HOW TO DETERMINE
PHYTOPLANKTON?
Silvana V. Rodrigues
Determination of phytoplynkton
composition 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 ?
peridinina
O
O
O
CH 3COO
OH
Dinoflagelados
Clorophyta
Cryptophyta
Cyanobacterias
OH
aloxanthin
HO
http: //oceancolor.gsfc.nasa.gov/.../BIOLOGY/
Importance of chlorophyll a
1.000 milhão tons produzidas por ano na terra e no mar
 indicator único da biomassa aquática
 parâmetro bioquímico mais freqüentemente medido
em oceanografia
Cloroplasto
struggle.net/history/images/
molecule.jpgwww.molecularexpressions.com
fig.cox.miami.edu/.../phts/c8.10x21.overview.jpg
Function of pigments in photosynthetic organisms
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 .
divisão/classe
nome comum
gên
espéc.
Algas marrons(clorofilas a e c)
Bacillariophyta
diatomáceas
210
Desconh.
Dinophyta
dinoflagelados
550
4000
Crysophyta:
Chrysophyceae
Rapidophyceae
Haptophyta
Primnesiophyceae
Xantophyta
flagelados marrom-amarel.
Crysophytas,silicoflagelados
raphydophytas (cloromonadas)
flagelados marrom-amarel.
cocolitoforídeos
algas verde-amareladas
120
4
1000
9
50
50
500
600
Cryptophyta
criptomonadas
8
>50
Eustigmatophyta
algas amarelo-esverdeadas
6
12
Chlorophyta
Clorophyceae
Algas verdes
Prasinophyceae
Flagelados verdes
Euglenophyta
Euglenoides
Algas vermelhas (clorofila a e biliproteínas)
350
13
43
2500
120
650-800
Rhodophyta
3
10
Algas verdes (clorofilas a e b)
Algas vermelhas
Algas azuis (Cyanobacteria) ( clorofila a e biliproteínas)
Cyanophyta
Prochlorophyta
Cianobactérias
proclorofitas
Characteristics which make it possible to use algal pigments
(chlorophylls, carotenoids and phycobiliproteins) as chemotaxonomic
markers
 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 phycobiliproteins
are fluorescent, what allows their detection with high sensitivity,
 Most of them are labile and esily dgraded after cell death, allowing to
distinguish living from dead cells
Hystorical overview
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
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)

Chlorophylls:
132 -Metilcarboxilates of Mg-phytoporphyrin (double bond in D ring): Cl c,
Mg-phytoclhorin: Cl a, Cl b
Phytil at C-173 (Cl a and b)
Acrílic acid at C17: Cl c
Propionic acid at C17: Cl a and b
Mg coordination complexes with cyclic tetra-pyrrols
Macrocicles with five member rings
Chlorophylls:
132 -Metilcarboxilates of Mg-phytoporphyrin (double bond in D ring): Cl c,
Mg-phytoclhorin: Cl a, Cl b
