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I am separating highly polar amino acids and their derivatives using paper electrophoresis. This requires pre-wetting a piece of filter paper with electrophoresis buffer (in this case, carbonate-bicarbonate buffer) and depositing spots of samples on the wet paper.

When I do so, the molecules in the samples instantly disperse on the wet paper, and form fairly wide spots (wider than on dry silica/cellulose TLC). This makes it hard to separate closely related molecules.

How can I minimize the dispersion of the molecules during the spotting step on wet paper?

I tried to make the samples slightly viscous by adding large amounts of poly(ethylene glycol) or glycerol, but it did not seem to make any difference in terms of spotting size. Concentrating the samples to reduce the loaded volume is not a viable solution, as I am already spotting very small volumes (1-2$\mu$L). Are there tricks used for example in TLC that could apply there?

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  • $\begingroup$ The classic way is to start with a dry stationary phase, spot a small volume, let the solvent evaporate, spot another small volume. This does not work if the protocol calls for wet paper. Another way is to minimize the volume applied while maximizing the sensitivity of detection (i.e. not look for color in daylight but fluorescence under UV light, or use a specific dye to stain after you run and dry the TLC). $\endgroup$
    – Karsten
    Commented Sep 27, 2022 at 22:26

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