Thin-layer chromatography of lipids

THIN-LAYER CHROMATOGRAPHY
OF LIPIDS
Abstract: There has been a tendency to discount thin-layer chromatography (TLC)
in recent years because of the availability of high-performance liquid
chromatography (HPLC) methods that appeared to offer greater versatility and ease
of quantification, amongst other reasons. However, TLC has made a comeback,
largely because of the high reproducibility and lower costs of commercial pre-coated
plates. It is the quality of the final result that matters and not how it is achieved.

TLC versus HPLC

In recent years, enormous strides have been made in the development of methods for the
analysis of lipids using high-performance liquid chromatography (HPLC). It is almost certainly
true to say that this technique has replaced preparative gas chromatography entirely. In
addition, it could be claimed that there is no type of lipid separation for which thin-layer
chromatography (TLC) was once favoured that cannot now be done by HPLC. The latter offers
great versatility in that it can be used in the adsorption, reversed-phase, ion exchange and silver
ion modes. It operates at room temperature, so is particularly suited to molecules containing
thermally labile functional groups. A host of bonded-phases, offering varying selectivities in
specific analyses, are available commercially and many have yet to be properly explored in lipid
applications. Many years ago, I used my TLC spreader for the last time and gave it away to a
colleague together with plates, tanks, spray guns and so forth. There must be many other
laboratories where this has occurred also. Does this mean that the technique of TLC can now
be considered obsolete?

There was a time when I would probably have answered yes to this question. I doubt whether I
will ever go back to making my own TLC plates, in spite of the variety this offers in terms of
thickness of layers and the capacity to incorporate complexing agents, such as silver nitrate,
into the adsorbent, not to mention costs. Freedom from silica gel dust in the laboratory has
turned out to be a considerable boon, and I would be reluctant to return to the times when a
grey patina covered all the glass-ware and furniture in the laboratory. On the other hand, I now
use commercial pre-coated plates. HPLC is a superb tool in research and micro-preparative
applications, but it also has high capital and running costs. Lack of a true and reliable universal
HPLC detector rules out the technique for many routine analytical purposes. Higher standards
in sample preparation may be necessary than with TLC, for example to eliminate minute
particulate contaminants.

At first, commercial pre-coated plates seemed expensive - a rich man’s luxury. Relatively
speaking, they do not seem as costly now as they were formerly. We were lured into using them
again, because of their convenience, for example in checking purity of a single sample where it
was tedious, time-consuming and costly in terms of solvents to do this by HPLC. For this
purpose, small pieces of aluminium-backed plates can be used to give an answer in a few
minutes. In the analysis of complex lipids, it is a simple matter to use specific spray reagents to
detect particular functional groups in lipids separated by TLC, but this is not possible with HPLC.

An unexpected bonus has proved to be that commercial pre-coated plates give much more
reproducible results in analysis of complex lipids than was ever possible with laboratory-made
plates in the bad old days. With the latter, it was necessary to use silica gel H, i.e. without binder

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Thin-layer chromatography of lipids

added, which made the layers less stable than was ideal. The brand and even batch of silica gel
H seemed to make a great difference to the quality of separation possible; some simply could
not be made to work. When a bottle was purchased that worked well, further material was
bought immediately and guarded jealously. Even then, resolution could be very variable and
may have been affected by such factors as temperature and humidity, or the presence of
differing amounts of ionic species in lipid extracts. It was necessary to be prepared to change
mobile phases, apparently arbitrarily, to compensate for these unquantifiable variables. In our
experience, this is less of a problem with modern pre-coated plates.

What counts is the quality of the end result and not how it is obtained, and in skilled hands TLC
also offers considerable versatility, in that it can be used in adsorption, reversed-phase and
complexation modes, and at low cost. In preparative applications, components of interest may
be contaminated with silica gel and spray reagents, but there are ways to overcome such
problems with a little effort. I do not discuss the IatroscanTM analyser in this brief overview of
TLC, as I have no recent experience of the technique. Applications of TLC in lipid analysis in
general have been reviewed [1-3].

Some Practical Examples

With TLC in the adsorption mode (silica gel), the principle application in lipid analysis is for the
separation of different lipid classes from animal and plant tissues. It is a relatively easy matter to
resolve each of the main simple lipids from a tissue in one step, i.e. cholesterol esters,
triglycerides, free fatty acids, cholesterol and diacylglycerols, using mobile phases consisting of
a mixture of hexane and diethyl ether, with a little formic acid to ensure that the free acids
migrate successfully. Complex lipids such as phospholipids and glycosphingolipids will remain
at the origin, and they can then be quantified as if they were a single lipid class. In the analysis
of plasma lipids from clinical experiments, such separations often afford adequate information
for diagnostic purposes, and there are many other circumstances where this type of separation
is sufficient. It is perhaps one of the disadvantages of HPLC that there is no analogous method
for the determination of complex lipids as a single entity.

