1. Brett Kelley, Senior Project Report
Analysis of Total Phosphorous Content by ICP-AES

2. Introduction:
The environmental engineering department runs a program called WESTT (Water, energy, and
sustainability training team) whose goal is to research biofuels and water recycling technology.
Many of the projects relate to the use of algae as a source of water treatment, this algae can
then be harvested from wastewater ponds and used as a form of biofuel. In order to determine
the ideal set of parameters for algae growth in these wastewater ponds, solid content, chemical
content, dissolved oxygen content, and biochemical oxygen demand must be tested for pond
samples on a regular basis. Determining the ideal set of parameters for algae growth is the
current focus of WESTT and several other research programs across the country that work with
WESTT. One of the chemical tests that we perform is for the total phosphorous content of a
pond sample, phosphorous is important to test for because it is a key nutrient for algae growth.
Although there is currently a successful assay already in use by the program, it is a long,
difficult, and relatively dangerous assay. This assay involves refluxing acidic solutions in the
fume hood and performing a titration on these refluxed samples before analyzing their
absorbance. I chose to attempt an alternate assay for total phosphorous content using an ICP-
AES (inductively coupled plasma atomic emission spectrometer). This assay involves heating
acidic samples on a hotblock and filtering before analysis by ICP-AES. This analysis also gives us
the advantage of being able to accurately test for a variety of metallic elements that may be
present in the samples. The data obtained for elements besides phosphorous is not being used
for any specific purpose; we just wanted to test the viability of this method for elements
besides phosphorous.
In order for this new assay to be used by the environmental engineering department, the
values I obtain for total phosphorous content must come close to the values obtained by the
spectroscopic method, and the data I obtain must pass the quality control standards in place by
the WESTT program. In considering whether or not this procedure will be adopted, the cost,
detection levels, speed, and skill required must be compared.

3. Experimental:
The digestion procedures used in this analysis are slightly modified versions of the digestion
procedures for total recoverable analytes in aqueous samples given by EPA method 200.7
(found in section 11.2.2, page 31). Modifications were made to account for a different sample
volume used.
All materials used in this analysis are supposed to be trace metal grade according to EPA 200.7
guidelines. This includes digestion tubes, filters, pipet tips, acids, and reagent water. Due to
monetary limitations we only utilized metal free digestion tubes, filters, and acids. Deionized
water was used instead of type 1 reagent water. Clear, plastic, disposable pipet tips were used.
Sample digestion
50 mL of homogenized sample were collected in a 50 mL digestion tube. 400 µL of 1:2 nitric
acid were added to each tube to preserve the sample. For the analysis of total recoverable
analytes, samples with low solids content (less than 1% undissolved solids), followed digestion
1, samples with high solids content (greater than 1% undissolved solids),followed digestion 2.
(Only the Digestate samples and LFB/LRB II underwent digestion 2 for this project) Procedures
are also listed for the analysis of acid extractable metals and dissolvable metals.
Digestion 1: 1 mL of 1:2 nitric acid and 0.5 mL of 1:2 hydrochloric acid were added to each
digestion tube. The digestion tube was put into the digestion block and allowed to evaporate at
approximately 95 degrees Celsius until a volume of 10mL remained. A watch glass is placed on
top of the digestion tube and the sample is allowed to reflux for 30 minutes. The digestion tube
is taken out of the digestion block, allowed to cool, and is re-diluted to 50 mL with deionized
water inside the digestion tube.
Digestion 2: The digestion tube was put into the digestion block and allowed to evaporate at 95
degrees Celsius until about 10 mL of sample remained. The tubes were removed from the
digestion block and allowed to cool. 2 mL of 1:2 nitric acid and 5 mL of 1:5 hydrochloric acid
were added to each digestion tube. The tubes were covered with watch glasses and allowed to

4. reflux at 95 degrees Celsius for 30 minutes. The samples were taken off of the digestion block,
allowed to cool, and re-diluted back to 50 mL.
After the digestion was completed, the samples were filtered using a disposable 0.45 µm filter
into a fresh digestion tube and left in the fridge until they could be analyzed (some samples
were in the fridge for a month before the analysis could be completed).
Acid Extractable Metals: 2.5 mL of 1:2 hydrochloric acid were added to the sample. The sample
was heated for 15 minutes on a hot block. The digestion tube is allowed to cool before the
solution is filtered through a 0.45 µm filter into a fresh digestion tube.
Dissolvable Metals: Filter homogenized sample through a 0.45 µm filter into a fresh digestion
tube.
ICP-AES analysis
Phosphorous was analyzed at 213.6nm and 214.9 nm, Calciumat 315.9 nm, Magnesium at
279.8 nm, and Potassium at 766.5 nm.
Before unknown samples are introduced to the machine, a calibration curve must be made for
each element that is to be analyzed. 5 multi-element standards were prepared, containing 10,
20, 50, 100, and 200 ppm each of Calcium, Potassium, and Magnesium. 5 phosphorous
standards that had undergone digestion 1 with concentrations of 2, 4, 7, 9, and 50 ppm were
used. LRB I was used as a blank.
Before running the standards, the concentrations of each element present in the different
standards must be entered into the computer. Once the instrument measures the intensities at
each requested wavelength for these standards, it is able to create a calibration curve for each
wavelength based on the information you entered. The instrument is then able to enter the
intensity it measures for each sample into its calibration curve and give you the calculated
concentration of the element corresponding to your selected wavelength. Once the calibration
curves had been created, the samples were analyzed by the instrument using an autosampler.
To introduce a sample to the instrument, you take the sample introduction straw, place it inside

