Determination of Sulfate Page 1 of 2 Reference: Reference : Methods for the Examination of Water Sulphate in Waters, Effluents and Solids 1988(second Edn) 0117522406 and EPA Method 375.4 Sulfate. Principle Sulphate ion is precipitated in a strongly acid medium with Barium Chloride. The resulting turbidity is measured photometrically at 405nm and compared with appropriate calibration standard solutions. Limit of Detection 0.05mg/l Solutions Precipitating Solution Dissolve 10.0g Barium Chloride [BaCl2], 10g Sodium Chloride [NaCl] and 0.25g Gelatin in 300ml of distilled water (requires prolonged stirring to dissolve). Carefully add 5ml conc. Hydrochloric acid and dilute to 1000ml with distilled water. This solution is stable for 1 month. Stock Sulphate Standard 10 mM Dissolve 1.421 g anhydrous Sodium Sulphate Na2SO4 in distilled water and dilute to 1000ml. This solution is stable for 6 months. Standard Curve Always run a standard curve alongside with your samples. For the standard curve, make dilutions from the Sulphate Standard Solutions to the following concentrations: 0mM, 2 mM, 4 mM, 6mM, 8 mM, 10 mM. Measurements should be done in triplicates Measurements Using the Spectramax • Take a 96 wells plate with flat bottom. • Add 125 µl water to the wells. • Add 25 µl of your samples, including the standards. • Measure the optical density at 420nm. This is your blank reading. • Add 40 µl of Precipitating Solution and mix well. • Wait 5 minutes to allow the reaction to develop. • Measure the OD again at 420nm. • OD values over 1.0 are not reliable. If your values are >1.0 dilute your original samples and restart protocol. Determination_of_sulfate.doc November 4, 2013 Determination of Sulfate Page 2 of 2 • Subtract the blank reading OD from the measurements. You now have the final OD values. • Put the standard curve values in a graph and add a trendline. The trendline should be linear and go through the origin of the graph (0,0). The formula obtained from the trendline can now be used to calculate the concentrations in your samples. Example: You obtain the formula y=0,01x. This translates as OD420=0,01*concentration. Invert the formula to obtain: Concentration (μM) = OD420 / 0,01 *or* Concentration (μM) = 100*OD420. • You can now calculate the concentrations of your samples. IMPORTANT NOTE FOR SULFATE REDUCING EXPERIMENTS: Keep in mind that sulphide reacts with oxygen and becomes again SO4, then be sure that SO4 is not overestimated! Determination_of_sulfate.doc November 4, 2013 .