High-performance thin-layer chromatography (HPTLC) is an enhanced form of thin-layer chromatography that provides improved resolution, sensitivity, and quantification abilities compared to traditional TLC. HPTLC uses plates with smaller particle sizes coated in a thin, uniform layer for separations over short distances. Samples can be applied manually or automatically using an instrument, then separated based on compound affinities to the stationary and mobile phases. HPTLC is useful for both qualitative and quantitative analysis in applications like pharmaceutical analysis and impurity detection.
Gas chromatography (GC) is a technique used to separate volatile organic compounds. It consists of a carrier gas, an injection port, a separation column, an oven, and a detector. Samples are vaporized and injected into the column where components separate based on interactions with the stationary phase. Separated components exit the column and are detected, producing a chromatogram. Common applications of GC include environmental monitoring, refinery and chemical plant process control, and analysis of biological samples.
High Performance Thin Layer Chromatography.pptxKartik Tiwari
This document provides an overview of High Performance Thin Layer Chromatography (HPTLC). HPTLC is an automated, sophisticated version of Thin Layer Chromatography that allows for both qualitative and quantitative analysis. It works on the principle of adsorption chromatography, separating components based on their varying affinities to the stationary phase. Key steps involve preparing samples, applying them to a silica gel plate, developing the plate in a solvent mobile phase, detecting and visualizing separated components, and scanning/documenting results. HPTLC is useful for applications like pharmaceutical analysis, forensics, and clinical/biomedical research.
The slides are informative of HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY & its thorough components further its advantages and applications. The comparison of HPLC and HPTLC is explained.
The document discusses various techniques for sample handling and preparation in infrared spectroscopy. It describes the different methods used for sampling solids, liquids, and gases. For solids, the key techniques discussed are running solids in solution, forming solid films, the mull technique, and pressed pellet preparation using KBr. For liquids, cells made of materials like NaCl, KBr, and ThBr are used. Gas samples utilize larger cells of the same materials to compensate for lower molecule numbers. Proper sample preparation is necessary to obtain infrared spectra without interference from cell materials.
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)Abhishek Gupta
HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way
It is also known as planar chromatography or Flat-bed chromatography.
This document discusses various concepts related to high performance liquid chromatography (HPLC) peak analysis including:
1. It describes factors that influence peak shape such as column packing, mobile phase composition, pH, and buffers which can improve peak symmetry and resolution.
2. Key parameters for characterizing chromatographic performance are discussed including retention factor (k), selectivity factor (α), plate number (N), and height equivalent of a theoretical plate (HETP).
3. Optimizing these parameters through adjusting mobile phase or column properties can enhance separation and analysis of chromatographic runs.
Gas chromatography (GC) is a technique used to separate volatile organic compounds. It consists of a carrier gas, an injection port, a separation column, an oven, and a detector. Samples are vaporized and injected into the column where components separate based on interactions with the stationary phase. Separated components exit the column and are detected, producing a chromatogram. Common applications of GC include environmental monitoring, refinery and chemical plant process control, and analysis of biological samples.
High Performance Thin Layer Chromatography.pptxKartik Tiwari
This document provides an overview of High Performance Thin Layer Chromatography (HPTLC). HPTLC is an automated, sophisticated version of Thin Layer Chromatography that allows for both qualitative and quantitative analysis. It works on the principle of adsorption chromatography, separating components based on their varying affinities to the stationary phase. Key steps involve preparing samples, applying them to a silica gel plate, developing the plate in a solvent mobile phase, detecting and visualizing separated components, and scanning/documenting results. HPTLC is useful for applications like pharmaceutical analysis, forensics, and clinical/biomedical research.
The slides are informative of HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY & its thorough components further its advantages and applications. The comparison of HPLC and HPTLC is explained.
The document discusses various techniques for sample handling and preparation in infrared spectroscopy. It describes the different methods used for sampling solids, liquids, and gases. For solids, the key techniques discussed are running solids in solution, forming solid films, the mull technique, and pressed pellet preparation using KBr. For liquids, cells made of materials like NaCl, KBr, and ThBr are used. Gas samples utilize larger cells of the same materials to compensate for lower molecule numbers. Proper sample preparation is necessary to obtain infrared spectra without interference from cell materials.
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)Abhishek Gupta
HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way
It is also known as planar chromatography or Flat-bed chromatography.
This document discusses various concepts related to high performance liquid chromatography (HPLC) peak analysis including:
1. It describes factors that influence peak shape such as column packing, mobile phase composition, pH, and buffers which can improve peak symmetry and resolution.
2. Key parameters for characterizing chromatographic performance are discussed including retention factor (k), selectivity factor (α), plate number (N), and height equivalent of a theoretical plate (HETP).
3. Optimizing these parameters through adjusting mobile phase or column properties can enhance separation and analysis of chromatographic runs.
HPLC is a chromatographic technique used to separate components of a mixture. The key components of an HPLC instrument are the pump, injector, column, and detectors. The pump forces the mobile phase through the column while the injector introduces the sample. Various detectors can then analyze the separated components as they elute from the column. HPLC is widely used in fields like biochemistry and pharmaceutical analysis for applications such as quantifying drug purity and identifying unknown compounds.
High Performance Thin Layer Chromatography (HPTLC) instrumentationMadhuraNewrekar
HPTLC is an advancement of TLC. It is a high performance liquid chromatography with automation compared to Thin Layer Chromatography(TLC).Speed, Efficiency and Accuracy are important advantages. Evaluation time is less due to updated automation in instrumentation.
Steps involved in HPTLC and the materials and instruments required in those steps are described in brief.
