This document provides tips and tricks for troubleshooting problems with HPLC systems. It discusses common issues like high pressure, peak shape problems, and retention time shifts. Potential causes are outlined for each problem, such as plugged frits or columns, leaks, incorrect flow rates, sample or method issues, column contamination, or extra-column volume. The document provides guidance on diagnostic steps to determine the root cause, such as checking for issues in different system components, reversing or washing the column. It also emphasizes prevention techniques like using inline filters to protect columns. Specific examples demonstrate how problems like peak splitting, tailing, or broadening can arise and how they can be addressed.
This document describes Millex® syringe filters for research, medical, and industrial applications. It provides details on various sterile and non-sterile syringe filter types including membrane material, pore size, diameter, housing material, and available pack sizes. Applications include tissue culture, buffers, drugs, vitamins, HPLC, IC, GC, particle removal, and more. Housings are made from modified acrylic, HDPE, PVC, or polypropylene and sterilized by radiation or ethylene oxide.
1. The document discusses troubleshooting strategies and common problems in HPLC.
2. It outlines a 5-step troubleshooting strategy of identifying the problem, determining the cause, isolating the exact cause, rectifying the problem if possible, and returning the system to use.
3. Common problems discussed include issues with the mobile phase, pump, injector, detector, and peaks/baseline, along with potential causes and solutions for each.
This document discusses decalcification, which is the process of removing calcium from bone and other calcified tissues prior to sectioning and microscopic examination. It defines decalcification and lists the criteria for an ideal decalcifying agent. Various factors that affect the rate of decalcification are described, including concentration, temperature, agitation, and suspension of the tissue. The main methods of decalcification are outlined as well as the principles, types, compositions, and procedures for different decalcifying agents such as acids, ion exchange resins, and chelating agents.
This document discusses the process of decalcification, which is the removal of calcium from tissues to make them suitable for section cutting. It outlines the key steps: selection of tissue, fixation, decalcification using mineral acids, chelating agents, or electrophoresis, detection of endpoint, neutralization, and washing. Common decalcifying agents discussed include Gooding and Stewart's fluid, von Ebner's fluid, citrate-citric acid buffer, and chelating agents like EDTA. The factors that influence decalcification speed and the importance of determining the endpoint are also summarized.
This document discusses methods for decalcifying bone and teeth for histological examination. It begins with an introduction to decalcification and describes the process of removing calcium ions from bone to make it flexible for pathological investigation. It then classifies common decalcifying agents such as acids, chelating agents, and other solutions. The document discusses factors that affect the rate of decalcification and tests used to determine when decalcification is complete. It provides details on processing and staining decalcified bone and teeth. The document concludes with precautions for decalcification and references.
High performance thin layer chromatography (HPTLC) is an automated and sophisticated form of thin layer chromatography that provides precise analytical results. HPTLC has several advantages over traditional TLC such as higher resolution, simultaneous processing of samples and standards, lower analysis times, and reduced solvent usage. The document outlines the basic principles, instrumentation, sample preparation steps, chromatographic development process, and applications of HPTLC for pharmaceutical analysis, food testing, and other areas. HPTLC is a versatile and cost-effective separation technique commonly used for quality control testing in industries such as pharmaceuticals, food, and forensics.
For HPLC, sample solvents that adequately dissolve target compounds are required. Therefore, sample solvents that contain a high concentration of organic solvent are often used for reversed phase chromatography. The problem is that these solvents sometimes cause peak broadening.
This presentation discusses techniques for reducing the effects of sample solvents on UHPLC analyses.
Euskadi Pais Estrategia vasca de envejecimiento activo 2015patxigalarraga
Este documento presenta la Estrategia Vasca de Envejecimiento Activo 2015-2020. Describe la situación demográfica actual en el País Vasco, donde el 20,2% de la población tiene más de 65 años. Se espera que estas cifras aumenten significativamente en los próximos años debido al envejecimiento de la población. La estrategia se centra en tres áreas principales: la adaptación de la sociedad al envejecimiento, la anticipación y prevención para un envejecimiento saludable, y la creación de una sociedad amigable y
This document describes Millex® syringe filters for research, medical, and industrial applications. It provides details on various sterile and non-sterile syringe filter types including membrane material, pore size, diameter, housing material, and available pack sizes. Applications include tissue culture, buffers, drugs, vitamins, HPLC, IC, GC, particle removal, and more. Housings are made from modified acrylic, HDPE, PVC, or polypropylene and sterilized by radiation or ethylene oxide.
1. The document discusses troubleshooting strategies and common problems in HPLC.
2. It outlines a 5-step troubleshooting strategy of identifying the problem, determining the cause, isolating the exact cause, rectifying the problem if possible, and returning the system to use.
3. Common problems discussed include issues with the mobile phase, pump, injector, detector, and peaks/baseline, along with potential causes and solutions for each.
This document discusses decalcification, which is the process of removing calcium from bone and other calcified tissues prior to sectioning and microscopic examination. It defines decalcification and lists the criteria for an ideal decalcifying agent. Various factors that affect the rate of decalcification are described, including concentration, temperature, agitation, and suspension of the tissue. The main methods of decalcification are outlined as well as the principles, types, compositions, and procedures for different decalcifying agents such as acids, ion exchange resins, and chelating agents.
This document discusses the process of decalcification, which is the removal of calcium from tissues to make them suitable for section cutting. It outlines the key steps: selection of tissue, fixation, decalcification using mineral acids, chelating agents, or electrophoresis, detection of endpoint, neutralization, and washing. Common decalcifying agents discussed include Gooding and Stewart's fluid, von Ebner's fluid, citrate-citric acid buffer, and chelating agents like EDTA. The factors that influence decalcification speed and the importance of determining the endpoint are also summarized.
This document discusses methods for decalcifying bone and teeth for histological examination. It begins with an introduction to decalcification and describes the process of removing calcium ions from bone to make it flexible for pathological investigation. It then classifies common decalcifying agents such as acids, chelating agents, and other solutions. The document discusses factors that affect the rate of decalcification and tests used to determine when decalcification is complete. It provides details on processing and staining decalcified bone and teeth. The document concludes with precautions for decalcification and references.
High performance thin layer chromatography (HPTLC) is an automated and sophisticated form of thin layer chromatography that provides precise analytical results. HPTLC has several advantages over traditional TLC such as higher resolution, simultaneous processing of samples and standards, lower analysis times, and reduced solvent usage. The document outlines the basic principles, instrumentation, sample preparation steps, chromatographic development process, and applications of HPTLC for pharmaceutical analysis, food testing, and other areas. HPTLC is a versatile and cost-effective separation technique commonly used for quality control testing in industries such as pharmaceuticals, food, and forensics.
For HPLC, sample solvents that adequately dissolve target compounds are required. Therefore, sample solvents that contain a high concentration of organic solvent are often used for reversed phase chromatography. The problem is that these solvents sometimes cause peak broadening.
This presentation discusses techniques for reducing the effects of sample solvents on UHPLC analyses.
