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conceptos de pigmentos y aplicación
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Determination of phytoplynkton Determination of phytoplynkton composition and biovolumecomposition and biovolume
Utermöhl method::
Advantage: asy sampling, long storage times
Disadvantage: requires a lot of time, and specialists
Results: relative contribution of algas classes x biovolume
HOW TO DETERMINE PHYTOPLANKTON?
Silvana V. Rodrigues
HOW TO DETERMINE PHYTOPLANKTON ?
O
CH3
COO OH
O
O
peridinina
Dinoflagelados
Cryptophyta
HO
OH
aloxanthin
Clorophyta
Cyanobacterias
http: //oceancolor.gsfc.nasa.gov/.../BIOLOGY/
1.000 milhão tons produzidas por ano na terra e no mar indicator único da biomassa aquática indicator único da biomassa aquática parâmetro bioquímico mais freqüentemente medido
em oceanografia
Importance of chlorophyll a
struggle.net/history/images/molecule.jpgwww.molecularexpressions.com
fig.cox.miami.edu/.../phts/c8.10x21.overview.jpg
CloroplastoCloroplasto
chlorophyll a:
light absorption (“Light harvesting complexes”)
electron donor and acceptor in reative centers
Carotenoids:
Light absorption
Protection of chlorophyll (“quenching “ of Chl photoinduced triplet
state ) and quenching of O2 singlet state .
Function of pigments in photosynthetic organisms
divisão/classedivisão/classe nome comumnome comum gêngên espéc.espéc.Algas marrons(clorofilas a e c)Algas marrons(clorofilas a e c)
Bacillariophyta diatomáceas 210 Desconh.
Dinophyta dinoflagelados 550 4000
Crysophyta:ChrysophyceaeRapidophyceae
flagelados marrom-amarel.Crysophytas,silicoflageladosraphydophytas (cloromonadas)
1204
10009
HaptophytaPrimnesiophyceae
flagelados marrom-amarel.cocolitoforídeos 50 500
Xantophyta algas verde-amareladas 50 600
Cryptophyta criptomonadas 8 >50
Eustigmatophyta algas amarelo-esverdeadas 6 12
Algas verdes (clorofilas a e b)Algas verdes (clorofilas a e b)
ChlorophytaClorophyceaePrasinophyceaeEuglenophyta
Algas verdesFlagelados verdesEuglenoides
3501343
2500120650-800
Algas vermelhas (clorofila a e biliproteínas)Algas vermelhas (clorofila a e biliproteínas)
Rhodophyta Algas vermelhas 3 10
Algas azuis (Cyanobacteria) ( clorofila a e biliproteínas)Algas azuis (Cyanobacteria) ( clorofila a e biliproteínas)
CyanophytaProchlorophyta
Cianobactériasproclorofitas
Characteristics which make it possible to use algal pigments (chlorophylls, carotenoids and phycobiliproteins) as chemotaxonomicmarkers
They are present in all photosynthetic algae, but absent in most bacteria, protozoa and detritus
Many occur only in specific classes or even genera, allowing the determination of phytoplankton taxonomic composition at least at class level, or better
They are strongly coloured, and in the case of chlorophylls and phycobiliproteinsare fluorescent, what allows their detection with high sensitivity,
Most of them are labile and esily dgraded after cell death, allowing todistinguish living from dead cells
Use of pigment chemotaxonomy for recognition, in field samples, of phytoplanktonic classes not detected since then, because of preservation problems or filtration losses.
»alloxanthin (Cryptophyta)
»chlor b (Chlorophyta and Prasinophyta)
»zeaxanthin (Cyanobacteria)
»19’-hexanoiloxifucoxanthin (Prymnesiophyta)
»divynil-chlorophyill a (Proclorophyta)
1952: chlorophyll was recognized as a selective phytoplankton marker, in the presence of other biological components (zooplankton, bacteria, detritus)
1984-1987: HPLC methods for the determination of chls, carotenoids and phytoplankton degradation products
Hystorical overview
