Three major classes of photosynthetic pigments occur among the algae: chlorophylls, carotenoids (carotenes and xanthophylls) and phycobilins. The pigments are characteristic of certain algal groups as indicated below. Chlorophylls and carotenes are generally fat soluble molecules and can be extracted from thylakoid membranes with organic solvents such as acetone, methanol or DMSO. The phycobilins and peridinin, in contrast, are water soluble and can be extracted from algal tissues after the organic solvent extraction of chlorophyll in those tissues.
The rationale behind the extraction techniques is to disrupt cell integrity as much as possible, thereby removing pigment molecules from intrinsic membrane proteins. Freezing the tissue with liquid nitrogen, and grinding the still frozen tissue in with a mortar and pestle or blender, overcomes some of the problems of working with material that produces large amounts of viscous polysaccharides. "Freeze-thawing" tissue also breaks down cellular membranes, but may liberate more polysaccharides. Finely ground tissue can be then homogenized in organic solvent to further disrupt cellular membranes, and to liberate pigment molecules from the light harvesting pigment protein complexes.
Once the pigments are extracted into appropriate solvents they can be separated chromatographically by TLC or HPLC for spectral analysis and identification. Pigment concentrations in hydrocarbon solvents can be estimated; however, these formulas are predictive, and may overestimate some pigment concentrations (see Seely et al. 1972 for the development of equations). Uncoupling pigments from the pigment binding proteins can change the absorption patterns of the pigments, resulting in shifts in maxima from 10 to 50 nm, when compared with spectra measured for intact tissues.
|Chlorophyta||Green algae||chlorophyll b|
|Phaeophyta||Brown algae||chlorophyll c1 + c2, fucoxanthin|
|Chrysophyta||Yellow-brown or golden-brown algae||chlorophyll c1 + c2, fucoxanthin|
|Pyrrhophyta||Dinoflagellates||chlorophyll c2, peridinin|
|Cryptophyta||Cryptomonads||chlorophyll c2, phycobilins|
|Rhodophyta||Red algae||phycoerythrin, phycocyanin|
|Cyanophyta||Blue-green algae||phycocyanin, phycoerythrin|
Pigments can be extracted from seaweeds by a variety of techniques though often, it is only through trial and error that an effective technique for a given species is found. Algae which produce large quantities of polysaccharide can be very difficult to work with. Choose your plants carefully!
It is important to note that light, heat, extremes of pH, and oxygen cause the destruction of pigment extracts. The extracts should be kept cold, wrapped in foil, and worked with in the lowest light possible throughout the procedure.
A. Acetone Extraction
This technique can be used for the green, brown, and red algae
as well as the seagrasses. Some of the greens and seagrasses may
be extractable without grinding in liquid nitrogen; for brown
and red algae more extreme measures may be necessary.
Note: Field collected tissue should be cleaned of epiphytes prior to the extraction.
NOTE: If there is color still remaining in the pellet, repeat steps 2 and 3 until the pellet is colorless.
(Samples can be stored in the dark at 4o C at this point for a limited period of time)
B. Thin Layer Chromatography
When handling the silica gel plates, care should be taken not to touch the face of the plate or damage the gel along the edges.
||100 % acetone|
Jeffrey, S.A. 1972. Biochem. Biophys. Acta. 279:15-33.
Jeffrey, S.A. and G. Hymphrey. 1975. Biochem. Physiol. Pflanzen. 167:191-194.
C. Determination of pigment concentration in organic solvents:
From Jeffery and Humphrey (1975).
