Page 1 of 10
v 2004
Photo of Brown’s River at Mill’s River Park, Jericho VT
Photo by Jack Cameron
How Much Stuff Is In There?: Quantitative Analysis of Phosphate In Natural
Waters
Objectives: .
Learn how to perform a quantitative spectrophotometric assay;
Analyze data using the statistical and graphing functions of Excel;
Determine the level of free phosphate in a pond water sample.
Introduction:
Scientists ask a lot of questions. We ask: “What kind of stuff is there in this piece of the universe we
are studying? Is that a neutrino being emitted from the sun? What compounds are present in drinking water?
Are there sodium ions in this softened water? Is there water on Mars? Are there anabolic steroids in the urine of
major-league baseball players? What elements are present? Is mercury present in this lake perch? Is this shiny
nugget gold or iron?” This is qualitative analysis.
Once you know what stuff is present, the next question you want to ask is: “How much stuff is there in this
piece of the universe we are studying? How many neutrinos are being emitted from the sun? How much
sodium ion is present in the softened water? How much phosphate is going into the lake from the new fish
hatchery?” This is quantitative analysis, from the Latin quantus?, “How much?”
In this lab you will quantitatively determine the amount of phosphate in a water sample taken from a
pond bordering the SMC campus or the Winooski River.
ON PHOSPHORUS IN WATER:
Phosphorus, the element, is non-metallic, and exists in three forms, or allotropes: These are red, white
and black phosphorus. Elemental phosphorus exhibits some rather interesting chemistry. For example, when
white phosphorus is placed in the dark and is exposed to air, it glows green and gives off white smoke. In
warm, humid air it will spontaneously burst into flames. Ingestion of as little as 50-100 mg of white
phosphorus may cause death. That being said, phosphorus-containing oxides, phosphates, (PxOyn-) are
absolutely necessary for life as we know it. As water runs over and through rocks it carries off small amounts
of phosphates. These inorganic phosphates (phosphates not attached to carbon-containing compounds) are a
plant nutrient and are taken in by plants with water and incorporated into organic phosphate compounds.
Animals obtain phosphorus from phosphates in water and plant material. Clean streams and lakes have a
phosphorus concentration of approximately 0.02 ug/L (parts per million, ppm). Phosphates are essential for
algae and plant growth, and at the 0.02 ug/L level, they are the limiting reagent for plant growth. Add more
Page 2 of 10
v 2004
nitrogen of potassium (also essential for plant growth) and plant and algae levels don’t change. Add more
phosphate, and you end up with a lot more green stuff in the lake.
Adding phosphates to lakes and stream increases eutrophication – that is, excessive growth of algae
and plants, resulting in the depletion of oxygen in the water. Eutrophication can lead to fish kills, degradation of
habitat, and loss of species. Large mats of algae can form and sometimes completely cover small lakes or
shallow bays. Sources of excess phosphate include human sewage, agricultural run-off from crops, sewage
from animal feedlots, fish hatcheries, the paper industry, vegetable and fruit processing, chemical and fertilizer
manufacturing, and detergents.
Phosphorus and Suspended Material
Phosphorus is present in streams and lake waters in several different forms:
orthophosphate, PO43-; polyphosphates [PnO3n+1(n+2)-] and metaphosphates
[(PO3)nn- ]; and organic phosphorus. Orthophosphate is the most biochemically
available form of phosphorus in natural waters, and microorganisms catalyze the
hydrolysis of condensed phosphates to orthophosphate. However, 30 to 60
percent of the phosphorus present in many natural waters is bound to organic
material.
