ORLANDO, Fla. Competition among nano-high-performance liquid chromatographer vendors was tight at this year's Pittcon, with Waters, Dionex, Shimadzu, and Agilent, among others, claiming their technology is the best at separating proteins and peptides within complex samples.
While speed is often highlighted when presenting HPLC systems, that attribute is more relevant for small-molecule analysis than for proteomics, vendors said. For proteomics, the most important attribute of an HPLC system is its peak capacity.
Waters
"People would argue that speed is relevant, but really peak capacity is the most import thing for proteomics," said Keith Compson, a life sciences business manager for Waters, which showcased its UPLC at the show this week. "You have a huge number of peptides in complex mixtures, and the better you can separate them, the better the mass spec can analyze them."
Waters' strategy for achieving maximum separation is to use small particles within its nano-columns. Waters' nano-Acquity Ultra Performance Liquid Chromatography uses 1.7-micron packing material.
"Smaller particles are what gives better separation," said Compson. "With smaller particles you also need higher pressures."
The Acquity UPLC system is designed to handle higher pressures than traditional HPLC systems, such as Waters' Alliance system, which generally can only handle a minimum particle size of 3 microns, according to Pat Young, product manager for nano-Acquity.
A nano-Acquity system costs 20 to 25 percent more than a capillary-LC Alliance system, Compson said.
Dionex
An alternative to using columns packed with small columns is to use monolithic technology a different way of making nano-particles in a column that also achieves good separation capability.
Dionex, which launched this week its UltiMate 3000 series of HPLC systems, uses monolithic columns to achieve efficient separations, said Mark Van Gils, manager of LC packings and the nano-capillary product line for Dionex.
The new Dionex HPLC system features a dual pump technology, Van Gils said.
"It's like having two HPLCs in one box," he said. "You could use one channel for sample preparation, and the other for [pre-] mass spec [separation.]"
Waters' Compson said that one disadvantage of monolithic columns is that researchers can only load about 20 percent as much sample onto a column as with an Acquity-packed column. However, Johahn Purkerson, Dionex's field engineer for LC packings, disagreed.
"You can actually load 20 times as much volume onto a monolithic column compared to a silica column," Purkerson said. "The disadvantage of a monolithic system is that it has a fast flow rate, so you need to make sure the mass spec can keep up with fast scanning times."
Shimadzu
Shimadzu also launched a new HPLC system this week, called the Prominence High-Throughput HPLC. Unlike Waters and Dionex, Shimadzu emphasizes multi-dimensional separations, rather than column packing material for achieving maximum peak capacity, said Terry Adams, marketing manager of HPLC, LC/MS, and data products for Shimadzu.
With the Prominence HPLC, samples are first separated out on one column using one technique, such as strong cation exchange, and then separated out on a second column using an orthogonal technique, such as reverse phase, Adams explained. The resulting peak capacity is equal to the first dimension multiplied by the second dimension.
"It's like a 1D versus a 2D gel," said Adams. "I don't care how good a resolution you have on a 1D. It's not going to be a 2D."
In terms of speed, a two-dimensional run might take about three hours, while a single-dimension run would take about a minute, Adams said. However, the difference in peak capacity is "10 versus 1,000," he said.
Agilent
Agilent, which showcased its HPLC this week, uses yet another strategy for achieving maximum separation of complex samples: It eliminates dead volume within the HPLC system by using microfluidics.
"You can take different paths to accomplish the same thing," said George Gauthier, product manager for HPLC chip technology at Agilent. "With microfluidics, you go after peak capacity by achieving a column's peak performance."
The connections within traditional nano-flow systems create dead volume, which results in band broadening, Gauthier explained.
"The principal culprit is between the end of the column and the spray emitter. That [dead space] causes band broadening," he said.
According to Gauthier, with Agilent's HPLC chip, a researcher can get 250 to 300 peaks within about 45 minutes.
"You can double the peak capacity by eliminating dead volume and band spreading," he said.
Another advantage of microfluidics is that plumbing is not a problem when making multi-dimensional separation systems, Gauthier said. Agilent already offers two-dimensional HPLC chips, and the company is working on developing three- and four-dimensional chips something that would be too complex if traditional plumbing had to be accounted for.
"Multi-dimensional chromatography is difficult in the nano world because it involves more plumbing," said Gauthier. "With an integrated chip surface, you're just adding two, three more layers onto the chip."
Size of column packing material is also something that Agilent is looking into to increase its peak capacity, Gauthier said. Currently, Agilent uses 5-micron packing material, but the company is looking into using 3.5 micron and sub-2-micron packing materials in the future.
"Five-micron material is pretty standard technology. It's tried, it's proven, it's reliable… it does the job," said Gauthier. "But we're also looking into how you could improve peak capacity with smaller packing material."
Asked to compare prices among different HPLC systems, vendors said that pricing is very competitive, and that it is difficult to quote numbers because each system is modular and the final price depends on which components the user wants.
"It depends on what is the value for what you buy," said Dionex's Van Gils. "The UltiMate 3000 starts from the low end at $25,000 to $30,000, and goes up to $130,000 to $140,000."
Shimadzu's HPLC product manager, Curtis Campbell, said that the new Prominence system costs between $21,000 for a unit with no temperature control, up to $50,000 to $60,000 for a unit with dual gradient capacity.
Tien Shun Lee ([email protected])