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Chapter 2 Classification of Electromigration Methods
Chapter 2
Classification of electromigration methods J. VACfK
CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zone electrophoresis. .................................. ..................... The moving boundary method . . . . . . . . . . . . . ............................... Isotachophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Focusingmethods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................... Isoelectric focusing . . . . . . . . . . . . . . ................................... Electrorheophoresis . . . . . . . . . . . . . .......................... Combined methods .................... .......................... Disc electrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................... Immunoelectrophoresis . . . . . . . . . . . .......................... References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
INTRODUCTION Electrophoretic methods can be classified according to different criteria, some of which are presented in Table 2.1. TABLE 2.1 CLASSIFICATIONS OF ELECTROPHORETIC METHODS Criterion of setmation
Electrophoretic method
Amount separated
analytical micropreparative preparative single-step continuous free on carriers capillary gel only migration is involved migration and interaction with carrier migration, interaction with carrier and diffusion low-voltage hig h-voIt age zone moving boundary isotachophoresis focusing methods
Type of procedure Method of stabilization
Separation principle
Potential gradient used Initial and side (marginal) conditions of separation (experimental arrangement)
23 24 26 27 29 31 33 33 33
35 37
24
CLASSIFICATION OF ELECTROMIGRATION METHODS
The criteria can be compared only with difficulty and their selection is determined by purpose of a particular classification. From the theoretical point of view, classification according to initial and marginal conditions of the separation is the most interesting. This makes it possible to characterize four basic types of electrophoretic methods - zone electrophoresis, moving boundary electrophoresis, isotachophoresis and focusing methods. For illustration in this chapter some combined methods will be described; these are procedures that exploit several electrophoretic principles or in w h c h the separations are carried out both on the electrophoretic and some other principle.
ZONE ELECTROPHORESIS Characterization of the method: The whole separation column is filled with a single basic electrolytic system with a specific conductivity K . A mixture of substances to be separated is applied to a certain location in the separation column (the start). The width of the mixed zone at the start (AL) is much smaller than the length of the separation column, L (ALQ L ) . After the application of an electric field to the system, individual substances migrate according to their effective mobilities towards the electrodes and form zones. For a velocity of the ith substance at a concentration cir eqn. 2.1 can be obtained by solving the continuity equations (eqn. 1.59) for all z components of the ith substance and by use of the definition of the effective mobility (eqn. 1.28):
ci
Fig. 2.1 shows a scheme of the course of the separation of three substances, A, B and C. The case when all three substances have a charge of the same sign and therefore migrate in the same direction was chosen. Three characteristic zone shapes, symmetrical (substance A), with a sharpened front* zone boundary (substance B) and with a sharpened back* zone boundary (substance C), are shown. The shapes of individual zones are influenced both by the distribution of the potential gradient and by the shapes of the adsorption isotherms (this holds for separations on carriers). In zone electrophoresis, the distribution of the potential gradient can have two characteristic courses: (a) The specific conductivity of the basic electrolyte is such that it is not influenced by the presence of the substance t o be separated. The potential gradient is then constant along the whole column. This is the most common type of zone electrophoresis. (b) The specific conductivity of the solution at a location in the zone K , differs from that of the basic electrolyte. Two possibilities are K , > K [usually if (UJefr > (Uel)eft] or
* The “front” refers to the concentration profile of the ith substance for which ( a q / a t ) , > 0; the “back” boundary is characterized by the inequality (aci/at), < 0.
25
ZONE ELECTROPHORESIS
I
I
x-
x-
-
Fig. 2.1. Schematic representation of the course of separation of three substances, A , B and C, by zone electrophoresis for (Ug)eff > (LIAIeff > (Uc),ff:(1) at the beginning of the experiment at zero time (i = t o ) ;(2) at time t l ( t l > t o ) ;(3) at time fZ(f1 > t , ) . Substance A ( H ) forms a symmetricalzone, forms a zone with a focused front and C(H) forms a zone with a focused end. The solid line represents the total concentration.
B(a)
K, < K [usually if (UJeir < (LleJeff]. In the former instance the potential gradient is higher at the site of the zone than outside it. This results in a situation where the back boundary of the zone becomes sharpened (ions that had been delayed enter a region of a higher potential gradient and catch up the zone), whereas the front boundary becomes extended (ions that had outrun the zone move to a region of a higher potential gradient and become even further removed). When K, < K the opposite situation occurs (the front boundary becomes sharpened and the back boundary becomes tailed). In a similar manner t o the distribution of the potential, the shape of the zones is also influenced by the participation of the ith substance in adsorption equilibria*. A linear
A detailed theoretical description of the effect of adsorption isotherms on the shape of zones was published for chromatographic methods, in which the difference in the adsorption properties of different substances is one of the basic separation principles.
26
CLASSIFICATION OF ELECTROMIGRATION METHODS
adsorption isotherm (a2ri/ac: = 0), in a similar manner to the constant potentialgradient, does not deform the zone, which remains symmetrical during the whole separation. The same “spreading” of both boundaries of symmetrical zones is caused both by participation of all z,i components of the ith substance in ionic equilibria and by diffusion. An adsorption isotherm concave with respect to the concentration axis (azri/ac;< 0, e.g., Langmuir’s isotherm), causes a sharpening of the front boundary; an adsorption isotherm convex to the concentration axis (a2Pi/ac? > 0) causes a sharpening of the back boundary of the zone. The time course of separation is also of importance. It can be seen that in symmetrical zones, the interzonal distances (determined according to the positions of maximal concentrations in the zones) increase. However, the width of the zone increases with increasing time (the zone spreads) and the maximal concentration of a substance in the zone decreases. With dilute samples a large amount of the sample becomes localized in places, where the concentration decreases below the limit of determination and the amount of sample apparently decreases. The widening of asymmetrical zones is even more pronounced, with the result that certain zones are incompletely separated. The characteristics of individual zones are presented in Table 2.2. Zone electrophoresis is analogous to elution chromatography. TABLE 2.2 CHARACTERISTICS OF ZONE ELECTROPHORESIS ~
Shape of zone Symmetrical Cause of zone shape
KC
=K
and
Sharp front boundary KC
01
a2ri
Maximal concentration in the zone Zone width
Sharp back boundary KC
>K
01
-a2ri >o ad
ionic equilibria and diffusion decreases permanently during separation increases permanently during separation
THE MOVING BOUNDARY METHOD
This method resembles closely the zone method, and can be characterized by the following parameters. The separation column is separated into three parts. Both side parts are filled with the same basic electrolytic system and the middle part contains, in addition to the same electrolytic system, a mixture of substances to be separated. The length of the middle part is usually comparable to the lengths of the side parts and, as a result, the
ISOTACHOPHORESIS
21
mixture cannot be separated into independent zones in a separation column of a given length. When the whole mixed zone is moved, only a gradual separation of substances from the front boundary occurs: the most mobile substance moves first, followed by a mixture of two most mobile substances, then a mixture of three most mobile substances, etc. The slowest substances behind the boundary of the mixed zone are delayed: the slowest substance comes last, preceded by a mixture of two slowest substances, etc. The solution of the equations for electrophoretic transport in the moving boundary method leads to a general equation for the regulating function (also known as the Kohlrausch function) for each location in the separation column:
which makes it possible to determine concentration profiles of individual substances related t o time and positional coordinates. The course of separation when adsorption is not involved and when all substances of the separated mixture migrate in the same direction is illustrated schematically in Fig. 2.2. In addition to the concentration profiles of individual substances and derivative curves, Fig. 2.2 also shows the distribution of the potential, which at each instant is proportional to the concentration distribution of the substances t o be separated. Also in this instance (if K , > K) a non-uniform distribution of the potential gradient causes desharpening and sharpening of the front and back boundaries respectively (when K, < K , the front and back boundaries would be sharpened and desharpened, respectively). The shape of the boundary is also influenced by diffusion and convection flows. In addition, flows brought about by the radial temperature gradient occur when using electrolytes with a higher ionic strength. The moving boundary method is analogous to the frontal chromatographic method. This analogy is particularly pronounced in arrangements of the electrophoretic experiment such that only “front” boundaries are collected (substances pass to the separation column from the reserve space) or only “back” boundaries are collected (substances from the separation column present there at the beginning are obtained). In this arrangement, the concentration profiles and distribution of the potential can differ from those shown in Fig. 2.2, as in this instance the basic electrolytic system need not be in the space in which the mixture of substances to be separated is localized.