The Nobel Prize in Chemistry 1952 was awarded jointly to Archer John Porter Martin and Richard Laurence Millington Synge for their invention of partition chromatography. They developed the plate theory of chromatography, which models a chromatographic column as being divided into theoretical plates with each plate representing equilibrium between the mobile and stationary phases. The number of theoretical plates is used to represent the efficiency and performance of the column.
High Performance Liquid Chromatography (HPLC) is a technique used to separate compounds dissolved in solution using high pressure to push a mobile phase through a column containing a stationary phase. HPLC instruments consist of pumps to deliver the mobile phase, an injector to introduce the sample, a separation column, and detectors. Compounds are separated based on differences in how they partition between the mobile and stationary phases. HPLC provides high resolution separation of complex mixtures and is characterized by its reproducibility, high pressure operation, and ability to use small particle sizes in the column.
Gas chromatography is a technique used to separate and analyze compounds that can be vaporized. It works by partitioning samples between a gas mobile phase and liquid stationary phase in a column. The separation depends on how the compounds partition between the phases. Key components of a GC system include the carrier gas, sample introduction system, column for separation, and detection system such as FID or TCD. Common applications include analysis of aromatics, hydrocarbons, flavors, and other volatile organic compounds.
Gas chromatography is a technique used to separate and analyze mixtures that can be vaporized without decomposition. It works by partitioning components to be separated between a stationary phase and a mobile gas phase. The key components of a gas chromatography instrument are the carrier gas, injection port, column, temperature control system, and detector. Factors like temperature, flow rate, column length, and amount of sample injected can influence separation of the components. Gas chromatography has applications in qualitative and quantitative analysis and is used in quality control of pharmaceuticals.
This document provides an introduction to gas chromatography including its components, advantages, and applications. It discusses the basic process of separating components using an inert gaseous mobile phase and immobilized liquid or solid stationary phase. Key components are described including the carrier gas, sample injection port, columns, and common detectors like FID and TCD. Applications include qualitative and quantitative analysis of compounds like oils, fatty acids, foods, drugs, and pollutants.
This document discusses various detectors used in gas chromatography including the flame ionization detector, thermal conductivity detector, flame photometric detector, photoionization detector, atomic emission detector, sulfur chemiluminescence detector, nitrogen chemiluminescence detector, and others. For each detector, it provides information on the basic principle of operation, components, applications, and limitations. The document focuses on explaining how each detector is able to detect and measure compounds eluting from the gas chromatography column based on their specific characteristics.
Gas chromatography and its instrumentationArgha Sen
This document provides an introduction to gas chromatography including a brief history and overview of the technique. It describes the basic components and instrumentation of a gas chromatography system including the carrier gas, sample injection systems, columns, temperature programming, and various detection systems. It also discusses different types of gas chromatography such as gas-solid, gas-liquid, and headspace GC. Finally, some common applications of gas chromatography are mentioned such as qualitative and quantitative analysis of compounds like fatty acids, foods, pollutants, and drugs.
This document outlines the five main steps for developing an analytical HPLC method: 1) selecting the initial HPLC method and conditions, 2) selecting the initial chromatographic conditions, 3) optimizing selectivity, 4) optimizing system parameters, and 5) validating the method. Key aspects of each step are discussed, including selecting the type of chromatography, column, detector, and mobile phase based on the analytes. The goal is to develop a validated method that provides adequate resolution and selectivity within a desired analysis time.
High performance thin layer chromatographySravani Ganti
This document provides an overview of high performance thin layer chromatography (HPTLC). It describes the advantages of HPTLC over HPLC and traditional TLC, such as its ability to process multiple samples simultaneously. The key steps of HPTLC are outlined, including plate selection, sample preparation, development, detection, and applications. HPTLC allows for enhanced separation resolution and automation compared to TLC. It is commonly used in pharmaceutical analysis and clinical testing due to its low cost, simplicity, and reproducibility.
a substance can absorb any visible light or external radiation and then again emit it. this called fluorescence and the process of reduction in fluorescence intensity is called quenching. this presentation is all about quenching of fluorescence.
Detectors in Gas Chromatography are devices used to detect and measure compounds eluting from the GC column. The document discusses several common detectors including:
- The Flame Ionization Detector (FID), one of the most widely used, responds to carbon-containing compounds. It is sensitive, destructive to samples, and provides a linear response.
- The Thermal Conductivity Detector (TCD) responds to differences in thermal conductivity between carrier gas and eluting compounds. It is non-destructive but has low sensitivity.
- Other detectors discussed are specific to certain functional groups like nitrogen/phosphorus (NPD), flame photometric (sulfur and phosphorus), electron capture (
HPTLC is an advanced form of thin layer chromatography with a thinner stationary phase layer and smaller particle sizes, allowing for faster separations, shorter migration distances, and quantitative analysis through scanning. Key differences between HPTLC and TLC include the thinner stationary phase, wider selection of phases, automated sample application, and use of scanning for quantitative and qualitative analysis. HPTLC provides advantages like simultaneous processing of samples and standards, lower analysis time and cost, simple sample preparation, and non-destructive detection methods.
This document provides an overview of gas chromatography (GC). It describes the basic components and principles of how GC works, including the carrier gas, injection port, separation column in an oven, and detector. It explains that GC separates volatile organic compounds based on how they partition between the mobile gas phase and stationary liquid phase. The document also outlines different types of columns, sample preparation procedures, common detectors like the flame ionization detector, and applications of GC in fields like environmental analysis and refineries.
This document provides an overview of gas chromatography. It discusses the history of gas chromatography, invented in 1901 by Mikhail Tswett. It then describes the basic principles, instrumentation, and applications of gas chromatography. The key components of a gas chromatograph are described, including the carrier gas, columns, injection port, temperature control, and detectors like the flame ionization detector and thermal conductivity detector. The document concludes by outlining some common applications of gas chromatography in fields like pharmaceuticals, petroleum, foods, and environmental analysis.
