Application Note 300

Rev. 8.0

The determination of Nitrogen according to Kjeldahl using Block Digestion and Steam Distillation
1. Introduction
The following information provides general guidelines for the analysis of Kjeldahl nitrogen using the FOSS Kjeltec Systems. The procedure described is applicable to all sample types, which may be analysed in a routine laboratory. Because Kjeldahl is a universal method no consideration is given to optimising the procedure described for a specific type of sample. This information can be found in FOSS Application Sub Note library for sample specific methodology. As FOSS supplies a complete range of Kjeltec systems, from manually operated to fully automatic, no reference is made to specific instrument settings. These can be found in the users manual for the individual systems and in the specific Application Sub Notes.

2. Safety
The Kjeldahl method requires the digestion of the sample using strong acid at high temperatures. Careful handling of the solutions is mandatory for laboratory safety. Refer to the appropriate material safety sheet for reagent handling instructions. For added protection, acid digestions should be performed in a fume hood with adequate ventilation. Eye protection should be worn at all times and care should be taken when handling hot digestion tubes.
10 35 002a

3. Sample preparation
Sample preparation for Kjeldahl analysis should be carefully considered to avoid errors in the final result. Obtaining a laboratory sample, which is representative of the bulk product, is the subject of well-documented sampling procedures and varies greatly across different product and industry types. It is not possible to cover all aspects of these procedures here. Obtaining the analytical sample from the laboratory sample of the bulk product may involve one or more treatments to ensure homogeneity. These may be simple physical treatments such as shaking, stirring, mortaring, riffling,

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coning and quartering, or mechanical treatments such as grinding blending, homogenising, or milling. The method or methods used will depend on the sample to be treated, whether it is a mixture or compound or a single substance. Generally samples can be divided in three groups, solids, pastes or semisolids, and liquids.

3.1 Solid Samples

Solid samples are normally treated by some form of grinding. This may be a simple coffee grinder or laboratory mill. The particular method selected is not critical. However, the consistency of the treatment is vital to obtain satisfactory results especially when the analytical method has been optimised to one set of conditions. For example the very fine particle size of a sample ground on a cyclone mill will enhance the efficiency of digestion. If a sample of the same substance is treated with a coffee grinder and then analysed under the same conditions the results may not be comparable because the digestion process may not be complete. As a recommendation the particle size should be equal to, or less than, 1 mm.

3.2 Semi Solid Samples

This category of sample is the most difficult to handle particularly when there is a wide variation in particle size and / or hardness of constituents. Depending on the particular sample type, homogenising, liquefying or ball milling may provide a suitable sample for analysis. In many cases a simple pestle and mortar may provide the best solution. As with all sample preparation, a combination of two or more treatments may be necessary to achieve the desired homogeneity. Nitrogen free paper can be used for simplifying weighing of the sample and the transfer to the digestion tube.

3.3 Liquid Samples

Liquid samples come in two basic forms, solutions containing fully solubilised constituents for example tap water, and solutions containing particulates or suspensions, for example milk. Fully solubilised solutions require no more than shaking or stirring to achieve a satisfactory sample. Suspensions on the other hand may require different treatments depending on whether the whole or part of the sample is of interest. Milk for example, will separate on standing into an aqueous phase containing suspended solids and a fat phase, the cream, which floats on the surface. Normally analysis is performed on the whole milk and therefore the sample requires homogenising. Alternatively, many water samples may contain precipitates, which are not required in the analysis or may be required for separate analysis. The analytical sample may then be obtained by filtration or sedimentation and the filtrate or supernatant liquid taken for analysis. Whichever sample type is to be analysed significant errors can be introduced unless satisfactory sample preparation is performed prior to analysis.

3.4 Sample Weight

For Kjeldahl analysis an analytical balance accurate to 0.1 mg should be used for weighing samples, which are quantitatively transferred to the digestion tube. The actual weight of sample required is dependent on Nitrogen content and homogeneity. In general the smaller the sample the faster the digestion will be completed. For selecting the size of sample a rough guide is given below.

