amateurs who wish to have a go at something more sophisticated may enjoy trying to separate blood proteins. This entails the sacrifice of a few drops of blood. You will also need access to a centrifuge (to extract the serum from the blood), a few grams of the barbiturate veronal and a liter of 95 per cent ethyl alcohol.

Dampen the filter paper with barbital buffer adjusted to pH 8.6. After blotting the paper, deposit five thousandths of a milliliter of serum on the ruled strip with a calibrated micropipette. Then clamp the paper between the plates and seal it with silicone grease

A potential of 15 volts per centimeter and a current of 15 milliamperes will resolve a specimen in five or six hours; however, the pattern may show traces of smearing. Four volts per centimeter and four milliamperes increases the time to 12 hours but yields sharper patterns. Blood-serum fractions are difficult to see. The albumin can be made more strikingly visible by labeling it with a few crystals of bromphenol blue. After the albumin has migrated an arbitrary distance, say seven centimeters, the paper is removed and dipped for two minutes into a solution of 95 per cent ethyl alcohol saturated with mercuric chloride, to which 1 per cent bromphenol blue is added. The strip will emerge from the stain a deep yellow. It is then washed repeatedly in water containing a thousandth part of acetic acid. On contact with water, the yellow changes to a deep blue. The color gradually disappears from the paper during washing but is retained by the protein fractions. The fourth and fifth patterns from the top on page 96 show typical separations of blood proteins taken from two individuals, one in normal health and the other diseased. The density of the spots in each pattern indicates the amount of protein in each fraction. From right to left the fourth pattern (normal serum) shows albumin, alpha-one globulin, alpha-two globulin, beta globulin and gamma globulin. The dense spot at the left in the fifth pattern is characteristic of the bone disease myeloma. Other diseases produce characteristic patterns which serve as valuable aids in diagnosis.

The result of an amateur's first attempt to fractionate albumin (the white of chicken egg) is shown in the bottom pattern on page 96. The smeared pattern explains this experimenter's passion for anonymity. Here the buffer was salt, baking soda and water.

A number of techniques have been devised for making quantitative measurements of protein patterns. In one the strip is sectioned into eighth-inch segments The dye in each is then quantitatively eluted in a two milliliter solution of 1 per cent N-sodium hydroxide and read, after an hour or so, on a colorimeter. The resulting values are plotted as points. The smooth curve drawn through them is equivalent to the curve derived by free electrophoresis.

As mentioned earlier, zone electrophoresis is not limited to filter paper. It is interesting to compare the behavior of a given test substance and buffer in media compounded of starch grains, silica gel, activated alumina and similar materials, as well as the reaction of various buffers with respect to a given medium. A slab of starch, for example, is easy to prepare. Put a pound of potato starch into a sieve lined with filter paper. Wash the starch for 30 minutes and pour it as a batter into a rectangular mold. The slab (about 3/8-inch thick) is then thoroughly blotted, and with suitable carbon electrodes it can be used in principle just like filter paper. The only limit to variations in the physical arrangement of the apparatus is set by the ingenuity of the worker. It is possible, for example, to adapt electrophoresis for the continuous separation of material in gross amounts. At least one amateur telescope maker prepares colloidal rouge by means of continuous electrophoretic separation. Buffer is allowed to flow down a wide strip of filter paper by capillary attraction. It drips from the bottom edge of the paper into a container below. The rouge mixture is fed onto the paper from a continuously flowing micropipette near the top. Electrical contact is made with the edges of the strip through wicks saturated by buffer. Fractions not ionized flow down the strip vertically. Ionized fractions take a diagonal course, the steepness of which depends upon the strength of the ionization. A scallop is cut into the bottom of the strip in line with each fraction, and collecting vessels are placed beneath the points. The collected fractions are then purified.

Bibliography

ELECTROPHORESIS. Dan H. Moore and H. A. Abramson in Medical Physics. Year Book Publishers, Inc., 1950.

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