- Troubleshooting Liquid Chromatographic Separations: Avoiding Common Issues through Informed Method Development Practices (Monday, 9:25-10:10 am), Room: Magnolia, 3rd floor of the Hilton
- Top Ten HPLC/UHPLC Column Myths (Monday, 11:10-11:55 am), Room: Magnolia, 3rd floor of the Hilton
- Column Efficiency (Monday, 1:55-2:40 pm), Room: Magnolia, 3rd floor of the Hilton
- Microfluidics: Basic Principles and Practical Applications (Tuesday, 8:55-9:40 am), Room: Magnolia, 3rd floor of the Hilton
- Synthesis Applications of Polymer Monoliths in Liquid Chromatography (Tuesday, 11:35am-12:20pm), Room: Magnolia, 3rd floor of the Hilton
- Ion Chromatography vs. Electrophoresis (Tuesday, 2:00-2:45 pm), Room: Magnolia, 3rd floor of the Hilton
- Theoretical and Practical Aspects of UHPLC (Wednesday, 8:55-9:40 am), Room: Magnolia, 3rd floor of the Hilton
- Dwell Volume: Hidden Aspects of UPLC Method Transfer (Wednesday, 11:35am-12:20pm), Room: Magnolia, 3rd floor of the Hilton
- Changes in the Role of Separation Scientists due to New Regulations (Wednesday, 1:55-2:40 pm), Room: Magnolia, 3rd floor of the Hilton
Monday Tutorial: Troubleshooting Liquid Chromatographic Separations: Avoiding Common Issues through Informed Method Development Practices
David S. Bell, Supelco/Sigma-Aldrich
Monday, 9:25-10:10 am, Magnolia Room, 3rd floor of the Hilton
Many of the difficult problems encountered in liquid chromatography are a result of method development practices rather than column or instrument issues. Unfortunately, the HPLC system is often treated as a black box. When issues arise practitioners are often left wondering what could have gone wrong. The objective of this tutorial session is to assist in the diagnosis of current issues and help prevent potential future problems through a discussion of the choices that are commonly made during method development. Intended for beginners to intermediate practitioners, the tutorial will center mainly on the impact of mobile phase and column parameters and how/why these choices may impact method reliability, reproducibility and operation. In addition, selected common issues pertaining to the use of modern instrumentation, column technologies and alternative modes of separation will be discussed. Attendees should come away armed with a better understanding of the role of method components as well as the potential problems that may arise from their use and potential misuse.
Monday Tutorial: Top Ten HPLC/UHPLC Column Myths
Ronald E. Majors, Columnist, LCGC No. America
Monday, 11:10-11:55 am, Magnolia Room, 3rd floor of the Hilton
In any field there are often “misconceptions” or “myths” that are perpetuated and passed on to the next generation. These myths are often driven by a lack of understanding by practitioners of the real issues. And these myths can change as time moves on. Since HPLC is approaching its 50th year, many column myths have already been passed down to 2 generations of liquid chromatographers. Recently, ultrahigh pressure liquid chromatography (UHPLC) has come into its own and a new set of myths are arising. The purpose of this tutorial is to try to dispel some of these myths before they get further perpetuated. Some of the myths to be discussed are: “all C18 columns are the same”; “it takes a minimum of 10 column volumes to re-equilibrate an LC column”; “you can’t reverse an HPLC column to flush out particulates”; “UHPLC packed columns plug easier than conventional HPLC packed columns” and the list goes on. With help of a team of myth-busters, notables in the chromatography field, I will provide proof statements and literature references that showed that these myths are unjustified. There are many myths that are still around but didn’t make the top ten. However, most likely old myths will continue, some will fade away, and new myths will arise as more practitioners enter the chromatography community. Those new arrivals to this community may have to discover these myths on their own (since they don’t necessarily listen to the old timers anyway). Perhaps new myth-busters will take on the next generation of myths.
