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Determination of Reactive Phosphorus

 

Parsons, T.R., Maita, Y., Lalli, C.M.,  1984.  A manual of chemical and biological methods for seawater analysis.  Pergamon Press, New York.  pp. 173.

 

Introduction

All methods for phosphate rely on the formation of a phospho - molybdate complex and its subsequent reduction to highly colored blue compounds.  The procedure given below is taken from Riley (Anal. Chim. Acta, 27:31, 1962).  In this method the sample is allowed to react with a composite reagent containing molybdic acid, ascorbic acid, and trivalent antimony.  The resultant complex heteropoly acid is reduced in situ to give a blue solution the extinction of which is measured at 885 nm.

 

Notes

Analysis should be performed as soon as possible after sample collection (0.5 - 2 hours).

Measurements should be taken within 2-3 hours after injection of reagents.

Reagent blank should not exceed 0.02, if it does then the ammonium molybdate is suspect.

If tubes are dried in an oven it should not be done in same oven as tubes used for Solarzano NH4 determination.

Dedicated volumetrics should be used for each standard.

DI water direct from Barnstead should be used for all reagents and standards.

All glassware should be rinsed after drying to remove any particulate contamination.

 

Capabilities

Range 0.03-5 uM in 5cm cells

 

Reagents

 

Reagent

Formula

VWR cat #

Reagent grade

Ammonium para molybdate

(NH4)6 Mo7 O24 •4H2O

EM-AX1310-3

Analytical

Ascorbic acid

C6H8O6

EM-AX1772-1

CRYSTALLINE USP GRADE

Sulfuric acid

H2SO4

EM-SX1244-14

ACS specifications

Potassium Antimonyl tartrate

C8H4K2O12Sb2•3H2O

JTO864-4

USP GRADE

 

Apparatus & supplies

Test tube racks (147919C) (holds 40)

Test tubes 13x 100 mm (25 ml) (14957J ) case of 72

500 ml volumetric (Ammonium molybdate solution)

5 ml pipette for aliquoting of sample

Re pipette set to .5 ml for mixed reagent

500 ml graduated cylinder (Ammonium molybdate solution)

plastic amber bottle (1 liter ) (Ammonium molybdate solution)

1 liter graduated cylinder (sulfuric acid solution)

200 ml graduated cylinder (sulfuric acid solution)

glass bottle (1 liter) (sulfuric acid solution)

500 ml beaker (ascorbic acid solution)

500 ml beaker (Potassium Antimonyl - tartrate solution)

500ml amber bottle for Potassium Antimonyl - tartrate solution


Preparation of Stock reagents

 

1.  Ammonium para molybdate

Dissolve 15 g Ammonium para molybdate (NH4)6 Mo7 O24 •4 H2O) in 500 ml DI water

Store in plastic amber bottle out of sunlight (stable indefinitely)

 

2.  Sulfuric acid solution

Gloves, goggles, and lab coat should be worn when mixing this reagent; reagent will get very hot.

 

In a 1 L beaker add 70 ml concentrated Sulfuric acid (4.8 N) to 450 ml DI water over an ice bath.  Allow solution to cool and store in an amber glass bottle.

 

3.  Ascorbic acid solution

Dissolve 2.7 g ascorbic acid in 50 ml DI water

This should be made daily

 

4.  Potassium Antimonyl - tartrate solution

Dissolve 0.34 g Potassium Antimony - tartrate in 250 ml DI water (warm if necessary)

Store in glass or plastic.  This solution is stable for many months.

 

Working solution:  Should be prepared fresh each day (stable for only 6 hours)

 

Mixed reagent

Add reagents to clean calibrated (500 ul) re-pipette

50 ml ammonium molybdate

125 ml sulfuric acid

50 ml ascorbic acid

25 ml Potassium Antimony - tartrate

250 ml (500 ul /sample) re-pipette contains enough for 500 samples

 

Procedure   (It is not necessary to add reagents in a hood)

 

1.  add 5 mls of filtered water/sample from sample bottle to lab tubes.

2.  add 500 ul mixed reagent

3.  vortex 

4.  cover with plastic (Saran®) wrap   *Do not use para-film since it contains Phosphorus and will contaminate samples*

5.  read on spectrophotometer @885 nm after 10 (preferably 30) min but within 2-3 hours.

 

Reading on the Spectrophotometer

                General notes on using the Spectrophotometer.  Never remove cell during a run, it is not necessary to ever open the door to the cell.  If there are bubbles in the line, they can be removed by either sipping air or cleaner. 

