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Determination of crude protein using the Kjeldahl method
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- čas přidán 20. 04. 2020
- Education movie about the Kjeldahl method and determination of total nitrogen / crude protein. The different steps of the Kjeldahl analysis performed and explained.
Steps:
• Weighing sample
• Adding catalyst, salt and sulphuric acid
• Digestion
• Distillation
• Titration
• Nitrogen is determined and crude protein is calculated
#proteinanalysis #kjeldahl #feedanalysis
I am studying veterinary medicine and we use the Kjeldahl method to determine the quantity of protein in a sample. Let me say, I always said I wouldn't work in a lab cause I wanna work in a clinic with actual animals, but daaaamn seeing your material and how equipped your lab is, I would work there anytime!!! We don't have any of these fancy machines at the faculty, for the distillation step we use a dispositive of Parnas Wegner. It helps us to visualise better but it's not as cool as your machine
Hey,
I am also a vet student 🤗
Very nice ❤️...best video for quick reference in an understandable way👌
Nice video learnt the Whole process by heart! Thanks 😁
Really informativ,thanks for sharing such a great video
It is highly educative, thanks a lot
Nice video very helpful
Thanks from Brazil!
It's so Amazing and I just get it so quickly
Thankyou so much for the video 🌸
Thank you for this video. My lab recently got a kjeltherm and vapodest 200, and I have been assigned to set it up and get it running even though I barely had experience with it in the past. So, just watching you go through the steps is a life save for me; however, I was wondering how you determined what quantity of sample to measure in gram. Thanks
This is excellent work
Thanks from Pakistan 🇵🇰
Väldigt informativt!
This is excellent, thank you very much! And I really like your hand-crank titration machine, much better than a buret
yeah that's 1130 euros I checked online
The steam generator- I’m unfamiliar with this method of distillation. What vapor is being funneled into the vessel?
Thanks 🙏
Hey, what are the concentrations that you use and how can you know that? I also have to do a labo about this but I have no idea what the concetration needs to be ( is for testing on beer)
Why does the solution turn black in colour when the distillation process start?
I'm getting problem in washing the Round bottom flask , can anyone please guide me it will be a very great help.
Merci think you 🇩🇿
how many g of samples were in there?
what is the concentration of all the reagents used in the experiment?
May I know this method you used by who? if it was by AOAC may I know the year?
Thank u sir.... here u use sample in powder form but if we want to determine the protein of spinach in raw form containing 90%moisture how is the procedure??
We also analyze raw samples and even liquids with our system. Typical raw samples are silage, fresh crops and fecal samples. Typical liquids are urine and buffer/water extracts from silage or other feeds. When a difference is required in the procedure, it is in the digestion step. If there is a large amount of liquid in the sample, a slower temperature increase may be needed to avoid boiling over of the sample. It is also helpful to let the sample rest overnight with the sulphuric acid before digestion, whereas Kjeltabs can be added just before digestion. Please note that raw samples are generally less homogenous than dried and milled (or liquid) samples, so replicates may be needed to compensate for that.
In the video the volume in your boric acid trap significantly increases between time of starting distilling and completion. Has this been diluted in preparation for titrating? Or is it increased from trapping Ammonia, though I wouldn't expect that it would increase the volume by that much.
Ammonia and water are transferred from the Kjeldahl tube to the boric acid flask during distillation. This is the reason why the volume increases. All ammonia is trapped in the boric acid flask and it is the moles of HCl required to neutralize it that gives the moles of N that were in the sample at the beginning.
Hi
@@app-animal-science-and-welfare The Catalyst tablet is made out of K2SO4 and CuSO4.
If i don't have that Tablet, but instead i had both ingredients what's the correct measurement (Weight) for each substance ??
And how much (Gram) should i put into the Sample as Catalyst
@@mr.z6252 The amounts of the Kjeltab reagents are actually visible in the video, when the label is displayed at 0:30. Each tablet contains 3.5 g K2SO4 which means 10.5 g with three Kjeltabs. Each tablet also contains 0.4 g CuSO4 • 5 H2O (copper sulfate pentahydrate), meaning a total of 1.2 g with three Kjeltabs. Copper concentration is 25.5% in this salt, so copper amount equals approx. 0.30 g with three Kjeltabs.
@@app-animal-science-and-welfare Yeah i just Notice it when i Replay the Video 😅😅
Same question for the BCG-MR indicator, how much gram/precentage needed for each of em
I read some articles that you need both BCG & MR at 0.1% (By 96% Ethanol) with BCG at 10ml & MR at 2ml.... is this correct ??
for silage total nitrogen estimation, should the sample be liquid or dry form?
Drying of silage usually cause loss of ammonia N. If the analysis is done for ranking several feeds, it may not be a problem. It is possible to compensate for this by assuming a standardized loss if a separate ammonia analysis has been done. If accurate (“true”) measurements are important, the silage can be analyzed fresh, without drying. Because homogeneity is not as good as with a dried and milled sample, it is commonplace to have replicate samples in this case.
