Pre-coated plates

Commercially available pre-coated plates with a variety of adsorbents are generally very good for quantitative work because they are of a standard quality. Plates of a standardised silica gel 60 (as medium porosity silica gel with a mean porosity of 6mm) released by Merck have a specific surface of 500 m /g and a specific pore volume of 0.75 mL/g. They are so efficient that they have been called high performance thin layer chromatography (HPTLC) plates (Ropphahn and Halpap J Chromatogr 112 81 1975). In another variant of thin layer chromatography the... 

Ready to use thin-layers (i.e. pre-coated plates or plastic sheets) are commercially available the chief advantage of plastic sheets is that they can be cut to any size or shape required, but they have the disadvantage that they bend in the chromatographic tank unless supported. 

TLC analysis indicated that the reaction was complete. TLC was performed on pre-coated plates with silica gel 60 F254 purchased from EM Sciences (Merck) using hexanes/ethyl acetate (1/1) as the eluent. 

Silica gel pre-coated plates. Silica gel 60 F-254, employing diethyl ether hexane (1 5) for the cinnamate and (1 1) for the nicotinate... 

Figure 5. Reflectance ultraviolet absorption spectrum of benzoic acid spotted on a silica gel F254 pre-coated plate.

A number of separations (of BHC isomers, of bromophos-ethyl and dimethoate. and of the active constituents of Trevespan 6038) were performed in order to provide a qualitative and quantitative comparison between TLC and HPTLC pre-coated plates, or between HPTLC plates coated with various sorbents, and in order to demonstrate their use in pesticide chemistry. 

Silica gel 60, the most versatile and most frequently used TLC sorbent, was taken as a basis. The mean particle size of this sorbent was optimized simultaneously, the particle size distribution was brought to within as narrow limits as possible (L- ) The sorbent material thus obtained was used t prepare HPTLC pre-coated plates silica gel 60, being followed subsequently by the development of other sorbents for processing into HPTLC pre-coated layers. The materials chosen were largely "reversed phase" sorbents, that is to say, chemically modified silica gels with a non-polar surface (, 8), as well as microcrystalline cellulose ( )""... 

Optimization of the mean particle size of silica gel 60 and the consequent reduction of the particle size distribution produced a number of advantages in HPTLC pre-coated plates silica gel 60 prepared from this improved material which the traditional TLC silica gel 60 pre-coated layers do not possess. These advantages are to be found not only in the already mentioned optimized mean particle diameter and the narrower particle size distribution, but also in the smoother suid more homogeneous surface of the plates, which leads to an increase in chromatographic performance. 

The improvements made in the HPTLC pre-coated plate silica gel 60 as compared with the TLC silica... 

In thTs paper ftie advantages of HPTLC pre-coated plates silica gel 60 (E. Merck, Darmstadt) as compared with TLC pre-coated plates silica gel 60 (E. Merck, Darmstadt), are presented in the separation of benzene hexachloride (BHC) isomers. 

Both for TLC and for HPTLC pre-coated plates silica gel 60 the best results were obtained with a solvent mixture consisting of cyclohexane and chloroform in a ratio of 65/35 by volume, with a 5 cm migration distance for HPTLC plates and a 7 cm migration distance for the TLC plates. In both cases the plates were adjusted prior to use to 20 relative humidity and were developed in normal chambers without chamber saturation. For both types of plates, 0.75 pi quantities of a test mixture of a-, B-, y-,... 

Table I shows that retention of the individual BHC isomers is virtually identical on the TLC and HPTLC pre-coated silica gel 60 plates. The hRf values of the BHC isomers on the HPTLC plates lie only slightly above those achieved on the TLC pre-coated plates.

Table I.Retention data (hRf values) of BHC isomers on TLC and HPTLC pre-coated plates silica gel 60 solvent system cyclohexane/chloroform 65/35...

It can be seen that in both cases a well correlating calibration curve in the shape of a straight line is obtained. It is also evident that for equal applied quantities the peak heights on the HPTLC plate are slightly more than twice as high as on the TLC plate. For this reason and on account of the fact that the baseline noise is markedly less on the HPTLC plate evaluated in reflectance mode as a result of its more homogeneous and smoother surface, the detection limit for S-BHC under the given conditions on the HPTLC pre-coated plate silica gel 60 is considerably less at 18 ng than on the TLC pre-coated plate silica gel 60 at 40 ng. 

