STUDIES INVOLVING POTENTIAL CHEMICAL ATTRACTANTS
FROM RUDBECKIA HIRTA INFLORESCENCES
A THESIS
SUBMITTED TO THE GRADUATE SCHOOL
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE
MASTER OF SCIENCE
BY
ASHLEY N. SIMPSON
DEPARTMENT OF CHEMISTRY
Committee Approval:
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Committee Chairman
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Committee Member
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Committee Member
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Committee Member
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Date
______________________________________
Head of Department
_________________
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______________________________________
Dean of Graduate School
_________________
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BALL STATE UNIVERSITY
MUNCIE, INDIANA
SUMMER 2010
STUDIES INVOLVING POTENTIAL CHEMICAL ATTRACTANTS
FROM RUDBECKIA HIRTA INFLORESCENCES
A THESIS
SUBMITTED TO THE GRADUATE SCHOOL
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
MASTER OF SCIENCE
BY
ASHLEY N. SIMPSON
ADVISOR – PATRICIA L. LANG
BALL STATE UNIVERSITY
MUNCIE, INDIANA
JULY 2010
Acknowledgements
I would like to thank Dr. Patricia L. Lang for allowing me to work with her on
this research project. She has taught me to have confidence in myself and in my abilities
as a chemist, and that is something I will carry with me throughout my life. She has
taught me to hold myself to high standards by seeking perfection in my work. Her
knowledge as a chemist and experience as a mentor were never unnoticed and greatly
appreciated.
I would like to thank Dr. Gary N. Dodson, Dr. James S. Poole, and Dr. Tykhon
Zubkov for taking the time to serve on my thesis committee. They were always there to
answer my questions and provide advice that was invaluable to this research project.
I would also like to thank my parents for teaching me to work hard and never give
up on my goals. Without my family’s continuous encouragement, love, and support, I
would not be who I am today. They have taught me to stay true to my morals and values,
and never change who I am for someone else. I am forever grateful to them for always
being there for me when I needed them most.
Lastly, I would like to thank my fiancé, Luke Versprille, for his continuous
encouragement, love, and helping me escape from chemistry at times when I needed it
most. He is truly my best friend, and I do not know what I would do without him by my
side. I greatly appreciate his support and always pushing me to my full potential.
iii
TABLE OF CONTENTS
Page
LIST OF TABLES………………………………………………....
vi
LIST OF FIGURES...........................................................................
vii
ABSTRACT……………………………………………………......
ix
Chapter 1
Introduction and Background......................................................
1
1.1
Purpose of Research.....................................................................
1
1.2
Experimental Approach for Bioassay Trials……………………
1
1.3
Results from Previous Researcher. …………………………….
4
1.4
Similar Studies in the Literature...………………….………......
10
1.5
Definition of Cuticular Wax……………………………………
11
1.6
Ideas for Removing Cuticular Wax ……………………………
12
1.7
Gas-Liquid Chromatographic Methods …………..……………
13
1.8
Questions To Be Answered…………………………………….
13
Olfactory Bioassay Studies of M. formosipes…………………..
15
2.1
Experimental……………………………………………………
16
2.2
Results and Discussion.................................................................
18
Chapter 2
Chapter 3
Chromatographic Separation of Components in the 100%
Dichloromethane Fractions..........................................................
iv
21
3.1
Preliminary Experimental............................................................
22
3.2
Flash Column Chromatography-Experimental……..……...……
23
3.3
Results and Discussion…………………………………………..
24
3.4
Solid Phase Extraction-Experimental…………...…….…………
26
3.5
Results and Discussion…………………………………………..
26
3.6
Modified Solid Phase Extraction-Experimental………………....
28
3.7
Results and Discussion…………………………………………..
29
Chapter 4
Identification of the Possible Attractants in the 100%
Dichloromethane Fractions Using Spectroscopic Methods….....
31
4.1
Spectroscopic Results of the 0.7 Rf Component……………..…
31
4.2
Infrared Spectroscopy Results of the 0.2 Rf Component…….…
39
4.3
Infrared Spectroscopy Results of the Component Identified
from the Methanol Wash………………………………………..
42
Flash Column Chromatography “Blank”………….……………
45
4.4.1
Experimental…………………………………………….
45
4.4.2
Results and Discussion…………………………………..
45
Solid Phase Extraction “Blank”…………………………………
46
4.5.1
Experimental…………………………………………….
46
4.5.2
Results and Discussion………………………………….
46
Conclusions..................................................................................
47
List of References……………………………………………………………….
50
4.4
4.5
4.6
v
LIST OF TABLES
Table
Page
2.1
Summary of Bioassay Trial Results from Summer 2008 and 2009.........
3.1
Thin Layer Chromatography Rf Values for Different Solvent
Fractions...................................................................................................
vi
20
25
LIST OF FIGURES
Figure
Page
1.1
Flowchart of Experimental Approach……………………………….….
3
1.2
Infrared Spectrum of Bulk R. hirta Extract Collected on 8/10/08……...
6
1.3
Infrared Spectrum of Bulk R. hirta Extract Collected on 8/20/08……...
7
1.4
Infrared Spectrum of 100% Dichloromethane Fraction 45……………..
9
1.5
Stem Testing Arena for Bioassay Trials Performed on Adult Beetles.....
10
2.1
Interface of Airstreams from Each Arm of the Y-tube………………....
17
3.1
Solid Phase Extraction Technique……………………………………....
22
3.2
Thin Layer Chromatography Results for Solid Phase Extraction…...….
27
3.3
Color of Solution in Each Vial Obtained From Solid Phase
Extraction…………………………………………………………...…..