5. a beaker of water that is to be used as a blank for several seconds (in order to clear out the
system), and place the sample introduction straw inside your sample. The autosampler is able
to switch between a rinse beaker and different samples.
Standards List
Phosphorous: 2,4,7,9, and 50 ppm, all taken through digestion 1.
Peach Leaves: 7 ppm P, taken through digestion 1
Laboratory Reagent Blank I/II: Deionized water taken through either digestion 1 or 2, no
standard added.
Laboratory Fortified Blank I/II: Deionized water with phosphorous standard added to create a
7ppm P solution, taken through either digestion 1 or 2.
Materials
Peach Leaf standard: 1370 ppm P (NIST# 1547)
Phosphorous standard: 1000 ppm phosphate form Fischer (Lot #1503A51)
500 mL trace metal grade hydrochloric acid, Sigma Aldrich, (231-595-7)
500 mL trace metal grade nitric acid, Sigma Aldrich, (7697-37-2)
Disposable watch glass, Environmental express, (SC505)
50 mL digestion tube, Environmental express, (SC475)
Disposable Syringe Filter (0.45 µm), Environmental express (SF045V)

6. Data:
Abbreviations
LRB I/II: Laboratory reagent blank (50 mL of deionized water) taken through digestion 1/2.
LFB I/II: Laboratory fortified blank (Deionized water with phosphorous standard added) taken
through digestion 1/2.
PL: Peach leaves with a known phosphorous content used to check the procedures ability to
determine the concentration of organically bound phosphorous.
AE: Indicates a sample has undergone the acid extractable metals procedure
DISS: Indicates a sample has undergone the dissolvable metals procedure
Data Tables
*Number listed in parenthesis after a sample name to distinguish between duplicate samples*
5/18/2015
Element Ca K Mg P(213.6) adj. P(213.6) P(214.9)
2ppm P -1.25 4.04 -0.65 3.04 1.96408 2.2
4ppm P -1.25 6.38 -0.64 6.41 4.24557 4.2
7ppm P -1.2 10.21 -0.65 11.06 7.39362 7.46
9ppm P -1.24 11.2 -0.64 12.49 8.36173 8.43
50ppm P -1.24 60.62 -0.65 76.47 51.67619 49.28
LFB I -1.24 10.43 -0.65 7.31 4.85487 7.59
LRB II -1.27 1.86 -0.65 0.17 0.02109 0.07
LFB II -1.26 10.6 -0.65 11.72 7.84044 7.77
Inf (1) 47.8 24.33 41.06 12.35 8.26695 8.08
Inf (2) 43.3 22.35 36.83 10.86 7.25822 6.99
Inf (3) 46.63 23.22 39.48 11.39 7.61703 7.8
P9(1) 43.86 23.74 48.61 9.69 6.46613 6.5

7. P9(2) 44.97 24.28 48.99 9.96 6.64892 6.49
P9(3) 47.42 25.42 51.64 10.31 6.88587 6.69
P9 spike 42.85 27.33 46.91 14.69 9.85113 9.28
Inf spike 38.73 24.58 33.12 15.09 10.12193 9.66
Inf Diss(1) 37.95 20.98 34.88 6.11 4.04247 3.95
Inf Diss(2) 38.24 21.23 34.46 6.91 4.58407 4.39
Inf Diss(3) 37.87 21.29 34.27 6.27 4.15079 3.97
P9 A.E. (1) 43.35 23.98 47.29 9.17 6.11409 5.82
P9 A.E. (2) 43.66 23.6 48.29 9.05 6.03285 6.17
P9 A.E. (3) 43.51 23.9 47.52 9.2 6.1344 6.05
P.L. 81.15 115.23 20.12 10.22 6.82494 6.76
Table 1: Data obtained for samples digested on 5/18/15. All numbers listed are concentrations
in ppm.
5/1/2015
Element Ca K Mg P(213.6) Adj P(213.6) P(214.9)
Inf:4/3(1) 47.39 22.37 40.64 10.18 6.79786 6.59
Inf:4/3(2) 44.04 20.99 38.17 9.49 6.33073 6.19
Inf:4/6(1) 46.4 20.77 46.08 8.98 5.98546 5.94
Inf:4/6(2) 46.1 20.47 45.68 8.86 5.90422 5.814
P6 :4/6(1) 52.69 23.62 51.99 10.68 7.13636 7.32
P6 :4/6(2) 51.44 23.07 50.68 10.4 6.9468 7.08
P9:4/13 52.84 23.79 57.49 9.59 6.39843 6.2
P9:4/13 Spike 52.99 28.05 57.21 14.93 10.01361 9.55
PL:5/1 79.98 112.89 19.8 10.31 6.88587 6.91
DGST:EDE:4/17 594.51 89.53 101.14 271.59 183.7724 179.68
DGST:REW:(1) 45.34 24.18 39.21 11.26 7.52902 7.39
DGST:REW(2) 44.9 23.61 38.17 10.93 7.30561 7.12
DGST:REW:(3) 43.45 23.07 37.32 10.5 7.0145 6.82