Euskadi Pais Estrategia vasca de envejecimiento activo 2015patxigalarraga
Este documento presenta la Estrategia Vasca de Envejecimiento Activo 2015-2020. Describe la situación demográfica actual en el País Vasco, donde el 20,2% de la población tiene más de 65 años. Se espera que estas cifras aumenten significativamente en los próximos años debido al envejecimiento de la población. La estrategia se centra en tres áreas principales: la adaptación de la sociedad al envejecimiento, la anticipación y prevención para un envejecimiento saludable, y la creación de una sociedad amigable y
Stars madrid 2015 es_guia para asesores de secundaria_starspatxigalarraga
Este documento proporciona una guía para implementar el programa STARS Europa de compromiso entre iguales en centros de secundaria. El objetivo del programa es aumentar el número de estudiantes que van en bicicleta a la escuela a través de la formación de un grupo de Embajadores de la Movilidad Juvenil en cada escuela. Estos embajadores diseñan y llevan a cabo actividades para promover el ciclismo y competir en el Reto Ciclista. La guía incluye plantillas y consejos para las encuestas, actividades y evaluación
Este documento presenta un manual de intervención en viviendas para mejorar la calidad de vida de personas con perfiles sociosanitarios en Ermua. El manual incluye fichas sobre diferentes tipos de obras e intervenciones en las viviendas, como modificación de tabiques, sustitución de bañeras por platos de ducha, e instalación de barras de apoyo y otros elementos. El objetivo es dar pautas para que las adaptaciones se realicen de acuerdo a las necesidades específicas de cada persona residente y mejorar su autonomía y calidad
Stars madrid 2015 es_guia para asesores de primaria_starspatxigalarraga
Este documento proporciona información sobre el programa STARS Europa, un sistema de
acreditación diseñado para promover formas sostenibles de viajar a la escuela como andar o ir en
bicicleta. El documento guía a los asesores de escuelas primarias sobre cómo implementar las
actividades necesarias para que las escuelas obtengan la acreditación STARS en niveles de
compromiso, bronce, plata u oro. Se explican las tareas de los asesores, el marco de acreditación,
cómo organizar un taller local y
La aportación de la UE al envejecimiento activo y a la solidaridad entre las ...patxigalarraga
Este documento describe la contribución de la Unión Europea al envejecimiento activo y la solidaridad entre generaciones. Explica que la población europea está envejeciendo rápidamente y que esto presenta desafíos sociales, económicos y de políticas públicas. Argumenta que fomentar el envejecimiento activo, permitiendo que las personas mayores permanezcan en el mercado laboral, participen en la sociedad y vivan de forma independiente, es fundamental para abordar estos desafíos. A continuación, resume los diferentes instrumentos y políticas de
High performance-liquid-chromatography-hplcShreya Feliz
Gas chromatography is a technique used to separate mixtures by exploiting differences in how compounds interact with a mobile phase (carrier gas) and a stationary phase. Samples are injected into a column containing a stationary phase and separated components emerge from the column over time, detected, and recorded as peaks. Common applications include analyzing volatile organic compounds, flavors, fragrances, and petrochemicals. Limitations include only being able to analyze volatile compounds.
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.
Chromatography separates components in a mixture using a stationary and mobile phase. High performance liquid chromatography (HPLC) is a type of chromatography that uses high pressure to force a liquid mobile phase through a column packed with solid particles. The document discusses various aspects of HPLC including separation modes, selecting stationary and mobile phases, HPLC system components, and applications.
This document provides information about handling and operating high performance liquid chromatography (HPLC). It discusses the basic components and setup of an HPLC instrument. It also compares HPLC to thin layer chromatography. The document explains reverse phase and normal phase HPLC, proposed mechanisms, column selection, mobile phase preparation including buffers, ion pair reagents, column properties, and common detectors. It provides guidelines for handling HPLC including preparation of samples, standards and mobile phases, and assessing system suitability.
The seminar document discusses key concepts in high performance liquid chromatography (HPLC) including resolution factor, theoretical plates, capacity factor, and tailing factor. It defines these terms and explains how they are calculated and their practical applications. Specifically, it covers how resolution factor, theoretical plates, and capacity factor are used to measure column performance and efficiency, and how tailing factor is important to avoid misinterpretation of peaks. The document provides examples of how these factors are determined from chromatograms and discusses optimal ranges.
Hplc (basic principles, operation and maintenance)alim125135
This document provides an overview of high performance liquid chromatography (HPLC), including basic principles, instrumentation, separation modes, detectors, maintenance, troubleshooting, and good laboratory practices. It discusses reversed phase and normal phase HPLC systems. Key topics covered include column configuration, peak parameters, solvent selection, converting between normal and reversed phase, various detectors such as UV-Vis and ELSD, individual module maintenance, common problems, and filtration best practices. The document emphasizes the importance of proper solvent and buffer preparation, filtration, and storage to minimize contamination and maximize system performance.
High performance liquid chromatography (HPLC) is summarized as follows:
HPLC is a technique used to separate mixtures by distributing the components between a stationary and mobile phase. It can be used for both qualitative and quantitative analysis. HPLC utilizes high pressure pumps to pass a mobile phase through a column packed with adsorbent particles, allowing separation of components based on differences in their partitioning behavior between the mobile and stationary phases. Common detectors used in HPLC include UV/Vis, refractive index, fluorescence, and mass spectrometry.
The document discusses high-performance liquid chromatography (HPLC). It defines HPLC and describes its basic principles, which involve separating mixtures by distributing components between a stationary and mobile phase under high pressure. The key components of an HPLC system are described, including pumps, injectors, columns, detectors, and data systems. Various modes, columns, and detectors are discussed. The document provides an overview of the technique of HPLC.
This document provides an overview of high performance liquid chromatography (HPLC) presented by Ravi Pratap Pulla. It introduces HPLC and its history. Key topics covered include HPLC components like columns and systems. Applications to pharmaceutical analysis are discussed. The document also reviews some basic HPLC terminology and concepts.
The document discusses rapid HPLC method development. It outlines four critical aspects of rapid development: sample preparation, column selection, column configuration, and instrument optimization. Choosing the right bonded phase is key to achieving resolution. Common phases like C18 and C8 are recommended initially, with specialized phases for difficult samples. The document provides examples of developing methods for various sample types using different columns and conditions based on a rapid development scheme. Developing at low pH first and using temperature, then moving to mid pH if needed, can reduce analysis time while maintaining resolution and reproducibility.
108 HPLC. Comparing Narrow Bore column with a standard Bore of 4.6 mm ID for ...Hossein Hodjat
The document compares the use of a narrow bore (2.1 mm ID) STYROS® 2R column to a standard bore (4.6 mm ID) STYROS® 3R column for the separation of protein standards. The narrow bore column uses only 2 μL of sample compared to 25 μL for the standard bore column, and requires less solvent at 1.6 mL compared to 17.5 mL. Both columns successfully separated the five protein standards.
Enhancing Sensitivities and Peak Capacities for UHPLC-MS Fast Gradient Analys...Sandy Simmons
When compared to 1.7 μm fully porous materials, the ultra-high
efficiency and low backpressures provided by Kinetex core-shell
2.6 μm columns, provides users opportunities to go beyond what
is traditionally accepted for UHPLC runs
This webinar will provide pesticides residue analysts with valuable information on the development and optimization of chromatographic separations and mass spectrometry methods for the analysis of pesticide residues in food. The expert speakers will share their knowledge in understanding the critical aspects of the method, assisting analysts in optimizing their methods for the most challenging analyses.
