Kjeldahl Nitrogen Analysis

~~red:__NOTE:__ Only trained personnel are authorised to undertake this process~~
__Before attempting to use this protocol please read ‘Health, safety & hazardous waste disposal considerations’ below.__

This protocol is used to determine the organic nitrogen content of a sample (e.g. for calculating protein content in a food resource or as a method of nitrogen analysis in water, fossil fuels, fertilizers and more). The Kjeldahl analysis underestimates values for total N because the technique does not measure nitrate-N or nitrite-N unless they are reduced before acid digestion. Although still a common analysis, combustion analyses have largely superseded the Kjeldahl technique in the last 25 years. The description that follows was written for the Tecator 2012 digestor and Gerhardt-5 distillation and titration apparatus. We have, however, digested samples and distilled and titrated them with much cruder systems so the description should aid anyone doing Kjeldahl analyses regardless of the apparatus. As a rough guide, with our Kjeldahl system one person can process 24 samples in duplicate per day of which about 10 % required repeat analyses. This proved too slow once we started measuring available nitrogen (DeGabriel et al. 2008), whereby each sample requires five nitrogen analyses prompting us to move to combustion analysis.

None other than Johan Kjeldahl presented the Kjeldahl nitrogen procedure to the Danish Chemical Society in 1883. All that has changed since this time is the apparatus. The system we have automates some of the steps, notably the distillation and titration.

”Note: Leaf material can be collected and prepared for Kjeldahl Nitrogen Analysis by following:” ((Collecting leaves for chemical analysis)).