Mg coordination complexes with cyclic tetra-pyrrolsMg coordination complexes with cyclic tetra-pyrrols
Macrocicles with five member ringsMacrocicles with five member rings
Chlorophylls:Chlorophylls:13132 2 -Metilcarboxilates of --Metilcarboxilates of -
Mg-phytoporphyrin (double bond in D ring): Mg-phytoporphyrin (double bond in D ring): Cl c,Cl c, Mg-phytoclhorin: Mg-phytoclhorin: Cl a, Cl bCl a, Cl b
Phytil at C-17Phytil at C-173 3 (Cl a and b) (Cl a and b)
Propionic acid at C17: Cl a and bPropionic acid at C17: Cl a and bAcrílic acid at C17: Cl cAcrílic acid at C17: Cl c
Chlorophylls:Chlorophylls:13132 2 -Metilcarboxilates of --Metilcarboxilates of -
Mg-phytoporphyrin (double bond in D ring): Mg-phytoporphyrin (double bond in D ring): Cl c,Cl c, Mg-phytoclhorin: Mg-phytoclhorin: Cl a, Cl bCl a, Cl b
Phytil at C-17Phytil at C-173 3 (Cl a and b) (Cl a and b)
Propionic acid at C17: Cl a and bPropionic acid at C17: Cl a and bAcrílic acid at C17: Cl cAcrílic acid at C17: Cl c
Oxo substituent at C-13Oxo substituent at C-1311
methyl-carboxilate groups at C-13methyl-carboxilate groups at C-132 2 --
N N
CH2
CH3
CH3CH3
N
H
O
N
O
CH3
H
COOCH 3
Mg
H
CH3
O
CH3 CH3H
CH3H
CH3
CH3
N N
CH2
CH3
CH3CH2
N
H
O
N
O
CH3
H
COOCH3
MgH
CH3
O
CH3 CH3H
CH3H
CH3
CH3
N N
CH2
CH3
CH3
N
H
O
N
O
CH3
H
COOCH3
MgH
CH3
O
CH3 CH3H
CH3H
CH3
CH3
OH
N N
CH2
CH3
CH2
N
H
O
N
O
CH3
H
COOCH 3
Mg
H
CH3
O
CH3 CH3H
CH3H
CH3
CH3
OH
chlorophyll a
DV-chlorophyll a
chlorophyll b
DV-chlorophyll b
Molecule drawings:N. Montoya
N NCH3
N
O
N
O
CH3
HCOOCH 3
Mg
CH3
OH
CH2 CH3 CH3
N NCH3
N
O
N
O
CH3
HCOOCH 3
Mg
CH3
OH
CH2 CH3 CH2
N NCH3
N N
O
CH3
HCOOCH 3
Mg
CH3
OH
CH2
CH2
COOCH 3
chlorophyll c1
chlorophyll c2 chlorophyll c3
Molecule drawings:N. Montoya
N N
CH2
CH3
CH3CH3
N
H
O
N
O
CH3
H
COOCH 3
Mg
H
CH3
O
CH3 CH3H
CH3H
CH3
CH3
Loss of metal
Chla Phaeophitin
in organic solvents
In dilute acids
under high intensity of light
Degradation by chemical processes:
Molecules become chemically and fotochemically
more labile in organic solvents
than in the cells
N N
CH2
CH3
CH3CH3
N
H
O
N
O
CH3
H
COOCH 3
Mg
H
CH3
O
CH3 CH3H
CH3H
CH3
CH3•Chl a 132 Hydroxiclhorophyll a•Chl a Cl a - Hyidroxilactone.
Allomerization (oxidation by O2):
In alcoholic or hydro-alcoholic solutions Specially in pH >7
Epimerization
(HPLC: in SiO2):
Cl enolate Cla’, b’
Both processes can be minimized
by decreasing the temperature
Degradation by chemical processes:
N N
CH2
CH3
CH3CH3
N
H
O
N
O
CH3
H
COOCH 3
Mg
H
CH3
O
CH3 CH3H
CH3H
CH3
CH3Loss of phytil group
Cl chlorophyillide
In methanol or ethanol in basic medium
Degradation by chemical processes:
N N
CH2
CH3
CH3CH3
N
H
O
N
O
CH3
H
COOCH 3
Mg
H
CH3
O
CH3 CH3H
CH3H
CH3
CH3
Biodegradation:
To cyclic tetra-pirrols
perifercally modified (enzymatically,
Specially in the absence of
light and O2):
Hydrolisis of the phytil
ester (chlorophyllase)
chlorophillide formation
Decarboximetilation Formation ofpirophaeophytins e pirophaeophorbides
Allomerization
Epimerization (Chl-oxidase)
Loss of metal:Mg-dequelataseFormation of phaeophytins
N N
CH2
CH3
CH3CH3
N
H
O
N
O
CH3
H
COOCH 3
Mg
H
CH3
O
CH3 CH3H
CH3H
CH3
CH3
Biodegradation:
To linear tetrapirrols
Normally by oxidative opening
of the macrocycle ring, between
C-4 and C-5,
C-5 stays as an aldehyde
45
Carotenoids
β- β- carotene
Derive from carotene:
-carotene: ,-carotene-carotene: ,-carotene-carotene: ,-carotene-carotene: ,-carotenelycopene: ,-carotene
Polyen:Absorbtion
of light.COLOUR
C40H56
Isoprenoidunits
Properties
More stable in phytoplankton and in plants than chlorophylls: they don‘t
have N, so can‘t be used in enzymatic amino-acid building.
