Caduceon Environmental Laboratories Page: 1 of 4 Total Kjeldahl Nitrogen/Total Phosphorus Revision Date: 26 Nov, 2019 Method TPTKN-001 Revision #: 2.6 Management Review: MD Quality Review: SKB Determination of Total Kjeldahl Nitrogen and Total Phosphorus in Waters, Liquid Sludge, Solid Sludge and Soil Samples 1.0 Scope and Application 1.1 This method is applicable to the analysis of Total Kjeldahl Nitrogen (TKN) and/or Total Phosphorus (TP) in drinking water, groundwater, surface water, domestic, industrial wastewater, soils and sludge’s (liquid and biosolids). The analytical ranges are: for TKN, 0.1 mg/L to 6.4 mg/L as N and for TP, 0.01 mg/L to 1.60 mg/L or 0.002-0.400mg/L(low level) as P. The upper limit of the analytical ranges can be extended by appropriate dilution of the water sample prior to digestion, or by dilution of the sample digestate prior to analysis. The digestion procedure described below is best used for samples containing up to 10 mg/L of TKN as N and TP as P in solution. If the analyzed digestate is determined to have more than 10.0 mg/L of analyte a smaller portion of the sample should be taken or diluted for digestion and re-digested. 1.2 This method may be applied to soils and other solid materials. The digestion of such samples must be monitored carefully to ensure that bumping or excessive foaming does not occur, and the digestate must be filtered to prevent plugging of the sampling tube and analytical cartridge. The recommended soil sample of 0.1 g yields analytical ranges of: for TKN, 5 µg/g to 640 µg/g as N and for TP, 0.5 µg/g to 160 µg/g as P. If the analyzed digestate is determined to have more than 10.0 mg/L of analyte a smaller portion of the sample must be used for digestion and re-digested. 2.0 Principle and Theory 2.1 Principle 2.1.1 Total Kjeldahl Nitrogen 2.1.1.1 An aliquot of the sample is digested in a block digester with Kjeldahl's reagents to convert trivalent organic nitrogen compounds to ammonium ions (highly acidic media). 2.1.1.2 The determination of TKN is based on the colourimetric method in which a blue colour is formed by the reaction of salicylate sodium hypochlorite, and sodium nitroprusside. 2.1.2 Total Phosphorus 2.1.2.1 An aliquot of the sample is acid digested in a block digester, converting all phosphorus compounds to orthophosphate by high temperature hydrolysis with sulphuric acid. 2.1.2.2 The determination of phosphorus is based on the colourimetric method in which a blue colour is formed by the reaction of orthophosphate, molybdate ion and antimony ion followed by reduction with ascorbic acid at an acidic pH. The absorbance of the blue phosphomolybdate complex is measured in the colourimeter using a 1.0cm flow cell and a 660nm filter. 2.2 Theory 2.2.1 Total Kjeldahl Nitrogen 2.2.1.1 The intensity of the blue colour varies linearly with the concentration of ammonium ions through the analytical range. The absorbance is measured using a 1.0cm flow cell and a 660nm filter. uncontrolled
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Caduceon Environmental Laboratories Page: 1 of 4 Total Kjeldahl Nitrogen/Total Phosphorus Revision Date: 26 Nov, 2019 Method TPTKN-001 Revision #: 2.6 Management Review: MD Quality Review: SKB Determination of Total Kjeldahl Nitrogen and Total Phosphorus in Waters, Liquid
Sludge, Solid Sludge and Soil Samples 1.0 Scope and Application 1.1 This method is applicable to the analysis of Total Kjeldahl Nitrogen (TKN) and/or Total
Phosphorus (TP) in drinking water, groundwater, surface water, domestic, industrial wastewater, soils and sludge’s (liquid and biosolids). The analytical ranges are: for TKN, 0.1 mg/L to 6.4 mg/L as N and for TP, 0.01 mg/L to 1.60 mg/L or 0.002-0.400mg/L(low level) as P. The upper limit of the analytical ranges can be extended by appropriate dilution of the water sample prior to digestion, or by dilution of the sample digestate prior to analysis. The digestion procedure described below is best used for samples containing up to 10 mg/L of TKN as N and TP as P in solution. If the analyzed digestate is determined to have more than 10.0 mg/L of analyte a smaller portion of the sample should be taken or diluted for digestion and re-digested.
1.2 This method may be applied to soils and other solid materials. The digestion of such samples
must be monitored carefully to ensure that bumping or excessive foaming does not occur, and the digestate must be filtered to prevent plugging of the sampling tube and analytical cartridge. The recommended soil sample of 0.1 g yields analytical ranges of: for TKN, 5 µg/g to 640 µg/g as N and for TP, 0.5 µg/g to 160 µg/g as P. If the analyzed digestate is determined to have more than 10.0 mg/L of analyte a smaller portion of the sample must be used for digestion and re-digested.
2.0 Principle and Theory 2.1 Principle
2.1.1 Total Kjeldahl Nitrogen
2.1.1.1 An aliquot of the sample is digested in a block digester with Kjeldahl's reagents to convert trivalent organic nitrogen compounds to ammonium ions (highly acidic media).
2.1.1.2 The determination of TKN is based on the colourimetric method in which a blue colour is formed by the reaction of salicylate sodium hypochlorite, and sodium nitroprusside.
2.1.2 Total Phosphorus
2.1.2.1 An aliquot of the sample is acid digested in a block digester, converting all
phosphorus compounds to orthophosphate by high temperature hydrolysis with sulphuric acid.
2.1.2.2 The determination of phosphorus is based on the colourimetric method in which a blue colour is formed by the reaction of orthophosphate, molybdate ion and antimony ion followed by reduction with ascorbic acid at an acidic pH. The absorbance of the blue phosphomolybdate complex is measured in the colourimeter using a 1.0cm flow cell and a 660nm filter.
2.2 Theory
2.2.1 Total Kjeldahl Nitrogen
2.2.1.1 The intensity of the blue colour varies linearly with the concentration of ammonium ions through the analytical range. The absorbance is measured using a 1.0cm flow cell and a 660nm filter.
