Stationary capillary electrophoresis system

Abstract

A system and method for capillary electrophoresis are provided for analyzing a macromolecule prepared from a complex liquid mixture. In particular applications, methods and apparatus are provided for separating and analyzing a solution containing a denatured macromolecule by employing a stationary capillary electrophoresis apparatus. An apparatus for capillary electrophoresis includes an inlet chamber and a capillary electrophoresis column. One end of the column is fixed at the interior of the inlet chamber. The column has a length of at least about 20 centimeters. Also included is a liquid source adapted for automatic control. The liquid source supplies a liquid sample through an input valve into the inlet chamber so that the sample is in fluid communication with the end of the column. A method for capillary electrophoresis includes automatically supplying the liquid sample to the apparatus.

1 . An apparatus for capillary electrophoresis, comprising:
 an inlet chamber;   a capillary electrophoresis column, having a length of at least about 20 centimeters, one end of the column being fixed at the interior of the inlet chamber; and   a liquid source adapted for automatic control, that supplies a liquid sample through an input valve into the inlet chamber, the sample supplied to be in fluid communication with the end of the column.   
     
     
         2 . The apparatus of  claim 1 , further comprising an outlet valve located at the inlet chamber. 
     
     
         3 . The apparatus of  claim 1 , wherein the sample source pressurizes the inlet chamber to create a pressure differential across the length of the column. 
     
     
         4 . The apparatus of  claim 1 , further comprising an outlet chamber, the other end of the column being fixed at the interior of the outlet chamber. 
     
     
         5 . The apparatus of  claim 4 , wherein the sample source pressurizes one chamber compared to the other chamber to create a pressure differential across the length of the column. 
     
     
         6 . The apparatus of  claim 5 , the liquid source further comprising a reservoir supplying a cleaning solution. 
     
     
         7 . The apparatus of  claim 6 , the liquid source further comprising a mechanical pump. 
     
     
         8 . The apparatus of  claim 4 , further comprising an output valve at each chamber that is controlled to independently remove liquid from each chamber. 
     
     
         9 . The apparatus of  claim 8 , further comprising at least one reservoir supplying a buffer, the liquid source independently supplying the buffer to the chambers. 
     
     
         10 . The apparatus of  claim 9 , further comprising a fluid level sensor at each chamber. 
     
     
         11 . The apparatus of  claim 10 , further comprising a filter to separate at least a portion of insoluble components from the liquid sample, the liquid source applying the liquid to the filter with a pressure differential across the filter. 
     
     
         12 . The apparatus of  claim 4 , further comprising electrophoresis electrodes coupled to an automatically controlled power supply. 
     
     
         13 . The apparatus of  claim 12 , further comprising a heat exchanger in thermal contact with the column. 
     
     
         14 . The apparatus of  claim 13 , further comprising a degas unit that removes at least a portion of gas dissolved in the liquid. 
     
     
         15 . The apparatus of  claim 14 , further comprising an automated detector that detects a molecular analyte in the liquid. 
     
     
         16 . The apparatus of  claim 15 , wherein the detector is located at the column. 
     
     
         17 . The apparatus of  claim 16 , further comprising an automated controller. 
     
     
         18 . An apparatus for capillary electrophoresis, comprising:
 a hydraulic system adapted for control by an automated controller, comprising a pump and one or more valves;   a rough filter selected to separate from a macromolecule in a liquid mixture, at least a portion of one or more rough components in the mixture that are larger than the macromolecule;   a fine filter selected to separate from the macromolecule, at least a portion of one or more fine components in the mixture that are smaller than the macromolecule;   an inlet chamber that receives a liquid sample filtered by the rough and fine filters, the sample comprising the macromolecule;   a capillary electrophoresis column, having a length of at least about 20 centimeters, one end of the column being fixed at the interior of the inlet chamber; and   the hydraulic system being controlled to create the liquid sample, the sample comprising the macromolecule, by applying the liquid mixture to each filter, with a pressure differential across each filter, and to supply the liquid sample to the inlet chamber.   
     
     
         19 . An apparatus for capillary electrophoresis, comprising:
 a hydraulic system adapted for control by an automated controller, comprising a pump and one or more valves;   a filter selected to separate, at least in part, a macromolecule in a liquid mixture from one or more salt components in the mixture;   an inlet chamber that receives a liquid sample, the sample comprising the macromolecule separated from the salt components;   a capillary electrophoresis column, having a length of at least about 20 centimeters, one end of the column being fixed at the interior of the inlet chamber; and   an automated controller that controls the hydraulic system to create the liquid sample, the sample comprising the macromolecule, by applying the liquid mixture to the filter, with a pressure differential across the filter, and to supply the sample to the inlet chamber.   
     