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Homogenous samples (excluding water) Non homogenous samples Water samples (dependant on N content)

0.1 - 1.0 g 1.0 - 3.0 g or more 1.0 - 100 ml

Where homogeneity of sample is not a controlling factor the sample weight can be selected relative to the Nitrogen content. Using a titrant concentration of 0.2 N the analytical sample should ideally contain 10 - 100 mg N and the appropriate sample Minimum weight in mg =

1000 x

where x = approximate % nitrogen anticipated. Liquid samples can be measured by weight or volume. When using volume the results are normally reported in w/v units for example mg N/100 ml or g N/l. This is because results reported in w/w units may differ significantly dependant on the specific gravity / density of the sample.

4. Digestion
During digestion the Nitrogen or Protein in the sample is converted to Ammonium Sulphate according to the formula Salt K2SO4 Protein + H2SO4 -------------> Catalyst (NH4)2SO4

In a Kjeltec system the controlled conditions during digestion eliminate the potentially large loss of acid, which might cause loss of nitrogen. Therefore the volume of acid required is generally less than that recommended in classical methods. Thus when a conventional method recommends 25 to 30 ml H2SO4 it is normally sufficient to use 10 to 15 ml in 250 ml tubes and only 2 - 5 ml in semi micro (100 ml) tubes in a Kjeltec system. Generally the 250 ml tubes give easier sample handling than the 100 ml tubes. 250 ml tubes give flexibility to handle the broadest range of samples, sample sizes and applications. They also handle foaming problems during the first part of the digestion better than the 100 ml tubes.

4.1 Salt
Since all compounds do not decompose at the boiling point of concentrated sulphuric acid it is necessary to increase the boiling point with a salt, usually potassium sulphate. This salt is incorporated in the Kjeltabs together with the catalyst. For some types of samples the decomposition takes a very long time due to low boiling point. In this case it might be necessary to add an extra Kjeltab. The ratio between salt and acid is now changed and the boiling point is increased. If samples with high fat or carbohydrate content are to be analysed, crystallisation can occur because it takes more acid to oxidise these constituents than protein, in which case add 2 - 3 ml extra acid at the start of the digestion.

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Note Crystallisation during digestion can cause nitrogen losses!

4.2 Antifoaming Agents

Hydrogen Peroxide is in widespread use in Kjeldahl laboratories throughout the world. This reagent has two functions: 1. As an oxidising agent to accelerate the decomposition of organic matter. 2. As an antifoam agent to control the tendency for foaming during digestion exhibited by various sample types. Use of hydrogen peroxide which is extremely reactive in the presence of sulphuric acid can cause loss of nitrogen as gas and in many cases makes no appreciable improvement to digestion times. Undoubtedly there are benefits as an antifoam particularly when the sample content of fats and / or carbohydrates is high. If hydrogen peroxide is used it should be added in small quantities < 5 ml slowly down the inner walls of the digestion tube. As a general rule unless experience shows an improvement in digestion conditions it is preferable not to use hydrogen peroxide. If foaming is the only problem to be overcome it is better to use 1 - 3 drops of octanol or a proprietary antifoam emulsion instead. Table 1 CATALYST AND DIGESTION TIMES Dog food
Digestion time (min)

% protein Hg 25.6+0.07 25.6+0.14 25.7+0.13 25.7+0.07 Se 25.1+0.10 25.4+0.15 25.4+0.16 25.6+0.11 Cu 24.8+0.19 25.2+0.22 25.4+0.11 25.5+0.12 Ti 25.0+0.17 25.3+0.27 25.5+0.25 25.6+0.22

10 20 30 45 60 Meat Min. 10 20 30 45 60

Hg 18.0+0.26 18.0+0.15 18.0+0.13 18.2+0.09

Se 17.7+0.33 17.7+0.27 17.9+0.17 17.9+0.24

Cu 17.4+0.23 17.8+0.22 17.8+0.24 18.1+0.23

Ti 17.2+1.07 17.9+0.37 18.0+0.32 18.0+0.06

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Fishmeal Min. 10 20 30 45 60 Hg 69.1+1.62 72.4+0.22 73.0+0.31 72.7+0.14 72.7+0.28 Se 65.0+0.75 70.1+0.66 71.0+0.23 71.7+0.19 72.2+0.15 Cu 64.7+1.21 72.6+2.83 70.6+0.74 71.4+0.39 72.3+0.21 Ti 64.8+1.15 69.5+0.54 70.4+0.34 71.4+0.43 72.3+0.45

