HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY .pptx

CHROMATOGRAPHY – Group of techniques used for separation of complex mixture of compounds by their distribution between two phases.
THIN LAYER CHROMATOGRAPHY – Technique in which finely divided adsorbent spread over a glass plate act as stationary phase using liquid as mobile phase.
HPTLC – Sophisticated and automated form of TLC.
High Pressure Thin Layer Chromatography/ Flat – Bed Chromatography.
Chemically well defined mostly porous inorganic substances with high surface area.
Small particle size increases separation efficiency, resolution and decreases analysis time.
Precoated plates using small quantities of very high molecular weight polymer as binder.
Silica gel GF with less polar mobile phase.
Solvent or solvent mixture moving through stationary phase on the HPTLC plate during development.
Depends on the chemical properties of analytes and sorbent layers.
Mobile phase optimization is necessary for HPTLC.
Trough chambers are used in which smaller volume (10-15ml) is required.
Pre-washing is required because sorbents with large surface area absorb not only water vapours and other impurities but also volatile substances from atmosphere which often condense particularly after the packing has been opened and exposed to atmosphere for a long time.
Plates are activated in drying cup-board to remove the washing solvent and are placed in oven at 110-120˚C for 15min (polyester sheets) or 30min (glass plate / aluminium sheets) prior to the sample application
Activation at higher temperature should be avoided – very active layer or decompose sample
Freshly opened box do not require activation
Quantitative sample identification by the scanning densitometer is performed by recording spectra of the analyzed components.
It measures the amount of light transmitted through or reflected by separated compounds on a TLC plate, allowing for the determination of their concentrations
It measures visible, UV absorbance and fluorescence quenching

1. HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY PRESENTED BY NAHIDA P H II SEM M.PHARM (2024 BATCH) DEPARTMENT OF PHARMACEUTICAL CHEMISTRY ST. JAMES’ COLLEGE OF PHARMACEUTICAL SCIENCES

2. CONTENTS INTRODUCTION PRINCIPLE METHODODLOGY SOFTWARES ADVANTAGES AND DISADVANTAGES APPLICATION

3. INTRODUCTION CHROMATOGRAPHY – Group of techniques used for separation of complex mixture of compounds by their distribution between two phases. THIN LAYER CHROMATOGRAPHY – Technique in which finely divided adsorbent spread over a glass plate act as stationary phase using liquid as mobile phase. HPTLC – Sophisticated and automated form of TLC. High Pressure Thin Layer Chromatography/ Flat – Bed Chromatography.

4. FEATURES Simple, rapid and efficient tool in quantitative analysis Simultaneous processing of sample and standard Simple sample preparation No prior treatment for solvents like filtration and degassing Mobile phase consumption is extremely low Hyphenation – HPTLC-IR, HPTLC-MS Lower analysis time Low maintenance cost, etc.

5. FEATURES TLC HPTLC Technique Manual Instrumental Plates Handmade/ precoated Precoated Mean particle size 10-12 μ m 5-6 μ m Stationary phase Silica gel, Alumina, Kiesulguhr (diatomaceous earth) Wide choice – Silica gel for normal phase and C8, C18 for reversed phase modes Efficiency Less High due to small particle size Sample spotting Manual Auto-sampler Shape of sample Circular/ spot Rectangular/ band Separation 10-15cm 3-5cm Analysis time 20-200min 1-3min Development chamber More amount of mobile phase Less amount of mobile phase Spectrum analysis/ Scanning Not possible Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram qualitatively and quantitatively

6. ADSORPTION CAPILLARY ACTION AFFINITY PRINCIPLE

7. METHODOLOGY

8. STATIONARY PHASE Chemically well defined mostly porous inorganic substances with high surface area. Small particle size increases separation efficiency, resolution and decreases analysis time. Precoated plates using small quantities of very high molecular weight polymer as binder. Silica gel GF with less polar mobile phase.

