How to Calculate Rf Values With Multiple Spots

Thin Layer Chromatography

Thin‑layer chromatography involves the same principles as column chromatography, it also is a form of solid‑liquid adsorption chromatography. In this case, however, the solid adsorbent is spread as a thin layer (approximately 250 um) on a plate of glass or rigid plastic. A drop of the solution to be separated is placed near one edge of the plate, and the plate is placed in a container, called a developing chamber, with enough of the eluting solvent to come to a level just below the "spot." The solvent migrates up the plate, carrying with it the components of the mixture at different rates. The result may then be a series of spots on the plate, falling on a line perpendicular to the solvent level in the container (see, for example, the following Figure). The retention factor (Rf) of a component can then be measured as indicated in the figure below.

�� This chromatographic technique is very easy and rapid to perform. It lends itself well to tine analysis of mixture composition and may also be used to advantage in determining the best eluting� solvent for subsequent column chromatography.� However, it should be borne in mind that volatile compounds (bp < 100EC) cannot be analyzed by TLC.

�� The same solid adsorbents used for column chromatography may be employed for TLC; silica gel and alumina are the most widely used. The adsorbent is usually mixed with a small amount of "binder"--for example, plaster of Paris, calcium sulfate, or starch--to ensure adherence of the adsorbent to the plate. The plates may be prepared before use, or commercially available prelayered plastic sheets may be used. In either case, the plates or sheets should be dried for an hour or more in an oven at 110EC before use.� This procedure removes moisture that is adsorbed on the adsorbent and produces a surface that is more effective in adsorbing and separating the components of the mixture being analyzed. The relative eluting abilities of the various solvents that may be used are the same as given in Section 6.1 of your text. It should be remembered that the eluting power required of the solvent is directly related to the strength of adsorption of the components of the mixture on adsorbent.

�� A distinct advantage of TLC is the very small quantity of sample required. A lower limit of detection of 10^‑9 g is possible in some cases. The spot of sample must be applied to the plate with care; it should not be large, for the same reason that the amount of sample placed at the top of a column should not be too large.� However, larger samples such as 0.5 mg may be used on larger TLC plates, which have thicker coats of adsorbent. In these�� cases isolation of the components may be achieved by scraping the separated spots from the plate and eluting (extracting) them with an appropriate solvent. It is usually necessary to repeat this process a number of times in order to obtain even several milligrams of material.� Such a procedure would offer a method of identification of the various components, however, because enough material could be collected to allow spectra to be obtained.

�� Often it is useful for the purpose of identification to run TLC chromatograms of knowns and unknowns side by side. Other useful applications involve running multiple aliquots of samples collected from a chromatographic column or samples taken from a reaction mixture in order to follow reaction progress as a function of time.

�� Detection of spots on the chromatogram is easy for colored materials, and a number of procedures are available for locating spots of colorless materials. For example, irradiation of the plate with ultraviolet light will permit location of the spots of compounds that fluoresce.� Alternatively, the solid adsorbent may be impregnated with an otherwise inert, fluorescent substance.� Spots of materials that absorb ultraviolet light but do not fluoresce will show up as black spots against the fluorescing background when the plate is irradiated with ultraviolet light.� Other detecting agents are more often used. These agents may be sprayed onto the chromatograms, causing the spots to become readily apparent. Examples of detecting agents used in this way are sulfuric acid, which causes many organic compounds to char, and potassium permanganate solution. Iodine is another popular detecting agent. In this case the plate is placed in a vessel whose atmosphere is saturated with iodine vapor. Iodine is adsorbed by many organic compounds, and their spots on the chromatogram become colored (usually brown). Since these spots usually fade, it is a good idea to circle the spots with a pencil while they are still visible in order to have a permanent record of the chromatogram.

�� Under a given set of conditions (adsorbent, solvent, layer thickness, and homogeneity) the rate of movement of a compound with respect to the rate of movement of the solvent front, R_f, is a property of that compound. The value is determined by measuring the distance traveled by a substance from a starting line in the before-mentioned figure.� The property has the same significance as retention time in a GC experiment.

A.�� Thin-Layer Chromatography:� General Instructions

�� Place a TLC strip ("Bakerflex" brand or equivalent) on a paper towel on the bench surface.

�� Handle the strip by the edges only and be sure the coated side is up.� Two coatings are

�� available:� Aluminum Oxide IB-F and Silica Gel IB-F.� (IB stands for inorganic binder, �and

�� F means that the coating contains a fluorescent indicator.)

�� At the top of the strip write the letter A or S (to identify the adsorbent); also identify the

�� solvent to be used for development (e.g., 1:3 EA/C for 25% ethyl acetate - 75% cyclohexane).

