KROMATOGRAFI LAPIS TIPIS (KLT)

THIN LAYER CHROMATOGRAPHY (TLC)

TLC
Thin layer chromatography (TLC) is an important technique for identification and separation of mixtures 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

Forensic Analysis using Thin Layer Chromatography

Ink analysis
Determines the specific chemicals Uses organic solvents Results are compared to a database of pen ink

Forensic Analysis using Thin Layer Chromatography

Dye analysis
Fibers Significant evidence Use thin layer chromatography to determine the different dyes in the fiber
See how the colors elute

Forensic Analysis using Thin Layer Chromatography

Pesticide analysis
Pesticides are a hazard to the environment Many deaths are the results of poisoning from pesticides Pesticides are classified by their use or chemical type Determination of organophosphorus compounds in pesticide

Forensic Analysis using Thin Layer Chromatography

Organic acid analysis
Separation of carboxylic acids Organic acids are in textile, food preservatives, and medical agents

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

TLC Procedures Plate preparation

Mix the absorbent, water and a binder such as calcium sulfate
Silica gel, paper and alumina

Spread a thin layer of absorbent on an unreactive hard surface

Glass, plastic, thick aluminum

Heat in oven at 110C for 30 mins to activate and dry the plate

TLC Procedure
Place a small amount of solvent in a beaker In pencil, draw a straight line across the plate about 1 cm from the end of the plate Place a drop of sample solution on the line

TLC procedure
Add filter paper Place in solvent

Sealed container

How TLC works

Sample solution is dissolved by solvent The solution sample will travel at different distances based on solubility, polarization, size Silica gel
Polar substances do not move far Non polar substances move farther up the plate
HO R O Si O R R OH Si O R OH Si O R

Calibration/Standards TLC
No calibration Standards
Compare to other known substances Rf value

Solvents
Choose a solvent depending on the polarity of the compound
Least Polar
Petroleum ether
Cyclohexane Toluene Chloroform Acctone

More polar

Ethanol Methanol

Solvents
The solvent can be a mixture of compounds but the polar solvent properties will over take the non-polar one.
10-30% Methly tert-butyl ether, MTBE, in hexane, C6H14, works well 10-30% Methylene chloride, CH2Cl2, in hexane, C6H14, for a less polar mixture 10-30% Acetone, CH3COCH3, in Methylene chloride, CH2Cl2, for a more polar mixture

Trial and error is the best way to approach which solvent to use.

Visualization
Destructive visualization
Spray plate with H2SO4, and then bake in the oven at 110C for 15-20 minutes. Compound is destroyed but all spots will be visible

Nondestructive visualization because of the use of a UV light the sample will not be destroyed. Although, not all of the spots on the plate will be visible.
Long wave UV Short wave UV Semi-destructive visualization

Visualization
A plate under a UV light to display the compounds after they were developed

Interpretation

Distance that the Spot Traveled R f Value Solvent Front Distance

Distance that theSpot Traveled R f Value Solvent Front Distance

Calculate Rf Value

Rf Value
The Rf value needs to be between 0.0 and 1.0
If the value is over 1.0 or less than 0.0, the calculation is wrong (you goofed)

If the Rf value is greater than 0.8 or lower than 0.2 the values are hard to interpret, thus creating a larger error The best Rf values are 0.3 to 0.6

Rf Value
The Rf value is not informative What affects the Rf value?
Temperature Solvent Thickness and amount of spot Other compounds

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
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 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 (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 is very low; the elution strength of ethyl acetate is higher; the elution strength of ethanol is even higher; e = 0.01. e = 0.45 e = 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 (e) 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: eonet = eoA (mole % A) + eoB (mole % B) mole % A = (moles A) / (moles A + moles B)

where:

Thus, to determine the eonet of a solvent mixture, the molar ratio of the solvents must first be calculated. For example, the eonet of a solvent mixture prepared from 1.0 mL of ethyl acetate plus 9.0 mL of hexanes is calculated as shown below:
eonet = eoEtOAc [(moles EtOAc)/(moles EtOAc+moles hexane)] + eohexane [(moles hexane)/(moles EtOAc+moles hexane)] where: moles EtOAc = [(volume EtOAc) (density EtOAc)] / [molecular weight of EtOAc]

thus: eonet = {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 eonet = 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 eonet (= 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

Pros for TLC

Sensitivity Speed Inexpensive

Cons for TLC

Too little of sample Too much of sample Subjective

KLT 2 DIMENSI

TLC Stationary Phases

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PREPARATIVE TLC (PTLC)

DIKEROK DILARUTKAN DIUAPKAN DIDAPAT ZAT MURNI

TLC - Optimizing for column chromatography

Optimum: 0.2 < Rf < 0.5