Food Lipids: Chemistry, Nutrition, and Biotechnology

Lipids detected by nondestructive methods can be recovered by scraping the
bands of interest and dissolving them in an appropriate solvent. Complex lipid
mixtures cannot always be separated by one-dimensional TLC; however, two-dimensional
TLC may resolve such mixtures.
The separation efficiency of TLC plates is affected by the degree of hydration
of the adsorbent. Therefore, activation of the adsorbent, which depends on both the
time and the temperature as well as the storage conditions of the plates and the
relative humidity of the atmosphere, must be considered. Use of authentic lipid
standards would allow direct comparison of the resolved lipids in an unknown mixture.
High-performance TLC (HP-TLC) plates have high resolving power and speed
of separation. They are available commercially as precoated plates with fine (5-�m)
and uniform particle size silica gel. However, the amount of sample that can be
applied to such HP-TLC plates is very small. TLC is a preferred method of purification
and separation of lipid classes before subjecting them to further separation of
individual components.
For quantification of TLC separated lipids, traditional scraping followed by
quantitation and in situ determination are available. The separated lipid classes on
silica gel can be scraped off, extracted by means of suitable solvents, and quantitated
gravimetrically, spectrophotometrically, or by GC. Determination of the phosphorus
content of the eluted phospholipids is a classic example for spectrophotometric quantitation.
GC can be employed to quantify separated neutral and phospholipid classes
by derivatizing the constituent fatty acids into their methyl esters. Densitometric
methods provide an in situ quantification method for lipids. Lipids are sprayed with
reagents and their absorption or fluorescence can be measured under UV or visible
light by densitometry.
The spots detected by charring can be measured by scanning photodensitometer
areas of peaks, which are proportional to the amount of original lipid present, are
recorded. Scintillation counting is also possible after introducing a correct scintillator
(e.g., mixture of 2,5-diphenyloxazole and 1,4-bis-2-(5-phenyloxazoyl)benzene, PPO,
and POPOP in toluene) into the lipid. Scanning fluorometry allows resolution of
lipids on an adsorbent containing a fluorescent dye or by spraying with such reagents.
TLC could be coupled with other methods to facilitate detection, quantitative
identification, or quantitation of separated lipids. These include coupling with HPLC
(HPLC/TLC), Fourier transform infrared (TLC/FTIR), nuclear magnetic resonance
(TLC/NMR), and Raman spectroscopy (TLC/RS) [119]. The Iatroscan (Iatron Laboratories
of Japan) applies the same principles of TLC to separate lipid mixtures
followed by their detection using FID. The TLC medium is silica (7.5 �m thick)
sintered onto quartz rods (0.9 mm in ID, 15 cm long, called Chromarods). Chromatographic
resolution of lipid classes on Chromarods and the composition of solvent
systems used are similar to those employed in classical TLC with modifications.
Copper sulfate, silver nitrate, boric acid, or oxalic acid impregnation has been reported
to produce better resolution and increased responses in the determination
[124]. Parrish and Ackman [125] have shown that stepwise scanning and developing
in solvent systems of increasing polarity resolves individual lipid components in
neutral lipids of marine origin. FID gives a linear response that is proportional to
the number of nonoxidized carbon atoms in the material entering the flame. The
Chromarods are reusable up to 100–150 times and require thorough cleaning after
each use. The main advantage of TLC-FID is the short analysis time, with the pos-
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