logging in or signing up HpTLC power point hakeemkhan Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 3241 Category: Science & Tech.. License: All Rights Reserved Like it (4) Dislike it (1) Added: November 10, 2010 This Presentation is Public Favorites: 1 Presentation Description No description available. Comments Posting comment... By: hakeemkhan (7 month(s) ago) Warm Greetings: Dear Dhammika as u required the other presentations. Can u mension the specific topics so i can guide u properly. Thanks for the complement..... Saving..... Post Reply Close Saving..... Edit Comment Close By: s.dhammika (7 month(s) ago) superb Saving..... Post Reply Close Saving..... Edit Comment Close Premium member Presentation Transcript Slide 1: (HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY) Presented by: ABDUL RAZZAQ M.Pharma in Pharmaceutical Chemistry LUQMAN COLLEGE OF PHARMACY GULBARGA HPTLC Definition: : Definition: 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. Introduction: : Introduction: 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. Differences between TLC and HPTLC: : Differences between TLC and HPTLC: Slide 5: SAMPLE AND STANDARD PREPARATION SELECTION OF CHROMATOGRAPHIC PLATES LAYER PRE-WASHING LAYER PRE-CONDITIONING APPLICATION OF SAMPLE CHROMATOGRAPIC DEVELOPMENT DETECTION OF SPOTS SCANNING AND DOCUMENTATION OF CHROMOPLATE USING PC CATS SOFTWARE Slide 6: Selection of HPTLC plates Hand plates were available which are made up of cellulose and other materials which are not used much now-a –days. Slide 7: Pre coated plates: The plates with different support materials and sorbent layers with different format and thickness are used. Plates with sorbent thickness of 100-250μm are used for qualitative and quantitative analysis. Supports : Supports Some of the sorbents used in HPTLC: : Some of the sorbents used in HPTLC: Slide 10: Some of the binders used: Gypsum (G) Starch (S) Layer containing fluorescent indicator (F) Plate size: : Plate size: 20X20cm 10X20cm 5X10 cm 5X7.5 cm Good cut edges of sheets is important to obtain constant Rf values. Slide 12: Pre washing of pre coated plates The main purpose of the pre-washing is to remove impurities which include water vapours and other volatile substances from the atmosphere when they get exposed in the lab environment. Silica gel 60F is most widely used sorbent. The major disadvantage of this sorbent is that it contain iron as impurity. This iron is removed by using Methanol : water in the ratio of 9:1.This is the major advantage of the step of pre-washing. : : : Some common methods involved in pre-washing: Ascending method: Dipping method: Continuous method: Solvents used for pre-washing : Solvents used for pre-washing 1.Methanol 2.Chloroform: methanol ( 1:1 ) 3.Choloroform: Methanol: Ammonia (90:10:1 ) 4.Methylene chloride: Methanol ( 1:1 ) 5.Ammonia solution (1%) Activation of plates: : Activation of plates: Freshly opened box of HPTLC plates doesn’t need activation. Plates exposed to high humidity or kept in hand for long time require activation. Plates are placed in oven at 110o-120oc for 30 min prior to the sample application. Activation at higher temperature for longer period is avoided as it may lead to very active layers and risk of the samples being decomposed. Sample Preparation: : Sample Preparation: Proper sample preparation is an important pre-requisite for success of TLC separation. For normal chromatography: Solvent should be non-polar and volatile. For reversed chromatography: Polar solvent is used for dissolving the sample Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution at starting zones. Application of sample: : Application of sample: The selection of sample application technique and device to be used depends primarily on: Sample volume No. of samples to be applied Required precision and degree of automation. Slide 18: Some applicators used for spotting are: a) Capillary tubes b) Micro bulb pipettes c) Micro syringes, d)Automatic sample applicator. The major criteria is that they shouldn’t damage the surface while applying sample. Slide 19: The sample should be completely transferred to the layer. Micro syringes are preferred if automatic application devices are not available. Volume recommended for HPTLC-0.5-5μl to keep the starting zone down to minimum of 0.5-1 mm in concentration range of 0.1-μg/ml Sample spotting should not be excess or not low. Problem from overloading can be overcome by applying the sample as band. Advantages of application of sample as band are : Advantages of application of sample as band are Better separation because of rectangular area. Response of densiometer is higher in case of band than that observed from an equal amount/equal volume of sample applied as a spot. Large quantiti Automatic applicators used: : Automatic applicators used: 1) CAMAG Nanomat: Samples applied in the form of spots. The volume is controlled by disposable platinum iridium of glass capillary which has volume of 0.1-0.2μl. 2) CAMAG Linomat : 2) CAMAG Linomat Automated sample application device. Sample is loaded in micro syringe (Hamilton Syringe) 1μl capacity. Sample can apply either as spot or band by programming the instrument with parameters like spotting volume ,band length etc. Slide 26: 3) CAMAG automatic TLC sampler III : Applies sample as spot or bands automatically from the rack of sample vials. Mobile phase : Mobile phase Mobile phase should be