Taper Bore Column Technology
UHPLC Performance at HPLC Flows and Pressures
The Van Deemter equation is an empirical formula describing the relationship between plate height (H, the length needed for one theoretical plate) which is a measure of column efficiency, and linear velocity (µ) (Figure 1). Smaller plate height values corresponds to greater peak efficiencies, as more plates, or analyte partitioning, can occur over a fixed length of column.
The Van Deemter equation is governed by three cumulative terms: (A) eddy diffusion, (B) longitudinal diffusion, and (C) mass transfer. A loss in peak efficiency can be observed as a wider analyte band, and therefore, these three terms can also be viewed as factors that contribute to band broadening. Figure 1 illustrates the effect of these terms, both individually and cumulatively. Eddy diffusion, the A term, is caused by a turbulence in the solute flow path and is mainly unaffected by flow rate. Longitudinal diffusion, the B, or difference, term, is the movement of an analyte molecule outward from the center to the edges of its band. Higher column velocities will limit this outward distribution, keeping the band tighter. Mass transfer, the C term, is the movement of analyte, or transfer of its mass, between the mobile and stationary phases. Through this type of diffusion, increased flows have been observed to widen analyte bands, or lower peak efficiencies.
Decreasing particle size has been observed to limit the effect of flow rate on peak efficiency—smaller particles have shorter diffusion path lengths, allowing a solute to travel in and out of the particle faster. Therefore the analyte spends less time inside the particle where peak diffusion can occur. Figure 2 illustrates the Van Deemter plots for various particle sizes. We notice that as the particle size decreases, the curve becomes flatter, or less affected by higher column flow rates. Smaller particle sizes yield better overall efficiencies, or less peak dispersion, across a much wider range of usable flow rates.
Figure 1.
Figure 2.
UHPLC utilizes sub 2 micron particles and high linear velocities to achieve high efficiency separations. For a 2.0 mm ID UHPLC column, optimum performance is achieved at linear velocities of 5-20 mm/sec, which translates to a flow range of 1000-4000 ul/min. Since LC-MS sensitivity is inversely proportional to flow rate, analysts must compromise between sensitivity and efficiency for UHPLC-MS.
Since the tapered bore of a WarpLCMS column provides a 16 fold increase in linear velocity from inlet to outlet, optimum UHPLC performance can be achieved at LC-MS flow rates of 200-800 ul/min without impacting MS sensitivity. Initial studies with sub 2 micron particles (1.0 - 1.9 micron) indicate that column efficiency continues to improve at even higher linear velocities, making the 25 mm length and the tapered bore design of the WarpLCMS column even more important (due to the higher pressures required by these smaller particles limiting the maximum flow that can be achieved with UHPLC systems).
WarpLCMS Column Design and Materials
For most quantitative LCMS applications, the analytes of interest represent a very small percentage of the total sample matrix (i.e. drugs in physiological fluids), requiring a high loading capacity at the column inlet. As the analytes of interest are separated from the sample matrix, this high column capacity is no longer necessary, making a tapered bore design a logical alternative to the conventional constant ID LC column.
Figure 3.
The unique design features of the Warp column as shown in Figure 3 are:
1. Injection molded carbon filled PEEK insert with a tapered bore ID and OD maintains a uniform thickness to insure high quality molded parts. This molded insert provides an even smoother inner wall than a highly polished SS LC column blank which is critical for UHPLC performance.
2. A stainless steel jacket that the taped bore insert is pressed into, to allow operation at pressures up to 20,000 PSI.
3. A 2.0 mm inlet ID and frit (0.5 micron porosity) with a distribution cone to insure the equivalent high loading capacity of a conventional 2.0 mm ID UHPLC column.
4. A 0.5 mm outlet ID and frit (0.5 micron porosity) with a 125 micron web to minimize band dispersion post column.
5. A 25 mm bed length to minimize backpressure when using 1-2 micron column packings and optimize throughput and resolution.
6. A 20 ul packed bed volume to allow displacement of 10-40 column volumes per minute at flows from 200-800 ul/min (versus 2.5-10 column volumes for a 2.0 x 25 mm column).
7. Tapering of the ID provides a 16 fold increase in linear velocity over the 25 mm length of the column, allowing optimum UHPLC velocities at conventional LCMS flows.
8. The tapered bore column provides both linear and axial compression of the analyte bands, resulting in higher analyte concentrations at the column outlet versus a conventional 2.0 mm ID column running at the same flow rate.
9. The reduction in column packing material over the length of the column results in smaller losses of trace analytes which may irreversibly adsorb to the column packing.
10. WarpLCMS columns can be packed with a variety of 1-3 micron totally porous and core shell column packings, providing optimum performance for most LCMS instrumentation and high throughput applications.