GRADIENT ELUTION MOVING BOUNDARY ELECTROPHORESIS 

Inventors: Jonathan G. Shackman and David Ross 

ABSTRACT 

A novel method for performing electrophoretic separation and/or purification of 

compounds in a mixture is described. The method utilizes the electrophoretic migration 

of chemical species in solution in combination with variable hydrodynamic bulk flow of 

the solution through a separation capillary or microfluidic channel. The method can be 

used with the bulk solution flow either in the same direction as the electrophoretic 

migration or in the opposite direction. Continuous sample introduction eliminates the 

need for a sample injection mechanism, allowing for implementation in much reduced 

areas. The method has been designated as gradient elution moving boundary 

electrophoresis (GEMBE). 

With the bulk flow and electrophoretic migration in opposite directions, the method is 

useful for providing improved separation resolution, particularly in very short separation 

channels. In this case, only analytes with electrophoretic velocities greater than the bulk 

fluid velocity can enter the separation column and be detected as boundary zones. As the 

hydrodynamic flow is swept from high to low, analytes of lower mobilities are 

introduced, allowing for separation of compounds based upon electrophoretic mobility 

variations. Slower sweeping rates allow for greater separation resolution. 

With the bulk flow and electrophoretic migration in the same direction, the species to be 

analyzed pass through the separation channel more rapidly, so that analysis times are 

reduced. The variation of the bulk flow velocity over time insures that the optimal bulk 

flow velocity is used for each portion of the separation. 

BACKGROUND OF THE INVENTION 

Field of the Invention 

The present invention relates in general to electrophoretic separations, both in capillaries 

and in microfluidic channels, in order to separate and/or purify ionic compounds from a 

mixture, and, more particularly, to the combination of electrophoresis in the presence of a 

variable hydrodynamic flow with continuous introduction of sample. It is expected that 

the primary application will be in a microfluidic embodiment in order to reduce the total 

footprint of such microfluidic devices. 

1. Brief Description of Related Art 

Electrophoresis is a well known analytical method for separation of ionic compounds 

through use of an applied voltage. The separation mechanism is based mainly on 

differences in analytes' size and charge, although other factors can be exploited such as 

shape, density, affinity, etc. Early electrophoresis work utilized continuous sample 

introduction into a relatively large glass U-tube across which a voltage was applied; 

analytes were detected in free solution as discrete boundaries throughout the tube [1]. 

Electrophoresis was then applied to hydrated gel supports (e.g., polyacrylamide gel 
electrophoresis, PAGE) and later to micron diameter capillary tubes (i.e.., capillary 

electrophoresis or CE). Typically, CE utilizes a discrete sample injection into the 

capillary in the presence of buffer electrolytes. A fixed detector is placed near the 

capillary outlet, past which analytes of highest mobility migrate first followed by species 

of slower mobility; individual analytes are typically detected as bands or peaks. For 

example, with a negative voltage applied to the capillary inlet, positively charged 

analytes will be detected first, followed by neutral species, and finally species exhibiting 

a negative charge. The detector response can then be correlated to analyte migration 

speed and, through the use of standard mixtures, be used to identify and/or quantify 

analytes. 

The two primary electromotive forces in electrophoresis in a confined separation medium 

(i.e., tubular, capillary, or microfluidic channel) are analyte electrophoretic mobility and 

electroosmotic flow (EOF). EOF arises when a surface charge is present along the 

separation column; in glass-based separations with solutions above pH of approximately 

2 the surface silanol groups exhibit net negative charges. Positively charged ions in the 

solution media (typically a buffered electrolyte solution) are drawn towards the negative 

surface forming an electrical double layer which can be mobilized axially under an 

applied voltage along the length the separation media. The mobile cations additionally 

cause movement of the bulk solution through viscous drag, referred to as EOF [2].

There are two primary disadvantages to the common method of performing CE. First, the 

method requires an injection to load the mixture to be analyzed into the capillary as a 

minimally wide plug. Second, long channels are required to produce high-resolution 

separations. Both of these disadvantages increase the space required to implement CE in 

a microfluidic chip format. Although long separation channels can be fit into a small area 

on a microfluidic chip using a serpentine pattern, every turn in the path of the separation 

channel degrades the separation resolution. Consequently, microchip CE still generally 

gives lower resolution separations than can be achieved with conventional capillarybased CE. 

Two sample loading techniques are in general use for CE: electrokinetic and 

hydrodynamic injections. The former relies upon electrophoresis to introduce the sample. 

The inlet is placed in the mixture media to be analyzed, a voltage is applied for a finite 

time, and the inlet is then placed in a blank solution containing only the background 

electrolytes. This method introduces preferential injection bias, as those analytes of 

highest mobility are injected to a greater extent than those of lower mobility. For 

example, when a negative voltage is applied to the inlet, positive species are injected both 

by electrophoresis and EOF, while neutral compounds are mobilized solely by EOF and 

negative species are pulled away from the inlet by electrophoresis while still being 

injected by EOF. 

Hydrodynamic injections can be accomplished similarly to electrokinetic methods by 

using a pressure differential across the separation column, as opposed to an electrical 

differential. Most commonly, this is employed using either a sample vial elevated relative 

to the column outlet (gravity fed) or by using a pump. While eliminating electrical 

injection bias, hydrodynamic injections suffer from possible sample injection plug 

broadening due to the laminar flow profile induced by the pressure differential, ultimately 
leading to analyte zone broadening and a loss in separation efficiency. Additionally, 

hydrodynamic injections are much more difficult to implement in a microfluidic format, 

either necessitating off-chip injection equipment or complicated fabrication procedures, 

as the gravity fed method of injection is not readily available. 

