Evaluation of HACH Procedures for the Determination of
Phosphate in Water
Orosz, Maria T., Mutti, Laurence J.
Department of Geology at Juniata College
Abstract
Phosphorous is a trace mineral occurring in water in both organic and inorganic forms. Since high levels suggest contamination, this nutrient is used to monitor water quality. Studies of Raystown Lake (Smith, 2001; Haley, 2002) revealed discontinuous trends
and instances in which reactive phosphate exceeded total phosphate. Their studies utilized EPA-certified procedures, performed on a HACH portable spectrophotometer using HACH reagents. This study was conducted to determine the reliability of HACH
procedures, identify variables that affect results, and develop a procedure to accurately assess total and reactive phosphate.
Seven standard solutions were digested in persulfate to convert all phosphate to orthophosphate. Solutions contained three phosphate sources: orthophosphate (PO4-3), metaphosphate (PO3-), and organic phosphate. Samples were treated to an assortment of heating
methods and were allowed to react for various times, to assess sensitivity to these variables. Samples digested on a hot plate produced more consistent analyses than those digested in a water bath. Tungsten carbide boiling chips had no effect on measured
concentrations or yield. Variations in reaction time produced no statistically significant differences. Concentrations between 0.3 – 2.0 mg/L exhibited a linear relationship; meanwhile concentrations below 0.3 mg/L were curvilinear and non-systematic.
Unfortunately, most analyses recorded by Smith and Haley, fall within this low concentration range in which both precision and accuracy of the HACH protocol are poor. Despite efforts to reduce error within the method, the source of variability remains obscure.
HACH procedures failed to accurately measure concentrations of standards. This was true for both those procedures intended to measure solely orthophosphate and those procedures designed to determine total phosphate.
Introduction:
Method:
Phosphorous is an essential trace mineral occurring in
water in both organic and inorganic forms. In animals, it is
needed to maintain metabolic reactions and in plants and
algae it is essential for growth. Inorganic phosphate
sources include household and industrial wastes,
agricultural runoff, and disturbances of land near bodies of
water. Since high phosphate levels indicate an influx of
this nutrient, it is commonly used to monitor water quality
for a given body of water. Raystown Lake is a large
reservoir in Huntingdon County, Pennsylvania surrounded
by predominantly agricultural land. Recent studies
conducted by Smith (2001) and Haley (2002) to monitor
nutrient loading within the lake, focused specifically on
nitrate and phosphate (total and reactive) concentrations.
These studies utilized EPA-certified analytical procedures,
performed in a laboratory setting on the HACH portable
spectrophotometer using HACH reagents. Although
nitrogen concentrations exhibited strong, consistent
geographical and seasonal trends and highly replicable
results, measured phosphate concentrations revealed
discontinuous trends and highly variable results as well
samplings in which measured reactive phosphate was
higher than measured total phosphate. This study was
conducted to determine the accuracy of standard phosphate
digestion methods, and identify variables within the
procedure that may be affecting results, with the hope of
developing a procedure that would accurately recover total
and reactive phosphate.
To obtain total phosphate, organic and inorganic phosphates must be
converted to orthophosphate through acid hydrolysis. The suggested
HACH method for phosphate digestion for concentrations in the
range between 0.02 – 2.50 mg/L, the range recorded by Smith (2001)
and Haley (2002), is the acid persulfate digestion method using
PhosVer 3 (ascorbic acid) as outlined in Figure 1. Specific HACH
methods used are Method 8048 for reactive phosphate and Method
8180 for total phosphate. Smith (2001) modified procedure 8180
slightly, introducing tungsten carbide boiling chips into the samples
during digestion in order to reduce boiling rates and prevent
excessive splashing of the sample. Some concerns subsequently
arose as to whether the boiling chips might produce interferences
with the phosphate test and be responsible for the highly variable
results she recorded. The experiments reported on in this paper
sought to assess which heating method produces the most accurate
and precise results during the digestion process. This study analyzed
samples using three types of heating methods: water bath, direct
heating on a hot plate without boiling chips, and direct heating with
boiling chips. Boiling chips were not placed in the samples digested
in a water bath because boiling and splashing were already at a
minimum. Following digestion, a spectrophotometer analysis was
used to determine orthophosphate (reactive phosphate)
concentrations. No changes were made to this HACH protocol. All
equipment and glassware was washed with hydrochloric acid and
rinsed with de-ionized water to avoid cross-contamination between
solutions during testing. Four series of solutions were analyzed using
samples with known and unknown concentrations.
