Planar Chromatography

Thin-Layer Chromatography
High-Performance Thin-Layer Chromatography
Over-Pressure Liquid Chromatography
TLC and HPTLC
Similarities

Adsorption Chromatography
Utilizes differences in the degree to which the components are adsorbed on to a solid support as a means of
separating them
The separation relies on the relative affinity of compounds towards both the phases
Types: Normal phase and Reverse phase

Retardation factor or Retention factor (Rf)

Rf= Distance of spot center from the start point
Distance of the solvent front from start point
Normal phase – Low Rf- polar compound
High Rf- less polar/non polar compound

Advantages
Fast and economical
Multiple samples can be analyzed simultaneously
Snapshot of all components present in the sample

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TLC and HPTLC
Sample application and Components

Sample Application Techniques

1. Contact spotting
2. Spray spotting
• Bands
• Rectangles
Sample Applicators
Nanomat
Linomat
Automatic TLC Sampler (ATS)
Components
1. Plate coater 2. Drying racks
3. Plate cutter 4. Immersion device
5. Plate heater 6. Sample applicators
7. Development Chamber 8. Derivatization process
9. Scanning
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TLC and HPTLC
Visualization and Detection

Visualization Detection and Scanning

• Densitometer-measures the degree of darkness (the
• UV-light-fluorescent-Spots of non fluorescent
optical density) of a photographic or
compounds can be seen fluorescent stationary phase
semitransparent material or of a reflecting surface
–silica GF
• It can performed in absorbance or fluorescence
• Iodine chamber-Unsaturated compounds
mode
• Chemical Stain/Derivatization agents: • For quantitative evaluation the measurement should
a) Aniline phthalate -reducing sugars
be performed at the individual absorption maximum
b) Bromocresol green-organic acids
of each compound
c) Dragendorff-alkaloids
• WinCATS offers the multi-wavelength option for
d) Ninhydrin-amino acids, amines
that purpose
e) Rhodamine B-lipids
• Converts spot or band to chromatogram

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 TLC and HPTLC
Sample Applicators
Manual sample application
For plates up to 20 x 20 cm
Nanomat 4 Application of spots by contact transfer
Disposable capillary pipette
Precise positioning to apply a defined pattern

Semi-automatic application
Application of spots or bands
Any plate format up to 20 x 20 cm
A special feature of ATS 4,and also the Linomat 5
Spray-on application
Linomat 5, is “over-spotting”: a sequential Data input through visionCATS software
application from different vials onto the same
position
This technique can be used in pre- Fully automated sample application
chromatographic derivatization or spiking Application of bands, spots or rectangles
Any plate format up to 20 x 20 cm
ATS 4 Spray-on application or by contact transfer
Software-controlled by visionCATS
Heated Spray Nozzle (option)

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TLC and HPTLC
Differences
Parameters HPTLC TLC
Type of plates Precoated Hand-made or precoated
Layer of Sorbent 100μm 250μm
Efficiency High Low
Particle size 5 μm-Smaller than TLC 20 μm Larger than HPTCL
Separations 3 - 5 cm 10 – 15 cm
Sample volume 0.1-1 μL 0.5-5 μL
Analysis Time Shorter Longer
Solid support Silica gel for normal phase and C8 , C18 for Silica gel , Alumina & Kieselguhr
reversed phase modes
Development chamber Require less amount of mobile phase More amount of mobile phase is required

Sample spotting Auto sampler/Semi-automatic Manual spotting

Scanning UV/ Visible/ Fluorescence scanner scans the Not possible
entire chromatogram qualitatively and
quantitatively and the scanner is an advanced
type of densitometer

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Over-Pressured Liquid Chromatography
A bridge between TLC and HPLC
OPLC: uses a planar two-dimensional (2-D) column format, is a
hybrid between conventional TLC and HPLC

TLC HPLC

OPLC

Modified TLC plate between metal Pressure applied to the layer and pumped-
or glass sheets flow chromatography system
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OPLC
Introduction

• It is an analytical and semi preparative technique developed by Tiyhak [Link] 1979