2.2.2.1 The intensity of the blue colour varies linearly with the concentration of Orthophosphate through the analytical range. The absorbance is measured using a 1.0cm flow cell and a 660nm filter.
2.3 Interferences
2.3.1 Total Kjeldahl Nitrogen
2.3.1.1 Nitrate in excess of 10mg/L can cause a negative interference. This occurs when nitrate oxidizes a portion of the ammonia from digested organic nitrogen producing N2O. In the presence of sufficient organic matter in a low state of oxidation, nitrate can be reduced to ammonia causing a positive interference. There is no proven way to eliminate this interference.
2.3.1.2 Samples which contain a very large quantity of salt or inorganic solids that dissolve during digestion, the temperature of the digestion may exceed 400oC. This will result in pyrolitic loss of nitrogen. Excess sulphuric acid may be added to maintain the proper acid-salt balance. Addition of too much acid will lower the digestion temperature below 360oC, resulting in incomplete digestion and poor recovery. This technique may be used should results be suspect or spike does not yield proper results
2.3.1.3 Large amounts of organic matter in the sample will consume too much acid. This will cause the same kind of overheating during digestion as outlined in section 2.3.1.2. To reduce this effect, addition of 10mL of concentrated sulphuric acid for each gram of organic matter, to the digestion tube. This technique may be used should results be suspect or spike does not yield proper results.
2.3.1.4 Many reagents may contain trace amounts of ammonia. Be sure to analyze digestion blanks with the same quantity of reagents as used for samples, to compensate for this effect.
2.3.2 Total Phosphorus
2.3.2.1 Ferric ion concentration above 50mg/L, copper ion concentration above 10mg/L
and silicate concentration above 10mg/L may cause interferences. These can be overcome by addition of sodium hydroxide to the digested solution to precipitate oxide salts of the interferents.
2.3.2.2 Arsenate concentration above 0.10mg/L may cause a positive interference. At this time, there is no known way to compensate for this interference.
2.3.2.3 Nitrite and sulphide interferences can be eliminated by adding excess bromine water or a saturated potassium permanganate solution.
2.3.3 The use of spike samples can be incorporated into the procedure to evaluate and overcome
interferences due to sample matrix. This is done by preparing a sample with a spike calculated to produce a response of the same magnitude as that produced by the sample alone. The spiked sample solution is analyzed along with a portion of the sample that has been diluted with reagent water at the same volume as the spike. In this manner the recoveries for TKN and/or TP may be calculated from the difference between the spiked and blank-spiked sample aliquots.
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Caduceon Environmental Laboratories Page: 3 of 4 Total Kjeldahl Nitrogen/Total Phosphorus Revision Date: 26 Nov, 2019 Method TPTKN-001 Revision #: 2.6 Management Review: MD Quality Review: SKB 3.0 Safety 3.1 See Caduceon Safety Manual for general safety information. Consult SDS prior to handling
chemicals. 3.2 Extreme caution should be exercised when preparing acidic and basic solutions. Safety goggles,
gloves and a lab coat should be worn when preparing these solutions. All reagent preparations should be performed in a fume hood with the sash set as low as possible to permit working with the solutions.
4.0 Sample Requirements 4.1 Water Samples
4.1.1 Samples may be collected in either plastic or glass bottles with plastic-lined caps, preserved with H2S04 at a pH <2 and stored at 4 ± 3C. Minimum sample volume is 100 mL. The holding time for samples preserved in this manner is 28 days. All drinking water samples falling under Ontario Regulations 170/03 and 252/05 should not be filtered at the time of sample collection.
4.1.2 Samples that are received in the Laboratory un-preserved (e.g. in 500 mL plastic jars to be
tested for a range of parameters) are stored at 4 ± 3C until analyzed. The holding time for unpreserved samples is 7 days.
4.2 Liquid Sludge Samples
4.2.1 Samples can be collected in either plastic or glass jars with plastic-lined lids. Use at least a 100 mL wide mouth jar. Store at 4 ± 3C. The holding time for sludge samples is 28 days.
4.3 Soils and Biosolids – (Dewaterd Sludges)
4.3.1 Samples may be collected glass jars with plastic-lined lids. Use at least a 100 mL wide mouth jar. Store at 4 ± 3C. The holding time for soil samples is 28 days.
5.0 Apparatus 5.1 Labware
5.1.1 Volumetric Flasks, Class A various sizes 5.1.2 Analytical Balance, accurate to 0.1mg Accuracy verified as per CP-021 5.1.3 Top-Loading Balance, accurate to 0.01g Accuracy verified as per CP-021 5.1.4 Pipettes, Syringes Various volumes 5.1.5 Auto-pipettors, 2 mL and 20 mL Accuracy verified as per CP-022 5.1.6 Glass Bottles, amber 500, 1000 mL 5.1.7 Specimen Cups 90mL 5.1.8 Drying Oven 103 ± 2 C 5.1.9 Graduated Cylinders 25 mL 5.1.10 Spatula 5.1.11 Aluminum Weighing Dishes 70 mm 5.1.12 Filter Paper Whatman #2, or equivalent 5.1.13 Anti-Bumping Granules Teflon chips
5.2.1 Digestion Apparatus 5.2.1.1 Block digester BD40 for 50 mL tubes 5.2.1.2 Digestion Rack For 50mL tubes 5.2.1.3 Digestion Tubes 50 mL 5.2.1.4 Anti-bumping granules
5.2.2 AA3 Autoanalyzer
5.2.2.1 Autosampler 5.2.2.2 Proportioning pump 5.2.2.3 Multitest MT8 Manifold TKN (see schematic) 5.2.2.4 Multitest MT8 Manifold TP (see schematic) 5.2.2.5 Dual Channel Colourimeter 660nm filter, 1.0 x 10mm flow cell 5.2.2.6 Pump Tubes 5.2.2.7 4 mL Sample Cups
5.2.3 Data Acquisition Interface Dual Channel
5.2.4 Computer System
5.2.5 Software Current Instrument Software 6.0 Reagents and Standards 6.1 Reagents
Document ordering, receiving and storage of Reagents, Supplies and Standards, using the appropriate forms, as directed in SOP-009 Receiving of Chemicals, Products and Essential Parts, and other applicable procedures.
