A Peak at HPLC

In 1906, Mikhail Tswett separated plant pigments on a chalk column. Alluding to the separated colors, he termed his technique chromatography.1 Nearly a century later, chromatography hasn't changed all that much; it still relies on the differential affinities of analyte components for the mobile and stationary phases of the separation. Yet in high-performance liquid chromatography (HPLC), the technique assumes a decidedly high-tech air, with gleaming stainless steel components, computer control, and advanced detectors. The stationary phase is typically packed into a small column through which the mobile phase migrates. An injector valve introduces the analyte into the mobile phase, and one or more detectors scan the column output. This general setup describes most medium-pressure and low-pressure systems, as well. Lou Bellafiore, president of TechniKrom in Evanston, Ill., points out that the basic distinction "is really the backpressure generated by the [stationary phase] particle size .... Ultimately the particle size dictates the flow pressure that will be observed." HPLC originally referred to high-pressure liquid chromatography, but was changed to high performance when it became apparent that the high pressure typical of HPLC separations was not necessarily responsible for the improved performance. Most HPLC separations fall into three modes: adsorption, ion exchange (IEC), and size exclusion (SEC). Adsorption chromatography includes normal-phase HPLC, with a polar stationary phase and nonpolar mobile phase, and reversed-phase HPLC, with a nonpolar stationary phase and a polar mobile phase. In IEC the charged stationary phase binds samples with opposing charge, while SEC relies on precisely controlled pore sizes in the stationary phase to separate analytes by molecular size. Other separation modes also exist, such as affinity chromatography, which relies on specific interactions between an immobilized ligand and the analyte, and chiral separations, which rely on preferential interaction of a chiral selector with one enantiomer.2 Most biological separations, though, use reversed-phase chromatography, because the samples are generally polar and in aqueous phase.3 PUMP DOWN THE VOLUME In biological research, scientists often separate mixtures using a mobile-phase gradient. "In a gradient," says TechniKrom's Bellafiore, "... the product sticks to the head of the column when you load it. As you approach a particular solvent or ionic strength that moves the product down the column, it moves in a tight band." To produce a gradient, differing proportions of two (binary) to four (quaternary) mobile phases are mixed upstream of the column, at either high or low pressure, using a pump. The pump then pushes the mobile phase through the column. HPLC systems usually employ either syringe or reciprocating piston pumps. In the former, a piston attached to a stepping motor empties the syringe's mobile phase at a constant rate. According to Richard Tobeau, fluidics pump manager for Harvard Apparatus, Holliston, Mass., "We use approximately 12,800 pulses to turn the motor once." The large number of steps delivers a smooth liquid flow. Reciprocating piston pumps are also common, especially at higher flow rates where syringe pumps would be inconvenient. In a reciprocating piston pump, one piston pumps while the other refills, dramatically reducing pulsations required to refill the piston chamber on the upstroke (a noticeable problem in many single-piston pumps). The lower limit of accurate solvent flow for smaller HPLC pumps has changed in recent years to meet the stringent requirements of ever-smaller columns and samples. Application chemist Luke Roenneburg at Gilson, Middleton, Wis., confirms that "scale-down is the major area [of current interest]." Gilson's newest pumps, he says, handle flow rates as low as 100 nanoliters per minute in gradient modes. STACKS OF COLUMNS Most HPLC columns contain hydrated porous silica particles, on which various bonded layers are immobilized via siloxane bonds. Supelco of Bellefonte, Pa., for example, offers butyl (C4), octyl (C8), octadecyl (C18), and diphenyl groups on a silica matrix for reversed-phase applications. MicroSolv Technology of Long Branch, NJ, makes columns with a more durable and pH-stable silicon- carbon bond, while Zirchrom Separations of Anoka, Minn., uses zirconia as a base. Some manufacturers carry polymer-based stationary phases, generally polystyrene-divinylbenzene copolymers or, as in the case of Bellingham, Mass.