3.4
TLC Results for Modified Solid Phase Extraction of Pooled
0.2 Rf Fractions........................................................................................
3.5
30
TLC Results for Modified Solid Phase Extraction of Methanol Wash
Fractions...............………………………………………………………
4.1
28
30
Proton NMR of Fraction 0.7 Rf Component from SPE of 100%
Dichloromethane Pooled Fractions……….….........................................
vii
33
4.2
Infrared Spectrum of 0.7 Rf Component from SPE of 100%
Dichloromethane Pooled Fractions...……….………………………......
36
4.3
Infrared Reference Spectrum of Di(2-ethylhexyl) Phthalate…………...
37
4.4
Proton NMR Predictor Spectrum of Di(2-ethylhexyl) Phthalate…….....
38
4.5
Infrared Spectrum of 0.2 Rf Component……….…………………….....
40
4.6
Infrared Reference Spectrum of Microcrystalline Hydrocarbon Wax.....
41
4.7
Infrared Spectrum of Component Identified in Methanol Wash…….....
43
4.8
Infrared Reference Spectrum of Erucamide……………………………
44
viii
ABSTRACT
THESIS:
Studies Involving Potential Chemical Attractants from Rudbeckia
hirta Inflorescences
STUDENT:
Ashley N. Simpson
DEGREE:
Master of Science
COLLEGE:
Sciences and Humanities
DATE:
July 2010
PAGES:
52
Our research involves the isolation and identification of the possible chemical
compounds in black-eyed Susans that may be responsible for the olfactory attraction of
the crab spider Misumenoides formosipes to the inflorescences of these plants.
In olfactometric bioassays, 80% of 30 male spiders moved towards olfactory-only
cues from R. hirta inflorescences over a water control (P = 0.0014). The bulk extract was
separated using flash column chromatography (silica column) with a series of solvents.
Spiders in olfactometer bioassays showed a significant preference for the fractions
collected using 100% dichloromethane over the solvent-only control (P=0.039).
The 100% dichloromethane pooled fractions were separated using solid phase
extraction (SPE). Three compounds were isolated and identified using TLC, infrared and
NMR spectroscopy. Two compounds were identified as contaminants, di(2-ethylhexyl)
phthalate and erucamide, found in the flash column chromatography apparatus and SPE
ix
apparatus, respectively. A long-chain crystalline hydrocarbon wax was extracted from R.
hirta inflorescences. Research shows that several insects use the lipids of the wax layer,
specifically various long-chain alkanes and alcohols, as cues in host plant selection or as
kairomones, chemical cues used in communication from one organism to another [3]. It
also shows that the waxes can act as absorbents or release agents for biologically active
material. Thus, the long-chain hydrocarbon wax interacting with the volatile components
could play a major role in attracting the male crab spiders to the R. hirta inflorescences.
x
Chapter 1
Introduction and Background
1.1 Purpose of Research
Our research in conjunction with Dr. Gary N. Dodson in the Biology Department
involves the isolation and determination of the possible chemical compounds in blackeyed Susans Rudbeckia hirta that may be responsible for the attraction of the crab spider
Misumenoides formosipes to the inflorescences of this plant.
1.2 Experimental Approach for Bioassay Trials
The research goal is to determine if the crab spider M. formosipes has an olfactory
response to R. hirta inflorescences, and the experimental approach is shown in Figure
1.1. If the crab spiders show a significant preference for the inflorescences as the
attractant in the olfactory bioassay trials then this would lead to bioassay trials performed
on the bulk extract components which were extracted from the inflorescences. Any
observed M. formosipes preference for the bulk extract over the control would result in
the extract being subjected to flash column chromatography to separate the components
using a series of solvents. Pooled fractions collected together would then be tested in the
bioassay.
2
Lastly, if M. formosipes males showed an attraction to one or more of the pooled
fractions, then further separation of those fractions would be necessary. By this process,
we hope to isolate and identify one or more potential chemical attractants in the
inflorescences of R. hirta.
3
= Spider bioassay trial
= Inflorescence
Figure 1.1: Flowchart of Experimental Approach
(Reproduced from Reference 1)
4
1.3 Results from Previous Researcher
A previous researcher [1] in our group performed olfactometer bioassay trials in
the summer of 2008 in which a significant proportion (15 of 19) of male crab spiders
moved towards olfactory-only cues from R. hirta inflorescences over a water control,
(exact binomial probability= 0.019, 2-tailed). An ultrasonic-assisted extraction was then
performed on R. hirta inflorescences using a hexane/diethyl ether mixture. Twelve of 17
male crab spiders in olfactometer bioassays moved towards the bulk extract from R. hirta
inflorescences; a significant preference (P=0.14) over a solvent-only control. Finally, the
bulk extract was separated using a silica column and a series of solvents. Again,
significantly more (8 of 9) male spiders moved towards the 100% dichloromethane
pooled fractions over the solvent-only control (P=0.039). In summary, the male crab
spiders chose the floral chemistry over the controls in all three versions of the
olfactometer trials.
One of the interesting results from many chemical analyses performed in her
study was that the infrared spectra of the ultrasonic bulk extracts from R. hirta
inflorescences revealed different amounts of esters and ketones [1]. The infrared spectra
in Figures 1.2 and 1.3 were taken of bulk extracts extracted on two different dates. In
Figure 1.2, the carbonyl stretch for an ester at 1736 cm-1 is more intense than the ketone
carbonyl stretch at 1707 cm-1. However, in Figure 1.3 the ketone carbonyl is more
intense than the ester carbonyl stretch. Also, notice the spectrum in Figure 1.2 shows a
weak O-H stretch at 3401 cm-1, but this alcohol group is not present in Figure 1.3.
