Thin Layer Chromatography (TLC)

By: AMIYA KUMAR GHOSHB.pharm,7th sem,4th yearRoll No. : 27701912002

Registration No. : 122770210002 OF 2012-2013

NSHM Knowledge Campus-Kolkata, Group of Institutions

Chromatography:

Chromatography is the physical separation of a mixture into its individual components.

We can use chromatography to separate the components of inks and dyes, such as those found in pens, markers, clothing, and even candy shells. Chromatography can also be used to separate the colored pigments in plants or used to determine the chemical composition of many substances.

Gas ChromatographyUsed to determine the chemical composition of unknown

substances, such as the different compounds in gasoline shown by each separate peak in the graph below.

Paper ChromatographyCan be used to separate the components of inks, dyes,

plant compounds (chlorophyll), make-up, and many other substances

Liquid ChromatographyUsed to identify unknown plant pigments & other

compounds.

Thin-Layer ChromatographyUses thin plastic or glass trays to identify the

composition of pigments, chemicals, and other unknown substances.

Examples of Chromatography

TLC:• Thin layer chromatography (TLC) is an important technique for

identification and separation of mixtures of organic compounds.

• 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.

Why use TLC:• 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.

Advantages:

Easy to useCheapPossible multiple analysisPossible recovery of the productsNo sample preparation required2-dimensional analysis

• Drawbacks:

• Slow (typically 30-60 minutes)• Limited quality of the separation• Limited reproducibility• Evaporation of the mobile phase

(composition varies during the analysis)

Difference between TLC and HPTLC

Principle: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”.

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.

STATIONARY PHASE

• Silica is commonly used as stationary phase• The separation of sample mixture will be depend on the polarity of sample.• Some modified silica is also used in certain purposes.

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 GF254 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• The ability of mobile phase to move up is depend on the polarity itself• Volatile organic solvents is preferably used as as mobile phase.

SOLVENT POLARITY INDEX

Hexane 0

Butanol 3.9

Chloroform 4.1

Methanol 5.1

Ethanol 5.1

Acetonitrile 5.8

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

• 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

Materials :• TLC plate• ‘Developing container’ - chamber/ jar/ glass beaker• Pencil• Ruler• Capillary pipe• Solvents / mobile phase

- organic solvents• UV lamp

Procedure:• 1.Developing Container Preparation:

Solvent is transferred into the container with 0.5-1cm in dept from the bottom

2. TLC Plate Preparation

Commercialy obtained with 5cm x 20cm in size

Prepare your size when neccesary Line 1 cm from the bottom with a pencil

as a part should be spotted.

Image Notes1. These were made with lab grade silica gel, on glass slides, with plaster of paris as the binder2. These were made with silica gel from dessicator packets, prepared in the same way as above but with less suspension to work with (hence the gaps near theedge)

3.Spotting’ TLC platesMake sure that your sample is liquified already. stick it using capillary pipe & spott onto the line

4.‘Develop the plate’after spotting, put the plate inside the chamber in the

ascendant position Make sure that the depth of solvent doesn’t touch the

spotsLet it develop up to the 1cm from the top of plateAfter that, pull out the plate from the chamber and let

the solvent be vaporized

5. Detection of spots• The color samples are easy to be seen and no need to

use UV lamp to detect them

Chromatogram of 10 essential oils,Stained with vanillin reagent.

Detection :Compound class Derivatizing agent

General Iodine vapor

General Sulphuric acid (50%)

Acids Bromo cresol green

Aldehyde and ketone 2,4 dinitro phenyl hydrazine

Amines and amino acid ninhydrin

Alkaloids Mercuric nitrate

Barbiturate Diphenylcarbazone

Lipids Bromo thymol blue

Steroids Antimony trichloride

carbohydrate Aniline phthalate

Chromatogram of 10 essential oils,Stained with vanillin reagent.

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.

Visualization

Absorption of UV radiation is proportional to concentration

Quantification is possible

Rf value:

solvent front

component B

component A

origin

dSdB

dA

Rf of component A =

dA

dS

Rf of component B =

dB

dS

The Rf value is a decimal fraction, generally only reported to two decimal places

solvent

solutef d

dR

THIN LAYER CHROMATOGRAPHY – Rf’s

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.

Mobile Phase Mixture Composition Number

% Ethyl Acetate

% Hexane

Rf

Salicylic Acid

Rf

Acetylsalicylic Acid

1 100 0 0.530 0.545

2 90 10 0.620 0.600

3 70 20 0.255 0.291

4 50 30 0.945 0.291

5 30 50 0.360 0.280

6 20 70 0.270 0.250

7 10 90 0.182 0.164

8 0 100 0 0

Mobile Phase Mixture combinations for the separation of Salicylic Acid (1%) and Acetylsalicylic acid (1%)

Time (minutes) Salicylic acid Rf

Acetylsalicylic acid Rf

Reaction Mixture Rf Lower Spot

Reaction Mixture Rf Upper Spot

15 0.385 0.346 0.385 0.481

30 0.320 0.340 0.360 0.500

45 0.264 0.321 0.321 0.491

Retardation factor (Rf) values for TLC of salicylic acid, acetylsalicylic acid, and reaction mixture in a 50/50 ethyl acetate/hexane solvent system over time.

Some problem:a. The spot shape is too broad

- Diameter is supposed to be < 1-2mmb. 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

EXAMPLE :Mobile phase Stationary phase Herbs and Herbal Products20 volumes of toluene+ 45 volumesof ethyl acetate,+20 volumes of glacial acetic acid + 5volumes of formic acid

silica gel Amalaki

100 volumes of ethyl acetate+11 volumes of formic acid,+11 volumes of acetic acid + 25 volumes of water.

silica gel Amra

Glacial acetic acid. kieselguhr G. Arachis Oil

A mixture of 1 volume of hexane and 1 volumeof diethyl ether.

silica gel GF254 Artemisia

Toluene silica gel GF254 Clove Oil

A mixture of 90 volumes of toluene and10 volumes of ethyl acetate

silica gel G Eucalyptus Oil

Derivatizing agent

anisaldehyde sulphuric acid reagent

vanilin sulphuric acid reagent

starch solution

1 volume of sulphuricacid and 0.5 volume of anisaldehyde

anisaldehyde solution,

anisaldehyde solution

Reference:1. R.A.Day, Jr. A.L.Undewood(1987). Analisis Kualititatif. Edisi ke Empat,

ms: 474-529.2. David G. Watson(2005). Pharmaceutical analysis. Edisi ke-2, ms 315-3313. http//orgchem.colorado.edu/handbooksupport/TLC/

TLCprocedure.html4. Indian pharmacopeia, volume 3, 20075. Kapp, Khail. Chapter 7, Thin Layer Chromatography, March 2, 20106. Kaine, Mary Ann. Chapter 7, Thin Layer Chromatography, March 2,

2010. 7. Young, Wei. Chapter 7, Thin Layer Chromatography, March 2, 2010.

Thank you

Contact: amiyaghosh94@gmail.com

• Contact: amiyaghosh94@gmail.com