1. ______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

2. NETCHEM Remote Access Laboratory Guide
Phosphorus analysis in natural waters
In this exercise, you will:
Learn about eutrophication as a process that result from
accumulation of nutrients in lakes or other water bodies.
Learn about determination of phosphorus in natural waters using ascorbic acid method.
Gain experience in practical application of analysis of phosphorus in natural waters.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

3. Background
Eutrophication is a natural process, but can be greatly accelerated by human activities that increase the rate at which nutrients enter the water.
If excessive amounts of these nutrients are added to the water, algae and aquatic plants can grow in large quantities.
Dissolved oxygen concentrations can drop too low for fish to breathe, leading to fish kills.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

4. Background Eutrophication
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

5. Background
Factors Affecting Phosphorus Concentrations: Wastewater and Septic System Effluent. Detergents Fertilizers Animal Waste Development/Paved Surfaces Forest Fires
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

6. Background
Phosphorus in the environment switches between organic and inorganic forms due to the action of bacteria and plants.
Orthophosphate is the only form of phosphorus that plants and micro-organisms can use.
Organic phosphorus is changed to inorganic by bacterial action.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

7. Background Ascorbic Acid Method:
- Orthophosphate reacts, in acid medium, with ammonium molybdate and potassium antimonyltartrate to form
phosphomolybdic acid.
- This is reduced by ascorbic acid to form highly coloured molybdenum blue.
- Measured spectrophotometrically at 880 nm.
- Arsenate, chromium (VI) and nitrite interfere.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

8. Expected concentration
Background
Expected concentration of total phosphorus concentration in different water samples.
Sample
Source
Expected concentration 1.
Polluted pond water
2-3 mg/l 2.
River water
mg/l 3.
Tap water
mg/l

9. Material
For determination of phosphorus, you will need the following material:
Apparatus
Hot plate,
Safety shields,
Safety goggles,
Glass cuvette,
Erlenmeyer flasks, 125-mL
Volumetric glass flask
Micropipettes ( µL)
Spectrophotometer UV VIS connected to computer
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

10. ii. Potasium persulfate (K2S2O8), iii. Phenolphthalein indicator,
REAGENTS
i. Sulfuric acid (H2SO4),
ii. Potasium persulfate (K2S2O8),
iii. Phenolphthalein indicator,
iv. Potasium antimonil tartrate K(SbO)C4H4O *0.5 H2O,
v. Ammonium molibdate (NH4)6Mo7O24*4 H2O,
vi. Ascorbic acid,
vii. Stock phosphate solution.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

11. Firt Step - Persulfate digestion
PROCEDURE
Firt Step - Persulfate digestion
Use 25 mL or a suitable portion of thoroughly mixed sample. Add 1 ml H2SO4 solution and either K2S2O8.
Boil gently on a preheated hot plate for 30 to 40 min or until a final volume of 10 ml is reached.
Cool, dilute to 20 ml with distilled water, add 0,05 mL phenolphthalein indicator solution and neutralize to a faint pink color with NaOH.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

12. Second step - Determination of phosphorus as orthophosphate
To 20 mL of sample and/or standards add 0.5 mL ascorbic acid and mix.
After 5 minutes add 1 mL of ammonium molybdate-antimony potassium tartrate (combined reagent) and mix well.
After minutes, measure the absorbance at 880 nm.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

13. Fill the table after proceeding with the procedure
Fill the table after proceeding with the procedure. Plot calibration curve and read concentration values for the samples. Report results as P, mg/L.
Standards & samples
Absorbance at
880 nm
Corrected absorbance
0 mg/L
0.15 mg/l
0.3 mg/l
0.6 mg/l
0.9 mg/l
Sample 1
Sample 2
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

14. REPORT
The aim of the investigation The results that you have produced The phosphorus concentration of the samples and what this means in terms of water quality.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

15. ______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

16. Remote Access Connection Instructions
What makes these labs different and unique from other classroom experiments is that we have incorporated a section in each activity to remotely characterize your samples from your classroom.
Request a remote lab session specifying information such as: the day, the time, and the instrument you are interested in using by visiting our web site:
You will see the list of partners with the instruments provided to chose from.
You will be contacted by a Remote Access staff member to set up a test run to ensure you are set up properly and have the required infrastructure.
Send samples or verify the in-house sample you would like us to prepare and load for characterization.
Send your samples to the Remote Access center that you chose on your request.
There are two communications soft-ware packages, that will allow us to communicate instructions and answer questions during the session.
- TeamViewer: You can obtain a free download at:
- Skype
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

17. Remote Access Connection Instructions
You will need:
Computer with administrator access to install plug-ins and software
An internet connection
Speakers
Microphone
Projector connected to the same computer
Web browser (Firefox preferred)
During the test run you can refer to this guide to perform the following steps, but it’s very important that you only proceed with these steps during your scheduled times. You may interfere with other remote sessions and potentially damage equipment if you log in at other times.
Open and logon to your Zoom/Team-viewer account. You will be given the access code to enter at the time of your test and then again during the remote session.
If you are using the Zoom software, Remote Access staff will give you the access code.
If you are using the Team-viewer software, Remote Access staff will give you the ID & password.
You should soon see the Remote Access desktop and at this point you can interact with the icons on the screen as if it were your desktop.
Switch to full screen mode by selecting the maximize screen option in the top right corner of the screen.
Upon completion of the session, move your mouse to the top right corner of the screen, and click on the X to disconnect the remote session. It will ask if you want to end the remote session. Click Yes.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

18. Author, Editor and Referee References
This remote access laboratory was created thanks to work done primarily at University of Niš. Contributors to this material were: Sonila Duka Date of creation: September, Refereeing of this material was done by: _____________________ Editing into NETCHEM Format and onto NETCHEM platform was completed by: ______________
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

19. References and Supplemental Material
The NETCHEM platform was established at the University of Nis in through the Erasmus Programme. Please contact a NETCHEM representatives at your institution or visit our website for an expanded contact list. The work included had been led by the NETCHEM staff at your institution.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.