Exp. 1.  Determination of nitrates in drinking water

            In a flow injection analysis the solution is pumped to the detector by a peristaltic pump. Several tubes can be handled simultaneously on the same pump. This feature permits to avoid the preliminary preparation of solutions of analyte and reagent needed for a specific determination, and so high analysis rate (about 100 samples per hour) can be reached.

            The sample and all reagents are introduced from separate channels through the same pump or through different pumps. Complete mixing is achieved before the combined solution reacts the detector. Constant flow through each channel is of paramount importance for stable proportioning of sample to reagents.

            Different modes of mixing the solutions of analyte and reagent are available. One of them, when two pumps are used, is shown on Fig.7-1.

Note: Peristaltic pump - device in which fluid is squeezed through plastic tubing by rollers.

The flow rate is controlled by the speed of the motor (greater than 30 rpm) and the inner diameter of the microbore tubing (0.2 - 3 mm).



Fig.7-1  Scheme of a flow system with two peristaltic pumps.

A timer controls the time of operation of pump A. Solutions (1) and (2) are injected via pump A, mixed in chamber (4) and flown to the detector. At the end of a predetermined injection time pump A stops and pump B starts to transfer solution (3) as long as needed up to next injection.


Preparation of the flow system

·        Do not operate the system without the assistance of the instructor.

·        Insert the tubes into the solutions before turning on the pumps. If this is not done, the air will penetrate into the tubes, distorting the smoothness of the flow.

·        Turn the pumps off before changing solutions.


1.  Set the plastic tubing on the peristaltic pumps as shown on Fig.7-1. The pressure on plates (5) and (6) is adjusted with the respective screws by the instructor.

2.  Ensure that the polarographic detector (Fig.7-2) is connected to the flow system.

3.  Test for proper operation of the flow system (use distilled water):

*   Check that there is free flow through each one of the three tubes. The flow may be obstructed due to blocking of the thin tubes.

*   Check that solution from pump A does not penetrate in tube (3). To perform the test, take out tube (3) from beaker (3) while pump A is operating. No drops should be formed at that end of the tube. Explain when such leakage is possible.

Repeat the same test with pump B and tubes (1) and (2).


Preparation of the polarographic detector

            1.  Check that the container for drainage of mercury and waste is in place.

            2.  Raise the level of the mercury reservoir and check that the mercury is flowing.

            3.  Nitrogen gas: Make sure that the fine needle valve, positioned on the polarographic stand, is closed. Open the main nitrogen valve and adjust the local pressure to about 0.4 bar. Turn on the peristaltic pump (use distilled water with a flow rate about 0.5 - 1 ml/min). In absence of nitrogen air bubbles are interlaced along the liquid flown through the degassing tube.

Note: BE AWARE! Mercury vapors are poisonous!

Notify the instructor in the event of mercury spill. Mercury should be cleaned up immediately. Do not throw it down the drain.



Fig.7-2  High-performance polarographic cell designed by Ch. Yarnitzky1.

The solution is forced in the capillary tube by a peristaltic pump. In absence of nitrogen air bubbles are interlaced along the degassing tube. When nitrogen gas introduced, the solution is pushed to the walls of the capillary and forms a thin film. The intimate solution-gas contact thus formed enables an efficient purging of oxygen. The oxygen-free solution slides from the end of degassing tube to the mercury capillary and is trapped by surface tension forces in the gap between the end of the mercury capillary and the ceramic separator. This gap comprises the entire electrochemical cell: the mercury electrode from above, the counter electrode from below and the reference Ag/AgCl/1 M KCl electrode at the end of the lower tube. The volume of the cavity is about 0.1 cm3.


            Carefully open the fine needle valve (positioned on the polarographic stand) and observe the flow of the liquid across the tube. The nitrogen gas pushes the liquid against the walls of the tube, and an invisible thin liquid film is formed. Increase the nitrogen flow up to the stage, at which the thin film is stable, as marked by a seemingly motionless state along the tube. Observe the flow of the liquid along the mercury capillary and in the cell cavity. It is smooth and steady. These are the right operating conditions. A further increase of the nitrogen flow distorts the smoothness of the flow in the cell cavity.


Chemicals       1.  0.1 M KCl

                        2.  0.2 mM UO2 (CH3COO)2, 0.02 M HCl, 0.2 M KCl

                        3.  5 mM KNO3



            1.  Prepare six standard solutions of KNO3 in the concentration range of 0.05 mM - 0.3 mM (0.05, 0.10, 0.15, 0.20, 0.25, 0.30 mM). You are provided with six 50 ml volumetric flasks and six plastic beakers, each marked with the respective concentration. The beakers are used for aspirating the standard solutions and the samples.

            2.  The principle of the experiment is demonstrated by recording DC polarograms in each of the following three solutions:

I. Nitrate and KCl (as supporting electrolyte).

II. Uranyl acetate and KCl (as supporting electrolyte).

III. Nitrate with uranyl acetate (as catalyzer) and KCl (as supporting electrolyte).

What do you expect to obtain in each one of the three cases in respect to limiting currents and E1/2?