Document all Reagent Preparations on the appropriate TP/TKN Digestion Reagent Preparation Data Sheet, following SOP-010 Chemical Preparation.
6.2.1 Sulphuric Acid Solution, 20%: Carefully add 200 mL of Concentrated Sulphuric Acid, H2SO4, to
about 600 mL of DW in a properly labelled 1000 mL amber glass bottle, cool to room temperature and dilute to 1000 mL. Prepare in a fume hood, using suitable protective equipment. This solution is stable indefinitely.
6.2.2 Mercuric Oxide Solution: Dissolve 20.0g of Mercuric Oxide, HgO, in 250 mL of 20% Sulphuric
Acid Solution (6.2.1). Prepare as needed and store in a properly labelled 500 mL amber glass bottle at room temperature. This solution is stable indefinitely.
6.2.3 Digestion Acid: Dissolve 133 g of Potassium Sulphate, K2SO4, in 600 mL DW and carefully
add 200 mL of Concentrated Sulphuric Acid, H2SO4. When the solution has cooled, add 50 mL of Mercuric Oxide Solution (6.2.2), dilute to 1000 mL with DW, mix well and store in a properly labelled 1000 mL amber glass bottle at room temperature. Prepare as needed. This solution is stable indefinitely.
6.2.4 Dilution Solution: Use Wash Solution 6.3.1
6.2.5 Anti-Bumping Granules (Cleaning): Soak in 10% sulphuric acid (H2SO4) for at least 24 hours,
rinse three times with DW.
6.3 Detection System Reagent Solutions
Document all Reagent Preparations on the appropriate TP/TKN Reagent Preparation Data Sheet, forms TPTKN-2, following SOP-010 Chemical Preparation. All Detection System Reagent Solutions may be prepared in the Reagent Containers in which they are stored.
6.3.1 Wash Solution: Dilute 30 mL of Concentrated Sulphuric Acid and 18.6 g Potassium Sulphate
to 1000 mL with DW. Prepare as needed and store in a properly labelled 1000 mL amber glass bottle at room temperature. This solution is stable indefinitely.
6.3.2 Buffer (TKN): Dissolve 50g of sodium potassium tartrate (C4H4KNaO6.4H2O) and 4-6 NaOH
pellets in 600mL of DW. **To prevent contamination, apply heat and maintain boiling temperature for 1 hour**. Add 27.2 g Disodium Hydrogen Phosphate-7-Hydrate, (Na2HPO4.,7H2O), 64 g of 50% w/w Sodium Hydroxide Solution (NaOH) and), and 4.0 mL of 15% Brij-35 solution. Dilute to 1000mL. Prepare as needed and store in a properly labelled 1000 mL amber glass bottle at room temperature. This solution is stable for one month. IMPORTANT NOTE: Do not allow Brij-35 to enter the TP manifold as it forms an insoluble compound with molybdate.
6.3.3 Sodium Salicylate Solution (TKN): Dissolve 20 g Sodium salicylate (Na2C7H5O3), in 300 mL
DW. Add 0.5g of Sodium nitroprusside (Na2[Fe(CN)5NO].2H2O, dilute to 500mL with DW and mix thoroughly Prepare as needed and store in a properly labelled 500 mL amber glass bottle at room temperature. This solution is stable for one week
6.3.4 Hypochlorite Solution (TKN): Dilute 17.5mL of Sodium Hypochlorite Solution, 5% Chlorine into
250mL of DW. Prepare as needed and store in properly labelled 500mL amber glass bottle at room temperature. Prepare solution daily.
6.3.5 Acid/Salt Diluent (TP): Dissolve 5 g of Sodium Chloride in approximately 700mL of DW, add 12 mL of Concentrated Sulphuric Acid and dilute to 1000 mL with DW. Add 2.0 g of sodium dodecyl sulfate and mix. Prepare as needed and store in a properly labelled 1000 mL amber glass bottle at room temperature. This solution is stable indefinitely.
6.3.6 Molybdate/Antimony Solution (TP): Dissolve 3.1 g of Ammonium Molybdate and 0.085 g of
Antimony Potassium Tartrate in approximately 300 mL of DW, using heat if necessary. Dilute to 500mL with DW, prepare as needed and store in a properly labelled 500 mL amber glass bottle at room temperature. This solution is stable for two weeks.
6.3.7 Ascorbic Acid Solution (TP): Dissolve 7.5 g of Ascorbic Acid, C6H8O6 in about 300mL of DW.
Dilute to 500mL with DW, prepare as needed and store in a properly labelled 500 mL amber glass bottle at room temperature. This solution is stable for one week.
6.3.8 15% Brij35 Solution (TKN Buffer): Dissolve 15g of Brij35 pellets in 100mL of DW. Prepare as
needed and store in properly labelled plastic bottle at room temperature. This solution is stable for one month. IMPORTANT NOTE: Do not allow Brij-35 to enter the TP manifold as it forms an insoluble compound with molybdate.
6.3.9 System Rinse Solution (Wash-Out – as required): Add 2g of sodium dodecyl sulphate to
approximately 700 mL of DW. Mix thoroughly and dilute to 1000mL. Prepare as needed and store in a properly labelled 1000 mL amber glass bottle at room temperature. This solution is stable indefinitely. Do not pump this solution through the wash line as surfactants are not to run through wash pot.
6.3.10 System Wash Solution (For Cleaning – as required): Dilute 60g of NaOH and 6g of
Tetrasodium EDTA up to 1000mL with DW. This solution is to be pumped through all tubing for 10 minutes every day or if the baseline and carryover increases.
6.3.11 System Acid Wash (For Cleaning – as required): Dilute 86mL of Concentrated Sulphuric Acid
in 1000mL DW. Prepare as needed and store in properly labelled 1000mL amber bottle. This solution is stable indefinitely. This solution is to be pumped through all tubes for 10 minutes following a pump tube change.