Stars madrid 2015 es_guia para asesores de secundaria_starspatxigalarraga
Este documento proporciona una guía para implementar el programa STARS Europa de compromiso entre iguales en centros de secundaria. El objetivo del programa es aumentar el número de estudiantes que van en bicicleta a la escuela a través de la formación de un grupo de Embajadores de la Movilidad Juvenil en cada escuela. Estos embajadores diseñan y llevan a cabo actividades para promover el ciclismo y competir en el Reto Ciclista. La guía incluye plantillas y consejos para las encuestas, actividades y evaluación
Este documento presenta un manual de intervención en viviendas para mejorar la calidad de vida de personas con perfiles sociosanitarios en Ermua. El manual incluye fichas sobre diferentes tipos de obras e intervenciones en las viviendas, como modificación de tabiques, sustitución de bañeras por platos de ducha, e instalación de barras de apoyo y otros elementos. El objetivo es dar pautas para que las adaptaciones se realicen de acuerdo a las necesidades específicas de cada persona residente y mejorar su autonomía y calidad
Stars madrid 2015 es_guia para asesores de primaria_starspatxigalarraga
Este documento proporciona información sobre el programa STARS Europa, un sistema de
acreditación diseñado para promover formas sostenibles de viajar a la escuela como andar o ir en
bicicleta. El documento guía a los asesores de escuelas primarias sobre cómo implementar las
actividades necesarias para que las escuelas obtengan la acreditación STARS en niveles de
compromiso, bronce, plata u oro. Se explican las tareas de los asesores, el marco de acreditación,
cómo organizar un taller local y
La aportación de la UE al envejecimiento activo y a la solidaridad entre las ...patxigalarraga
Este documento describe la contribución de la Unión Europea al envejecimiento activo y la solidaridad entre generaciones. Explica que la población europea está envejeciendo rápidamente y que esto presenta desafíos sociales, económicos y de políticas públicas. Argumenta que fomentar el envejecimiento activo, permitiendo que las personas mayores permanezcan en el mercado laboral, participen en la sociedad y vivan de forma independiente, es fundamental para abordar estos desafíos. A continuación, resume los diferentes instrumentos y políticas de
High performance-liquid-chromatography-hplcShreya Feliz
Gas chromatography is a technique used to separate mixtures by exploiting differences in how compounds interact with a mobile phase (carrier gas) and a stationary phase. Samples are injected into a column containing a stationary phase and separated components emerge from the column over time, detected, and recorded as peaks. Common applications include analyzing volatile organic compounds, flavors, fragrances, and petrochemicals. Limitations include only being able to analyze volatile compounds.
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.
Chromatography separates components in a mixture using a stationary and mobile phase. High performance liquid chromatography (HPLC) is a type of chromatography that uses high pressure to force a liquid mobile phase through a column packed with solid particles. The document discusses various aspects of HPLC including separation modes, selecting stationary and mobile phases, HPLC system components, and applications.
This document provides information about handling and operating high performance liquid chromatography (HPLC). It discusses the basic components and setup of an HPLC instrument. It also compares HPLC to thin layer chromatography. The document explains reverse phase and normal phase HPLC, proposed mechanisms, column selection, mobile phase preparation including buffers, ion pair reagents, column properties, and common detectors. It provides guidelines for handling HPLC including preparation of samples, standards and mobile phases, and assessing system suitability.
The seminar document discusses key concepts in high performance liquid chromatography (HPLC) including resolution factor, theoretical plates, capacity factor, and tailing factor. It defines these terms and explains how they are calculated and their practical applications. Specifically, it covers how resolution factor, theoretical plates, and capacity factor are used to measure column performance and efficiency, and how tailing factor is important to avoid misinterpretation of peaks. The document provides examples of how these factors are determined from chromatograms and discusses optimal ranges.
Hplc (basic principles, operation and maintenance)alim125135
This document provides an overview of high performance liquid chromatography (HPLC), including basic principles, instrumentation, separation modes, detectors, maintenance, troubleshooting, and good laboratory practices. It discusses reversed phase and normal phase HPLC systems. Key topics covered include column configuration, peak parameters, solvent selection, converting between normal and reversed phase, various detectors such as UV-Vis and ELSD, individual module maintenance, common problems, and filtration best practices. The document emphasizes the importance of proper solvent and buffer preparation, filtration, and storage to minimize contamination and maximize system performance.
High performance liquid chromatography (HPLC) is summarized as follows:
HPLC is a technique used to separate mixtures by distributing the components between a stationary and mobile phase. It can be used for both qualitative and quantitative analysis. HPLC utilizes high pressure pumps to pass a mobile phase through a column packed with adsorbent particles, allowing separation of components based on differences in their partitioning behavior between the mobile and stationary phases. Common detectors used in HPLC include UV/Vis, refractive index, fluorescence, and mass spectrometry.
The document discusses high-performance liquid chromatography (HPLC). It defines HPLC and describes its basic principles, which involve separating mixtures by distributing components between a stationary and mobile phase under high pressure. The key components of an HPLC system are described, including pumps, injectors, columns, detectors, and data systems. Various modes, columns, and detectors are discussed. The document provides an overview of the technique of HPLC.
This document provides an overview of high performance liquid chromatography (HPLC) presented by Ravi Pratap Pulla. It introduces HPLC and its history. Key topics covered include HPLC components like columns and systems. Applications to pharmaceutical analysis are discussed. The document also reviews some basic HPLC terminology and concepts.
The document discusses rapid HPLC method development. It outlines four critical aspects of rapid development: sample preparation, column selection, column configuration, and instrument optimization. Choosing the right bonded phase is key to achieving resolution. Common phases like C18 and C8 are recommended initially, with specialized phases for difficult samples. The document provides examples of developing methods for various sample types using different columns and conditions based on a rapid development scheme. Developing at low pH first and using temperature, then moving to mid pH if needed, can reduce analysis time while maintaining resolution and reproducibility.
108 HPLC. Comparing Narrow Bore column with a standard Bore of 4.6 mm ID for ...Hossein Hodjat
The document compares the use of a narrow bore (2.1 mm ID) STYROS® 2R column to a standard bore (4.6 mm ID) STYROS® 3R column for the separation of protein standards. The narrow bore column uses only 2 μL of sample compared to 25 μL for the standard bore column, and requires less solvent at 1.6 mL compared to 17.5 mL. Both columns successfully separated the five protein standards.
Enhancing Sensitivities and Peak Capacities for UHPLC-MS Fast Gradient Analys...Sandy Simmons
When compared to 1.7 μm fully porous materials, the ultra-high
efficiency and low backpressures provided by Kinetex core-shell
2.6 μm columns, provides users opportunities to go beyond what
is traditionally accepted for UHPLC runs
This webinar will provide pesticides residue analysts with valuable information on the development and optimization of chromatographic separations and mass spectrometry methods for the analysis of pesticide residues in food. The expert speakers will share their knowledge in understanding the critical aspects of the method, assisting analysts in optimizing their methods for the most challenging analyses.
