Recent developments in the pharmaceutical industry and in combinatorial chemistry have exponentially increased the number of potentially useful compounds, each of which must be characterized in order to identify their active components and/or establish processes for their synthesis. To more quickly analyze these compounds, researchers have sought to automate analytical processes and to implement analytical processes in parallel.
One useful analytical process is chromatography, which encompasses a number of methods that are used for separating ions or molecules that are dissolved in or otherwise mixed into a solvent. Liquid chromatography (“LC”) is a physical method of separation wherein a liquid “mobile phase” (typically consisting of one or more solvents) carries a sample containing multiple constituents or species through a separation medium or “stationary phase.” Various types of mobile phases and stationary phases may be used. Stationary phase material typically includes a liquid-permeable medium such as packed granules (particulate material) disposed within a tube (or other channel boundary). The packed material contained by the tube or similar boundary is commonly referred to as a “separation column.” High pressure is often used to obtain a close-packed column with a minimal void between each particle, since better resolution during use is typically obtained from more tightly packed columns. As an alternative to packed particulate material, a porous monolith or similar matrix may be used. So-called “high performance liquid chromatography” (“HPLC”) refers to efficient separation methods that are typically performed at high operating pressures.
Typical interactions between stationary phases and solutes include adsorption, ion-exchange, partitioning, and size exclusion. Examples of types of stationary phases to support such interactions are solids, ionic groups on a resin, liquids on an inert solid support, and porous or semi-porous inert particles, respectively. Commonly employed base materials include silica, alumina, zirconium, or polymeric materials. A stationary phase material may act as a sieve to perform simple size exclusion chromatography, or the stationary phase may include functional groups (e.g., chemical groups) to perform other (e.g., adsorption or ion exchange separation) techniques.
Mobile phase is forced through the stationary phase using means such as, for example, one or more pumps, gravity, voltage-driven electrokinetic flow, or other established means for generating a pressure differential. After sample is injected into the mobile phase, such as with a conventional loop valve, components of the sample will migrate according to interactions with the stationary phase and the flow of such components are retarded to varying degrees. Individual sample components may reside for some time in the stationary phase (where their velocity is essentially zero) until conditions (e.g., a change in solvent concentration) permit a component to emerge from the column with the mobile phase. In other words, as the sample travels through voids or pores in the stationary phase, the sample may be separated into its constituent species due to the attraction of the species to the stationary phase. The time a particular constituent spends in the stationary phase relative to the fraction of time it spends in the mobile phase will determine its velocity through the column. Following separation in an LC column, the output or eluate stream contains series of regions having an elevated concentration of individual component species. Thus, HPLC acts to provide relatively pure and discrete samples of each of the components of a compound. Gradient separations using conventional HPLC systems are typically performed within intervals of roughly five to ten minutes, followed by a flush or rinse cycle before another sample is separated in the same separation column.
Following chromatographic separation in the column, the resulting eluate stream (consisting of mobile phase and sample) contains a series of regions having elevated concentrations of individual species, which can be detected by various flow-through techniques including spectrophotometric (e.g., UV-Vis), fluorimetric, refractive index, electrochemical, or radioactivity detection. Liquid chromatography with flow-through detection generally provides signal response that is proportional to analyte amount or concentration. As a result, LC is well suited for quantitative analysis, but it is difficult to identify or characterize individual components using only LC, particularly when novel or previously uncharacterized compounds are used.
Another important analytical technique that can complement LC analysis is mass spectrometry (“MS”), which is widely used in many industrial and academic settings. MS permits molecular mass to be measured by determining the mass-to-charge ratio (“m/z”) of ions generated from target molecules. A mass spectrometer typically includes a source for generating ions from a sample and delivering them into the gas phase, an analyzer for separating and sorting the ions, and a detector for sensing the ions as they are sorted. MS is a fast analytical technique that typically provides an output spectrum displaying ion intensity as a function of m/z. The benefit of using MS is that it can provide unique information about the chemical composition of the analyte—information that is much more specific than that can be obtained using flow-through detectors used with most conventional LC systems. Knowing the mass and composition of a desired molecule is especially important for pharmaceutical research, particularly in the synthesis of novel and uncharacterized molecules. The ability to qualitatively identify molecules using MS complements the quantitative capabilities of LC, thus providing a second dimension to the chromatographic analysis.
Various mass spectrometric techniques are known, including time-of-flight (“TOF”), quadrupole, and ion trap. In a TOF analyzer, ions are separated by differences in their velocities as they move in a straight path toward a collector in order of increasing mass-to-charge ratio. In a TOF MS, ions of a like charge are simultaneously emitted from the source with the same initial kinetic energy. Those with a lower mass will have a higher velocity and reach the detector earlier than ions with a higher mass. In a quadrupole device, a quadrupolar electrical field (comprising radio frequency and direct-current components) is used to separate ions. An ion trap (e.g., quadrupole-based) can trap and mass-analyze ions using a three-dimensional quadrupolar radio frequency electric field. In ion trap instruments, ions of increasing mass-to-charge ratio successively become unstable as the radio frequency voltage is scanned.
