Abstract:
An on-column detector for electrophoresis samples based on the principles of potential gradient detection, in which the electrodes for detection are physically isolated from the electrophoretic separation process, but maintains the same electrical potential as the corresponding interior of the electrophoretic separation channel. Potential gradient detection is used to measure the applied electrical field at two points within the electrophoretic channel during electrophoresis. When sample components with conductivity different from the electrophoretic medium passes between these two points, it causes a change in the potential gradient between the two points, which would be sensed by the sensing electrodes of the detector and registered by a data acquisition system. The apparatus can make use of conventional separation channel as well as separation channels on microchips. In accordance with the present invention, a sensor with electrically conductive medium is added and connected to the separation channel via a conductive element on the surface of the separation channel.

Description:
FIELD OF THE INVENTION 
   The present invention is related to sample detection in electrophoresis. In particular, the present invention is related to conductivity detection in electrophoresis. 
   BACKGROUND OF THE INVENTION 
   Capillary electrophoresis (CE) is a powerful analytical separation technique for the analysis of complex mixtures. In CE, an unknown sample is introduced at an Inlet of a capillary channel filled with a buffer solution, and a high voltage is applied across a section of the capillary. Different constituents of the sample migrate through the capillary at different rates depending on their electrophoretic mobility&#39;s, and are separated into different zones. By detecting the chemicals passing through a part of the capillary or its outlet as a function of time, and knowing the of the possible constituents, the chemical composition of the sample can be determined. A number of detectors have been developed for CE, including optical and electrochemical methods. Electrochemical detection can be classified into three main categories: amperometry, voltammetry and conductivity measurements. Conductivity detection is a non-selective detection mode and universally applicable. Analytes are detected because of their different conductivities to that of the background electrolyte. 
   One method to measure conductivity during electrophoresis is potential gradient detection, which is accomplished by putting two electrodes in the applied electric field for electrophoresis and detecting sample components by measuring potential changes between these two electrodes when sample components are passing by. This method has been used for isotachophoresis (U.S. Pat. No. 3,941,678, 20 Feb. 1975 and U.S. Pat. No. 3,932,264, 13 Jan. 1976) and it has been mentioned that such a method can be used in modem CE (F. Foret, L. Krivankova and P. Bocek, Capillary Zone Electrophoresis, chapter 7, p147-150). There are, however, problems for using this method in electrophoresis. Firstly, the sensing electrodes need to be inserted into the separation column or capillary. The procedures are troublesome and tedious, especially if the inner diameter of the capillary is small, for example in the case of capillary electrophoresis (usually between 10-100 μm). The more serious problem is that the sensing electrodes are polarized during electrophoresis. In order to prevent formation of bubbles and deposits on the electrodes so that the electrophoresis processes can be performed under stable conditions and high sensitivity can be obtained, special means have to be used, such as adopting v/F and F/v converters in the instrumental design, reducing the areas of electrodes contacting with the buffer solutions and adding nonionic surfactant. However, all these means can only serve to alleviate, but can&#39;t eliminate completely the problems encountered. 
   Therefore, conductivity detection is usually accomplished by measuring the potential difference (signal) between two electrodes while passing through a small constant current (excited source). Several designs are used for conductivity detection in CE, i.e., on-column, end-column and contactless structures. On-column detection cells (Anal. Chem., 1987, 59, 2747-2749, U.S. Pat. No. 5,223,114, 29 Jun. 1993 and U.S. Pat. No. 5,580,435, 1994) are usually made by inserting two sensing platinum wires into the separation capillary so that the sensing electrodes can directly contact the electrolyte solution in the capillary. Although on-column conductometric detection works well, the question arises as to how to produce such structures reliably and inexpensively. The more common practices are the use of end-column detectors (such as those disclosed in Anal. Chem. 1991, 63, 189-192, J. of Capillary Electrophoresis, 1996, 1:1-11, U.S. Pat. Nos. 5,298,139, and 5,126,023), which have the advantage that the sensing electrode can be constructed directly at the outlet of the separation capillary. For end-column detection, the correct alignment of the sensing electrode with the outlet of the separation capillary is critical for success. However, the alignment is usually difficult due to the small inner diameter (10 μm-100 μm) of the capillary. 