-based Jordi Associates, divinylbenzene-based polymers. These polymer-based columns are most commonly used for IEC and SEC.2,4 Monolith columns are a recent innovation. According to Darryl Glover, manager of international marketing at BIA Separations in Slovenia, "A monolithic column is a solid piece of material with much better contact between the active groups on the column and the analyte. [You] can get excellent separations in less than a minute ... [and] put multiple discs in one column" to use separate chemistries in a single run. Additionally, monolith uniformity simplifies scaling up protocols. Because contamination by metal ions is a concern during protein separations, some companies offer biocompatible systems that replace stainless steel components with polyaryl ether ether ketone (PEEK) or other inert materials. Such concerns may be unnecessary, however. Derek Southern, vice president of Walnut Creek, Calif.-based LC Resources, notes that years ago, separations could take up to 20 hours, time enough to denature or chemically alter proteins. "Now with 20- to 30-minute separations, problems are less likely," he says. "A very few compounds may have an adverse reaction but I really haven't seen very many." NONDESTRUCTIVE DETECTORS HPLC is primarily a separation technique, and until recently the detector's role was mainly to signal the elution of an analyte. Increasingly, however, detectors do more than signal an analyte's presence; they also provide vital chemical information. HPLC detection methods can be nondestructive or destructive. Common nondestructive detectors measure absorbance, radiation, refractive index, fluorescence, conductivity, chemiluminescence, and other attributes. Complex HPLC systems aimed at identification of unknown samples will often include one or more nondestructive detectors followed by a destructive detector. Ultraviolet and visible absorbance, still the most common HPLC detection methods, includes the familiar variable- and fixed-wavelength instruments as well as diode array detectors (DADs), which allow simultaneous monitoring of a large number of wavelengths, but at a substantial cost. Other less-expensive alternatives exist. Grenville Chamberlain, general manager at Cambridge, UK-based Cecil Instruments, notes that his company's £6,000 CE4200 dual-channel detector is capable of a "full-range UV scan per quarter second." Tokyo-based Jasco's circular dichroism (CD) detector, says chromatography product manager Bill Hedgepeth, identifies chiral molecules by detecting absorption differences in left- and right-handed, circularly polarized light. Jasco's detector, Hedgepeth notes, "gives a simultaneous UV and CD signal," enabling immediate discrimination of the absorbance peaks of chiral molecules. A viscosity detector (viscometer) "measures a pressure differential between the mobile phase and the sample to determine viscosity," says Bill Willebey, regional representative for Houston-based Viscotek. "From this information plus molecular weight we can calculate the hydrodynamic size," which is a measure of the size of a molecule in solution. Classical light-scattering detectors, says Wyatt Technology president Geofrey Wyatt, are used to examine the solution properties of large molecules. Trevor Havard, president of Precision Detectors in Bellingham, Mass., explains the two types: "Static light scattering measures the scatter of light from large molecules, which lets you obtain molecular weight [given a concentration]. ... Dynamic light scattering measures the translational diffusion constant of the molecules in solution. From that, you can obtain the particle-size distribution and hydrodynamic radius." DESTRUCTIVE DETECTORS Destructive detectors can provide a wealth of information about the identity and characteristics of an analyte. Evaporative light-scattering detectors (ELSD), for instance, measure concentration. "ELSD is a universal detector whose primary objective is to detect and measure components," says Havard. "It nebulizes [the eluate], evaporates the mobile phase, and produces a fine, evaporated solid particle which passes a light source and scatters the light in a particular direction." Electrochemical detectors (ECD) look at "a chemical reaction, usually an oxidation, of the sample" notes Elizabeth Robertson, product manager of detection systems at Milford, Mass.-based Waters. "You set the potential as high as possible ... to push an electron onto the compound." In ECDs with amperometric electrodes, "this reaction affects only about 5% of the [reactive species], and compounds often revert to their original state," Robertson says. With a coulometric electrode, such as that manufactured by ESA in Chelmsford, Mass., nearly all the applicable molecules are reacted. Antek Instruments, Houston, manufactures a destructive nitrogen detector, which burns the eluate in a high-temperature environment, generating nitric oxide (NO) from any nitrogen present. In the presence of ozone, NO converts to an excited NO2 molecule, and the detector monitors its near-infrared emissions. The Unimas201 detector from UK-based Scientific Detectors takes another approach to analyte detection. Small amounts of eluate are continuously deposited on a moving oxidized-titanium tape. The solvent is then evaporated and the dried sample pyrolized. Theoretically, at this point any gas chromatography detector could be used, though an argon