IMPROVING PROTEIN IMPROVING PROTEIN ANALYSIS RESULTS BY ANALYSIS RESULTS BY KJELDAHL PROCEDUREKJELDAHL PROCEDURE

1616thth Annual ASAAnnual ASA--IM Sea Feed IM Sea Feed Technology and Nutrition Workshop Technology and Nutrition Workshop

May 28, 2008May 28, 2008SingaporeSingapore

Pinky Pinky PePe TobianoTobiano

IntroductionIntroductionImportance of crude protein values for Importance of crude protein values for

feed ingredientsfeed ingredients

Diet formulationDiet formulation

Monitoring quality of raw Monitoring quality of raw materials and mixed feedsmaterials and mixed feeds

Government regulation and Government regulation and control control

What is What is KjeldahlKjeldahl analysis?analysis?It is an analytical method to determine It is an analytical method to determine

quantitatively nitrogen in the quantitatively nitrogen in the trinegativetrinegative state in certain organic state in certain organic compounds.compounds.

The method was developed in 1833 by The method was developed in 1833 by Johan Johan KjeldahlKjeldahl, a Danish Chemist., a Danish Chemist.

This involves 3 main steps:This involves 3 main steps:Digestion, Digestion, Distillation, and Distillation, and Titration.Titration.

Principle of Principle of KjeldahlKjeldahl Nitrogen Nitrogen AnalysisAnalysisThe sample is digested to convert the The sample is digested to convert the protein nitrogen into ammonium sulfateprotein nitrogen into ammonium sulfate

The ammonium sulfate is treated with The ammonium sulfate is treated with an alkali, thus liberating ammonia an alkali, thus liberating ammonia

The ammonia is received by an acid and The ammonia is received by an acid and quantified through titrationquantified through titration

3 Different Steps of 3 Different Steps of KjeldahlKjeldahlMethodMethod

Digestion Digestion –– conversion of nitrogen conversion of nitrogen in the sample to ammonium in the sample to ammonium sulfatesulfate

ProteinProtein--N + HN + H22SOSO44 (NH(NH44))22SOSO44

DistillationDistillation-- separation of ammonia separation of ammonia from from digestatedigestate using alkali (using alkali (NaOHNaOH) ) and collection of ammonia using a and collection of ammonia using a receiverreceiver

(NH(NH44))22SOSO44 + 2NaOH 2NH+ 2NaOH 2NH33 + H+ H22O+ NaO+ Na22SOSO44

NHNH33 + H+ H33BOBO33 (Boric Acid) NH(Boric Acid) NH44 BorateBorate

Or, if the receiver used is hydrochloric acid:Or, if the receiver used is hydrochloric acid:

NHNH33 + + HClHCl NHNH44ClCl+ excess + excess HClHCl

TitrationTitration -- quantification of ammonia and quantification of ammonia and calculation of the initial protein calculation of the initial protein concentrationconcentration

NHNH44 Borate + Borate + HH22SOSO44 (NH(NH44))22SOSO4 4 ++ Boric AcidBoric Acid

The amount of NHThe amount of NH33 evolved is directly evolved is directly proportional to the amount of Hproportional to the amount of H22SOSO44 used used as as titranttitrant..

Or: NHOr: NH44ClCl+ excess + excess HClHCl + + NaOHNaOH NaClNaCl + H+ H22OO

Table 1. Crude protein content based on 6.25 Table 1. Crude protein content based on 6.25 factor factor vsvs specific factorspecific factor

Sample %N CP Specific % Crude Sample %N CP Specific % Crude using 6.25 factor Pusing 6.25 factor Proteinrotein

Skimmed 5.23 32.69 6.38 33.3Skimmed 5.23 32.69 6.38 33.388MilkMilk

Rice 1.57 9.78 5.95 Rice 1.57 9.78 5.95 9.319.31Oats 1.95 12.19 5.83 1Oats 1.95 12.19 5.83 11.371.37Soya Bean 7.43 46.44 5.71 42.42Soya Bean 7.43 46.44 5.71 42.42

MealMealWheat Bran 2.57 16.04 5.70 14.63Wheat Bran 2.57 16.04 5.70 14.63Safflower 3.35 20.94 5.30 17.7Safflower 3.35 20.94 5.30 17.755Sunflower 3.30 20.60 5.30 17.47Sunflower 3.30 20.60 5.30 17.47

FACTORS AFFECTING KJELDAHL FACTORS AFFECTING KJELDAHL TEST RESULTSTEST RESULTS

1. Sample Preparation1. Sample Preparation2. Sample Amount2. Sample Amount3. Catalyst3. Catalyst4. Digestion Time4. Digestion Time

Sample PreparationSample PreparationSample should be carefully prepared Sample should be carefully prepared

to avoid errors in the final result.to avoid errors in the final result.

This procedure may involve one or This procedure may involve one or more treatments to homogenize the more treatments to homogenize the sample. sample. Example: The particle size of the Example: The particle size of the sample sample must be reduced to a size must be reduced to a size < 1mm.< 1mm.

