Hach Company TNTplus 880 Total Kjeldahl Nitrogen 

Method 10242 

Simplified Spectrophotometric Measurement of Total 

Kjeldahl Nitrogen in Water and Wastewater 
Hach Company TNTplus 880 s-TKN Method 10242 

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Hach Company TNTplus 880 Method 10242 

Simplified Spectrophotometric Measurement of Total Kjeldahl Nitrogen 

 in Water and Wastewater 

Revision 1.1 

January 10, 2013 

1.0 Scope and Application 

1.1 This method is for the measurement of total Kjeldahl nitrogen (TKN) in water and wastewater. 

 

1.2 The method may be used as an alternative to other methods approved at 40 CFR 136, Table IB for 

the purposes of regulatory reporting of TKN. 

1.3 The method is for use in the United States Environmental Protection Agency’s (EPA’s) survey and 

monitoring programs for the measurement of TKN under the Clean Water Act. 

1.4 The method is applicable in the range from 1.0 to 16.0 mg/L TKN. The range of detection can be 

extended with dilution of samples that exceed 16.0 mg/L TKN. 

2.0 Summary of Method 

2.1 Total Kjeldahl nitrogen is the sum of free-ammonia and organic nitrogen compounds which are 

converted to ammonium sulfate (NH4)2SO4 and subsequently analyzed colorimetrically using 

salicylate or phenate chemistry with nitroprusside. Organic Kjeldahl nitrogen is the difference 

obtained by subtracting the free-ammonia value from the total Kjeldahl nitrogen value. Total 

nitrogen (TN) is the sum of TKN (free-ammonia and organic nitrogen) plus nitrate and nitrate. 

[TKN] = [(NH3 +NH4

+

)] + [Nitrogenorg] 

[Nitrogenorg] = [TKN] - [(NH3 +NH4

+

)] 

[TN] = [TKN] + [NO3

-

] + [NO2

-

] 

2.2 Hach Method 10242, also known as TNTplus 880 is a simplified green chemistry alternative to 

other methods approved at 40 CFR 136 for the purposes of regulatory reporting of TKN. In the 

simplified total Kjeldahl (s-TKN) method, inorganic (NH3, NH4

+

, NO2

-

) and organic nitrogen are 

oxidized to nitrate by digestion with peroxodisulfate. The nitrate ions react with 2,6-dimethylphenol 

in a solution of sulfuric and phosphoric acid to form a nitrophenol. Oxidized forms of nitrogen in the 

original sample (nitrite + nitrate) are determined in a second test vial and then subtracted, resulting 

in TKN. 

[TKN] = [TN] – [NO3

-

 + NO2

-

] 

3.0 Interferences 

3.1 High levels of oxidizable organic substances (COD) affect the reagent color and give high results. 

3.2 Nitrite concentrations of more than 2.0 mg/L interfere (high-bias results). Add 50 mg of sulfamic 

acid to 5.0 mL of sample, dissolve, and wait for 10 minutes. Analyze the prepared sample as 

described in Section 11. 
Hach Company TNTplus 880 s-TKN Method 10242 

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4.0 Safety 

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely 

determined; however, each chemical should be treated as a potential health hazard. Exposure to 

these chemicals should be reduced to the lowest possible level. It is suggested that the 

laboratory perform personal hygiene monitoring of each analyst using this method and that the 

results of this monitoring be made available to the analyst. 

4.2 Unknown samples may contain high concentrations of volatile toxic compounds. Sample 

containers should be opened in a hood and handled with gloves to prevent exposure. 

4.3 This method does not address all safety issues associated with its use. The laboratory is 

responsible for maintaining a safe work environment and a current awareness file of OSHA 

regulations regarding the safe handling of any chemicals specified in this method. A reference 

file of material safety data sheets (MSDSs) should be available to all personnel involved in these 

analyses. Additional information on laboratory safety can be found in Sections 16.3 and 16.4. 

5.0 Equipment 

Note: Brand names, suppliers, and part numbers are for illustrative purposes only. No endorsement is 

implied. Equivalent performance may be achieved using apparatus and materials other than 

those specified here, but demonstration of equivalent performance that meets the requirements of 

this method is the responsibility of the laboratory. 

