This slideshow contains a short overview of importance of total nitrogen determination, traditional Kjeldahl method, its improvements and Dumas method of total nitrogen determination.

1. TOTAL NITROGEN DETERMINATION
TRADITIONAL AND MODERN METHODS

2. OUTLINE
1. What is total nitrogen
2. Importance of total nitrogen determination
3. Kjeldahl method
4. Improvements to the Kjeldahl method
5. Dumas method
6. Comparison between Kjeldahl method and Dumas
method
2

3. WHAT IS TOTAL NITROGEN?
• Total Organic Nitrogen (Derived from protein, urea,
nucleic acids etc.)
• Total Ammonia
• Nitrate
• Nitrite
3

4. WHY IS TOTAL NITROGEN
DETERMINATION IMPORTANT?
• Food Industry – The standard method for estimating the protein
content in foods and food products
• Agriculture – Determination of nitrogen content in soils and
fertilizers/manure samples
• Dissolved nitrogen content is an indicator of water quality
• Determination of nitrogen content in atmospheric nitrogen
depositions
• Nitrogen percentage is one of the specifications in the quality
control of nuclear fuels 4

5. THE K JELDAHL METHOD
• The Danish chemist Johan Kjeldahl (1849 – 1900)
• In 1883
• For determining nitrogen in organic substances
• Satisfactory for levels of N as low as 1.0 mg/L
5

6. THREE MAIN STEPS
1) Digestion 2) Distillation 3) Titration
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7. DIGESTION
• Goal – Quantitative conversion of organic N to ammonia
Organic N + H2SO4 (NH4)2SO4 + H2O + CO2
• Initially, conc. H2SO4 was used alone for the digestion
• Problem – Very long digestion times resulted
• Solution – Addition of an inorganic salt to elevate the boiling
point of H2SO4 (e.g. K2SO4)
• Shorten the length of time required for digestion
7

8. DIGESTION (CONT.)
• Salt addition – Several Precautions
• Temperature depends on salt to acid ratio
• If temperature goes above 400 °C, volatile N compounds may
be lost
• If salt/acid ratio is too high, material will solidify upon cooling
• Concentrated acid pockets can be contained within the solid
• Digest must contain residual acid to retain NH3 as NH4
+
8

9. DIGESTION (CONT.)
• The amount of acid required – Several factors
• Takes at least 20-30 minutes up to several hours
• Catalyst – To increase the rate of organic break down during the
digestion
• HgO – Most effective
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10. PRE-DISTILLATION STEPS
1. Cooling
2. Dilution – with ammonia-free water
• Prevents/ minimizes solidification and reduces the likelihood of
bumping
• Solidification cause low N recoveries and entrapped acid react
with base during distillation
3. Preparation of receiving vessel – To capture distilled NH3
• The tip of the condenser is submerged in the flask
• Either standard acid or boric acid solution
10

11. DISTILLATION
• Goal – Separation of NH3
• Conc. NaOH (usually 50% solution) is added
• Make the digestion mixture strongly alkaline ( pH > 11)
(NH4)2SO4 + 2NaOH NH3 + NaSO4 + H2O
• The flask is connected to the condenser and mixed before
heating and distillation begins
• The rate of distillation is affected by condenser cooling capacity
and cooling water temperature
11

12. DISTILLATION (CONT.)
• Connecting bulbs/ expansion chambers – To prevent carryover of
the alkaline digestion mixture
• When very low levels of nitrogen – “Precondition” the distillation
apparatus
12

13. 13

14. TITRATION
• Goal – Indicate the amount of ammonia present in the distillate
• Two types:
1. Back titration
2. Direct titration
14

15. TITRATION (CONT.)
Nitrogen Determination by Back Titration
• Capture Ammonia by excess of a standardized acid solution
2NH3(aq) + 2H2SO4(aq) (NH4)2SO4(aq) + H2SO4(aq) (excess)
• Excess acid solution is neutralized by standardized alkaline
solution
H2SO4(aq) + 2NaOH(aq) Na2SO4(aq) + 2H2O(l)
15

16. TITRATION (CONT.)
Nitrogen Determination by Direct Titration
• Boric acid (H3BO3) is used
NH3(aq) + H3BO3(aq) NH4
+ :H2BO3
- + H3BO3(aq) (excess)
• Standardized H2SO4 neutralizes the ammonium borate complex
NH4
+:H2BO3
- + H2SO4(aq) (NH4)2SO4(aq) + 2H3BO3(aq)
• Only one standard solution is necessary for the determination
• The combination of methyl red and methylene blue indicator (Tashiro's
indicator) is frequently used 16

