Smaller is Better

Smaller Columns and Particle Size in HPLC Systems Offer Higher Throughput and Require Less Solvents

While the fundamental components of a high performance liquid chromatography (HPLC) system—pumps to deliver solvent, an injector, a column for separating the constituents of a sample, a detector and computing software—have remained the same, there continues to be innovations in their design and capabilities. The selection of an HPLC system is predominantly driven by the end users’ needs; however, the availability of specialized, customizable platforms has given rise to many more options. Preparative HPLC, for instance, is ideal for large-scale purifications of small molecules or peptides, while high-throughput HPLC systems, optimized for short run times and integrated with autosampler units, allow for rapid analysis of large numbers of samples.

HPLC accessories such as columns and detectors also can be modified to suit the application. While a fluorescent, UV or visible light detector is often a standard component in an HPLC system, customizable platforms include other detection technologies ranging from radiometric to electrochemical to mass spectrometry (MS). Multiple detectors also can be integrated, such as tandem MS/MS systems that offer more focused, quantitative analyses. Two major variations in the pump design include high-pressure and low-pressure gradient systems. High-pressure gradient systems mix the solvents after reaching the pump and are more suitable for low flow–rate applications, such as high throughput sampling. In contrast, low-pressure gradient systems mix solvents before the pump inlet and may operate in a higher flow-rate range. Additional components, such as column ovens and autosamplers, also can be integrated, depending on the customers’ needs.

There also has been significant innovation in column technology. Newer columns, such as the monolith, amide, polar embedded and fused particle, are more resistant to changes in temperature, pH and flow rates, and they allow users to explore new methodologies and applications. The recent shortage in acetonitrile, the most common solvent for HPLC analysis, also is causing people to reevaluate their protocols and chromatography systems. “We have gotten used to using acetonitrile as a solvent, but there are many other options,” says George Limpert, advisory scientist for the Analytical Services Division at Celsis International plc. “You can solve some problems with what we have on hand without investing in very expensive equipment.” Some of the new column technologies, for instance, are certainly amenable to the use of other solvents like water, methanol and THF.

Using columns with smaller particle size not only reduces costs but also improves the throughput. “Smaller particle size is where the industry is headed,” says Elizabeth Hodgdon, senior product manager in the Waters Division of Acquity UPLC Systems. Ultra performance liquid chromatography (UPLC) systems use columns with polymeric particles less than two microns in size that allow rapid analysis of samples at sub-micromolar flow rates. “We have always focused on chemistry, and UPLC is really a chemistry change,” says Hodgdon. Although the separation principle for UPLC is exactly the same as for HPLC, the differentiation is in the design of the system, which takes advantage of the smaller particle size. “We found that we could reduce the particle size and yet have a particle that was robust enough to withstand high pressures and could be suitably packed in a column. Then we realized that in order to truly reap the benefits of the increase in efficiency with using a smaller size particle, we needed to redesign the system.”

Besides the systems and the accessories, the software programs for HPLC also are getting more sophisticated in order to handle and organize the large and complex data files generated. Web-based operations now allow data sharing across multiple users and multiple sites, while enabling complete automation and access. While these new technologies do exist, users have to carefully evaluate what they need. “Lab managers need to evaluate their lab procedures and hone in on processes or products that are slowing down their work flow, and find ways to improve their efficiency and performance in critical areas,” says Hodgdon. For their part, vendors and service providers are becoming more proactive in sharing information and offering technical support. Companies are becoming increasingly aware of the need for customer service and periodic monitoring and troubleshooting. “We can’t always predict when maintenance will be needed, but we can certainly plan for it,” says Hodgdon.

Tanuja Koppal, PhD, is a freelance science writer and consultant based in Randolph, N.J.

Cecil Instruments
AutoQuest LC autosamplers are designed for use with the Adept HPLC, Q-Adept HPLC and IonQuest ion chromatography systems. They may also be used with third party LC systems. The autosamplers are available with 50 or 100 sample positions and provide for ultra low carryover, ultra high injection precision, priority sampling, replicate injections and sample volumes from 5 μL to 2 mL. The ultra low carry-over provides for the ultimate in effective sampling for trace residue analysis, impurity determinations and contaminant levels. The autosamplers fit neatly with the Adept HPLC, Q-Adept quaternary HPLC and IonQuest ion chromatography systems, each of which is designed to achieve long-term, fast and reliable measurements. Because the systems are modular and versatile, different detectors may be accommodated depending on analytical requirements. These detectors include variable wavelength UV/ Visible, WaveQuest UV/Visible, electrochemical, refractive index, conductivity and fluorescence.
www.cecilinstruments.com