5.1 Sampling equipment 

5.1.1 Sample collection bottles – Preferably use polyethylene bottles for collecting and storing 

samples for nitrate analysis. Glass bottles are satisfactory if previously they have not 

contained high nitrogen containing solutions. 

5.1.2 Cleaning 

5.1.2.1 All glassware used should be washed with hot 1:1 HCl and rinsed with distilled 

water. Preferably, this glassware should be used only for the determination of 

nitrate and after use it should be rinsed with distilled water and kept covered until 

needed again. If this is done, the treatment with 1:1 HCl is only occasionally 

required. 

 

6.0 Equipment for Sample Analysis

6.1 Hach Company DR 6000, DR 5000, DR 3900, DR 3800, or DR 2800 spectrophotometer, or 

equivalent 

6.2 Block Digester, capable of heating to 100o

 C. Hach Catalog Number LTV082.53.30001, or 

equivalent 

6.3 Light shield for Hach DR2800 spectrophotometer, Hach Catalog Number LZV646, or equivalent 

6.4 Pipette, variable volume, 1 to 5 mL, Hach Catalog Number BBP065, or equivalent 

6.5 Pipette tips, for BBP065 pipette, Hach Catalog Number BBP068, or equivalent 

6.6 Pipette, variable volume, 0.2 to 1.0 mL, Hach Catalog Number BBP078, or equivalent 
Hach Company TNTplus 880 s-TKN Method 10242 

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6.7 Pipette tips, for BBP078 pipette, Hach Catalog Number BBP079, or equivalent 

7.0 Reagents and Standards

7.1 Reagent water – Water in which TKN is not detected at or above the method level of this method. 

Bottled distilled water, or water prepared by passage of tap water through ion exchange and 

activated carbon have been shown to be acceptable sources of reagent water. 

7.2 TNTplus Simplified TKN (s-TKNTM) Reagent - Hach Catalog Number TNT880. 

7.3 Sulfuric Acid, ACS - Hach catalog Number 97949, or equivalent 

7.4 Sodium Hydroxide, 5N - Hach Catalog Number 245053, or equivalent 

7.5 Method Detection Limit Ammonia Standard Solution – 1.0 mg/L as NH3-N, Hach Catalog Number 

189149, or equivalent 

7.6 Initial Precision and Recovery Ammonia Standard Solution – 1000 mg/L as NH3-N, Hach Catalog 

Number 2354153, or equivalent 

8.0 Sample Collection, Preservation and Storage

8.1 Samples may be collected in clean glass or plastic bottles. 

8.2 Analyze samples as soon as possible. If immediate analysis is not possible, store at ≤6o C and 

analyze within 28 days of collection. 

8.3 Adjust sample pH to less than 2 with sulfuric acid (about 2 mL per liter). 

9.0 Quality Control

9.1 It is recommended that each laboratory that uses this method be required to operate a formal 

quality assurance program (16.1). The minimum requirements of this program consist of an initial 

demonstration of laboratory capability and ongoing analyses of laboratory prepared water 

standards as a test of continued performance to assess accuracy and precision. Laboratory 

performance is compared to established performance criteria to determine if the results of 

analyses meet the performance characteristics of the method. 

9.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable 

accuracy and precision with this method. This ability is established as described in 

Section 9.2. The laboratory shall, on an ongoing basis, demonstrate through analysis of 

the ongoing precision and recovery sample that the analysis system is in control. 

9.1.2 Accompanying QC for the determination of nitrate is required per analytical batch. An 

analytical batch is a set of samples processed during a contiguous 8-hour period. Each 

analytical batch must be accompanied by a laboratory reagent blank (LRB), an ongoing 

precision and recovery sample (OPR), a matrix spike sample (MS), and a matrix spike 

duplicate sample (MSD) resulting in a minimum of five analyses (1, LRB, 1 OPR, 1 

sample, MS, and MSD). 

