Sample application 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. 

FIGURE 3.4 Stages of the development of PLC plates silica gel 60 with concentrating zones separation of lipophilic dyestuffs with toluene as the mobile phase, (a) Sample application by dipping in the sample solution, (b) dot-like sample application. 

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. 

Samples may be applied as bands to TLC plates using either specially prepared plates with a concentrating zone (see section 7.3.1) or by usin a band applicator such as the Linomat IV,... 

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. 

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. 

Improvements in the separating performance achieved with HPTLC pre-coated plates silica gel 60 with concentrating zone (E. Merck, Darmstadt) compared with HPTLC pre-coated plates silica gel 60 without concentrating zone, taking as an example the application of a relatively large sample volume of 0.75 ill (in HPTLC sample volumes of between 0.02 and 0.1 pi are common practice ), is demonstrated by the separation of Trevespan 6038, as already described. 

It is possible to apply sample substances by immersing the concentrating zone of the plate into a dilute sample solution. This method can replace a multiple, streak-wise application of very dilute sample solutions using specialised apparatus. 

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. 

Quantitative evaluation of thin-layer chromatograms can be performed by direct, in situ visual, and indirect elution techniques. Visual evaluation involves comparison of the sizes and intensities of color or fluorescence between sample and standard zones spotted, developed, and detected on the same layer. The series of standards is chosen to have concentrations or weights that bracket those of the sample zones. After matching a sample with its closest standard, accuracy and precision are improved by respotting a more restricted series of bracketing standards with a separate sample spot between each of two standard zones. Accuracy no greater than 5-10% is possible for trained personnel using visual evaluation. The determination of myco-toxins in food samples is an example of a practical application of visual comparison of fluorescent zones. 

Application llOXfor 30 min. The samples were applied to the concentrating zone as bands in the direction of chromatography. The zones were concentrated hv brief develonment in the mobile nhase described in situ quantitation The fluorimetric determination was carried out in UV light (Aexc = 313 nm, An > 390 nm). 

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]. 

A suitable technique for this purpose (concentration of trace components and their isolation in a new reaction) involves the use of a liquid bubbler. The applicability of this technique in analytical reaction gas chromatography was shown earlier [15] using as an example the preliminary concentration of hydrogen sulphide and carbon dioxide by means of an alkaline solution of sodium hydroxide through which a flow of the sample gas was passed. When the solution was acidified, the acidic traces formed a concentrated zone, and were separated on a chromatographic column. A liquid bubbler as illustrated in Fig. 8.3 was used to concentrate the trace components. 

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. 

FIGURE 38.14 (See color insert following page 810.) CCD camera images of on-chip sample peaks of AlexaFluor 488 at the LE/TE interface in two different ITP experiments. In (a) there is finite (nonuniform) EOF and the sample peak streamwise dimension is on the order of channel width or larger. In (b) EOF is suppressed and the sample is concentrated in narrower zone ( 5 jjim) at relatively high electric field. While Taylor dispersion based analysis is probably applicable in the first case, more comprehensive modeling is required for case (b). 

Preadsorbent zones This is a thin-layer plate designed with an area below the silica or bonded silica sorbent that is made up of diatomaceous earth or a wide pore silica gel. The purpose of this preadsorbent area is to allow fast sample application even with a crude spotting device with no absorption or separation of the sample components. After drying, when the development begins, the sample dissolves and concentrates onto itself to form a narrow band before it moves onto the active sorbent for separation. These sample bands improve the resulting separation compared to spots placed on the active layer, as streaking the sample does on any TLC plate. These plates are particularly well suited for dirty or biological samples where this area acts to preclean the sample, rather like an initial filtration. 

Botz et al. (1990) and Nyiredy (1996) described a solid-phase sample application method and device that is useful for both capillary-and forced-flow PLC. The initial zone produced is homogeneous within the entire cross section of the preparative layer and has an extremely sharp edge leading to the chromatographic layer, with the advantage of in situ sample concentration and cleanup. 

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