There are many examples in the prior art of the use of bulk flow in conjunction with 

electrophoresis for enhanced resolution or reduced analysis times. The most common 

example is the use of the native, unmodified EOF of the silica capillary to induce a bulk 

flow in the direction opposite to the direction of migration of anionic analytes. The time 

required for the migration of analytes through the length of the separation capillary and 

the resolution of the separation are thus increased. In addition, there are many examples 

of the use of coatings [3;4] or external electric fields [5;6] to vary the EOF of silica 

capillaries so that the bulk flow is set to the value desired for a given separation problem. 

In addition to controlling bulk flow through modifications of the EOF, pressure-driven 

flow has also been used in conjunction with EOF as a means of better regulating bulk 

flow during electrophoresis. In this manner, the bulk flow rate can be decoupled from the 

electrical characteristics of the system and employed in a way to generate a constant, bulk 

flow for CE using discrete injections [7-l0]. U.S. Pat. Nos. 5,429,728 and 5,482,608 are 

illustrative of the method. 

Generally, methods to control the bulk flow are used to either increase analyte residence 

time in the electric field in order to enhance the separation or to decrease the residence 

time in order to reduce analysis times. Use of a bulk counter flow and continuous 

introduction of the sample can also allow for the purification of species [11-13], provided 

that the mobility of the species of interest is either the greatest or least of all mixture 

components. 

SUMMARY AND DESCRIPTION OF THE INVENTION 

The present invention is a method for the separation of the constituents of a sample 

mixture. The present invention incorporates a process for coupling continuous sample 

introduction with a variable hydrodynamic bulk flow for electrophoretic separations in a 

capillary, microfluidic channel, or other separation column. By changing the bulk flow 

velocity over time during the separation, analytes are sequentially eluted onto the 

separation column from the sample vessel where they can individually be detected as 

zone boundaries. 

The method can be applied to a capillary or micro fluidic system. A typical apparatus 

includes a sample reservoir, a separation column, an outlet reservoir, and a detector for 

detecting the presence of analyte at some point along the length of the column. Both the 

inlet and outlet ends of the separation column are connected electrically to a voltage 

control device. Also included in the apparatus is a means for controlling the variable bulk 

flow velocity. For example, a regulated pressure control device could be connected to 

either the inlet reservoir or outlet reservoir. Schematic representations are shown in 

Figure 1. The pressure differential across the inlet and outlet of the separation column 

(and, hence, the bulk flow rate) can be made to vary with time, either linearly, or nonlinearly (e.g., exponentially or step-wise) throughout the electrophoretic separation. 
The primary advantage of this invention over prior art methods of electrophoresis is that 

it requires much shorter separation lengths and a much smaller total area or footprint on a 

microfluidic chip. The use of continuous sample introduction and variable bulk flow has 

the major advantage over conventional CE methods of not requiring any injection 

mechanism, eliminating the associated costs and footprints of such mechanisms. 

Consequently, much smaller chips can be used, reducing the cost of fabrication. 

Additionally, reduction of the area required for electrophoretic separation allows for the 

integration of more analysis elements (sample preparation, chemical reaction, etc.) into a 

fixed chip area. 

Prior art electrophoresis methods have generally employed a constant bulk flow during 

the separation, either arising solely from native EOF or through the use of controlling 

mechanisms. A modified constant bulk flow, such as a pressure induced flow counter to 

EOF, can be used to provide increased resolution for a separation of two closely 

migrating analytes. However, it is of limited utility for separations involving more than 

two analytes. For example, if three analytes of interest are present, a constant counter 

bulk flow can be set to provide enhanced resolution of the first two analytes; however the 

third analyte will then likely move through the column in the opposite direction, away 

from the detector. In a defined injection electrophoretic method as is commonly 

practiced, the third analyte peak would likely migrate out of the column and never be 

detected. On the other hand, if the constant bulk flow is chosen so that the third analyte 

does migrate in the correct direction towards the detector, the desired resolution may not 

be achieved between the first two analytes. 

In the method of the current invention, the bulk flow is varied over time during the 

separation in a systematic way to provide the desired resolution between all analyte 

zones. In the three-analyte example, the bulk flow would be varied so that early in the 

separation, it would provide the desired resolution between the first two analytes, and 

then later in the separation it would provide the desired resolution between the second 

and third analytes. Furthermore, the fact that the third analyte moves away from the 

detector early in the separation is used to advantage in that the third analyte does not 

enter the column until after the first two analytes have been resolved and detected. In the 

commonly used defined injection methods, should the analyte zone migrate out of the 

inlet, the analyte zone would be diluted within the blank electrolyte reservoir to which the 

inlet is connected, likely making it wholly undetectable. This is in contrast to the method 

described herein, which utilizes a continuous injection, whereby during the portion of the 

separation that the third analyte migrates away from the detector, it remains in the sample 

reservoir at an effectively constant concentration, available to be eluted into the column 

later in the separation. 

Even for separations of just two analytes, the method of the current invention has 

advantages over prior art electrophoretic methods employing constant bulk flow. With a 

constant bulk flow, in order to achieve a desired resolution between two closely 

migrating analytes requires accurate and precise control of the value of the bulk flow 

velocity. In many situations (because of variation of the electroosmotic flow, for 

example), it is difficult or impossible to control a fixed bulk flow velocity to the required 

accuracy and precision; this difficulty is exacerbated when using surface modifications to 
control EOF, which can be irreproducible in production and can degrade over time within 

a given column. With the current invention, the desired resolution is set by the choice of 

the rate of change of the bulk flow velocity, or the bulk flow acceleration. Because 

column-to-column or run-to-run variations in the electroosmotic mobility do not effect 

the bulk flow acceleration, it is easier to set the acceleration with the necessary level of 

precision. 

