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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: ThP-LB1
Session: LATE-BREAKING/Proteomics: Quantification
Presentation date: Thu, Aug 31, 2006
Presentation time: 09:50 – 11:20


Edmund T Bergstrom1, David M Goodall1, David R Knight2, Kevin J Moon3, Noumie L Suragau1, Mebs A Surve3, Pawel L Urban1

1 University of York, York, United Kingdom
2 University of Manchester, Manchester, United Kingdom
3 Paraytec Ltd, York, United Kingdom

Correspondence address: Mebs A Surve, Paraytec Ltd, Business Development, 1a St George's Place, York, YO24 1GN United Kingdom.

Web site: http://www.paraytec.com

Keywords: Chromatography, Liquid -, Nanoscale Capillary; Detector, Array; Electrophoresis Capillary; Mass Spectrometry, Liquid Chromatography.

Novel aspect: Capillary Based UV Imaging Detector


Fused silica capillaries have two key features that make them ideal for development of in-line detectors. Firstly, their inner core enables sample streams to flow through them; secondly, they are UV transparent thus enabling direct quantification of components within the stream in real time. A further feature is that a liquid-filled capillary acts as a cylindrical lens, and this allows the capillary to combine the dual functions of sample vessel and optical element. For a light beam incident on the capillary, for certain ratios of internal to external diameters, rays travelling through the centre are separated from those travelling through the walls without traversing the sample. The different classes of rays may be directed onto separate regions of a pixellated sensor, with those travelling through the walls acting as reference for those through the sample [Patent reference US 10/524779]. In our work, an active pixel sensor is used for area imaging; active pixel sensors combine process and control functions into the integrated circuit of the sensor enabling individual pixel elements to be addressed independently, facilitating real time imaging and analysis.

Data will be presented to show performance of the in-line UV detector (ActiPix D100, Paraytec Ltd) with capillary LC and CE separations to give high sensitivity detection without sacrificing spatial resolution. The high spatial resolution offers particular benefits with high resolution capillary separations, e.g. complex peptide mixtures for proteomics. A dynamic range of 5 orders of magnitude will be shown as well as very low noise and drift characteristics. This allows major components and trace levels of impurities to be quantified simultaneously. The miniature size of the detector makes it ideal for coupling with LC-MS and mounting close to an MS spray tip. The measured noise is less than 7 microabsorbance units at 200 nm (10 nm bandpass filter) with a 1 s risetime. Results will be presented to demonstrate performance for a protein digest and correlated with MS data. The UV peak areas generated from the in-line detector when combined with mass and fragmentation data allow quantification at the same time as peak characterization.

Imaging of contents of multiple capillaries is readily achieved on a single sensor, unlike existing UV detectors. Results with test peptide mixtures and protein digests will be shown using multiplexed capillaries. The monitoring of nanoscale biocatalytic reactions is illustrated in electrophoretically mediated microanalysis (EMMA). Side-by-side imaging of capillaries with and without enzyme allows ready visualization and quantification of reactants and products.