November 1, 2007 (Vol. 27, No. 19)
Vendors Are Striving to Meet Customer Demands for Greater Reliability and Cost Savings
Liquid chromatography is an essential analytical tool for molecular biologists and biotechnologists around the world. Combined with mass spectrometry, the pair form a necessary part of a laboratory’s capability to characterize complex molecular mixtures and ensure their consistency for either basic research or product evaluation. They are invaluable for food safety and agricultural quality control, and their unrivaled sensitivity makes them important in the detection of environmental contaminants.
Recent improvements in instrumentation, multidimensional chromatographic separation, software programs, and column design have made LC technology even more important in sample preparation.
HPLC and UPLC
“HPLC has been around for a long time, but aside from minor refinements, there weren’t many advances in the field until UPLC® came along,” alleges John Gebler, Ph.D., director of biopharmaceutical sciences at Waters (www.waters.com). “We saw improvements in column design and improved lab software over the years, but these were incremental in nature and not groundbreakers.”
In conventional HPLC, samples are forced through the column under pressure, which decreases their diffusion, allowing sharper resolution of peaks as they elute from the column.
In UPLC, much smaller particles, less than 2.0 mm, are used to pack the column and a greater pressure is applied, resulting in improvements in separation profiles, according to Dr. Gebler. “The 1.7 micron particles are composed of silica in the form of a bridged ethyl hybrid, an appropriately durable material for the separation procedures. Our UPLC systems are designed to work under pressures of up to 15,000 psi, so this increased pressure provides greater speed, resolution, and sensitivity.”
Waters offers two options, the Acquity UPLC System and the nanoAcquity UPLC System. Both can be coupled to a mass spectrometer to give the investigator increased peak concentrations in less time. “With UPLC you increase sensitivity and you maximize the signal-to-noise ratios of your mass spec instrument.”
The Waters system can be employed in research protocols for antibody characterization in which translational modifications are of special interest. According to Dingyi Wen, Ph.D., principle scientist at Biogen Idec (www.biogenidec.com), “We use the Waters Acquity system for determining the primary sequences of antibodies as well as glycosylation and other secondary modifications. The UPLC separation provides sharp peaks, even when the quantities available are small.”
Dr. Wen investigates antibody candidates by digesting them with endoproteases, followed by separation, and finally identification of peptides in the mass spectrometer. HPLC does not provide the resolution or sensitivity needed for the research protocols, and its use of splitting can result in loss of material, she explains.
Dr. Wen has two Waters UPLC systems in her laboratory and plans to add a third. She finds the Acquity UPLC HSS T3 columns for retaining and separating polar compounds via reversed-phase HPLC and UPLC useful.
HPLC Techniques for Rapid Analysis
According to Curtis Campbell, liquid chromatography product manager for Shimadzu (www.shimadzu.com), injection of plasma samples directly into a liquid chromatography system without any sample preparation is now a popular high-throughput strategy. “Our systems are totally modular, so the user can custom assemble components to fit his protocol,” Campbell states. “There are numerous valving options available to match the needs of the investigator.”
As an example, Campbell describes the quantitation of the gastric proton pump inhibitor rabeprazole in rat and dog plasma using the technique of online sample cleanup. This approach involves the use of Shimadzu’s MAYI™ pretreatment columns where the drug is concentrated and the protein washed out. The valves are then switched and the material introduced into the analytical column. The basis of this approach resides in the design of the silica gel that packs the column, which is coated with a hydrophilic polymer, while the pore interior is chemically modified with an octadecyl group.
Proteins cannot penetrate into the small pores and are easily removed by the washing procedure. On the other hand, the low-molecular weight analytes such as rabeprazole permeate the pore interior and remain bound until they are eluted with the appropriate mobile phase. The sample can then be moved into the mass spectrometer for identification and quantification.