6.3.12 10% Bleach Solution (For Cleaning – as required): Dilute 100mL of Concentrated Bleach in
100mL of DW. Prepare as needed and store in 1000mL amber glass bottle at room temperature. This solution is stable indefinitely. This solution is to be pumped through all tubes for 10 minutes following a pump tube change.
6.4 Calibration Standards
Document all Calibration Standard Preparations on the appropriate TP/TKN Standards Preparation Data Sheets TPTKN-3, following SOP-021 Inorganic Calibration and Quality Control Standard Preparation.
6.4.3 Calibration Intermediate Standard, 80 mg/L N, 20 mg/L P: Pipette 8.0 mL of Nitrogen Calibration Stock Standard (6.4.1) and 2.0 mL of Phosphate Calibration Stock Standard (6.4.2) into a 100 mL Class A volumetric flask containing about 80 mL of DW and 1.0 mL of 20% Sulphuric Acid Solution (6.2.1), mix thoroughly and dilute to volume with DW. Prepare as needed and store in a properly labelled 90 mL Specimen Cup at room temperature. This solution is stable for two months. 6.4.3.1 Low Level TP Calibration Intermediate Standard, 10mg/L P: Pipette 1mL of Phosphate Calibration Stock Standard (6.4.2) into a 100mL Class A volumetric flask containing about 80mL of DW and 1,0mL of 20% Sulphuric Acid Solution (6.2.1), mix thoroughly and dilute to volume with DW. Prepare as needed and store in a properly labelled 90mL Specimen Cup at room temperature for up to two months.
6.4.4 Calibration Working Standards, 0.0-2.0 mg/L P and 0-8.0 mg/L N: Pipette the appropriate
amount of the Calibration Intermediate Standard (6.4.3) into TKN/TP digestion tubes (see Section 7.2, below. Prepare fresh Working Standards for each run.
Standard Volume of Intermediate
Solution (mL)Final Concentrations,
mg/L as PFinal Concentrations
mg/L as N
S1 0 0.00 0.00
S2 0.05 0.04 0.16
S3 0.10 0.08 0.32
S4 0.50 0.40 1.60
S5 1.0 0.80 3.20
S6 2.0 1.60 6.40
6.4.4.1 Low Level TP Calibration Working Standards, 0.0-0.400 mg/L P: Pipette the
appropriate amount of the Low Level Calibration Intermediate Standard (6.4.3.1) into TKN/TP digestion tubes (see Section 7.2, below)
Standard Volume of Intermediate
Solution (mL) Final Concentrations,
mg/L as P
S1 0 0.00
S2 0.025 0.001
S3 0.05 0.02
S4 0.1 0.04
S5 0.2 0.08
S6 1.0 0.4
6.5 Quality Control Standards
Document all Quality Control Standard Preparations on the appropriate TP/TKN Standards Preparation Data Sheets TPTKN-3, following SOP-021 Inorganic Calibration and Quality Control Standard Preparation.
6.5.1 Quality Control Stock Standard, 8000 mg/L N, 2000 mg/L P: Dissolve 15.28 g of Ammonium
Chloride, NH4Cl, and 4.39 g of Potassium Dihydrogen Phosphate, KH2PO4, in about 400 mL of DW in a 500 mL Class A volumetric flask, mix well and dilute to volume with DW. Store in a properly labelled amber glass bottle at 4 ± 3C. This solution is stable for six months.
6.5.2 Quality Control Intermediate Standard, 80 mg/L N, 20 mg/L P: Pipette 1.0 mL of QC Stock
Standard (6.5.1) into about 80 mL of DW and 1.0 mL of 20% Sulphuric Acid Solution (6.2.1), in a 100 mL Class A volumetric flask, mix well and dilute to volume with DW. Prepare as needed and store in a properly labelled 90 mL Specimen Cup at room temperature. This solution is stable for two months.
6.5.3 Quality Control Working Standards, 0.08-1.20 mg/L P and 0.32-4.8 mg/L N: Pipette the
appropriate amount of the Quality Control Intermediate Standard (6.5.2) into TKN/TP digestion tubes (see Section 7.2, below)
QC Standard Volume of QC Intermediate Solution (mL)
Final Concentrations, mg/L as P
Final Concentrations, mg/L as N
QC1 0.10 0.08 0.32
QC2 1.00 0.80 3.2
QC3 1.5 1.20 4.8
6.5.3.1 Low Level TP Quality Control Working Standards, 0.008-0.020 mg/L P: Pipette
the appropriate amount of the Quality Control Intermediate Standard (6.5.2) into TKN/TP digestion tubes (see Section 7.2, below)
QC Standard Volume of QC Intermediate Solution (mL)
Final Concentrations, mg/L as P
QC1 0.01 0.008
QC2 0.1 0.08
QC3 0.25 0.20
6.5.4 Recovery Stock Standard (Water), 566.5 mg/L N: Dissolve 1.00 g of Glycine p-Toluenesulfonate (5.665% N) in approximately 80 mL of DW in a 100 mL Class A volumetric flask, mix well and dilute to volume with DW. Prepare as needed and store in a properly labelled 90 mL Specimen Cup at room temperature. This solution is stable for six months.
6.5.5 Recovery Intermediate Standard (Water), 5.665 mg/L N: Pipette 1.0 mL of Recovery Stock Standard (6.5.3) into approximately 80 mL of DW in a 100 mL Class A volumetric flask, mix well and dilute to volume with DW. Prepare as needed and store in a properly labelled 90 mL Specimen Cup at room temperature. This solution is stable for two months.
6.5.6 Method Recovery Working Standard, 1.14 mg/L N (RecN)
6.5.6.1 Waters: Pipette 5.0 mL of Recovery Intermediate Standard (6.5.4) into a
TKN/TP digestion tube (see Section 7.2, below). Prepare fresh for each digestion.
6.5.7 Spiking Solution: Same as QC Intermediate Standard (6.5.2)
6.5.8 Blank: The digestate of an aliquot of DW, or an aliquot of Blank Solution (6.2.4), is analysed after
each group of ten Samples, to monitor possible cross-contamination.