of high graded. Chemical properties ,analytes and sorbent layer factors should be considered while selection of mobile phase. Use of mobile phase containing more than three or four components should normally be avoided as it is often difficult to get reproducible ratios of different components Slide 29: Mobile phase optimization is necessary while performing HPTLC. Various components of MP should be measured separately and then placed in mixing vessel. This prevents contamination of solvents and also error arising from volumes expansion or contraction on mixing. Trough chambers are used in which smaller volumes of MP usually 10-15 ml is required. Slide 30: Different components of MP are mixed first in mixing vessel and then transferred to developing chambers. Chambers containing multi component MP are not generally used for re-use for any future development , due to differential evaporation and adsorption by layer and also once the chamber is opened , solvents evaporate disproportionally depending on their volatilities. Development of chambers: : Development of chambers: 1.Twin trough chamber. 2.Rectangular chambers : 2.Rectangular chambers Slide 33: 3. V-shaped chambers 4.Sandwitch chamber 5.Horizontal development chamber 6.Automatic development chamber Pre-conditioning : (Chamber Saturation) : Pre-conditioning : (Chamber Saturation) Chamber saturation has a pronounced influence on the separation profile. Time required for the saturation depends on the mobile phase. If plates are introduced into the unsaturated chamber ,during the course of development , the solvent evaporates from the plate mainly at the solvent front and it results in increased Rf values. Development and Drying: : Development and Drying: The different methods used for development of chambers are like-Ascending , descending .2-dimentional, horizontal , multiple overrun , gradient ,radial ,anti-radial ,multimodal ,forced flow planar chromatography. Plates are spotted with sample and air dried and placed in the developing chambers. After the development plate is removed from chamber and mobile phase is removed under fume cup-board to avoid contamination of laboratory atmosphere. The plates should be always laid horizontally because when mobile phase evaporates the separated components will migrate evenly to the surface where it can be easily detected Drying : : Drying : Drying of chromatogram should be done in vacuum desiccators with protection from heat and light. If hand dryer is used there may be chances of getting contamination of plates ,evaporation of essential volatile oils if any present in the spot or compounds sensitive to oxygen may get destroyed due to the rise in temperature. Factors influencing separation and resolution of spots: : Factors influencing separation and resolution of spots: Type of stationary phase Type of pre-coated plates Layer thickness Binder in layer Mobile phase Solvent purity Size of developing chamber Sample volume to be spotted Size of initial spot Solvent level in chamber Slide 39: Gradient Relative humidity Temperature Flow rate in solvent Separation distance Mode of Derivatization Greater the difference between two spots and smaller the initial spot diameter of sample and better will be the resolution Detection and visualization : Detection and visualization One of the characteristic feature of HPTLC is the possibility to utilize post-chromatographic off line derivatization Detection are of two types: Qualitative Quantitative Slide 41: Qualitative detection; HPTLC is routinely used for qualitative analysis of raw materials , finished products ,plant extracts etc. It involves the identification of unknown sample mixture by comparing the Rf values of the sample components with the standards. Quantitation Evaluation: Quantitative of the chromatogram by HPTLC basically involves direct and indirect methods; Densiometry; : Densiometry; Documentation: : Documentation: 1. Documentation is important because labeling every single chromatogram can avoid mistake in respect of order of application. 2. Type of plate, chamber system, composition of mobile phase, running time and detection method should be recorded. 3. TO assist the analysts and researchers E .merck has introduced HPTLC pre-coated plates with an imprinted identification codes. 4. Suppliers name, item number, batch no. , individual plate no. are imprinted near upper edge of pre-coated plates. This will not only help in traceability of analytical data, but will also avoid manipulation of data at any stage as coding will automatically get recorded using photo-documentation. Applications of HPTLC: : Applications of HPTLC: Pharmaceutical industry: Quality control, content uniformity, uniformity test, identity/purity check. Food Analysis: Quality control , additives , pesticides ,stability testing ,analysis of sub-micron levels of aflotoxins etc Clinical Applications: Metabolism studies , drug screening ,stability testing etc Industrial Applications; Process development and optimization, In-process check ,validation etc. Forensic : Poisoning investigations References: : References: HPTLC- Quantitative analysis of pharmaceutical Formulations by P.D. Sethi www.pharmainfo.net http://images.google.co.in/images?q=hptlc+plates&ie=ISO-8859-1&hl=en http://images.google.co.in/images?svnum=10&hl=en&lr=&ie=ISO-8859-1&q=linomat www.camag.com http://www.infoexpo.ch/abstract Slide 46: THANK YOU You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.
