UNIVERSITY OF THE SOUTH PACIFIC

MARINE CHEMISTRY
AKARSANA KUMAR S11132245

Sterol Extraction and Identification from Seawater Samples By TLC

Abstract

The experiment focuses on determination if major sterol- cholesterol is found suspended in the surface
sea water. First part of this experiment discussed on sea water reaction with dichloromethane that
forms the organic layer containing sterols within. Sterol analyses, both for particulate and for dissolved
sterols, were carried out by thin layer chromatography for seawater samples. The rf values of standard
cholesterol obtained on the thin layer chromatography paper is compared with that of the extracted
compound, confirming the presence of cholesterol in seawater and its predominance over other sterols.
An infrared radiation is run to analyses the cholesterol structure. Different peaks produced on the ir
spectrum represents the different types of vibrations between the various bonds contributing to the
structure of cholesterol, where it is hypothesized that cholesterol is present in the seawater and will
produced a similar Rf value to that of the standard cholesterol.

Introduction

Cholesterol is one such seawater sterol and the total amount of this sterol in seawater is enormous if
you consider the fact that the earth is about two thirds Ocean. But the amount of cholesterol in a given
sample of seawater is usually very small. Sea water sterols either dissolved or associated with
suspended matter. Due to their high degree of stability in the marine environment and to their diversity,
sterols are useful ecological markers. (Eddy, 2009). Thin-layer chromatography (TLC) is a
chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is
performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent
material, usually silica gel, aluminium oxide (alumina), or cellulose. This layer of adsorbent is known as
the stationary phase. Thin-layer chromatography can be used to monitor the progress of a reaction,
identify compounds present in a given mixture, and determine the purity of a substance. To quantify the
results, the distance traveled by the substance being considered is divided by the total distance traveled
by the mobile phase. (The mobile phase must not be allowed to reach the end of the stationary phase.)
This ratio is called the retardation factor (Rf).

Infrared (IR) spectroscopy is one of the most common and widely used spectroscopic techniques.
Absorbing groups in the infrared region absorb within a certain wavelength region. The absorption
peaks within this region are usually sharper when compared with absorption peaks from the ultraviolet
and visible regions. In this way, IR spectroscopy can be very sensitive to determination of functional
groups within a sample since different functional group absorbs different particular frequency of IR
radiation. Also, each molecule has a characteristic spectrum often referred to as the fingerprint. A
molecule can be identified by comparing its absorption peak to a data bank of spectra. IR spectroscopy
is very useful in the identification and structure analysis of a variety of substances, including both
organic and inorganic compounds. It can also be used for both qualitative and quantitative analysis of
complex mixtures of similar compounds.

The use of infrared spectroscopy began in the 1950's by Wilbur Kaye. He had designed a machine that
tested the near-infrared spectrum and provided the theory to describe the results. Karl Norris started
using IR Spectroscopy in the analytical world in the 1960's and as a result IR Spectroscopy became an
accepted technique. There have been many advances in the field of IR Spec, the most notable was the
application of Fourier Transformations to this technique thus creating an IR method that had higher
resolution and a decrease in noise. The year this method became accepted in the field was in the late
1960's

This experiment aims to determine the presence of sterol components in sea water sample with the
assumption that the dominating sterol is cholesterol, hence using the standard of cholesterol to
compare the Rf values.

Methodology

The extraction and identification of sterol in provided sea water was carried out as follows:

Extraction using dichloromethane

100 cm3 of provided sea water sample was pipette out and added to the separating funnel that was set.
To the separating funnel 20 cm3 of dichloromethane was added, which was followed by the shaking of
the mixture in the funnel. After shaking the content was allowed to settle for a few minutes forming two
layers of liquid. The organic layer was then separated into a pre- weighed round bottom flask, re-
extracting the aqueous layer with another portion of 20 cm3 of dichloromethane. The extraction with
dichloromethane was repeated once more. The organic layer was dried using anhydrous sodium
sulpahte and concentrated in a rotavapor. The mass of the extracted product was weighed and
recorded.

