Narrow bore layer open tube capillary column and uses thereof

Abstract

A polymer-based PLOT 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. Styrenic based monomers such as styrene and divinylbenzene or meth/acrylic based monomers such as butyl or stearyl methacrylate and ethylene glycol dimethacrylate, are preferred. 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, high column-to-column reproducibility and relatively high loading capacity. When these columns are coupled on-line with, e.g., ESI-MS detection, the resulting systems provide high sensitivity for analysis of complex proteomic samples, even down to the low attomole to sub-attomole level.

1 . A porous layer open tube 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; and   a rigid porous layer separation medium comprising a highly crosslinked, macroporous, organic polymeric stationary phase layer attached covalently to the inner wall surface of said column or channel, wherein said organic polymeric stationary phase layer comprises styrenic, methacrylic or acrylic monomeric units, or combinations thereof; wherein said organic polymeric stationary phase layer is from 0.5-3 μm in thickness; wherein said organic polymeric stationary phase layer is thermally stable to 250° C.; and wherein the reproducibility of retention time on comparable said columns or channels during use varies less than 10%.   
   
   
       2 . A capillary column or channel according to  claim 1 , wherein the reproducibility of retention time on comparable said columns or channels during use varies less than 5%. 
   
   
       3 . A capillary column or channel according to  claim 1 , wherein, further, said column or channel during use has a flow rate at 6000 psi or less of 5-50 nL/min. 
   
   
       4 . A capillary column or channel according to  claim 1 , wherein said column or channel has an i.d. of 10 μm or less. 
   
   
       5 . A capillary column according to  claim 1 , wherein said column has a length of greater than or equal to one meter. 
   
   
       6 . A capillary column according to  claim 1 , wherein said column has a length of greater than or equal to three meters. 
   
   
       7 . A capillary column or channel according to  claim 1 , wherein said organic polymeric stationary phase layer is poly(styrene-divinylbenzene). 
   
   
       8 . A capillary column or channel according to  claim 1 , wherein said organic polymeric stationary phase layer comprises (C4-C18) alkyl methacrylate monomer units. 
   
   
       9 . A method of preparing a separation capillary column or channel in a microfabricated device, said column or channel comprising a porous layer open tube separation medium comprising a macroporous, organic polymeric stationary phase layer, said method comprising the steps of:
 providing an unfilled capillary column, or channel in a microfabricated device, said column or channel open at both ends thereof and having an i.d. of 15 μm or less, the inner wall surface of said column or channel comprising a bifunctional anchoring or coupling agent suitable for covalent attachment of a macroporous, organic polymeric stationary phase layer as a porous layer open tube separation medium;   adding to said column or channel a mixture comprising a functional monomer selected from the group consisting of styrenic, methacrylic and acrylic monomers, and combinations thereof; a crosslinker compatible with said functional monomer, said crosslinker being capable of providing extensive crosslinking; a polar porogenic solvent; and an initiator for thermal or UV induced polymerization; and   polymerizing said mixture in said column to form said macroporous, organic polymeric stationary phase layer as said porous layer open tube separation medium attached to the inner surface of said column or channel.   
   
   
       10 . The method of  claim 8 , wherein the inner wall surface of said column or channel is silica and wherein said bifunctional anchoring or coupling agent contains at one end a functional group reactive with silica and at the other end a functional group reactive with said functional monomer. 
   
   
       11 . The method of  claim 10 , wherein said bifunctional anchoring or coupling agent is 3-(trimethoxysilyl)propyl methacrylate. 
   
   
       12 . The method of  claim 9 , wherein said functional monomer in said polymerization mixture is styrene. 
   
   
       13 . The method of  claim 12 , wherein said crosslinking agent is divinylbenzene. 
   
   
       14 . The method of  claim 9 , wherein said functional monomer in said polymerization mixture is methacrylate. 
   
   
       15 . The method of  claim 14 , wherein said functional monomer is (C4-C18) alkyl methacrylate. 
   
