SUMMARY: Seawater samples are collected at discrete depths in 12-liter Niskin bottles. The water samples are prefiltered (202 µm) and transferred to specially designed, precalibratedfiltration bottles, pressure filtered through combusted acid-rinsed GF/F filters and stored frozen for subsequent analysis. In the laboratory, the filters are combusted at 450-500°C and the concentration of the resulting inorganic phosphorus is determined by colorimetric analysis.
The procedure presented here is a modification of one used by the Hawaii Institute of Marine Biology Analytical Services laboratory at the University of Hawaii. It is a method pioneered and used by soil scientists and marine chemists for particulates which can be homogenized into a fine powder.
The method relies on the release of organically-bound phosphorus compounds as orthophosphate, by high temperature combustion at 450-500°C. The orthophosphate is then extracted with 0.5 N HCl at 90°C. The liberated orthophosphate is reacted with a mixed reagent of molybdic acid, ascorbic acid and trivalent antimony to form phosphomolybdic acid. This heteropoly acid is then reduced to the colored molybdenum blue complex by ascorbic acid and the solution is measured spectrophotometrically.
This procedure measures all forms of phosphorus which can be released by combustion and acid hydrolysis.
Contamination is the primary problem to be avoided with these samples. Combusted acid rinsed filters are used. All sampling bottles, forceps, tubing and filtration bottles are also acid rinsed.
|3.1.||Seawater samples are collected in 12-liter Niskin bottles and
transferred directly to acid-cleaned filtration bottles. The
samples are transferred via Tygon tubing which incorporates an
in-line 202 µm Nitex screen prefilter to remove zooplankton or
any other rare particles which might otherwise affect the
precision of the estimate. The filtration bottles are 4- and
12-liter polyethylene aspirator bottles fitted with a valve
assembly and tubing connection.
Once the rosette/CTD unit is on deck, the vent valve from each Niskin bottle is opened and one end of the drawing tube is attached to the outflow spigot of the sampling bottle and the other end to the tubing connector on the cap of the filtration bottle. Particular attention is paid to the orientation of the in-line screened drawing tube (the shorter, larger bore section is attached to the Niskin bottle). The filtration bottle valve is opened and 100-200 ml is run through the transfer tube and valve assembly to rinse the sampling bottle. The bottle and cap are rinsed 3 times in this fashion. During the rinsing and filling operation, the filtration bottle cap valve is used to control and direct the sample flow. After rinsing, the cap is placed on the bottle mouth (without tightening), the valve is opened and the polyethylene bottle is filled to the calibration mark.
|3.2.||After filling, the filtration bottles are inverted and placed into the filtration rack. The contents are then pressure filtered (4-7 psi nitrogen gas) through combusted in-line acid-rinsed 25 mm GF/F filters.|
|3.3.||Following filtration, clean forceps are used to transfer each filter to a combusted 16 x 100 mm glass test tube which is then covered with a 3.5 cm square piece of combusted foil. Each sample is labeled and stored frozen (-20°C). Any water remaining in the carboy is measured to calculate the volume filtered. This information and any other appropriate data are entered on the data sheet.|
Standards are corrected for reagent blanks while samples are corrected for field filter blanks.
|4.1.||Standards: At least 2 reagent blanks are prepared and individual standard absorbances are corrected by the mean blank value.|
|4.2.||Samples: The mean absorbance from three field filter blanks, stored and processed in the same manner as samples, are used to correct individual sample absorbances for filter, reagent and systematic procedural contamination.|
|5.1.||Samples are combusted in 16 x 100 mm test tubes at 450°C for 4.5 hours in a muffle furnace. The samples are then allowed to cool and are immersed in 10 ml of 0.5 M HCl. The test tube is then heated for 60 minutes at 90°C in a heating block.|
|5.2.||The samples are allowed to cool, and centrifuged for 30 min at 2800 g. 5 ml of the supernatant is volumetrically subsampled into another combusted acid washed 16 x 100 mm test tube.|
|5.3.||One half ml of mixed reagent is added to samples and standards and mixed thoroughly. Color is developed for 60 minutes and absorbance is read at 880 nm against a DDW reference. Standards are corrected for absorbance of reagent blanks and samples are corrected for absorbance of filter or procedural blanks.|
Niskin bottles and rosette/CTD unit
low pressure filtration apparatus (4-7 psi)
combusted, acid-rinsed GF/F filters
acid-rinsed vacuum filtering assembly
spectrophotometer (Perkin-Elmer Lambda 3B) and 1-cm cuvette
combusted (450°C, 3 hours), acid-washed 16 x 100 mm glass tubes
Glass distilled deionized water (DDW)
0.5 M HCl
HCl for cleaning (1 M)
Ammonium molybdate solution: Dissolve 15 g of ACS grade ammonium paramolybdate [(NH4)6 MO7O24 . 4H2O], in 500 ml DDW. Store in plastic bottle in the dark. Solution is stable indefinitely.
Sulfuric acid solution (5 N)
Ascorbic acid solution: Dissolve 0.54 g of ACS ascorbic acid in 10 ml DDW (5.4% wt/vol). Prepare fresh.
Potassium antimonyl-tartrate solution: Dissolve 0.34 g of ACS potassium antimonyl-tartrate (tartaremetic), in 250 ml DDW. Solution is stable for many months.
Mixed reagent: Mix together 10 ml ammonium molybdate, 25 ml 5 N sulfuric acid, 10 ml 5.4% ascorbic acid and 5 ml potassium antimony tartrate. Prepare fresh.
Stock phosphate standard solution (1000 µM): Dissolve 0.1361 g of dry KH2PO4, in 1000 ml of DDW. Store in a dark bottle with 1 ml of chloroform.
Working phosphate standard (100 µM): Dilute 10 ml of the stock standard to 100 ml, using a volumetric flask.
Dilute the working standard to prepare a series of standards to cover the range from 0.05 - 10 µM.
Strickland, J. D. H. & T. R. Parsons. 1972. A Practical Handbook of Seawater Analysis. Fisheries Research Board of Canada, 167 p.