Unit 4 of the Soil Chemical Analysis

Analysis of Soil Nitrogen

土壤中氮的分析

Sheng-Bin Ho
Department of Agricultural Chemistry
National Taiwan University

2019/10/18 1
 Forms of Nitrogen in Soils

 Organic nitrogen:
Soluble (Non-humic substances)
Insoluble (Humic substances)
 Inorganic nitrogen:
Nitrate ( NO -3 ), Nitrite ( NO -2 )
Ammonium( NH 4+ )
Exchangeable form
Non-exchangeable or fixed form
 Gaseous nitrogen: N2, N2O, NH3, …..

耕地土壤表土的總氮含量約在0.06~0.5%之間，其中90%以上
的氮是以有機態氮存在，十分複雜。 2
 Determination of Total Nitrogen in Soils

 The Kjeldahl method (a wet oxidation procedure)

Conc. H2SO4 (b.p. 327 oC)
+ Promoters
Organic N −−−−−−−−−−−−−→ NH4+
(320~370 oC)
(Digestion)

 The Dumas method (a dry oxidation procedure)

O2, CuO, Cu
Total N −−−−−−−−−−−−−→ N2
(>600 oC) 3
  The Kjeldahl method (a wet oxidation procedure)
Conc. H2SO4 (b.p. 327 oC)
+ Promoters
Organic N −−−−−−−−−−−−−→ NH4+ −−−→ (usually determined by steam
(320~370 oC) distillation method)

(Step I: Digestion) (Step II: Determination of NH4+-N)

Promoters are used to increase the speed and the completeness of

conversion of organic N to NH4+-N by digestion with H2SO4.
Salts: raise the temperature of digestion, e.g., K2SO4, Na2SO4.
Catalysts: increase the rate of oxidation of organic matter by H2SO4,
e.g., Se, HgO, CuSO4, TiO2.

K2SO4:CuSO4•5H2O:Se = 100:10:1(w/w/w)
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Table 4-1. Amounts of H2SO4 consumed by various materials
during Kjeldahl digestion (Bremmer, 1960)
Material Acid Consumption
mL of 18 M H2SO4/g of material
Soil organic C 10.0
Soil organic matter 5.8
Al2O3 1.63
Fe2O3 1.04 (Fe2(SO4)3 as the product)
Clay 0.60
CaCO3 0.55
Silt 0.30
Sand 0
Salicylic acid 6.76
Na2S2O3．5H2O 0.58
Reduced Fe 1.50 (Fe2(SO4)3 as the product)
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Table 4-2.

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Table 4-3.

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 Compounds containing N-N and N-O linkages (e.g., azo-, nitroso-, and
nitro- compounds, hydrazines, hydrazones, oximes, pyrazolones,
isooxazoles, 1,2-diazines, triazines, nitrites, nitrates) can not be
completely recovered by the two-step Kjeldahl procedure.

R-N=N-R’ R-N=O R-NO2

Azo compounds Nitroso compounds Nitro compounds

R1R2CNOH
H2N-NH2 Oximes

Hydrazines
Hydrazones

2,4-Dinitrophenylhydrazine 8
1,2-diazines triazines

isooxazoles
9
 Pyrazolones

Pyrazole

Ampyrone Phenazone, or phenazon

Metamizole
Phenylbutazone
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 Kjeldahl N vs. Total N

Pretreatment of Sample
 Grinding soil samples. (to pass through a 0.15-mm(100-mesh)
screen)
 Treating soils with water before digestion.
 Hydrofluoric acid (HF) modification of the Kjeldahl method to
include clay-fixed NH4+-N.
Regular
Kjeldahl
Soil sample
5 M HF-1 M HCl 4.5 M H2SO4 Heat to method
mixture remove water
(24 hours)

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 Pretreatment of Sample
 Methods used to include NO3--N and NO2--N
Salicylic acid-thiosulfate modification of Kjeldahl method

Soil sample containing Soil + Soil + Regular

NO3--N + NO2--N Nitro Amino Kjeldahl
Salicylic acid- Thiosulfate,
method
sulfuric acid mixture compounds compounds
(Overnight)

Permanganate-reduced iron modification of Kjeldahl method

(Recommended for 15N trace study)
Regular
Soil sample containing
Soil + Soil Kjeldahl
NO3--N + NO2--N 5% KMnO4, Reduced Fe, +NH +-N
- method
1:1 H2SO4 NO3 -N 4

(5 min.)

