With Automation, HPLC Systems Tailored To Research Needs

Sidebar: It's Okay To Be Fast High-performance liquid chromatography (HPLC) is a mainstay of biological and chemical research, analytical, and production laboratories. After a researcher loads a sample mixture, the instrument pumps mixtures of solvents-usually at high pressures-through a column packed with a material that slows individual compounds to different degrees, separating the substances as they pass through the column. Once the journey is complete, a detector and often an automatic fraction collector are charged with redirecting the solvent stream into separate, purified fractions. BRAINY: Isco's Foxy 200 collects HPLC fractions by time, volume, or peak. "Ultimately these systems are a set of pumps, a controller, and a fraction collector," says Roland Strong, a protein crystallographer at the Fred Hutchinson Cancer Research Center in Seattle who studies protein interactions involved in the mucosal immune system. Strong observes that "there must be hundreds of different types" of separation columns that users can plug in. That versatility allows researchers with varying needs to share similar setups by simply exchanging columns and solvent types, easing the strain on everyone's budget. For example, Strong says, "there are three protein crystallography labs here: We each have a different company's [instrumentation] system, and we swap columns back and forth." Columns may separate on the basis of a sample's size, ionic character, hydrophobicity, affinity for another molecule, or isoelectric point (the pH at which a compound is neutral), to name some of the more popular separation methods. HPLC lends itself to separation of just about every kind of compound, including (but not limited to) peptides, proteins, oligonucleotides, carbohydrates, lipids, and small organic molecules. Perhaps the greatest concern in purchasing HPLC equipment is reliability. The commonly used HPLC techniques have been around for decades, and probably aren't going to disappear over the horizon any time soon. Operators hope their equipment will last, too. COMPACT: Isco's Foxy Jr. fraction collector holds up to 144 tubes in a square foot of bench space Flexibility is also a big plus for any HPLC equipment. Isco Inc. of Lincoln, Neb., sells the popular Foxy 200 and Foxy Jr. fraction collectors. "Fraction collectors with brains are highly desired," says Vikas Padhye, program manager for HPLC at Isco. The Foxy models can collect a wide range of volumes into many kinds of containers. And they can be programmed to collect only selected peaks, determined by retention time, threshold level, and slope of the signal from an ultraviolet or other detector. On the other end, autosamplers keep the process going by dipping into samples to analyze the contents. Autosamplers may allow control of injection time, number of injections per vial, rinses, and other parameters. "I like my autosampler-I do my method development by day and my routine work overnight. [It is] very handy from an efficiency standpoint," says Christopher Judd, a chemist at Collaborative Laboratories, an East Setauket, N.Y.-based company that develops encapsulation and delivery systems for a variety of applications. PROGRAMMABLE INJECTION: Alcott Chromagography’s model 718 variable-volume autosampler The 708 Series autosampler, available from Alcott Chromatography Inc. of Norcross, Ga., offers control of all parameters and is said to fit any HPLC or ion chromatography setup. If fitted with Alcott's optional FilterCap, the 708 will auto-filter samples, reportedly saving time and increasing productivity. Alcott is evaluating the potential of FilterCap to help automate sample processing in the blood plasma industry, according to Lou Leazenbee, marketing manager at Alcott. It can filter blood plasma down to a 2 mm pore size. Alcott's model 718 autosampler uses one of three high-resolution syringes to give a programmable injection volume from 1 ml to 2,000 ml. The Series 200 autosampler, available from Perkin-Elmer Corp. of Norwalk, Conn., was designed to lessenthe headaches of those running quality-control samples. The 200 offers precision and ease of use and can process up to 225 samples at a time, according to the company. The instrument comes with complete installation and operational qualification protocols to assist in generating an internal performance-verification system. And that's no small feat, says Judd. "Instrument certification is increasingly necessary in my lab, and the first time I did it, it was a chore just to find out exactly what the relevant tolerances were on all the instruments." PREVENTS 'TRACKING NIGHTMARES': The Gilson Combinatorial Chromatography system comes with sample-tracking software The 215 Liquid Handler-sold by Gilson Inc. of Middletown, Wis.