NITROGEN, TOTAL, Test N Tube * Method 10071 TNT Persulfate Digestion Method (0 to 25.0 mg/l N) Scope and Application: For water and wastewater; digestion is required for determining total nitrogen. The digestion procedure is included in the method. 1. Turn on the COD Reactor. Heat to 103 106 C (best temperature is 105 C). Place the plastic shield in front of the reactor. Note: Ensure safety devices are in place to protect the analyst from splattering should leakage occur. Note: For proof of accuracy, run a 20 mg/l NH 3 N standard through digestion and analysis. 2. Using funnels, add the contents of one Total Nitrogen Persulfate Reagent Powder Pillow to each of two Total Nitrogen Hydroxide Reagent vials. Note: Wipe off any reagent that may get on the lid or the tube threads. Note: One reagent blank is sufficient for each set of samples. 3. Add 2 ml of sample to one vial. Add 2 ml of organic-free water to another vial (the reagent blank). Cap both vials. Shake vigorously to mix (more than 30 seconds). Place the vials in the COD Reactor. Heat for 30 minutes. Note: The persulfate reagent may not dissolve completely after shaking. Note: Alternative water must be free of all nitrogen-containing species. 4. Using finger cots or gloves, remove the hot vials from the reactor and allow to cool to room temperature. Note: It is very important to remove the vials from the COD Reactor after exactly 30 minutes. * TenSette, Test N Tube, and Voluette are trademarks of Hach Company.
410 nm 5. Enter the stored program for Test N Tube Total Nitrogen. Press: 3 5 0 READ/ENTER The display will show: Dial nm to 410 Note: DR/2000s with software version 3.0 and greater will not display Dial nm to message if the wavelength is already set correctly. The display will show the message in step 7. Proceed with step 8. Note: For DR/2000s without this stored program, see Instrument Setup following these steps. 6. Rotate the wavelength dial until the display shows: 410 nm Note: For DR/3000s, set the wavelength to 410 nm and press CLEAR. 7. Press: READ/ENTER The display will show: mg/l N TN TNT 8. Remove the caps from the cooled, digested vials and add the contents of one TN Reagent A Powder Pillow to each vial. Cap vials and shake for 15 seconds. Press: SHIFT TIMER after shaking. A three-minute reaction period will begin. Note: For DR/3000s, press 3 TIMER. 2
9. After the timer beeps, uncap the vials and add one TN Reagent B Powder Pillow to each vial. Cap vials and shake for 15 seconds. Press: SHIFT TIMER after shaking. A two-minute reaction period will begin. Note: The reagent will not completely dissolve. The solution will begin to turn yellow. 10. After the timer beeps, remove the caps from two TN Reagent C Vials and add 2 ml of digested, treated sample to one vial. Add 2 ml of the digested, treated reagent blank to the second TN Reagent C Vial. 11. Cap both vials and invert 10 times to mix. Use slow, deliberate inversions for complete recovery. The vials will be warm. Note: Hold the vial vertical with the cap up. Invert the vial and wait for all of the solution to flow to the cap end. Return the vial to the upright position and wait for all of the solution to flow to the vial bottom. Pause. This is one inversion (10 inversions = 30 seconds). 12. Press: SHIFT TIMER A 5-minute reaction period will begin. Do not shake the vial again. Note: The yellow color will intensify. Note: For DR/3000s, press 5TIMER. Note: For DR/3000s, press 2 TIMER. 13. Insert the COD Vial Adapter into the cell holder with the marker to the right. Note: For DR/3000s, the groove in the adapter faces the front of the instrument. 14. When the timer beeps, wipe the outside of both vials. Note: Wiping with a damp towel then a dry one will remove fingerprints or other marks. 15. Place the vial containing the reagent blank into the adapter with the Hach logo toward the front of the instrument. Place the cover on the adapter. Note: The reagent blank is stable when stored in the dark; see Blanks for Colorimetric Measurement on page 10 following this procedure. 16. Press: ZERO The display will show: then: WAIT 0.0 mg/l N TN TNT Note: For DR/3000s, press MANUAL PROGRAM. Then press ZERO. Press ZERO again if the display does not show 0.000. Enter the concentration factor (50.05) and press CONC FACTOR. Then press 1 CONC. 3