ISOTACHOPHORESIS It is a characteristic feature of this method that it is not possible to separate simultaneously substances that carry both positive and negative charges. The separation of anions will be described here (analogous conditions are used for the separation of cations). The whole separation compartment is divided into three unequal parts. One part of the anodic compartment and the separation column is filled with the leading electrolyte. A second part is formed by a compartment into which a mixture of substances to be separated is introduced. The third part represents the compartment filled with a terminating electrolyte (the cathode compartment in this instance). The leading electrolyte contains anions with an effective mobility higher than that of any of the anions in the
28
CLASSIFICATION OF ELECTROMIGRATION METHODS
I
4 I
I
c
A+B+C
X -
I
2
-x
3
dc
A+0
I
A
x-
dx 4
I
I
I
Fig. 2.2. Schematic representation of the course of separation of three substances by the moving boundary method: (1) at the beginning of the experiment ( t o = 0); (2) at time t , ( t , > t o ) ;( 3 , 4 and 5 ) at time t , ( t , > t i ) . The solid tine designates the total concentration. Substances: R,A ; B; C; basic electrolyte.
m,
m, m,
mixture and the cations, the buffering capacity of which can be utilized. The terminating electrolyte contains an anion with an effective mobility lower than that of any anion in the mixture to be separated. Cations of the terminating electrolyte are not important for the separation. On applying an electric field, separation proceeds until a steady state is established. This steady state is characterized by the fact that individual substances are separated, according to their effective mobilities, into independent sharp, yet close zones. The so-called separated boundaries are localized among the zones. The separated substance is localized only at one side of the boundary. This steady state, during which
FOCUSING METHODS
29
all substances (zones, zone boundaries) move with the same velocity (hence the name of the method) is characteristic of isotachophoresis. Prior to establishment of the steady state, individual zones are also mutually separated; however, in addition t o zones that contain only one of the separated substances (pure zones), zones with more separated substances (mixed zones) are present. The solution of the equations of the electrophoretic separation for the steady isotachophoretic state leads t o certain important conclusions: (a) The potential gradient in the ith zone is determined by the effective mobility of anions in this zone, as
v
=
(Ul)eff- E l = (U2)eff*E2= (Ui)eff.Ei = constant
(2.3)
where ZI is the velocity of movement of zone boundaries. (b) The concentration of any separated anion B with charge zB is determined by the concentration of the leading anion A (with charge zd,by the mobilities of both anions and by the mobility of a common counter ion C. This relationship is expressed by means of the Kohlrausch regulating function in the form This feature of isotachophoresis is particularly important for dilute solutions, which are “concentrated” to a concentration corresponding t o eqn. 2.4, which represents a considerable difference to zone electrophoresis. The separation of three substances is illustrated schematically in Fig. 2.3. In addition to the concentration profiles of individual substances, the distribution of the potential gradient is also presented. After establishment of the steady state, the distribution has a characteristic stepwise course. In isotachophoresis the self-sharpening effect of the electric field on all zone boundaries is of considerable importance. (When an ion is delayed and therefore enters the following zone with a higher potential gradient, its velocity increases, the ion catches up “its” zone and enters again. On the other hand, when an ion outruns “its” zone, it moves to a zone with a lower potential gradient and its velocity therefore decreases and the ion is overrun by “its” zone.) Diffusion flows and flows brought about by pressure or temperature gradients act against the self-sharpening effect. The shape of zone boundaries can also be influenced by phase equilibria. Isotachophoresis is analogous to displacement chromatography.
FOCUSING METHODS The methods mentioned above are based on the fact that in the substance flow every substance is characterized by a non-zero migration term, oriented in one direction during the whole separation. Also, the substance flow of all components is non-zero at any location in the separation column. In methods in which the substance flow of each substance decreases from a maximal positive value at one end of the separation column, through a zero value to a maximal value at the other end of the column, the course and particularly the result of the separation are completely different. A number of procedures that make it possible to demonstrate in practice this
CLASSIFICATION OF ELECTROMIGRATION METHODS
L ct
-X
I L
-x
Fig. 2.3. Schematic representation of the separation of three substances by isotachophoresis: (1) at the beginning of the experiment; (2) beginning of separation (mixed zone of B and C still exists); (3) steady state; (4) distribution of the potential gradient. leading electrolyte (L); 81, terminal substance B; substance C. The solid line represents the total electrolyte (T); H,substance A; concentration.