HpTLC power point hakeemkhan Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 3241 Category: Science & Tech.. License: All Rights Reserved Like it (4) Dislike it (1) Added: November 10, 2010 This Presentation is Public Favorites: 1 Presentation Description No description available. Comments Posting comment... By: hakeemkhan (7 month(s) ago) Warm Greetings: Dear Dhammika as u required the other presentations. Can u mension the specific topics so i can guide u properly. Thanks for the complement..... Saving..... Post Reply Close Saving..... Edit Comment Close By: s.dhammika (7 month(s) ago) superb Saving..... Post Reply Close Saving..... Edit Comment Close Premium member Presentation Transcript Slide 1: (HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY) Presented by: ABDUL RAZZAQ M.Pharma in Pharmaceutical Chemistry LUQMAN COLLEGE OF PHARMACY GULBARGA HPTLC Definition: : Definition: 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. Introduction: : Introduction: 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. Differences between TLC and HPTLC: : Differences between TLC and HPTLC: Slide 5: SAMPLE AND STANDARD PREPARATION SELECTION OF CHROMATOGRAPHIC PLATES LAYER PRE-WASHING LAYER PRE-CONDITIONING APPLICATION OF SAMPLE CHROMATOGRAPIC DEVELOPMENT DETECTION OF SPOTS SCANNING AND DOCUMENTATION OF CHROMOPLATE USING PC CATS SOFTWARE Slide 6: Selection of HPTLC plates Hand plates were available which are made up of cellulose and other materials which are not used much now-a –days. Slide 7: Pre coated plates: The plates with different support materials and sorbent layers with different format and thickness are used. Plates with sorbent thickness of 100-250μm are used for qualitative and quantitative analysis. Supports : Supports Some of the sorbents used in HPTLC: : Some of the sorbents used in HPTLC: Slide 10: Some of the binders used: Gypsum (G) Starch (S) Layer containing fluorescent indicator (F) Plate size: : Plate size: 20X20cm 10X20cm 5X10 cm 5X7.5 cm Good cut edges of sheets is important to obtain constant Rf values. Slide 12: Pre washing of pre coated plates The main purpose of the pre-washing is to remove impurities which include water vapours and other volatile substances from the atmosphere when they get exposed in the lab environment. Silica gel 60F is most widely used sorbent. The major disadvantage of this sorbent is that it contain iron as impurity. This iron is removed by using Methanol : water in the ratio of 9:1.This is the major advantage of the step of pre-washing. : : : Some common methods involved in pre-washing: Ascending method: Dipping method: Continuous method: Solvents used for pre-washing : Solvents used for pre-washing 1.Methanol 2.Chloroform: methanol ( 1:1 ) 3.Choloroform: Methanol: Ammonia (90:10:1 ) 4.Methylene chloride: Methanol ( 1:1 ) 5.Ammonia solution (1%) Activation of plates: : Activation of plates: Freshly opened box of HPTLC plates doesn’t need activation. Plates exposed to high humidity or kept in hand for long time require activation. Plates are placed in oven at 110o-120oc for 30 min prior to the sample application. Activation at higher temperature for longer period is avoided as it may lead to very active layers and risk of the samples being decomposed. Sample Preparation: : Sample Preparation: Proper sample preparation is an important pre-requisite for success of TLC separation. For normal chromatography: Solvent should be non-polar and volatile. For reversed chromatography: Polar solvent is used for dissolving the sample Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution at starting zones. Application of sample: : Application of sample: The selection of sample application technique and device to be used depends primarily on: Sample volume No. of samples to be applied Required precision and degree of automation. Slide 18: Some applicators used for spotting are: a) Capillary tubes b) Micro bulb pipettes c) Micro syringes, d)Automatic sample applicator. The major criteria is that they shouldn’t damage the surface while applying sample. Slide 19: The sample should be completely transferred to the layer. Micro syringes are preferred if automatic application devices are not available. Volume recommended for HPTLC-0.5-5μl to keep the starting zone down to minimum of 0.5-1 mm in concentration range of 0.1-μg/ml Sample spotting should not be excess or not low. Problem from overloading can be overcome by applying the sample as band. Advantages of application of sample as band are : Advantages of application of sample as band are Better separation because of rectangular area. Response of densiometer is higher in case of band than that observed from an equal amount/equal volume of sample applied as a spot. Large quantiti Automatic applicators used: : Automatic applicators used: 1) CAMAG Nanomat: Samples applied in the form of spots. The volume is controlled by disposable platinum iridium of glass capillary which has volume of 0.1-0.2μl. 2) CAMAG Linomat : 2) CAMAG Linomat Automated sample application device. Sample is loaded in micro syringe (Hamilton Syringe) 1μl capacity. Sample can apply either as spot or band by programming the instrument with parameters like spotting volume ,band length etc. Slide 26: 3) CAMAG automatic TLC sampler III : Applies sample as spot or bands automatically from the rack of sample vials. Mobile phase : Mobile phase Mobile