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(In the name of GOD)
HIGH PERFORMANCE THIN
LAYER
CHROMATOGARPHY(HPTLC)
1 Dr. A.R.Bekhradnia
HIGH
PERFORMANCE THIN LAYER
CHROMATOGARPHY
(HPTLC)
2 Dr. A.R.Bekhradnia
THIN LAYER CHROMATOGRAPHY (TLC)
3 Dr. A.R.Bekhradnia
Dr. A.R.Bekhradnia 4
Chromatography is a physical process of
separation in which the components to be
separated are distributed between 2 immiscible
phases a stationary phase which has a large
surface area and mobile phase which is in
constant motion through the stationary phase.
THIN LAYER CHROMATOGRAPHY (TLC)
5 Dr. A.R.Bekhradnia
• Mikhail Tsvet
• Born 14 May 1872
• Asti, Italy
• Died 26 June 1919 (age 47)
• Nationality Russia
• Fields botany
• Mikhail Semyonovich Tsvet (Михаи́л Семёнович Цвет, also spelled Tsvett, Tswett, Tswet, Zwet, and Cvet) (1872–1919) was a Russian-Italian botanist who invented adsorption chromatography. 6 Dr. A.R.Bekhradnia
LAAQ-B-LC001B 7
Invention of Chromatography by M. Tswett
Ether
CaCO3
Chlorophyll
Chromatography
Colors
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LAAQ-B-LC001B 8
Comparing Chromatography to the Flow of a River...
Base
Water flow Light leaf
Heavy stone
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LAAQ-B-LC001B 9
Chromato-graphy / -graph / -gram / -grapher
Chromatography: Analytical technique Chromatograph: Instrument Chromatogram: Obtained “picture” Chromatographer: Person
Dr. A.R.Bekhradnia
LAAQ-B-LC001B 10
Three States of Matter and Chromatography Types
Mobile phase
Gas Liquid Solid
Stationary phase
Gas
Liquid
Solid
Gas chromatography
Liquid chromatography
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LAAQ-B-LC001B 11
Liquid Chromatography
Chromatography in which the mobile phase is a liquid. The liquid used as the mobile phase is
called the “eluent”. The stationary phase is usually a solid or a
liquid. In general, it is possible to analyze any
substance that can be stably dissolved in the mobile phase.
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LAAQ-B-LC001B 12
Interaction Between Solutes, Stationary Phase, and Mobile Phase
Differences in the interactions between the solutes and stationary and mobile phases enable separation.
Solute
Stationary phase Mobile phase
Degree of adsorption, solubility, ionicity, etc.
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LAAQ-B-LC001B
Classification
According to the force of separation: Adsorption chromatography Partition chromatography Ion exchange chromatography Gel filtration chromatography Affinity chromatography
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LAAQ-B-LC001B 14
Column Chromatography and Planar Chromatography
Separation column
Packing material
Column Chromatography
Paper or a substrate coated
with particles
Paper Chromatography Thin Layer Chromatography (TLC)
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O
utpu
t co
ncen
tratio
n
Time
Chromatogram
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tR
t0
Inte
nsity
of d
etec
tor s
igna
l
Time
Peak tR : Retention time
h
A
t0 : Non-retention time
A : Peak area h : Peak height
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LAAQ-B-LC001B 17
Separation Process and Chromatogram for Column Chromatography
Out
put
conc
entra
tion
Time
Chromatogram
Dr. A.R.Bekhradnia
Once the solvent is within ~1-2 cm of the top of
the TLC sheet, the TLC is removed from the
developing chamber and the farthest extent of
the solvent (the solvent front) is marked with a
pencil.
The solvent is allowed to evaporate from the
TLC sheet in the hood.
The spots are visualized using a UV lamp.
A fluorescent compound, usually Manganese-
activated Zinc Silicate, is added to the adsorbent
that allows the visualization of spots under a
blacklight (UV254). The adsorbent layer will
fluoresce light green by itself, but spots of analyte
quench this fluorescence and appear as a dark spot.
