Chlorophyll Analysis - Methods?

john markwell markwell at unlinfo.unl.edu
Wed Jul 27 07:52:49 EST 1994

jboulton at TrentU.CA writes:

>	Hi...

>	I am looking for any suggestions on the best way to sample for 
>Chlorophyll a.  I am looking at the amount of chlorophyll in a sand crust 
>which was previously innoculated with different algae.  
>	I have run across some problems trying to determine which is the 
>best method (namely: solutuion) to use for breaking the cells which are 
>bound to the sand grains.  I have been using DMSO4 + 90% acetone (1:4) 50 
>ml blending in a waring blender for 4 min.s.  These are then filtered, and 
>then analyzed for chlorophyll a.  
>	The problem I am having (I think) is that during analysis, the 
>volatile acetone isevaporating from the blank (control) cuvette, and thus 
>changing the reference value of the test sample.  

>	Any suggestions...  I have read two articles saying that DMSO4 + 
>acetone is better and two that say that just acetone is accurate enough.  
>Both have valid arguments and so I am at a loss as to which may be better.  
Stuff Deleted

>                                                  /        
>      JBOULTON at TRENTU.CA                        / |

I have three comments:
1)  Be VERY CAREFUL when using acetone in a Waring Blendor.  A fire 
may result.  Also, I try to avoid using DMSO whenever possible because
of the hazards of this solvent.

2)  The trick in Chl extraction seems to have the solvent 
concentration high enough to extract the pigments from the protein 
complexes in the membrane and to inactivate the chlorophyllase enzyme,
but not to get the solvent concentration too high, as this will reduce
the efficiency of extraction.  When M. Tswett (inventor of 
chromatography) was fooling around with chlorophylls about 90 years 
ago, he noted that he could extract them from leaves with methanol, 
but not toluene or ligroin.  After extraction into the methanol they 
were then soluble in the latter two solvents.  Thus, extraction can be
impaired by making the initial exposure too nonpolar.

3)  I recommend that you consult a chapter by Margret Holden 
"Chlorophylls" in Chemistry and Biochemistry of Plant Pigments, T.W. 
Goodwin, ed., 2nd Ed, Vol 2, pp. 1-37.

Good Luck

John Markwell			Phone: 402-472-2924
Dept. Biochemistry		FAX:   402-472-7842
University of Nebraska		Internet: markwell at unl.edu
Lincoln, NE  68583-0718

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