Determination of Common Counterions and Impurity Anions in Pharmaceuticals Using a Capillary HPIC System with Suppressed Conductivity and Charge Detection
Recently, identification and quantification of ions in early stage drug development has gained increasing attention, because the APIs maybe contaminated with different counter ions from synthesis steps, and because selecting the counter ion to enhance APIs’ solubility and stability is becoming a key step in formulation development. This presentation demonstrates the identification and quantification of 22 commonly found anions in pharmaceuticals in a single run using a high-pressure capillary IC system (HPIC) with 4-μm particle ion –exchange column, and CD-QD dual detectors.
HPLC Problems, Probable Causes, and Remedies2020.pptxutpalanand7
The document discusses common problems encountered with HPLC, their probable causes, and recommended remedies. It addresses issues such as no peaks, no flow, variable retention times, loss of resolution, tailing peaks, fronting peaks, baseline noise, negative peaks, and ghost peaks. The remedies include checking detector lamps, pump flow, column connections, mobile phase composition, column contamination, and adjusting operating parameters.
The document provides information on Waters CORTECS UPLC and HPLC columns including ordering details and specifications. It lists the available chemistries (C18, C18+, etc.), dimensions (2.1mm x 50mm, etc.), and part numbers. The document explains that CORTECS columns are available with 1.6um and 2.7um particles to enable methods transfer between UPLC, UHPLC, and HPLC systems for improved speed, resolution, and solvent usage. A total of 25 column configurations are available across the different chemistries and dimensions.
114 Application note to highlight the characteristics of STYROS® simulated-mo...Hossein Hodjat
This document compares the separation of four paraben compounds using a capillary column with 0.5 mm internal diameter and a micro bore column with 1 mm internal diameter, both packed with STYROS® 2R polymeric stationary phase. The capillary column requires only 50% of the eluent and sample volume compared to the micro bore column, while achieving similar separation. As a simulated monolith, STYROS® 2R also allows for high linear velocities and efficient column regeneration. Chromatograms are provided showing the separations on each column type.
This document discusses chromatography and PCR techniques. It provides details on:
- The principles and types of chromatography including TLC, HPLC, and their components and procedures. HPLC allows for quantitative analysis and is commonly used for pharmaceutical quality control.
- PCR amplification which uses DNA polymerase to exponentially replicate DNA sequences. It requires template DNA, primers, nucleotides, and DNA polymerase. Repeated heating and cooling cycles allow for target DNA replication.
- Applications of chromatography and PCR include pharmaceutical analysis, forensic analysis, detection of genetic disorders, microbial detection, and molecular biology research techniques. Both provide powerful tools for separation, detection, and analysis of biological molecules.
110 Separation of 5 peptides on Narrow Bore column of 2.1 mm ID. Acidic and ...Hossein Hodjat
This document describes the separation of 5 peptide standards on a narrow bore column with 2.1 mm internal diameter packed with STYROS® 2R polymeric reversed phase material. The separation was run with acidic and basic mobile phase gradients at flow rates over 4,000 cm/hr. The narrow bore column and polymeric packing allow for minimal solvent and sample usage while maintaining high efficiency separations suitable for mass spectrometry detection.
This document provides tips and tricks for troubleshooting problems with HPLC systems. It outlines a 5-step troubleshooting strategy of identifying the problem, determining the cause, isolating the exact cause, rectifying the problem if possible, and getting help from maintenance. Common problems like no peaks, no flow, pressure issues, and peak issues are described along with probable causes and remedies. The guidelines provide a logical process for isolating and resolving chromatography issues.
Flash chromatography is a technique that uses positive air pressure to force solvent through a column of adsorbent material, allowing for faster separation of compounds compared to traditional column chromatography. It uses shorter columns packed with smaller silica gel particles. The document discusses the principles, components, instrumentation, sample loading, applications in natural products and pharmaceuticals, and differences between flash chromatography, column chromatography, and HPLC.
This document describes Hydranautics' Low Differential (LD) membrane technology, which aims to minimize costs from fouling. The key features of the LD technology include:
1. Enhanced membrane chemistry for increased chemical resistance and higher salt rejection.
2. An innovative spacer design that prevents small particles from being trapped to reduce fouling and differential pressure.
3. A proprietary vented seal carrier that prevents pressure shock damage during system startups.
The benefits of the LD technology are longer membrane life through reduced cleaning needs, lower cleaning costs, increased productivity, improved water quality, and reduced operating costs due to lower fouling and differential pressure.
The document introduces the Nexera UHPLC system developed by Shimadzu to maximize sample throughput. It aims to expand the capabilities of UHPLC, UFLC and HPLC through the highest pressure range and widest flow rate range allowing for ultra-high speed and high resolution analysis. Key features include the world's highest maximum pressure of 130MPa, fastest autosampler injection time of 10 seconds, and largest sample capacity of 4600 samples. These features work together to provide ultra-fast injection speeds, minimized delay volumes, and maximized sample throughput for high productivity bioanalysis.
This document provides tips and recommendations for maintaining chromatography systems to reduce problems and ensure smooth operation. It discusses best practices for solvents, pumps, autosamplers, columns, and sample preparation. Key points covered include filtering solvents, maintaining inlet valves, replacing seals, removing particulates from samples, and using solid phase extraction or other cleanup methods to reduce matrix interference. Maintaining instrumentation properly and preparing samples carefully can help optimize system performance and productivity.
The new LV1 Microfluidizer processor allows for high pressure processing of small sample volumes between 1-20ml with near total sample recovery. It is well-suited for applications in research and development, discovery, exploratory studies, and small clinical studies due to its scalability and ability to process small volumes. The LV1 provides consistent shear rates and particle size reduction performance comparable to larger Microfluidizer systems.
High performance liquid chromatography (HPTLC) presentation by using case study "Bioautographic method for selective description of the antioxidant and alpha amylase inhibitory in activity inplant extracts".
All the basic thing of hptlc is explained. The diffrences between tlc ,hplc and hptlc is expalin.All tools of hptlc with images is explained.
Similar to Tips and tricks_hplc_troubleshooting (20)
Building a Raspberry Pi Robot with Dot NET 8, Blazor and SignalRPeter Gallagher
In this session delivered at NDC Oslo 2024, I talk about how you can control a 3D printed Robot Arm with a Raspberry Pi, .NET 8, Blazor and SignalR.
I also show how you can use a Unity app on an Meta Quest 3 to control the arm VR too.
You can find the GitHub repo and workshop instructions here;
https://bit.ly/dotnetrobotgithub
"IOS 18 CONTROL CENTRE REVAMP STREAMLINED IPHONE SHUTDOWN MADE EASIER"Emmanuel Onwumere
In iOS 18, Apple has introduced a significant revamp to the Control Centre, making it more intuitive and user-friendly. One of the standout features is a quicker and more accessible way to shut down your iPhone. This enhancement aims to streamline the user experience, allowing for faster access to essential functions. Discover how iOS 18's redesigned Control Centre can simplify your daily interactions with your iPhone, bringing convenience right at your fingertips.
1. 1
Page 1
Tips and Tricks of HPLC System
Troubleshooting
Agilent Technologies, Inc.
LC Tips And Tricks Seminar Series
Page 2
Trouble Shooting Steps
You Have Recognized There is a Problem!
How Do You Fix It?
•1st Did System Suitability or Sample Fail?