Example:
Leaves lose the green colour in autumn (chlorophyll),But don‘t lose colours due to carotenoids
Polyene chain is responsible for instability:
Oxidation by air or peroxides
Electrophyle addition ( H+ and Lewis acids)
Isomerization E/Z caused by heat, light or chemicals,
Undergo reactions at the ends of the molecules
Production of artefacts
Phytoene
Lycopene
Dessaturation
, -carotene , -carotene
Ciclization
Zeaxanthin
Hydroxilation
lutein
Hydroxilation
Anteraxanthin
Violaxanthin
Neoxanthin
Epoxidation
Epoxidation
Deepoxidation
Deepoxidation
Rearrangement
VIOLAXANTHIN CICLE
Acetil-CoA Geranylgeranyldiphosphate
Biosynthesis:occurs in thylakoid
membranes
Can occur in the darkDepends a lot on light
Light
Light Dark
Dark
Geranylgeranyldiphosphate
DIADINOXANTHIN CICLE
Diadinoxantin
Diatoxanthin
LIGHTDARK + 2H - H2O+ 2H + O2 - H2Oepoxidation
Carotenoids
β- β- carotene
C40H56
Enzimatic hydroxilation
Epoxidation
Carboxi (CO2H), carbometoxi (CO2Me)ou metoxi (OMe)
Hydroxi-carotenoidsas fatty acid esters,or asGlycosides or glycosylesters, others as sulphates
Acetates (OCOMe)e lactones
Aldehydes,ketones
Xantophylls
Isoprenoids
Zeaxanthin
Lutein
isomers
Acetilenic
Alenic
fucoxanthin
Norcarotenoids
( skeleton C37)
Peridinin
C39H50O7
Diatoxanthin
In acid mediumEpoxides rearrange (5,6 to 5,8 form)
5
7
68
58
6
7
violaxanthin
neoxanthin
In basic medium:
In general stable
exception: esters are hydrolysedsome compounds suffer structural change (fucoxanthin, peridinin)
fucoxanthin
Division or class
/
Pigment
Cyan
op
hyta
Pro
chlo
rop
hyta
Rh
od
op
hyta
Cry
pto
ph
yta
Ch
loro
ph
yceae
Prasin
op
hyc
eae
Eu
glen
op
hy
ta
Eu
stimato
ph
yta
Bacillario
ph
yta
Din
op
hyta
Prym
nesio
ph
yceae
Ch
rysop
hycea
e
Rap
hid
op
hycea
e
Chl a
Chlb
Chl c1
Chl c2
Chl c3
Tipo pyhtilat. Chlc
MgDVP
DVchla
DVChlb
Distribution of chlorophylls among divisions/classes of phytoplankton
Division or class
/
Pigment
Cyan
op
hyta
Pro
chlo
rop
hyta
Rh
od
op
hyta
Cry
pto
ph
yta
Ch
loro
ph
yceae
Prasin
op
hyc
eae
Eu
glen
op
hy
ta
Eu
stimato
ph
yta
Bacillario
ph
yta
Din
op
hyta
Prym
nesio
ph
yceae
Ch
rysop
hycea
e
Rap
hid
op
hycea
e
,
,
,
,
,
Distribution of carotenes among divisions/classes of phytoplankton
Division or class
/
Pigment
Cyan
op
hyta
Pro
chlo
rop
hyta
Rh
od
op
hyta
Cry
pto
ph
yta
Ch
loro
ph
yceae
Prasin
op
hyc
eae
Eu
glen
op
hy
ta
Eu
stimato
ph
yta
Bacillario
ph
yta
Din
op
hyta
Prym
nesio
ph
yceae C
hryso
ph
yceae
Rap
hid
op
hycea
e
Aloxanthin
Anteraxanthin
Astaxanthin 2 2 2
19‘-Butanoil-
fucoxanthin
Cantaxanthin 2
Crocoxanthin
Diadinoxanthin
Diatoxanthin
Dinoxanthin
Echinenona 2 2
Fucoxanthin 1
Distribution of xantophylls among divisions/classes of phytoplankton
Division or class
/
Pigment
Cyan
op
hyta
Pro
chlo
rop
hyta
Rh
od
op
hyta
Cry
pto
ph
yta
Ch
loro
ph
yceae
Prasin
op
hyc
eae
Eu
glen
op
hy
ta
Eu
stimato
ph
yta
Bacillario
ph
yta
Din
op
hyta
Prym
nesio
p.
Ch
rysop
hycea
e
Rap
hid
op
hycea
e
19‘hexanoilfuco 1
Luteína
Monadoxanthin
Neoxanthin
P457+P468
Peridinina
Peridininol
Prasinoxanthin
Pirroxanthin
Sifonaxanthin 14 14
Sifoneina
Ést. Vaucheriax
Violaxanthin
Zeaxanthin
Distribution of xantophylls among divisions/classes of phytoplankton
Amphidinium carterae (Dinophyta)
peridinin
dinoxanthin
diadinoxanthin
chlorophyll c2
Rz =[peridinin]/[chlorophyll a]Rz =[peridinin]/[chlorophyll a]RzRzii =[lpigm =[lpigmii]/[chlorophyll a]]/[chlorophyll a]
chlorophyll a
neoxanthin
chlorophyll a
chlorophyll b
luteinviolaxanthinanteraxanthin
Dunaliella tertiolecta (Chlorophyta)
Rz =[lutein]/[chlorophyll a]Rz =[lutein]/[chlorophyll a]RzRzii =[lpigm =[lpigmii]/[chlorophyll a]]/[chlorophyll a]
Pigment Significance Chl a: an index of total algal biomass, excluding
prochlorophytes.
Unambiguous markers for algal types DV-Chl a: an index of prochlorophyte biomass DV-Chl b: unambiguous marker for prochlorophytes Siphonaxanthin esters: unambiguous marker for Type 2 prasinophytes
(Egeland et al., 1997) Prasinoxanthin: unambiguous marker for Type 3 prasinophytes Peridinin: Type 1 dinoflagellates Alloxanthin: Cryptophytes Gyroxanthin diester: Dinoflagellates Type 2 Chl c2 MGDG [14:0/14:0]: Chrysochromulina spp. (Haptophyte Type 7, Zapata
et al., 2004)
S. Wright, Class notes
Hierarchical guide to the use of pigments
Peak no.