The method of the invention is based upon balancing of electrophoretic velocities and 

bulk fluid velocity in order to discriminate different constituents within a mixture. 

Typically, the analyte electrophoretic velocities and the bulk velocity are in opposite 

directions in the separation column in order to increase species' resolutions. The 

magnitude of the bulk flow velocity is initially greater than the magnitude of the 

electrophoretic velocity of any of the analyte species, and analyte is therefore excluded 

from entering the column. The bulk flow is then decreased while electrophoresis remains 

constant. When the magnitude of the bulk flow velocity becomes less than the magnitude 

of the electrophoretic velocity of an analyte, that analyte will enter the column and 

migrate towards the detector. As the analyte moves past the detector it will be detected as 

a step or moving boundary. Analytes with different electrophoretic mobilities will be 

resolved because they will enter the column at different times (as the bulk velocity is 

decreased) and because, once in the column, they will migrate towards the detector at 

different speeds. Because different analytes are made to enter the capillary at different 

times, very short separation columns can be used to produce high-resolution separations. 

The method has been designated as gradient elution moving boundary electrophoresis 

(GEMBE). 

The resolution of the separation can be increased by reducing the rate at which the bulk 

velocity is decreased (the bulk flow acceleration) in order to increase the time between 

the points at which each analyte enters the capillary. As an example, two similar 

fluorescent dyes, fluorescein and carboxyfluorescein were separated in a GEMBE 

apparatus at different rates of bulk flow acceleration. The apparatus used was similar to 

that depicted in Figure 1a. The separation column was a 3 cm length capillary with an 

inner diameter of 30 μ m and an outer diameter of360 μm. The inlet end of the capillary 

was connected via a 360 μm i.d. hole to a 150 μL volume sample reservoir. The outlet 

end of the capillary was connected via a silicone septum to a sealed, pressure-controlled 

waste reservoir. A sample solution containing 1 μmol/L each of fluorescein and 

carboxyfluorescein in 0.5 mol/L Tris borate buffer was placed in the sample reservoir and 

the waste reservoir and capilary were filled with blank 0.5 mol/L Tris borate buffer. A 

voltage of -3000 V was applied to the sample reservoir, while the waste reservoir was 

electrically grounded. During the separation, the bulk flow was comprised of a 

combination of electroosmotic flow resulting from the applied voltage and pressuredriven flow resulting from the applied pressure at the waste reservoir. The bulk flow 

velocity was varied by changing the pressure applied to the waste reservoir. Detection of 

the analytes was performed at a distance of 1 cm from the sample inlet using a 

fluorescence microscope and color CCD camera. The applied pressure at the start of the 

separation was made great enough to exclude both analytes from entering the separation 

capillary, typically on the order of 1000 Pa. The pressure was then swept at a fixed rate 

(producing a fixed bulk flow acceleration) to sequentially introduce the analytes, 
typically on the order of 1 Pa/s. The detector response produced a stepwise pattern, 

similar to Figure 2a. The derivative of the detector response with respect to time shows a 

pattern of peaks similar to a conventional electropherogram, as in Figure 2b. Figure 3a 

demonstrates the effect of different pressure sweep rates (or bulk flow acceleration rates) 

on the resolution between the analytes in a GEMBE separation while Figure 3b shows the 

relationship between resolution and total analysis time. The separations were performed 

with the detector only 1 cm from the capillary inlet. 

As an alternative to increasing resolution between species, the analysis time ofa 

separation can be shortened in instances when more than sufficient resolution is obtained 

with conventional electrophoresis, leading to faster analysis times and higher throughput. 

An example of decreasing analysis time is shown in Figure 4, wherein five amino acids 

are separated by GEMBE. In this example, five fluorescent dansyl-labeled amino acids 

(tryptophan, phenylalanine, serine, glycine, and aspartic acid) in 0.5 mol/L Tris borate 

buffer were placed in the sample reservoir at equal concentrations of 1 mmol/L. Other 

experimental conditions were the same as the example of Figure 3, above. Figure 4a 

shows the results of a linear pressure sweep (constant bulk flow acceleration), while 

Figure 4b shows the results of a two-step gradient to elute the four fastest migrating 

amino acids at one sweep rate, followed by a faster rate to elute the slowest amino acid, 

aspartic acid. 

With GEMBE, the analyst is afforded all the experimental parameters available to 

traditional capillary electrophoresis separations, including electric field strength, 

separation column dimensions, temperature, background electrolyte composition, etc., as 

well as the new parameters of bulk flow velocity and acceleration. All these parameters 

can be adjusted to realize a separation of analytes. For a given separation column and 

background electrolyte, the voltage range can be determined through the use of Ohm's 

Law plots, which present the relationship between observed current and applied voltage. 

Positive deviations from linearity at higher voltages indicate Joule heating of the 

capillary, which can degrade the separation, and establish an upper voltage limit (along 

with physical limitations of available voltage sources). Common field strengths are 

between 100 and 1000 V cm. As with conventional CE, temperature control of the 

separation column can be utilized to extend the working voltage range, especially in the 

cases of highly conductive solutions. Either polarity of applied voltage can be used, so 

that the direction of electrophoretic migration of the analytes can be from inlet to outlet 

or from outlet to inlet. The magnitude and direction of bulk flow would then be set 

accordingly so that during the separation the analytes would move from the inlet to the 

outlet. 

