Thin Layer Chromatography: Separation / Identification of compounds based on differences in their polarity
Introduction * Procedure

(Adapted from McMaster University's Experiments: http://www.chemistry.mcmaster.ca
and the University of Colorado, Boulder:
http://orgchem.colorado.edu/hndbksupport/ochemlabtech.html )


TLC of Analgesic Drugs - Introduction (Read Lab Text/Guide pp.165-172 2nd ed.; pp.153-160, 1st ed.)

General Principles of Chromatography

Chromatography may be divided broadly into three kinds: adsorption, partition and ion-exchange; the simplest type is adsorption chromatography. In this method, the compound or compounds of interest is (are) adsorbed onto a solid support (the stationary phase), such as alumina (aluminum oxide) or silica gel (silicon oxides). Separation is achieved by elution ("washing" with a moving solvent that is either gravity fed or pressurized, Flash Chromatography). Adsorption chromatography may be carried out in two ways, by column (for relatively large samples, which is also referred to as a "preparative method"), or by thin layer which is most often used for identification and done with very, very small amounts. Thin layer chromatography most often uses alumina or silica gel that is spread out in a thin layer over a glass, aluminum, or plastic sheet. The compounds of interest are placed on this layer in the form of a separate spot for each compound. A suitable solvent is then allowed to run up the sheet by capillary action.

The order in which the compounds are eluted will depend on how strongly they are adsorbed on the surface of the stationary phase. Alumina, unless specially pretreated, is slightly basic and hence strongly adsorbs acidic substances or materials capable of forming hydrogen bonds to the basic oxygen atoms of the alumina. Compounds without the ability to form hydrogen bonds, but with substantial dipole moments, will be somewhat less strongly adsorbed due to electronic interactions between their dipoles and those of the alumina. Compounds with neither acidic hydrogens nor dipole moments are only very weakly adsorbed due to dipole-induced dipole interactions.

Silica gel (SiO2) and alumina (Al2O3), the two adsorbents commonly used for column chromatography are sold in different mesh sizes, as indicated by a number on the bottle label: “silica gel 60” or “silica gel 230-400” are a couple examples. This number refers to the mesh of the sieve used to size the silica, specifically, the number of holes in the mesh or sieve through which the crude silica particle mixture is passed in the manufacturing process. If there are more holes per unit area, those holes are smaller, thus allowing only smaller silica particles go through the sieve. The relationship is: the larger the mesh size, the smaller the adsorbent particles.

Adsorbent particle size affects how the solvent flows through the column. Smaller particles (higher mesh values) are used for flash chromatography, larger particles (lower mesh values) are used for gravity chromatography. For example, 70–230 silica gel is used for gravity columns and 230–400 mesh for flash columns.

Alumina is used more frequently in column chromatography than it is in TLC. Alumina is quite sensitive to the amount of water which is bound to it: the higher its water content, the less polar sites it has to bind organic compounds, and thus the less “sticky” it is. This stickiness or activity is designated as I, II, or III, with I being the most active. Alumina is usually purchased as activity I and deactivated with water before use according to specific procedures. Alumina comes in three forms: acidic, neutral, and basic. The neutral form of activity II or III, 150 mesh, is most commonly employed.

The choice of solvents used to elute the various components of the mixture from the column will depend upon the components in the mixture. For a very weakly adsorbed component a very non­polar solvent such as petroleum ether (a mixture of hydrocarbons) would be used. For more strongly adsorbed components, a more polar solvent such as ether might be used. For very strongly adsorbed components, a very polar solvent such as ethanol, water or even acetic acid might be required to displace the material from the column.

In Thin layer chromatography the compound of interest is spotted on a thin layer of adsorbent coated onto a glass, aluminum or plastic sheet. A suitable solvent is then allowed to run up the sheet by capillary action, as shown below:

where,
x = distance (in cm) from origin to the sample spot = 2.1 cm
y = distance (in cm) from origin to solvent front = 2.8 cm

and Rf = x / y

NOTE: If too much sample is applied, the spot can be very large and not symmetrical, therefore the measurement is usually made to the front of the sample spot.

During the elution with the solvent, the sample will partition itself between the stationary phase (the adsorbent layer) and the moving phase (the solvent) so that the distance which the sample moves up the plate is characteristic of that substance and will differ from one substance to the next. The distance moved by the spot of sample divided by the distance moved by the solvent is known as the Rf value and is characteristic of that compound for the solvent system used. A mixture of substances will thus give rise to a series of spots, one corresponding to each component. This technique is extremely useful for analysis on a micro scale and for the purification of small quantitites (usually less than 0.1 g) of material. In preparative work, a substance is recovered from the plate after development by removing the particular region of the adsorbent layer containing that substance from the plate, followed by the removal of the substance from the adsorbent layer by extraction with a suitable solvent.

