This document summarizes high performance thin layer chromatography (HPTLC) and its advances. It discusses the principles, instrumentation, applications and comparison to traditional thin layer chromatography. The key steps in HPTLC are described including sample preparation, development, detection and documentation. Recent developments coupling HPTLC to mass spectrometry are also summarized, including elution-based and desorption-based techniques. HPTLC provides high resolution, sensitivity and reproducibility for analysis of compounds in various fields such as pharmaceuticals, cosmetics and forensics.

1. A seminar on
HIGH PERFORMANCE THIN LAYER
CHROMATOGRAPHY AND ITS ADVANCES
Presented by : Ms. Sonali R. Phadke
M.Pharm-III (QAT)
Roll No. 636
Guided by: Dr. R. P. Bhole
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2. Contents
• Introduction
• principle
• Comparison between HPTLC and TLC
• Features of HPTLC
• Steps involved in HPTLC
• Instrumentation
• Applicability
• Introduction to TLC-MS
• Other developing TLC-MS techniques
• Conclusion
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3. Principle
• It has same approach and physical principle of separation as TLC i.e.
adsorption.
• Mobile phase runs by capillary action.
• The separation of components is based on its affinity towards mobile phase
i.e. the one having more affinity towards stationary phase runs slowly than
the having less affinity, thus the separation occurs.
Typical HPTLC chromatogram
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4. Comparison of TLC and HPTLC
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5. Features of HPTLC:
 Simultaneous processing of sample and standard - better analytical precision and
accuracy.
 Several analysts work simultaneously.
 Lower analysis time and less cost per analysis.
 Low maintenance cost.
 Simple sample preparation.
 No prior treatment for solvents like filtration and degassing
 Low mobile phase consumption per sample
 No interference from previous analysis - fresh stationary and mobile phases for each
analysis - no contamination
 Visual detection possible - open system
 Non UV absorbing compounds detected by post-chromatographic derivatization
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6. Steps involved in HPTLC
Selection of chromatographic layer
Sample and standard preparation
Layer pre-washing
Layer pre-conditioning
Application of sample and standard
Chromatographic development
Detection of spots
Scanning
Documentation of chromatic plate 6

7. Instrumentation
Camag Sample semi automatic
applicator ( Linomat 5)
•Sample volume : 0.1-5 µl
Camag Nanomat 4
•Sample volume : 0.5, 1,2, 5 µl
Camag automatic TLC sampler 4
• Sample application is automatic only
changing syringe.
Camag automatic TLC
sampler 4
Camag Nanomat 4
Camag Linomat 5
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8. Camag
Development
twin trough
chamber
•It’s a development chamber
with the unique feature i. e.
twin trough.
•Available with different
dimensions, for
20 x20 cm
20x10 cm
10x10 cm plates
•Better pre-conditioning by
solvent vapours.

9. CAMAG Automatic
Developing Chamber ADC2
•ADC2 is the heart of HPTLC
system
•Fully automatically,
reproducible and dependent of
environmental effects
•Pre-conditioning chamber
saturation, developing solvent,
drying can be done & further
automatically monitored.
Camag ADC 2
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10. Camag TLC scanner and
software for documentation
•Camag TLC scanner 3 and
TLC scanner 4 are
sophisticated instruments for
densitometric evaluation of
TLC/HPTLC chromatograms.
•‘WINCATS’ is the software
used to operate the whole
HPTLC system.
•Camag TLC visualizer 2
Professional imaging and
documentation system for
TLC/HPTLC chromatograms
with a new digital CCD
camera, connected by USB
Camag TLC scanner 4
Camag TLC scanner
3
Camag TLC visualizer 2 10

