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Bachelor Thesis Chemistry
A new High-Performance Liquid Chromatography
method for purifying and quantifying Heterocyst
Glycolipids out of Heterocystous Cyanobacteria
By
Steven de Vries
30 June 2016
Student number Research group
10616950 Mariene Microbiologie en Biogeochemie
Research-institute
NIOZ
Tutor
Dr. Ir. Ellen C. Hopmans/Dr. Nicole J. Bale
Responsible docent
Prof. Dr. Ir. Stefan Schouten
Contents 1 List of abbreviations .................................................................................................................... 1
2 Abstract .......................................................................................................................................... 2
3 Wetenschappelijke samenvatting .............................................................................................. 3
4 General introduction ................................................................................................................... 5
4.1 Cyanobacteria ....................................................................................................................... 5
4.2 Heterocyst glycolipids ......................................................................................................... 6
4.3 Potential biomarker ............................................................................................................. 7
4.4 Relevance and scope of this research ................................................................................ 8
5 Analytical methods for analyzing/purifying heterocyst glycolipids .................................. 10
5.1 Bauersachs et al. .................................................................................................................. 10
5.2 Gambacorta et al. ................................................................................................................ 10
5.3 L. Wörmer et al. ................................................................................................................... 11
5.4 Discussion of the methods ................................................................................................ 11
6 Experimental section ................................................................................................................. 12
6.1 Extraction procedure ......................................................................................................... 12
6.2 Orbitrap–MS analysis ........................................................................................................ 13
6.3 1st round of semi-preparative HPLC ............................................................................... 13
6.4 HPLC/MSD analysis .......................................................................................................... 14
6.5 2nd round of semi-preparative HPLC .............................................................................. 14
6.6 Ion-trap MS analysis .......................................................................................................... 15
6.7 Response curves ................................................................................................................. 15
6.8 NMR ..................................................................................................................................... 16
7 Results and Discussion .............................................................................................................. 17
8 Conclusion .................................................................................................................................. 29
9 Future prospects ......................................................................................................................... 30
10 Acknowledgement ..................................................................................................................... 30
11 References .................................................................................................................................... 31
12 Appendix ..................................................................................................................................... 33
1
1 List of abbreviations
°C Degrees Celsius
BDE Bligh-Dyer Extraction
BDS Bligh-Dyer extraction Solvent
CHCl3 Chloroform
DCM Dichloromethane
Et al. And others
GC Gas Chromatography
H Hour
HCl Hydrochloric acid
HG Heterocyst glycolipid
HPLC High Pressure Liquid Chromatography
IPL Intact Polar Lipid IS Internal Standard
KOH Potassium hydroxide
M Molarity
MeOH Methanol
Mg Milligram
Min Minutes
mL Milliliter
MS Mass Spectrometer
N2 Nitrogen
NH3 Ammonium
NIOZ Royal Netherlands Institute for Sea Research
p-buffer Phosphate buffer
Psi Pound-force per square inch
QToF-MS Quadrupole Time-of-Flight Mass Spectrometer
SIM Selected Ion monitoring
sp. Species
SRM Selected Reaction monitoring
UHPLC Ultra-High Pressure Liquid Chromatography
δ13CTOC Ratio of stable isotopes 13C /12C from the Total Organic Carbon
δ15N Ratio of stable isotopes 15N /14N
μL Microliter
Μm Micrometer
2
2 Abstract
N2-fixing cyanobacteria are known to differentiate vegetative cells, under nitrogen
depletion, into so-called heterocysts, which have thick cell walls consisting of
glycolipids for oxygen impermeability. These heterocyst glycolipids (HGs) consist
of a sugar moiety, either a hexose or pentose, and a long chain, C26-C32, with
different alcohol or ketone groups. Several reports have been published
describing analysis of these HGs, determining the structure and quantifying the
HGs as the integrated peak area response. In this research a method is shown for
purifying and quantifying pure heterocyst glycolipids with several analytical
techniques, providing an external standard for reliable quantitative analysis of
HGs in samples from the natural environment. Accurate quantification of HGs
will potentially make it possible to determine the amount of N2-fixation and to
reconstruct a model for past nitrogen fixation.
Two different separation methods are used to isolate the HGs, beginning with
normal–phase liquid chromatography (NP-LC), followed by reversed–phase LC
(RP-LC), which resulted in the successful isolation of four different kinds of HGs.
The purity of the isolated compounds was validated by MS and MS2. The
response factor of the HG was compared to an already available glycolipid
internal standard. Nuclear Magnetic Resonance (NMR) spectra were taken from
the heterocyst glycolipid obtained in the highest amount.
3
3 Wetenschappelijke samenvatting
In de oceaan komen bacteriën voor die
stikstofgas omzetten in voedingsstoffen,
zogenoemde cyanobacteriën, die ook zuurstof
produceren bij fotosynthese. Omdat het enzym
dat aanwezig is in cyanobacteriën voor het
fixeren van stikstof, gedeactiveerd wordt door
zuurstof, moet deze beschermd worden.
Hiervoor is een aparte cel (de heterocyst) met
een celwand die niet doordringbaar is voor
zuurstof. In deze speciale celwand zijn vetten
aanwezig die specifiek zijn voor stikstof-
fixerende cyanobacteriën, en in dit onderzoek
wordt er gefocust op een soort; de heterocyste glycolipiden (Figuur A). Deze hebben een
suiker kopgroep en een lange koolstof keten met een alcohol groep aan het einde en een
alcohol of keton groep aan het begin van de keten. Deze verbindingen worden geanalyseerd
in oceaanwater maar deze analysetechnieken zijn niet kwantitatief wegens het ontbreken
van zuivere standaarden van heterocyste glycolipiden. Daarom is het doel van dit project
om zuivere hoeveelheden glycolipiden te isoleren. De onderste twee glycolipiden in Figuur
A, zijn aanwezig in een zodanig lage concentratie in cyanobacteriën, dat deze niet in
voldoende hoeveelheid geïsoleerd kunnen worden, daarom ligt de focus op de glycolipiden
(1–4).
