Thin Layer Chromatography - Sample Application 1
Samples should be applied to a TLC plate as a spot that must have as small a diameter as possible. The sample volume employed with normal TLC plates is usually 1 to 2 ml. However, the so-called HPTLC (High Perfomance TLC) plates are coated with particles 5-7 mm in diameter and these will have a maximum loading of about 100-200 nl. Preferably, on any HPTLC plate, the sample should occupy a circular area on the plate that is no greater than 1 mm in diameter. Unfortunately, as most samples will also require the use of samples volumes only a few nanoliters in volume, it is likely that the sample will require to be concentrated. Manually, the sample is often applied with a micro-pipette which is filled by touching the end of the pipette with the sample solution and then discharging the contents of the pipette by surface tension, touching the surface of the plate. If the solvent is then allowed to evaporate, a second sample can be placed on the top of the first and by a sequence of such operations a sufficiently concentrated sample can be placed on the plate.
It is important to make sure that the solvent used to dissolve the sample does not contain components that will effect the elution of the solutes and, thus, the resolution obtainable from the plate. For instance, consider the case where a plate, coated with silica, is being developed using a mixture of n-hexane and methylene dichloride as the mobile phase and the sample is made up in a solvent containing significant amounts of methanol. The strong polar solvent would be carried along by the mobile phase and progressively deactivate the silica which would result in the rapid migration and poor resolution of the components of the mixture. This situation must be avoided and appropriate solvents must be carefully selected for sample dissolution so that the activity of the stationary phase is not effected.
Micro-syringes are also often employed to place a sample on to the plate and sample concentration can be accomplished by employing a little local heating,. The contents of the syringe are slowly, but continuously, discharged onto the plate and at the same time the solvent is progressively evaporated. This procedure can be automated, using computer controlled syringes and in this way samples can be focused onto the plate and constrained to a very small area (achieving a very small spot diameter). It is clear that the sample must be placed very carefully on the plate in order to realize best resolution. It also must be realized that due to spot measurement limitations there will also be a limited accuracy that will be obtainable.
In general glass pipettes are inexpensive, but need considerable skill in their use and, even then, are not particularly precise. Alternatively, micro syringes, and sampling systems employing micro-syringes are far more precise but are also far more expensive. microsyringes are also a little fragile in the sense that the needles of the syringes easily become blocked.