Chapter 3. Thin Layer and Paper Chromatography Analysis

SGARIGLIA, M. A.; SOBERÓN, J. R.; SAMPIETRO, D. A.; VATTUONE, M. A.

Soil Allelochemicals

Studium Press LLC

Lugar: Houston; Año: 2011; p. 55 - 86

Paper Chromatography (PC) and Thin Layer Chromatography (TLC) are separation techniques for preliminary identification of organic compounds in mixtures. They are based on planar or open bed chromatography. This means that components of a sample are separated between a liquid mobile phase and a stationary phase, which is presented as or on a plane. The stationary phase is a strip or piece of paper (PC) or a layer of solid particles spread on a support (TLC), e.g. a glass, aluminum or plastic plate. Compounds can be separated according to different separation principles defined by characteristics of both mobile and stationary phases. Types of chromatography are as under: (i). Adsorption Chromatography: The separation involves competing interactions between the adsorption at the solid sorbent surface and dissolution in a liquid mobile phase. Substances are separated as per polarity of their functional groups, when they migrate on and interact with the sorbent surface; (ii). Partition Chromatography: The separation occurs in a liquid-liquid phase system where sample components are separated according to their differential solubilization in the mobile and stationary phases. This principle governs the PC and TLC in cellulose plates when using an aqueous mobile phase. Partition chromatography can be achieved in TLC based on other stationary phases by their impregnating with liquids such as ethylene glycol, water or acetic acid; (iii). Ion-Exchange Chromatography: Separation is based mainly on the differences in ion exchange affinities of sample components; (iv). Exclusion Chromatography: Separation is based mainly on exclusion effects (differences in molecular size and/or shape, or in charge). The term Size-Exclusion Chromatography may also be used when separation is based on molecular size. The term Ion-Exclusion Chromatography is specifically used for the separation of ions in an aqueous phase. (v). Affinity Chromatography: This expression characterizes a particular variant of chromatography in which the unique biological specificity of the analyte and ligand interaction is used for the separation.       PC was the first chromatographic technique developed in organic chemistry and is still used, to isolate flavonoids because it allows an inexpensive, highly reproduced and powerful qualitative analysis with small quantities of a sample. Nevertheless, PC has been largely replaced by TLC. The main advantages of TLC is the wide array of compound polarities in which it can be used, short analysis time, moderate price per analysis and multiple fractions/compounds can be analyzed in parallel on a single TLC plate. It only requires small quantities of compound (~ng) and is much faster than other chromatographic procedures. This chapter provides procedures and criteria needed for use of planar chromatography in separation, identification and characterization of soil allelochemicals and related compounds.