Gas chromatography is a technique used to separate components of a mixture based on how they partition between a mobile gas phase and a stationary liquid phase. It involves vaporizing the sample and injecting it into a column containing a stationary phase, where components are separated as they are transported through the column by the mobile gas phase at different rates depending on their partition coefficients. Common detectors used at the end of the column include the thermal conductivity detector, electron capture detector, and flame ionization detector, which produce signals proportional to the concentration of eluting components. Gas chromatography has many applications in fields like pharmaceutical analysis, food testing, and environmental analysis.
This document provides an overview of high performance thin layer chromatography (HPTLC). It discusses the principle, steps involved, applications, and references. HPTLC is an automated form of TLC that allows for the separation and analysis of multiple samples simultaneously. Key steps include selecting plates and mobile phases, applying samples, developing the plate, and detecting and visualizing results. HPTLC has various applications in pharmaceutical analysis, food testing, herbal product identification, and clinical and forensic analysis. It can be used to quantify compounds and perform multi-component analyses of drugs, food contaminants, and herbal constituents.
This document provides an overview of high performance thin layer chromatography (HPTLC). It describes the principles, methodology, and applications of HPTLC. Some key points include:
- HPTLC is an improved version of thin layer chromatography (TLC) that allows for more optimized separation of analytes.
- Samples are applied to HPTLC plates manually or automatically and developed in a chamber using a mobile phase solvent.
- Separated components are detected visually or using a densitometer, and may require derivatization for improved detection.
- HPTLC is used in pharmaceutical analysis, clinical testing, food testing, and other applications due to its high resolution, sensitivity, accuracy and reproducibility.
HPLC is a chromatographic technique used to separate components of a mixture. The key components of an HPLC instrument are the pump, injector, column, and detectors. The pump forces the mobile phase through the column while the injector introduces the sample. Various detectors can then analyze the separated components as they elute from the column. HPLC is widely used in fields like biochemistry and pharmaceutical analysis for applications such as quantifying drug purity and identifying unknown compounds.
High Performance Thin Layer Chromatography (HPTLC) instrumentationMadhuraNewrekar
HPTLC is an advancement of TLC. It is a high performance liquid chromatography with automation compared to Thin Layer Chromatography(TLC).Speed, Efficiency and Accuracy are important advantages. Evaluation time is less due to updated automation in instrumentation.
Steps involved in HPTLC and the materials and instruments required in those steps are described in brief.
The Nobel Prize in Chemistry 1952 was awarded jointly to Archer John Porter Martin and Richard Laurence Millington Synge for their invention of partition chromatography. They developed the plate theory of chromatography, which models a chromatographic column as being divided into theoretical plates with each plate representing equilibrium between the mobile and stationary phases. The number of theoretical plates is used to represent the efficiency and performance of the column.
High Performance Liquid Chromatography (HPLC) is a technique used to separate compounds dissolved in solution using high pressure to push a mobile phase through a column containing a stationary phase. HPLC instruments consist of pumps to deliver the mobile phase, an injector to introduce the sample, a separation column, and detectors. Compounds are separated based on differences in how they partition between the mobile and stationary phases. HPLC provides high resolution separation of complex mixtures and is characterized by its reproducibility, high pressure operation, and ability to use small particle sizes in the column.
Gas chromatography is a technique used to separate and analyze compounds that can be vaporized. It works by partitioning samples between a gas mobile phase and liquid stationary phase in a column. The separation depends on how the compounds partition between the phases. Key components of a GC system include the carrier gas, sample introduction system, column for separation, and detection system such as FID or TCD. Common applications include analysis of aromatics, hydrocarbons, flavors, and other volatile organic compounds.
Gas chromatography is a technique used to separate and analyze mixtures that can be vaporized without decomposition. It works by partitioning components to be separated between a stationary phase and a mobile gas phase. The key components of a gas chromatography instrument are the carrier gas, injection port, column, temperature control system, and detector. Factors like temperature, flow rate, column length, and amount of sample injected can influence separation of the components. Gas chromatography has applications in qualitative and quantitative analysis and is used in quality control of pharmaceuticals.
This document provides an introduction to gas chromatography including its components, advantages, and applications. It discusses the basic process of separating components using an inert gaseous mobile phase and immobilized liquid or solid stationary phase. Key components are described including the carrier gas, sample injection port, columns, and common detectors like FID and TCD. Applications include qualitative and quantitative analysis of compounds like oils, fatty acids, foods, drugs, and pollutants.
This document discusses various detectors used in gas chromatography including the flame ionization detector, thermal conductivity detector, flame photometric detector, photoionization detector, atomic emission detector, sulfur chemiluminescence detector, nitrogen chemiluminescence detector, and others. For each detector, it provides information on the basic principle of operation, components, applications, and limitations. The document focuses on explaining how each detector is able to detect and measure compounds eluting from the gas chromatography column based on their specific characteristics.
Gas chromatography and its instrumentationArgha Sen
This document provides an introduction to gas chromatography including a brief history and overview of the technique. It describes the basic components and instrumentation of a gas chromatography system including the carrier gas, sample injection systems, columns, temperature programming, and various detection systems. It also discusses different types of gas chromatography such as gas-solid, gas-liquid, and headspace GC. Finally, some common applications of gas chromatography are mentioned such as qualitative and quantitative analysis of compounds like fatty acids, foods, pollutants, and drugs.