THIN LAYER CHROMATOGRAPHY
http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html
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THIN LAYER CHROMATOGRAPHY - 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
Chromatogram of 10 essential oils, Stained with vanillin reagent.
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THIN LAYER CHROMATOGRAPHY Calculation 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 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
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THIN LAYER CHROMATOGRAPHY Calculation 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 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
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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
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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 = 0.45 the 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
(ε)
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
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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: εo
net = εoA (mole % A) + εo
B (mole % B) where: mole % A = (moles A) / (moles A + moles B) Thus, to determine the εo
net of a solvent mixture, the molar ratio of the solvents must first be calculated. For example, the εo
net of a solvent mixture prepared from 1.0 mL of ethyl acetate plus 9.0 mL of hexanes is calculated as shown below: εo
net = 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: εo
net = {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 εo
net = 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
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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, 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
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HIGH
PERFORMANCE THIN LAYER
CHROMATOGARPHY
(HPTLC)
Dr. A.R.Bekhradnia 28
Dr. A.R.Bekhradnia 29
Introduction of HPTLC
• HPTLC is the improved method of TLC which utilizes the
conventional technique of TLC in more optimized way.
• HPTLC takes place in highspeed capillary flow range of the mobile phase.
• There are three main steps HPTLC procedure, they are
1] Sample preparation, volume precision and exact position are achieved by use of suitable instrument.
2] Solvent (mobile phase) migrates the planned distance in layer (stationary phase) by capillary action. In this process sample separated into it’s components.
3] Separation tracks are scanned in densitometer with light
beams in visible or uv region
Dr. A.R.Bekhradnia 30
Steps Involving in HPTLC
Sample Preparation Selection of
chromatography layer
Pre-washing
Pre-conditioning
Application of sample
Chromatography development
Detection of spots
Scanning & documentation
Dr. A.R.Bekhradnia 31
Sample preparation
• Normal phase chromatography: non polar solvent
• Reversed phase chromatography: polar solvent
Selection of chromatography layer
Depends on nature of material to be separated Commonly used(silica gel, alumina)
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Pre-washing
• It is purification step
• Mainly methanol is used
• Essential for quantitative evaluation
Linomat lV applicator
Dr. A.R.Bekhradnia 33
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Selection of HPTLC plates
• Previously hand made plates is used in TLC for both
qualitative and quantitative work. Certain drawbacks with
that is nonuniform layer, formation of thick layer, paved for
advent of precoated plates.
• Nowadays precoated plates are available in different format
and thickness by various manufactures. Precaoted plates can
be used for both qualitative and quantitative work in
HPTLC, they are
• GLASS PLATES
• POLY ESTER/POLYETHYLYNE
• ALUMINIUM PLATES
Dr. A.R.Bekhradnia 36
Glass Plates: Offers superior flat and smooth surface.
- fragile
- high weight
- higher production cost
Polyester/polyethylene plates:
Thickness of plate is 0.2mm.
- It can be produced in roll forms.
- Unbreakable.
- Less packing material is required.
- Development of plate cann’t be above temperature
1200 c loses its shape.
Aluminium plates: Thickness of plate is 0.1mm.
- It can be produced in roll forms.
- Unbreakable.
- Less packaging material is required.
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SORBENTS USED IN HPTLC PLATES:
sorbents which are used in convential TLC are also used in HPTLC with or without modification.
- silica gel 65F
- highly purified silicagel 60
- aluminium oxide
- cellulose microcrystalline
- silica gel
- reversed stationary phase
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The layer thickness in HPTLC is around 100-
200cm,in conventional it is 250mm.
Layer prewashing: Ascending method
- Dipping method
- Continuous method
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The plates are activated by placing in an oven at 1101200 C for 30 min, this step will removes water that has been physically absorbed on surface at solvent layer.
Freshly opened box of HPTLC plates usually does not require activation.
