José L. Garrido

Influence of injection conditions in reversed-phase high-performance liquid chromatography of chlorophylls and carotenoids

SummaryThe RP-HPLC analysis of chlorophylls, their degradation products (chloropigments) and carotenoids is very sensitive to the nature of the injection solvent. The effect of sample-solvent interaction can result in the production of distorted, or even false, peaks that could be erroneously interpreted as “pigment like” or as poor chromatographic resolution. The previously suggested theoretical explantion, based on differences in solvent characteristics as expressed by the Polarity Index (P′) or even by the more precise solvent strength parameter for reversed-phase systems (S) we use, was unsatisfactory. The problem seems more complex, with other parameters such as injection volume and solute concentration or solvent selectivity also playing a role. As a practical consequence, however, suggesions are made for optimum injection conditions. Also, the presence of ion-pairing reagents in the injection solvent is demonstrated to be unnecessary.

Separation of chlorophylls and their degradation products in marine phytoplankton by reversed-phase high-performance liquid chromatography

SummaryAn RP-HPLC procedure which separates chlorophylls and their degradation products is described. By employing an amonium acetate buffered mobile phase and a linear gradient elution, complex mixtures of chloroand phaeopigments are separated in less than 30 minutes. The method has been applied to the control of chlorophyll enzymatic degradation in cultures of two algal species, and has also been successfully used in the analysis of algal xanthophylls.

The geographic mosaic in predispersal interactions and selection on Helleborus foetidus (Ranunculaceae)

We examine the hierarchical geographic structure of the interaction between a plant, Helleborus foetidus, and its floral herbivores and pollinators (interactors). Six populations from three distant regions of the Iberian Peninsula were used to examine intra‐ and inter‐regional variation in plant traits, interactors and plant fecundity, and to compare, through selection gradient and path analyses, which traits were under selection, and which interactors were responsible for differential selection. Geographic and temporal congruency in interactor‐mediated selection was further tested using a recent analytical approach based on multi‐group comparison in Structural Equation Models. Most plant traits, interactors and fecundity differed among regions but not between populations. Similarly, the identity of the traits under selection, the selection gradients (strength and/or the direction of the selection) and the path coefficients (identifying the ecological basis for selection) varied inter‐ but not intra‐regionally. Results show a selection mosaic at the broad scale and, for some traits, a link of differential selection to trait differentiation.

Losses of chlorophylls and carotenoids in aqueous acetone and methanol extracts prepared for RPHPLC analysis of pigments

SummaryRPHPLC methods for analysis of photosynthetic pigments (chlorophylls and carotenoids) usually require addition of water to methanol or acetone extracts to prevent distortion of early-eluting peaks corresponding to the more polar compounds. In this work we have investigated the short-(<2 min) and long-term (up to 48 h) effect of adding water to acetone and methanol extracts from two marine phytoplankton species,Emiliania hyxleyi andDunaliella tertiolecta. Solvent extracts were prepared and separated into fractions that were subsequently diluted with water to 90%, 80%, 70%, 60%, 50%, and 40% for methanol, and the same range extended to 30% and 20% for acetone. Changes in pigment concentration with time were followed spectrophotometrically and chromatographically. Losses of pigments as a result of precipitation were clearly observed immediately after dilution of acetone extracts to 60% or less and methanol extracts to 80% or less. For chlorophyll a the most substantial losses were recorded for 50% acetone (up to 27% decrease) and for 70% methanol (31% decrease). This effect increased considerably with time. Only for 90% and 80% acetone were the initial concentrations of all the pigments unchanged after 24h, and even up to 48 h. In contrast, more than 60% and 57% of the initial amounts of chlorophyll a were lost after 24 h in 50% acetone and 70% methanol extracts, respectively. These losses increased to 83% and 60% after 48 h. There was a clear correlation between the polarity of a pigment and the polarity of the solvent at which maximum precipitation occurred. Losses of pigment from pure acetone and methanol extracts with time were also observed, although we attribute these to pigment degradation rather than precipitation. Some of the losses occurring with time can be avoided by use of autosamplers in which the sample can be mixed with water immediately before injection.

