Thin Layer Chromatography

Experiments can be sensitive to slight sample impurity, or sample degradation.  Thin Layer Chromatography (TLC) is a cheap, quick way to confirm the presence of impurities.  A positive TLC result saves you from more involved tests on your sample, while a negative TLC tells you nothing obvious is wrong with your sample.  The book, "Lipid Analysis : A practical approach" by R.J. Hamilton and S. Hamilton (Oxford University Press, 1992) has a good description in chapter 3.  TLC is very flexible, although each sample type requires a specific solvent and visualization method.

TLC for PE and PC lipids.
            Theory -   In TLC, solvent travels up a thin solid substrate via capillary action.  Each species in the sample is drawn up the substrate by the solvent.  However, species affinity for the substrate may differ and this affects the speed at which they travel.  A species with a high affinity for the substrate will bind and unbind many times, slowing its travel.  In contrast, a species with a low affinity for the substrate will travel quickly.  For a given solvent, substrate and species, the ratio of the distance travelled by the species compared to the solvent is called the Rf value.  TLC separates species on the basis of their Rf value.  The final step is to visualize the species by colouring them in some way.

For a sample consisting of a hydrocarbon, DOPE lipid, DEPE lipid and DOPC lipid, all will dissolve in the solvent. The TLC plate is coated in silica beads that have highly polar surfaces.  The hydrocarbon has a low affinity for the substrate so it should travel quickly and have an Rf value near 1.  The DOPC lipid's polar headgroup likes the silica beads giving it a high affinity for the substrate and a low Rf.  While the PE lipids headgroup is polar the affinity for the substrate is lower an intermediate Rf value.  TLC should show three spots, with the hydrocarbon on top, the DOPC at the bottom and the DEPE and DOPE probably forming a single spot in the middle.

A common reason for TLC failing is if the species have very similar Rf values, as illustrated by the DEPE and DOPE above.  Adjusting the substrate and solvent properties can help.  Another problem is if a species does not respond to the visualization.  There are a wide range of visualization techniques, some of which respond to only particular chemical species.  This selectivity is a powerful tool.

Commonly TLC is quoted as being sensitive to 1% impurities although this depends on the purity of TLC plates, solvents and the skill of the operator.  You can easily benchmark your setup using a pure standard with known amounts of impurities added to it.

        Materials and Equipment -         1.  PC or PE lipid dissolved in chloroform or cyclohexane at the ratio of 1mg:10mL to 1mg:30mL.
                                                                  2.  10 microL Drummond syringe and tweezers.
                                                                  3.  Approximately 2mL of chloroform:methanol:water in the volume ratio (1.2 : 0.6 : 0.08), stored  in a secure screw-top 100mL bottle.  Check the bottle and amount of solvent by placing a microscope slide vertically inside the bottle.  The solvent should cover the bottom 5 to 10mm of the slide and you must be able to screw the lid of the bottle on while the slide is inside. (Caution - chloroform is dangerous.  Read the MSDS.   Use the chlorinated waste bottle.)
                                                                  4.  A set of Silica gel 60 plates.  The usual specifications are 2.5cm*7.5cm slides precoated with a silica-gel 60 thickness of 0.25mm and they are sold by Sigma among other vendors.  TLC plates should be stored in a desicator
                                                                  5.  10mL to 50mL of phosphomolybdic acid (5-10% in ethanol).  (Caution - avoid skin contact.  Use the chlorinated waste bottle.)

Note, many impurities arise from improper sample handling.  Use the same proceedures you use for generating samples when handling TLC equipment.  The presence of impurities may reflect on your technique rather than your supplier.  Gloves, clean surfaces, properly cleaned glassware and tools (using organic and polar solvents) are all important.

           Method -        1.  Use a 10microL Drummond microsyringe to place 1microL to 2microL dots of each sample on the TLC plate.  The sample dots should be in a line roughly 15mm from one end of the plate.  For a 25mm wide plate you can fit 4 dots comfortably.  Each sample dot should be as small as possible.
                                     2.  Give the solvent a minute or so to evaporate.  You can probably see the lipid samples as shiny spots on the plate.
                                     3.  Unscrew the cap of the jar containing the solvent.  Lower the TLC plate (lipid dots end first) into the solvent so the bottom edge is covered to a depth of about 5mm.  Be careful not to make a splash so only the bottom 5mm is directly exposed to solvent.  Screw the cap of the jar on tightly.  Before your eyes, the solvent will climb up the plate via capillary action.  The solvent makes the white silica appear pale or almost transparent.
                                     4.  When the solvent line has almost reached the top of the plate (<10mm) pull it out of the solvent with tweezers and lie it on a tissue under the hood to dry.  Wait 5 minutes for all the solvent to evaporate.
                                     5.  Visualize the sample.  Phosphomolybdic acid works well for lipids.  Quickly dip the TLC plate into the phosphomolybdic acid solution so the entire surface is covered.  Pull it out and holding the plate horizontally with the silica surface upright, wait for the ethanol to evaporate.  Place the dry plate on a tissue and heat it to 100C for approximately 5 minutes.  A hairdryer works, but an oven or hot plate is much better.  Lipid spots should turn dark green.  You can photocopy the plate to keep in your records.

Immersing the lipid in Phosphomolybdic acid seems to cause smears in the direction the TLC plate was pulled from the acid.  An aerosol of acid may work better.

Another way to visualize lipids is to place them in a bottle with a few crystals of iodine and wait an hour or so.  Spots of lipid should turn a sickly orange colour.  To work, the crystals must have been in the bottle for a few days to equilibrate.  Once the TLC plate is removed from the iodine rich environment, the spots will fade so you must mark them with a pencil.  Another way to mark with iodine is to place a few crystals in the top of a 5 ml Pasteur pippette to blow a stream of iodine rich air at the plate surface.  This works surprisingly well.  Iodine isn't as effective as Phosphomolbdic acid.  Many other visualizers exist for specific tasks.

             Conclusion -    TLC is a rapid, easy method for checking sample purity.  The references below describe the tremendous power and flexibility of this technique.

            References -   Hamilton R.J., Hamilton S. "Lipid Analysis : A practical Approach" (1991) Oxford University Press. Chapter 3.
                                    Williamson K.L "Macroscale and Microscale Organic Experiments" (2nd Ed.) Chapter 10.
                                    Kirchner, J.G. "Techniques of Chemistry" (1978)
                                    Kates, M. "Techniques of Lipidology" p231.

November 2000.

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