Wheat Min. 10 20 30 45 60 Lysine-HCl Min. 10 20 30 45 60 Hg 14.8+0.18 15.3+0.12 15.3+0.16 15.3+0.09 Se 13.0+1.00 13.3+0.72 13.9+0.56 14.2+0.27 Cu 12.6+0.60 13.0+0.19 13.4+0.56 13.8+0.27 Ti 12.5+0.32 12.9+0.48 13.7+0.32 14.2+0.55 Hg 11.4+0.31 11.7+0.06 11.7+0.08 11.7+0.07 11.7+0.05 Se 11.2+0.24 11.6+0.07 11.6+0.03 11.6+0.04 11.7+0.07 Cu 11.0+0.19 11.6+0.06 11.6+0.07 11.6+0.05 11.7+0.04 Ti 11.0+0.29 11.0+0.03 11.7+0.22 11.7+0.06 11.7+0.05

The results are based on 5 - 7 subsamples. The results obtained obviously show that mercury is an outstanding catalyst. According to the results in Table 1, 30 minutes duration of digestion should be sufficient for common food products if mercury is used as catalyst. The other catalysts need considerably longer digestion times particularly if they are supposed to be used as general catalysts. Only for special applications e.g. for digestion of cereals; copper, titanium or selenium may be considered for rapid digestion procedures if 100 percent recovery is the ultimate goal. However, this may not always be the case. In practice a recovery close to 100 percent within reasonable time, may be sufficient. As shown in Table 1, there is a general tendency that shorter digestion periods in combination with incomplete digestion tend to reduce the reproducibility. One of the reasons for this is likely to be variations in salt acid ratio causing variations in the initial speed of the digestion.

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5. Mains Voltage
The mains voltage determines the time needed to reach any preselected temperature setting of the digestor. It has also a pronounced effect on the time required for the digestor to return to its original working temperature after a set of digestion tubes with samples and reagents have been inserted. These factors may have a measurable effect on the time needed to complete a digestion. In particular, voltage supply should be kept in mind when a short digestion time is preferred. Note Unless otherwise stated, the digestion time as indicated in Tecator application notes is determined at 230 V.

6. Exhaust System
To be able to evacuate the SO2 and SO3 fumes coming from the digest, a fume exhaust manifold is necessary. Not only to evacuate the fumes but also to prevent excessive acid losses. 1. 2. 3. Place the samples in the digestor with the exhaust manifold on top. The water aspirator must operate at full flow the first five minutes of the digestion. This is done to evacuate moisture, etc. After five minutes it is essential to decrease the aspirating effect to a minimum to prevent acid losses. To simplify this procedure and secure good working conditions a Flow Regulator can be used.

7. Heat Shields
Note Heat shields are integrated in current models. Since the short sides of the digestion rack are open, cold air will pass around the test tubes and cool them. This will create a low condensation point and samples stuck to the test tube walls will not be rinsed down to the bottom of the test tube. This sample will not be completely decomposed. To ensure correct refluxing of the acid the heat shields must be in place (See Fig)

B L O C 0 0 1 a

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A. Condensation point low due to cooling of the test tube (no heat shield in position) B. Correct position of condensation ring. (Heat shield in position).

8. Distillation
Since all samples after the digestion will form ammonium sulphate (NH4)2SO4, it can be used as a standard to check the recovery of the distilling units. The way to use this chemical will be described in the following. The distillation principle is to convert ammonium (NH4+) into ammonia (NH3) by using an alkali (NaOH) and thereafter steam distil it into a receiver flask containing boric acid and titrate with standard acid solution using colorimetric end-point detection.

8.1 Chemical Check

1. Use ammonium sulphate (NH4)2SO4 > 99.5 % Mol. weight = 132.14 g/mol. Dry Ammonium sulphate for 4 hours at 102C, and store in a desiccator % Nitrogen in ammonium sulphate (99.5 %) = 21.09 Weigh 0.15 g ammonium sulphate into a tube Add 75 ml distilled water and 50 ml 40 % NaOH and distil

% Nitrogen =

ml

sample

mlblank N 14,007 100 mg sample

N = Normality of titrant to 4 places of decimal.

% Recovery =

% Nitrogen 100 21.09

2.

