3.1.1 Standardization of Hydrochloric Acid Standard Solution >>

3.1A Addendum to Nitrogen Determination by Kjeldahl (Rack): Options for Experienced Laboratories

References:
Protein (Crude) Determination in Animal Feed: Copper Catalyst Kjeldahl Method. (984.13) Official Methods of Analysis. 1990. Association of Official Analytical Chemists. 15th Edition.

Protein (Crude) in Animal Feed: CuSO4/TiO2 Mixed Catalyst Kjeldahl Method. (988.05) Official Methods of Analysis. 1990. Association of Official Analytical Chemists. 15th Edition.

Nitrogen (Total) in Fertilizers: Kjeldahl Method (Mercury). (955.04) Official Methods of Analysis. 1990. Association of Official Analytical Chemists. 15th Edition.

Nitrogen (Total) in Milk. (991.20) Official Methods of Analysis. 2nd supplement. 1991 Association of Official Analytical Chemists. 15th Edition.

Scope:
A number of alternatives to the original procedure are described; alternatives used will depend on sample type, the user of analytical data, equipment availability, and environmental considerations.

Basic Principle:
A number of alternatives to the method have evolved: several catalysts other than copper are available for digestion. Choice of catalyst will depend on the difficulty of breakdown of the peptides in the sample protein to be analyzed and environmental problems associated with the disposal of the waste containing the catalyst. If necessary, the digestion can also be modified to reduce nitrates to ammonia, which might otherwise not be recovered, by the addition of salicylic acid and a reducing agent.

A distillation/titration alternative utilizes a weak acid (boric acid) as the trapping solution. The ammonia is then quantified by titration with a standard strong acid (hydrochloric acid).

Additional Equipment:
None.

Additional Reagents:
Alternative catalyst Mercury catalyst, Mercuric oxide (HgO) or metallic mercury (Hg), reagent grade, N-free Potassium sulfate (K2SO4) or anhydrous sodium sulfate (Na2SO4), reagent grade, N-free

Alternative trapping solution (with indicator)/titrant Boric acid solution, 4% dissolve 400 g boric acid (H3BO3) in distilled water containing 70 mL 0.1% alcoholic solution of methyl red and 100 mL 0.1% alcoholic solution of bromocresol green and dilute to 10 L with distilled water. Standard hydrochloric acid solution, 0.2 N Prepare by diluting 172 mL 36.5 to 38% HCl to 10 L with distilled water and standardize by method 3.1.1

Option for nitrate containing samples: Salicylic acid, reagent grade, N-free Reducing agent (one of the following) a) Sodium metabisulfite, (Na2S2O3.5H20) b) Zinc dust, impalpable powder Potassium nitrate, NIST SRM 193, dried at 110oC for 2 hr (used to check nitrate reduction)

Sulfide or thiosulfate solution (when mercury catalyst is used) Dissolve 40 g commercial potassium sulfide (K2S) in 1 L water (solution of 40 g Na2S or 80 g sodium thiosulfate (Na2S2O3.5H20) in 1 L water may be used)

Safety Precautions:

• Handle acid safely: Use acid-resistant fumehood; always add acid to water unless otherwise directed in method; wear face shield and heavy rubber 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.
• Mercury in contact with ammonia, halogens and alkali can produce extremely toxic and cumulative vapors. Regard spills as extremely hazardous and clean up promptly. Powdered sulfur sprinkled over spilled mercury can assist in cleaning up spills. A high degree of personal cleanliness is necessary for persons who use mercury. Use skin and respiratory protection when dry mercuric salts are to be used.
• The sulfur oxide fumes produced during digestion are hazardous to inhale.
• 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.

Procedure:

Digestion Alternatives

1. Weigh approximately 1 g ground sample into digestion flask, recording weight (W) to nearest 1.0 mg. Weight range should depend on nitrogen content of sample. Weigh a second subsample for laboratory dry matter determination.
2. Add one of the following catalysts:
   Option a) Mercury: add 0.7 g HgO or 0.65 g Hg, 15 g K2SO4 or anhydrous Na2SO4
   Option b) CuSO4/TiO2: add 16.7 g K2SO4, 0.01 g anhydrous CuSO4, 0.6 g TiO2
   Option c) Copper: add 15 g K2SO4, 0.04 g anhydrous CuSO4
3. Add 3 g pumice or 0.5 to 1.0 g alundum granules, and 20 mL concentrated sulfuric acid. (Add additional 1.0 mL sulfuric acid for each 0.1 g fat or 0.2 g other organic matter if sample weight is >1 g.)
4. Place flask on preheated burner (adjusted to bring 250 mL at 25oC water to rolling boil in 5 min).
5. When white fumes clear bulb of flask swirl gently and continue heating
   • (for option a) until 30 min after clearing.
   • (for option b) 40 min.
   • (for option c) 90 min.
6. Cool, cautiously add 250 mL distilled water and cool to room temperature (25oC). Note: add water as soon as possible to reduce amount of caking.