•2nd Review Method for Compliance
– Is The Procedure Being Followed Properly?
– Are Instrument Settings Correct?
•3rd Ask More Questions!
– When Did the System Last Function Properly?
– Has Anything Been Changed?
•4th Review ALL parameters!
– The Obvious Is Not Always the Cause
– Was There More Than One Change?
3. 3
Page 5
Column Observations Potential Problems
High pressure - Plugged frit
- Column contamination
- Plugged packing
Low Pressure - Leak
- Flow Incorrect
Pressure Issues
Page 6
Determining the Cause and Correcting
High Back Pressure
• Check pressure with/without column - many pressure
problems are due to blockages in the system or guard col.
•Remove Column - Pressure Still High?
•Remove Guard – Pressure Still High?
•If Column pressure is high:
• Back flush column – Clear “dirty” frit surface
• Wash column – Eliminate column contamination
and plugged packing
– high molecular weight/adsorbed
compounds
– precipitate from sample or buffer
Change frit – Clear plugged frit PREVENT THIS!
4. 4
Page 7
Column Cleaning
Use at least 25 mL of each solvent for analytical columns
Flush with stronger solvents than your mobile
phase.
Reversed-Phase Solvent Choices
in Order of Increasing Strength
• Mobile phase without buffer salts
• 100% Methanol
• 100% Acetonitrile
• 75% Acetonitrile:25% Isopropanol
• 100% Isopropanol
• 100% Methylene Chloride*
• 100% Hexane*
*Tip: When using either Hexane or Methylene Chloride the column must be flushed
with Isopropanol before returning to your reversed-phase mobile phase.
Must Reverse
to
Re-Equilibrate
This Is Time Consuming
Often Performed Offline
Page 8
Changing a Frit May Not Be a Good Idea
May not be possible with new generation columns
May damage high performance columns
Column
Inlet Frit
Compression
Ferrule
Column Body
Female End Fitting
Male End Fitting
Wear gloves
Do not allow bed to dry
Do not touch the column -
body heat will extrude packing
Do not overtighten
Tip: Prevention is a Much Better Idea!
5. 5
Page 9
The Trick:
Prevention Techniques - A Better Choice!
• Use column protection
- In-line filters
- Guard columns
• Filter samples
• Filter buffered mobile phases
• Sample clean-up (i.e. SPE)
• Appropriate column flushing
Easy
Not As Easy
Page 10
Inexpensive Filters Prevent Column Frit Plugging
Regenerated Cellulose (RC) Recommended
•Universal hydrophilic membrane, compatible
with most solvents - aqueous and organic
•High purity, extremely low extractables and
binding
•More Uniform Surface
•Different than Other Cellulose Filters!!
In-line Filters Easy to Use and replace
Frits Available in 0.2,0.5 and 2.0µ Porosity
Much Less expensive than a Column
Easier and Faster to Replace than a Column Frit
6. 6
Page 11
What Are Common Peak Shape Issues?
1. Split peaks
2. Peak tailing
3. Broad peaks
• Many peak shape issues are also combinations - i.e. broad and tailing
or tailing with increased retention
•Symptoms do not necessarily affect all peaks in the chromatogram
•Each of these problems can have multiple causes
Page 12
Peak Splitting Caused By Disrupted Sample Path
Split or Double Peaks
Normal Double
Peaks
Tip: Similar Effect Can be Caused by Partially Plugged Frit
•Flow Path Disrupted by Void
•Sample Allowed to Follow Different Paths
Through Column
•Poorly Packed Bed Settles in Use
•High pH Dissolves Silica
7. 7
Page 13
0 5 10 15
1
3
4
2
Time (min)0 5 10 15
1
3
4
2
Time (min)
0 5 10 15
1
3
4
2
Time (min)
Split Peaks from Column Contamination
Column: StableBond SB-C8, 4.6 x 150 mm, 5 μm Mobile Phase: 60% 25 mM Na2HPO4, pH 3.0 : 40% MeOH Flow Rate: 1.0 mL/min
Temperature: 35°C Detection: UV 254 nm Sample: Filtered OTC Cold Medication: 1. Pseudoephedrine 2. APAP 3. Unknown 4. Chlorpheniramine
Injection 1 Injection 30
Injection 1
After Column Wash
with 100% ACN
Tip: Column washing eliminates the peak splitting, which resulted from a contaminant on the column
How could this be prevented? (Guard Column, SPE clean up of samples, Periodic column wash)
Group/Presentation Title
Agilent Restricted
September 10, 2008Month
##, 200X
Page 14
Split Peaks from Injection Solvent Effects
Column: StableBond SB-C8, 4.6 x 150 mm, 5 μm Mobile Phase: 82% H2O : 18% ACN
Injection Volume: 30 μL Sample: 1. Caffeine 2. Salicylamide
A. Injection Solvent
100% Acetonitrile
B. Injection Solvent
Mobile Phase
Tip: Injecting in a solvent stronger than the mobile phase can cause peak shape
problems such as peak splitting or broadening
Trick: Keep Organic Concentration in Sample Solvent < Mobile Phase
0 10
Time (min)
1
2
0 10
Time (min)
1
2
8. 8
Page 15
Peak Tailing, Broadening
and Loss of Efficiency
May be caused by:
• Column “secondary
interactions”
• Column contamination
• Column aging
• Column loading
• Extra-column effects
Page 16
Normal Tailing
Normal Tailing
Symmetry > 1.2
All Peaks Tail:
Extra-Column Effects.
Build up of Contamination on Column
Inlet.
Heavy Metals.
Bad Column.
Causes
Some Peaks Tail:
Secondary - Retention Effects.
Residual Silanol Interactions.
Small Peak Eluting on Tail of Larger Peak.
Peak Shape: Tailing Peaks
9. 9
Page 17
Peak Tailing
Identifying Column “Secondary Interactions”
Tip: Mobile phase modifier (TEA) competes with Sample for surface ion exchange
sites at mid-range pH values
Column: Alkyl-C8, 4.6 x 150 mm, 5μm Mobile Phase: 85% 25 mM Na2HPO4 pH 7.0 : 15% ACN Flow Rate: 1.0 mL/min
Temperature: 35°C Sample: 1. Phenylpropanolamine 2. Ephedrine 3. Amphetamine 4. Methamphetamine 5. Phenteramine
No TEA
USP TF (5%)
1. 1.29
2. 1.91
3. 1.63
4. 2.35
5. 1.57
10 mM TEA
USP TF (5%)
1. 1.19
2. 1.18
3. 1.20
4. 1.26
5. 1.14
TIme (min) Time (min)
0.0 2.5 5.0
5
4
32
1
5
4
3
2
1
0.0 2.5 5.0
Page 18
Peak Tailing
Low pH Minimizes “Secondary Interactions”
for Amines
Column: Alkyl-C8, 4.6 x 150 mm, 5μm Mobile Phase: 85% 25 mM Na2HPO4 : 15% ACN Flow Rate: 1.0 mL/min
Temperature: 35°C Sample: 1. Phenylpropanolamine 2. Ephedrine 3. Amphetamine 4. Methamphetamine 5. Phenteramine
pH 3.0
USP TF (5%)
4. 1.33
pH 7.0
USP TF (5%)
4. 2.35
Tip: Reducing mobile phase pH reduces interactions with silanols and peak tailing.