Now that reversed phase column packing materials have been used successfully in column liquid chromatography for a number of years, success has also been achieved in thin-layer chromatography in that it is now possible to prepare HPTLC pre-coated plates with the same types of surface-modified sorbents and to use them for separations (2 8, 1, 20). 

The basic skeleton o7 tEe modified sorbents consists of a surface-active silica gel. Modification is in the form of a surface reaction with specially active silanes at the silanol groups of the silica gel. Following elimination of these silanol groups, new siloxane groupings are formed, on which aliphatic hydrocarbon groups are chemically bound by silicon-carbon bonds to the silica gel skeleton. Pre-coated plates with these modified silica gels are designated ... 

Figure 1. Calibration curves for 8-BHC on a TLC pre-coated plate silica gel 60 ( leftj and on an HPTLC pre-coated plate silica gel 60 fright) (abscissae applied quantity (ng) ordinates peak height (mV))...

Figure 2. Linear chromatograms for the separation of bromophos-ethyl and dimethoate on an HPTLC pre-coated plate silica gel 60 Cleft) and an HPTLC pre-coated plate RP-18 fright) (solvent system on silica gel 60 n-heptane/acetone 65/35 on RP-18 acetone/water 80/20 application from left to right—(1st band) bromophosethyl (2nd band) dimethoate (3rd-5th band) mixture migration distance 5 cm applied quantities 0.75 fiL =-- 750 ng detection spraying with PdCl2 in ethanol heating to 120°C)...

HPTLC pre-coated plate RP-2, RP-8 and RP-18. The figure following the RP abbreviation refers to the chain length of the aliphatic hydrocarbon. 

The solvent system most suitable for separating bromophos-ethyl and dimethoate is an 80/20 mixture of acetone/water for the HPTLC pre-coated plate RP-18, and a 65/35 mixture of n-heptane/acetone in the case of the HPTLC pre-coated plate silica gel 60. The optimum migration distance was 5 cm in both cases. 

Table IV Chromatographic data for the separation of bromophos-ethyl and dimethoate on HPTLC pre-coated plates silica gel 60 and HPTLC pre-coated plates RP-18...

Apart from the sorbents already mentioned in connection with HPTLC pre-coated layers, a micro-crystalline cellulose has also been produced in an average particle size and a narrow particle size distribution suitable for HPTLC (9.). HPTLC pre-coated plates cellulose P 254 s (E. Merck, Darmstadt), composed of this microcrystalline cellulose, were used to separate Trevespan 6058. This is a mixture of the substances ioxynil (3,5-diiodo-4-hydroxy-benzo-nitrile), flurenol (9-hydroxyfluorenecarboxylic acid) and MCPA (2-methyl-4-chlorophenoxyacetic acid). For comparison purposes Trevespan 6038 was also separated on HPTLC pre-coated plates silica gel 60 F 254 (E. Merck, Darmstadt). 

The solvent system most suitable for separating Trevespan on HPTLC pre-coated plates cellulose consists of tert. butanol/water/25 ammonia in a ratio of 80/20/1 by volume, whilst for HPTLC pre-coated plates silica gel 60 the best mixture is n-hexane/ethyl acetate/formic acid in the ratio 40/60/0,5. Prior to the separation proper, the HPTLC pre-coated plates were adjusted to a relative humidity of 20. Quantities of o.73 pi of a "test solution in methanol, composed as follows, were applied to the plates and migrated over 5 cm ... 

Although the migration distance was the same in both cases, namely 5 cm, the development time for the HPTLC pre-coated plate silica gel 60 is considerably shorter than for the HPTLC pre-coated plate cellulose on account of the higher flow rate. In both cases the three substance components were completely separated, though the R values achieved on the HPTLC silica gel plate were considerably higher for this particular separation than the values obtained on the HPTLC cellulose plate. 

Well correlating calibration curves in form of straight lines are obtained on both types of HPTLC plate for all three sample substances in the range of quantities examined. Although the applied quantities are identical, the peak heights on the HPTLC pre-coated plate cellulose are somewhat higher than those obtained on the HPTLC pre-coated plate silica gel 60. The detection limits determined by the peak heights and by the baseline noise for the three substances examined are listed in Table VII. 

HPTLC pre-coated plates silica gel 60 with concentrating zone... 

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. 

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