5
These data suggest that individual plants of the same species can have some
inconsistencies in the quantity and types of volatile compounds. These differences can
possibly be attributed to slight differences in extraction procedure, slight differences in
flash column chromatography separation technique, plant defense mechanisms,
atmospheric CO2 levels, soil and air humidity, temperature, light intensity, age of plant,
and plant-plant interactions [2].
cm-1
Figure 1.2: Infrared Spectrum of Bulk R. hirta Extract Collected on 8/10/08
(Reproduced from Reference 1)
6
cm-1
Figure 1.3: Infrared Spectrum of Bulk R. hirta Extract Collected on 8/20/08
(Reproduced from Reference 1)
7
8
Another important finding [1] is that a long-chain hydrocarbon was the main
component isolated in the 100% dichloromethane fraction after the extract from the
inflorescences had been separated using flash column chromatography. The hydrocarbon
present in the infrared spectrum of the isolated fraction 45 (Figure 1.4) could be cuticular
wax from the plant. Notice the intense C-H stretching absorptions compared to the
relatively weak carbonyl stretches, which collectively indicate the presence of a longchain hydrocarbon.
Unfortunately, the previous research indicated that it was not easy to separate the
hydrocarbon from the volatile components that are likely to be the attractants. It is not
likely that this is the volatile component, and it seems to be eluted with the volatile
compounds, making spectral identification and chromatographic separating difficult. If
the previously isolated long-chain alkanes are a main component of cuticular wax, one of
the research goals will be to separate the long-chain hydrocarbon from the volatile
components.
cm-1
Figure 1.4: Infrared Spectrum of 100% Dichloromethane Fraction 45
(Reproduced from Reference 1)
9
10
1.4 Similar Studies in the Literature
Heisswolf et al. studied the response of an adult beetle to extracts from its host
plant in a testing arena where the attractant and control were dropped onto cigarette filters
(simulating stems as shown in Figure 1.5) [3]. The time the beetles spent on the
attractant or control stems was recorded continuously for 8-10 minutes.
Figure 1.5: Stem Testing Arena for Bioassay Trials Performed on Adult Beetles
(Reproduced from Reference 3)
11
The beetles showed a significant preference for the n-hexane extract over the
control, but no preference for the other two extracts (dichloromethane and methanol) [3].
Given the positive response in the n-hexane extract, they separated the extract using a
silica solid-phase extraction column and eluted separate fractions in (A) 100% n-hexane,
(B) 90% n-hexane+10% dichloromethane, (C) 50% n-hexane+50% dichloromethane, (D)
90% dichloromethane+10% methanol, and (E) 100% methanol. Beetle preferences were
then tested for these fractions with the same arena methodology. The fractions A-E of
the n-hexane extract did not reveal a significant response; however, a slight tendency of a
preference was observed for fractions A (100% n-hexane) (P=0.289) and C (50% nhexane, 50% dichloromethane) (P=0.196). The beetles also showed a significant
preference for a mixture of fractions A and C over the control. The researchers
speculated that typical components of cuticular waxes were present in the n-hexane
extract.
1.5 Definition of Cuticular Wax
The beetle study results are relevant to current research since the spectral
evidence from a past researcher indicated that long-chain alkanes may be present in the
pooled fractions to which the spiders were attracted. The cuticle covers aerial parts of
higher plants (i.e., stems, leaves, petals, and fruits) and the cuticular waxes are a complex
mixture of long-chain aliphatic and cyclic compounds [4].
Muller and Riederer [3] reported that the aliphatic fraction of plant cuticular
waxes consists of a complex mixture of compounds having a hydrocarbon backbone with
12
21 to >40 carbon atoms. Smaller proportions of alcohols (C22-C40), fatty acids (C20-C24),
aldehydes (C24-C36), ketones (C21-C35), and n-alkyl esters (C36-C60) are present. Various
amounts of cyclic compounds, cinnamic acid derivatives, flavonoids, and their respective
glycosides, may also be present in the wax.
1.6 Ideas for Removing Cuticular Wax
Our group has several different approaches for removing the cuticular wax from
the bulk extract. In the experimental approach described herein, we performed an
ultrasonic-assisted extraction on the inflorescences of R. hirta using 1:2 hexane to diethyl
ether solution as the extracting solvent. Next, a bioassay was performed on the bulk
extract in order to determine if the chemical components could be possible attractants.
Therefore, one approach is to try to eliminate the wax from the bulk extract by using
dichloromethane as the extraction solvent, rather than 1:2 hexane to diethyl ether mixture.
Another clean-up procedure that might be useful was reported by Schinor et al.
[5] in which they studied the ultrasound-assisted extractions of steroids and triterpenoids
from stem, leaves, and flowers of three Chresta species. A 10 mg aliquot of each hexane
extract was re-suspended in analytical grade chloroform (3 ml) and percolated through a
sep-kap column. The column was eluted with hexane (10 ml) and chloroform (10 ml).
Fractions were collected separately and evaporated to dryness at room temperature.
Chloroform phases were analyzed by high resolution gas chromatography in duplicate.
Another option is to use a reverse-solid phase extraction; thereby, eliminating the
long -chain hydrocarbon from the eluate. In the research by Vazquez et al. [6], solid-
13
phase extraction followed by gas chromatography coupled to mass spectrometry was
used to analyze the volatile compounds in eucalyptus honey. Lastly, one could try using
a cold finger trap and distill any volatiles away from the wax.