6.5.9 Duplicate Sample: A second aliquot of a randomly chosen Sample is digested and analysed after each group of ten samples. Duplicates must not be run consecutively.
6.5.10 Spiked Sample- Water: A third aliquot of the Sample chosen for the Duplicate (6.5.8) is spiked
and then digested and analysed after every second group of ten samples. The Duplicate and
6.5.10.1 Spiked Sample, 3.20 mg/L N and 0.80 mg/L P: Pipette 1.0 mL of Quality Control Intermediate Standard (6.5.2) into a TKN/TP digestion tube (see Section 7.2, below) containing an appropriate aliquot of sample.
6.5.10.2 Low Level TP Spiked Sample, 0.20 mg/L P: Pipette 0.25 mL of Quality Control Intermediate Standard (6.5.2) into a TKN/TP digestion tube (see Section 7.2, below) containing an appropriate aliquot of sample.
6.5.11 Method Recovery Working Standard (Dewaterd Sludge)
6.5.11.1 Liquid Sludge/Biosolid CRM 545 - 41400 mg/L as N, 19100 mg/L as P Cat# 545: Accurately weigh out 0.10g of the CRM. Follow soil sample preparation digestion instructions starting at 7.2.4.3.
6.5.12 Method Recovery Working Standard (Biosolid/Soil)
6.5.12.1 Soil CRM 542 – 911 ug/g as N, 1710 ug/g as P CAT#542: Accurately weight
out 0.10g of the CRM. Follow soil sample preparation digestion instructions starting at 7.2.4.3
7.0 Test Procedures
Refer to the Instrument manuals and the Instrument Software documentation for details of setting up the Instrument and Software for a sample run.
7.1 Instrument Settings
7.1.1 Channel 1 (TP) 7.1.1.1 Filters, nm: 660 7.1.1.2 Flow Cell: 1.0 x 10mm 7.1.1.3 Chemistry: Direct
7.1.2 Channel 2 (TKN)
7.1.2.1 Filters, nm: 660 7.1.2.2 Flow Cell: 1.0 x 10mm 7.1.2.3 Chemistry: Direct
Samples and Method Recovery Spiked Samples must be included in each tray of digestion tubes. Treat the samples, standards and blanks identically. The setup shown in Table 1 of Appendix A is recommended.
7.2.4 Sample Preparation
7.2.4.1 Water Samples: Mix each sample well and transfer 25 mL to the digestion
tube, if no dilution is required. Samples known to be high in TKN and/or TP may be diluted directly in the digestion tube by transferring a lesser amount.
7.2.4.2 Liquid Sludge Samples: Mix each sample well and accurately transfer 5.0 mL into a 50 mL volumetric flask. Dilute to mark with DW. Mix thoroughly, and quantitatively transfer 2.5 mL of this solution to the digestion tube. This gives a dilution factor of 100 times on the original sample.
7.2.4.3 Biosolid-Dewatered Sludge/Soil Samples: Dry a sufficient quantity of soil in the drying oven at 105 ± 3 ºC and mix well. (If % Solids is run on the soil sample, the dried material from that test may be used.) Weigh 0.10 g of dried sample and quantitatively transfer to the digestion tube, rinsing with DW and diluting to 25 mL.
7.2.4.4 Record the sample ID, and dilution in the appropriate position on the TP/TKN
Digestion Data Sheet.
7.2.5 Using a pre-calibrated dispenser, add 5.0 mL of the Digestion Acid (6.2.3) to each digestion tube.
7.2.6 Add a few acid-washed boiling chips to each tube. 7.2.7 Place the rack containing the tubes in the block at 200°C and hold the temperature for 80
minutes. See Digestion Parameter Table 7.2.12. Note: Until all samples start to boil, do not leave the area in case one of the tubes bumps or boils over. If the digestion batch includes sludge or soil samples, the whole digestion process should be monitored. If one of the tubes does boil over turn off the digester and allow block to cool. Discard all samples and repeat the digestion procedure. Do not handle the tubes until they have cooled.
7.2.8 When the water has completely boiled away (after 80 minutes at 200C), place in 380°C digester. Hold the temperature at 380 C for 40 minutes. See Digestion Parameter Table 7.2.12
7.2.9 Remove the rack from the digestion block and allow the tubes to cool in the fume hood for 15
minutes. Cautiously add 25.0 mL of DW to each tube using a precalibrated auto-pipettor and vortex to mix thoroughly. Transfer digested samples to 80mL sample cup. Digested samples may be kept for up to two weeks.
7.2.10 Digestates containing solids may be filtered to remove particulate prior to transfer to the
sample cups. Filter the digestate through a folded Whatman #2 filter paper into a properly labelled 90 mL Specimen Cup. Digestion tubes should not be allowed to air dry prior to cleaning.
7.2.11 When one or more digestates are filtered, prepare a filter Blank using Blank Solution (6.2.4).
7.3.1 Ensure that each Reagent Container contains sufficient solution to complete a sample run. Prepare fresh reagents if needed. Consult the Instrument log book to ensure that all scheduled maintenance has been performed.
7.3.2 Place all the reagent lines and the Sample Wash line in the System Wash container. Insert the
platen on the proportioning pumps and turn on the pump, heaters and colourimeters, and flush the system for 10 minutes, then run reagent lines through the system rinse container for 10 additional minutes. When changing chemistries, flush the system with DW for 10-15 minutes.
7.3.3 Place the reagent lines into their respective containers and allow the system to equilibrate for
15-20 minutes. Add Salicylate last
7.3.4 Turn on the computer and load the Instrument Software. Set up a Sample Table File named ‘YYMMDDNP’ and Sample Data Files named ‘YYMMDDN’ and ‘YYMMDDP’.
7.3.5 Start the software and view the baselines for several minutes. Once stable set the base line,
right click the mouse after channel 1 has been selected and press “set baseline”. Perform the same procedure for channel 2. This can be performed multiple times in the beginning of the run.
7.3.6 The software will set the base line to approximately 5%. If a level baseline is not achieved on
each system, confirm that all reagents are being delivered through the pump, that all reagent lines are in the proper Reagent Containers and that there are no leaks in the system.
