SCHA 319 Separations Lab
SCHA 319 Chemical Separations
"Separation of Food Dyes by TLC (Thin Layer Chromatography)"
Thin-layer chromatography is a liquid-solid (adsorption) or liquid-liquid (partition) separation technique that takes advantage of equilibration of the solute between the mobile phase and the stationary phase. In this experiment you will use silca gel, coated on a flexible polymer backing, as your stationary phase. This will be a form of liquid-solid chromatography.
The TLC plate is spotted with the sample at the base and placed in a shallow reservoir of the appropriate mobile phase. The solvent rises up the plate by capillary action, this elution of the solute up the plate hopefully achieves an adequate separation. The plate is then removed and allowed to dry. A vertical series of spots will be apparent. (They appear in this experiment because we are using solutes which absorb light in the visible region of the electromagnetic spectrum.) TLC experiments sometime need to have their separated compounds developed, This can be done by applying a color forming spray, which is often bound irreversibly, i.e. ninhydrin. Some plates are manufactured with a fluorescent dye that will appear dark when observed under UV light if a compound is present. Many compounds will form a colored adduct with iodine so the plates are placed in chamber saturated by iodine vapor and colored spots will develop when compound is present. This process is reversible so the spot must be marked with pencil so a permanent record is established. Finally since the TLC stationary phase is often inorganic the plates can be sprayed with hot concentrated sulfuric acid. The separated spot will charred to carbon. This type of detection is very sensitive but destructive. Hardware has been developed that allow the color intensity of the separation track to be recorded and to allow for plotting of chromatograms similar to an HPLC output. It is possible to recover solutes from the planar surface but this is a difficult process.
The separation is based on the many phase equilibria that exist between the solid stationary phase and the liquid phase. THE MORE POLAR THE COMPOUND THE STRONGER IT WILL ADSORB TO THE STATIONARY PHASE. This is true in adsorption chromatography, when you use silica gel, alumina, cellulose, charcoal, etc. The distribution of the solute between the solid and liquid is a dynamic equilibrium which depends on both the polarity of the solute and the polarity of the solvent. The mobile phase and the solute are actually in competition for the active adsorption sites of the stationary phase. In conclusion, separation occurs because the different polarity solutes will have different solvent - stationary phase equilibria values and hence elute up the TLC plate at different rates.
TLC Chromatography of some dye mixtures.
In this experiment, you will separate a unknown dye mixture prepared for you by the stockroom. You will also be given a series of standard compounds from which to develop the proper composition of your mobile phase so that you can identify the dyes in your unknown. In this lab you will examine the effect of your solvent strength (εo) for your separation.
The solvent systems that you will use for this lab are listed in the Table below.
Table I. Mobile Phase Composition
Mobile Phase Solvent / 4 Parts / 1 PartA / 50:50 v/v methanol and isopropanol / Aqueous Ammonia
B / 100 % methanol / Aqueous Ammonia
C / 50:50 v/v isopropanol and ethyl ether. / Aqueous Ammonia
D / 100% Isopropanol / Aqueous Ammonia
The dyes, with one alternate name, that are in the solutions are
FDC Red 3 or Erythrosin B
FDC Blue 2 or Indigo Carmine
FDC Green 3 or Fast Green FCF
FDC Yellow 6 or Sunset Yellow FCF
Parts of the volumes of the mobile phase mixtures are listed in the solvent compositions Table since you will need to determine the amount of mobile phase to prepare. Graduate cylinder accuracy should be sufficient to prepare these solutions. You should strive to minimize evaporation losses due to the fact that it will change your solvent composition as the lab progresses.
General Procedure.
For you TLC chambers you should use beakers. Watch glasses should not be used as covers since they do not make a tight seal. It is important the vapor phase over the plates be saturated with the mobile phase vapors. In addition, if you do not have an air tight seal the composition of your mobile phase will change during the course of the lab due to evaporation of the more volatile constituent. Parafilm is the better choice for your covers.
TLC sheets will be available in the lab. Good laboratory practice would call for you to record product details including the lot number of all the materials that you use in the lab. You will cut the sheets to a size that will fit into your beaker. Your sheet edges should not rest against the side walls since this might cause uneven migration of you mobile phase front. Your TLC plates should be wide enough to allow for the spotting of all your standards and your unknown on each sheet in each mobile phase composition. Make sure to label your sheets in some way (pencil).
Mark you TLC starting line with a pencil - (not ink) . Your spots, placed on the starting line, will be introduced by using the microcaps in the lab and you should try to have as small a spot as possible but as much material as possible. This will require some practice. Several spottings, allowing the solvent to dry after each spot works best. Transfer the spotted TLC sheet to your solvent beaker and then seal it up with the parafilm. When you place your TLC plate in TLC chamber you should make sure your spot ‘starting line’ is above the solvent level. Do not ‘drown’ your spots. You will most likely want to carry our your plate development and solution preparation in the hood. Ether is very flammable and aqueous ammonia has a very noxious smell. Allow the solvent front to migrate to about 80% of the distance to the top. When the solvent front has reached this point, remove the parafilm, remove the plate and mark the position of the wet solvent front. This will vanish when the solvent evaporates. Allow the plates to dry in the hood. Reseal your beaker with parafilm. You might need to do another separation and you want to be sure the solvent composition is the same for that next run.
For your report. Short Format
Prepare a proper abstract.
Address these questions in order.
1) The key element of your write up is to report which dyes are in your unknown? Which solvent system worked best? Why did it work best?
2) Discuss the mechanism of separation for this type of TLC separation.
3) You should provide scanned images of your plates and include them in your report. This should be properly labeled as Figures. .
4) Provide a Table(s) that have the calculated Rf values for each dye and unknown in each of the mobile phases.
5) Discuss the solvent strength of each mobile phase and the impact it has on the migration of each dye. See page 563 of your text (Harris 7e) to place your solvents in solvent strength order. Give the structures of each of the dyes (Chemfinder.com from the lab computers is a good start). Give the CAS numbers and one additional alternate name for each dye. Table will work well for this.
Select one of your spots on one of the plates. Estimate the number of plates for this spot. Determine the Resolution between two of the spots in your unknown mixture for a system that has some resolution.
TLC Lab Printed 3/12/2007 Page 3