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hi....

Postby aparna » Mon Nov 26, 2007 5:58 am

I have a doubt regarding the purification of plant extracts before doing HPLC. Is it necessary to remove the chlorophyll ?
One more thing, which solvent system is most suitable for doing TLC for the separation of plant extracts ?
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Re: hi....

Postby Louise » Mon Nov 26, 2007 7:08 am

aparna wrote:I have a doubt regarding the purification of plant extracts before doing HPLC. Is it necessary to remove the chlorophyll ?
One more thing, which solvent system is most suitable for doing TLC for the separation of plant extracts ?


This forum is intended for school children doing science fair projects. Your question sounds like it is for a college or graduate level course or project. (I don't know many high schools with access to an HPLC!)

As for the best conditions for separation, that depends very much on the system, the type of column you have, and other conditions you can set in the HPLC (such as gradient, pressure, or temperature). Method developement is not trivial, and determining appropriate conditions for the best separation of your compounds requires some experimentation. There are also numerous books on this topic.

You can use TLC to estimate conditions for the HPLC run, but the conditions are different, so they should just be a starting point. If you want to remove a fraction (such as chlorophyll) before HPLC, I would recommend that you use a column and not a plate, otherwise you'll spend days running TLC, since the quantity you can load on a plate it limitted.


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Hi

Postby aparna » Mon Nov 26, 2007 7:13 pm

Thanks a lot for your suggestion.But i thought of doing TLC inorder to check the suitable solvent system so that it will be easier for me to run a Column Chromatography.
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Re: Hi

Postby Louise » Tue Nov 27, 2007 7:36 am

aparna wrote:Thanks a lot for your suggestion.But i thought of doing TLC inorder to check the suitable solvent system so that it will be easier for me to run a Column Chromatography.


Right, as I said, you can use TLC to estimate solvent conditions, but usually the column in an HPLC behaves a little differently than the TLC plate (and that is assuming you use the same separation material). Even changing from a silica TLC plate to a regular silica gravity column can produce different results, so you always need to do some trials when you switch systems.


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hi

Postby aparna » Thu Dec 13, 2007 5:07 am

What reaction occurs when a plant methanol extract with dilute HCl is heated with activated charcoal ? i doubt it because before treatment with charcoal, i had a light green colored solution.After that, it turned to dark red colored clear solution.
Please do help.
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Re: hi

Postby Louise » Thu Dec 13, 2007 7:15 am

aparna wrote:What reaction occurs when a plant methanol extract with dilute HCl is heated with activated charcoal ? i doubt it because before treatment with charcoal, i had a light green colored solution.After that, it turned to dark red colored clear solution.
Please do help.


Hello again.

Could you please provide some more information about your science fair project, hypothesis, and procedure? Additionally, could you tell me a little about the lab you are working in and what type of mentoring you are receiving? I'm afraid I don't really understand what you are doing and how it relates to your first question about running HPLC and TlC. More information will help me come up with answers that are grade appropriate. I just don't understand why you would do what you are doing to optimize HPLC conditions.

Lastly, the green solution... is it green colored liquid, or clear liquid with greasy green chunks?

Your project sounds very interesting, and I hope to learn more about it soon.

Louise
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Postby barretttomlinson » Thu Dec 13, 2007 11:53 pm

You seem to be dealing with a very complex set of chemical reactions in a very complex mixture. Acidifying a methanol extract of plant leaves apparently destroys chlorophyl, causing spectral changes. I searched Google for the terms chlorophyl, HCl, methanol (all one search term), and several papers popped up. One of them is:

http://www.aslo.org/lo/toc/vol_23/issue_5/1059.pdf

I do not know if you have had enough chemistry, or have good enough mentor support, to decipher this stuff, but it definitely looks interesting enough to try.

You may also wish to research chlorophyl a and b, both of which sound like they were originally present in your methanol extract.

I agree with Louise that I have no idea what you are trying to do, or how this relates to your question.
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Hi

Postby aparna » Fri Dec 14, 2007 4:47 am

Thanks for your suggestions.Well, let me explain my project.My aim is to check the anti inflammatory component present in a particular medicinal plant.For that, i need a good protocol to obtain the plant extract without the presence of chlorophyll. Only if i get a pure sample, i can go for an HPLC if possible.Otherwise i will end up in doing TLC and Column chromatography.To find the exact component, more experiments has to be worked out as per my professor says.My lab has got average requirements to do my work.But i hope i get more exposure to other labs.Now i am trying out different protocols to isolate the alkaloids and also organic components present in that plant.

Hope you understand my work now.I am following the basic protocols seen in google for isolation.
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Re: Hi

Postby Louise » Fri Dec 14, 2007 8:23 am

aparna wrote:Thanks for your suggestions.Well, let me explain my project.My aim is to check the anti inflammatory component present in a particular medicinal plant.For that, i need a good protocol to obtain the plant extract without the presence of chlorophyll. Only if i get a pure sample, i can go for an HPLC if possible.Otherwise i will end up in doing TLC and Column chromatography.To find the exact component, more experiments has to be worked out as per my professor says.My lab has got average requirements to do my work.But i hope i get more exposure to other labs.Now i am trying out different protocols to isolate the alkaloids and also organic components present in that plant.

Hope you understand my work now.I am following the basic protocols seen in google for isolation.


So you are a graduate student working with a professor in college lab? Do you have access to a UV-VIS? Are there analytical service labs available? I have no idea what "average requirements for your work" means. When you refer to HPLC, you are meaning analytical or preparatory?

Nor is "basic protocols seen in google" a useful citation. You either need to provide the key words you used to search or (better) the link to the protocols.

I agree with the previous expert about chlorophyll. Acid can both destroy the macrocycle and demetallate the ring. Both will lead to color change. I'm not sure why the activated charcol is needed in the heating step. I would expect the protocol to be: 1) heat with acid 2) filter through characol. Charcol is freqeuntly used to remove metal ions, so I would guess you are demetallating.

As I've mentioned before, this forum is intended for school children to get help with science fair projects. This is not the place to meet people working in different labs or "get exposure to other labs". Most of the answer you are going to get are based on people googling based on what you post here, and not because they are actively researching this area. Most of the experts on this forum are not even chemists. I strongly recommend you go to the library and ask the librarian how to do a literature search on your topic. The information that you get from the scientific literature will be MUCH better than any information you can get here or on google searching.

Good luck.

Louise
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hi

Postby aparna » Sun Dec 16, 2007 9:45 pm

Thanks again.........Sorry for the trouble.

bye.
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Re: hi

Postby Louise » Mon Dec 17, 2007 6:39 am

aparna wrote:Thanks again.........Sorry for the trouble.

bye.


Aparna,
No trouble at all. I asked specific question about you and your lab so I can give better answers. It is difficult to help with a complicated chemistry without knowing what you have access too, and what your educational level is. The more information you give us, the better our answers are. But, you also do have to understand the purpose of this forum. People will probably help with your project even if you are not a school kid working on a science fair project. However, the advice won't be as 'expert' and you won't be making professional contacts. If you don't mind this, then post away.

One of the hardest lessons is learning _where_ to find information. [The experts here are good at knowing where to find answers, which is why we can answer many different types of questions.] When you are doing university level research, searching the scholarly literature is usually the best choice (hence my recommendation). At a university you have access to material that is not freely found on the web. You can find detailed papers and procedures that we could never find (or if we did find, it would be illegal for us to share with you). A good librarian can teach you how to use the many wonderful tools available to find the answers you need. These skills will be important for the rest of your career.

Best of luck with your project.

Louise
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