Homogeneity of the analytical Homogeneity of the analytical sample improves the sample improves the reproducibility of the method reproducibility of the method and also offers the possibility to and also offers the possibility to reduce the sample size used, reduce the sample size used, without sacrificing the quality of without sacrificing the quality of the final results.the final results.

Physical treatments used to Physical treatments used to homogenize samples:homogenize samples:

shakingshakingstirringstirringmortaringmortaringrifflingrifflingconing and quarteringconing and quarteringgrindinggrindingblendingblendinghomogenizing homogenizing millingmilling

Sample WeightSample Weight

The actual weight of sample The actual weight of sample required for analysis depends required for analysis depends primarily on the homogeneity primarily on the homogeneity of the sample.of the sample.

For For nonnon--homogeneous homogeneous samplessamples, high precision of the , high precision of the measurements cannot be measurements cannot be obtained using small sample obtained using small sample sizes.sizes.

For For homogeneous sampleshomogeneous samples, , amount is not as critical and amount is not as critical and can be optimized to give a can be optimized to give a suitable final titration volume. suitable final titration volume.

Table 2. Rough Guide for Table 2. Rough Guide for selecting the sample sizeselecting the sample sizeProtein Content mg sampleProtein Content mg sample

Up to 5% 1000Up to 5% 1000-- 5000500055--30% 50030% 500-- 15001500

more than 30% 200more than 30% 200-- 10001000

Note: the analytical sample should ideally Note: the analytical sample should ideally contain 10 contain 10 --100mg N100mg N

CatalystCatalystThe speed and efficiency of the digestion The speed and efficiency of the digestion

is not only influenced by the is not only influenced by the temperature used but can also be temperature used but can also be improved by the addition of a suitable improved by the addition of a suitable catalyst.catalyst.

Catalyst is used to hasten the chemical Catalyst is used to hasten the chemical reaction (oxidation) by increasing reaction (oxidation) by increasing boiling pointboiling pointSelenium MercurySelenium MercuryCopper TitaniumCopper Titanium

Factors in choosing a suitable Factors in choosing a suitable catalyst:catalyst:

1. Cost1. Cost2. Availability2. Availability3. Disposal3. Disposal

Usually, catalysts are composed of a Usually, catalysts are composed of a mixture of salts:mixture of salts:

Example:Example:

Potassium Sulfate plus Copper SulfatePotassium Sulfate plus Copper Sulfate

Sodium Sulfate plus Copper SulfateSodium Sulfate plus Copper Sulfate

Digestion TimeDigestion Time

Digestion time Digestion time -- the time it takes until the the time it takes until the digestatedigestate has cleared or become colorless.has cleared or become colorless.

Factors that affect the total Factors that affect the total digestion timedigestion time::1. Type of sample1. Type of sample2. Volume of acid2. Volume of acid3. Amount of catalyst used3. Amount of catalyst used4. Oxidizing agent ( Sulfuric Acid)4. Oxidizing agent ( Sulfuric Acid)5. Temperature of block 5. Temperature of block digestordigestor

TitrationTitrationIndicatorIndicator-- included in the receiver. Examples:included in the receiver. Examples:

1. Methyl red (from light green to light pink) 1. Methyl red (from light green to light pink) 2. 2. BromcresolBromcresol green (from light green to green (from light green to

light blue or light gray to very light pilight blue or light gray to very light pink) nk)

TitrantTitrant –– exact concentration and volume (titration reading) is exact concentration and volume (titration reading) is necessarynecessary

End pointEnd point-- achieved when the color of achieved when the color of digestatedigestate changes changes ex. The end point is achieved when the ex. The end point is achieved when the light light

green clear green clear digestatedigestate changes to light gray to very changes to light gray to very light pink. light pink. The The digestatedigestate is over titrated when the color is over titrated when the color becomes reddish pink.becomes reddish pink.

20 placer Kjeldahl Unit

6 placer Kjeldahl unit

Conventional Kjeldahl Apparatus

Digested at 4200C for 1 hr

Distilled with 50ml 40% NaOH

End-point- light gray to very light pink

Over titrated- pink color

Verification of Verification of KjeldahlKjeldahl TestTestUsing standard sample to check Using standard sample to check accuracy of result accuracy of result ex. ex. glycineglycine or or tryptophantryptophan

ureaurea

Using natural ingredient (ex. Using natural ingredient (ex. soyasoya or or corn) for the reproducibility of test resultcorn) for the reproducibility of test result

PER SYSTEM VERIFICATIONPER SYSTEM VERIFICATION

1.1. Digestion SystemDigestion System

In order to ensure high quality of the In order to ensure high quality of the analytical results produced in routine analytical results produced in routine work, it is essential to include check work, it is essential to include check sample in all batches digested and sample in all batches digested and analyzed.analyzed.

