Kromatografi Lapis Tipis

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


• Dye analysis– Fibers

• Significant evidence • Use thin layer

chromatography to determine the different dyes in the fiber

– See how the colors elute


• 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


• 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 CU D

A B CU

filter paper

D

TLC ProceduresPlate 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 110°C 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

RO

SiO

SiO

SiO

R

OH OH OH

R RR

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

• More polar

Petroleum ether

Cyclohexane

Toluene

Chloroform

Acctone

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

110ºC 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

V alu e R D is tan ceF ro n t S o lv en t

T rav e ledS p o t th a t th eD is tan cef

Value R DistanceFront Solvent

TraveledSpot that theDistancef

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 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

http://orgchem.colorado.edu/hndbksupport/

Pros for TLC

• Sensitivity • Speed• Inexpensive

Cons for TLC

• Too little of sample• Too much of sample• Subjective

KLT 2 DIMENSI

TLC – Stationary Phases

www.vwr.com

www.vwr.com

PREPARATIVE TLC (PTLC)

DIKEROK

DILARUTKAN

DIUAPKAN

DIDAPAT ZAT MURNI

TLC - Optimizing for column chromatography

Optimum: 0.2 < Rf < 0.5

Kromatografi Lapis Tipis

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