m,
a,
m,
relationship between the substance flow and the positional coordinate have been described. Two methods that differ in the way in which they bring about the required course of the substance flow, isoelectric focusing and electrorheophoresis, will be described here as examples. The former method utilizes the relationship between the effective mobility and the
0
-
.(pH)A
B
Fig. 2.4. Dependence of the velocity, i?, on thesstance x in the column. (A) g i s determined by the U e f f * E )a; linear gradient of pH occurs in the column. effective velocity of the ampholyte=;( the migration velocity is constant. (B) G'= +
zmie
Electrorheophoresis utilizes the dependence of the convection flow on the spatial coordinate and can be used for any substance. When the convection flow is caused by a sucking flow, the velocity of this flow is characterized by eqn. 1.48 and the relationship between the substance flow and distance has a course as shown schematically in Fig. 2.4B, provided that the value of the migration term is constant. The course of separation of three substances in an experimental arrangement with the above dependence of the substance flow on distance is shown schematically in Fig. 2.5. It can be seen that the established dynamic equilibrium rather than the course of separation plays an important role in this experimental arrangement. Individual substances are focused at locations at which values of the substance flow of any given substance are zero, irrespective of the starting point and width of the initial mixed zone of the sample. Isoelectric focusing This technique is usually used for the separation of ampholytes, mostly macromolecular. The separation compartment is formed by three parts: two electrode compartments and the separation column. A stable gradient of pH values, limited by minimal and maximal pH values in the anode and cathode spaces, respectively, is formed in the column. In this gradient ampholytes, the pHis,, of which is within the range pHmi, < < pHiso < pH,, can be focused. When a mixture of ampholytes is applied at an arbitrary location in the column and an electric field is applied to the system, the ampholytes
32
CLASSIFICATION OF ELECTROMIGRATION METHODS
X-
X-
X-
A
X-
B
Fig. 2.5. Schematic representation of the separation of three components by the focusing method: (A) mixture applied to the middle part of the column; (B) mixture applied to the whole column. ( 1 ) Beginning of the experiment; (2) focusing occurs; (3) end of focusing. The solid line represents the total concentration. and H, concentrations of individual substances.
a,
begin to migrate either towards the cathode or the anode, according to their effective mobilities. The velocity of movement of all substances decreases during the separation. After a certain time, a dynamic equilibrium is established, which is characterized by the fact that each ampholyte has already moved to a position at which its effective mobility is zero, i.e., to a position where the pH is identical with the pHis,, of a given ampholyte. A similar dependence of Ueffon pH also occurs with certain complex substances. Hence these substances can also be separated as mentioned above. A stable pH gradient in the column is essential for isoelectric focusing and is usually produced as described below. The column is filled with a mixture of special ampholytes, the so-called ampholyte carrier. Each of the ampholytes in this mixture must have a certain buffering capacity near to its isoelectric point, and adequate conductivity and solubility. The mixture should contain such ampholytes in relative proportions such as to cover the required pH range by its pHiso values. The pH gradient is first formed as a result of different pH values in the anode compartment, the column and the cathode compartment. As the electric current passes through the column, the substances of the
33
COMBINED METHODS
ampholyte carrier, originally homogeneously distributed along the whole column, begin to move according t o their pHis,, values as a result of isoelectric focusing. The distribution originating in this way, in addition t o the buffering capacity of individual components of the ampholyte carrier, secure and stabilize the required pH gradient.
Electrorheophoresis A gradient of hydrodynamic flow of the electrolyte occurs as a result of sucking flows caused by evaporation of the electrolyte from the surface of the stabilizing porous medium. The velocity of a substance at any location X in the column (usually a paper strip) is determined by the sum of the migration velocity (eqns. 1.22 and 1.28) and the velocity of the electrolyte flow (eqn. 1.48). Thus, ( v , ) ~ = sgnz*(UJeti-E+ rn*(X-OSL).(pSd)-'
(2.5)
Focusing of the i t h substance occurs at a site Xi:
It is apparent that in a given gradient of hydrodynamic flow it is possible to focus substances the migration velocity of which is lower than velocity of the electrolyte flow. Also in this method the formation of a stable velocity gradient is a necessary condition, and can be achieved by using a suitable composition of the basic electrolyte system, in which all substances have approximately the same volatility. In the opposite case, volatile substances (solvent) are preferentially evaporated, so that the electrolyte concentrates in the porous carrier. The evaporation of the electrolyte from the whole surface and its supplementation from the electrode spaces leads to concentration of the electrolyte in the direction of the point of zero hydrodynamic flow. The formation of the concentration gradient is also reflected in a non-uniform distribution of the potential gradient (a decrease in the middle and an increase at the sides of the porous carrier). The increase in the potential gradient at both ends of the carrier can cause an increase in migration velocity such that a substance focused at the beginning, near one of the ends, starts to move towards it and finally leaves the carrier completely. Characteristic features of the above electrophoretic methods are compared in Table 2.3.
COMBINED METHODS Of combined methods, which have increased in importance in recent years, we can consider as a representative example of the combination of two different methods the so-called disc electrophoresis and immunoelectrophoresis.
Disc electrophoresis In this method the experimental arrangement (which also gave the name to the discontinuous method) allows the consective application of isotachophoretic and zone principles .
TABLE 2.3 CHARACTERISTIC FEATURES OF BASIC ELECTROPHORETIC METHODS Parameter
Method Zone
Moving boundary
Isotachophoresis
elution
frontal
displacement
particles with charges of both signs
basically particles with charges of both signs, practically particles with charges of one sign
particles with charges of only one sign
particles with charges of both signs
Zone composition
zones of individual substances in the basic electrolyte
only a zone of the fastest substance in the basic electrolyte; other zones are mixed
each zone contains only a given substance
Concentration in the zone
decreases permanently during separation
remains constant during separation
after establishment of the steady state each zone contains only one separated substance can both increase and decrease during separation; after establishment of the steady state it is constant and is determined by a regulating function
Zone boundary
one sharp, the other spread or both spread
a l l boundaries sharp
both boundaries sharp
Zone width
increases permanently during separation
one sharp, the other spread (second boundary need not occur) increases permanently during separation
increases or decreases during separation; remains constant after establishment of the steady state
decreases during focusing; remains constant after establishment of dynamic equilibrium
Velocity of zone movement
different for different substances; can change during separation
different for different substances; remains constant during separation
after establishment of the steady state identical for all substances and constant with time
during focusing it changes with distance (to a different extent for different substances); after establishment of dynamic equilibrium the velocity of all separated substances is zero
Analogous chromatographic method Simultaneous separation
Focusing methods
increases during focusing, remains constant after establishment of dynamic equilibrium
COMBINED METHODS
35
