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Author: Subject: The best TLC plate and solvent/solvents for Chlorophyl
Little_Ghost_again
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[*] posted on 12-11-2014 at 06:04
The best TLC plate and solvent/solvents for Chlorophyl


Hi
I would like to separate chlorophyll into A and B for a reference sample, I have so many different types of the Whatmann TLC plates there are too many to list, I have tried to extract and separate chlorophyll from grass (the lawn type) using K5 and K6 plates with IPA and chloroform (90/10), I have also tried reverse phase plates. I cant get the chlorophyll to separate out into A and B.
As this is for a school project it dosnt really matter what plate I need to use (the school will supply), the same is true of solvent or solvent pair. I did search and found several methods described, but couldnt get it to work.
So rather than post a link to what I used I wanted to ask what you have found to work best (I want to see if you used the same method I did, if so then its me cocking up).
I also need to separate different plant sugars but that dosnt matter yet.
The obvious solution is to ask my dad, but I am asked by my Biology teacher if my dad has helped when I do extended projects, and they rightly mark me down if he has. There is no penalty for asking a public available resource like a forum though.

I was given a hint so the following questions are related to that.

There is polar and non polar, protic and A protic

To separate something that is polar you use a less polar solvent on a non polar plate.
Have I got this correct?

Also when looking at if chlorophyll was polar or not I got two opposite answers!! So now utterly confused.
The reason for all this is I need to measure and separate amounts of chlorophyll, this is being used as a bench mark in my experiment with plants (grass), Basically I am growing grass (lawn type) using hydroponics and feeding each try with a nutrient solution, each solution for each try is exactly the same except that each one Lacks one of the components, so for example one solution lacks all magnesium, I have 22 trays (12cm x 8cm). The idea is to find which of the grass trays will only produce Chlorophyll A and which will only produce Chlorophyll B.
The simple solution would have Been Gas Chromatography as i have 7 more weeks until the end of the project, but I am not allowed to use it due to it being seen as a unfair advantage.

Second problem

How best to titrate to determine the amount of starch present? Is it just the same as the basic starch test done via burette?
So sorry for such dumb ass questions but not fireing on all cylinders at the mo
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veganalchemist
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[*] posted on 12-11-2014 at 13:05


Hi,

we did the chlorphyll extraction yesterday at work.

I work as a lab tech at a local Uni and we had a class extracting chlorphyll from spinach.

We test frozen and fresh to see which is best. The students use silica TLC plates and I think the solvent is

acetone:pet ether (60-80) 7:3 or it may be 3:7 can't remember off hand.

I can check the script tomorrow. Works really well and get about 5 coloured bands.

G
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Little_Ghost_again
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[*] posted on 12-11-2014 at 13:06


Ok looks like I need to ask a more simple question.......................

One source have read says to use 9 parts petroleum ether to 1 part acetone, when I search petroleum ether on ebay I get more than one substance based on different boiling points, so with this in mind can I just use petrol and acetone to develop the plates? I am not so bothered about carotene at the moment (maybe later), but what I need to find is which chemical deficiencies will reduce what chlorophyll.
The nutrient mixture being used is based on a recipe widely used for planted aquariums with supplemental CO2 added, I am not adding CO2 but the recipe for the nutrients has always been pretty good when I have used it in aquariums, so I thought it would be good for this kind of hydroponics.
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Mailinmypocket
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[*] posted on 12-11-2014 at 14:42


Try to find camping fuel made with naphtha, this can be considered petroleum ether... Petroleum (petrol) will not work as it is a mixture of components plus other things for automotive engines!
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Ozone
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[*] posted on 12-11-2014 at 14:50


Chlorophyll A and B can be readily distinguished by UV-VIS absorption spectoscopy. Any scanning instrument should give you a quick answer.

e.g. https://www.google.com/search?q=absorbance+spectra+of+chloro...

IIRC, the separation also works well on paper: http://www.chem.purdue.edu/teacher/table_of_contents/uvvus/u...

Cheers,

O3




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Metacelsus
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[*] posted on 12-11-2014 at 17:41


Ethyl acetate / hexanes is a common solvent pair. It should work pretty well.



As below, so above.
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Little_Ghost_again
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[*] posted on 13-11-2014 at 02:31


Hi thanks for the answers. We can get a camping fuel called Coleman. That should do it.
As its a School project I have to play by certain rules. Paper I have done but its too hard to get a sample from. Vis etc is breaking rules.
Shame as I would just use a GC and MS. I sort of wish I had made the project easier. But it sounded simple enough at the time
LG
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Ozone
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[*] posted on 13-11-2014 at 05:29


Hmm...It should be quite easy to recover your sample from paper--just cut your band out and rinse it with acetone. Evaporate the acetone, which is easy, and you should have your fraction.

I always found scraping spots off of TLC plates to be a bigger pain.

O3




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Little_Ghost_again
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[*] posted on 13-11-2014 at 07:20


I will try it(cant hurt), so far I have never got chlorophyll to separate! I was hoping the whatman plates would do it. I have always found the non F versions of there plates great to get the bands from.
I dont see why the GC is considered an unfair advantage, they have one at school, but there you go arbutary rules yet again from Mrs X (Richards!!!).
I had originally found a really good recipe for hydroponics on an indoor gardening site, when I looked more in dpth at the site it was a specialized type of indoor gardening! I had to provide a reference source for the fertilizer when I put my project proposal in. I couldnt find the recipe ANYWHERE other than the site with stinky plants on, so I had to go with the tropical aquarium one instead.
In some ways it makes it easier as there is less in the recipe.
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Ozone
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[*] posted on 13-11-2014 at 15:34


I don't think GC will work for compounds like these, anyway. They are too big and, if they don't decompose, probably won't make it past the injection liner. You would need to use liquid chromatography (or, of course, TLC/paper). I've had good results running porphyrins via flash on silica with something like chloroform, but I can't recommend it, here.

I used to take a round filter paper, poke a hole in the center. Cut a rectangular piece of filter paper and roll it up tightly. It should be slightly larger in diameter than the hole in the round paper. Poke the wick into the hole; it should fit tightly. Heavily spot the top of the wick with you mixture and let it dry (you can dip the wick and dry it prior to insertion, if you prefer). Place the whole over a container of your solvent with the bottom of the wick dipped in the solvent, and watch her go. When complete, let the whole dry, and cut out your bands. You can get more per-run this way than with a regular sheet.

It's also fun for kids--separating marker inks, etc. to make colored objects you can tack to the refrigerator. Methanol worked very well for this, IIRC.

O3




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Little_Ghost_again
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[*] posted on 14-11-2014 at 04:49


TLC appeals because I have the plates and uv ligbt source. I will try the method you mention.
I chose the project because next year I can do it again using the improvements I will have in results discussion this year.
I need to read up and understand chelation.At present I dont think the iron oxixe mentioned in the recipe is the best source of available plant iron
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