Biology Reference
In-Depth Information
After extraction by the classic Folch method, lipids are distributed between 3 fractions:
the methanol/water upper phase containing gangliosides and other very polar lipids;
the lower chloroform phase containing the membrane phospholipids; and the acetone-
extracted phase containing non-polar lipids like triacylglycerols and cholesterol. Each of
these fractions can be dried and subjected to additional fractionation. Although there
are a variety of methodologies used to separate membrane lipid fractions, the two
most common are thin layer chromatography (TLC) and high-performance liquid chro-
matography (HPLC)
[17]
.
TLC
TLC was discovered by Ukranian scientists N.A. Izmailov and M.S. Shraiber in 1938
[18]
.
At first, TLC was difficult to use since the TLC plates all had to be home made. Now, a wide
variety of pre-made TLC plates are commercially available. TLC is so fast, simple, and inex-
pensive it is routinely employed and is considered to be 'a poor man's HPLC' (discussed
below). TLC plates consist of a supporting sheet of glass, aluminum or plastic, to which is
adhered a thin coating of a solid adsorbent, usually silica or alumina. Aluminum or plastic
plates have the advantage of being easily cut to a desired shape by a pair of scissors. A small
amount of a volatile solvent containing the dissolved membrane lipid mixture is spotted near
the bottom of the plate
[19,20]
. The spot is dried and the plate then placed inside a sealed
developing chamber containing a shallow pool of a developing solvent known as the eluent.
The eluent constitutes the mobile phase and percolates up through the stationary phase (the
adsorbant that is used for lipid separations is usually silica), by capillary action. The spotted
lipids are bound to a different extent to the stationary silica depending on their physical
properties of polarity and hydrophobicity. As the eluent passes through the bound lipids,
an equilibrium is established for each lipid in the mixture between the stationary silica
and the mobile liquid phase. The lipids that are most soluble in the mobile phase will spend
more time in that phase and move farther up the TLC plate. When the mobile solvent front
approaches the end of the plate, the plate is removed from the developing chamber and
dried. The separated lipids are then visualized by a number of agents that can be sprayed
on the plate
[21]
.
Table 13.2
lists some of the most commonly used sprays to visualize lipids
on TLC plates. The relative distance the lipid travels from the original spot (the origin) is
quantified by its R
f
value (retention factor). R
f
is defined as the distance the lipid travels
up the plate divided by the total distance the eluent travels (from the origin to the front).
Determination of R
f
values is depicted in
Figure 13.7 [20]
.
Lipids can be separated by 1-dimensional or 2-dimensional TLC as shown in
Figure 13.8
[22]
. Sequential 1-dimensional TLC is shown in the left panel. The polar lipids were first
separated by two successive runs with chloroform-methanol-water (60:30:5 by volume).
In the third run, the non-polar lipids were separated using hexane-diethyl ether-acetic
acid (80:20:1.5 by volume). In the first two runs the non-polar lipids ran at the front and
were not separated. In the third run, the polar lipids did not migrate, but the non-polar
lipids did, resulting in their separation. The right panel of
Figure 13.8
demonstrates a 2-
dimensional TLC lipid separation. In the first direction, the eluent was chloroform/meth-
anol/concentrated ammonia (65/35/4, by volume). This was followed by a second eluent,
2, butanol/acetic acid/water (60/20/20, by volume) run at right angles to the first. Better
