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Protein Determination

Nitrogen (Total)  in Milk - Kjeldahl Methods

Protein Nitrogen Content of Milk - Kjeldahl Method (Direct Method)

NonProtein Nitrogen in Whole Milk - Kjeldahl Method

Protein Nitrogen Content of Milk - Kjeldahl Method (Indirect Method)

Prepared by
Test Procedures Committee of Market Administrators, June 1991

Preface
    The following  procedures for determining the protein content of milk have been approved by the Market Administrators' Milk Test  Procedures Committee. 
These procedures involve the following Kjeldahl methods:
    1. Nitrogen (Total)
    2. Protein Nitrogen
    3. Nonprotein Nitrogen
    4. Protein Nitrogen (Indirect Method)

    These methods resulted from collaborative studies which were presented to the Association of Official Analytical Chemists (AOAC) in September 1989 and were published in "Changes in Official Methods of Analysis", 1991 journal.
    Nine laboratories analyzed nine pairs of blind duplicate raw milk samples for percent protein by these prescribed methods.  The goals of these collaborative studies were to develop testing procedures for protein determinations that would yield highly accurate results and ones which could be uniformly adapted and applied throughout the dairy industry.
    These procedures when properly followed will yield comparable results between and within laboratories.
    The full report for each of these protein determination procedures by the Kjeldahl method is available from the office of any Federal Milk Market Administrator or from the USDA, Ag. Mkt. Service, Dairy Division, P.O. Box 96456, Washington, DC 20090-6456.

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.
    Trichloroacetic acid (TCA).  TCA is very corrosive and can cause burns.  Wear eye protection and plastic gloves when working with TCA.  If TCA is spilled on the skin, wash immediately with large amounts of cool water.  NEVER pipet TCA solutions by mouth.
    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!

Nitrogen (Total) in Milk Kjeldahl Methods

Principle
    Milk is digested in H2SO4, using CuSO4 . 5H20 as catalyst with K2SO4 as boiling point elevator, to release nitrogen from protein and retain nitrogen as ammonium salt.  Concentrated NaOH is added to release NH3, which is distilled, collected in H3BO3 solution, and titrated.

Traditional Method

Apparatus

  1. Digestion flasks.  Kjeldahl.  Hard, moderately thick, well annealed glass.  Total capacity is ca 500 or 800 mL.

  2. Distillation flasks.  Same as Kjeldahl flask as in (a), fitted with rubber stopper through which passes lower end of efficient connecting bulb or trap to prevent mechanical carryover of NaOH during distillation.  Connect upper end of bulb to condenser tube with rubber tubing.  Use graduated 500 mL Erlenmeyer titration flask to collect distillate.  Trap outlet of condenser in manner to ensure complete absorption of NH3 distilled into boric acid solution.

  3. Digestion/distillation system.  Traditional apparatus with adjustable controls for individual flasks.

  4. Titration buret.  50 mL.  Class A or equivalent.

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.  50% 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).

  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.  Prepare as in Association of Official Analytical Chemists method number 936.15 or use pre-made solution of certified specification range 0.0995-0.1005N and use 0.1000N for calculation.

  9. Ammonium sulfate.  99.9% (NH4)2SO4.

  10. Tryptophan or lysine hydrochloride.  99% C11H12N2O2 or C6H15ClN2O2.

  11. Sucrose.  Nitrogen free.

Sample Preparation
    Add 15 g K2SO4, 1 mL CuSO4 . 5H2O catalyst solution and 8-10 boiling chips to digestion flask.  Warm milk to 38+10C and mix thoroughly.  Weigh warm sample (5+0.1 mL) and immediately place in digestion flask.  (Note: Weights mist be recorded to nearest 0.0001 g.)  Add 25 mL H2SO4, rinsing any milk on neck of flask down into bulb.  Flask may be stoppered and held for digestion at later time.  Digest and distill a blank (all reagents and no sample) each day.

Determination

  1. Digestion burner setting.  Conduct digestion over heating device that can be adjusted to bring 250 mL H2O at 250 to rolling boil in ca 5-6 min.  To determine maximum heater setting to be used during digestion, preheat 10 minutes (gas) or 30 minutes (electric) at burner setting to be evaluated.  Add 3 or 4 boiling chips to 250 mL water at 250C and place flask on preheated burner.  Determine heater setting that brings water from 250 to rolling boil in 5-6 minutes on each burner.  This is maximum burner setting to be used during digestion.

