Thin Layer Chromatography

A little history To write with colors -- literally translated from

its Greek roots chroma and graphein , chromatography was first developed by the Russian botanist Mikhail Tswett in 1903 as he produced a colorful separation of plant pigments through a column of calcium carbonate, particularly the carotenoids and the chlorophylls.

Early work consisted of dropping dyes onto filter paper and seeing the development of concentric rings with different colors: this technique was called “capillary analysis”.

More history Enter Martin and Synge: these two British

gentlemen were pioneers in early separation science. They used silica as a stationary phase with chloroform as the mobile phase to separate amino acids in wool. They called their idea “partition chromatography” and moved into the theoretical by suggesting that the mobile phase could well be a gas . . . Initial gas chromatography worked was started during WW II.

For their efforts, they were awarded the Nobel Prize in Chemistry and Physics in 1952.

More history Problems still were present: reproducibility

and lack of uniformity of silica production made the techniques less than desirable.

Paper Chromatography was suggested as a way to separate materials: cellulose with tightly adsorbed water molecules would make a very uniform polar stationary phase with less polar mobile phases. This technique revolutionized planar chromatographic methods but had limitations.

More history To overcome these limitations (stronger

solvent systems and developing agents), two Soviet pharmacists, Nikolay A. Izmaylov and Maria S. Shrayber, coated films of silica onto glass slides and used in a similar fashion to paper chromatography.

In the 1950’s, a German chemist named Egan Stahl did a HUGE amount of research into this field and produced the “bible” of thin layer chromatography. Stahl’s book emphasized applications and different reagents that could be used to indicate types of compounds that were being analyzed. He also helped standardize silica gel production, which improved reproducibility!

So what’s going on in TLC? We have a backing – plastic, glass, aluminum We have a stationary phase – usually silica,

but occasionally alumina, zirconium oxide or cellulose.

A binder holds the stationary phase to the backing (as an example, gypsum is used to bind silica to the plate).

Within the stationary phase, there is usually a fluorescent material added to allow easier visualization of separated materials once the plate has been developed.

Theory If we thought about how a solute compound would interact

with such a solid stationary phase, we would realize that it must essentially “stick” to the surface by some intermolecular (van der Waal) forces. This sticking process in chromatography is known as adsorption.

If a solute molecule dissolves into a liquid stationary phase in a chromatographic system, we say that the molecule partitions between the two phases.

These two terms are so important in chromatographic systems that it is worth summarizing them again:

Adsorption – describes the process of a solute molecule adhering to a solid surface

Partition – describes the process of a solute molecule dissolving into a liquid stationary phase

Theory The van Deemter equation relates the resolving power (HETP,

height equivalent to a theoretical plate) of a chromatographic column to the various flow and kinetic parameters which cause peak broadening, as follows:

HETP = A + B/u + C*u Where HETP = height equivalent to a theoretical plate, a measure

of the resolving power of the column [m] A = Eddy-diffusion parameter, related to channeling through a

non-ideal packing [m] B = diffusion coefficient of the eluting particles in the longitudinal

direction, resulting in dispersion [m2 s-1] C = Resistance to mass transfer coefficient of the analyte between

mobile and stationary phase [s] u = Linear Velocity [m s-1] In TLC, B predominates. u is really small (5-10 minutes to develop

a 7.5 cm plate!)

Theory Smaller uniform particles provide lots of

surfaces for the two processes (adsorption and partition) to occur.

These processes combined with the capillary action of the plate interacting with the solvent allows for “random walk”.

The primary process at work for this kind of separation is polarity: the stationary phase is usually polar and the mobile phase is less polar (hexanes methanol).

Theory to Practical As with most liquid chromatographic

techniques, this technique is SCALABLE! If you have a solvent system that works for separating materials on an analytical level, using a larger (preparative) plate or a column with a similar silica (size of particle and pore size) will allow you to scale from micrograms to 10-100’s of milligrams and in some cases, gram separations.

Preparative TLC plates use larger tanks.

Prep TLC

Practical Considerations TLC plates need to be kept DRY. Remember

that silica gel is in those little packets you get with your new shoes or electronics that says “do not eat!”.

Gentle heating (80 oC will remove any extra adsorbed water).

TLC plates should only be handled on the edges since your fingers contain oils which will interact/interfere with the desired separation.

Proper application of the material for separation is REALLY important.

How-to guide Prepare the solvent tank:

Get your solvent system measured out. Add a piece of filter paper to the back of the beaker/tank. Add the solvent and tightly cover the tank and let the

system sit for a few minutes: this allows the tank to become saturated with solvent vapors .

Prepare the TLC plate. Get a TLC plate and mark a line WITH PENCIL about 6-8

mm above the depth of the solvent in the tank. This is the origin

Using a capillary tube, apply the materials to be separated on the origin.

Dry off any solvent and GENTLY place the TLC plate into the tank.

TLC chamber for

development e.g. beaker

with a lid or a closed jar

after ~5 min after ~10 min after drying

After the TLC plate has been run . . . Visualize the spots

Depending upon the materials present, you could try UV light (254 nm and/or 365 nm)Reagent Detection of

Aniline phthalate reducing sugars, oxohalic acidsBromocresol green organic acids2',7'-Dichlorofluorescein lipids (saturated, unsaturated)4-(Dimethylamino)-benzaldehyde terpenes, sugars, steroidsReagent according to alkaloids + other nitrogen

compoundsDragendorff-MunierIron(III) chloride acetylsalicylic acid, paracetamolPotassium hexacyanoferrate(III) lipids, sterols, steroids,Molybdatophosphoric acid reducing compounds

Ninhydrinamino acids, amines, amino sugars

Rhodamin B lipidsRubeanic acid heavy metal cations

http://www.mn-net.com/tabid/5578/default.aspx

Measuring the compounds The distance the solvent travels is measured

from the origin to the solvent front, which is marked as soon as the TLC plate is removed from the TLC tank. This is Z.

The distance from the origin to the center of a spot is X.

Rf = X / Z, which is a unitless quantity. Each compound should have a different Rf

Troubleshooting if your separation wasn’t great Streaking of material: two usual causes. Either

over spotting/exceeding capacity of what plate can separate OR adjusting the pH. Amines can streak less if a little ammonium hydroxide is added to the solvent system.

Spots too faint: several possible causes. The material spotted was too dilute, so concentrate and run another TLC plate. Using the wrong reagent to visualize the spots or this is the wrong solvent system for this plate.

Spots narrow as the plate develops: usually indicates that the TLC plate was touching the filter paper/another plate so capillary action wasn’t uniform.

So who uses TLC? Researchers – great for monitoring chemical

reactions in real time. As a reaction proceeds, the product appears on the plate as a spot with a different Rf and the product becomes more faint as time moves forward.

Forensic Analysts - TLC is useful in detecting chemicals of forensic concern, including chemical weapons, explosives, and illicit drugs. Unknown samples are compared to a reference on the same TLC plate. If the unknown compound has the same Rf and responds to a developing reagent in the same manner as a known reference compound like cocaine, a more careful analysis would be warranted. The TLC could be run in 30 minutes!

So who uses TLC? Environmental Analysis

Groundwater analysis, determination of pollutants from abandoned armaments in soils and surface waters, decomposition products from azo dyes used in textiles.

http://www.chem-ilp.net/labTechniques/TLC.htm

Other Planar Techniques Chromatotron