This document outlines the five main steps for developing an analytical HPLC method: 1) selecting the initial HPLC method and conditions, 2) selecting the initial chromatographic conditions, 3) optimizing selectivity, 4) optimizing system parameters, and 5) validating the method. Key aspects of each step are discussed, including selecting the type of chromatography, column, detector, and mobile phase based on the analytes. The goal is to develop a validated method that provides adequate resolution and selectivity within a desired analysis time.
High performance thin layer chromatographySravani Ganti
This document provides an overview of high performance thin layer chromatography (HPTLC). It describes the advantages of HPTLC over HPLC and traditional TLC, such as its ability to process multiple samples simultaneously. The key steps of HPTLC are outlined, including plate selection, sample preparation, development, detection, and applications. HPTLC allows for enhanced separation resolution and automation compared to TLC. It is commonly used in pharmaceutical analysis and clinical testing due to its low cost, simplicity, and reproducibility.
a substance can absorb any visible light or external radiation and then again emit it. this called fluorescence and the process of reduction in fluorescence intensity is called quenching. this presentation is all about quenching of fluorescence.
Detectors in Gas Chromatography are devices used to detect and measure compounds eluting from the GC column. The document discusses several common detectors including:
- The Flame Ionization Detector (FID), one of the most widely used, responds to carbon-containing compounds. It is sensitive, destructive to samples, and provides a linear response.
- The Thermal Conductivity Detector (TCD) responds to differences in thermal conductivity between carrier gas and eluting compounds. It is non-destructive but has low sensitivity.
- Other detectors discussed are specific to certain functional groups like nitrogen/phosphorus (NPD), flame photometric (sulfur and phosphorus), electron capture (
HPTLC is an advanced form of thin layer chromatography with a thinner stationary phase layer and smaller particle sizes, allowing for faster separations, shorter migration distances, and quantitative analysis through scanning. Key differences between HPTLC and TLC include the thinner stationary phase, wider selection of phases, automated sample application, and use of scanning for quantitative and qualitative analysis. HPTLC provides advantages like simultaneous processing of samples and standards, lower analysis time and cost, simple sample preparation, and non-destructive detection methods.
This document provides an overview of gas chromatography (GC). It describes the basic components and principles of how GC works, including the carrier gas, injection port, separation column in an oven, and detector. It explains that GC separates volatile organic compounds based on how they partition between the mobile gas phase and stationary liquid phase. The document also outlines different types of columns, sample preparation procedures, common detectors like the flame ionization detector, and applications of GC in fields like environmental analysis and refineries.
This document provides an overview of gas chromatography. It discusses the history of gas chromatography, invented in 1901 by Mikhail Tswett. It then describes the basic principles, instrumentation, and applications of gas chromatography. The key components of a gas chromatograph are described, including the carrier gas, columns, injection port, temperature control, and detectors like the flame ionization detector and thermal conductivity detector. The document concludes by outlining some common applications of gas chromatography in fields like pharmaceuticals, petroleum, foods, and environmental analysis.
Gas chromatography is a technique used to separate components of a mixture based on how they partition between a mobile gas phase and a stationary liquid phase. It involves vaporizing the sample and injecting it into a column containing a stationary phase, where components are separated as they are transported through the column by the mobile gas phase at different rates depending on their partition coefficients. Common detectors used at the end of the column include the thermal conductivity detector, electron capture detector, and flame ionization detector, which produce signals proportional to the concentration of eluting components. Gas chromatography has many applications in fields like pharmaceutical analysis, food testing, and environmental analysis.
This document provides an overview of high performance thin layer chromatography (HPTLC). It discusses the principle, steps involved, applications, and references. HPTLC is an automated form of TLC that allows for the separation and analysis of multiple samples simultaneously. Key steps include selecting plates and mobile phases, applying samples, developing the plate, and detecting and visualizing results. HPTLC has various applications in pharmaceutical analysis, food testing, herbal product identification, and clinical and forensic analysis. It can be used to quantify compounds and perform multi-component analyses of drugs, food contaminants, and herbal constituents.
This document provides an overview of high performance thin layer chromatography (HPTLC). It describes the principles, methodology, and applications of HPTLC. Some key points include:
- HPTLC is an improved version of thin layer chromatography (TLC) that allows for more optimized separation of analytes.
- Samples are applied to HPTLC plates manually or automatically and developed in a chamber using a mobile phase solvent.
- Separated components are detected visually or using a densitometer, and may require derivatization for improved detection.
- HPTLC is used in pharmaceutical analysis, clinical testing, food testing, and other applications due to its high resolution, sensitivity, accuracy and reproducibility.
This document provides an overview of High Performance Thin Layer Chromatography (HPTLC). It discusses that HPTLC is an advanced form of TLC with greater separation efficiency and detection limits. The document then covers the basic principles of HPTLC, key features such as automated sample application and scanning, differences between TLC and HPTLC as well as HPLC, and the experimental procedures involved in HPTLC including sample preparation, development, detection, and derivatization.
HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way.
It is also known as planar chromatography or Flat-bed chromatography.
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.
This document provides information on high performance thin layer chromatography-mass spectrometry (HPTLC-MS). It begins with introducing HPTLC-MS, including its history and principles. It then discusses the steps to perform HPTLC-MS, including sample preparation, chromatography development, and various interface techniques to couple HPTLC with mass spectrometry. Finally, it provides examples of applications of HPTLC-MS, such as analysis of acetylcholinesterase inhibitors and Cyclanthera pedata. In summary, the document outlines the technique of HPTLC-MS, from its background and methodology to examples of its applications in chemical analysis.
