Choice of Adsorbents - Thin Layer Chromatography (TLC)

CHOICE OF ADSORBENTS

The choice of proper adsorbent in TLC plays a vital role in the separation of components either belong-ing to natural origin or to purely synthetic origin. It is chiefly based on certain crucial informations like :

(i) Solubility of the substance e.g., hydrophilic and lipophilic,

(ii) Nature of the compound i.e., whether it is acidic/basic/neutral/amphoteric

(iii) Reactivity of compound with either the solvent or the adsorbent, and

(iv) Chemical reactivity of compounds with the binders.

In actual practice, the adsorbents are of two types : firstly the inorganic, and secondly, the organic adsorbents. A host of substances from each type are used in TLC and these shall be discussed briefly as below :

1. Inorganic adsorbents

These are namely :

(i) Aluminium oxide- (Al₂O₃) : The alkali (Na₂CO₃ ; NaHCO₃) present in alumina very often gives rise to secondary reactions that may be eliminated by washing with dilute mineral acid or with water, followed finally by methanol and ultimately by heating at 200 °C.

Note : Justisaz and Teichner* in 1947 suggested that 1 g of alumina for TLC has 90 sq. M surface area and the one having less than 6 sq. M is useless. Alumina is usually available in three grades :

(a) acidic (pH ~    4.0) ; (b) basic (pH ~      9.0) ; and (c) neutral (pH ~ 7.5)

(ii) Aluminium Silicate : It permits the adsorption of sterols and sterol glycosides from oils without the use of solvent.

(iii) Bauxite (aluminium oxide ore) : Zechmeister used bauxite for the separation of enzymic hydrolysates of chitin (a nitrogen-containing polysaccharide found in certain fungi e.g., ergot) ; whereas La Lande employed it for the refining of sugar.

(iv) Bentonites : It is used mostly for the separation of Vitamin D from vitamin A and sterols and 2,4-dinitrophenyl hydrazones of aldehydes and ketones.

(v) Calcium Carbonate : It is used as such for the separation of xanthophylls and napthaquinones or other pigments and elution is done with dilute acid to isolate the various components present.

Note : Vaterite-the unstable crystalline modification of calcium carbonate has much greater adsorbent ca-pacity than aronite or calcite.

(vi) Calcium Hydroxide : It is used as an adsorbent for the separation of carotenoids.

(vii) Calcium Oxalate : It is used for the separation of anthraquinones and related hypericins (i.e., a dianthrone pigment found in the leaves and petals of Hypericum perforatum, Family ; Guttiferae).

(viii) Calcium Silicate : It is employed frequently for the separation of carbohydrates and the corre-sponding phenylosazones.

(ix) Calcium Sulphate : It is found to be suitable for the separation of steroids and lipids.

(x) Dicalcium Phosphate : It is used for the purification of carotene-the natural red pigment.

(xi) Fuller’s Earth : It is hydrous magnesium aluminosilicate which is employed extensively in the petroleum industry for the decolaration of oils. It is also employed for the separation of amino acids and pteridines.

(xii) Hydoxyl-Apatite : It is a complex calcium phosphate hydroxide which is used for the separation of proteins and glycerides. In may be used with/without binder.

(xiii) Kieselguhr (Diatomaceous Earth) : (pH 7.0) : It is available both with and without a binder. Its capacity of resolving constituents is less than either silica gel or alumina.

(xiv) Magnesium Silicate (Magnesol : MgO 2.5 SiO₂.H₂O) : It is usually employed for the separation of sugar acetates ; whereas, magnesium trisilicate is used for the separation of steroids, acetylate gycosides, esters, glycerides, lactones etc.

(xv) Silica Gel : (pH 6.0) : It is used extensively for the separation of sterols, fatty acids, glycerides, azoated carbohydrates, sugar acetates, amino acids.

(xvi) Tri-calcium Phosphate : It is mostly used for the separation of enzymes.

(xvii) Water-soluble salts : A number of water-soluble salts are used in TLC for affecting separation of constituents, namely :

CuSO₄ (anhydrous) :for azobenzene derivatives,

CuSO₄.5H₂O :found to be better than alumina,

ZnSO₄ ; MnSO₄ ; Al₂(SO₄)₃ and MgSO₄ : anhydrous salts good for azobenzene derivatives,

Al₂(SO₄)₃:for hydroxyl anthraquinones, and

Na₂CO₃:for Vitamin A

(xviii) Zinc Carbonate : It is used for the separation of carotenoids and coloured derivatives of amino acids.

2.2. Organic Adsorbents

The organic adsorbents are known for their relatively milder action for the separation of good number of components, namely :

(i) Cellulose and Acetylated Cellulose : These adsorbents are commercially available in various forms e.g., particle size, degree of acetylation, with or without binders like starch or Plaster of Paris.

(ii) Charcoal and Activated Carbon : Tiselius used charcoal for the frontal analysis of sugars, amino acids and other substances. Charcoal absorbs strongly aromatic substances, such as : amino acids, which may be explained by virtue of the fact that the carbon-carbon spacings in graphite are almost of the same order as those present in benzene. Charcoal is also employed for the adsorption of fatty acids.

Weiss* used impregnated activated carbon with fatty acid or non-electrolyte thereby modifying and attributing special and improved adsorption characteristics.

(iii) Dextran Gels : Proteins and nucleotides can be separated by using cross-linked dextran gels available in various types and particle sizes. The molecular weight of dextran-gels vary considerably depending upon the extent of cross-linked nature.

(iv) Cellulose Ion-Exchange Powder : Interestingly, the cellulose powder have been modified by state-of-the-art technique that they invariably mimic as real ion-exchangers, namely :

DEAE-Cellulose             = Diethaminoethyl cellulose,

ECTEOLA-Cellulose  = Epichlorhydrin linked triethanolamine cellulose, and

PEI-Cellulose                  = Polyethylenimine cellulose.

Note : These absorbents may be used both with or without binders, such as : colloidion.

(v) Ion-Exchange Resins : Nucleic acids and their respective derivatives may be separated either by using ion-exchange resins alone or in conjunction with cellulose powder.

(vi) Polyamide : Flavanoids-the phenolic substances may be separated effectively using polyamide as such or with a binder, for instance : plaster of Paris or starch.

(vii) Polyethylene Powder : Fatty acids and their corresponding esters are separated by using polyethylene powder.

(viii) Sucrose : Both xanthophylls and chlorophylls (i.e., chlorophyll-a and -b) are separated by using sucrose powder effectively.