Figure 8 : Sampling fundamentals. How to use brushes and why not syringes ? The round brown spot above can be changed into a circle or a line. The round spot with a diameter of about 6 mm results on the HPTLC plate by an enough long lasting touch with a micro brush of size 00 after its saturation with a sample solution. What is micro brush saturation ? The micro brush is first dipped and rotated in a cleaning solvent - preferably methanol. Several times dipping in and removing the taken solvent on a piece of dry soft well soaking paper cleans the brush. The final removal of the solvent should be done for some seconds and a little pressure around the hairs. The same procedure is used to stabilize the brush in the sample solution. Let us assume, some substances chemisorb or just strongly adsorb on the brush hairs: often enough dipping, a little bit shaking in the sample solution and drying on let say a soft handkerchief paper allows to take and give a correct sample solution to the stationary phase of the HPTLC layer. NOTE: not so save is this sample stabilization process inside a syringes or a capillary, however a fused silica capillary will much faster reach an equilibrium than the steel needle of a syringe. The inner part of a steel needle looks like an adsorption column causing sorption, chemi sorption, displacement chromatography.... Now the brush is brought to its correct volume by a short move along the inner surface of the bottle holding the sample volume. That means: there is no solution droplet hanging on the tip of the brush. Each water color artist knows, this hanging droplet would cause some precision trouble in his artwork. So it is easy to learn how to avoid overfilling and under filling a brush. A soft tip for about 10...12 seconds time - and no local movement - will transfer the sample solution onto and inside the HPTLC layer. The transfer amount is reproducible by around +- 5% for the smallest brushes (type 00) and better than +- 2% for a larger brush (type 5). See for accurate data figure 10, click the right arrow on top of this figure twice. Let the spot dry. Set the cleaned but now methanol or an other focussing liquid containing micro brush into the center of the round brown spot. This produces a sharp sample circle. Or draw a line stepwise next to the round spot. It will produce a sample line. Why to treat sample spots by focussing liquids ? See figure 9 (next page) and the figures 18 and 19. Back to the first line above: Why not using syringes ? 1. it is nearly not possible to transfer a liquid sample onto / into the layer without a layer damage. 2. Layer damage as such is not an analytical disaster, but the layer particles which go into the needle tip cause not only mechanical problems but can drastically falsify the next sample composition - to see this, click HERE and then go to figure 10 in the TLC/HPTLC pictogram page. The click onto HERE brings you to this page. For technical reason the figure numbers in both pictogram µ-PLC- and TLC - types do NOT differ digitally but only by color. µ-PLC = blue; TLC = violet. 3. Besides the delicate mechanical character and the unwanted chromatographic behavior of precision syringes there is an economical argument. A micro brush is available for 50 cents and often reusable. 4. µ-PLC takes nano liter sample volumes - which can be given easily by fused silica capillary pieces. Or takes many micro liters. No problem with micro brushes of a higher type number. Micro brushing never damage layers. Micro brushes allow to draw bows and linear lines. For what long sample lines are good is shown in the figures 18, 19 and here . Syringes can do similar tasks but with utmost expensive electronic devices only (by now - 2009).