9.2 Initial demonstration of laboratory capability. 

9.2.1 Method Detection Limit (MDL) - To establish the ability to detect nitrate the analyst shall 

determine the MDL per the procedure in 40 CFR 136, Appendix B (16.2) using the 
Hach Company TNTplus 880 s-TKN Method 10242 

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apparatus, reagents, and standards that will be used in the practice of this method. The 

analyst also shall calculate the Minimum Level (ML) of quantitation by multiplying the 

MDL by 3.18 and rounding to the number nearest to (1,2 or 5) x 10n, where n is a 

positive or negative integer. The calculated MDL should be less than or equal to the MDL 

in Section 13.0 prior to the practice of this method. Similarly, the calculated ML should be 

less than or equal to the ML in Section 13.0 

9.2.1.1 Obtain the Method Detection Limit Ammonia Standard Solution in Section 7.5. 

The concentration of the MDL samples should be 1.0 mg/L NH3

-N. Analyze a 

minimum of seven replicate of this standard according to the procedure in 

Section 11. 

9.2.2 Initial Precision and Recovery (IPR) - To establish the ability to generate acceptable 

precision and accuracy, the analyst shall perform the following operations: 

9.2.2.1 Obtain the Initial Precision and Recovery Limit Ammonia Standard Solution in 

Section 7.6. 

9.2.2.2 Add 0.5 mL of the Initial Precision and Recovery Ammonia Standard Solution into 

a 100-mL volumetric flask and bring to volume with reagent water. The 

concentration of the IPR solution should be 5 mg/L NH3-N. Analyze four replicate 

volumes of this standard according to the procedure in Section 11. 

 

9.2.2.3 Using the results of the set of four analyses, compute the average percent 

recovery (x) and the standard deviation of the percent recovery (s) for nitrate. 

Use the following equation for calculation of the standard deviation of the percent 

recovery: 

 

1

2

2







 

n

n

x

x

s

i

i

 where: 

 n = Number of samples 

 xi

 = % recovery in sample i 

9.2.2.4 Using the values of x and s calculated in the previous step, calculate the Relative 

Standard Deviation (RSD) using the equation below: 

 *100%

X

s RSD 

9.2.2.5 Compare RSD and x with the corresponding limits for initial precision and 

recovery in Table 2. If RSD and x meet the acceptance criteria, system 

performance is acceptable and analysis of samples may begin. If, however, the 

RSD exceeds the precision limit or x falls outside the range for recovery, system 

performance is unacceptable. In this event correct the problem, and repeat the 

test. 

9.3 Laboratory Reagent Blank (LRB) – The laboratory reagent blank (LRB) is an aliquot of reagent 

water that is treated exactly as a sample including exposure to all glassware, equipment and 

reagents that are used with other samples, The laboratory must analyze at least one LRB with 

each batch of samples. Data produced are to asses contamination from the laboratory 
Hach Company TNTplus 880 s-TKN Method 10242 

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environment. Values that exceed the MDL indicate laboratory or reagent contamination should 

be suspected and corrective action should be taken before continuing the analysis. 

9.4 Ongoing Precision and Recovery (OPR) - To demonstrate that the analysis system is in control, 

and acceptable precision and accuracy is being maintained with each analytical batch. An 

analytical batch is a set of samples processed during a contiguous 8-hour period, not to exceed 

20 samples. The analyst shall perform the following operations: 

9.4.1 Prepare a precision and recovery standard following the procedure in Section 9.2.2 and 

analyze at the end of each analytical batch according to the procedure in Section 11. 

9.4.1.1 If the recovery is within the acceptable range of 90 -110%, measurement process 

is in control and analysis of samples may proceed. If, however, the recovery is 

not in the acceptable range, the analytical process is not in control. In this event, 

correct the problem, re-analyze analytical batch, repeating the ongoing precision 

and recovery test. 

9.4.1.2 The laboratory should add results that pass to IPR and previous OPR data and 

update QC charts to form a graphic representation of continued laboratory 

performance. The laboratory should also develop a statement of laboratory data 

quality for each analyte by calculating the average percent recovery (R) and the 

standard deviation of the percent recovery (sr). Express the accuracy as a 

recovery interval from R - 2sr to R + 2sr. For example, if R = 95% and sr = 5%, 

the accuracy is 85% to 105%. 

9.4.1.3 Depending upon specific program requirements, field replicate spikes may be 

required to assess the precision and accuracy of the sampling and sample 

transporting techniques.

9.5 Matrix Spike and Matrix Spike Duplicate Precision and Recovery (MS/MSD) – The laboratory 

must, on an ongoing basis, spike at least 5% of the samples from each analytical batch as 

defined in Section 9.4. 