__Three distinct steps__
There are three distinct steps to a Kjeldahl nitrogen analysis, namely digestion of the sample, distillation of the ammonia and titration to determine how much boric acid was used to capture the ammonia and thus the amount of nitrogen in the sample.
Problems frequently arise when using the equipment and usually appear as poor duplication. In other words, two identical samples give vastly different results. You will solve these problems in minimum time and with minimum loss of samples only if you understand the chemistry involved in the Kjeldahl analysis. Following is a brief description:
#__Digestion:__ %%%The purpose of the digestion is to convert most* of the nitrogen in the sample into NH{SUB()}4{SUB} ions. We do this by heating the sample to high temperature (420C) with concentrated sulphuric acid (98%) in the presence of a catalyst. There are many possible catalysts but we use tablets containing selenium and potassium sulphate. These catalysts increase the digestion temperature and the rate of breakdown of organic matter.^Organic N + H{SUB()}2{SUB}SO{SUB()}4{SUB} (NH{SUB()}4{SUB}){SUB()}2{SUB}SO{SUB()}4{SUB} + H{SUB()}2{SUB}O + CO{SUB()}2{SUB}^ %%%*”Nitrate and nitrite ions are not measured by the Kjeldahl analysis unless they are reduced before acid digestion. There are various procedures to do this, most using salicylic acid followed by sodium thiosulphate. Both nitrate and nitrite ions occur at low concentrations in most of our samples so we do not bother with this step.”%%% %%%
#__Distillation and ammonia capture:__ %%%The Tecator system automates the distillation and digestion. However, in order to understand the analysis, these steps must be considered separately. In the distillation the acid digestion mixture is diluted with H{SUB()}2{SUB}O, neutralised and then made strongly alkaline with concentrated (ca 40-50%) NaOH, thus liberating ammonia. The formula below shows "the neutralisation rather than the excess OH-".^(NH{SUB()}4{SUB}){SUB()}2{SUB}SO{SUB()}4{SUB} + 2NaOH 2NH{SUB()}3{SUB} + Na{SUB()}2{SUB}SO{SUB()}4{SUB} + 2H{SUB()}2{SUB}O ^%%%The ammonia released upon addition of the NaOH is transferred by steam distillation to the receiving vessel containing acid. We use a weak boric acid solution. Because hydrogen ions are used to catch the ammonia by converting it to ammonium ions, the pH of the mixture in the receiving vessel increases as the distillation proceeds:^NH{SUB()}3{SUB} + H{SUP()}+{SUP} NH{SUB()}4{SUB}{SUP()}+{SUP}^ %%%The entire reaction is: ^NH{SUB()}3{SUB} + H{SUB()}3{SUB}BO{SUB()}3{SUB} NH{SUB()}4{SUB}{SUP()}+{SUP}:H{SUB()}2{SUB}BO{SUB()}3{SUB}{SUP()}-{SUP} + H{SUB()}3{SUB}BO{SUB()}3{SUB} (reaction yields a NH{SUB()}4{SUB}{SUP()}+{SUP}-borate complex and excess boric acid)^ %%%Clearly, if the system leaks or if there is insufficient boric acid then some of the ammonia will be lost to the air and your analysis will suggest a lower concentration of nitrogen than there really is. Similarly, a deficit of NaOH will leave the digestion contents unneutralised and there will be little or no NH{SUB()}3{SUB} to distil. The pH of the boric acid in the receiving vessel should be the same at the start of each run. A fluctuating pH often occurs if the apparatus has not been used recently. It also occurs if the level of boric acid is too low. Sometimes topping up the level with distilled H{SUB()}2{SUB}O solves the problem. %%% %%%