8. LFB I: -1.25 10.27 -0.64 10.3 6.8791 7.04
LRB I: -1.26 1.75 -0.65 0.174 0.023798 0.08
LRB II: -1.25 1.78 -0.65 0.2 0.0414 0.1
LFB II: -1.21 6.13 -0.64 5.36 3.53472 3.61
Table 2: Data obtained for samples digested on 5/1/15, the date next to the sample name
refers to the sample collection date, if different from the digestion date. All numbers listed are
concentrations in ppm.
Sample name Sample Date Concentration of P (ppm)
Inf 3/30 4.76
Inf 4/6 5.91
P6 4/6 7.65
Digestate (EDE) 4/17 115.63
Inf 5/11 5.76
P9 5/11 6.75
Inf 5/25 6.3
P9 5/25 6.3
Table 3: Total phosphorous data measured by other members of the WESTT program utilizing
the traditional Total Phosphorous analysis. These values are assumed to be true values for
phosphorous.

9. Calibration Curves
y = 16582x + 1897.6
R² = 0.9999
0
200000
400000
600000
800000
0 10 20 30 40 50
Intensity
Concentration (ppm)
P (213.6nm), un-adjusted
y = 24494x + 4200.4
R² = 0.9996
0
200000
400000
600000
800000
1000000
1200000
0 10 20 30 40 50
Intensity
Concentration (ppm)
P (213.6nm), adjusted
y = 25786x + 6979.1
R² = 1
0
200000
400000
600000
800000
1000000
1200000
0 10 20 30 40 50
Intensity
Concentration (ppm)
P (214.9nm)

10. y = 13618x + 8679.2
R² = 0.9989
0
500000
1000000
1500000
2000000
2500000
3000000
0 50 100 150 200
Intensity
Concentration (ppm)
Mg
y = 2035x + 936.87
R² = 0.9992
0
50000
100000
150000
200000
0 20 40 60 80 100
Intensity
Concentration (ppm)
K
y = 12362x + 17113
R² = 0.9978
0
500000
1000000
1500000
2000000
2500000
0 50 100 150 200
Intensity
Concentration (ppm)
Ca

11. Appendices attached to back of report:
Appendix 1: Shows data obtained from the standards used to create the calibration curves for
each element. Includes the 3 separate intensity measurements taken by the machine, the
average intensity, and measurement standard deviations.
Appendix 2: Shows the average intensity measurement, calculated concentration, and
measurement standard deviation for every sample at all 5 wavelengths.

12. Discussion:
Before any samples could be analyzed by the ICP-AES, an ideal spectral line had to be
determined for phosphorous. EPA method 200.7 states that phosphorous should be analyzed at
a wavelength of 214.9 nm, and that Copper and Molybdenum are both spectral interferents at
this wavelength. Mr. Stubler informed me that there were several wavelengths that have been
shown to obtain accurate results for phosphorous on the ICP-AES instrument in building 180,
they are 177.4, 178.2, 213.6, and 214.9 nm.
To determine the ideal wavelength, we tested several standards containing phosphorous by
itself and several standards containing phosphorous along with its 2 spectral interferents.
Several runs using the 177.4 nm wavelength did not appear to give good quantitative results so
it was removed from consideration. Results using 178.2 nm were good, Copper and
Molybdenum are also not spectral interferents at this wavelength. Unfortunately good results
at this wavelength seemed to depend on a nitrogen purge between runs (analyses at 213.6 and
214.9nm didn’t require a nitrogen purge); the nitrogen purge adds an additional cost to the
analysis, so this wavelength was removed from consideration. Results obtained at 213.6 nm
and 214.9 nm both seemed accurate, but at 213.6nm the spectral interferents had a diminished
effect. Although we don’t expect significant contaminations from Copper or Molybdenum in
our pond samples, we selected 213.6 nm as our ideal wavelength, but decided to run analyses
at both 213.6nm and at 214.9nm to be safe.
After we had completed the analyses of all the samples, the data obtained from the 213.6nm
runs appeared to be completely off, the results obtained for calibration verification standards
were all about 1.5 times higher than expected. When looking at the data obtained for the
calibration curves, I noticed that all the data points for the 213.6 nm Phosphorus curve had
exceptionally high measurement standard deviations (standard deviation between the multiple
measurements the instrument takes of a single sample). Upon looking at the raw intensities for
each of the 3 measurements’, I noticed that for every single data point, the first measurement
was significantly lower than the following 2 measurements. The unknown samples did not