1.7 Gas-Liquid Chromatographic Methods
Once the cuticular wax is removed from the volatile compounds that are potential
attractants, then we must identify the components in the dichloromethane fractions that
showed attraction using various spectroscopic and chromatographic methods. In addition
to infrared and NMR spectroscopy, GC/MS can be used to help identify the compounds.
The column currently in our GC and GC/MS that could be used is a fused silica capillary
column with 5%-phenyl-dimethypolysiloxane/ (DB-5) film.
Alternatively, one could use a different column, such as that reported by Cisowski
et al. [7] in which their group used a Carbowax 20M column to analyze fatty acids
obtained from the fruit of Asteraceae plants. A carbowax column is a polar column
suitable for analyses of alcohols, amines, carboxylic acids, diols, esters, ethers, ketones,
and thiols. A carbowax column may be needed in order to achieve better separation in
the volatile compounds.
1.8 Questions To Be Answered
The research reported in this thesis addressed most of the following questions that
remained after our group’s preliminary experiments.
Does the whole inflorescence of the R. hirta exhibit strong attraction?
14
Is it specifically the chemistry of the R. hirta plant that attracts the spiders to
them, or could it be any plant? For example, are the spiders attracted to mulberry
leaves?
Are the spiders attracted to the overall chemistry of a R. hirta plant, or is it
specifically something in the inflorescence? For example, are the spiders
attracted to the leaves of the R. hirta?
What volatile compounds are in the dichloromethane fraction?
Is there a way to remove the cuticular wax during the extraction or after the
extraction?
Do the R. hirta inflorescences contain the same chemistry as other species of
Rudbeckia where crab spiders have been found such as R. triloba?
How is the chemistry different in the leaves vs. inflorescences?
Regardless of their first choice of olfactometer arms, do the male spiders spend
more time in the treatment arm than in the control arm?
Do the male spiders spend more time in their first choice arm than in their second
choice?
Chapter 2
Olfactory Bioassay Studies of M. formosipes
Olfactory bioassay trials using the inflorescences of R.hirta were performed
during the summer of 2009 to determine if the crab spiders showed an attraction to
the inflorescences in the same manner as the previous summer. This was deemed an
important confirmation for two reasons. The sample size of trials from 2008 alone
was considered too small for complete confidence in the interpretation. We also
wished to confirm that the experimental apparatus was operating in the same manner
as the previous year.
Likewise bioassay trials using mulberry (M. rubrus) leaves needed to be
increased in number from the previous summer’s trials. These trials were performed
to test whether M. formosipes males were as attracted to another type of organic plant
matter as they were to R. hirta inflorescences.
For the first time within this research program, leaves from R. hirta plants
were tested as a potential attractant. The purpose was to test the hypothesis that M.
formosipes males would be attracted to volatiles from any part of R. hirta plants and
not just the inflorescence.
16
2.1 Experimental
Y-tube olfactometer bioassay trials using parameters reported by Judkins [1]
commenced on July 30, 2009 and concluded on August 13, 2009, spanning the
majority of M. formosipes’ mating season. Male crab spiders, R. hirta inflorescences,
R. hirta leaves, and M. rubrus leaves were collected daily from the area of an
established M. formosipes population within the Cooper Farm wildlife area (Muncie,
IN) and used within 24 h after collection.
In each bioassay trial, 2 mL of distilled water was added to a 150 mL flask
along with one of the following: an entire inflorescence from R. hirta, a single M.
rubrus leaf, or two R. hirta leaves. Concerted effort was made to use roughly
equivalent volumes of the plant tissues for each trial. The attractant flask was then
connected to one arm of the olfactometer. A control flask containing 2 mL of
distilled water alone was then connected to the opposite arm. A single adult male M.
formosipes was released into the bottom of the y-tube apparatus and allowed freedom
of movement for the duration of the trial.
All bioassay trials lasted 10 h and three trials were run simultaneously in
identical olfactometer set-ups. A set of trials was started each morning and another
each evening, and every trial was recorded using a video camera. From the digital
recordings, the following intervals were acquired: 1) the time at which the initial
choice was made to enter the attractant or the control arm, and 2) the time for any
subsequent moves out of and/or into either arm. This allowed for the total time spent
17
in the attractant and the control arms (as well as in the central tube) to be calculated
for future reports. In order to qualify as time spent in an arm, the spider had to have
crossed the interface of the air streams as shown in Figure 2.1. The y-tube was
washed with a soapy solution, rinsed, and over dried at a temperature of 55 degrees
Celsius between each trial [1].
Interface
of air
streams
If a spider crossed this line a
choice for the right arm was
recorded.
If a spider crossed this line a
choice for the left arm was
recorded.
Figure 2.1: Interface of Airstreams from Each Arm of the Y-tube.
(Reproduced from Reference 1)
18
2.2 Results and Discussion
Olfactory responses from the male crab spiders across treatment categories are
shown in Table 2.1. Exact binomial probabilities of obtaining the results for each
category in relation to random selection were calculated using the following equation:
n!
(pk)(qn-k)
P(k out of n) =
k!(n-k)!
where k is the number of times the spider chose the “attractant” for a particular trial
type, p is the probability that the spider would choose the “attractant” by chance (0.5),
q is the probability that the spider would choose the “control” by chance (0.5), and n
is the number of trials in which a spider made a choice [1]. A low Ρ value therefore is
indicative of a low probability that the spiders made their choice due to random
movement. A P value of less than 0.05 is considered significant.