            The arrangement of the solutions for this experiment is shown in Fig.7-3. Only pump A is used, and the timer is set to a long enough time to permit the recording of a whole polarogram. The advised order for recording the polarograms is III, II, I. Record the three polarograms at the same sensitivity. What are your conclusions from the above polarograms?

            Determine the value of the potential corresponding to the limiting current of the catalyzed nitrate wave. At this potential the current-time curves of the samples and the standard solutions, flown through the detector, are recorded.



Fig.7-3  Arrangement of solutions for demonstrating the principle of the nitrate determination.

Only pump A is used. Sets I, II and III - different experimental configurations of solutions.

UO22+ - solution of uranyl acetate with HCl and KCl;

NO3- - solution of nitrate ion; KCl - supporting electrolyte.


            3.  The analytical determination of nitrates. Set the potential to the value corresponding to the limiting current of the catalyzed nitrate wave.

            The arrangement of the solutions is given in Fig.7-4. Tube (1) is dipped in the same uranyl acetate solution throughout the entire analysis. The samples and the standard solutions are aspirated from tube (2). For building the calibration curve inject each of the standard solutions and record the current signal. The injection time is controlled by pump A (20 sec). At the end of this time pump B starts to operate as long as needed for rinsing the detector. Repeat several times the injection of the analyte, record the current and collect for each solution at least 3 - 4 signals with good reproducibility. Plot the calibration curve.

            Repeat the procedure with the drinking water samples. If the signal is higher than that of the most concentrated standard, dilute the analyte (why?). Calculate the concentration of nitrates in the sample.



Fig.7-4  Arrangement of solutions for the routine analysis of nitrates. Both pumps are used. Pump A injects the sample or standard solution of nitrates and the make up solution of uranyl acetate for a predetermined time. Pump B transfers the supporting electrolyte as long as needed.


            4. At the end of the experimental session:

·        Rinse the system (tubing and cell cavity) by passing distilled water for about two minutes.

·        Release the tension applied on the peristaltic pump tubes.

·        Lower the mercury reservoir.

·        Nitrogen gas: Close the fine needle valve located on the polarographic stand and turn off the main nitrogen valve.



1.  M.Noufi, Ch.Yarnitzky and M.Ariel, Anal. Chim. Acta, 1990, 228, 117.



Exp. 2.  Determination of fluoride in drinking water with ion-selective electrode

Fluoride ion-selective electrode

            Electrodes employing ion-selective membranes can display high selectivity toward certain ions. No electrode, however, responds exclusively to one kind of ion. The sensitivity of an electrode to a competing ion, N, is characterized by the selectivity coefficient kM,N, defined at equal concentrations of M and N.

kM,N = response to M / response to N

            The response of an electrode specific to an ion M in presence of a competing ion N is:


where CM and CN are the concentrations of ions M and N, z is the charge of ion. The value s is in the range 54 - 60 mV/decade at 25°C and at constant ionic strength.

            The fluoride ion-selective electrode is a solid-state electrode employing a crystal of LaF3, doped with Eu(II). The doping of the crystal enables the fluoride ion to migrate throughout the membrane.

            The response of the fluoride electrode in the presence of a competing anion N is given by equation:


where CF and CN are the concentrations of fluoride and of the competing species N.

            The fluoride electrode yields a nearly Nernstian response over a concentration range of 1 - 10-6 M F- at a constant ionic strength and at a constant temperature.

            The electrode operates with relatively few interferences. Interfering species are:

·        hydronium ions, which below pH 5 form HF and HF2- complexes;

·        iron(III), silicon, aluminum and other polyvalent cations, which also form complexes with fluoride ion;

·        hydroxide ions, which compete with the fluorides with a selectivity coefficient kF,OH = 0.1.

            The degree of interference caused by the competing ion OH-  is determined by the term kF,OH·COH / CF:

            for kF,OH·COH / CF << 1, the interference is negligible;

            for kF,OH·COH / CF >>1, the effect of the competing ion is predominant.

            The effect of interference at low-level concentrations of fluoride at different pH values is given in table below. In order to ensure negligible effect of the OH- competing ion, the pH should be lower than 7.


Degree of interference of OH-, expressed as kF,OH·COH / CF




CF = 10-5 M

CF = 10-6 M

















            The time response of the electrode (the time required to reach 99% of the stable potential reading) varies from several seconds in concentrated solutions to several minutes near the limit of detection.

            Measurements with a fluoride electrode are performed in presence of TISAB (Total Ionic Strength Adjusting Buffer). TISAB should provide constant ionic strength of the solution, adjusting the pH and complex the interfering species.