NOTE: If recovery of nitrate nitrogen is of concern, replace steps 2 and 3 with 2A and 3A:

• 2A) Add 40 ml H2SO4 containing 2 g dissolved salicylic acid to each flask. Shake until thoroughly mixed and let stand, with occasional shaking, at least 30 min. Transfer to a fume hood and then add either 5 g Na2S2O3.5H20 or 2 g zinc dust (as impalpable powder, not granulated zinc or filings). Shake and let stand 5 min, then heat slowly until frothing ceases and white fumes appear (ca 10 min).
• 3A) Turn off heat and add boiling chips (3 g pumice or 0.5 to 1.0 g alundum granules) and one of the following catalysts:
  • Option a) Mercury: add 0.7 g H2O or 0.65 g Hg, 15 g K2SO4 or anhydrous Na2SO4
  • Option b) CuSO4/TiO2: add 16.7 g K2SO4, 0.01 g anhydrous CuSO4, 0.6 g TiO2
  • Option c) Copper: add 15 g K2SO4, 0.04 g anhydrous CuSO4

Alternative Distillation and Titration

1. (For boric acid receiving solution) Place 250 mL titrating flask containing 25 mL boric acid solution with mixed indicator so that tube of condenser is immersed below surface of absorbing solution.
2. Add 2 to 3 drops of tributyl citrate to digestion flask to reduce foaming.
3. Add another 0.5 to 1.0 g alundum granules.
4. If mercury catalyst was used, add 25 mL of the sulfide or thiosulfate solution and mix to precipitate mercury.
5. Slowly down side of flask, add sufficient 45% sodium hydroxide solution (approximately 80 mL) to make mixture strongly alkali. Do not mix.
6. Immediately connect flask to distillation apparatus, swirl to mix contents and distill at about 7.5 boil rate until ³ 150 mL distillate is collected in titrating flask. If excessive bumping occurs during distillation, increase dilution water from 250 mL to 300 mL.
7. Remove titrating flask from unit, rinsing the condenser tube with distilled water as the flask is being removed.
8. Titrate with 0.2 N HCl to neutral gray endpoint and record volume to nearest 0.1 mL (VA). Titrate reagent blank (VB) similarly. (Color change is green to gray to purple.)

Comments:

• Reagent proportions, heat input and digestion time are critical factors - do not change.
• Choice of catalyst will depend on the difficulty of peptide breakdown in the sample protein, environmental problems, and costs associated with disposal of the catalyst.
• Commercial preparations are available for the 4% boric acid indicator solution and HgO/K2SO4 , CuSO4/TiO2/K2SO4 and CuSO4/K2SO4 catalysts.
• The sulfuric acid/salicylic acid solution is sensitive to light and air and has a very short shelf life (approximately 2 days).
• Include a reagent blank and at least one sample of high purity lysine hydrochloride in each day's run as check of correctness of digestion parameters. If digestion is not complete, make appropriate adjustments. A standard, such as NIST Standard Reference Material No 194, ammonium phosphate (NH4H2PO4), certified 12.15% nitrogen should also be included. If nitrate recovery is critical, a sample of potassium nitrate (KNO3) should also be added.

Following is a list of some standards available to include in Kjeldahl runs:

The ammonium salts and glycine p-toluenesulfonate serve primarily as a check on distillation efficiency and accuracy in titration steps because they are digested very readily. Lysine and nicotinic acid are difficult to digest, therefore serve as a check on digestion efficiency.

Calculation: Percent Nitrogen (N)
For boric acid trapping solution/standard HCl titrant:
% N (DM basis) = [(VA - VB) xNHCl x 1.4007] / W x Lab DM/100

• VA = Volume, in mL, of standard HCl required for sample
• VB = Volume, in mL, of standard HCl required for blank
• NHCl = Normality of standard HCl
• 1.4007 = milliequivalent weight of N X 100
• W = sample weight in grams

Calculation: Percent Crude Protein (CP)
CP (DM basis) = % N (DM basis)X F

• F = 6.25 for all forages and feeds except wheat grains
• F = 5.70 for wheat grains

Quality Control:
Include a reagent blank, one sample of high purity lysine hydrochloride, and one or more quality control (QC) samples in each run, choosing QC samples by matching analyte levels and matrices of QC samples to the samples in the run. Include at least one set of duplicates in each run if single determinations are being made. An acceptable average standard deviation among replicated analyses for crude protein ranges from about ±0.10 for samples with 10% CP to ±0.20 for samples with 20% CP, which results in warning limits (2s) ranging from ±0.20 to 0.40 and control limits (3s) ranging from ±0.30 to 0.60. Plot the results of the control sample(s) on an X-control chart and examine the chart for trends. Results outside of upper or lower warning limits, ±2s (95 percent confidence limits), are evidence of possible problems with the analytical system. Results outside of upper or lower control limits, ±3s (99 percent confidence limits), indicate loss of control and results of the run should be discarded. Two consecutive analyses falling on one side of the mean between the warning limits and the control limits also indicate loss of control.

3.1.1 Standardization of Hydrochloric Acid Standard Solution >>

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