#__Titration:__ %%%The titration is the final step in the analysis. We use automatic direct titration of the contents of the receiving vessel. Rather than using an indicator, the apparatus uses a pH meter. In theory, this should give a perfect titration but there is a tendency for the machine to add too much acid and overshoot the endpoint. However, this error is minor. %%%As pointed out above, the capture of ammonia uses H{SUP()}+{SUP} ions, resulting in a large increase in pH by the end of the distillation. The titration works by adding enough H{SUP()}+{SUP} ions to return the pH to that recorded for the boric acid at the beginning of the sample analysis (see equation below). A programme runs the titration adding progressively less acid as the titration gets closer to the endpoint. Thus, by altering the speed at which acid is added one can fine-tune the titration. Typically, I slow the titration down. ^NH{SUB()}4{SUB}{SUP()}+{SUP}:H{SUB()}2{SUB}BO{SUB()}3{SUB}{SUP()}-{SUP} + HCL NH{SUB()}4{SUB}Cl + H{SUB()}3{SUB}BO{SUB()}3{SUB}^ %%% %%%
#__Calculation:__ %%%The calculation is simple and if you enter your sample weights into the analyser then it will give you a result at the end of each analysis. But remember that this result will be %N on an air-dry (AD) basis unless you dried samples before analysis or have otherwise accounted for residual moisture.^% Nitrogen (AD) = (mL standard acid – mL blank) x normality of acid x 1.4007 / Sample mass (g)^ %%%The multiplier 1.4007 comes from the atomic mass of nitrogen and the factor to convert the result to a % or g N per 100 g of sample.

*Digestion apparatus (e.g., Foss Tecator{SUP()}TM{SUP} 2012 digestor, Sweden)
*Glass digestion tubes (e.g., for the Vapodest system 290 x 25 mm)
*Concentrated (98 % w/v) analytical grade sulphuric acid
*Catalyst tablets (e.g., Kjeltab catalyst tablet containing 3.5 g K{SUB()}2{SUB}SO{SUB()}4{SUB} and 3.5 mg Se; Foss Tecator{SUP()}TM{SUP}, Sweden cat no 1527-0003).
*Filter paper – Whatman No. 1 nine cm
*Acid scrubber to neutralise acid fumes during the digestion (e.g., Foss Tecator{SUP()}TM{SUP} scrubber)
*Distillation system
*Concentrated sodium hydroxide solution (ca 40 % w/v). Note this can be industrial grade NaOH.
*Ammonium sulphate standard
*Hydrochloric acid standard (0.025 and 0.05 N) for titration
*Boric acid solution (about 2 % w/v)
*Deionised water for distillation
*MilliQ{SUP()}TM{SUP} water or similar for solutions
*pH meter or indicator if doing manual titrations

!!Units, terms, definitions
Digestion = hydrolysis

((Hydrolysis)): In chemistry, a double decomposition reaction with water as one of the reactants. In this case, protein is hydrolysed using a strong acid in the presence of heat and a catalyst.