Table 2.1 provides the y-tube olfactometer bioassay results from 2009 both in
isolation and combined with those obtained in 2008. Given the clearly similar pattern
between the 2009 results and those reported on page 4 from 2008, it is reasonable to
focus on the combined results for interpretation. M. formosipes males exhibited a
clear preference for R. hirta inflorescences over the control as their first choice of
arms. They did not, however, differ significantly from random choices of the arms
with either R. hirta or M. rubrus leaves relative to their controls. These results are
consistent with the hypothesis of Stellwag and Dodson [8] that these spiders respond
19
to chemical cues unique to the inflorescences of R. hirta (and perhaps the
inflorescences of other species in their habitat) that are not found as volatiles within
all plant tissue. Such specificity would serve as efficient navigational cues, directing
the males to more likely sites of receptive adult females [8].
20
Table 2.1: Summary of Bioassay Trial Results from Summer 2008 and 2009
Chapter 3
Chromatographic Separation of Components in the 100%
Dichloromethane Fractions
Ultrasonic bulk extracts obtained from the R. hirta inflorescences gathered from
summers of 2007, 2008, and 2009 were subjected to flash column chromatography. Thin
layer chromatography was then performed on each pooled fraction to determine if the
chemistry of R.hirta bulk extracts from different dates and years showed the same
chemistry. For example, do the same number of components elute in each pooled
fraction regardless of the date?
The 100% dichloromethane pooled fraction became the focus because it was the
fraction that appeared to be the most attractive fraction to the crab spiders [1].
Significantly, eight of nine male crab spiders chose the 100% dichloromethane pooled
fraction over the solvent control (P=0.039).
In order to find the possible attractants in the 100% dichloromethane fraction, a
separation technique was developed using solid phase extraction (SPE), as shown in
Figure 3.1. This technique was chosen because it was quicker and less expensive than
flash column chromatography, and it allowed us to get smaller vial fractions which in
turn, increased the concentration of the compounds of interest. To examine the efficiency
22
of the developed separation technique, thin layer chromatography was performed on the
vials that were collected.
Figure 3.1 Solid Phase Extraction Technique
3.1 Preliminary Experimental
The inflorescences of R. hirta were collected from Cooper Farm wildlife area
(Muncie, IN).
Ten grams of finely cut R. hirta inflorescences were used for an
ultrasonic-assisted extraction within approximately 24 hours of being collected. The
inflorescences were placed into a 125 mL Erlenmeyer flask along with 60 mL of 1:2
hexane (Fisher Scientific):diethyl ether (Sigma-Aldrich,
99.5%) solution as the solvent.
After 30 minutes the ultrasonic-assisted extract was separated from the plant material by
gravity filtration. The solvent was removed from the filtrate by using a gentle stream of
argon.
23
3.2 Flash Column Chromatography-Experimental
The bulk extracts from these summers were each separately injected into a silica
flash chromatography column (Analogix SF25-40g, Sepra Si 50, average particle size: 50
μm, pore size: 60 Å) using the solvent system developed by Judkins [1]. The flash
column chromatography method was slightly modified by collecting a total of 39
fractions which included thirteen 18 mL-fractions obtained using 95% hexane: 5%
dichloromethane, thirteen 18 mL-fractions obtained using 95% dichloromethane: 5%
hexane, seven 9 mL-fractions obtained using 100% dichloromethane, and six 9 mLfractions obtained using methanol. Lastly, the fractions obtained from each solvent
system were pooled together, for example, the 95% hexane: 5% dichloromethane
fractions were pooled; the 95% dichloromethane: 5% hexane fractions were pooled, etc.
Then, the solvent from each of the pooled fractions was evaporated off overnight. Next,
the pooled fractions were re-dissolved to ensure a consistent concentration using 3-5 mL
of the solvent used during the separation process. Finally, thin layer chromatography was
performed on each fraction (Baker-flex 2.5 × 7.5 cm, silica gel IB-F) by spotting each
TLC plate with 5 drops of the fraction. To develop the plates, 95% dichloromethane: 5%
hexane was used as the mobile phase and phosphomolybdic acid (PMA) (1:25 PMA:
95% ethanol) as the developing stain.
24
3.3 Results and Discussion
It is important to note when looking at the results that these are the same species
of inflorescences, R hirta, and these flowers were gathered on different dates and
different years. One of the most interesting results one can observe in Figure 3.2 is that
for the most part the same number of components elutes in each particular solvent system
elutions regardless of the date. For instance in the 100% dichloromethane pooled
fractions, one can observe two components at roughly the Rf distance, a spot between
0.2-0.3, and one between, 0.6-0.8. The higher Rf value component is UV active and
infrared spectra (to be discussed later) indicate that it is the same compound in each
pooled fraction. In order to separate these two components, a solid phase extraction
procedure was developed.
25
Table 3.1 Thin Layer Chromatography Rf Values for Different Solvent Fractions
26
3.4 Solid Phase Extraction-Experimental
A solid phase extraction procedure was developed which consisted of
conditioning, loading, washing, and eluting steps using a Grace Normal-Phase ExtractClean SPE column (4.0 mL silica column, bed weight: 200 mg, average particle size: 50
μm, pore size: 60 Å, catalog ID: 209250) [9]. Each step was performed at a flow rate of
about 1 mL every 2 minutes using a 5-mL plastic syringe and syringe adapter connected
to the silica column. The first step of the SPE procedure was to condition the column
using 3 mL of isopropanol followed by 3 mL of hexane. The second step was to load 1
mL of the sample (in which we dissolved the residue from the 100% dichloromethane
pooled fractions with 5 mL of dichloromethane). The third step was to wash the column
of impurities with 5 mL of hexane. Lastly, we eluted the components of interest using 5
mL of 100% dichloromethane.
A total of seven vials were collected using a silica gel column for SPE. One vial
was collected as we loaded the sample. The second vial was collected during the
washing of the column with hexane. Lastly, five vials were collected when each 1 mL of
100% dichloromethane was added to elute the different components.
