Lipid analysis：Thin layer chromatography

Lipid analysis

Thin layer chromatography is based on the separation of a mixture of compounds as it migrates with the help of a suitable solvent through a thin layer of adsorbent material which has been applied to an appropriate support. Thin layer chromatogram can be looked upon as being "open column" system. Following the development of a relatively simple apparatus for preparing chromatoplates with uniformly thin layers of adsorbent by Stahl (1964), TLC became very popular. Nowadays, ready-to-use TLC plates are normally used, since they offer greater reproducibility and higher mechanical strength.
Different stationary phases are available that allow to separate by partition, ion-exchange, adsorption or a combination of any of these phenomena. Adsorption is the most common mechanism. In adsorption TLC the sample is continually fractionated as it migrates through the adsorbent layer. Competition for active adsorbent sites between materials to be separated and the developing solvent produces continuous fractionation. A portion of the material to be separated will be found in the mobile phase and a portion will be adsorbed to the solid adsorbent particles. As the process continues the various components move different distances, depending on their relative affinities for the adsorbent as compared with the migrating solvent. In general, the more polar compounds are held back by the adsorbent while less polar material advance further. Polar solvent effect the most movement of sample material over the adsorbent.

The migration of a compound in a given TLC system is described by its Rf value:

distance traveled by compound Rf = distance of the solvent front from the origin

i. Adsorbent:

The most common adsorbent used is Silica gel (silicic acid combined with a small amount of gypsum as a binding agent). Many other material have also proven useful for specific purposes; for example, alumina for the separation of steroids and water soluble vitamins and Kieselgur for separation of sugars.

ii. Solvents:

Although it is frequently possible to select a good solvent system by the empirical methods, some time and effort may be saved by consideration of some principles of the chromatographic separation. Of particular importance in TLC are the elutropic series of solvents. This is a series of solvents arranged in the order of their eluting power; that is having increasing ability to remove compounds from an adsorbent. A knowledge of the relative adsorbent characteristics of the class of compounds to be separated is also important. For example, saturated hydrocarbons are adsorbed poorly while unsaturated hydrocarbons are adsorbed according to the increase in number of their double bonds.

iii. Development:

The apparatus used for development must have certain features:

a) be capable of retaining any of a multitude of mixtures of original solvents

b) be air tight, to insure maintenance of a solvent saturated atmosphere

c) be relatively easy to handle

iv. Visualization:

Compounds separated can be visualized by

a) natural colour

b) spray reagents; yields coloured derivatives on reaction with the separated spots.

c) iodine vapour

d) fluorescence and UV absorption

e) autoradiography

EXPERIMENTAL PROTOCOL

Handle the plates carefully. NO FINGER PRINTS. These will be shown after exposing to I2 vapour.

1. Using the template provided, mark the plates with a sharp pencil, as shown here

2. Line the chamber with chromatography paper. Prepare 202 ml of solvent system (Hexane:Ether:Acetic acid 60:40:1) in a 500 ml Erlenmeyer flask. Mix and pour ~150 ml into the chamber. Cover and let the chamber saturate while loading the plates.

3. With a 10 µl capillary pipette, spot 1-2 µl of phospholipids standard onto the TLC plate, as shown. Make sure the spot remains smaller than 4 mm in diameter. Move on to the other standards. After the spots have dried, repeat loading each standards until you have loaded approx. 10 µl each. Also, load 10 µl of your lipid extract on one spot, and then the remainder of the extract as a line (i.e., a series of spots).

4. Let dry the spots. Make sure that the loading area is above the solvent. Place the plates in the chamber to develop.

5. Immediately close the cover and let run for approximately 30 min, until the solvent front has reached the upper line.

6. Remove the plate and leave to dry in the rack in the fume hood. Discard the solvent in the waste container provided, remove the chromatography paper and leave in the chamber. Leave the chamber in the fume hood to dry.

7. Now place the plate in the iodine tank in the fume hood. You will see the lipids as yellow spots after about 5 min or so.

8. Mark the edges of the spots with a pencil. Make a tracing on onion skin paper for a record.

9. Scrap off lipid fractions as shown, place in weighing paper, fold and roll to grind the clumps.

10. Meanwhile prepare columns to elute the lipids from silica gel. To do this insert a small amount of glass wool into a pasteur pipette, label the pipette and leave on the stand provided.

11. Carefully transfer the silica gel having different lipids into appropriate columns. Keep 7 ml glass vials beneath the columns.

12. Drip 1 ml of chloroform into each column except for the phospholipid and monoacylglycerol columns. To the latter, add >1 ml of 100 % methanol.

13. Shake the vials well. Place them in the fume hood evaporating set-up and evaporate off the solvent under a stream of N2. (Ask the instructor before using the N2 tank).

14. Store the dry lipids under N2.

15. Label and leave the separated lipid in the -20 °C freezer until transmethylation (day 2).

Results

 You should have obtained a good separation of the lipid classes, similar to the plate shown below.

However, differences in the solvent composition will lead to altered mobility of the different components. A common problem occurs when the TLC tank is not completely sealed, and the solvents can slowly evaporate. Since evaporation rate depends on the boiling point, solvents like hexane will evaporate faster. As a result, the mobile phase becomes more polar. Nevertheless, a positive identification of your spots should always be possible since you ran standards on the same plate as well.