   Another solution offered in the prior art is contactless conductivity detection (Anal. Chem., 1998, 70, 563-567). In this method, two electrodes are laid on the outside wall of the separation capillary. Therefore, no electrode is in contact with electrolyte solution. However, it is generally accepted that the contactless detection is not sensitive enough. Although their structures are varied, all the prior designs should use their own excited source and considered the high voltage applied for electrophoresis as a noise source. 
   Although the three techniques described above (i.e. potential gradient detection, potential difference detection, and contactless conductivity detection) are all based on the difference in conductivity between the electrophoretic medium and the samples, potential difference detection is the most widely used technique in capillary electrophoresis. Therefore, commercially available and commonly described conductivity detection systems typically employ the potential difference detection method. 
   OBJECT OF THE INVENTION 
   It is therefore an object of the present invention to provide a conductivity detection system which obviates the need to insert electrodes into the separation channel. 
   It is another object to provide a conductivity detection system which is effective and sensitive. 
   SUMMARY OF THE INVENTION 
   The present invention provides an on-column electrochemical detector based on the principle of potential gradient detection for electrophoresis of samples in which the electrodes for detection are physically isolated from the electrophoretic separation channel, but maintain the same electrical potential as the corresponding interior of the electrophoretic separation channel. Since the sensing electrodes are not in direct contact with the electrophoretic medium within the electrophoretic channel, problems due to bubble and deposit formation are eliminated. 
   The apparatus can make use of conventional separation channel with an inlet end connected to an inlet reservoir and an outlet end connected to an outlet reservoir. In accordance with the present invention, a sensor reservoir with electrically conductive medium is added and connected to the separation channel via a conductive element on the surface of the separation channel. A sensing electrode is submerged in the electrically conductive medium within the sensing reservoir. The conductive element allows electrical potential from the interior of the separation channel to be transferred to the sensor reservoir without detectable bulk flow of electrophoretic medium or samples. Detectable bulk flow refers to the movement of solute or sample to an extent that there is detectable interference or disruption to migration of the sample. This detection may be based standard detection methods or the method described in the present invention. In one embodiment, the conductive element is a fracture in a separation channel made of fused silica tubing. In another embodiment, the conductive element is a thin layer of porous glass on the wall of a capillary channel electrophoretic chip. 
   During electrophoresis, the channel and reservoirs are filled with electrophoretic medium, and the ground and power electrodes from a power supply are connected to the outlet and inlet reservoirs respectively. Sample detection is achieved by sensing the potential gradient between the conductive element and the outlet end where the sensing and reference electrodes are respectively connected in the preferred embodiment. The distance between the element and the outlet end is also preferably as small as the length of the sample plug in order to maximize sensitivity of detection and resolution. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIGS. 1A and B  are typical detection methods described in the prior art. 
       FIG. 2A  is a schematic diagram of a capillary electrophoresis system as described in one embodiment of the present invention. 
       FIG. 2B  is an enlarged view of area G as shown in FIG.  2 A. 
       FIG. 3A  shows the electric field as detected by the data acquisition system using the capillary electrophoretic system shown in  FIG. 2A  without sample. 
       FIG. 3B  shows the electric field as detected by the data acquisition system using the capillary electrophoretic system shown in  FIG. 2A  when sample is injected and separated into zones. 
       FIG. 4  is a schematic diagram of the microchip CE system to illustrate another embodiment of the invention. 
       FIG. 5  is an enlarged view of area H as shown in FIG.  4 . 
       FIG. 6  is a block diagram of a circuit for the detector to illustrate yet another embodiment of the present invention. 
       FIG. 7  is an electropherogram obtained using the system shown in FIG.  2 A. 
       FIG. 8  is an electropherogram obtained on microchip CE using the system shown in FIG.  4 . 
       FIG. 9  is a schematic diagram of yet another embodiment of the present invention. 
   