O
O
P
Phosphate ion,
“orthophosphate”
O-
-O
O-
O
O
O
P
P
P
O
O
O-
O
-O
OO-
O
O-
P
P
P
OO-
O-
O
-O
O
O-
A polyphosphate
H3O+
O
-O
OO
P
P
O
O
O-
P
3
O-
-O
O
C
N
C
H
O
-O
N
P
O
O-
O-
CH 2
O-
O
C H
H
H C
C
C
O
H
P
O-
-O
O-
NH
C
O
P
OO-
O-
O-
P
H
C
C
N
NH 2
Hydrolysis of a
polyphosphate to
orthophosphate
Page 3 of 10
v 2004
One may analyze phosphorus content in water several ways, and get very different results. A Total Phosphorus
analysis, for example, will measure phosphate, polyphosphates, metaphosphates, and organic phosphates, both
dissolved in the water and adsorbed onto suspended solids. This is a somewhat time-consuming procedure,
requiring reaction of the samples with strong acids, and heating. In this experiment, we will test only for
soluble orthophosphate, that is, PO43- dissolved in the water sample.
See: Merck Index, 9th Edition, 1976. M. Windholtz, ed. Merck and Company, Rahway, NJ,
Groundwater Contamination: Sources, Control, and Preventative Measures. 1989. Chester D. Rail, ed.
Technomic Publishing Co., Inc., Lancaster, PA.
http://va.water.usgs.gov/chesbay/RIMP/glossary.html#concentration
Standard Methods For The Examination,of Water and Wastewater, 14th ed. 1975. Rand, M.C., WPCF, Chair,
Joint Editorial Board. Published by APHA-AWWA-WPCF, Washington, DC.
Page 4 of 10
PHOSPHATE ANALYSIS PROTOCOL:
v 2004
This method follows the protocol for orthophosphate analysis published in: Standard Methods For The
Examination,of Water and Wastewater, 14th ed. 1975. Rand, M.C., WPCF, Chair, Joint Editorial Board.
Published by APHA-AWWA-WPCF, Washington, DC. Orthophosphates (PO43-) are used as fertilizers, and
enter lakes and streams by storm run-off and melting snow. DO NOT REFERENCE THIS UNLESS YOU GO
GET IT AND READ IT (AND YOU NEED NOT DO THAT!)
In this experiment you will:
1.
2.
3.
4.
5.
6.
7.
Obtain a water sample from the small pond behind the townhouses, or from a stream or river;
Treat the pond water with decolorizing charcoal, and filter it;
Make up several solutions containing known amounts of sodium hydrogen phosphate (Na2HPO4);
Obtain a “Quality Control” phosphate solution;
React all samples with molybdate-vanadate reagent and obtain absorbance readings at 400 nm.
Construct a standard curve of absorbance vs. [PO43-] using data from the known phosphate samples;
Determine [PO43-] in your pond water and “Quality Control” samples.
You will need the following glassware and equipment:
PHOSPHOROUS SET UP:
[per pair unless noted otherwise]
Sample bottle, acid washed, 2 dram (or
scintillation vial)
1 L bottle to make up 1 M HCl + stopper
[Under sinks in lab]
Ehrlenmeyer flask, 125ml (for filtering
into)
Volumetric Flask, 10.00 mL
Volumetric pipet, 1.00 mL
Volumetric pipet, 2.00 mL
Volumetric pipet, 3.00 mL
Volumetric pipet, 5.00 mL
Blue pipet bulb/handle
Glass funnel, long stemmed
Filter paper, qualitative (to fit above)
Waste Jug (“Phosphorous”)
Ring Stand w/ 1 ring (to hold funnel)
Spatula
1 per student
1
1
7
2
orange tape on one
of the two
1
1
3
Red & blue tape
on one of the 3
2
2
2 boxes per riser
1 in Hood
1
1 per charcoal set
up
Page 5 of 10
v 2004
You will need the following reagents:
CHEMICAL PREP:
12 M HCl
Molybdate-vanadate reagent
0.02 M ammonium molybdate
(VI)/0.01 M ammonium
metavanadate in 4.0 M HCl*
Stock: 36.75ug/mL [PO4 ]
Quality Control: 14.70 ug/ml
Decolorizing charcoal
83ml per pair of
concentrated 12M HCL
10 mL per pair:
1 set up per hood:
2x125ml flask w/ CC
10ml grad cyl. ORANGE
20 ml per pair
1 set up per hood
2x 250ml flask w/ CC
10ml grad cyl. RED
10 ml per pair
1 per hood
2 x Tall form beaker
w/CC 10ml grad cyl.