17. Place the prepared sample into the cell holder with the Hach logo toward the front of the instrument and place the cover on the adapter. Note: Multiple samples may be read after zeroing on one reagent blank. 18. Press: READ/ENTER The display will show: WAIT then the result in mg/l total nitrogen will be displayed. Note: If the test overranges, repeat the digestion and measurement with diluted sample. The entire digestion must be repeated for accurate results. Note: For DR/3000s, pressing READ is not necessary. The instrument will automatically display the results. Instrument Setup DR/2000 with software versions 2.0 and 2.2. Enter the calibration as an update to Hach-stored programs. 1. Press I/O. 2. Press SHIFT CONFIG/METH. 3. Press: 4. Within 3 seconds, press SHIFT PROG/3 CONFIG/METH. The display will show: ENTER nm Note: If you make an error, press SHIFT CLEAR and reenter the number. When the number is correct, press READ/ENTER. 5. Press: The display will show: Decimal? 00.00 4
6. Use the arrow keys to correctly position the decimal point. Press the down arrow key once. The display will show: DECIMAL? 000.0. Press: READ/ENTER 7. The display will show: UNITS? 8. Use the arrow keys to select the appropriate unit of measure. Press the down arrow key twice. The display will show: mg/l 9. Press READ/ENTER when the correct unit of measure is displayed. This display will show: SYMBOL? 10. Construct the correct symbol display. N TN TNT a. Select letters and numbers by scrolling to the correct character with the arrow keys. b. To make a letter uppercase, press the SHIFT key. c. To enter a subscript number, enter the digit on the numeric keypad. d. To enter a superscript number, enter the digit on the numeric keypad then press SHIFT. e. The space is the character displayed after one press of the down arrow key. f. Make sure to enter the display line EXACTLY as shown, including the spaces. Do not enter trailing spaces. g. Accept each symbol by pressing READ/ENTER. h. To end symbol entry, press READ/ENTER a second time. 11. When the instrument is out of symbol entry mode, the display will read: TIMER? 12. This method has 3 timed steps, so press SHIFT TIMER. The display will show: MM:SS TIME 1? 13. Enter a timer value of 3 minutes. Press: 14. Press READ/ENTER to accept the timer value. The display will show: MM:SS TIME 2? 15. Enter a timer value of 2 minutes. Press: 16. Press READ/ENTER to accept the timer value. The display will show: MM:SS TIME 3? 17. Enter a timer value of 5 minutes. Press: 18. Press READ/ENTER to accept the timer value. The display will show: MM:SS TIME 4? 5
19. Press READ/ENTER to complete the timer entry. The display will show: #1 Data 20. Enter the following 12 numbers as shown. Complete each number by pressing the READ/ENTER key. # 1 Data 0 # 2 Data 7968 # 3 Data 7968 # 4 Data 7968 # 5 Data 7967 # 6 Data 8447 # 7 Data 65535 # 8 Data 65535 # 9 Data 65535 #10 Data 10922 #11 Data 544 Checksum 13755 The final number is a check value which determines if the data sequence was correctly entered. If an error was made during number entry, the display will return to the prompt for data #1 and the entire sequence must be re-entered. If all numbers are correctly entered, the display will return to the method prompt and is ready for use. DR/2000 with software version 3.0 and higher 1. Turn the instrument on. Press SHIFT METHOD to enter the configuration mode. The display will show: MOMENTARY or CONSTANT ON. 2. Press the up arrow key twice to select HACH UPDATE. Press READ/ENTER. The display will show: ENTER #: Note: If you make an error, press SHIFT CLEAR and reenter the number. When the number is correct, press READ/ENTER. 