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Author: Subject: extraction of Chlorophyll with acetone"requesting"
calamus
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[*] posted on 3-5-2010 at 08:12
extraction of Chlorophyll with acetone"requesting"


hi all,
i extract chlorophyll with acetone :
0.5g+100 acetone with morteor and sand and filtrate
for measuring on spectrophotometer
that is the method of extraction
problem:
the amount of acetone used
i wanna to use only 20 ml acetone for extraction and
taking 1 ml of stock (20 ml) and dilute it to last equal
that 100 ml in the method
need someone to explain me how to do that

[Edited on 3-5-2010 by calamus]
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bahamuth
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[*] posted on 3-5-2010 at 12:33


Extraction of chlorophyll.

You need it dry, not the substrate but the extract.
Chlorophyll degrade in water and light so work in dim light.

How to:

Extract chlorophyll with acetone in freezer overnight, decant and filter off extract and add more acetone for a second overnight freezer extraction, keep first extract in freezer as well. Do with second extraction as with first. All this preferably under an inert atmosphere.
Combine extracts and concentrate on a rotary evaporator. Don't try to get it dry, water is hard to rotavap, add enough acetone to dissolve sample completely and add some solvent which have a strong azeotrope with water, like ethanol (we use benzene) and rotavap once more. If sample does not look completely dry, repeat ethanol/acetone drying.

When completely dry, dissolve in acetone and run it through a 0.2 um filter if not done on such a fine filter in the extraction phase. (we sometimes use a glass syringe and syringe filters which withstand acetone).

PS. If substrate is fibrous as bark or the like, grind as you do in a mortar or any other sufficient way.

Hope this helps.




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JohnWW
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[*] posted on 4-5-2010 at 15:59


I think that you could also equally extract chlorophyll from leaves with diethyl ether, which is much less miscible with water; and the likes of other ethers like dioxan, tetrahydrofuran, tetrahydropyran, etc. are also possibilities. I once did this in a lab class years ago, using, if my memory serves me correctly, diethyl ether. The extract from the leaves was used in a chromatographic experiment to separate the types of chlorophyll present by thin-film chromatography, and examine them spectroscopically.
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Reduce-Me
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[*] posted on 5-5-2010 at 13:18


Are you measuring the existence or are you quantifying?

Because you really don't even need to do an extraction to do either on a spec, you should just be able to stick cells in it and quantify from the peaks, the extinction coefficients are available.
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bahamuth
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[*] posted on 5-5-2010 at 13:23


If calamus is out to get analytical results he needs to kill of the enzymes, and remove the water as fast as possible.

Have had good luck with this method.

But some ether would work fine too, like THF..




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Reduce-Me
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[*] posted on 5-5-2010 at 13:27


THF is awful IMO, just smelly and gross. Ethanol/Acetone/diethyl ether are all common to use, as well as mixtures thereof.

Good luck.
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calamus
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[*] posted on 9-5-2010 at 02:12


thanks bahamuth ,
for your method but i want to quantify the chlorophyll (chlorophyll a, chlorophyll b & carotenoid)in the fresh leaves

thanks JohnWW ,your method is good but we did not have chromatography

thanks Reduce-Me , i quantify the chlorophyll
could you explaine that"the extinction coefficients are available"

thanks all for helping me
i reach to a solution for that by taking any volume of the extract and divide it by
total/initial and complete the equation of Metzner, et al.,
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bahamuth
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[*] posted on 9-5-2010 at 04:13


If you want to quantify the different pigments you have to run it on a HPLC, since the chlorophylls have very similar absorbance, & exitation if you plan to use florescence, same goes for the carotenoides...

But anyway which extraction method you plan to use, you have to make it free from water as fast as possible, keep it under an inert atmosphere and keep it away from light since all these will kill you pigments (chlorophyll & carotenoid).




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Reduce-Me
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[*] posted on 10-5-2010 at 08:48


Quote: Originally posted by calamus  
thanks bahamuth ,


thanks Reduce-Me , i quantify the chlorophyll
could you explaine that"the extinction coefficients are available"




This includes extraction ideas as well.



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calamus
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[*] posted on 12-5-2010 at 10:52


thanks bahamuth, you are so kind really thanks for your advise
the hplc is not available at this time

thanks Reduce-Me ,you are helpful thanks for your papaer

i used that equation for calculations :
mg chl “a”/g tissue = (10.3 D663 – 0.918 D644)
mg chl “b”/g tissue = (19.7 D644 – 3.87 D663)
mg carotenoid/g tissue = 4.2 D452.5 – (0.0264 chl.a + 0.426 chl.b)

really thanks for you help

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