  2. Digestion.  Place flask in inclined position with fume ejection system on.  Start with setting low enough so that sample does not foam up into neck of Kjeldahl flask.  Digest at least 20 minutes or until white fumes appear in flask.  Next, increase burner setting half way to maximum setting determined in (a) and heat for 15 minutes.  Next, increase heat to maximum setting determined in (a).  When digest clears (clear with light blue-green color), continue to boil 1-1.5 hr at maximum setting (total time ca 1.8-2.25 hr).
        To determine specific boil time needed for analysis condition in your laboratory, select a high protein, high fat milk sample and determine protein content using different boil times (1-1.5 hr) after clearing.  mean protein test increases with increasing (0-1.5 hr) boil time, becomes constant, and then decreases when boil time is too long.  Select boil time that yields maximum protein test.
        At end of digestion, digest should be clear and free of undigested material.  Cool acid digest to room temperature (ca 25 min.).  Cooled digest should be liquid or liquid with few small crystals.  (Large amount of crystallization before addition of water indicates too little residual H2SO4 at end of digestion and can result in low test values.)  After digest is cooled to room temperature, add 300 mL H2O to flask and swirl to mix (for 800 mL flasks add 400 mL H2O).  When room temperature water is added some crystals may form and then go into solution; this is normal.  Let mixture cool to room temperature before distillation.  Flasks can be stoppered for distillation at a later time.

  3. Distillation.  Turn on condenser water.  Add 50 mL H3BO3 solution with indicator to graduated 500 mL Erlenmeyer titration flask and place flask under condenser tip so that tip is well below H3BO3 solution surface.  To room temperature diluted digest, carefully add 75 mL 50% NaOH down sidewall of Kjeldahl flask with no agitation.  NaOH forms clear layer under the diluted digest.  Immediately connect flask to distillation bulb on condenser.  Vigorously swirl flask to mix contents thoroughly; heat until all NH3 has been distilled (>150 mL distillate; >200 mL total volume).  Do not leave distillation unattended.  Flasks (500 mL) may bump at this point (ca 150 mL distillate; 200 mL total volume).  Lower receiving flask and let liquid drain from condenser tip.  Turn off distillation heater.  Titrate H3BO3 receiving solution with standard 0.1000N HCL solution to first trace of pink.  Lighted stir plate may aid visualization of end point.  Record mL HCL to at least nearest 0.05 mL.

Nitrogen Recovery Verification

Run nitrogen recoveries to check accuracy of procedure and equipment.

  1. Nitrogen loss.  Use 0.12 g ammonium sulfate and 0.85 g sucrose per flask.  Add all other reagents as stated in Sample Preparation.  Digest and distill under same conditions as for a milk sample.  Recoveries shall be at least 99%.

  2. Digestion efficiency.  Use 0.16 g lysine hydrochloride or 0.18 g tryptophan, with 0.67 g sucrose per flask.  Add all other reagents as stated in Sample Preparation.  Digest and distill under same conditions as for milk sample.  Recoveries shall be at least 98%.

Calculations

Calculate results as follows:

                        1.4007 x (mL HCL, sample - mL HCL, blank ) x normality HCL
Nitrogen, % = --------------------------------------------------------------------
                                                            g sample

Multiply percent nitrogen by factor 6.38, to calculate percent "protein."  this is "protein" on a total nitrogen basis.
Maximum recommended difference between duplicates is 0.03% "protein."

Repeatability and Reproducibility Values
    For method performance parameters obtained in collaborative study of this method Sr = 0.014, SR = 0.017, RSDr = 0.385%, RSDR = 0.504%, r value = 0.038 and R value = 0.049.

Reference: D.M. Barbano, J.L. Clark, C.E. Dunham, and J.R. Fleming.  1990.
    Kjeldahl Method for Determination of Total Nitrogen Content of Milk:
    Collaborative Study.  Journal of Association of Official Analytical Chemists
    73: 849-859.

Block Digestor / Steam Distillation Method

Apparatus

  1. Digestion block.  Aluminum alloy block or equivalent apparatus, with adjustable temperature control and device for measuring block temperature.