((Steam distillation)): is a special type of distillation (a separation process) for temperature sensitive materials. In this case, ammonia is vapourised with steam and then captured by condensing the steam into a weak acid (boric acid).

((Titration)): a measured amount of a solution of unknown concentration is added to a known volume of a second solution until the reaction between them reaches an endpoint.
In this case, we add hydrochloric acid of known concentration to the captured ammonia (now ammonium) until the pH of the solution reaches the original pH of the boric acid solution.

”===Note=== that these instructions are not an instruction manual on how to do Kjeldahl nitrogen analyses. Instead their purpose is twofold. First, the instructions are a guide when you are being shown how to operate our apparatus. Secondly, they are hopefully useful to refresh your memory. In both cases, it is necessary to understand the preceding chemistry.”

Here the analysis is considered in two parts: (A) the digestion and (B) the distillation and titration.

__Note: if no one has used the distillation apparatus for some time (weeks/months) then it is wise to check its operation before digesting samples. This involves replacing the water, preparing new acid for titration, checking that pumps work and then running blanks and standards.__

__===A. Digestion===__
#__Laboratory book__%%%Create a databook for recording nitrogen data. Rule up a page into six columns labelled “Sample ID”, “Tube No.”, “Sample mass”, “Titre #”, “Titre” and “%N”. Analyse samples in duplicate so that aliquots of sample 1 will go into tubes 1 and 2, sample 2 into tubes 3 and 4, etc.
#__Turning on the digester__%%%Turn on the digester and set the temperature to 250C so that it is warmed up by the time you have your samples ready. The digestion must be done in the fumehood.
#Weighing the sample%%%Before weighing the sample it is necessary to select and number the glass digestion tubes and place them in a rack. Use only the wide-diameter tubes with a noticeable rim and check them carefully for cracks and for damage to the rim. If the rim is chipped or the tube is cracked then it is ”useless”. Also, don’t ever use the thin tubes. I typically number the tubes sequentially from 1 to 36 or 48 so that batch number 1 will have tubes 1 to 12.%%%The mass of sample depends on its nitrogen content and on the apparatus (tube size, distillation apparatus, etc). In other words, it is sensible to weigh out a given mass of nitrogen (__but see Note Jan 2008 below__). This is wonderful in theory but fails somewhat in practise because you might end up with miniscule amounts of very high nitrogen materials and then the error of the balance becomes important. For eucalypt leaves, which typically contain 1-2% N, we weigh about 250 mg (250-260 mg). The sample is weighed directly into a paper holder made by folding a ¼ sheet of Whatman No. 1 nine cm filter paper into a cone-shape. This is the easiest way to get the sample to the bottom of the tube in a dry climate where static electricity is a problem. %%%First, tare the paper. Then add the correct amount of sample. Fold the paper so that the sample cannot escape and then place the paper+sample into a glass digestion tube.%%%To avoid getting the balance dirty, always take the holder off the balance to add the sample. With practice you will soon find that weighing samples is quick. %%%__Note Jan 2008.__ Samples from the ”in vitro” digestion (de Gabriel et al 2008) tend to swell and rise up the tube. By far the best way of averting this problem is to weigh out less material. I have got excellent results with 150-200 mg, even though the samples may contain little N. Something that also helps to alleviate the problem is to add the acid 15-30 minutes before starting the digestion.
#__Adding the catalyst tablet and acid__%%%Place a catalyst tablet in each tube using a pair of forceps. ===Never touch the catalyst tablets with bare hands.===%%%Now, in the fumehood add 6-7mL (more if you have more sample) of concentrated sulphuric acid to each tube. Set the dispenser at 3.5 mL and put two squirts into each tube. This is best done by leaving the tubes in the rack and moving the rack to position tubes under the dispenser. Do the tubes in one side of the rack and then the other. __Make sure the tip of the dispenser is over a tube before you lift the plunger.__ Lightly shake each tube after adding the acid.
#__Placing tubes in the digester__%%%When the temperature is at 250C place the heat shield on top of the digester and then place the tubes into the digestion block. Be careful to ensure that the heat shield is correctly aligned with the block so that the tubes sit on the bottom of the digester. The tops of all tubes should be in roughly the same plane. This ensures that no tube is “sitting up” and likely to fall and possibly break during the digestion.%%%Watch the tubes for a few minutes to check for sample rising up the tube. This is easiest if you remove the front plate of the heat shield. If a sample does rise, put on the acid-resistant gloves, remove the tube and lightly shake it, pointing the mouth of the tube away from you. If a sample rises more than half way up the tube then it is useless. Remove it from the digestion block and leave it to cool in the fumehood before flushing it down the sink. Immediately weigh a replacement.%%%After 10 minutes at 250C place the scrubbing manifold (Section 6) on the tubes, turn on the tap so that there is moderate suction as indicated by a steady bubbling, and increase the temperature to 420C. Digestion takes 60 minutes from when the unit gets to temperature (ca 75 min from 250C).
#__Operating the Foss Tecator{SUP()}TM{SUP} acid scrubber__ %%%The purpose of this apparatus is to capture the acid fumes so that they don’t escape up the fumehood exhaust and corrode any metal they meet on the way. The apparatus is simple with fumes passing first through a strong (ca 20%) NaOH solution to neutralise the acid fumes and then through water. ===But a word of warning:=== acid condenses in the black tube that runs from the collector that sits on the digestion tubes. Before removing this tube (for example to wash out the collection manifold) make sure that you have a container in place for it to drain into. Check the pH of the NaOH and replace when necessary. Likewise, replace the water regularly, filling both containers roughly 2/3-3/4 full. When turned on both the NaOH and the water should bubble. If not the containers are not sealed. Lightly press down on the lid to seal them.
#__Removing tubes from the digester__%%%~~red:__This is undoubtedly the most dangerous part of the analysis.__~~ Take care and make sure the fumehood door is down so that you can get your arms in the fumehood but the rest of your body is protected. Wearing a full face shield and acid resistant gloves, grasp the handles on each side of the heat shield and lift the samples directly from the block. Gently place the heat shield and samples down in the fumehood and leave them to cool. %%%I often digest samples in the afternoon and then analyse them the next morning. In this case, I wait for the samples to cool and then cover them with paper.