Time (min)
0.0 2.5 5.0
5
4
32
1
5
4
3
2
1
Time (min)
0.0 2.5 5.0
10. 10
Page 19
0 5
1
2,3
4
5
Time(min)
7
6
0 5 10
1
2
3
4
Time(min)
6
5
7
Peak Tailing
High pH Eliminates “Secondary Interactions” for
Amines
Peak Shape and Retention of this sample of basic compounds improves
at high pH where column has high IEX activity. Why?
Column: ZORBAX Extend-C18, 4.6 x 150 mm, 5 m m Mobile Phase: See Below Flow Rate: 1.0 mL/min Temperature: RT
Detection: UV 254 nm
Sample: 1. Maleate 2. Scopolamine 3. Pseudoephedrine 4. Doxylamine 5. Chlorpheniramine 6. Triprolidine 7. Diphenhydramine
pH 7
30% 20 mM Na2HPO4
70% MeOH
pH 11
30% 20 mM TEA
70% MeOH
tR = 8.5 tR = 11.4
Page 20
0.0 2.5 5.0
2
4
1
3
Time (min)
2
4
1
3
0.0 2.5 5.0
Time (min)
2
41
3
0.0 2.5 5.0
Time (min)
Peak Tailing - Column Contamination
Column: StableBond SB-C8, 4.6 x 250 mm, 5μm Mobile Phase: 20% H2O : 80% MeOH Flow Rate: 1.0 mL/min
Temperature: R.T. Detection: UV 254 nm Sample: 1. Uracil 2. Phenol 3. 4-Chloronitrobenzene 4. Toluene
Plates TF
1. 7629 2.08
2. 12043 1.64
3. 13727 1.69
4 13355 1.32
Plates TF
1. 7906 1.43
2. 12443 1.21
3. 17999 1.19
4 17098 1.25
Plates TF
1. 7448 1.06
2. 12237 1.21
3. 15366 1.11
4 19067 1.17
QC test forward
direction
QC test reverse direction
QC test after cleaning
100% IPA, 35°C
Tip: Quick Test to Determine if Column is Dirty or Damaged
Trick: Reverse Column and Run Sample –If Improved, Possible Cleaning Will
Help -No improvement-Column Damaged and Needs to be Replaced
11. 11
Page 21
Causes:
Column Overload
Normal Fronting
Symmetry < 0.9
2000
1500
1000
500
0
0 5 10 15 20 25
Time (min)
mAU
Peak Shape: Fronting Peaks
Group/Presentation Title
Agilent Restricted
September 10, 2008Month
##, 200X
Page 22
Peak Tailing/Broadening
Sample Load Effects
Columns: 4.6 x 150 mm, 5μm Mobile Phase: 40% 25 mM Na2HPO4 pH 7.0 : 60% ACN Flow Rate: 1.5 mL/min
Temperature: 40°C Sample: 1. Desipramine 2. Nortriptyline 3. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine
Broadening
Competitive C8
Plates
A.
B.
C.
D.
High Load
x10
Low Load
C D
1. 850 5941
2. 815 7842
3. 2776 6231
4. 2539 8359
5. 2735 10022
6. 5189 10725
Tailing
Eclpse XDB-C8
USP TF (5%) i
A B
1. 1.60 1.70
2. 2.00 1.90
3. 1.56 1.56
4. 2.13 1.70
5. 2.15 1.86
6. 1.25 1.25
0 5 10
Time (min)
0 5 10
Time (min)
0 5
Time (min)
0 5
Time (min)
Tip: Evaluate Both Volume and Mass Loading
12. 12
Page 23
Peak Shape: Broad Peaks
All Peaks Broadened:
• Loss of Column Efficiency.
• Column Void.
• Large Injection Volume.
Some Peaks Broadened:
• Late Elution from Previous Sample
(Ghost Peak).
– High Molecular Weight.
– Sample - Protein or Polymer.
Page 24
Time (min)
3
1
2
0 5 10 15
Unknown “Phantom” Peaks
Column: Extend-C18, 4.6 x 150 mm, 5 μm Mobile Phase: 40% 10 mM TEA, pH 11 : 60% MeOH Flow Rate: 1.0 mL/min
Temperature: R.T. Detection: UV 254 Sample: 1. Maleate 2. Pseudoephedrine 3. Chlorpheniramine
Plates
1. 5922
2. 9879
3. 779
Tip: The extremely low plates for moderately retained peaks are an indication of a
very late eluting peak from a preceding run.
Time (min)
1
0 5 10
Sample 1: Chlorpheniramine maleate
Peak 1: maleate
Sample 2 : Chlorpheniramine
maleate
and Pseudoephedrine
Peak 1: maleate
Peak 2: pseudoephedrine
Peak 3: chlorpheniramine (from 1st
injection)
“Phantom” peak from
first injection
13. 13
Page 25
Use short, small internal diameter tubing between the injector and
the column and between the column and the detector.
Make certain all tubing connections are made with matched
fittings.
Use a low-volume detector cell.
Inject small sample volumes.
Increasing Extra-Column Volume
Extra-Column Dispersion
Page 26
Peak Broadening
Extra-Column Volume
Column: StableBond SB-C18, 4.6 x 30 mm, 3.5 μm Mobile Phase: 85% H2O with 0.1% TFA : 15% ACN Flow Rate: 1.0 mL/min
Temperature: 35°C Sample: 1. Phenylalanine 2. 5-benzyl-3,6-dioxo-2-piperazine acetic acid 3. Asp-phe 4. Aspartame
10 mL extra-column
volume
50 mL extra-column
volume (tubing)
Time (min)
0.0 0.5 1.0 1.5 2.0
4
3
2
1
Time (min)
0.0 0.5 1.0 1.5 2.0
4
3
2
1
14. 14
Page 27
Tip: Poorly Made HPLC System Connections
Can Cause Peak Broadening
The System Has Been Optimized and :
– All Tubing Lengths Are Minimum
– Smallest Diameter Tubing Used
– Proper Flow Cell Volume
Symptom Still Seems to Have Too Much Extra-Column
Volume
What Is Wrong?
Have You Made the Connections Properly?
Page 28
Column Connectors Used in HPLC
0.090
in.
0.130
in.
0.090
in.
0.170
in.
Swagelok Waters
Parker Rheodyne
Valco Uptight
0.090
in.
0.080
in.
Troubleshooting LC Fittings, Part II. J. W. Dolan and P. Upchurch. LC/GC Magazine 6:788 (1988)
15. 15
Page 29
What Happens If the Connections Poorly Made ?
If Dimension X is too long, leaks will occur
Ferrule cannot seat properly
Mixing Chamber
If Dimension X is too short, a dead-volume,
or mixing chamber, will occur
Wrong … too long
Wrong … too short
X
X
Page 30
Stainless Steel and Polymer Fittings
Which type is used and when?