3. Press: The display will show: P350 ENTER nm 4. Press: The display will show: P350 Decimal? 00.00 5. Use the arrow keys to correctly position the decimal point. Press the down arrow key twice. The display will show: DECIMAL? 000.0 Press: READ/ENTER. The display will show: P350 UNITS? 6. Use the arrow keys to select the appropriate unit of measure. Press the down arrow key twice. The display will show: P350 mg/l 6
7. Press READ/ENTER when the correct unit of measure is displayed. This display will show: P350 mg/l _ 8. Construct the display to read the correct symbol. The symbol must be entered exactly as shown including spaces between characters: N TN TNT a. Select letters and numbers by scrolling to the correct character with the arrow keys. b. To make a letter uppercase, press the SHIFT key. To make numbers and symbols superscript, subscript, or regular, press SHIFT until the display is correct. c. The space is the character displayed after one press of the down arrow key. d. Make sure to enter the display line EXACTLY as shown, including the spaces. Do not enter trailing spaces. e. Accept each symbol by pressing READ/ENTER. f. To end symbol entry, press READ/ENTER a second time. Alternatively, use this procedure: To enter N, press 7 8 READ/ENTER READ/ENTER (a space) 3 2 READ/ENTER READ/ENTER T N 8 4 READ/ENTER READ/ENTER 7 8 READ/ENTER READ/ENTER (a space) 3 2 READ/ENTER READ/ENTER T N T 8 4 READ/ENTER READ/ENTER 7 8 READ/ENTER READ/ENTER 8 4 READ/ENTER READ/ENTER 9. When the instrument is out of symbol entry mode, the display will read: P350 TIMER 10. This method has 3 timed steps, so press SHIFT TIMER. The display will show: MM:SS TIME 1? 11. Enter a timer value of 3 minutes. Press: 12. Press READ/ENTER to accept the timer value. The display will show: MM:SS TIME 2? 13. Enter a timer value of 2 minutes. Press: 7
14. Press READ/ENTER to accept the timer value. The display will show: MM:SS TIME 3? 15. Enter a timer value of 5 minutes. Press: 16. Press READ/ENTER to accept the timer value. The display will show: MM:SS TIME 4? 17. Press READ/ENTER to complete the timer entry. The display will show: #0 STANDARD 18. Press READ/ENTER to display the zero data pair. The display will show: 0.000 Abs 000.0 mg/l 19. Press READ/ENTER. The display will show: #1 STANDARD 20. Press READ/ENTER. The display will prompt for entry of the first concentration point: #1 000.0 mg/l 21. Enter concentration point #1 by pressing 0028 so that the display shows: #1028.0mg/l 22. Press READ/ENTER. The display will prompt for entry of the first absorbance point: # 1 0.000 Abs 23. Enter the absorbance point #1 by pressing 0557. The display will show: # 1 0.557Abs 24. Press READ/ENTER. The display will show the first data pair: 0.557 Abs 028.0 mg/l 25. Press READ/ENTER to accept the first data pair. The display will show: #2 STANDARD 26. The data pair values from the table below are now entered. Standard Concentration Absorbance # 0 0.0 mg/l 0.000 Abs # 1 28.0 mg/l 0.557 Abs 27. The data pair values are now entered. The display will show: #2 STANDARD 28. Press SHIFT READ/ENTER to complete data point entry. The display will show: #: 29. Enter the validation number 2749. The display will show: #: 2749 30. Press READ/ENTER. The display will show: COMPLETED then: P350 mg/l N TN TNT Note: If the display shows INCORRECT #, then prompts for the validation number, a mistake may have been made during data entry. Make sure the validation number is correct. If so, the error occurred during some other portion of the method entry. Press METH and respond to the ABORT? message by pressing READ/ENTER, then re-enter the method. The instrument is now ready for use with method 350. 8
