Kjeldahl Method
Kjeldhal Digestion unite
Kjeldhal Destilation unite
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Principle
Nitrogen is one of the five major elements in organic materials such as protein.
This fact was recognized by a Danish chemist, Johan Kjeldahl, who used it as a
method of determining the amount of protein In 1883. The central basis used in
this procedure is the oxidation of the organic compound using strong sulfuric acid.
As the organic material is oxidized the carbon it contains is converted to carbon
dioxide and the hydrogen is converted into water.
The nitrogen, from the amine groups found in the peptide bonds of the polypeptide
chains, is converted to ammonium ion, which dissolves in the oxidizing solution,
and can later be converted to ammonia gas.
Safety Precautions
Sulfuric acid. Sulfuric acid can cause severe burns. Wear eye protection and acid
resistant gloves. If acid is spilled on skin, wash immediately with large amounts
of cool water. NEVER NEUTRALIZE ACID ON SKIN WITH BAKING SODA.
Sodium Hydroxide. Alkalies can cause severe burns. Violent boiling can occur
when sodium hydroxide is added to the digest (especially if there is too much
residual acid). Wear eye protection and heavy rubber gloves when working with
sodium hydroxide. If sodium hydroxide is spilled on skin, wash immediately with
large amounts of cool water.
0.1N and .01N Hydrochloric acid (HCL). HCL solutions can cause burns. Wear eye
protection. Wearing acid resistant gloves when handling HCL is recommended
but may not be practical during titration. If HCL is spilled on the skin, wash
immediately with large amounts of cool water.
Digestion. Check the bottom of the Kjeldahl flasks for star cracks prior to adding
the sample and reagents. Discard cracked flasks. Acid fumes are generated
during digestion. Make sure the digestion apparatus (traditional or block)
effectively removes fumes. Wear eye protection and heavy rubber gloves when
handling Kjeldahl flasks.
Distillation. Distillation involves the heating of an acid digest plus sodium
hydroxide with the release of ammonia gas. Care should be taken under
conditions such as these. Always wear eye protection and heavy rubber gloves.
Most Kjeldahl distillation units have a protective glass (or plastic) barrier that is
used as protection for the operator during distillation. If your unit has this, use it!
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A three step procedure
The Kjeldahl method consists of three steps, which have to be carefully carried out
in sequence: digestion, distilation ,titration
1. the sample is first digested in strong sulfuric acid in the presence of a
catalyst, which helps in the conversion of the amine nitrogen to ammonium
ions,
2 NH3 + H2SO4 → (NH4)2SO4
2. the ammonium ions are then converted into ammonia gas, heated and
distilled. The ammonia gas is led into a trapping solution where it dissolves
and becomes an ammonium ion once again,
(NH4)2SO4 + 2 NaOH → Na2SO4 + 2 NH3
H3BO3 + 3 NH3 → (NH4)3BO3
3. finally the amount of the ammonia that has been trapped is determined by
titration with a standard solution, and a calculation made.
(NH4)3BO3 + 3HCl→ H3BO3 + 3 (NH4)Cl
Reagents
1. Sulfuric acid. 95-98% H2SO4. Nitrogen free.
2. Copper catalyst solution. CuSO4 . 5H2O. Nitrogen free. Prepare solution
0.05 g/mL H2O.
3. Potassium sulfate. K2SO4. Nitrogen free.
4. Sodium hydroxide solution. 35 % w/w nitrate-free NaOH.
5. Boiling chips. Mesh size 10 suggested. High purity, amphoteric alundum
granules, plain.
6. Methyl red/bromocresol green indicator solution. Dissolve 0.2 g methyl red
and dilute to 100 mL in 95% ethanol. Dissolve 1.0 g bromocresol green and
dilute to 500 mL in 95% ethanol. Mix 1 part methyl red solution with 5 parts
bromocresol green solution (combine all of both solutions).
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7. Boric acid solution. 4%, with indicator. Dissolve 40 g H3BO3 and dilute to
1 L in water and add 3 mL methyl red/bromocresol green indicator solution,
(f). Solution will be light orange color.
8. Hydrochloric acid standard solution. 0.1000N pre-made solution of certified
specification range 0.0995-0.1005N and use 0.1000N for calculation.
Procedure
1.
2.
3.
4.
5.
Take 2.5 ml of sample which contain 0.1g of dry matter,
add 7.5 ml d. w. & 20 ml 35% NaOH mix in Kjeldhal flask ,
put 15 ml boric acid in 250 ml conical flask, soon befor dis.
add to Boric 3 drops Tashero indicator
As soon aspossible Perform Distilation,until distelate becomes 100ml, wash tip
of plastic tube withwater, cool .
6. titrate with HCl .1N using washing bottle till first faint pink colour.
7. Perform a blank experiment using water instead of sample
Calculation
Con. of N% = 0 .00014 X (titre test- titre blank) X 100 = x
ML taken of ( sample)
Con. of N% in dry sample = X*2.5
the amount of "crude protein" (CP) can be found by multipling the percent
Nitrogen by a factor (usually 6.25).
CP = %N x 6.25
References
http://www.google.com/images?hl=&q=Johan+Kjeldahl&sourceid=navclientff&rlz=1B7GGHP_enSA455&ie=UTF-8
http://www.brooklyn.cuny.edu/bc/ahp/SDKC/Chem/SD_KjeldahlMethod.html
http://www.fmmaseattle.com/laboratory.htm
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