Various conventional ionization techniques may be used with mass spectrometry. One prevalent technique is electrospray ionization (ESI), which is a “soft” ionization technique. That is, ESI does not rely on extremely high temperatures or extremely high voltages to accomplish ionization, which is advantageous for the analysis of large, complex molecules that tend to decompose under harsh conditions. In an ESI interface, highly charged droplets of analyte dispersed from a capillary in an electric field are evaporated (typically assisted by the application of a drying gas), and the resulting desolvated charged ions are drawn into a MS inlet. Other known ionization techniques include: chemical ionization (which ionizes volatilized molecules by reaction with reagent gas ions); field ionization (which produces ions by subjecting a sample to a strong electric field gradient); spark-source desorption (which uses electrical discharges or sparks to desorb ions from samples); laser desorption (which uses a photon beam to desorb sample molecules); matrix-assisted laser desorption ionization or “MALDI” (which produces ions by laser desorbing sample molecules from a solid or liquid matrix containing a highly UV-absorbing substance); fast atom bombardment or “FAB” (which uses beams of neutral atoms to ionize compounds from the surface of a liquid matrix); and plasma desorption (which uses very high-energy ions to desorb and ionize molecules in solid-film samples).
By coupling the output of an HPLC system to a MS system, it becomes possible to both quantify and identify the components of a sample. There exist challenges, however, in providing efficient integrated HPLC/MS systems. Conventional MS systems are capable of much faster sample analysis than HPLC systems, and are much more expensive by a factor of roughly four to five times the cost of a single-column HPLC system. Integrated HPLC/MS systems including a single HPLC column coupled to a MS by way of an ESI interface are known, but they suffer from limited utility since the overall system throughput is limited by the HPLC column, which requires several minutes to separate a single sample. In other words, a HPLC/MS system having only a single HPLC column fails to efficiently utilize the rapid analytical capabilities of a mass spectrometer.
High throughput HPLC/MS systems having multiple HPLC columns coupled to a single MS are also known and provide greater separation efficiency compared to single-column HPLC/MS systems. Such systems, however, still suffer from limited utility. Examples are provided in U.S. Pat. No. 6,410,915 to Bateman et al.; U.S. Pat. No. 6,191,418 to Hindsgaul et al.; U.S. Pat. No. 6,066,848 to Kassel et al.; and U.S. Pat. No. 5,872,010 to Karger et al., each showing some variation of a multiplexed HPLC/MS system where the outputs of multiple simultaneously-operated separation columns are periodically sampled by a single MS device. However, in such real-time multiplexed HPLC/MS systems, the MS can sample an eluate stream from only one LC column at a given time. While one stream is being analyzed, the others must continue to flow, as these systems have no storage capacity. The streams that are not being directed to the MS at any point in time are directed to waste, inherently resulting in data loss. To mitigate this data loss, MS sampling must occur very quickly. The MS instrument thus receives very small plugs of sample, reducing the ability of the instrument to integrate data in order to eliminate noise and resulting in reduced signal clarity.
Another staggered ‘parallel’ approach is described in U.S. Pat. No. 6,318,157 to Corso et al (“Corso”). Corso describes a multiplexed HPLC/MS device where gradient separations are performed by staggering the initiation of separations in four separate columns by using input lines of varying length. In this manner, each output stream may be analyzed continuously by the MS instrument. The staggering technique taught by Corso effectively acts as four serial separations. While some efficiencies are gained by not having to prepare a single column four times, the overall run time of the four columns run in a stagger is much longer than the run time of four columns run simultaneously. Additionally, the necessary amount of stagger (i.e., the length of each input line) must be calculated in advance to insure that regions of interest have no temporal overlap, which may be difficult when characterizing unknown compounds. Corso also suggests that the staggering of inputs is not necessary for isocratic separations; however, Corso does not indicate how overlap of regions of interest can be avoided. Presumably, a sampling technique is used, thus creating the same data loss and signal clarity issues discussed above.
Accordingly, there exists a need for improved HPLC/MS systems that permit parallel analysis of multiple samples. Advantageous system characteristics would include scalability to permit a large number of samples to be analyzed simultaneously at a low cost per analysis with minimal loss of data and/or signal clarity. Ideally, an improved system would operate rapidly and be comparatively simple and inexpensive to build and operate.
None of the figures are drawn to scale unless indicated otherwise. The size of one figure relative to another is not intended to be limiting, since certain figures and/or features may be expanded to promote clarity in the description.