Using Raman Spectroscopy to

investigate the ackee (Blighia

Sapida) seed for Hypoglycin and
other chemicals.

Author: T. Strachan
Supervisor: Dr. K. Edward

Author Information
Name: Theodore S. Strachan
ID Number: 620033391
Major: Medical Physics

Author Note
This report was prepared for the course Phys2200 Practices in
Med. Physics 1, University of the West Indies Mona, Science &
Technology Faculty; Physics Department
Date of Submission: April 25 2014

History of Raman Spectroscopy

Raman spectroscopy was first proposed in1923 by Alfred Smekal, it was based on the idea of
the inelastic scattering of light[2,3]. It was later proved and verified by CV Raman and
Krishna in 1928[2,3-1]. He received the noble prize later in 1930 for this work[2,3-2]. Raman
spectroscopy has since flourished with advancements in tissue, cell and diseases study;
especially considering its advantages against the in vivo1 fluorescence techniques .[4] Some
of these advancements include the use near-infrared Fourier transform (NIR-FT)2 in the
regions of (780nm-900nm) and more recently CCD(charged-couple device) systems allow
faster scan times.[4-1]

Abstract

The problem that we are faced with is obtaining a Raman spectra that can be utilized to
determine chemicals in our sample of the seed of the Blighia Sapida1 (ackee). Tests have been
done on the ackee seed prior to this research publication [1] but never before with the use of an
advanced Raman system. What this research seeks to investigate is if we do have Raman signals
can we discern a chemical such as hypoglycin from the spectra and if not what does the spectra
indicate. The experiment was done on several ackees and from the spectra chemicals were
discovered but nothing concrete that depicted the presence of Hypoglycin.

1.
2.

In vivo stands for invasive studies of biological samples(living samples)

The Fourier transformation is named after Joseph Fourier which made a transfer between spatial time and
frequency possible. It has many applications in physics and engineering.

Introduction

Raman spectroscopy is a useful tool for us to examine the nature of any small substance or
object. Even so it is not just limited to these uses as it has a wide range of applications across
Biology, Chemistry and Physics as well as any combination of those fields. In this case we are
using this system to investigate the Hypoglycin content and any other chemical that could help to
improve the knowledge of the ackee seed as well as future research. Hypoglycin is a dangerous
toxin that if consumed can cause an illness known as Jamaica vomiting sickness symptoms
which vary from hypoglycemia, vomiting, coma and the illness can result in fatalities[1,6,7].
Previous research has shown that there are significant levels of hypoglycin in the ackee seed [1].
The ackee seed contains both components of hypoglycin; Hypoglycin-A and Hypoglycin-B, with
HG-B being the larger amount [1-1]. Hypoglycin-B is also contained in larger amounts from
regular to medium sized seeds than in small seeds. However it was the reverse for Hypoglycin-A
as higher levels were contained in the smaller seeds than regular to medium sized seeds [1-2].
Raman Spectroscopy has some advantages over previous techniques. For example Chemistry
techniques are long and drawn out and require several processes before the sample (ackee seed)
is ready for testing. Raman offers faster scans and less invasive work. Previous work requires the
sample to be destroyed, centrifuged and then brewed in chemicals such as methanol, water or
triethylamine [1-3]. Without a Proper lab setup this experiment becomes quite difficult, while our
Raman system can do a complete scan of the seed in just a few minutes. How accurate our
results versus previous work1 depends on what we can discern from the spectra.
We yielded good results in terms of our spectrum and we had many peaks that related
various chemical bonds to the composition/content of the ackee. Specifically many bonds were
located but none that gave a direct clue to the presence of Hypoglycin (A or B). Our results also
showed us that in general seed size was directly related to the Intensity level of chemicals, with
larger intensity going to the larger seed size.

