Reagents:

1. Carefully open an oral rehydration sachet and empty the contents into a clean 250 ml beaker. Add about 150 ml distilled water and gently swirl the contents until dissolved.
2. Pour the solution into a 200 ml volumetric flask and rinse out the beaker with small amounts of distilled water, adding the washings to the flask. Finally, make up the flask to exactly 200 ml and mix thoroughly.
3. Make a 1/50 dilution of the redissolved sachet solution by accurately pipetting 2 ml of the solution into a 100 ml volumetric flask and making up to 100 ml with distilled water. Retain the undiluted solution for Experiment B.

Instructions for use of the flame photometer:

4. Ensure that the photometer drain is leading into a sink and that the instrument is connected to gas, air and electricity supplies. Ensure the mains supply gas tap is off.
5. Turn the "Sensitivity" and instrument "Gas" controls control fully counterclockwise (towards you).
6. Insert the sodium optical filter.
7. Switch on the instrument and unclamp the galvanometer by turning counterclockwise.
8. Open the mica window, turn on the mains gas supply, light the gas and close the window.

(CAUTION: DO NOT LEAN OVER THE INTRUMENT OR YOU WILL SET YOUR HAIR ALIGHT)

9. Turn on the air supply control and adjust the air pressure to 10 lb/in². Leave for 1-2 minutes to stabilise.
10. Place a beaker of distilled water into position at the left hand side of the instrument and insert the narrow draw tube into it to allow water to pass through the photometer. (NOTE: once set up, the photometer must have water running through it at all times when a salt solution is not being measured. The rate of uptake is fast, so make sure there is always enough water in the beaker).
11. Adjust the gas control to give a flame with a large central blue cone then, with water passing through the instrument, slowly close the gas control until ten separate blue cones just form.
12. Set the galvanometer to zero using the "Set zero" control.
13. Replace the distilled water with the 5 mM NaCl standard and adjust the "Sensitivity" control till the galvanometer reads 100.
14. Quickly but carefully, replace the 5 mM NaCl standard with standards of decreasing concentration from 4 mM to 0.25 mM and note the readings in the Table below.
15. Run water through the instrument again for 1-2 min then place the draw tube into a beaker containing the 1 in 50 diluted rehydration sachet solution and note the galvanometer reading.
16. Run water through the instrument again and replace the sodium with the potassium filter.
17. Repeat the above procedure with the KCl standards, setting to 100 with 2.0 mM KCl, then reading the others in reverse order. Then read the 1 in 50 diluted rehydration sachet solution.
18. Finally, run water through the instrument until the flame appears free of colour again.
19. When the instrument is no longer required, switch off in the following sequence:

Reagents:

Technical note : In order to measure the change in potential difference across the ion-selective membrane as the ionic concentration changes, it is necessary to include in the circuit a reference electrode which acts as a half-cell from which to measure the relative deviations.

1. Ensure that the multimeter (yellow box) and electrode amplifier (grey box) are connected properly with the black cable in the lowest socket (COM) and the red cable in the middle socket (VWmA) and that the silver BNC connector from the electrode head is plugged into the back of the amplifier.
2. With the electrodes immersed to a depth of about 3-4 cm in NaCl storage solution, switch on the multimeter to the 2000 mV position (4 clicks counterclockwise) and the amplifier to position I (down). Wait until the reading has stabilised (up to 5 min.) then remove the electrodes from the storage solution, wipe the bodies (but NOT the bottoms) with a tissue, transfer to a beaker of distilled water and rinse with swirling for about 30 sec.
3. Remove the electrodes from the water, wipe as before, then transfer to the 10 mM NaCl standard. Swirl the solution for 15 sec to help the ISE membrane to equilibrate with the solution, then leave undisturbed for a further minute. Note the multimeter reading (this is millivolts, mV) (the meter may fluctuate if you wave your hand over it so ensure that the environment is undisturbed when taking the reading).
4. Remove, wipe and transfer the electrodes to the 20 mM standard and measure as before, waiting one minute before taking the reading.
5. Repeat the procedure for the 40 mM and 80 mM standards.

[Na+] (mM)	10	20	40	80
Log10 [Na+]
Meter reading (mV)

 

6. Finally, take a reading in the same way from the undiluted redissolved sachet from Experiment A.
7. Transfer the electrodes back to distilled water and, if nobody else is going to use the system, switch off the multimeter and amplifier (position O).
8. Plot mV (y-axis) against log₁₀ [Na⁺] (x-axis) and from this graph determine the apparent [Na⁺] in the redissolved sachet solution.

2.5 Results:

1. From the information provided on the sachet packaging, calculate the expected concentrations of Na⁺ and K⁺ ions in the solution you made.

[Na+] (mM)	[K+] (mM)

2. How do your own values for Na⁺ and K⁺ determined by flame photometry and for Na⁺ determined with the ISE compare to the expected values?
3. The class data for sodium determination only by both methods will be collected and tabulated on the blackboard in the class. Collect these data in the table below before you leave (12 sets maximum). Using a calculator, calculate the mean and standard deviation (S.D.) for the two data sets and compare these for accuracy and precision. What do you conclude?

	1	2	3	4	5	6	7	8	9	10	11	12
[Na+](flame)
[Na+] (ISE)

 

Sodium concentration (mean ± S.D, n = ) by flame photometry = ……………… (mM)

 

Sodium concentration (mean ± S.D, n = ) by ISE = ……………… (mM)

Accuracy: Assuming the pipette you use has been accurately calibrated (unfortunately not guaranteed), the closeness of the values you measure to the expected values will show how accurate your technique is. Any significant inaccuracy with a low standard deviation would indicate a consistent error in your technique, e.g. always (wrongly) pushing the pipette button fully down before drawing liquid into the pipette (or an improperly calibrated pipette).

Precision: Even though your mean value may appear accurate, if your individual values vary all over the place (high standard deviation), then your technique obviously suffers from a variety of errors (i.e. general carelessness!).

       Procedure:

1. Read the instructions on the use of pipettes in Appendix 2and watch and listen carefully to the demonstration that will be given.
2. Practice pipetting water out of and into a beaker, watching all the time what you are doing and checking the water in the tip for air spaces, drops of water left at the top of the tip after expelling etc. Show the demonstrator your technique before proceeding to the next step.
3. Place a plastic weighing boat on the balance and tare the balance so that it reads zero (this will be demonstrated).
4. Using a "blue button" pipette, pipette 1.0 ml (1000 µl) distilled water into the weighing boat and record the weight. If the pipette has been accurately calibrated, what should the reading be?
5. Pipette another 1.0 ml water into the boat and record the weight again.
6. Repeat this until you have taken 10 readings (10 ml total pipetted) then calculate the weight of each individual 1.0 ml sample.
7. Calculate the mean and standard deviation of your 10 measurements.

      Mean weight  = .................. g    Standard deviation =  ± .................. g

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