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Determination of Inorganic Nitrogen (exchangeable and
soluble forms) in Soils

Determination of inorganic nitrogen in soils is complicated:

 Extensive spatial variability

 Be subject to microbial transformations

Analyses of inorganic N in soils should be carried out

immediately after sampling.

Extraction: 2 M KCl, 10:1 solution/soil ratio, shaking for 1 hour.

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Table 4-4.

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Table 4-4.

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 Steam Distillation Method
Alkali-distillation vs. MgO distillation

Steam distillation of 2 M KCl soil extracts with MgO and Devarda’s alloy
have advantages over colorimetric methods：
 These procedures are accurate and reliable.
 The interference by alkali-labile organic-N compounds, commonly
present in soil extracts, in alkali-distillation methods of determining
NH4+ is eliminated.
 Analyses may be performed on colored or turbid extracted without
difficulty.
 They permit isotope-ratio analysis of exchangeable NH4+-N, NO3--N,
and NO2--N in tracer studies concerning the fate of labile N applied in
soil.
Devarda's alloy is an alloy of aluminum (44% – 46%), copper (49% – 51%)
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and zinc (4% – 6%).
 Splash
Head
Electric
power Condenser
supply

Steam

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Figure 4-1 Semi-micro Kjeldahl Distillation Apparatus (Parnas-Wagner type).
Table 4-5.

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 Conway’s Microdiffusion Method

4-2.

19
 4-3.

4-4.
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Receiving Acids:
 Standardized HCl or H2SO4 solution.
No indicator available.
Need both standardized acid and base solutions.
Exact volume of standardized acid solution is needed.
 Boric acid solution with methyl red- bromcresol green mixed
indicator.
NH 3 + H 3 BO 3 → NH 4+ + H 2 BO -3

(purple) (blue green)

H + + H 2 BO -3 → H 3 BO 3
(standardized acid) (purple) 21
 Colorimetric Methods

NH4+ by Nessler method or Indophenol blue method (Phenate

method).
NO3- by Griess-Ilosvay method after reducing to NO2-.
NO2- by Griess-Ilosvay method

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內斯勒法 (Nessler method)(適用範圍
適用範圍： ～10 mg NH4+-N L-1)
適用範圍：0.1～

Nessler試劑： 2KI + HgI 2 → K 2 HgI 4

(KOH)

呈色反應：

+ (10% tartrate) and/or gum acacia

2K 2 HgI 4 + 3KOH + NH 3 → Hg 2O(NH 2 I) + 7KI + 2H 2O
(Orange-brown)
410-490 nm

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 酚藍法 (Indophenol blue method; Phenate method)
適用範圍：
適用範圍：0.005～
(適用範圍 ～20 mg NH4+-N L-1)

Berthelot reaction:
• pH 11~13 (buffered)
• Oxidizing agent
• Catalyst
Phenate + NH 3 → Blue color ( λ = 625 ~ 670 nm)

Oxidizing agent: Sodium hypochlorite (NaOCl) or dichloroisocyanurate

Catalyst: Sodium nitroprusside, acetone, or manganous ions

The optimum conditions for analysis of NH4+ by the Berthelot reaction

depend upon the reagents employed.
pH: phenol (pH11-12), salicylate (pH 12-13)
Temperature: gentle heating can reduce analysis time and increase
sensitivity.
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NaC3N3O3Cl2 + 2H2O ↔ NaH2C3N3O3+ 2HOCl

25
4-5.

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 格里斯-伊洛斯維法
格里斯 伊洛斯維法 (Griess-Ilosvay method)
適用範圍：
適用範圍：0.01～
(適用範圍 ～12 mg NO2-N L-1)

Soil extracts
containing Diazonium Azo
NO2- Diazonium reagent salt Coupling reagent compound
(sulfanilamide in HCl (N-(1-naphthyl)-
solution) ethylenediamine)) (λmax= 540 nm)

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Diazotization reaction

重氮化試劑 重氮鹽
(Diazotizing reagent) (Diazonium salt)
R= −SO2NH2(Sufanilamide)

Coupling reaction

偶合試劑 偶氮化合物
(Coupling reagent) Azo compound
R'= −NHCH2CH2NH2 (Redish purple)(540 nm)
[N-(1-naphthyl)-ethylenediamine)] 28
 Methods by Using Ion-Selective Electrodes

4-6.

