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A seminar on

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY AND ITS ADVANCES

Presented by : Ms. Sonali R. PhadkeM.Pharm-III (QAT)

Roll No. 636

Guided by: Dr. R. P. Bhole

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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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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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Comparison of TLC and HPTLC

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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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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

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InstrumentationCamag 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

Camag Development twin trough chamber

•It’s a development chamber with the unique feature i. e. twin trough.•Available with different dimensions, for20 x20 cm 20x10 cm10x10 cm plates•Better pre-conditioning by solvent vapours.

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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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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

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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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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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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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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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Other developing TLC-MS techniques

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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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TLC-MALDI-MS

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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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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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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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Comparison of the major TLC-MS coupling techniquesTechnique 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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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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• 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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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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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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THANK YOU