Watershed Management Research SOP 028
TN-TDN -Nitrite-Nitrate (NO2-3) Wet Chemistry
Version Date 02/18/11
Page 1 of 15
TITLE: Total Nitrogen and Total Dissolved Nitrogen Wet Chemistry
SOP No. ESF-SOP 0028
Last Revision Date: 2/18/2011
Last Review Date/Initials: ______/______
PI Approval for Use: ______Date: ______
QA Concurrence: ______Date: ______
Watershed Management Research SOP 028
TN-TDN -Nitrite-Nitrate (NO2-3) Wet Chemistry
Version Date 02/18/11
Page 1 of 15
Revision Summary
Revision / Name / Date / Description of Change0 / Christopher Nietch, Ph.D. / 3/15/2005 / Developed SOP
1 / Christopher Nietch, Ph.D. / 02/21/2006 / Reformat SOP
2 / Don Brown / 2/18/2011 / Editorial clarification
TABLE OF CONTENTS
SECTION NUMBER / SECTION TITLE / PAGE NUMBER1.0 / Scope and Applicability / 4
2.0 / Summary of Method / 4
3.0 / Definitions / 4
4.0 / Health and Safety / 6
5.0 / Cautions / 6
6.0 / Interferences / 7
7.0 / Personnel Qualifications / 7
8.0 / Equipment and Supplies / 7
9.0 / Procedures / 8
10.0 / Calculation / 11
11.0 / Data and Records Management / 11
12.0 / Quality Assurance/Quality Control / 11
13.0 / References / 12
Table 1. Nitrate-Nitrite-TDN/TN Wet Chemistry Reagent , Standard, and Spike Recipes, Holding Times, Container Specs, Disposal Method, Notes, and Tracking
Appendix A: Analytical Schedule Worksheets for TDN and TN Analysis / 12-13
1.0 Scope and Applicability
1.1 Total Nitrogen (TN) and/or Total Dissolved Nitrogen (TDN) in surface water and intercobble (pore) water is analyzed as Nitrate after oxidation of all nitrogen species in the sample under an alkaline persulfate digest procedure. Once samples have been digested this standard operating procedure is equivalent to ESF-SOP-026 where nitrate is quantitatively reduced to nitrite by passage of the sample through a copperized cadmium column. The nitrite is then determined by diazotizing with sulfanilamide followed by coupling with N-(1-naphthyl)ethylenediaminedihydrochloride. The resulting water soluble dye has a magenta color which is read at 520 nm.
1.2 The applicable concentration range for this SOP is 0 to 5 mg N/L as Nitrate. The method detection limit was determined at 0.008 mg/L for a 0.16 mg/l Nitrate standard. The SOP is also applicable over an equivalent range for wastewater and groundwater samples. The SOP throughput is approximately 50 injections per hour.
2.0 Summary of Method
2.1 This SOP covers procedures that take place in the wet chemistry laboratory after samples have been processed according to ESF-SOP-007. The TN and TDN determinationsare made on raw or filteredfiltered water samples, respectively after sample digestion using the Latchat Quickchem 8000, Flow Injection Analysis, Autoanalyzer (Hach Co.).
2.2 The wet oxidation digestion procedure is designed to convert all organic nitrogen species quantitatively to Nitrate with and alkaline digestion reagent. The nitrate is quantitatively reduced to nitrite by passage of the sample through a copperized cadmium column. The nitrite then determined by diazotizing with sulfanilamide followed by coupling with N-(1-naphthyl)ethylenediaminedihydrochloride. The resulting water soluble dye has a magenta color which is read at 520 nm. Nitrite alone also can be determined by removing the cadmium column.
2.3 The project principle investigator has optimized the Latchat instrument for all nutrient analyses required. Four independent channels are set with mixing manifolds in line with a colorimeter (spectrophotometer). For each sampling event analytical schedules are run that cover all nutrient species of interest. For the TN-TDN determination the second schedule is of interest and is set to analyze these constituents on a 5 ml sample split (5 ml each). A mixed standard seriesis prepared and used to calibrate the instrument for both TP/TN and TDP/TDN analyses. (seeTable 1). The digestion reagent may be adjusted chemically to analyze these nutrients simulatenously following the method of Patton and Kyrskalla, 2003 with volumetric adjustments made based on sample volumes.
2.4 Sampling preparation, processing, and analytical blanks are included in each schedule, as are Matrix spikes, calibration check standards, recovery standards, and analytical duplicates as indicated in the example sample schedule provided in Appendix A.