 9.5.1. The concentration of the spike in the sample should be determined as follows: 

9.5.1.1 If, as in compliance monitoring, the concentration of TKN in the sample is being 

checked against a regulatory concentration limit, the spike should be at that limit 

or one to five times higher than the background concentration determined in 

Section 10, whichever concentration would be larger. 

9.5.1.2 If the concentration of a TKN in the sample is not being checked against a 

regulatory concentration limit, the spike should be at 5 mg/L or one to five times 

higher than the background concentration determined in Section 11, whichever 

concentration would be larger. 

9.5.2 Analyze one sample aliquot to determine the background concentration (B). 

9.5.3 Analyze the spiked MS and MSD aliquots following the procedure in Section 11. 

9.5.5 Calculate each percent recovery (P) as 100 (A-B)%/T, where A is the concentration of 

 TKN in the spiked samples and T is the known true value of the spike. 

9.5.5.1 Compare the percent recovery (P) TKN with the corresponding 

 QC acceptance criteria found in Section 17, Table 3. 
Hach Company TNTplus 880 s-TKN Method 10242 

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9.5.6 Calculate the relative percent difference (RPD) between two sample results using the 

following equation: 

 x 100

( 1 2)/ 2

1 2

D D

D D

RPD 

 

Where, D1 = Concentration of analyte in the MS, D2 = Concentration of analyte in the 

MSD. 

9.5.6.1 Compare the calculated RPD with the corresponding QC acceptance criteria 

found in Section 17, Table 3. 

10.0 Calibration and Standardization

10.1 The Hach DR series spectrophotometers have a built-in calibration that is automatically used 

when the TNTplus nitrate vial is inserted into the instrument. No further initial calibration is 

required. However, the instruments have the capability of developing a user-calibration. See 

manufacturer’s manual for instructions. 

10.2 Calibration Verification 

10.2.1 To verify that the instrument is measuring TKN properly, analyze a 1.0 mg/L and 10.0 

mg/L NH3-N standard. Results shall be within 10 percent of the actual value. Perform 

this calibration verification daily while instrument is in use. 

11.0 Sample Analysis Procedure

11.1 Instrument Setup – follow the instrument manufacturer’s instructions for instrument setup. 

11.2 Heating Block - Turn on heating block and heat to 100 °C. 

11.3 Analysis – Using TNTplus s-TKN Reagent Set 

11.3.1 Add 1.3 mL of sample, 1.3 mL of Solution A and 1 Reagent B tablet in quick 

succession to a dry 20-mm reaction tube. Close the reaction tube immediately. Do not 

 Invert tube.

11.3.2 Insert the reaction tube in the reactor and heat for one hour. 

11.3.3 After one hour, remove the reaction tube from the heating block and cool to room 

temperature (15-20o C). 

11.3.4 Remove the cap from the reaction tube and add 1 Micro Cap C to the tube. Cap and 

invert reaction tube 2-3 times until no more streaks can be seen in the reaction tube 

solution. 

11.3.5 Pipette 0.5 mL of the digested sample from the reaction tube into a Test Vial 1 (red 

label). 

11.3.6 Pipette 0.2 mL of Solution D into the test vial. Quickly cap and invert the test vial 2-3 

times until no more streaks can be seen in the vial solution. Immediately proceed to step 

11.3.7. 

11.3.7 Pipette 1.0 mL of undigested sample into a Test Vial 2 (green label). 
Hach Company TNTplus 880 s-TKN Method 10242 

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11.3.8 Pipette 0.2 mL of Solution D into the test vial. Quickly cap and invert the test vial 2-3 

times until no more streaks can be seen in the vial solution and let react for 15 minutes. 

11.3.9 After 15 minutes, wipe the Test Vial 1 with a clean tissue or cloth and insert the prepared 

vial into the cell holder of the spectrophotometer. The instrument will read the barcode 

on the Test Vial 1 and display E1. Remove the vial and proceed immediately to step 

11.3.10. 

11.3.10 Wipe the Test Vial 2 with a clean tissue or cloth and insert the prepared vial into the cell 

holder of the spectrophotometer. The instrument will read the barcode on the Test Vial 2. 