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Ammonium Gas-sensing Electrode

(Reference electrode)

(0.1 M)

(0.1 M HCl)

Figure 4-6. Schematic of a gas-sensing probe for ammonia. 30

 Hydrophobic
gas-permeable
membrane

Internal NH4Cl solution (0.1 M)

NH3 in sample solution
NH 3 + H 2O ↔ NH 4+ + OH -

( NH +4 )(OH − )
= K ''' (Constant)
( NH 3 )
[OH − ] '' K '''
= K (= +
)
[ NH 3 ] [NH 4 ]

Eobs = K ' − S log(OH − )

= K − S log( NH 3 ) [ K = K '− S log( K '' )]

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Eobs = Eind − Eref + E j

 RT + 
= k + ln ( H )  − E ref
 nF 
RT
(
= k − E ref ) +
nF
ln ( H + )

RT Kw
(
= k − E ref ) +
nF
ln
(OH − )
 RT  RT
=  k − E ref + ln( K w )  − ln(OH − )
 nF  nF
= K '− S log(OH − ) [OH − ] K '''
= K '' ( = )
= K '− S log[(NH 3 ) • K '' ] [ NH 3 ] [NH 4 ]+

= ( K ' − S log K '' ) − S log( NH 3 )

= K − S log( NH 3 )

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 NH 3 + H 2O ↔ NH 4+ + OH -

( NH +4 )(OH − )
= K b = 1.8 × 10 − 5
( NH 3 )
( NH +4 ) Kb 1.8 × 10 −5 9 + 9.25 − pH
= = = 1. 8 × 10 × ( H ) = 10
( NH 3 ) (OH − ) 10 −14
(H + )

(The exact value of Kb will vary with both temperature and

ionic strength.)

 Ionic Strength Adjustor (ISA) :

5M NaOH/0.05M Disodium EDTA/10% Methanol with Thymolphthalein.

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 The ammonia electrode responds to the partial pressure of
dissolved ammonia gas.
 The partial pressure of dissolved ammonia gas is related to
the ammonia concentration by Henry’s Law:

[NH 3 ]
Kh = = 56 moles/liter - atm (at 25 oC)
PNH 3
(The Henry’s Law constant, Kh, varies both with temperature and
the level of dissolved species.)

  To keep the Henry’s Law constant close to the same value,

standards and samples should contain the same level of
dissolved species and be about the same temperature.

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 Determination of Nonexchangeable Nitrogen in Soils

(Remove
Soil exchangeable Steam distillation
sample NH4+ and with 10 M KOH
KOBr-KOH organic-N) Washed with 0.5 M
KCl and shaken
with 5M HF-1M HCl
solution for 24 h

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 Determination of Organic Nitrogen in Soils

4-7.

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《品質管制》

⼀、 總氮含量或凱達氏氮含量之測定應注意土樣之製備、取樣、及
/或前處裡。消化分解之過程應監控。
二、 以蒸餾法進行各種型態可溶性氮的測定時，應使用各型態氮之
標準溶液做回收率的測定，影響回收率的主要因子有蒸餾裝置
組裝恰當與否，蒸餾條件最佳化情況，氮標準溶液之濃度正確
與否，滴定用酸之濃度正確與否，硼酸指示劑配製是否恰當等
。如果上述因子都能控制好，回收率應達98%以上。
三、 進行田間土壤可交換性氮的測定時，土壤樣本之運送及儲存須
在4 oC及暗處下進行，並且應儘速於採樣後24小時內完成分析
測定。
四、 每⼗個樣本溶液或每批次樣本溶液⾄少執⾏⼀次添加標準溶液
分析，其回收率應在 85 ~115 % 範圍內。
五、分光光譜法之檢量線其相關係數應大於或等於0.995。
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