Smita,

Of course you also take 2ml of your proline standards and put thru the whole process as well , including a reagent blank of 2 ml DI water only. By taking only 2ml of your 5ml 3% sulfosalicylic acid, you are simply sub-sampling your extract. The concentration determined from your calibration curve as to what was in this 2ml is representative of the total 5ml.

Example, lets say that for the sub sampled 2ml going thru the whole method (ninhydrin, boiling, etc) you determine from your calibration curve the concentration of proline to be 10 ug/ml.

So that's 10 ug proline per ml of your 3% sulfosalicylic acid extract, and you had 5ml of this 3% sulfosalicylic acid extract, so a total of 50 ug proline. This 50 ug proline was from your initial 0.2 g plant material homogenised, so 50ug/0.2 g = 250ug/g. Of course these numbers are for example only and may bear no resemblance to what you are truly measuring. Dividing this 250 ug proline by molecular weight 115.5 will then give you 2.165 umoles proline per gram sample

you put 0.2g of sample weight as a gram of sample. By using standard curve u can calculate ug proline/ml. Multiplication of absorbance with standard value is not proper method for calculation. so u just follow according to standard curve.

Let's say you extracted 0.2 g of plant tissue in final volume of 10 ml toluene. You also prepare a series of proline standards (in toluene) to calibrate the spectrophotometer with, i.e. come up with a standard curve, linear regression with proline concentration on the x-axis and the measured absorbance at 520 nm on the Y-axis.

From the measured absorbance of your toluene extract, you can determine the concentration of proline in that extract form the linear regression, let's say your x-axis is in units of ug proline/ml toluene. Once you know the concentration of proline (ug/ml) in your extract you then simply multiply by 10 for the 10 ml of toluene extract you have, this then gives you the mass (ug) of proline in your toluene extract. this mass of course is from your 0.2 g plant tissue extracted. The equation converts the mass (ug) of proline to umoles with the molecular weight for proline term of 115.5

Thank u so much Nitesh Litoriya and Greg Cawthray. But I have a doubt. I have homogenised 0.2g of sample in 5 ml of 3% sulfosalicylic acid and from that I have taken 2ml of extract and mixed with 2ml acid ninhydrin and 2 ml glacial acetic acid.The reaction mixture was boiled in a test tube for 1 hr at 100 c and then extracted with 4 ml toluene. So my doubt is 5ml of sulfosalicylic acid contain 0.2 g of sample. As I have taken only 2 ml extract according to that I have to calculate i.e. 0.8 in place of g sample or 0.2 (total sample i have homogenised with sulfosalicylic acid) in place of gram sample. Please clarify this thing. Again thank u so much for clearing my doubt about ug prolin/ml.

Smita,

Of course you also take 2ml of your proline standards and put thru the whole process as well , including a reagent blank of 2 ml DI water only. By taking only 2ml of your 5ml 3% sulfosalicylic acid, you are simply sub-sampling your extract. The concentration determined from your calibration curve as to what was in this 2ml is representative of the total 5ml.

Example, lets say that for the sub sampled 2ml going thru the whole method (ninhydrin, boiling, etc) you determine from your calibration curve the concentration of proline to be 10 ug/ml.

So that's 10 ug proline per ml of your 3% sulfosalicylic acid extract, and you had 5ml of this 3% sulfosalicylic acid extract, so a total of 50 ug proline. This 50 ug proline was from your initial 0.2 g plant material homogenised, so 50ug/0.2 g = 250ug/g. Of course these numbers are for example only and may bear no resemblance to what you are truly measuring. Dividing this 250 ug proline by molecular weight 115.5 will then give you 2.165 umoles proline per gram sample

Thank u so much Greg Cawthray for clearing my doubt...

Swapnil,

ideally you would dissolve your proline standards in the same solvent as what your samples are in, this is matrix matching.  if that's not possible not easy to do, then dissolve the proline standards in milli-Q/deionised water.  Make at least 5 different proline standards. Once you make up your standards, take these thru the colorimetric method and then plot concentration of standard (x-axis) versus the absorbance read at 520 nm, this is of course for the Bates (1973) methodology.

Cheers

Greg

i have the same problem with this formula.i dissolved proline in deionised water to make the standard curve.i made 4 proline standards.then i followed the collorimetric method so as to plot concentration (x-axis)(after dilution) versus the absorbance read at 520 nm.my question is : at  x axis we put  μg proline/ml from the first concentration or after the dilution?And what is the concentration after dilution?the μg of proline containing at the solutionof  2ml of extract and mixed with 2ml acid ninhydrin and 2 ml glacial acetic acid or the μg of proline containing at 4 ml toluene?

Giannis

You may consider the concentration of proline in your standards as values to be used in X axis. Of course when you use, for example, a 6 micro gram/mili liter proline as a standard, you would have a concentration of 2 microgram/ mili liter after adding 2ml acid ninhydrin and 2 ml glacial acetic acid. BUT you have the same process for your leaf samples and their proline concentration.  So,  the initial concentrations of proline standards are just okay to be used.

Swapnil

getting a standard curve for  proline is a little bit tricky and you just need to repeat it carefully.