9. MOBILE PHASE Solvent or solvent mixture moving through stationary phase on the HPTLC plate during development. Depends on the chemical properties of analytes and sorbent layer s . Mobile phase optimization is necessary for HPTLC . Trough chambers are used in which smaller volume (10-15ml) is required .

10. PRE WASHING Pre-washing is required because sorbents with large surface area absorb not only water vapours and other impurities but also volatile substances from atmosphere which often condense particularly after the packing has been opened and exposed to atmosphere for a long time.

11. ACTIVATION OF PLATES Plates are activated in drying cup-board to remove the washing solvent and are placed in oven at 110-120˚C for 15min (polyester sheets) or 30min (glass plate / aluminium sheets) prior to the sample application Activation at higher temperature should be avoided – very active layer or decompose sample Freshly opened box do not require activation

12. SAMPLE APPLICATION NANOMAT 4 LINOMAT 5 AUTOMATIC TLC SAMPLER 4

13. TYPES OF CHAMBERS Twin Trough Chamber Flat Bottom Chamber Horizontal Development Chamber Sandwich Chamber V-shaped Chamber Automatic Development Chamber PRE-CONDITIONING (Chamber Saturation) Chamber saturation influences separation

14. AUTOMATIC DEVELOPING CHAMBER HORIZONTAL DEVELOPING CHAMBER TWIN TROUGH CHAMBER

15. DEVELOPMENT METHODS Ascending method Descending method Horizontal method Radial method Gradient method, etc. DEVELOPMENT AND DRYING

16. DEVELOPMENT METHODS

17. DETECTION

18. DENSITOMETER Quantitative sample identification by the scanning densitometer is performed by recording spectra of the analyzed components. It measures the amount of light transmitted through or reflected by separated compounds on a TLC plate, allowing for the determination of their concentrations It measures visible, UV absorbance and fluorescence quenching TLC SCANNER 4

19. DETECTING AGENTS Iodine chamber Sulphuric acid spray reagent Ferric chloride Ninhydrin in acetone Dragendroff’s reagent 3,5-dinitro benzoic acid, etc. DERIVATIZATION Derivatization is a technique used to enhance the visibility and detectability of compounds on a thin layer chromatographic plate. It is essentially required for detection when individual compound does not respond to UV or does not have intrinsic fluorescence DERIVATIZER It is of two types: - Pre-chromatographic Post-chromatographic

20. DOCUMENTATION Documentation is important because labelling every single chromatogram can avoid mistake in respect of order of application Type of plate, chamber system, composition of mobile phase, running time and detection method should be recorded E-Merck has introduced HPTLC precoated plates with an imprinted identification codes A reprography illumination chamber is used for photo-documentation PHOTO-DOCUMENTATION SYSTEM

21. SOFTWARES winCATS visionCATS winCATS was CAMAG’s windows–based software for controlling instruments and analyzing chromatographic data, particularly in HPTLC and TLC applications, but it is now end-of-life and unsupported. It has been succeeded by the more user-friendly and feature-rich visionCATS software, which offers advanced data analysis, a modern user interface and features like client-server architecture and comprehensive reporting

22. APPLICATIONS HPTLC is one of the most widely applied methods for the analysis in pharmaceutical industries, clinical chemistry, forensic chemistry, biochemistry, cosmetology, food and drug analysis, environmental analysis and other areas. PHARMACEUTICALS Quality control – Verify the identity and purity of drug substances and formulation Active ingredient analysis – Quantification of active pharmaceutical ingredients in various dosage forms Fingerprinting – Generate unique fingerprints of herbal medicines, aiding in quality control and identification Detecting adulterants – Identify and quantify adulterants in drug formulations