===__B. Distillation and Titration__===
Confucius says {QUOTE()}"There is nothing worse with a Kjeldahl analysis than weighing and digesting the last quarter gram of sample and then losing it through an act of gross stupidity."{QUOTE}
__Setting up the machine__
#Check fluid levels in all containers (i.e. NaOH, Boric Acid, Distilled H{SUB()}2{SUB}O and waste). Always replace the distilled H{SUB()}2{SUB}O if the machine has not been used for several days. The NaOH is roughly a 40% w/v solution using NaOH obtained from the hardware in 2 kg containers. Seek assistance unless you have considerable experience mixing these large volume solutions. %%%The boric acid solution contains 10 g of boric acid per litre of 20% ethanol in distilled H{SUB()}2{SUB}O. (I typically make 10 L of the solution by first dissolving 100 g of boric acid in about 2L of H{SUB()}2{SUB}O using a heated magnetic stirrer and then making it to volume.) %%%In some labs it is acceptable to flush the waste down the sink but do this with large volumes of H{SUB()}2{SUB}O. %%%The HCL for titration is mixed from Volucon standards using milliQ H{SUB()}2{SUB}O. The standards we purchase make 1 L of 0.05N HCl or 2 L of 0.025N. If you do not know or have forgotten how to prepare this acid then get advice from someone who does know.
#Place the large grey drainage hose in the sink.
#Attach H{SUB()}2{SUB}O hose from the machine to the tap over the sink and turn it on.
#Wash the rubber tube supports in distilled H{SUB()}2{SUB}O and place them on the tube holder. Insert an empty tube (blank) by pushing down the lever on the left hand side of the machine.
#Put about 3 cm of distilled H{SUB()}2{SUB}O in the glass receiving cup and then put it in place.
#Attach burette (= syringe) with an upward right screwing action. Turn on the machine at the switch on the right hand side. Select YES for steam and YES for FLUSH burette. Secure the burette to the arm with the thumbscrew when the arm reaches it. Flush the burette if the machine has not been used for some days. Turn off the machine, if necessary, to stop the arm and hence flushing. Make sure there is no air in the burette.
#Remove red cap and plastic vial from the pH probe. Check that the electrolyte level is near the opening covered by the red cap. If not add some more AgCl using a Pasteur pipette. Place some pH standard solution (pH 4 and 7) in scintillation vials ready for calibration.
#Calibrate pH probe. Press CAL, select NO until pH flashes, then select YES. Follow directions using the green pH 7 and the red pH 4 bottles. Rinse probe at every stage, being careful not to touch the delicate tip of the probe with anything other than light tissue wipe. The slope should be about 58 ∓ 1. Press ENTER.
#Calibrate the machine by running three blanks. Press CAL, select NO until BLANK flashes, and then press YES. Enter Program # (1 uses 0.025M HCl, 2 uses 0.05M HCl). Press ENTER then RUN. Record values for all blanks in the laboratory book. Press NO to load the value and repeat three times. Press YES to load the third value if appropriate (i.e. ~0.3 for program 1, ~0.15 for program 2). Repeat with blank until appropriate. Often the first blank is poor, probably due to the boric acid not being refreshed. Thus, before doing any blanks press the boric acid switch to renew the contents of the receiving vessel. It often helps to add some distilled H{SUB()}2{SUB}O to the vessel to increase the volume.
#Enter sample weights. Press WEIGHT, enter SERIES number, ENTER, 1ST sample weight, ENTER, etc, followed by ENTER, ENTER after the last sample.
#You are now ready to run the first sample.
__Starting samples__
#Place the first sample tube on the machine.
#Press SAMPLE, press program number (see above), press ENTER, press series number 1 then ENTER, press sample number 1 then ENTER. Before pressing RUN to start the analysis, check that the pH is appropriate (i.e. ~ 4.5). If it is not, press BORIC ACID to flush the receiving vessel. Perhaps add some distilled H{SUB()}2{SUB}O too. If the pH is appropriate and stable, press RUN. Note that results are often much better if you place a new sample on the machine as soon as the previous sample finishes.
#After the sample is run, record the titre and %N in the laboratory N databook. Remove the tube and drain the contents into the sink with copious running H{SUB()}2{SUB}O. Replace tube with the second tube and press SAMPLE and RUN.
#Wash tubes with three flushes of tap H{SUB()}2{SUB}O and three of distilled H{SUB()}2{SUB}O. Place upside down on the drying rack above the sink.
__Calibration standards__
There are several parts of the procedure to calibrate. Running blanks on the machine has already been mentioned. It is sensible also to run blanks through the digestion phase. In this case, a tube contains filter paper, catalyst tablet and acid only. Run this as a normal sample on the machine but enter a small value (eg 0.01 g) for sample mass. Ideally, the result will be 0 % N.

The third calibration is to distil and titrate samples of dry (NH{SUB()}4{SUB}){SUB()}2{SUB}SO{SUB()}4{SUB}. You should do this with every batch of chemicals i.e. whenever you replace the boric acid or NaOH, or when the machine has not been used for some time. Also, this is a good troubleshooting technique when results are poor. Place a small amount (ca 1 g) in a scintillation vial and drying it overnight at 45 C works. Weigh 100 mg into paper cones and place them in digestion tubes but instead of digesting them simply enter the sample mass on the machine and then distil and titrate them. The result should be 21.2% N.