   
       16 . The method of  claim 15 , wherein said functional monomer is butyl or stearyl methacrylate. 
   
   
       17 . The method of  claim 15 , wherein said crosslinking agent is ethylene glycol dimethacrylate. 
   
   
       18 . The method of  claim 12  or  claim 14 , wherein said porogenic solvent is C n H 2n+1 OH, wherein 1≦n≦4). 
   
   
       19 . The method of  claim 18 , wherein said porogenic solvent is ethanol. 
   
   
       20 . The method of  claim 12  or  claim 14 , wherein said porogenic solvent is acetonitrile. 
   
   
       21 . The method of  claim 9 , wherein, in said polymerization mixture, the ratio of total monomer (functional monomer plus crosslinking monomer) to total porogenic solvent varies between 10-40% (V/V). 
   
   
       22 . The method of  claim 9 , wherein, in said polymerization mixture, the ratio of functional monomer to crosslinking monomer varies between 1:1 to 1:3. 
   
   
       23 . A process of carrying out a chemical analysis method comprising the steps of:
 providing the separation capillary column, or channel in a microfabricated device, according to  claim 1 ;   coupling said column or channel to a concentration sensitive detector; and   carrying out said chemical analysis method.   
   
   
       24 . A process of carrying out a chemical analysis method comprising the steps of:
 providing a separation capillary column, or channel in a microfabricated device, prepared according to the method of  claim 9 ;   coupling said column or channel prepared by said method to a concentration sensitive detector; and   carrying out said chemical analysis method.   
   
   
       25 . A system for carrying out a chemical analysis method comprising:
 a porous layer open tube, separation capillary column or channel in a microfabricated device, said separation column or channel comprising:
 a column or channel having an i.d. of 15 μm or less, an entrance end and an exit end; and 
 a rigid porous layer separation medium comprising a highly crosslinked, macroporous, organic polymeric stationary phase layer attached covalently to the inner wall surface of said column or channel, wherein said organic polymeric stationary phase layer comprises styrenic, methacrylic or acrylic monomeric units, or combinations thereof; wherein said organic polymeric stationary phase layer is from 0.5-3 μm in thickness; wherein said organic polymeric stationary phase layer is thermally stable to 250° C.; and wherein the reproducibility of retention time on comparable said columns during use varies less than 10%; and 
   a concentration sensitive detector coupled with an interface to the exit end of said separation column or channel.   
   
   
       26 . The system of  claim 25 , wherein said concentration sensitive detector is a mass spectrometer, a fluorescence detector, an electro-chemiluminescence detector or a nuclear magnetic resonance detector. 
   
   
       27 . The system of  claim 25 , wherein said interface is an electrospray ionization (ESI) interface or a matrix assisted laser desorption ionization (MALDI) interface. 
   
   
       28 . The system of  claim 25 , wherein said organic polymeric stationary phase layer attached to the inner wall surface of said column or channel comprises styrene and divinylbenzene monomer units. 
   
   
       29 . The system of  claim 25 , wherein said organic polymeric stationary phase layer attached to the inner wall surface of said column or channel comprises (C4-C18) alkyl methacrylate monomer units. 
   
   
       30 . The system of  claim 25 , wherein said column or channel has an i.d. of 10 μm or less. 
   
   
       31 . The system of  claim 25 , wherein the reproducibility of retention time on comparable said columns or channels during use varies less than 5%. 
   
   
       32 . The system of  claim 25 , wherein, further, said column or channel during use has a flow rate at 6000 psi or less of 5-20 nL/min. 
   
   
       33 . The system of  claim 25 , wherein said column has a length of greater than or equal to one meter. 
   
   
       34 . The system of  claim 25 , wherein said column has a length of greater than or equal to three meters. 
   
   
       35 . The system of  claim 25 , said system further comprising a preparatory precolumn coupled to the entrance end of said separation column or channel.