Phytil at C-173 (Cl a and b)
Acrílic acid at C17: Cl c
Propionic acid at C17: Cl a and b
Oxo substituent at C-131
methyl-carboxilate groups at C-132 -
H2C
H2C
CH3
CH3
H3C
N
N
N
H
Mg
chlorophyll a
N
O
H
H
O
O
COOCH 3
CH3
H2C
H3C H
CH3
H3C
H3C
H
N
N
CH3
H
H3C
H
CH3
DVchlorophyll b
H
O
CH3
Molecule drawings:N. Montoya
N
H
H3C
O
COOCH 3
H3C H
CH3
O
Mg
DV-chlorophyll a
O
H
CH2
N
H
H3C
H
H3C
CH3
H
H3C
CH3
CH2
N
O
H3C H
H2C
Mg
N
CH3
H3C
N
N
O
COOCH 3
O
O
H3C
chlorophyll b
N
CH3
H3C
H
H
N
H
CH3
H3C
N
N
Mg
H3C
O
CH3
H3C
H
H
O
COOCH 3
O
CH3
H3C
H3C H
H3C
H
CH3
H2C
CH3
CH3
H3C
N
N
Mg
N
N
H3C
CH3
H
O
OH
O
COOCH 3
chlorophyll c1
H2C
CH3
H2C
H3C
H3C
N
N
Mg
N
H3C
CH3
H
O
OH
N
CH3
H
OH
chlorophyll c2
N
H3C
O
COOCH 3
Molecule drawings:N. Montoya
N
N
Mg
N
COOCH 3
CH2
CH2
chlorophyll c3
O
COOCH 3
Degradation by chemical processes:
Molecules become chemically and fotochemically
more labile in organic solvents
than in the cells
H2C
CH3
H3C
Loss of metal
CH3
Chla

Phaeophitin
N
N
Mg
N
H
H3C
 in organic solvents
N
CH3
In dilute acids
H
H
O
 under high intensity of light
O
COOCH 3
O
CH3
H3C
H3C H
H3C
H
CH3
Degradation by chemical processes:
H2C
CH3
H3C
CH3
N
N
Mg
N
H
Allomerization
(oxidation by O2):
H3C
Epimerization
(HPLC: in SiO2):
N
CH3
H
H
O
Cl  enolate  Cla’, b’
O
COOCH 3
O
•Chl a  132 Hydroxiclhorophyll a H3C
•Chl a  Cl a - Hyidroxilactone.
In alcoholic or hydro-alcoholic solutions
Specially in pH >7
CH3
H3C H
H3C
H
CH3
Both processes
can be minimized
by decreasing the temperature
Degradation by chemical processes:
H2C
CH3
H3C
CH3
N
N
Mg
N
H
H3C
N
CH3
H
H
O
COOCH 3
O
O
Loss of phytil group
Cl  chlorophyillide
CH3
H3C
H3C H
H3C
H
CH3
In methanol or ethanol in basic medium
Biodegradation:
To
cyclic tetra-pirrols
perifercally modified
(enzymatically,
Specially in the absence of
light and O2):
H2C
(chlorophyllase)
chlorophillide formation
CH3
CH3
H3C
N
N
Mg
N
H
Hydrolisis of the phytil
ester
Loss of metal:
Mg-dequelatase
Formation of phaeophytins
H3C
Decarboximetilation
N
Formation of
CH3 pirophaeophytins e
pirophaeophorbides
H
H
O
O
COOCH 3
O
CH3
H3C
H3C H
Allomerization
Epimerization (Chl-oxidase)
H3C
H
CH3
Biodegradation:
To
linear tetra
pirrols
H2C
CH3
5
4
H3C
N
N
Normally by oxidative opening
of the macrocycle ring, between
C-4 and C-5,
C-5 stays as an aldehyde
CH3
Mg
N
H
H3C
N
CH3
H
H
O
O
COOCH 3
O
CH3
H3C
H3C H
H3C
H
CH3
Carotenoids
Derive from carotene:
C40H56
β- β- carotene
Isoprenoid
units
Polyen:
Absorbtion
of light.