�� Dip a Strumia tube into the solution of the material to be analyzed and then very carefully

�� touch the end of the vertically held capillary tube to the strip at a point about 10 mm from the

�� bottom edge.� Immediately make a mark at the side of the strip in line with the center of the

�� spot.� Allow the solvent to evaporate completely from the spot.� Waving the strip gently in the

�� air hastens the evaporation.

�� Obtain a 4-oz. round screw-cap bottle, containing a whole or half disk of 7-cm filter paper.

�� The cap should be Teflon or aluminum foil lined.� Pour solvent into the bottle to a depth of

�� no more than 5 mm.� Cap the bottle tightly and shake it vigorously to saturate the space inside

�� with the solvent vapor and to thoroughly wet the paper.

�� Carefully place the strip (spotted end down) inside the bottle, and allow it to lean against the

�� wall.� Close the cap tightly and let the chromatogram develop.� Do not disturb the bottle

�� during the development.

�� When the solvent front has reached a point about 1 cm from the top edge of the strip,

� �� carefully open the bottle and remove the strip.� Immediately mark the position of the solvent

�� front with a pencil.� Wave the strip vigorously in the air to evaporate the solvent from the

�� surface quickly.

�� Examine the strip under ultraviolet light (in the special viewer) and outline with a pencil any

�� spots that are detected.�

�� When you are finished, do not empty the 4-oz. round screw cap bottle.� Cap the bottle tightly,

�� see that it is properly labeled as to which solvent it contains, and return it to the storage area.

B.�� Thin-Layer Chromatography:� Analysis of a Commercial Analgesic

Most non-prescription pain-relieving remedies contain aspirin (o-acetylsalicylic acid) or

acetaminophen - and some contain both.� Many also have some auxiliary compounds with analgesic properties.� In addition, caffeine is included in some preparations.� Caffeine is not an analgesic, but aids in the relief of certain types of headaches because of its stimulant effect (CNS and cardiac).

You will receive as an "unknown" a tablet of a commercial analgesic, to be analyzed by TLC for

the presence or absence of four specific constituents:� acetaminophen, aspirin, phenacetin, and caffeine.� Some of the products also have other active ingredients, but no attempt will be made to identify these other substances.�

PROCEDURE

�� Crush the tablet to a powder on a piece of glassine weighing paper.� Put half of the powder in

�� a 5 mL plastic beaker and add 2 mL of methanol and 1 mL of dichloromethane.

�� Stir the mixture with a glass rod for about one minute to allow the solvent to extract the active

�� ingredients.� Allow the insoluble matter to settle.� Carefully decant the supernatant liquid into

�� a clean, dry 5-mL beaker.

�� On a TLC strip coated with Silica Gel IB-F, apply a spot of the solution of "unknown" and two

�� of the four known solutions.� On a second Silica Gel strip, spot the "unknown" solution and the

�� remaining two known solutions.� Be sure to mark the identity of each spot on each strip.

� �Develop each chromatogram in a bottle containing 100% ethyl acetate.

�� Examine each strip under the ultraviolet light viewer (short wave UV light).� Compare the spot

�� positions (the Rf values) of the constituents of the tablet with those of the individual authentic

�� substances.� If very little movement of components has occurred on a strip, do not mark any

�� visible spots with a pencil.� Replace the strip in the bottle and repeat the development.� Make

�� a table recording the distance each substance moved from the origin and the distance the

�� solvent moved.

Che 317�� Sp�2005

Lab Report

Title: Thin Layer Chromatography pp. 144-147

Name :

Date:

In the following table, enter the distance traveled by each spot, along with its' corresponding R_f

Sample	Distance Front Traveled cm.	Distance Component Traveled �cm.	Silica Gel TLC Plate in 100 % Ethyl Acetate Solvent
Aspirin			R_f _Aspirin �� = __________
Acetaminophen			R_f _{Acetominophen} �� = __________
Caffeine			R_f _Caffeine �� = __________
Phenacetin			R_f _Phenacetin �� = __________
Unknown Tablet		1_________ 2_________ 3_________ 4_________	R_f ₁ �� = __________ R_f ₂ �� = __________ R_f ₃ �� = __________ R_f ₄ �� = __________

The components in your tablet were (Justify your conclusions):

How to Calculate Rf Values With Multiple Spots

Source: http://chemistry.csudh.edu/faculty/noel/CHE317L/Thin%20Layer%20Chromatorgaphy%20Experiment.htm

Buzacott Hings1995

How to Calculate Rf Values With Multiple Spots

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