In November 2010, Lab Manager Magazine organized a first-of-its-kind “Product Showcase” webinar, featuring a panel of seven experts representing some of the leading vendors in the ultra high performance liquid chromatography (UHPLC) market; these experts discussed their perspectives on the new applications and trends in the field. This online event attracted a large international audience from diverse backgrounds and with various levels of expertise who were looking for an opportunity to interact with the panelists in real time, and to get their insights and advice on issues that were important and pertinent. Each panelist gave a brief presentation to outline the benefits of using UHPLC, and to help users decide if making the transition from traditional HPLC to UHPLC is right for them. Below are answers to attendees’ follow-up questions, provided by panelists Diab Elmashni, senior marketing manager, LC and LC/MS, Thermo Fisher Scientific; Fraser McLeod, senior director of product marketing, Dionex Life Sciences Business Unit; and Wilhad Reuter, technology specialist, PerkinElmer.
Q: With all the different UHPLC systems available, how does one choose the right system for his or her laboratory needs?
Elmashni: One should look at the features that are important to him or her. For example, do you need a quaternary pumping capability? How important is ease of use or are multiple detection technologies, or the ability to scale up to LC/MS? These are some important considerations.
McLeod: The key questions are: Will the system run the methods you use right now and run the future methods that you will develop? For your current methods, you should essentially look for the same kind of system, but with UHPLC compatibility. For example, if your current method uses a quaternary pump, you should be looking for a system with a UHPLC quaternary pump. With regard to future methods, you should look for a UHPLC system with a lot of flexibility. Some systems are very restricted in things like flow rate and injection volume range. Such systems may severely restrict your future application needs, so it is always best to look for systems that provide wider operational ranges. Finally, ease of use is a key selection criterion. Here you need a system that makes it easy to perform day-to-day tasks (such as changing over columns), and you need to be able to use control software that makes creating, evaluating, processing, and reporting your data as straightforward as possible.
Reuter: One should decide what columns he or she wishes to use and the overall flow rate requirements. This combination will then determine the highest pressure limit required. He or she should also have an idea of the expected injection volume ranges. From knowing the desired flow, pressure, and injection limits/ranges, one should select a system that meets these needs. Almost all the available UHPLC systems from reputable vendors that meet these requirements should be quite suitable.
Q: What are the key differences between sample and mobile phase preparations for HPLC and UHPLC?
Elmashni: UHPLC samples will need to be cleaner, because you are dealing with a smaller particle size, and dirty samples can easily plug up the sub-2-micron columns, whereas with a 5-micron column it will not be a problem. You will also be injecting nearly 80 percent less sample than before. With regard to the mobile phase, you will be using a lot less solvents, and these solvents should also be HPLC grade to ensure that they are clean for both the integrity of the analysis, the repeatability, and the lifetime of the column.
McLeod: UHPLC uses columns with smaller particle sizes (less than 3 μm instead of 5 μm). This requires that the mobile phase is filtered before use, with filters of 0.45 μm mesh size or, better, 0.2 μm. In some cases, it will also be necessary to filter samples before injection, particularly if the samples contain a lot of undissolved particles. UHPLC typically requires less sample volume, enabling volume reduction in sample preparation steps by a factor of 4 to 5. This can save a lot of time, and also saves on solvent costs. Finally, if the high-resolution power of UHPLC is fully exploited, the removal of matrix compounds, as normally required in conventional LC, can sometimes be skipped.
Reuter: There are two primary requirements for UHPLC. Always filter your samples and solvents through 0.2 μm filters, and typically keep the injection volumes under 5 to 6 μL to avoid overloading UHPLC columns.
Q: What are the cost savings resulting from the use of UHPLC? What are the incremental costs involved in the routine maintenance of UHPLC systems compared to conventional HPLC?