High Performance Thin Layer Chromatography- Harsh Wardhan BilloreHarsh Billore
HPTLC is an improved form of TLC that provides higher resolution, shorter development times, and less solvent consumption compared to conventional TLC. It utilizes pre-coated plates with smaller adsorbent particles and pore sizes. HPTLC is useful for both qualitative and quantitative analysis in applications such as pharmaceutical quality control, food analysis, and clinical and forensic studies. The basic steps in HPTLC include sample preparation and application, chromatographic development and separation, and detection and visualization of spots.
High performance thin layer chromatography (HPTLC) is an advanced form of thin layer chromatography that provides high resolution separation and quantitative analysis of compounds. HPTLC uses plates coated with a thin and finely divided stationary phase allowing for shorter run times and better separation efficiency compared to traditional TLC. The document discusses the principles, instrumentation, applications, and factors affecting HPTLC analysis.
HPTLC is an improved version of TLC that provides better resolution and allows for quantitative analysis. It uses plates with finer silica gel particles between 5-7 micrometers compared to 10-25 micrometers for regular TLC. This allows for faster development times of 3-20 minutes for HPTLC versus 30-200 minutes for TLC. HPTLC also has automated instrumentation for precise sample application and development as well as densitometric scanning for quantification. It has various applications in pharmaceutical analysis, clinical analysis, food and environmental testing by providing fingerprints to identify compounds and allowing quantification of biomarkers.
This document provides an overview of high performance thin layer chromatography (HPTLC). It begins with an introduction to HPTLC, describing it as an automated and sophisticated form of thin layer chromatography. The document then covers the principles, instrumentation, differences from TLC, steps involved in HPTLC including sample preparation and detection, and applications such as qualitative and quantitative analysis, stability indicating assays, drug analysis in biological samples, and herbal drug analysis. Examples are given of HPTLC methods developed for analysis of various pharmaceutical drugs and determination of compounds in herbal formulations.
High Performance Thin Layer Chromatography (HPTLC) - Dr. P. Saranraj, Assistant Professor, Department of Microbiology, Sacred Heart College (Autonomous), Tirupattur, Vellore District, Tamil Nadu, India.
HPTLC, or high performance thin layer chromatography, is an enhanced form of TLC that allows for more accurate quantitative measurements and higher resolution separation of mixtures compared to traditional TLC. Key features of HPTLC include use of pre-coated plates with smaller particle sizes, semi-automated or automatic sample application, shorter development times, and scanning densitometry for quantification and fingerprint analysis. The process involves selection of plates and mobile phase, sample preparation and application, chromatographic development and detection, followed by scanning and documentation.
This document provides information on high-performance thin layer chromatography (HPTLC). It discusses the basic principles of TLC and how HPTLC enhances it with automation. The key steps of HPTLC are described, including sample preparation, application, development, detection, and documentation. Various instrumentation, plates, mobile phases, and detection methods used in HPTLC are also outlined. HPTLC is highlighted as a useful technique for pharmaceutical analysis, food testing, clinical applications, forensics, and industrial processes.
Plants contain thousands of compounds and are a valuable source of new drugs. High-performance thin-layer chromatography (HPTLC) is a simple and economical analytical method useful for characterizing herbal compounds. HPTLC provides better separation and repeatability compared to traditional thin-layer chromatography. It can be used to develop standardized herbal extracts, isolate pure compounds, and determine compound structures. HPTLC is also used to create chemical "fingerprints" of herbal products for quality evaluation and authentication. The HPTLC process involves selecting a stationary phase, applying samples, developing the plate, detecting and quantifying separated compounds, and documenting results.
HPTLC is a sophisticated form of thin layer chromatography that allows for efficient separation and analysis of samples in a short period of time. The key steps in HPTLC include sample preparation, selecting a chromatographic layer, applying the sample, developing the plate in a mobile phase, detecting spots on the plate, and scanning/documenting results. HPTLC offers advantages over other chromatography methods like simultaneous processing of samples and standards, lower analysis times, and less cost per analysis. It has applications in fields like pharmaceutical analysis, biochemistry, and pharmacokinetic studies.
This document discusses High Performance Thin Layer Chromatography (HPTLC). It begins by defining HPTLC and noting that it is a sophisticated, automated form of TLC. The key differences between TLC and HPTLC are described, including HPTLC using smaller layer thicknesses and particle sizes for higher efficiency. The principles, advantages, steps and applications of HPTLC are summarized. Specific examples discussed include the separation of analgesics and determination of caffeine content using HPTLC.
This document discusses High Performance Thin Layer Chromatography (HPTLC). It begins by defining HPTLC and noting that it is an automated form of TLC that uses instruments for application, development, documentation, and densitometry. The key differences between TLC and HPTLC are described, including HPTLC using smaller layer thicknesses and particle sizes for higher efficiency. The principles, advantages, steps, and applications of HPTLC are then outlined in detail. Specific examples provided include the separation of analgesics and determination of caffeine content using HPTLC.
High performance thin layer chromatography(HPTLC)GOPAL KUMBHANI
This document provides an overview of high performance thin layer chromatography (HPTLC). It begins by explaining that HPTLC is an enhanced version of thin layer chromatography (TLC) that allows for more accurate and higher resolution separations. The document then covers the basic principles, instrumentation, steps involved in HPTLC including sample preparation and development, and factors that can affect separations. Finally, some common applications of HPTLC are discussed such as use in pharmaceutical quality control and clinical analysis.
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High Performance Thin Layer Chromatography
1. High-Performance Thin-layer
Chromatography (HPTLC)
Dept. Of Pharmaceutical Sciences & Technology
BIRLA INSTITUTE OF TECHNOLOGY, MESRA
- Presented by-
- PROTTAY DUTTA(MPH/10055/20)
Facilitated to-
Dr. B.N.SINHA
Professor
Dept. Of Pharmaceutical Sciences & Technology
BIT Mesra, Ranchi
2. CONTENTS
• Introduction
• Principle
• Comparison Of TLC , HPLC, HPTLC
• Steps involved
• Instrumentation
• factors influencing separation and resolution of spots
• Applications
• References
3. INTRODUCTION
• High-performance thin-layer chromatography (HPTLC) is an enhanced form
of thin-layer chromatography (TLC).