17. TITRATION (CONT.)
• Calculation
• When standard acid is used (Back titration):
%𝑁𝑖𝑡𝑟𝑜𝑔𝑒𝑛 =
𝑚𝐿 𝑠𝑡𝑑. 𝑎𝑐𝑖𝑑 × 𝑀 𝑜𝑓 𝑎𝑐𝑖𝑑 − 𝑚𝐿 𝑠𝑡𝑑. 𝑏𝑎𝑠𝑒 × 𝑀 𝑜𝑓 𝑏𝑎𝑠𝑒 × 14.007
𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑔
× 100%
• When boric acid is used (Direct titration):
%𝑁𝑖𝑡𝑟𝑜𝑔𝑒𝑛 =
𝑚𝐿 𝑠𝑡𝑑. 𝑎𝑐𝑖𝑑 × 𝑀 𝑜𝑓 𝑎𝑐𝑖𝑑 × 14.007
𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑔
× 100%
• To determine % protein, the calculated % N is multiplied by a factor, the
magnitude of which depending on the sample matrix
17

18. IMPROVEMENTS TO THE K JELDAHL
METHOD
• The Kjeldahl procedure is hazardous, lengthy, and labor
intensive
• The original Kjeldahl method has been continuously improved
• Enhanced environmental and personnel safety aspects
• Increased speed and versatility
• Simplified the analytical procedure
18

19. IMPROVEMENTS (CONT.)
• Will not recover 100% of organic N in most samples
• Three modifications used to recover nitrate and nitrite nitrogen
1. Salicylic acid modification – the sample is preheated with salicylic
acid and sodium thiosulfate
2. Alkaline reduction modification – Devarda’s alloy and alkali are
used to reduce nitrate and nitrite to ammonia
3. Permanganate-reduced iron modification – KMnO4 is used to
oxidize nitrite to nitrate-nitrogen, which is then reduced to
ammonium by reduced iron 19

20. IMPROVEMENTS (CONT.)
• Mercury is very toxic and expensive to use
• Environmental concerns over the handling and disposal of mercury
• Distillation of ammonia is incomplete as mercury forms a complex
with ammonia; sodium sulfide, sodium thiosulfate, or zinc dust is
added to decompose the complex
20

21. IMPROVEMENTS (CONT.)
• Many researches to find an efficient substitute of low toxicity and
cost
• Selenium (1931)
• Copper (1935)
• Copper sulfate for the determination of nitrogen content in milk
• Copper sulfate and Titanium oxide in combination
• Mixed Na2SO4-CuSO4 catalyst in plant tissue digestion for
macronutrient analysis
• Zirconium dioxide
21

22. IMPROVEMENTS (CONT.)
• In classical method, a relatively large sample (1–2 g) was used
• Require large amounts of acid
• Micro Kjeldahl methods – Commonly used
• Produce a reduced amount of acid fumes and also require less
acid and catalyst mixture
• Environmental considerations – To safe disposal of mercury
(when used as catalyst) and, to minimize acid usage
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23. IMPROVEMENTS (CONT.)
Block digesters
• High temperature ceramic or aluminum blocks
• Take less time than classical macro Kjeldahl digestions
• Possible to improve the speed and accuracy of the
digestion, as well as save space, chemicals and energy
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24. IMPROVEMENTS (CONT.)
Steam distillation units
• Simple and efficient
• Faster than classical distillation
• Appropriate for micro Kjeldahl analyses
• Block digestions followed by steam distillation are called
Rapid Kjeldahl
• Semi-automated/fully automated systems based on the
classical procedure – Cut cost and save time 24

25. IMPROVEMENTS (CONT.)
Hach Method (1985)
• H2O2 – A rapid and powerful oxidizer of organic matter
• Used in Kjeldahl digestion to decompose organic samples
• To achieve acceleration and completion of the decomposition
step
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26. IMPROVEMENTS (CONT.)
• Extracted N can be determined by several other methods
• Spectrophotometry
• Potentiometry with ion-selective ammonium electrode
• Flow Injection Analysis (FIA)
• Ion chromatography
• Near-infrared reflectance (NIR) spectrophotometry
• Chemiluminescent method
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27. DUMAS METHOD
• The French Chemist Jean-Baptiste Dumas (1800-1884)
• In 1831
• Older than the Kjeldahl method
• Developed recently as an alternative
• Detect total nitrogen content
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28. THREE MAIN STEPS
1) Combustion
2) Reduction and
Separation
3) Detection
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29. COMBUSTION
• Heat in a high temperature furnance
• Rapid combustion in the presence of pure O2
Sample + O2 CO2 + H2O + NxOY + O2 + Other
oxides
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30. REDUCTION AND SEPARATION
• O2 is removed by passing over hot Cu
• Converts nitrogen oxides into molecular N
• Passes through traps that remove H2O and CO2
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31. DETECTION
• Measures signal by thermal conductivity or gas
chromatography
• Detector converts into total N
• Expensive instruments and require skilled operators
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32. K JELDAHL VS. DUMAS
Kjeldahl Method Dumas Method
Entire time of analysis for one sample
several hours
Entire time of analysis for one sample
approx. 3-4 minutes
Large quantities of concentrated acids
and a lot of flasks to wash
No concentrated acids, no toxic
substance, sample prepare in tin foils.
No flasks
Sample size: 100 mg – many grams Sample size: mg – 5g
Very low initial price. Semi-automatic
analysis with automatic distillation and
titration devices available but
expensive
Low price for fully automated unit
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33. 33
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