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Author Topic: ion exchange chromatography  (Read 1116 times)

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fa911662

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ion exchange chromatography
« on: March 28, 2017, 09:34:20 AM »

Hello everyone,

I did an ion chromatography on dowex resin ( with sulfonyl group SO3H) to separate lysin from glutamic acid. After the separation we put few quantity of ninhydrine in every tube to detect the proportion of amino acids that was in the initial sample. I have a question about ninhydrine reaction with lysin. Because lysin has two amine groups (RNH2) on on the chain and one on the alpha carbon, does ninhydrine react with the two amin function or only with that is on the alpha carbon ?

 sorry for my english I'm from france :)
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Babcock_Hall

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Re: ion exchange chromatography
« Reply #1 on: March 28, 2017, 10:58:01 AM »

My understanding is that ninhydrin reacts with any primary amine; therefore, I think it will react twice.  However, I don't see that it matters for you application, unless you are trying to quantitate by using a ninhydrin test.  That is another matter entirely.
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fa911662

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Re: ion exchange chromatography
« Reply #2 on: March 28, 2017, 11:19:42 AM »

After the chromatography, we had to determine the proportion of lysine and glutamic acid in the sample that the teacher gave to us. We put ninhydrin in every tubes and after that we measured the absorbance of every tube.After that we draw a chromatogram. I think we have to do the ratio of areas but if we have two ninhydrin for 1 lysine Do we have to include a factor 2 somwehere. Just I precise we don't have to measure the concentrations but only the propotion of the two amino acid that the teacher gave to us
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Babcock_Hall

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Re: ion exchange chromatography
« Reply #3 on: March 28, 2017, 03:00:45 PM »

I would be tempted to divide the absorbances for lysine-containing fractions by two.  However, we are making an assumption about the linearity of the assay that may or may not be true.  That is why I tried to draw a distinction between a qualitative assay and a quantitative assay in my earlier comment.
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fa911662

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Re: ion exchange chromatography
« Reply #4 on: March 28, 2017, 07:31:22 PM »

I understand, I would be tempted to do the same. The problem is that in the teacher's book, we have and example and he don't divide by two. That's why I asked if the lysine react with two molecules of ninhydrine. In the book, the teacher wrote that ninhydrine react with the amine fonction on the alpha carbon of the amino acid but like you said I read some text that say the ninhydrine react with all primary amine functions. I didn't find the specific reaction of lysine with ninhydrin so I'm a bit blocked now ..
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Babcock_Hall

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Re: ion exchange chromatography
« Reply #5 on: March 29, 2017, 04:30:21 AM »

P. 230 of Robyt and White's textbook states, "The color yields for the different amino acids, however, are not the same and must be taken into consideration when different amino acids are being quantitatively determined."  Sounds to me that even if you were comparing, say alanine and leucine, you could not make a comparison of the two, unless you prepared a standard curve for each.
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fa911662

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Re: ion exchange chromatography
« Reply #6 on: March 29, 2017, 09:34:24 PM »

Thank you for your help !!! JusI I have another question on the same topic :
The second amino acid was retained on the column and it was the last to exit (lysine with positive charge). We worked with a citrate buffer to have pH 6. But what I don't understand is that we didn't change the buffer, we worked with the citrate buffer all the time so how the citrate push the lysine out from the column. Is it possible that Na+  ion take the place of the lysine on the SO3- resine ??
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Babcock_Hall

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Re: ion exchange chromatography
« Reply #7 on: March 30, 2017, 03:10:39 AM »

Sodium ions can certainly compete versus ammonium groups of amino acids.
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