For more than 20 years, the ongoing evolution of high-performance liquid chromatography (HPLC) has been a constant reminder of how intimately scientific progress depends on analytic technology. HPLC has kept up with the complexity of biological investigations and in many cases has enabled it.
According to Curtis Campbell, HPLC product manager at Shimadzu Scientific Instruments (www.ssi.shimadzu.com), the most significant trend in HPLC instrumentation has been the clash of user levels and the breadth of applications for which they purchase HPLC systems. Some users want the instrument to be more sensitive, faster, and higher-performing, while others want a big green button on the front that they can push to get their data out. Our goal is to balance the highest performance with instruments easy enough for a technician to operate.
A majority of HPLC systems today are purchased by quality laboratories, which view HPLC as a commodity service. The average lab technician has about two years of college-level education and considerably less training than their predecessors of a decade ago. At the same time, instrument developers must be cognizant of the chromatographer market consisting of scientists and engineers who ponder the nature of internal standards and retention times.
Top-tier vendors acknowledge and embrace this dichotomy. Shimadzus instruments are as comfortable in QA/QC environments as in R&D circles as the front end for triple quadrupole or ion trap MS. Modular design assures that users can custom-tailor a system based on any combination of dozens of mechanically compatible pumps, columns, detectors, and autoinjectors, or build redundant systems with multiple columns or detectors.
Shimadzu introduced its Prominence HPLC line in 2004 and promises upgrades at this years event. Prominence is a network-ready system that serves as a front-end to leading MS instruments. Improvements are expected including reduced delay volume, quicker reaction to gradients, faster injections, and optimization of tubing and delay volumes to lessen peak spreading and band broadening.
Economic and Scientific Drivers
To Jasmine Gray, Ph.D., marketing director for protein discovery at GE Healthcare (www.gehealthcare.com), proteomics is driving demand for HPLC products. The transition from genomics to proteomics, and all its omic subspecialties, is causing great interest in systems that deliver high sensitivity, reproducibility, and the ability to analyze membrane proteins and post-translational modifications, says Dr. Gray. Our users are also looking for ways to handle the mountains of data proteomics experiments generate.
Proteomics differs from traditional biochemistry in several ways. Complex tissue samples strain detectors and columns to their limits, since high-abundance proteins, such as albumin, are present along with proteins of interest. The concentration dynamic range for proteomic studies can be as high as 1012 and, as Dr. Gray notes, Researchers are usually interested in target proteins that are in lowest abundance.
For analyzing the prodigious quantity of proteomics data, GE Healthcare continues to build on its DeCyder differential analysis software package. DeCyder 2D analyzes data from fluorescence-difference 2-D gel electrophoresis (DIGE) experiments. A new component of this software platform, DeCyder EDA (extended data analysis), provides detailed statistical and cluster analysis, according to GE.
A third component launched last year, DeCyder MS, takes the retention times from the chromatography, and mass/ charge ratios from the mass spectrometer, and plots these as 2-D and 3-D intensity maps to quantify differential protein expression without the use of molecular weight tagging. According to Dr. Gray, the plots present data in a more meaningful way and facilitates trouble-shooting.
GE Healthcare inherited (from its previous existence as Amersham) chromatography products that employ bio-inert, metal-free contact surfaces. Interactions between metals and phosphorous can complicate analysis of phosphorylated proteins and peptides.
GEs Ettan nanoLC is a dedicated 1-D HPLC system that allows users to step back from complex, multidimensional experiments and work out HPLC separations in simpler form. Once theyre comfortable with their model system, they can move forward with 2-D or 3-D approaches and transition to the Ettan MDLC (multi-dimensional liquid chromatography), says Dr. Gray.
Helmut Schulenberg-Schel, Ph.D., HPLC marketing manager at Agilent (www.agilent.com), sees productivity as the most significant economic driver for HPLC. Users want to streamline their workflow by spending less time on an experiment or getting more or better results using the same resources.
Globalization has accelerated standardization of HPLC methods, especially for organizations and collaborators that share information or protocols among multiple sites. Based on these needs, end-users now choose suppliers with the full range of hardware, software, instrument, and service capabilities.
Although speed and productivity matter to everyone, the omics disciplines are forcing users to dig more deeply into samples as well. It all depends on where the customer fits into the value chain, says Dr. Schulenberg-Schel. In many applications, for example in quality labs, standardization and methods optimization are everything. In QA you know what the goal is, but in research one must remove the onion skin one layer at a time.
While customers value bells and whistles, they also appreciate backward compatibility to help protect their instrument investment. Agilents 1200 Series LC is a case in point. The 1200 builds on the 1100 series model, which has sold 60,000 units worldwide. Customers can combine modules from both systems and continue to use methods developed on the 1100 without revalidation or retraining.
