Steven Wolk, PhD, associate director of analytical chemistry at SomaLogic Inc., discusses the use of high-performance liquid chromatography (HPLC) and ultra high-performance (or pressure) liquid chromatography (UHPLC) technologies for analytical work with contributing editor, Tanuja Koppal, Ph.D. He reflects on the changes taking place in the liquid chromatography market, on both the hardware side and the software side, and comments on the incremental improvements that can be made.
Q: Why did you transition from a traditional HPLC to a UHPLC?
A: In terms of improving resolution using LC systems, I don’t believe there is anything better than a UHPLC, and for us, resolution was the number one priority. The beauty of UHPLC is that along with increased resolution you also get a reduction in run times. As the run times get shorter, the throughput goes up. So it’s like getting the best of both worlds. We have seen resolution improve by two to four times and run times reduced by two to eight times, or even more in some cases. At SomaLogic we purify our modified oligonucleotides (proprietary molecules called SOMAmers) via large-scale preparative HPLC purification, and all the analytical characterization is done using UHPLC.
Q: What other advantages have you found using a UHPLC?
A: With UHPLC you use less solvent, so it’s a cheaper and greener technology. UHPLC also offers a lot more flexibility. The fundamental principle of UHPLC is that you use a column with smaller particle size (resolution. However, the smaller particle size leads to an increase in column pressure, and the pumps have to be designed to handle these high pressures. So now, you have a much broader pressure range that you can operate under. You can use a normal HPLC column and operate it under regular pressures and flow rates or with a UHPLC column, you can operate in the higher pressure range. Other advantages are that the UHPLC systems have been designed with a much smaller system volume and hardware components that minimize dispersion, which results in lower band broadening and more precise mixin of the mobile phases when generating gradients. These factors also improve resolution.
Q: What is the disadvantage to having an increased back pressure in your system?
A: I haven’t found any downsides to the higher pressure. However, there are minor issues like having a smaller column capacity. People who are used to HPLC often overload their UHPLC columns. With UHPLC columns you have to adapt to smaller sample volumes and smaller amounts of material. Overall, our columns and our systems are very robust and not affected by the high pressure. As the hardware and software for the LC systems get better, there is also better monitoring of the pressure. In our UHPLC system we get a readout of the pump pressures, and once, when we were experiencing a problem, I just took a printout of the monitoring screen and e-mailed it to the service representative. Just by looking at the diagnostic pressure response readout, he was able to tell me that there was a check valve problem on the lead pump, which saved me time in terms of getting someone to come in and fix the problem.
Q: What is involved with routine care and maintenance of these systems?
A: Our company uses service contracts. So we do the basic maintenance ourselves but let the vendors take care of the in-depth maintenance. With the stuff that we do in-house I have not noticed any increased problems when compared to HPLCs. That being said, our people are well trained to take good care of these systems. We don’t do anything special but use good common sense that any well-trained chromatographer would use. UHPLC is not that different from HPLC, but there are some minor exceptions. For instance, if you leave aqueous buffers in contact with the system for a long time, it will eventually grow bugs and clog the head of the column. This bioburden in the buffer will affect a UHPLC faster than it would an HPLC column because of the smaller packing in the column. Very salty buffers, like those used for size exclusion or anion exchange chromatography, can affect the pumps. Hence, you need to take extra care to make sure that al the salt gets flushed out of the system.
Q: What about sample preparation for UHPLC? Any special concerns there?
A: You have to be a little more careful about filtering your samples to remove insoluble materials because of the smaller packings, which may cause the column to get clogged. Typical commercial systems come equipped with autosamplers that can handle about 100 vials, and most modern systems can also accommodate 96-well plates.
Q: Where and what types of improvements would you like to see in the UHPLC technology?
A: There is always room for incremental improvements in the particle size, the system volume, and the flow rate. As particle size gets smaller, the resolution gets better but you have to deal with higher pressure, and there’s room for improvement there. There is still some catching up to do in terms of the types of columns and solid supports that you can buy. However, it’s getting better all the time, and every few months there are new columns being offered. I’ll challenge the vendors to try and find a way to make cheaper columns, as the columns are still reasonably expensive. Sample prep still requires some work. If there is a way to automate the sample prep, perhaps by filtering samples within the autosampler or something else, that would be an advantage.
Q: How easy is it to justify the return on investment?
A: It depends on what you are trying to achieve. For us, that extra resolution helps s determine the quality of our reagents (SOMAmers). If you are making an oligonucleotide therapeutic then quality is of huge importance. So it’s difficult to put a number on the quality of science that you can do. In addition to that, the savings in solvent costs and increase in throughput over time make things easy to justify.
Q: If you had the resources, would you replace your HPLCs with UHPLCs over time?
A: If I were going to buy a new instrument then there would be no reason to buy an HPLC. I would just buy a UHPLC. I don’t see any advantage to buying the old-style systems. However, there are instances, especially in the pharmaceutical industry, where methods need to be validated. If a technology changes then the method has to be revalidated and that can be very time consuming and expensive. That’s the only situation I can think of where I wouldn’t buy a UHPLC. Or in some rare case where your samples are particularly dirty and there is no way to filter them, then you are likely to continually clog your UHPLC columns.
Q: What advice do you have for lab managers looking to invest in the UHPLC technology?
A: There is no reason to hesitate when looking to invest in a UHPLC. Whenever a new technology comes on the scene, you hear the hype. Then reality sets in and it doesn’t quite live up to the hype. UHPLC has been an exception, in that it has lived up to the hype and delivered on all its promises. UHPLC does everything your HPLC did and more.