“The Shimadzu systems can also be used in environmental toxicology to measure trace pesticides and chemicals in water,” Campbell notes. “The samples are concentrated via a loading pump onto a trap column and subsequently eluted by backflushing onto the analytical column. The amount of water passed through the trap prior to elution determines the degree of concentration. The ultimate sensitivity of the measurement then depends on the mass spectrometer and the loading capacity of the trap column.”
Since this approach does away with the complex sample preparation associated with classical methods, sample processing time and manpower are saved, as well as solvent costs and extraction cartridges.
The Shimadzu Prominence HPLC system is also capable of being integrated into automated online sampling devices such as the ARS from Groton Biosystems (www.grotonbiosystems.com). This combination allows sterile sampling from a process stream like a fermentation unit. “The speed and simplicity of this approach means that we can save time and efficiently monitor ongoing protocols,” Campbell states.
Shimadzu offers another feature—isothermal temperature control. Commonly, block heaters, which offer only limited temperature regulation, are used to control column temperatures. The Shimadzu forced air circulating heating units control this factor much more tightly, which greatly decreases variability from run to run, Campbell says. The heating units are designed to accommodate switching valves, mixers, and some detector cells for better reproducibility and higher stability of the analytical data.
Automated Separation
Thermo Fisher Scientific (www.thermo.com) also provides high-speed U-HPLC systems and 2-D LC systems including EQuan and TurboFlow® systems.
“A key feature of the Thermo-Fisher technology is the TurboFlow system, a bimodal or dual-separation technology employing a highly turbulent TurboFlow column in the first dimension,” reports Chris Loran, business director of R&D and marketing for HPLC at Thermo. “Using high velocity, a powerful turbulence is generated in the columns, which increases the rate of trapping of the small molecules, while the larger proteins are rapidly passed through the column unretained.”
The Accela U-HPLC provides chromatographic separations over an expansive range of flow rates and pressures. Accela is designed to optimize performance of sub-two micron particle columns, providing operational capability spanning conventional LC pressures from short LC columns up to 15,000 psi for U-HPLC long-column separations of complex biomixtures.
The EQuan system consists of a 2-D chromatograph designed for trace component analysis in environmental and food safety applications. The first dimension comprises a large particle column, typically a 12 micron Hypersil Gold with a high carbon load providing high-loading capacity for online sample enrichment. In the EQuan system, up to 20 milliliters of water are injected onto the loading column, trapping trace levels of pesticides among other analytes. The enriched sample is then eluted onto the 1.9 micron Hypersil Gold column in the second dimension.
Loran argues that the Thermo Scientific technology speeds analysis. “This means that in food safety monitoring and environmental sampling, a lot more material can be processed in a shorter time, which provides the laboratory with more robust data,” he states.
“Multiple LCMS applications continue moving toward automated chromatographic sample cleanup, eliminating a significant amount of sample preparation steps and time. By combining online matrix cleanup offered with TurboFlow systems with runtime reductions of U-HPLC, significant advantages in throughput can be realized,” Loran adds.
Rapid Separation LC
According to Frank Arnold, director of product marketing for the life sciences business unit at Dionex (www.dionex.com), “The UltiMate 3000 Series of LC Systems are smart HPLC solutions that don’t sacrifice reliability or ease of use to achieve marginal improvements in performance.
“Our UltiMate 3000 x2 Dual LC systems are designed for advanced chromatographic techniques in addition to standard LC applications. The x2 Dual LC systems feature 2-D LC, online SPE-LC, automated method scouting and a variety of column-switching techniques that increase sample throughput and instrument utilization,” he adds. “In addition to x2 Dual LC systems, Dionex offers solution kits that make advanced chromatographic techniques truly turnkey and ready for widespread use.”
Recently Dionex added two new kits to its product line. One is designed for automated method scouting, exploring various combinations of columns, eluents, and other method parameters, in an automated fashion. The other provides a solution for automated off-line 2-D LC of peptides and proteins.”
