SOP For Nitrogen Determination of Kjeldahl

PATHGENE
HEALTH CARE PRIVATE LIMITED
STANDARD OPERTING PROCEDURES

Document No.:
Document Name:- SOP for Nitrogen
PGHPL/ AMH/SOP/0553
Determination of Kjeldahl
SCOPE
The methods described are applicable for determination of nitrogen (N) in forages.
BASIC PRINCIPLE
The Kjeldahl method is the standard method of nitrogen determination dating back to
its development in the late 1800's. The method consists of three basic steps:
1) Digestion of the sample in sulfuric acid with a catalyst, which results in
conversion of nitrogen to ammonia;
2) Distillation of the ammonia into a trapping solution; and
3) Quantification of the ammonia by titration with a standard solution.
The sample is digested by sulphuric acid in the presence of a catalyst. The acid
solution is made alkaline with sodium hydroxide solution. The ammonia is distilled
and collected an excess of boric acid solution, followed by titration with standard
sulphuric acid solution.
EQUIPMENT
 Kjeldahl flasks, 500 to 800 mL
 Kjeldahl digestion unit with fume removal manifold
 Kjeldahl distillation apparatus - Kjeldahl flask connected to distillation trap by
rubber stopper. Distillation trap is connected to condenser with low-sulfur tubing.
 Erlenmeyer flask, 250 ml
 Analytical balance, sensitive to 0.5 mg
Reagents
Issue No. : 01 Amendment No. : 00 Reviewed Date : 01/12/2019
Issue Date : 01/12/2017 Amendment Date : -- Page No. : 1 of 5
Prepared by Reviewed & Approved by Issued by
Technical Manager Quality Manager Laboratory Director

PATHGENE HEALTH CARE PRIVATE LIMITED

Document No.:
PGHPL/ AMH/SOP/0553
 Sulfuric acid, concentrated, 95-98%,

 reagent grade Sodium hydroxide
 Potassium sulfate (K2SO4), anhydrous
 Copper sulfate (CuSO4), anhydrous
 boric acid, 2% (w/v)
 Standardized HCl (C= 0.1000 mol/l)
SAFETY PRECAUTIONS
 Handle acid safely: use acid resistant fume hood. Always add acid to water
unless otherwise directed in method. Wear face shield and heavy gloves to
protect against splashes. If acids are spilled on skin, immediately wash with
large amounts of water. Sulfuric acid and sodium hydroxide can burn skin,
eyes and respiratory tract severely.
 Wear heavy rubber gloves and face shield to protect against concentrated acid or
alkali.
 Use effective fume removal device to protect against acid fumes or alkali
dusts or vapors. Always add concentrated sulfuric acid or sodium hydroxide
pellets to water, not vice versa. Concentrated sodium hydroxide can quickly
and easily cause blindness. If splashed on skin or in eyes, flush with copious
amounts of water and seek medical attention.
 Digests must be cool before dilution water is added to avoid a violent
reaction during which the acid can shoot out of the flask. Likewise, the
diluted digest must be cool before sodium hydroxide is added to avoid a
similarly violent reaction.



Document No.:
PGHPL/ AMH/SOP/0553
PROCEDUE
DIGESTION
 Weigh 0.5 g of the sample to the nearest 0.001 g and transfer the sample to the
Kjeldahl flask of the digestion apparatus.
 Add 15g of mix of potassium sulphate and catalyst (copper (II) sulphate
pentahydrate), 12 ml of sulphuric acid and mix. Heat the Kjeldahl flask moderately
at first, swirling from time to time if necessary until the mass has carbonized and
the foam has disappeared;
 Then heat more intensively until the liquid is boiling steadily.
 Heating is adequate if the boiling acid condenses on the wall of the flask. When
the solution becomes clear and light green continue to boil for another two hours,
then leave to cool
DISTILLATION INTO BORIC ACID

o Where the distillation unit is fully automated to include titration of the
ammonia content of the distillate, follow the manufacturer's instructions for
operation of the distillation unit. In a semi- automatic distillation unit, the
addition of excess sodium hydroxide and the steam distillation are performed
automatically.
o Place a collecting flask containing 60 ml of the boric acid solution under the
outlet of the condenser in such a way that the delivery tube is below the
surface of the excess boric acid solution. Operate the distillation unit in



Document No.:
PGHPL/ AMH/SOP/0553
accordance with the manufacturer's instructions and distil off the ammonia
liberated by the addition of the sodium hydroxide solution. Collect distillate
in the boric acid receiving solution.
TITRATION
 Titrate the contents of the collecting flask with the sulphuric acid standard
volumetric solution using a burette and read the amount of titrant used.
 When colorimetric end-point detection is applied, the end-point is reached
when color of the solution changes from green to red. Estimate the burette
reading to the nearest 0, 01 ml.
Blank test
To confirm that the reagents are free from nitrogen, carry out a blank test (digestion,
distillation and titration) using only reagents (no sample is added).
CALCULATION OF RESULTS
Nitrogen content in the sample:
(Va -Vb ) .cHCl .M N
mN (g / kg) = .1000
mvz
Va = volume of standard HCl solution when titrating sample (l)

Vb = volume of standard HCl solution when titrating blank (l)
CHCl = concentration of HCl (mol/l)



Document No.:
PGHPL/ AMH/SOP/0553
mN = nitrogen molar mass (g/mol)

mvz = weight of sample (g)
Crude protein (CP) in sample:
CP(g / kg) = mN (g / kg) . F
mN = nitrogen content in sample (g/kg)

F = factor (6.25 for feed samples)
Quality control
Maximum allowed differences between parallel determinations:
Crude protein content (g/kg) Allowed differences
less than 160 ± 4.0 g/kg (A)
160.0 – 320.0 ± 2.5% (R)
more than 320.0 ± 8.0 g/kg (A)


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PATHGENE

HEALTH CARE PRIVATE LIMITED

STANDARD OPERTING PROCEDURES

Technical Manager Quality Manager Laboratory Director

STANDARD OPERTING PROCEDURES

 Sulfuric acid, concentrated, 95-98%,

Issue No. : 01 Amendment No. : 00 Reviewed Date : 01/12/2019

Technical Manager Quality Manager Laboratory Director

STANDARD OPERTING PROCEDURES

DISTILLATION INTO BORIC ACID

Issue No. : 01 Amendment No. : 00 Reviewed Date : 01/12/2019

Technical Manager Quality Manager Laboratory Director

STANDARD OPERTING PROCEDURES

Va = volume of standard HCl solution when titrating sample (l)

Issue No. : 01 Amendment No. : 00 Reviewed Date : 01/12/2019

Technical Manager Quality Manager Laboratory Director

STANDARD OPERTING PROCEDURES

mN = nitrogen molar mass (g/mol)

Crude protein (CP) in sample:

CP(g / kg) = mN (g / kg) . F

mN = nitrogen content in sample (g/kg)

Issue No. : 01 Amendment No. : 00 Reviewed Date : 01/12/2019

Technical Manager Quality Manager Laboratory Director

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