Pigment resolution

Pigment resolution

When importing a data base, is important to recognize the HPLC method used and the the pigments that are not resolved by the method (co-elution). This allow proper interpretation of data into the correct pigment or pigment sums in the database. In many cases this is not important for HPLC determinations of unialgal cultures, since overlapping pigments do not co-occur in a taxonomic groups (e.g, Fuco and derivatives only present in the red lineage and Neo only present in the green lineage). The possible overlap is checked against a pigment distribution table, available on the ancillary (anc) directory. For application of chemical taxonomy, however, the resolution is important since in environmental samples likely many groups are present. A R script will be available in a future version to aggregate the database to the resolution of the most common HPLC methods in phytoplankton pigment research.

The description of the limitations and comparative performance of different HPLC methods was taken from Wright & Jeffrey (2006).

The HPLC method of Wright et al. (1991), recommended in Jeffery et al. (1997) does not separate monovinyl from divinyl forms, therefore those pigments are summed (if co-occuring):

• Chl a, DVChl a (not known to co-occur in the same organism);
• Chl b, DVChl b (= TChl b, but only co-occur in CYANO_4);
• Chl c₁, Chl c₂, MgDVP (= Chl c_1,2, except for PRASINO_3);
• Chl c₃, MVChl c₃ (only known to co-occur in HAPTO_6 and possibly Chrysophytes);
• But-Fuco, Siph (not known to co-occur in the same organism);
• c-But-Fuco, t-Neo (not known to co-occur in the same organism);
• Hex-Fuco, Hex-kFuco, c-Neo (the first two not known to co-occur with c-Neo in the same organism).

Not much information is available on the resolution of Garrido & Zapata (1997). It allows separation of Chl c forms but at expense of resolution in the carotenoids.

The HPLC method of Zapata et al. (2000) separate many forms of Chl c, which is particularly important for Haptophytes. The few unresolved pigments are:

• Chl b, DVChl b (= TChl b, but only co-occur in CYANO_4);
• Micral, Diadchr (both pigments only occur in PRASINO_3);
• Loro, c-Neo (both pigments can occur in some CHLORO_1, CHLORO_2 and PRASINO_1).

The method of van Heukelem and Thomas (2001) does not separate the following pigments. Note, however, that Chl c₁ and Chlide a can be separated with a dichromatic equation and that MgDVP is present in trace concentrations for most organisms, being abundant only in PRASINO_3. The list of co-eluted pigments is:

• MgDVP, Chl c₂ (= Chl c₂, except for PRASINO_3);
• Chl c₁, Chlide a;
• Hex-kFuco, c-Neo (not known to co-occur in the same organism);
• Pras, Microl (both pigments only co-occur in PRASINO_3B);
• β,ε-Car, β,β-Car (= Car, but many algal groups present only one of the two).