Basic Principles of Extraction and Analysis of Lipids
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jkjeon882Basic Principles of Extraction and Analysis of Lipids
Uploaded by
jkjeon882Basic principles of extraction and analysis of lipids
INTRODUCTION
Lipids are immiscible in water; hence organic solvents are used to extract and fractionate lipids from
a mixture of lipids. The source of lipids could be a cell or a tissue from an organism. After extraction,
the separation of lipids is done by different methods based on the polarity or solubility of lipids in
the organic solvents.
The steps include:
1. Extraction of lipids – By organic solvents
2. Separation of lipids – By adsorption chromatography methods such as HPLC and thin layer
chromatography
3. Analysis of fatty acid composition – By gas liquid chromatography, specific hydrolysis
methods, mass spectrometry
EXTRACTION USING ORGANIC SOLVENTS
The method of extracting lipids using organic solvents is referred to as the “solvent extraction
method”. After extraction of lipids, the weight of the lipid is measured either by weighing the
remaining sample after extracting lipids or by weighing the extracted lipid mixture after evaporating
the organic solvent.
Different classes of lipids are extracted by using different combinations of organic solvents in a
certain ratio:
1. Triacylglycerols, waxes etc, classified as neutral lipids are generally extracted from
cells/tissues using ethyl ether, chloroform or benzene. All these are solvents that will not
allow the lipids to cluster due to hydrophobic interactions.
2. For membrane lipids, polar organic solvents are used such as ethanol, methanol.
Hydrophobic interactions between the lipids, ionic and hydrogen interactions between lipids
and membrane proteins are weakened by these solvents.
SEPARATION OF LIPIDS
ADSORPTION CHROMATOGRAPHY
The general principle of adsorption chromatography is to have a matrix to which analytes can be
adsorbed. This matrix is usually insoluble but polar in nature such as silica gel, which is packed into a
glass column. The lipid mixture, which is dissolved in an organic solvent like chloroform is then
applied onto the glass column.
High-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) are two
different types of adsorption chromatography. Both these techniques make use of silica gel as the
supporting matrix, to which polar lipids will bind. In HPLC there is column that is packed with silica
while in TLC, a slurry of silica gel is evenly spread onto a glass plate.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
The small particle size of the stationary phase is able to withstand high pressures. This results
in faster and better resolution, making HPLC the most powerful form of chromatography. In
HPLC, the diameter of the column is very small hence the lipid sample is forced through the
column under high pressure. Gradient elution is generally best suited for eluting different
types of lipids. The neutral lipids will pass through the column and will be the first ones to
elute. The polar lipids that remain bound to the polar silica column are eluted in order of
increasing polarity by washing the column with solvents of increasing polarity.
The material applied to HPLC is very small, therefore it is mandatory to have detector
systems that are highly sensitive and stable. Fluorescence detectors, mass spectrometer
detectors etc are commonly used for determining the nature of analytes in a given sample.
HPLC coupled to a mass spectrometer is used to detect lipids in a sample.
THIN LAYER CHROMATOGRAPHY
The principle behind TLC is largely similar to that of techniques involving columns for
separation of analytes. The difference lies in its practical format, wherein the stationary
phase is coated as a thin layer on a glass plate or a metal foil plate. The samples to be
separated are applied on this plate as a spot or a band near one end of the plate, marked as
the origin. The plate is placed in a glass chamber/tank that contains the mobile phase. The
mobile phase is a mixture organic solvent mixed in a certain ratio. The mobile phase passes
through the TLC plate by capillary action very rapidly. As a consequence, it carries analytes in
the sample with it at a rate that is dependent on the partition coefficient of the analyte
between the stationary and the mobile phase. The movement of the analyte stops once the
mobile phase reaches the end of the plate. The plate is then removed and air dried before
detection. The movement of a given analyte is determined by calculating the retardation
factor (Rf).
Rf = distance moved by the analyte from the origin / distance moved by the mobile phase
from the origin
Fig. Setup for TLC
ANALYSIS
Analytes separated in the TLC plate can be detected by the following methods:
a. the plate is sprayed with a specific reagent that will react with the analyte to give a coloured
product that would be visible to the eye. One such example is iodine. The plate is exposed to iodine
vapours, which reacts reversibly with the unsaturated fatty acids in a way that the spots will be seen
as yellow or brown in colour.
b. the plate can be examined under ultraviolet light, if the analytes are absorbing in this region.
c. a fluorescent dye like rhodamine can be incorporated in the stationary phase. When these plates
are visualized under ultraviolet light, the analytes will appear as blue, green or black spots.
GAS LIQUID CHROMATOGRAPHY
GLC is used to separate volatile components in a given lipid mixture. Their separation is determined
by the partition coefficients between a stationary liquid phase and an inert mobile carrier gas phase.
The use of GLC is therefore limited to compounds that are volatile but thermally stable. The partition
coefficients and volatility of a compound is inversely related to each other.
GLC is considered ideal for identifying fatty acids with varying chain lengths and with varying degree
of unsaturation.
Many different types of detectors are used in conjunction with GLC. Of all mass spectrometers is the
universally used detector alongside a GC. When a mass spectrometer is attached to the GC machine,
the technique is referred to as GC-MS.
Mass spectrometry therefore enables the user to completely identify the lipid structure. The
chemical properties of two lipids of same chain length and same degree of unsaturation but with
placement of double bonds in different positions will be very similar to each other. They will elute
together from a GC column and hence will not be differentiated. In this situation, GC coupled with
mass spectrometry (GC-MS) will help in the identification of these two similar lipids by their unique
fragmentation pattern.