• It is also known as planar or flat bed chromatography.
• HPTLC is a powerful analytical method equally suitable for qualitative and
quantitative analytical tasks.
4. HPTLC is popular for many reasons such as :-
visual chromatogram,
Multiple sample handling,
Enables the most complicated separation
Detection limit in nano gram range with UV-absorption detection and in picogram range with
fluorometric detection
Large number of theoretical plates in minimum area of plates.
Analysis time greatly reduced in HPTLC due to short migration distant.
Simplicity
high efficiency due to small particle size
Fast and inexpensive
5. Principle
• HPTLC have similar approach and employ the same physical principles of TLC
(Adsorption chromatography) i.e. the principle of separation is adsorption.
• The mobile phase solvent flows through because of capillary action.
• The components move according to their affinities towards the adsorbent.
• The component with more affinity towards the stationary phase travels slower,
component with lesser affinity towards the stationary phase travels faster.
6. Parameter for comparison TLC HPLC HPTLC
AVERAGE PARTICLE SIZE 19-15µm 3-5µm 5-7µm
PARTICLE SIZE DISTRIBUTION wide Normal Normal
LAYER THICKNESS 250µm Not applicable 100-200µm
NUMBER OF SAMPLES Max 12 One sample at a time 36-72 on a 20cm plate
SEPARATION DISTANCE 100-150µm Not applicable 30-70mm
RUNNING TIME 30-200 minutes 10-30 minutes or longer 3-10 minutes
SOLVENT CONSUMPTION 50ml 1ml/minute For a typical run time of
30minutes , 30ml of solvent is required.
5-10ml
DETECTION LIMIT (ABSORBANCE
MODE)
100-1000ng ng/ml to mg/ml 10-100ng
DETECTION LIMIT (FLOURESCENCE
MODE )
1-100ng ng/ml 0.1-10ng
7. Parameter for comparison TLC HPLC HPTLC
Usage - Column life of 1000-2000
samples
Single use plates
Sample application - Auto-sampler Manual/ Auto-sampler
Elution Technique - Isocratic / gradient Isocratic / gradient
Development - Single dimension only
possible
2 dimensional development
possible which can separate
a complex mixture .
Dimensions - Column length – 5-30cm
Diameter- 4.6mm
Plate size of
5x10cm,10x10cm &
20x20cm.
Separation Type - Normal / Reversed Phase Normal / Reversed Phase
Material - Silica gel / NH2
Diol / CN
Silica gel 60 F254 / NH2
Diol / CN , Cellulose
9. STATIONARY PHASE:
HPTLC can be regarded as the most advanced form of modern TLC.
It uses HPTLC plates featuring small particles with a narrow size distribution. As a result,
homogenous layers with a smooth surface can be obtained.
HPTLC uses smaller plates (10 × 10 or 10 × 20 cm) with significantly decreased
development distance (typically 6 cm) and analysis time (7–20 min).
HPTLC plates provide improved resolution, higher detection sensitivity, and improved in
situ quantification and are used for industrial pharmaceutical densitometric quantitative
analysis.
Normal phase adsorption TLC on silica gel with a less polar mobile phase, such as
chloroform– methanol, has been used for more than 90% of reported analysis of
pharmaceuticals and drugs.
Lipophilic C-18, C-8, C-2; phenyl chemically-modified silica gel phases; and hydrocarbon-
impregnated silica gel plates developed with a more polar aqueous mobile phase, such as
methanol–water or dioxane–water, are used for reversed-phase TLC.
10. Other precoated layers that are used include aluminum oxide, magnesium silicate,
magnesium oxide, polyamide, cellulose, kieselguhr, ion exchangers, and polar modified
silica gel layers that contain bonded amino, cyano, diol, and thiol groups.
Optical isomer separations that are carried out on a chiral layer produced from C-18
modified silica gel impregnated with a Cu (II) salt and an optically active enantiomerically
pure hydroxyproline derivative, on a silica layer impregnated with a chiral selector such as
brucine, on molecularly imprinted polymers of a-agonists, or on cellulose with mobile
phases having added chiral selectors such as cyclodextrins have been reported mostly for
amino acids and their derivatives.
Mixtures of sorbents have been used to prepare layers with special selectivity properties.
HPTLC plates need to be stored under appropriate conditions. Before use, plates should be
inspected under white and UV light to detect damage and impurities in the adsorbent.
It is advisable to prewash the plates to improve the reproducibility and robustness of the
results.
12. MOBILE PHASE:
The selection of mobile phase is based on adsorbent material used as stationary phase and
physical and chemical properties of analyte.
General mobile-phase systems that are used based on their diverse selectivity properties
are diethyl ether, methylene chloride, and chloroform combined individually or together
with hexane as the strength-adjusting solvent for normal-phase TLC and methanol,
acetonitrile, and tetrahydrofuran mixed with water for strength adjustment in reversed-
phase TLC.
Accurate volumetric measurements of the components of the mobile phase must be
performed separately and precisely in adequate volumetric glassware and shaken to ensure
proper mixing of the content.
Volumes smaller than 1 ml are measured with a suitable micropipette. Volumes up to 20 ml
are measured with a graduated volumetric pipette of suitable size. Volumes larger than 20
ml are measured with a graduated cylinder of appropriate size. To minimize volume errors,
developing solvents are prepared in a volume that is sufficient for one working day.