Based on LC technology, Dionex also offers Rapid Separation LC (RSLC) solutions, which comprise a dedicated UltiMate 3000 system, a Chromeleon Method Speedup calculator, and a series of Acclaim RSLC columns. “Like all other LC solutions, Rapid Separation LC solutions are optimized to provide the most equitable balance between performance, reliability, and ease of use,” Arnold states.
Fighting Flow Splitting
Eksigent’s(www.eksigent.com) HPLC technology includes the NanoLC Proteomics system for separation of peptides before analysis by a mass spectrometer. According to Remco Van Soest, product manager for Eksigent, “Most HPLC pumping systems are designed to deliver flow rates of 0.1–5 mL/min and are incompatible with the demands of nanoLC.”
In order to deliver gradients at nanoliter per minute flow rates, conventional pumps may be equipped with a splitter, so 0.1% of the total flow is routed through the column and the rest of the sample is discharged as waste. “Flow splitting is an attempt to use existing hardware to generate low flow rates, which are essential for present-day proteomics studies,” Van Soest continues.
This inelegant solution suffers, however, from variations in split ratio when the column backpressure changes, so that the flow rate through the nanocolumn is varying and retention-time reproducibility suffers. “The Eksigent NanoLC system’s flow control runs precise HPLC gradients at flow rates as low as 100 nL/min, while at the same time the amount of solvent used is reduced with a factor up to 1,000,” Van Soest adds.
The flow-control capability allows the flow rate to be changed rapidly for higher-speed sample loading or for extended MS/MS analysis to identify coeluting and low abundance peptides at lower flow rates (peak parking), without the need for additional valves or pumps. After the transit of the sample through the gradient, Eksigent flow-control technology can automatically increase the flow rate to accelerate flushing and reduce the lag time between sample runs.
The repeatability of the system is established by experiments performed on the Eksigent NanoLC-2D in which Cytochrome C digests were analyzed. The 17 major peaks seen in the digest were analyzed in multiple runs for repeatability. The standard deviations of the repeated samples were very tight, in the range of 0.16–0.37%, according to Van Soest.
“The Eksigent NanoLC system, in combination with mass spectrometry, provides more precise data, while saving solvent compared with split-based systems,” Van Soest concludes.
HPLC Chips
Agilent Technologies (www.agilent.com) is one of a number of companies that have approached chromatography issues through the introduction of microfluidic chip technology. “With chip technology you just plug it in,” says Keith Waddell, LC/MS applications solutions manager. “It’s built for ease of use.”
The company’s HPLC Chip is composed of laminated polyimide and contains features such as an enrichment trap column, an analytical column, and a microvalve to switch LC flow. The end of the chip is laser ablated to form a nanoelectrospray tip. The design allows the device to be electronically recognized by the system when inserted. Tight fluid connections are established by sandwiching the chip between the rotor and the stator of the built-in multiport microvalve. The chips are reusable, about the size of a microscope slide, and created with a variety of features and functions.
The various chip configurations include the classic reverse-phase C18 columns, a C8 version for proteins, a Glycan chip for pulling out glycosylated species, and choices of enrichment columns. Also under consideration is a new Phospho-chip, which acts by pulling phosphopeptides onto a titanium dioxide surface.
All of these chips integrate the separation columns of a nanoflow system with intricate connectors that eliminate traditional fittings and reduce the possibility of leaks, says Waddell. The system allows for the identification of additional compounds in complex samples. “Chips with specific functions are easily changed out, allowing each researcher to have his own personalized resource.”
Another Agilent product for use in proteomics is the Human Plasma Hu-14 column for removal of highly abundant proteins from plasma. Albumin, IgG, transferrin, haptoglobin, IgA, antitrypsin, and fibrinogen comprise 90% of the proteins in plasma and make the analysis of less abundant species difficult.
