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Figure 1a: Focus-Mode: Loading Step. Turbulent flow sweeps debris from the sample matrix through the Turboflow column while analyte(s) are retained.
Evolution of the TFC Platform The theory of turbulent flow in open tubes has been discovered and studied for decades; however its application to LC packed columns was only patented in 1997 by Quinn and Takareski [1]. The challenge at the time was to design a chromatographic platform that would utilise turbulent flow properties to isolate small analytes from macromolecules present in complex matrices such as biological fluids. The study and understanding of the limitations associated with this initial setting were key to the learning’s and innovative solutions behind the evolution of the commercially available platform as we know it today. The various ways in which the challenges associated with on-line extraction techniques in general were addressed through refining and re-designing the different components of the TurboFlow platform are discussed next.
Figure 1b: Focusing/Transfer Step. The flow fromboth pumps is combined (and hence diluted) through the T rotor seal, thus allowing the loop contents to transfer the analytes retained on the Turboflow column into a stacked band on the analytical column
Figure 1c: FocusMode - Eluting Step. The analytes are removed from the analytical column via isocratic or gradient elution. The Turboflow column is washed and the loop is filled and closed in preparation for the next injection.
are then focused into a sharp band at the head of the HPLC column (Figure 1b). When the transfer is completed, both valves turn to isolate each column, thereby permitting washing of the TurboFlow
column and filling of the loop for the next injection. A ‘regular’ gradient or isocratic elution can then take place in parallel on the analytical column into the MS detector (Figure 1c).
On-line extraction systems are generally using a 2-D chromatography concept where the extraction happens in the first dimension and the chromatographic separation in a second dimension. The key challenge is to ensure the different chromatographic conditions of the two dimensions remain compatible in order to optimise the analyte transfer. Switching from a 5.0 mL/min extraction flow rate to a typical 1.0 mL/min chromatographic flow rate (50 x 4.6 mm i.d. analytical column) meant that refocusing on an analytical column post extraction could be difficult. For some applications where chromatographic resolution was not crucial, a dilute and shoot approach (Quick elute mode [2]) was used to redirect the flow, after splitting, towards the MS detector. Very quickly, ionisation effect issues were reported due to co-elution of the peak of interest and endogenous materials. Therefore, the need for a chromatographic separation or at least a refocusing step before entering the MS became pivotal to build up analysis quality. The focus mode approach (Figure 1a, 1b and 1c) was then introduced to allow the analyte to be transferred on a suitable HPLC column before detection. Further evolution of the column design, essentially a reduction of the column diameter to 0.5 mm i.d., allowed a decrease in the flow rate required to achieve turbulence (~ 1.5 – 2.0 mL/min). Typically a flow rate above 1.2 mL/min is sufficient to obtain clean extraction and good recovery. Lower extraction flow rate and smaller column dead volume facilitated the analyte transfer and resulted in lower peak dispersion. In addition to improving chromatographic resolution (Figure 2), this also considerably
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