Figure 1. Flow chart to determine the correct phosphate method to be used for the
desired concentration within a given sample.
For Series #1 two sets of standard solutions, as outlined in the HACH Standard Methods, were prepared and digested, incorporating all three sources of phosphate: orthophosphate (PO4-3), metaphosphate (PO3-), and organic phosphate. Orthophosphate was obtained
from Na3PO4 ·12 H2O, metaphosphate from NaPO3, and organic phosphate from adenosine 5+ - monophosphoric acid (ADP). Phosphate sources were weighed (to a precision of milligrams) and mixed with one liter of de-ionized water to prepare a stock solution and
were then diluted with de-ionized water (milliliter) in ratios of 100 (stock): 900 (DI) (Run #1) and 10(P): 990 (DI) (Run #2).
Series #2 was designed to test only for reactive phosphorous, requiring only a source of orthophosphate. KH2PO4 (potassium phosphate monobasic) was substituted for Na3PO4 ·12 H2O in this and all subsequent tests because of uncertainties about the amount of
potential absorbed water present in off-the shelf Na3PO4 ·12 H2O. Seven standard solutions were prepared using a series of dilutions composed of KH2PO4 and de-ionized water. For each of the seven samples, five runs were preformed in which each run was allowed
to react for two, four and six minutes prior to measurement to assess the sensitivity of the technique to the time variable.
The third series of analyses tested for both reactive and total phosphate. Reactive phosphate and total phosphate were recovered using the aforementioned methods. Originally, eight standard stock solutions were prepared and diluted using all three sources of
phosphate combined with de-ionized water. However, only two stock solutions (B and D) were actually processed.
For Series #4, five river water samples from five different locations containing unknown phosphate concentrations were tested for both total and reactive and reactive phosphate. The only heating method utilized during digestion was a hot plate without boiling chips.
In addition two standard solutions were prepared by diluting a known amount of KH2PO4 with a volume of river water to test for recovery of reactive phosphorous and the presence in natural waters of possible interfering solutes. Each test was performed under a range
of pH conditions, to assess the sensitivity of the technique to these variables.
For all of these analyses either four or five replicate samples were prepared and analyzed to assess analytical precision. Following the completion of all four series of analyses, each variable was evaluated to determine the most accurate method.
1
Run Number
Phosphate
Concentration (mg/L)
Graph 1. Series #1 Heating Methods: High Concentrations for Run #1
0.38
0.36
0.34
0.32
0.3
0.28
0.26
B
Stock
Phosphate Concentration
(mg/L)
Graph 3. Series #1 Heating Methods: Intermediate Concentrations for Stock B
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
2 min
4 min
6 min
0
0.5
1
1.5
2
Target
Graph 5. Series #2 Actual Concentrations vs Experimental Concentrations
The variables within the total phosphate digestion process of Series 1 were variations in heating method and the use of
tungsten carbide boiling chips. Graphs 1 and 2 depict comparisons among the heating methods for standard samples at
comparatively high concentration (Run #1) and intermediate concentration (Run #2). In Graph 1, phosphate
concentrations often exceeded the detection limit of the spectrophotometer at 2.75 mg/L. Standard deviation was the
lowest for samples digested directly on the hot plate with boiling chips. Variation for these measurements was only onehalf that of the samples directly heated without boiling chips and one-quarter of that for samples digested in a boiling
water bath. There were complications using the water bath technique in that buoyancy tended to cause the flasks to tip
and spill. For the intermediate concentration standards (Run #2) direct heating on a hot plate without boiling chips
produced the lowest standard deviation. Since Graph 2 represents concentrations within the range recovered by Smith
(2001) and Haley (2002), it is thought to most accurately represent our data. Heating method was also tested during
Series #3 for Stock Solutions B and D. This data is illustrated in Graphs 3 and 4. In Graph 3, phosphate concentrations
within the prepared solutions were similar to the intermediate concentration standards of Run #2. The water bath and
the hot plate without boiling chips had equal standard deviations, while the hot plate with boiling chips exhibited the
highest standard deviation. Graph 4, which depicts concentrations within the low part of the testing range, illustrates a
very high standard deviation for the water bath. No phosphate at all was detected for those samples heated on hot plate
without boiling chips, but these analyses are represented in the graph at concentration of 0.001. The standard error at
concentrations this low is such as to make the analyses meaningless. For Series #3 as a whole, the method of digesting
on a hot plate without boiling chips produced the most consistent results. In addition, careful observation during
digestion noted very little splashing with or without the tungsten carbide boiling chips. Therefore since the original
procedure does not instruct the use of boiling chips, and measurements were more consistent in their absence, we
conclude that they are unnecessary. There is, however, no evidence that boiling chips have a systematic effect on
measured concentration levels, as had been feared.