Rt Pigment identification
Observed λmax
Published λmax
Reference
(min)
(nm) (nm)
1 11.4 Erythroxanthin sulfate
465 469 Takaichi et al. (1991)
2 18.4 Bacteriorubixanthinal
513 510 Takaichi et al. (1988)
3 19.1 Zeaxanthin (428), 454, 482
(428), 454, 481
Jeffrey et al. (1997)
4 20.4 Bacteriochlorophyll a
359, 580, 771
358, 577, 773
Scheer (1991)
5 23.4 Bacteriophaeophytin a
358, 525, 750
357, 525, 749
Scheer (1991)
6 25.4 β,β-carotene (426), 454, 478
(426), 454, 480
Jeffrey et al. (1997)
Retention times and mean absorption properties (inHPLC eluant) of the major pigments detected in Erythrobacter longus (ATCC 33941) and isolates NAP1, MG3, and NJ3Y. Peak numbers correspond to those indicated in Fig. 5. Solvents and caroteneid band ratios from the literature data: 1 solvent=methanol+ water (4:1) containing 40mM NH4OH, %(III/II)=0; 2 solvent= methanol, %(III/II)=0; 3 solvent=acetone, %(III/II)=33; 4, 5 solvent=diethyl ether; 6 solvent=acetone, %(III/II)=21
Michal Kobližek Arch Microbiol (2003) 180 : 327–338
Reverse-phase HPLCchromatograms (360 nm) foracetone extracts prepared fromwhole cell pellets of a Erythrobacterlongus ATCC 33941,b NAP1, c MG3, and d NJ3Y.Peak identities: 1 erythroxanthinsulfate, 2 bacteriorubixanthinal,3 zeaxanthin, 4 bacteriochlorophylla, 5 bacteriophaeophytina, and 6 β,β-carotene
Michal Kobližek Arch Microbiol (2003) 180 : 327–338
HPLC chromatogram of fuorescent pigments from a surface sample(2 m depth) collected at station C354-004. Excitation was at 365 nm,emission at 780 nm, with 20-nm slits. These wavelengths were chosen tomaximize the signal from BChla, while minimizing the signal from the moreabundant pigments, Chla and Chlb. (Inset) Fluorescence emission spectrum ofthe peak eluting at 16.7 min in (A). Excitation was at 365 nm and slits were20 nm.
Zbigniew S. Kolber et al, Science 292, 2492-2495; 2001.
PIGMENTS IN SEDIMENTSPIGMENTS IN SEDIMENTS
Pigmentos Pigmentos Em geral são moléculas lábeis, atingem o sedimento em vários estágios de degradação.
Na água: rápida e extensa(≤95 % dos compostos em poucos dias) • digestão por herbívoros,• enzimática, na senescência celular• oxidação química, microbiológica e pela luz.
Nos sedimentos: taxa de degradação menor, especialmente em condições anóxicas. Depende de:• intensidade de luz e da• bioturvação invertebrada
Degradação dos pigmentos originais Degradação dos pigmentos originais
principalmente na água e na superfície do sedimento, durante a deposição (Hodgson et al., 1997)
Fatores que afetamFatores que afetam a taxa a taxa
de degradação:de degradação:
• Tempo para chegarTempo para chegar ao fundoao fundo
• Tipo de pigmentoTipo de pigmento
• Grau de ataque Grau de ataque químico e biológicoquímico e biológico
Separation and quantification of pigments in sedimentsSeparation and quantification of pigments in sediments
More complex than in phytoplankton samples, due to the variety of degradation or transformation products (Mendes et al. 2007) .
DEGRADATIN PRODUCTS: DEGRADATIN PRODUCTS:
• degradation to uncoloured compounds• conversion to cis-carotenoids and phaeopigments more difficult to identify (Steenbergen et al., 1994 apud Hodgson et al., 1997).
Chl a‘ and phaeophytin:Chl a‘ and phaeophytin:degradação products due todegradação products due toEnvironmental stressEnvironmental stress
Pirophaeophitins and steril Pirophaeophitins and steril Chlorins: degradationChlorins: degradationproducts due to zooplanktonproducts due to zooplankton
Phaeophorbides:Phaeophorbides:Degradation products due Degradation products due to zooplanktonto zooplankton
Jeffrey, 1997 apud Kowalewska et al., 2004).
Kowalewska et al., 2004.
Chlorophyll b: occurs mainly ingreen algae and vascular plants, Chlorophylls c: in diatoms, dinophlagellates and some brown algae
Chlorophylls :Chlorophylls :
More labile than carotenoids , but phaephitins are persistent in sedimentary records
Carotenoids:Carotenoids:
Stability depends on structure (decreases with the increase of the number of functional gruoups).