Use ammonium iron (II) sulphate (NH4)2 Fe(SO4)2 x 6H2O Mol. weight = 392.14 g/mol. % Nitrogen in ammonium iron (II) sulphate = 7.145 Weigh 0.5 g ammonium iron (II) sulphate into a tube. Add about 75 ml distilled water, and 50 ml 40 % NaOH

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% Nitrogen =

ml

sample

mlblank N 14,007 100 mg sample

N = Normality of titrant to 4 places of decimal.

% Recovery =

% Nitrogen 100 7,145

Note Please also note that the above calculations must be adjusted if other purity levels of ammonium salts are used. For a full check of your chemistry, a substance like glycine can be digested and used as a test sample for recovery. See also specific application notes and sub-notes regarding quality control routines.

8.2 Titrant Concentration

To be able to achieve accurate nitrogen / protein results, one must be quite sure that the HCl (hydrochloric acid) concentration is what it is supposed to be. Incorrect HCl concentration can otherwise cause substantial errors. For routine use pre-made solutions of certified specification range 0.0995-0.1005N may be used. Apply 0.1000N for calculation. For prepared solutions (and as a quality check of pre-made solutions) a titration against a predetermined solution of sodium carbonate as described below is necessary. a) Standard substance Weigh approx. 10 g of anhydrous sodium carbonate (Na2CO3). Use a mortar to make a fine powder. Dry it for 1 h at 265oC or 2 h at 200oC. After cooling in a desiccator, transfer the sodium carbonate to a beaker with a tight lid. Store it in a desiccator. b) Indicator solutions Dissolve 0.1 g methyl red in 100 ml 95% methanol or ethanol. Dissolve 0,1g bromocresol green in 100 ml 95% methanol or ethanol.

c)

Procedure Weigh approx. 0.4 g of the standard substance a), using an analytical balance, note the weight (W1). Transfer the sodium carbonate to a receiver flask and add 40 ml of H2O (distilled or deionised). Add 8 drops from each of the indicator solutions. Titrate to pink with the acid of which the titre has to be determined. Note the amount in ml used (A1). Boil this solution for a few minutes. The solution will turn green. Cool rapidly to room temperature under running water. Continue the titration until the next pink colour change occurs. Note also this volume

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(A2). Boil the solution again for a few minutes. Cool rapidly to room temperature under running water. Continue the titration until the next pink colour occurs. Note also this volume (A3) Remark! Temperature changes will influence the volume and the concentration of the titrant solution. The working temperature of the titrant should approximate that of its temperature during standardization. If temperature corrections are necessary, sufficient accuracy may be obtained by use of a correction table. (AOAC 942.25)

8.3 Calculation
Molarity (M) = Note Concentration must be accurate to four digits, i.e. 0.2000 M. Remark! The colour change of this official procedure (AOAC 936.15) may be difficult to see, therefore a pH meter or a mixed indicator (e.g. 0.1 g Methyl red and 0.1 g Bromocresol green in 100 ml methanol or ethanol) will make it much easier to perform.

18,870 W1 A1 A2 A3

8.4 Alkali
To convert ammonium into ammonia an excess of sodium hydroxide is necessary. In our application notes an amount of 50 ml 40 % NaOH is stated. This is based upon an acid digestion volume of 12 ml H2SO4.

8.5 Sodium Hydroxide

Use 400 g NaOH per litre of solution. Commercially available in concentrations up to 50%. Do not use concentrations above 40% as this will lead to crystal formation impairing the function of the pumps. If you can only buy concentrations > 40%, dilute it before use.

8.6 In General
4 times the amount of acid = the amount of alkali to use with the concentration 40 %. Example When 20 ml concentrated H2SO4 is used during digestion the amount of alkali used (40 %) should be: 20 x 4 = 80 ml 40% NaOH. When HgO is used as a catalyst, mercury has to be complexed by using sodium thiosulphate Na2S2O3 x 5H2O. This can be mixed directly into the alkali, 60 g to 1 l of alkali. Note It is very important that alkali is present in excess, otherwise hydrogen sulphide (H2S) will be formed. Hydrogen sulphide is a strongly acidic gas which will make the indicator turn red and no result will be achieved.

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9. Receiver Solution
Boric acid 1% with bromocresol green / methyl red indicator solution - for auto titration in Kjeltec models 1030, 1035, 2300, 2400 & 8400 Boric acid 4% with bromocresol green / methyl red indicator solution - for manual titration In order to obtain accurate results the receiver solution is adjusted so that a small (0.05-0.15 ml) positive blank is obtained when running a blank sample.

