The line appears always in the penultimate run. We try to decrease the volume of the solvent system, wich is composed of butanol:acetic acid:water (2:1:1) (76 mL) but the line remains. Below I attach an image. What is this error due to?
Good TLC technique! Round spots and each one neither over- nor under-loaded! To answer your question, it's water or an aqueous solution of some sort that causes that line. The water-rich solution is immiscible with the butanol-rich phase, while both are on the plate, and the water-rich phase is both more polar (more attracted to the stationary material on the plate) and more viscous and slower than the butanol-rich phase. The irregularity at the top is "viscous fingering" of the more viscous (thicker) water-rich phase trying to displace the butanol-rich phase from the plate, and not being perfectly successful (see spot 3 from left; water didn't quite finish displacing the butanol from the place where the spot used to be, so there's a little left behind between the "water fingers"). If you try to "fix" the line (phase-separation) problem, you might lose the separation you are getting, or it might not work at all anymore. If you want to try something, you could substitute n-butanol (I suppose that's what you are using) with another isomer of butanol. n-Butanol is the least soluble (approx. 7.3 g will dissolve in 100 mL of water), sec-butanol is the next more soluble (8.7 g per 100 mL of water), isobutanol is still better (29 g in 100 mL of water), and tert-butanol is best (miscible with water, solubilities may not be exact and are from Wikipedia), but tert-butanol is a solid just above room temperature (m.p. 25-26 deg. C), so I thaw it on top of a drying oven or other heat source like a radiator in winter, and use it as a liquid, because weighing the solid out is like trying to weigh out ice (it starts melting and gets all over the place). In biochem. lab in the 80's we (in the stockroom) mixed together several butanols so we didn't get that line in the middle, but I don't remember the recipe.
The take-home message, though, is there's nothing wrong with the TLC. It's doing just what it is expected to do and the line in the middle usually doesn't hurt the separation you see, and in truth may be responsible for the good separation. Best wishes and keep on science-ing!
Potential Causes and Solutions
Sample Overloading:
Cause:
If too much sample is applied, it can cause streaking and lead to the formation of a horizontal line.
Solution:
Reduce the amount of sample applied to the TLC plate.Solvent Front Contamination:
Cause:
If the solvent front contains contaminants, it can leave a residue line on the plate.
Solution:
Ensure that the solvent system is fresh and properly mixed.
Clean the developing chamber before use.
Impurities in Solvent:
Cause: Impurities in the solvent can precipitate out as the solvent front moves up the plate.
Solution: Use high-purity solvents and ensure they are properly mixed.
Poor Plate Preparation:
Cause: If the TLC plate is not properly pre-treated or stored, it can lead to artifacts.
Solution: Use a fresh, high-quality TLC plate and ensure it is pre-treated if necessary (e.g., drying or activation).
Uneven Application of Sample:
Cause: Uneven application of the sample can cause irregular spots and lines.
Solution: Apply the sample evenly using a proper capillary or micropipette technique.
Development Chamber Issues:
Cause: Uneven saturation of the development chamber can lead to inconsistent solvent front movement.
Solution: Ensure the development chamber is evenly saturated with solvent vapor.
Use a proper lid to maintain saturation.
Plate Handling:
Cause: Touching the TLC plate with bare hands or allowing dust to settle on it can lead to contamination.
Solution: Handle the TLC plate with gloves and keep it in a clean environment.
Troubleshooting Steps
Verify Solvent System: Ensure that the butanol:acetic acid:water mixture is freshly prepared and mixed thoroughly.
Check Sample Application: Apply a smaller amount of sample and ensure even application across the spot.
Use Fresh Plates: Use a new TLC plate and avoid touching the surface.
Control Development Environment: Make sure the developing chamber is clean and evenly saturated with the solvent system.
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