Cheers

Dear Greg Sir, greetings from priyanka...I went through your proline estimation answer, which is well defined and easy to understand also but it is not as per bates formula. For ex if i have 40 ug /ml proline so as per you its should be 40*10 (salicylic acid)=400/0.5 g (tissue taken) ..so its 800 ug/g fm my final answer..... but bates formula is differnt

"µmoles per gram tissue =[(µg proline/ml) x ml toluene)/115.5 µg/µmole] / [(g sample)/5]

so according to him ...it should be 40* 4 (toluene vol)=400

400/.1= 40 ug/g fm...

now both answers are different???

Hi,

I want to share my experience with making the proline standard curve using the Bates et al. 1973 method, and to also ask some questions.

I used L-proline dissolved in 3% 5-sulphosalicylic acid in the following concentrations: 20,40,60,80,100 ug/ml (http://www.scientistsolutions.com/a7356-protocol-free+proline+estimation+in+plants.aspx  ;   http://www.personal.psu.edu/svr11/9-1-2011/Journal%20Club/Journal%20Club/monica_effi.pdf). However, after two attempts, I got very high absorbance readings - i.e. the 40 - 100 ug/ml standards had an absorbance higher than 1.0 and the 80 and 100 ug/ml standards had an absorbance reading of +/- 3.0. It is my understanding that in order to have accurate results, your absorbance readings should be between 0.1 - 1.0. Therefore, my questions are: has anyone else made a standard curve using similar concentrations to what I have used? And if so, were your absorbance readings as high as mine? And did you have linearity throughout the entire concentration range or just from 20-80 ug/ml? 

If the concentrations I used are incorrect, what is the ideal concentration range I should be using when working with plant material?

Dear Priyanka

According to the formula, I think 5 is sulfosalicylic acid vol. used for extraction.   

"µmoles per gram tissue =[(µg proline/ml) x ml toluene)/115.5 µg/µmole] / [(g sample)/5]

Also we can write the formula like this: 

"µmoles per gram tissue =[(µg proline/ml) x ml toluene x ml salicylic acid] /(115.5 µg µmole x sample (g))

Thank you so much to all of you for your valuable answers and the discussion.

Mrs Marzieh Etehadpour this calculation gives you proline / 1 gram tissue or proline / gramms of sample (f.ex 0,2)

Can some one answer this question: If we did standard by the same way as sample measurement, Why we multiply in toluene volume 4 ?.

Dear Marzieh Etehadpour, I don't think that 5 is the extraction volume because Bates used 10 ml of 3% sulfosalicylic acid.

Dear Ali Hassan Ibrahim , when you use toluene to extract proline, you may convert the unit to µg proline by eliminating the volume of toluene (4ml). that's why you need to multiply in toluene volume 4.

Cheers

Noureddine

I think that if we carried out the standard curve using toluene as the sample, there is no need to multiply in toluene volume (4). I f we carried out the standard without toluene extraction, but extracted the samples with toluene we must multiply in toluene volume which is 4.

Dear Noureddine I agree with you that if we extracted the standards with toluene as samples, there is no need to multiply in toluene volume which is 4. with regards.

Dr.Ali H Ibrahim

Hello everyone,

I got confused with the 'ml of toluene' part in the bates equation to express proline in terms of µ moles/g FW . Should we simply multiply the 'µg proline/ml' with 4 ml of toluene(as per the protocol) and proceed further with the equation? And is it okay to just let go of the volume of sulfosalicylic acid, ninhydrin and glacial acetic acid used in the process? Or, otherwise what is the value normally used in the 'ml toulene' part of the equation?

I would really appreciate the help. Thank you.

Dear Lushan

Hello, I think the volume of sulfosalicylic is taken in consider the nomber 5 used in equation result in 10 ml of sulfosalicylic used to extract plant sample/ 2ml volume taken = 5

Also, toluene volume should be taken in consider (it is the solvent of proline to give the color which measured on spectrophotometer), it is better to use 4 ml of toluene as Bates but sometimes you get high color intensity may be over the standard range, so you repeat the experiment with high volume of tulene (like a diluting factor), then it is necessary to put in the equation.

Hallo everybody. I think that I am a little bite confused with this protocol. I am following Bates protocol with modifications. For make standar curve I added 9 ml sulfosalicylic acid 3% to 1 ml of proline known-concentration (25-1000ug/ml). Then I took 1 ml of this solution and I added 1 ml of glacial acetic acid and 1 ml of ninhydrin 2.5% (prepared by warming in glacial acetic acid and orthofosforic acid 85%, 60/40 (v/v)). I boil 1 hour at 100°C the test tube, and I terminated the reaction in an ice bath. Finally I add 2 ml of toluene, mix by vortex ten seconds and I took the superior fase to measure the absorbance in spectophotomer at 520 nm.

For process my fresh leaves samples I did the same but I homogenized 1g of leaves in 9 ml of sulfosalicylic acid, I centrifugate 10 min, 4C, 2500g and I took one ml of extract and mix with glacial acetic acid and ninhydrin following the protocol describe above. I am observed absorbance between 0.150-0.600

It is correct my process?

Thanks very much in advance.