     
         20 . An apparatus for capillary electrophoresis, comprising:
 a hydraulic system adapted for control by an automated controller, comprising a pump and one or more valves;   a lysis unit that lyses cells in a liquid mixture comprising cells and a macromolecule;   a filter selected to separate from the macromolecule, at least a portion of components in the mixture that are larger than the macromolecule, the components comprising insoluble lysed cell components;   an inlet chamber that receives a liquid sample, the sample comprising a macromolecule separated from the insoluble lysed cell components;   a capillary electrophoresis column, having a length of at least about 20 centimeters, one end of the column being fixed at the interior of the inlet chamber; and   an automated controller that controls the hydraulic system to create the liquid sample, the sample comprising the macromolecule, by applying the liquid mixture to the filter, with a pressure differential across the filter, and to supply the sample to the inlet chamber.   
     
     
         21 . An apparatus for capillary electrophoresis, comprising:
 an inlet chamber and an outlet chamber, the chambers each comprising an inlet valve, an output valve, a fluid level sensor, and an electrode, the electrodes coupled to a power supply;   a capillary electrophoresis column, having a length of at least about 20 centimeters, the opposite ends of the column being fixed at the interior of the respective chambers;   a liquid source comprising a pump and at least one valved reservoir supplying a buffer, the liquid source being coupled to the input valves; and an automated controller that controls:   the liquid source and at least one valve to create a pressure differential across the length of the column by pressurizing or depressurizing at least one chamber;   the liquid source, the output valves, the valved reservoir, and the level sensors to independently:   drain the chambers;   supply the chambers with liquid to place the liquid in fluid communication with the end of the column in each chamber, including supplying:   the buffer to the outlet chamber; and   independently to the inlet chamber; the buffer and a liquid sample, the liquid sample comprising a macromolecule; and   a power supply to apply a voltage differential across the column to cause electrophoresis of the macromolecule in the column.   
     
     
         22 . The apparatus of  claim 21 , further comprising an automated detector that detects the macromolecule. 
     
     
         23 . The apparatus of  claim 22 , the liquid source further comprising:
 a hydraulic system adapted for control by an automated controller, comprising a pump and one or more valves;   a rough filter selected to separate from the macromolecule in a liquid mixture comprising the macromolecule, at least a portion of one or more rough components in the mixture that are larger than the macromolecule;   a fine filter selected to separate from the macromolecule from the macromolecule in a liquid mixture comprising the macromolecule, at least a portion of one or more fine components in the mixture that are smaller than the macromolecule; and   the hydraulic system being controlled to create the liquid sample, the liquid sample comprising the macromolecule, by applying the liquid mixture to each filter, with a pressure differential across each filter.   
     
     
         24 . A method for capillary electrophoresis, comprising automatically supplying a liquid sample through a valve to an inlet chamber to place the sample in fluid communication with a capillary electrophoresis column, the chamber having one end of the column fixed at the interior of the chamber, and the column having a length of at least about 20 centimeters. 
     
     
         25 . The method of  claim 24 , further comprising pressurizing the inlet chamber to create a pressure differential across the length of the column. 
     
     
         26 . The method of  claim 24 , the other end of the column being fixed at the interior of an outlet chamber, further comprising directing fluid through the column by creating a pressure differential between the chambers. 
     
     
         27 . The method of  claim 26 , further comprising creating a pressure differential by electro-kinetic pumping. 
     
     
         28 . The method of  claim 26 , further comprising creating a pressure differential by mechanical pumping. 
     
     
         29 . The method of  claim 26 , further comprising independently directing liquid from each chamber to a waste site. 
     
     
         30 . The method of  claim 29 , further comprising independently supplying a buffer to each chamber. 
     
     
         31 . The method of  claim 30 , further independently sensing the fluid level in at least one chamber. 
     
     
         32 . The method of  claim 31 , further comprising separating at least a portion of insoluble components from the liquid sample by applying the liquid to a filter with a pressure differential across the filter. 
     
     
         33 . The method of  claim 26 , further comprising applying a voltage differential across the column to create electrophoretic flow in the column. 
     
     
         34 . The method of  claim 33 , further comprising cooling the column. 
     
     
         35 . The method of  claim 34 , further comprising degassing at least a portion of gas dissolved in the liquid sample and the buffer. 
     
     
         36 . The method of  claim 26 , further comprising detecting a molecular analyte in the liquid sample. 
     
     
         37 . The method of  claim 36 , the molecular analyte being a macromolecule. 
     
     
         38 . A method for capillary electrophoresis comprising automatically:
 acquiring a liquid mixture, the mixture comprising a macromolecule, one or more rough components that are larger than the macromolecule, and one or more fine components that are smaller than the macromolecule;   creating a liquid sample, the sample comprising the macromolecule, by separating from the macromolecule at least a portion of the components by applying the mixture to each of a plurality of filters, with a pressure differential across each filter, the filters comprising a rough filter selected to separate at least a portion of the rough components and a fine filter selected to separate at least a portion of the fine components; and   supplying the liquid sample through a valve to an inlet chamber to place the sample in fluid communication with a capillary electrophoresis column, the chamber having one end of the column fixed at the interior of the chamber, and the column having a length of at least about 20 centimeters.   
     
