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Chromatography

Thin Layer Chromatography

Kompetensi Yang diharapkan:

• Mengetahui prinsip dasar dari kromatografi

lapis tipis

• Mengetahui jenis-jenis fasa gerak dan fasa

diam dalam kromatografi lapis tipis

• Mengetahui cara mendeteksi spot dalam

kromatografi lapis tipis

• Mampu menganalisa secara kualitatif dan

kuantitatif kromatogram KLT

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Milestone of TLC

• Schraiber in 1939 : discovered TLC and first to employfluorescence as a method of detecting the spots of theseparated components

• Kirchner in 1951 : the use of starch as a binder in theproduction of thin layer plates (5) and determined thatnecessary starch content could be reduced to between2% and 5% and still mainyain suitable resolution

• Stahl in 1956 invented an automatic spreader for TLCplates and convinced Merk, a company thatmanufactured silica gel, to produce TLC platescommercially and make them generally available

•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,) and a

solvent ( the mobile phase) which flows through the adsorbent.

WHAT IS THIN LAYER CHROMATOGRAPHY

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- 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 acceptinginteractions 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/

Advantages of TLC

• Cheap

• Simple

• The developing can be monitored visually

• Able to use various chemical as a detector

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Stationary Phase

• Silika

• Alumina

• Magnesia

• Polyamides

• Selulose

• Kiesl guhr

Alumina

• Sifat kimia hampir mirip dengan silika, namun

tidak memberikan effisiensi plate sebagus silika

(pada umumnya)

• Alumina is prepared by heating aluminum

hydroxide precipitated from aluminum chloride

solution to moderate temperatures. Initially,

alkali metals are removed by washing with dilute

aqueous acids, then with water, then dried with

methanol and finally heated to about 800oC

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Ada 3 jenis alumina:

Alumina Asam

Alumina Basa

Alumina Netral

Magnesia

• The use of magnesia is relatively rare today

• the early days of TLC it was employed for the separation of thecarotenoids and other plant materials.

• Magnesia is manufactured by heating magnesium hydroxide toabout 350oC. If heated to 500oC or above, the retentive activity ofmagnesia starts to fall and at 1000oC it is rapidly transformed intoinactive magnesium oxide.

• The chromatographic characteristics of magnesia are similar tothose of silica gel but the material is basic in nature as opposed tosilica gel which is acidic.

• The surface moieties of magnesia that are interactive are againhydroxyl groups consequently, the molecular forces involved insolute retention are largely polar in nature.

• Magnesia interacts with solvents in much the same way as silica gel.

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Kieselguhr• Kieselguhr is a purified, thermally treated,

diatomaceous earth that has been ground to a particlesize of about 5-40 mm. Kieselguhr has very large poresand, as a consequence, has a relatively small surfacearea (1-5 m2/g).

• In TLC, it is used largely as a support for liquidstationary phases and not as an adsorbent orstationary phase.

• The use of kieselguhr as a support in TLC is afforded bythe separation of the phenols in tobacco smokecondensates.

• supports generally are not well-liked and adsorbentstationary phases such as silica and silica-basedbonded phases are far more popular.

cellulose

• There are two kinds of cellulose commonly used in TLC, native cellulose that has between 400 and 500 units per chain and micro-crystalline cellulose that is prepared by the partial hydrolysis of regenerated cellulose and has between 40 to 200 units per chain.

• Employing a polar solvent mixture (e.g. acetonitrile/water) the more polar component (water) is absorbed into the cellulose that functions as a liquid stationary phase. If the cellulose is acetylated or esterified, dispersive groups are introduced on to the cellulose support and more dispersive solvents can be absorbed that act as a stationary phase.

• Cellulose is not commonly employed in general TLC separations but has been used for the separation of a number of amino acid mixtures and, in the form of ion exchange cellulose, for the separation of inorganic ions.

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Polyamide

• There are a number of polyamides that have been employed as stationaryphases in TLC, (e.g. polyamide 6,6 (Nylon 6,6) polyhexamethylenediamine,polyamide 6, (Nylon 6), aminopolycapro-lactame and polyamide 11 (Nylon11) and polyaminoundecanoic acid. Polyamides can be produced. bypolycondensing a dicarboxylic acid with a diamine and they can berepresented by the following formula.

