DR/4000 PROCEDURE SELENIUM. 4. Measure 100 ml of sample into a second 500-mL erlenmeyer flask (label the flask sample ).

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1 Method 8194 DR/4000 PROCEDURE Diaminobenzidine Method* (0 to mg/l) Scope and Application: For water and wastewater; distillation is required for determining total selenium. See the Distillation procedure following the Colorimetric procedure. The estimated detection limit for program number 3300 is mg/l Se. * Adapted from Standard Methods for the Examination of Water and Wastewater. HACH PROGRAM HACH PROGRAM: 3300 Selenium ENTER 1. Press the soft key under HACH PROGRAM. Select the stored program for selenium (Se) by pressing 3300 with the numeric keys. Press: ENTER Note: If samples cannot be analyzed immediately, see Sample Collection, Storage and Preservation following these steps. Adjust the ph of preserved samples before analysis. 2. The display will show: HACH PROGRAM: 3300 Selenium The wavelength (λ), 420 nm, is automatically selected. Note: To determine total selenium, perform a distillation. See Distillation, after this procedure. Use this distillate in Step 4. See Summary of Method following these steps. 3. Measure 100 ml of deionized water into a 500-mL erlenmeyer flask (label the flask blank ). 4. Measure 100 ml of sample into a second 500-mL erlenmeyer flask (label the flask sample ). Note: For proof of accuracy, use a 0.5 mg/l selenium standard solution (see Accuracy Check) in place of the sample. Note: The Flow Cell and Sipper Modules cannot be used with this procedure. Selenium_None_Other_DAB_Eng_4000.fm Page 1 of 10

2 , continued 5. Add a 0.2-g scoop of TitraVer Hardness Reagent to each flask. Swirl to mix. 6. Add 0.05-g scoop of diaminobenzidine tetrahydrochloride to each flask. Swirl to mix. 7. Add 5.0 ml of Buffer Solution, sulfate type, ph 2.0, to each flask. Swirl to mix. Note: If the sample has been distilled as described under Distillation, omit the Buffer Solution. Adjust the ph of the sample distillate to 2.7 (±0.2) using 5 N Sodium Hydroxide Standard Solution. Adjust the deionized water blank to the same ph value using 5.25 N Sulfuric Acid Standard Solution. 8. Heat each flask on a hot plate or over a flame, bringing the contents to a gentle boil. START TIMER 9. Press the soft key under START TIMER. A 5-minute reaction period will begin. Continue to boil the contents gently during this time period. Note: A yellow color will develop if selenium is present. 10. When the timer beeps, remove both flasks. Cool to room temperature using a water bath. Note: Do not boil more than one minute after the timer beeps. 11. Transfer the contents of each flask to separate 250-mL separatory funnels. Label the funnels blank and sample. 12. Add 2.0 ml of 12 N Potassium Hydroxide Standard Solution to each funnel using a calibrated 1.0-mL plastic dropper. Stopper. Shake each funnel to mix. Page 2 of 10 Selenium_None_Other_DAB_Eng_4000.fm

3 , continued START TIMER START TIMER START TIMER 13. Add 30 ml of toluene to each funnel. Stopper. Swirl and invert each funnel, then open the stopcock to vent the funnel. Close the stopcock. Repeat twice with each funnel. 14. Press the soft key under START TIMER. A 30-second period will begin. Shake the funnel containing the blank vigorously for the 30-second period. 15. Press the soft key under START TIMER. A 30-second period will begin. Shake the funnel containing the sample vigorously for the 30-second period. 16. Press the soft key under START TIMER. A 4-minute reaction period will begin. Note: Use toluene only with adequate ventilation. ZERO 17. When the timer beeps, drain the lower water layer from each funnel and discard. 18. Insert a cotton plug into the delivery tube of each separatory funnel. Slowly drain the toluene into respective sample cells labeled blank and sample. Stopper the sample cells. Note: Do not wait more than 5 minutes after the timer beeps before completing steps 19 through 21. Note: Filtering the toluene through dry absorbent cotton will remove any water or suspended particles. 19. Place the blank into the cell holder. Close the light shield. 20. Press the soft key under ZERO. The display will show: mg/l Se Note: For alternate concentration units press the soft key under OPTIONS. Then press the soft key under UNITS to scroll through the available options. Press ENTER to return to the read screen. Note: The developed color is stable but should be measured as soon as possible. Note: Glass stoppered sample cells can be used to prevent evaporation. Selenium_None_Other_DAB_Eng_4000.fm Page 3 of 10

