Stationary Phases for Modern Thin-Layer Chromatography

May 31, 2012

The stationary phases currently in use for modern thin-layer chromatography (TLC) are reviewed here, together with traditional and newer detection techniques. Advantages of the planar, off-line format of TLC relative to on-line column high performance liquid chromatography (HPLC), the steps in TLC analyses, and future prospects of TLC are also discussed. A limited number of applications are presented throughout the article, and many others can be found in the books, book chapters, and review articles cited.

This review discusses the current status of thin-layer chromatography (TLC), focusing mainly on commercial precoated layers that are in popular use. Bulk sorbents, for those who prefer to make their own TLC plates, are available but will not be covered.

TLC is an increasingly widely used analytical method for the determination of many types of analytes in a variety of sample matrices. Its theory, practice, and applications have been documented in biennial reviews written by Sherma since 1970, the latest of which will be published in the Journal of AOAC International (1). An article in LCGC North America (2) gave the three largest industrial applications for TLC as clinical, pharmaceutical, and food testing. The most active research areas today are in the analysis of pharmaceutical bulk materials and dosage forms (3,4) and in natural phytochemical medicines (for example, traditional Chinese medicines and nutritional supplements [5,6]).

The techniques and instruments of modern, instrumental TLC allow it to meet the guidelines for validated analytical methods in current good laboratory practice (cGLP) and current good manufacturing practice (cGMP); pharmacopeias of China, India, and the United States (U.S. Pharmacopeia and American Herbal Pharmacopeia); international food regulations; official methods of AOAC International; International Conference on Harmonization (ICH) pharmaceutical analyses (7); and international food regulations (8,9). Here, we cover some special attributes of TLC relative to column high performance liquid chromatography (HPLC), briefly describe the steps of TLC analyses, focus on the stationary phases (layers) in use today, and speculate on prospects for TLC in the future.

TLC Compared to HPLC

The use of a planar stationary phase rather than a column leads to a number of significant contrasts between TLC and HPLC. HPLC is a closed, on-line method with dynamic detection of eluted solutes, usually by UV absorption or mass spectrometry (MS). The predominant mode of HPLC is reversed phase on a C18 bonded silica gel column, and the predominant mode of TLC is normal phase on a silica gel layer; these diverse mechanisms allow TLC and HPLC to serve as methods with complementary selectivity for separations and analyte identification. An HPLC column is used for many analyses, but each thin-layer plate is used only once. This often leads to the need for less sample preparation (cleanup) for TLC compared to HPLC, because impure samples can be applied without concern for carryover and cross-contamination of samples that leads to additional (ghost) peaks in column chromatograms.

Although it is not yet fully automated like HPLC, many of the steps in the TLC procedures are automated for faster sample analyses. TLC has the advantage of very high sample throughput because multiple standards and samples can be applied to a single plate and separated at the same time under essentially identical conditions. Each track on a TLC plate contains a complete chromatogram of an entire sample, including irreversibly sorbed substances (at the origin). On the other hand, in HPLC each chromatogram requires a separate injection and the chromatogram contains only those sample components that happen to be eluted and detected. Modern computer controlled scanning densitometers and automated sample application and layer development instruments allow validated quantification that is in many cases equivalent to HPLC (10). As shown later, TLC has the widest choice of stationary phases and mobile phases, making it the most versatile and flexible LC method. Solvents that can interfere with HPLC UV detection can be used because the TLC mobile phase is removed before detection. Mobile-phase consumption is low, minimizing solvent purchase and disposal costs, thereby making TLC a "green" method. The wide choice of development methods and pre- and postchromatographic detection methods leads to unsurpassed selectivity in TLC. Because TLC is an off-line method, the various steps can be carried out independently without time constraints; as an example of an advantage of this approach, zones can be scanned repeatedly with a densitometer using different parameters that are optimum for individual sample components. In addition, a TLC plate can be stored and reinvestigated at a later date if additional questions arise.