Examples of various methods of treating chromatograms have already been discussed in the foregoing Sections, e.g. heating, described in Section 6.4.2, and intensification of fluorescence by treatment with Macrogol 400, described in Section 6.3. Whereas in the latter case the brilliance of the fluorescence colors and hence the detection sensitivity of the flavonoids could be increased, i.e. there was an improvement in qualitative in- formation, the minutely described technique of causing a reaction to occur by heating the plate obtained with bronchial herbal tea is followed by a quantitative evaluation, i.e., special attention is given to the aftertreatment in this case. In Vol. 1a of their re- agent books, Jork et al. describe in detail aftertreatments by changing the pH, applying heat, and stabilizing colored and fluorescent zones, both from the point of view of the theoretical background and also with the aid of examples given in Tables. In the pre- sent book, therefore, only the two most commonly used methods of aftertreatment of derivatized chromatograms are described.
6.5.1 Effect of Heat
To enable the desired chemical reactions in the layer of sorbent to proceed as rapidly as possible or to enable them to proceed at all, it is advisable to heat the TLC plates.
Formerly, infrared heaters, drying ovens and early forms of hotplates were used, but these are now largely replaced by modern plate heaters with a Ceran®ceramic heating surface (CAMAG) as shown in Fig. 91. These are not only stable towards all com- monly used reagents but are also easy to clean. The temperature range, 25–200 °C, is sufficient for most applications. According to the manufacturers, the set temperature is maintained over the whole area of the plate. Heat treatment at 100–120 °C for 5–10 min, as is often specified in the literature, will often give complete reaction, but experi- ment sometimes shows that temperatures of 160 °C and above are necessary.
The use of microwave equipment to promote reactions on TLC plates has also been reported [115]. However, this is not yet widely used in the laboratory, as, apart from the fact that equipment with direct fume extraction is not yet available, aluminum foil has to be ruled out as a support for sorbents!
148 6 Derivatization
Figure 91. Plate heater with Ceran®ceramic heating surface (CAMAG)
Practical Tipsfor heating:
The TLC plate heater must always be operated in an efficient fume cupboard, as the vapors from the reagents must always be assumed to be highly toxic.
For every job, and especially for quantitative work, it should be determined whether better results are obtained with a cold or a preheated plate.
The set temperature and heating time of the equipment should be exactly specified in the testing procedures.
When using a plate heater with an aluminum surface (e.g. the Thermoplate S of DESAGA), it is advisable to place a clean glass plate in direct contact with the alu- minum block and to place the TLC plate on this. If this is not done and the equip- ment is preheated, the TLC plate, because of the difference between the thermal expansion coefficients of aluminum and glass, becomes warped, and the outer edge lifts slightly. This leads to poorer heat transfer and hence nonuniform heating.
Quantitative work is then impossible! A further reason for having the protective plate is to avoid contaminating the surface of the aluminum with reagent.
If chromatograms on aluminum foil supports are derivatized by the action of heat, an additional glass plate or a metal frame ca. 1 cm wide will prevent the foil from curling up. Of course, if a metal frame is used the reaction vapors will be able to es- cape more easily.
Because of the high rate of heat loss in a fume cupboard, it is better to set the tem- perature of the plate heater at 120 °C rather than 100 °C.
149 6.5 Further Treatment of Derivatized Chromatograms
Example of the use of heat for derivatization:
stress tests of dexpanthenol ointment
The solvent system specified in the DAC monograph for dexpanthenol was used. After the sam- ple treatment process the ointment is in the form of an aqueous solution, and a water-resistant sil- ica gel layer (DC-KG 60 WF254s, Merck 16485) was therefore chosen. Because of the low sensitiv- ity of the method, the derivatization was not performed by ninhydrin reagent (as specified in the DAC), but as a reaction sequence with the 2,5-dimethoxytetrahydrofuran/4-(dimethylamino)- benzaldehyde reagent (Jork et al, Vol. 1a, p. 265). It should be noted here that the traces of am- monia that come from the solvent mixture must be completely removed from the chromatograms before using the reagent, as the background colors can otherwise by too intense.
Reagents: 2,5-Dimethoxytetrahydrofuran Merck 802961
4-(Dimethylamino)-benzaldehyde Merck 103058
Acetic acid, 100 % Merck 100063
Ethyl acetate Merck 109623
Hydrochloric acid, 32 % Merck 100319
Reagent solution A: 2,5-Dimethoxytetrahydrofuran (0.3 ml) with ethyl acetate (24 ml) and acetic acid (6 ml).
Reagent solution B: 4-(Dimethylamino)benzaldehyde (600 mg) is dissolved in a mixture of 25.5 ml acetic acid and 4.5 ml hydrochloric acid.
Storage: Solution A can be kept for a few days and solution B for a few weeks at room temperature.
Procedure: After complete removal of the ammonia (ca. 30 min heating by a hot air blower), the plate is sprayed with solution A and then heated at 160 °C for 10 min. After cooling the plate to room temperature, it is then sprayed with solution B.
Safety precautions: When applying and handling these reagents, appropriate safety precau- tions must be taken, including personal protection by use of disposal gloves, protective goggles and breathing masks!
Figure 92 shows the chromatogram of stressed samples of dexpanthenol ointment after derivatization with the reagent known internally as “JFFW 1a/265”.
Figure 92:see Photograph Section.
6.5.2 Stabilization of Colored and Fluorescent Zones
The colored and/or fluorescent chromatogram zones obtained on derivatization should be stable for at least 30 min to enable documentation and/or quantitative evalu- ation to be performed.
With fluorescent substances, this is usually very satisfactorily achieved by the use of fluorescence intensifiers (FITs). Vol. 1a of the reagent books of Jork et al. gives tables of lipophilic FITs (mainly mineral oils) and hydrophilic FITs such as polyethylene glycols, triethylamine, triethanolamine and especially Triton X-100, including areas of use. Treatment by spraying and dipping in various solvents is recommended. In gen- 150 6 Derivatization
eral, this increases the fluorescence yields in comparison with those of untreated chromatograms by a factor of 5–10. However, the physicochemical processes that lead to these improvements in stability and intensity are not yet fully understood.
Example of an intensification of fluorescence: caffeic acid derivatives
See Section 6.3.1, Fig. 84: various species of Echinacea derivatized by the flavone re- agent according to Neu and stabilized with a 2 % ethanolic solution of Magrocol 400.
There are no general recommendations for stabilization of the color of colored chromatogram zones. Jork et al. recommend storage in a nitrogen atmosphere in the absence of light until the plates are measured quantitatively, and they suggest suitable methods of stabilization in the case of some reagents, e.g. by the addition of salt to the reagent or by treatment of the plates with ammonia vapor.
Practical Tip for stabilizing colored zones: On their way from the spray cabinet or plate heater to a documentation system, colored chromatograms should be covered with a clean glass plate to minimize reactions with oxygen.
151 6.5 Further Treatment of Derivatized Chromatograms
7 Evaluation After Derivatization
It would have been possible to combine Chapter 5 “Evaluation without Derivatiza- tion” with the present Chapter 7 to form a single Chapter “Evaluation”, as the earlier Chapter is mainly of general application. However, evaluationbeforederivatization in the course of the TLC process needed to be clearly emphasized. As the use of a TLC scanner was described in detail in Section 5.2, it would be a good idea to reread this Section at this point in the book.
Evaluation after derivatization does not only involve assessment of the chromato- gram in daylight or measurement in light of wavelength 400 nm; there are also a large number of reactions whose products are detected in 254-nm or 365-nm UV light.