7.3.7 If a level baseline cannot be achieved, it may be necessary to prepare new Detection System
Reagent Solutions. Consult the Instrument log book, the Non Conformity log and the instrument manuals for further information.
7.3.8 Go into the XY2 sampler 1 Icon and select the high standard (S5) to sample, after a few minutes select wash and wait for the peak to appear on the screen (approx. 12 min) Once the peak is about 80-95% select a channel and right click mouse and select “set gain”. Repeat this process with the other channel.
7.4 Calibration and QC Checks
7.4.1 While the Instrument equilibrates, fill the Sample Table File with the Sample ID’s, dilutions and types to control the sample run. Refer to Table 2 in Appendix A for a sample sequence that satisfies the QC requirements of the method. The table includes entries for Baseline (B) and Drift (D) samples to adjust for minor shifts in baseline or sensitivity.
7.4.2 Prepare either the 4 mL autosampler cups or the 15.5mL tubes containing Blanks, Calibration
Standards, QC Samples, Client Samples, Duplicates and Spikes and place them in the autosampler tray in the order indicated by the Sample Table File. Record Sample ID’s, dilutions and cup locations on the Analysis Sample Log.
7.4.4 Monitor the run carefully during analysis, using the SEAL Program to plot the Standard Curves for each analytical system. If calibrations are satisfactory, proceed to the QC Checks in step 7.4.9.
7.4.5 If the R2 value for either calibration does not exceed 0.995, restart the run.
7.4.6 If a satisfactory Calibration is still not achieved, confirm that all reagents are being delivered
through the pumps, that all reagent lines are in the proper Reagent Containers and that there are no leaks in the system. Correct any faults, and restart the run. (It may be necessary refill the other autosampler cups which have already been sampled).
7.4.7 If this Calibration run fails, stop the run and repeat the Digestion Procedure (Section 7.2) for
all Standards and Samples in the batch, using freshly prepared Calibration Intermediate Standard (6.4.3 or 6.5.3) to prepare the Calibration Working Standards.
7.4.8 If a satisfactory Calibration is still not achieved, cleaning or re-tubing of one or both of the
analytical cartridges, or other equipment adjustments may be required. Refer to the Instrument Manuals for further details of maintenance and repair procedures.
7.4.9 Monitor the run to view the results for the QC Samples. If the calculated values meet the
current internal limits of acceptability found in the method QC log, proceed to Section 7.5 Analysis.
7.4.10 If the result for a QC Sample falls outside acceptable limits, prepare a new autosampler cup of
that QC Sample digestate and insert it in the next available position of the tray and the Sample Data File. If the result for the new QC Sample meets the current internal limits of acceptability found in the method QC log, proceed to Section 7.5 Analysis.
7.4.11 If all QC requirements are met for only one of the analyses (channels), the run may be
continued (section 7.5 Analysis) and, if QC results are acceptable throughout the run, results calculated and reported for that analyte only. The colourimeter and cartridge heater for the other channel may be turned off and the reagent lines transferred to the DW rinse container. The pump on the second channel must continue to run to maintain the sample split ratio.
7.4.12 If the new QC Sample result falls outside acceptable limits, stop the run and repeat the
digestion procedure (Section 7.2) for all Standards and Samples in the batch, using freshly prepared Quality Control Intermediate Standard (6.6.2 and 6.7.2) to prepare the Quality Control Working Standards.
7.4.13 Record all Calibration and QC failures, and sample run restarts and re-digestions in the Non
Conformity Log. 7.5 Analysis
7.5.1 The Sample Table File, and the cups in the Autosampler tray, must be arranged so that a Blank, a Duplicate Sample, one of the QC samples used alternately and a Drift Standard are analysed following each 10th sample. Analyse a Spiked Sample with every other set of ten samples. The suggested order is shown in Table 2 of Appendix A.
7.5.2 The remainder of each digestate should be retained in the cup until the sample run is
completed and checked. If further storage is needed, digestates can be stored in properly labelled 80 mL sample cups at room temperature for up to two weeks.
Note: At least one Duplicate Sample, and one Spiked Sample must be run with each set of Samples.
7.5.3 If the result for a Duplicate, Spiked or QC Sample falls outside acceptable limits, prepare a new autosampler cup of the digestate of that Sample and insert it in the next available position of the tray and the Sample Data File. If the result for the new Sample meets the current internal limits of acceptability found in the method QC log, the sample run may be continued.
7.5.4 If a second failure occurs during the run, stop the run and repeat the Digestion Procedure
(Section 7.2) for all Samples and Standards in the batch.
7.5.5 If all QC requirements are met for only one of the analyses (see Step 7.4.11, above), results may be calculated and reported for that analyte only.
7.5.6 Any over-range samples must be re-analysed after appropriate dilution with Wash Solution.
7.5.7 Record all QC failures in the Non Conformity Log.
7.5.8 After all sample digestates and dilutions are analyzed, shut down the system as described in
the operation manual. Place all reagent lines and the sample Wash line in the DW rinse container and pump DW through the lines for about 15 minutes to flush reagents. Turn off the pumps and remove the platens.
7.6 Documentation Procedure
7.6.1 Document all Sample ID’s, dilutions on the Analysis Sample Digestion Data Sheet TPTKN-1. Print out the calibration curves, print the results for the current group and store the data in the appropriate data binder. Printouts need to be stamped with the Data Validation stamp and signed off and dated by the analyst when the all data has been validated.
7.6.2 Document all preparations of Calibration and QC Standards using the Standards Preparation
Logs.
7.6.3 Document all Calibration and QC failures, sample run restarts and re-digestions in the Non Conformity Log.
7.6.4 Document all maintenance, comments and any changes in the Instrument log book.
7.6.5 QC data must be entered into the QC Log files after each batch of analysis by the analyst.
7.6.6 The QC data files are stored on the Caduceon File Server.
7.6.7 Open these files and enter the data (Dates, Expected and Found Concentrations for the QC samples, Duplicate results, Sample Spike results and Blanks) in the appropriate fields.
