New instruments are providing higher levels of throughput, while improved column technology is yielding better peak shapes, less carryover
Patrick McGee
Senior Editor
It was just over a century ago that one of the earliest uses of chromatography was described by Russian botanist Mikhail Tsvett, and over the years new technologies have refined the technique. High-pressure liquid chromatography (HPLC), which emerged in the 1970s, provided an effective and widely adopted analytical and preparative technique, and the technology has continued to advance.
The development of new column-packing technologies played a key role in the evolution of this tool. That has remained true over the last two to three years, as advances in column technology have provided researchers with improved peak shapes and carryover. Other newer technologies include ultra high pressure liquid chromatography, which is touted as providing greater sensitivity and higher levels of throughput. Some researchers single out advances in coupling liquid chromatography with mass spectrometry as a promising tool for identifying new compounds.
 click the image to enlarge This chromatogram shows a separation of a mixture of benzo- diazepines attained using Hypersil Gold, a newer column based on a highly pure silica that is designed to provide improved peak shape. (Source: Thermo Electron Corp.) |
“We’re always striving to have better column technology and that technology is advancing greatly,” says Chris Loran, the LC and LCMS business director for Thermo Electron Corp., San Jose, Calif. “At one point, that was one of the things that was sort of holding back the whole industry. People wanted better capacity, better resolution, and advances in column technology have really come a long way. They’ve given us really clean phases, great reproducibility and lower carryover.”
Last spring, Thermo Electron unveiled Hypersil Gold LC and liquid chromatography/mass spectroscopy (LC/MS) columns, new offerings based on highly pure silica that are available in 3 µm or 5 µm columns. They are designed to provide improved peak shape, even when analyzing compounds that traditionally yield poor peak shape on traditional C18 or L1 chemistries.
“This was all about enhancing the peak shapes for basic compounds,” says Mark Woodruff, the product manager for LC and LC/MS columns for Thermo Electron in Runcorn, UK. “Most of the pharmaceutical drug entities appear to have the same basic functionality, and if you don’t have the best peak shapes possible, then you’re not going to make the best use of sensitivity and efficiencies that the whole system, meaning the column, the instrumentation, and the mass spectroscopy, are going to offer you.”
Woodruff says Hypersil Gold was developed in collaboration with AstraZeneca, which did the beta site testing. Hypersil Gold was designed to ensure better peak shape for basic analytes than common stationary phases, and to improve angles of sensitivity, resolution, and throughput for pharmaceutical industry users. Some pharmaceutical researchers like Wil Bartolini, PhD, believe improved peak shape is not the only attractive feature of Thermo’s new columns.
Reducing carryover
“It certainly has improved peak shape, but the most impressive thing about this column is this: We had some assays where carryover was a significant problem. It was obviously not part of the auto sampler and this carryover was on the column, and we were having to do some funny things with the HPLC gradients to get rid of it. In the case of the Hypersil Gold, we noticed that in just a single injection, there was no carryover at all, there was no memory effect from the column itself. That is a huge improvement over any other column chemistry that I’ve seen so far,” says Bartolini, senior scientist at Microbia Inc., Cambridge, Mass.
Sample carryover is a vexing problem in drug discovery and development, particularly with compounds that have a tendency to adsorb to system components. Woodruff says Thermo Electron has been investigating the use of smaller particles for columns and will launch a 1.9-µm version of Hypersil Gold in March. “We believe this will offer people even higher throughput and greater efficiencies.”
Bartolini says the impact of the improved column technology in his lab has been clear. One column can handle a significant portion of their needs, they don’t have to switch columns out as often, and their run times are getting shorter. “We’re dropping our run times by 30%, which is huge, because that cycle time on the instruments is very valuable. Time is our most valuable thing right now.”
Bartolini’s lab has also been using the new columns in preclinical work and has cut run times there nearly in half, allowing them to run entire assays overnight. “It got the data in our hands quicker, and we were able to move the instruments into other assays in a shorter time.”
Another recent advance in columns has been monolith technology pioneered by BIA Separations, Ljubljana, Slovenia. Beads or other materials traditionally used for packing do not completely fill the space in columns, a factor that contributes to peak broadening and lower column efficiency, the developers of the technology argue. Monolith supports consist of homogenous blocks of polymer with highly interconnected channels, a structure that reduces separation time and minimizes column back pressure while allowing for flow rates as high as five to 10 bed volumes per minute, they say. Critics contend that while the technology demonstrates great promise in certain application areas, problems with reproducibility and scalability limit its use.
Turbocharged HPLC
Other advances in chromatography have come in the arena of HPLC, where several companies are working to make their systems more powerful and efficient. Last year, Waters Corp., Milford, Mass., introduced the Acquity UPLC (ultra performance liquid chromatography) system, basically a turbocharged HPLC system. While most conventional technology features particulates in the 3- to 5-µm range and pressures in the 6,000 psi range, which has been the standard for years, Acquity features 1.7-µm particles and pressures of up to 15,000 psi.