Stainless Steel (SS) fittings are the best choice for
reliable high pressure sealing
• Agilent uses Swagelok type fittings with front and back
ferrules – which give best sealing performance –
throughout all our LC systems
PEEK (<400b bar System Pressure) fittings are
ideal where:
• Connections are changed frequently, i.e. connecting
columns
• Pressure is less critical
PolyKetone
• Easy, hand tighten column connection
• 600 bar Pressure Rating PN: 5042-8957 (10/pk)
• Fits to SS Tubing
16. 16
Page 31
Changes in Retention Can Be Chemical or Physical
May be caused by:
• Column aging
• Column contamination
• Insufficient equilibration
• Poor column/mobile phase combination
• Change in mobile phase
• Change in flow rate
• Different Gradient Delay Volumes
Group/Presentation Title
Agilent Restricted
September 10, 2008Month
##, 200X
Page 32
Column Aging/Equilibration Causes
Retention/Selectivity Changes
Column 1 - After Cleaning
with 1% H3PO4
/Equilibration
• The primary analyte was sensitive to mobile phase aging/
conditioning of the column
• The peak shape was a secondary issue (metal chelating
compound) resolved by “de-activating” the active metal
contamination
Column 1 - Next DayColumn 1 - Initial
0 3 5 9 12 15
Time (min)
2
1
0 3 5 9 12 15
Time (min)
2
1
17. 17
Page 33
Metal Sensitive Compounds Can Chelate
C O
O
H
H
M+2
OH
::
M+2
C O
OHC N
N: M+2
:::
O
:
OH + M+2
CH
:::
Hint: Look for Lone Pair of Electrons on :O:
or N Which Can Form 5 or 6 Membered
Ring with Metal
:
Salicylaldehyde 6-membered ring complex
8-hydroxyquinoline
5-membered ring complex
a-benzoinoxomine
5-membered ring complex
Page 34
Acid Wash Can Improve Peak Shape
OH
OH
OHHOOHHO OH
OH1. 2. 1. 2.
Columns: ZORBAX SB-Phenyl
4.6 x 150 mm
Mobile Phase: 75% 25 mM
ammonium phosphate buffer
25% ACN
Flow Rate: 1.0 mL/min.
Temperature: RT
Sample Size: 5 mL
1
1
2
2
Tf: 1.2Tf: 3.7
Before Acid Wash After Acid Wash
50 – 100 mLs 1% H3PO4
• A 1% H3PO4 solution is used on SB columns, 0.5 % can be used on endcapped columns.
18. 18
Group/Presentation Title
Agilent Restricted
September 10, 2008Month
##, 200X
Page 35
Example: Change in Retention/Selectivity
Unintended Mobile Phase Variation
Tip: The Source of the Problem is Often Not the Obvious Change
“I have experimented with our mobile phase, opening new bottles of all mobile phase
components. When I use all fresh ingredients, the problem ceases to exist, and I
have narrowed the problem to either a bad bottle of TEA or phosphoric acid. Our
problem has been solved.”
Column 1 Column 2 - Fresh
mobile phase
Column 2
0 4 6
Time (min)
0 2 3 4 5 6 7
Time (min)
0 4 6
Time (min)
Page 36
0 10 20 30 40
Tip: Dwell Volume Differences Between Instruments
Can Cause Changes in Retention and Resolution
0 10 20 30 40
VD = 0.43 mL Column: ZORBAX Rapid Resolution
Eclipse XDB-C8
4.6 x 75 mm, 3.5 µm
Mobile Phase: Gradient, 0 - 100 %B in 52.5 min.
A: 5/95 methanol/ 25 mM
phosphate
pH 2.50
B: 80/20 methanol/25 mM
phosphate
pH 2.50
Flow Rate: 0.5 mL/min
Temperature: 25°C
Injection: 5 μL
Detection: 250 nm
Sample: Mixture of antibiotics and
antidepressants
Upper trace simulates actual run
data entered into DryLab® 3.0
software
Lower trace is simulated
chromatogram for larger VD
VD = 2.0 mL
19. 19
Page 37
If VD1 > VD2
Compensate for longer VD1 by adding
an isocratic hold to VD2, such that
Hold + VD2 = VD1
If VD1 < VD2
Delay injection, such that VD2 - delay = VD1
001014P1.PPT
Trick: Measure and Correct for Dwell Volume (VD)
Page 38
Mobile Phase pH and pH Buffers
Why Are These So Important in HPLC?
•pH Effects Ionization
– Silica Surface of Column
– Sample Components of Interest
• Buffers
– Resist Changes in pH and Maintain Retention
– Improve Peak Shape for Ionizable Compounds
• Effects Column Life
– Low pH strips Bonded Phase
– High pH Dissolves Silica
20. 20
Page 39
Minimize Change in Retention/Selectivity
Lot-to-Lot
• All causes of column-to-column change*
• Method ruggedness (buffers/ionic strength)
• pH sensitivity (sample/column interactions)
*All causes of column-to-column change should be considered first,
especially when only one column from a lot has been tested.
Evaluate:
Group/Presentation Title
Agilent Restricted
September 10, 2008Month
##, 200X
Page 40
0 2 4 6 8 10 12 14 16 18
Time (min)
2-Base
3
4-Base
1
0 2 4 6 8 10 12 14 16 18
Time (min)
2
3
4
1
Lot-to-Lot Selectivity Change Related to pH Choice
• pH 4.5 shows selectivity change from lot-to-lot for basic compounds
• pH 3.0 shows no selectivity change from lot-to-lot
• Indication of poorly controlled ionization
pH 4.5 - Lot 1 pH 3.0 - Lot 1
pH 4.5 - Lot 2 pH 3.0 - Lot 2
0 2 4 6 8 10 12 14 16 18
Time (min)
2-Base
3
4-Base
1
0 2 4 6 8 10 12 14 16 18
Time (min)
2
3
4
1
21. 21
Page 41
Why Worry About pH?
pH, pKa and Weak Acids
At pH 4.2 – the sample exists as benzoic acid and the benzoate ion in a ratio
of 1:1. Peak shape can be poor
At pH 5.2 – 91% of the sample exists as the benzoate ion. RP retention decreases.
At pH 3.2 – 91% of the sample exists as benzoic acid. RP retention increases.
RCOOH RCOO- + H+
Ka = 6.4 x 10-5
pKa = 4.2
Ka =
[RCOO-][H+]
[RCOOH]
+ H+
COOH COO
_
Page 42
Effect of pH on Peak Shape at or
Near the Sample pKa
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
Time (min) Time (min)
Column: ZORBAX SB-C8 4.6 x 150 mm, 5 mm Mobile Phase: 40% 5 mM KH2PO4: 60% ACN
Flow Rate: 1.0 mL/min. Temperature: RT
pH 4.4 pH 3.0
Ibuprofen
pKa = 4.4
CH3CHCOOH
CH2CH(CH3)2
• Inconsistent and tailing peaks may occur when operating close to an analyte
pKa and should be avoided.
22. 22
Page 43
Why Worry About pH?
pH, pKa and Weak Bases
At pH 9 – the sample exists as protonated and unprotonated diphenhydramine
in a ratio of 1:1. Peak shape can be poor.
At pH 10 – 91% of the sample exists as unprotonated diphenhydramine.
At pH 8 – 91% of the sample exists as protonated diphenhydramine.