Separation column cross sectional areas are typically less than 0.1 mm2

 in order to allow 

rapid heat transfer and prevent Joule heating of the capillary. The lower limit of cross 

sectional area is dictated both by availability of substrate and, when using pressure to 

control the bulk fluid flow, the limitation of the pressure control device to affect bulk 

flow against high back pressures. Commonly 100 to 20000 μm2 cross sectional areas can 

be used with good effect. As one major benefit of GEMBE is the use of short separation 

columns, separation lengths are typically limited by geometrical constraints including 

electrical and/or hydrodynamic connections to the apparatus, as well as having available 
room for detection instruments. Using a standard fluorescent microscope as a detector, 

column lengths on the order of 1 cm can be utilized without great difficulty. 

Background electrolyte compositions are typically dependent upon the analytes of 

interest, and are most commonly conductive aqueous buffered ionic solutions. The 

background electrolyte pH and ionic strength are two primary factors in modifying 

electroosmotic flow and realizing separation of two distinct analytes. Additionally, 

additives, such as organic modifiers or polymers, can be used to enhance selectivity. A 

buffer usually will have a pKa at or near the desired working pH and usually wil be less 

than 100 mmol/L in order to avoid excessive conductivity. 

When using an applied pressure to control the bulk flow rate, the initial pressure is 

typically chosen to exclude entrance of any detectable analytes onto the separation 

column. In a standard linear method (constant bulk flow acceleration), the pressure is 

decreased in a linear fashion until all analytes have entered the column for detection. The 

pressure sweep rate (acceleration) can be reduced in order to increase the resolution of 

analytes or increased in order to decrease analysis time. Commonly 0.1 Pa/s to 10 Pa/s 

rates can be utilized on a standard pressure control system (whether gravity, gas, or piston 

driven) with the other separation parameters similar to that described above. 

Alternatively, the pressure can be held constant, stepped, or varied in other non-linear 

manners throughout the separation. 

It should be noted that this method can be used for the separation of both anionic and 

cationic species in the same separation with analysis times greatly reduced as compared 

to conventional methods. For example, if a negative voltage is applied to the inlet end of 

the separation column, anions will electrophoretically migrate from the inlet to the outlet, 

and cations will electrophoretically migrate from the outlet to the inlet. The bulk flow 

velocity will initially be set in the direction from outlet to inlet and at a magnitude that is 

greater than the electrophoretic migration speed of the anionic analytes. As the bulk flow 

velocity is reduced each of the anionic analytes will be eluted into the column and 

detected as above. The bulk flow velocity would then be varied further until it changes 

direction (from inlet to outlet). The magnitude of the bulk velocity would then increase 

with time, and each cationic analyte would be pulled into the column when its 

electrophoretic velocity is overcome by the increasing bulk flow velocity. 

As in prior art electrophoretic methods, it is also possible to use this method in 

conjunction with additives to modify the electrophoretic mobility of analytes and provide 

different separation selectivity. Examples of additives include cyclodextrins or other 

chiral selectors for chiral separations, micelles, microemulsions, liposomes, dendrimers 

or other additives that act as a pseudo stationary phase to provide selectivity based upon 

the strength of interaction between the additives and the analytes. Note that with 

appropriate additives, this method can be used to separate neutral molecules. 

The steps required to implement the method of the present invention are: 

providing a reservoir of solvent or solution in fluid contact with the outlet end of the 

separation column; 
filling the separation column with the solvent or solution; 

introducing a sample solution to the inlet end of the separation column; 

producing a bulk flow of the solvent or solution through the separation column; 

applying a voltage along the length of the separation column to drive the electrophoretic 

motion of analyte(s) through the column; 

changing the bulk flow velocity over time, so that each of the analytes of interest will 

sequentially be eluted from the sample solution into the column. 

A possible additional step is: 7) detecting the analytes as they move through the column. 

A plot of detector signal vs. time will have steps similar to the result of prior art moving 

boundary electrophoresis methods. Each step will correspond to an analyte, and the 

height of each step will correspond to the concentration of that analyte in the sample. Any 

detector usable with prior art electrophoresis methods could be used. 

The solvent or solution used in step 1 can be any solvent or solution useable for 

electrophoretic separations: aqueous or non-aqueous buffer, with or without a 

pseudostationary phase or other additives. 

Examples of appropriate separation columns would be capillaries or microchannels, made 

from glass or polymer or any other electrically insulating material. The cross-sectional 

size of the inside of the column would typically be less than 0.1 mm2

, but could be larger 

as in prior art of tubular free zone electrophoresis [14]. The column could also contain a 

stationary phase, present either as a packed bed or wall coating or any other form used for 

capillary electrochromatography [15; 16]. 

The sample solution of step 3 can be prepared in a solvent or buffer that is similar or 

dissimilar to the solvent or buffer of step 1. The types of analytes that can be separated 

include any analytes that can be separated by conventional CE methods (small molecules, 

large molecules, particles, amino acids, nucleic acids, carbohydrates, proteins, peptides, 

pesticides, pharmaceuticals, cells, viruses, bacteria, etc.) 

The bulk flow of step 4 can be driven either by electroosmosis (using the same applied 

voltage as step 5) or by applied pressure gradients or both. The direction of the bulk flow 

can be in either direction through the column, from inlet to outlet or from outlet to inlet. 

