System and method for the separation of analytes
View Patent ↗A separation module operates to fractionate or separate an analyte into fractions according to pI, i.e., pI bands, utilizing capillary isoelectric focusing (“CIEF”) within a first microchannel. The fractions are stacked to form plugs, the number of which is determined by a number of parallel second microchannels integrally connected to the first microchannel, into which the fractions are directed according to the buffer characteristics found in each of the individual microchannels. Within the microchannels the plugs are separated into proteins according to a different chemical property, i.e., “m/z,” utilizing capillary electrophoresis (“CE”).
1. A system for separating a sample comprising:
a module containing a first microchannel;
a first and a second reservoir located at opposite ends of the first microchannel;
a first electrode in electrical contact with the first reservoir and a second electrode in electrical contact with the second reservoir; and
an isoelectric focusing buffer having a first ionic strength, which isoelectric focusing buffer is located within the first microchannel;
a plurality of second microchannels integrated with the first microchannel on the module along the length of the first microchannel at a first end of each of the plurality of second microchannels;
an electrophoresis buffer having a second ionic strength different from the first ionic strength of the isoelectric focusing buffer, which electrophoresis buffer is located within the plurality of second microchannels;
a first detector, which first detector is positioned to scan the first microchannel, which first detector is operative to detect isoelectric separation of the sample within the first microchannel; and
a third microchannel integrated with the first microchannel and containing a third reservoir at one end thereof, the third reservoir being in electrical contact with a third electrode.
2. The system according to claim 1 , wherein the module is comprised of at least one of silicon and polydimethylsiloxane.
3. The system according to claim 1 , wherein at least the third reservoir contains a buffer.
4. The system according to claim 1 , wherein the first detector is an UV-Vis photodetector array.
5. The system according to claim 1 , wherein each of the plurality of sub-samples contains at least one known component and at least one unknown component.
6. The system according to claim 5 , wherein the at least one known component is an internal standard and the at least one unknown component is a protein component.
7. The system according to claim 6 , wherein the protein component is at least one of an amino acid, a peptide, a polypeptide or a protein.
8. The system according to claim 1 , wherein each of the plurality of sub-samples is further separated into a plurality of protein components within one of the plurality of second microchannels.
9. The system according to claim 8 , wherein each of the plurality of protein components are at least one of an amino acid, a peptide, a polypeptide or a protein.
10. The system according to claim 9 , further including at least a second detector for detecting the plurality of protein components in order to identify the composition of the sample.
11. The system according to claim 10 , wherein the second detector is a mass spectrometer for producing a spectrum indicative of each of the plurality of protein components.
12. The system according to claim 10 , wherein the second detector is a Raman spectrometer.
13. The system according to claim 1 , wherein the first detector is mounted on an X-Y translation stage.