14. LAYER PREWASHING:
Plates are generally handled only at the upper edge to avoid contamination. Usually
plates are used without pretreatment unless chromatography produces impurity fronts
due to contamination of the plate.
For reproducibility studies and quantitative analysis, layers are often prewashed using
20 ml methanol (generally, methanol is used as a prewashing solvent; however, a
mixture of methanol and ethyl acetate or even mobile phase of the method may also be
used) per trough in a 20 × 10 cm twin-trough chamber (TTC).
Up to two 20 × 10 cm or four 10 × 10 cm plates can be developed back-to-back in each
trough of the TTC.
Remove the plate and dry it for 20 min in a clean drying oven at 120°C. Equilibrate
plate with laboratory atmosphere (temperature, relative humidity) in a suitable container
providing protection from dust and fumes.
15. PREPARATION OF PLATE:
Precoated layers: TLC plates can be made in any lab with suitable apparatus. However
such layers do not adhere well to the glass support.
Precoated plates that use small quantities of very high molecular weight polymer as binder
overcomes most limitations of a home-made layer.
Precoated layers are reasonably abrasion resistant, very uniform in layer thickness,
reproducible, preactivated, and ready to use. They are available with glass or aluminum or
polyester support.
Aluminum foil plates are less expensive to buy, cheaper, can be cut, and therefore easy to
carry around or transport or mail.
Glass plates are the best for highest quality of results. Most often, layers containing a
fluorescent indicator F 254 are used. This enables the visualization of samples in a UV
cabinet very simply, instantly, and in a nondestructive manner.
Commonly used size of plates in TLC is 20×20 cm and in HPTLC 20 × 10 cm or 10 × 10
cm is widespread.
16. SAMPLE PREPARATION AND APPLICATION:
Sample preparation
• It’s important to prepare proper sample for successful separation.
• Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution
at starting zones.
• It needs a high concentrated solution, as very less amount of sample need to be applied.
Sample application
• In Thin-Layer Chromatography manual sample application with capillaries is usually performed for simple
analyses.
• Sample volumes of 0.5 to 5 μL can be applied as spots onto conventional layers without intermediate
drying. HPTLC layers take up to 1 μL per spot.
• More demanding qualitative, quantitative, and preparative analyses or separations are made possible only
by instruments for band wise application of samples using the spray-on technique. Particularly HPTLC
takes full advantage of the gain in separation power and reproducibility available by precise positioning and
volume dosage.
17. AUTOMATIC TLC SAMPLER
Automatic sample application is a key factor for productivity of
the HPTLC laboratory.
The requirements for an instrument serving this purpose, i.e.
precision, robustness during routine use and convenient
handling are fully met by the Automatic TLC Sampler 4.
The ATS 4 offers fully automatic sample application for
qualitative and quantitative analyses as well as for preparative
separations.
It is best suited for routine use and high sample throughput in mass analysis. Samples are
either applied as spots through contact transfer (0.1–5 μl)or as bands or rectangles (0.5 to >
50 μl) using the spray-on technique.
Starting zones sprayed on as narrow bands offer the best separation attainable with a given
chromatographic system.
18. Key features:
• Fully automatic sample application, suitable for routine.
• Application of spots, bands, or rectangles.
• Data input and monitoring through WINCATS.
• Application of solutions onto any planar medium.
• Application of sample volumes between 0.1 and 5 μl by contact transfer.
• spray-on application of sample volumes between 0.5 and > 50 μl.
Application in the form of rectangles allows precise application of large volumes without
damaging the layer. Prior to chromatography, these rectangles are focused into narrow
bands with a solvent of high elution strength. The ATS 4 allows “over spotting”, i.e. a
sequential application from differentials onto the same position. This technique can be used
e.g. in pre chromatographic derivatization, spiking, etc.
19. DEVELOPMENT OF CHROMATOGRAM:
Thin-layer chromatography differs from all other chromatographic techniques in the fact
that in addition to stationary and mobile phases, a gas phase is present. This gas phase can
significantly influence the result of the separation.
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.
20. Provided the chamber is closed, four partially competing processes occur:
1. Between the components of the developing solvent and their vapor, an
equilibrium will be established eventually . 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.
2 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 .
3 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 . Unlike this process is not as much
governed by vapor pressure as by adsorption forces.
4 During migration, the components of the mobile phase can be separated by the
stationary phase under certain conditions, causing the formation of secondary
fronts.
21. DERIVATIZATION:
Post chromatographic Derivatization
It is an inherent advantage of Thin-Layer Chromatography that fractions remain stored on the plate and can
be derivatized after chromatography. By derivatization substances that do not respond to visible or UV light
can be rendered detectable. In many cases, substances or classes of substances can be identified by specific
reagents.
1. Changing non-absorbing substances into detectable derivatives
2. Improving the detectability (lowering detection limits)
3. Detecting all sample components
4. Selectively detecting certain substances
5. Inducing fluorescence
QUANTIFICATION
Most modern HPTLC quantitative analysis are performed in situ by measuring the zones of samples and
standards using a chromatogram spectrophotometer usually called a densitometer or scanner with a fixed
sample light beam in the form of a rectangular slit.
Generally, quantitative evaluation is performed with the TLC Scanner 3 using WINCATS software. It can
scan the chromatogram in reflectance or in transmittance mode by absorbance or by fluorescent mode;
scanning speed is selectable up to 100 mm/s.
22. DOCUMENTATION
Each developed plate is documented using digital documentation system under UV light at 254 nm, UV light at 366
nm, and white light. If a type of light does not produce usable information, that fact must be documented. If a plate is
derivatized, images are taken prior and after derivatization.
Factors affecting HPTLC
• Types of stationary phase.