3.5 Results and Discussion
The TLC plates of the contents in the seven vials collected using the SPE
procedure are shown in Figure 3.2. The TLC plates were developed using the same
technique described previously in section 3.2. The TLC results show that there could be
2 or 3 potentially different compounds which are consistent with the results from the
27
flash column chromatography. The upper spot component has an Rf value of
approximately 0.7 which is an UV active spot, and the lower spot component has an Rf
value of approximately 0.2. As one can see from the observations in Figure 3.3, the
lower spot on the TLC plate is responsible for the yellow component, and the upper spot
is responsible for the colorless component. This shows that the 0.7 and 0.2 Rf
components are in fraction 2 and 3, whereas the other vials contain only the 0.7 Rf
component. Thus, good separation was obtained in fractions 1, 4, 5, 6, and 7. It appears
that there may be two spots very close together in fraction 2 and 3 at between 0.2-0.3 Rf
values.
Figure 3.2 Thin Layer Chromatography Results for Solid Phase Extraction
28
Figure 3.3 Color of Solution in Each Vial Obtained From Solid Phase Extraction
3.6 Modified Solid Phase Extraction-Experimental
Since fractions 2 and 3 contain both the 0.2 and 0.7 Rf components, the procedure
was modified to try to separate the two components. First, the 100% dichloromethane
pooled fraction was further separated into smaller vial fractions to possibly increase the
concentration of the components of interest. Then, cyclohexane was used in place of
hexane because cylcohexane is less polar. The solvents used in conditioning the column
were changed to those used in the elution procedure, 100% dichloromethane and
cyclohexane. The elution step was changed to using 9 mL of 100% dichloromethane
instead of 5 mL of 100% dichloromethane. Lastly, a methanol elution was added to the
end of the procedure to elute anything that might remain on the column.
A total of twelve vials were collected using a silica gel column for SPE. One vial
was collected as we loaded the sample. The second vial was collected during the
washing of the column with cyclohexane. Nine vials were collected when each 1 mL of
29
100% dichloromethane was added to elute the different components. Lastly, the twelfth
vial was collected during the methanol elution.
3.7 Results and Discussion
TLC results shown in Figure 3.4 show that, in fact, good separation was obtained
using the SPE procedure developed. Fractions 5 and 6 were all colorless solutions and
UV active, and their UV spots showed up at the Rf value of 0.7. However, only one
noticeably stained spot showed up at 0.7 Rf value. The 0.7 Rf component appears to be
spread out amongst several fractions, and it is now too weak to stain. With better
separation from the SPE, it helped in isolating the 0.7 Rf component. Fraction 7, 8, 9
were all yellow solutions and showed a spot at the Rf value of 0.2. The TLC plates
showed a good separation between the 0.2and 0.7 Rf components.
However, in order to better purify and concentrate the 0.2 Rf component and
methanol wash component, all of the vials containing the 0.2 Rf component from several
SPE trials were pooled, and all of the methanol elution vials were pooled. Then, the
modified SPE procedure was repeated on each. See Figures 3.4 and 3.5.
30
Figure 3.4 TLC Results for Modified Solid Phase Extraction of Pooled 0.2 Rf
Fractions
Figure 3.5 TLC Results for Modified Solid Phase Extraction of Methanol Wash
Fractions
Chapter 4
Identification of the Possible Attractants in the
100% Dichloromethane Fractions Using Spectroscopic
Methods
NMR and/or FT-IR spectra were obtained on fractions from the SPE procedures
that showed TLC spots. Spectra were obtained on the 0.7 Rf component, the 0.2 Rf
component, and the unknown component from the methanol wash. Three components
were identified. Proton NMR spectra were obtained in d-CH2Cl2 using a Jeol 400 MHz
NMR spectrometer with 64 co-added scans. Infrared spectra were obtained using a
Perkin-Elmer Spectrum 100 with 4.00 cm-1 resolution and 1 scan. All spectra were
obtained by transmission through a thin film of the sample on a NaCl plate with the
exception of a AgCl plate used for methanol fraction samples.
4.1 Spectroscopic Results of the 0.7 Rf Component
The NMR spectrum of the colorless component with 0.7 Rf value was obtained
from fraction 4, (vial #4 described in sections 3.4 and 3.5) and is shown in Figure 4.1.
The spectrum showed peaks between 7.5-7.7 ppm which are assigned to aromatic
protons. The peak around 7.2-7.3 ppm is from the NMR solvent, deuterated chloroform.
The peaks at 4.2 ppm region are assigned to protons next to an ester group (R-CO2-CH2).
32
The resonances at 0.8 ppm are in the methyl region, and those at 1.3 ppm are in the
methylene region. The peak at 1.7 ppm is assigned to the proton on a C-H group.
Figure 4.1: Proton NMR of 0.7 Rf Component from SPE of 100% Dichloromethane
Pooled Fractions
33
34
The infrared spectrum of the 0.7 Rf component which is a colorless, UV active
component is shown in Figure 4.2. One may observe that the relative intensities of the
aliphatic stretches to the carbonyl stretch are about the same. This indicates that the
spectrum is not overwhelmed by a long-chain hydrocarbon component that a previous
researcher had found which is shown in Figure 1.4 [1].
The infrared spectrum showed a methyl asymmetric stretching frequency at 2958
cm-1, methylene asymmetric stretching frequency at 2928 cm-1, and then the methyl and
methylene symmetric stretching frequencies are overlapping at 2872 cm-1. There is also a
peak around 3072 cm-1 which is the C-H stretching on an unsaturated carbon. The peak
at 1729 cm-1 is an ester C=O stretch. The peak at 1286 cm-1 is the C-O stretching in the
ester which shows we could have an aryl conjugated ester such as benzoate or phthalate.