COLOUR
-carotene:
-carotene:
-carotene:
-carotene:
lycopene:
,-carotene
,-carotene
,-carotene
,-carotene
,-carotene
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
Acetil-CoA
Geranylgeranyldiphosphate
Geranylgeranyldiphosphate
Biosynthesis:
occurs in
thylakoid
membranes
Phytoene
Dessaturation
Lycopene
Ciclization
,  -carotene
,  -carotene
Hydroxilation
lutein
Zeaxanthin
Deepoxidation
Dark
Light
Anteraxanthin
Dark
Light
Deepoxidation
Violaxanthin
Epoxidation
Epoxidation
VIOLAXANTHIN
CICLE
Rearrangement
Neoxanthin
Hydroxilation
Can occur in the dark
Depends a lot on light
DIADINOXANTHIN CICLE
Diadinoxantin
epoxidation
+ 2H + O2 - H2O
Diatoxanthin
DARK
LIGHT
+ 2H - H2O
Carotenoids
C40H56
β- β- carotene
Enzimatic
hydroxilation
Epoxidation
Carboxi
(CO2H),
carbometoxi
(CO2Me)
ou metoxi
(OMe)
Acetates
(OCOMe)
e lactones
Aldehydes,
ketones
Hydroxicarotenoids
as fatty acid esters,
or as
Glycosides or
glycosylesters,
others as
sulphates
Xantophylls
Isoprenoids
Zeaxanthin
isomers
Lutein
Acetilenic
Diatoxanthin
Alenic
fucoxanthin
Norcarotenoids
( skeleton C37)
Peridinin
C39H50O7
In acid medium
Epoxides rearrange (5,6 to 5,8 form)
7
6
8
5
violaxanthin
7
6
8
5
neoxanthin
In basic medium:
In general stable
 exception:
esters are hydrolysed
some compounds suffer structural change (fucoxanthin, peridinin)
fucoxanthin
Distribution of chlorophylls among divisions/classes of phytoplankton
Raphidophyceae
Chrysophyceae
Prymnesiophyceae
DVChlb
Dinophyta
DVchla
Bacillariophyta
MgDVP
Eustimatophyta
Tipo pyhtilat.
Chlc
Euglenophyta
Chl c3
Prasinophyceae
Chl c2
Chlorophyceae
Chl c1
Cryptophyta
Chlb
Rhodophyta
/
Pigment
Cyanophyta
Chl a
Prochlorophyta
Division or
class
Distribution of carotenes among divisions/classes of phytoplankton
Raphidophyceae
Chrysophyceae
Prymnesiophyceae
Dinophyta
Bacillariophyta
Eustimatophyta
Euglenophyta
,
Prasinophyceae
,
Chlorophyceae
,
Cryptophyta
,
Rhodophyta
/
Pigment
Cyanophyta
,
Prochlorophyta
Division or
class
Distribution of xantophylls among divisions/classes of phytoplankton
Astaxanthin
19‘-Butanoilfucoxanthin
Cantaxanthin
2
Crocoxanthin
Diadinoxanthin
Diatoxanthin
Dinoxanthin
Echinenona
Fucoxanthin
2
2
1
Raphidophyceae
Anteraxanthin
Chrysophyceae
Aloxanthin
Prymnesiophyceae
2
Dinophyta
Euglenophyta
2
Bacillariophyta
Prasinophyceae
2
Eustimatophyta
Chlorophyceae
Cryptophyta
Rhodophyta
Pigment
Cyanophyta
/
Prochlorophyta
Division or class
Distribution of xantophylls among divisions/classes of phytoplankton
Peridininol
Prasinoxanthin
Pirroxanthin
Violaxanthin
Zeaxanthin
14
14
Raphidophyceae
Peridinina
Chrysophyceae
P457+P468
Prymnesiop.
Neoxanthin
Ést. Vaucheriax
Dinophyta
Monadoxanthin
Sifoneina
Bacillariophyta
1
Luteína
Sifonaxanthin
Eustimatophyta
19‘hexanoilfuco
Euglenophyta
Prasinophyceae
Chlorophyceae
Cryptophyta
Rhodophyta
Pigment
Prochlorophyta
/
Cyanophyta
Division or
class
Amphidinium carterae (Dinophyta)
Rzi =[lpigmi]/[chlorophyll a]
Rz =[peridinin]/[chlorophyll a]
chlorophyll c2
chlorophyll a
dinoxanthin
peridinin
diadinoxanthin
Dunaliella tertiolecta (Chlorophyta)
Rz =[lutein]/[chlorophyll a]
Rzi =[lpigmi]/[chlorophyll a]
chlorophyll b
chlorophyll a
neoxanthin
violaxanthin
anteraxanthin
lutein
Hierarchical guide to the use of pigments
Pigment
Chl a:
Significance
an index of total algal biomass, excluding
prochlorophytes.