Howmuch wieght can take from wet sample 🙏
Can someone please tell me what errors can be made by this experiment?
Best vedio .colud you plz mention any refernce document about this method
Analyzed as Kjeldahl-N x 6,25. We use copper sulphate as catalyst. Reference: Nordic committee on food analysis, 1976. Nitrogen. Determination in food and feed according to Kjeldahl, No 6, third edition.
@@app-animal-science-and-welfare Thankyou ❤️❤️❤️
Can I use a bunsen burner instead of heating block, what sort of temperature is necessary for digestion
Yes, bunsen burners are commonly used for digestion when a heating block is not at hand. The temperature in our system is 420 °C. Experienced lab technicians may be able to judge completeness of digestion from the color of the solution.
what can we do if we don't have this distillation matching in our lab?
You can make a simple distillation device / apparatus. You will need a heat-resistant flask, heat source, distillation column, condenser, collection bottle.
In Wikipedia, there is a good sketch of the method: en.wikipedia.org/wiki/Kjeldahl_method
It is also seen in several videos available on CZcams. For example this one: czcams.com/video/DHVWkSU9Oyk/video.html
What does the blank tubes contain and why do we use blank samples
The blank tube contains only the reagents (sulphuric acid and Kjeltabs) and goes through the whole procedure. This is because there is a small contribution to the final result from those reagents and the blank sample is used to correct for that by subtracting the blank value. It is common in many different analyses to have blank samples for that reason.
@@app-animal-science-and-welfare thank you so much 👍
How much ml of each indicator do you use for boric and what color it appears?
Boric acid 1% with Bromocreosol green / Methyl red indicator.
Indicator: 1g of Bromocresole green and 1g of Methyl red, respectively, are dissolved in 1000ml of 95% Ethanol.
Add 2.5L 4% (or 5L 2%) Boric acid to a 10L flask. Add deionized water to approx. 9L. Add 100ml Bromocreosole and 70ml Methyl red. Add 3ml 1M NaOH. Adjust volume to 10L and mix. The solution should now shine in green.
The NaOH is added because we want a slight response for the blank sample.
(Alternatively, but not recommended because > 5.5% Boric acid is classified as cancerogenic, mutagenic, toxic to reproduction :
Weigh 100g Boric acid into a 10L flask, add 2L warm de-ionized water, and mix until it has dissolved. Add water to 9L and proceed as above).
@@app-animal-science-and-welfare thank you so much , what if you dont add NaOH ? The solution appears in what color? Is it purple? Does % concentration of solution effect the color of indicator for example 1% boric vs 1.5% boric
How can the experiment be adjusted for a methyl orange indicator instead of methyl red?
As methyl orange has a lower pKa, more acid will be needed to get the color change.
Do I need Kjedahl cubes? can I just use a solution ? Im trying to use the method to calculate protein concentration in milk for a school experiment
You need copper (or an alternative catalyst) and you need potassium sulfate. Some decades ago our lab used two small copper nails and a spoon of potassium sulfate for each sample. Appropriate weights to add may be calculated from the specifications of Kjeltabs. Calculation factor for milk is 6.38.
Just wanted to ask if your initial sample was an ash residue?
No, it was a forage sample that had been dried and milled through a 1 mm screen.
what is the weight of the sample inside the glass tubes?
It depends on the expected nitrogen content, but it is usually around 1 g for samples with an expected crude protein content of 10-25%.
What 1401 for in the equation
Can you write the steps that you wrote in the video below with comments?
Because it is not clear to me because of the weakness of the Internet
thank you
Steps
:
• Weighing sample
• Adding catalyst, salt and sulphuric acid
• Digestion
• Distillation
• Titration
• Nitrogen is determined and crude protein is calculated
@@app-animal-science-and-welfare
How to test blank ?
I stop at endpoint just like you, but the staff from the lab that offer this service ask me to do it more darker. I'm so confused why they did like that.
If you continue titration beyond the equivalence point, it is a risk that more moles of HCl than the moles of nitrogen in the sample will be added and a falsely high N result may be obtained. A good practice for maintaining similar endpoint color throughout is to compare the current sample with the blank that was titrated first and also to keep a few of the most recent tubes for comparison when working through the batch.
Can you provide the calculation part?
Calculation of % nitrogen:
((ml HCl sample - ml HCl blank) * conc HCl (moles per L) * 14.01 (molecular weight nitrogen) * 100 ) / (1000 * weight of sample (gram))
Calculation of % crude protein:
% nitrogen * conversion factor for crude protein (commonly 6.25)
There are other conversion factors (depending on the amino acid composition of the protein) but 6.25 is most commonly used.
may I know the brand of Boric acid you used?
*why for the alternative you did not use ethanol ?*
Boric acid 1% with Bromocreosol green / Methyl red indicator.
Indicator: 1g of Bromocresole green and 1g of Methyl red, respectively, are dissolved in 1000ml of 95% Ethanol.