9.1 Preparation
The 4% boric acid receiver solution is prepared by dissolving 400 g of boric acid in about 5-6 l very hot deionised water. Mix and add more hot deionised water to a volume of about 9 l.

9.2 Indicators
Cool the solution to room temperature and add 100 ml of bromocresol green solution (100 mg in 100 ml 95% methanol or ethanol) and 70 ml of methyl red solution (100 mg in 100 ml of 95% methanol or ethanol). Dilute to 10 l with deionised water and mix carefully. The 1 % boric acid receiver solution is prepared by directly dissolving 100 g Boric acid in 10 l deionised water, followed by addition of the two indicators as above.

9.3 Adjustment
Adjustment of the boric acid 1% or 4% is made by the following procedure: 1. Transfer 25 ml boric acid solution to a receiver flask and add 100 ml of distilled water. If the solution in the flask is still red, titrate with 0.1 M sodium hydroxide solution until a neutral grey colour is obtained. Calculate the amount of sodium hydroxide solution necessary to adjust the boric acid solution in the 10 l flask with the formula: ml 0,1 M alkali = ml titrant x 400 or 1,0 M alkali = ml titrant x 40 2. Add the calculated amount of NaOH solution to the boric acid solution. Mix. When using 0,2 N HCl titrant acid the addition of 30 ml 0,1M NaOH usually gives a good adjustment , when using 0,1 N HCl titrant acid the addition of 15 ml 0,1 M NaOH. The lower concentration of titrant acid used the less addition (adjustment) with NaOH needs to be done. 3. To check proceed as follows using 25 ml of the boric acid solution. Run a blank. If the value of this blank is high (0.5 ml of 0.2 M HCl) the boric acid is incorrectly adjusted. This might create irregular blanks. For correction add HCl directly into the boric acid tank, mix it carefully and repeat until a reading of 0.05 - 0.15 ml titrant for blank values is obtained. If a positive blank is not achieved, add further small quantities of NaOH and repeat the check until a satisfactory value is achieved.

Note The addition of alkali is to achieve a positive blank value. This should, however, be kept between 0.05 - 0.15 ml titrant, to obtain good repeatability when testing blanks.

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10. General Summary

The following text describes a step by step procedure for Kjeldahl analysis which can be successfully applied to a wide range of samples. The procedure can of course be optimised to suit the requirements of a particular laboratory and types of sample.

1. Prepare a representative sample and weigh 1 gram to an accuracy of 0.1 mg into a digestion tube. NOTE! When using Kjeltec 1035, 2300, 2400 or 8400 these weights can be entered into the system's memory for automatic result calculation. 2. Add two Kjeltabs Cu 3.5 (alternatively 7 g K2SO4 and 0.8 g CuSO4 x 5H2O). 3. Carefully add 12 ml of concentrated H2SO4 and gently shake to "wet" the sample with the acid. NOTE! Samples containing high- fat (>10% fat) or carbohydrate levels use 15 ml H2SO4. 4. Attach the exhaust system to the digestion tubes in the rack and set the water aspirator to full effect. NOTE! The exhaust is automatically attached when using a Lift System. A Controller and Flow Regulator will set the water aspirator to full effect. 5. Load the rack with exhaust into a preheated digestion block (420 C). NOTE! This is done automatically when using a Lift System. 6. After about 5 minutes turn down the water aspirator until the acid fumes are just contained within the exhaust head. NOTE! This is done automatically when a Controller and Flow Regulator is used. 7. Continue to digest until all samples are clear with a blue / green solution. This will normally be after 30 - 60 minutes depending on the sample type. 8. Remove the rack of tubes with exhaust still in place and put in the stand to cool for 10 - 20 minutes. Using a commercial air blower can increase cooling. NOTE! Using a Lift System, the rack together with the exhaust move to the cooling position. The Controller and Flow Regulator will turn off the water aspirator when samples have cooled down. 9. Carefully add 80 ml deionised water to the tubes. NOTE! This can be done automatically at the start of the distillation cycle when using Kjeltec 1026, 1030, 1035, 2200, 2300, 2400, 8100, 8200 & 8400. 10. Add 25-30 ml of receiver solution to a conical flask and place it into the distillation unit and raise the platform so that the distillate outlet is submerged in the receiver solution.

