Thin Layer Chromatography in the
Purification of Lipids
Dr. Gary Witman,
Dr. Mark Moskovitz
The purification of lipids has
become simple and convenient. Some twenty years ago
HPLC replaced preparative gas chromatography as the
method of choice for the analysis of lipids. But
these days commercially prepared pre-coated TLC
plates do just as good a job as HPLC and are much
more convenient to use. Additionally thin layer
chromatography is frankly and inexpensive analytical
tool, a feature having great merit in times of
budgetary constraint. High performance TLC plates
manufactured using silica gel or alumina prepared
with uniform small particle size material for the
stationary phase permits excellent separations with
short elution times. As explained below, in the
analysis of complex lipids by TLC it is simply a
matter of using specific spray reagents to detect
particular functional groups in lipids: this
simplicity in performance is not possible with HPLC.
TLC is incredibly flexible in that it can be used in
adsorption, reversed phase or complexation modes,
such as for the separation of complex lipid
components integrated into mammalian cell membranes
or in vegetable oils. When performing a quantitative
analysis of these lipid separations it is
recommended that the thickness of the adsorbent
placed on the prepared plate be greater than for
qualitative analysis.
There is no lower cost high
resolution technology available for the detection of
lipids. Indeed, there are reports that some
researchers substitute institutional size mayonnaise
jars as TLC chambers which are then covered by a
flat glass plate in which to perform experiments.
Talk about being able to keep the costs of research
down! Standard TLC plates are 20 cm tall with
varying widths. The width of a TLC plate depends
upon the number of samples to be chromatographed. We
recommend use of a standard size commercially
prepared TLC plate, which is 20 x 20 cm.
Thin layer chromatography (TLC)
is currently used for two different methods of lipid
analysis. In the first approach the different
classes of lipids are separately extracted and then
each class of lipid is analyzed via unique TLC
methodology. In the second approach complex mixtures
of lipids are separated on TLC plates and then
further characterized. The lipid classes are divided
into neutral lipids such as triglycerides (which is
formed from one molecule of glycerol and three fatty
acids), polar lipids such as phospholipids, and
cholesterol. Ideally lipids are chromatographed on a
single alumina or silica gel TLC plate using
sequential solvent systems running in the same
dimension. Relatively nonpolar lipids such as
neutral lipids, fatty acids and cholesterol migrate
to unique positions in the upper half of the
chromatogram, whereas relatively polar lipids like
phospholipids and sphingolipids are separated on the
lower half of the chromatogram.
In the adsorption mode (either
silica gel or alumina is an excellent sorbent agent)
one principal application is for the separation of
different lipid classes from animal and plant
tissues. Through the use of a mobile phase
consisting of hexane and diethyl ether it is simple
to resolve simple lipids such as cholesterol esters,
triglycerides, free fatty acids, cholesterol and
diacylglycerols. When performing a separation using
reverse phase TLC plates everything except the flow
of the solvent is backwards. Using the reverse phase
technique polar compounds move faster than non polar
and the more polar the solvent the less things move
up the plate.
In a standard adsorption run
complex lipids such as phospholipids and
glycosphingolipids remain at the origin, and these
can be quantified as if they are a single lipid
class. This offers benefits over lipid purification
using HPLC. If two dimensional TLC procedures are
used with complex lipids better resolution may be
possible than can be achieved in a single HPLC run.
When performing two dimensional TLC excellent
resolution can be achieved using either aluminum or
glass backed plates.
For routine analytical work ten
or more samples can be applied to a 20 x 20 cm plate
and then the amount of lipid present can be
quantitated by charring-densitometry, which consists
of spraying the plate with an oxidant and heating to
carbonize the lipid. Prior to use it is recommended
that the plates be heat activated at a temperature
of 110 F for an hour. The lipids migrate along the
plate forming unique and separate spots on the
plate. Then for the detection of spots after a run,
in which the selected solvent front is able to
migrate half way up the plate (approximately 30
minutes) the plates are air dried in a chemical hood
at room temperature for 20 minutes. All lipids can
be easily visualized with a single water soluble dye
such as amido black 10B. This dye preferentially
interacts with relatively nonpolar entities and when
present in a 1 M sodium chloride solution will
associate with lipid spots on the chromatogram.
The use of this amido black dye
is generalized and does not help to differentiate
the separate lipid compounds. As an aid to
identification of separate lipid classes the TLC
plates can be treated with a variety of specific
reagents for specific lipid types. The following
aids are currently recommended:
-
Ninhydrin reagent shows up
lipids which contain amino acids, useful for
identification of phosphatidylethanolamine and
phosphatidylserine.
-
Iodine vapor, or ammonium molybdate-perchloric
acid spray or sulphuric acid spray can all be used
to reveal the presence of all lipid materials
-
Dragendorff reagents are specific for choline
-
Dinitrophenylhydrazine and the Schiff reagent are
specific for plasmalogens (any of a group of
glycerol-based phospholipids in which a fatty acid
group is replaced by a fatty aldehyde).
-
Hydroxylamine ferric acid chloride spray are
specific for esterified fatty acids
The power of TLC technology for
quantitative lipid detection is impressive. It is
possible to detect as little as 25 ng of
phospholipids, 25 ng of cholesterol, and 50 ng of
neutral lipids and fatty acids.
Using these simple tools it is
possible to identify lipid and lipid components
quickly, and with high specificity.
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