Series #2 was designed to test the accuracy of the HACH technique across the recommended concentration range, and to
assess whether the results would behave in a sufficiently consistent fashion to enable the construction of standardization
curves. Another variable was simultaneously evaluated during Series #2, that being the sensitivity of the technique to
reaction time prior to analysis (the HACH procedure specifies a precise time of two minutes). We monitored this
sensitivity by taking replicate readings on the same samples following reaction times of two, four, and six minutes.
Graph 5 illustrates our results. The most important results pertain to the accuracy of the technique across range. At
concentrations levels above 0.03 mg/L all of the measurements are co-linear and therefore could in principle be
corrected for systematic error; however, Graph 6, which expands the view for concentrations less than 0.03 mg/L,
reveals a non-systematic relationship between the observed concentrations and target values. The HACH procedure is,
therefore, inadequate to accurately determine phosphate at concentrations below approximately 0.03 mg/L. We are
unclear about the source of the deviation between measured and target concentrations in the linear range. Some
possibility of error exists in the preparation of the standards, given that the quantities of reagent required to make
accurate standards at these low concentrations demands extremely precise weighing. There were no significant
differences in measured concentrations given an extended reaction time. Therefore, extreme care in timing during the
analytical process is not essential.
0.60
0.40
0.20
0.00
2
Run Num ber
Graph 2. Series #1 Heating Methods: Intermediate Concentrations for Run #2
0.06
0.04
0.02
0
D
Stock
Graph 4. Series #1 Heating Methods: Low Concentrations for Stock D
0.15
Phosphate Concentration
(mg/L)
4.00
3.00
2.00
1.00
0.00
Discussion:
Phosphate
Concentration (mg/L)
Hot Plate w ithout Boiling Chips
Phosphate
Concentration
(mg/L)
Hot Plate w ith Boiling Chips
Phosphate
Concentration (mg/L)
Water Bath
0.13
0.11
0.09
0.07
0.05
0.03
0.01
-0.01
0
0.05
0.1
0.15
0.2
Target
Graph 6. Actual Concentrations vs Experimental Concentrations below 0.2 mg/L
Conclusion:
Samples digested directly upon a hot plate produced somewhat more consistent analyses than did those digested in a boiling water bath. Additionally, the use of tungsten carbide boiling chips had no effect on measured phosphate concentrations, neither increasing nor
decreasing yield. Although measured concentrations were slightly more accurate given a prolonged reaction time, there were no statistically significant differences among results. Graphs were constructed to examine the relationship between actual concentrations and
standard concentrations. Concentrations between 0.03 – 2.0 mg/L exhibited a linear relationship which could be modified to fit standard curves; concentrations below 0.03 mg/L have to be considered below reliable measurement. Unfortunately, many of the phosphate
analyses recorded by Smith and Haley for samples from Raystown Lake, fall within this extremely low concentration range in which both precision and accuracy of the HACH protocol are poor.
References
American Public Health Association et al., 1981, Standard Methods for the Examination Water and Wastewater: Washington, D.C., American Public Health Association.
HACH Company, 1994, HACH Water Analysis Handbook: Loveland, CO, HACH Company, 2nd Ed., p. 487-488, 509-511.
Haley, C. J., 2002, Nutrient Concentrations and Temporal Variations: A Study of Phosphates and Nitrates in Raystown Lake, PA, Juniata College Undergraduate Research Sympsium.
Smith, J.L. 2001, Nutrient Concentrations in Raystown Lake, Juniata CollegeUndergraduate Research Symposium.
Wetzel, R. G., 2001, Limnology: Lake and River Ecosystems: San Diego, CA, Academic Press, 3rd Ed., p. 239-288.