Saturation of the chamber

The kinetics of eluent migration will depend on the degree of saturation of the chamber. The eluent front runs faster in a saturated chamber than in an unsaturated one. The problem of the reproducibility of Rf values can be seriously affected by an unsaturated chamber. 

There is another type of S chamber which permits prior saturation of the chamber atmosphere, simultaneously with the preconditioning of the adsorbent, before starting the chromatographic process (Fig. lb). For this purpose, the adsorbent is removed (a 1 cm width) from all the sides of the plate (1). The opposite frame plate (2) contains, on all of its surface, a thin layer of sorbent. The two plates are fixed by two clamps (3). The chamber is introduced into the trough (4) through a slit. At the beginning, the level of the eluent (5) does not touch the bottom of adsorbent on the thin-layer plate (1). When saturation of the atmosphere is achieved, a volume of eluent is added to the eluent in the trough until the adsorbent is wetted. At this moment, the separation can be started. 

The newly introduced HPTLC-LiChrospher Si 60 F254S precoated plates in many cases show a still better separation efficiency than the HPTLC silica gel 60 precoated plates. For example. Fig. 12a (HPTLC silica gel 60) and Fig. 12b (HPTLC-LiChrospher Si 60254s) how the scanned chromatograms of eight pesticides, all in the solvent system petroleum ether 40-60 °C + acetone (70 + 30 ml, without saturation of the chamber) [13a]. Also in the analysis of frankincense (olibanum), better separations were obtained on plates precoated with spherical silica gel than on those precoated with irregularly shaped particles [13b-dj. 

Pre-loading means, in general terms, i.e. irrespective of the degree of saturation of the chamber and layer, any sorptive uptake of gas molecules from the chamber atmosphere by the imwetted TLC layer (see also Section 3.4 Conditioning ). 

To produce saturation of the chamber, circular filter papers must never be used, as these would not take up solvent over the whole width of the chamber. 

The Rf-values were determined on a 8 cm-flow distance with saturation of the chamber ... 

Note Since differences in adsorbent and solvent affect the reproducibility of i /-values, it is a good idea to run at the same time one or more alkaloids or even dyes (rhodamine B or butter yellow). Their E/-values then serve as references for those of the unknown alkaloids. The saturation of the chamber also has a powerful influence on i /-values. Development only at chamber saturation is thus recommended this improves the reproducibility of the alkaloid J /-values, if sometimes at the expense of resolution sharpness. It must consequently be stressed here also that all i /-values quoted in this chapter are to be regarded only as approximate, guide values. Solvent mixtures must be prepared with care and prepared freshly before each use, especially if they contain volatile components. They should preferably contain 1—3 solvent components too many components militate against good reproducibility of the i /-values. 

With the help of this approach the chamber type can be characterized for a certain separation (X in Figure 3) without specification of the vapor phase. Obviously, the separation can be influenced by changing the saturation of the vapor phase. The example depicted in Figure 3a, shows that increasing the degree of saturation of the chamber used for separation X would reduce the hRy values of the compounds separated and reduce the resolution obtained. Figure 3b shows that use of a less saturated vapor phase increased the resolution obtained at lower Ry values. 

There is no other facet where thin-layer chromatography reveals its paper-chromatographic ancestry more clearly than in the question of development chambers (Fig. 56). Scaled-down paper-chromatographic chambers are still used for development to this day. From the beginning these possessed a vapor space, to allow an equilibration of the whole system for partition-chromatographic separations. The organic mobile phase was placed in the upper trough after the internal space of the chamber and, hence, the paper had been saturated, via the vapor phase, with the hydrophilic lower phase on the base of the chamber. 

In the case of thin-layer chromatography there is frequently no wait to establish complete equilibrium in the chamber before starting the development. The chamber is usually lined with a U-shaped piece of filter paper and tipped to each side after adding the mobile phase so that the filter paper is soaked with mobile phase and adheres to the wall of the chamber. As time goes on the mobile phase evaporates from the paper and would eventually saturate the inside of the chamber. 

Ascending, one-dimensional development at 10 —12 °C in a twin-trough chamber with 5 ml ammonia solution (25%) in the second part of the chamber chamber saturation for 15 min. 

Development of plates. The chromatogram is usually developed by the ascending technique in which the plate is immersed in the developing solvent (redistilled or chromatographic grade solvent should be used) to a depth of 0.5 cm. The tank or chamber used is preferably lined with sheets of filter paper which dip into the solvent in the base of the chamber this ensures that the chamber is saturated with solvent vapour (Fig. 8.6). Development is allowed to proceed until the solvent front has travelled the required distance (usually 10-15 cm), the plate is then removed from the chamber and the solvent front immediately marked with a pointed object. 

Inside newiy formed caverns, calcium carbonate precipitates from water dripping from the ceilings of the chambers. This happens when water that is saturated with carbon dioxide and calcium hydrogen carbonate comes into contact with air. Some of the dissolved CO2 escapes into the gas phase. This shifts the two equilibria to the left, and solid calcium carbonate precipitates ... 