Activation at higher temp and for longer time is avoided which leads to very active layer and there is risk of sample being decomposed
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- Methanol (commonly used)
- Chloroform:methanol:ammonia(90:10:1)
- Chloroform:methanol(1:1)
- Methylene chloride:methanol(1:1)
- Ammonia(1%)solution
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Usual concentration range is 0.1-1µg / µl,above
this causes poor separation.
Linomat IV (automatic applicator) - nitrogen gas
sprays sample and standard from syringe on TLC
plates as bands.
Band wise application - better separation - high
response to densitometer.
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Processes in the Developing Chamber The «classical» way of developing a chromatogram is to place the plate in a chamber, which contains a sufficient amount of developing solvent. The lower end of the plate should be immersed several millimeters. Driven by capillary action the developing solvent moves up the layer until the desired running distance is reached and chromatography is stopped. The following considerations primarily concern silica gel as stationary phase and developments, which can be described as adsorption chromatography.
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Provided the chamber is closed, four partially competing processes occur: Between the components of the developing solvent and their vapor, an equilibrium will be established eventually (1). This equilibrium is called chamber saturation. Depending on the vapor pressure of the individual components the composition of the gas phase can differ significantly from that of the developing solvent. While still dry, the stationary phase adsorbs molecules from the gas phase. This process, adsorptive saturation, is also approaching an equilibrium in which the polar components will be withdrawn from the gas phase and loaded onto the surface of the stationary phase (2). Simultaneously the part of the layer which is already wetted with mobile phase interacts with the gas phase. Thereby especially the less polar components of the liquid are released into in the gas phase (3). Unlike (1) this process is not as much governed by vapor pressure as by adsorption forces. During migration, the components of the mobile phase can be separated by the stationary phase under certain conditions, causing the formation of secondary fronts.
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• Also called Chamber Saturation
• Low polarity mob. Phase:- no need
• High polar mob. Phase:- desirable
• For reverse phase saturate chamber with polar solvent
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LOW SOLVENT
CONSUMPTION
pre-equilibration
with
solvent vapor
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Start of
development
CAMAG Twin Trough Chambers offer several ways to improve the results of TLC/HPTLC developing techniques. It allows low solvent consumption, reproducible pre-equilibration with solvent vapor, equilibration performed with any liquid and for any period of time, and development is started only when developing solvent is introduced into the trough with the plate. Twin Trough Chambers are available with stainless steel lid or as a Light-Weight Twin Trough Chamber made from highly transparent sheet glass with a glass lid.
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After development, remove the plate and
mobile phase is removed from the plate - to avoid
contamination of lab atmosphere.
Dry in vacuum desiccator - avoid hair
drier because essential oil components may
evaporate.
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Detection under UV light is first choice - non destructive.
Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length).
Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF.
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CATS STANDARD PROGRAM.
CATS PROGRAM OPTIONS
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Non UV absorbing compounds like
ethambutol, dicylomine etc - dipping the plates in
0.1% iodine solution.
When individual component does not
respond to UV - derivatisation required for
detection .
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HPTLC 100µm High due to smaller particle
size generated
3 - 5 cm
Shorter migration distance and the analysis time is greatly reduced
Wide choice of stationary phases like silica gel for normal phase and C8 , C18 for reversed phase modes
New type that require less amount of mobile phase
Auto sampler
Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram qualitatively and quantitatively and the scanner is an advanced type
of densitometer
TLC
250µm
Less
10 - 15 cm
Slower
Silica gel , Alumina
More amount
Manual spotting
Not possible
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Pharmaceutical Researches Biomedical Analysis Clinical Analysis Environmental Analysis Food Industry Therapeutic drug monitoring to determine concentration of drug and it’s metabolite in blood, urine etc Analysis of environmental pollutions levels Quantitative determination of prostaglandin’s and thromboxanes in plasma Analysis of nitrosoamines in food and body fluids Determination of sorbic acid in wine Characterization of hazards in industrial waste
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