     
         39 . A method for capillary electrophoresis, comprising automatically:
 acquiring a liquid mixture, the mixture comprising a macromolecule and one or more salt components;   creating a liquid sample, the sample comprising the macromolecule, by separating the macromolecule from at least a portion of the salt components, by applying the mixture to a filter with a pressure differential across the filter and   supplying the liquid sample through a valve to an inlet chamber to place the sample in fluid communication with a capillary electrophoresis column, the chamber having one end of the column fixed at the interior of the chamber, and the column having a length of at least about 20 centimeters.   
     
     
         40 . A method for capillary electrophoresis comprising automatically:
 acquiring a liquid mixture, the mixture comprising a macromolecule and one or more cells;   lysing at least a portion of the cells; and   creating a liquid sample, the sample comprising the macromolecule, by separating from the macromolecule at least a portion of components larger than the macromolecule, the components comprising insoluble lysed cell components, by applying the mixture to a filter with a pressure differential across the filter; and   supplying the liquid sample through a valve to an inlet chamber to place the sample in fluid communication with a capillary electrophoresis column, the chamber having one end of the column fixed at the interior of the chamber, and the column having a length of at least about 20 centimeters.   
     
     
         41 . A method for capillary electrophoresis, comprising automatically:
 supplying a liquid sample through a valve to an inlet chamber to place the sample in fluid communication with a capillary electrophoresis column:   the chamber having one end of the column fixed at the interior of the chamber;   the column having a length of at least about 20 centimeters; and   the sample comprising a macromolecule;   directing fluid through the column by creating a pressure differential between the inlet chamber and an outlet chamber, the other end of the column being fixed at the interior of the outlet chamber;   independently controlling the fluid lever in each chamber by:   sensing the fluid level in each chamber;   supplying a buffer to each chamber; and   directing liquid from each chamber to a waste site; and   electrophoretically separating the macromolecule in the column by applying a voltage differential across the column.   
     
     
         42 . The method of  claim 41 , further comprising detecting the macromolecule. 
     
     
         43 . The method of  claim 42 , further comprising:
 acquiring a liquid mixture, the mixture comprising the macromolecule, one or more rough components that are larger than the macromolecule, and one or more fine components that are smaller than the macromolecule;   creating the liquid sample, by separating from the macromolecule at least a portion of the components by applying the mixture to each of a plurality of filters, with a pressure differential across each filter, the filters comprising a rough filter selected to separate at least a portion of the rough components and a fine filter selected to separate at least a portion of the fine components.   
     
     
         44 . An apparatus for capillary electrophoresis, comprising:
 means for automatically supplying a liquid sample through a valve to an inlet chamber to place the sample in fluid communication with a capillary electrophoresis column, the chamber having one end of the column fixed at the interior of the chamber, and the column having a length of at least about 20 centimeters; and   means for causing electrophoresis in the column.   
     
     
         45 . An apparatus for electrophoretic separation of a macromolecule, comprising:
 a plurality of valves;   a rough separation circuit comprising a rough pump, a first stage rough filter selected to separate rough components, and a second stage rough filter selected to separate rough components that pass through the first stage rough filter;   a fine/desalination circuit, comprising a fine pump, a reservoir that supplies a desalination buffer, and a fine filter s selected to separate fine components from the macromolecule;   a denaturation circuit comprising a denaturation pump, a denaturing vessel comprising a heating element and a cooling element, a precipitation pump, a reservoir supplying a denaturation buffer, a reservoir supplying a pH buffer, a pH sensor and a precipitation filter selected to separate insoluble denaturation precipitate components;   a capillary electrophoresis circuit comprising   an inlet chamber and an outlet chamber, the chambers each comprising an inlet valve, an output valve, a fluid level sensor, and an electrode, the electrodes coupled to a power supply;   a capillary electrophoresis column, having a length of at least about 20 centimeters, the opposite ends of the column being fixed at the interior of the respective chambers;   a buffer reservoir supplying the chambers;   an automated controller in electronic communication with the pumps, the elements, the valves, the sensors and the power supply that controls the apparatus to:   acquire a liquid mixture from a sampling site, the mixture comprising a macromolecule, rough components, and fine components;   separate at least a portion of rough components from the macromolecule in the rough separation circuit;   separate at least a portion of fine components from the macromolecule in the fine/desalination separation circuit, the fine components comprising salt components;   denature the macromolecule in the denaturation circuit; and   electrophoretically separate the denatured macromolecule from other components by employing the capillary electrophoresis circuit.