phase should be of high graded. Chemical properties ,analytes and sorbent layer factors should be considered while selection of mobile phase. Use of mobile phase containing more than three or four components should normally be avoided as it is often difficult to get reproducible ratios of different components Slide 29: Mobile phase optimization is necessary while performing HPTLC. Various components of MP should be measured separately and then placed in mixing vessel. This prevents contamination of solvents and also error arising from volumes expansion or contraction on mixing. Trough chambers are used in which smaller volumes of MP usually 10-15 ml is required. Slide 30: Different components of MP are mixed first in mixing vessel and then transferred to developing chambers. Chambers containing multi component MP are not generally used for re-use for any future development , due to differential evaporation and adsorption by layer and also once the chamber is opened , solvents evaporate disproportionally depending on their volatilities. Development of chambers: : Development of chambers: 1.Twin trough chamber. 2.Rectangular chambers : 2.Rectangular chambers Slide 33: 3. V-shaped chambers 4.Sandwitch chamber 5.Horizontal development chamber 6.Automatic development chamber Pre-conditioning : (Chamber Saturation) : Pre-conditioning : (Chamber Saturation) Chamber saturation has a pronounced influence on the separation profile. Time required for the saturation depends on the mobile phase. If plates are introduced into the unsaturated chamber ,during the course of development , the solvent evaporates from the plate mainly at the solvent front and it results in increased Rf values. Development and Drying: : Development and Drying: The different methods used for development of chambers are like-Ascending , descending .2-dimentional, horizontal , multiple overrun , gradient ,radial ,anti-radial ,multimodal ,forced flow planar chromatography. Plates are spotted with sample and air dried and placed in the developing chambers. After the development plate is removed from chamber and mobile phase is removed under fume cup-board to avoid contamination of laboratory atmosphere. The plates should be always laid horizontally because when mobile phase evaporates the separated components will migrate evenly to the surface where it can be easily detected Drying : : Drying : Drying of chromatogram should be done in vacuum desiccators with protection from heat and light. If hand dryer is used there may be chances of getting contamination of plates ,evaporation of essential volatile oils if any present in the spot or compounds sensitive to oxygen may get destroyed due to the rise in temperature. Factors influencing separation and resolution of spots: : Factors influencing separation and resolution of spots: Type of stationary phase Type of pre-coated plates Layer thickness Binder in layer Mobile phase Solvent purity Size of developing chamber Sample volume to be spotted Size of initial spot Solvent level in chamber Slide 39: Gradient Relative humidity Temperature Flow rate in solvent Separation distance Mode of Derivatization Greater the difference between two spots and smaller the initial spot diameter of sample and better will be the resolution Detection and visualization : Detection and visualization One of the characteristic feature of HPTLC is the possibility to utilize post-chromatographic off line derivatization Detection are of two types: Qualitative Quantitative Slide 41: Qualitative detection; HPTLC is routinely used for qualitative analysis of raw materials , finished products ,plant extracts etc. It involves the identification of unknown sample mixture by comparing the Rf values of the sample components with the standards. Quantitation Evaluation: Quantitative of the chromatogram by HPTLC basically involves direct and indirect methods; Densiometry; : Densiometry; Documentation: : Documentation: 1. Documentation is important because labeling every single chromatogram can avoid mistake in respect of order of application. 2. Type of plate, chamber system, composition of mobile phase, running time and detection method should be recorded. 3. TO assist the analysts and researchers E .merck has introduced HPTLC pre-coated plates with an imprinted identification codes. 4. Suppliers name, item number, batch no. , individual plate no. are imprinted near upper edge of pre-coated plates. This will not only help in traceability of analytical data, but will also avoid manipulation of data at any stage as coding will automatically get recorded using photo-documentation. Applications of HPTLC: : Applications of HPTLC: Pharmaceutical industry: Quality control, content uniformity, uniformity test, identity/purity check. Food Analysis: Quality control , additives , pesticides ,stability testing ,analysis of sub-micron levels of aflotoxins etc Clinical Applications: Metabolism studies , drug screening ,stability testing etc Industrial Applications; Process development and optimization, In-process check ,validation etc. Forensic : Poisoning investigations References: : References: HPTLC- Quantitative analysis of pharmaceutical Formulations by P.D. Sethi www.pharmainfo.net http://images.google.co.in/images?q=hptlc+plates&ie=ISO-8859-1&hl=en http://images.google.co.in/images?svnum=10&hl=en&lr=&ie=ISO-8859-1&q=linomat www.camag.com http://www.infoexpo.ch/abstract Slide 46: THANK YOU