Om de glycolipiden te isoleren werd eerst de biomassa, die bestond uit verschillende
soorten cyanobacteriën, geëxtraheerd, en geanalyseerd, waarbij bleek dat er heel veel
soorten vetten aanwezig waren. De glycolipiden werden hieruit geïsoleerd met behulp van
semi-preparatieve High Performance Liquid Chromatografie (HPLC), waarbij de stof door
een kolom wordt geleid met behulp van een loopmiddel, en dan in fracties wordt
opgevangen. Als eerste werd normale–fase chromatografie (NP-LC) toegepast, wat betekent
dat er een polaire kolom is en een apolair loopvloeistof, hexaan in dit geval. Dit leidde tot
een goede scheiding van de verschillende vetten en de glycolipiden werden in twee fracties
gevonden met behulp van massa spectrometrie, en deze fracties werden verzameld. Na
analyse bleek dat er nog veel andere soorten vetten dan de glycolipiden aanwezig waren en
dat er dus nog een scheiding moest plaatsvinden. Dit keer was het gewenst om een andere
scheidingsmethode te gebruiken op de semi-preparatieve HPLC, dus werd er in plaats van
normale–fase chromatografie, reversed–fase chromatografie (RP-LC) gebruikt, wat betekent
dat er een apolaire kolom wordt gebruikt en een polair loopmiddel, water in dit geval. De
verschillende glycolipiden (1–4) kwamen nu terecht in verschillende fracties en konden
apart verzameld worden. De verschillende glycolipiden werden geanalyseerd en het bleek
Figuur A: zes verschillende heterocyste glycolipiden
4
dat er van glycolipide (1) het meest was verzameld. Hiervan werden Nuclear Magnetic
Resonance (NMR) spectra geproduceerd, maar de hoeveelheid bleek te laag om een goed
spectrum te verkrijgen. Desalniettemin kunnen deze geïsoleerde glycolipiden nu als
authentieke standaard gebruikt worden, en kan de hoeveelheid glycolipiden in onbekende
monsters vastgesteld worden. De gehele procedure is weergegeven in Schema A.
Dit onderzoek werd uitgevoerd onder leiding van Dr. Ir. Ellen. C. Hopmans en Dr. Nicole J.
Bale, op het Koninklijk Nederlands Instituut voor Onderzoek der Zee (NIOZ) wat het
oceanografische instituut voor Nederland is. Het instituut verricht fundamenteel en frontier
toegepast wetenschappelijk onderzoek om kennis te verzamelen en te verspreiden over
belangrijke processen in deltagebieden, kustzeeën en open oceanen. Het instituut fungeert
tevens als nationale faciliteit voor het universitaire zeeonderzoek in Nederland en
ondersteunt het onderzoek en onderwijs in de mariene wetenschappen op nationaal en
Europees niveau.
Schema A: de methode voor het scheiden van de glycolipiden (2–5)
5
4 General introduction
4.1 Cyanobacteria
Marine organisms are highly dependent on the nitrogen cycle in the ocean, which includes
fixation of N2 from the atmosphere and production of different compounds as nitrate (NO3),
nitrite (NO2), nitrous oxide (N2O), ammonium (NH4+), urea and dissolved organic nitrogen.1
Nitrogen fixation is primarily performed by cyanobacteria, which play an important role in
the history of life, and are reported to be 3500 Ma years old.2 There are a wide range of
known cyanobacteria, divided into five morphological groups; Chroococcales (I),
Pleurocapsales (II), Oscillatoriales (III), Nostocales (IV) and Stigonematales (V).3 The first
two groups are unicellular, which means their systems consist of only one cell, whereas
groups III, IV and V are filamentous, which means they consist of a strand of cells that are in
contact. Example of a unicellular (A) and a filamentous cyanobacterium (B) are depicted in
Figure 1.
Figure 1: photomicrographs of cyanobacteria; Cyanothece sp. (A) and Anabaene spp. (B) from http://www-
cyanosite.bio.purdue.edu/images/images.html
Cyanobacteria that fixate N2 need a defense system to keep oxygen out of the inner cell, one
strategy is the formation of a heterocyst, which forms an anaerobic environment for the
nitrogenase enzyme complex, which reduces nitrogen into NH4+. Heterocystous cells are
found in morphological group IV and V. These groups differentiate heterocyst under
nitrogen depletion, maintaining the energy for the biological system.4 This heterocyst is
formed via an intermediate, called a proheterocyst, which can return to the earlier state or
form a heterocyst. Group IV and V can be divided in several species, where not all species
are cultivated and examined. For further reading on cyanobacteria the following biological
papers are advised.5,6
6
4.2 Heterocyst glycolipids
Heterocysts are characterized by a thick cell wall, impermeable to oxygen, consisting of
three extra layers in comparison to the vegetative cell. The focus in this paper lies on the
most inner layer, which consist of glycolipids, being specific to heterocystous cyanobacteria
and being therefore good biomarkers for these microbes. Morphological groups IV and V
can be divided into several species with different kinds of heterocyst glycolipids (HGs), of
which examples are given in Figure 2, and the structures (1–6) are of interest in this research.
Every structure consist of a sugar moiety, either a hexose or a pentose, and a long chain, C26
to C32, with several alcoholic or ketone groups.
Figure 2: The six common heterocyst glycolipids with a hexose head group (1–6) and a heterocyst glycolipid with a pentose–
headgroup (7) ; 1-(O-hexose)-3,25-hexacosanediol (1); 1-(O-hexose)-3-keto-25-hexacosanol (2); 1-(O-Hexose)-3,27-
octacosanediol (3); 1-(O-Hexose)-3-keto-27-octacosanol (4); 1-(O-Hexose)-3,25,27-octacosanetriol (5); 1-(O-Hexose)-27-
keto-3,25-octacosanediol (6) and 1-(O-ribose)-3,27,29-triacontanetriol (7)
Various species from the Nostocaceae and Rivulariaceae, both families of the Nostocales (IV)
division were analyzed by Bauersachs et al. (2009a) together with non-heterocystous and
7
unicellular species, confirming that non-heterocystous and unicellular species do not contain
glycolipids.7 In the Nostocales (IV) division five kinds of glycolipids were found, examples
are structure (1–6), (Fig. 2), where the chain is characterized by an alcohol or a ketone on the
C-3. Earlier research on the Rivulariaceae, was performed by Gambacorta et al. (1998), who
determined, which sugar was present in the HGs, for instance α-Glucose, α-Galactose or β-
Glucose, and that the long chain was characterized by a ketone on the ω-3 group.8
The HG composition of endosymbiotic heterocystous cyanobacteria, which are found inside
living diatom cells, were also analyzed by Schouten et al. (2013), and were found to be
structurally different to those detected in free-living heterocystous cyanobacteria.9
Glycolipids with pentose instead of hexose were found and the long chain was 2 till 4
carbons longer, an example is structure (7) (Fig. 2).
In conclusion, different HGs are present in different cyanobacterial species, and further
researches may shed light on a wider range of HGs in different species.