High-performance TLC makes use of silica gel of a very uniform and small particle size,
permitting excellent separations with comparatively short elution times. For example, most of
the important lipid classes in clinical samples can be achieved by one-dimensional TLC in a
single chromatographic run. An example of what can be achieved, taken from the literature, is
illustrated in Figure 1 [4].

Y CE

TG Figure 1. Schematic separation of simple lipids and phospholipids

of plasma by high-performance TLC (1); two developments as far
as "X" with chloroform-methanol-water (60:30:5 by volume), and a
FFA third development to "Y" with hexane-diethyl ether-acetic acid
C (80:20:1.5 by volume). Abbreviations: CE, cholesterol esters; TG,
X triacylglycerols; FFA, free fatty acids; C, cholesterol; PE,
phosphatidylethanolamine; PC, phosphatidylcholine; SPH,
PE sphingomyelin; LPC, lysophosphatidylcholine.

PC
SPH
LPC

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Thin-layer chromatography of lipids

Commercial HP-TLC plates were utilized and they were first given a double development for
part of the way in a polar solvent to separate the phospholipids, and then for the full length of
the plate with a less polar mobile phase in order to resolve each of the simple lipids. The total
elution time was only 30 minutes, in spite of the triple development. In routine analytical work,
ten or more samples can be applied to a 20 x 20 cm plate and then quantified by charring-
densitometry. Although this methodology has been employed mainly in the medical field, I can
envisage many similar applications in industrial laboratories.

HP-TLC plates have proved of particular value to research workers who deal with complex
glycosphingolipids, such as gangliosides, as the extra resolving power is invaluable. A further
advantage in this instance is that analysis times can be reduced from 8 to10 to 1 to 2 hours [5].

Of course, it is possible to isolate the complex lipids from TLC plates and then re-
chromatograph them with more polar solvents (and with silica gel without added binder) to
isolate each of the individual phospholipid classes say with comparable resolution to HPLC.

If two-dimensional TLC procedures are used with complex lipids, better resolution may be
possible than in a single HPLC run. It should also be noted that we can be sure that we see
every lipid in the sample using this technique, but with HPLC it is always possible that some
components may be strongly adsorbed and not eluted. Aluminium-backed plates (10 cm x 10
cm) are ideal for analytical purposes with 2-D TLC, but in micro-preparative applications (up to 5
mg lipid) glass-backed plates are preferred (20 cm x 20 cm). Figure 2 illustrates the nature of
the separation for leaf lipids from Arabidopsis thaliana, a plant species much used in molecular
biology. Each of the main individual phospholipids and glycolipids is clearly resolved.

simple lipids

MGDG
first direction

DGDG
PE
SQDG
PG DPG
PC
PI
PS

second direction

Figure 2. Schematic representation of two-dimensional TLC separation of complex lipids

from A. thaliana. Abbreviations, MGDG, monogalactosyldiacylglycerols; DGDG,
digalactosyldiacylglycerol; SQDG, sulfoquinovosyldiacylglycerol; DPG,
diphosphatidylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PI,
phosphatidylinositol; PS, phosphatidylserine; and PC, phosphatidylcholine.

In this instance, plant phospholipids and glycolipids are separated by first developing the plate
in chloroform-methanol-water (75:25:2.5, by volume) in the first direction. After allowing

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Thin-layer chromatography of lipids

sufficient time for drying, the plate is developed, at right angles to the first development, in
chloroform-methanol-acetic acid-water (80:9:12:2, by volume).

For analytical purposes, all components can be detected by spraying the plate with ethanolic
phosphomolybdic acid reagent (commercially available as a 20% (w/v) solution), followed by
charring in an oven, and they appear as dark spots on a light yellow-green background. We feel
that this is less hazardous than the charring reagents containing concentrated sulphuric acid
that are often used for universal detection. Alternatively, as an aid to identification, plates can be
treated with a variety of specific reagents for different lipid types. Spraying with α-naphthol-
sulphuric acid reagent followed by charring is useful for detecting glycolipids, and the phosphate
reagent of Dittmer and Lester is specific for the phosphorus moiety in lipids. Ninhydrin reagent
shows up lipids containing amino groups, so it is particularly useful for verification of
phosphatidylethanolamine and phosphatidylserine. For non-destructive detection of individual
polar lipids separated micro-preparatively as above, we have found primulin (0.01% (w/v)
solution in acetone-water (60:40, v/v)) to be preferable to the commonly used Rhodamine 6G.
Lipids are observed under UV light, and give very clear spots even with light spraying with the
primulin reagent. The components are extracted from the silica with polar solvent mixtures, prior
to analysis by other means.

My colleagues and I have been in the forefront of developing methods for separating complex
phospholipids and glycolipids from plant leaf tissue by HPLC methods, and we have used these
with thousands of samples for screening purposes. However, when we require a precise
detailed analysis of a sample, we go to TLC. This is tedious as it requires that we carry out a
separation on the 5 mg scale, locate the spots with the spray reagent, isolate the lipids (with an
internal standard added) and then transesterify for gas chromatographic analysis. The important
point is that we can get higher accuracy as well as much more information than by HPLC.

