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The CHROMacademy Essential Guide Webcast:
UHPLC Troubleshooting - How U is Your UHPLC?

Thursday 21st May 2015,
8:00am PDT / 11:00am EDT / 16:00 BST / 17:00 CEST

Since its inception UHPLC has gained popularity for enabling fast and efficient chromatography which allows the demands of modern day analyses to be met; namely increased sensitivity and higher throughput. However, moving from conventional HPLC systems to UHPLC is not without its problems and the requirement to adjust to a new way of working. Fundamental practices such as careful sample and solvent preparation and the use of appropriate fittings and tubing to reduce extra column volume and maximize peak efficiency will be discussed. We will also consider important principles such as pressure/selectivity effects, the practical implication of frictional heating, and instrument parameters such as system solvent wash out volume (as opposed to gradient dwell volume). Finally, initial conditions for method development in UHPLC and some simple, easy to use equations which can be utilized to facilitate method transfer from HPLC to UHPLC will be provided.

Presented by Dr. Dwight R. Stoll (Assistant Professor of Chemistry, Gustavus Adolphus College) and Tony Taylor (Technical Director, Crawford Scientific).

  • Critical differences in UHPLC
  • Making the right connections - fittings, ferrules, and tubing
  • Extra column volume – you CAN make a difference
  • Column selection – small particles or core shells – the debate
  • Eluent preparation for UHPLC
  • Selectivity and Pressure – they are related
  • Frictional heating  - practical implications
  • Practical detector considerations
  • Taking the guess work out of initial method development
  • Problem free method transfer – including wash out volume
  • Assessing performance improvements when moving to UHPLC – just how U is your UHPLC?

Key Learning Objectives:

  • Understand the key differences between conventional HPLC and UHPLC
  • Understand the requirements for sample and solvent preparation with UHPLC
  • Learn how to correctly plumb a UHPLC instrument to avoid band broadening effects
  • Choosing column dimensions to take advantage of higher pressure and linear velocity
  • Gain knowledge of how pressure and frictional heating can change a separation when transferring to UHPLC
  • Improve method transfer from HPLC to UHPLC by using simple equations and taking into account system effects such as wash out volume
  • Assessing performance improvements when moving to UHPLC – just how U is your UHPLC?

Who Should Attend:

  • All analytical scientists
  • Anyone using or beginning to use UHPLC
  • Anyone requiring higher throughput
 
Biopharmaceutical Analysis
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Find out more about this Month's Essential Guide Webcast »

 
 

UHPLC Troubleshooting - How U is Your UHPLC? - Tutorial

Since its inception UHPLC has gained popularity for enabling fast and efficient chromatography which allows the demands of modern day analyses to be met; namely increased sensitivity and higher throughput. However, moving from conventional HPLC systems to UHPLC is not without its problems and the requirement to adjust to a new way of working. Fundamental practices such as careful sample and solvent preparation and the use of appropriate fittings and tubing to reduce extra column volume and maximize peak efficiency will be discussed.

We will also consider important principles such as pressure/selectivity effects, the practical implication of frictional heating, and instrument parameters such as system solvent wash out volume (as opposed to gradient dwell volume). Finally, initial conditions for method development in UHPLC and some simple, easy to use equations which can be utilized to facilitate method transfer from HPLC to UHPLC will be provided.

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eLearning Modules


Fast HPLC Fast HPLC

'Fast HPLC' has come to refer to the technique of decreasing analysis time using a variety of novel column and instrument technologies. Speed increases are usually measured using 'Peak Capacity' - the number of compounds separated with satisfactory resolution per unit time. For example a separation of ten compounds in five minutes is more efficient ('faster') than a separation of five compounds in the same timescale.

Increasing Resolution via Improved Efficiency | Speeding up HPLC Separations | HPLC Method Conversion

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Quick Guides

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Fast HPLC is where we obtain the same (or better) separation, but with a shorter runtime.

This is obtained simply by exploiting certain parameters (the column particle size, internal diameter, length and particle morphology) to increase the efficiency of the separation. If the column and conditions are more efficient we can do the same job in less time.

