Monolithic columns are an alternative to traditional particulate solid phases for capillary LC.  Made by polymerizing and precipitating the stationary phase within the separation capillary, the resulting flow channels have both a higher permeability with the stationary phase and a lower impedance to bulk flow, than the stacked spheres of particulate materials.  This enables capillary LC to be performed using smaller i.d. (20 micron) columns, which increases the mass sensitivity of nano-ESI-MS by approximately 10-fold, over using 75 µm columns.  This is a crucial step towards proteomic assays of, for example, phosphorylation states of receptors and their activation cascades from limited biological samples such as biopsy tissues.

SEM Micrographs of a Monolithic Stationary Phase
(click for larger image)

      Magnification × 3500                                Magnification × 8000

Background

 LC-MS using an electrospray ionization (ESI) interface is a well-established analytical tool with strong applications in proteomic and metabolomic research.  HPLC column technology is driven by demand for high sensitivity detection, as well as fast, high-resolution separations of complex samples.   It is well-known that the analyte concentration is much higher in narrow columns (given the same separation efficiency, linear velocity and mass loaded) and so, because ESI is a concentration-sensitive technique over a wide range of flow rates, miniaturizing the separation column diameter greatly increases the LC-MS detection sensitivity (as 1/r²).  Traditional particulate stationary phases, however, do not miniaturize well below the 3 µm bead sizes used in 75 µm i.d. columns.  Restricted mass transport between the interstitial flow (between the beads) and the stagnant mobile phase (within the pores of the beads) causes peak broadening and low efficiency.   Smaller (1 µm) bead sizes require high operating pressure (> 10000 psi), which requires new higher-strength packing materials to be developed. 

Monolithic Columns

An alternative approach is the development of monolithic columns, in which the stationary phase is a continuous interconnected skeleton with large through-pores.  This structure reduces the diffusion path and provides high permeability, resulting in excellent separation efficiency.  The integral structure enhances the mechanical strength, while the large through-pores (a few mm) have very low flow impedance.  This combination allows smaller diameter monolithic columns to be operated at higher flow rates, simultaneously increasing both sensitivity and throughput.  

Synthetic polymer monolithic columns are made by in situ polymerization of mixtures of monomers and porogens within fused-silica capillaries which have been functionalized with vinyl groups.  The resulting monolithic polymer bed is a uniformly porous piece integrated with the quartz capillary wall.  The strength of this structure is very high, with no need for frits or encapsulation.   A typical structure is depicted in the SEM picture above of a 25 mm i.d. column.   After polymerization, any of the many surface types (C-4, C-18, etc.) can be applied.   Our team at the Barnett Institute is developing capillary monolithic column materials and methods for a variety of bioanalytical applications.

Overview of LC-MS Proteomics

Detecting minor components in complex protein mixtures requires starting with sample preparation methods that can concentrate large volume samples (SDS-PAGE, immuno-precipitation, protein chromatography, proteolytic digestion) and ending with the miniaturized ionization sources that provide the highest MS sensitivity.  There is a particular demand for general methods which can survey large numbers of proteins, and quantify changes in their expression levels or post-translational modifications.  Capillary chromatography techniques are the most promising for analyses of low copy-number proteins extracted from small tissue samples.  In addition to providing the higher chromatographic resolution needed for analysis of complex mixtures, gradient modes of chromatography can pre-concentrate and clean up solutes prior to separation and detection of sample components: while the sample is being loaded onto the column, salts and many other contaminants wash through.  Loading samples initially onto a wide-diameter precolumn permits fast loading of large sample volumes.  After the sample is trapped on the precolumn, its outlet is switched to the narrow-bore separation column and the separation gradient is started.  Performing the separation at flow rates in the low nanoliter per minute range permits the capillary LC column to be eluted directly into an ESI emitter, reducing or eliminating deleterious signal suppression effects commonly encountered in complex biological matrices.

Monolithic Columns in LC-MS Proteomics

We have developed improved capillary HPLC columns using monolithic packings with very high separation efficiencies.  Larger precolumns of 150-mm i.d. are effective for concentrating microliter-volume samples, while narrow separation columns of 25-mm i.d. maintain high analyte concentration for optimal LC-MS detection sensitivity.  Monolithic columns have exceptional chromatographic resolution and can operate at low nanoliter-per-minute flow rates, which enhances ionization efficiency when transferring poorly-ionized or low-abundance samples into the ESI emitter.  The both simplifies LCMS data and provides the highest mass spectrometer sensitivity.

A typical LCMS chromatogram obtained using this tandem monolithic column system is shown below.

Figure 2)  Tandem monolithic nano-LC / ion-trap-MS   analysis of 5-fmol HSSPHQSEDEEEPR (mixed a-, mono- and triphospho) mixed with 10-amol β-lactoglobulin (BLG) tryptic digest, in a background of 100-fmol of a BSA tryptic digest.  The gradient profile (from 5% to 40% acetonitrile, in water) is overlaid on the chromatogram.