12.0 Data Analysis and Calculations

12.1 The sample result will be displayed in mg/L Total N, mg/L NO3-N + NO2-N and mg/L TKN. 

13.0 Method Performance

 Acceptance Criterion Section Limit 

 

Method Detection Limit 9.2.1, Table 1 0.43 mg/L NH3

-

 N as TKN 

Minimum Level 9.2.1, Table 1 1 mg/L NH3

-

 N as TKN

 Initial Recovery Range 9.2.2, Table 2 90% - 110% 

 

 Matrix Recovery Range 9.5, Table 3 90% – 110% 

 

14.0 Pollution Prevention

14.1 Follow guidelines in Section 15. 

15.0 Waste Management

15.1 It is the laboratory’s responsibility to comply with all federal, state, and local regulations governing 

waste management, particularly the hazardous waste identification rules and land disposal 

restrictions, and to protect air, water, and land by minimizing and control all releases from fume 

hoods and bench operations. Compliance with all sewage discharge permits and regulations is 

also required. 

15.2 For further information on waste management, consult “The Waste Management manual for 

Laboratory Personnel”, and “Less is Better: Laboratory Chemical Management for Waste 

Reduction”, both available from the American Society’s Department of Government Relations and 

Science Policy, 1155 16th Street N.W., Washington, D.C. 20036. 

16.0 References

16.1 “Handbook of Analytical Quality Control in Water and Wastewater Laboratories,” USEPA, EMSLCI, Cincinnati, OH 45268, EPA-600-4-79-019, March 1979. 

16.2 40 CFR 136, Appendix B. 

16.3 “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and 

Health Administration, OSHA 2206 (Revised, January 1976) 
Hach Company TNTplus 880 s-TKN Method 10242 

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16.4 “Safety in Academic Chemistry Laboratories,” American Chemical Society, Committee on 

Chemical Safety, 3rd Edition, 1979. 

16.5 “Water Analysis Handbook,” Hach Company, 5th Edition, 2008. 

16.6 Nitrogen, Simplified TKN (s-TKNTM), DOC316.53.01258, Hach Company, Loveland, Colorado 

USA. http://www.hach.com/quick.search-quick.search.jsa?keywords=DOC316.53.01258. 

16.7 Protocol for EPA Approval of New Methods for Organic and Inorganic Analytes in Wastewater 

and Drinking Water, United States Environmental Protection Agency, Office of Water, 

Washington, D.C., EPA-821-B-98-003, March 1999. 

17.0 Tables

17.1 Acceptance Criteria for Performance Tests 

Table 1. Method Detection Limit (MDL) & Minimum Level (ML) of Quantitation 

MDL Test 

Concentration MDL ML ML Rounded 

1.0 mg/L NH3-N 

as TKN 

0.430 mg/L NH3-N as 

TKN 1.35 mg/L NH3-N as TKN 1 mg/L NH3-N as TKN 

 

Table 2. Initial Precision and Recovery QC Acceptance Criteria 

IPR Spike 

Concentration 

Maximum 

RSD (%) 

Lower Limit 

of Recovery 

(%) 

Upper Limit of Recovery (%) 

5.0 mg/L NH3-N as 

TKN 10 90 110 

Table 3. Matrix Spike (MS) / Matrix Spike Duplicate (MSD) QC Acceptance Criteria 

MS/MSD Spike 

Concentration Maximum RPD (%) 

Lower Limit 

of Recovery 

(%) 

Upper Limit of Recovery 

(%) 

5.0 mg/L NH3-N 

as TKN 20 90.0 110 
Hach Company TNTplus 880 s-TKN Method 10242 

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18.0 Glossary of Definitions and Purposes

The definitions and purposes are specified to this method but have been conformed to common 

usage as much as possible. 

18.1 Units of weight and measure and their abbreviations 

18.1.1 Symbols 

 o C: degrees Celsius 

18.1.2 Alphabetical characters 

 mg/L: milligram per liter 

18.2 Definitions, acronyms, and abbreviations 

18.2.1 TKN – Total Kjeldahl Nitrogen 

18.2.2 s-TKN – simplified Total Kjeldahl Nitrogen 

18.2.3 LRB – Laboratory Reagent Blank 

18.2.3 MDL: Method detection limit 

18.2.4 ML: Minimum level of quantitiation 

18.2.5 IPR: Initial precision and recovery 

18.2.6 OPR: On-going precision and recovery 

18.2.7 MS: Matrix spike 

18.2.8 MSD: Matrix spike duplicate 

18.2.9 RSD : Relative Standard Déviation