THE MAIN NONPOLAR CHLOROPHYLL c FROM EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) IS A CHLOROPHYLL c 2-MONOGALACTOSYLDIACYLGLYCERIDE ESTER: A MASS SPECTROMETRY STUDY

The main nonpolar chlorophyll c ‐like pigment was extracted from Emiliania huxleyi (Lohm.) Hay et Mohler (strain CCMP 370) cultures and isolated by preparative column chromatography and HPLC. The pigment, whose visible spectrum closely resembled that of chlorophyll c 2, was studied by low‐resolution fast atom bombardment mass spectrometry, showing a very high mass molecular ion (m/z 1313). The fragment ions, either in the direct spectrum or obtained by tandem mass spectrometry with collision‐induced dissociation of the molecular ion, were compatible with the consecutive losses of two fatty acids (14:0 and 18:4), glycerol, and a hexose, leaving a chlorophyll c 2 backbone, suggesting the molecule consists of a chlorophyll c 2 residue linked, via an ester bond, to the sugar moiety of a monohexosyldiacylglycerol. The identities of the two fatty acid residues (14:0 and 18:4n‐3) were subsequently corroborated by gas chromatography of the corresponding methyl esters. Chemical hydrolysis–derivatization–gas chromatography–mass spectrometry demonstrated the occurrence of glycerol and that galactose is the constituent sugar. The porphyrin obtained on acid hydrolysis showed chromatographic and visible spectral properties identical to pheoporphyrin c 2. This evidence led us to propose a tentative structure whose molecular formula, C76H96O14N4Mg, was supported by the values of exact mass measurements by high‐resolution fast atom bombardment mass spectrometry. This novel structure represents the highest molecular weight natural chlorophyll described to date.

One-step conversion of fatty acids into their 2-alkenyl-4,4-dimethyloxazoline derivatives directly from total lipids

Abstract 2-Alkenyl-4,4-dimethyloxazoline derivatives of fatty acids were obtained by direct reaction of 2-amino-2-methylpropanol with oils or total lipid extracts. The yields were similar to those obtained by the formation of oxazoline derivatives from fatty acid methyl esters after transesterification of the starting lipids. The procedure is especially useful in the analysis by gas chromatography-mass spectrometry of samples rich in lipids containing α-unsaturated ethers (alkenylglycerols), as it avoids the formation of the corresponding dimethylacetals that can complicate the chromatograms and the mass spectra of the fatty acid oxazoline derivatives.

DETECTION OF NEW PIGMENTS FROM EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY, LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY , VISIBLE SPECTROSCOPY, AND FAST ATOM BOMBARDMENT MASS SPECTROMETRY

Pigment extracts from Emiliania huxleyi (Lohm.) Hay et Mohler (strains CCMP 370, CCMP 373, and NIOZ CH 24) were analyzed using high‐performance liquid chromatography (HPLC) on highly efficient monomeric and polymeric octadecylsilica columns using either ammonium acetate or pyridine containing mobile phases. Both systems showed chromatographic profiles with peaks corresponding to pigments of uncertain structure: those of the polar and nonpolar chlorophyll c forms and one peak whose on‐line diode array spectrum resembled that of the fucoxanthin acyloxy derivatives. Liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry gave a molecular mass of 786 units for the unknown carotenoid. The pigments corresponding to each of these fractions were isolated and their visible spectra recorded in various solvents. Samples of the isolated pigments were subjected to analysis by fast atom bombardment mass spectrometry that confirmed a molecular mass of 786 for the unknown carotenoid and gave a mass of 654 units for the polar chlorophyll c3, compatible with the monovinylic structure previously suggested. The detection of these new pigments calls for attention on the use of correct methodologies when HPLC pigment signatures are used to study the taxonomic composition of natural phytoplankton populations.