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NOTE! This process is handled automatically when using Kjeltec 1030, 1035, 2200 (Option), 2300, 2400, 8200 & 8400. 11. Place the digestion tube in the distillation unit and close the safety door. NOTE! Dilution water is automatically added on the Kjeltec 1026, 1035, 2200, 2300, 2400, 8100, 8200 & 8400 if the option is selected. 12. Dispense 50 ml of 40 % NaOH into the tube. NOTE! On Kjeltec 1002 is this done by manually operating the alkali pump on all other Kjeltecs this is automatic providing the correct settings have been made. On all Kjeltecs a delay time can be used between the addition of alkali and the start of the steam generator. The strong reaction between added alkali and the acid in the tube is reduced. On Kjeltec 2100, 2200, 2300, 2400, 8100, 8200,& 8400 a SAfE(patented) function is available which further reduces the intensity of this reaction. 13. Open the steam valve on the Kjeltec 1002 and distil for approximately 4 minutes. NOTE! The distillation cycle is controlled automatically on Kjeltec 1026, 1030, 1035, 2100, 2200, 2300, 2400, 8100, 8200 & 8400. 14. Kjeltec 2100, 2200, 8100 & 8200 have a separate valve to prevent suction of liquid back into the system when distillation is finished. At about 90 % of distillation time lower the distillate platform on the Kjeltec 1002. On the Kjeltec 1026 this will happen automatically. NOTE! On the Kjeltec 1030, 1035, 2300, 2400 & 8400 the complete distillation, titration and calculation of result is performed automatically. 15. At the end of the distillation cycle when using Kjeltec 1002 close the steam valve. NOTE! This is done automatically on Kjeltec 1026, 1030, 1035, 2100, 2200, 2300, 2400, 8100, 8200 & 8400 16. For Kjeltec 1002, 1026, 2100, 2200, 8100 & 8200 only; the receiver solution in the distillate flask will now be green indicating the presence of an alkali - Ammonia. 17. Titrate the distillate with standardised HCl (usually 0.1000 N or 0.2000 N) until the blue / grey end point is achieved. Note the volume of acid consumed in the titration.

11. Blank Determinations

Full chemical blanks should be run before each batch of analyses to compensate for any contribution from the reagents used. Blanks should be treated identically to samples to be meaningful. In this example 12 ml of H2SO4 and 2 Kjeltabs Cu 3.5 would be digested and subsequently treated exactly as samples.

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12. Calculation of Results

The most common ways of reporting Kjeldahl results are as % Nitrogen, % Protein, mg N/l (ppm), g N/l and mg N/100 ml. The calculations are as follows

%N

T B N 14,007 100
weight sample (mg )

% Pr otein N F

mgN / l

T B N 14,007 1000
volumesample (ml )

gN / l

T B N 14,007
volumesample (ml )

mgN / 100ml

T B N 14,007 100
volumesample (ml )

T= B= N= F=

Titration volume for sample (ml) Titration volume for blank (ml) Normality of acid to 4 places of decimal Conversion factor for Nitrogen to Protein. There are many factors for converting Nitrogen to Protein depending on the specific commodity. Those most commonly used are as follows: 6.25 for General Food and Feed Applications 5.70 for Wheat 6.38 for Dairy

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13. Revision History

Revision Rev. 5.0 Rev 6.0 Description of Change Added definition of High fat content (>10% fat) Rewritten the calculation formulas Added another indicator at adjustment of titrant 2002-07-02 acid Corrected the instruction for recovery tests, none of the suggested ammonium salt salts need to be dried. Rev. 7.0 Changed back to former recommendations; the ammonium sulphate needs to be dried. Made the adjustment procedure for boric acid clearer. Rev. 8.0 8000 series units added, plus minor spelling corrections 2008-12-04 SOn 2003-03-05 LLg LLg Date 2002-04-08 Sign LLg

Dedicated Analytical Solutions

P/N No. 1026245 Issue No. 8 February 2009 FOSS Analytical A/S Slangerupgade 69 DK-3400 Hilleroed Tel: Fax: E-mail: Web: +45 7010 3370 +45 7010 3371 info@foss.dk www.foss.dk

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