The final calibration is to take a sample of known protein content and run it through the entire analysis. This is not as simple as it seems and to be done effectively probably requires standard protein or a sample stored under ideal conditions (evacuated and stored at low temperature)).

__Shutting down the machine__
#Remove rubber stoppers from the tube holder.
#Fill 2L plastic measuring cylinder with boiling H{SUB()}2{SUB}O. Place the sample tube hose in the boiling H{SUB()}2{SUB}O and press and hold EMPTY. Top up cylinder with tap H{SUB()}2{SUB}O and press and hold EMPTY again.
#Turn off machine.
#Remove and rinse pH probe, replace red cap and plastic vial making sure that the AgCl in the vial covers electrode’s tip. Be careful not to damage the probe on the bottom of the vial.
#Remove and rinse receiving cup and burette. You may need to turn the machine off and on again to move the burette arm.
#Turn off H{SUB()}2{SUB}O tap.
#Wash all tubes in sink and place on drying rack.
#Check and fill any fluid containers if you intend running samples the next day.
#Wash the plastic receptacle that sits under the sample and receiving vessel. Place some paper under the sample apparatus to catch any fluids that may drain out.
#Wipe down the rest of the machine.

!!Other resources
!!Notes and troubleshooting tips
__Typical problems__
*If the machine has not been used for some time expect many problems, especially with pumps not primed, strange values for blanks and the empty function failing at the end of a run. In this case it is wise to start the machine before you digest samples. Check each step before you even do a blank by placing a tube in position and pressing the NaOH button; remove the receiving vessel, fill it with water and then press the boric acid button. Watch fluids move in the lines and check for air-blocks. Check that the burette is free of air bubbles. On the first blank, check that some distilled water dispenses before the NaOH. Do not start running hydrolysed samples until you are sure that the distillation/titration machine is functioning properly.
*No boric acid in receiving vessel. This is most likely caused by an air block. You will need to prime the tube using a large syringe and tubing. The same applies to the other solutions. This is a common problem when the machine has lain idle for a long time. If you do not know how to prime a pump then see someone who does.
*Fluctuating pH at the start of a run. The most likely explanation is that the boric acid level is too low. The problem often disappears as you run more samples. First, remove the receiving vessel, add some distilled water and then replace the receiving vessel. The vessel still contains all of the boric acid – sufficient to react with the distilled NH{SUB()}3{SUB}. If the problem persists it may be necessary to alter the programme so that the machine delivers more boric acid. Minimising the delay between samples often solves this problem. Note that whenever you remove the receiving vessel while the pH meter is in place you need to be careful not to damage the meter.
*No H{SUB()}2{SUB}O for distillation: the plastic distilled water vessel is empty or the tube is not below the surface. Apart from not replenishing the steam vessel (think boiling jug running out of water!!!!), this will mean that during a sample analysis no dilution H{SUB()}2{SUB}O is added resulting in a more violent reaction upon addition of NaOH. NOTE: pump vibration often causes tubes to rise above the liquid in vessels. Keep an eye on this or you will lose valuable samples. Stuffing a wad of paper into the top of each vessel is a good way to solve the problem
*The titration overshoots the endpoint. You may need to alter the programme to slow the addition of acid. However, in the first instance determine how much it overshoots and what result this has.
*The sample contains no nitrogen. This is hopefully true of a digested piece of filter paper but should not occur with a sample. The likely explanation is that there was insufficient NaOH to neutralise the sample. Causes: too much acid, not enough NaOH, tube above the level of liquid in the NaOH vessel.
*Blanks: sometimes with blanks a problem occurs such that the machine takes its initial pH reading (say 4.7) and then the pH suddenly drops (this is an apparent pH drop caused by a low fluid level in the receiving vessel. The machine is then in a hopeless position: at the end of the distillation the pH will still be below the starting pH but the machine will still add acid in an attempt to increase the pH! Press the STOP button, then the EMPTY, remove the receiving vessel and wash it thoroughly with distilled water. Fill it to about two-thirds with distilled water, place it back in position and then press the BORIC ACID button. The vessel will empty and then refill. You are now ready to start a new blank.