  2. Digestion block tubes.  250 mL capacity.

  3. Distillation unit.  For steam distillation.  To accept 250 mL digestion tubes and 500 mL titration flasks.

  4. Titration buret.  50 mL.  Class A or equivalent

Reagents
    See Reagents (a)-(k) for the Traditional Method.
    Note: 40% w/w NaOH may be used instead of 50% w/w.  Boiling chips should not be used if equipment manufacturer does not recommend such use.

Sample Preparation
    Add 12 g K2SO4 and 1 mL CuSO4 . 5H2O catalyst solution to digestion tube.  Warm milk to 38+1oC and mix thoroughly.  Weigh warm sample (5 + 0.1 mL) and immediately place in digestion tube.  (Note: weights must be recorded to nearest 0.0001 g.)  Add 20 mL H2SO4.  Tube may be stoppered and held for digestion at later time.  Digest and distill a blank (all reagents and no sample) each day.

Determination

  1. Digestion.  Set block at al low initial temperature to control foaming (ca 180-230oC).  Place tubes with aspirator connected in block digestor; suction should be just enough to remove fumes.  Digest 30 minutes or until white fumes develop.  Increase temperature to 410-430oC and digest until clear.  It may be necessary to increase temperature gradually over ca 20 minutes to control foaming.  Do not let foam in tube rise higher than ca 4-5 cm below surface of fume collection device inserted into top of tube.  After digest clears (clear with light blue-green color), continue to boil (H2SO4 must be boiling) for at least 1 hr, total digestion time ca 1.75-2.5 hr.
        To determine specific length of boil time needed for analysis conditions in your laboratory, select high protein, high fat milk sample and determine protein content using different boil times (1-1.5 hr) after clearing.  Mean protein test increases with increasing (0-1.5 hr) boil time, becomes constant, and then decreases when boil time is too long.  Select boil time that yields maximum protein test. (Note: before removing hot tubes from block, make sure there is no condensate layer in aspirator manifold.  If there is a liquid layer, increase aspiration to remove liquid.)
        At the end of digestion, digest should be clear and free of undigested material.  Cool digest to room temperature (ca 25 min).  Cooled digest should be liquid or liquid with a few small crystals at bottom of tube.  (Excessive crystallization indicates too little residual H2SO4 at end of digestion and may cause low results.  To reduce acid loss during digestion, reduce fume aspiration rate.)  After digest has cooled to room temperature, add 85 mL H2O (blanks may require 100 mL) to each tube, swirl to mix, and let cool to room temperature.  When room temperature water is added some crystals  may form and then go into solution; this is normal.  Tubes can be stoppered for distillation at a later time.

  2. Distillation.  Place 50% (or 40%) NaOH in alkali tank of distillation unit.  Adjust volume dispensed to 55 mL (65 mL for 40% NaOH).  Attach digestion tube containing diluted digest to distillation unit.  Place graduated 500 mL Erlenmeyer titration flask containing 50 mL H3BO3 solution with indicator on receiving platform, with tube from condenser extending below surface of H3BO3 solution.  Steam-distill until >150 mL distillate is collected (> 200 mL total volume).  Remove receiving flask.  Titrate H3BO3 receiving solution with standard 0.1000N HCL to first trace of pink.  Lighted stir plate may aid visualization of end point.  Record mL HCL to at least nearest 0.05 mL. 

Nitrogen Recovery Verification

Run nitrogen recoveries to check accuracy of procedure and equipment.

  1. Nitrogen loss.  Use 0.12 g ammonium sulfate and 0.85 g sucrose per flask.  Add all other reagents as stated in Sample Preparation.  Digest and distill under same conditions as for a milk sample.  Recoveries shall be at least 99%.

  2. Digestion efficiency.  Use 0.16 g lysine hydrochloride or 0.18 g tryptophan, with 0.67 g sucrose per flask.  Add all other reagents as stated in Sample Preparation.  Digest and distill under same conditions as for a milk sample.  Recoveries shall be at least 98%.

Calculations
See Calculations from Traditional Method.

Repeatability and Reproducibility Values
See Traditional Method.  Values are the same.

Reference:  D.M. Barbano, J.L. Clark, C.E. Dunham, and J.R Fleming.  1990. Kjeldahl Method for Determination of Total Nitrogen Content of Milk:  Collaborative Study.  Journal of Association of Official Analytical Chemists 73:849-859.

