Routine proteomic analysis requires methods for sampling low abundance proteins, peptides and digests that would be difficult to accomplish using standard HPLC systems. Modern bioanalytical demands are moving towards diminished injection volumes, flow rates, inner-column diameters and UV path lengths. For such 'Capillary LC' protocols, it makes sense for the instrumentation industry to provide a package that addresses the needs for protocol miniaturization for scientists.
Agilent has engineered a capillary HPLC system that not only meets such demands, but is modular enough to retain many of the same parameters available in the classic Agilent 1100 system. For example, in addition to restrictor-less flow (< 2.5ml/min), two flow restrictor modules are available and easily interchangeable. One module permits flow rates under 20uL/min for capillary-chromatography, while a second module permits flow rates under 100uL/min which works best for micro-LC applications. This interchangeable system provides a diverse flow range while assuring precision for both isocratic and gradient delivery (dual binary pump mode, four channels total). In addition, the modules are electronically detected by both the controller unit and the software, linking the module in use to the detection system. The recommended range for the instrument is pH 1.0-8.5 and 20-400 bar (5880 psi) of pressure.
The autosampler of this HPLC system has been engineered to deliver a range of volumes as well. It claims to be able to deliver volumes as small as 10nL from a 5uL sample or as high as 1800 mL with an optional multi-draw kit installed. The lowest volume attempted in our laboratory was 60nL and we obtained only mediocre results (may be attributed to the analyte injected), but the claimed RSD is typically <3% for the 0.2-1mL range. The autosampler can be connected to a regulating module for maintaining samples either below (10C below room temperature) or above (up to 80C) ambient temperatures for bio-reaction analyses. Several tray options are available as well, including 100x2 mL vials and microvials (100, 300uL) and a 96-well autosampler. Due to the nature of the capillary system involved, there is a tendency to block the sample needle. Regular inspection of carry-over is highly recommended. The autosampler is the weakest link in an otherwise strong product.
Also included in the package is a degasser (invaluable with low flow buffering systems and small i.d. columns), an upgradeable column oven that can include a sample valve in close proximity to the column (tubing can get cramped with this configuration, but this is a convenient option), and a DAD detector with a 500nL flow cell and a wavelength accuracy of +/-1nm (190-950nm, maximum pressure is 50 bar). There is a compact, mobile control module available that is connected by a communications cable. The control module allows for quick changes, assay building and system monitoring off-line using any computer-based system.
All-in-all, I would recommend this system for its universal application abilities alone. If you are in the market for an HPLC system that will compliment electrospray mass spectrometry, then this system is close to ideal. The miniaturization of the Agilent 1100 capabilities enables reproducible HPLC analyses, as well as being a wise investment for building a nanospray-LC/MS/MS system.
Ronald A. Miller
Research Biochemist
Merck Research Laboratories
Department of Alzheimer's Disease Research