µ-PLC helps HPTLC

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Inst.f.Chromatography


	µ-PLC concepts can help TLC and HPTLC users who still need the application of existing tools

Line Sampling and Focussing:
After a 100 x 100 mm, or 100 x 200 mm or a 200 x 200 mm plate has been cleaned and made dry it is easy to draw a thin line by a particularly soft black graphite pencil at about 10 mm above the lower edge. Now sample solutions are “written” very near above this graphite line in about 6..to..10 mm long lines using a micro brush no 1. Sample line to sample line should differ by 3...5 mm, but of course in enough accurate geometry - see figure 1 below. The sample line length and the distance from line to line depends on the amount of samples one wants to have on one single plate.

Figure 1
First clean the plate, but if you use a mobile phase mix take care, that the final clean up run is made with dry methanol.
Draw a fine pencil line using the softest graphite pencil and no mechanical forces. Take a micro brush no. 1 and the sample solutions. Six lines of sample solutions, 10 mm each plus 5 mm distance from line to line are “written” onto the layer along the graphite line. If one line should have a double sample concentration draw above the dry first one the same sample a second time. By this way you can even “write” a calibration line.

Figure 2
“Write” with a micro brush no. 6 or higher a methanol “line” near to the sample lines. Do not move over parts of the sample position. The brush edge must remain about half to one mm apart from the sample lines. The methanol phase line will broaden quickly. The speed with which you “write” the line is about 2 mm / sec but a few training minutes will give you the necessary feeling. You will have optimized this type of an easy procedure (compared to Chinese hand writing) soon after some tests.

Figure 3
The lower part of all sample line pieces are already focussed. Now the plate should already be made dry with a hair dryer BUT NOT with hot air. Clever hair dryers have temperature levels adjustment and correct is room temperature. This one works in a few minutes. If the line pieces are still wet from methanol, focussing will not succeed into a very sharp sample line as seen in Figure 5 below.

Figure 4
This is just the same procedure as used in figure 2 but now methanol is focussing the sample lines from the lower position. If everything works as it should, you have all six sample lines compressed to sharp lines and pushed correctly into the chromatography start position. Now the plate must again be blown totally dry using room temperature air from the cold hair dryer. Than the plate should look like shown in figure 5. If the focussed sample lines are NOT accurately positioned, a final focus run is possible in a TLC tank with methanol.

Figure 5:
What have we achieved ? The same result could be expected by using a sample line spraying system as commercially available ?
Probably not. Spraying may cause electric fields which may result in substance specific losses at sampling. Even a soft air movement causes specific sample losses. The sprayed line is definitively very much thicker than the double focussed line reducing drastically the separation efficiency especially in the lower Rf range. And what about the economy ?

The procedure discussed above works also for aquatic samples onto silica plates. Multi sampling by micro brushes at the same position caused no damage to the stationary phase but spraying did destroy the layer structure causing drastic systematic quantitative errors. Even if everything works fine, the inhomogeneity of a spray sampled line is critical. The sample line thickness is always too large. Click HERE and have a look onto the first two figures you will see by using the scroll bar. To return to this page click onto the left arrow in the upper browser line.

How data for a calibration line are in easy reach by the simple procedure described above will be shown now.

We sampled an accurately produced calibration mixture the way shown in the following figure 7 and proceeded as discussed above in figure 1 to 6.

Figure 6:
First 6 lines have been “written” as discussed in figure 1 above. The layer was blown dry. Then from position x6 to x2 the same volume of calibration sample solution was “rewritten” a second time. After drying a third time the calibration solution was “rewritten” from position x6 to x3 .... and so on until only one line was “rewritten” onto position x6. The plate was now treated with methanol according to the figures 2 till 5. As micro brush sampling is quantitatively reproducible by about +- 2 % RSD, the separation of the six sample lines resulted in a calibration line of the quality shown in the next figures and tables

First lets have a look onto the picture of the 1...6 calibration data as evaluated by the winCATS Planar Chromatography Manager. The chromatogram picture has been printed onto plain paper, therefore the strong structure signal. This is not the stationary phase structure but plain paper structure. We found by studies with computer made test chromatograms (Macintosh color graphics) a simple further way to reduce PLC structure problems. The following figure 7 is the practical example made along the figure 1 to 6 procedure discussed above. The test substances are a CAMAG test color mix on a HPTLC F254 silica glass plate 100 x 100 mm. Mobile phase was toluene : methanol / 99:1.