Fossile Pigments:Fossile Pigments:
Used in paleoclimatic and paleoenvironmental issues
Pigmento Grupos Funcionais
Afinidade taxonômica
b,b-caroteno 0 Cianobactérias, algas eucarióticas e plantas vasculares
b,e-caroteno 0 Criptofitas
Aloxantina 2 Cryptofitas
Luteina 2 Clorófitas
Neoxantina 4 Clorófitas
Violaxantina 4 Chrisofitas e Clorófitas
Fucoxantina 5 Chrisofitas e Diatomáceas
Diatoxantina 2 Diatomáceas
Diadinoxantina 3 Dinoflagelados, Crisofitas e Diatomáceas
Peridinina 6 Dinoflagelados
Dinoxantina 4 Dinoflagelados
Zeaxantina 2 Cianobactérias, Clorófitas
Myxoxantofila 3 Cianobactérias
Echinenona 1 Cianobactérias e zooplâncton (Cladocera)
Cantaxantina 2 Cianobactérias e zooplâncton (Cladocera)
Astaxantina 4 Zooplâncton (Crustacea)
Okenona 2 Bactérias fotossintéticas (Chromatiaceae)
Scytonemina-1, -2 4 Organismos fotossintéticos expostos a alta radiação UV
(adaptado de Buchaca & Catalan 2008)
Carotenoids:Carotenoids:
Estáveis, abundantes
Pigmento Afinidades taxonômicas
Bacteriofeofitina-a Bactérias fotossintéticas (Rodospirillaceae e Chromatiaceae)
Bacterioclorofila-e Bactérias fotossintéticas (variedades marrons de Chlorobiaceae)
Clorofila-a Razão molar Cl-Cl-aa/forbinas /forbinas aa como indicador de preservação
Chlorofilídeo-a Produto de degradação da Cl-a, abundante em Diatomáceas
Cl-a (alômero) Produto de degradação da Cl-a
Cl-a (epímero) Produto de degradação da Cl-a
Feofitina-a1, -a2 Produto de degradação da Cl-a (senescência)
Feoforbídeo-a1, -a2, Produto de degradação da Cl-a („grazing“)
-a3, -a30, -a4
Clorofila-b Clorófitas
Feofitina-b1, -b2 Produto de degradação da Cl-b
Clorofila-c1 Crisofitas e Diatomáceas
Clorofila-c2 Crisofitas, Diatomáceas, Criptofitas e Dinoflagelados
Clorofila-c3 Crisofitas e Diatomáceas
(adaptado de Buchaca & Catalan 2008)
Chlorophylls :Chlorophylls :
UV/VIS absorption of pigments
Phaeophytin a
Pirophaephytin a
Chlorophyll a
Phaephorbide a
Chlorophylls
Jeffrey et al.;1997
- Mg
- Mg, -COOMe- Mg - Phytil
UV7VIS: Electronic transitions
Polyene chain: chromophore
Maintransition
Vibrationalfine structure
00IIIII
Calculation of % III/II for a caroteneidCalculation of % III/II for a caroteneid
Vibrationalfine structure
Molecular structure x spectroscopic properties
Chromophore (polyene chain): Lenght
carotenoid Conjug. db. bonds
max (hexane)
phytoene 3 276 286 297
-carotene 7 378 400 425
lycopene 11 444 470 502
Molecular structure x spectroscopic properties
Geometrical cis-trans isomers: small hypsochromic effectSignificant hypochromic effectReduction of vibrational fine structureAppearance of a cis-peak (≈ 142 nm below the longest maximum of the all-rans,measurd in hexane
Beta-Rings: fine structure much reduced, max shorter than in the acyclic
Acetylenic groups: replacement of d.bond to triple bond - 15-20 nm shorter wavelength
Allenic groups
Carbonyl groupsBritton, 1995, Carotenoids,3 vol, Birkhäuser
Molecular environment x spectroscopic properties
Solvent Approx. bathochromic shift1
Hexane, light petroleum, ethanol, diethylether, acetonitrile
0
acetone 2-6
chloroform 10-20
dichlorometane 10-20
benzene 18-24
toluene 18-24
pyridine 18-24
Carbon disulphide 18-24
1: displacement of max to longer wavelength
Identification of pigments by Mass Spectrometry
HPLC method with improved resolution, LC–MS analysis and the automated acquisition of MS/MS data for pigments
extracts from a sediment (Priest Pot, Cumbria, UK),
a microbial mat (les Salines de la Trinital, South Catalonia, Spain)
a culture (C. phaeobacteroides):
SEPARATION OF A GREAT NUMBER OF PIGMENTS, INCLUDING NOVEL BACTERIOCHLOROPHYLL DERIVATIVES.
Airs, 2001
QuickTime™ and a decompressor
are needed to see this picture.
Airs, 2001
More than 60 pigments during the run:
QuickTime™ and a decompressor
are needed to see this picture.
QuickTime™ and a decompressor
are needed to see this picture.
Frassanito 2005
HPLC coupled both to UV photodiode array detection and to atmospheric pressure mass spectrometric techniques (HPLC–DAD-APIMS)
Pigments ( chlorophylls, carotenoid), galactolipids, alkaloids, sterols and mycosporine-like amino acids,
QuickTime™ and a decompressor
are needed to see this picture.
Extraction and separation
of pigments
Chemotaxonomic estimation of phytoplankton communities in aquatic and
sedimentary environments involves not only the choice of marker
pigments, but also efficient extraction and separation procedures and a
reasonable treatment of the data obtained.