Unambiguous markers for algal types
DV-Chl a:
DV-Chl b:
Siphona xanthin esters:
an index of prochlorophyte biomass
unambiguous marker for prochlorophytes
unambiguous marker for Type 2 prasinophytes
(Ege land et al., 1997)
Prasinoxanthi n:
unambiguous marker for Type 3 prasinophytes
Peridinin :
Type 1 dinoflagell ates
Alloxanthi n:
Cryptophytes
Gyroxanthi n diester:
Dinoflagell ates Type 2
Chl c2 MGDG [14:0/14:0]: Chrysochromulina spp. (Haptophyte Type 7, Zapata
et al., 2004)
S. Wright, Class notes
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
Pea
k
no.
Rt
Pigment identification
(min)
Observed
λmax
Published
λmax
(nm)
(nm)
Reference
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
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
Michal Kobližek Arch Microbiol (2003) 180 : 327–338
Jeffrey et al. (1997)
Jeffrey et al. (1997)
Reverse-phase HPLC
chromatograms (360 nm) for
acetone extracts prepared
from
whole cell pellets of a
Erythrobacter
longus ATCC 33941,
b NAP1, c MG3, and d
NJ3Y.
Peak identities: 1
erythroxanthin
sulfate, 2
bacteriorubixanthinal,
3 zeaxanthin, 4
bacteriochlorophyll
a, 5 bacteriophaeophytin
a, 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 to
maximize the signal from BChla, while minimizing the signal from the more
abundant pigments, Chla and Chlb. (Inset) Fluorescence emission spectrum of
the peak eluting at 16.7 min in (A). Excitation was at 365 nm and slits were
20 nm.
Zbigniew S. Kolber et al, Science 292, 2492-2495; 2001.
PIGMENTS IN SEDIMENTS
Pigmentos
Em geral são moléculas lábeis, atingem o sedimento em vários estágios de degradação.
Degradação dos pigmentos originais
principalmente na água e na superfície do sedimento, durante a deposição
(Hodgson et al., 1997)
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
Fatores que afetam
a taxa
de degradação:
• Tempo para chegar
ao fundo
• Tipo de pigmento
• Grau de ataque
químico e biológico
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).
Separation 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) .
Chlorophyll b: occurs mainly ingreen algae and vascular plants,
Chlorophylls c: in diatoms, dinophlagellates and some brown algae
Kowalewska et al., 2004.
Phaeophorbides:
Degradation products due
to zooplankton
Chl a‘ and phaeophytin:
degradação products due to
Environmental stress
Pirophaeophitins and steril
Chlorins: degradation
products due to zooplankton
Jeffrey, 1997 apud Kowalewska et al., 2004).
Fossile Pigments:
Used in paleoclimatic and paleoenvironmental issues
Chlorophylls :
More labile than carotenoids , but phaephitins are persistent in
sedimentary records
Carotenoids:
Stability depends on structure (decreases with the increase of the number
of functional gruoups).
Carotenoids:
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
Estáveis, abundantes
(adaptado de Buchaca & Catalan 2008)
Chlorophylls :
Pigmento
Afinidades taxonômicas
Bacteriofeofitina-a
Bactérias fotossintéticas (Rodospirillaceae e Chromatiaceae)
Bacterioclorofila-e
Bactérias fotossintéticas (variedades marrons de Chlorobiaceae)
Clorofila-a
Razão molar Cl-a/forbinas a 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)
UV/VIS absorption of pigments
Chlorophylls
Phaeophytin a
Chlorophyll a
- Mg
- Mg - Phytil
- Mg, -COOMe
Phaephorbide a
Pirophaephytin a
Jeffrey et al.;1997
Polyene chain: chromophore
UV7VIS: Electronic transitions
Main
transition
Vibrational
fine
structure
Calculation of % III/II for a caroteneid
II
0
0
III
Vibrational
fine
structure
Molecular structure x spectroscopic properties
Chromophore (polyene chain):
Lenght  
Conjug.
