64
Phosphorus Determination using the Colorimetric
Ascorbic Acid Technique
Introduction
Phosphorus has been identified as a prime nutrient needed for algae growth in
inland environments. In 1992, the EPA reported that accelerated eutrophication was
one of the leading problems facing the Nation's lakes and reservoirs. Eutrophication
caused by the overabundance of nutrients in water can result in a variety of waterquality problems, including fish kills, noxious tastes and odors, clogged pipelines,
and restricted recreation. In freshwater, phosphorus is often the nutrient responsible
for accelerated eutrophication. Many algae blooms in rivers and lakes are attributed to
elevated phosphorus concentrations resulting from human activities. Phosphorus
enters surface waters from agricultural and urban runoff as well as from industrial and
municipal wastewater treatment plant effluent.
No national criteria have been established for concentrations of phosphorus
compounds in water; however, to control eutrophication, the EPA makes the
following recommendations:
• Total phosphates should not exceed 50 µg/L (as phosphorus) in a stream at a
point where it enters a lake or reservoir.
• Total phosphorus should not exceed 100 µg/L in streams that do not discharge
directly into lakes or reservoirs.
Municipal wastewater treatment plants in many areas are required to remove
phosphorous in their treatment process. While the biological treatment process
removes some phosphorus, in most cases precipitation as an insoluble metal
phosphate is required to meet discharge regulations. This precipitation step is
normally accomplished with a metallic salt such as ferric sulfate, ferric chloride or
aluminum sulfate. This precipitation step may be accomplished in the primary or
secondary clarifiers.
Phosphorus Quantification Techniques
Quantification of phosphorous requires the conversion of the phosphorus to
dissolved orthophosphate followed by colorimetric determination of dissolved
orthophosphate. The analysis of different phosphorous forms (e.g. particulate or
organic-P) is obtained by various pretreatment steps. Pretreatment may consist of
filtering to remove suspended matter or various digestion techniques designed to
oxidize organic matter.
Phosphorus can be present in surface waters as organic phosphorus,
orthophosphate (an inorganic form of PO4 ), or as condensed (solid) phosphates. The
phosphorus may be in solution or as a component of suspended particulates. The wet
chemical colorimetric analysis of phosphorus only works for orthophosphates and
thus other forms of phosphorus must be converted to this form if they are to be
analyzed. Organic phosphorus can be oxidized (digested) using perchloric acid, nitric
acid-sulfuric acid, or persulfate with the persulfate technique being the safest and
least time consuming. The digestion methods are detailed in APHA method 4500-P
B.
CEE 453: Laboratory Research in Environmental Engineering
Spring 2001
65
Three techniques for colorimetric analysis of phosphorus are available. The
technique most commonly used is the ascorbic acid method, which can determine
concentrations of orthophosphate in most waters and wastewater in the range from 2200 µg P/L. Ammonium molybdate and antimony potassium tartrate react in an acid
medium with dilute solutions of orthophosphate-phosphorus to form an intensely
colored antimony-phospho-molybdate complex. This complex is reduced to an
intensely blue-colored complex by ascorbic acid. The color is proportional to the
phosphorus concentration. The complex is not stable and thus analysis must be
performed within 30 minutes of adding the ammonium molybdate and antimony
potassium tartrate.
Barium, lead, and silver interfere by forming a precipitate. The interference from
silica, which forms a pale-blue complex is small and can usually be considered
negligible. Arsenate is determined similarly to phosphorus and should be considered
when present in concentrations higher than phosphorus.
Method Detection Limit
"Method detection limit" is the smallest concentration that can be detected above
the noise in a procedure and within a stated confidence level. Several types of
detection limits are used including instrument detection limit (IDL), method detection
limit (MDL), and practical quantitation limit (PQL). The IDL is strictly instrument
noise and does not include variability due to sample preparation steps. The MDL
includes both instrument noise and sample preparation variability. The MDL is
obtained by making a standard that is near the MDL and dividing it into at least 7
portions. Each of the portions is then processed through all sample preparation steps
and then analyzed. The MDL is calculated using the following equation.
MDL = st n−1,α
6.1
where n is the sample size and α=0.01 is generally the required confidence. The
student t distribution function is available in Excel as a two sided test statistic (so use
TINV(2α,n-1)) and the standard deviation, s, can be computed in Excel as STDEV().
The PQL is about five times the MDL and represents a practical and routinely
achievable detection limit with reasonable assurance that any reported value greater
than the PQL is reliable. According to Standard Methods the method detection limit
for phosphorus when using a 1 cm light path is approximately 150 µg P/L.