• Mobile phase
• Layer thickness
• Temperature
• Mode of development
• Amount of sample
• Dipping zone
23. Isolation of Drug from excipients
We can understand this by taking an example –
Determination of Paracetamol, Pseudoephedrine and Loratidine in Tablets.
A sensitive, accurate and selective high performance thin layer chromatography (HPTLC)
method was developed and validated for the simultaneous determination of paracetamol
(PAR), its toxic impurity 4-aminophenol (4-AP), pseudoephedrine HCl (PSH) and
loratidine (LOR).
After the completion of the process we obtain the following graphs :-
24. HPTLC chromatogram of (A) blank plasma at 254 nm, (B) blank plasma at 208 nm, (C) a mixture of 4 μg/band PAR, 0.8 μg/band LOR and
2 μg/band pseudoephedrine in spiked human plasma at 254 nm and (D) a mixture of 4 μg/band PAR, 0.8 μg/band LOR and 2 μg/band
pseudoephedrine in spiked human plasma at 208 nm
25. 1. Pharmaceutical applications
Quality control
Content Uniformity Test (CUT)
Identity- and purity checks
Stability tests, etc.
3. Cosmetics
Identity of raw material
Preservatives, colouring materials, etc.
Screening for illegal substances, etc.
6. Industrial applications
Process development and optimization
Process monitoring
Cleaning validation, etc.
2. Clinical applications
Lipids
Metabolism studies
Drug screening
Doping control, etc.
4. Herbal medicines and botanical dietary supplements
Identification
Stability tests
Detection of adulteration
Assay of marker compounds, etc.
5.Food and feed stuff
Quality control
Additives (e.g. vitamins)
Pesticides
Stability tests (expiration), etc.
7. Forensics
Detection of document forgery
Investigation of poisoning
Dyestuff analyses, etc.
Applications of HPTLC
26. • Foods usually originate as botanical products and therefore are naturally variable as well as complex.
HPTLC can confirm identities of complex mixtures as well as detect adulteration.
• HPTLC is the first method of analysis because it is simple, risk free, fast, economical and analyses 100-120
samples a day, without producing much waste.
HPTLC-FOOD ANALYSIS
27. HPTLC- HERBAL APPLICATIONS
• HPTLC Fingerprint is a technique adopted by the US & European Pharmacopoeias recently for the purpose
of identification of “botanical materials”, all of which are very complex in nature.
• HPTLC Fingerprint is the representation of the phytochemical composition of a plant extract or
formulation, in the form of a conventional image i.e. a photograph.
• Fingerprint can also be used to monitor batch to batch consistency and stability studies of herbal medicines,
dietary supplements etc.
HPTLC-FORENSIC ANALYSIS
• Forensic analysis is the multi-disciplinary application of scientific knowledge and sophisticated
instruments for investigating crime related materials and biological samples.
• A frequent but challenging aspect of forensic toxicology is the identification of unknown poisonous
substances in lethal intoxication cases.
• HPTLC offers identification as well as qualitative and quantitative analysis for toxic substances, CAMAG
HPTLC offers rapid identification of such toxins for antidote administration.
28. • High Performance Thin Layer Chromatography (HPTLC) is a valuable tool to check purity, impurity of any
non-volatile organic industrial materials such as dyes, surfactants, pesticides, perfumery compounds,
intermediates etc. It is far simpler, cheaper and easier to understand than other similar methods of analysis.
Chemical reactions can be studied very quickly e.g. within 2 hours.
• Complex mixtures like biological samples, reaction mixtures, fermentation broth can be easily
chromatographed without much sample preparation.
HPTLC- DYES AND INTERMEDIATES ANALYSIS
• Dyes and intermediates are non-volatile organic substances and so best suited for HPTLC analysis. HPTLC
analysis is very low cost and results are “visible”. Colored substances are therefore particularly ideal for
HPTLC analysis.
• HPTLC enables comparison of different samples or with standard, analysis of competition samples apart
from purity and impurity determination. All kinds of optical brightner, intermediates, dyes, banned amines
can be analyzed by HPTLC.
HPTLC-SPECIALITY CHEMICALS ANALYSIS
29. HPTLC-BIOTECHNOLOGY APPLICATIONS
• HPTLC is a most versatile analytical technique which offers a great separation power using precise sample
application, software-controlled chromatographic steps, chromatogram development and scanning and photo
documentation.
• Separated samples can be visually checked on the plate is an unique aspect of HPTLC. Biotechnology
industry is considered as one of the most research-intensive sectors in the world. Therefore, shorter analysis
time, low sample analysis cost per sample, minimal contamination possibilities and reliable accurate results
are essential which are provided by HPTLC.
• HPTLC can analyse different samples simultaneously with zero risk of cross contamination.
• HPTLC also offers the advantage of evaluating a plate by any specific process using different detection
modes (UV, fluorescence, etc.) By coupling HPTLC with a MS or other suitable methods such as NMR,
FTIR, ESI, MALDI, one can identify and or confirm the chemical structures of analytes under study.
30. REFERENCES :-
CAMAG, 2010-2011. Instrumental thin layer chromatography. Switzerland: Camag.
Available from: camag.com/downloads/free/brochures/CAMAG_TLC10-11_E.pdf
Patel, R.B. and Patel, M.R. and Patel, B.G. (2011) Experimental Aspects and
Implementation of HPTLC. In: Shrivastava, M.M. HPTLC. New York: Springer, pp. 41- 54.
https://academic.oup.com/chromsci/article/54/4/647/2754774
https://www.pharmatutor.org/articles/high-performance-thin-layer-chromatography-
hptlc-instrumentation-overview
A Textbook of Pharmaceutical Analysis By Dr. S . Ravi Shankar