db. bonds
 max
phytoene
3
276 286 297
-carotene
7
378 400 425
lycopene
11
444 470 502
carotenoid
(hexane)

Molecular structure x spectroscopic properties
Geometrical cis-trans isomers:
small hypsochromic effect
Significant hypochromic effect
Reduction of vibrational fine structure
Appearance 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 groups
Britton, 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
More than 60 pigments during the run:
QuickTime™ and a
decompressor
are needed to see this picture.
Airs, 2001
QuickTime™ and a
decompressor
are needed to see this picture.
HPLC coupled both to UV photodiode array detection
and to atmospheric pressure mass spectrometric
techniques (HPLC–DAD-APIMS)
QuickTime™ and a
decompressor
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Pigments ( chlorophylls, carotenoid), galactolipids, alkaloids, sterols and
mycosporine-like amino acids,
Frassanito 2005
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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 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
Separation
(HPLC)
GF/F 47mm
Extration:
Methanol: NH4Ac 0,5M
(98:2) +
Sonification, ice-bath
(30 s) +
Centrifugation (5 min,
4800 rpm)
Chromatographic separation of
Phytoplankton pigments
Separation with C30 columns: Development of a
Fase estacionária:
C30 (YMC, C30, 5µm,
polimérica
250x4,6 mm ID
computer-assisted method (Software Dry Lab)
c3
Fase móvel:
c1c2
alo-, diato-xanthins
e luteína
DV, MV cl b
DV, MV cl a
A:CH3OH:TBA (28 mM)
70:30 (v/v)
B: CH3CH2OH
pH 6,5
Resolution:
otimization
for chlorophylls
And for carotenoids in
Sparate runs
luteína
chlorophylls:
Fig A:30-100 % B, 50 min
Vazão: 1,2 ml/min
T: 47 oC
Carotenóides:
Fig B:25-63 % B, 35 min,
63-100%B/13 min
Vazão: 1,4 ml/min
T: oC
aloxanthin
diatoxanthin
Gradiente:
Mistura-teste
Van Heukelem e Thomas, Journal of Chromatography A, 910 (2001) 31-49
Resolution: separation mono/divynil clh a, b
They don‘t separate in C18 !! (depends on aliphatic chain?)
Separation with C8 columns:
1) Development of a computer-assisted method (Software Dry Lab)
C8 (Eclipse XDB, 3,5 µm
150x4,6 mm ID
Fase móvel:
C2 +MgDVP
Fase estacionária:
c3
DV, MV cl b
c1 +clorofilídeo a
Zeaxanthin, luteína,
DV, MV cl a
A:CH3OH:TBAA (28 mM)
70:30 (v/v), pH 6,5
B: CH3OH
Mistura-teste.Van Heukelem e Thomas, Journal of Chromatography A, 910 (2001) 31-49
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)
C8, Zapata
R=0,8
Zeaxanthin,
dihidroluteína
Clor c2
R>1
4k Hex
/9‘cis Neo
R> 1,25
MgDVP
R< 0,5
C8, Van Heukelem
cl b/DV cl b
R< 0,5
R=1
cl b/DV cl b
R= 0,8
4k Hex
/9‘cis Neo
Não resolve
Pigment mixture, S. Wright, Course Notes
C8: better for chlorophyll c family
Comparison of method sensitivity with C18 and C8 columns
Fases estacionárias:
C8 (Symmetry C8, 3,5 µm
150 x 4,6 mm)
C18 (Supelcosil L-C18, 5 µM
250 x 4,6 mm)
Fase móvel:
Coluna C18:
adap. Kraay, 1992
A:CH3OH:H2O (85:15)
B: CH3CN.H2O (90:10)
C: Acet. Etila
(vazão 0,6 ml/min)
Coluna C8:
Zapata, 2000
Mendes et al., Limnol. Oceanogr. Methods 5, 2007, 363-370
C18: More sensitivity
Lower limit of detection
Better for low concentration pigments
Separation of complex samples, method
compatible with LC/MS
Método SCOR 1997
Fase estacionária:
2 colunas „in line“
Waters Spherisorb ODS2
3 µM
150 x 4,6 mm)
Fase móvel:
A: NH4Ac 0,01M
B: CH3OH
C: CH3CN
D: 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
Método Airs et Al.