4.3 Potential biomarker
HGs have been found in samples, 49 million years old, which indicates that these glycolipids
are preserved over a long timescale and can thus make a potential biomarker, which is a
measurable indicator of a biological state or condition, for paleo-research.10 Through
comparison of N2 fixation and concentrations of HGs of heterocystous cyanobacteria, some
conclusions can be drawn about their biomarker potential for nitrogen fixation. Additional
evidence of nitrogen fixation is given with stable nitrogen isotopes, where δ15N values range
from +1‰ to -3‰, and depleted values for δ15N are evidence for nitrogen fixation.11 By
measuring the natural abundancy of 15N in ancient samples, the presence of N2 fixating
bacteria can be indicated, and the biomarker potential of HGs was confirmed.
The diversity of the HGs has been examined by a range of studies, together with their
potential as biomarkers.12,13 The diversity is considerable within the divisions (c.f. Fig. 2), a
property which makes this suit of biomarkers valuable for identification of specific
heterocyst-forming cyanobacterial species. Glycolipids with pentose head groups have been
found in endosymbiotic heterocystous cyanobacteria9 and in freshwater environments in
Spain8. However, those HGs associated with endosymbiotic heterocystous cyanobacteria
8
have to date, been found to exhibit a combination of an extended chain-length and a pentose
headgroup, thought to be specific to these species.8,9 The HGs with a hexose group ( e.g. as
determined by Bauersachs et al. (2009a) and Wörmer et al. (2012)12) are found in a wide range
of cyanobacteria in different environments, such as microbial mats and fresh water lakes. A
study on the HG composition of microbial mats was carried out on the island of
Schiermonnikoog, where twelve samples were taken at the coast at different locations, and
the lipid biomarker and stable isotopes (δ13CTOC, δ15N) were studied.14 At the intertidal zone
no HGs were detected, only in higher laminated parts of the beach different HGs were
detected. These detected HGs show remarkable differences in structure at different places,
as 1-(O-Hexose)-3,25-hexacosanediol (1) was found in the direction of the sea and 1-(O-
hexose)-3,25,27-octasanetriol (5) was found near the dunes, making the HGs excellent
biomarkers for community changes.
Also temperature changes can be determined, using the HG26 index (Formula 1), which is
based on the fact that a higher amount of the ketone HG26 is present at a higher
temperature.7
𝐻𝐺26 =𝐶26 𝑘𝑒𝑡𝑜 − 𝑜𝑙
(𝐶26 𝑑𝑖𝑜𝑙 + 𝐶26 𝑘𝑒𝑡𝑜 − 𝑜𝑙(Formula 1)
HG26 values range from 0.18 to 0.31 at 14 °C and from 0.05 to 0.11 at 27 °C. At higher growth
temperature, more HGs were found with alcohol groups than ketone groups.15
In conclusion HGs are excellent biomarkers for different divisions of heterocystous
cyanobacteria in different times, places and temperatures. Furthermore is it important to
focus on a pattern of HGs instead of just one specific HG.7
4.4 Relevance and scope of this research
As stated in sections 4.2–4.3, HGs are excellent biomarkers for different divisions of
heterocystous cyanobacteria, whereas other parts of N2 fixing cyanobacteria are poorly
preserved and lack the potential of a biomarker. However, the quantification of HGs is
problematic as an authentic standard is not available. An authentic standard would give
more insight in the amount of HGs and consequently the amount of N2 fixation, which will
help in reconstructing the past, whereupon models can be made about the future, and
conclusions can be drawn about the atmospheric composition in the future. The nitrogen
9
cycle is of great interest, mostly because human activity is changing this cycle, for example
with the Haber-Bosch process. This fertilization is causing problems in aquatic ecosystems,
as are other processes, which indicate the importance of the nitrogen cycle, and more
research has to be conducted to model the future N2–cycle. The results are directly relevant
for policy making decisions regarding the impact of human activity on the natural
environment.
The scope of this research is to develop a new method for obtaining HGs out of
cyanobacterial cultures using semi-preparative High Performance Liquid Chromatography.
Due to the lack of an authentic standard, HGs have, to date, been, quantified as the
integrated peak area response, keeping the data treatments semi-quantitative. The material
used in this study is a mixture of different cyanobacteria of the Anabeana sp. and Nostoc sp.,
both of the Nostocaceae family, which are known to have traces of six different HGs, which
are depicted in Figure 2.7 HGs (1) and (2) are present in Anabeana sp. and Nostoc sp. and are
similar to HGs (3) and (4), having elongated chains of two carbons. The triol or keto-diol
chains, (5) and (6), are not expected, demonstrated that these chains are found
predominantly in Rivulariaceae, like Calothrix sp.7,16
HG (1) is detected in a higher amount than HG (3) and the two other elongated structures,
and is expected to be obtained as the purest compound.7 The research-question is: Which
analytical methods can be used for purifying and quantifying heterocyst glycolipids? The
research goal is to obtain at least 1 milligram with a purity of 80%.
First, previous methods for analysis of HGs from cyanobacteria will be summarized and
discussed, because these methods can be helpful for method development of the purification
of HGs. Subsequently the methods for purifying and quantifying HGs will be explained and
the results will be presented. Thereafter the results will be discussed and a conclusion will
be made about the used method and the purity of the obtained HG as external standard.
10
5 Analytical methods for analyzing/purifying heterocyst
glycolipids
There are several methods published for analyzing cyanobacterial HGs of which three
methods will be described, from researchers in this field; the method from Bauersachs et al.
(2009b) (NIOZ), the method from Gambacorta et al. (1992) and the method from Wörmer et
al. (2012). In all these researches no authentic standard was present, but the HGs were
quantified as the integrated peak area response, keeping the data treatments semi-
quantitative.
5.1 Bauersachs et al. (2009b)16
Bauersachs et al. (2009b) used semi-preparative HPLC, to separate individual HGs for
further analysis by gas chromatography-mass spectrometry (GC-MS). Extract of biomass,
from the Culture Collection Yerseke (CCY), were isolated by semi preparative HPLC with a
LiChrospher DIOL column, with a method first performed by Sturt et al. (2004)17, using a
linear gradient of 3 mL min-1: with eluent A as hexane/isopropanol/formic acid/14.8 M
aqueous NH3 (79:20:0.12:0.04, v:v:v:v) and eluent B as isopropanol/water/formic acid/14.8 M
aqueous NH3 (88:10:0.12:0.04, v:v:v:v). The flow scheme was 90% A→70% A in 10 min,
which was maintained for 20 min. subsequently the flow went from 70% A to 35% A in 15
min, and was maintained for 15 min.