The second general type of chromatography is partition chromatography, the three sub-types: gas-liquid chromatography, liquid-liquid chromatography and paper chromatography, which is an application of liquid­liquid chromatography. In the first two cases, the stationary phase commonly consists of a liquid which is bonded to an inert solid substance. For liquid chromatography, eluting the system with a moving liquid leads to separation of the components of a mixture by the partitioning of these components between the stationary and moving liquid phases so that the components will move at differing rates through the column. Separation is thus achieved by collecting the moving liquid phase in different fractions as it leaves the column. However, in gas chromatography, the moving phase is a stream of an inert gas, such as nitrogen or argon. The sample under study is vaporized, and the compounds, which are in the gas phase, are swept through a column of the packed stationary phase by the inert carrier gas. Partition of the sample between the stationary and moving phases will occur. The sample may be recovered from the gas as it leaves the column by condensing it at a low temperature. Paper chromatography is analogous to thin layer chromatography except that in this case, the support material consists of a sheet of specially prepared paper, and the stationary phase is considered to be water adsorbed on the paper. Elution with a solvent, measurement of the Rf value and preparative work is carried out in the same way as for thin layer chromatography.

The third type of chromatography, ion­exchange chromatography, is of somewhat more limited application. Here, the solid support consists of a resin which can have either basic or acidic properties, and mixtures of acidic or basic substances can be separated using these resins by eluting with buffers of different pH's.


Procedure

SEE: http://orgchem.colorado.edu/Technique/Procedures/TLC/TLC.html

In this experiment, TLC will be used to examine the composition of various over the counter analgesic (pain relieving) drugs. The best known of these is aspirin, but several other chemically similar compounds are (or were) also used as analgesics. Among them are salicylamide and acetaminophen. Caffeine is not an analgesic but it is sometimes added to their formulations to overcome drowsiness. In prescription analgesics, several other compounds may be added such as codeine, N-cinnamylephedrine (cinnamedrine) and diphenylpyrilene. They are included for other therapeutic effects, such as antispasmodic or mild sedative effects. In addition to the active ingredients, the tablets can contain starch, lactose, other similar compounds, and sometimes inorganic bases. These additives are non-active and serve as binders and to improve dissolution, uptake and transport. The objective of this experiment is to identify a commercial unknown by identifying the active ingredients present in an unknown sample using TLC. Budget 60-90 minutes to complete the procedure once you have prepared the prelab materials.

The reference compounds are:
ASP - Aspirin (acetylsalicylic acid)
ACE - Acetaminophen (4-acetamidophenol)
CAF - Caffeine
IBU - Ibuprofen
SAL - Salicylamide

The unknown will be one of the brand names listed below. The structures for the active ingredents are provided. The check marks indicate what active ingredient(s) are present in each product.

 
Anacin
x
x
Excedrin
x
x
x
Midol
x
x
Tylenol
x
Oradine
x
x
Advil
x
BC Tablets
x
x
x
Bayer Aspirin
x


SEE: http://orgchem.colorado.edu/Technique/Procedures/TLC/TLC.html


You will have five solutions (5 reference compounds) to examine relative to your unknown which is a solid. You should use two or three TLC plates. They should be spotted on the coated side in a line ~1 cm from one end of the sheet, equally spaced apart, with the outer two spots about 0.75 cm from the edge of the sheet. The unknown should be placed in the centre, with one or two reference compounds on each side. You should use one plate to run your unknown plus two of the reference samples, and the other plate to run the unknown plus the remaining three reference samples. Depending on your technique you may want to spread the reference samples over three plates being sure to include the unknown on each plate.
------------------------------------------------------------------------
Sample Preparation/ Plate Application
Dissolve all of your unknown sample in ~3-5 mL of 1:1 ethanol:dichloromethane solution.

Apply the reference solutions and the unknown solution by touching the microcap tube to the solution, and then gently touch the Silica Gel plate at the proper spot. Use the microcap tube that is dedicated for each reference sample. (BE VERY ATTENTIVE. DO NOT CONTAMINATE the reference solutions!!) The spots should not be larger than 2 - 3 mm diameter.
------------------------------------------------------------------------
Developing the Chromatogram
When each plate has been prepared, place the plate, spotted end down, in the developing chamber. Make sure that spots are above the level of the 200:1 ethyl acetate: acetic acid solvent pool in the bottom of the developing chamber. Cover and allow about 5 - 10 minutes for the solvent to rise to within about 1 cm from the top of the sheet - do not allow the solvent to run all the way to the top of the plate! Remove the sheet and immediately mark the solvent front. Allow the sheet to dry.
------------------------------------------------------------------------
Visualization
The colorless compounds are visualized by illumination of the plate with an ultraviolet lamp. Many substances, particularly aromatic compounds, will show a bright fluorescence, which may have a characteristic color. The thin layer plates used contain a trace of fluorescent dye. Compounds which are fluorescent show up as bright spots on a light background; any others appear as a dark spot since they quench the fluorescence of the background dye. Circle the spots lightly in pencil, and note any distinctive colors. After circling the spots, place the plates in an iodine chamber to confirm the UV spot analysis.
------------------------------------------------------------------------
Interpretation & Results
(a) Calculate the Rf values of the reference compounds and the components of the unknown.
(b) Neatly draw the chromatogram to scale in your lab book; identify and label the spots in the chromatogram, identifying the spots in the unknown.
(c) From the number, position and appearance of the spots in the unknown, and the composition of the possible unknowns, identify the commercial trade name your unknown analgesic.
(d) Answer the post lab questions.