Extraction must be quantitative for all pigments
HPLC separation must be able to: separate simultaneously groups of molecules of very different polarities
Resolve very similar compounds, for instance isomers
Extraction of phytoplankton pigmentsExtraction of phytoplankton pigments
Solvents: Acetone 90 %
Acetone 100 %
Methanol
Acetone :Methanol ( 1:1)
N,N-dimetilformamide (DMF)
Buffered Methanol ( 2% NH4Ac 0,5 M)
Procedure: Sonication or criogenic homogenization„overnight“ or immediate extraction
Filtration
GF/F 47mm
Extration:
Methanol: NH4Ac 0,5M (98:2) +
Sonification, ice-bath (30 s) +
Centrifugation (5 min, 4800 rpm)
Separation (HPLC)
Chromatographic separation of
Phytoplankton pigments
Fase estacionária: C30 (YMC, C30, 5µm, polimérica250x4,6 mm ID
Fase móvel: A:CH3OH:TBA (28 mM) 70:30 (v/v)B: CH3CH2OHpH 6,5Gradiente: chlorophylls:Fig A:30-100 % B, 50 minVazão: 1,2 ml/minT: 47 oCCarotenóides:Fig B:25-63 % B, 35 min, 63-100%B/13 minVazão: 1,4 ml/minT: oC
Separation with C30 columns:Separation with C30 columns: Development of a computer-assisted method (Software Dry Lab)
Mistura-testeVan Heukelem e Thomas, Journal of Chromatography A, 910 (2001) 31-49
Resolution: otimization
for chlorophyllsAnd for carotenoids in
Sparate runs
Resolution: separation mono/divynil clh a, bThey don‘t separate in C18 !! (depends on aliphatic chain?)
c3
c1c2
DV, MV cl bDV, MV cl a
alox
anth
indi
atox
anth
in
lute
ína
alo-, diato-xanthinse luteína
Separation with C8 columnsSeparation with C8 columns:
Fase estacionária: C8 (Eclipse XDB, 3,5 µm150x4,6 mm ID
Fase móvel: A:CH3OH:TBAA (28 mM) 70:30 (v/v), pH 6,5B: CH3OH
Mistura-teste.Van Heukelem e Thomas, Journal of Chromatography A, 910 (2001) 31-49
c3
C2
+M
gDV
P
c 1 +
clor
ofilí
deo
a
DV, MV cl b
DV, MV cl aZeaxanthin, luteína,
2) Zapata et al., 2000 Mar. Ecol Progr. Ser. 195: 29-45, 2000
Fase móvel: A: CH3OH : CH3CN : pirid.acet. (50:25:25); B: CH3OH : CH3CN : acetona (20:60:20)
1) Development of a computer-assisted method (Software Dry Lab)
MgDVP
Clor c2
Zeaxanthin, dihidroluteína
C8, Zapata
C8, Van Heukelem
Pigment mixture, S. Wright, Course Notes
R>1
R< 0,5
R=0,8
R=1
cl b/DV cl bR< 0,5
cl b/DV cl bR= 0,8
4k Hex/9‘cis NeoR> 1,25
4k Hex/9‘cis NeoNão resolve
C8: better for chlorophyll c family
Fases estacionárias: C8 (Symmetry C8, 3,5 µm 150 x 4,6 mm)C18 (Supelcosil L-C18, 5 µM250 x 4,6 mm)
Fase móvel: Coluna C18: adap. Kraay, 1992A:CH3OH:H2O (85:15) B: CH3CN.H2O (90:10)C: Acet. Etila(vazão 0,6 ml/min)
Coluna C8: Zapata, 2000
Comparison of method sensitivity with C18 and C8 columnsComparison of method sensitivity with C18 and C8 columns
Mendes et al., Limnol. Oceanogr. Methods 5, 2007, 363-370
C18: More sensitivityLower limit of detection
Better for low concentration pigments
Fase estacionária:2 colunas „in line“Waters Spherisorb ODS23 µM150 x 4,6 mm)
Fase móvel: A: NH4Ac 0,01M B: CH3OHC: CH3CND: Acet. Etila
Gradiente:5%A, 85% B, 15 % C isocr.5 min,0%A, 20% B,15%C,65% D,95 min, 0%A, 1%B, 1%C, 98%D, 5 min,isocr. 5 min
Adequado para LC/MS
Método SCOR 1997
Método Airs et Al.
Extrato de amostra de sedimento (Priest Pot) Airs et al.; Journal of Chromatography a 917 (2001) 167-177
Separation of complex samples, methodSeparation of complex samples, methodcompatible with LC/MScompatible with LC/MS
Labor. UFF, Cromatógrafo Bischoffanalysentechn., Mistura-teste (DHI), 100µL injetados na fase A,
Cl
c3C
l c2
peri
dini
na19
,-bu
tan
oilfu
cofu
coxa
nthi
nne
oxa
nth
inpr
asin
oxa
nth
in
diad
inox
anth
in
viol
axan
thin
diat
oxan
thin
dino
xant
hin
lute
ina
alox
anth
in
zeax
anth
in
Cl b
+ D
V c
lb
Cl a
+ D
V c
la
Fase estacionária:Spherisorb ODS1/ C18 250 x 4,6 mm – 5 m
Fase móvel: A: CH3OH 0,3 M em NH4Ac : ACN : H20 (51:36:13) B: AcetEtila: ACN (70:30)Vazão: 1,2 ml/min
Gradiente:0 a 25 % B em5 min, isocr.5 min,25% a 100% Bem 20 min.
