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CHM 312 Fall 2008

CHROMATOGRAPHY

THIN LAYER CHROMATOGRAPHY (TLC)

THIN LAYER CHROMATOGRAPHY


Thin layer chromatography (TLC) is an important technique for identification and separation of mixtures of organic compounds. It is useful in:
Identification of components of a mixture (using appropriate standards) following the course of a reaction, analyzing fractions collected during purification, analyzing the purity of a compound.

In TLC, components of the mixture are partitioned between an adsorbent (the stationary phase, usually silica gel, SiO2) and a solvent ( the mobile phase) which flows through the adsorbent.

THIN LAYER CHROMATOGRAPHY


In TLC, a plastic, glass or aluminum sheet is coated with a thin layer of silica gel. A very small amount of a solution of the substance to be analyzed is applied in a small spot with a capillary tube, ~1cm from the bottom of the TLC plate The TLC is developed in a chamber which contains the developing solvent (the mobile phase). A truncated filter paper placed in the chamber serves to saturate the chamber with mobile phase.
A B U C D

B U

C D

filter paper

THIN LAYER CHROMATOGRAPHY


As the mobile phase rises up the TLC plate by capillary action, the components dissolve in the solvent and move up the TLC plate. Individual components move up at different rates, depending on intermolecular forces between the component and the silica gel stationary phase and the component and the mobile phase. The stationary phase is SiO2 and is very polar.
http://www.instructables.com/id/EW1YDCYF4REC0IU/

It is capable of strong dipole-dipole and H-bond donating and accepting interactions with the analytes (the components being analyzed). More polar analytes interact more strongly with the stationary phase in move very slowly up the TLC plate. By comparison, the mobile phase is relatively nonpolar and is capable of interacting with analytes by stronger London forces, as well as by dipoledipole and H-bonding. More nonpolar analytes interact less strongly with the polar silica gel and more strongly with the less polar mobile phase and move higher up the TLC plate.

THIN LAYER CHROMATOGRAPHY


Once the solvent is within ~1-2 cm of the top of the TLC sheet, the TLC is removed from the developing chamber and the farthest extent of the solvent (the solvent front) is marked with a pencil. The solvent is allowed to evaporate from the TLC sheet in the hood. The spots are visualized using a UV lamp.
A fluorescent compound, usually Manganeseactivated Zinc Silicate, is added to the adsorbent that allows the visualization of spots under a blacklight (UV254). The adsorbent layer will fluoresce light green by itself, but spots of analyte quench this fluorescence and appear as a dark spot.
http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html

THIN LAYER CHROMATOGRAPHY - Visualization


As the chemicals being separated may be colorless, several methods exist to visualize the spots:
Visualization of spots under a UV254 lamp. The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence. Iodine vapors are a general unspecific color. Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate. Once visible, the Rf value of each spot can be determined

Chromatogram of 10 essential oils, Stained with vanillin reagent.

THIN LAYER CHROMATOGRAPHY


Calculation of Rfs
Rf (A) =
Solvent Front

2.0 cm 5.0 cm

= 0.40

Rf (B) = 3.0 cm
Distance solvent migrated = 5.0 cm 4.0 cm Distance A migrated = 3.0 cm

5.0 cm

= 0.60

Rf (C) = 0.8 cm = 0.16


5.0 cm

Distance B migrated = 2.0 cm

3.0 cm

Rf (D) = 4.0 cm = 0.80


5.0 cm

Distance C migrated = 0.8 cm


Origen

0.8 cm

x A

x x x x B U C D

Rf (U1) = 3.0 cm = 0.60


5.0 cm

Rf (U2) =

0.8 cm 5.0 cm

= 0.16

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)

THIN LAYER CHROMATOGRAPHY


Calculation of Rfs
Rf (A) =
Solvent Front

2.0 cm 5.0 cm

= 0.40

Rf (B) = 3.0 cm = 0.60


Distance solvent migrated = 5.0 cm 4.0 cm Distance A migrated = 3.0 cm

5.0 cm

Rf (C) = 0.8 cm = 0.16


5.0 cm

Distance B migrated = 2.0 cm

3.0 cm

Rf (D) = 4.0 cm = 0.80


5.0 cm

Distance C migrated = 0.8 cm


Origen

0.8 cm

x A

x x x x B U C D

Rf (U1) = 3.0 cm = 0.60


5.0 cm

Rf (U2) =

0.8 cm 5.0 cm

= 0.16

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)

THIN LAYER CHROMATOGRAPHY Rfs

Rf values can be used to aid in the identification of a substance by comparison to standards. The Rf value is not a physical constant, and comparison should be made only between spots on the same sheet, run at the same time.