5.2 Gambacorta et al. (1992)18
A sample of Nodularia harveyana was obtained from Sammlung von Algenkulturen
Gottingen and was lyophilized for usage. These lyophilized cells were extracted with 0.9 l of
CHCl3/i-PrOH (1:1, v:v) for five days, subsequently the extract was dissolved in
CHCl3/MeOH (2:1, v:v) and was injected on a Sephadex column and was eluted with
MeOH/CHCl3 (9:1, v:v). The separation was monitored by TLC (CHCl3:MeOH, 8:2, v:v), and
the fraction were again dissolved in CHCl3/MeOH (2:1, v:v), put on a RP-8 column and
extracted with MeOH/CHCl3 (9:1, v:v). Repeatedly the product fractions were dissolved in
CHCl3/MeOH (2:1, v/v) and were put on preparative TLC, at which the final HGs were
11
eluted with CHCl3/MeOH (8:2, v/v). The HGs were tested on 1H-NMR and the HGs were
successfully detected.
5.3 L. Wörmer et al. (2012)12
Microbial mat samples from Antarctica and water-samples from Spanish lakes were
extracted using a modified Bligh and Dyer method,19 followed by solvation in 0.5 mL
DCM/MeOH (9:1, v/v). The samples were injected on a UHPLC coupled with a QToF-MS,
and two eluents were used, with eluent A as acetonitrile/isopropanol/formic
acid/ammonium hydroxide (46:15:1:1, v/v/v/v) and eluent B as methanol/water/formic
acid/ammonium hydroxide (15:15:1:1, v/v/v/v). The flow scheme began with 100% eluent A
for 3 min, decreasing to 94% eluent A-6% eluent B during 11 min, followed by washing with
40% eluent A-60% eluent B for 2 min, all with a flow rate of 0.4 mL min-1.
5.4 Discussion of the methods
These different methods were compared to deduce which method would be best, for
application. Wörmer et al. (2012) used an UHPLC, which is only used for analyzing and not
for purification. The method of Gambacorta et al. (1992) is a method utilizing column
chromatography, which may be of interest for purification steps in this study, e.g. raising
the purity above the 80%. However this process takes a lot of time and requires a lot of
eluent in comparison to Bauersachs et al.16 Bauersachs et al. (2009b) used semi-preparative
HPLC to separate individual HGs for further analysis by GC-MS. The semi-preparative
method utilized by Bauersachs et al. (2009b) would be an appropriate initial point for
method development for this research.
The chosen approach for purification of HGs is: use first HPLC with the same eluent A and
B as Bauersachs et al. (2009b), coupled to MS, to detect the target analyte, followed by using
semi-preparative HPLC to obtain this target analyte and analyze again with MS to verify the
presence of the target compound in the fractions. If additional purification is required,
different HPLC methods or the method of Gambacorta et al. (1992) can be used.
12
6 Experimental section
6.1 Extraction procedure
The sample of cyanobacterial biomass that were analysed contained several kinds of
cyanobacteria, which are given in Table 1.
Table 1: The different samples that were mixed together.
Species Number related to NIOZ Yerseke Amount (g)
Anabaena spp. CCY 9922 0.0325
Nostoc spp. CCY 1933 0.0159
Anabeana variabilis CCY 9922 0.0655
Residue from previous BDE # 1.5251
The total amount of sample was 1.639 g (0.1374 g dry weight). Subsequently a modified
Bligh-Dyer extraction (BDE) was performed, with MeOH/DCM/P-buffer (pH 7–8) (2:1:0.8
v:v:v) as Bligh-Dyer solvent (BDS).19 First all the amount of sample was dissolved in 20 mL
BDS, followed by stirring with a spatula and sonicating in a sonic bath for 10 minutes. Then
the sample was centrifuged (3000 rpm, 2 min) and the BDS was decanted, keeping the
residue which was extracted with BDS two more times. The decanted BDS was obtained in a
large centrifuge tube, DCM and P-buffer were added to obtain a final solvent ratio
(MeOH/DCM/P-buffer 1:1:0.9, v:v:v) and centrifuged (1000 rpm, 2 min), followed by
collection of the DCM layer with a Pasteur pipette. The remaining MeOH/p-buffer layer was
extracted two more times with DCM by centrifuging (1000 rpm, 2 min). The combined DCM
fractions were evaporated under nitrogen and the Bligh-Dyer extract was dissolved in 2 mL
IPL solvent (Hexane:IPA:H2O, 718:271:10, v:v:v), transferred to a centrifuge tube and
centrifuged (3000 rpm, 2 min). Subsequently the supernatant was filtered through 0.45 μm
mesh True Regenerated Cellulose syringe filters (4 mm diameter; Grace Alltech) and the
filtrate was dried under nitrogen.
13
6.2 Orbitrap–MS analysis
Initial analysis of the extract was carried out according to Sturt et al. (2004)17 with some
modifications and the extract was dissolved in IPL solvent (Hexane:IPA:H2O, 718:271:10,
v:v:v). An Ultimate 3000 RS UHPLC, equipped with thermostatted auto-injector and column
oven, coupled to a Q Exactive Orbitrap MS with Ion Max source with heated electrospray
ionization (HESI) probe (Thermo Fisher Scientific, Waltham, MA), was used. Separation was
achieved on an Acquity UPLC® BEH HILIC column (150 x 2.0 mm, 2.1 µm particles, pore
size 12 nm; Waters, Milford, MA) maintained at 30 °C. The following elution program was
used with a flow rate of 0.2 mL min-1: 100% A for 5 min, followed by a linear gradient to 66%
A: 34% B in 20 min, maintained for 15 min, followed by a linear gradient to 40% A: 60% B in
15 min, followed by a linear gradient to 30%A:70%B in 10 min, where A = hexane/2–
propanol/formic acid/14.8 M NH3 (aq) (79:20:0.12:0.04, v:v:v:v) and B = 2-
propanol/water/formic acid/ 14.8 M NH3 (aq) (88:10:0.12:0.04, v:v:v:v). Total run time was 70
min with a re-equilibration period of 20 min in between runs. HESI settings were as
follows: sheath gas (N2) pressure 35 (arbitrary units), auxiliary gas (N2) pressure 10
(arbitrary units), auxiliary gas (N2) T 50 ˚C, sweep gas (N2) pressure 10 (arbitrary units),
spray voltage 4.0 kV (positive ion ESI), capillary temperature 275 °C, S-Lens 70 V. The
Orbitrap-MS analysis was performed in positive-ion mode (m/z 300–2000), for MS2 analysis.