Protein Nitrogen Content of Milk Kjeldahl Method (Direct Method)

Principle
    Protein is precipitated from milk by trichloroacetic acid (TCA) solution.  Precipitation must be done in Kjeldahl flask or tube.  Final concentration of TCA in mixture is ca 12%.  The 12% TCA solution, which contains nonprotein nitrogen components of a sample, is separated from protein precipitate by filtration.  Nitrogen content of protein precipitate is determined as in method titled Nitrogen Total in Milk - Kjeldahl Methods.

Apparatus
   
See method titled Nitrogen (Total) in Milk - Kjeldahl Methods for both traditional and block digestor systems. 

Reagents
    See method titled Nitrogen (Total) in Milk - Kjeldahl Methods for both traditional and block digestor systems, and in addition:
(a) Trichloroacetic acid solution.  15% w/v, analytical grade CCl3COOH.  (Caution:  see safety note on trichloroacetic acid.)  TCA is a soft, white, deliquescent crystal, which should be stored in a container protected from light and moisture.

Preparation of Sample
    Warm milk to 38+1oC and mix thoroughly.  Immediately place weighed sample (5 + 0.1 mL) in Kjeldahl digestion flask.  Record all weights to nearest 0.0001 g.  Add 5 + 1 mL H2O rinsing any milk on the neck of the flask into the bulb.  Add 40 + 0.5 mL 15% TCA solution to flask.  Swirl mixture.  Let precipitate settle (ca 5 minutes).  Pour mixture from Kjeldahl flask through filter paper (Whatman No. 1, 15 cm, N-free; or equivalent) and collect filtrate.  (Some protein precipitate will remain in Kjeldahl flask and some will be collected on paper.  It is not necessary to remove precipitate from flask.)
    Immediately after pouring mixture (do not let precipitate dry on neck of Kjeldahl flask), use pump dispenser to add 10+0.5 mL 15% TCA to Kjeldahl flask and rinse any precipitate on neck of flask down into bulb.  Swirl to mix.  Pour mixture from Kjeldahl flask through same filter paper, and add filtrate to that previously collected.  Immediately rinse neck of Kjeldahl flask with another 10+0.5 mL rinse of TCA solution.  Swirl to mix and pour mixture from flask through same filter paper used earlier.  Collect entire filtrate.  Filtrate should be clear and free of particulate matter.  At this point.  filtrate is no longer needed and may be discarded in the appropriate manner.
    Wearing TCA-resistant gloves, pick up filter paper; take care not to lose any precipitate.  Pinch paper at top and twist sides and bottom to form oblong shape.  If any precipitate remains on either inner or outer lip of Kjeldahl flask, wipe with filter paper so precipitate adheres to paper.  Drop filter paper in Kjeldahl flask.  Add boiling chips, K2SO4, CuSO4 . 5H2O catalyst solution, and H2SO4 as in method titled Nitrogen (Total) in Milk - Kjeldahl Methods.  Flask may be stoppered and held for digestion at a later time.  Digest and distill a blank (filter paper) each day that samples are analyzed.  Keep record of blank values.  If blank values change, identify cause.

Determination
    Proceed as in Method titled Nitrogen (Total) in Milk - Kjeldahl Methods.

Calculation
   
Calculate protein nitrogen in milk as in method titled Nitrogen (Total) in Milk - Kjeldahl Methods.

Repeatability and Reproducibility
   
For method performance parameters obtained in collaborative study of this method, Sr = 0.008, SR = 0.021, RSDr = 0.285%, RSDR = 0.702%, r value = 0.024 and R value = 0.059.

Reference: D.M. Barbano, J.M Lynch, and J.R. Fleming.  1991.  Direct and Indirect Determination of True Protein Content of Milk by Kjeldahl Analysis: Collaborative Study.  Journal of Association of Official Analytical Chemists. 74:281-288.

Nonprotein Nitrogen in Whole Milk Kjeldahl Method

Principle
    Protein is precipitated from milk by addition of trichloroacetic acid (TCA) solution.  Final concentration of TCA in the mixture is about 12%.  Precipitated milk protein is removed by filtration.  Filtrate contains nonprotein nitrogen components of milk.  Nitrogen content of filtrate is determined as in method titled Nitrogen (Total) in Milk - Kjeldahl Methods.

Apparatus
   
See method titled Nitrogen (Total) in Milk - Kjeldahl Methods.