The separation column, packed with a suitable (usually polyacrylamide) gel, is divided into two parts: the focusing part and the separation part. In each of these parts there is a different separation medium and different electrophoretic arrangements apply. The focusing part of the column is shorter, and is packed with dilute gel, the internal structure of which has virtually no effect on the mobility of individual substances, which is thus determined solely by the effective mobilities of the substances in question. The separation part of the column is considerably longer, the gel density is higher and, with the use of a higher content of cross-linking agent, its internal structure must be considered during the separation. Equally, the composition of the electrolyte system and its pH are different in the separation and focusing parts. An appropriate choice of the electrolyte systems in the cathodic and anodic compartment and in the focusing and separation parts of the column can result into concentration and ordering of the individual substances into narrow, closely contacted zones on the border between the dilute and solid gel by an isotachophoretic mechanism. In the separation part the substances are separated into individual zones according to the laws of zone electrophoresis in solid carriers (the sieving effect of the polyacrylamide gel occurs in this stage). Substances are detected by staining after the separation is completed. The advantage of this method is that optimal starting conditions for the zone electrophoretic separation are produced by the preceding isotachophoresis resulting in a concentrated narrow band. Immunoelectrophoresis This is an electrophoretic procedure that exploits the highly sensitive and highly specific immunochemical precipitation of the antigen-antibody system for detection. If both parts of the complex, e.g., antigen and antibody, come into contact at optimal concentrations, characteristic precipitation lines are formed (the complex, however, is soluble in the excess of both the antigen and antibody). The shape of the precipitation lines is also determined by the way in which both antigen and antibody come into contact. For this purpose differently oriented diffusion and migration flows are made use of. In the classical version of the method, the mixture of antigens is first separated in agarose gel by the zone electrophoresis. Serum antigens can be considered as an example. Then a suitable antibody (e.g., rabbit or horse polyspecific antiserum) is applied to a channel located parallel to the direction of the separation in the gel plate, so that the conditions for the occurrence of diffusion flows are thus fulfilled. Antigens diffuse almost radially around the locations that they have reached during the zone electrophoretic separation. Antibodies migrate laterally with respect to the channel, e.g., to meet the antigens. After a period of time (which is usually long, of the order of days, because of the slowness of the diffusion flows), a series of radial precipitation lines are formed in places where both antibodies and antigens have met (Fig. 2.6A). In other modifications of immunoelectrophoresis, the slow diffusion flow of antigens against antibodies is replaced with a much faster migrational flow. A method analogous to the classical version is the so-called “crossed electrophoresis”. In the first stage (separation of antigens by zone electrophoresis on agarose gel) the method is coincident with the classical version. Then a new separation medium is formed in such a way that another
36
CLASSIFICATION OF ELECTROMIGRATION METHODS
A
Fig.2.6. Schematic representation of the immunoa ctropherogram.( Classical arrangement: 1location of antigens after electrophoretic separation;6 , antibody; n, precipitation lines. (B) “Laurell
rocket” method; 1-3, comparative antigens of known concentration; 4-8, unknown samples; n, precipitation lines; h , height of precipitation peak.
layer of agarose gel is added t o the strip of agarose gel with separated antigens. This part, in addition to the electrolyte system, also contains the polyspecific antiserum. The composition and pH of the electrolyte are such as to make the effective mobility of antibodies zero. If an electric field is imposed on the system in a direction perpendicular to the direction of separation, antigens migrate into the antibody-containing media. Equally in this case in areas where antigens meet antibodies at optimal concentrations characteristic precipitation lines are formed. The migration of antigens against non-migrating antibody is also made use of in the “Laurell rocket” method, which is used in clinical biochemistry for the determination of a single antigen. In this instance agarose gel (with the exception of a narrow strip at the edge of the plate where the samples are applied) is saturated with the basic electrolyte together with the monospecific antibody which has a zero effective mobility. After a series of samples has been applied (at least three are usually employed as standards of known concentration and are used for designing the calibration line), the electric field is applied and most substances from individual samples pass through the agarose carrier. The estimated antigens, however, form characteristic precipitation lines (Fig. 2.6B). The size (height) of the precipitation arcs is proportional to the antigen concentration in the sample. A more generally applicable method is the “fused rocket” method. The basic
REFERENCES
37
electrolyte system (which penetrates the agarose gel plate with the exception of a narrow strip at the ed@ where the samples are applied) also contains the polyspecific antiserum with a zero effective mobility of antibodies. To this plate are applied a series of samples that have been obtained as fractions in a prior separation of the original mixture (e.g., by chromatography, preparative electrophoresis, centrifugation or isotachophoresis). The precipitation lines obtained after an immunoelectrophoretic separation can be intensified by staining, using dyes suitable for the particular type of substances separated. Sometimes two or more dyes are used for different species present in the sample.
REFERENCES More detailed information regarding the topics discussed throughout this chapter can be found in more specialized papers or books devoted to electrophoretic and related techniques. Some of these are Listed below. 1
M. Bier (Editor), Electrophoresis. Theory, Methods and Applications, Academic Press, New York,
2
R. J. Block, E. L. Durrum and G. Zweig, A Manual of Paper Chromatography and Paper Electrophoresis, New York, 1955. H. Bloemendal, Zone Electrophoresis in Blocks and Columns, Elsevier, Amsterdam, 1963. H. G. Cassidy, Adsorption and Chromatography,Interscience, New York, 1951. H. G. Cassidy, Fundamentals of Chromatography,Interscience, New York, 1957. L. P. Cawley, Electrophoresis and Immunoelectrophoresis, Little and Brown, Boston, Mass., 1969. H. J . McDonald, R. J. Lappe, E. Marbach, R. H. Spitzer and M. C. Urbin,lonography, Electrophoresis in Stabilized Media, Chicago, Ill:, 1955. F. M. Everaerts, J . L. Beckers and T. P. E. M. Verheggen, Isotachophoresis;Theory, Instrumentation and Applications, Elsevier, Amsterdam, 1976. A. H. Gordon, Electrophoresis of Proteins in Polyacrylamide and Starch Gels, North-Holland, Amsterdam, 1969. C. J . Gidding, Dynamic of Chromatography.Part 1 . Principlesand Theog', Marcel Dekker, New York, 1965. S. Hjerten, Free Zone Electrophoresis,Almquist and Wiksell, Uppsala, 1967. M. Lederer, Introduction to Paper Electrophoresis, Elsevier, Amsterdam, 1955. M. Lederer (Editor), ChromatographicReviews (Progress in Chromatography.Electrophoresis and Related Methods), Vol. 1-3, Elsevier, Amsterdam, 1960. H. R. Maurer, Disc-Elektrophorese,Theorie und Praxis der diskontinuierlichen Polyakrylamidgel Elektrophorese, De Gruyter, Berlin, 1968. G. Sohay, Theoretische Grundlagender Gaschromatographie,VEB Deutscher Verlag der Wissenschaften, Berlin, 1960. D. J. Shaw, Electrophoresis, Academic Press, New York, 1969. J . Smith (Editor), Chromatographicand Electrophoretic Techniques. Vol. 2. Zone Electrophoresis, Interscience, New York, 1968. K. E. Stensio and G. Ekedahl, Chromatography and Electrophoresis, Norstedt, Stockholm, 1969. H. Svensson, Electrophoresis by the Moving Boundary Methods. A Theoretical and Experimental Study, Almquist and Wiksells, Stockholm, 1946. Ch. Wunderly, PIinciples and Applications of Paper Electrophoresis, Elsevier, Amsterdam, 1961. G. Zweig and J. R. Whitaker, Paper Chromatographyand Electrophoresis, Academic Press, New York, 1967.
3 4 5 6 7 8.
9 10 11 12 13. 14 15 16 17 18 19 20 21
1959.