The terms 'inlet' and 'outlet' refer to the net direction of motion of the analyte molecules. 

During a separation, analytes move from the sample solution through the column inlet, 

traverse the column length, and exit the column through the column outlet. 

The sign of the applied voltage depends on the direction of bulk flow and the sign of the 

charge of the analyte molecules. For example, if the analyte molecules are negatively 

charged in solution (or if they partition into a negatively charged pseudostationary phase) 

and the bulk flow is from outlet to inlet, then the voltage applied to the inlet will be 

negative relative to the voltage applied to the outlet. In most cases, when the desired 

result is high resolution in a short column, the bulk flow of the solvent and the 

electrophoretic motion of the analytes should be in opposite directions. However, in cases 
where the resolution is more than adequate, the analysis time can be reduced with this 

method by using a bulk flow that is in the same direction as the electrophoretic motion. 

The key step to this process (and the one that makes it distinguishable from the prior atr) 

is step 6. The bulk flow is initially set so that all or nearly all of the analytes of interest do 

not enter the column. During the separation, the bulk flow is changed over time so that 

each of the analytes of interest will sequentially move into the column. 

There are different ways that the bulk flow rate could be varied over time: In the 

reduction to practice described above, the bulk flow is driven by a combination of 

electroosmosis and externally applied pressure difference between the column inlet and 

outlet. The applied pressure is varied over time to vary the bulk flow rate. 

The bulk flow could also be driven entirely by electroosmosis and the electroosmotic 

mobility could be varied over time to vary the bulk flow rate. One example of this would 

be the use of a voltage applied across the walls of the separation column as described in 

ref. [5;6] 

Reference List 

1. Tiselius A (1937) Transactions of the Faraday Society 33:524-531 

2. Foret F, Krvanova L, and Bocek P (1993) Capillary Zone Electrophoresis. VCH, 

Weinheim. 

3. Dougherty AM, Cooke N, and Shieh P (1996) Handbook of Capillary 

Electrophoresis. CRC Press, Boca Raton. 

4. Pallandre A, de Lambert B, Attia R, Jonas AM, and Vio.vy JL (2006) 

Electrophoresis 27:584-610. 

5. Lee CS, Blanchard WC, and Wu CT (1990) Analytical Chemistry 62:1550-1552. 

6. Lee CS, Mcmanigil D, Wu CT, and Patel B (1991) Analytical Chemistry 

63:1519-1523. 

7. Kutter J and Welsch T (1995) Hrc-Journal of High Resolution Chromatography 

18:741-744. 

8. Kar S and Dasgupta PK (1999) Microchemical Joural 62:128-137. 

9. Kok WT (1993) Analytical Chemistry 65:1853-1860. 

10. Iwata T and Kurosu Y (1995) Analytical Sciences 11:131-133. 

11. Chanvetadze B, Burjanadze N, Bergenthal D, and Blaschke G (1999) 

Electrophoresis 20:2680-2685. 

12. McLaren DG and Chen DDY (2003) Electrophoresis 24:2887-2895. 

13. McLaren DG and Chen DDY (2004) Analytical Chemistry 76:2298-2305. 
14. Hjerten S (1967) Chromatographic Reviews 9:122-219. 

15. Knox JH and Grant IH (1991) Chromatographia 32:317-328. 

16. Jiskra J, Claessens HA, and Cramers CA (2003) Journal of Separation Science 

26:1305-1330. 

Moving Boundary Electrophoresis

1. A Tiselius. A new apparatus for electrophoretic analysis of colloidal mixtures. 

Transactions of the Faraday Society 33:524-531, 1937. 

2. Tiselius. Electrophoresis of serum globulin I. Biochemical Journal 31 :313-317, 

1937. 

3. Tiselius. Electrophoresis of serum globulin II. Electrophoretic Analysis of Normal 

and immune sera. Biochemical Journal 31: 1464-1477, 1937. 

4. Tiselius and E. A. Kabat. An electrophoretic study of immune sera and purified 

antibody preparations. Journal of Experimental Medicine 69 (1): 119-131, 1939. 

5. H Rilbe. Basic theory of electrophoresis: Definitions, terminology, and 

comparison of basic techniques. In: Electrophoretic Techniques, edited by C. F. 

Simpson and M Whittaker, London:Academic Press, 1983, p. 1-25. 

6. L. M. Hjelmelandand A. Chrambach. Formation of natural pH gradients in 

sequential moving boundary systems with solvent counterions .1. Theory. 

Electrophoresis 4 (1):20-26, 1983. 

7. Z. Buzas, L. M. Hjelmeland, and A. Chrambach. Formation of natural pH 

gradients in sequential moving boundary systems with solvent counterions .2. 

Predicted and experimental properties. Electrophoresis 4 (1):27-35, 1983. 

8. Chrambach and T. M. Jovin. Selected buffer systems for moving boundary 

electrophoresis on gels at various pH values, Presented in a simplified manner. 

Electrophoresis 4 (3):190-204, 1983. 

9. D.Lan, R. Horu, J. V. Sullivan, and A. Chrambach. An improved and simplified 

apparatus for protein extraction and concentration from gel slices using moving 

boundary electrophoresis. Electrophoresis 4 (5):335-337, 1983. 

10. Chrambach. Unified view of moving boundary electrophoresis - Practical 

implications (Plenary Lecture). Journal of Chromatography 320 (1):1-14, 1985. 

11. E. Gombocz, D. Tietz, and A. Chrambach. Moving boundary electrophoresis on 

agarose-gel of plant-viruses and polystyrene microspheres. Electrophoresis 8 

(6):286-293, 1987. 

