CPSA Digest 2002

Proceedings -Tuesday, October 8, 2002

TuOC3

Advances in Low Volume, High-Throughput Turbulent Flow Chromatography, Including Parallel Approaches Preparation

Traditional laminar flow chromatography uses 1.0 mm i.d. columns packed with 5um particles; typical flow rates are 0.05 mL/min (velocity about 0.14 cm/s) with pressure from 100 to 200 bar. Turbulent flow chromatography uses 1.0 mm i.d. TurboFlow columns that are packed with larger 60um particles and use larger pore size end column frits; typical flow rates are much faster at 4.0 mL/min (velocity about 11.2 cm/s) with pressure less than 20 bar. The combination of the large pore size of the frits and the large particle size packing materials make it possible for large molecules in biological fluids to pass through without plugging the column. The sample debris fraction is sent to the waste container while a second pump elutes the analyte fraction to a selective detector such as a mass spectrometer. This technique has been successfully used for the on-line extraction of analytes from biological fluids and other complex matrices, with subsequent introduction into a mass spectrometer (MS) using an appropriate interface.

The rapid separation of sample debris from analytes results in high sample throughput. Although the savings in time and effort is quite significant over conventional sample preparation techniques, the 4.0 mL/min flow rate requires large volumes of mobile phase over the course of each day. By decreasing the inside diameter of the TurboFlow column from 1.0 mm to 0.5 mm, turbulent flow can be achieved at a flow rate of just 1.0 mL/min. Thus, the eluting flow rate is reduced by a factor of 4, and the result is a reduction in total solvent consumption by about a factor of 4.

In order to properly operate the 0.5 mm i.d. column, it is necessary to reduce the dead volume of the overall system by using smaller diameter tubing, valve stators and rotors (all of the diameters are cut in half). Since the lower column and system volume results in less dilution of the analyte fraction, an increase in sensitivity is gained. The 0.5 mm column with this reduced volume system has provided more than twice the sensitivity of the 1.0 mm column using the standard volume system. With the added sensitivity smaller sample volumes (i.e., 10 uL rather than 20 uL) can be used.

Up to four TurobFlow systems can be combined to further improve sample throughput for LC-MS analysis. When the 0.5 mm Cyclone column is used, the solvent reduction becomes more dramatic. Using a TX4 system running an average of 30 samples per hour, switching from the 1 mm Cyclone column to the 0.5 mm column reduces the average solvent consumption from about 500 mL to less than 130 mL per 8 hr duration. This TX4 system consists of a CTC Twin PAL Autosampler with four injection valves, four loading pumps to run TFLC columns, four eluting pumps to run HPLC columns, and an Aria Valve Interface Module; all components are controlled by Aria software.

Application Note: "Reducing solvent consumption and improving sensitivity with 0.5mm HTLC columns"
Company website: www.cohesivetech.com

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