-is designed to help the user eliminate exposure to potentially harmful liquid samples and can pierce thick septa. The 215 accommodates up to 12 standard or deep-well microplates. The increased throughput and efficiency free up technicians to perform other tasks, claims Gilson's director of marketing, Gordon Hunter. In many cases, "speed is the issue," he says. As in basketball, track, and sneaker commercials, speed counts in HPLC analysis. Fast liquid chromatography (FLC) takes advantage of smaller particle size or reduced pore volume to achieve faster performance. Perkin-Elmer's Fast LC columns are packed with 3-mm particles, resulting in shorter equilibration times and lower solvent usage. The columns can enhance productivity two to five times over that afforded by conventional 15-cm HPLC columns packed with 5-mm particles, the company says. MICRA Scientific Inc. of Northbrook, Ill., has developed a manufacturing process that eliminates pores in the silica and prepares very pure, highly uniform 1.5-mm non-porous silica (NPS) microspheres. NPS columns speed up the analysis of analytes and minimize the diffusion that interferes with separation. "You get much higher productivity because run times are three to 60 times shorter than with porous columns, and this frees up your instrument for additional analysis," says Theresa Dawson, marketing specialist at MICRA. Support stability and sample recovery also benefit because internal pore blockage and silica breakdown are greatly reduced or eliminated. The column also uses 75 percent to 90 percent less solvent than traditional, porous silica. NPS can be used for reversed-phase, normal phase, and chiral applications, according to Dawson. MICRA sells several non-porous minibore, low-pressure, extended-chain, capillary, and chiral columns. Lower solvent usage and faster run times are attractive in these days of high-throughput applications, making fast LC worth a long look. -J.K. Alltech Associates Inc. of Deerfield, Ill., sells the Model 570 autosampler, which is available in refrigerated, large-volume, partial loop-fill, and biocompatible versions. It draws solvent from a reservoir to flush the needle and connecting lines after each injection to minimize sample carryover. It also comes with either total or partial-loop filling, which reportedly helps avoid sample waste. The one part of the HPLC setup that is not optional-aside from the column itself, of course-is the pump. Not the most glamorous piece of the puzzle, the pump must reliably mix and drive the solvents through the column at the chosen flow rate. If it balks, analysis grinds to a halt. One- and two-piston pumps are available. Single-piston pumps are much cheaper, easier to prime, and less expensive to maintain, but they suffer from surges in the flow rate that result from the single driving force, while double-piston pumps even out the surges by pumping consecutively. Although single-piston surges can interfere with the detector, these pumps do have their place; "they are [useful for] some applications where the sensitivity of the detectors is very low," says Alcott's Leazenbee. Perkin-Elmer's Series 200 LC pumps are available in isocratic, binary, and quaternary versions to handle a variety of different solvent-mixing needs. The isocratic and binary pumps can be field upgraded to quaternary capability. Pressure pulsations are minimized by dual-piston design. Isco's Model 2350 programmable pump allows the user to change the flow rate after an elapsed time and delivered volume. Its flow rate stays constant within 0.5 percent over the entire flow range, which should ensure reproducible retention times, according to the company. Isco's Models 2360 and 2361 gradient programmers can feed the pump precisely mixed gradients from up to three different solvent sources, or they can proportion isocratic solvent mixtures online to reduce preparation effort and solvent waste. The Gold Nouveau Solvent Delivery modules sold by Beckman Instruments Inc. of Fullerton, Calif., have a patented floating piston and reciprocating shaft assembly that increases the reliability and extends the seal's lifetime, according to Beckman. The company's Gold Nouveau Microbore Pump uses high-pressure mixing to ensure that even difficult-to-mix gradients such as TFA/Acetonitrile/water mix thoroughly and reproducibly. Of course, a steady stream trickling from the end of the column doesn't do anybody much good unless there is a detector in place to alert the fraction collector that a substance of interest has arrived. A variety of detectors are on the market, but none fits every possible HPLC application; instead, most specialize in the detection of certain classes of