There are two ways in which sensitivity of pFI-SE method can be further enhanced. By optimization of the performance of the detection system and by increasing accumulation of the analyte on the microcolumn.
Spectrophotometric detection can be enhanced by optimizing selection of reference and monitoring wavelengths, and also by extending the light path of the flow cell. Spectrum of PMoB in the alkaline eluate (A) was obtained by processing 10 ppB P sample by using the assay protocol (3.2.3.) where the volume of eluant was reduced to 60 microliters, followed by 2 seconds stop flow period. This arrested centroid of eluted zone in the middle of the light path of flow cell, thus allowing spectrum of PMoB to be recorded and compared with spectrum of eluate obtained with 0 ppB P sample (BLANK) as shown in two duplicate runs (B) Note: While the highest sensitivity would have been obtained by monitoring at 910nm, 880nm was selected in this work, because weak intensity of tungsten light source at longer wavelengths, causes a noisy response. Use of another light source may reveal continuation of PMoB spectrum beyond 910nm limit, imposed by the light source, and allow selection of wavelength that will yield more sensitive measurement. Curiously, in previous publications 710nm Heckermann (2000) and 635nm (Ma 2017) was used to monitor eluted PMoB. While use of 20cm long flow cell enhanced sensitivity of iron by pFI-SE (1.3.14.C.), use of cell longer than 10 cm did not enhance sensitivity of phosphate measurement, because all of PMoB is eluted from the column within a 50 microliter band, a volume that is entirely accommodated within 10 cm long flow cell.
Optimization of Phosphate Assay by Sorbent Extraction
3.2.6.
Because sensitivity of SE based assay increases with the quantity of analyte retained on the column, increasing sample volume is obviously the most efficient approach. In this work the sample volume processed within one cycle was 800 microliters, being limited by the volume of the holding coil.
Although increasing sample volume by repeating the sampling cycle increases sensitivity of assay, use of more than two repeats is deemed impractical, because it reduces sampling frequency below 12s/hr. Therefore modifying the instrument by increasing volume of the holding coils is the best approach for increasing sensitivity of pFI SE based assay.
The renewable microcolumn when fully packed with sorbent (Strata XL, 100 microns size, column volume 20 microliters) retained PMoB efficiently even at a high on column flowrate. However column size and loading flowrate were not systemically optimized in this work, and therefore there is, without doubt, a room for improvement that will result in more efficient retention of the analyte.
In summary, phosphate assay in pFI-SE format can be further optimized, possibly reaching LOD at 1 nmP level.
NOTE: Assistance of Gabrielle Weiss and Mariko Hatta in recording spectra of PMoB is much appreciated.