Quantitative Analysis by Densitometry
Most reported quantitative analyses by TLC involve reflectance scanning in the absorbance or fluorescence mode using a Camag
TLC Scanner 3, a Desaga CD-60 densitometer, or a J&M diode array detector scanner. Standard zones are applied to a plate to
create a calibration curve of peak area or height versus weight through linear, nonlinear, polynomial, or Michaelis–Menten
regression, and weights of bracketed sample zones on the plate are interpolated from the curve. Videodensitometers with a
visible or UV light source, camera, and software for image capture and quantification are also available from Camag and Desaga,
but these do not allow measurement at an optimal wavelength as the slit scanning densitometers with a monochromator do.
The chronology of the introduction of different TLC sorbents, plate formats, and instruments was reviewed in an historical
article (22). As in column chromatography, the chemistry and size of the particles as well as the thickness of the layer affects
the speed and resolution of the separations that are possible. Silica gel is the predominant TLC layer because of its high
selectivity for a large range of compounds with different functional groups. Supposedly, similar layers according to the label
from different manufacturers (for example, the most widely used layer by far, silica gel 60 F254) with the same layer thickness can give quite different results, perhaps because of the unstated binder type and content,
and should be comparatively evaluated before substitution of plates in a given method.
In addition to the chosen sorbent, precoated plates usually contain a binder to help the particles adhere to the support that
serves as the backing for preparation of the layer. The most widely used commercial TLC and high-performance TLC (HPTLC) plates
contain an organic polymeric binder, such as polymethacrylate at a concentration of 1–2%; these are harder, smoother, more
durable, and generally give better separations than G layers. The classical TLC plates designated as "G" for gypsum (calcium
sulfate) binder are still available from EMD/Millipore, Macherey-Nagel, and Analtech. The nature of the binder in a plate
can affect separations and detection based on color reactions.
Layers containing an indicator that fluoresces when irradiated with 254- or 366-nm ultraviolet light are designated as "F"
or "UV" layers. These layers are used to facilitate the detection of compounds that absorb at these wavelengths and give dark
zones on a bright background (fluorescence quenching). F254 indicators can give green (zinc silicate) or blue (magnesium tungstate) fluorescence. F366 indicators can be an optical brightener, fluorescein, or a rhodamine dye (23). Some precoated plates have both indicators
to detect compounds that absorb at both wavelengths (F254+366 plates). Plates designated with an "s" have a UV indicator that is acid stable (F366s plates). Lux TLC plates (EMD/Millipore) and Adamant TLC and Nano-SIL-HPTLC plates (Macherey-Nagel) have enhanced brightness
because of increased levels of the F254 indicator.
Table I: A comparison of the physical and operational parameters of TLC and HPTLC
TLC versus HPTLC
The differences between TLC and HPTLC can be seen in Figure 3 and Table I. The effect of the smaller particle size and the
shortened development distances combine to give higher efficiency, shortened development times, increased sensitivity, and
a larger number of samples processed simultaneously.
Figure 3: Comparison of the separation of dansyl amino acids on (a) a classical TLC silica gel 60 plate and (b) an HPTLC silica
gel 60 plate under identical conditions. Samples (bottom to top): N-α-dansyl-L-asparagine (lowest Rf), α-dansyl-L-arginine, dansyl-L-cysteic acid, N-dansyl-L-serine, dansyl-glycine, and N,N-didansyl-L-tyrosine (highest Rf). Sample volume: TLC, 4 µL; HPTLC, 0.3 µL; mobile phase: ethyl acetate–methanol–propionic acid (22:10:3,v/v/v); migration
distance: TLC, 10 cm; HPTLC, 5 cm; analysis time: TLC, 42 min; HPTLC, 13 min, 45 s; detection: UV at 366 nm. (Chromatograms
courtesy of Merck KGaA, Darmstadt, Germany.)
TLC and HPTLC silica gel 60 plates with etched identification codes, spherical particle plates (not irregular silica gels
as used in most standard layers), and thinner layers for special applications are also available. These can be checked out
on the manufacturers' web sites (EMD/Millipore, Macherey-Nagel, and Analtech).