Sampling and Storage Collect samples in clean plastic or glass bottles. Best results are obtained with immediate analysis. Preserve the sample by reducing the ph to 2 or less with concentrated sulfuric acid (at least 2 ml). Store at 4 C (39 F) or less. Preserved samples may be stored up to 28 days. Warm samples to room temperature and neutralize with 5 N sodium hydroxide before analysis. Correct the test result for volume additions; see Correcting for Volume Additions in Section 1 of the Procedures Manual. Accuracy Check This method generally yields 95 100% recovery on organic nitrogen standards. For proof of accuracy, Hach offers a set of 3 Primary Standards for Kjeldahl Nitrogen. 1. Prepare one or more of the following 3 solutions. Each preparation is for an equivalent 25 mg/l N standard. Use water that is free of all nitrogen-containing species. a. Weigh 0.3379 g of Ammonium p-toluenesulfonate(ptsa). Dissolve in a 1000-mL volumetric flask with deionized water. Add deionized water to the 1000-mL mark. b. Weigh 0.4416 g of Glycine p-toluenesulfonate. Dissolve in a 1000-mL volumetric flask with deionized water. Add deionized water to the 1000-mL mark. c. Weigh 0.5274 g of Nicotinic p-toluenesulfonate. Dissolve in a 1000-mL volumetric flask with deionized water. Add deionized water to the 1000-mL mark. 2. Analyze each of these solutions using the test procedure above. Calculate the percent recovery for each using this formula: measured concentration % recovery = --------------------------------------------------------------- 100 25 The percent recovery is listed in Table 1: Hach analysts have found Ammonia-PTSA to be the most difficult to digest. Other compounds may yield different percent recoveries. Table 1 Recoveries For Primary Standards Compound Standard Solution Method For proof of accuracy, substitute 2 ml of a 20-mg/L ammonia nitrogen standard solution for the sample in the procedure. To prepare a 20-mg/L ammonia nitrogen standard, use a 20-mL Class A pipet to add 20 ml of a 100-mg/L Ammonia Nitrogen Standard (see Optional Reagents) to a 100-mL Class A volumetric flask. Dilute to the line with deionized water that is free of all nitrogen-containing species. A single analyst should obtain less than 5% variation on replicates. 9 Lowest Expected% Recovery Ammonia-PTSA 95% Glycine-PTSA 95% Nicotinic-PTSA 95%
Standard Additions Method a. Fill three, 25-mL graduated mixing cylinders with 25 ml of sample. b. Snap the neck off an Ammonia Nitrogen Standard Solution Voluette Ampule, 160 mg/l as NH 3 N. c. Use a TenSette Pipet to add 0.3, 0.6 and 0.9 ml of standard, respectively, to the three mixing cylinders. d. Stopper each cylinder and mix thoroughly. e. Add 2 ml of each prepared solution, respectively, to three Total Nitrogen Hydroxide Reagent Vials. f. Analyze each standard addition sample as described in the procedure. The nitrogen concentration should increase by about 1.9, 3.8 and 5.6 mg/l N, respectively. g. If these increases do not occur, see Standard Additions in Section 1 of the Procedures Manual for troubleshooting information. Blanks for Colorimetric Measurement The reagent blank may be used repeatedly for measurements using the same lots of reagents. Store the reagent blank in the dark at room temperature (18 25 C) for a maximum of 7 days. If a small amount of white floc appears prior to the end of one week, discard the reagent blank and prepare a new one. Interferences Table 2 lists substances that resulted in a concentration change of ± 10%: Table 2 Interferences that Result in a Concentration Change of ± 10% Substance Level Effect Cl - > 1000 ppm positive interference Br - > 60 ppm positive interference Table 3 lists substances that have been tested and do not interfere up to the indicated levels (in mg/l): Substance Table 3 Substances that Do Not Interfere Maximum Level Tested (mg/l) Barium 2.6 Calcium 300 Chromium (3+) 0.5 Iron 2 Lead 6.6 ppb Magnesium 500 Organic Carbon 150 ph 13 ph units Phosphorus 100 Silica 150 Silver 0.9 Tin 1.5 10
