Stationary Phases for Modern Thin-Layer Chromatography - - Chromatography Online
Stationary Phases for Modern Thin-Layer Chromatography

LCGC North America
Volume 30, Issue 6, pp. 458-473

Cyano bonded layers: Cyano layers can act as a normal or reversed phase, depending on the characteristics of the mobile phase, with properties similar to a low capacity silica gel and a short-chain alkylsiloxane bonded layer, respectively.

Amino bonded layers: Amino layers are used in normal-phase, reversed-phase, and weak basic anion-exchange modes. In normal-phase TLC, compounds are retained on amino layers by hydrogen bonding using less polar mobile phases than for silica gel. Activity is less than silica gel, and selectivity is different.

A special feature of amino precoated layers is that many compounds (for example, carbohydrates, catecholamines, and fruit acids [24]) can be detected as stable fluorescent zones by simple heating of the plate at 105–220 C (thermochemical activation).

Sugars, sugar alcohols, barbiturates, steroids, carbohydrates, phenols, and xanthin derivatives have been separated on amino layers in various aqueous and nonaqueous mobile phases (24).

Diol bonded layers: Diol plates have functional groups in the form of alcoholic hydroxyl residues and unmodified silica gel has active silanol groups. The vicinal diol groups are bonded to silica with a nonpolar alkyl ether spacer group. Diol layers can operate with normal-phase or reversed-phase TLC mechanisms, depending on the mobile phase and solutes.

Polar compounds show reasonable retention by hydrogen bond and dipole-type interactions in the former mode, and in the reversed-phase mode retention with polar solvent systems is low but higher than with amino layers. A study of mixed mechanisms on cyano, amino, and diol layers was reported (24).

Nonsilica Sorbents


TLC cellulose consists of long chains of polymerized β-glucopyranose units connected at the 1–4 positions. Precoated TLC and HPTLC crystalline cellulose (Avicel, Analtech) layers (400–500 glucose units) and native fibrous cellulose TLC layers (40–200 glucose units) without binder are used for the separation of polar substances, such as amino acids and carbohydrates. The mechanism is normal-phase partition with sorbed water as the stationary phase, although adsorption effects cannot be excluded in cellulose separations. Zones are generally less compact and development times are much longer than on silica gel.

Modified Celluloses

Cellulose has been surface modified to produce reversed-phase (acetylated cellulose), basic anion exchange (polyethyleneimine [PEI], aminoethyl [AE], diethylaminoethyl [DEAE], and ECTEOLA), or acidic cation exchanger (cellulose phosphate [P] and carboxymethylcellulose [CM]) layers. Acetylated cellulose has been used mostly as a chiral layer for separation of enantiomers (26). The cellulose ion exchangers have open structures that can be penetrated by large hydrophilic molecules such as proteins, enzymes, nucleotides, nucleosides, nucleobases, and nucleic acids.


Alumina (aluminum oxide) is a polar adsorbent that is similar to silica gel in its general chromatographic properties, but it has an especially high adsorption affinity for carbon–carbon double bonds and better selectivity toward aromatic hydrocarbons and their derivatives. It is available in basic (pH 9–10), neutral (pH 7–8), and acid (pH 4–4.5) form. The most used TLC aluminas are alumina 60 (type E), 90, and 150 (type T). Other properties of alumina can be found in reference 28. Alumina is not yet available as an HPTLC plate.


Polyamide 6 (nylon 6; polymeric caprolactam) is a synthetic organic sorbent that shows high affinity and selectivity for polar compounds that can form hydrogen bonds with the surface amide and carbonyl groups. Reported applications of polyamide TLC include separations of flavonoid aglycones, flavonol glycosides, leaf flavonoids, and phenols (24).


Kieselguhr is natural diatomaceous earth consisting mostly of SiO2. It has high porosity and is completely inactive, used as a support for impregnated reagents (for example, ethylenediaminetetraacetic acid for separation of tetracycline broad spectrum antibiotics) and as a preadsorbent zone. Macherey-Nagel manufactures precoated 0.25-mm kieselguhr layers with and without UV254 indicators.

ProteoChrom Plates

ProteoChrom plates were especially developed by EMD/Millipore for fast and easy separations of peptide and protein digests. The silica gel 60 F254 plates are for one dimensional (1D) separations and the 10 10 cm microcrystalline cellulose plates are for 2D separations to produce peptide maps.


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