An integrated gradient capillary HPLC/MS system incorporating photodiode array detection

Library Number:
Cohen, Steven A.;Holyoke, J.;Dourdeville, T.;DellaRovere, D.
Presented at HPLC ''99, Granada Spain
Content Type:
Content Subtype:
Bovine Cytochrome c
Symmetry300(™) C18 300 Å 5 µm Steel 3.9 mm 150 mm
Recently, the popularity of capillary separations has been rapidly expanding, largely driven by the need to analyze increasingly smaller sample amounts. Capillary HPLC (CLC) is an attractive choice for these tasks, giving high resolution separations with flow rates and sample capacity suited for facile interfacing to mass spectrometers. CLC also has sufficient sample capacity to serve as a micropreparative technique for subsequent offline analyses. The challenges of designing an optimized CLC system, however, are numerous. Each component of the system, (solvent delivery, autosampler, fluidic connections, detection and interfacing) must be appropriately engineered. Component integration is essential to limit extra-column bandbroadening. Here we describe results obtained with an integrated CLC system consisting of a binary solvent delivery system, autosampler and photodiode array (PDA) detector suitable for analysis with columns 0.18 - 0.5 mm ID diameter. Key improvements in flow control technology have been incorporated. Each solvent is delivered by a pair of high resolution syringe pumps, where one syringe refills and is brought up to system pressure during the delivery cycle of the other. This allows for continuous, positive displacement flow with excellent flow accuracy and reproducibility. The solvent delivery system is thus capable of submicroliter per minute flow rates without need for problematic stream splitting schemes. Highly reproducible gradient separations are achieved with low microliter per minute flow rates. Typical relative standard deviations for retention time are less than 0.5%, even with rapid gradient analysis. Tight control over system bandspread typically yields peak volumes less than 5 microliters. A unique detector design with an extended path flow cell permits very high sensitivity detection (estimated detection limits less than 50 fmol for many peptides). Some unique design features include a fiber optic based light path, in conjunction with a flow cell design that provides for nearly quantitative light throughput. The low flow rates permit easy interfacing with an electrospray mass spectrometer without need for sample splitting, thus maximizing sensitivity. Detection by mass spectrometry has similar sensitivity to the PDA detector, and thus each detector provides confirmation of the presence of components observed by the other. Instrument performance will be illustrated with examples of peptide and drug analysis.

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