The discussed effects, such as evaporation and adsorptive saturation, are prevented by placing a counter plate at a distance of one or a few millimeters from the chromatographic layer. The development with such a reduced vapor phase in the so-called sandwich chambers (S-chambers) can improve the separation. The glass-backed 20 X 20 cm plate forms one wall of the chamber with the adsorbent facing inward. A glass plate with spacers, called counter plates, is clamped to this plate and forms the other wall of the chamber (Figure 5.31, left [32]). 

FIGURE 6.8 Horizontal DS-II chamber (a) before development, (b) during development 1 — cover plate of the mobile phase reservoir, 2 — mobile phase reservoir, 3 — chromatographic plate facedown, 4 — body of the chamber, 5 — main cover plate, 6 — cover plates (removable) of the troughs for vapor saturation, 7 — troughs for saturation solvent, 8 — mobile phase, 9 — mobile phase distributor. 

Referring to Figure 8, temperature Tc is the chamber temperature and Ts is the surface temperature at the salt solution/vapor interface. The temperature of the chamber is well defined and is an experimental variable, whereas Ts must be higher than Tc due to condensation of vapor on the saturated solution surface. We can determine Ts by applying the Clausius-Clapeyron equation to the problem. Assume that the vapor pressures of the surface and chamber are equal (no pressure gradients), indicating that the temperature must be raised at the surface (to adjust the vapor pressure lowering of the saturated solution) to Pc (at Tc) = Ps (at Tc). However, there is a difference in relative humidity between the surface and the chamber, where RHC is the relative humidity in the chamber and RH0 is the relative humidity of the saturated salt solution, and we obtain... 

A small 3 cm x 3.5 cm section of the catalyst-coated desiccant wheel (25 cm diameter) was cut and placed in specially made holder shown in Fig. 12.9-6a. The piece of sample was tested in a 0.2 m3 environmental chamber at Chiaphua Industries Ltd. (Fig. 12.9-6b) for reduction of airborne VOC. The chamber was filled with the target VOCs through two stage saturators shown in Fig. 32b. Once the VOC level in the chamber stabilized, the fan was turned on to circulate the air through the sample. Three sets of sensors were located at the inlet and outlet of the holder, as well as in the center of the chamber. The chamber temperature and relative humidity were kept constant during the test. Figure 12.9-6c shows the results for VOC levels of 4000, 2000 and 1000 ppb at room temperature. The reduction rate was slower because of the low VOC concentration and the poor air circulation in the chamber. Also unlike the Prototype Unit, the catalyst was kept at room temperature throughout the test. 

The equilibration of the chamber or chamber-saturation is a vital factor to obtain reproducible Rf values. It may be achieved by allowing the solvent system to remain in the chamber for at least 1 to 2 hours so that the vapours of the solvent(s) would pre-saturate the latter adequately. This is done to obtain distinct separation of constituents, uniform solvent from and prevent evaporation of the solvent on TLC-plates. 

A wide selection of solvent systems is available in the biochemical literature. If a new solvent system must be developed, a preliminary analysis must be done on the sample with a series of solvents. Solvents can be rapidly screened by developing several small chromatograms (2X6 cm) in small sealed bottles containing the solvents. For the actual analysis, the sample should be run on a larger plate with appropriate standards in a development chamber (Figure 3.3). The chamber must be airtight and saturated with solvent vapors. Filter paper on two sides of the chamber, as shown in Figure 3.3, enhances vaporization of the solvent. 

The decisive data during main drying (MD) is the temperature of the ice at the sublimation front, Tice, which can only be measured by barometric temperature measurement (BTM). Figure 1.77 shows the principle the valve between the chamber and condenser is closed for less than 3 s, to fill the chamber with water vapor of saturation pressure p., corresponding to Tke as shown in Table 1.11. The two conditions for the use of BTM a leak rate of the chamber below a certain limit and enough ice subliming during the time the valve is closed, are described in detail in Section 1.2.1. 

During the run the paper is heated above room temperature, and buffer solvent must evaporate in proportion to the size of the chamber and the quantity of condensate. This evaporation is greatest at the beginning of the run, but in a smaller chamber it may become so slight that rheophoresis falls to nil and the fraction travels into the buffer vessel (Fig. 23). Most commercial forms of apparatus are not vapor saturated before the run begins and evaporation remains throughout the experiment at a sufficiently high level to cause immobilization of the fractions at the place where buffer flow and electrophoretic velocity neutralize each other. 

As soon as the electrophoretic chamber is saturated with water vapor and under good working conditions, the horizontal evaporation flow practically disappears and only the vertical evaporation flow remains to compensate for condensation of water on the walls of the chamber. 

A small chamber is more rapidly saturated. This is a reason for keeping all accessory mechanisms, such as the actual fraction collector, out of the chamber. 

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