Chemical Instrumentation S. 2. LEWIN, New York University, Woshington Square, New York 3, N. Y.
T h i s series of articles presents a survey of the basic principles, chmacteriftics, and limitations of those instruments which find important applicatimts i n chemical work. The emphasis i s on cmnmercially available equipment, and approximate prices are quoted to show the order of magnitude of cost of the various types of design and construction.
14. Chromatographic The application of an electric potential to s solution containing dissolved or suspended particles which interact with (i.e., feel a force due to) the field is a general technique for producing directed migration of those particles. The term ionophoresis was originally ooined to refer to the movement of small inorganic ions under the influence of an electric field, and electrophoresis was later defined as designating the analogous motion of colloidal particles. If the conditions in the fluid are so arranged that a, aone, interface or boundary layer is initially formed by the specimen in the liquid medium, and if there are present in the specimen particles having different mobilities, the application of the eleotric field oiluses each t,ype of particle to migrate a t a characteristic rate, and a set of mare-or-less distinguishable zones is formed. The resolution of a mixtureinto its components in this manner is called zone eleetrophomis. There are two types of zone electrophoresis; the classical, free liquid technique, and the stabilized medium technique. The classical technique involves differential eleotromigrstion in a free (i.e., unstabiliaed) liquid. In order to achieve effective separations hy this technique, great care must be taken to eliminate the effects of mechanical vibration, agitation, or convective disturbances. Even with these factors under control, free liquid zone electrophoresis is generally successful only with high molecular weight samples, since the random diffusional processes which oppose the directed migration and smear out the electrophoretic pattern are faster, the smaller the migrating species. The substitution of a. stitbilised medium for the free liquid has produced s. significant advance in technique, for it has made possible many types of quick analyses and separations with simple, inexpensive, and efficient equipment. The types of st* bilieed media which have been employed include: liquid supported on paper, liquid held by capillary forces in packed columns of cellulose powder, liquid supported on glass powder or beads, agar gel, silica, gel, starch gel, and polyacrylamide gel. Electromigration in one of these stabilized media is often a much more complex mechanism than free electrophoresis, for i t involves adsorption and partition effects superimposed upon the electric field effeots. Hence, the terms paper (or
starch, or agar, etc.) electrophoresis, which are widely used, have been criticized because they do not emphasize the important roles which may he played by adsorption and partition. Consequently, the term eleetmeh~omatographyhas k e n proposed to cover all types of electrophoretic techniques in stabilized media. Some authors prefer to use pherographv to denote these techniques.
Design Considerations for Stabilized Medium Pherographs When a voltage is applied across a length of one of the stabilized liquid media described above, the ensuing flow of current produces the following effects: ( 1 ) electromigration of charged particles in the direction of the oppositely charged electrode; ( 8 ) heating of the migration track, which results in evaporation of liquid and a consequent flow of liquid from the wetter to the dryer regions, and (5) endosmotic flow of the liquid in the direction of one of the electrodes. In the case of mast of the supparting phases used in electrophoretic work, the solid tends to acquire a negative charge when it is in contact with an aqueous solutian containing an electrolyte. When a potential is applied, this charge causes the supporting phase to want to migrate toward the anode. Since it is rigid and cannot so migrate, the same relative motion is produced b , ~a streaming of the liquid phase in the opposit,~direction, is., toward the cathode. This electroosmotic flow is greatest in ground glass. and decreases in the order agar > starch > paper. On paper, the rate of streaming increases with increasing pH. These factors imposo n practical limitation on the length of the migration path for which an instrument of this type can he designed. If the linear distance between the ends of the migration path is too large, the passage of current may dry out t,he central part of the supported medium faster than liquid can Ron- to i t from the buffer reservoirs a t the ends. Even nit,h short path instruments, there is a considerable buffer flow from both ends toward the center of the migration path. The net result is s. nan-uniform rate of fluid flow as tl function of distance along the path, as indicated schemstically in Figure 34. Aa a migrating component
moves along, it may find itself traveling against the current of buffer flow arising from factom (2) and (3) above. When it reaches a place where its migration velaeity is equal and opposite to the rstc of flow of the medium, it no longer advances, and nothing is gained by lengthening the migration path or prolonging the time of electrophoresis. In general, the mobility of an ion is inversely proportional to the square root of the ionic strength of the solution. Hence, the rate of electrophoresis can be speeded up by working in solutions of low ionic strength. However, rednction in the ionic strength causes the resistance of the medium to increase, and in order to gain in speed of migration, it is necessary to increase the applied voltage in proportion to the rise in resistance. The en8rg.v dissipated per unit time in the medium due to thc Ron of current is:
P
=
EP/R
=
E XI
If R is constant, as in the case of a medium of constant ionic strength, the power increases as thesquare of the applied voltage, If I is constant, as in the rsse where the applied voltage is adjusted to offset increases in R, the pos-er increases linearly with the applied voltage. The practical limitation to the amount of power input that can be tolerated is the efficiency of cooling of the migration path by gas convection, evaporation, and/or a sold tahle support for the medium.
t'
5.
Figure 34A. During e1edrophare.i~ with a supported liquid, the buffer streams across the rupport or a consequence of endormoris plus the effect of evaporation from the central portion of the migration poth. B. The net flow of liquid is not uniform from one end d t h e poth to the other.
Each zone in an electrophoretoeram (pherogram) tends to spread out due to thermal diffusion. At s. given tempemture, this spreading is proportional to the square root of the time. Hence, an electrophoresis run should be completed in as
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