Add 2.5L 4% (or 5L 2%) Boric acid to a 10L flask. Add deionized water to approx. 9L. Add 100ml Bromocreosole and 70ml Methyl red. Add 3ml 1M NaOH. Adjust volume to 10L and mix. The solution should now shine in green.
The NaOH is added because we want a slight response for the blank sample.
(Alternatively, but not recommended because > 5.5% Boric acid is classified as cancerogenic, mutagenic, toxic to reproduction :
Weigh 100g of Boric acid into a 10L flask, add 2L warm de-ionized water, and mix until it has dissolved. Add water to 9L and proceed as above).
While calculating N, why we are dividing with 1000 but ofcourse sample weight is acceptable. So it's causing problem in converting the percent in 100gram
The equation will give an answer as “% N of sample weight”. If you prefer “N, g/kg sample”, you can omit both the “100” in the numerator and the “1000” in the denominator.
What’s the use of the boric acid please 🙏
It functions as a protolyte (acid/base) and forms anions (base) proportional to the amount of ammonia that was distilled to the flask. Those anions are then neutralized in the final titration step and the moles of acid needed corresponds to the moles of nitrogen that was in the sample from the beginning.
is it necessary to standardize boric acid?
We use 4% boric acid, which we buy ready-made and dilute to 1% and add an indicator to. To get a reasonable gloss value, we add a little NaOH.
For 10 liters of 1% boric acid, we use 100 ml of bromocresol green and 70 ml of methyl red and 3 ml of 1M NaOH. You can check that the blank value is "appropriate" (around 0.08 ml).
@@app-animal-science-and-welfare are there alternatives to using boric acid?
Can you provide a procedure for preparation of boric acid indicator?
Boric acid 1% with Bromocreosol green / Methyl red indicator.
Indicator: 1g of Bromocresole green and 1g of Methyl red, respectively, are dissolved in 1000ml of 95% Ethanol.
Add 2.5L 4% (or 5L 2%) Boric acid to a 10L flask. Add deionized water to approx. 9L. Add 100ml Bromocreosole and 70ml Methyl red. Add 3ml 1M NaOH. Adjust volume to 10L and mix. The solution should now shine in green.
The NaOH is added because we want a slight response for the blank sample.
(Alternatively, but not recommended because > 5.5% Boric acid is classified as cancerogenic, mutagenic, toxic to reproduction :
Weigh 100g Boric acid into a 10L flask, add 2L warm de-ionized water, and mix until it has dissolved. Add water to 9L and proceed as above).
@@app-animal-science-and-welfare thank you so much.
Thank you for your response.
Can you suggest; how long we can store boric acid solution once after preparation?
@@tulasachaudhary8044 It is possible to store it for a long time. We have used 6 months old boric acid solution, with no difference in blank value. We usually make new every 3 months.
@@app-animal-science-and-welfare thank you so much😊
Do we need a
blank solution for this experiment?
You need a blank sample where you use all chemicals but no sample.
Where are the black one in the last
All nitrogen has been distilled over as ammonia to the flask, so the remaining dark liquid in the tube is discarded.
Fuck my life! I have to do it like 1890 in Iran...
My Instructor told me that back titration can only done with alkali Not with Acid.Kindly verify
I think in the video she meant to call that "titration" rather than "back-titration" because back-titration means quantifying the excess amount of a reagent that was added intentionally excessively. In this video, there was a reagent added in excess intentionally (NaOH), but that reagent is not in the flask for the titration. That reagent was left in the big test tube. So I think this is properly called just "titration" rather than "back-titration". But back-titration does not need to be done with alkali all the time. For example, in a redox back-titration, neither the titrant nor the analyte need be acid nor alkali: one would be the reducing agent and the other the oxidizing agent. There is an example of such back-titrating near the bottom of the following web page (search for the words "back titrating" on the page):
chem.libretexts.org/Bookshelves/Ancillary_Materials/Demos_Techniques_and_Experiments/General_Lab_Techniques/Titration/Redox_Titration
I mean, if we were titrating to determine how much excess NaOH was left over in the big test tube, then yes, that would be called "back-titration", even though the titrant is an acid. So I do not agree that back titration is only done with alkali.
@@trysomechemistry2287 👏
@@trysomechemistry2287 how to get blank sample please?
I believe the blank sample is done with every reagent as usual, except there is no protein sample. The purpose of the blank in this case is just to measure the residual nitrogen that might be present in the reagents. When calculating moles of nitrogen present in any given sample using the data obtained by the titration, we don't just use the volume of titrant directly; instead, we subtract the volume of titrant for the sample MINUS the volume of titrant for the blank. So, the blank just has to be treated the same as any protein sample, except that it cannot contain any actual protein sample. See the calculation shown in the "Titration" step on the following web page: people.umass.edu/~mcclemen/581Proteins.html
Wijs method is better
Seeing this, I can realize how poor our lab is.....🥲🥲
It is highly educative, thanks a lot
Thank you for your appreciation!