2.5 This nutrient analysis protocol represents a unique application of the Latchat Flow Injection Analysis System and was developed specifically for the ESF-East Fork Watershed Study to meet the sample handling and loading requirements.
2.6 This TN-TDN SOP was adopted and slightly modified from the EPA approved Latchat QuikChem Method 10-107-04-4-A (Smith and Borgren, 2003), which was developed from AWWA standard method 4500-Norg D, 1995).
3.0. Definitions
Dissolved (nutrient form)Dissolved analytes are qualified based on a glass fiber filter size of .47 um (Whatman GF/F).
Total (nutrient form)Total analytes are qualified based on an unfiltered aliquot of the original environmental sample. The sample collection bottle is shaken vigorously for homogenization before the aliquot is taken for analysis.
Organic NitrogenShould Ammonium and nitrate-nitrite be determined individually, “organic nitrogen” can be determined by difference.
Nitrite-Nitrate (NO2-3)The analytical endpoint includes both Nitrate and Nitrite forms of Nitrogen dissolved in the sampled water. Based on the PI’s experience in natural and wastewaters the nitrite fraction of inorganic nitrogen represents a small and insignificant amount that the majority of the time resides at the method MDL. This SOP optimizes on sample throughput, therefore the two constituents are effectively lumped in the analysis, but for all practical purposes the endpoint can be considered as Nitrate.
DI- waterWater taken from a Reverse Osmosis, Ion Exchange Resin, or Distillation system and then passed through a second ion exchange system. Water of this quality should be no more than 18 ohm resistivity. The water meets ASTM II criteria and is considered reagent grade.
Analytical Batch The set of samples extracted/distilled/or digested at the same time.
Calibration StandardA solution prepared from the primary dilution standard solution or stock standard solutions. The solutions are used to calibrate the instrument response with respect to analyte concentration. Calibration standards are run through the digestion procedure thereby implicitly accounting for dilution of the original sample volume with the addition of the digestion reagent.
Field BlankAn aliquot of reagent water or equivalent neutral reference material treated as a sample in all aspects, including exposure to a sample bottle holding time, preservatives, and all preanalysis treatments. The purpose is to determine if the field or sample transporting procedures and environments have contaminated the sample.
Field Duplicate Two samples taken at the same time and place under identical circumstances which are treated identically throughout field and laboratory procedures. Analysis of field duplicates indicates the precision associated with sample collection, preservation, and storage, as well as with laboratory procedures.
Lab BlankAn aliquot of reagent water or equivalent neutral reference material treated as a sample in all aspects, except that it is not taken to the sampling site. The purpose is to determine if the analytes or interferences are present in the laboratory environment, the reagents, or the apparatus.
Laboratory DuplicateTwo aliquots of the same environmental sample treated identically throughout a laboratory analytical procedure. Analysis of laboratory duplicates indicates precision associated with laboratory procedures but not with sample collection, preservation, or storage procedures.
Method Detection LimitThe lowest level at which an analyte can be detected with 99 percent confidence that the analyte concentration is greater than zero.
Recovery StandardA solution prepared containing a known quantity of an organic nitrogen species. In this case Adenosine Triphosphate and/or Glutamic Acid are used. The recovery standard tests the efficiency of the digest procedure to convert the organic nitrogen species to nitrate.
4.0 Health and Safety Warnings
4.1Standard laboratory personal protective equipment (i.e., lab coat, gloves, safety glasses, and safety shoes) are required inwet chemistry lab where analysis is performed.
4.2The toxicity or carcinogenicity of each reagent used in this method has not been fully established. Each chemical shall be regarded as a potential health hazard and exposure shall be as low as reasonably achievable. Cautions are included for known extremely hazardous materials.
4.3Each laboratory is responsible for maintaining a current awareness file of the Occupational Health and Safety Act (OSHA) regulations regarding the safe handling of the chemicals specified in this method. A reference file of Material Safety Data sheets (MSDS) shall be made available to all personnel involved in the chemical analysis.
4.4The following chemicals have the potential to be highly toxic or hazardous, for detailed explanation consult the MSDS.