4 , continued 21. Place the prepared sample in the cell holder. Close the light shield. Results in mg/l selenium (or chosen units) will be displayed. Note: Acetone is a suitable solvent for removing toluene from glassware. Interferences Table 1 Interfering Substances and Suggested Treatments Interfering Substance Ferric iron Manganese Strong oxidizing agents (i.e., iodine, bromine or chlorine) Interference Level and Treatment Up to 2.5 mg/l. Distill sample to eliminate interference. Will not interfere Can react with the indicator to give low results. Distill sample to eliminate interference. There are no positive inorganic interferences with this method. Sample Collection, Storage and Preservation Collect samples in clean glass or plastic containers. Adjust the ph to 2 or less with nitric acid (about 1.5 ml per liter). Preserved samples can be stored for up to six months at room temperature. Correct the test result for volume additions; see Section Correcting for Volume Additions. Distillation Always perform this procedure under a fume hood! This distillation involves the use of a strong acid and oxidizer at high temperatures. To avoid personal injury, observe all laboratory safety precautions when operating the distilling apparatus. a. Measure 500 ml of sample into a 1000-mL beaker. b. Add 1 ml of Methyl Orange Indicator Solution. Stir with a glass rod. c. Using a dropper, add 0.1 N Hydrochloric Acid Standard Solution dropwise until the solution becomes pink. Then add an additional 2 ml. d. Pipet 5.0 ml Calcium Chloride Solution. Mix well. Page 4 of 10 Selenium_None_Other_DAB_Eng_4000.fm

5 , continued e. Using a dropper, add 1 g/l Potassium Permanganate Standard Solution drop-wise until the solution is purple. f. Place the beaker on a hot plate. Evaporate the solution to approximately 250 ml. Periodically add 1 g/l Potassium Permanganate Solution to keep the solution purple. Note: Any precipitate formed at this step is manganese dioxide and may be ignored. g. Cool the solution. While cooling, set up the distillation apparatus for the general purpose distillation as shown in the Hach Distillation Manual. h. Pour the treated sample solution into the distillation flask. Add a stirring bar to the flask. i. Pipet 5.0 ml of 0.1 N Sodium Hydroxide Standard Solution into the flask. Turn the stirrer power switch to ON. Set the stir control to 5. j. Turn on the water and adjust so a constant flow is maintained through the condenser. Set the heat control to 10. k. When only a few milliliters are left in the distillation flask, turn the power switch off. The distillate in the erlenmeyer flask may be discarded. l. Perform this step under a hood. When the flask has cooled, add 50 ml of 19.2 N Sulfuric Acid Standard Solution to the flask. Add the contents of one Potassium Bromide Powder Pillow to the flask. m. Fill a 250-mL beaker to the 75-mL mark with deionized water. Place it under the drip tube. Elevate the beaker with a laboratory jack so the tube extends below the level of the water. n. Add 1.0 ml of 30% Hydrogen Peroxide Solution to the flask. Turn the stir control to 5 and the heat control to 10. Cap the distillation flask. o. Heat the distillation flask until the yellow color is gone from the complete distillation apparatus, including the J-tube and condenser. Remove the beaker from under the drip tube. p. Turn off the heater switch. When the J-tube and condenser has cooled, rinse them with deionized water. Add the washings to the 250-mL beaker. Total volume in the beaker should be approximately 100 ml. q. Add the Phenol Solution drop-wise to the distilled sample to discharge the bromine color (a white precipitate of tribromophenol will form.) r. Allow the precipitate to settle. Using a dropper, collect about 5 ml of the clear, colorless distillate and transfer to a test tube. s. Test the solution for completeness of precipitation by adding 2 drops of Phenol Solution. If the solution becomes cloudy or white precipitate forms, residual bromine is still present (proceed to next step). If no cloudiness occurs, the sample is ready for analysis. t. Transfer the 5-mL aliquot back to the beaker and continue to add Phenol Solution until no turbidity is formed in subsequent 5-mL aliquots. u. Transfer the sample into a 500-mL volumetric flask. Rinse the beaker with deionized water and add to the flask. v. Dilute to volume with deionized water, stopper and mix well. The distillate is now ready for analysis. Selenium_None_Other_DAB_Eng_4000.fm Page 5 of 10