2 50ml beaker: ½ full w/
a spatula
Place on back bench on
paper towels.
Page 6 of 10
v 2004
STOCKROOM STAFF PREP DIRECTIONS:
TO MAKE UP MOLYBDATE-VANADATE REAGENT: IN THE HOOD!!!!!!!!!!!!
1.
2.
3.
4.
5.
**
DISSOLVE 25.0 G OF AMMONIUM MOLYBDATE (VI) [(NH4)6MO7O24.4H2O; MW 1235.86 G/MOL] IN 400
ML OF DISTILLED WATER IN AN ACID-WASHED 500 ML EHRLENMEYER FLASK.
DISSOLVE 1.25 G OF AMMONIUM METAVANADATE [NH4VO3, MW 116.98 G/MOL] BY HEATING TO BOILING
IN 300 ML DISTILLED WATER, IN AN ACID-WASHED 1.00 L VOLUMETRIC FLASK. COOL TO RT
THEN, IN INCREMENTS WITH STIRRING CAREFULLY ADD 330 ML OF CONCENTRATED HCL TO THE ABOVE
SOLUTION (#2). COOL. PLACE SET UP IN A BUCKET UNDER HOOD WHEN ADDING HCL.
POUR THE AMMONIUM MOLYBDATE [VI] SOLUTION INTO THE AMMONIUM VANADATE SOLUTION, AND
DILUTE TO 1.00 L.
COLOR CODE: ORANGE
TO MAKE UP STOCK PHOPSHATE SOLUTION: IN THE HOOD!
1. DISSOLVE 0.0534 G (NOTE EXACT MASS USED!!!!) OF SODIUM HYDROGEN PHOSPHATE [NA2HPO4.H20:
MW: 137.99 G/MOL] IN 900 ML OF DISTILLED WATER IN AN ACID-WASHED 1.00 L VOLUMETRIC FLASK, AND
THEN DILUTE TO 1.00 L. RECORD EXACT CONCENTRATION ON FLASK!
2. COLOR CODE: RED
3. MATH: MASS OF SOLID/ MW OF SOLID X 94,97G/MOL [MW OF PHOSPHATE ONLY]=_____PO4 G/LITER
________G/LITER X 106UG/G X 1L/1000ML =______UG/ML(LABEL AS…)
*** TO MAKE QUALITY CONTROL SOLUTION: IN THE HOOD!
1. DISSOLVE .0214 GRAMS (NOTE EXACT MASS!!!!) OF SODIUM HYDROGEN PHOSPHATE
[NA2HPO4.H20: MW: 137.99 G/MOL] IN 900 ML OF DISTILLED WATER IN AN ACID-WASHED
1.00 L VOLUMETRIC FLASK, AND THEN DILUTE TO 1.00 L. RECORD EXACT CONCENTRATION ON
FLASK!
2. COLOR CODE: BLUE
3. MATH: SAME AS ABOVE
Page 7 of 10
v 2004
PRELAB:
A. OBTAIN A WATER SAMPLE.
`You will need about 5 mL of water. Collect the water (fill the vial completely!) in the clean, acidwashed flask provided, seal it, and label it with your name, the date and time of
collection, and the site of collection. Describe the exact time, location, and conditions
under which you collected your sample. (This is part of your grade!) e.g. “The water
sample I obtained was from the Winooski River delta at the Burlington Bike Path Bicycle
Ferry landing. Due to severe drought (only 0.07 “ of rain in the last 30 days, according to
www.NOAA.gov. ” Bring your sample and collection data to lab.