Elmashni: The cost savings will be realized in using fewer solvents, less instrument time, and less labor to achieve the same amount of samples analyzed. There will be an increase in revenue if the number of instruments and labor are maintained, because the sample-per-hour ratio will dramatically increase. Sample runs that used to take 10 minutes now take 1 minute. In some cases, run times of 50 to 60 minutes have been reduced to 3 to 4 minutes. The cost in routine maintenance on a per-sample basis does not increase; UHPLC is just as robust as HPLC on a per-sample basis. You cannot measure the maintenance cost on a time basis, because you are running a lot more samples in the same time frame. For example, if you used to measure maintenance cost on a monthly basis, you are running 10 times the samples you used to run in that same month with UHPLC, so you will go through more columns and replaceable parts, but that is because you drastically increased your throughput. If you calculate the maintenance cost on a per-sample basis, you may even see a decrease.
McLeod: The main cost savings is the amount of time it takes to perform an analysis. Analysis times are reduced by a factor of between 5 and 50, providing labs with faster data and allowing instruments to run many more samples per day. In many cases, labs will find that they can run their current workloads with fewer UHPLC systems than they have HPLC systems. Another saving is solvent costs. UHPLC uses 80 to 95 percent less solvent than HPLC. There are no significant extra costs for UHPLC maintenance, as the mechanical robustness of wear parts in the latest-generation UHPLC systems comes close to that of conventional LC. If maintenance costs per analysis are considered rather than maintenance costs per time, UHPLC provides significant savings on maintenance costs.
Reuter: UHPLC has considerably reduced costs related to solvent consumption/disposal, besides being more environmentally friendly. There are very little, if any, incremental maintenance costs between UHPLC and HPLC.
Q: How does one assess the return on investment when comparing costs versus performance for UHPLC and HPLC systems?
Elmashni: This is always best to do on a per-sample basis, because that is the most accurate scale to use.
McLeod: The key metric is cost per analysis, which is calculated from the initial cost of the system, the cost of columns and the average number of injections per column, the costs for consumed mobile phase per analysis, the costs for sample prep per analysis (solvents, solid phase materials, labor), and the costs for analyst labor time. With UHPLC, you will generally find that the cost per analysis is lower than it is for HPLC.
Q: Can all conventional HPLC analyses be transferred to UHPLC?
Elmashni: As long as either the detection technology for the particular application is available in a UHPLC system or if using an HPLC detector with a UHPLC pump and autosampler provides the proper level of detection, then all analyses can be transferred.
McLeod: If there is a UHPLC column that provides a similar selectivity as the original conventional column, you will find that transferring is very easy. There are many software tools available to help calculate the new parameters, and that will give you a UHPLC method that separates all your analytes with the same resolution, but in less time. There are a wide range of UHPLC columns and selectivities now, so you will find that most methods can be transferred. In the rare cases where a highly dedicated separation column is required (e.g., enantiomeric separation), transfer to UHPLC conditions may not be possible. However, these columns can still be used on a UHPLC system, as these systems also support running HPLC methods.
Q: What are some of the improvisations and modifications being worked on for the new UHPLC systems entering the market?
Elmashni: Companies are always working on higher pressures, increased sensitivity, new column phases, and increased ease of use.
McLeod: Compatibility with conventional methods; reduced maintenance requirements and effort; assistance for method transfer; further extensions for performance specs like pressure, flow, and temperature; an increasing range of UHPLCcompatible detectors; and a better integration of mass spectrometers are some aspects that are being worked on.
Reuter: Vendors are looking to further reduce mixing delay volumes and IBW [instrumental bandwidth] to make improvements in detector sensitivity/flow cell design and, though not specifically hardware oriented, to continue making improvements in column performance/robustness.
Other panelists from the webinar included:
• Eric Denoyer, Ph.D., Product Planning and Portfolio Management for Chromatography, PerkinElmer
• Curtis Campbell, Ph.D., HPLC business development manager, Shimadzu Scientific Instruments
• Stefan Schuette, Ph.D., senior marketing director, Liquid Phase Separations Business, Agilent Technologies
• John Burchell, chromatography business manager, JASCO
• Remco van Soest, HPLC product manager, Eksigent
• Moderator: Tanuja Koppal, Ph.D.
If you missed the Product Showcase webinar “UHPLC Systems - Making the Impossible, Possible!”, originally broadcast on Thursday November 18, 2010, Click here to watch the archived video.