Adequado para
LC/MS
Extrato de amostra de sedimento (Priest Pot)
Airs et al.; Journal of Chromatography a 917 (2001) 167-177
Cl a + DV cla
Cl b + DV clb
diadinoxanthin
dinoxanthin
aloxanthin
diatoxanthin
luteina
zeaxanthin
peridinina
19,-butanoilfuco
fucoxanthin
neoxanthin
prasinoxanthin
violaxanthin
Cl c2
Cl c3
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 em
5 min, isocr.5 min,
25% a 100% B
em 20 min.
Labor. UFF, Cromatógrafo Bischoffanalysentechn., Mistura-teste
(DHI), 100µL injetados na fase A,
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
Based on the contribution, in terms of chlorophyll a,
of each group of taxonomical class (Chl a)c
to total chlorophyll a in the sample (Chl a)t :
(Chl a)t = (Chl a)c1 + (Chl a)c2 + (Chl a)c3 + ...... + (Chl a)cn
Easy !
Calculation of (Chl a)cn ?
METHOD 1:
Calculation of (Chl a)c by the choice of one marker pigment
for each class
Class
Marker pigment (Pm)
Pm/Cla ratio in the class
Cianobactérias
zeaxanthin
Clorophyta
luteína
Rzea/cla
Rlut/cla
Dinophyta
peridinina
Rper/cla
Cryptophyta
aloxanthin
..........................
.......................
Bacyllariophyta
fucoxanthin
Problem:
Ralo/cla
Fixed R
not necessarily
....................... Corresponds
Rfuco/cla
To the ratios
In the samples
{
(Chl a)t = Rzea/cla x (Zea) + Rlut/cla x (Lut) + .........+ Rfuco x (Fuco)
Fixed
(Chl a)c and
% of each class
sample
METHOD 2:
Multilinear regression
Sample 1:
Sample 2:
(Chl a)t1 = Rzea/cla x (Zea)1 + Rlut/cla x (Lut)1 + .........+ Rfuco x (Fuco)1
(Chl a)t2 = Rzea/cla x (Zea)2 + Rlut/cla x (Lut)2 + .........+ Rfuco x (Fuco)2
....................................................................................................................................
Sample n:
(Chl a)tn = Rzea/cla x (Zea)n + Rlut/cla x (Lut)n + .........+ Rfuco x (Fuco)n
Unknown Rs, determined by pela resolution of a system
of n equations and n unknowns

(Chl a)cn
% of ech class
Rs are determined, but
many classes don‘t
have a specific
pigment
MÉTODO 3:
Determinação da composição fitoplanctônica por análise fatorial
(MACKEY et al., 1996)
„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
MATRIZ F: Razões Ri =[lpigmi]/[chlorophyll a para cada classe
PER
BUT
FUC
HEX
NEO
PRA
VI0L
ALO
LUT
ZEA
CLB
CLA
Prasinophyt
a
0
0
0
0
0,061
0,127
0
0,004
0
0
0,381
Dinophyta
0,515
0
0
0
0
0
0
0
0
0
0
0,403
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
C: contribuição de cada
MATRIZ S: experimental
Amostra 1:
Amostra 2:
..................
Amostra n:
(Chl a)t1 (Zea)1 (Lut)1 ....... (Fuco)1
(Chl a)t2 (Zea)2 (Lut)2 ....... (Fuco)2
............