   DESCRIPTION OF THE INVENTION 
   The following detailed description describes the preferred embodiment for implementing the underlying principles of the present invention. One skilled in the art should understand, however, that the following description is meant to be illustrative of the present invention, and should not be construed as limiting the principles discussed herein. In addition, certain terms are used throughout the following description and claims to refer to particular system components. As one skilled in the art will appreciate, companies may refer to a component by different names. This document does not intend to distinguish between components that differ in name but not in function. For example, the pair of electrodes for electrophoresis are referred to herein as “ground” and “power” electrodes for clarity of description. It is understood by one skilled in the art that the ground electrode may be at zero volts or floating, and that the power electrode may be of positive or negative polarity. For the same reason of clarity of description, the pair of electrodes for potential gradient detection are referred to herein as “sensing” and “reference” electrodes. It should be understood that the “sensing” electrode could be the same type as the “reference” electrode. Their positions can be exchanged with each other without affecting detection results. When performing electrophoresis on microchip, at least four electrodes are often needed for sample introduction and separation. For ease of understanding, these electrodes are classified as “power”, “ground”, “sample” and “waste” electrodes. It should be understood that the exact potential on these electrodes are not fixed, and may be set up according to the needs of the user. The reservoirs on the microchip have also been given the names “inlet”, “outlet”, “sample” and “waste” reservoirs for clarity of description. It should also be understood that the reservoirs can be used to contain different medium depending on the experimental conditions required. Furthermore, no particular inlet and outlet reservoir structures are required if microinstruments are used to load small quantities of samples directly into the inlet end. 
   In the following discussion, and in the claims the terms “including”, “having” and “comprising” are used in an open-ended fashion, and thus should be interpreted to mean “including but not limited to . . . ”. Also, the term” or “couples” is intended to mean either an indirect or direct electrical connection. Thus if a first device “couples” to a second devices, that connection may be a direct electrical connection or through an indirect electrical connection via other devices, electrical conductive medium or connections. Capillary electrophoresis is used for purposes of illustration. It should be understood by one skilled in the art that the same principles may be applied to other types of electrophoretic separations, using the teaching provided herewith. 
     FIGS. 1A and B  show a section of an electrophoretic capillary tube, illustrating the principles of potential gradient and potential difference detection systems respectively as known in the art. In the potential gradient detection system as shown in  FIG. 1A , the two sensing electrodes  10 A and  10 B come into contact with the electrophoretic medium at two non-parallel positions along the longitudinal axis of the electrophoretic channel  12 , which is connected to a power source generating an electrical potential between ends  12   a  and  12   b . The portions of the electrodes in contact with the solution inside the capillary tube is shown in dotted lines. In the potential difference detection method, the two sensing electrodes  14 A and  14 B have to be in contact with the electrophoretic medium at exactly the same cross-sectional plane of channel  15  having an electrophoretic potential between ends  15   a  and  15   b.  The resistance of the electrophretic medium may be monitored using a small a.c. current between the sensing electrodes. Because the sensing electrodes are within high electric field during electrophoresis, bubble and deposit form on the surface of the sensing electrodes due to electrochemical reactions, which would affect the electrophoretic process, and decrease the detection sensitivity since the sensing electrodes are directly within the electrophoretic channel. 
     FIGS. 2A and 2B  show one setup of capillary electrophoresis (CE) based on potential gradient detection constructed in accordance with the present invention. A 50 μm inner diameter fused silica capillary is used as the separation capillary  32 . A fracture  58  is made before the outlet of the separation capillary  32 . The distance L between the fracture  58  to the outlet, usually between 0.1 mm to 5 mm, should be near or smaller than the length of the sample plug injected into the separation capillary  32  in order to obtain maximum resolution between separated peaks. The capillary  32  is then inserted into the buffer reservoirs so that the outlet  37  of the capillary is connected to outlet reservoir  38 , fracture  58  is submerged in sensor reservoir  34 , and inlet  31  of the capillary is inserted into inlet reservoir  30 . Good insulation between reservoirs  34  and  38  is made by using an insulation layer  54 . Running buffer solutions for electrophoresis are filled into the three buffer reservoirs as well as the bore of the separation capillary  32 . The ground and power electrodes  46  and  26  are connected with the high voltage power supply  20  to apply high voltages needed