Identification of the extracted compound:

Preparation of the TLC Chamber

Developing solvent that completely covers the beaker to a depth of approximately 0.5 cm was added.
The beaker the covered with aluminum foil. The chamber was kept covered to avoid evaporation from
changing the composition of the solvent. The chamber was saturated with vapor.

Preparation and separation of the NP-TLC plate

While holding only the sides, a normal phase TLC plate was obtained. Using a pencil, a faint line 2 cm
from the bottom of the plate was drawn. The extract dried in the rotavapor was dissolved in 10 ml of
dichloromethane. The organic layer from the flask was transferred into a beaker using a pasture pipette.
Using a capillary tube, drops of organic layer from the beaker was applied onto the drawn line and the
spot was allowed to dry. The procedure was repeated until a dark spot was obtained which was smaller
than 4 cm in diameter. Using another capillary tube, the standard cholesterol solution was spotted on
the other side of the TLC plate on the drawn line and allowed it to dry. The standard solution of
cholesterol was concentrated by several spotting of the solution. The cover of the chamber was
removed and the TLC plate was carefully loaded into the chamber, ensuring that the bottom of the plate
is in contact with the solvent. The TLC chamber was covered and left undisturbed while the movement
of the solvent was monitored. When the solvent has reached approximately 1 cm from the top of the
plate, the plate was removed and the solvent line was marked. The developed plate was allowed to dry
in the fume hood.

Identification of the Separated Compounds

The developed plate was sprayed with 5% ethanoic phosphomolybdic acid reagent solution in the fume
hood. The sprayed plate was dried in the oven. The spots developed on the plate after spraying was
recorded. The distance moved by each pigment was measured and recorded and the Rf values were
calculated. The compound extracted was identified using the Rf values of the sample and the standard.

Result and Discussion

Mass (g)
Mass of empty flask 124.9016
Mass of flask + product 125.8576
Mass of product 0.9560
Table 1.0 shows the mass of the reside obtained from drying in the rotavapor.

Rf value calculations

For qualitative results, thin- layer chromatography provides a chromatographic measurement known as
Rf value. The Rf value is the retardation factor or the ratio-to-front value expressed in decimal fractions.
The Rf values can be calculated as:

Rf = distance travelled by the spot

Distance travelled by the solvent

This number is calculated for each spot observed on the TLC plate. Essentially it describes the
distance travelled by the individual components. Of the two spots travel the same distance or
have the same Rf value then it can be concluded that the two components are the same
molecule. For the Rf values to be valid, however, TLC plates must run under the same
conditions. These conditions includes the stationary phase, the mobile phase, and temperature.
Rf of Unknown Compound

Rf = a/b

= 1.3/7

= 0.18 cm
Rf of Standard Compound

Rf = a/b

= 1.3/7

= 0.18 cm

Chromatography is simply the separation of two or more compounds by distribution between two
phases., one which is moving and the other which is stationary. In thin layer chromatography, the
stationary phase is a polar absorbent, usually finely ground alumina or silica particles. TLC is used for
analyzing colorless compounds, identifying compounds and mixture components, preparative
separation and purification od compounds. This solvent was coated on the glass slide creating a thin
layer of particular stationary phase. For the mobile phase almost, all mixtures of solvents in this case sea
water was used. By manipulating the seawater, organic compounds of cholesterol were separated. The
electropositive character of the TLC plate and the electronegative oxygen created a very polar stationary
phase. Since cholesterol contains a polar hydroxyl group, on the one hand, and a non-polar steroid ring
structure and hydrocarbon tail, on the other, it has both a water-soluble region and a fat-soluble region.
Molecules that have both water-soluble and fat-soluble properties are called amphipathic. Cholesterol's
properties as an amphipathic molecule are important to its function in the marine animal body.