compounds. The Mariner ESI-TOF MS detector system, from PerSeptive Biosystems Inc. of Framingham, Mass., is a benchtop time-of-flight/mass spectroscopy detector that uses electrospray technology to determine the mass of compounds as they come off the column, with minimal mass consumption, according to the company. "Mariner [is] ideally suited for high-throughput applications in characterizing proteins and peptides, in metabolic studies, in combinatorial chemistry strategies, and in high-throughput screening," says Brian Musselman, director of worldwide marketing for biospectrometry products at PerSeptive. Probably the most widely used detection method is measuring the absorbance of ultraviolet (UV) light or visible light by compounds exiting the column in the eluant flow. Isco's V4 variable UV/vis detector uses a deuterium lamp that has a life of more than 10,000 hours, according to the company. It has a range of 190 nm to 750 nm. Full sensitivity over the entire UV/vis range is ensured by using two lamps to heighten the performance at higher wavelengths. Isco also offers the Linear Model 205 programmable, dual-wavelength UV/vis detector, which allows detection at two wavelengths simultaneously. Dual-wavelength detection can help the researcher evaluate peak purity, quantitate overlapped peaks, or just compare and optimize chromatograms. Alltech's fixed-wavelength Model 450 UV detector incorporates what the company calls "indestructible" high-pressure flow cells, and its long-life lamps reportedly eliminate cell repairs and reduce maintenance. The Linear Model 2000 variable-wavelength UV/vis detector offers simple analog controls and interchangeable flowcells and lamps, while the company's Linear Model 205 and 201 programmable UV/vis detectors are dual-wavelength and single-wavelength, respectively. Diode array detectors scan an entire spectrum continuously, making them powerful tools for detecting complex sample mixtures. Beckman's Gold Nouveau 168 Diode Array Detector includes a real-time peak purity assessment feature. To determine a peak's purity and get an estimate of the number of compounds present in the peak, the researcher need only count the "purity bars" on the chromatogram. Perkin-Elmer's Model 785A programmable absorbance detector features a wavelength scanning range of 190 nm to 700 nm. The instrument offers user-selected wavelength, range, rise time (the time it takes for the output signal to rise from 10 percent of its final value to 90 percent of its final value-a larger rise time allows the recorder to average the noise frequencies to eliminate some of the short-term noise), and autozero (resetting the detector to baseline). The system also shuts down whenever it detects a solvent leak. The 500 ELSD evaporative light-scattering detector, available from Alltech, works with any volatile mobile phase, regardless of its optical properties. Inside the flow cell, the detector hits sample particles with light from a laser diode, and scattered light that results is detected by a silicon photodiode, which generates an electric signal. With the 500 ELSD, researchers no longer need to avoid mobile phases that absorb in UV or have refractive indices near that of the sample, the company says. For those working with fluorescent compounds or tags, Alltech sells its Linear Model 305 fluorescence, phosphorescence, and chemiluminescence detector. High-efficiency holographic diffraction gratings are optimized for high sensitivity at both lower and higher excitation wavelengths. INTERCHANGEABLE LIGHT SOURCES: McPherson’s model 750B fluorescence detector includes dual monochromator, xenon light source, and HPLC flow cell. The model 750B HPLC-plus variable-wavelength fluorescence detector-offered by McPherson Inc. of Acton, Mass.-has options of three interchangeable light sources: deuterium (30W), xenon (150W), and xenon/ mercury (200W). Additionally, it features interchangeable flow cells and a dual excitation and emission monochromator, which can also be used for scanning spectrofluorometry via 10 x 10 mm cuvettes. Other multimode fluorescence sampling accessories are available, as well. McPherson's new model 660 HPLC chemiluminescence detector allows HPLC or flow-through chemiluminescence detection via a flow cell design. The analytical reagents are mixed directly in front of the detector for maximum chemiluminescence signal, says Brian O'Flaherty, applications chemist at McPherson. Users can employ existing pumps, plumbing, and data-acquisition devices to construct a novel detector. Owing to its low background signals, chemiluminescence detection can achieve a 1,000-fold improvement in sensitivity over fluorescence (J. Kling, The Scientist, May 12, 1997, page 16). Despite the wide range of detectors available, one need not always make a big investment in new equipment to improve detection. "Adding a chromophore to a compound via derivatization may improve your detection limit," says Barbara Olson, senior product development chemist at Pierce Chemical Co. of Rockford, Ill. The HPLC derivatization reagents sold by Pierce can be used to covalently modify compounds either before or after they are separated on a column. Fluoraldehyde o-Pthalaldehyde, one of Pierce's biggest derivatization sellers, is frequently used for amino acid analysis. Ambient conditions are not always ideal for running HPLC analyses and separations, and to get around that limitation, companies like Sanyo Scientific of Itasca, Ill.