Classification of electromigration methods J. VACfK
CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zone electrophoresis. .................................. ..................... The moving boundary method . . . . . . . . . . . . . ............................... Isotachophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Focusingmethods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................... Isoelectric focusing . . . . . . . . . . . . . . ................................... Electrorheophoresis . . . . . . . . . . . . . .......................... Combined methods .................... .......................... Disc electrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................... Immunoelectrophoresis . . . . . . . . . . . .......................... References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
INTRODUCTION Electrophoretic methods can be classified according to different criteria, some of which are presented in Table 2.1. TABLE 2.1 CLASSIFICATIONS OF ELECTROPHORETIC METHODS Criterion of setmation
Electrophoretic method
Amount separated
analytical micropreparative preparative single-step continuous free on carriers capillary gel only migration is involved migration and interaction with carrier migration, interaction with carrier and diffusion low-voltage hig h-voIt age zone moving boundary isotachophoresis focusing methods
Type of procedure Method of stabilization
Separation principle
Potential gradient used Initial and side (marginal) conditions of separation (experimental arrangement)
23 24 26 27 29 31 33 33 33
35 37
24
CLASSIFICATION OF ELECTROMIGRATION METHODS
The criteria can be compared only with difficulty and their selection is determined by purpose of a particular classification. From the theoretical point of view, classification according to initial and marginal conditions of the separation is the most interesting. This makes it possible to characterize four basic types of electrophoretic methods - zone electrophoresis, moving boundary electrophoresis, isotachophoresis and focusing methods. For illustration in this chapter some combined methods will be described; these are procedures that exploit several electrophoretic principles or in w h c h the separations are carried out both on the electrophoretic and some other principle.
ZONE ELECTROPHORESIS Characterization of the method: The whole separation column is filled with a single basic electrolytic system with a specific conductivity K . A mixture of substances to be separated is applied to a certain location in the separation column (the start). The width of the mixed zone at the start (AL) is much smaller than the length of the separation column, L (ALQ L ) . After the application of an electric field to the system, individual substances migrate according to their effective mobilities towards the electrodes and form zones. For a velocity of the ith substance at a concentration cir eqn. 2.1 can be obtained by solving the continuity equations (eqn. 1.59) for all z components of the ith substance and by use of the definition of the effective mobility (eqn. 1.28):
ci
Fig. 2.1 shows a scheme of the course of the separation of three substances, A, B and C. The case when all three substances have a charge of the same sign and therefore migrate in the same direction was chosen. Three characteristic zone shapes, symmetrical (substance A), with a sharpened front* zone boundary (substance B) and with a sharpened back* zone boundary (substance C), are shown. The shapes of individual zones are influenced both by the distribution of the potential gradient and by the shapes of the adsorption isotherms (this holds for separations on carriers). In zone electrophoresis, the distribution of the potential gradient can have two characteristic courses: (a) The specific conductivity of the basic electrolyte is such that it is not influenced by the presence of the substance t o be separated. The potential gradient is then constant along the whole column. This is the most common type of zone electrophoresis. (b) The specific conductivity of the solution at a location in the zone K , differs from that of the basic electrolyte. Two possibilities are K , > K [usually if (UJefr > (Uel)eft] or
* The “front” refers to the concentration profile of the ith substance for which ( a q / a t ) , > 0; the “back” boundary is characterized by the inequality (aci/at), < 0.
25
ZONE ELECTROPHORESIS
I
I
x-
x-
-
Fig. 2.1. Schematic representation of the course of separation of three substances, A , B and C, by zone electrophoresis for (Ug)eff > (LIAIeff > (Uc),ff:(1) at the beginning of the experiment at zero time (i = t o ) ;(2) at time t l ( t l > t o ) ;(3) at time fZ(f1 > t , ) . Substance A ( H ) forms a symmetricalzone, forms a zone with a focused front and C(H) forms a zone with a focused end. The solid line represents the total concentration.
B(a)
K, < K [usually if (UJeir < (LleJeff]. In the former instance the potential gradient is higher at the site of the zone than outside it. This results in a situation where the back boundary of the zone becomes sharpened (ions that had been delayed enter a region of a higher potential gradient and catch up the zone), whereas the front boundary becomes extended (ions that had outrun the zone move to a region of a higher potential gradient and become even further removed). When K, < K the opposite situation occurs (the front boundary becomes sharpened and the back boundary becomes tailed). In a similar manner t o the distribution of the potential, the shape of the zones is also influenced by the participation of the ith substance in adsorption equilibria*. A linear
A detailed theoretical description of the effect of adsorption isotherms on the shape of zones was published for chromatographic methods, in which the difference in the adsorption properties of different substances is one of the basic separation principles.
26
CLASSIFICATION OF ELECTROMIGRATION METHODS
adsorption isotherm (a2ri/ac: = 0), in a similar manner to the constant potentialgradient, does not deform the zone, which remains symmetrical during the whole separation. The same “spreading” of both boundaries of symmetrical zones is caused both by participation of all z,i components of the ith substance in ionic equilibria and by diffusion. An adsorption isotherm concave with respect to the concentration axis (azri/ac;< 0, e.g., Langmuir’s isotherm), causes a sharpening of the front boundary; an adsorption isotherm convex to the concentration axis (a2Pi/ac? > 0) causes a sharpening of the back boundary of the zone. The time course of separation is also of importance. It can be seen that in symmetrical zones, the interzonal distances (determined according to the positions of maximal concentrations in the zones) increase. However, the width of the zone increases with increasing time (the zone spreads) and the maximal concentration of a substance in the zone decreases. With dilute samples a large amount of the sample becomes localized in places, where the concentration decreases below the limit of determination and the amount of sample apparently decreases. The widening of asymmetrical zones is even more pronounced, with the result that certain zones are incompletely separated. The characteristics of individual zones are presented in Table 2.2. Zone electrophoresis is analogous to elution chromatography. TABLE 2.2 CHARACTERISTICS OF ZONE ELECTROPHORESIS ~
Shape of zone Symmetrical Cause of zone shape
KC
=K
and
Sharp front boundary KC
01
a2ri
Maximal concentration in the zone Zone width
Sharp back boundary KC
>K
01
-a2ri >o ad
ionic equilibria and diffusion decreases permanently during separation increases permanently during separation
THE MOVING BOUNDARY METHOD
This method resembles closely the zone method, and can be characterized by the following parameters. The separation column is separated into three parts. Both side parts are filled with the same basic electrolytic system and the middle part contains, in addition to the same electrolytic system, a mixture of substances to be separated. The length of the middle part is usually comparable to the lengths of the side parts and, as a result, the
ISOTACHOPHORESIS
21
mixture cannot be separated into independent zones in a separation column of a given length. When the whole mixed zone is moved, only a gradual separation of substances from the front boundary occurs: the most mobile substance moves first, followed by a mixture of two most mobile substances, then a mixture of three most mobile substances, etc. The slowest substances behind the boundary of the mixed zone are delayed: the slowest substance comes last, preceded by a mixture of two slowest substances, etc. The solution of the equations for electrophoretic transport in the moving boundary method leads to a general equation for the regulating function (also known as the Kohlrausch function) for each location in the separation column:
which makes it possible to determine concentration profiles of individual substances related t o time and positional coordinates. The course of separation when adsorption is not involved and when all substances of the separated mixture migrate in the same direction is illustrated schematically in Fig. 2.2. In addition to the concentration profiles of individual substances and derivative curves, Fig. 2.2 also shows the distribution of the potential, which at each instant is proportional to the concentration distribution of the substances t o be separated. Also in this instance (if K , > K) a non-uniform distribution of the potential gradient causes desharpening and sharpening of the front and back boundaries respectively (when K, < K , the front and back boundaries would be sharpened and desharpened, respectively). The shape of the boundary is also influenced by diffusion and convection flows. In addition, flows brought about by the radial temperature gradient occur when using electrolytes with a higher ionic strength. The moving boundary method is analogous to the frontal chromatographic method. This analogy is particularly pronounced in arrangements of the electrophoretic experiment such that only “front” boundaries are collected (substances pass to the separation column from the reserve space) or only “back” boundaries are collected (substances from the separation column present there at the beginning are obtained). In this arrangement, the concentration profiles and distribution of the potential can differ from those shown in Fig. 2.2, as in this instance the basic electrolytic system need not be in the space in which the mixture of substances to be separated is localized.