for electrophoresis. Sensing electrode  44  is put in electrically conducting solution  36  contained in sensor reservoir  34 , and a reference electrode  42  is put in the solution  40  contained in outlet reservoir  38 . Between electrodes  44  and  42 , two resistors  48  and  50  are used to sample the potentials between electrodes  44  and  42  to the data acquisition system  52 . For sample separation, the sample can be injected by hydrodynamic injection or electrokinetic injection methods into capillary  31 , and a high voltage applied between the ground and power electrodes. Sample detection is achieved by sensing the potential difference between the reference electrode  42  and the sensing electrode  44  over time. These techniques are described by S. F. Y. Li in  Capillary electrophoresis: Principles, Practice and Applications,  Elsevier Science Publications, 1992. 
   The embodiment shown in the above figures can be used for conductivity detection in many methods of electrophoresis. For simplicity, capillary zone electrophoresis (CZE) is chosen for explaining the principle of the present invention.  FIGS. 3A and B  show a theoretical electric field across the corresponding section of capillary tube  32 .  FIG. 3A  shows buffer  33  alone.  FIG. 3B  shows buffer  33  with samples X and Y being separated by CZE. When a high voltage is applied, a straight baseline of electric field across the whole capillary  32  as shown in  FIG. 3A  is theoretically obtained because the running buffer is homogeneous during CZE. However, some difference in the electric field will exist if a sample is injected into the capillary. If the sample component&#39;s mobility, for example X, is larger than that of the running buffer, the electric field in the plug of the sample component will be lower than that of the running buffer as shown in FIG.  3 B. Conversely, if the sample component&#39;s mobility, for example Y, is smaller than that of the running buffer, the electric field in the plug of the sample component will be larger than that of the running buffer (FIG.  3 B). When the sample components are passing by the region between the fracture  58  and the outlet of the capillary, the potential between electrodes  44  and  42  will change and the analytes A or B can be detected. 
   A similar design can be used for microchip CE as shown in FIG.  4  and FIG.  5 . In this embodiment, only one capillary channel is shown for ease of illustration. It is understood that a CE chip may have numerous channels with various designs. The microchip CE in this example is made of two glass plates  60  and  64 . On bottom glass plate  60  is fabricated separation channel  78 , injection channel  80  connected to sample reservoir  82  and  62 , and sensor channel  68  connected to sensor reservoir  66  and  70 . Sample loading electrode  86 , waste electrode  90 , power electrode  88 , sensing electrode  72  and ground/reference electrode  74  are fabricated to connect to sample reservoir  82 , waste reservoir  62 , inlet reservoir  84 , sensor reservoir  70 , and outlet reservoir  76  respectively. On the top glass plate, access holes (not shown) are drilled to access the corresponding reservoirs and channels on the glass plate  60 . The two glass plates are bonded together during fabrication. The thickness L 1  of conductive wall  71  between the detection channel  68  and the separation channel  78  is less than 40 μm, preferably less than 30 μm for borate silicate glass. Samples are loaded using the loading and waste electrodes according to standard methods. It has been shown that a thin layer of glass is ion conductive. Based on the same principle described above for CE, sample components in microchip CE can be detected by measuring the potential between the electrodes  72  and  74  during electrophoresis. The distance L 2  from the detection channel to the outlet of the separation channel  78  is near or less than the length of the sample plug. For a channel made of glass, this thickness is preferably several tens of micrometers. For microchips made from other types of glass or from other material, the thickness of the conductive wall may be determined by one of ordinary skill in the art without undue experimentation. 
   Experiments have been done in the laboratory to test the feasibility of the present invention. To separate and detect K +  and Na + , 50 mM triethanolamine (pH 6.5, adjusted by adding HCl) was used as running buffer for CE. Platinum electrodes were used for applying high voltages. The sensing electrodes were Ag/AgCl wire (diameter, 1 mm) electrodes. Gigaohm (GΩ) resistors were chosen for the resistors  48  and  50 . Data acquisition was obtained through a microprocessor.  FIGS. 7 and 8  show typical electropherogram obtained. We can see that K +  and Na +  ions can be well separated and detected using the present invention. 
   From the above explanation, we can expect that noise will exist if high voltage is used for electrophoresis, and the voltage is not stable during electrophoresis, as can be seen in the baselines in  FIGS. 7 and 8 . To improve signal /noise ratio (S/N), the ratio of the potential measured to the current generated during CE can be measured using a noise reducing circuit  94 . One embodiment is shown in FIG.  6 . The voltage S 1  collected from the sensing electrodes and the voltage S 2  due to the current I are amplified by A 2  and A 1 . Then the signal S is obtained by dividing the output S 1 ′ from A 2  by the output S 2 ′ from A 1  through a divider A 3 . From  FIG. 6  it can be shown that:
 
