Flow injection Batch analysis
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The determination of phosphate in samples can be done manually, by ion
chromatography, flow injection analysis, or other instrumental
In manual procedures, the samples are prepared in defined volumes,
usually 25ml, with the same volumes of each reagent being added to each
solution. A period of at least 2 hours is then allowed for the color to
develop fully before measurement.
Similarly, the measurement stage usually requires the rinsing, filling
and wiping of a cuvette; measurement of the signal; emptying and
rinsing the cuvette. This process is repeated for each solution and
normally requires about 1 to 2 mins.
In normal flow injection analysis, the sample or standard solution is
injected into the reaction manifold, in which the reagents are
In this experiment, phosphate will be determined by batch development
of the color, and the color intensities of solutions measured by rapid
flow injection through a flowcell in a spectrophotometer.
(1) Stock solution of PO4 in distilled water: 50ug/ml.
(A) Potassium antimony tartrate: 1.3715 g in 50ml distilled water (Soln. A)
(B) Ammonium molybdate: 10g in 250ml (Soln. B)
(C) Ascorbic acid: 1.76g in 100 ml distilled water. Prepare fresh daily. (Soln. C)
(D) 5N sulphuric acid: 35ml to 250ml with distilled water (Soln. D)
(2)Mixed reagent: (Mix fresh daily)
50ml D + 5ml A + 15ml B + 30ml C
Standard and Sample Preparation
(a) PO4 standards: (mot more than 25ml each)
(1) Use the 50 ug/ml stock solution provided to prepare the following
phosphate standard solutions, using distilled water as diluant:
0.5 1.0, 2.0. 4.0, and 6.0 ug/ml.
(2) Pipette 5ml from each standard solution into clean dry test tubes,
and add 0.8ml of mixed reagent.Stopper and allow to stand for about 1
hr. for color to fully develop.
(1) Dissolve triplicate aliquots (0.100g) of salt provided in 100 ml volume
(2) Dilute 0.5 ml of each solution to 50 ml with distilled water (100x
(3) Pipette 5ml of each solution into clean dry test tubes, and add
0.8ml mixed reagent
(4) Stopper and allow color to develop
Prepare a standard blank using 5ml distilled water with 0.8ml mixed
reagent, and prepare a sample blank using 5ml sample and 0.8ml mixed
Allow to stand alongside the other solutions.
Record the time
taken for this phase of the experiment.
Flow Injection analysis
(1) Use pump tubing about 2.75 mm id. Set flow of distilled water carrier to 5ml / min.
(2) Injection loop: attach 60cm x 0.8mm id PTFE tubing to loop injector.
(3) Manifold connection: approx. 30cm length of 0.8mm id PTFE tubing between injector and flow cell.
Wavelength of measurement set at 825nm for phosphate
(a) Inject standard blank and then PO4 solutions into FIA
manifold and record peak heights and the maximal absorbance readings
for each solution
Inject sample blank and then sample solutions
Record total analysis times for all analyses
(b) Plot peak heights or absorbance values (after blank correction) vs
PO4 standard concentrations
(c) Determine the sample solution PO4 concentrations as
ug/ml, then calculate the PO4% in the salt provided
Use the salt formula weight (Na2HPO4 = 141.96) to calculate the
percentage purity of the salt provided
Provide answers to the following:
(1) Explain the need for standard and sample blanks
(2) Explain the rationale of this experiment, compared to the FIA procedure used previously for phosphate determination
(3) What possible advantages does this experiment possess over the previous FIA procedure used, in time and reagent consumption?