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The CHROMacademy Essential Guide Webcast:
What HPLC Operators Need to Know Part II
Everything Needed to Understand Column Selection, Mobile Phase Gradients and Detectors
 

Thursday 17th September 2015,
8:00am PDT / 11:00am EDT / 16:00 BST / 17:00 CEST

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.

In part one we discussed how and why mobile phases are chosen and prepared and the different variations of HPLC pumps and autosamplers which are available.

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.

Presented by Dr Paul Ferguson (Associate Principal Scientist, Astra Zeneca) and
Scott Fletcher (Technical BD Manager, Crawford Scientific).

Column Selection

  • What are the most important physicochemical parameters of the support material
  • What are the differences and relative merits of fully and superficially porous particles and what role does particle size play?
  • What are the most common stationary phases available and how do they influence selectivity – when are they best employed?

Mobile Phase Gradients

  • Are mobile phase gradients always required?
  • How is a gradient formed and what are the most important parameters to control
  • How are gradients optimized during method development and effectively transferred from HPLC to UHPLC, or vice versa.

Detectors

  • Role and function of (U)HPLC detectors?
  • Physicochemical analyte properties which influence detector choice
  • Working principles of Variable Wavelength and Diode Array UV detectors with key operational variables
  • Why, when and how would refractive index (RI), evaporative light scattering (ELSD), fluorescence (FLD) or charged aerosol detectors (CAD) be used?

Mobile Phase (Part I)
Pumping Systems (Part I)
Autosamplers (Part I)


Key Learning Objectives:

  • Understand the HPLC process and which parameters can and should be considered
  • Learn how to select and prepare mobile phases appropriately
  • Appreciate the variations in HPLC pumps and their relative benefits
  • Gain knowledge on HPLC column particles and stationary phases
  • Understand how injections are made and how the autosampler can be optimized
  • Appreciate how and why gradients are used and how they can be effectively scaled and transferred
  • Understand how HPLC detectors operate and the specific analyte properties which lend themselves to the various detection techniques

Who Should Attend:

  • All analytical scientists
  • Anyone using HPLC or UHPLC
 
Biopharmaceutical Analysis
Sponsored by
 
 

Find out more about this Month's Essential Guide Webcast »

 
 

What HPLC Operators Need to Know Part II
Everything Needed to Understand Column Selection, Mobile Phase Gradients and Detectors

All operators of (U)HPLC instrumentation should possess significant knowledge and understanding in order to operate their instrumentation in the most efficient and effective manner. This mini-series of two webcasts will detail all critical parameters in setting up the instrument for operation, performing the analysis and ensuring the system has been left in a fully functioning capacity and ready for its next user.

There are a vast array of variables associated with (U)HPLC columns and an effective (U)HPLC operative should be familiar with all so that they make informed choices when deciding on a column. They can range from purely physical factors such as column dimensions and particle size, shape and morphology but also include modifications such as incorporation of bridging organic groups in order to make the support more stable at elevated pH values. They will also vary in terms of the type of stationary phase bonded onto the surface but also in terms of the concentration of the bonded phase or whether it has been bonded in a mono-functional or a di/tri-functional manner. The type, nature and degree of endcapping used as well as the nature and amount of remaining/residual silanols may also be critical in separations.

The use of gradients to develop methods and in routine use has massively increased over the last decade. Which almost every method now developed using a gradient mobile phase, even if the final methods is run isocratically. There are some critical factors that a (U)HPLC operator should be aware of when developing or operating a gradient to ensure that they generate the most effective and robust chromatographic separations. Factors such as how the gradient is developed, the system dwell volume and the washout volume should all be understood.

There are an increasing number of detectors at the disposal of the practising liquid chromatographer meaning that unparalleled levels of sensitivity and selectivity can be generated. However, it is of paramount importance that there relative advantages and disadvantages are fully and properly understood so that they can be used in correct setting and the individual parameters specifically optimized.

Are you ‘fit for purpose’? This series of webcasts will ensure you are!

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eLearning Modules


Fast HPLC HPLC Columns

The (U)HPLC column is often the most important parameter to optimize in any method development. The variable range from the hardware physical dimensions through to the solid support particle morphology and size and also include the type of stationary phase ligand incorporated and type/nature of any end-capping employed. This is only a portion of the variables available and it therefore vital that all parameters are appreciated so that logical and effective choices can be when choosing the right column for you separation.

