Conclusion
Theory guides. Experiment decides.
I.M. Kolthoff
Flow Analysis techniques are based, in theory, on mass transport in laminar flow conditions, a type of flow that would prevent resolution of peaks in chromatography and cause carryover in reagent-based methods. In chromatography the laminar pattern is deconstructed by obstructing the flow by stationary phase. In continuous flow analysis (CFA), laminar flow is disturbed by air segmentation that promotes circular motion within segments of liquid. In continuous flow injection (cFI), distortion of flow channel and merging of streams destroys the bullet shaped sample zone formed by laminar flow. Flow programming offers additional tools: flow reversal and flow acceleration.
To optimize FI based assay, radial dispersion should be promoted by placing confluence point as close as practical to point of injection in order to mix sample with reagents right at the beginning of assay cycle. The volume of flow path between injector and flow cell should be minimized, albeit must be large enough to provide incubation time long for chemical reactions to generate a monitored species. In continuous flow format slowing flow rate is an option, albeit limited by need to flush the channel with carrier stream in a timely fashion. The obvious solution, stopping the flow was suggested in 1979, [D 46] but this first attempt at flow programming was far ahead of its time. (out of 23.000 publications listed in Hansen’s Database, on FI only 252 used stop flow so far.
There are two reasons why flow programming took many years to come to fruition.
First, stopping the flow addresses only a single component for FI optimization; the length of incubation time available for reactions to proceed. It does not address the need for accelerated mixing of sample with reagents in precise and known ratios. Next, miniaturization of flow channel was limited because manifold was assembled from individual components; injection valve, T-connectors, mixing coil(s) and flow cell mounted in this sequence to form a flow path. It is the integration of these components within lab-on-valve [D 10565] platform in a novel design of miniaturized flow path, where sample and reagent zones move upstream from injection point and several times through the same confluence point and into the flow cell, that makes pFI concept wholesome.
While conceivable, in theory, long time ago, it took 40 years [D 1] to make pFI feasible [D 23277] because the key hardware and software components were not available. The milliGAT pumps which are crucial for reliable flow programming are based on a unique, ingenious design of four pistons moving on a rotating cam, where one piston is filling, one is delivering, and the other two pistons are moving between the filling and emptying ports. Conceived by Duane Wolcott in 1995, patented in 2000 (D.K. Wolcott 2000) it took another 15 years to develop milliGAT pump into a reliable commercial product. The lab-on-valve manifold was conceived in 2000 [D 10565} into the present design with an interchangeable flow cell, only recently. The Linear Light Path flow cell (Hatta 2020) invented fifteen years ago by G. Klein, lingered in obscurity until it became part of commercially available programmable flow FIA instrument controlled by FLoZF software.
This presentation offers two concepts made real within two scales: traditional FI in milliliter scale and the novel one in microliter scale. There is no doubt that there is room for further development between these two extremes. And there are, of course many reasons why continuous Flow Injection will be used and will dominate the field of routine, applied, and serial assay. But we hope that examples of measurements performed in pFI format the flow-batch technique, such as single line calibration, and single standard calibration, which can not be performed in any other way, will lead to wider acceptance of this versatile technique.
D.K. Wolcott, G.D. Marshall, (2000) “Pulseless, reversible precision piston-array pump” 2000. USP 6,079,313
M. Hatta, J.Ruzicka, Ch. I. Measures “The performance of a new linear light path flow cell is compared with a liquid core waveguide and the linear cell is used for spectrophotometric determination of nitrite in sea water at nanomolar concentrations” Talanta, 219, Nov. 2020 121240