6 , continued Accuracy Check Method Performance Standard Additions Method a. Leave the unspiked sample in the sample compartment. Verify that the units displayed are in mg/l. Select standard additions mode by pressing the soft keys under OPTIONS, (MORE) and then STD ADD. b. Press ENTER to accept the default sample volume (ml), c. Press ENTER to accept the default standard concentration (mg/l), d. Press the soft key under ENTRY DONE. e. Prepare a 100-mg/L selenium standard solution by pipetting 10.0 ml of Selenium Standard Solution, 1000-mg/L, into a 100-mL volumetric flask. Dilute to volume with demineralized water and mix thoroughly. f. Use the TenSette Pipet to add 0.1, 0.2 ml and 0.3 ml of standard, respectively to three 100-mL samples and mix each thoroughly. g. Analyze each standard addition sample as described above. Accept the standard additions readings by pressing the soft key under READ each time. Each addition should reflect approximately 100% recovery. h. After completing the sequence, the display will show the extrapolated concentration value and the best-fit line through the standard additions data points, accounting for matrix interferences. i. See Section Standard Additions for more information. Standard Solution Method Prepare a 100-mg/L selenium standard solution as described in Standard Additions Method, step e, above. Prepare a 0.5-mg/L selenium standard solution by pipetting 1.00 ml of the selenium standard solution, 100-mg/L, into a 200-mL volumetric flask. Dilute to volume with deionized water. Transfer 100 ml of the standard into a 500-mL erlenmeyer flask. Perform the test as described above. Precision Standard: mg/l Se Program For more information on determining precision data and method detection limits, refer to Section 1.5. Estimated Detection Limit 95% Confidence Limits mg/l Se Program EDL mg/l Se For more information on derivation and use of Hach s estimated detection limit, see Section To determine a method detection limit (MDL) as defined by the 40 CFR part 136, appendix B, see Section Page 6 of 10 Selenium_None_Other_DAB_Eng_4000.fm

7 , continued Sensitivity Program Number: 3300 Portion of Curve Abs Concentration Entire Range mg/l See Section Sensitivity Explained for more information. Calibration Standard Preparation To perform a selenium calibration using the diaminobenzidine method, first prepare a 10 mg/l working standard by diluting 10 ml of 1000-mg/L Selenium Standard Solution (Cat. No ) to 1000 ml using Class A glassware. Prepare calibration standards containing 0.050, 0.080, 0.500, and mg/l selenium as follows: a. Into four different 1000 ml Class A volumetric flasks, pipet 5.00, 8.00, 50.00, and ml of the 10-mg/L selenium working standard using Class A glassware. b. Dilute to the mark with deionized water. Mix thoroughly. c. Using the diaminobenzidine method and the calibration procedure described in the User-Entered Programs section of the DR/4000 Spectrophotometer Instrument Manual, generate a calibration curve from the standards prepared above. Summary of Method An EDTA masking agent is added to the sample to remove interferences such as iron prior to the test. The addition of a sulfate buffer adjusts the sample to the optimum ph of 1 to 2. Under these conditions, diaminobenzidine reacts with all selenium present as selenite (Se 4+ ) to give a yellow-colored piazselenol complex which is extracted and the color intensity measured colorimetrically. Selenium present as Se 2 and Se 6+ is not detected unless the sample is distilled. Safety Good safety habits and laboratory techniques should be used throughout the procedure. Consult the Material Safety Data Sheet for information specific to the reagents used. For additional information, refer to Section 1. Pollution Prevention and Waste Management Toluene (F005) solutions are regulated as hazardous waste by the Federal RCRA. Do not pour these materials down the drain. Water saturated with toluene, toluene solutions, and the cotton plug used in the delivery tube of the separatory funnel should be collected for disposal with laboratory solvent wastes. See Section 1 for more information on proper disposal of these materials. Selenium_None_Other_DAB_Eng_4000.fm Page 7 of 10