6.3 1st round of semi-preparative HPLC
For semi-preparative HPLC two eluents were prepared following the procedure of Sturt et
al. (2004)17, with upscaling from Bauersachs et al (2009b)16. The composition of the two eluents
was the same as for the Orbitrap analysis (Section 6.2). For semi-preparative HPLC an
Agilent 1100 series (Agilent, San Jose, CA) was used, with a termostatted auto injector and a
Foxy Jr. fraction collector (Teledyne Isco, Lincoln, NE). The chemicals were minimally of
HPLC grade: with picograde Hexane (LGC Promochem, Wesel, Germany), isopropanol and
formic acid (Biosolve bv , Valkenswaard, The Netherlands), water (MilliQ) and ammonium
formate 14.8 M (Fluka chemie, Zwijndrecht, The Netherlands). The eluents were collected in
1 minute fractions after eluting through a LiChrospher DIOL column (250 x 10 mm, 5 μm;
Alltech, Deerfield, IL). Different injection volumes were examined and 100 μL was optimal
in terms of backpressure and sample throughput. The flow was 3 ml min-1 with an elution
14
program of 10% B to 30% B in 10 min, maintenance for 20 min, followed by a linear gradient
of 65% B in 15 min, which is maintained for 15 min, followed by 10% B for 10 min, for
washing the column.
6.4 HPLC/MSD analysis The fractions were analyzed by HPLC/MSD by Flow Injection Analysis (FIA), and the
composition of the eluents was the same as the Orbitrap analysis (Section 6.2). The direct
injections were performed with an HPLC of the Agilent 1100 series (Agilent, San Jose, CA)
coupled to an Agilent 1100 series (Agilent, San Jose, CA) MSD with an API-ES source. The
fractions 10 to 25 were analyzed using this technique to check whether the target
compounds were present, this was performed with a 0.2 ml/min flow. The conditions for the
API-ES source were; nebulizer pressure 60 psi, capillary voltage 3000 V, drying gas (N2) 6 L
min-1 with a temperature of 300 °C. Injections of 10 μL were performed in a stream of 50%
Eluent A and 50% Eluent B. The presence of the different m/z values were monitored using
selected ion monitoring (SIM) of the six ammoniated adducts of the six different HGs.
Subsequently the three fractions containing the target compound in the highest
concentration were pooled together in a round bottom flask and the eluents were
evaporated. The two tubes that surrounded the tubes with the highest concentration target
compound were also pulled together in another round bottom flask and the solvent was
evaporated. The residue was dissolved in DCM/MeOH (9:1, v:v) to prevent degradation.
6.5 2nd round of semi-preparative HPLC
To achieve further purification a second round of semi-preparative HPLC was carried out,
to accomplish purification and separation between the different kinds of HGs. Two eluents
were prepared for this RP-LC method following Wörmer et al. (2013)20, with eluent A as
MeOH:H2O (85:15 v/v) and eluent B as MeOH/IPA (50:50, v:v), with a flow of 3 mLmin-1 and
using the same system as described in section 6.3. A Symmetry prep C18-Column (250 x 10
mm, 7 μm, Waters, Milford, MA) was used, and 200 μL injections were performed. The
elution program was slightly modified; 100% A for 2 min, then an increase to 15% B in 0.1
min, followed by a linear gradient to 43.3% B in 13 min, and subsequent washing with 100%
B for 5 min and equibrilating with 100% A for another 5 min. The fractions were collected
15
every 15 sec between 7 and 15 min.
6.6 Ion-trap MS analysis
The collected fractions from the second round of semi-preparative HPLC were analyzed
using an LTQ XL linear ion trap with Ion Max source with electrospray ionization (ESI),
with Flow Injection Analysis (FIA) and the settings were; capillary temperature 275 °C,
sheath gas (N2) pressure 25 (arbitrary units), sweep gas (N2) pressure 20 (arbitrary units),
spray voltage 4.5 kV (positive ion ESI), auxiliary gas (N2) pressure 15 (arbitrary units). The
samples were analyzed using selected reaction monitoring (SRM) a dual-stage tandem MS
(MS2), with the parent mass and the first fragmentation of the four most abundant HGs
given (Table 2) (Normalized collision energy, 25; isolation width, 5.0; activation Q 0.175).
The composition of the two eluents was the same as for the Orbitrap analysis (Section 6.2).
The HGs (1–4) were identified and the fractions containing the highest concentration of
either (1), (2), (3) or (4), were pooled together.
Table 2: The masses, which were scanned for using selected reaction monitoring (SRM) during dual-stage tandem mass
spectrometry (MS2).
Heterocyst glycolipid Parent Mass Fragment Mass
(1) 577 415
(2) 575 413
(3) 605 443
(4) 603 441
6.7 Response curves
After collecting the fractions, the 4 separate HGs were analyzed by HPLC coupled to a
Thermo LTQ XL linear Ion trap MS, with the same settings as in section 6.6, using a normal
phase DIOL-120 column (250 x 2.1 mm, 5 μm, GL Science Inc. USA, Torrance, CA). The
composition of the two eluents was the same as for the Orbitrap analysis (Section 6.2). HG
(1) was used to generate a relative response curve with a synthesized internal standard (n-
16
Dodecyl β-D-glucopyranoside, ≥98% Sigma-Aldrich, Zwijndrecht, The Netherlands), by
injecting a mixture of HG (1) with the IS (1:1 w:w), in amounts ranging from 1 to 100 ng.
6.8 NMR
HG (1) gave the highest signal on the Thermo LTQ XL ion trap and was isolated in the
highest amount, making it the most suitable HG for NMR studies. This was performed on
the 500 MHz NMR, with the sample dissolved in deuterated DCM/deuterated MeOH (1:1
v/v) , creating a 1H-NMR spectrum.
17
7 Results and Discussion First the mix of cyanobacteria was extracted to obtain the Intact Polar Lipids (IPLs), which
was successfully performed with a Bligh-Dyer extraction,19 showing a broad scale of
different IPLs in the full MS chromatogram (Fig. 4). The MS chromatogram was obtained
using HPLC-ESI-Orbitrap-MS, with the standard approach, which was performed by
Bauersachs et al. (2009b), well suited for lipids with glycosidically bound sugar moieties.21
Six different HGs were identified as a component of the intact polar lipid extract (Fig. 3), by
their exact masses. 1-(O-hexose)-3,25-hexacosanediol (1), was present at the highest
concentration, which revealed a protonated molecule at m/z 577.4678 and showed the loss of
the hexose moiety at m/z 415.4145 ([M+H-C6H12O5]+), giving a loss of 162.0533 Da (Fig. 3, a).