The column uses specific antibody affinity resin to trap the 14 most abundant proteins, allowing the flow through to be subsequently analyzed using the HPLC-chip technology combined with mass spectrometry. “This is a very reproducible approach that allows us to filter out major proteins to be able to deal with these complex samples,” Waddell states. “It can be easily coupled to other separation techniques.”
Cooperation Is the Name of the Game
A number of the companies dealing with HPLC and mass spec technologies have entered into cooperative agreements, rather than duke it out in the marketplace.
Last year, Waters and Agilent announced that their HPLC systems will be programmed to interface with the Applied Biosystems (www.appliedbiosystems.com)/MDS SCIEX (www.mes-sciex.com) mass spectrometers. The two partnering entities are integrating the software controls for the Acquity UPLC system with Applied Biosystems’ Analyst Software—their goal being the improvement of the level of output for workers who are coupling the systems.
According to Joseph Anacleto, senior director of applied markets and clinical research for Applied Biosystems, the decision to collaborate with HPLC vendors is driven by customer demand. “Customers who use Applied Biosystems/MDS SCIEX’s mass spectrometry instruments want to know that it will be compatible with the LC front end of their choice,” states Anacleto. “Providing this choice to customers is Applied Biosystems/MDS SCIEX’s overreaching strategy.”
In a similar vein, Agilent Technologies has configured its 1200 Series Rapid Resolution Liquid Chromatography (RRLC) system to be compatible with Applied Biosystems/MDS SCIEX mass spec instrumentation. The Applied Biosystems Analyst 1.4.2 LC/MS control software will integrate the two companies’ entire line of triple quadrupole and linear ion trap mass spectrometers as well as the 1200 series. The system, while designed for the rapid resolution system, will also perform as a conventional HPLC system.
Dirty Samples Come Clean
“The major concern of our customers is compressing analytical time,” says Alessandro Baldi, Ph.D., product and marketing manager of chromatography at PerkinElmer (www.perkinelmer.com). “Ourline of products is designed to reduce the analytical cycle and streamline work flow.”
Dr. Baldi asserts that for many Perkin-Elmer clients, the sample-preparation step has become the bottleneck, slowing the analysis of biological samples for agricultural or environmental monitoring. Since many starting materials are extremely complex and dirty mixtures, the best approach is to do a prerun cleanup to remove the molecules that could disrupt the accuracy of the results and lengthen the analysis time.
In general, there are three approaches to sample preparation. “In some cases, investigators are looking for molecules in plant extracts or waste water such as the pesticide atrazine that are present in concentrations of parts per billion,” Dr. Baldi explains. For these, solid-phase consumable cartridges are frequently used for sample concentration. A second approach is to do a solvent extraction to focus and concentrate the molecules that are being analyzed, while a third approach is the use of techniques such as flash chromatography or gel permeation for a preliminary cleanup especially for large-scale samples.
It is essential to develop a procedure with a high level of repeatability and for this reason automation is essential. One or several internal standards are added during the sample cleanup steps, greatly enhancing the overall accuracy of the final results.
“Using an internal standard, you can follow the flow of the sample from beginning to the end, automatically tracking it with an information management system,” Dr. Baldi says. “This allows data traceability that will fill the final report.
“Data traceability is a major concern, as the target compounds must be tracked from the day they enter the lab until the final report is filed,” Dr. Baldi continues. “For these reason a laboratory information management system is required.”
LC Rules the Day
According to Campbell, “More people are looking at LCMS for more routine analysis; many forensics labs are looking to get away from GCMS and moving to LCMS due to the fact that they get defensible data in just one run.”
Campbell believes that LCMS provides the answer faster, with more sensitivity, and less human chance for error. No longer is the investigator faced with lengthy sample preparations, derivitizations followed by endless repetitions, and confirmations. “In reality, it is a dilute and shoot scenario,” he concludes.
K. John Morrow Jr., Ph.D., is
president of Newport Biotech and contributing editor for GEN.
Web: www.newportbiotech.com. Phone: (513) 237-3303.
E-mail: [email protected]
