Lecture 11 Chromatography 2 Thin Layer Chromatography (TLC)

Thin Layer Chromatography: Thin-layer chromatography (TLC) is a chromatography technique used to separate moderately volatile or nonvolatile mixtures Is a method for identifying substances and testing the purity of compounds. TLC is a useful technique because it is relatively quick and requires small quantities of material

A. Thin Layer Chromatography: The stationary phase: is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate. The mobile phase: is a developing liquid which travels up the stationary phase, carrying the samples with it. Components of the samples will separate on the stationary phase according to how much they adsorb on the stationary phase versus how much they dissolve in the mobile phase.

A. Thin Layer Chromatography: Faster moving component partitioned more into mobile phase Solvent movement Slower moving component adsorbed more into stationary phase Two component mixture applied to TLC plate Components of TLC

Identifying the Spots (visualization) If no spots are obvious, the most common visualization technique is to hold the plate under a UV lamp. Many organic compounds can be seen using this technique, and many commercially made plates often contain a substance which aids in the visualization of compounds. thin-layer chromatography plate viewed under UV light Use of visualizing agents For example: for Alkaloids the agent is Dragendorff’s reagent For Sugar: Aniline phthalate For Amino acids: Ninhydrin

TLC Steps 1- Prepare a plate for spotting with the origin marked in pencil 2-Spot all samples in the plate by microcapillary

3- Develop the plate using the solvent of choice: 3a-Pour solvent in the jar 3b. When the solvent front is seen to reach within 1-2 cm of the top of the plate, the plate is removed & the solvent front is marked

TLC Steps 4- measure Rf values : first draw a line across the origin then measure distances to the solvent front and the "center of gravity" of the individual spots

Interpreting the Data The Rf (retention factor) value for each spot should be calculated. Rf is characteristic for any given compound on the same stationary phase using the same mobile phase for development of the plates. Hence, known Rf values can be compared to those of unknown substances to aid in their identifications.

Interpreting the Data Rf values often depend on the temperature and the solvent used in the TLC experiment. The most effective way to identify a compound is to spot known substances next to unknown substances on the same plate. In addition, the purity of a sample may be estimated from the chromatogram. An impure sample will often develop as two or more spots, while a pure sample will show only one spot

Determination of Rf Values Less polar! More polar!

Determination of Rf Values The Rf is defined as the distance the center of the spot moved divided by the distance the solvent front moved (both measured from the origin)

Determination of Rf Values

Thin Layer Chromatography: Absorption of Solutes The adsorption strength of compounds increases with increasing polarity of functional groups, as shown below: -CH=CH2,-X,-OR,-CHO,-CO2R,-NR2,-NH2,-OH,-CONR2,-CO2H. (weakly adsorbed) (strongly adsorbed) (nonpolar) (more polar)

Elution Strength of Mobile Phase (e) Elution strength is generally considered to be equivalent to polarity. A solvents elution strength depends on Intermolecular Forces between the solvent and the analytes and between the solvent and the stationary phase. A more polar (or more strongly eluting solvent) will move all of the analytes to a greater extent, than a less polar, weakly elution solvent. For example, the elution strength of hexane (non polar) is very low; e = 0.01. the elution strength of ethyl acetate (moderately polar) is higher; e = 0.45 the elution strength of ethanol (very polar) is even higher; e = 0.68

Thin Layer Chromatography: The moving strength of compounds increases with increasing polarity of the mobile phase

Thin Layer Chromatography: Drawbacks: *Limited quality of the separation * Limited reproducibility *Evaporation of the mobile phase Advantages: *Easy to use *Cheap *Possible multiple analysis * Small volumes *No sample preparation required

TLC Analysis of an Analgesic “Analgesia”: Greek for the deadening or absence of pain without loss of consciousness Analgesics: compounds that relieve pain Range from aspirin to morphine, and other related narcotics Many (over the counter) analgesics -Fevadol, Paracetamol, Panadol(acetaminophen) Nurofen (ibuprofen, codeine) Excedrin acetaminophen, caffeine, aspirin

Identification unknown drugs using standard Reference(s) The purpose of this experiment is to use (TLC) to determine the composition of the analgesic tablet (X) Substance Rf Aspirin 0.76 Acetaminophen 0.52 Caffeine 0.22 Ibuprofen 0.91 Unknown Spot #1 0.24 Unknown Spot #2 0.58 What are Unknown spots ? Which substance is the most polar?