5.3.1.Cadmium
5.3.2.Ammonium Chloride
5.3.3.Sodium Hydroxide
5.3.4.Concentrated Hydrochloric Acid
5.3.5.Sulfanilamide
5.3.6. Potassium Persulfate
5.3.7. Boric Acid
4.5Safety precautions must be taken into consideration for operating an autoclave. The culture tubes used in the digest are capped tightly. Therefore, an appropriate time for cooling of the samples after autoclaving must be given to avoid an explosion hazard. Typical precautions for handling hot liquids must also be taken into consideration.
5.0 Cautions
5.1Because the digestion procedures requires an alkaline matrix samples can not be preserved with acid as recommended for Nitrate stabilization. This method can only be used if samples can be digested within 24 hours of collection.
5.2The nitrite standard for column efficiency check should be run with each analytical batch to insure cadmium column reduction efficiency is above 75%. If it falls below this value the cadmium needs to be reconditioned or changed according to Standard Method 4500-N03-E (AWWA 1995).
5.3If the pH of the sample is below 5 or above 9, adjust to between 5 and 9 with either conc. HCl or conc. NH4OH.
5.4Due diligence should be taken to minimize the entry of air bubbles into the cadmium column. This decreases column efficiency and smooth flow through the column. If this should occur it can often be remedied by holding the column in a vertical position and tapping on the outside with a spatula. If too much air enters the column it will have to be repacked.
6.0 Interferences
6.1Residual chlorine can interfere by oxidizing the cadmium column.
6.2Low results would be obtained for samples that contain high concentrations of iron, copper or other metals. In this method, EDTA is added to the buffer to reduce this interference.
6.3Samples that contain large concentrations of oil and grease will coat the surface of the cadmium. This interference is eliminated by pre-extracting the sample with an organic solvent.
6.4Samples preserved with acid cannot be analyzed with this procedure.
7.0 Personnel Qualifications
7.1Each scientist/technician is responsible for following this SOP as written and notifying the author, QA officer, and or the Project Leader, as appropriate, of equipment or procedural problems found during the use of this SOP.
7.2The techniques of a first-time scientist/technician shall be reviewed by a senior scientist/technician prior to initiating this SOP alone.
8.0Equipment and Supplies
8.1.Balance -- analytical, capable of accurately weighing to the nearest 0.0001 g.
8.2.Glassware -- Class A volumetric flasks and pipettes or plastic containers as required. Samples may be stored in plastic or glass. 13 ml borosilicate glass, screw top vials, with Teflon faced phenolic caps are used for sample digests.
8.3.Flow injection analysis equipment designed to deliver and react sample and reagents in the required order and ratios.
8.3.1.Sampler
8.3.2.Multichannel proportioning pump
8.3.3.Reaction unit or manifold
8.3.4.Colorimetric detector
8.3.5.Data system
8.4.Special Apparatus
8.4.1.Cadmium-Copper Reduction Column (Lachat part # 50237).
8.5. Vortex Mixer
8.6.Digital Pippettor for dilution of stock standard solutions and delivery of spike solutions
8.7.Helium tank, regulator, and attached degassing tube.
8.8.Disposable glass culture tubes; serve as Latchat system sampling ‘cups’. See ESF-SOP-007 for specifications. After digestion the sample is transferred from the digestion tube to the sample cup.
8.9Chemicals used to make reagents and standards in Table 1.
9.0Procedure
9.1. Prepare Reagents
9.1.1.Use ASTM Type II water for all reagents and standard solutions.
9.1.2.Degas reagents with helium, except for the digestion solution and the borate buffer solution:
To prevent bubble formation, degas all solutions except the standards with helium. Use He at 140kPa (20 lb/in2) through a helium degassing tube (Lachat Part No. 50100.). Bubble He through the solution for one minute.
9.1.3.Reagent 1.Sodium Hydroxide (See Table 1)
CAUTION: This solution gets very hot and is difficult to dissolve.
9.1.4. Reagent 2.Ammonium Chloride Buffer (See Table 1)
9.1.5.Reagent 3.Sulfanilimide (See Table 1)
9.1.6.Reagent 4. Di Water Carrier (See Table 1)
9.1.7.Reagent 5. Digest Reagent (See Table 1)
9.1.8.Reagent 6. Borate Buffer Solution (See Table 1)
9.2. Prepare Calibration Standards, Recovery Standards and Spike Solution as described in Table 1. 5 ml of calibration and recovery standards are pipetted into digestion tubes.
9.3. Prepare samples for analysisfollowing sample processing procedures outlined in ESF-SOP-007. At the end of this step there should be a ‘digestion’ tray of culture tubes ready for addition of digestion reagent (See example sample schedule in Appendix A).
