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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 employ fluorescence as a method of detecting the spots of the separated components Kirchner in 1951 : the use of starch as a binder in the production of thin layer plates (5) and determined that necessary starch content could be reduced to between 2% and 5% and still mainyain suitable resolution Stahl in 1956 invented an automatic spreader for TLC plates and convinced Merk, a company that manufactured silica gel, to produce TLC plates commercially and make them generally available

WHAT IS 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,) and a solvent ( the mobile phase) which flows through the adsorbent.

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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.

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 Netral

Alumina Asam

Alumina Basa

Magnesia
The use of magnesia is relatively rare today the early days of TLC it was employed for the separation of the carotenoids and other plant materials. Magnesia is manufactured by heating magnesium hydroxide to about 350oC. If heated to 500oC or above, the retentive activity of magnesia starts to fall and at 1000oC it is rapidly transformed into inactive magnesium oxide. The chromatographic characteristics of magnesia are similar to those of silica gel but the material is basic in nature as opposed to silica gel which is acidic. The surface moieties of magnesia that are interactive are again hydroxyl groups consequently, the molecular forces involved in solute 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 particle size of about 5-40 mm. Kieselguhr has very large pores and, as a consequence, has a relatively small surface area (1-5 m2/g). In TLC, it is used largely as a support for liquid stationary phases and not as an adsorbent or stationary phase. The use of kieselguhr as a support in TLC is afforded by the separation of the phenols in tobacco smoke condensates. supports generally are not well-liked and adsorbent stationary phases such as silica and silica-based bonded 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 microcrystalline 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 stationary phases in TLC, (e.g. polyamide 6,6 (Nylon 6,6) polyhexamethylenediamine, polyamide 6, (Nylon 6), aminopolycapro-lactame and polyamide 11 (Nylon 11) and polyaminoundecanoic acid. Polyamides can be produced. by polycondensing a dicarboxylic acid with a diamine and they can be represented 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 Nylon 6,10 etc.). The polyamide material can exhibit dispersive interactions with its aliphatic chains, polar characteristics due to interactions with the CO and the amino groups and even offer ion exchange capabilities if appropriately buffered. Nevertheless, the polar characteristics of the polyamides are the most commonly exploited and such materials have been used successfully for the separation of phenols, sulfonic acids, anilines, carbohydrates and a number of substances of biotechnological interest (e.g. nucleotides and nucleosides).

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 Silica gel G

Description
Silica gel with average particle size 15m containing ca 13% calcium sulfate binding agent Silica gel G with fluorescence added

Application
Used in wide range pharmacopoeial test

Silica gel G254

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

Cellulose

Identification of Cellulose powder of less than 30m particle tetracyclines size.

Mobile Phase
SOLVENT Heksana Butanol Chloroform Methanol Ethanol Acetonitrile Air POLARITY INDEX 0 3.9 4.1 5.1 5.1 5.8 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
Solvent Front

Rf (A) =

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)

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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 () 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; = 0.01. = 0.45 = 0.68

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Solvent Properties and Elution Strengths

Solvent MF MW C6H14 86.17 C7H8 92.13 C4H10O 74.12 CH2Cl2 84.94 C4H8O2 88.10 C3H6O 58.08 C4H8O 72.10 C4H10O 74.12 C3H8O 60.09 C2H6O 46.07 CH4O 32.04 H2 O 18.02 Bp (oC) Density (g/mL) 68.7 0.659 110.6 0.867 34.6 0.713 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 Hazards* Dipole Elution Stength ( ) 0.01

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

0.31

0.22

1.15

0.29

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: onet = oA (mole % A) + oB (mole % B) mole % A = (moles A) / (moles A + moles B)

where:

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

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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 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 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.

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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 Rf value of each spot can be determined

Detection of UV spots
The Iodine Reagent- forms brown/ yellow complexes with organic compounds The Sulfuric Acid Spray Heatpermanent 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 Lightsome 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

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