I can remember when silver ion TLC was viewed by industrial analysts as a rather esoteric
technique, but it has come to be the standard method for the determination of cocoa butter
equivalents in confectionery fats, for example. Whether the HPLC or high-temperature gas
chromatography methods will supplant it remains to be seen. Silver ion chromatography
methods are reviewed in detail elsewhere on this website.

Reversed-phase TLC has been relatively little used for the analysis of triacylglycerols, mainly
because it has been considered a messy technique and because the separated components
are not easily visualised. Methods are available which overcome many of these difficulties, and
excellent separations of triacylglycerols have been reported [6]. The recommended stationary
phase is a silanized kieselguhr, with a mobile phase consisting of acetonitrile-acetone-water
mixtures. Charring densitometry is preferred for detection and quantification. However, I have
little doubt that in this instance HPLC techniques are much better when the necessary
equipment is available.

Quantification

The disadvantages of various detectors in HPLC have been debated ad nauseam in recent
years. What of quantification in TLC? The generally accepted method for multiple samples
involves spraying with a corrosive reagent (usually containing concentrated sulphuric acid),
charring at high temperature for a set period, and finally densitometry of the carbon produced.
Any experienced analyst coming fresh to lipid methodology would probably look askance at
such a procedure. Yet it can be made to work, if great care is taken to standardise the charring
conditions and to calibrate for each of the lipid classes with authentic standards, i.e. with a
similar degree of unsaturation to the samples. Modern scanning densitometers can give
excellent results in skilled hands. However, there is considerable scope for inexperienced
analysts to compound the errors. In research applications with relatively few samples, I prefer to

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Thin-layer chromatography of lipids

transesterify the separated lipids in the presence of an internal standard and use gas
chromatography of the methyl ester derivatives of the fatty acids for quantification purposes [1].

Conclusions

What of those of us that have ready access to HPLC? I mentioned earlier that I had resurrected
TLC in my own laboratory but with commercial pre-coated plates for the sake of a cleaner
environment. TLC has been missed most often when I have had a single sample whose purity
or identity needed checking. It is tedious, time-consuming and costly in terms of solvents to do
this by HPLC often. In the analysis of complex lipids, it is possible to use specific spray reagents
to detect particular functional groups in lipids separated by TLC. I recall listening to a lecture
many years ago in which the speaker described a chromatography-tandem mass spectrometry
system in which phosphatidylcholine was eventually identified by the presence of a specific ion
relating to the choline moiety; the same could have been done with a simple TLC spray. We
lack simple colorimetric methods for the identification of components separated by HPLC.

What counts is the quality of the end result and not how it is obtained, and in skilled hands TLC
offers considerable versatility and precision in lipid analysis with relatively low capital costs. Of
course, there are many parts of the world where HPLC is not available to analysts. If scientists
in these countries can obtain equally good results by TLC, they should be encouraged to do so.
For the moment, suffice it to say that traditional TLC is not dead, but alive and well, and likely to
be around for some time to come.

References

1. Christie, W.W. Lipid Analysis. 3rd Edition (Oily Press, Bridgwater) (2003).
2. Henderson, R.J. and Tocher, D.R. Thin-layer chromatography. In Lipid Analysis. A Practical
Approach (edited by R.J. Hamilton & S. Hamilton, IRL Press, Oxford), pp. 65-111 (1992).
3. Touchstone, J.C. Thin-layer chromatographic procedures for lipid separation. J. Chromatogr. B, 671,
169-195 (1995).
4. Kupke, I.R. and Zeugner, S. Quantitative high-performance thin-layer chromatography of
lipids in plasma and liver homogenates after direct application of 0.5-µl samples to the silica-gel
layer. J. Chromatogr. B, 146, 261-272 (1978).
5. Yu, R.K. and Ariga, T. Ganglioside analysis by high-performance thin-layer chromatography.
Methods Enzymol., 312, 115-134 (2000).
6. Amidzhin, B. and Nikolova-Damyanova, B. Densitometric identification of triglycerides separated by
reversed-phase thin-layer chromatography. J. Chromatogr. A, 446, 259-266 (1988).

This article has been updated appreciably from two earlier papers (now amalgamated) by the
author that first appeared in Lipid Technology (Christie, W.W. Has thin-layer chromatography
had its day? Lipid Technology, 2, 22-23 (1990); Christie, W.W. and Dobson, G. Thin-layer
chromatography-revisited. Lipid Technology, 11, 64-66 (1999)).

W.W. Christie
Scottish Crop Research Institute (and Mylnefield Research Services Lipid
Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland

Last updated 26/11/2007

© W.W. Christie www.lipidlibrary.co.uk 5