• -[NH-(CH2)6-NH-CO-(CH2)x-C)]n

• (if x = 4 then the product is Nylon 6,6, if x = 8 then the product is Nylon6,10 etc.).

• The polyamide material can exhibit dispersive interactions with itsaliphatic chains, polar characteristics due to interactions with the CO andthe amino groups and even offer ion exchange capabilities if appropriatelybuffered. Nevertheless, the polar characteristics of the polyamides are themost commonly exploited and such materials have been used successfullyfor the separation of phenols, sulfonic acids, anilines, carbohydrates and anumber of substances of biotechnological interest (e.g. nucleotides andnucleosides).

Stationary Phase

• A plate of TLC can be made from aluminium or glass which is coated by a solid matter as a stationary phase.

- The coated material has 0.1-0.3mm in thickness

-some of them has been added by fluorescent indicator that will make it florescence during the UV light exposure.

• Silica is commonly used as stationary phase• The separation of sample mixture will be depent on the polarity of sample.

• Some modified silica is also used in certain purposes.

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Stationery phase Description Application

Silica gel G Silica gel with average particle size 15µm containing ca 13% calcium sulfate binding agent

Used in wide range pharmacopoeial test

Silica gel G254 Silica gel G with fluorescence added

Same application with Silica gel G where visualization is to be carried out under UV light.

Cellulose Cellulose powder of less than 30µm particle size.

Identification of tetracyclines

Mobile Phase

SOLVENT POLARITY INDEX

Heksana 0

Butanol 3.9

Chloroform 4.1

Methanol 5.1

Ethanol 5.1

Acetonitrile 5.8

Air 9.0

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TLC Development Setup

TLC apparatus: Chamber

Normal Method of Thin

Layer Plate development

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Pre-equilibrium TLC plate

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Effect of saturation

Continuous Plate development

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Forced Flow

Two dimensional forced Flow

development TLC

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Rf

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 cm0.8 cm

3.0 cm

Rf (A) =

Rf (B) =

Rf (C) =

Rf (U1) =

Rf (U2) =

2.0 cm

5.0 cm= 0.40

= 0.60

= 0.16

= 0.60

= 0.16

3.0 cm

5.0 cm

0.8 cm

5.0 cm

3.0 cm

5.0 cm

0.8 cm

5.0 cm

D

x

Rf (D) = = 0.804.0 cm

5.0 cm

4.0 cm

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)

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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 (ε)

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; ε = 0.01.the elution strength of ethyl acetate is higher; ε = 0.45the elution strength of ethanol is even higher; ε = 0.68

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Solvent MF MW

Bp (oC)

Density (g/mL) Hazards* Dipole Elution

Stength

(εεεε)

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:

εonet = εo

A (mole % A) + εoB (mole % B)

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

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

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

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

εonet = εoEtOAc [(moles EtOAc)/(moles EtOAc+moles hexane)] +

εohexane [(moles hexane)/(moles EtOAc+moles hexane)]

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

thus: εonet =

{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 εonet = 0.067

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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 εonet (= 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.

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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 Rfvalue of each spot can be determined

Detection of UV spots

• The Iodine Reagent- forms brown/ yellow complexes with organic compounds

• The Sulfuric Acid Spray Heat—permanent charred spots are produced

• Chromic-Sulfuric Acid Spray

• Fluorescence- compounds fluoresce when placed under UV light

• Silver Nitrate Spray (for Alkyl Halides)—dark spots form upon exposure to light

• Ultraviolet Light—some organic compounds illuminate or fluoresce under short-wave UV light

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Densitometric TLC

Single and Double Beam Densitometric Scanner

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Problems dalam KLT

a. The spot shape is too broad

- Diameter is supposed to be < 1-2mm

b. The movement of solvent

- should be straight up

- unproportionality in stationary phase surface will inhibit the movement of solvent

c. streaking formation

- caused by too concentrated sample