 I=V/R   0   (1)
 
 S   1   =I×R   S ×( R   2 /( R   1   +R   2))   (2)
 
 S   2   =I×R   3   (3)
 
 S   1   ′=S   1 ×(1+ R   4   /R   5 )  (4)
 
 S   2   ′=S   2 ×(1+ R   7   /R   6 )  (5)
 
 S=k   1 ( S   1   ′/S   2 ′)  (6)
 
From Eq. 1-6
 
 S=k   1 ( S   1   ′/S   2 ′)= kR   S   (7)
 
Where k=k 1 ×{(1+R 4 /R 5 )×R 2 }/[R 3 ×(1+R 7 /R 6 )×(R1+R 2 )]=constant
 
   From the above results, one can see the signal S is proportional to R S  only and not affected by voltage, current or the resistance of the circuit. In other words, this improved circuit can remove the effects of ripple of the high voltage power supply. Therefore, the baseline noise can be reduced and the ratio of signal to noise will improve. 
   Those skilled in the art will know that many variations of design can be realized based on the same principle as described above. Although a separate noise reduction circuit  94  is shown in  FIG. 6 , it should be understood by one skilled in the art that other equivalent interfaces are possible. For instance, the noise reducing function of circuit  94  can be incorporated into a sophisticated data acquisition system  52  as part of its internal submodules. 
   As mentioned above, the distance between the two points where potential difference is measured (e.g. L and L 2  in  FIG. 2B and 5  respectively) is preferably smaller than the length of the sample plug injected in order to obtain maximum resolution. For capillary electrophoresis, the length of the sample plug Injected is typically around 1 mm. Therefore, L and L 2  are preferably less than 1 mm in order to achieve high resolution and sensitivity. Thus good electrical insulation would have to be provided between the two measuring points. Alternatively, a channel with a smaller diameter than that of the separation channel may be provided between the two measuring points such that the distance therebetween may be lengthened without compromising resolution and sensitivity. One example is shown in FIG.  9 . In this example, the inlet  101  and outlet ends  107  of a capillary tube  102  is shown. The tube  102  is separated into two parts. Section S 1 , used for separation, has a larger diameter D 1 , while section S 2 , proximate the outlet end in this example, has a smaller inner diameter of D 2 . A sample  109  of length L 3  is shown to migrate from the inlet to the outlet end. As the sample moves towards section S 2 , the length of the sample would be lengthened due to the smaller diameter of the channel. If the two measuring points for potential gradient detection (which are fracture  103  and the outlet end in this example) is provided at section S 2 , it is clear that the distance between these two measuring points may also be proportionately lengthened. 
   Capillaries with varying diameters can be made by normal commercial machines for making capillaries or pulling one end of a capillary tube with uniform diameter to produce one end with a small diameter after heating the tube. Commercially available machines include Laser-based micropipette pullers, for example the P2000 from Sutter Instrument Co. Channels on microchips having varying sizes can be easily produced through different mask design and performing the appropriate photolithography known in the art. The electrically conductive medium contained within the various sensing, outlet and inlet reservoirs may be the same or different, depending on the applications. 
   Although fused silica and glass substrates are commonly used as separation channels in CE and microchip CE, other substrates, such as poly(dimethylsiloxane) (PDMS) and PMMA, can be used also. 
   The present invention can be applied to existing electrophoretic channels by providing conductive elements on them, for example, by bonding some filters on them. The bonding method could be, for example, thermal bonding for many plastics, oxygen plasma bonding for PDMS. For a fused silica capillary, well-known techniques such as fracturing, making a frit (U.S. Reissued Pat. 035102) and applying polymers after fracturing (U.S. Pat. No. 5,169,510) may all be applied. For glass channels, a thin wall of 1-40 μm, preferably 1-20 μm, may be used. The most effective thickness is dependent on the quality of the glass, and may be determined by one of ordinary skill in the art by routine experimentation. 
   The detection channel on microchip CE could be on the top or the bottom of the separation channel rather than lying adjacent to the separation channel. The electrodes for sensing can be other electrodes, such as calomel electrode, platinum and gold. The reference electrode In the outlet reservoir can be combined with the ground electrode. For microchip CE, both sensing electrodes and the electrophoresis electrodes can be microfabricated on the chips or just inserted directly in the buffer reservoirs. It is also possible to create two or more conductive elements on the capillary or the separation channel in order to detect sample components at different places. For example, by having two factures along two different points of a capillary tube. The reference electrode may also be positioned away from the outlet end by creating an additional conductive element and the corresponding reservoir for connection to the reference electrode.