Furthermore the loss of 18 Da (water) was observed three times, at m/z 397.4040, 379.3934
and 361.3828, revealing the loss of two alcohol groups and the oxygen that was present for
binding the hexose. The 1-(O-hexose)-3-keto-25-hexacosanol (2) was present with a slightly
lower relative response, showing the protonated molecule at m/z 575.4515, and the loss of
the hexose group at m/z 413.3986 ([M+H–C6H12O5]+) together with the same losses observed
for HG (1), at m/z 395.3881, 377.3775 and 359.3669 (Fig. 3, b). Furthermore the HGs with a
slightly longer chain, C28 instead of C26, 1-(O-Hexose)-3,27-octacosanediol (3) and 1-(O-
hexose)-3-keto-27-octacosanediol (4), were present as expected (Fig. 3, c–d), based on the
protonated molecule peaks at m/z 605.4988 and 603.4831, which are masses of 28 Da higher,
showing the elongation of the chain by two carbons. In both MS spectra of the C28 chains,
three times the loss of 18 Da was observed, indicating a diol and a keto-ol structure as
described in the introduction. The triol (5) and keto-diol (6) chains were also detected,
whereas they were absent in the previous studies, probably due to the higher detection
capability of the Orbitrap–MS.7,16 The triol and keto-diol, were indicated by the protonated
molecule peaks at m/z 621.5106 and 619.5076, together with the loss of the hexose group at
m/z 459.4583 and 457.4409 ([M+H-C6H12O5]+). In both spectra four times the loss of 18 Da was
observed, confirming that three alcohol/ketone groups are present. These two forms of HGs
were present in such a low concentration that the spectra are not depicted in Figure 3,
because these were very unclear. The retention time of the target analytes with the UHPLC-
column was taken as an indication for their retention time on the semi-preparative HPLC.
The base peak chromatogram is depicted in Figure 4, with the HGs eluting between the 16
18
and 24 minutes. The ion chromatograms show split peaks, indicating the presence of
isomers of the individual HGs. A separation was deemed possible between HGs (1-2) and
HGs (3-4), and also between the HGs (5-6). While the presence of the HGS was confirmed, a
wide range of other IPLs were also present, such as (PC) at 25 and 26 min, diacylglyceryl
carboxyhydroxymethylcholine (DGCC) at 21 min, and many times the loss of hexoses were
present, indicating a wide range of IPLs with a sugar head group.
Figure 3: Full MS spectra of target compounds in Bligh-Dyer extracted biomass, measured with the Orbitrap-MS: (a) 1-(O-
Hexose)-3,25-hexacosanediol (1); (b) 1-(O-hexose)-3-keto-25-hexacosanol (2); (c) 1-(O-Hexose)-3,27-octacosanediol (3); (d)
1-(O-hexose)-3-keto-27-octacosanediol (4)
19
Figure 4: Orbitrap MS chromatogram a) Base peak chromatogram of BD extract, The extracted ion chromatograms of
individual HGs: b) m/z 577.4 (1), c) m/z 575.4 (2), d) m/z 605.5 (3), e) m/z 603.5 (4), f) m/z 621.5 (5), g) m/z 619.5 (6)
After confirmation that HGs were present in the extracted biomass, a separation method
was set up, using semi-preparative HPLC. The separation of the IPLs was obtained using the
aqueous Normal Phase liquid chromatography (NP-LC) conditions, using a polar stationary
phase to separate polar compounds, which is suited for separating small polar components
like IPLs.22 A DIOL-column was used for getting the polar groups of the compounds
connected to the silica, with the polar group of the stationary phase depicted in Figure 5.
The hexane in eluent A is apolar, leaving the polar compounds in the column and eluting
the apolar compounds out, which happens at the beginning of the separation when eluent A
is present at high concentrations. Eluent B is more polar as it contains water, solvating the
polar lipids in the column, which results in them eluting off the column. By slowly
increasing eluent B in the eluent A and B ratio, the polar lipids will start to elute and the
different IPLs can be collected with a fraction collector. The formic acid (pKa 3.75) and
ammonium hydroxide (pKa 9.25) were added to the eluents as a buffer, to control the mobile
phase and the ionic strength, resulting in a decrease in electrostatic interactions between the
20
polar compounds and the dissociated particles (silanol groups) from the column.23 The
formic acid and ammonium hydroxide form together ammonium formate (NH4HCO2),
which helps the analytes evaporate, due to the volatility of this adduct. The buffer is slightly
acidic with positive ions and produces only positive ions, in combination with the positive
voltage on the MS,making MS measuring easier.24
Figure 5: Stationary phase of the DIOL column
In aqueous NP-LC several interactions are observed between the compounds and the
mobile/stationary phase; the chemical interaction as hydrogen bonding and donor acceptor
interactions (between Lewis acid and Lewis base), the intermolecular interaction also known
as the Van der Waals force, the hydrophobic interactions and the physical interactions as the
Ion—Dipole, Dipole—Dipole and the Dipole—Induced Dipole.25 The mechanism for elution
of the different compounds is well understood, the most contributing forces are probably
Dipole—Dipole and hydrogen bonding and a absorption mechanism can be subscribed to
this method, whereby a competition is present between the water/IPA phase and the other
stationary DIOL column. When the concentration of the water/IPA phase is favorable the
water will take the place of the analyte molecules and the target compound will start
eluting26. The retention time is proportionate to the strength of the solvent, which is low for
hexane as the interaction with the column is zero, and high for water, the strongest normal-
phase eluent. The amount of water could affect the column, but as the target analyte
consistently eluted in the same tubes, this was not the case.
After the first round of semi-preparative HPLC the fractions 10–25 were analyzed using
HPLC-MSD with two different methods; Full Scan and Selected Ion Monitoring (SIM). The
Full scan was performed with the first semi-preparative HPLC for verification of the
presence of the target compound, which was successful and showed peaks at the correct
mass of 575.5 and 603.5 together with the expected fragmentation (Fig. I, Appendix 1).
