Narrow bore porous layer open tube capillary column and uses thereof
A polymer-based PLOT capillary column prepared by in situ copolymerization of a functional monomer, which usually contains the retentive chemistries, and a crosslinking monomer, which enhances the strength of the polymer matrix, is disclosed. Also disclosed is a system comprising the polymer-based PLOT column coupled to a mass flow or concentration sensitive detector, for carrying out a chemical analysis method on samples separated by liquid chromatography using the column, and a process for using the system. Columns of the invention can be prepared in a robust fashion with a very narrow i.d., e.g., 5-15 μm. Thus, they are suitable for commercial use in ultratrace LC/MS proteomic analysis. Columns according to the invention are characterized by high resolving power and high column-to-column reproducibility. When these columns are coupled on-line with, e.g., ESI-MS detection, the resulting systems are capable of detecting the component parts of complex proteomic samples down to the low attomole to sub-attomole level.
1. A process for carrying out a chemical analysis method on a sample, said method comprising the steps of:
providing a porous layer open tube (PLOT) capillary column, or channel in a microfabricated device, said column or channel comprising:
a capillary column or channel having an i.d. of 15 μm or less, an entrance end and an exit end;
a rigid porous layer separation medium comprising a highly crosslinked, macroporous, organic polymeric stationary phase layer covalently attached to the inner wall surface of said column or channel, wherein said organic polymeric stationary phase layer is from 0.5-3 μm in thickness, and wherein the reproducibility of retention time on comparable said columns or channels during use in high performance liquid chromatography varies less than 10%; and
an open unfilled bore inside said rigid porous layer, wherein said open bore is disposed around a central axis of said column or channel and extends the length of said column or channel;
coupling a mass flow or concentration sensitive detector via an interface to the exit end of said column or channel, wherein said interface is an electrospray ionization (ESI) interface or a matrix assisted laser desorption ionization (MALDI) interface;
applying an aliquot of a liquid sample to said column or channel;
conducting a high performance liquid chromatographic separation procedure on said applied sample; and
detecting said separated sample with said detector to carry out said chemical analysis method.
2. The process of claim 1 , wherein the reproducibility of retention time on comparable said columns or channels during use for said high performance liquid chromatographic separation procedure varies less than 5%.
3. The process of claim 1 , wherein said high performance liquid chromatographic separation procedure is conducted at a flow rate of 5-50 nL/min.
4. The process of claim 1 , wherein said column or channel during said high performance liquid chromatographic separation procedure has a flow rate of 5-50 nL/min. at 6000 psi or less.
5. The process of claim 1 , wherein said column has a length of greater than or equal to three meters.
6. The process of claim 1 , wherein, in said column or channel, said organic polymeric stationary phase layer attached to the inner wall surface of said column or channel comprises styrene and divinylbenzene monomer units.
7. The process of claim 1 , wherein, in said column or channel, said organic polymeric stationary phase layer attached to the inner wall surface of said column or channel comprises (C4-C18) alkyl methacrylate monomer units.
8. The process of claim 1 , wherein said detector is a mass spectrometer, a fluorescence detector, an electro-chemiluminescence detector or a nuclear magnetic resonance detector.
9. The process of claim 1 , wherein said interface is an electrospray ionization (ESI) interface and said detector is a mass spectrometer.
10. The process of claim 1 , wherein said interface is a matrix assisted laser desorption ionization (MALDI) interface and said detector is a mass spectrometer.
11. The process of claim 1 , said process further comprising coupling a preparatory precolumn to the entrance end of said separation column or channel and applying said aliquot of said liquid sample to said preparatory precolumn.
12. The process of claim 1 , wherein said organic polymeric stationary phase layer comprises styrenic, methacrylic or acrylic monomeric units, or combinations thereof.
13. The process of claim 1 , wherein said organic polymeric stationary phase layer is thermally stable to 250° C.