13. share this problem since the measurement standard deviations at this wavelength were all low
(less than 5% for most samples).
To account for this first skewed first measurement that likely resulted from some timing delay
on the sample introduction, I created an adjusted calibration curve by averaging the final 2
measurements. The measurement standard deviations between the final 2 measurements were
low for every data point on the calibration curve, a good indication that these adjusted results
should be more reliable. By setting the two calibration curve equations equal to one another, I
was able to come up with a simple equation to convert from unadjusted concentration to
adjusted concentration. All of the adjusted concentrations for 213.6nm come very close to their
corresponding value for 214.9 nm, there is an exception with one of the laboratory fortified
blanks, but this inconsistency is explained by a very high relative measurement standard
deviation of 85%. In the absence of the instruments’ error, both wavelengths are able to
produce accurate results.
One of the goals of the experiment was to compare the Total Phosphorous values obtained
from the ICP-AES analysis to those obtained from the traditional spectroscopic analysis. The
results obtained from the spectroscopic analysis is considered to be the true result for total
phosphorous and if our measurements are able to consistently match with the true results then
the experiment can be regarded as a success. Unfortunately there are only several data points
that we are able to compare to our data, several sets of samples that I collected and analyzed
never ended up being analyzed by the traditional method so there is no data to compare results
against for those points.
The three data points that we are able to make a direct comparison with are the 4/6/15 Inf and
P6 samples, and the 4/17/15 Digestate (EDE) sample. The ICP-AES results match very closely to
the true results for the Inf and P6 samples, but the Digestate samples results are significantly
far from the true results. The Digestate sample underwent digestion 2, this suggests that the
results obtained for digestion 2 can vary significantly from results obtained from digestion 1.
For the 5/18/15 pond samples, we can compare the results obtained from our analysis to the
true results. The P9 ICP-AES readings are very close to the true values while the Inf ICP-AES

14. readings are a little far from the true values, however, phosphrous levels can vary significantly
from one week to another, the concentrations obtained from the ICP-AES method are atleast
relatively near to the actual concentration.
Overall the results obtained for this data set seems good. The grouping of duplicate samples
seems very tight, the calibration verification standards all had calculated concentrations that
were very close to their actual values. The Dissolvable Metals influent samples showed
phosphorus values that were about half the value of the total recoverable analytes
concentration, showing that this method will not give a reasonable estimation for total
recoverable analytes for Phosphorous. The dissolvable metals values for Calciumwere also not
close to the true values, but the dissolvable metals concentration of both Potassium and
Magnesium were relatively close to the true values, so this technique can give a reasonable
estimation of the true value for some elements. The Acid Extractable Metals P9 sample showed
results very similar to the total recoverable analytes P9 sample at all 5 wavelengths, showing
that this technique gives a good estimation of the true value.

15. Conclusion
Overall this experiment appears to have been successful. All the calibration curves had high R2
values, the phosphorous concentrations obtained by the 214.9 nm line and the adjusted 213.6
nm line are very similar, and the calibration verification standards all have calculated
concentrations that are very close to their actual concentrations. The dissolvable metals
technique provides a reasonable estimate for the concentration of total recoverable analytes
for only some of the metals tested. The acid extractable metals technique seems to provide a
reasonable estimate for the concentration of total recoverable analytes for all of the metals
tested. The discrepancy between the true value and our calculated value for the digestate
sample as well as the poor results for obtained for one of the LFB II samples suggest that
digestion 2 is not effective at determining total recoverable analyte concentration, this may be
fixed by putting a set of phosphorous standards through digestion 2 and using these values to
create a second calibration curve exclusively for samples undergoing this digestion. To get a
better idea of the precision of this test, more samples with comparable true values from the
traditional spectroscopic method are needed.
Special thanks to Braden Crowe and Craig Stubler, without whom, this project would not have
been possible.
References
1: Determination of metals and trace elements in water and wastes by inductively coupled
plasma-atomic emission spectroscopy. Retrieved June 12, 2015, from
http://water.epa.gov/scitech/methods/cwa/bioindicators/upload/2007_07_10_methods_meth
od_200_7.pdf