Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
2000-05-23
2002-12-10
Tung, T. (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C204S612000, C204S409000, C422S082020
Reexamination Certificate
active
06491805
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to analytical chemistry and, more particularly, to conductivity detectors for electrophoresis and other analysis methods using sample flow channels. A major objective of the invention is to provide for improved contactless conductivity detection for electrophoretically separated sample components.
Much of modern progress in the medical, environmental, forensic, and other sciences can be attributed to advances in analytical chemistry. One important class of analytical tools separates sample components by moving them at different rates along a separation channel. Of primary interest herein is electrophoresis in which an electric field moves sample components along a separation channel; the components are separated according to their electrophoretic mobilities (roughly corresponding to charge-to-mass ratios).
There are two prevalent types of electrophoresis. In capillary zone electrophoresis (CZE) a sample is dissolved in an otherwise uniform buffer. A constant voltage potential is applied along the separation channel so that ions move at rates corresponding to their electrophoretic mobilities. Since different ionic species have different charge-to-mass ratios, they separate as they migrate along the channel.
In isotachophoresis (ITP), the separation channel is initially filled by a “leading” buffer, with the sample introduced at one end of the channel. An electric potential gradient along the channel causes the sample ions to migrate according to their electrophoretic mobilities. As the sample moves along the channel, it is followed by a trailing buffer having a lower electrophoretic mobility than the leading buffer. Sample components with intermediate electrophoretic mobilities remain between the buffers, forming into adjacent bands.
Once the sample components are separated it is usually desirable to identify and, perhaps, quantify the components. This typically requires detection of the components. Detectors are available that detect components by monitoring certain parameters, such as conductivity, fluorescence or absorption of ultra-violet (UV) electromagnetic energy as sample components pass.
Conductivity detection is appealing for electrophoresis since it operates on the same parameter used to separate the components. In other words, sample components that cannot be detected by monitoring conductivity are those unlikely to be separated by electrophoresis. Separated components necessarily have a measurable conductivity associated with their electrophoretic mobilities.
Contact conductivity detection can be implemented by locating electrodes on the interior channel walls of an electrophoretic channel. Typically, electrodes can oppose each other across a transverse width or diameter of the electrophoretic channel. An alternating current can be applied to a drive electrode, while the potential at a detection electrode (arranged as an intermediate node in a voltage divider) can be monitored to provide an indication of sample conductivity. However, since the electrodes are in contact with the sample fluid, chemical reactions at the electrodes can affect both the electrodes and the sample. Such interaction can cause undesirable artifacts within a run and undermine repeatability between runs.
Contactless conductivity detection typically involves forming electrodes on the outside walls that define the electrophoresis channel. Electrodes can be electrically coupled (i.e., an electrical signal on one can be detected by the other) to each other through the channel. Since the sample does not contact the electrodes, the problem of chemical interaction between sample and electrodes is effectively addressed.
Changes in sample conductivity as components pass the electrodes cause changes in impedance between the electrodes. This impedance can be monitored using a voltage divider arrangement. However, the impedance variations are relatively small due to the constant capacitance contribution of the channel walls to the impedance. Small signal-to-background ratios result in reduced sensitivity to conductivity changes. The output gain of the detector can be increased to amplify the effect of conductivity changes on the detector output. However, undesirable artifacts, such as variations in the AC drive amplitude due to power source fluctuations, are amplified as well.
There are further problems in the case of isotachophoresis. The conductivity profile of a typical sample separated by isotachophoresis is a step function. This means that the background signal increases as the sample component bands progress past the detector. This further reduces the signal-to-background ratio of the detector output.
Finally, the contributions of individual sample components are not readily read from a step function. A more readable function is obtained by differentiating the step function to obtain a profile of the rate of conductivity change over time. This produces a relatively readable series of peaks at the boundaries between component bands. However, the mathematical differentiation introduces an additional step in the procedure and introduces computation errors into the final data.
What is needed is a conductivity detector that is more sensitive than the foregoing contactless conductivity detectors, but more reliable than foregoing contacting conductivity detectors. Furthermore, in the case of isotachophoresis, the problems with the step function should be addressed.
SUMMARY OF THE INVENTION
The present invention provides sample-analysis systems with antisynchronously driven contactless conductivity detectors. The invention has particular applicability to electrophoresis because of its amenability to conductivity detection. However, in its most general aspect, the invention is not dependent on the separation technology.
The sample-analysis system includes a sample-component separator and a sample-component detector. The separator provides a channel along which sample components move past the detector. The detector includes an AC source, at least two drive electrodes, at least one detection electrode, and a signal processor. The AC source drives two drive electrodes antisynchronously (180°+/−45° out of phase, the closer to 180° the better). A detection electrode is electrically coupled to both drive electrodes so that the drive signals tend to cancel; the degree of cancellation varies according to the local conductivity in the separation channel. The signal processor provides a readout that represents the degree of cancellation, and thus conductivity.
So that conductivity changes can be detected, at least one drive electrode is electrically coupled to the detection electrode through the separation channel. In differential realizations of the invention, both antisynchronously driven electrodes are coupled to the detection through the channel, while in direct realizations of the invention, one of the antisynchronously driven electrodes is electrically coupled to the detection electrode but not through the channel. The direct realizations provide a direct readout of local conductivity, while the differential realizations provide a direct readout of changes in local conductivity. Accordingly, the direct realizations provide a desired “series-of-peaks” readout for CZE, while the differential realizations provide a “series-of-peaks” readout for ITP. The direct realization provides superior spatial resolution, while the differential realization provides superior background signal cancellation.
The invention provides for hybrid-detection analytical systems that implement both differential and direct detector modes. For example, three drive electrodes can be used, two of which are coupled to the detection electrode through a separation channel, and one of which is coupled to the detection electrode but not through the separation channel. A switch can be used to select whether the AC power source is coupled for differential detection or direct detection. Such hybrid-detection systems can provide a desired series-of-peaks readout for both CZE and ITP
Gordon Gary B.
van de Goor Tom A.
Agilent Technologie,s Inc.
Noguerola Alex
Tung T.
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