I. Filtering and Decolorizing
PROCEDURE:
If your water sample has color to it, add a small spatula-tip full of activated charcoal to it – THINK TINY
AMOUNT (about 20 mg. DO NOT WEIGH ON THE BALANCE). Swirl it around, let it sit for 5 min. and filter
the sample through a moistened filter to remove the carbon.
II.
The Phosphate Assay
To be done by TA: Make a stock solution of phosphate according to the procedure on page 5.
Record the exact concentration on the container.
To be done by the student: RECORD the exact concentration of the phosphate standard solution in
your notebook.
Before you come to lab: Calculate the volume of 12 M HCl you will need to dilute to
1 L to make a 1 M solution.
ACID WASH ALL YOUR GLASSWARE WITH 1 M HCL, and then rinse with distilled water.
Now that you have squeaky clean dishes:
Making calibration standards & solutions for analysis:
1. Obtain 7 10.00 mL volumetric flasks.
2. Label the volumetric flasks "0" through "6".
3. Add 5 mL of deionized water to the "0" flask..
4. To flask "1" carefully pipet in 1.00 mL of the stock phosphate solution using a volumetric
pipet.
5. Following the chart on the following page, add 2.00 mL of the stock phosphate to flask 2, 3.00
mL of the stock phosphate to flask 3, and so on. To flask 5 add 5.00 mL of the Quality Control
solution. To flask 6 add 5.00 mL of your water sample.
6. Add 1.00 mL of the vanadate-molybdate reagent to each flask, & dilute to the mark on the
neck with deionized water. The bottom of the meniscus should be resting on the mark.
Remember to thoroughly mix each flask.
7. Let the yellow color develop for 10 minutes or more. Read and record the absorbance of each
sample at a wavelength of 400 nM.
Page 8 of 10
v 2004
SET UP THE FOLLOWING CHART IN EXCEL BEFORE YOU COME TO LAB. PRINT OFF A
HARD COPY AND BRING IT WITH YOU.
Assay #
mL stock PO43-
0
1
2
3
4
0
1.00
2.00
3.00
5.00
Final volume
(mL)
10.00
10.00
10.00
10.00
10.00
5 quality control
6 water sample
5.00
5.00
10.00
10.00
[stock PO43-]
ug/mL
A480
For best results, calibration standards should be prepared fresh each time a batch of samples is analyzed.
Page 9 of 10
v 2004
Calculations:


Calculate the [PO43-] in each of your standards.
Generate a standard curve of absorption vs. concentration for your standards. Indicate
what wavelength you used. Calculate the best fit straight line for your data. See chapter 4 in
the Excel Manual to find out how to do this, at http://academics.smcvt.edu/chemistry/CHEM
103/week_2.htm Using your standard curve best fit line, calculate the [PO43-] in your water
sample.
 Also calculate [PO43-] in your quality control.

Calculate a % absolute error between what you KNOW your quality control [PO43-]
content is and your experimental value.
WASH & RETURN ALL GLASSWARE!
400 nm
Slope of best fit straight
line= 4.111 x 10-6
Y –int = 2.63 x 10-3
The equation of the best fit straight line is: Y = mx + b, where Y = absorbance, m = slope, x = [PO4-3], and b =
Y-intercept.
Thus, if your water sample has an absorbance of 0.0151 at 400 nm, the [PO43-] is:
0.0151 = 4.111 x 10-6([PO43-]) + 2.63 x 10-3
[PO43-] = (0.0151 – 2.63 x 10-3)/ 4.111 x 10-6 = 3030 ug/L
Page 10 of 10
You originally used 5.00 mL of water (0.005 L) and diluted it to 10.00 mL (0.0100 L).
Thus, the original [PO43-] in your sample was:
v 2004
(3030 ug/L )(0.0100 L/0.005 L) = 6060 ug/L
You can use this as a suggested format to set up your data in Excel…Notice that this data is different than
yours!
ALL WASTE FROM THIS EXPERIMENT GOES IN THE JUG LABELLED
“PHOSPHATE WASTE” IN THE HOOD.