Plates with Channels and Preadsorbent Zone
Kieselguhr (diatomaceous earth) and silica 50,000 are small surface area, weak adsorbents that are used as the lower 2–4 cm
inactive preadsorbent (also called concentration zone) in the manufacture of silica gel and C18 preadsorbent plates. Samples
applied to the preadsorbent zone run with the mobile-phase front and reform into sharp, narrow, concentrated bands at the
preadsorbent–analytical sorbent interface. This leads to efficient separations with minimum time and effort in manual application
of samples, and possible sample cleanup by retention of interferences in the preadsorbent, which can be modified by dipping
(then drying) into reagents that can capture unwanted impurities once spotted.
Channeled (laned) TLC and HPTLC plates have tracks scribed in the layer that are 9 mm wide with 1 mm between channels (19
useable channels across a 20-cm wide plate). Advantages include no possible cross-contamination of zones during development;
exact location of zones during application, which facilitates alignment of a densitometer light beam for automated scanning;
and easier removal of separated zones by scraping, before recovery by elution, without contaminating or damaging adjacent
Pretreatment of Plates
To remove extraneous materials that may be present because of manufacture, shipping, or storage conditions, it is recommended
to preclean plates before use by development to the top of the plate with methanol (24). The plate is then dried in a drying
oven or plate heater for 30 min at 120 °C. If the plate is being used immediately, it will equilibrate with the laboratory
relative humidity (which should be controlled to 40–60% and recorded regularly) during sample application. Plates that are
prewashed do not need activation by heating unless they have been exposed to high humidity. More complete suggestions for
initial treatment, prewashing, activation, and conditioning of different types of glass- and foil-backed layers have been
Glass plates can be cut to a smaller size using a commercial glass cutter and broken with grozier pliers for better results
(Figure 4). Scissors, a razor knife, razor blade, or circular cutter (as used to cut fabric) can all be used to cut plastic
or aluminum sheets. A straightedge should be used, and care must be taken to avoid damaging the sorbent layer and your fingers.
Figure 4: Cutting tools for scoring and breaking glass TLC plates: (a) pistol-grip glass cutters and (b) grozier pliers. These
items are available from hobby or stained glass stores. Images from author’s collection (F.R.).
Silica gel (silica) is by far the most frequently used layer material in TLC. The structure is held together by bonded silicon
and oxygen (siloxane groups), and separations take place primarily because of differential migration of sample molecules caused
by selective hydrogen bonding, dipole–dipole interaction, and electrostatic interactions with surface silanol groups (Si-OH).
The intensity of these adsorption forces depends on the number of effective silanol groups, the chemical nature of the sample
molecules to be separated, and the elution strength of the mobile phase. Differences in the type and distribution of silanol
groups for individual sorbents may result in selectivity differences, and separations may not be exactly the same for brands
of silica gel layers from different manufacturers. Typical properties of silica gels suitable for planar chromatography are
as follows (25): mean hydroxyl (silanol) group density, approximately 8 µmol/m2 (independent of silica gel type); mean pore diameter between 40 and 120 Å (4–12 nm); specific pore volume (v
) between 0.5 and 1.2 mL/g; mean particle size and particle size distribution are shown in Table I; and specific surface area
(SBET) between 400 and 800 m2 /g. Silica gel 60 has a 60-Å pore diameter and is the most commonly used type in TLC and HPTLC. Precoated plates used in
a laboratory with 40–60% relative humidity and temperature of 20 °C will become equilibrated to a hydration level of 11–12%
water, will not require preactivation unless earlier exposed to high humidity, and will give consistent R
f values within this moisture range.
Figure 5 shows a typical high-resolution separation with symmetrical, narrow densitometer peaks obtained on a silica gel HPTLC
Figure 5: Densitogram of a neutral lipid standard mixture showing peaks of (left to right) cholesterol, oleic acid, triolein,
methyl oleate, and cholesteryl oleate, which serve as marker compounds for free sterols, free fatty acids, triacylglycerols,
methyl esters, and steryl esters, respectively. 4 µL of a solution containing 0.2 µg/µL solution (0.8 µg) of each compound
was applied to a laned Analtech HPTLC silica gel preadsorbent plate, which was developed with petroleum ether–diethyl ether–glacial
acetic acid (80:20:1) in a vapor-saturated Camag HPTLC twin-trough chamber. The lipid bands were detected by spraying the
plate with 5% ethanolic phosphomolybdic acid solution and heating at 110 °C for 10 min, and densitometry was carried out at
610 nm using a Camag Scanner 3 with winCATS software.