Reagents
   
See method titled Nitrogen (Total) in Milk - Kjeldahl Methods, and in addition:
(a) Trichloroacetic acid solution.  15% w/v, analytical grade CCl3COOH.  (Caution:  See safety note on trichloroacetic acid.)  TCA is a soft, white, deliquescent crystal, which should be stored in a container protected from light and moisture.
(b) Hydrochloric acid standard solution.  0.0100N HCL.  Prepare as in Association of Official Analytical chemists method number 936.15.  Alternatively, use pre-made solution of certified specification range 0.0101-0.0099N and use 0.010 for calculation.

Preparation of Sample
    Warm milk to 38+1oC and mix thoroughly.  Immediately pipet milk (10+1 mL) into preweighed 125 mL Erlenmeyer flasks and weigh.  Record all weights to nearest 0.0001 g.  Add 40 + 0.5 mL 15% TCA solution to flask.  Weigh flask and contents, swirl to mix.  Let precipitate settle (ca 5 min).  Filter (Whatman No. 1 paper, 15 cm, N-free; or equivalent) and collect entire filtrate.  Filtrate should be clear and free of particulate matter; if it is not, repeat sample preparation.  Swirl filtrate to mix.  Pipet 20 + 0.2 mL filtrate into a 50 mL beaker and weigh.  Pour filtrate from beaker into Kjeldahl digestion flask that contains boiling chips, Cu2SO4, and CuSO4 . 5H2O catalyst solution as in method titled Nitrogen (Total) in Milk - Kjeldahl Methods. Immediately reweigh empty beaker.  Add H2SO4 as in method titled Nitrogen (Total) in Milk - Kjeldahl Methods.  Flask may be stoppered and held for digestion at a later time.  Digest and distill a blank solution 916 + 0.5 mL 15% TCA and no sample) each day that samples are analyzed.  Keep record of blank values.  If a blank value changes, identify the cause.

Determination
    Proceed as in method titled Nitrogen (Total) in Milk - Kjeldahl Methods, substituting 0.0100N HCL solution for 0.1000N HCL solution as a titrant.

Calculation
    Calculate as follows:

                        1.4007 x (Vs - Vb) x N
Nitrogen % = ----------------------------------
                    (Wf x Wm)/[Wt - (Wm x 0.065)]

Where:

    Vs  = mL titrant used for sample
    Vb  = mL titrant used for blank
    N    = normality of HCL solution
    Wf  = weight, g, of 20 mL filtrate
    Wm = weight, g, of milk
    Wt  = weight, g, of milk plus 40 mL 15% TCA solution

Note: Factor 0.065 in denominator assumes that milk contains about 3.5% fat and 3.0% true protein (i.e., 0.035 + 0.030).  Factor may need to be adjusted if liquid diary products of different composition are analyzed (i.e., concentrated or fractionated skim or whole milk products, etc.). 

                "protein equivalent, " % = nitrogen x 6.38
which is nonprotein nitrogen expressed as protein equivalent.

Repeatability and Reproducibility
    For method performance parameters obtained in collaborative study of this method, Sr = 0.0006, SR = 0.012, RSD r = 2.817%, RSDR = 5.707%, r value = 0.016, and R value = 0.033.

Reference: D.M> Barbano, J.M. Lynch, and J.R. Fleming.  1991.  Direct and Indirect Determination of True Protein Content of Milk by Kjeldahl Analysis: Collaborative Study.  Journal of Association of Official Analytical Chemists 74:281-288.

Protein Nitrogen Content of Milk Kjeldahl Method (Indirect Method)

Principle
    Total nitrogen and nonprotein nitrogen contents of milk sample are determined separately.  Difference between results of these 2 determinations is protein nitrogen content of milk.

Determination
(a) Total nitrogen.  Determine as in method titled Nitrogen (Total) in Milk - Kjeldahl Methods.
(b) Nonprotein nitrogen.  Determine as in method titled Nitrogen (Total) in Milk - Kjeldahl Methods.

Calculation
    Subtract nonprotein nitrogen content from nitrogen content of milk sample and multiply result by 6.38.

Repeatability and Reproducibility
    For method performance parameters obtained in collaborative study of this method, Sr = 0.014, SR = 0.031, RSDr = 0.483%, RSDR = 1.051%, r value = 0.040, and R value = 0.088.

Reference: D.M Barbano, J.M. Lynch, and J.R. Fleming.  1991.  Direct and Indirect Determination of True Protein Content of Milk by Kjeldahl Analysis: Collaborative Study.  Journal of Association of Official Analytical Chemists 74:281-288.


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