            The type of TISAB depends on the composition of the sample. Drinking water contains less than 1.5 ppm F- (7.5·10-5 M) and negligible amounts of interfering species. A suitable composition of TISAB for drinking water is 0.01 M acetate buffer pH 5 - 5.5 in 1 M NaCl and 4 g/l CDTA. The TISAB is added to the sample in volume ratio 1:1. The acetate buffer keeps the pH at the optimal value, at which the formation of HF and HF2- is negligible, and the response of the electrode to hydroxides is virtually zero. NaCl is added to maintain constant ionic strength, independent of the composition of the sample. For the specific configuration of the reference electrode (AgCl-coated silver wire), used in this experiment, the constant concentration of chlorides is needed to ensure the constant value of the electrode potential. CDTA, chelating agent similar to EDTA, complexes polyvalent ions, which otherwise complex with F-.


The effect of Fe(III)-F complexes on the determination of fluorides in drinking water

            Fe(III) forms three complexes1 with F-: FeF2+, FeF2+, FeF3.

Fe3+ + F- = FeF2+

Kfl = 105

FeF2+  + F- = FeF2+

Kf2 = 103.9

FeF2+ + F- = FeF3

Kf3 = 103

Only uncomplexed, free fluoride is measured by the fluoride selective electrode. In order to illustrate the effect of complexation, the concentration of the different complexes as function of the total concentration of F- is shown in Fig.7-5(a). The calculations are performed for 2·10-5 M Fe(III), concentration considered as the highest permissible in drinking water. The concentration of uncomplexed iron decreases with increasing concentration of fluoride. Around [F-]total = 5·10-5 M the predominant complex is FeF2+. Beyond [F-]total = 1·10-4 M the predominant species is FeF2+.

            The data from Fig.7-5(a) are also presented as , the fraction of free fluoride to the total concentration of all fluoride species, as a function of the total concentration of fluoride (Fig.7-5(b)). The value of  is considerably lower than unity in the typical concentration range of fluoride in drinking water. The lower the total concentration of Fe3+, the higher .

            For the determination of the total F- concentration, the chelating agent CDTA (forming a complex with Fe(III) with a high formation constant) is added. This allows the determination of F- concentration, independent of the level or nature of dissolved minerals. In a 1 ppm fluoride sample the TISAB complexes about 5 ppm iron or aluminum.



Fig.7-5  Concentration of the Fe(III)-F complexes (a) and  (b) as function of the total concentration of F-.The calculations are performed for [Fe3+ ]total = 2·10-5 M. The dashed area represents the concentration range of fluorides, typical to drinking water.


Experimental setup

            The experiment can be performed in a batch mode or in a flow system. The flow system shown in Fig.7-6 is highly efficient, saving reagents and time of operator.



Fig.7-6  A flow system for determination of fluorides.


Chemicals       1.  10-2 M NaF

                        2.  10-3 M NaF

                        3.  TISAB containing 0.01 M acetate buffer pH 5-5.5, 1 M NaCl and 4 g/l

                             CDTA (1,2-diaminocyclohexane N,N,N',N'-tetraacetic acid)


Electrodes:     Fluoride ion-selective electrode and Ag/AgCl reference electrode (silver wire coated with AgCl)



            Check the connection of the electrodes to the pH-meter. Set the meter to voltage reading.

            Calibration curve for the fluoride standards is constructed in the range 10-2 - 5·10-6 M. Prepare standard solutions of 10-4 M, 3·10-5 M, 10-5 M and 5·10-6 M NaF in 50 ml volumetric flasks. Transfer each of the fluoride solutions (standards and analytes) and the TISAB via the flow system (cf. Fig.7-5) at a flow rate of 5 to 10 ml/min. It is recommended to perform the measurements in decreasing order of concentrations. Record stable voltage reading of the potential between the electrodes for each fluoride solution. One minute or more should be allowed to reach steady state readings, depending on the concentration of F-. The lower the concentration, the longer the time.

            At the end of the working session rinse the system with distilled water.

            Construct a calibration curve (potential vs concentration of fluoride). Mark the X-axis in mole/l and ppm. Report the concentration of the fluorides in drinking water samples in mole/l and ppm.



1.  “Stability constants of metal-ion complexes”, L. G. Sillen and A. E. Martell, Special Publication #17, London: The Chemical Society, Burlington House, 1964.


Recommended Literature

Exp. 1. Determination of nitrates in drinking water

1.         H. H. Willard, L. L. Merritt, J. A. Dean and F. A. Settle, Instrumental Methods of Analysis.

2.         D. A. Skoog and D. M. West, Principles of Instrumental Analysis.

3.         D. A. Skoog and J. J. Leary, Instrumental Analysis.

4.         I. M. Kolthoff, W. E. Harris and G. Matsuyama, J. Am. Chem. Soc., 66 (1944), 1782.

5.         H. Hemmi, K. Hasebe, K. Ohzeki and T. Kambara, Talanta, 31 (1984), 319.

6.         M. Noufi, Ch. Yarnitzky and M. Ariel, Anal. Chim. Acta, 234 (1990), 475.

Exp. 2. Determination of fluorides in drinking water

1.         D. C. Harris, Quantitative Chemical Analysis.




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