This instruction should be clearly displayed in every TLC laboratory, so that it may ul- timately put an end to the bad habits of using solvent systems many times over, adding used solvent to fresh solvent, or storing solvents in bottles for many months and then helping oneself at irregular intervals. Stahl has pointedly remarked on p. 70 of the 2nd edition of his 1967 laboratory handbook: “Mixtures of different solvents should only be used a small number of times for development, and the chamber should never at any time be opened for any longer than necessary.” Thirty years have now gone by, but notwithout leaving their mark on TLC! Objections are sometimes made to the sys- tematic use of fresh solvent for each plate, with the claim that reproducible hRf values should be obtainable even with multiple use of the solvent. Facts in support of these assertions have never been produced, so that the best principle must always be that, at least in experiments performed in compliance with GMP/GLP, solvent systems should be used onlyonce. The exception to this, of course, is the single-component system, but these are seldom used in TLC.
An example of the harmful effects of the multiple use of the solvent system is shown in Fig. 51. Four plates, each with two 10-mm lanes, each containing 20 g metoc- lopramide hydrochloride, were developed successively with the solvent system “chlo- roform + methanol + concentrated ammonia solution” (56 + 14 + 1 ml). The continu- ous downwards shift of the hRf value can be clearly seen, combined with an increasing broadening of the spots of the substance during the chromatography. These effects were caused by the reduction in the amount of base in the remaining solvent.
Figure 51:see Photograph Section.
79 4.1 Solvent Systems
Practical Tipsfor the preparation and storage of solvents:
Single components should be measured out separately and then well mixed. How- ever, this should not be done in the TLC chamber itself! Suitable mixing vessels are conical flasks with ground-glass stoppers, or mixing cylinders, which have the ad- vantage that the main components can be measured out into these.
The term “mixing cylinders” means glass vessels with ground glass stoppers and not
“measuring cylinders”, where the palm of the hand can replace the stopper, a com- mon occurrence!
Further solvent components are added by pipette, and this can cause heating of the mixture. In this case, the mixture should be cooled before adding acidic or volatile components.
As a rule it is not important to follow any particular order of addition of solvent components. However, every rule has its exception! The DAB gives an example in which the order of addition of solvent components starts from that with the smallest volume and highest polarity and ends with the component with the largest volume and lowest polarity. In this case, the prescribed order of addition should be strictly adhered to, as otherwise a cloudy solvent system will be obtained (as we have dem- onstrated many times!). In the DAB monographs for fructose, glucose, lactose and sucrose, the following solvent system composition is given:
10 volumes of water 15 volumes of methanolR
25 volumes of anhydrous acetic acidR 50 volumes of dichloroethaneR
It is recommended in the monographs that the solvent systems should be measured accurately, as a small excess of water can cause the mixture to be cloudy. Chroma- tography with this solvent system is described in Section 4.3.1.1 “Vertical Develop- ment”.
Many methods prescribed for TLC include the addition of concentrated ammonia solution. Here, the following is recommended: Only use ammonia solution of the highest purity (e.g. Suprapur, Merck No. 1.05428), order the smallest package size (250 ml) and always store bottles that have been opened in the refrigerator. Left- over amounts of ca. 20 ml should be used for purposes other than TLC; it is much better to open a fresh bottle of solvent. If there is too little ammonia gas dissolved in the aqueous phase, experimental results will not be reproducible.
If several plates have to be developed with the same solvent system in one day, the total amount required can be prepared in one batch provided that the solvent com- positions are uncritical. This is recommended even for quantitative work in order to maintain reproducible hRf values. Critical solvent systems are discussed in Section 4.1.3 “Problematical Solvent System Compositions”.
Solvent system compositions are often given in the literature such that the total equals 100, e.g. 60 + 38 + 2. This can facilitate both the comparison of one devel- opment method with another and the calculation of amounts required in a given ex- 80 4 Solvent Systems, Developing Chambers and Development
Table13:SolventrequirementsfordifferentTLCseparationchambers Manufacturerof chamberTypeofchamberDimensions[cm]BottomLidVolumeofsolvent[ml] WithoutCSWithCS BaronAutomaticSolventboxof PTFEMax.20Additionally10–20a) CAMAGVertical20×20FlatbottomMetal110110 Vertical20×20TwintroughMetal3570 Vertical20×20TwintroughGlass4080 Lightchamber Vertical20×10TwintroughMetal3570 Vertical10×10TwintroughMetal10–1220–25 Horizontal10×10PTFEGlasseachsideapprox.1,5a) Horizontal20×10PTFEGlasseachsideapprox.3a) AutomaticADC20×10max.12a) AutomaticADC20×20max.25a) AutomaticAMD8/development,gradientwith20steps approx.200incl.rinsingstepsa) DESAGAVertical20×20FlatbottomGlasswithknob155 Vertical20×10ConicalMetal70 Vertical10×20FlatbottomGlass90 Roundchamberapprox.12 Horizontal5×5PTFEGlass33+2forCS Horizontal10×10PTFEGlass66+3forCS Macherey-NagelVertical4×8SlightlycurvedPlasticmaterialApprox.10 Glasswithscrewcap MerckVertical20×10TwintroughGlass3570 SchottVertical7×7FlatbottomGlassApprox.10b) GlaswerkeStainingtroughacc. toHellendahl a) Acc.tothemanufacturer b)Acc.toliterature Inall“vertical”chambersthequotedquantityofsolventisper1cmheightofsolutioninthetank.
81 4.1 Solvent Systems
periment. To minimize the consumption and hence the cost of solvents as well as the amounts of used solvents to be disposed of, only the minimum amount of freshly prepared solvent system should be used. The amount of solvent required depends on the type of developing chamber in which the development is to be performed.
TLC developing chambers have been discussed in Section 4.2, and the amounts of solvent needed for the various types of chamber are listed in Table 13.
Storage of solvents is unnecessary if they are used in a TLC chamber immediately after they have been prepared. However, it is sometimes stated in the literature that certain solvent systems can be stored for several months [50]. In this case, the best advice is to store them in a dark bottle in a cool place. The “daily quota” of a sol- vent system should also be kept cool in the summer, e.g. if laboratory temperatures exceed 25 °C. Care must be taken to adjust the temperature to room temperature before the development.
The storage behavior of solvent systems, as described in the last paragraph, was checked by Wagner using the example of the “classical” mixture used for flavo- noids. The mixture of ethyl acetate, formic acid, glacial acetic acid and water (100 + 11 + 11 + 27 ml) was used, and a reference plate was prepared using the fresh sol- vent. After 17 days, the same standard solution and sample solution were applied to two plates and developed in parallel, on one hand in the stored solvent and on the other hand in freshly prepared solvent. The development time for a migration dis- tance of 7.5 cm was 43 min with the stored solvent and only 37 min at 23 °C room temperature with the freshly prepared solvent. The chromatograms obtained in this experiment were different, as shown in Fig. 52a,b. The recommendation only to use freshly prepared solvents is therefore well justified.
Figure 52:see Photograph Section.