23. APPLICATIONS FOOD ANALYSIS Food authentication – Detect adulterants in food products like honey, spices and edible oils Quantifying additives – Determine the levels of preservatives, colorants and other additives in food Analysis of natural products – Analyze natural sweeteners, like steviol glycosides, in food matrices FORENSIC SCIENCE Drug analysis – Identify and quantify illicit drugs, drug metabolites and related substances in forensic samples Analysis of ink and dyes – Analyze inks and dyes in documents and other evidences Detection of explosives – Identify explosive residues

24. APPLICATIONS ENVIRONMENTAL ANALYSIS Pollutant detection – Analysis of pollutants like pesticides and herbicides in environmental samples Monitoring contaminants – Monitor the presence and levels of various contaminants in water and soil BIOTECHNOLOGY AND BIOCHEMISTRY Biomolecule analysis – Analyze biomolecules like proteins, peptides, nucleic acids, lipids and carbohydrates Metabolite profiling – Analyze metabolic profiles in biological samples COSMETICS Ingredient analysis – Analyze the components of cosmetic products Quality control – Verify the quality and purity of cosmetic ingredients

25. RECENT ADVANCEMENTS Green High-Performance Thin-Layer Chromatography: A Step Towards Eco-Friendly Analysis – The integration of these green solvent-based HPTLC methods marks a significant step towards sustainable and environmentally conscious analytical practices. Versatile Platforms based on HPTLC: Multimodal and Green Solutions for Food and Herbal Quality Assurance – Integration with high-end techniques like Mass Spectrometry (MS) and Surface-Enhanced Raman Spectroscopy (SERS). Automation and artificial intelligence are improving data processing efficiency through techniques like Convolutional Neural Networks (CNNs) for automated spot recognition. Instrumentation and Future Prospects of HPTLC – A Review – The detection capabilities of HPTLC have evolved with the introduction of fluorescence quenching and digital imaging techniques, allowing for enhanced visualization of separated compounds. By employing different wavelengths of UV light, various compounds on the chromatographic plate can be identified based on their fluorescence properties.

26. REFERENCES ManMohan Srivastava. High-Performance Thin-Layer Chromatography (HPTLC). 2011. 25-45. Dharmamoorthy G, Saiteja K, Keerthana J, Dinesh Ajay L, Ganesh A, Balaji A. An Overview on High-Performance Thin-Layer Chromatography (HPTLC). Vol. 17, Asian Journal of Pharmaceutics. 2010. 1-15. Kumar Vishwakarma S. HPTLC (High Performance Thin Layer chromatography) ADVANCED SPECTRAL ANALYSIS (MPC201T) UNIT-IV_CHROMATOGRAPHY (GC-AAS). 1-10. Attimarad M, Mueen Ahmed KK, Aldhubaib BE, Harsha S. High-performance thin layer chromatography: A powerful analytical technique in pharmaceutical drug discovery. Pharmaceutical Methods. 2011 Apr;2(2):71–5. Mukherjee PK. High-Performance Thin-Layer Chromatography (HPTLC) for Analysis of Herbal Drugs. In: Quality Control and Evaluation of Herbal Drugs. Elsevier; 2019. 377–420.

27. REFERENCES Sethi PD. HPTLC: High-Performance Thin-Layer Chromatography. In Quantitative Analysis of Pharmaceutical Formulations 3 Volume set. CBS Publications. 2013. 124-140. Ristivojević P, Trifković J, Andrić F, Milojković-Opsenica D. Recent trends in image evaluation of HPTLC chromatograms. Journal of Liquid Chromatography & Related Technologies. 2020 Jun 14;43(9–10):291–9. Dhull P, Dunuweera S, Bietsch J, Bandu R, Wannere C, Achanta S, et al. Recent advances and application of liquid chromatography in pharmaceutical industry. Journal of Liquid Chromatography & Related Technologies. 2025 Jun 15;48(6–10):168–87. Singh S, Khan N, Sawant T, Raheja R. Green High-Performance Thin-Layer Chromatography: A Step Towards Eco-Friendly Analysis. Chromatographia . 2025 Apr 14;88(4):287–302.

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