ISOTACHOPHORESIS It is a characteristic feature of this method that it is not possible to separate simultaneously substances that carry both positive and negative charges. The separation of anions will be described here (analogous conditions are used for the separation of cations). The whole separation compartment is divided into three unequal parts. One part of the anodic compartment and the separation column is filled with the leading electrolyte. A second part is formed by a compartment into which a mixture of substances to be separated is introduced. The third part represents the compartment filled with a terminating electrolyte (the cathode compartment in this instance). The leading electrolyte contains anions with an effective mobility higher than that of any of the anions in the
28
CLASSIFICATION OF ELECTROMIGRATION METHODS
I
4 I
I
c
A+B+C
X -
I
2
-x
3
dc
A+0
I
A
x-
dx 4
I
I
I
Fig. 2.2. Schematic representation of the course of separation of three substances by the moving boundary method: (1) at the beginning of the experiment ( t o = 0); (2) at time t , ( t , > t o ) ;( 3 , 4 and 5 ) at time t , ( t , > t i ) . The solid tine designates the total concentration. Substances: R,A ; B; C; basic electrolyte.
m,
m, m,
mixture and the cations, the buffering capacity of which can be utilized. The terminating electrolyte contains an anion with an effective mobility lower than that of any anion in the mixture to be separated. Cations of the terminating electrolyte are not important for the separation. On applying an electric field, separation proceeds until a steady state is established. This steady state is characterized by the fact that individual substances are separated, according to their effective mobilities, into independent sharp, yet close zones. The so-called separated boundaries are localized among the zones. The separated substance is localized only at one side of the boundary. This steady state, during which
FOCUSING METHODS
29
all substances (zones, zone boundaries) move with the same velocity (hence the name of the method) is characteristic of isotachophoresis. Prior to establishment of the steady state, individual zones are also mutually separated; however, in addition t o zones that contain only one of the separated substances (pure zones), zones with more separated substances (mixed zones) are present. The solution of the equations of the electrophoretic separation for the steady isotachophoretic state leads t o certain important conclusions: (a) The potential gradient in the ith zone is determined by the effective mobility of anions in this zone, as
v
=
(Ul)eff- E l = (U2)eff*E2= (Ui)eff.Ei = constant
(2.3)
where ZI is the velocity of movement of zone boundaries. (b) The concentration of any separated anion B with charge zB is determined by the concentration of the leading anion A (with charge zd,by the mobilities of both anions and by the mobility of a common counter ion C. This relationship is expressed by means of the Kohlrausch regulating function in the form This feature of isotachophoresis is particularly important for dilute solutions, which are “concentrated” to a concentration corresponding t o eqn. 2.4, which represents a considerable difference to zone electrophoresis. The separation of three substances is illustrated schematically in Fig. 2.3. In addition to the concentration profiles of individual substances, the distribution of the potential gradient is also presented. After establishment of the steady state, the distribution has a characteristic stepwise course. In isotachophoresis the self-sharpening effect of the electric field on all zone boundaries is of considerable importance. (When an ion is delayed and therefore enters the following zone with a higher potential gradient, its velocity increases, the ion catches up “its” zone and enters again. On the other hand, when an ion outruns “its” zone, it moves to a zone with a lower potential gradient and its velocity therefore decreases and the ion is overrun by “its” zone.) Diffusion flows and flows brought about by pressure or temperature gradients act against the self-sharpening effect. The shape of zone boundaries can also be influenced by phase equilibria. Isotachophoresis is analogous to displacement chromatography.
FOCUSING METHODS The methods mentioned above are based on the fact that in the substance flow every substance is characterized by a non-zero migration term, oriented in one direction during the whole separation. Also, the substance flow of all components is non-zero at any location in the separation column. In methods in which the substance flow of each substance decreases from a maximal positive value at one end of the separation column, through a zero value to a maximal value at the other end of the column, the course and particularly the result of the separation are completely different. A number of procedures that make it possible to demonstrate in practice this
CLASSIFICATION OF ELECTROMIGRATION METHODS
L ct
-X
I L
-x
Fig. 2.3. Schematic representation of the separation of three substances by isotachophoresis: (1) at the beginning of the experiment; (2) beginning of separation (mixed zone of B and C still exists); (3) steady state; (4) distribution of the potential gradient. leading electrolyte (L); 81, terminal substance B; substance C. The solid line represents the total electrolyte (T); H,substance A; concentration.