7.6.8 The Excel program will calculate the % recoveries of the QC samples and Spikes, the %
difference of the Duplicate samples and the average concentration of the blanks. Where appropriate, points are added to Control Charts automatically.
7.6.9 The Excel program files will be maintained by the QA officer. Control Charting and trend
analysis are performed using CP-031. 8.0 Calculations and Reporting
The software program will automatically perform the concentration of N or P in the solution analyzed. The analyst will have to calculate the true concentration of the sample if there were any dilutions or any sample preparations. See Sections 8.1 and 8.2. Alternatively, the analyst can enter all dilution factors and weights into the software and allow the software to calculate the final concentration. Note that units will not be properly displayed for solid samples.
where A = Final Dilution Volume (mL) B = Sample Volume taken (mL) 8.2 Sample Concentration
8.2.1 Water mg/L:
Concentration (mg/L) = C * DF
where C = Result from Instrument Software (mg/L) DF = Dilution factor
8.2.2 Liquid Sludge/Biosolid:
Concentration (mg/L) = 20 * C * DF / mL of sample digested
where C = Result from Instrument Software (mg/L) DF = Dilution factor
8.2.3 Soil, Dewatered Biosolid
Concentration (µg/g) = 20 * C * DF / W
where C = Result from Instrument Software (mg/L) DF = Dilution factor W = Weight of Solid, g
8.2.4 Organic nitrogen can be calculated from the following formula having an MDL of 0.1mg/L:
Org-N, mgN/L = TKN, mgN/L – NH3(N), mg/L
Where TKN value is the final calculated concentration from 8.1
NH3(N) is the final calculated concentration from Caduceon
method A-NH3-01
8.3 Refer to Caduceon CP-012 LIMS Training for details of the recording of results in the Laboratory Information Management System (LIMS).
8.3.1 The details of any sample pre-treatment (e.g. soil or sludge dilution) are entered in the
Laboratory Job module of the LIMS Job Book. written on the bench sheet, form TP/TKN-1.
8.3.2 The Total Kjeldahl Nitrogen (TKN as N) results and digestate dilution factors, if any, are entered into the Instrument Run module of the LIMS Job Book. onto the bench sheet, form TP/TKN-1.
8.3.3 The Total Phosphorus (TP as P) results and digestate dilution factors, if any, are entered into
the Instrument Run module of the LIMS Job Book. onto the bench sheet, form TKN/TP-1.
9.1.11.1 The Method Performance is monitored by the results of PT sample analysis. Each round’s reported results and consensus values are entered in the Method QC log. The % Recovery is calculated on a minimum of 8 sample results. The maximum allowable deviation of a PT result is based on 3sd.
9.1.12.1 The expanded uncertainty is determined as per CP-026. The data
accumulated in the QC log is used to calculate the expanded uncertainty detailed in the tables below.
9.1.12.2 Total Kjeldahl Nitrogen (TKN as N)
Typical Concentration
(ug/g) Combined Uncertainty
(ug/g) Expanded Uncertainty *
(Percent)
10 7 67
100 58 58
600 346 58
* Expanded uncertainty at 95% confidence interval (coverage factor k=2).
9.1.12.3 Total Phosphorus (TP as P)
Typical Concentration (ug/g)
Combined Uncertainty (ug/g)
Expanded Uncertainty * (Percent)
10 2.7 27
50 13 26
200 51 25
* Expanded uncertainty at 95% confidence interval (coverage factor k=2).
9.1.13 Method Performance
9.1.13.1 The Method Performance is monitored by the results of PT sample analysis. Each round’s reported results and consensus values are entered in the Method QC log. The % Recovery is calculated on a minimum of 8 sample results. The maximum allowable deviation of a PT result is based on 3sd.
9.1.13.2 Total Kjeldahl Nitrogen (TKN as N) – Insufficient data available.
9.1.13.3 Total Phosphorus (TP as P) – Insufficient data available.
Mean % Recovery 102.4
Standard Deviation (sd) 5.4
3sd (99% Confidence Level) 16.2
Data Points 40 Updated March 24, 2017
9.1.14 Method Uncertainty – Biosolids
9.1.14.1 The expanded uncertainty is determined as per SOP-056. The data accumulated in the QC log is used to calculate the expanded uncertainty detailed in the tables below.
9.1.14.2 Total Kjeldahl Nitrogen (TKN as N)
Typical Concentration
(µg/g) Combined Uncertainty
(µg/g) Expanded Uncertainty *
(Percent)
10000 1370 27
25000 3420 27
50000 6840 27
* Expanded uncertainty at 95% confidence interval (coverage factor k=2).
9.1.14.3 Total Phosphorus (TP as P)
Typical Concentration (µg/g)
Combined Uncertainty (µg/g)
Expanded Uncertainty * (Percent)
10 1.57 31
20 2.70 27
30 3.91 26
40 5.15 26
* Expanded uncertainty at 95% confidence interval (coverage factor k=2).
9.1.15 Method Performance
9.1.15.1 The Method Performance is monitored by the results of PT sample analysis. Each round’s reported results and consensus values are entered in the Method QC log. The % Recovery is calculated on a minimum of 8 sample results. The maximum allowable deviation of a PT result is based on 3sd.
9.1.15.2 Total Kjeldahl Nitrogen (TKN as N) – Insufficient data available.
9.1.15.3 Total Phosphorus (TP as P) – Insufficient data available.
Mean % Recovery 107
Standard Deviation (sd) 22.5
3sd (99% Confidence Level) 67.5
Data Points 7 Updated March 03, 2017
9.2 Based on the method validation data supplied above, this method has been deemed as
fit for its intended use (as stated in Section 1 of this document).
9.3 Unless otherwise indicated all tables found in section 9 of this method contain original validation data and are present to support the statements made in section 9.2. All analysts must refer to the relevant QAQC logs for the current limits when verifying data is acceptable for reporting.
10.0 References
10.1 Analytical Methods
10.1.1 Seal Method No. G-189-97 Rev.3 Total Phosphorus in Acid Digests 10.1.2 Seal Method No. G-188-97 Rev.6 Total Kjeldahl Nitrogen in Acid Digests 10.1.3 Ontario Ministry of the Environment and Climate Change, Laboratory Services
Branch 2017. E3516 Rev 2 10.1.4 USEPA, Methods for Chemical Analysis of Water and Wastes, Method 351.2,
March 1983.