Finally, there are C=C stretches around 1580 and 1600 cm-1 for the aromatic ring
stretching.
The infrared spectrum of 0.7 Rf component look exactly band for band, including
peak frequencies and relative intensities, like the infrared spectrum of the compound
shown in Figure 4.3. This compound is di(2-ethylhexyl) phthalate which is a common
plasticizer.
This is consistent with the NMR spectrum. The 0.8 ppm peaks can be assigned to
methyl protons and the methylene protons on the third carbon of the hexyl group. The
peak at 1.3 ppm is assigned to the rest of the methylene protons. The NMR spectrum
also matches the predictor spectrum of di(2-ethylhexyl) phthalate [11] which is shown in
35
Figure 4.4. The proton ratios are consistent with assigned. For example, the integration
of the 1.3 ppm peak to the 0.8 ppm peak is 0.87 to 1.21 which is very close to a 3:4 ratio
expected for di(2-ethylhexyl) phthalate. Also, the integration of the 7.5-7.7 ppm peaks to
the 4.3 ppm peaks is 0.18 to 0.19 which is very close to a 1:1 ratio expected for di(2ethylhexyl) phthalate.
Figure 4.2: Infrared Spectrum of 0.7 Rf component from SPE of 100% Dichloromethane Pooled
Fractions
36
Figure 4.3: Infrared Reference Spectrum of Di(2-ethylhexyl) Phthalate
[10]
37
[11]
Figure 4.4: Proton NMR Predictor Spectrum of Di(2-ethylhexyl) Phthalate
38
39
4.2 Infrared Spectroscopy Results of the 0.2 Rf Component
The infrared spectrum of 0.2 Rf component is shown in Figure 4.5. It is a light
yellow oily solution. This spectrum showed the methylene asymmetric stretching
frequency at 2917 cm-1 and the methylene symmetric stretching frequency at 2849 cm-1.
There are also peaks at 1472 and 1463 cm-1 which are assigned to methylene bending
frequencies. The peaks at 730 and 719 cm-1 are methylene rocking frequencies. The
doublet splitting of the methylene rocking and bending frequencies are due to the longchain crystallinity [10].
The 0.2 Rf component spectrum looks exactly band for band, including peak
frequencies and relative intensities, like the infrared spectrum of the compound shown in
Figure 4.6, a microcrystalline hydrocarbon wax. As the flash column chromatography
tubing is made of Teflon, a cuticular wax has actually been isolated and extracted from
the R. hirta inflorescences.
Figure 4.5: Infrared Spectrum of 0.2 Rf Component
40
Figure 4.6: Infrared Reference Spectrum of Microcrystalline Hydrocarbon Wax
[10]
41
42
4.3 Infrared Spectroscopy Results of the Component Identified from the Methanol
Wash
The infrared spectrum of the compound identified from the methanol wash is
shown in Figure 4.7. This spectrum showed the methylene asymmetric stretching
frequency at 2922 cm-1, and the methylene symmetric stretching frequency at 2852 cm-1.
There are also peaks at 3359 and 3192 cm-1 which are the NH2 asymmetric and
symmetric stretching frequencies, respectively. There is a peak around 3003 cm-1 which
is assigned to a C-H stretching absorption on an unsaturated carbon. The doublet near
1660 and 1633 cm-1 involves the C=O stretch and NH2 deformation, respectively [12].
There is also the methylene bending frequency around 1469 cm-1. Lastly, the peak at
1411 cm-1 is the C-N stretch.
The infrared bands of the unknown component identified from the methanol wash
match the frequencies of those in the infrared spectrum of erucamide, a fatty acid
derivative used in the most common slip agents. Figure 4.8 shows the infrared spectrum
of erucamide. The unknown compound could also possibly be oleamide, another
common slip agent with a near identical infrared spectrum. The only different between
erucamide and oleamide is that oleamide (C18H35NO) has 18 carbon atoms and erucamide
(C22H43NO) has 22 carbon atoms; both contain one double bond.
Figure 4.7: Infrared Spectrum of Component Identified in Methanol Wash
43
Figure 4.8: Infrared Reference Spectrum of Erucamide
[13]
44
45
4.4 Flash Column Chromatography “Blank”
A “blank” flash column was necessary to run due to the identification of a
phthalate using thin layer chromatography, NMR spectroscopy, and infrared
spectroscopy in the first flash column chromatography separation. It was important to
determine whether the phthalate came from contamination off the column, or if it was
actually extracted from the R. hirta inflorescences in which the plant’s root had possibly
absorbed the plasticizer from its environment [14].
4.4.1 Experimental
The same solvent system described previously was used to run a “blank” on the
flash column [1]. No sample was loaded onto the column. The same procedure was used
as before in which thirty-nine total fractions were collected as described in section 3.2.
4.4.2 Results and Discussion
The TLC plates showed that there were UV and stained spots between Rf values
of 0.6 -0.8 for each of the four solvents used in the flash column chromatography.
Infrared spectra obtained on pooled fractions from each solvent system matched that in
Figure 4.2 and 4.3. The unknown compound has been identified as di(2-ethylhexyl)
phthalate. This compound is a well-known plasticizer which shows it is clear that there is
a contaminant from the flash column chromatography apparatus. Infrared spectra were
obtained from each solvent (95% hexane: 5% dichloromethane, 95% dichloromethane:
46
5% hexane, 100% dichloromethane, and methanol) used in the flash column
chromatography to check for any contaminant; no contaminants were found.