12. W. Brzezinski, W. M. J. Vangelder, P. Mendelewski, and P. Kolster. 

Polyacrylamide-gel electrophoresis of wheat gliadins - the use of a moving 

boundary for improved resolution. Euphytica 40 (3):207-212, 1989. 
13. J. Pawliszyn and J. Q. Wu. Moving boundary capillary electrophoresis with 

concentration gradient detection. Journal of Chromatography 559 (1-2):111-118, 

1991. 

14. S. Yefimov, C. Sjomeling, A. L. Yergey, and A. Chrambach. Stacking of 

unlabeled sodium dodecyl sulfate-proteins within a fluorimetrically detected 

moving boundary, electroelution and mass spectrometric identification. 

Electrophoresis 22 (6):999-1003, 2001. 

15. Z. Buzas and A. Chrambach. Moving boundaries in sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Electrophoresis 25 (7-8):970-972,2004. 

16. E. X. Vrouwe,R. Luttge, W. Olthuis, and A. van den Berg. Microchip analysis of 

lithium in blood using moving boundary electrophoresis and zone electrophoresis. 

Electrophoresis 26 (15):3032-3042, 2005. 

17. P. Gebauer and P. Bocek. A new type of migrating zone boundary in 

electrophoresis: 1. General description of boundary behavior based on 

electromigration dispersion velocity profiles. Electrophoresis 26 (2):453-462, 

2005. 

18. P. Gebauer, Z. Mala, and P. Bocek. A new type of migrating zone boundary in 

electrophoresis: 2. Transient sample zone shapes. Electrophoresis 27 (3):519-525, 

2006. 

19. P. Gebauer, Z. Mala, and P. Bocek. A new type of migrating zone boundary in 

electrophoresis: 3. The hybrid boundary and stacking criteria. Electrophoresis 27 

(5-6):962-967,2006. 

Tubular Zone Electrophoresis

20. S. Hjerten. Free zone electrophoresis. Chromatographic Reviews 9 (2):122-219, 

1967. 

21. R. Virtanen and P. Kivalo. A new quantitative high-voltage zone electrophoresis 

method. Suomen Kemistilehti 42 (4): 182-&, 1969. 

22. S Hjerten. Free zone electrophoresis. Theory, equipment, and applications. 

Methods of Biochemical Analysis 18:55-79, 1970. 

23. P. R. Dijksterhuis and R. Rust. Device for electrophoretic analysis using a 

capillary tube with detection means. U.S. Patent 3,620,958. 1971. 

24. R. Virtanen. Zone electrophoresis in a narrow-bore tube employing 

potentiometric detection - Theoretical and experimental study. Acta 

Polytechnica Scandinavica-Chemical Technology Series (123):1-67, 1974. 

25. L. A. Lewis and J. J. Opplt. CRC Handbook of Electrophoresis: Lipoproteins: 

Basic Principles and Concepts, Boca Raton:CRC Press, 1979. 
Flow Controlled Electrophoresis

26. F. Everaerts. Method in counterflow isotachophoresis. U.S. Patent 3,705,845. 

1972. 

27. B. F. Sunden. Method in counter flow isotachophoresis. U.S. Patent 3,869,365. 

1975. 

28. P. H. O'Farell. Method and apparatus for dynamic equilibrium electrophoresis. 

U.S. Patent 4,323,439. 1982. 

29. P. H. O'Farrell. Separation techniques based on the opposition of two 

counteracting forces to produce a dynamic equilibrium. Science 227 

(4694):1586-1589, 1985. 

30. M. Bier and G. E. Twitty. Process and apparatus for recycling isoelectric 

focusing and isotachophoresis. U.S. Patent 4,897,169. 1990. 

31. S. Lee, C. T. Wu, T. Lopes, and B. Patel. Analysis of separation efficiency in 

capillary electrophoresis with direct control of electroosmosis by using an 

external electric-field. Journal of Chromatography 559 (1-2):133-140, 1991. 

32. D. Schran, S. Morkowski, E. R. Karamova, and G. 1. Abelev. Counterflow 

affinity isotachophoresis on cellulose-acetate membranes. Electrophoresis 12 

(6):414-419, 1991. 

33. N. B. Egen, G. E. Twitty, D. W. Samons, and R. Sharnez. Multi-modality 

electrical separator apparatus and method. U.S. Patent 5,173,164. 1992. 

34. C. F. Ivory and W. A. Gobie. Electrophoretic processor and methods. U.S. 

Patent 5,200,050. 1993. 

35. Keely, R. R. Holloway, T. A. A. M. van de Goor, and D. McManigill. 

Dispersion in capillary electrophoresis with external flow-control methods. 

Journal of Chromatography A 652 (1):283-289, 1993. 

36. W. T. Kok. Off-column detection with pressure compensation in capillary 

electrophoresis. Analytical Chemistry 65 (14):1853-1860, 1993. 

37. R. R. Deshmukh and M. Bier. Counterflow in isotachophoresis - Computersimulation and experimental studies. Electrophoresis 14 (3):205-213, 1993. 

38. N. B. Egen, G. E. Twitty, and D. W. Sammons. Electrical separator apparatus 

and method of counterflow gradient focusing. U.S. Patent 5,336,387.1994. 

39. T. Culbertson and J. W. Jorgenson. Flow counterbalanced capillary 

electrophoresis. Analytical Chemistry 66 (7):955-962, 1994. 