Agilent offers more than 60 instrument modules with the 1200, including a new rapid-resolution format as well as prep-scale, standard, narrow, capillary, nanoflow, and chip-based microfluidic HPLC. Agilent offers chips for small molecules as well as genes and proteins. According to Dr. Shculenberg-Schel, his companys nanospray HPLC-Chip/MS technology provides sensitivity gains 1,000-fold higher compared with conventional LC/MS.
Building on the success of the Zorbax Rapid Resolution High Throughput (RRHT) columns introduced three years ago, Agilent plans to debut 70 new advanced RRHT columns ranging from 1-mm to 4.6-mm internal diameter and from 20- to 150-mm length, with bonded phase particle sizes of 5, 3.5, and 1.8 mm.
Rising interest in sub 2-micron particles in solid phases allows a wider range of chromatography options, including greater efficiency over a wider linear dynamic range of flowrates. We see that across the board, whether its for standalone HPLC systems, LC/MS, or LC/MS/MS in front of triple quads or linear ion trap detectors, says Chris Loran, LC and LC/MS business director for Thermo Electron (www.thermo.com). The companys 1.9-m Hypersil GOLD columns, installed into Thermos Surveyor HPLC system, minimizes delay volumes to get the gradient from the pump, onto the column, and into the detector in less time, translating to shorter runs, according to Loran. Users can gain the advantages of small particles in a conventional system.
HPLC hardware and software continue to evolve toward greater simplicity and standardization. All Thermo systems use standard or open-source software, which Thermo says enables even novices to get up and running with a minimal learning curve.
As vendors strive to simplify HPLC operations, instruments IT requirements have become more demanding. Early HPLC instruments consisted of a pump, autosampler, column, injector, and UV detector. Todays photodiode array and MS detectors churn out more data than anyone can process by hand. A computer is essential for working with data from both types of detector, says Eksigent Technologies (www.eksigent.com) senior product manager Phil Deland. Temperature control is also becoming de rigueur, as peak widths of one to two seconds are susceptible to temperature artifacts. We dont see 20-second peaks any more, says Deland.
Waters (www.waters.com) continues to innovate on its UPLC (Ultra Performance LC) family of products. Introduced in 2004, UPLC features small stationary-phase particle sizes, and with smaller particles very high pressureup to 15,000 PSI. To achieve the higher resolution and efficiency of smaller particles you need higher pressure, says Jeffrey Mazzeo, Ph.D., director of applied technology. Thats UPLC.
UPLC was originally based on reverse-phase C18 chemistry, but in 2005 Waters debuted a C8 phase, phenyl, and what it terms shield chemistry or embedded polar phase, a variant of C18 featuring a carbamate group that provides unique selectivity. The company also introduced, for its Acuity UPLC product line, next-generation ultraviolet detectors with lower noise levels than traditional UV cells.
Normally, smaller particle size stationary phases require not just high pressure but minimal volume in the fluid path. Otherwise you get band broadening, losing all the benefits of the small particles, Dr. Mazzeo explains. Flow cell design must therefore trade off between an illumination volume large enough for stable detection, but not so large that it affects chromatographic behavior. Waters uses total internal reflectance to maximize the amount of sample the detector sees, while keeping the absolute volume low, in the hundreds of nanoliters.
Smaller is Better
Eksigent, a relative newcomer to HPLC, was formed about five years ago by a group of nanofluidics experts from Sandia National Laboratory. The company sells two HPLC systems based on the Sandia technology. The nanoscale LC/MS uses columns with internal diameters of 75 and 150 microns, with flow rates in the hundreds of nanoliters per minute. The nano system employs either ion trap or triple quadrupole mass spectrometry as the detector. The larger-scale system uses 300-micron internal diameter columns and operates in the microliter per minute range. This system, which is targeted to small molecule drug discovery, uses traditional ultraviolet detection as well as evaporative light scattering and mass detection.
With solvent throughputs just 1% of those of conventional 4.6-mm columns, fully functional micro- and nano-HPLCs save bundles on solvent costs and qualify as environmentally conscious systems. Many end-users consider the green angle when planning projects and purchases.
Five years ago nobody cared, muses Deland. Today they mention their environmental efforts in their annual reports.
According to Deland, the tiny devices provide better selectivity and resolution than conventional HPLCs as well. Everything is engineered into one small box, and since the components are closely connected there is little band spreading or peak dispersion. So far Eksigents strategy bet on nano is working. The company has shipped and installed more than 150 LC systems, mostly in North America.
As HPLC systems shrink, the next big thing, says Deland, will be sample-preparation systems that operate at this scale and do not become the rate-limiting step in chromatography methods. Novel approaches to sample prep must remain user-friendly, however. More and more HPLC operators are not chromatographers. They dont want to see chromatograms or discuss resolution between peaks, they want to see a report that tells them if their run was successful.