Spectrophotometer Limitations
The diode array spectrophotometer has 316 diodes that cover the wavelength range
of 190 nm to 820 nm. Each diode generates a voltage output that is proportional to the
number of incident photons. The voltage is then digitized, but the manufacturer of the
instrument in the Cornell Environmental Laboratory, Hewlett-Packard, doesn't report
the resolution of the analog to digital converter. At very low concentrations the
difference between the intensity of light transmitted through the reference and the
intensity of light transmitted through the sample approaches zero. At some low
concentration the difference in light intensity approaches the resolution of the analog
to digital converter. Another source of instrument error is drift in lamp intensity over
Phosphorus Determination using the Colorimetric Ascorbic Acid Technique
66
time. The lamp intensity is measured when a reference sample is made. The light
intensity recorded by the diodes will vary proportionally to any lamp intensity drift.
The IDL should decrease as the number of diodes used in the analysis increases (as
in Spectral analysis) for the same reason that replicate analysis of samples decreases
the standard deviation. The "Spectral analysis" feature, which can be used to measure
either single or multiple components, uses as much of the spectrum as the user desires
and thus potentially decreases the IDL. Spectral analysis uses general least squares
regression to add multiples of extinction coefficient arrays for each component to
obtain the best curve fit for the sample. The extinction coefficient arrays are obtained
from the slope of the linear regression line for absorbance as a function of
concentration at each wavelength.
Experimental Objectives
1) Measure the concentration of phosphorus in several samples to test the precision
of the ascorbic acid technique.
2) Compare the results obtained using conventional analysis at a single wavelength
with spectral analysis.
3) Analyze the data using spectrophotometer software outside the lab.
4) Analyze multiple samples so that confidence intervals can be calculated.
5) Estimate the method detection limit (MDL).
6) Discuss methods to improve the method detection limit.
7) Discuss method automation.
Experimental Procedures
Standards Preparation Method
1) Use 100 µg P/L stock.
2) Use a digital pipet and prepare 1 mL of each standard.
3) Use E-pure water to dilute the 100 µg P/L stock.
Reagent Addition for Samples and Standards
1) Pipette 1 mL sample into a disposable microcuvet using a 1 mL digital pipette.
2) Add 160 µL combined reagent and mix thoroughly by swirling.
3) After at least 10 minutes but no more than 30 minutes, measure absorbance of
each sample using a reagent blank as the reference solution.
Samples and Standards to Prepare
1) Reagent blank to be used as reference samples.
2) Prepare 6 standards containing 0 (reagent blank), 1, 3, 10, 30, 100 µg P/L.
3) Prepare 6 additional 10 µg P/L standards.
CEE 453: Laboratory Research in Environmental Engineering
Spring 2001
67
4) Prepare 5 samples such as Cayuga Lake water, tap water, Chem lawn runoff, local
creeks…
Spectrophotometer Method
1) Use Sample Cuvettes. (Make sure to orient all cuvettes with the arrow on the left
because the cuvettes are not symmetrical and have different absorbance when
turned 180°.)
2) Use the reagent blank as the reference sample for all samples.
3) Use units of µg P/L.
4) Label all samples with descriptions that your classmates will understand!
5) Fill in the general description with your initials and a description of the type of
samples
Samples Analysis
1) Measure the reference using a reagent blank. (The reagent blank is also the 0
mg/L standard.)
2) Analyze the reagent blank as a sample and verify that the absorbance deviates less
than 0.004 AU (absorbance units) from zero. If the absorbance deviates more than
0.004 AU reanalyze the reference sample.
3) Analyze 6 standards as standards using the spectrophotometer and save the data as
\\Enviro\enviro\Courses\453\phosphorus\netid_Pstd.
4) Analyze 6 standards as samples using the spectrophotometer and save the data as
\\Enviro\enviro\Courses\453\phosphorus\netid_Pstdsam.
5) Analyze 7 10-µg P/L standards as samples and save the data as
\\Enviro\enviro\Courses\453\phosphorus\netid_10Pstdsam.
6) Analyze 5 samples as samples using the spectrophotometer and save the data as
\\Enviro\enviro\Courses\453\phosphorus\netid_Psam.
Prelab Questions
1) You will be creating 1 mL standards by diluting a stock of 100 µg P/L. Create a
table showing how you will prepare 1 mL of each of the standards.
2) All of the samples are diluted with a small amount of combined reagent. How is
this dilution accounted for when calculating the concentration of samples?
Data Analysis
1) Plot the absorbance spectra of the standards. What is happening in the UV region?
Are there any absorbance peaks?
2) Choose an appropriate wavelength (perhaps an absorbance peak) and use Excel to
create a calibration curve. For the calibration curve, absorbance should be a
function of phosphorus concentration.
Phosphorus Determination using the Colorimetric Ascorbic Acid Technique
68
3) Use the 10-µg/L standards that were analyzed as samples to evaluate the Method
Detection Limit using single wavelength analysis. Use your Spreadsheet to
calculate the concentration of each of the replicates.