Separates:,-carotene, ,-carotene, Aloxanthin,Lutein, Neoxanthin, Violaxanthin, Fucoxanthin, Diatoxanthin,Diadinoxanthin, Peridinina,Dinoxanthin, Zeaxanthin, Mixoxantophyll, Equinenone, Cantaxanthin,Astaxanthin, Okenone, Scytonemin-1, -2, Bacteriophaeophytin-a,Bacteriochlorophyll-e, chlorophyll-a, Chlorophilide-a, Chl-a Allomer and Epimer, phaeophytin- a1, a2, phaeophorbide -a1, -a2, -a3, -a3’, -a4, chlorophyll b,phaeophytin -b1, -b2, chlorophyll –c1, -c2, -c3
Buchaca e Catalan (2008)
HOW TO DETERMINE PHYTOPLANKTON ?
ESTIMATION OF THE ABUNDANCE OF PHYTOPLANKTONIC
COMMUNITY BY PIGMENT MARKERS
Calculation ofCalculation of (Chl a)(Chl a)cn cn ??
Based on the contribution, in terms of Based on the contribution, in terms of
chlorophyll chlorophyll aa, ,
of each group of taxonomical class (Chl a)of each group of taxonomical class (Chl a)cc
to total chlorophyll a in the sample (Chl to total chlorophyll a in the sample (Chl
a)a)tt : :
(Chl a)(Chl a)t t = (Chl a)= (Chl a)c1c1 + (Chl a) + (Chl a)c2c2 + (Chl a) + (Chl a)c3c3 + + ...... + (Chl a)...... + (Chl a)cncn
Easy !
Calculation ofCalculation of (Chl a)(Chl a)c c by the choice of one marker pigmentby the choice of one marker pigment
for each classfor each class
Class Marker pigment (Pm)
Pm/Cla ratio in the class
Cianobactérias zeaxanthin Rzea/cla
Clorophyta luteínaRlut/cla
Dinophyta peridininaRper/cla
Cryptophyta aloxanthinRalo/cla
.......................... ....................... .......................
Bacyllariophyta fucoxanthinRfuco/cla
(Chl a)(Chl a)t t = = RRzea/cla zea/cla x (Zea) x (Zea) + + RRlut/cla lut/cla x (Lut) + x (Lut) + ......... .........
+ + RRfuco fuco x (Fuco)x (Fuco)
METHOD 1:
{
(Chl a)(Chl a)c c andand% of each class% of each class
Fixed sample
Problem:Problem:Fixed RFixed R
not necessarilynot necessarilyCorresponds Corresponds To the ratiosTo the ratios
In the samplesIn the samples
METHOD 2:
Multilinear regressionMultilinear regression
Sample 1: (Chl a)Sample 1: (Chl a)t1 t1 = = RRzea/cla zea/cla x (Zea)x (Zea)11 + + RRlut/cla lut/cla x (Lut)x (Lut)11
+ + .........+ .........+ RRfuco fuco x (Fuco)x (Fuco)11
Sample 2: (Chl a)Sample 2: (Chl a)t2 t2 = = RRzea/cla zea/cla x (Zea)x (Zea)22 + + RRlut/cla lut/cla x (Lut)x (Lut)22
+ + .........+ .........+ RRfuco fuco x (Fuco)x (Fuco)22
..........................................................................................................................................
..............................................................................................................................Sample n: (Chl a)Sample n: (Chl a)tn tn = = RRzea/cla zea/cla x (Zea)x (Zea)nn + + RRlut/cla lut/cla x (Lut)x (Lut)nn
+ + .........+ .........+ RRfuco fuco x (Fuco)x (Fuco)nnUnknown Rs, determined by pela resolution of a system of n equations and n unknowns
(Chl a)(Chl a)cncn
% of ech class% of ech class
Rs are determined, but many classes don‘t have a specific pigment
„„Software „CHEMTAX: problema de análise fatorial:Software „CHEMTAX: problema de análise fatorial:
matriz de dados S: concentrações encontradas para os pigmentos
no ambiente num conjunto de amostras
fatorizada em matrizes
F : matriz das razões dos pigmentos para as diferentes classes
de algas puras e
C : abundâncias de cada classe de alga em cada amostra
MÉTODO 3:
Determinação da composição fitoplanctônica por análise fatorialDeterminação da composição fitoplanctônica por análise fatorial((MACKEY et al., 1996) MACKEY et al., 1996)
Amostra 1: (Chl a)Amostra 1: (Chl a)t1 t1 (Zea)(Zea)11 (Lut) (Lut)11 ....... ....... (Fuco)(Fuco)11
Amostra 2: (Chl a)Amostra 2: (Chl a)t2t2 (Zea)(Zea)22 (Lut) (Lut)22 ....... ....... (Fuco) (Fuco)22
.................. ............ ......... ........ ......................... ............ ......... ........ .......Amostra n: (Chl a)Amostra n: (Chl a)tn tn (Zea)(Zea)nn (Lut) (Lut) ....... ....... (Fuco) (Fuco)nn
PER BUT FUC HEX NEO PRA VI0L ALO LUT ZEA CLB CLA
Prasinophyta 0 0 0 0 0,061 0,127 0 0,004 0 0 0,381 0,403
Dinophyta 0,515 0 0 0 0 0 0 0 0 0 0 0,485
Cryptophyta 0 0 0 0 0 0 0 0,186 0 0 0 0,814
Haptophyta3 0 0 0 0,630 0 0 0 0 0 0 0 0,370
Haptophyta4 0 0,104 0,247 0,227 0 0 0 0 0 0 0 0,422
Chorophyta 0 0 0 0 0,040 0 0,035 0 0,127 0,006 0,165 0,628
Synecho. 0 0 0 0 0 0 0 0 0 0,258 0 0,742
Diatomaceas 0 0 0,430 0 0 0 00 0 0 0 0,570
MATRIZ F: Razões RRii =[lpigm =[lpigmii]/[chlorophyll a para cada classe]/[chlorophyll a para cada classe
MATRIZ S: experimental
Clpras
ClDin
ClCryp
ClHapt3
ClHapt4
ClChlor
ClSyn
ClDiatom
C: contribuição de cada classe (a ser determinada)
F x C = SF x C = S
Para uma fatorizaPara uma fatorizaçção de S que tenha um significado fão de S que tenha um significado fíísico:sico:F : variável, Fo: dados da literatura (normalizados/Cl a)
Estimativa inicial da matriz de abundâncias das classes (CEstimativa inicial da matriz de abundâncias das classes (Coo):): calculada resolvendo-se a equação de mínimos quadrados:
Um algoritmo de Um algoritmo de „„decrdecrééscimo mscimo mááximoximo““ do res do resííduo foi usadoduo foi usado(varia(variaçção dos elementos de F, 10% a cada iteraão dos elementos de F, 10% a cada iteraçção)ão)
minimizar: S – Co Fo ,
sob as condições: [Co]ij 0 i, j [Co]ij = 1 j
O resíduo é expresso por: o = S – Co Fo
Juturnaíba reservoir as a study model
Rio de JaneiroState
42°
23°
Marcelo Marinho e Silvana V. Rodrigues
QuickTime™ and a decompressor
are needed to see this picture.