8 , continued REQUIRED REAGENTS AND STANDARDS Selenium Reagent Set (100 tests) Includes: (1) , (1) , (2) , (1) , (1) Quantity Required Description per test Unit Cat. No. Buffer Solution, sulfate type, ph ml ml Diaminobenzidine, tetrahydrochloride g... 5 g Potassium Hydroxide Standard Solution, 12 N... 4 ml ml TitraVer Hardness Reagent, ACS g g Toluene, ACS ml... 4 liters Water, deionized ml... 4 liters REQUIRED EQUIPMENT AND SUPPLIES Cotton Balls, absorbent /pkg Cylinder, graduated, 50-mL each Cylinder, graduated, 100-mL each DR/ Inch Cell Adapter each Dropper, 0.5 & 1.0 ml marks, glass /pkg Dropper, 0.5 & 1.0 ml marks, plastic /pkg Flask, erlenmeyer, 500-mL each Funnel, separatory, 250-mL each Pipet, volumetric, 5-mL each Pipet Filler, safety bulb each Ring, support, (3") 83-mm each Sample Cells, matched pair, 1-inch, glass, with stopper pair Spoon, measuring, 0.05-g each Spoon, measuring, 0.2-g each Support, ring stand, (5 x 8") 127 x 203mm each Select one based on available voltage: Hot Plate, 4" diameter, 120 VAC each Hot Plate, 4" diameter, 240 VAC each OPTIONAL REAGENTS AND STANDARDS Description Unit Cat. No. Acetone, ACS ml Calcium Chloride Solution ml Hydrochloric Acid Standard Solution, 0.1 N ml Hydrogen Peroxide, 30%, ACS ml Methyl Orange Indicator Solution, (0.50-g/L) ml Nitric Acid, ACS ml Phenol Solution, 30-g/L...29 ml Potassium Bromide Powder Pillows /pkg Potassium Permanganate Standard Solution, 1-g/L ml Selenium Standard Solution, 1000-mg/L ml Sodium Hydroxide Standard Solution, N ml Sodium Hydroxide Standard Solution, 5.0 N ml MDB Sulfuric Acid Standard Solution, 5.25 N ml MDB Sulfuric Acid Standard Solution, 19.2 N ml Page 8 of 10 Selenium_None_Other_DAB_Eng_4000.fm

9 , continued OPTIONAL EQUIPMENT AND SUPPLIES Beaker, 250-mL... each Beaker, 1000-mL... each Bottle, wash, 500-mL... each Clippers, for opening powder pillows... each Cylinder, graduated, 50-mL... each Cylinder, graduated, 500-mL... each Distillation Apparatus Set, general purpose... each Distillation Apparatus Heater, 115 VAC... each Distillation Apparatus Heater, 230 VAC... each DR/4000 Carousel Module Kit... each Dropper, 0.5- and 1-mL marks, glass... 6/pkg Flask, volumetric, Class A, 1000-mL... each Jack, laboratory... each ph Meter, EC10, portable... each Pipet, serological, 10-mL... each Pipet, TenSette, 0.1 to 1.0 ml... each Pipet Tips, for Pipet... 50/pkg Pipet, volumetric, Class A, 1.00-mL... each Pipet, volumetric, Class A, 5.00-mL... each Pipet, volumetric, Class A, 8.00-mL... each Pipet, volumetric, Class A, mL... each Pipet, volumetric, Class A, mL... each Pipet, volumetric, Class A, mL... each PourRite Ampule Breaker... each Rod, stirring, glass... 3/pkg Stoppers, for cells, hollow, size #2... 6/pkg Selenium_None_Other_DAB_Eng_4000.fm Page 9 of 10

10 FOR TECHNICAL ASSISTANCE, PRICE INFORMATION AND ORDERING: In the U.S.A. Call toll-free Outside the U.S.A. Contact the HACH office or distributor serving you. On the Worldwide Web HACH COMPANY WORLD HEADQUARTERS Telephone: (970) FAX: (970) Hach Company, All rights reserved. Printed in the U.S.A. 6/03 11ed Page 10 of 10 Selenium_None_Other_DAB_Eng_4000.fm