CHM 312Fall 2008

CHROMATOGRAPHY

THIN LAYER CHROMATOGRAPHY(TLC)

•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

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 CU D

A B CU

filter paper

D

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.

THIN LAYER CHROMATOGRAPHY

The stationary phase is SiO2 and is very “polar”.

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 dipole-dipole 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.

http://www.instructables.com/id/EW1YDCYF4REC0IU/

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 Manganese-activated 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.

THIN LAYER CHROMATOGRAPHY

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 CHROMATOGRAPHYCalculation of Rf’s

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)

A B CU

x xx x

Solvent Front

Origen

Distance solvent migrated = 5.0 cm

Distance A migrated = 3.0 cm

Distance B migrated = 2.0 cm

Distance C migrated = 0.8 cm

0.8 cm

3.0 cm

Rf (A) =

Rf (B) =

Rf (C) =

Rf (U1) =

Rf (U2) =

2.0 cm5.0 cm

= 0.40

= 0.60

= 0.16

= 0.60

= 0.16

3.0 cm5.0 cm

0.8 cm5.0 cm

3.0 cm5.0 cm

0.8 cm5.0 cm

Dx

Rf (D) = = 0.804.0 cm5.0 cm

4.0 cm

THIN LAYER CHROMATOGRAPHYCalculation of Rf’s

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)

A B CU

x xx x

Solvent Front

Origen

Distance solvent migrated = 5.0 cm

Distance A migrated = 3.0 cm

Distance B migrated = 2.0 cm

Distance C migrated = 0.8 cm

0.8 cm

3.0 cm

Rf (A) =

Rf (B) =

Rf (C) =

Rf (U1) =

Rf (U2) =

2.0 cm5.0 cm

= 0.40

= 0.60

= 0.16

= 0.60

= 0.16

3.0 cm5.0 cm

0.8 cm5.0 cm

3.0 cm5.0 cm

0.8 cm5.0 cm

Dx

Rf (D) = = 0.804.0 cm5.0 cm

4.0 cm

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 – Rf’s

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)

THIN LAYER CHROMATOGRAPHY – Rf’s

Elution Strength of Mobile Phase (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; = 0.01. the elution strength of ethyl acetate is higher; = 0.45 the elution strength of ethanol is even higher; = 0.68

Solvent MF MW

Bp (oC) Density (g/mL)

Hazards* Dipole Elution Stength

() Hexane CH3(CH2)4CH3

C6H14 86.17

68.7 0.659

Flammable Toxic

0.08 0.01

Toluene C6H5CH3

C7H8

92.13 110.6 0.867

Flammable Toxic

0.31 0.22

Diethyl ether CH3CH2OCH2CH3

C4H10O 74.12

34.6 0.713

Flammable Toxic, CNS Depressant

1.15 0.29

Dichloromethane CH2Cl2

CH2Cl2 84.94

39.8 1.326

Toxic, Irritant Cancer suspect

1.14 0.32

Ethyl Acetate CH3CO2CH2CH3

C4H8O2 88.10

77.1 0.901

Flammable Irritant

1.88 0.45

Acetone CH3COCH3

C3H6O 58.08

56.3 0.790

Flammable Irritant

2.69 0.43

Butanone CH3CH2COCH3

C4H8O 72.10

80.1 0.805

Flammable Irritant

2.76 0.39

1-Butanol CH3CH2CH2CH2OH

C4H10O 74.12

117.7 0.810

Flammable Irritant

1.75 0.47

Propanol CH3CH2CH2OH

C3H8O 60.09

82.3 0.785

Flammable Irritant

1.66 0.63

Ethanol CH3CH2OH

C2H6O 46.07

78.5 0.789

Flammable Irritant

1.70 0.68

Methanol CH3OH

CH4O 32.04

64.7 0.791

Flammable Toxic

1.7 0.73

Water HOH

H2O 18.02

100.0 0.998

1.87 >1

Solvent Properties and Elution Strengths

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:

onet = o

A (mole % A) +oB (mole % B)

where: mole % A = (moles A) / (moles A + moles B)

Thus, to determine the onet of a solvent mixture, the molar ratio of the solvents must first

be calculated. For example, the onet of a solvent mixture prepared from 1.0 mL of ethyl

acetate plus 9.0 mL of hexanes is calculated as shown below:

onet = oEtOAc [(moles EtOAc)/(moles EtOAc+moles hexane)] +

ohexane [(moles hexane)/(moles EtOAc+moles hexane)] where: moles EtOAc = [(volume EtOAc) (density EtOAc)] / [molecular weight of EtOAc]

thus: onet = {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 onet = 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 Rf’s of both compounds are between 0.2 and 0.5

* To Improve Rs, change the elution strength of the solvent to optimize Rf’s

• change onet (= 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 Rf’s

TLC – Stationary Phases

www.vwr.com

www.vwr.com

PREPARATIVE TLC (PTLC)

TLC - Optimizing for column chromatography

Optimum: 0.2 < Rf < 0.5