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The CHROMacademy Essential Guide Webcast:
Developing Better HPLC MS Methods

Thursday 28th July 2016,
8:00am PDT/ 11:00am EDT/ 4:00pm BST/ 5:00pm CEST

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.  

Presented by Prof. Kevin Schug
(Professor and the Shimadzu Distinguished Professor of Analytical Chemistry, Department of Chemistry and Biochemistry, The University of Texas at Arlington)

Dr. Dawn Watson
(CHROMacademy Technical Expert, Crawford Scientific).

Topics Covered

  • What target specifications should be set for an HPLC method
  • How sample preparation can impact on the sensitivity and robustness of an HPLC method
  • Stationary phase chemistry and important column properties
  • Designing ‘smart’ eluent solutions
  • Detector parameters to improve quality and robustness
  • Developing inherently robust methods
  • Aspects of LC-MS methods which can (and should!) be optimized


Presenter Information »


Developing Better HPLC MS Methods - Tutorial

This month’s webcast focused on developing better HPLC (MS) methods.  The CHROMacademy material provided in this month’s tutorial will help you understand some of the basic principles behind HPLC so that you can ask the big questions like, what do I want from my method, what limits do I want to set on my chromatographic parameters, and how do I optimize my mass spec detector to give the best possible results?

Our Quick guides, CHROMacademy course content, and archived webcasts and tutorials will provide you with the knowledge you require to design better HPLC (MS) methods.  From considering the sample diluent to choosing an HPLC column and finally detecting your analytes there is a wealth of knowledge required as a chromatographer, but we have it all here at your fingertips.

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

Chromatographic Parameters

Understanding the fundamental chromatographic parameters associated with HPLC is the first step to being able to manipulate them to produce better chromatographic methods. 

How to change Retention (Capacity) Factor | How to change Selectivity | How to change Efficiency

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

Fundamental LCMS

The following pages will examine how to optimize LC-MS interface parameters in ESI, APCI, and APPI.

Optimsing the Sprayer Position | Electrospray - Eluent Flow Rate | Nebulisation - Applied Potential Difference
Source Heating | APCI Source Parameter Optimisation | APPI Source Parameter Optimisation

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? »

My LC-MS Isn’t Behaving. Where Do I Start?

Instrument manufacturers try to convince us that mass spec is just another detector. Most of us who work with LC-MS know that’s simply not the case – they can be maintenance intensive, unforgiving and generate complex information. When they’re not working it can be difficult to work out exactly where the problem lies. Here’s some advice to point you in the right direction.

My LC-MS Isn’t Behaving. Where Do I Start? »

Does Mass Spectrometry Need Chromatography?

Prof. Kevin Schug (University of Texas at Arlington) discusses the merits of combining chromatography and mass spectrometry.

Does Mass Spectrometry Need Chromatography? »


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 »


Setting HPLC Chromatographic Parameters

Whether you work in a regulated environment or not, setting chromatographic performance targets can help to keep us focused.  Let’s consider how to set these targets and examine some real life examples that may not always follow the rules.

Setting HPLC Chromatographic Parameters »


Sample Diluent Effects in HPLC

Most of us will know that the solvent (diluent) used to prepare HPLC samples can have an effect on HPLC peak shapes.  The following discussion highlights some facts, figures and tips and tricks that can help in a practical situation.

Sample Diluent Effects in HPLC »

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 »

What LC-MS Operators Need to Know

Many people use HPLC with Mass Spectrometric Detection without properly understanding the working principles or key parameters.

If you are using HPLC-MS instruments, and would like to know more about how they work and how to get the best from them, this webcast is for you!

What LC-MS Operators Need to Know »

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Prof. Kevin Schug (Professor and the Shimadzu Distinguished Professor of Analytical Chemistry, Department of Chemistry and Biochemistry, The University of Texas at Arlington)

Kevin A. Schug is Professor and the Shimadzu Distinguished Professor of Analytical Chemistry in the Department of Chemistry and Biochemistry at The University of Texas at Arlington (UTA). 

He received his B.S. degree in Chemistry in 1998 from the College of William and Mary, and his Ph.D. degree in Chemistry from Virginia Tech in 2002 under the supervision of Prof. Harold M. McNair. 

From 2003-2005, he performed post-doctoral research in the laboratory of Prof. Dr. Wolfgang Lindner at the University of Vienna in Austria. Since joining UTA in 2005, his research has been focused on the theory and application of separation science and mass spectrometry for solving a variety of analytical and physical chemistry problems.  He has over 100 peer-reviewed publications and 400 presentations, posters, and invited talks to his group’s credit. 

He has been the primary mentor and research advisor to more than 20 graduate and 50 undergraduate students.  Dr. Schug has received the 2009 Emerging Leader in Chromatography award given by LCGC magazine, an NSF CAREER award, the 2009 Eli Lilly and Company ACACC Young Investigator Award in Analytical Chemistry, and the 2013 American Chemical Society Division of Analytical Chemistry Young Investigator in Separation Science Award.

For his teaching, he received the 2014 University of Texas System Regents’ Outstanding Teaching Award and was named in 2016 as a Fellow of the University of Texas System Academy of Distinguished Teachers. He is a member of the Editorial Advisory Board of LCGC Magazine (Advanstar), Analytica Chimica Acta (Elsevier), and the Journal of the American Society for Mass Spectrometry (Springer). He is a Senior Editor for Journal of Separation Science (Wiley).

Dr Doug Carlton

Dr. Dawn Watson (CHROMacademy Technical Expert, Crawford Scientific)

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. 

The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands.  As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis. 

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450.  In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.