11. Applications
• This technique is clearly the most important in providing the Sensitivity & speed
of analysis.
• Determination of active ingredients in herbal and pharmaceutical formulations.
• Cosmetic and environmental analysis
• Clinical applications i.e. lipids, metabolites studies
• Detection of document falsifications, Poisoning investigations, Dye stuff analysis.
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12. Introduction to TLC-MS
• Hyphenating TLC and mass spectrometry opens new application areas for Planar
Chromatography.
• The techniques for coupling TLC with mass spectrometry can be divided into elution-based, or
desorption-based.
• In elution based TLC-MS, The analyte on the TLC plate is first scraped, extracted, purified, and
concentrated, then transferred in the liquid phase to the mass spectrometer’s ion source for
further analysis.
• In desorption based TLC-MS, analyte is vaporized from the silica, and transferred to the mass
spectrometer in the gas phase.
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13. Camag TLC-MS interface 2
• Rapid and contamination-free elution of selected
zones
• Online transfer to the mass spectrometer
• Plug & play installation
• Compatible with any LC-MS system
• Confirmation of known substances within a minute
• Highly effective backwashing function prevents the
elution path from becoming blocked
• Easy handling ensures accurate and reproducible plate
positioning
• Low solvent consumption
Camag TLC-MS interface 2
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14. Characterization of separated compounds by mass spectrometry ( Steviol
Glycosides)
Chromatogram for
localizing the zones
(derivatized with ß-
naphthol reagent)
HPTLC plate after
elution of zones with
the TLC-MS
Interface
HPTLC-ESI-MS spectra of Rebaudioside A, m/z
989.6 [M+Na]+
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15. Other developing TLC-MS techniques
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16. Other developing TLC-MS techniques
1.TLC-MALDI-MS
• Bruker Daltonics introduced an adapter that allows
you to directly insert your TLC plate into a MALDI
instrument.
• The fully automated measurement process allows
an entire plate to be scanned and produces a visual
representation of separations.
• However, the data evaluation software enables so
called MALDI chromatograms that plot molecular
mass against TLC position, producing a two-
dimensional view; analytes that overlap on the TLC
plate are separated by mass and shown in a different
color [5,6]
MTP sized TLC-MALDI Adapter
Target
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17. TLC-MALDI-MS
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18. Other developing TLC-MS techniques
2.TLC-DART-MS and TLC-DESI-MS
• Like MALDI, direct analysis in real time
(DART) and desorption electro spray
ionization (DESI) are general surface analysis
techniques and therefore also lend themselves
to TLC analysis.
• The mode of operation at the surface of the
plate is somewhat similar, but in DART-MS a
gas stream is focused on the TLC plate; in
DESI, the stream hitting the surface is a
solvent mixture.
• The AccuTOF-DART 4G is a powerful
problem-solving tool for a wide range of
applications.
TLC-MS-DART
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19. Other developing TLC-MS techniques
3.Liquid Extraction Surface Analysis
• LESA™ technology was originally developed to
investigate tissue slices, but it can analyze almost any
surface with its nano-robotic ESI source – and that
includes TLC plates.
• The TriVersa® NanoMate (Advion) automatically
works its way across the plate, taking a fresh pipette
tip to analyze each “zone,” which practically
eliminates carry over.
• The robot picks up a pipette tip, draws extraction
solvent from a reservoir, moves to the zone of interest,
allows a small droplet of solvent to mix with the
sample spot for a preset time, and draws up the
mixture before nanospray injection into any high-end
MS system.
Advion TriVersa NanoMate 5
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20. Other developing TLC-MS techniques
4.Elution-based TLC-MS
• Morlock introduced the concept to CAMAG’s
Interface in the article noted earlier, and very
recently Advion released its Plate Express.
• Plate Express operates on a similar principal to
CAMAG’s Interface – both utilize a head that
seals to the plate with a cutting edge.
• A capillary channel introduces solvent to the
spot within the seal, and another channel
extracts the analytes and solvent for
introduction into a mass spectrometer.
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21. Comparison of the major TLC-MS coupling techniques
Technique Features
Elution-based TLC-
MS
Can be connected to any LC-MS system Most types of TLC
layers can be used Extraction into vials is also possible
TLC-MALDI-MS High degree of automation Scan mode (imaging) Large mass
range (up to 20 kDa) Requires 5 x 7.5 cm plates with
aluminum backing
Liquid Extraction
Surface Analysis
(LESA™)
High degree of automation High sensitivity with nanospray
source Requires hydrophobic TLC layer
TLC-DART-MS and
TLC-DESI-MS
Scan mode Fast measurements
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22. conclusion
• Coupling TLC plates with mass spectrometry (TLC-MS) is a new field of high interest in
planar chromatography.
• Excellent Efficiency and Resolution
Detection limit of HPTLC-MS-grade plates is in the lower nanogram range
Silica gel matrix of MS-grade plates has considerably fewer impurities than standard plates
Targeted recording of mass spectra on zones or lines of interest is performed after
development, thus ensuring high efficiency
Mass spectra are obtained quickly by direct sample access on TLC plates at room temperature
• Flexible Choice of Mobile Phase
In contrast, standard LC-MS coupling using HPLC is not compatible with certain mobile
phases (e.g. inorganic buffers)
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23. • Easy Method Transfer
Similar separation performance to standard TLC and HPTLC plates allows direct transfer
of methods
• High Sensitivity with Low Background
MS-grade plates are much cleaner than standard TLC or HPTLC plates, thus ensuring
superior sensitivity with extremely lower background signals
MS-grade plates are packed in aluminum foil to prevent contamination
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24. References
1. P. D. Sethi, Sethi’s HPTLC Quantitative Analysis of Pharmaceutical Formulations, vol-
1, first edition, pg no. 3-64.
2. CAMAG manual for Instrumental Thin Layer Chromatography.
3. https://theanalyticalscientist.com/issues/0715/state-of-the-art-tlc-ms/
4. http://www.merckmillipore.com/
5. B. Fuchs, J. Schiller, R. Süß, M. Schürenberg and D. Suckau 2007 A direct and simple
method of coupling matrix-assisted laser desorption and ionization time-of-flight mass
spectrometry (MALDI-TOF MS) to thin-layer chromatography (TLC) for the analysis of
phospholipids from egg yolk Anal. Bioanal. Chem.389:827–834.
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25. 6. B. Fuchs, J. Schiller, R. Süß, A. Nimptsch, M. Schürenberg and D. Suckau 2008
Capabilities and drawbacks of combined matrix-assisted laser desorption and ionization
time-of-flight mass spectrometry (MALDI-TOF MS) and high-performance thin-layer
chromatography (TLC): Analysis of egg yolk lipids JPC J. Planar Chromatogr. (in
press).
7. Bruker Daltonics Technical Note 18, A New Matrix Application Device for MALDI
Tissue Imaging 2008.
8. Katerina Matheis, Beate Fuchs, Katharina Lemmnitzer, Rosmarie Süß, Hans Griesinger,
Susanne Minarik, Michaela Oberle, Michael Schulz and Jurgen Schiller (2015)
Combining TLC Separation with MS Detection - A Revival of TLC: J Glycomics
Lipidomics,vol.5,issue 1,1-5.
9. http://www.clubdeccm.com/PDF/20081023/20081023_03bis.pdf
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26. THANK YOU
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