Last revised July 22, 1997
Page contents (click to skip down):
[Introduction]
[Equipment]
[Reagents]
[Procedure]
[Post-analysis calculations]
[Comments]
2) 13x100 mm glass screw-cap culture tubes with teflon-lined caps. (Tubes are acid-washed in 20% HCl and muffled at 500 degrees C for two hours. Caps are acid-washed in 50% HCl)
3) autoclave-safe test tube racks
4) 100 ml, 200 ml or 500 ml acid-washed volumetric flask for oxidizing reagent (depending on how much reagent is needed)
5) 500 ml acid-washed volumetric flask for 3.75M NaOH stock
6) 1000 ul and 5000 ul automatic pipetters
7) weigh boats and clean chemical spatula
2) low-N potassium peroxydisulfate (e.g. Fisher P282-100)
3) boric acid (e.g. Baker 0084-01)
4) low-N NaOH if stock is needed
5) EPA-certified Nutrient 2 quality control digest standard
100 ml 200 ml 250 ml 500 ml
persulfate 5.2 g 10.4 g 13 g 26 g
boric acid 3.12 g 6.24 g 7.8 g 15.6 g
NaOH stock 10 ml 20 ml 25 ml 50 ml
(This reagent may be stored 7 days at room temperature. Crystalizes when refrigerated.)
2) make up fresh digest reagent, and NaOH stock if needed
3) obtain acid-washed, muffled digest tubes; label them.
4) for unknowns and EPA2 samples:
5) For reagent blanks, pipette only 1 ml oxidizing reagent into tube and cap tightly. (N.B., take care! Qualls (5) p.136: "For low level samples the variability in the reagent blanks determines the limit of detection, not the error associated with the NO3 and PO4 analyses themselves.")
6) Place capped tubes in autoclave, 30 minutes on liquid cycle. (= 30 minutes on "sterilize" in addition to all other cycle segments. If using pressure-cooker field method, time 30 minutes after coming to canning temperature in addition to warmup and cooldown times.)
7) After tubes are cool, add 5 ml diH2O to all reagent blank tubes so that the total volume of liquid in these tubes is the same as in the others. (N.B. Qualls (5) p.136: "Since distilled or deionized water contains significant N, the dilution water [for the blanks] is added after the digestion.")
8) Analyze digest-tube contents on Alpkem using the nitrate-nitrite and orthophosphate manifolds.
2) To compensate for color absorption by the digest reagents, subtract the mean reagent blank N and P values from the Alpkem determined values for each unknown or EPA2 sample.
3) The effect of diluting the samples by the addition of digest reagents must be reversed:
sample volume + reagent volume
df = --------------------------------------
initial sample volume
In the case of the above procedure, where initial sample volume is 5 ml and reagent volume is 1 ml,
5 ml sample + 1 ml reagent
df = ---------------------------------- = 1.2
5 ml sample
Find the actual value of the undiluted sample by multiplying the determined value (after reagent blank subtraction) by the df.
True analyte concentration
=
(Alpkem raw determined value - rblank value)*(df)
2) Instead of using reagent blanks, it is possible to digest the calibration standards (including water blanks, i.e. calibration standards of content zero), the D3 (recalibrant) and ref3 (reference check) and the W (baseline drift correction) cups. Thus with digest reagent in both samples and calibants, the reagent's contribution to total absorbance will be compensated for automatically. (This strategy is of course useable only if all samples in the run are using the same reagent/diluent ratio.)
It may be necessary to have different dilutions of the EPA2 QC standards for nitrate and phosphate to get both into the optimum manifold range (e.g. if the PO4 manifold range is 0.2-1 ppm while the expected TP content of EPA2 is 1.5 ppm PO4, the QC digests will be offscale for phosphate unless diluted.) Perform these dilutions before digestion and then use the same reagent/diluent ratio for everything.
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