Two substances that have the same Rf value may be identical; those with different Rf values are not identical.

THIN LAYER CHROMATOGRAPHY Rfs 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 (I 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 is very low; the elution strength of ethyl acetate is higher; the elution strength of ethanol is even higher; I = 0.01. I = 0.45 I = 0.68

Solvent Properties and Elution Strengths


Solvent MF MW C6H14 86.17 C7H8 92.13 C4H10O 74.12 Bp ( C) Density (g/mL) 68.7 0.659 110.6 0.867 34.6 0.713
o

Hazards*

Dipole

Hexane CH3(CH2)4CH3 Toluene C6H5CH3 Diethyl ether CH3CH2OCH2CH3 Dichloromethane CH2Cl2 Ethyl Acetate CH3CO2CH2CH3 Acetone CH3COCH3 Butanone CH3CH2COCH3 1-Butanol CH3CH2CH2CH2OH Propanol CH3CH2CH2OH Ethanol CH3CH2OH Methanol CH3OH Water HOH

Flammable Toxic Flammable Toxic Flammable Toxic, CNS Depressant Toxic, Irritant Cancer suspect Flammable Irritant Flammable Irritant Flammable Irritant Flammable Irritant Flammable Irritant Flammable Irritant Flammable Toxic

0.08

Elution Stength (I) 0.01

0.31

0.22

1.15

0.29

CH2Cl2 84.94 C4H8O2 88.10 C3H6O 58.08 C4H8O 72.10 C4H10O 74.12 C3H8O 60.09 C2H6O 46.07 CH4O 32.04 H2O 18.02

39.8 1.326 77.1 0.901 56.3 0.790 80.1 0.805 117.7 0.810 82.3 0.785 78.5 0.789 64.7 0.791 100.0 0.998

1.14

0.32

1.88

0.45

2.69

0.43

2.76

0.39

1.75

0.47

1.66

0.63

1.70

0.68

1.7

0.73

1.87

>1

Elution Strength of Mixed Solvents


The elution strength of the mixture is assumed to be the weighted average of the elution strengths of the components: Ionet = IoA (mole % A) + IoB (mole % B) mole % A = (moles A) / (moles A + moles B)

where:

Thus, to determine the Ionet of a solvent mixture, the molar ratio of the solvents must first be calculated. For example, the Ionet of a solvent mixture prepared from 1.0 mL of ethyl acetate plus 9.0 mL of hexanes is calculated as shown below:
Ionet = IoEtOAc [(moles EtOAc)/(moles EtOAc+moles hexane)] + Iohexane [(moles hexane)/(moles EtOAc+moles hexane)] where: moles EtOAc = [(volume EtOAc) (density EtOAc)] / [molecular weight of EtOAc] thus: Ionet = {0.45[(1.0mLEtOAc)(0.902g/mL)/(88.11g/mole)]+0.01[(9.0mLhexane)(0.659g/mL)/86.18g/mole)]} {(1.0 mLEtOAc)(0.902g/mL)/88.11g/mole) + (9.0 mLhexane)(0.659g/mL)/86.18g/mole)} and Ionet = 0.067

Resolution
The separation between two analytes on a chromatogram can be expressed as the resolution, Rs and can be determined using the following equation: Rs = (distance between center of spots) (average diameter of spots) In TLC, if the Rs value is greater than 1.0, the analytes are considered to be resolved.
x x

Improving Resolution:
For two closely migrating components, optimum resolutions are usually obtained when the Rfs of both compounds are between 0.2 and 0.5 * To Improve Rs, change the elution strength of the solvent to optimize Rfs change Ionet (= in capacity factor), all compounds will be effected similarly. Alter the composition of the solvent system so that the components affinity for the mobile phase vs. the solid phase are differentially changed (= change in selectivity).
Changing the chemical nature of the solvent system, such as changing a hydrogen bonding solvent to a solvent which cannot hydrogen bond to the analyte, is often the most effective.

** Improve Rs by decreasing the diameter of the analyte spots. This can be achieved by applying smaller and less concentrated spots.

http://orgchem.colorado.edu/hndbksupport/ TLC/TLCprocedure.html

Optimize Rfs

TLC Stationary Phases

www.vwr.com

www.vwr.com

PREPARATIVE TLC (PTLC)

TLC - Optimizing for column chromatography

Optimum: 0.2 < Rf < 0.5