(Chl a)tn
......... ........ .......
(Zea)n (Lut) ....... (Fuco)n
FxC=S
classe (a ser determinada)
Clpras
ClDin
ClCryp
ClHapt3
ClHapt4
ClChlor
ClSyn
ClDiatom
Para uma fatorização de S que tenha um significado físico:
F : variável, Fo: dados da literatura (normalizados/Cl a)
Estimativa inicial da matriz de abundâncias das classes (Co):
calculada resolvendo-se a equação de mínimos quadrados:
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 
Um algoritmo de „decréscimo máximo“ do resíduo foi usado
(variação dos elementos de F, 10% a cada iteração)
Juturnaíba reservoir as a
study model
42°
23°
Rio de Janeiro
State
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Marcelo Marinho e Silvana V. Rodrigues
OBJETIVOS
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
METODOLOGIA
Fitoplâncton
–Coletas quinzenais - jun/96 - mai/97 (estação central)
–Biovolume
• método de sedimentação (Utermöhl, 1958)
Pigmentos
Amostra
(0,25 - 1,8 L)
• Filtração (GF/C)
• Congelamento
(CO2 sólido)
Injeção e análise
HPLC
CONDIÇÕES
CROMATOGRÁFICAS
• Coluna C18 - fase reversa
• Gradiente alta pressão
(modificado de Garrido & Zapata, 1993)
Extração
Metanol 100%
• Detecção - 440nm
Biomass (chlorophyll a)
Contribution calculated by marker pigments
Razão Xan/Chl-a
150
250
200
150
100
100
50
50
0
0
100
100
Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr Aprb)May
1996
1997
80
60
40
20
0
Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr Apr May
1996
1997
relative contribution
relative contribution
µg L
-1
200
300
a)
-1
250
Cyanobacteria
Chlorophyceae
Cryptophyceae
Bacillariophyceae
Dinophyceae
µg L
300
CHEMTAX
Cyanobacteria
Chlorophyceae
Cryptophyceae
Bacillariophyceae
Dinophyceae
a)
Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr Aprb)
May
1996
1997
80
60
40
20
0
Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr Apr May
1996
1997
Biovolume
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)
Biomass (Biovolume)
90
mg/L
cyanobacteria
diatoms
cryptomonads
60
dinoflagellates
green algae
30
others
0
Jun
Jul
Aug
Sep Oct
1996
Nov
Nov
Dec
Jan
Feb
Mar
1997
Apr
Microcystis aeruginosa
May
20 mg/L
Anabaena spiroides
Cylindrospermopsis raciborskii
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
Correlations between contributions of the c lasses found by
pigment data and by biovolume calculation (significant *p
< 0.05, **p < 0. 01; n = 25).
Ratio Xan/Chl-a
Dinophyceae
Bacillariophyceae
Cryptophyceae
Chlorophyceae
Cyanobacteria
Biovolume total
0.20
0.64*
0.39
0.39
0.89**
0.97**
CHEMTAX
0.27
0.76**
0.73**
-0.35
0.97**
0.97**
Biomass (CHEMTAX) x Biomass (biovolume)
2 periods in both methods
CHEMTAX:
Period 1 (June - November 96):
3.7 - 36.4 mg/L chl a
Chlorophyceae, Cyanobacteria, Cryptophyceae
Period 2 (December 96- May 97):
46.9 - 254.4 mg/L chl a
81% to 99 % Cyanobacteria.
CONCLUSIONS
• 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/Chla 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.
12 SAMPLING SITES:
SAMPLING FREQUENCE:
- 12 CAMPAIGNS
- JANUARY TO AUGUST
(SUMMER/AUTUMN) 2006
GUANABARA
BAY
RJ/BRAZIL
HOMOGENEITY OF SAMPLES
WITHIN EACH DATA MATRIX
Data processing:
CHEMTAX:
Samples divided in 5
environmentally
different groups
5
3
4
1
2