Method Performance Hach chemists tested this chemistry on standard nitrogen solutions prepared from the following compounds with 95% or better recovery: Ammonium Acetate Ammonium Chloride Ammonium Sulfate Glycine Urea Ammonium chloride or nicotinic-ptsa spikes in domestic influent, effluent, and the ASTM standard specification for substitute wastewater (D 5905-96) also resulted in 95% recovery. The large amounts of nitrogen-free organic compounds in some samples may decrease digestion efficiency by consuming some of the persulfate present. Samples known to contain high levels of organics should be diluted and re-run to verify digestion efficiency. Precision A Hach chemist analyzed two independent nutrient standards. The lowest average percent recovery was 95% with a standard deviation of ± 2%. DR/2000: In a single laboratory, using a standard solution of 15 mg/l NH 3 N and two lots of reagent with a DR/2000 Spectrophotometer, a single operator obtained a standard deviation of less than 1.0 mg/l N. DR/3000: In a single laboratory, using a standard solution of 15 mg/l NH 3 N and two lots of reagent with a DR/3000 Spectrophotometer, a single operator obtained a standard deviation of less than 1.0 mg/l N. Summary of Method An alkaline persulfate digestion converts all forms of nitrogen to nitrate. Sodium metabisulfite is added after digestion to eliminate halogen oxide interferences. Nitrate then reacts with chromotropic acid under strongly acidic conditions to form a yellow complex with an absorbance maximum at 410 nm. 11
REQUIRED REAGENTS Description Cat. No. Test N Tube Total Nitrogen Reagent Set (50 tests)... 26722-45 Includes: (4) 26717-25*, (4) 26721-25*, (1) 26718-49, (1) 26719-49, (1) 26720-49 Quantity Required Description Per Test Unit Cat. No. Total Nitrogen Hydroxide Reagent Vials, 0.1 N... 2...25/pkg...* Total Nitrogen Persulfate Reagent Powder Pillows... 2...100/pkg... 26718-49 Total Nitrogen Reagent A Bisulfite Powder Pillows... 2...100/pkg... 26719-49 Total Nitrogen Reagent B Indicator Powder Pillows... 2...100/pkg... 26720-49 Total Nitrogen Reagent C Acid Solution Vials... 2...25/pkg...* REQUIRED APPARATUS COD Reactor, 115/230 V, North American Plug... 1...each... 45600-00 COD Reactor, 230 V, European Plug... 1...each... 45600-02 COD Vial Adapter, DR/2000-3000... 1...each... 44799-00 Funnel, micro... 1...each... 25843-35 Pipet, TenSette, 1.0-10.0 ml... 1...each... 19700-10 Pipet Tips for 19700-10... 2...50/pkg... 21997-96 Pipet, TenSette, 0.1-1.00 ml... 1...each... 19700-01 Pipet Tips for 19700-01... 2...50/pkg... 21856-96 Safety Shield, for COD Reactor... 1...each... 23810-00 Test Tube Cooling Rack... 1...each... 18641-00 OPTIONAL REAGENTS Nitrogen, Ammonia, 100 mg/l NH 3 N... 500 ml... 24065-49 Nitrogen, Ammonia, Voluette Ampules, 160 mg/l NH 3 N, 10 ml...16/pkg... 21091-10 Primary Standards for Kjeldahl Nitrogen...3/pkg... 22778-00 Sodium Hydroxide Standard Solution, 5.0 N... 59 ml MDB... 2450-26 Sulfuric Acid, ACS... 500 ml... 979-49 Water, organic-free... 500 ml... 26415-49 OPTIONAL APPARATUS Balance, analytical, 110/220 V...each... 26103-00 Flask, volumetric, Class A, 1000 ml (3 required for accuracy check)...each... 14574-53 * Not available separately. Hach Company, 1996 1999. All rights reserved. Printed in the U.S.A. 1891 3/96 1ed aa/dp rev. 1 8/99