1.  Make sure Spec is set to sip 3ml.

2.  Set wavelength to 885 nm and make sure the spec is reading absorbance.

3.  Make sure the waste tube enters the phosphorus waste container.

4.  Sip DI water and 0 the reading.

5.  Read standards in order of concentration (i.e. low standards are read first (start w/ blanks and work way up).  After each standard, sip water through and re-zero if necessary.  Sip water through between high standard and first sample to remove any residual PO4.  Re-zero if necessary. 

6.  Before re-zeroing, sip again to make sure all residual material is removed and the reading is stable.

7.  Wipe the sipper tube on spec w/ kimwipes to dry off the outside of the tube in between samples so that contamination is minimized.

8.  If absorbance is higher than highest standards then you will need to dilute your sample.

 


Standard Preparation

 

Stock A Solution-1000mM PO4-P

 

Dissolve 0.136 g of anhydrous potassium dihydrogen phosphate (KH2PO4, m.w. = 136.07) in approximately 900ml of deionized water contained in a 1 L volumetric flask (note all salts should be dried in oven prior to weighing).  Dilute the solution to the mark with deionized water and mix it well.  Transfer this solution to an amber bottle.  Add 1ml chloroform for preservation.  This solution is stable for many months.  Refrigerate when it is not in use.  

 

Stock B Solution-100mM PO4-P

 

Prepare this solution weekly.  Using a volumetric pipet or a calibrated automatic pipet, add 10.0ml of Stock A to approximately 90 mL of deionized water contained in a 100ml volumetric flask.  Dilute the solution to the mark with deionized water and mix it well.   Add 1ml chloroform for preservation and store in an amber bottle.

 

 

Working Standards

Prepare these solutions daily.  Use adjustable, micro liter pipettes to add the designated volumes B listed in the following table.  Calibrate the pipet for each required volume.  (NOTE:  The standards for each day are presented in bold typeface.  The other concentrations are included for reference, if needed.)  Prepare each standard by adding the required amount of stock to the required volume volumetric flask containing deionized water.  Standards for both Ammonium and Phosphate can be made in the same volumetrics.  After adding standards for both methods, dilute each to the mark with deionized water and mix well.  Keep these solutions tightly sealed. 

 

Standard Concentration (uM)

Total

Volume

(ml)

Calibrant

Volume

(ml)

Stock

Standard

0.05

100

0.05

B (100uM)

0.1

100

0.1

B

0.2

100

0.2

B

0.3

500

1.5

B

0.4

100

0.4

B

0.5

100

0.5

B

0.6

100

0.6

B

0.7

100

0.7

B

0.8

100

0.8

B

0.9

100

0.9

B

1.0

100

1.0

B

2.5

100

2.5

B

5.0

100

5.0

B

 


Record the absorbance for each standard and spike sample in the nutrient log book and in a spreadsheet to keep track of trends and calculate standard deviation mean detection limit.  Determine the standard curve by plotting absorbance (y-axis) versus standard concentration (x-axis).  We have created an Excel workbook, entitled nutrients.xls, for data input.  Put a copy of the daily Excel worksheet on a diskette and a copy of the curves on the door to the spec room. 

                The daily absorbance for each should be fairly consistent from day to day.  If the absorbance is significantly different, there is a problem with either the standards or the stock solutions.  Data from 1998 measurements show the following mean values for each absorbance. 

 

Standard

Mean

- 1 Std Dev

+ 1 Std Dev

Std Dev

Std error

Min

Max

Number

Blank

.001

-.001

.002

.001

.0002

-.001

.006

47

.05 mM

.006

.004

.008

.002

.0003

.003

.013

48

.1 mM

.012

.010

.014

.002

.0004

.009

.015

16

.3 mM

.029

.026

.032

.003

.0004

.025

.038

46

.4 mM

.040

.037

.043

.003

.0004

.036

.047

48

.7 mM

.071

.067

.075

.004

.001

.064

.079

48

1 mM

.102

.098

.106

.004

.001

.094

.112

48

 

 

Method Detection Limit

 

The detection limit for this method should be determined daily.  If the detection limits are consistent for a couple of weeks, then it will be necessary to perform this task weekly. 

 

Spike DI water with 2-3 times the estimated instrument detection limit .  This should be around 0.2 to 0.3 mM.  (Use the 0.2 or 0.3 mM standard as the spike.)  You must run at least 7 spikes as samples after the standard curve has been run.  Fewer than 7 will result in the calculations being wrong.  Calculate the method detection limit (MDL) by 

MDL= [t(7, 0.01) * s]

where t=t statistic for 7 reps (t=3.14) with 99% confidence and s=standard deviation of the calculated concentration. 

 

 

Please contact arc_im@mbl.edu with questions, comments, or for technical assistance regarding this web site.