High-performance TLC

The separation capacity of a TLC method can be easily improved by use of a two-dimensional high performance TLC technique (2D HPTLC). Various plant essential oils (menthae, thymi, anisi, lavandulae, etc.) have been analysed by 2D TLC with florisil (magnesium silicate) as the adsorbent, using dichloromethane/ -heptane (4 6) in the first direction and ethyl acetate/n-heptane (1 9) in the second direction (69). 

FIGURE 6.19 Circular chromatography of dyes on precoated silica gel high-performance TLC plate lipophilic dyes, mobile phase hexane-chloroform-NH3, 70 30. (From Ripphahn, J. and Halpaap, H., HPTLC High Performance Thin Layer Chromatography, Zlatkis, A. and Kaiser, R. E., Eds., Elsevier, Amsterdam, 1977, pp. 189-221. With permission.)... 

PLC is used for separations of 2 to 5 mg of sample on thin-layer chromatography (TLC) plates (0.25-nun layer thickness) or high-performance TLC (HPTLC) plates (0.1-mm thickness). In these instances, the method is termed micropreparative TLC. The isolation of one to five compounds in amounts ranging from 5 to 1000 mg is carried out on thicker layers. PLC is performed for isolation of compounds to be used in other tasks, i.e., further identification by various analytical methods, such as ultraviolet (UV) solution spectrometry [1] or gas chromatography/mass spectrometry (GC/MS) [2], obtaining analytical standards, or investigations of chemical or biological properties [3]. 

Valproic acid was determined in plasma by treatment with 2,4-dibromoacetophe-none or 2-bromoacetonapththalone and with dicyclohexano-18-crown-6 and heated at 70 °C for 40 min. The solution was subjected to TLC on C8F octyl plates or to high performance TLC on RP 8 254 S or Kieselgel 60 F254 plates with developing solvents of aq. 63% ethanol, aq. 73% ethanol or CHCl3-cyclohexane (2 1), respectively, with detection at 280 or 254 nm for the naphthoylmethyl or phenacyl derivative, respectively. The limits of detection were 9.7 and 4.9 pg/mL of valproic acid for the TLC RP 8 and HPTLC RP 8 plates, respectively. Recovery was 84-92.74% [28],... 

The number of theoretical plates available for separation is limited in routine TLC systems, although high performance TLC (HPTLC) plates can offer nearly the same efficiency in a 10 cm distance as an HPLC column of the same length... 

A mobile-phase developing distance of approximately 10-15 cm is typical, but some chromatographers prefer developing their plates 15-20 cm. For high-performance TLC (HPTLC) plates, which typically have particle sizes of 5 nm versus 20 ftm for conventional plates, a developing distance of 3-6 cm is typical. Interestingly, similiar efficiencies are obtained when both types are developed 8 cm [29]. 

Quantitative thin-layer chromatography (TLC) has progressed considerably and has been adapted to forensic chemistry of pesticide poisoning incidents. High-performance TLC (HPTLC) with precoated plates is discussed separately. 

The availability of new materials, methods, and instrumentation over the last 10 years has so greatly altered TLC, that the new form is now called high performance TLC (HPTLC). The HPTLC plates are far superior to the conventional type, having much smaller particles (2 to 7 pm) as well as a very narrow particle size distribution. This makes HPTLC faster, more reproducible, more sensitive, and more accurate for quantitative work. Its growth in drug applications has been greatly speeded by the use of automated sample application devices and accurate densitometers. A comparison of HPTLC and TLC is given below. 

Comparative studies have often found that high performance TLC (HPTLC) is superior to HPLC in terms of total cost and time required for pharmaceutical analyses. " The Bibliography contains sources of general information on the principles, theory, practice, instrumentation, and applications of TLC and HPTLC. Detailed information on the subjects mentioned above as well as on additional topics and applications that could not be covered because of lack of space will be found in these references. 

Fig. 1 Camag chromatogram immersion device set for a 10-cm dipping depth with high-performance TLC plates. Vertical dipping and removal rates and the residence time in the reagent can be preselected. [Photograph (left) supplied by D. Jaenchen, Camag, Muttenz, Switzerland schematic diagram (right) reprinted from Handbook of Thin Layer Chromatography (J. Sherma and B. Freid, eds.), p. 146, with permission of Marcel Dekker, Inc.]...

Layers often require heating after applying the reagent in order to complete the reaction upon which detection is based and ensure optimum color development. Typical conditions are 10-15 min at 105-110°C. If a laboratory oven is used, the plate should be supported on a solid metal tray to help ensure uniform heat distribution. A plate heater (Fig. 2), which contains a 20 X 20-cm flat, evenly heated surface, a grid to facilitate proper positioning of TLC and high-performance TLC plates, programmable temperature between 25°C and... 

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