m,
a,
m,
relationship between the substance flow and the positional coordinate have been described. Two methods that differ in the way in which they bring about the required course of the substance flow, isoelectric focusing and electrorheophoresis, will be described here as examples. The former method utilizes the relationship between the effective mobility and the
0
-
.(pH)A
B
Fig. 2.4. Dependence of the velocity, i?, on thesstance x in the column. (A) g i s determined by the U e f f * E )a; linear gradient of pH occurs in the column. effective velocity of the ampholyte=;( the migration velocity is constant. (B) G'= +
zmie
Electrorheophoresis utilizes the dependence of the convection flow on the spatial coordinate and can be used for any substance. When the convection flow is caused by a sucking flow, the velocity of this flow is characterized by eqn. 1.48 and the relationship between the substance flow and distance has a course as shown schematically in Fig. 2.4B, provided that the value of the migration term is constant. The course of separation of three substances in an experimental arrangement with the above dependence of the substance flow on distance is shown schematically in Fig. 2.5. It can be seen that the established dynamic equilibrium rather than the course of separation plays an important role in this experimental arrangement. Individual substances are focused at locations at which values of the substance flow of any given substance are zero, irrespective of the starting point and width of the initial mixed zone of the sample. Isoelectric focusing This technique is usually used for the separation of ampholytes, mostly macromolecular. The separation compartment is formed by three parts: two electrode compartments and the separation column. A stable gradient of pH values, limited by minimal and maximal pH values in the anode and cathode spaces, respectively, is formed in the column. In this gradient ampholytes, the pHis,, of which is within the range pHmi, < < pHiso < pH,, can be focused. When a mixture of ampholytes is applied at an arbitrary location in the column and an electric field is applied to the system, the ampholytes
32
CLASSIFICATION OF ELECTROMIGRATION METHODS
X-
X-
X-
A
X-
B
Fig. 2.5. Schematic representation of the separation of three components by the focusing method: (A) mixture applied to the middle part of the column; (B) mixture applied to the whole column. ( 1 ) Beginning of the experiment; (2) focusing occurs; (3) end of focusing. The solid line represents the total concentration. and H, concentrations of individual substances.
a,
begin to migrate either towards the cathode or the anode, according to their effective mobilities. The velocity of movement of all substances decreases during the separation. After a certain time, a dynamic equilibrium is established, which is characterized by the fact that each ampholyte has already moved to a position at which its effective mobility is zero, i.e., to a position where the pH is identical with the pHis,, of a given ampholyte. A similar dependence of Ueffon pH also occurs with certain complex substances. Hence these substances can also be separated as mentioned above. A stable pH gradient in the column is essential for isoelectric focusing and is usually produced as described below. The column is filled with a mixture of special ampholytes, the so-called ampholyte carrier. Each of the ampholytes in this mixture must have a certain buffering capacity near to its isoelectric point, and adequate conductivity and solubility. The mixture should contain such ampholytes in relative proportions such as to cover the required pH range by its pHiso values. The pH gradient is first formed as a result of different pH values in the anode compartment, the column and the cathode compartment. As the electric current passes through the column, the substances of the
33
COMBINED METHODS
ampholyte carrier, originally homogeneously distributed along the whole column, begin to move according t o their pHis,, values as a result of isoelectric focusing. The distribution originating in this way, in addition t o the buffering capacity of individual components of the ampholyte carrier, secure and stabilize the required pH gradient.
Electrorheophoresis A gradient of hydrodynamic flow of the electrolyte occurs as a result of sucking flows caused by evaporation of the electrolyte from the surface of the stabilizing porous medium. The velocity of a substance at any location X in the column (usually a paper strip) is determined by the sum of the migration velocity (eqns. 1.22 and 1.28) and the velocity of the electrolyte flow (eqn. 1.48). Thus, ( v , ) ~ = sgnz*(UJeti-E+ rn*(X-OSL).(pSd)-'
(2.5)
Focusing of the i t h substance occurs at a site Xi:
It is apparent that in a given gradient of hydrodynamic flow it is possible to focus substances the migration velocity of which is lower than velocity of the electrolyte flow. Also in this method the formation of a stable velocity gradient is a necessary condition, and can be achieved by using a suitable composition of the basic electrolyte system, in which all substances have approximately the same volatility. In the opposite case, volatile substances (solvent) are preferentially evaporated, so that the electrolyte concentrates in the porous carrier. The evaporation of the electrolyte from the whole surface and its supplementation from the electrode spaces leads to concentration of the electrolyte in the direction of the point of zero hydrodynamic flow. The formation of the concentration gradient is also reflected in a non-uniform distribution of the potential gradient (a decrease in the middle and an increase at the sides of the porous carrier). The increase in the potential gradient at both ends of the carrier can cause an increase in migration velocity such that a substance focused at the beginning, near one of the ends, starts to move towards it and finally leaves the carrier completely. Characteristic features of the above electrophoretic methods are compared in Table 2.3.
COMBINED METHODS Of combined methods, which have increased in importance in recent years, we can consider as a representative example of the combination of two different methods the so-called disc electrophoresis and immunoelectrophoresis.
Disc electrophoresis In this method the experimental arrangement (which also gave the name to the discontinuous method) allows the consective application of isotachophoretic and zone principles .
TABLE 2.3 CHARACTERISTIC FEATURES OF BASIC ELECTROPHORETIC METHODS Parameter
Method Zone
Moving boundary
Isotachophoresis
elution
frontal
displacement
particles with charges of both signs
basically particles with charges of both signs, practically particles with charges of one sign
particles with charges of only one sign
particles with charges of both signs
Zone composition
zones of individual substances in the basic electrolyte
only a zone of the fastest substance in the basic electrolyte; other zones are mixed
each zone contains only a given substance
Concentration in the zone
decreases permanently during separation
remains constant during separation
after establishment of the steady state each zone contains only one separated substance can both increase and decrease during separation; after establishment of the steady state it is constant and is determined by a regulating function
Zone boundary
one sharp, the other spread or both spread
a l l boundaries sharp
both boundaries sharp
Zone width
increases permanently during separation
one sharp, the other spread (second boundary need not occur) increases permanently during separation
increases or decreases during separation; remains constant after establishment of the steady state
decreases during focusing; remains constant after establishment of dynamic equilibrium
Velocity of zone movement
different for different substances; can change during separation
different for different substances; remains constant during separation
after establishment of the steady state identical for all substances and constant with time
during focusing it changes with distance (to a different extent for different substances); after establishment of dynamic equilibrium the velocity of all separated substances is zero
Analogous chromatographic method Simultaneous separation
Focusing methods
increases during focusing, remains constant after establishment of dynamic equilibrium
COMBINED METHODS
35