Monday Tutorial: Column Efficiency
Fabrice Gritti, University of Tennessee
Monday, 1:55-2:40 pm, Magnolia Room, 3rd floor of the Hilton
This tutorial is addressed to a general audience willing to familiarize itself with the fundamental factors that control the efficiency of modern chromatographic columns (fully porous particles, core-shell particles, monolithic columns) in liquid chromatography. The experimental methods developed for the determination of the universal reduced HETP plots, h(v), and its three main contributions are presented and explained in details. They will include the peak parking method for the determination of the longitudinal diffusion HETP term, B/v, the selection of the most relevant model of effective diffusion in chromatographic beds for the determination of the solid-liquid mass transfer resistance, Cv, and the numerical peak integration method for the measurement of the eddy diffusion term, A(v). The results will be discussed for standard fully porous particles, sub-3 µm core-shell particles, and for the monolithic columns of the first and second generation. Eventually, it is shown how the accurate knowledge of the h(v) plots can be used in practice to rapidly assess the expected change in column efficiency when changing the physical state of the fluent (HPLC to SFC), the size of the packed particles (HPLC to vHPLC), or the molecular weight of the analytes (from MW=100 Da to MW=50 kDa). Finally, it is revealed how much room is left for further improvement of the current column technology in terms of generating higher plate counts.
Tuesday Tutorial: Microfluidics: Basic Principles and Practical Applications
Steven Soper, University of North Carolina, Chapel Hill
Tuesday, 8:55-9:40 am, Magnolia Room, 3rd floor of the Hilton
Microfluidics is rapidly becoming an enabling tool for a variety of applications like medical diagnostics, environmental monitoring, DNA sequencing, proteomics and drug discovery to name a few. Indeed, microfluidics has been deployed in a number of high profile commercial products such as Illumina (HiSeq instrument flow cell for DNA sequencing), Fluidigm (digital PCR), Fluxion (circulating tumor cell isolation), RainDance Technologies (droplet microfluidics) and Advanced Liquid Logic (digital microfluidics for sample preparation). In this tutorial, a brief overview of fabrication technologies for microfluidic chips fabricated in glass, PDMS and thermoplastics (PMMA) will be discussed with a comparison made between these microfluidic platforms in terms of their fabrication modalities, surface chemistry, electrical behavior, optical detection and solvent compatibility. Information will also be provided on different types of microfluidic operational modes including centripetal, digital and droplet microfluidics and conventional flow-based microfluidics. A survey of different applications enabled by microfluidics will be presented as well. Some examples include microfluidic devices for the solid-phase extraction and purification of targets from biological samples (isolation of RNA/DNA from clinical samples), PCR amplifications, electrophoresis for DNA fragment sizing, isolation of rare cells (circulating tumor cells), fluidic interfacing between micro-scale separations and mass spectrometry and integrated systems for carrying out point-of-care diagnostics. Special attention to scaling effects (what does miniaturization offer) and fabrication/material selection on device performance will be offered. Finally, a brief introduction to nanofluidics and its future applications will be provided.
Tuesday Tutorial: Synthesis and Applications of Polymer Monoliths in Liquid Chromatography
Emily Hilder, University of Tasmania
Tuesday, 11:35 am-12:20 pm, Magnolia Room, 3rd floor of the Hilton
Polymer monoliths were first introduced over 20 years ago as an alternative to packed particle columns for chromatography. Numerous publications in the literature have since demonstrated that polymer monoliths can offer significant advantages over particle packed columns, particularly for the separation of large biomolecules. This tutorial is suitable for those completely new to the field who want to learn something about polymer monoliths for the first time, and also those wishing to extend their knowledge about the latest developments in this area. The tutorial will cover practical aspects including the synthesis of polymer monoliths in different formats including capillaries, columns and microdevices, characterization and performance evaluation of polymer monoliths, strategies to achieve best performance for monolithic columns and design and application of polymer monoliths for a range of separations from small to large molecules using different separation modes (e.g. reversed phase, HILIC, ion-exchange, HIC). The advantages and disadvantages of polymer monoliths will be discussed and examples will be included to illustrate when the performance of polymer monoliths can be superior to packed particle columns.
Tuesday Tutorial: Ion Chromatography vs. Electrophoresis
Christopher Pohl, Thermo Scientific
Tuesday, 2:00-2:45 pm, Magnolia Room, 3rd floor of the Hilton
This tutorial will provide an overview of the relative strengths and weaknesses of both Ion Chromatography and Electrophoresis when applied to the analysis of small ionic analytes. This overview will take a look at the two techniques from both a historical and a modern day point of view. Various analytical figures of merit such as speed, resolution, sensitivity, precision and accuracy will be covered. In addition, more subjective aspects such as popularity will also be reviewed.
