TLC Sample Application:

Sample overloading is a common cause for poor chromatographic spot resolution on TLC layers. An overloaded spot migrates and diffuses into the surrounding in the direction of migration depending on the amount of overloading results streak overlaps and poor resolution. 

It is sometimes difficult to determine how much sample to load due to a number of variables, such as the sample matrix, the layer thickness, sorbent nature and the sample solvent.

Figure below shows the correlation between the sample volume and spot shape during 18F- FDG QC testing. After making an appropriate sample volume adjustment, the peak shape has improved. Molpure TL-100 exhibited nearly twice faster migration run time than Merck TLC SG 60 tested by a third party Cleveland Clinic cyclotron facility. 

Spot Control:

Other poor chromatographic separation results are often due to improper sample solvents, pipette and capillary syringes damage to the sorbent layer.

Too polar solvents such as water may not completely wet the sorbent layer and get with a star-shaped spot. To obtain small spot size, the least polar solvent should be used. With the least polar solvent the sample on the sorbent layer is immediately adsorbed onto the porous surface leaving the solvent to diffuse out into the layer. Conversely, for the more polar solvent the sample is carried out with it and diffuses out into a much larger spot.

To obtain small spot size, the less polar solvent should be used. Using the less polar solvent will result in obtaining a small spot size. The less polar solvent will also cause the sample on the sorbent layer to be absorbed onto the porous surface. The solvent will expand into the sorbent layer. Conversely, for the more polar solvent, the sample is carried out with it and diffuses out into a much larger spot.

To avoid the spot distortion produced by initial spot sizes greater than 1.1 mm, it is important to apply sample volumes, such that the spot diameter is 1 mm or less.

The initial spot size is important, and it can have a dramatic effect of the separation number or spot capacity. The smaller the spot size the higher the spot capacity.

Reference: The Thin-Layer Chromatography A modern practical approach book by Peter Wall