We are developing a spectrophotometric analysis lab for Gen Chem looking at nitrate and phosphate concentrations in water samples from different locations in the Willamette River.
For the phosphate analysis, we are using the ammonium molybdate/ascorbic acid spectrophotometric assay, working under very dilute concentrations for the standards (~0.05 ppm). Has anyone done this analysis before and have any key procedural tips? The issue is we are not getting particularly linear results for our standard curves. One problem might be the cheap, plastic cuvettes we are using (the analysis wavelength is >800 nm), so we are going to switch to optical glass or quartz to see if that makes a difference.
Someone also mentioned being sure to do a dilute acid rinse of glassware to remove phosphate contamination from detergents? Suggestions on a concentration for the acid? And is this really crucial? Remember we are trying to do this in Gen Chem. Other tips?
I'm also curious as to the molybdenum chemistry that is going on in solution (a beautiful blue color). Can anyone describe the absorbing species or point me in the right direction?
Thanks!
my students do it in the fishtank lab based on that in the literature: (Hughes, K. D. Anal. Chem. 1993, 65, 883A-889A, DOI: 10.1021/ac00068a001).
based on running the experiment for 2 years (prepping for 3rd year today!) I have some suggestions. The book "Standard methods for the examination of water and wastewater" is the standard reference for this procedure.
don't contaminate your reagents with phosphate (soaps). Acid wash the glassware and keep it foil wrapped. Hot, dilute HCl is called for in the standard reference book. The direct quote is "clean all glassware with hot dilute HCl and rinse well with distilled water. Preferably, reserve the glassware only for phosphate determination, and after use, wash and keep filled with water until needed. If this is done, acid treatment is required only occasionally." We typically use hot 0.5 M HCl.
Make the standards by serial dilution and not fresh samples. We start with about 2 mg/L standard phosphate and dilute 4-15 in 25 mL. these are ca. 0.02-0.05 ppm
We use a 5 cm cell (more b in Beer’s law), but that shouldn't really matter, though natural phosphate is quite dilute and you may have a hard time seeing it in a 1 cm cell.
Finally, we have found that its pretty imporant to take the measurements at the same time; say, exactly 10 minutes after the addition of tin or ascorbic acid (reductant) as the color development reaction is time dependent (for stannous chloride reduction, the book says to measure between 10 and 12 minutes; for the ascorbic acid reaction, it says to measure between 10 and 30 minutes).
The colored species is a molybdenum blue polyoxometalate (formed from reduction of the heteropoly acid - phosphomolybdic acid) of uncertain stoichiometry.
hope that's helpful!
In reply to tips by Adam Johnson / Harvey Mudd College
Well, with a bit more research, I answered my own question on the structure of the blue compound used for the colorimetric detection of phosphate. It turns out that good 'ole Wikipedia has a very nice entry describing the chemistry!
http://en.wikipedia.org/wiki/Molybdenum_blue
We don't use this reaction for gen chem/ inorganic chem anymore, but a couple years ago we developed an experiment with it for a local high-school chemistry course doing watershed water chemistry. The teacher then got it to go microscale and uses it with Vernier colorimeters with some nice results.
I agree with Adam's comments...it is critical to keep the glassware phosphate free. We do a 2% HCl rinse (but not heated) and cover everything with parafilm. Soaps are banned from the room. We use the citric acid/ammonium molybdate reaction and timing can be anywhere between 10-20 minutes reliably, but you have to use exactly the same amount of time per run...within a minute is critical to get the reproducibility you want. Last, we have really phosphate-rich surface water around here which works like a charm, but our standards in distilled water were originally much lower than many of the water samples we collected, and less linear because of the cheap colorimeters used...the Verniers wouldn't measure anything reliably below 0.05 ppm PO4, so we do calibration curves (and samples) on top of a 0.2 ppm PO4 standard (a method of standard additions) and the students are getting very linear results, they just have to extract the absolute concentration from their plots a little more carefully...it's a teaching moment. I should probably check to see if my memory of 0.2 ppm is correct...it might be 0.1 ppm, but if the solutions aren't turning really deep blue by the time you measure them, you might get linearity problems that way.
Hope this helps - good luck.
Graham,
May I ask why you use citric acid for the phosphate analysis procedure you describe? Do the citric acid molecules act as the reducing agent, or does the citric acid material contain some ascorbic acid? I imagine that many commercial-grade citric acid materials contain some ascorbic acid as a nutritional-supplement (Vitamin C).
Brendon