Layers concentrating zones

Samples and reference substances should be dissolved in the same solvents to ensure that comparable substance distribution occurs in all the starting zones. In order to keep the size of the starting zones down to a minimum (diameter TLC 2 to 4 mm, HPTLC 0.5 to 1 mm) the application volumes are normally limited to a maximum of 5 xl for TLC and 500 nl for HPTLC when the samples are applied as spots. Particularly in the case of adsorption-chromatographic systems layers with concentrating zones offer another possibility of producing small starting zones. Here the applied zones are compressed to narrow bands at the solvent front before the mobile phase reaches the active chromatographic layer. 

The samples were applied to the concentrating zone as bands in the direction of chromatography. The zones were concentrated by brief development in the mobile phase described below almost to the junction between the concentrating zone and the chromatographic layer, followed by drying for 5 min in a stream of warm air. The actual chromatographic separation was then carried out. 

Layer HPTLC plates Silica gel 60 with concentrating zone... 

The most-nsed stationary phase in PLC is sihca gel, with type 60 taking preference. In the fnture, other sorbents snch as the RP materials will also most probably be increasingly nsed. This will also be trae for the case of special PLC plates consisting of layer combinations snch as precoated plates with concentrating zones, resnlting in simphfication of sample application as well as an increase in the efficiency of separation. 

A very helpful tool for manual application can be the employment of layers with a concentrating zone. The so-called concentrating or preadsorbent zone is a small part of the plate that is covered with an inert but highly porous adsorbent such as diatomaceous earth. Various precoated preparative layers with a preadsorbent zone are commercially available. The effect of the concentrating zone is depicted elsewhere in detail (see Chapter 3, Figure 3.4). In brief, the preadsorbent zone serves as a platform for manual application of any desired performance quality. When development starts, soluble components migrate with the mobile phase front and are... 

FIGURE 12.2 Diagrammatic representation of the effect of the concentration zone in preparative separations, (a) Precoated layer without concentrating zone, (b) Precoated layer with concentrating zone. (From Nyiredy, S., Preparative layer chromatography, in Handbook of Thin Layer Chromatography, 3rd ed., Vol. 89, Sherma, J. and Fried, B., Eds., Marcel Dekker, New York, 2003, pp. 99-133. With permission.)... 

Tapered plates, prepared with a gradual increase in thiclcness of the layer from 0.3 nm to 1.7 am, can be used to improve resolution of the sample [215]. On the tapered layer the solvent front velocity decreases as the thickness of the layer increases. This results in the formation of a negative velocity gradient in the direction of solvent migration. As a result the lower portion of a zone moves faster than the top portion, keeping each component focused as a narrow band. Plates with concentrating zones are useful for optimizing sample application. 

F. Rabel and K. Palmer, Advantages of using thin-layer plates with concentration zones, Am. Lab., 77 20BB (1992). 

The HPTLC pre-coated layers examined in this paper, namely silica gel 60, RP, cellulose and silica gel 60 with concentrating zone, are new developments or modifications of existing TLC pre-coated layers offering new potential for pesticide analysis by thin-layer chromatography. 

In order to improve the separating performance of HPTLC pre-coated plates silica gel 60 even at larger applied volumes, as may be necessary at low sample concentrations, and with a rapid and simple technique of application, HPTLC pre-coated plates silica gel 60 with so-called "concentrating zones" were developed (10, 11, 12). This type of plate consists of two distinct layer sections, namely the separating layer proper consisting of silica gel 60 and a concentrating zone composed of an inert, porous silicon dioxide. These two sorbent materials pass into one another at a clearly defined boundary-line in such a way that the eluant is offered no resistance as it passes through. 

One zone is normally kieselguhr, 3 cm long and 150 pm thick, which has comparatively poor ad-sorptive properties. Thus, any size of spot placed on tiiis layer and run in the mobile phase will become a sharp band before it gets to the analytical silica gel layer. Anotiier form of plate for special applications is one with a pre-concentration zone of octadecyl-silica and an analytical layer of silica. These plates simplify sample application and improve sensitivity, but are very expensive compared with conventional plates. Approximately the same effect can be obtained using conventional plates and running them first in methanol for 0.5 cm. This converts all the spots to thin bands which can then be run in the solvent of choice. 

Description. The model organism is a free-floating unicellular sphere with characteristics selected, where possible, to match those of a phytoplankton cell. The organism and its environment (Figure Ic) are divided into four concentric zones -the bulk solution, the diffusion layer, the containing membrane and the cell concents. We will assume that the species taken up by the cell is the free metal ion since most of the studies of the uptake of B-subgroup metals by organisms support this hypothesis Z . 5 steady-state transport processes are... 

TLC. Silica gel 60 layer with concentrating zone, ethyl acetate-methanol-acetic acid (8 1 1) mobile phase. 

Macherey-Nagel, and HPTLC-Chir, Merck, Germany) for ligand-exchange chromatography. The chiral layer on the latter plates is combined with a so-called concentrating zone. ... 

Since the introduction of commercial precoated plates in the mid-1960s, continual developments with regard to the increase of selectivity and improvement of separation efficiency were pursued [i.e., ready-to-use layers suitable for high-performance thin-layer chromatography (HPTLC), polar and hydrophobic bonded phases, plates with concentrating zones],... 

Subsequently, thin layer chromatography (TLC) methods have been extensively evaluated to optimize the detection of laxatives/ The most important findings for the successful detection of laxatives by TLC were (1) the skill and experience of the operator, (2) the choice of the mobile phase (ethyl acetate toluene glacial acetic acid [4 16 1] and hexane toluene glacial acetic acid [3 1 1] gave the best results), and (3) use of high-performance TLC plates with a concentrating zone. 

Fig. 3.6 Conical concentration zone of a thin-layer plate according to Stahl (1967).

Layer HPTLC plates Silica gel 60 F254, with concentrating zone wavelength UV light (A = 365 nm). In each case the detection limits were 2 ng... 

Standard and nano silica gel, also with concentrating zone Normal phase chromatography Most frequent apphcation of all TEC layers... 

At this point, there usually follows a chapter about the pretreatment of the samples. However, in contrast to HPLC/GC, sample preparation for TLC is not considered to be quite as critical. As well as the use of precoated layers with a concentration zone (e.g. an application zone consisting of silica 50 000 and a separation zone of sihca gel 60 or RP-18 material) upon which the matrix constituents can often be held back by suitable choice of solvent system, a chromatogram that is imusable for lack of sample preparation is more rapidly rectified (use a different preparation method and a new plate ) than an irreversibly destroyed column. A detailed treatment of the subject of sample preparation would exceed the scope of the present book. In Section 9.4, rm-der the title Examples of GMP/GLP-Conforming Testing Procedures , we describe the extraction of a pharmaceutically active substance from a tablet and the working up of plant components from dry extracts. The reader is referred to other TLC textbooks [2,21] and to literature and brochures produced by manufacturers of articles for sample preparation [28, 29]. 

The use of commercially available preparative plates with a concentration zone will enhance separation. This zone is a layer of inert large-pore silica at the bottom of the plate onto which the sample is applied. As the solvent migrates through this zone, the mixture is unretained and focuses at the interface between the zone and normal sorbent. Uneven applications of mixtures are focused as discrete lines, and this will greatly improve separation/resolution. 

---