Subsequently SIM was performed on after every semi-preparative HPLC (injections) run to
confirm at which fractions the target compounds were present. The ammonium adduct was
21
included in the SIM, present at a high concentration due to the presence of ammonium
formate, with a typical spectrum (Fig. II, Appendix 1). The target analyte was mostly found
in fractions 16–19 (Table 3). This corresponds with the used method of Sturt et al. (2004)17, as
was used by Bauersachs et al. (2009b)16, with the HGs eluting out at 18 to 20 min (fractions
18-20)
After the first semi-preparative HPLC, the two fractions with the highest signals as
determined by SIM were collected in a round bottom flask and the two surrounding
fractions were collected in another round bottom flask. Subsequently the fractions were
dried under nitrogen and the amount after one purification step was roughly determined,
although it was assumed to be inaccurate, due to the presence of ammonium formate, at 9.1
mg for fractions (17–18) and 8.6 mg for fractions (16) and (19). The fractions (16–19) were
analyzed with the Orbitrap–MS, with the resulting spectra in Figure 6 and the
chromatogram of the retention time in Figure 7. The MS2 spectra are given in Figure 8.
Table 3: The distribution of the HGs in the fractions as determined by SIM
Fractions 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
HG (1–4) — — — — — — + +++ ++ + — — — — — —
Figure 6: Full MS spectra after the first round of semi-preparative HPLC over peak (RT 18-20 min), measured with the
Orbitrap MS; (a) 1-(O-Hexose)-3,25-hexacosanediol (1); (b) 1-(O-hexose)-3-keto-25-hexacosanol (2); (c) 1-(O-Hexose)-3,27-
octacosanediol (3); (d) 1-(O-hexose)-3-keto-27-octacosanediol (4)
22
Figure 7: Orbitrap MS chromatogram a) Base peak chromatogram after semi-preparative HPLC 1. Extracted ion
chromatograms after semi-preparative HPLC 1: b) m/z 577.4 (1), c) m/z 575.4 (2), d) m/z 605.5 (3), e) m/z 603.5 (4), f) m/z
621.5 (5), g) m/z 619.5 (6)
Figure 8: Mass spectra of HGs after the first round ofsemi- preparative HPLC. (a) MS2 of the [M+H]+ of HG (2) and (b)
MS2 of the [M+H]+ of HG (1) and (c) MS2 of the [M+H]+ of HG (4) and (d) MS2 of the [M+H]+ of HG (3)
23
Regarding to the chromatogram (Fig. 7); the retention time was constant and the signals of
the HGs (5) and (6) were reduced. The signals of HGs (1) and (2) were present in a high
enough concentration that they were visible in the base peak chromatogram. After the HGs
a further peak is present, which is probably a phosphatidic acid, determined by the
observed mass loss of 80, typical for the loss of a phosphoryl group. When the base peak
chromatogram from after the semi-preparative HPLC (Fig. 7) is compared with that before
the semi-preparative HPLC (Fig. 4), a few differences could be determined; the major peaks
at 24 minutes had disappeared, as had the peaks at 10 and 34 minutes, but a new signal at 3
minutes had appeared, which is the injection peak, full of apolar compounds. The base peak
chromatogram showed the presence of other IPLs and therefore a second purification step is
required.
As the 1st round of semi-preparative HPLC was carried out in normal phase to separate the
bulk of polar lipids from the HGs, the 2nd round of semi-preparative HPLC was carried out
in reverse phase. Resulting in a different separation by using the hydrophilic or
hydrophobic properties of the analyte, making hydrogen bonding the strongest factor of
retention, as the sample partitions between the polar mobile phase and the apolar stationary
column. There are three explanations for the retention in RP-LC, the hydrophobic theory,
partitioning and absorption; the hydrophobic theory is based on the cavities that arise from
the hydrophobic groups of the analyte, and as the surface tension of the eluents increase, the
retention decreases. The partitioning theory is based on the hydrogen bonding as explained
above, and the absorption theory is based on the absorption of the solvents, but is lacking to
explain the discontinuities between the retention and the chain lengths of the silica,
therefore, the partitioning theory is the most likely.26
Figure 9: stationary phase of a C-18 column
A RP-LC method was used by Wörmer et al. (2013), for the separation of general IPLs for
MS-detection, with MeOH:H2O (85:15) and MeOH/IPA (50:50) as eluents and a C18-column
as stationary phase (Fig. 9).20 The HGs have an intermediate polarity, as they have a sugar-
24
head group and alcohol groups, but also a long apolar chain, and are expected to elute in the
middle of the chromatogram. An advantage of this method is the short time to run a full
method, 20 minutes, making it possible to take 30 second fractions for a better separation of
the HGs and the other IPLs. The fractions were analyzed with the Ion-trap MS, with an ESI
source and SIM at a mass of 577.5, resulting in a high signal at fraction 18 (9 min). This
retention time is similar to the retention time of the phosphatidyl-glycerol, which also has a
long chain and the number of alcohol groups similar to HGs, and therefore elutes first off
the column, due to it being the more polar compound.27 Therefore, the fractions between 7
and 15 minutes were collected with a time interval of 15 seconds and were checked on the
Ion-trap MS, for presence of the four most abundant HGs. This was performed using
selected reaction monitoring (SRM), whereby the MS scanned for the parent mass and the
loss of hexose (-162), resulting in the confirmation that the HGs with m/z 577 and 575 eluted
at 9.5 min, and the HGs with m/z of 605 and 603 eluted at 12 min. An excellent separation
between the two different kinds of HGs is achieved and in the following step the diol and
the keto-ol structures have to be separated, which can be achieved by performing the same
semi-preparative HPLC method several times on collected fractions. This is based on the
distribution of the different HGs (Table 4 and Table 5), with the keto-ol HG eluting before
the diol HG, thereby, collecting the fractions containing only one kind of HG will lead to
pure isolation of the compounds. Following from this distribution, the fraction containing
only HG (1) was collected together with the fraction containing only HG (2), with the
fractions containing (1) and (2) pooled together and run again. Likewise, the fraction with
alone HG (3) and the fraction containing solely HG (4) were collected and the other fractions
containing both (3) and (4) were run again.