Methodology

To approach this experiment we had an

expensive but fairly compact Raman
system to do our tests on the ackee seed.
Ackee fruit (arilli) from 5 different trees
were picked. The samples from those
trees were termed t1-t5 respectively and
each individual seed placed into a
category of small, medium and large.
These ranges were <2cm, 2cm-2.5cm
and >2.5cm accordingly. The Raman
system that used was comprised of a
high powered laser(monochromatic light
source) with probe, a box that would
hold the samples in place and keep them
at a consistent distance from the laser
light , an advanced spectrometer(qe65
pro) and a computer with a special
program to represent the spectra from
the spectrometer. Fig. 1 shows us a xi
Fig. 2 shows the setup that was used to
the ackee seeds.
We set our integration time1 to 45
seconds this allowed us to have a deep
scan of the ackee seed. Next we set our
scan to average at 5, allowing us to get
more precise readings. We recorded
several spectra that gave us images
similar to that of Fig. 1.Gloves were
applied when dealing with the immature
ackee seed to reduce our contact with the
toxins of the seed. Each ackee seed was
separated from the flesh and placed in
our white box(fig. 2). We kept our seeds
in an environment that is as dark as
possible to reduce excess light (noise).
The CCD detector in the spectrometer
recorded the movements of the excited
molecules and formed a spectra that
could then be seen on our computer
through the program2.
1.
2.

Fig. 1 Showing a sample spectra

Fig. 2 Setup of our Experiment

Fig. 3 showing a medium-sized ackee and a

cross-section
___________________________________

Integration time is a image technique that captures the movement of very small objects in our case the
longer integration time yields better results.[5]
The program used was ocean optics spectra suite.

Theory

Raman Spectroscopy bases its technology on the scattering of light. A molecule is able to
be excited if enough energy is fire onto it, when light photons are scattered off of it they
do so with a wavelength and frequency [3,4] . Most of the light scattered will contain the
same amount of energy and wavelength as the incident light source this is Rayleigh
scattered light; a small amount however (1 in 107 of the scattered) will do so with a
different frequency and wavelength (often less). The process that leads to this is termed
Raman Scattering. This can occur with a change in vibrational rotational or electrical
energies [2,3,4..]. When light is elastically scattered it is termed Rayleigh1 scattering. Any
inelastic scattering is termed Raman scattering[4].

Fig. 42 showing stokes and anti-stokes scattering

The arrows in Fig. 4 represent the difference in energies of the incident and scattered
photons. Raman spectroscopy is associated with both stokes and anti-stokes scattering but
more so with the former.
E=h v ..eqn. (1) This is the energy of a photon where h is
plancks constant, E is energy and v the velocity[3].

hvo - hvr = hvf - hvieqn. (2) Represents a relation between incident and scattered
photons.[4]
hvo represents the scattered photons and hvr represent the incident photons. The law of
energy must be conserved so that whenever one is less hvo or hvr the change in energy
must be the same. This equation represents the change in frequency of the photons.
The motion of the scattered photons are collected by a CCD system and transformed into
a spectra. In detail spectrometers can detect these low level wavelengths associated with
and transform these light energies into electrical signals[2]. Our spectrometer reads the
change in this wavelength as cm-1 a formula relates the frequency to these raman shifts
cm-1[8].
v

1
inc

1
scattered eqn (3)[4-2] this equation derives our Raman

shifts(cm-1) that we see on our spectra. These shifts also represent the chemical bonds
that are located in a test substance.
1.
2.

___________________________________________________________________________
Rayleigh scattering was discovered by British physicist Lord Rayleigh
Picture taken from source[..]

Presentation of Data

Spectra for medium Mature ackee t4

Fig. 5 Showing raman spectra for

A Small Ackee vs Large Ackee
from the same fruit.

Spectra for a medium Immature Ackee seed t4

Raman Spectra -Small Ackee seed t3

Fig. 6 showing a comparison

Raman Spectra-Large Ackee seed t3 between medium sized mature
and immature ackee seeds.

