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The CHROMacademy Essential Guide Webcast:
Critical Evaluation of HPLC Methods


Thursday 16th June 2016,
8:00am PDT/ 11:00am EDT/ 4:00pm BST/ 5:00pm CEST

There are numerous factors that affect the quality of your HPLC separation; including mobile phase composition, temperature, ionic strength, pH, column dimensions, stationary phase chemistry and support material characteristics.  These can all be manipulated to change and improve your HPLC analyses.  However, what are the changes we should expect?  Will retention improve if another solvent is used and would we expect selectivity to change?  Will certain analytes elute in the same order if the mobile phase pH, ionic strength, additives or temperature are changed?  This Essential Guide webcast will evaluate many of the aspects of the HPLC process giving you useful practical information on how to use these parameters to alter your HPLC method, resulting in better and more robust chromatography.

Topics Covered

  • The effect of mobile phase polarity on HPLC separations
  • The role of a buffer in an HPLC separation and how it should be chosen
  • The effect of temperature on analyte retention and selectivity
  • Additives for improved chromatography
  • Stationary phase and support material chemistry
  • The effect of column dimensions on gradient elution
  • Why and when you would use a guard column or in-line filter


Presenter Information »


Critical Evaluation of HPLC Methods - Tutorial

This month’s webcast focused on the critical evaluation of HPLC methods. Every facet of the HPLC method can impact your chromatography, therefore, using the CHROMacademy material provided in this month’s tutorial you can fully understand how you can use each parameter of an HPLC method to improve resolution or produce alternate chromatographic selectivity and results.

Our Quick guides, CHROMacademy course content, and archived webcasts and tutorials will provide you with the knowledge you require to apply this powerful analytical technique to your applications. Our content will provide you with the basic theory behind the separation mechanism, teach you how to alter mobile phases to control retention of ionizable analyte, give you an understanding of the effect of stationary phases on chromatography, and impart practical knowledge on aspects such as column selection.

Watch the webcast

eLearning Modules

Reversed Phase HPLC

Reversed phase HPLC is one of the most utilized modes of chromatography. This comprehensive module will provide you with the knowledge and understanding you need to apply this method, design or improve mobile phases through solvent choice or pH adjustment, and which parameters can be altered to change selectivity and retention and improve resolution.

Analyte Retention in Reversed Phase HPLC | Changing the Organic Modifier
Reversed Phase HPLC of Ionizable Samples
| Controlling the Extent of Ionization
Basic Analytes and Ion Suppression

Column Chemistry

Column stationary phase chemistry has the largest impact on selectivity which in turn affects resolution to the greatest degree. Understanding the phase chemistries as well as the influence of the packing material can help to produce robust chromatography with optimum resolution.

Chemically Bonded Phases | End capping | Reverse Phase Stationary Phases | Silanol and Separation

Mobile Phase Considerations

The chemical nature of the mobile phase can be altered to affect the retention and separation of analyte molecules. However, there are some physical parameters which must be considered such as, viscosity, UV cut off, and mobile phase preparation (degassing, filtering etc.) in order to obtain successful analyses.

Analyte Retention Processes | Solvent Type and Selectivity | Optimising Selectivity using Retention
Solvent Miscibility | UV Cut Off

Quick Guides

Why Is pH Important for HPLC Buffers?

The ionization state of an analyte affects its retention in HPLC. Ionization is controlled by buffers in the mobile phase pH, therefore, understanding the role of pH and how it affects analyte ionization and retention gives you an excellent tool for manipulating separations.

Why Is pH Important for HPLC Buffers? »


Is the Art of HPLC Method Development Dead?

What do you think? Is there an art to method development? Read this article and see if you agree or find out if there’s anything you could be doing to improve your method development.

Is the Art of HPLC Method Development Dead? »


How to Choose an HPLC Column

Tony Edge (Agilent Technologies) shares some great general hints and tips for HPLC column selection.

How to Choose an HPLC Column »


HPLC Column Dimensions

HPLC columns are manufactured in a variety of different internal diameter and length combinations, as well as having an assortment of particle sizes. HPLC column dimensions will affect the efficiency sensitivity, and speed of an analysis. The choice of column dimensions will depend on the chromatographic application; analytical, semi-preparative, preparative, number of analytes in the mixture etc. However, the dimensions can also be altered to improve chromatography by achieving more efficient, sensitive, and faster analyses.

HPLC Column Dimensions »


HPLC Mobile Phases - 10 Bad Habits to Avoid

How many of our 10 bad habits are you guilty of?

Find out how many? »

What HPLC Operators Need to Know Part I

During this two part event we will look at the entire HPLC separation process from mobile phase selection and preparation, to injection, separation, and detection, from the perspective of the laboratory practitioner. We will provide practical hints and tips for the essential HPLC parameters in order to make your use of this powerful technique straightforward and successful.

What HPLC Operators Need to Know Part I »

What HPLC Operators Need to Know Part II

Part two starts with column selection probably one of the most important choices in any chromatographic method. We will share our insights on how to select the correct column each time covering particle size, morphology, physicochemical properties as well as stationary phase selection. Finally, we will detail how to utilize and optimize HPLC gradients and review the various detectors available and when and how they are best employed.

What HPLC Operators Need to Know Part II »

Critical Choices in HPLC - Selecting Column Stationary Phase and Dimensions

How do you currently select a column for an HPLC application? Do you just use the one that is on the instrument, root around in an old drawer and hope that you pull out one that works, use your favorite C18 or ODS column, or borrow one from a colleague who’s chromatography is currently working particularly well? These are probably not ideal situations but there will be many of us who are guilty of these exact scenarios...

Critical Choices in HPLC - Selecting Column Stationary Phase and Dimensions »

Developing Better HPLC (MS) Methods

Where do you start when you want to develop a better HPLC method? Whether you work in a regulated environment or not, setting specifications for your HPLC protocols is a good practice. In this Essential Guide we will set out guidelines for chromatographic parameters such as retention, resolution, and efficiency that can be used to improve your HPLC methods prior to method validation. We will explain how mobile phase design and instrument parameters can be used to achieve improvements in method robustness and what to look for when a method is failing. Practical tips for sample and eluent preparation, and the correct detector settings to use will also be discussed.

Developing Better HPLC (MS) Methods »

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Tony Edge ACG R&D
Leader for Church Stretton
Centre of Excellence
Agilent Technologies

Tony Edge is a Research Manager at Agilent technologies, with more than 25 years of experience in chromatography gained in manufacturing and in the pharmaceutical industry.

He is the author of more than 25 papers and has written two book chapters.

In 2008, he was fortunate enough to be awarded the Desty memorial lecture for his contributions to innovating separation science, and in the same year also won a clinical excellence award from AstraZeneca.

Tony is the Vice-President of The Chromatographic Society in the UK and a contributing editor for the Chromatography Today magazine. Tony is also part of the Reid Bioanalytical conference organising committee, and a permanent member of the scientific committee for the International Symposium on Chromatography.

Dr Doug Carlton

Tony Taylor Technical Director, Crawford Scientific

Tony comes from a pharmaceutical background and has many years' research and development experience in small molecule analysis and bio analysis using LC, GC and hyphenated MS techniques.

Tony is actively involved in method development within the analytical services laboratory at Crawford Scientific and continues to research in LC-MS and GC-MS methods for structural characterisation.

Tony is the Technical Director of the CHROMacademy and has spent the past 12 years as a trainer and developing on-line education materials in analytical chemistry techniques