Wednesday Tutorial: Theoretical and Practical Aspects of UHPLC
Monika Dittmann, Agilent Technologies
Wednesday, 8:55-9:40 am, Magnolia Room, 3rd floor of the Hilton
The increasing need for higher speed and resolution in analytical separations drives the trend towards moving from HPLC (ca. 400 bar max. pressure) to UHPLC (>1000 bar). This tutorial will cover the basic principles of UHPLC and discuss various aspects that need to be considered to successfully apply this technology in method development and routine work. In the first section the basic theory of mass transfer in columns packed with different particle types will be discussed. It will be demonstrated how the increase in operation pressure and temperature can result in enhanced resolution and/or speed of separation. In the second section optimization strategies for UHPLC separations for different types of applications will be discussed using the concept of kinetic plots. The kinetic plot (also known as Poppe plot) model uses column efficiency along with column permeability to determine the number of plates generated per unit time. This is a much more useful measure for column performance than using column efficiency or plate number alone. The kinetic plot model will be explained in detail and examples for different particle sizes and morphologies will be shown. The third section will focus on instrumental aspects of UHPLC such as dwell volumes and system dispersion, where the requirements are much more stringent as in HPLC instruments due to the smaller column diameters used in UHPLC. The impact of extra-column volume on the efficiency for small bore columns will be demonstrated for isocratic and gradient separations. Another topic is the transfer of methods between HPLC and UHPLC instruments where differences in system properties (dwell volume and mixing behavior) can lead to changes in retention times and selectivity. Finally some of the side effects of high pressure separations will be discussed such as frictional heating, possible selectivity changes and changes of physical properties of eluents at high pressures.
Wednesday Tutorial: Dwell Volume: Hidden Aspects of UPLC Method Transfer
Eduard Rogatsky, Daniel Stein, Albert Einstein College of Medicine
Wednesday, 11:35 am-12:20pm, Magnolia Room, 3rd floor of the Hilton
Method transfer from HPLC to UHPLC is not a straightforward task despite the apparent simplicity of the task. Among with simple parameters such as column dimensions and flow rate, it is essential to account for system dwell volume for both systems. Surprisingly, we found that dwell volume assessment is not a simple task. Different manufacturers measure dwell volume using different methodologies. As a result, the calculated values may, and will vary, and even be misleading. Currently, there is no consensus on the standard method to use. Therefore, a uniform protocol for pump volume measurement is essential. We have investigated pump dead volume using UV-based techniques (slope intercept and half-max signal) and compared this with absolute measurements, and with standard volume addition. We found that different approaches and conditions of measurement generate different results. Surprisingly, we found that the gradient delay volume is different from the physical volume stored inside of the pump. The next paradoxical finding is that gradient delay volume is strongly dependent upon pump hardware and especially – mixer design, rather than actual pump internal volume. As a consequence, 2 identical internal volumes may result in different gradient delay volumes. Our next important finding was that the use of a linear gradient provides more accurate information compared to a step gradient for delay volume evaluation. On the other hand, we found that a step gradient is an excellent source of information about pump equilibration dynamics (we have termed this the dispersion factor), which can be an additional characteristic of pump performance. Successful HPLC to UHPLC (or backwards) method transfer requires a detailed understanding of dwell volume assessment. It is of great importance to alert both end-users and manufacturers, that one of the key parameters (dwell volume) in method transfer may not be reliable as stated.
Wednesday Tutorial: Changes in the Role of Separation Scientists due to New Regulations
Lois Ann Beaver, LAB Enterprises
Wednesday, 1:55-2:40 pm, Magnolia Room, 3rd floor of the Hilton
The new internationally accepted regulatory approach for the manufacture of pharmaceuticals places greater responsibility on the scientist. An understanding of and ability to communicate the scientific basis for choices made in performing separations is required. While international guidance is mainly concerned with processing pharmaceuticals and related chemicals, other chemical production areas are under consideration. This session will focus on aspects of recent domestic and international regulations and guidance of interest to separation scientists involved with pharmaceuticals and biopharmaceuticals. Participants will be provided a summary of freely available information sources to obtain guidance as needed.