“We looked at the entire process—instrumentation, packing material, column technology, down to detectors and injectors—and optimized all of that to capitalize on small particle separations,” says Warren Potts, senior business development manager for the pharmaceutical business, Waters Corp. “What we’ve done here is realized the theoretical advantages of small-particle separations. . . . You need to be able to operate at higher pressures and you need to be able to detect things more quickly. Your detectors need to keep up with the chromatographic peak widths because they’re very narrow. That includes not only optical detection, but also mass spectrometry.”
Ian Wilson, PhD, a principal scientist at AstraZeneca, Charnwood, UK, believes the introduction and adoption of HPLC-MS/MS is a key chromatography advance, and he has recently begun using Acquity. “The system offers improved resolution for complex mixtures, and because of the very sharp peaks, [gives] better sensitivity and probably also reduces ion suppression in LC-MS. If you don’t need the resolution, you do get the speed,” he says.
Shimadzu Corp., Kyoto, Japan, is set to introduce a high-throughput HPLC autosampler this month at Pittcon in Orlando, Fla. The LC-20A Prominence series will build on and improve the features of the SIL-HT, which was introduced three years ago, says Curtis Campbell, HPLC product manager at Shimadzu Scientific Instruments Inc., Columbia, Md. The SIL-HT is a medium capacity autosampler that holds four titer plates and can perform a 10-µL injection from any one of the plates in about 15 seconds with a carryover performance that Campbell calls “phenomenal.”
The SIL-HT was the result of work done in Japan to remove all active sites from the needle with a very thin layer of platinum alloy coating and to redesign the valve-closure mechanism so it could use a peak rotor. “Previously, peak was too soft to use in that high-stress environment, but they redesigned the closure so they can still have a good seal on the high-pressure valve yet have the chemical inertness of the peak,” Campbell says.
Advancing the technology
The LC-20A Prominence advances that technology by making the internal valve passages smaller and making the shape and design of the needle closure better so there are less unswept areas. The result, Campbell says, is a system that can perform a 10 µL injection from any plate in any position in about 10 seconds with excellent carryover. Other features of the LC-20A include a dual-rinse phase for the autosampler, a temperature-controlled flow cell, internal mercury lamp for detector calibration, and new optics and electronics for improved detector performance.
 click the image to enlarge A standard sample mix was analyzed using the Acquity UPLC System. The extracted ion chromatogram of theophylline illustrates the high resolving power of the system captured within Waters eLab Notebook software. (Source: Waters Corp.) |
Xiangyu Jiang, PhD, is associate director of bioanalytical chemistry at Covance Laboratories, Madison, Wis. He has not yet used the new LC-20A system because it was beta-tested in Japan, but he is very familiar with the SIL-HT, which he says was the best system of its kind when it came on the market. Jiang said speed is one of the reasons he likes the SIL-HT. “When we have a lot of samples to run 24 hours a day, seven days a week, if we can shorten the run time, we can save time and money,” Jiang says. Most importantly, the SIL-HT decreased carryover significantly, by about two to three times less than other autosamplers to which Jiang compared it.
Campbell says another advantage of the LC-20A is that it is a totally modular system, so it allows Shimadzu to customize it to meet the needs of different customers. “Whether it’s multiplexing or fast analysis or rapid serial analysis, there are all kinds of ways that people are doing things now,” he says. “What we’re trying to do is look at taking standard equipment and making it as flexible as possible so that when someone comes to us and says, ‘I want to run 10 µL per minute on this system, they can do it.'”
Campbell says many standard systems on the market don’t allow for that. If a researcher runs at a rate of 10 µL per minute, but then needs to step up to 1.5 mL per minute, they are often unable to do this. “Our system will do both, because we’re looking at keeping everything as flexible as possible.”
LC and MS
In addition to the development of new technologies, coupling existing tools with chromatography systems is helping advance the field as well. “The addition of a mass spectrometer as the standard detection technique in the chromatographic system is certainly one of the key advances in chromatographic drug discovery and development that we’ve seen over the past few years,” says Thermo Electron’s Loran. The use of mass spectrometry and other optical detectors has become common, and “a lot of the credit is due not just to the hardware, but the software systems that drive the hardware.” Campbell agrees and says he has seen major improvements in the sensitivity and selectivity of mass spectrometers. “They can look at smaller and smaller and lower and lower volumes and impurities, which forces chromatography manufacturers to step up their game as well, as far as flow precision, reproducibility, and carryover especially.”
As for HPLC column technology, Thermo Electron’s Woodruff says it has leveled out. “It has reached a pretty stable state at the moment and it is possibly going to take some shock to the system to really reveal the next technology that’s going to take things to the next stage. . . . It remains to be seen if the newer technologies being talked about have the potential to become commercially viable to the point that they get put into that sort of system.”
Bartolini says the ideal system of the future would combine the strengths of the tools already on the market. “You have the Monoliths, which can basically give up no back pressure. You have these high-pressure systems like the Waters Acquity system, and then you have the excellent chemistry, peak shape and carryover that Hypersil has. If they could converge all of these together where you could run higher flows or even shorten your runs, that would be an improvement.”
AstraZeneca’s Wilson believes the technology will continue to shrink as it advances. “Attempts at further miniaturization, being largely led by academics, will probably continue, but I think it’s going to take a while for pharma to buy into them.”
Filed Under: Drug Discovery