Eksigent
The ExpressHT™-Ultra HPLC system provides short cycle times and high throughput for LC/MS studies of pharmacokinetics and drug metabolism. With cycle times as short as 60 seconds, the ExpressHT-Ultra can run six 96-well sample plates overnight. Running at a maximum pressure of 10,000 psi, it enables the use of small particle stationary phases to deliver narrow peaks to the mass spectrometer. Employing columns with diameters of 1 mm or smaller, the system uses up to 95 percent less solvent per analysis than traditional HPLC systems, helping customers meet green initiative goals and reduce purchase and disposal costs. Proprietary Microfluidic Flow Control (MFC) technology generates accurate and reproducible flow rates from nanoliters per minute to hundreds of microliters per minute. Control software includes integrated drivers for the most widely used mass spectrometry programs including Applied Biosystems/MDS SCIEX Analyst™, Thermo Scientific Xcalibur™ and Bruker Daltonics HyStar™.
www.eksigent.com

SMI-Labhut Ltd.
The HT800L HPLC autosampler handles up to 110 autosampler vials or two microplates in an ambient or cooled environment with no re-calibration. Complete flexibility of sample handling ensures that up to 10 reagents can be added for derivitization, addition or dilution. The contents of any vial may be added to any other vial. Samples can be combined, mixed, and injected in microliter or full/partial loop volumes through the integral injection valve, with precise accuracy and exceptional reproducibility. For simple applications, the user sees only the required options in the control software, thus making the HT800L very simple to use. Thoughtful features such as internal lighting and a special safety mode of operation when the door is open, ensure its usefulness for retrofit and Private Label applications.
www.labhut.com

Waters
The ACQUITY UPLC® system eliminates time and cost per sample from analytical processes while improving the quality of results. By outperforming traditional or optimized HPLC, the system allows chromatographers to work at higher efficiencies with a much wider range of linear velocities, flow rates, and backpressures. UPLC® technology is a highly robust, dependable, and reproducible system. What differentiates the system’s holistic design is the patented sub-2 μm hybrid particle chemistry, offering significant benefits over today’s HPLC systems equipped with standard 5 μm particle chemistries. The ACQUITY system, used on its own or paired with proprietary optical and MS detection technologies, provides endto- end solutions for applications including ADME screening, food safety, bioanalysis, clinical, metabolite identification, and more. The system also routinely handles demanding applications such as the turn-key solutions built for amino acid and peptide analyses.
www.waters.com

Restek
Ultra II™ LC columns are specifically designed for universal application—optimal chromatography on any system. This column line is built on highly inert, high surface area silica that is completely Restekmanufactured, providing excellent column-to-column reproducibility. The columns are available in a comprehensive range of particle sizes (1.9μm, 2.2μm, 3μm, and 5μm), creating truly scalable chromatography on any type of LC instrument, from conventional to ultra-high pressure systems. Ultra II™ columns are available in traditional phases (C18, Aqueous C18, Silica) and unique chemistries that provide alternate selectivity (Biphenyl, Aromax, PFP-Propyl).
www.restek.com/hplc

Büchi
Designed and developed with chemical, biochemical and pharmaceutical research laboratories, SepacoreControl software controls all Buchi chromatography systems, from simple Flash Systems to advanced Preparative workstations. Intuitive and easy to use, the software allows quick and efficient setup of general chromatography parameters. This method management enables simple programming and operation of purification parameters such column conditioning, separation, column cleaning and system purging. A database of column specifications, suggested flow rates and maximum pressure ratings for the extensive range of proprietary cartridges and columns helps set up the run quickly and efficiently. Peak tracking, the ability to highlight fractions of interest and the generation of user-specific flow charts are definable advantages. Operation panels can be adjusted and an export function allows detailed reports to be created.
www.buchi.com

Shimadzu
Prominence nano HPLC supports high-precision gradient analysis by providing advanced flow-rate precision in the nano flow-rate range. It delivers exact flow rates via two LC-20ADnano pumps that are independently controlled by feedback from integrated high-precision nano flow sensors. The instrument’s Reflux Flow Control System (patent pending) stabilizes solvent delivery and ensures low solvent consumption with no disposal of split flow, minimizing environmental impact. Prominence nano is configured with a proprietary FCV nano switching valve to optimize bio sample analysis in the nL/min level for trap injection and 2-D LC applications. With the low dead volume FCV nano, the volume between ports is as low as 25 nL, so there is virtually no peak broadening in the nano flow-rate range.
www.ssi.shimadzu.com

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