Changes in fatty acids, amino acids and carbon/nitrogen biomass during nitrogen starvation of ammonium- and nitrate-grown Isochrysis galbana

Growth of cells ofIsochrysis galbana with either nitrate or ammonium as the N-source, and the effects of subsequent N-starvation of these cells, were compared. During exponential N-sufficient growth nitrate-grown cells had double the fatty acid content of the ammonium-grown cells but lower concentrations of a few amino acids. Following resuspension in N-free medium the fatty acid content of the ammonium-grown cells increased to that of the nitrate-grown cells, but there was no further increase in fatty acid content on a C-biomass or cellular basis during the following 4 days for either culture. Fatty acid synthesis was continuous during N-starvation, while it occurred during the light-phase only in exponential growth. The proportion of 18:1n9 fatty acid increased from 10 to 25% total fatty acids during N-starvation. Intracellular free amino acid content decreased in a similar manner in both cultures on N-starvation, the ratio of intracellular free amino-N/cell-C falling more rapidly than overall cellular N/C. It was concluded that optimal amino acid and fatty acid content would be attained by growth in the presence of excess nitrate. Measurements of chlorophyll and carotenoid content and ofin vivo fluorescence indicated that these parameters had potential for monitoring the C and N biomass in cultures grown under relatively constant (not necessarily continuous) illumination.

Spatial genetic structure of Aquilegia taxa endemic to the island of Sardinia

BACKGROUND AND AIMS The Mediterranean Basin is one of the most important regions for the Earths plant biodiversity; however, the scarcity of studies on fine scale patterns of genetic variation in this region is striking. Here, an assessment is made of the spatial genetic structure of all known locations of the three Sardinian endemic species of Aquilegia in order to determine the relative roles of gene flow and genetic drift as underlying evolutionary forces canalizing the divergence of Sardinian Aquilegia taxa, and to see if the spatial genetic structure found fits the current taxonomic differentiation of these taxa. METHODS DNA from 89 individuals from all known locations of Aquilegia across Sardinia was analysed by means of amplified fragment length polymorphism (AFLP) markers. Both principal co-ordinates analysis (PCoA) and Bayesian clustering analyses were used to determine the spatial genetic structure irrespective of any taxonomic affiliation. Historical effects of gene flow and genetic drift were assessed by checking for the existence of isolation-by-distance patterns. KEY RESULTS STRUCTURE and PCoA analyses revealed a pattern of genetic variation geographically structured into four spatial genetic groups. No migration-drift equilibrium was detected for Aquilegia in Sardinia, when analysed either as a whole or in individual groups. The scenario approached a Case III pattern sensu Hutchinson and Templeton, which is associated with extreme isolation conditions where genetic drift has historically played a dominant role over gene flow. CONCLUSIONS The pattern of genetic variation of Sardinian taxa of Aquilegia indicates that genetic drift has been historically more influential than gene flow on population structure of Sardinian species of Aquilegia. Limited seed dispersal and divergent selection imposed by habitat conditions have been probably the main causes reinforcing post-Pleistocene geographical isolation of Aquilegia populations. The spatial genetic structure found here is not fully compatible with current taxonomic affiliations of Sardinian Aquilegia taxa. This is probably a consequence of the uncoupling between morphological and genetic patterns of differentiation frequently found in recently radiated taxa.

SPECTRAL CHARACTERIZATION OF NEW CHLOROPHYLL C PIGMENTS ISOLATED FROM EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) BY HIGH‐PERFORMANCE LIQUID CHROMATOGRAPHY1

Pigment extracts from the prymnesiophyte Emiliania huxleyi (Lohm.) Hay & Mohler (CCMP 370) were analyzed using a new chromatographic method based on the use of polymeric octadecylsilica columns, which split both the chlorophyll c3 peak and phytol‐substituted chlorophyll c‐like peak into two fractions. The pigments corresponding to each of these fractions were isolated. The spectra and chromatographic behavior suggest that chlorophyll c3 from E. huxleyi is a mixture of two different compounds whereas the splitting of the phytol‐substituted c‐like pigment could be the result of the equilibrium of two forms showing the same absorption spectrum. The method allowed, as well, the detection of magnesium 3,8‐divinylpheoporphy‐rin a, monomethyl ester in the prymnesiophytes E. huxleyi (CCMP 370) and Prymnesium parvum Carter (CCMP 708) and the chrysophyte Pelagococcus subviridis Norris (CCMP 1429).