4) Use the 10-µg/L standards that were analyzed as samples to evaluate the Method
Detection Limit using spectral analysis. Report the wavelength range used for
your analysis. You will need to analyze each sample and copy the results into a
spreadsheet. (Note that within a data file you change which sample the software is
analyzing with the sample number control.)
5) Use the method with the lowest MDL to analyze your samples. Create a bar graph
showing the concentration of each of the samples. Report the MDL in the figure
caption.
Spreadsheet requirements
Your spreadsheet must contain all of the analysis requested above as well as the
following capabilities:
1) A well-marked cell containing the analytical wavelength for single wavelength
analysis. Changes to this cell must be reflected in all calculations and graphs.
2) The graph showing the absorbance spectra of the standards must have a vertical
line indicating the analytical wavelength.
3) All of the graphs must be on the same page as the analytical wavelength control
so the effect of changing the wavelength can be easily observed.
4) A separate sheet where you answer the 4 questions.
Hints
If you haven't already learned how to use Vlookup() now is the time!
The row() function returns the number of the row. I found it useful for this analysis!
The slope() and intercept() functions eliminate the need to type equations off of
graphs!
Questions
1) Which analysis technique gave the best results? Explain why. If the analytical
technique didn't significantly affect the MDL, explain why not.
2) What types of errors dominated your ability to measure phosphorus?
3) What method modifications do you propose to improve phosphorus
measurements?
4) Total phosphorus concentration in Cayuga Lake varies between 10 and 50 ppb.
Would the techniques used in this lab be able to measure these phosphorus levels?
References
http://wwwrvares.er.usgs.gov/nawqa/circ-1136/h6.html#PHOS
http://www.epa.gov/glnpo/lmmb/methods/index.html#Volume 3
Standard Methods for the Examination of Water and Wastewater.
CEE 453: Laboratory Research in Environmental Engineering
Spring 2001
69
Lab Prep Notes
Reagents
Table 1. Reagents
A Sulfuric acid solution, 4.9 N: Add
136 mL concentrated H2 SO4 to
800 mL E-pure water. Cool and
dilute to 1 L with E-pure water.
B Ammonium molybdate solution:
Dissolve 40 g of (NH4 )6
Mo7 O24 •4H2 O in 900 mL E-pure
water and dilute to 1 L. Store at
4°C.
C Ascorbic acid: Dissolve 9 g of
ascorbic acid (C 6 H8 O6 ) in 400 mL
E-pure water and dilute to 500
mL. Store at 4°C. Keep well
stoppered. Prepare fresh monthly
or as needed.
D Antimony
potassium
tartrate:
Dissolve
3.0
g
of
K(SbO)C 4 H4 O6 •½H2 O in 800 mL
E-pure water and dilute to 1 L.
Store at 4°C.
Combined color reagent: Combine
the following solutions in order,
mixing
Description
Supplier
concentrated
H2 SO4
(NH4 )6
Mo 7 O24 •4H2 O
C6 H8 O6
K(SbO)C4 H4 O6 •
½H2 O
sodium lauryl
sulfate
KH2 PO4
Fisher Scientific
Catalog
number
Fisher Scientific
Fisher Scientific
Fisher Scientific
Fisher Scientific
Table 2. Equipment list
Description
100-1095 µL
pipette
10-109.5 µL pipette
Disposable cuvets
Cuvet holder
UV-Vis
spectrophotometer
Supplier
Fisher Scientific
Catalog #
13-707-5
Fisher Scientific
Fisher Scientific
Fisher Scientific
Hewlett-Packard
Company
13-707-3
14-385-942
14-385-939
8452A
(but do not entrain air as oxygen oxides the ascorbic acid)
after
each addition: (Prepare fresh weekly. Store at 4°C)
Stock A, (4.9 N H2 SO4 ) 50 mL
Stock B, (Ammonium molybdate solution) 15 mL
Stock C, (Ascorbic acid solution) 30 mL
Stock D, (Antimony-tartrate solution) 5 mL
Water diluent solution: Add 4.0 g sodium lauryl sulfate and 5 g NaCl per L of E-pure
water.
Stock phosphorus standard: Dissolve 0.4394 g of Potassium phosphate monobasic
(KH2 PO4 ) (dried at 105°C for one hour) in 900 mL E-pure water. Add 2 mL of
concentrated H2 SO4 and dilute to 1 L. 1.0 mL = 0.100 mg P (100 mg P/L).
Standard phosphorus solution: Dilute 1 mL of stock solution to 1 L. (1.0 mL = 0.1 µg
P) (100 µg P/L).
Phosphorus Determination using the Colorimetric Ascorbic Acid Technique