TLC Fundamentals – How to make a successful TLC plate (part 3)

1. Deposit line & Solvent front

Plot the deposit line with a pencil at 3mm above the mobile phase level.
Recommended migration height is at least 8cm.
Make the solvent front migrate at up to 1cm from the top edge of the TLC plate.

2. How to make the deposit

a) Sample concentration it is necessary to avoid over-loading State of the art => for a standard: concentration of 0.1mg/L for the sample: concentration must not exceed 2%

b) Deposit size

The deposit must be spreaded at the minimum possible (smallest possible volume) otherwise there is a loss of separation.
If the chemist wants to make several deposits, it is necessary to dry the plate between the successive deposits. The plate must be left to dry for 10min at room temperature before its development.

Influence of the size of the deposit on the separation – Dimensions of the spots according to their Rf.

3. Deposit of the raw sample

a) Spot mode: By capillarity using an HPLC syringe or a capillary, there is a more or less deep penetration of the solute in the adsorbent layer. The finest deposit spots are obtained with a syringe rather than a capillary. Using this mode of deposition mode, spots are more or less deformed. Load the smallest drop as possible to avoid dispersion caused by overloading which could affect the compounds resolution. Avoid touching the TLC plate with the deposit tool to limit compounds penetration deep in the layer of the plate. Spot density and dispersion may vary with solvent polarity. Do not place the spot too close to the edges of the plate.

b) Spray mode: By using a spray bottle, the spray speed must be slower than the evaporation velocity to avoid any projection. Using this mode, there is little penetration of the solute into the adsorbent layer staying on the surface. The eluted spots are less spreaded out. Using this spray mode the separation of the migration spots is optimal.

Solvent of dilution
It must have a weak elution force, be very pure.
It must be sufficiently polar to dissolve the sample (but not too much to be easily eliminated).
The use of bases and acids should be limited.
Avoid viscous and high-boiling solvents (N,N-Dimethylformamide, DMSO, BuOH, water), as the migration time of the solvent will be longer.
It is necessary to dry the plate between two successive deposits.

Solvent of elution
Adapt the elution strength according to the compounds polarity to keep Rf in the optimal zone (0.15 – 0.35).
The mobile phase velocity is not constant over the entire length of the plate.
Use the same mobile phase for TLC & Flash purification.

Preparation of the mobile phase to ensure a perfect transfer to Flash purification =>
Solvents must be measured precisely in volume using separated flask (check the precision of the flask).
Low volume in % can be measured using syringe to ensure greater precision.