You Need a Fancy System to Run Fast HPLC – or do you? »

 
Speeding up your HPLC Speeding up your HPLC

Cars, internet, airplanes, people, everything is getting faster, including chromatography.  The ultimate goal for chromatographers is to be able to achieve analyses in shorter run times without compromising the resolution, efficiency, and sensitivity that is strived for during method development.  The wide availability of modern stationary phase packing materials and column dimensions makes this goal achievable to all chromatographers without, necessarily, the need for expensive UHPLC equipment.

Speeding up your HPLC »

 
HPLC Column Dimensions 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.

HPLC Column Dimensions »

 
Superficially Porous or Sub 2µm Stationary HPLC Phases Superficially Porous or Sub 2µm Stationary HPLC Phases

The ability to produce rugged reproducible column packing materials below 2.0μm in diameter has been an important and relatively recent breakthrough. Efficiency increases markedly when using particles less than 2.5μm in diameter due to the reduction in the Mass Transfer contribution to band broadening.

Smaller particles have shallower pores (including through pores) which ultimately results in a smaller distribution of analyte 'retention' times within each pore during the 'diffuse in / adsorb / desorb / diffuse out' process.

Superficially Porous or Sub 2µm Stationary HPLC Phases »

 
Core Shell Particles – Present and Future Core Shell Particles – Present and Future

In this session we highlight the benefits of core-shell particle technology and the reasons behind their surprisingly high efficiency. We will discuss where core-shell particles fit against other efficiency improving measures such as the use of sub 2μm particles with high pressures and also narrow bore HPLC columns. Also discussed are the benefits of using larger and smaller particle size core-shell particles and how particle size and particle morphology might be used to achieve separation requirements. The interesting topic of core to shell thickness ratio will be investigated which will in turn lead to a discussion of current limitations and future possibilities with core-shell technology.

Core Shell Particles – Present and Future »

 
Gradient HPLC Gradient HPLC - 10 Things You Absolutely Need to Know

Everyone loves a top ten list !
So here is our list of “Top Ten Things You Absolutely Need to Know About Gradient HPLC”. 

Whether you are a novice or seasoned gradient HPLC user there is something in here for everyone.

Gradient HPLC - 10 Things You Absolutely Need to Know »

 
HPLC – Selecting Column Stationary Phase and Dimensions 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. Instead, why not join us for this webcast on HPLC column selection in which we will look at the available column stationary phases and support materials (including conventional fully porous and core-shell/superficially porous material), column dimensions and the effect that they can have on your chromatography, and how to optimize and improve your current chromatographic separations.

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

 
Translating and Transferring HPLC Methods Translating and Transferring HPLC Methods

An informative session on method translation and transfer in HPLC. In this session, we present a definitive guide on everything that one would need to consider for translating and transferring HPLC methods. The session includes in-depth consideration of the hardware and data acquisition requirements for simple method transfer or translation to different column / particle size and morphology. We present many worked examples of calculations, approaches and pitfalls associated with method scaling and transfer and include up to date concepts such as frictional heating and pressure based selectivity to ensure you are fully up to date in this subject area. A must see for everyone translating or transferring HPLC methods.

Translating and Transferring HPLC Methods »

 
 
 
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  • Understand the key differences between conventional HPLC and UHPLC
  • Understand the requirements for sample and solvent preparation with UHPLC
  • Learn how to correctly plumb a UHPLC instrument to avoid band broadening effects
  • Choosing column dimensions to take advantage of higher pressure and linear velocity
  • Gain knowledge of how pressure and frictional heating can change a separation when transferring to UHPLC
  • Improve method transfer from HPLC to UHPLC by using simple equations and taking into account system effects such as wash out volume
  • Assessing performance improvements when moving to UHPLC – just how U is your UHPLC?

Who Should Attend:

  • All analytical scientists
  • Anyone using or beginning to use UHPLC
  • Anyone requiring higher throughput

Dr. Dwight R. Stoll
Assistant Professor of Chemistry, Gustavus Adolphus College

 

Tony Taylor
Technical Director
Crawford Scientific