The separation of dichloromethane and the seawater in the separating funnel in water-organic phase
extraction was dependent on the density of the two liquids, where the organic mixture was on top and
the aqueous was on the bottom. The quantitative analysis of sterols was performed on TLC plate with
80% Hexane: 20% Diethyl Ether: 1.5% Acetic acid mobile phase. The distance travelled by both the
sample and the standard was 0.18 cm, hence it can be said that cholesterol is the major sterol extracted
from the sea water after reaction with dichloromethane. As mentioned before, the Rf value of
cholesterol in seawater sample is constant since the following experimental conditions are kept
unaltered:

• Temperature

• Chromatography medium, i.e. same type and grade of Chromatography Paper

• Solvent concentration and purity

• Amount of sample spotted on Chromatography medium

The TLC was run in a closed chamber and the interior was saturated with the solvent vapor to ensure
maximum resolution between components and the to prevent the mobile phase solvent from
evaporating off which could have result in lower Rf value.

If the same grade of Chromatography medium is used (typically Grade 1 CHR or 3 MM CHR) and the
room temperature of the experiment does not fluctuate too much, the remaining critical variable to be
observed is the amount of dye spotted. Large amounts tend to form elongated zones with uneven
distribution of dye along its zone. Too much dilute spots makes visibility of separated dye poor. Trial and
error is involved to find the ideal proximate amount to be spotted.
The sterols present in sea water comes from the synthesis by marine organisms, while some originate
from exogenous sources. A research done by Mathews and Smith (1998) analyzed that a part of the
sterols detected in sea water might be derived from diatoms and planktons. Sterols is mostly composed
of cholesterol and small fraction of other varying sterols.

Infrared spectroscopy also known as vibrational spectroscopy involves the interaction of infrared
radiation with matter, used to identify and study chemicals. The analysis of a compound is conducted
using infrared spectrometer, which produces an infrared spectrum. Infrared spectroscopy exploits the
fact that molecules absorb frequencies that are characteristic of their structure. These absorptions occur
at resonant frequencies, i.e. the frequency of the absorbed radiation matches the vibrational frequency.
The energies are affected by the shape of the molecular potential energy surfaces, the masses of the
atoms, and the associated vibronic coupling.

The major band identified for cholesterol molecule were between

-1,
2800-3000 cm are characterized due to symmetric and asymmetric stretching vibrations of CH2 and
CH3 groups. The observed broad and intense band close to 3400 cm−1 is attributed to OH
stretching.1417 The characteristic strong peak at 2899 cm−1 is due to CH2 symmetric stretching
vibration. Cholesterol has one double band (C = C) in the second ring. This was prominently shown at
1674 cm−1. This peak assignment of 1674 cm−1 achieved for the double bond in the second ring of
cholesterol is in good agreement with the results reported by Zheng et al.19 and also with the 1693–
1671 cm−1 assignment for cyclopentane. The band at 1464 cm−1 is due to asymmetric stretching
vibrations of CH2 and CH3 groups. The sharp peak at 1055 cm- can be attributed to ring deformation of
cholesterol.

It is concluded that the major sterol present in seawater is in form of cholesterol. Treatment with
dichloromethane separates the organic in the seawater and a rotavapor can be used to dry the obtained
sterol. In order to confirm that the major compounds in seawater is cholesterol a thin layer
chromatography can be used while comparing to a standard solution of cholesterol. An infrared
radiation further confirms the component as cholesterol as the peaks in the spectrum of this experiment
matches with those in the literature findings.
Reference

• S. Zheng and A. T. Tu, Appl. Spectroscopy. 40, 1099 (1986).

• https://www.bing.com/images/search?q=structure+of+cholesterol HYPERLINK
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• Eddy, S. (2009). Steroid geochemistry in the oxygen minimum. Marine Chemistry. 1998, 60, 111-
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• https://www.researchgate.net/publication/262416524_Spectroscopic_Studies_of_Cholesterol_
Fourier_Transform_Infra-Red_and_Vibrational_Frequency_Analysis

• http://www.cholesterol-and-health.com/Hydrocarbon-Tail.html