The separation column, packed with a suitable (usually polyacrylamide) gel, is divided into two parts: the focusing part and the separation part. In each of these parts there is a different separation medium and different electrophoretic arrangements apply. The focusing part of the column is shorter, and is packed with dilute gel, the internal structure of which has virtually no effect on the mobility of individual substances, which is thus determined solely by the effective mobilities of the substances in question. The separation part of the column is considerably longer, the gel density is higher and, with the use of a higher content of cross-linking agent, its internal structure must be considered during the separation. Equally, the composition of the electrolyte system and its pH are different in the separation and focusing parts. An appropriate choice of the electrolyte systems in the cathodic and anodic compartment and in the focusing and separation parts of the column can result into concentration and ordering of the individual substances into narrow, closely contacted zones on the border between the dilute and solid gel by an isotachophoretic mechanism. In the separation part the substances are separated into individual zones according to the laws of zone electrophoresis in solid carriers (the sieving effect of the polyacrylamide gel occurs in this stage). Substances are detected by staining after the separation is completed. The advantage of this method is that optimal starting conditions for the zone electrophoretic separation are produced by the preceding isotachophoresis resulting in a concentrated narrow band. Immunoelectrophoresis This is an electrophoretic procedure that exploits the highly sensitive and highly specific immunochemical precipitation of the antigen-antibody system for detection. If both parts of the complex, e.g., antigen and antibody, come into contact at optimal concentrations, characteristic precipitation lines are formed (the complex, however, is soluble in the excess of both the antigen and antibody). The shape of the precipitation lines is also determined by the way in which both antigen and antibody come into contact. For this purpose differently oriented diffusion and migration flows are made use of. In the classical version of the method, the mixture of antigens is first separated in agarose gel by the zone electrophoresis. Serum antigens can be considered as an example. Then a suitable antibody (e.g., rabbit or horse polyspecific antiserum) is applied to a channel located parallel to the direction of the separation in the gel plate, so that the conditions for the occurrence of diffusion flows are thus fulfilled. Antigens diffuse almost radially around the locations that they have reached during the zone electrophoretic separation. Antibodies migrate laterally with respect to the channel, e.g., to meet the antigens. After a period of time (which is usually long, of the order of days, because of the slowness of the diffusion flows), a series of radial precipitation lines are formed in places where both antibodies and antigens have met (Fig. 2.6A). In other modifications of immunoelectrophoresis, the slow diffusion flow of antigens against antibodies is replaced with a much faster migrational flow. A method analogous to the classical version is the so-called “crossed electrophoresis”. In the first stage (separation of antigens by zone electrophoresis on agarose gel) the method is coincident with the classical version. Then a new separation medium is formed in such a way that another
36
CLASSIFICATION OF ELECTROMIGRATION METHODS
A
Fig.2.6. Schematic representation of the immunoa ctropherogram.( Classical arrangement: 1location of antigens after electrophoretic separation;6 , antibody; n, precipitation lines. (B) “Laurell
rocket” method; 1-3, comparative antigens of known concentration; 4-8, unknown samples; n, precipitation lines; h , height of precipitation peak.
layer of agarose gel is added t o the strip of agarose gel with separated antigens. This part, in addition to the electrolyte system, also contains the polyspecific antiserum. The composition and pH of the electrolyte are such as to make the effective mobility of antibodies zero. If an electric field is imposed on the system in a direction perpendicular to the direction of separation, antigens migrate into the antibody-containing media. Equally in this case in areas where antigens meet antibodies at optimal concentrations characteristic precipitation lines are formed. The migration of antigens against non-migrating antibody is also made use of in the “Laurell rocket” method, which is used in clinical biochemistry for the determination of a single antigen. In this instance agarose gel (with the exception of a narrow strip at the edge of the plate where the samples are applied) is saturated with the basic electrolyte together with the monospecific antibody which has a zero effective mobility. After a series of samples has been applied (at least three are usually employed as standards of known concentration and are used for designing the calibration line), the electric field is applied and most substances from individual samples pass through the agarose carrier. The estimated antigens, however, form characteristic precipitation lines (Fig. 2.6B). The size (height) of the precipitation arcs is proportional to the antigen concentration in the sample. A more generally applicable method is the “fused rocket” method. The basic
REFERENCES
37
electrolyte system (which penetrates the agarose gel plate with the exception of a narrow strip at the ed@ where the samples are applied) also contains the polyspecific antiserum with a zero effective mobility of antibodies. To this plate are applied a series of samples that have been obtained as fractions in a prior separation of the original mixture (e.g., by chromatography, preparative electrophoresis, centrifugation or isotachophoresis). The precipitation lines obtained after an immunoelectrophoretic separation can be intensified by staining, using dyes suitable for the particular type of substances separated. Sometimes two or more dyes are used for different species present in the sample.
REFERENCES More detailed information regarding the topics discussed throughout this chapter can be found in more specialized papers or books devoted to electrophoretic and related techniques. Some of these are Listed below. 1
M. Bier (Editor), Electrophoresis. Theory, Methods and Applications, Academic Press, New York,
2
R. J. Block, E. L. Durrum and G. Zweig, A Manual of Paper Chromatography and Paper Electrophoresis, New York, 1955. H. Bloemendal, Zone Electrophoresis in Blocks and Columns, Elsevier, Amsterdam, 1963. H. G. Cassidy, Adsorption and Chromatography,Interscience, New York, 1951. H. G. Cassidy, Fundamentals of Chromatography,Interscience, New York, 1957. L. P. Cawley, Electrophoresis and Immunoelectrophoresis, Little and Brown, Boston, Mass., 1969. H. J . McDonald, R. J. Lappe, E. Marbach, R. H. Spitzer and M. C. Urbin,lonography, Electrophoresis in Stabilized Media, Chicago, Ill:, 1955. F. M. Everaerts, J . L. Beckers and T. P. E. M. Verheggen, Isotachophoresis;Theory, Instrumentation and Applications, Elsevier, Amsterdam, 1976. A. H. Gordon, Electrophoresis of Proteins in Polyacrylamide and Starch Gels, North-Holland, Amsterdam, 1969. C. J . Gidding, Dynamic of Chromatography.Part 1 . Principlesand Theog', Marcel Dekker, New York, 1965. S. Hjerten, Free Zone Electrophoresis,Almquist and Wiksell, Uppsala, 1967. M. Lederer, Introduction to Paper Electrophoresis, Elsevier, Amsterdam, 1955. M. Lederer (Editor), ChromatographicReviews (Progress in Chromatography.Electrophoresis and Related Methods), Vol. 1-3, Elsevier, Amsterdam, 1960. H. R. Maurer, Disc-Elektrophorese,Theorie und Praxis der diskontinuierlichen Polyakrylamidgel Elektrophorese, De Gruyter, Berlin, 1968. G. Sohay, Theoretische Grundlagender Gaschromatographie,VEB Deutscher Verlag der Wissenschaften, Berlin, 1960. D. J. Shaw, Electrophoresis, Academic Press, New York, 1969. J . Smith (Editor), Chromatographicand Electrophoretic Techniques. Vol. 2. Zone Electrophoresis, Interscience, New York, 1968. K. E. Stensio and G. Ekedahl, Chromatography and Electrophoresis, Norstedt, Stockholm, 1969. H. Svensson, Electrophoresis by the Moving Boundary Methods. A Theoretical and Experimental Study, Almquist and Wiksells, Stockholm, 1946. Ch. Wunderly, PIinciples and Applications of Paper Electrophoresis, Elsevier, Amsterdam, 1961. G. Zweig and J. R. Whitaker, Paper Chromatographyand Electrophoresis, Academic Press, New York, 1967.
3 4 5 6 7 8.
9 10 11 12 13. 14 15 16 17 18 19 20 21
1959.