Forced-Flow Planar Chromatography in the Rear View Mirror

Journal of Chromatographic Science 2015;53:436– 442 doi:10.1093/chromsci/bmu225 Review Forced-Flow Planar Chromatography in the Rear View Mirror Huba Kalász* Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary *Author to whom correspondence should be addressed. Email: Received 7 October 2014; revised 12 December 2014 Mobile phase progress in planar stationary phase can be evoked by either external or internal forces. An internal force is capillarity, while gravity, electric field, a pump and centrifugal forces belong to external forces. Overpressured layer chromatography gives a widely used special chapter of forced-flow planar chromatography, a special bridge between high-performance liquid column chromatography and thin-layer chromatography (TLC). A simple and special rule characterizes the progress of mobile phase. Optimal efficiency is composed by the doubled effect of flow resulting from the pump-forced mobile phase (convex profile of laminar flow) and capillary forces on the dry stationary phase (concave laminar flow). This review describes the most important aspects of forced-flow TLC, including how the setups are developed and also the progress of detection methods used. Introduction Chromatographic processes are differentiated based on the physical state of the mobile phase as liquid chromatography and gas chromatography. The mode of development is characteristic of the separation and behavior of the individual spots, and is called as elution, frontal and displacement development. Based on the geometrical arrangement of the stationary phase, we distinguish planar and column chromatographies (1). Planar chromatography is operated by liquid mobile phase, which progresses to the open dry stationary phase. The separated bands/spots can be either visually followed or detected using specific and sensitive color reagents. A wide scale of specific contact detection can be easily done, such as using X-ray sensors, digital autoradiography, enzyme assay and bioautography using microorganisms. Several samples can be separated at the same time on the same plate, or two-dimensional developments can be easily performed, simply turning the plates by 908 following the first-dimensional development and drying it. Mobile phase progress takes place as a consequence of several types of either internal or external forces. The internal force is capillarity, which means forward movement of the solvent system (mobile phase) to the dry stationary mobile phase. The speed of front movement is determined by capillarity, and counterbalanced by viscosity of the gross path of mobile phase in the stationary phase. The gross weight of the mobile phase on the plate gives an additional factor either accelerating or decelerating mobile phase velocity depending on the mode of development (downward or upward). These cases mean the addition of gravity forces to capillarity. Mobile phase components are constantly evaporating from the TLC plate and condensing to the TLC plate (and from and to the open surface of the mobile phase at the bottom of TLC chamber) as shown in Figure 1. Forced-flow liquid chromatography has always been characteristic of high-performance liquid column chromatography (HPLC). Gravity has widely been used for the development of mobile phase in classical liquid chromatography. Electric field is utilized in electro-chromatography (either capillary or column) and planar electro-chromatography. Cvet (2, 3) used transparent columns, and planar (both paper and thin-layer) chromatography utilized the fact that the entire chromatographic process, and also its final stage separation can be visually followed. Moreover, a wide choice of specific color reagents could also be used to detect the separated spots. Just following the so-called dormant period of liquid column chromatography, the very first separations of thin-layer chromatography (TLC) were done. Its origin goes back to the experiment of Ismailov and Shraiber (4), just after the present arrangement of column chromatography, the flow-through technique was introduced. The progress of TLC has been detailed in several publications (5–12). The features of TLC include its easy handling, separation of several samples at the same time, direct visual observation of both the development and the results of separation, easy and simple use of specific and sensitive color reagents. Moreover, TLC is a technique, which offers a wide scale of instrumentation, from the simplest arrangement (with no or minor technical support) to the highly supported sample applications, forced-flow development, sophisticated detection of separations and computer-aided evaluation and documentation. While tools and instruments of sample load and detection devices were mainly developed by instrumental companies, a unique method to generate mobile phase front with linear front with optimized flow velocity was made possible by a simple tool, called overpressured layer chromatography (OPLC). The elution type of developments was used in the overwhelming majority of OPLC. Forced-flow displacement chromatography as well as elution-displacement development was used by Kalász et al. (13) to separate and identify ecdysteroids of plant origin. This paper reviews the progress and application of the history of the technique of forced-flow development of mobile phase on planar stationary phase. History of forced-flow planar chromatography A relatively compact set-up for OPLC was patented in 1976 (14), although the idea of forced-flow thin-layer chromatography (FFTLC) was initiated to cover the stationary phase, thereby to eliminate the vapor phase (14) (Figure 2). Modes of forced-flow planar chromatography Forced-flow planar chromatography with circular front of mobile phase that is propagated by a pump Izmailov and Shraiber (4) pioneered planar (thin-layer) chromatography using circular arrangement of mobile phase front. # The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: Figure 3. Generation of linear front of mobile phase. Arrows indicate the direction of mobile phase. Figure 1. A constant evaporation (open arrow) and condensation (solid arrow) of volatile components of the mobile phase is characteristic of TLC. Figure 4. Time versus distance curves for (1) saturated chamber, (2) unsaturated chamber and (3) FFTLC. FFTLC performs constant flow velocity of mobile phase. Figure 2. The scheme of a chamber for FFTLC. 1: plastic base plate, 2: stationary phase, 3: covering membrane, 4: mobile phase inlet, 5: gas or liquid pressing the covering membrane to the stationary phase, 6: inlet of gas or liquid pressing the covering membrane to the stationary phase. Kalász et al. (15) published their paper on forced-flow planar chromatography developed with a circular front of mobile phase. Forced-flow planar chromatography with linear mobile phase front of mobile phase propagated by a pump The mob (...truncated)