The advantage of programmable Flow Injection compared to Sequential injection is that pFI can
process large sample volumes. Thus SE in pFI format achieved sensitivity enhancement of spectrophotometric determination of iron over 1000x (A) by monitoring pre concentrated eluate form 4000 microliters of sample. The enhancement was calculated by comparing slope of calibration curve with the value of molar extinction coefficient of the measured complex, Fe(II) ferrozine (see 1.3.14.A.)
Protocol for SE-pFI assay, depicted in diagram (B) and detailed in software script (C) comprises five steps:
1) 1000mcrL of sample is aspirated into holding coil (HC1)
2) valve is turned to reagent port and 1400mcrl is aspirated into the second holding coil (HC2), while 1200mcrL are delivered from HC1 trough the confluence point resulting in 6+1 sample+reagent mixture in HC2. The mixture is incubated for 3 seconds.
3) Valve is turned towards microcolumn and flow cell and reacted sample is perfused through microcolumn at flowrate of 10mcrL/sec. and column is washed by carrier solution.
4) Eluant is aspirated into HC1,
5) Target analyte is eluted from microcolumn into the flow cell for monitoring.
The software script is set here for one repeat loading which includes steps 1,2 and 3. Thus Fe-ferrozine assay (A see details in next slide) was enhanced 200x using one loading and 500x with two loadings. Beyond two loadings the sampling frequency drops under 10s/hr, which is too slow for practical purposes, unless the slowest step, perfusion of the microcolumn (3), could be performed at a higher flowrate.
This protocol can be further modified for two reagent assays, and longer incubation times as discussed in section 1.2.27. Examples of spectrophotometric assays of iron and phosphate enhanced by SE are in the following sections.