4.5 Solid Phase Extraction “Blank”
A “blank” SPE column was performed due to the identification of a phthalate
plastic contaminant.
4.5.1 Experimental
The same procedure described previously in section 3.6 was used to run a “blank”
on SPE column. No sample was loaded onto the column, and 12 total vials were
collected.
4.5.2 Results and Discussion
The TLC plates showed a stained spot from the contents of fraction 12 which was
obtained during the methanol elution. An infrared spectrum confirmed that it was
identical to Figure 4.7 and 4.8 which indicated that the erucamide was a contaminant
coming from the SPE apparatus.
The TLC also showed weak spots at about 0.3 Rf value in fractions 7-10. The
identity of this compound was not conclusive, but the infrared spectrum did not match
that of the long-chain microcrystalline hydrocarbon wax. In conclusion, the
microcrystalline hydrocarbon wax was confirmed to not be a contaminant from the SPE;
therefore, in fact, the wax was actually extracted from the R. hirta inflorescences.
47
Infrared spectra obtained from the bulk extracts of R. hirta inflorescences from each
summer showed evidence of a long-chain hydrocarbon wax.
4.6 Conclusions
The olfactometry data show strong evidence that the male crab spiders, M
formosipes are attracted to R. hirta inflorescences, the bulk extracts, and the 100%
dichloromethane pooled fractions. A long-chain crystalline hydrocarbon wax was
successfully isolated and identified from R.hirta inflorescences, as shown in Figure 4.5
and 4.6. This finding is consistent with reports of long-chain hydrocarbon compounds
extracted from Echinacea species [15]. In addition, there were two contaminants isolated
and identified: di(2-ethylhexyl) phthalate from the flash column chromatography
apparatus and erucamide from the SPE apparatus.
Although the phthalate is a common contaminant, in general, it is one that has
been over-looked by previous researchers. In a 2005 publication [14], the di(2ethylhexyl) phthalate was isolated from the petroleum ether extract from roots of
Eupatorium odoratum L. (Asteraceae). The researchers reported that it was the “first
time ever being isolated” from this plant [14]. However, with the phthalate clearly
showing up in our experimental apparatus, we speculate that this compound might have
been a contamination in their apparatus, too, rather than a compound extracted from the
plant’s roots for the first time.
In our research, the volatile compounds in the R. hirta inflorescences have been
difficult to isolate and identify because they seem to stay with the cuticular waxes. The
48
volatile components seem to also be less concentrated than the long-chain hydrocarbon
wax. Volatile compounds can be released from plant tissues and accumulate in the
epicuticular waxes of a plant [4]. Research shows that several insects use lipids of the
wax layer, specifically various long-chain alkanes and alcohols, as cues in host plant
selection; however, in some species, lipids act only as kairomones which act as chemical
cues used in communication from one organism to another [4]. Lastly, “the waxes which
act as absorbents or release agents for biologically active material in the chemical
ecology have been underappreciated because the interaction of epicuticular wax
components with exogenous and endogenous substances make the plant surface a
chemical mosaic of signals that may evoke reactions in potential receivers such as insect
herbivores, carnivores, or microorganisms” [4]. Thus, the long-chain hydrocarbon wax
interacting with the volatile components could play a major role in attracting the male
crab spiders to the R. hirta inflorescences.
One suggestion to improve the chances of isolating the volatile components might
be to use 50-100 grams of R. hirta inflorescences instead of 10 grams in the ultrasonicassisted extraction step thus, making the bulk extract more concentrated and perhaps the
identification possible. Another suggestion might be to eliminate using the flash column
chromatography altogether and just subject the bulk extract to SPE because this could
eliminate using large amounts of solvents and increase the concentration. Lastly, one
might suggest using liquid nitrogen to pulverize the inflorescences rather than finely
cutting them thus, possibly releasing more chemicals from the inflorescences.
49
Although the phthalate is definitely from the flash column chromatography
apparatus, future researchers will need to reconfirm that the phthalate is coming from the
tubing. If the tubing is in fact causing the contamination then the tubing needs to be
replaced with new tubing which is more resistant to the solvent series. Lastly, a future
researcher should perform the SPE using a glass syringe instead of a plastic syringe to see
if the unidentified contaminant in the SPE apparatus and/or the erucamide is coming from
the syringe or the SPE column itself. Although the erucamide being in the methanol
wash is not as much of a concern since the erucamide has been identified as a
contaminant and elutes in the methanol wash.
Finally, the olfactory bioassay trials that were videotaped from summer 2008 and
2009 are in the process of being analyzed by others to help answer some of the questions
after preliminary experiments. Specifically, keeping track of the trial type, whether the
trial started during the day or night, the position of the attractant whether left or right arm,
the time in which the trial began (when the spider was first introduced into the
olfactometer), the time the spider made its first choice, the latency (difference between
spider’s first choice and time the trial began), whether spider chose attractant or control,
and time spent in attractant arm vs. control arm. Again, the visual data is in the process
of being converted into these values, and then a Student’s t-test, which assesses whether
the means of two groups are statistically different from each other, will be used to address
some of the questions in section 1.8.
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Inflorescences.” MS Thesis. Ball State University, (2009).
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by Gas Chromatography Coupled to Mass Spectrometry.” European Food Res
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11. Proton NMR Predictor Spectrum of Di (2-ethylhexyl) phthalate.
<http://www.chemexper.com/chemicals/supplier/cas/103-23-1.html>
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52
13. Infrared Reference Spectrum of Erucamide. FT-IR, Perklin Elmer. Peak Match
Hit List from Spectra Library: Euclidean Search Hit Lit, LB0131, 92%.
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