10.2 Caduceon Standard Operating Procedures
10.2.1 Caduceon CP-024 Preparation of Reagent-Grade Water. 10.2.2 Caduceon SOP-009 Receiving of Chemicals, Essential Parts and Equipment. 10.2.3 Caduceon SOP-010 Chemical Preparation. 10.2.4 Caduceon SOP-017 Calibration and Method Performance Verification. 10.2.5 Caduceon CP-031 Control Charting. 10.2.6 Caduceon CP-022 Volumetric Calibration and Verification. 10.2.7 Caduceon CP-021 Balance Calibration and Verification. 10.2.8 Caduceon SOP-021Inorganic Calibration and Quality Control Standard
Preparation. 10.2.9 Caduceon SOP-027 Inorganic Quality Control Analysis. 10.2.10 Caduceon CP-032 Non Conformity Logs. 10.2.11 Caduceon CP-026 Uncertainty in Measurement. 10.2.12 Caduceon CP-012 LIMS Training. 10.2.13 Caduceon Safety Manual.
10.3 Instrument Manuals
10.3.1 Seal AA3 Instrument Manuals. 10.3.2 Seal Software Manual 10.3.3 Block Digester Manual.
Appendix A: Recommended Setup for Digestion and Analysis Sequence
Table 1 – Digestion Set up
S1 S2 S3 S4 S5
S6 Blank QC1 QC2 QC3
Sample Sample Sample Sample Sample
Sample Sample Sample Sample Sample
Duplicate 1 Spike 1 or CRM Sample Sample Sample
Sample Sample Sample Sample Sample
Sample Sample Duplicate 2 Sample Sample
Sample Sample Sample Sample RECN
(One of the sample positions may be left open for a thermometer to monitor the digestion block temperature.) Note: Only the first rack will contain the calibration standards. All other racks will still require the appropriate QC (recovery check and sufficient duplicates and spikes if digested on the same day)
Figure 1 - Analysis Sequence
1 S5 to trigger Data Acquisition – Labelled as Primer 2 S5 – High – Carry Over Correction 3 S1 – Low – Carry Over Correction 4 S1 – Low – Carry Over Correction 5 B - Baseline Correction – Wash 6 S1 7 S2 8 S3 9 S4 10 S5 11 S6 12 Blank – Digested Blank 13 B - Baseline Correction – Wash 14 QC1 15 QC2 16 QC3 17 Null – Wash 18 Drift - Drift Correction S5 19 Blank – Digested Blank 20 Baseline – Baseline Correction – Wash 22-31 Samples 1-10 32 Duplicate of one sample from 1-10 33 Spike of the duplicate sample (water matrices only) or CRM for Soil/Biosolids 34 Blank – Digested Blank 35 Baseline – Baseline Correction – Wash 36 Drift - Drift Correction S5
36 QC1 37 Null - Wash 38-48 Samples 11-20 49 Duplicate of one sample from 11-20 50 Blank – Digested Blank 51 Baseline - Baseline Correction – Wash 52 Drift - Drift Correction S5 52 QC2 53 Null – Wash 54-64 Samples 21-30 65 Duplicate of one sample from 21-30 66 Spike of the duplicate sample (water matrices only) or CRM for Soil/Biosolids 67 Blank – Digested Blank 68 Baseline – Baseline Correction – Wash
69 Drift - Drift Correction S5 69 QC3 70 Null 71 Repeat Pattern end run with
Blank Baseline – Baseline Correction – Wash Drift - Drift Correction S4 QC – Calibration Check Sample End Run
1 The Standard S5 is run first to trigger the collection of data by the Software program. 2 The baseline correction for TKN/TP is sampled from the wash pot labelled 0. 3 The digested blank labelled 901 is run to correct for baseline drift when running Low Level TP. 4 D5 is the same as S5 and is used to correct for sensitivity drift and is run after the standards, after
QC checks and at the end of the run. 5 The last three samples of any sequence must be a baseline to correct for baseline drift, QCNP-0?
Caduceon Environmental Laboratories Page: 24 of 25 Total Kjeldahl Nitrogen/Total Phosphorus Revision Date: 26 Nov, 2019 Method TPTKN-001 Revision #: 2.6 Management Review: MD Quality Review: SKB Log of Revisions
Date Rev Description Author
13-Jun-2016 2.4 Section 6.1 updated to include missing chemical Section 6.3 updated to correct or include missing solution preparations Section 6.5.11 updated to reflect current CRM information Section 7.1.3.2 updated to correct ratio Section 7.2 updated to reflect current practice Section 7.5.2 updated to update storage conditions Appendix A update to correct sequence
AB
05-Jul-2018 2.5 Updated to include Low Level run option for TP Section 3.1 updated to SDS Section 6.3.2 updated to correct amount of NaOH added Section 6.4 update to correct supplier Section 6.5.10 and 6.5.11 updated to correct matrices Section 7.5.2 updated to correct temperature Section 9 updated to include additional PT data and statement about validation data
SB
26-Nov-2019 2.6 Section 6.3 updated to include form TPTKN-2 Section 6.2.2 updated mercuric oxide preparation Section 6.2.5 updated cleaning process Section 6.3.2 updated buffer preparation Section 6.3.3, 6.4.4 updated holding times Section 6.5.10.2 updated solution name Section 7.3.2 updated procedure Section 8.3 updated to include form TPTKN-1 Section 10.1.3 updated method reference
AB/SB
uncontrolled
Caduceon Environmental Laboratories Page: 25 of 25 Total Kjeldahl Nitrogen/Total Phosphorus Revision Date: 26 Nov, 2019 Method TPTKN-001 Revision #: 2.6 Management Review: MD Quality Review: SKB Document Review This document was last reviewed and authorized by: Laboratory Branch Manager Date Laboratory Branch Manager Date Laboratory Branch Manager Date Laboratory Branch Manager Date