ionization detector is currently recommended. A mass spectrometer (MS) measures the mass of ionized molecules using the mass-to-charge (m/z) ratio of the ions formed. The LC eluate is converted to a gas, ionized, and run continuously through the MS during the separation. The MS continuously scans the m/z range of interest, and elution is monitored as peaks at particular m/z values. LC-MS is also an extremely powerful technique for analyzing complex mixtures because of its highly specific separations at different m/z ratios, even when multiple compounds are eluting at the same time. In proteomics particularly, separations have become complex enough that instruments have been developed to provide a second dimension of LC separation. According to Jon S. Gingrich, marketing and sales manager at Michrom BioResources, Auburn, Calif., these two-dimensional HPLC systems recognize the elution of a peak and divert the flow to a second column, usually with a different chemistry, where it is further fractionated before being run on an MS. Companies addressing multidimensional HPLC include Agilent Technologies, LC Packings, Michrom BioResources, and Micro-Tech Scientific. PURCHASING DECISIONS More than 90 companies sell HPLC hardware and accessories, including fluidics, columns, and detectors. Of these, the largest manufacturer is Waters (with approximately 18% market share overall in 2001, according to Eric Gay, drug discovery industry analyst at Frost & Sullivan). Other major players include Agilent, Amersham Biosciences, Applied Biosystems, Bio-Rad Laboratories, Dionex, Hitachi, Perkin-Elmer, Shimadzu, Supelco, and Thermo Finnigan. Some 30 companies offer integrated packages, but full systems are still largely customized products. A major advantage of purchasing a full system from a single vendor is that the integrated components are tested together, resolving some of the inevitable interface and control glitches. The most important question when purchasing an HPLC system, according to LC Resources' Southern, is "Does the company have a good service department in your area? Find the company that has the best service team and buy it from them." David Keller of Sonntek, Upper Saddle River, NJ, echoes Southern's advice, noting that the "relationship between the user and the two to four people they are going to interact with from the company ... is an important factor in the decision." The company's policies on support and service are also extremely important. Keller continues, "There is a tremendous amount of ignorance as to what after-sales costs really are, and there is a blurring of up-front costs and total cost of owning the system." Cost of ownership is even more important for preparative systems. As TechniKrom's Bellefiore points out, "Fewer fractions necessary to analyze [means] a lower resulting total cost of separation." Since preparative separations tend to involve large amounts of material, the cost of suboptimal separations can easily outweigh even large differences in initial equipment outlay. Jeremy Peirce (jlpeirce@princeton.edu) is a freelance writer in Jersey City, NJ. References 1. M.S. Tswett, "Adsorption analysis and chromatographic method. Application to the chemistry of chlorophyll," Berichte der Deutschen Botanischen Gesellschaft, 24:385, 1906 (translated in M. Teich, A Documentary History of Biochemistry, 1770-1940, Rutherford, NJ: Fairleigh Dickinson Press, 1992). 2. J.R. Larson et al., "Introduction," in HPLC: Practical and Industrial Applications, J. Swadesh, ed., New York: CRC Press, pp 1-55, 1997. 3. D. Swanson, "Six degrees of separation: HPLC separates mixtures of biomolecules to allow detailed studies," The Scientist, 14[7]:32, April 3, 2000. 4. Y. Kazakevich, H. McNair, Basic Liquid Chromatography: Textbook on High Performance Liquid Chromatography, available online at hplc.chem.shu.edu/HPLC. THE OEM GAME Many producers of high-performance liquid chromatography (HPLC) systems specialize in the manufacture of a small number of components but would like to offer a more comprehensive product line for marketing purposes. A common solution to this dilemma exists in the HPLC market, with an original equipment manufacturer (OEM) selling components, assemblages, or even complete pumps, detectors, and more, to another company, which may or may not modify the OEM's product to suit its own needs. The second company then sells the item under its own name. It is not uncommon for companies to sell to both OEM and retail markets. As an anonymous representative of a company with a large OEM and retail business quipped, "Our competitors are our biggest customers." Often the retail version of an OEM product will differ significantly in software, supplemental hardware, support, and so on. But if the buying decision depends on the hardware, a cost comparison between the retail and OEM versions may well save considerable expense. --Jeremy Peirce

A Peak at HPLC

Interested in reading more?

Become a Member of