The following may be used for verifying The following may be used for verifying accuracy of the digestion procedure. accuracy of the digestion procedure. A A standard substance with known standard substance with known nitrogen contentnitrogen content

1. 1. GlycineGlycine purepure, use 500mg, use 500mgNitrogen content= 18.66%Nitrogen content= 18.66%

2. 2. TryptophanTryptophan purepure, use 500mg, use 500mgNitrogen content = 13.72 %Nitrogen content = 13.72 %

Table 3. Results of Table 3. Results of KjeldahlKjeldahlVerification TestVerification TestStandard Sample %, N %,N % N % NStandard Sample %, N %,N % N % N

GlycineGlycine 18.69 18.66 18.64 8.6618.69 18.66 18.64 8.66

Urea 45.10 45.23 45.16 45.05Urea 45.10 45.23 45.16 45.05

Note : % N= Nitrogen ContentNote : % N= Nitrogen Content

2. Distillation System2. Distillation SystemDistillation principle is to convert ammonium Distillation principle is to convert ammonium

(NH4) into ammonia (NH3) by adding alkali, (NH4) into ammonia (NH3) by adding alkali, and steam distilling into a receiver flask and steam distilling into a receiver flask containing boric acid with mixed containing boric acid with mixed indicators.indicators.

Since all nitrogen in the samples after Since all nitrogen in the samples after digestion form digestion form ammonium sulfateammonium sulfate, it can , it can be used as a standard to check the be used as a standard to check the recovery of the distilling unit.recovery of the distilling unit.

The following procedures can be used:The following procedures can be used:1. Use ammonium sulfate > 99.5%1. Use ammonium sulfate > 99.5%

% Nitrogen (NH% Nitrogen (NH44))22SOSO44 = 21.09 = 21.09 weight to use = 0.15gweight to use = 0.15g7575--100 ml distilled water, 50ml 100 ml distilled water, 50ml

NaOHNaOH (40%)(40%)2. Ammonium Iron (II) Sulfate2. Ammonium Iron (II) Sulfate

% Nitrogen = 7.145% Nitrogen = 7.145weight to use = 0.5gweight to use = 0.5g7575--100ml distilled water, 50ml 100ml distilled water, 50ml

NaOHNaOH 40%40%

3. Inter3. Inter--laboratory Verificationlaboratory VerificationParticipate in Participate in ““round robinround robin””

sample tests with other testing sample tests with other testing laboratorieslaboratoriesPrepare reference sample within Prepare reference sample within

the laboratory and verify versus the laboratory and verify versus other laboratories.other laboratories.

Note:Note: Always use this sample as internal Always use this sample as internal reference in daily routine use.reference in daily routine use.

When comparing results with other When comparing results with other laboratories, it is important to know the laboratories, it is important to know the moisture content of the sample.moisture content of the sample.

This can either be done by correcting for This can either be done by correcting for the moisture content and reporting the moisture content and reporting results on results on dry basis,dry basis, or by always or by always analyzing analyzing predriedpredried samples.samples.

Content on dry basis = Ax100 /(100Content on dry basis = Ax100 /(100--B)B)A= % Protein of sampleA= % Protein of sampleB= % Moisture of sample when B= % Moisture of sample when

analyzed for proteinanalyzed for protein

Table 4A. Common ingredients and their Table 4A. Common ingredients and their observed values (CP%, observed values (CP%, meanmean±±stdstd) as ) as analyzed by analyzed by KjeldahlKjeldahl ProcedureProcedure

Ingredient N Ingredient N As AnalyzedAs Analyzed As Dry BasisAs Dry Basis% (mean) Std. D % (me% (mean) Std. D % (mean) an) Std.DStd.D

Fish Meal 15 62.67 1.91 68.98Fish Meal 15 62.67 1.91 68.98 2.552.55Rice Bran 6 12.65 0.23 13.9Rice Bran 6 12.65 0.23 13.99 0.33 9 0.33 Soya Bean 12 45.47 1.09 51.75 Soya Bean 12 45.47 1.09 51.75 1.55 1.55

Meal Meal Yellow Corn 4 7.56 0.36 8.8Yellow Corn 4 7.56 0.36 8.83 0.233 0.23

Table 4B. Different Qualities of Soya Bean Meal and their observed Crude Protein

value as analyzed by Kjeldahl Procedure

IngredientN

(No. of samples)

As Analyzed% (mean) SD

Soya Bean MealLow Protein

(44.0% to 46.0%)

57 45.45 0.87

Soya Bean Meal High Protein

(47.0 to 48.0 %)

9 47.41 0.40

SUMMARY/ CONCLUSIONSUMMARY/ CONCLUSIONThe key to successful The key to successful KjeldahlKjeldahl analysis analysis

can be found:can be found:1. In the sample preparation step;1. In the sample preparation step;2. By validating the test procedure that 2. By validating the test procedure that

includes use of catalyst, digestion time, includes use of catalyst, digestion time, amount of acid; amount of acid;

3. By verifying the digestion procedure;3. By verifying the digestion procedure;4. By monitoring regularly the 4. By monitoring regularly the

performance of distillation system.performance of distillation system.

A SUCCESSFUL KJELDAHL A SUCCESSFUL KJELDAHL ANALYSIS WILL HAVE ANALYSIS WILL HAVE

THE RIGHTTHE RIGHTKJELDAHL TEST RESULT.KJELDAHL TEST RESULT.

THANK YOU!THANK YOU!

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