40. P. Welch and B. M. Bredt. Capillary electrophoresis method and apparatus. U.S. 

Patent 5,302,264. 1994. 
41. M. Mazereeuw, U. R. Tjaden, and J. Vandergreef. In-line isotachophoretic 

focusing of very large injection volumes for capillary zone electrophoresis using 

a hydrodynamic counterflow. Journal of Chromatography A 677 (1):151-157, 

1994. 

42. T. Iwata and Y. Kurosu. Enhancement of the sensitivity and resolution by flowcounterbalanced capillary electrophoresis. Analytical Sciences 11 (1): 131-133, 

1995. 

43. J. Kutter and T. Welsch. The effect of electroosmotic and hydrodynamic flow 

profile superposition on band broadening in capillary electrophoresis. HrcJournal of High Resolution Chromatography 18 (12):741-744, 1995. 

44. G. B. Gordon. Electroosmotic flow control using back pressure in capillary 

electrophoresis. U.S. Patent 5,429,728. 1995. 

45. Keely, D. McManigil, and R. R. Holloway. Capillary electrophoresis flow 

control system. U.S. Patent 5,482,608. 1996. 

46. J-L. Liao, S. Hjerten, and C. Siebert. Concentration of biological samples on a 

microliter scale and analysis by capillary electrophoresis. U.S. Patent 5,505,831. 

1996. 

47. J. A. Kinzer, J. W. Olesik, and S. V. Olesik. Effect of laminar flow in capillary 

electrophoresis: Model and experimental results on controlling analysis time 

and resolution with inductively coupled plasma mass spectrometry detection. 

Analytical Chemistry 68 (18):3250-3257, 1996. 

48. Wiliams and G. Vigh. The use of hydrodynamic counterflow to improve the 

resolution and detection of the minor component in the capillary electrophoretic 

analysis of enantiomers. Enantiomer 1 (3):183-191, 1996. 

49. D. W. Sammons, G. E. Twitty, J. G. Utermohlen, and R. Sharnez. Method for 

separation of nucleated fetal erythrocytes from maternal blood samples. U.S. 

Patent 5,662,813. 1997. 

50. D. W. Sammons, M. Manley, J. G. Utermohlen, and G. E. Twitty. Method for 

enrichment of fetal cell population from maternal whole blood samples. U.S. 

Patent 5,676,849.1997. 

51. Williams and G. Vigh. Determination of effective mobilities and chiral 

separation selectivities from partially separated enantiomer peaks in a racemic 

mixture using. pressure mediated capillary electrophoresis. Analytical 

Chemistry 69 (21):4410-4418, 1997. 

52. H. S. Chen and H. T. Chang. Capillary electrophoretic separation of 1 to 10 kbp 

sized dsDNA using poly(ethylene oxide) solutions in the presence of 

electroosmotic counterflow. Electrophoresis 19 (18):3149-3153, 1998. 
53. Soontornniyomkij, S. Chen, E. S. Minnoor, and D. J. Pietrzyk. Application of an 

electroosmotic flow gradient in capillary zone electrophoresis separations. 

Journal of Liquid Chromatography & Related Technologies 21 (19):2957-2976, 

1998. 

54. S. Kar and P. K. Dasgupta. Improving resolution in capillary zone 

electrophoresis through bulk flow control. Microchemical Journal 62 (1):128-

137, 1999. 

55. Chanvetadze, N. Burjanadze, D. Bergenthal, and G. Blaschke. Potential of flowcounterbalanced capillary electrophoresis for analytical and micropreparative 

separations. Electrophoresis 20 (13):2680-2685, 1999. 

56. R. Bowerban and M. L. Lee. Comprehensive isotachophoresis-capillary zone 

electrophoresis using directly inserted columns having different diameters with 

a periodic counterflow and dual ultraviolet detectors. Journal of Microcolumn

Separations 13 (8):361-370,2001. 

57. C. F. Ivory, Z. Huang, and F. J. Schuetze. Device and method for focusing 

solutes in an electric field gradient. U.S. Patent 6,277,258. 2001. 

58. Z. J. Jia, T. Ramstad, and M. Zhong. Determination of protein-drug binding 

constants by pressure-assisted capillary electrophoresis (PACE)/frontal analysis 

(FA). Journal of Pharmaceutical and Biomedical Analysis 30 (3):405-413, 

2002. 

59. P. H. Rhodes and R. S. Snyder. Method and apparatus for electrophoretic 

focusing. U.S. Patent 6,478,942. 2002. 

60. G. McLaren and D. D. Y. Chen. A quantitative study of continuous flowcounterbalanced capillary electrophoresis for sample purification. 

Electrophoresis 24 (17):2887-2895, 2003. 

61. L. K. Ista, G. P. Lopez, C. F. Ivory, M. J. Ortiz, T. A. Schifani, C. D. 

Schwappach, and S. S. Sibbett. Microchip countercurrent electroseparation. Lab 

on A Chip 3 (4):266-272, 2003. 

62. J. Astorga-Wells and H. Swerdlow. Fluidic preconcentrator device for capillary 

electrophoresis of proteins. Analytical Chemistry 75 (19):5207-5212, 2003. 

63. O. V. Oshurkova, A.I. Gorshkov, and V. P. Nesterov. Use of counterflow in 

bidirectional capillary isotachophoresis. Russian Journal of Electrochemistry 40 

(5):516-520, 2004. 

64. McLaren and D. D. Y. Chen. Continuous electrophoretic purification of 

individual analytes from multicomponent mixtures. Analytical Chemistry 76 

(8):2298-2305, 2004. 
65. O. V. Oshurkova, A. I. Gorshkov, and V. P. Nesterov. Use of counterflow in 

bidirectional capillary isotachophoresis. Russian Journal of Electrochemistry 40 

(5):516-520,2004. . 