Ka =
[R3N][H+]
[R3NH+]
Ka = 1 x 10-9
pKa = 9
R3NH+ R3N + H+
CHOCH CH N2 2
CH3
CH3
∅
∅
+
H
CH3
CH3
∅
∅
CHOCH CH N2 2
+ H+
Page 44
pH vs. Selectivity for Acids and Bases
5
SCD
+
+
2 +
1
1.5
1.0
0.5
0.0
-0.5
logk«
3 4 5 6 7 8 pH
A B C
2
4
5
3
3 5 7 9
ELUENT pH
40
30
20
10
10
Retention
+
++
3
1
7,12 - OC
UDC
SOC
4
6
C
12-OC
J.C. 268(1983) 1
J.C. 111(1975) 149
Column: Nucleosil-C18
Mobile Phase: 45% ACN/55% phosphate buffer
Sample: Bile Acids
Column: mBondapak-C18
Mobile Phase: 60% 25 mM phosphate buffer
40% Methanol
1. Salicylic acid
2. Phenobarbital
3. Phenacetin
4. Nicotine
5. Methamphetamine
• Retention and selectivity can change dramatically when pH is changed.
23. 23
Page 45
Importance of pH and Buffers
A Practical Example
•Why the Sample Dictates Use
•What Happens When Buffer Used Effectively
•What Happens When Buffer Ignored or Used Improperly
Page 46
Importance of pH and Buffers - A Practical Example
Optimized Isocratic Conditions for Cardiac Drugs
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (min)
5
4
3
2
1
Column: StableBond SB-C18, 4.6 x 150
mm, 5 mm
Mobile Phase: 45% 25 mM
NaH2PO4, pH 3.0
55% MeOH
Flow Rate: 2.0 mL/min.
Temperature:35°C
Detection: UV 254 nm
Sample: Cardiac Drugs
1. Diltiazem
2. Dipyridamole
3. Nifedipine
4. Lidoflazine
5. Flunarizine
24. 24
Page 47
I Don’t Have Time to Make Buffers or Adjust pH …
0 5 10 15 20 25
Time (min)
Column: StableBond SB-C18
4.6 x 150 mm, 5 mm
Mobile Phase: A: 20% H2O
B: 80% MeOH
Flow Rate: 1.0 mL/min.
Temperature:35°C
UV Detection: 254 nm
Sample: Cardiac Drugs
• Buffers are critical to good retention and peak shape in many separations.
Even at very high % MeOH Most Components
Strongly Retained with Poor peak Shape Due to
IEX at Surface
Page 48
What If You Work Outside the Buffer Range?
0 5 10 15 20 25
5
1
2
3
4
Time (min)
Columns: StableBond SB-C18
4.6 x 150 mm, 5 mm
Mobile Phase: A: 30% 25 mM NaH2PO4, pH
4.8 unbuffered
B: 70% MeOH
Flow Rate: 1.0 mL/min.
Temperature:35°C
UV Detection: 254 nm
Sample: Cardiac Drugs
1. Diltiazem
2. Dipyridamole
3. Nifedipine
4. Lidoflazine
5. Flunarizine
Unsuitable Peak Shape
25. 25
Page 49
Don’t Forget - Match Column to pH of Mobile Phase
for Maximum Column Lifetime
low pH and high temperature (pH 0.8, 90°C)
000023P1.PPT
Purge Solvent:
50% methanol/water with
1.0% TFA
Solute: Toluene
Kirkland, J.J. and J.W. Henderson, Journal of Chromatographic Science, 32 (1994) 473-480.
Page 50
Don’t Forget - Match Column to pH of Mobile Phase
for Maximum Column Lifetime
High pH and Room Temperature (pH 11 RT)
Tip: Use Columns Designed for chosen pH
Mobile Phase: 50%ACN: 50% Water : 0.2% TEA
(~ pH 11)
After 30 injections
Initial
26. 26
Page 51
Detection Issues
Recognize Where the Problem Originates
• Is it a consequence of technique?
• Is It expected due to use of certain mobile phase
components?
• Can it be corrected by adjusting detector
parameters?
• Answers Will Help Find a Solution!
Let’s Explore Some Problems and Solutions
Page 52
Causes:
Absorbance of sample is less than the mobile phase.
Equilibrium disturbance when sample solvent passes through the
column.
Normal with Refractive Index Detectors.
Normal Negative
Peak Shape: Negative Peaks
27. 27
Page 53
Ghost Peaks
20% - 100%
MeOH Gradient
No Sample Injected
Ghost Peaks - Peaks which appear even when no sample
is injected.
Problem - Dirty Mobile Phase
60
15
30
15
0
3 7 15 17
Page 54
Noisy Baselines
Possible Causes:
Dirty Flow Cell
Detector Lamp Failing
Pulses from Pump if Periodic
Temperature Effects on Detector
Air Bubbles passing through Detector
Time
(min.)
28. 28
Page 55
Gradient Elution
Temperature Unstable (Refractive Index Detector)
Contamination in Mobile Phase
Mobile Phase Not in Equilibrium with Column
Contamination Bleed in System
Drifting Baselines
Page 56
Chromatographic Results with “Wrong” Lamp at
214 nm Wavelength
OEM LampOEM Lamp
Lamp from Generic SourceLamp from Generic Source
Tip: Could also be a symptom of aging lamp
29. 29
Page 57
Expanded View of Chromatographic Results
Generic Source Lamp at 214 nm Wavelength
OEM LampOEM Lamp
Lamp from Generic SourceLamp from Generic Source
Peak 1Peak 1 S/N = 150S/N = 150
Peak 2Peak 2 S/N = 400S/N = 400
Peak 3Peak 3 S/N = 300S/N = 300
Peak 1Peak 1 S/N = 15S/N = 15
Peak 2Peak 2 S/N = 50S/N = 50
Peak 3Peak 3 S/N = 50S/N = 50
Tip: Poor S/N makes it difficult to detect low level impurities
Page 58
Effect of Detector Response Time
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Time(min)
1.0
Agilent 1100 DAD
Agilent 1100 WPS with ADVR
Column: Poroshell 300SB-C18
2.1 x 75 mm, 5 mm
Mobile Phase:
A: 95% H2O, 5% ACN with 0.1% TFA
B: 5% H2O, 5% ACN with 0.1% TFA
Flow Rate: 2 mL/min
Temperature:70°C
Detector: UV 215 nm
Piston stroke: 20
Sample:
1. Neurotensin3. Lysozyme
2. RNaseA 4. Myoglobin
0.1 sec
0.2 sec
0.5 sec
1.0 sec
1st peak = 1.2 sec
At 20 pts/sec = 24 pts/sec
Response Time
2.0 sec
1st peak = 1.2 sec
At 5 pts/sec = 6 pts
• Tip: Adjust the response rate of your detector for best peak detection.
The System is operating well-the settings were poorly made!
Slow Data Rates Can Hinder Impurity Detection and Reduce Sensitivity
30. 30
Page 59
Conclusions
• High pressure (prevention better than the cure)
• Undesirable peak shape
• Changes in retention/selectivity
Often these problems are not associated with the column and
may be caused by instrument and chemistry issues.
•pH of mobile Phase
•Instrument Connections
•Detector Settings
•Metal Contamination
Start With the Correct Questions
•Find the Answers
•The Answers will Lead to Solutions
HPLC column problems are evident as