-a division of Sanyo Sales and Supply Corp.-offer refrigerator environments for HPLC. Sanyo's MPR-1410 large-capacity enviro-center can maintain a temperature range of 2°C to 23°C, with an effective capacity of 1,370 liters (48.36 cubic feet). The MPR-1410R has similar features, but includes drawers instead of the MPR-1410's movable shelves. For those in need of decreased mobile-phase viscosity and increased mass transfer and sample solubility, elevated column temperatures may do the trick. Controlled, elevated temperatures, provided by products like Perkin-Elmer's Series 200 LC column oven, can give better resolution and greater speed of analysis. The 200 eliminates thermal gradients along the column by preheating the mobile phase and sample before they reach the column, to temperatures from 30°C to 100°C. Sampler's Little Helpers Automation is a major trend in HPLC. The software that comes with these systems varies widely, from very simple setups to the highly complex, and Strong says users should be careful to pick a system with software that suits them. He suggests assessing "not only [that] you are comfortable with it, but does it do what you want to do? Some labs have very simple needs, while others need more flexibility." SELECTED SUPPLIERS OF HPLC PRODUCTS: Alcott Chromatography Inc. Alltech Associates Inc. Beckman Instruments Inc. Gilson Inc. Isco Inc. McPherson Inc. MICRA Scientific Inc. Perkin-Elmer Corp. PerSeptive Biosystems Pierce Chemical Co. Sanyo Scientific Waters Corp. Perceiving a need among researchers to run isocratic and gradient separations with columns ranging from 1 mm to 4.6 mm internal diameter, Waters Corp. of Milford, Mass., designed the Alliance automated HPLC system. The system features a new, patented fluid delivery technology, which is optimized for flow rates from 50 mm to 5,000 mm per minute. "Combining the fluidics with improved solvent proportioning has resulted in more accurate, reproducible gradient performance," says Steven A. Cohen, principal research chemist at Waters. For those researchers in search of a system that will keep working while they are home sleeping, Perkin-Elmer offers its Nightshift system. Equipped with the PE Nelson Turbochrom Workstation, the system can handle up to eight complete HPLC or gas chromatograph systems. It also includes Perkin-Elmer's new Smart Suitability software, with decision-making intelligence, to ensure that the samples run only when the system passes preset criteria. "Smart Suitability knows that your calibration is out of range or that your column is failing and either continues, reinjects the standard or group of standards, or stops the system, not wasting precious samples," says Frank Vandemark, liquid chromatography product manager at Perkin-Elmer. Gilson sells the Gilson Combinatorial Chromatography system to meet the needs of high-throughput combinatorial chemistry-a technique by which thousands of related compounds are synthesized and separated automatically. The Gilson system comes with sample-tracking software that matches samples, peaks, and fractions, saving technicians from the tedium of manual tracking. "It can be a tracking nightmare trying to know where your purified peaks are relative to the initial sample," says marketing director Hunter. Sample injection and fraction collecting are handled by the same instrument, allowing it to automatically reinject collected samples for reanalysis. Beckman and Isco offer customized HPLC systems, designed to fit the researcher's individual needs. Automation is attractive, indeed, for any user with a big enough budget. Features and capabilities abound, but all available models still lack one critical function, says Judd. "I'd like one that makes a good double espresso on Monday morning." Well, that perk may not be coming anytime soon, but researchers can always dream. James Kling, a science writer based in Bellingham, Wash., is online at jkling@nasw.org.

With Automation, HPLC Systems Tailored To Research Needs

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