Doering 6/2004

Thin Layer Chromatography

Overview: This is the general technique used for thin layer chromatography (TLC) in our lab. We generally
use this for analysis of glycolipids, lipids, monosaccharides, and small polysaccharides. Several common
solvent systems we use are listed as well.
Reagents/equipment:
TLC plates: we most often use Kieselgel 60, a silica gel plate with no added dyes; 10 x 20 or 20 x 20 cm
TLC tank and lid
Organic solvents for running plate
An oven that can reach ~ 70 C
Samples to run, dried down, including all appropriate standards.
Protocol:
1. Set up the TLC tank. Make sure you have a clean glass lid, which will fit tightly - do not use grease!
It is helpful to line the tank with filter paper. For a standard tank, simplest is to cut a sheet of 3MM
paper lengthwise, then fold it in half and cut a notch (~1-2 cm square in from one edge at the halfway
fold. This sheet will now fit nicely down inside the tank, wrapping horizontally around the front, one
side, and the back, slide it down to touch the bottom. (The notch allows the paper to fit over the glass
spine in the base of the tank.)
2, Equilibrate the TLC tank with your developing solvent for ~ one hour. Typically you will use 80-100 mls
of solvent. For lipids we often use 10:10:3 (chloroform:methanol:water); for monosaccharides 5:4:2 (npropanol:acetone:water).
Mix the solvent in a beaker to make sure it forms one phase (and you havent made a measuring error),
then pour into the tank.
3. Mark and dry the TLC plate. (Use gloves any time you handle the TLC plate.) Using a soft pencil, draw
a light line along the plate 2 cm from one edge (short edge for a 10 x 20 cm plate). You will spot samples
along this line. Then mark darker 0.5 cm lines on this line to indicate sample position. Label the marks for
which sample to load, and label the plate with date and other details (e.g. solvent, experiment ID). Then put
the plate in an oven ~70 C for ~15 min.
Marks for samples should be at least 8 mm apart (1 cm is convenient) and should start 2 cm in from the
edges of the plate to avoid edge effects. This will allow you to run up to 12 samples on a 20 x 20 plate.
It is also convenient to draw a line for the solvent front 2 cm from the top of the plate. This will allow
you to run separate plates very similarly and make comparisons easy.
4. Load your samples. Resuspend the dried samples in appropriate solvent (e.g. 2:1 chloroform:methanol
for lipids or 40% n-butanol for sugars). Try to minimize volume and water, 15 l for a sample dried in a
microfuge tube works well. Vortex well, and spin down to the bottom of the tube. Load with a capillary
tube or pipetman tip evenly along the 0.5 cm line you marked, under a gentle flow of air to help drying. Be
patient, slowly load material all along the mark, then let it dry completely before loading more. Avoid
gouging the plate surface.
For air flow, ideal is a base mounted hair dryer, or put a regular hair dryer on a ring stand. You want
the flow more down towards the plate than across the face of it, or it can distort your loading. How you
load the sample has a lot to do with how good your spots will look, so take your time

Doering 6/2004

5. Run the plate. Open the tank and gently put your plate in, samples down. Avoid splashing into the
solvent, and prop the bottom edge against the glass spine and the top edge against the filter paper liner. Keep
an eye on the solvent front until you get to know the system, and remove the plate when it reaches your
solvent front line.
Some protocols call for several developments, to improve separation near the origin. In this case remove
the plate, place in a hood or under a hair dryer until it is completely dry, and then put it back in the tank.
(Keep the tank closed in between.)
6. Analyze the data. As appropriate scan the plate and/or spray with EnHance and expose to film for hot
samples. For non-radioactive samples spray with appropriate stain and visualize as directed in your protocol.
For hot samples you may want to draw small (~1 mm diam) circles at two places on the plate, then spot a
l of leftover hot liquid in that point after running but before spraying. These hot spots will show on
the film and allow you to align the film with the TLC plate to know where samples and standards were.