Spectrophotometric determination of Fe(II) based on its reaction with ferrozine was automated by programmable Flow Injection method,( Hatta 2018) The stop in holding coil method (SHC) had detection limit of 55nM Fe, while the stop in flow cell method had limit of detection of 10nM Fe.
Sorbent Extraction enhanced this assay by using the instrumental setup (3.1.6. Fig.A), reagents and standards described in (A) and the assay protocol (3.2.1. Fig. B and C.). The reaction mixture was heated by thermostating both holding coils at 35 C. Because reaction of Fe(II) with ferrozine is fast the incubation time was short, set to 3 seconds. Elution of the complex from microcolumn packed with OASIS HLB sorbent was carried out at flow rate of 10mcrL/sec. Spectrophotometry at 560nm was performed using 20 cm long flow cell ( internal volume 100mcrL), with data collection window (WIN) set up to capture peak maximum, while reference baseline readout (BS) was set ahead of sample elution from the column (B). The data and resulting calibration graph (C) obtained with 1000mcrL sample yield limit of detection 1nM Fe. The sensitivity of this as assay, computed from the slope of calibration curve (0.09AU/ppB Fe), and the molar extinction coefficient of Fe(II) ferrozine complex (0.0005 AU/ppB Fe) is enhanced 180x. Because molar extinction coefficient is defined for 1cm long light path the enhancement obtained here is combination of analyte preconcentration and of the length of the flow cell (20 cm). However, the flow cell was not entirely filled with Fe-ferrozine complex, because peak width, at baseline (B) eluted at 10mcrL/s corresponds to 40mcrlL – less than half of flow cell volume.
This example shows interdependence of factors influencing the outcome of optimization: even if analyte is efficiently captured on microcolumn, the rate of its release from sorbent, its elution and subsequent dilution on the way to optical path, have to be considered and optimized.
For comparison, spectrophotometric assay of Fe by ferrozine (Hatta 2018) was enhanced by sorbent extraction 55 times (SHC) or 10 times (SFC) by loading the column with 1 mL of sample. Double loading will enhance the detection limit correspondingly, and because the same instrument can be used for pFI as well as for pFI-SE, it is demonstrated that automated sorbent extraction in pFI format is a viable method, suitable to enhancement of sensitivity of a spectrophotometric assay.
M. Hatta, C.I.Measures, J.Ruzicka, “Programmable Flow Injection. Principle, methodology and application fortrace analysis of iron in sea water matrix. Talanta 178 (2018) 698.