Thanks Maik. I know how the SPAD works for chlorophyll measurements. I guess, then, the method you use doesn't work for dry samples and is closer to the approach of LLC mentioned by Ruxandra, right?. I'm looking for the way to determine N at lower costs than using CHN analyzers. I know that many other nutrients can be determined from dry plant tissue by colorimetric techniques (extraction and subsequent determination by spectrophotometric reading).

Hi Frida, it´s very easy to determine N concentration with spectrophotometry using NIRS. Is such a device availble in your Lab?

Hi Sebastian, Many thanks for your response. We don't have a NIRS, but we have a multiplate spectrophotometer that may read in in the infrared (I've to check the manual). Do you have some paper to recommend, describing the technique? Thanks again. Frida.

Digestion of the tissue in hot sulphuric acid at 360°C in the presence of some catalysts using a Kjeldahl apparatus. The N gets reduced to ammonium which can then be detected colorimetrically using Nessler' reagent. This method is cheap and simple if you have access to a Kjeldahl apparatus. Please contact me if you want the analytical protocol.

thanks Michael. Unfortunately, I don't have access to Kjeldahl apparatus.

Yes Marcello, that's an option that I always consider. However, when it is possible, I like to do some analyses by myself. My lab has some good equipments. From your response and the previous ones, however, it seems that the equipments we have are not the appropriate to measure N.

Hi Frida,

Oser (1965) method using Nessler reagent is simple and convenient for determination of nitrogen.If interested I can give the protocol.

Thanks Bijendra. I'd appreciate if you can send me the protocol. Frida.

Hi Frida,

The protocol is as follows.By this you can estimate protein as well as non protein (soluble nitrogen).

Crush desired amount (50 mg) of plant material with 5.0ml distilled water.Take 2.0 ml from this and add 3.0 ml 10% cold TCA and centrifuge.Supernatant and residue are used for non protein and protein nitrogen respectively.Digest supernatant and residue separately with 2.0 ml conc.sulphuric acid and a pinch of catalyst (CuSO4 :K2SO4:SeO2 :: 1:8:1).Appearance of apple green colour confifms digestion.Make the volume of the digest to 15 ml with distilled water.This will act as stock solution.

Now,take 1.0 ml of stock solution and to this add 14 ml distilled water and 2.0 ml of 6 N NaOH and keep in an ice bath for 30 minutes.After 30 minutes add freshly

prepared Nessler reagent and read the O D at 490 nm with help of spectrophotometer.

Caliberate the nitrogen content against standard curve. Prepare the standard curve for nitrogen by dissolving 66.0 mg of Ammonium sulphate in 100.0 ml water.

Preparation of Nessler reagent -----

Dissolve 100 g mercuric iodide and 70 g potassium iodide in about 400 ml water (solution A) .A separate solution of NaOH is prepared by dissolving 100 g in about 300 ml water (solution B).Now mix solution A and B and make the volume

to 1000 ml.When the small amount of brownish red ppt. settles down,pour of the

supernatant and use.

Many thanks for the protocol (and sorry for the delay to answer)

Bijendra Saran Thank you so much for your protocol. I would like to know how much concentration of Nessler reagent you add into the mixture. And, may I know the reference if it is possible, please.

Hi Myo, Add 3.0 ml of freshly prepared Nessler's reagent.The reference for the protocol is :

Oser,B.L.(1965) Blood Analysis.Hawk"s Physiological Chemistry.(ed) B.L.Oser.The

Blckiston Division,McGraw Hill Book Company,New York,pp 875 -1152.

Hope this will fulfill your requirement.

Bijendra Saran Thank you so much for your kind reply. I will try it. Thanks again.

Myo, The catalyst is in powder form.Take crystals of the three components in the

ratio mentioned and make them powder.From this mixture add a pinch of this mixture.

Bijendra Saran Thank you Sir. I really appreciate it.