!!Links to resources and suppliers
[http://www.foss.com.au/Solutions/ProductsDirect/SamplePreparationMills/Cyclotec1093SampleMill.aspx|Cyclotec 1093 sample mill (Tecator{SUP()}TM{SUP}, Sweden)]
[http://www.gerhardt.de/en/Products/Distillation.html|Vapodest 5 analyser (Gerhardt, Germany)]

!!Description for publication
We determined nitrogen concentrations in foliage on duplicate samples (0.2500 ∓ 0.0100 g) of freeze-dried leaf using the semi-micro-Kjeldahl technique. Before analysis, all samples were ground to pass a 1 mm screen in a Cyclotec 1093 sample mill (Tecator{SUP()}TM{SUP}, Sweden) before weighing them into vessels made by folding quarter sections of 9 cm qualitative filter papers. The vessel containing the sample was dropped to the bottom of a glass digestion tube (250 x 20 mm). We then added a Kjeltab catalyst tablet containing 3.5 g K{SUB()}2{SUB}SO{SUB()}2{SUB} and 3.5 mg Se (Foss Tecator{SUP()}TM{SUP}, Sweden cat no 1527-0003) followed by 6 mL of AR concentrated sulphuric acid. The tubes were gently shaken and then placed in a Tecator{SUP()}TM{SUP} 2012 Digestor and digested at 250C for 10 min before raising the temperature to 420C, where it was held for 1 h. Upon cooling, we steam-distilled and titrated (0.025 M HCl) the samples on a Vapodest 5 analyser (Gerhardt, Germany). Blanks were calculated by digesting filter paper alone, while the analyser was calibrated by distilling and titrating both empty tubes and samples (0.025 g) of dried (NH{SUB()}4{SUB}){SUB()}2{SUB}SO{SUB()}4{SUB}. A shorter version appears in Wallis et al (2002).

!!Literature references
DeGabriel JL, Wallis IR, Moore BD, Foley WJ. 2008. A simple, integrative assay to quantify nutritional quality of browses for herbivores. ”Oecologia” 156:107-116.

Wallis IR, Watson ML and Foley WJ (2002). Secondary metabolites in ”Eucalyptus
melliodora”: field distribution and laboratory feeding choices by a generalist herbivore, the common brushtail possum. ”Australian Journal of Zoology” 50: 507-519

!!Health, safety & hazardous waste disposal considerations
~~red:__NOTE:__ Only trained personnel are authorised to undertake this process~~
*__Sulphuric acid heated to 420C is not only an incredibly effective skin remover but it removes underlying muscle too.__ If spilt on the skin it will convert the protein to NH{SUB()}4{SUB}{SUP()}+{SUP} ions. Tiny droplets on clothing will reveal themselves as large holes after the next wash.
*Remember too, that __concentrated (40%) NaOH neutralises the acid.__ Hydroxide of this strength is nasty stuff as are the catalyst tablets. Thus, safety is paramount when doing Kjeldahl nitrogen analyses.
*Read all material safety data sheets (MSDS) before you start. If you have any doubts about what you are doing then ask someone who knows. A good way to view MSDSs is by doing a Google search eg MSDS sulphuric acid.

__TREATMENT:__ The treatment for both NaOH and H{SUB()}2{SUB}SO{SUB()}4{SUB} spilled on the skin is the same: wash with copious cold running water, remove contaminated clothing. Seek medical advice. See MSDS for other injuries, eg eye injuries.

!!Search terms and classification

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