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:
means for introducing a sample into a first microchannel having an isoelectric focusing buffer with a first ionic strength therein formed in a module;
means for separating the sample into a plurality of sub-samples based on varying isoelectric points of the plurality of sub-samples;
means for causing a first of the plurality of sub-samples to enter a first of a plurality of second parallel microchannels integrally connected to the first microchannel, the plurality of second parallel microchannels having an electrophoresis buffer with a second ionic strength therein, wherein the second ionic strength of the electrophoresis buffer is different from the first ionic strength of the isoelectric focusing buffer;
means for separating the first of the plurality of sub-samples into a plurality of protein components according to electrophoresis;
means for applying opposite charges to either end of the first microchannel to facilitate separating the sample into the plurality of sub-samples;
means for applying a first charge to the non-intersecting end of a third microchannel integrally connected to the first microchannel and applying a second opposite charge to the non-intersecting end of the first of the plurality of second microchannels to facilitate the first of the plurality of sub-samples entering the first of the plurality of second microchannels and further separating according to electrophoresis;
means for aligning a first detector with the first microchannel in order to detect the separation of the sample into the plurality of sub-samples based on varying isoelectric points of the plurality of sub-samples within the first microchannel;
means for aligning a second detector with the non-intersecting end of the first of the plurality of second microchannels; and
means for detecting the plurality of protein components for the first of the plurality of sub-samples.
2. The system according to claim 1 , wherein the means for detecting comprises UV-Visible (“UV-Vis”) and photodiode (“PDA”) arrays.
3. The system according to claim 2 , wherein the UV-Vis and PDA arrays continuously monitor the separation of the sample into the plurality of sub-samples.
4. 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.
5. The system according to claim 4 , wherein the at least one known component is an internal standard and the at least one unknown component is a protein component.
6. The system according to claim 5 , wherein the protein component is at least one of an amino acid, a peptide, a polypeptide or a protein.
7. The system according to claim 1 , wherein the second detector is a mass spectrometer that produces a spectrum indicative of each of the plurality of protein components.
8. The system according to claim 1 , wherein the means for detecting the plurality of protein components comprises a mass spectrometer for producing a spectrum indicative of each of the plurality of protein components.
9. The system according to claim 1 , wherein the means for aligning the first detector with the first microchannel comprises an X-Y translator.