algae pigment extraction

Han Asard hanasard at RUCA.UA.AC.BE
Mon Jan 8 04:10:04 EST 1996



On 5 Jan 1996, Kathleen Archer wrote:

> Dear Folks,
> I thought I remembered the posting of a simple protocol for the extraction
> and comparison of pigments from red, brown and green algae in this venue
> some time ago.  However, my search of the archives turned up nothing, so
> I'll just ask:
> 
> Does anyone have a simple procedure for students that lets them extract and
> compare the different pigments from the major groups of algae?
> 
> Secondly, I'm trying to think how to impress on my students that the first
> type of photosynthesis to evolve did not produce oxygen.  Is there a
> non-oxygenic but photosynthetic bacterium out there that that one could use
> in a lab exercise to show students there are others ways of doing
> photosynthesis?  I'm totally ignorant here and at your mercy.
> 
Dear Kathleen

We have successfully been using two methods to extract pigments from algae
and higher plants. However, both procedures result in total extracts and
in order to identify individual pigments these extracts are submitted to a
simple partitioning chromatography on a starch column. The great advantage
of this is that students are also introduced in what looks to be a
'sophisticated biochemical method'. 

Some details:

Procedure 1: a relatively small amount of tissue (0.2 to 1 grams) is
submerged in 10 ml of dimethylformamide in a small flask. This is a rather
toxic chemical and some care should be taken in handling the solution
(using a dispenser works fine). The mixture is kept for 24 to 48 hours in
a cold room in the dark. After extraction spectrophotometry can easily be
performed. We have not used this extract for further separation. 

Procedure 2: here we first lyophilize the tissue to reduce water content.
Extraction is performed by grinding the thalli (or plant leaves) in a
mixture of toluene:petroleumether:methanol (50:50:1). Grinding can be done
most blenders I guess but we use an Ultraturrax homogenizer. The
homogenate is kept in the dark for 10 to 30 minutes for further extraction
and a clear supernatant is obtained after sedimentation or filtration.
This extract may be directly applied to a starch column prepared with
toluene:petroluemether (1:1) as the liquid phase. This column results in
the separation of chlorophylls and carotenes. The coloured starch bands
are excised and redissolved in diethylether which rapidly liberates the
pigments. 

Running the columns always is a great way to impress the students because
of the colourful spectacle. However TLC with these extracts would work
too. 

Best regards, Han Asard



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