Avaliar a aplicabilidade do método de análise de pigmentos por HPLC
para detecção das variações na biomassa e composição do fitoplâncton,
comparando com os dados obtidos por microscopia
OBJETIVOSOBJETIVOS
Fitoplâncton–Coletas quinzenais - jun/96 - mai/97 (estação central)
–Biovolume
• método de sedimentação (Utermöhl, 1958)
Pigmentos
METODOLOGIA
Amostra(0,25 - 1,8 L)
• Filtração (GF/C)• Congelamento
(CO2 sólido)
ExtraçãoMetanol 100%
Injeção e análiseHPLC
• Coluna C18 - fase reversa
• Gradiente alta pressão (modificado de Garrido & Zapata, 1993)
• Detecção - 440nm
CONDIÇÕES CROMATOGRÁFICAS
-1
Contribution calculated by marker pigments-1
Razão Xan/Chl-a CHEMTAX
Biomass (chlorophyll a)
BiovolumeBiovolume
0,2 L +Lugol’s solution
sedimentation method (Utermöhl, 1958)
biomass: product of population and mean unit
volume of each species
(specific density of cells = 1 g/cm3,
cell size = mean of at least 30 measurements)
20 mg/LMicrocystis aeruginosa
Anabaena spiroides
Cylindrospermopsis raciborskii
Jun Jul Aug Sep Oct Nov Nov Dec Jan Feb Mar Apr May1996 1997
0
30
60
90mg/L
others
green algae
dinoflagellates
cryptomonads
diatoms
cyanobacteria
Percentages of phytoplankton assemblages as dominant groups
of species, by period in Juturnaíba Reservoir.
Period 1 Period 2a Period 2b
12 Jun - 10 Dec 26 Dec - 17 Apr 30 Apr - 28 May
24% A. distans 72% M. aeruginosa 46% C. raciborskii
21% Cryptomonas sp. 11% A. spiroides 42% A. spiroides
Biomass (Biovolume)
Correlations between contributions of the classes found by pigment data and by biovolume calculation (significant *p < 0.05, **p < 0.01; n = 25).
Ratio Xan/Chl-a CHEMTAX
Dinophyceae 0.20 0.27 Bacillariophyceae 0.64* 0.76** Cryptophyceae 0.39 0.73** Chlorophyceae 0.39 -0.35 Cyanobacteria 0.89** 0.97** Biovolume total 0.97** 0.97**
Biomass (CHEMTAX) x Biomass (biovolumeBiomass (CHEMTAX) x Biomass (biovolume)
2 periods in both methods
CHEMTAX: CHEMTAX:
Period 1 (June - November 96): 3.7 - 36.4 mg/L chl aChlorophyceae, Cyanobacteria, Cryptophyceae
Period 2 (December 96- May 97): 46.9 - 254.4 mg/L chl a81% to 99 % Cyanobacteria.
• High correlation between biovolume and Chl-a. Chl-a can
be used as a parameter to estimate biovolume.
• Interpretation of pigment data with CHEMTAX: better
correlation with biovolume than that based on Xan/Chl-a
ratios from unialgal cultures.
• Only Chlorophyceae and Dinophyceae did not present
significant correlation with cell count.
• Similar general pattern of the phytoplankton community
dynamics by cell count and pigment analysis: two periods
and the Cyanobacteria bloom recorded.
CONCLUSIONS
12 SAMPLING SITES:
SAMPLING FREQUENCE:
- 12 CAMPAIGNS
- JANUARY TO AUGUST (SUMMER/AUTUMN) 2006
GUANABARABAY
RJ/BRAZIL
1
2
3
4
5Data processing:CHEMTAX:Samples divided in 5environmentallydifferent groups
HOMOGENEITY OF SAMPLESWITHIN EACH DATA MATRIX
