The theoretical advantages of microLC are no longer just theories. Starting with a non-splitting solvent delivery system based on Microfluidic Flow Control, Eksigent reinvented microLC to bring the practice of this powerful method in-line with its promise.
Scientists were excited when the first micro HPLC systems became available. Those on the lookout for ways to increase lab productivity understood the theoretical advantages of capillary chromatography -- faster throughput, better resolution, low sample volume requirements, and greatly reduced solvent consumption.
However, the manufacturers of early microLC systems did not rethink chromatography for capillary columns. Most systems they installed were based on modifications to conventional-scale HPLC systems and did not perform as expected. The effects of pulsing pressure sources were magnified, causing losses in flow rate precision and retention time reproducibility. Large dead volumes in the flow path degraded resolution and decreased sensitivity. Flow-splitting systems, added in an effort to adapt conventional-scale pumps to capillary flow rates, reduced gradient formation accuracy, added connections and complexity, and wasted liters of solvent every day. In addition, there were a limited number of capillary columns to choose from and the ones that were available did not always work the same way each time. No wonder researchers were disappointed.
Here are some of the ways that Eksigent reinvented micro HPLC with new technological advantages that fulfill on the promise of superior results and increased productivity:
Microfluidic Flow Control (MFC)
MFC works by continuously measuring the flow rate of each mobile phase. An embedded processor uses the flow rate measurements to control each pneumatically driven pump. Full gradients can be run as rapidly as 10 seconds with excellent reproducibility. MFC improves separation speed and flow stability.
Microscale Mixing
The advantages of microLC over conventional HPLC disappear unless there's a proportional reduction in fluid mixing volume. Eksigent's microscale mixer has a volume of just 300 nL, and a mixing delay of only 2 seconds at 10 uL/min. This rapid mixing virtually eliminates the gradient delays inherent in conventional systems at the start of the method and at re-equilibrations. The rapid “deterministic” mixing leads to extremely precise gradients, rapid system response, and high gradient linearity.
Variable Injection System
A conventional-scale HPLC injection volume of 10 µL becomes a 40 nL injection on a capillary system. Conventional autosamplers cannot deliver precise 40 nL injections, but our design philosophy required that the system provide injection precision of 1.5% RSD. Eksigent developed a new technology to make the scale-down simple.
Our variable injection system utilizes an electronically controlled injection valve coupled with the MFC flow delivery system to deliver precise nanoliter injection volumes. A standard autosampler fills a sample loop (typically 250 nL) with sample. The valve is then toggled from load to inject and back to the load position, based on integrating the flow rate over time to reach the desired injection volume. This solution provides high precision, software-selectable injection volume control, and compatibility with the popular CTC Analytics autosampler system.
Microscale UV Flow Cell Detection
Simply scaling down a conventional UV absorbance detection system to capillary levels results in increased dispersion and reduced detection sensitivity. Eksigent utilized its expertise in microfluidic chip development to develop a sensitive, non-dispersive UV absorbance flow cell. The detection system brings the dynamic range and linearity of conventional detection systems to the capillary format. Furthermore, it offers fully dispersed array-based detection from 200 nm to 380 nm, providing full spectral information for each peak.