Figure 7:
Manual line sampling of CAMAG test color solution. The “written” sample lines have been focussed by a long methanol line from both sides as described above in the figure series 1 to 6
Evaluation by winCATS PLC Manager and CAMAG TLC scanner 3
With special thanks to CAMAG
NOTE the remarkable improvement of the separation efficiency caused by correct sample line focussing. The improvement is strong in the linear
Rf range from start
to 0.3. Even SPOT sampling is improved by focussing-
see figure 10 below.

1 sample line “written”	signal 16085 violet	calculated 16195	deviation - 0.67 %
2 lines	20787	20369	2.01
3	22292	22869	- 2.59
4	24599	24280	1.29
5	25157	25186	- 0.12
6	26153	26173	- 0.08
third degree polynom	B = 0.9909	data quality 1.13 %	+- 1.03 %


	NOTE: all data measured showed a third degree polynom with an unexpectedly good data quality. Note that the “line length written” was hand made - no any mechanical tool was used. Just the line length fluctuation would cause errors, as the CAMAG scanner light window has a constant length.

Spot Sampling and Focussing:
Spots are either too small (by standard procedures one must use sharp small spots in order to keep the separation power of the linear spot chromatogram) or one can use the µ-PLC procedure for spot sampling by micro brushes, dry the then quite large spots and focus them to sharp short lines as shown in figure 8 below.
The benefit of brush spotting is given by the following facts:
The single spot volume reproducibility is in the range of about +- 2 % standard deviation. Multiple spotting is possible without any damage of the stationary phase layer if drying steps are done in between multiple spotting at a fixed position. Spots can easily be focussed to sharp lines, see the the small line positioned right of the circle in figure 8 below Both, the circle and the line have been focussed from round brown spot.

Figure 8:
We changed the brown spot with a focus liquid - again methanol in this case - into a sharp circle or into a short line. Just by spotting methanol with a micro brush no.5 positioned near to the border of the spot. A little movement with the methanol wetted brush finishes the deformation from a round spot to an enough linear but sharp line. If now a fully cleaned dry plate is sampled, we may end up with a plate shown in figure 9. After the round spot deformation into short lines we dry carefully.




	Figure 9: Samples spotted - two on left - by micro brush no. 1, the second is twice spotted. Two in the middle: spotted by a fused silica needle spotter. Three to the right: Higher concentration, smaller volume - about 300 nl.




		Figure 10: Next to the original sample spots we spotted methanol and moved the micro brush toward the spots - one after the next in order to deform the round sample spots into sharp short lines. The plate was dried.


	Figure 11 is the linear chromatogram of a NON deformed spotted plate as shown in figure 9 above. - 100 x 100 mm HPTLC silica F 254, toluene as mobile phase. Figure 12 is the linear chromatogram after DEFORMATION according to figure 10 above.




	Figure 11 The separation efficiency is - as it is. In the low Rf region we see the sample volume problems. If the spots are too large, there is no acceptable separation.




		Figure 12: The separation efficiency is improved, especially in the Rf range below 0.5, the sensitivity is higher. Systematic error ? YES, the deformation is NOT optimal. Some training will help. But the whole concept is easy !

Drying errors:
Sample solvents may hang in the layer much longer than expected or known from too simple test runs. If for instance in a sample we have polar substances like glycoles and used ethanol as sample solvent, ethanol may need a much longer drying time than normal. One can easily see in the chromatogram of a test color next to an ‘A’- or ‘Q’-samples. The latter are only visible under UV. There are three differing. The figures below show only one quarter of the circular chromatogram. Focussing was done with ethanol but the last drying step was too short. The figure below left side shows a selectivity shift. The figure right below shows that the selectivity shift can even be quantitized by multi integration. Selectivity shifts are soft as the solvent substances are wide distributed over a sample line. Such findings underline the importance of long enough drying times to get rid of a sample and focus solvents. May be 30 minutes flush time with 2 L/min air into the plate center at room temperature does it under the conditions shown although a 10 to 15 minute drying time may be enough normally. Under UV one can check drying times with toluene as sample solvent.




	See the selectivity shift of the blue-grey and the blue substance bows within the borders of red and yellow




	The selectivity shifts can easily be quantitized by a multi integration run. Shift size is larger than 10 %.


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