66. 66. Gillogly and C. E. Lunte. pH-mediated acid stacking with reverse pressure 

for the analysis of cationic pharmaceuticals in capillary electrophoresis. 

Electrophoresis 26 (3):633-639, 2005. 

Frontal Analvsis CapilarvElectrophoresis 

67. T. Hattori, R. Hallberg, and P. L. Dubin. Roles of electrostatic interaction and 

polymer structure in the binding of beta-lactoglobulin to anionic 

polyelectrolytes: Measurement of binding constants by frontal analysis 

continuous capillary electrophoresis. Langmuir 16 (25):9738-9743, 2000. 

68. T. Hattori, K. Kimura, E. Seyrek, and P. L. Dubin. Binding of bovine serum 

albumin to heparin determined by turbidimetric titration and frontal analysis 

continuous capillary electrophoresis. Analytical Biochemistry 295 (2):158-167, 

2001. 

69. Zhang, T. Hattori, and P. L. Dubin. Observation of compositional heterogeneity 

in poly(styrene sulfonate) using frontal analysis continuous capillary 

electrophoresis. Macromolecules 34 (19):6790-6794, 2001. 

70. Zhang, G. F. Kirton, and P. L. Dubin. Compositional heterogeneity in 

anionic/nonionic mixed micelles observed by frontal analysis continuous 

capillary electrophoresis. Langmuir 18 (12):4605-4609, 2002. 

71. J. Backhouse, H. J. Crabtree, and D. M. Glerum. Frontal analysis on a 

microchip. Analyst 127 (9):1169-1175, 2002. 

72. J. Ostergaard and N. H. H. Heegaard. Capillary electrophoresis frontal analysis: 

Principles and applications for the study of drug-plasma protein binding. 

Electrophoresis 24 (17):2903-2913, 2003. 

73. T. Le Saux, A. Varenne, and P. Gareil. Determination of the aggregation 

threshold of non-UV - absorbing, neutral or charged surfactants by frontal- and 

vacancy-frontal analysis continuous capillary electrophoresis. Journal of 

Chromatography A 1041 (1-2):219-226, 2004. 

74. T. Le Saux, H. Hisamoto, and S. Terabe. Measurement of monomolecular 

binding constants of neutral phenols into the beta-cyclodextrin by continuous 

frontal analysis in capillary and microchip electrophoresis via a competitive 

assay. Journal of Chromatography A 1104 (1-2):352-358, 2006. 

Cyclic Capilarv Electrophoresis

75. J. G. Zhao, T. Hooker, and J. W. Jorgenson. Synchronous cyclic capillary 

electrophoresis using conventional capillaries: System design and preliminary 

results. Journal of Microcolumn Separations 11 (6):431-437, 1999. 
76. J. G. Choi, M. Kim, R. Dadoo, and R. N. Zare. Electrophoretron: a new method 

for enhancing resolution in electrokinetic separations. Journal of 

Chromatography A 924 (1-2):53-58, 2001. 

77. Griess, H. Choi, A. Basu, J. W. Valvano, and P. Serwer. Cyclic capillary 

electrophoresis. Electrophoresis 23 (16):2610-2617, 2002. 

78. J. C. T. Eijkel, A. van den Berg, and A. Manz. Cyclic electrophoretic and 

chromatographic separation methods. Electrophoresis 25 (2):243-252, 2004. 

79. Capillary Electrochromatographv 

80. Ericson and S. Hjerten. Reversed-phase electrochromatography of proteins on 

modified continuous beds using normal-flow and counterflow gradients. 

Theoretical and practical considerations. Analytical Chemistry 71 (8):1621-

1627, 1999. 

81. Ericson, J. Holm, T. Ericson, and S. Hjerten. Electroosmosis- and pressuredriven chromatography in chips using continuous beds. Analytical Chemistry 72 

(1):81-87, 2000. 

82. Rimmer, S. M. Piraino, and J. G. Dorsey. Gradient elution techniques for 

capillary electrochromatography. Journal of Chromatography A 887 (1-2):115-

124, 2000. 

83. Steiner and B. Scherer. Instrumentation for capillary electrochromatography. 

Journal of Chromatography A 887 (1-2):55-83, 2000. 

84. Vanoenacker, T. Van den Bosch, G. Rozing, and P. Sandra. Recent applications 

of capillary electrochromatography. Electrophoresis 22 (19):4064-4103, 2001. 

85. T. B. Stachowiak, F. Svec, and J. M. J. Frechet. Chip electrochromatography. 

Journal of Chromatography A 1044 (1-2):97-111, 2004. 

86. T. H. Henley, R. T. Wilburn, A. M. Crouch, and J. W. Jorgenson. Flow 

counterbalanced capillary electrophoresis using packed capillary columns: 

Resolution of enantiomers, and isotopomers. Analytical Chemistry 77 (21 

):7024-7031, 2005. 

87. Nilsson and S. Nilsson. Nanoparticle-based pseudo stationary phases in 

capillary electrochromatography. Electrophoresis 27 (1):76-83, 2006. 
Figure 1a - capillary GEMBEF 
Figure 2a - GEMBE raw data 

Figure 2b – GEMBE derivative 
Figure 3a - RS vs Rate 

Figure 3b – Time vs Rate 

Figure 3c – RS vs rate 
Figure 4a - linear aa 

Figure 4b – stepped aa