Silica as a Packing Material | Surface Treatment - End capping | Reversed Phase Stationary Phases

 
Fast HPLC Gradient HPLC

The use of gradients can not only vastly reduce the time taken to develop methods but can also build in time saving, resolution improvements and robustness gains. However there are a number of critical steps that a competent (U)HPLC operator must follow in order to develop, transfer and translate gradients effectively. This module details all such steps and demonstrates the advantages and pitfalls that one may encounter

Gradient HPLC Analysis | Gradient Elution Principles | Peak Shape in Gradient HPLC

 
 
Quick Guides

Fast HPLC HPLC Mobile Phases – 10 bad habits to avoid

Measuring the pH of the mobile phase after the organic has been added pH meters are calibrated to give the correct pH readback in aqueous solution – the buffers you verify this with are aqueous. If you measure the pH with the organic added, the pH will be different to that of measuring before organic addition. However, the most important point is to be consistent. If you do always measure pH after the organic is added, make sure you state this in the method so that everyone does it the same way. It won’t be 100% accurate, but at least it will be consistent. This is probably more important than having the exact pH.

HPLC Mobile Phases – 10 bad habits to avoid »

 
Fast HPLC HPLC Troubleshooting Guide to Cycling Baselines and Pressure Fluctuations

The Problem

A short term cycling baseline is seen along with pressure fluctuations...

Cycling Baselines and Pressure Fluctuations »

 
Fast HPLC HPLC Troubleshooting Guide to Peak Splitting Problems

The Problem

The analysis of an α2 adrenergic agonist and its main impurity are showing peak splitting that was not previously seen (Figure 1).  A moderate increase in system backpressure has also been observed.
The analyte is fairly hydrophobic with a log P value of 2.0.  The analyte possesses a number of basic nitrogen groups within its structure; however, the only significant pKa is at 7.49. 

Peak Splitting Problems »

 
Fast HPLC HPLC Filtration - Rocks, Boulders and Sand

First – what’s the point of filtration? Mainly to protect the HPLC system and analytical column from damage due to the build-up of particulate materials. Particulate build up usually results in an increase in system back pressure, to the point where the maximum system operating pressure is exceeded and the system shuts down...

HPLC Filtration »

 
Fast HPLC Setting HPLC Chromatographic Parameters

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

HPLC Chromatographic Parameters »

 
Fast HPLC Checklists are only for pilots?

How many times have you been in too much of a hurry to get to get a sequence running, set one tiny instrument parameter wrongly and had to spend the whole of the next day repeating the analysis...

HPLC Checklist »

 
 
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 »

 
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 the CHROMacademy Essential Guide 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. This practical guide will give you the tools required to make an informed choice every time you develop a new HPLC method.

Selecting Column Stationary Phase and Dimensions »

 
HPLC Detectors - What, Where, When, and Hows

The selection of a detector for HPLC is based on the chemical nature of the analytes and any potential interferents that may be present, the limit of detection required, and often the availability and cost of the detector. This month’s Essential Guide will focus on the operating principles of the most common HPLC detectors. The parameters that should be optimized for each detector type will be examined to give the optimum performance from the chosen detector. The advantages, disadvantages, and application areas for each detector type will also be discussed. This in depth review of HPLC detectors will result in a better understanding of the operation, optimization, and detector choice for every application.

HPLC Detectors - What, Where, When, and Hows »

 
HPLC Troubleshooting - Autosampler, Column & Detector Issues

This session examines different autosampler designs, injection valve operations and common problems associated with modern HPLC autosamplers. Sample solvent and injection volume effects on chromatography will also be discussed in detail. We will consider column hardware design, materials of construction and associated issues – including minimizing dead volume and avoiding / troubleshooting column blockage issues, especially with the popular Sub 2µm column packing materials. We will also investigate the importance of controlling the temperature of the column during analysis and the potential chromatographic effects of poor temperature control. We will conclude by reviewing common HPLC detector hardware problems and the chromatographic issues associated with detector hardware and acquisition settings. We will build a checklist of suggested maintenance operations as well as outlining common diagnostic tests.

HPLC Troubleshooting - Autosampler, Column & Detector Issues »

 
 
 
 
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Key Learning Objectives:

  • Understand the HPLC process and which parameters can and should be considered
  • Learn how to select and prepare mobile phases appropriately
  • Appreciate the variations in HPLC pumps and their relative benefits
  • Gain knowledge on HPLC column particles and stationary phases
  • Understand how injections are made and how the autosampler can be optimized
  • Appreciate how and why gradients are used and how they can be effectively scaled and transferred
  • Understand how HPLC detectors operate and the specific analyte properties which lend themselves to the various detection techniques

Who Should Attend:

  • All analytical scientists
  • Anyone using HPLC or UHPLC

Dr. Paul Ferguson
Associate Principal Scientist
Astra Zeneca

 

Scott Fletcher
Technical BD Manager
Crawford Scientific