Biomedical Engineering Reference
In-Depth Information
3.1.5 Lipid Extraction and TLC of Lipids
A
chloroform/methanol 1:1 (v/v)
Solutions/Reagents
B
chloroform/methanol 3:1 (v/v)
C
chloroform/methanol/1,2 N HCl 10:10:1 (v/v/v)
D
3% copper acetate (w/v), 8% phosphoric acid (w/v) in ddH
2
O
1
chloroform/methanol/4.3 M ammonia 90:65:20 (v/v/v)
Mobile solvents
2
n-propanol/4.3 M ammonia 65:35 (v/v)
3
n-butanol/chloroform/glacial acetic acid/ddH
2
O 60:10:20:10
(v/v/v/v)
4
isopropylether/glacial acetic acid 96:4 (v/v)
5
petrol ether/diethylether/glacial acetic acid 90:10:1(v/v/v)
6
chloroform/methanol/ddH
2
O 65:25:4 (v/v/v)
7
n-butanol/glacial acetic acid/ddH
2
O 60:20:20 (v/v/v)
8
chloroform/methanol/acetone/glacial acetic acid/ddH
2
O 75:
15:30:15:7.5 (v/v/v/v/v)
Extraction
Freeze the tissue, which should be extracted, with liquid nitrogen,
and pulverize it in a mortar under liquid nitrogen.
Extract the tissue powder with 1 ml
/
g of Soln. A in a glass/Teflon
homogenizator at RT.
Centrifuge at room temperature, decant the liquid, extract with
10 vol. of Soln. B, and centrifuge again and homogenize with 2.5 vol.
of Soln. C again.
Combine the liquid extracts and vaporize the solvent in a ni-
trogen atmosphere.
Thin-Layer Chromatography
Activate the silica gel TLC plates for 3 h at 120
◦
C and store them in
adesiccator.
Apply the lipid-containing solution as a short line with a mi-
croliter syringe or a pipet. The start zone should be as thin as
possible. If larger volumes have to be applied, pipet several times
onto the same position with intermediate drying. For drying the
starting area as well as the plate after chromatography use a stream
of nitrogen instead of air to avoid oxidation of unsaturated lipids.
Develop the chromatogram one- or two-dimensionally using
the solvents 1 - 8. The choice of solvents depends on the lipids to be
analyzed and has to be checked experimentally.
Phosphatidylinositols are separated on a gypsum-free silica
plate, saturated with a 1% potassium oxalate solution in water, and
activated as described above. The recommended solvent is Soln. 2.
A second run with the same solvent after an intermediate drying
increases the separation performance.
A general chromatographic system for separation of lipids does
not exist. Whereas sophisticated systems for groups of lipids are
described in literature, do not hesitate to try other solvent systems,