9.4. Prepare Matrix Spikes (See Table 1). After the spike has been added to the sample, vortex the sample cup for 5 seconds and return to the digestion tray.
9.5. Add specified amount of digestion solution to each 5 ml sample aliquot and QC samples. Cap the each sample tightly with a Teflon faced phenolic screw cap, and invert to mix.
9.6. Autoclave the unknown samples and QC samples at 110oC for at least 45 minutes. Allow time for the samples to cool. If using the Community Autoclaves in the AWBERC facility they are programmed to allow for appropriate cool down.
9.7.Transfer all digested samples into autosampler sample cups following the sequence specified in the analytical schedule (See Appendix A for an example).
9.85.Set up Nitrate-Nitrite manifold as specified in Diamond 2001. At the start of a new ESF experiment, after approximately 1000 sample runs, or if column efficiency checks returns a lower than expected conversion rate on the Nitrite standard the cadmium column should be re-packed with freshly conditioned cadmium granules following the procedures outlined in AWWA Standard Method 4500-NO3-E. (AWWA 1995 or later edition).
9.9. Turn on the Latchat Quickchem Colorimeter Unit.
9.10.Input analytical system parameters in the latchat software program as specified in Diamond, 2001. This occurs upon first application of the method. If the manifold specifications do not change or the manifold is not switched out between runs then this information remains saved in the software system.
9.11. Attach reagent lines and condition pump tubes by rolling on a flat surface for several seconds. Replace pump tubes every 6 months or earlier as required.
9.12. Fill a container with fresh DI-water or AWBERC house RO water. The reagent lines are placed in this container to serve as the pre-analysis rinse of the manifold. Fill a separate container with DI-water and use this for the probe rinse.
9.13.Turn pump on and select the “manual” button. If needed spray rollers with silicone and wipe with kim-wipe or equivalent. The pump rollers should be greased at least quarterly.
9.14. Attach pump compression brackets to each reagent line, probe rinse line, and sample line, and then to the pump.
9.15. Pump DI water through all reagent lines and check for leaks and smooth flow. Switch lines to the reagent bottles, making sure effluent line from the colorimeter is directed into the appropriate waste container. Allow reagent to pass through the manifold for at least 3 minutes, and then switch the valve on the manifold to put the cadmium column in-line. Allow the system to equilibrate until a stable baseline is achieved. Baseline stability is viewed by selecting the “preview” button in the software system.
9.16.Turn on the autosampler.
9.17. Place standards and samples in the autosampler. Input the schedule information required by the software system (See Hach 2003). Follow the sample ID scheme outlined in ESF-SOPs-007 and 010. See Appendix A for an example. Sample Schedules can be imported from an excel file saved in csv format. The format for the csv file is: sample number, sample id, cup number.
9.17.1.Digested Calibration standards are scheduled to begin each run. The software system will then associate the concentrations with the peak area for each standard to determine the calibration curve. Calibration curves with r-squared values less then 0.95 are un-acceptable. If this occurs standards need to be prepared again and re-run.
9.17.2.Digested Quality control Blanks are scheduled to run immediately after the calibration curve is verified. Blanks returning concentrations greater than 2.5 times the method MDL (0.008 mg/l) are considered contaminated and a correction of the final concentration will have to be applied to the sample concentrations. The equation for the blank correction factor is:
Blank Corrected Concentration = D-Bave
Where D = Determined concentration of analyte and Bave = is the average concentration of blank replicates. Blanks are run at 2% of an analytical batch.
9.17.3.Quality control Analytical Duplicates are scheduled to run on every 10th sample. After a run compute the relative percent difference (RPD) between two sample results using the following equation:
Where, D1 = Concentration of analyte in the sample, D2 = Concentration of analyte in the second (duplicate) sample. RPDs for nitrate-nitrite wet chemistry falling outside of ±7% fail QC and the data must be flagged accordingly before submission to the ESF database administrator.
9.17.4.Analyses of matrix spike samples are required to demonstrate the method accuracy and to monitor matrix interferences (interferences caused by the sample matrix). The laboratory must spike a minimum of 10% of all samples (one sample in each batch of ten samples) in an analytical batch. The concentration of the spike in the sample shall be determined as 1 to 5 times higher than the background concentration (see Table 1).
Calculate the percent recovery (P) of the analyte in each aliquot using the following equation.