1. Higher plants and algae having chlorophyll a and b.
In 80% acetone:
Total chlorophyll (a and b) (mg/L) = 20.2(A645) + 8.02(A663)
OR Total chlorophyll (mg/L) = A652/36
Chlorophyll a (mg/L) = 12.7(A663) - 2.69(A645)
Chlorophyll b (mg/L) = 22.9(A645) - 4.68(A663)
In 90% acetone:
Chlorophyll a (mg/L) = 11.93(A664) - 1.93(A647)
Chlorophyll b (mg/L) = 20.36(A645) - 5.50(A664)
2. Diatoms, chrysomonads, and brown algae containing chlorophylls a, c1 and c2 in actual proportions.
In 90% acetone:
Chlorophyll a (mg/L) = 11.47(A664) - 0.4(A630)
Chlorophyll c1 + c2 (mg/L) = 24.36(A630) - 3.73(A664)
A. Extraction of Pigments from Brown Algae
Extraction of pigments from brown algae, particularly the larger macrophytes, can be difficult because of the rubbery nature of the thalli, and the large amounts of polysaccharides in the tissue. The most commonly used methods for extraction of chlorophyll a, c, and fucoxanthin are described below (Seely et al. 1972). The formulas presented do not include a correction for carotenoid absorption, and therefore, may overestimate the amount of fucoxanthin present.
NOTE: This is only a recommendation. The amount of DMSO required should be determined empirically.
NOTE: Use 4:1 DMSO:H2O and 3:1:1 Acetone:Methanol:H2O as blanks.
Chl a = A665/ 73.6
Chl c = (A631 + A581 - 0.3A664)/62.2
Fucox.= (A480 - 0.772(A631 +A582 - .297A665) -.049A665)/130
Chl a = A664/73.6
Chl c = (A631 + A581 - 0.3A664)/62.2
Fucox.= (A470 - 1.239(A631+ A581- 0.3A664) -.0275A664)/141
B. Extraction of Phycobilin Pigments
Phycobilin pigments are water soluble and therefore are not well extracted by organic solvents. Phycobilin pigments may be extracted from the pellet of an organically extracted pellet (although some loss may occur into the organic phase) or from fresh thalli using the following protocol (Evans 1988).
1. Weigh out 0.05 - 0.5 g of thallus. Transfer to mortar and grind in 5 mL 0.1M phosphate buffer, pH 6.8, with acid-washed sand.
2. Centrifuge for 10 min at 1,000 x g.
3. Transfer to 25 mL volumetric flask and bring up to volume.
4. Determine phycobilin concentration using following formula:
R-PE = [(A564- A592) - (A455 - A592)0.20]*0.12 C/R-PC = [(A618 - A645) - (A592 - A645)0.51]*0.15
OR FOR PLANTS CONTAINING C-PC (from Kursar and Alberte 1983)
C-PC = 166(A618 )- 108(A650)
R-PE = 169(A498)- 8.64(A615) - 1.76(A650)
TLC and in vivo spectral analysis and pigment identification:
Owens, T.G., J.C. Gallagher, and R.S. Alberte. 1987. Photosynthetic light-harvesting function of violaxanthin in Nannochloropsis spp. (Eustimatophyceae). J. Phycol. 23:79-85.
Determination of pigment concentration in solvents:
A. Green algae and seagrasses:
Jeffrey and Humphrey. 1975. Biochem. Biophys. Phlanz. 167:191-194.
B. Brown algae and diatoms:
Duncan, M.J. and P.J. Harrison. 1982. Bot. Mar. 25:445-447.
Seely et al. 1972. Mar. Biol. 12:184-188.
C. Red algae:
O'Carra. 1965. Biochem. J. 94:171-174.
Kursar, T. and R.S. Alberte. 1983. Plant Phys. 72:409-414.
Evans, L.V. The effects of spectral composition and irradiance level on pigment levels in seaweeds. In: Experimental Phycology. Lobban, C.S., D.J. Chapman and B.P Kremer. Eds. New York. pp 123-134.
Dring, M.J. 1982. The Biology of Marine Plants. Edward Arnold. pp 1-8.
SOLUTIONS AND MATERIALS
A. Solutions and chemicals:
100 % Acetone (reagent grade)
Petroleum ether:Acetone (7:3 v:v) for 1 L : 700 ml petroleum ether and 300 ml acetone. This solution is highly flammable, use with caution.
- Mortar and pestle (place into freezer well in advance of the use)
- Tissue homogenizers
- Silica gel plates
- Developing Tanks
- Centrifuge tubes
- Eppendorf tubes
- Filter paper
- Razor blades