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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
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