General Raman Spectra

Fig. 7 showing a general Raman Spectra of A medium Ackee Seed from t5

T-TEST for Figure 5 =1.9016x10-256

T-TEST for Figure 6 = 0.20489

Discussion & Analysis

A t-test was done on the Raman Intensities(y values) fig.5 and a result of
1.9016x10-256 was obtained this is <0.5 and therefore statistically significant. From
Figure 5 it is clear that a smaller ackee seed(<2cm) holds less chemical content
than a larger ackee seed(>2.5cm), specifically the highest peak/intensity on small
ackee seed t3-figure 5 is around the 4,500 while on the corresponding large
ackee seed t3-figure 5 its highest peak is above 9000. So if there is some
hypoglycin content then it can be said that it(HG-A or HG-B) exists at higher
levels in the larger ackee seed.

A t-test was completed on the Raman intensities for an immature ackee vs a

mature ackee of the same tree and a result of 0.20489 was obtained. This is <0.5
and therefore statistically significant . Because of the close proximity of peak
values between the mature seed and the immature seed assumptions were made.
The first is that the time of capturing samples was relatively close(less than a
week) so this could explain why the chemical levels are around the same. What it
does not explain is the idea that the seed should lose some of its toxicity when
exposed to the open air[1,6]. From observation fig.6 it would seem that between
the shifts of 1500-1900cm-1 that the peaks are higher in comparison to the mature
seed. This could indicate presence of higher chemical levels in that region.

Figure 7 shows us the general Raman spectra for a medium sized immature ackee
seed. There are several peaks on this graph that could hint at chemical bonds
including the general formula of Hypoglycin C7H11NO2. Hypoglycin-B is what we
are really interested in as it is the largest component of HG in the seed[1] and
easier to detect, chemical bonds of CO2H are a major component this is
carboxylic acid and from our research[7] it lies in the raman shifts of 920-955cm-1
. From all figures we can see peaks around the 900cm-1 band and this could be an
indicator of the presence of this bond. NH2, H, O and NH are also present within
the chemical make-up of Hypoglycin-B. Hydrogen and oxygen bands occur and
overlap right across the shifts so their presence is more than likely. NH or amino
group occur shifts between 3000 and 3500cm-1, unfortunately none of our spectra
allows us to see shifts/bands in that range. An NH bond may exist somewhere
below 3000 but none of the resources[7-2] has allowed us to find that range.

Conclusion
There isnt enough evidence for us to make the claim that by using Raman spectroscopy
we can detect the presence of any Hypoglycin component. However the peaks do point
out the presence of several chemical components. Whether these peaks from our spectra
relate to other useful or harmful chemicals; they can be used to facilitate further research
using the Raman system on the ackee seed.

References
1.) Dundee J.S. S. & Minott A. D.(2011). Impact of seed size on residual hypoglycin
levels in ackee. Food Research International 47. 306309
2.)Colthup, N. B., Daly, L. H., & Wiberley, S.E.(1990). Introduction to infrared and
Raman spectroscopy. Elsevier.
3.) Smith W.E. and Dent G.(2005). Modern Raman Spectroscopy A Practical
Approach. John Wiley & Sons. P.1-17
4.) Horn R.(2009) Modern Methods in Heterogeneous Catalysis: Script to Lecture
Raman Spectroscopy.Fritz-Haber-Institute of the MPG
Department of Inorganic Chemistry
5.) Boyle, W. S., & Smith, G. E. (1970). Charge coupled semiconductor devices. Bell
System Technical Journal, 49(4), 587-593.
6) Kean, E. A. (1975). Hypoglycin, proceedings of a symposium. New York: Academic
Press.
7.) Socrates, G., & Socrates, G. (2001). Infrared and Raman characteristic group
frequencies: tables and charts (Vol. 245). Chichester: Wiley. Pg.125-128
8.) Hager R., Anderson JR. and R, (1970). Theory of the Derivative Spectrometer, J. Opt.
Soc. Am. 60, 1444-1449
9.) Bressler, R., Corredor, C., & Brendel, K. (1969). Hypoglycin and hypoglycin-like
compounds. Pharmacological reviews, 21(2), 105-130.

Acknowledgements

I would like to thank my classmates in particular Cheddi Grierson and Shanice Whyte for
their contributions.

I would also like to thank Dr. K. Edward, without his contributions the research could not
have been completed

And finally I would like to thank the Chemistry Department for their very useful
information as well.