Table 4: The distribution of the HGs in the fractions containing C26-HGs checked with the Ion-trap MS on SRM mode
Fractions 5 6 7 8 9 10 11 12 13 14 15 16 17 18
HG (1) — — — — — + ++ +++ ++ + — — — —
HG (2) — — — — + +++ +++ + — — — — — —
Table 5: The distribution of the HGs in the fractions containing C28-HGs checked with the Ion-trap MS on SRM mode
Fractions 19 20 21 22 23 24 25 26 27 28 29 30 31 32
HG (3) — — — — + ++ +++ + — — — — — —
HG (4) — — — + +++ + — — — — — — — —
25
After collecting the fractions with the highest concentration of a single compound, the four
different compounds were run separately on the Ion-trap with column chromatography, to
see if the compounds were pure. When 100 ng of the different HGs were injected, a signal
was showing in the mass spectrum of HG (1), (2) and (3), resulting in 3 spectra (Fig. 10–12)
The different HGs, showed the total mass [M+H]+, the [M+H-C6H12O5]+ signal, and the
signals corresponding to the loss of the alcohol groups on MS2. When 100 ng of purified HG
(1) was injected on column (Fig. 10), still traces of HG (2) could be observed, having a 1000
times lower intensity, proving that only traces of HG (2) are present. When 100 ng of
purified HG (2) was injected (Fig. 11), a lower intensity was observed, concluding that HG
(2) is not pure or ionizes with higher difficulty. As HG (1) and (2) are almost similar in
structure, it can be concluded that HG (2) is not purified successfully, and can only be used
as the ionization of the compound is constant. Also HG (3) and (4) were injected in an
amount of 100 ng on column, but only HG (3) gave response (Fig. 12), which is very low,
concluding that both HG (3) and (4) are not purified enough. In conclusion, only HGs (1)
and (2) are pure enough for usage, and HGs (3) and (4) need more purification, that is
difficult considering the small amount isolated, as the HGs (3) and (4) were also detected in
low concentration in the Bligh-Dyer extract and after the first semi-preparative HPLC run.
26
Figure 10: a) Base Peak chromatogram of HPLC/MS analysis of HG (1), and extracted ion chromatograms of b) m/z 577 and
c) m/z 575
Figure 11: a) Base Peak chromatogram of HPLC/MS analysis of HG (2), and extracted ion chromatograms of b) m/z 577 and
c) m/z 575
27
Figure 12: a) Base Peak chromatogram of HPLC/MS analysis of HG (3), and extracted ion chromatograms of b) m/z 605 and
c) m/z 603
In order to quantify HG in samples from the natural environment, relative response curves
were produced and a mixture of HG (1) or HG (2) and IS (n-Dodecyl β-D-glucopyranoside)
1:1 (w:w) was injected in amounts ranging from 1 to 100 ng, to examine the ionization
difference of the two compounds. When the areas of the two compounds in different
concentrations are plotted, a trend line can be drawn, of which the slopes of the trend line
give a relation of the ionizing ability. It is expected that the isolated HGs will ionize better as
the IS, because more alcohol groups are present on the HGs. The two dilution series of HG
(1) and (2) are given in Figure 14, showing that HG (1) ionizes better than HG (2), which
indicates that HG (1) is isolated in purer form.
28
Figure 14: a) The peak area’s of HG (1) and b) the peak area’s of HG (2) compared with the Internal Standard (n-dodecyl-β-
D-glucopyranoside)
Hg (1) was isolated with the highest yield (0.5 mg), which makes it possible to analyze this
compound with NMR, resulting in a 1H and Heteronuclear single quantum coherence
(HSQC) NMR spectra. HSQC makes it possible to distinguish the different hydrogen atoms
from each other, and detect the alcohol groups on the long carbon chain to make sure that an
alcohol group is present on the C-3 and the ω-2 position. The resulting spectra were unclear,
due to the amount of isolated HG (1), which lays below the limit of detection. Some peaks
could be assigned to the long carbon chain, but peaks of the sugar head group were not
identified. Nonetheless the isolated HG (1) is considered pure, based on the MS
chromatogram, with only traces of HG (2), and the response curve.
29
8 Conclusion
HGs (1-3) were successfully extracted and isolated out of the biomass (which consisted of
different cyanobacteria) by using two different separations on the semi-preparative HPLC
(Scheme 1). First a normal phase LC was performed, which resulted in the separation of the
HGS and the bulk of IPLs, followed by a reversed phase LC, separating the different kinds
of HGS. The purity of the isolated HGs were verified by MS and MS2 and response curves
were made, comparing the isolated HGs with an internal standard, n-dodecyl-β-D-
glucopyranoside. HG (1) ionizes better than the IS, due to the elongated chain and the
presence of more alcohol groups, which would also be expected when the IS is compared
with HG (2), only now the IS ionizes better, concluding that HG (2) contains impurities. To
verify the purity of HG (1) any further, NMR was performed on the sample, only the
amount of sample was below the limit of detection, resulting in incomplete spectra.
Especially for the 13C-NMR spectrum, which measures only the 13C, an isotope of carbon that
is only 1 percent of all the carbon atoms, and resulted in an unclear spectrum. The 1H-NMR
spectrum showed traces of a aliphatic chain, but the signals for the sugar-head group,
common around 3.5–4 ppm, were not present, probably because the sugar-head groups did
not dissolve in the deuterated DCM or the deuterated methanol and were still attached to
the glass.
Scheme 1: Summary of the different steps in the developed method for purifying HGs (2-5)
30
9 Future prospects
A method was developed to purify HGs (1–6), with HG (1) in the highest amount, which can
be used as an authentic standard. The next step would be to use this external standard to
quantify the heterocyst glycolipids present in sediment and oceanic samples taken by the
NIOZ. To state something about the purity of the HGs a NMR could be taken, but a higher
amount of HGs is desired. After the quantification of the HGs with the produced authentic
standard, models of the nitrogen fixation rate could be made, creating models of the past
oceanic nitrogen fixation. Subsequently the nitrogen fixation rate could be used to predict
the future nitrogen fixation, making it possible to predict the future atmospheric
composition.
10 Acknowledgement
I would like to thank Prof. Dr. Ir. Stefan Schouten from the NIOZ, who made it possible to
perform this research. I would also like to thank Dr. Ir. Ellen C. Hopmans and Dr. Nicole J.
Bale for helping me, guiding me and teaching me about the techniques used in this research.
Furthermore I liked to thank Prof. Dr. Jan van Maarseveen for being my second reviewer,
and making it possible to analyse my sample on the NMR, which was performed by Ed
Zuidinga en Jan–Meine Ernsting. I also would like to thank all the people who made me feel
very welcome on the NIOZ, and who made my research time at the NIOZ enjoyable.
31
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12 Appendix
Figure I: SIM MSD chromatogram after the 1st round of semi-preparative HPLC; fractions 10–25, a) base peak
chromatogram, b) pressure profile of each injection, c-f) extraction ion chromatograms of individual HGs
Figure II: Full MS spectrum after the 1st round of semi-preparative HPLC; fraction 17, integrated over the peak.