Chapter 1

Milestones, Core Concepts, and Contrasts

Colin F. Poole     Department of Chemistry, Wayne State University, Detroit, MI, USA

Abstract

Thin-layer chromatography and column chromatography are complementary separation techniques based on liquid chromatography. Although their application domains overlap, there is generally good reason to select one method over the other for particular applications. Capillary-controlled flow and the development mode are widely used in thin-layer chromatography. This restricts its kinetic performance compared to forced-flow techniques. Multiple development and multidimensional strategies increase the separation potential when using capillary-controlled flow. Incremental multiple development facilitates the use of solvent-strength gradients for the separation of mixtures with a wide range of retention properties. In this chapter, the general parameters used to describe separations in thin-layer chromatography are described and compared with the equivalent terms employed in column chromatography with a view to establishing the similarities and differences for the two techniques. This approach also affords general framework for method selection and to establish expectations.

Keywords

Capillary-controlled flow; Column chromatography; Forced flow; High-pressure liquid chromatography; History; Multiple development; Plate height; Plate number; Resolution; Retardation factor; Solvent-strength gradients; Spot capacity; Thin-layer chromatography; Two-dimensional chromatography

1.1. Introduction

Column chromatography and thin-layer chromatography are alternative formats for liquid chromatography [1]. Both formats exist as simple laboratory tools requiring little instrumentation and also as fully instrumental techniques. In both the cases, the stationary phase consists of a sorbent bed containing homogeneously packed particles or as a porous monolith. When movement of the mobile phase through the sorbent bed is controlled by capillary forces, the separation performance is suboptimal but requires little instrumentation affording a convenient and flexible arrangement for simple separations at the analytical or preparative scale. For faster separations, or separations with a higher peak capacity, a mechanism is required to enhance the mobile phase velocity. This requires instrumentation to pressurize the mobile phase and is the basis of high-pressure (or high-performance) liquid chromatography (HPLC) for columns and forced flow (or overpressured layer chromatography) for layers [2-4]. Although forced-flow instrumentation for thin-layer chromatography is commercially available, it is not in common use. Thus, whereas the practice of HPLC is a forced-flow technique, the practice of thin-layer chromatography is predominantly a capillary-controlled flow technique. In the latter case, instrumentation is required to optimize the various steps in the separation process and is referred to as high-performance thin-layer chromatography (HPTLC), or instrumental thin-layer chromatography, to distinguish the technique from conventional thin-layer chromatography (TLC) performed with much simpler equipment [5,6]. The general advantages of utilizing HPLC conditions versus conventional column chromatography are well known. The same argument cannot be made for conventional TLC versus HPTLC, and the general migration of separations from conventional TLC practices to HPTLC has not been universal. In fact, one might say that conventional TLC thrives in the laboratory environment as a quick, inexpensive, flexible, and portable method for surveying the composition of simple mixtures while only a few laboratories are equipped to perform more complex and quantitative analyses by HPTLC.

1.2. Milestones

The origins of thin-layer chromatography can be traced to the experiments on drop chromatography performed by Izmailov and Shraiber in the late 1930s [7]. From this beginning, thin-layer chromatography evolved into a fast and more powerful tool than gravity flow column chromatography for analytical separations. Thin-layer chromatography, as we know it today, was established in the 1950s due in large part to the efforts of Stahl and Kirchner on different continents. Their main contribution was the development of standardized materials and procedures that led to improved performance and reproducibility, as well as popularizing the technique by contributing many new applications [8]. At about the same time, commercialization of materials and devices commenced making the technique accessible to all laboratories. This ushered in the golden era of thin-layer chromatography where it quickly displaced paper chromatography as the main analytical liquid chromatographic method. By the 1970s, high-pressure liquid chromatography was becoming firmly established as an alternative approach for liquid chromatography and eventually grew to eclipse thin-layer chromatography for analytical applications. Thin-layer chromatography did not disappear in subsequent years but became less well known to those working in analytical laboratories where its strengths were often under appreciated. Developments continued in thin-layer chromatography as indicated by the time line Figure 1.1 [6,9]. First the development of high-performance thin-layer chromatography in the late 1970s is discussed. Layers coated with smaller particles of a narrow size distribution required the development of instruments for their convenient use. This was achieved by the early 1980s and so began the second era of thin-layer chromatography, known as modern or instrumental thin-layer chromatography. The evolutionary changes during this second era are captured in a series of books, which if ordered chronologically, represent the state-of-the-art at different times during this period to the present [5,10-15]. The main characteristic features of modern thin-layer chromatography are the use of fine particle layers for fast and efficient separations; sorbents with a wide range of sorption properties to optimize selectivity; the use of instrumentation for convenient and usually automated sample application, development and detection; and the accurate and precise in situ recording and quantification of chromatograms. Improvements in virtually all aspects of thin-layer chromatography continued over the next quarter century as indicated in Figure 1.1 and form the basis of subsequent chapters in this book. This period also marks the beginning of the philosophical division between conventional and high-performance thin-layer chromatography that has not been crossed by all those who use thin-layer chromatography. Expectations in terms of performance, ease of use, and quantitative information from the two approaches to thin-layer chromatography are truly opposite (see Section 1.1). As an example of expectations for a separation by modern thin-layer chromatography, see the chromatogram in Figure 1.2 for structurally similar ethyl estrogens (steroids used for birth control) [2]. Because of the small structural differences for these compounds, a high selectivity is required for their separation. Baseline separation is obtained with a short migration distance typical of fine particle layers and scanning densitometry provides a conventional record of the separation in the form of a chromatogram, as well as quantification of individual steroids after calibration. The quantitative results for tablet analysis are as accurate and precise as other chromatographic methods and the method is suitable for high-throughput routine tablet conformity analysis in which sample preparation requires no more than dissolution and filtration. Some specific reasons for choosing thin-layer chromatography for quantitative analysis are outlined in the next section.
FIGURE 1.1 Time line depicting important developments in the evolution of modern thin-layer chromatography. FFD = forced-flow development in an overpressured development chamber; AMD = automated multiple development chamber; AMC = automatic development chamber; and PPEC = pressurized planar electrochromatography.
FIGURE 1.2 Separation of ethynyl steroids by modern thin-layer chromatography. Two 15 min developments with the mobile phase hexane-chloroform-carbon tetrachloride-ethanol (7:18:22:1) on a silica gel HPTLC plate. The chromatogram was recorded by scanning densitometry at 220 nm. Reproduced with permission from Ref. [2].

1.3. Attributes of a Planar Format

Columns afford a better arrangement for operation at high pressures and for variation of the separation conditions by the control of external parameters. The thin-layer format provides a better arrangement for high sample throughput, flexible detection strategies, and a greater tolerance of samples with a high-matrix burden [2,16]. The throughput advantage is a consequence of the possibility of separating multiple samples in parallel with each sample occupying a single lane (or track) on the layer and several samples assigned to different lanes for simultaneous separation. Column chromatography is inherently a sequential separation process in which the separation time for a group of samples is the product of the...