Biomedical Engineering Reference
In-Depth Information
multiple developments or a 2D chromatography to solve your dis-
tinct separation problem.
Identify the unknown lipids by comparison with a set of known
lipids (standards) of the same type, chromatographed in the same
run on the same plate.
A versatile detection reagent for lipids is iodine vapor. The dry
Lipid detection
plate is placed into a chamber containing one or two Petri dishes
with crystals of solid iodine. Lipid-containing spots are colored
yellow to brownish after few minutes. The color disappears quickly
incontactwithair.
An additional general detection method is the oxidation by
concentrated sulfuric acid. Spray the dry plate in a hood with con-
centrated sulfuric acid and then heat it in a drying oven to 130
◦
C.
Organic substances yield dark brown spots.
Lipids are visualized by spraying the plate with Soln. D and
heating to 180
◦
C 15 min.
For identification of phospholipids use Hanes' reagent as de-
scribed in Protocol 3.1.4.
For quantification of phospholipids identify the spots with io-
dine vapor or Soln. D. Mark the spots with a pencil, and cut the
spotsaswellasapartoftheplatewiththesamesizebutfree
of lipids. Transfer the material into a test tube and determine the
amount of phosphate as described in Protocol 1.3.3.
References
Lowenstein JM (ed.) (1969) Lipids. Meth Enzymol 14
Neuhoff V et al. (ed.) (1973) Micromethods in molecular biology. Springer,
Berlin
Touchstone JC, Dobbins MF (1983) Practice of thin layer chromatography,
2nd ed., Wiley, New York
3.2 Hints for Column Chromatography of Proteins
Column chromatography is used to analyze and prepare biomole-
cules. Chromatography techniques separate them according to dif-
ferences in their specific properties as size, charge, hydrophobicity,
and biospecific interactions. All chromatographic techniques use
these properties and are different only with respect to instrumenta-
tion. The following hints are made for chromatography on columns
of packed beds (matrix, stationary phase, support) passed by sol-
vents (mobile phase) driven by low or medium pressure. The main
difference to high-performance liquid chromatography (HPLC) is
given by particle size and hydrodynamic resistance resulting in
much higher backpressures in the latter case. Theoretical consid-
erations are the same in low-pressure chromatography and HPLC,
but because HPLC needs much more complex technical equipment,
