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Comparison of Phosphate Analysis Methods

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    Lab Activity: Statistical Comparison of two Methods for Phosphate Detection: Hach TNT 843 vs. Standard Method 4500-PE

    Pre-Lab Exercise:

    Prior to coming to lab, you will need to find a procedure for phosphorus detection for Standard Method 4500-PE (Ascorbic Acid Method) from the Standard Methods for the Examination of Water and Wastewater manual. Text for this method can be found in electronic form by performing a simple google search. In addition, you will need to perform a literature search for two methods for phosphorus detection in water published in a reputable, peer-reviewed journal. More information about the literature search will be presented in class. For your pre-lab chemical information, you will need to use the reagents listed in this procedure.  Failure to complete this pre-lab exercise will result in a loss of all of the pre-lab exercise points.

    This is a three-week lab experiment. Preliminary Work: As stated above, you will need to perform a literature search for phosphorus detection procedures. More information will be presented in class prior to this lab experiment. Week 1 will involve finalizing your procedure upon instructor approval. Week 2 will involve preparing your standards for analysis and Week 3 will involve performing the measurements of your prepared standards.


    Phosphorus is an important nutrient in the life cycle of plants, and it can have significant effects on the ecosystem as a whole. Right here at SVSU, much research by the Saginaw Bay Environmental Science Institute (SBESI) has focused on phosphorus levels in the Saginaw Bay Watershed. The importance of maintaining proper phosphorus levels is several-fold; however direct implications lie in the growth of algae. As phosphorus levels in water rise, this provides extra nutrients for algal growth. As bacteria decompose algae, oxygen is consumed, creating a high oxygen demand on the surrounding area. This depletion of oxygen can result in hypoxic (low oxygen) or anoxic (oxygen depleted) zones, where many organisms cannot survive, most notably benthic species. Due to the importance of phosphorus, it is inherent that its runoff is limited. Runoff of phosphorus into water sources can occur due to leaky septic tanks, fertilizer containing phosphorus, and livestock feces.

    This lab refers to the determination of phosphates in water, rather than the determination of phosphorus in water, simply due to the fact that the phosphorus is present in the chemical form as phosphates. There are different types of phosphates, known as orthophosphates, condensed phosphates, and organic phosphates. Orthophosphates are what we commonly refer to as phosphate ion (PO43-), in which all three acidic protons of phosphoric acid have been donated. Condensed phosphates result when condensation produces polyphosphates. Organic phosphates form through biological processes and are commonly present in fecal matter.

    Due to the fact that the Kawkawlin River flows directly into the Saginaw Bay, heightened phosphorus levels have resulted in increased algal growth at the Kawkawlin’s mouth, which can possibly be a component of the resulting muck on the shores of the Bay City State Recreation Area beach. Not only is the increased algal growth an aesthetic problem, but it can wreak economic havoc through decreased property values, and may be a harbinger of bacteria, which can directly affect the health of those who venture into the coastal waters of the Saginaw Bay. The latter is a point of contention among environmental scientists however, as sometimes muck will contain detectable levels of bacteria, while other times it will not. For the sake of time, we will not discuss it further here.

    There are many colorimetric methods for the detection of phosphorus in water samples, most of which are described in Standard Method 4500-P in the Standard Methods for the Examination of Water and Wastewater1. This lab will focus on Standard Method 4500-PE, which is the detection of orthophosphate using the ascorbic acid reduction method. In this method, sample containing orthophosphate reacts with a combined reagent containing sulfuric acid, ammonium molybdate, and potassium antimonyl tartarate to form an antimony phosphomolybdic complex. This product then reacts with ascorbic acid, resulting in reduction to molybdenum blue, which can be detected at 880 nm using an absorbance spectrophotometer. Different companies employ variations on how these compounds are mixed, as well as the amount of each compound that is mixed.

    As analytical chemists, you should be aware that different analytical methods may produce results that do not statistically agree with one another due to various reasons. These reasons can include differences in reagent concentrations, differences in reagent delivery order, differences in reaction time, differences in ambient conditions, etc. In this lab experiment, we will be statistically comparing results from two phosphate detection methods: Standard Method 4500-PE, and the Hach Test-in-Tubes (TNT) method, which is a variation of Method 4500-PE. Upon collecting your data, each group of two will be responsible for constructing a calibration curve for your Standard Method 4500-PE samples to determine the concentration of your samples. The Test-N-Tube sample concentrations will be determined by the Hach DR 6000, which has a built-in calibration. Once the concentrations are determined, statistical analysis will be performed.



    This lab experiment will be performed in groups of two or three.

    The procedure for this lab is more self-guided than your other lab experiments and will require some digging on your part. You will write your own procedure for the standard method 4500-PE test. For the Hach Test-N-Tubes method, you will perform the procedure written on the product box, modifying for the ultra-low range method which can also be found at, searching for “Ultra low range total and reactive phosphorus”, and downloading the application note.

    Each group will prepare four phosphorus standards for testing. Upon analysis of the samples, each group will write their resulting phosphorus concentrations on the group spreadsheet. Each group will need to take other groups’ data into account when writing their report.


    • For the TNT method, you will be performing the Reactive Method (Not the Total Phosphorus Method).
    • You will still need to write your procedure in your lab notebook apart from your pre-lab exercise!

    Method 4500-PE

    First, you will generate an external standard calibration curve from potassium phosphate monobasic. With this in mind, you will need to write a procedure for the analysis of phosphorus in water samples. Things that you should think about are the following:

    • What external standard concentrations will be prepared and how much volume?
    • What is an appropriate blank?
    • What glassware will I need?
    • How many replicate analyses should I perform?
    • What are the detection limits for the methods?
    • What wavelength do we need to perform our analysis?

    Hach Test-N-Tube Method

    Before performing this method, you have to ask yourself the following questions:

    • How do you plan on performing your statistical analysis between methods?
    • How many replicate analyses will you perform?

    Data Recording and Reporting

    Once you have collected your absorbance readings for your Method 4500-PE samples, they must be recorded in the class spreadsheet at the instructor’s bench. The included information will be:

    • Theoretical phosphorus concentration of standard
    • Wavelength and absorbance collected at each standard
    • Concentration of samples as determined by Method 4500-PE
    • Concentration of samples as determined by Hach Test-N-Tube

    Data Analysis:

    Using the data that you have collected and that other groups in your lab section have collected, you will need to perform a statistical analysis comparing the two methods.

    Report Instructions:

    One report per group will be turned in. The report for this experiment will be different than normal. In order to prepare you for formal lab reports, you will be turning in a procedure, along with a summary of your statistical analysis, and answering a set of questions, all of which should be typed. Tips for writing your procedure are included below the reference.

    Items to be included in your summary of statistical analysis:

    • Include a spreadsheet with calculated phosphorus concentrations of each sample using the Method 4500-PE and Hach Test-N-Tube method.
    • Include a paragraph describing what statistical method(s) was/were used. This will include sample calculations of the math that was used to perform the statistical analysis.
    • In addition, provide typed answers to the following questions:
      • Which method is considered more precise and which method is considered more accurate? Be sure to explain your answer.
      • Looking at your least precise method, give a reason or reasons why that particular method is not as precise as the other method. (i.e., what do you think your major source(s) of error is/are?) How could you change the procedure to limit this source of error?


    1. Rice, Eugene W., Roger B. Baird, Andrew D. Eaton, Lenore S. Clesceri, eds. “Standard Methods for the Examination of Water and Wastewater”, 22nd Edition (2012) American Public Health Association

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