• OPLC instrument comprising of separation chamber and solvent delivery pump
• The basis of OPLC is similar to that of other chromatographic techniques in that a pump is used to force a liquid
mobile phase through a stationary phase, such as silica or a bonded-phase media
• The uniqueness of OPLC lies in its column housing structure that allows flat planar columns to be used in the same
way as cylindrical glass or stainless steel ones
• The structure of column consists in planar stationary phase layer on and aluminum glass plate as for TLC but is
manufactured to be used like cylindrical HPLC column
• The column is inserted into a cassette between support and teflon foil
• The assembly is inserted in separation chamber and submitted to pressure

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Instrumentation
OPLC

 In OPLC the vapor phase is completely eliminated,

the chromatographic plate being covered with an
elastic membrane under external pressure, and the
separation can thus be performed under controlled
conditions
 Advantage: OPLC enables the prediction of elution
time and the quality of separation of compounds to
be isolated using FC, LPLC, MPLC, or semi-
preparative HPLC techniques from data from
analytical OPLC separations
• Direct transfer of the mobile phase from OPLC to
different preparative column chromatographic
methods (FC, LPLC, MPLC, semi-preparative
HPLC) is also possible

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Instrumentation
OPLC Separation chamber

1 = support of the instrument

2 = chromato-plate
3 = inner part of the instrument
4 = spring
5 = cassette system for the chromatoplate between two teflon sheets
6 = teflon sheets
7 =mobile phase inlet
8 =mobile phase outlet
9 = hydraulic system
10 = polymer suspension for chromato-plate protection

(a) Schematic diagram of the commercially available cassette-type OPLC apparatus

(b) Prepared plate for OPLC separation

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Modes of injection and detection
OPLC-Online and Offline modes

• Offline describes the way sample is s applied to the

column and online describes the way compound are
detected after their separation
• In offline mode the column is handled outside the
development chamber for the application and the
detection of samples
• In online mode the column is first equilibrated
inside the chamber and samples are injected with
valve and detected by UV/ELSD
• It is possible to combine online and offline modes

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2D TLC
2-Dimention Thin-Layer Chromatography

2D-in-time 2D-in-space

• In this method the analyzed sample is spotted at the

corner of the stationary phase and developed in the first
dimension. Then it is carefully dried, rotated at 90◦ and
redeveloped in the second direction
Compounds are subjected to a Separation is performed
1D separation (normally on a over a two-dimensional • Reacting the separated solutes before re-chromatography
LC column) and then planar surface, e.g. a with the same/different solvent in 2nd direction
individual fractions from the chromatographic plate
first column undergo the (planar chromatography) • Use of bonded plates that separate according to different
analysis in a second 1D mechanisms depending on the solvent is advantage of
separation
2D TLC
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Application
TLC, HPTLC, OPLC

TLC HPTLC OPLC

• Reaction monitoring • Separation of • Determination of
• Purity and identification components obtained impurities
of given sample from natural products/ • Isolation of metabolites
complex matrices in biological fluids
• Quantification and • Separation of oligomers
identification of given of natural (e.g. peptides)
sample and synthetic polymers
(e.g. polystyrene)
• Determination of toxins
in foodstuffs

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References
1 Bryson, N. & Papillard, D. An introduction to OPLC operation and applications. LC GC North
America, 88-94 (2004).
2 Cieśla, Ł. & Waksmundzka-Hajnos, M. Two-dimensional thin-layer chromatography in the analysis
of secondary plant metabolites. Journal of Chromatography A 1216, 1035-1052 (2009).
3 Mincsovics, E. & Tyihák, E. Overpressured Layer Chromatography (OPLC)–A Flexible Tool of
Analysis and Isolation. Natural product communications 6, 1934578X1100600528 (2011).
4 Sherma, J. Planar chromatography. Analytical chemistry 72, 9-26 (2000).
5 Nyiredy, S. The bridge between TLC and HPLC: overpressured layer chromatography (OPLC). TrAC
Trends in Analytical Chemistry 20, 91-101 (2001).
6 [Link]
7 [Link]
8 [Link]

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 Thanks for
Listening with
great patience

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