27 Octobre 2006-10-27

Toward the Future:

Tandem and Hyphenated Methods

Through combinations with analytical techniques, chromatographic methods are enhanced.

Cullen T. Vogelson

Over the past 30 years or so, the need for chromatographic techniques that provide increased resolution, molecular structure elucidation, sensitivity, and separation power, along with decreased analysis times and detection limits, has become critical for applications ranging from environmental analyses, chemical syntheses, and polymer characterizations to toxicological investigations and pharmaceutical development. To meet this need, techniques that combine various chromatographic methods, as well as ones that incorporate analytical (primarily spectroscopic) techniques into traditional chromatography systems, have evolved. The result of this wide range of pairings is a new world of separation and analysis technology that is simultaneously as flexible, efficient, and precise as any specific application may necessitate.

Chromatography in Tandem

Beginning in the late 1960s, efforts to improve separation technologies focused on methods that involved the sequential use of different chromatographic techniques. This approach to isolating multiple components in a complex mixture of materials is still used today and is commonly referred to as a multidimensional, two-dimensional, "coupled column," or tandem chromatographic system.

The coupling idea itself is an evolution of flatbed chromatography and essentially involves separating a mixture of compounds first along one direction and then along a different, perpendicular direction . Although this may be accomplished using a continuous flow, flatbed approach, today the goal is achieved most commonly by physically transferring partially resolved sample components from one separation system of a given selectivity to another of differing selectivity (a process referred to as "heartcutting"). Methods that combine LC systems (or HPLC columns) with GC columns in multiple combinations are also common. Further, modern sample processing may be conducted simply by using multiple, thin-layer chromatographic developments, or complexly by coupling supercritical fluid columns with standard HPLC or GC instruments.

At present, multidimensional methods are conducted either on- or off-line. Off-line techniques evolved first, and the term refers to the manual collection of chromatographically separated fractions followed by the processing of each individual fraction by a second instrument. The problem with this approach, however, is that contamination of the fractions is possible, concentration issues may become significant, and the precise timing of the collection may be both necessary and difficult to control. As a result, on-line methods, in which multiple chromatographic instruments are directly connected to one another, have been developed to resolve these issues.

Specific Applications

Tandem methods, used in all manner of organic and inorganic syntheses, are increasingly used in the environmental and biotechnology fields. The total range of applications is too lengthy and diverse to summarize. However, three recent literature examples illustrate the importance of this separation and analysis approach.

Two Examples from Environmental Science and Medical Biotechnology. The pyrolysis of organic matter leads to the production of polycyclic aromatic hydrocarbons (PAHs). This fact is an environmental and health concern because the pollutants are commonly found in coal tar, crude oil, creosote, and automobile exhaust, and are used in the manufacture of some dyes, plastics, and pesticides. Additionally, food production is an issue, given that the process of smoking and grilling meats, poultry, and fish can result in pollutant concentrations significant enough to endanger human health.

Several methods for determining PAH levels in smoked foodstuffs have recently been developed. In general, however, these techniques require extensive solvent use, expensive equipment, and lengthy analysis periods. Researchers at the University of Udine in Italy have recently shown that an offline HPLC-HPLC method can be used to assess the PAH content of smoked fish (Moret et al., 1999). Specifically, Sabrina Moret and co-workers extracted the PAHs, fat, and other contaminants from insoluble fish samples and then used a large-scale, silica HPLC column to separate the triglycerides and other contaminants from the PAHs. The eluted PAH fraction was subsequently injected into a reversed-phase HPLC column for further separation and eventual quantitation. At present, the scientists are working to develop an on-line approach that would be even simpler to conduct.

The second example concerning tandem methods and PAHs is an environmental one. Environmental analyses of PAHs are complicated because of a combination of low analyte concentration levels; the presence of additional, interfering components; and the fact that PAHs may be chemically bound to many sample matrices. Currently available analysis techniques often lack the appropriate levels of selectivity and sensitivity, and they uniformly begin with time-consuming extractions that require the use of significantly large, organic solvent volumes.

Attempts to use LC and GC individually to analyze PAH contamination of soil samples, for instance, have generally failed, but researchers at the University of Helsinki have developed an on-line, coupled, LC-GC technique that appears to be successful (Hyotylainen et al., 2000). In their process, pressurized hot water extraction is used to remove insoluble components from the sedimentary samples. The extracted materials are then passed through an LC column that elutes the various hydrocarbons and transfers them to a GC instrument for final separation and quantitation. This LC-GC approach takes advantage of the large sample capacity of LC and the high separation and selectivity of GC. As a result, the entire process is faster, less expensive, and more reliable than other methods. Further, the repeatability of the measurements and the lower solvent volumes required by the analyses add considerable value.

An Example of a Nonmedical Biotechnology Use. During the 18th century, the bergamot plant was first cultivated in Italy. Today, it is also found in West Africa, Brazil, Argentina, and Uruguay, and it is of commercial importance because its oils are used as raw materials by the cosmetics and food industries. The assessment of bergamot’s essential oil quality is complicated, however, as genuine bergamot oil contains both R- and S-linalool with the S-enantiomeric contribution constrained to not more than 1% of the total linalool concentration. The complexity arises because single chromatographic techniques, although capable of detecting an excess of S-linalool, cannot discern between genuine oil and a reconstituted one that happens to contain the proper enantiomeric linalool ratios.

To address this problem, Luigi Mondello and co-workers at the University of Messina in Italy developed an on-line, GC-GC method for determining the enantiomeric distribution of the various flavor components in bergamot oils (Mondello et al., 1998). Specifically, using a self-engineered, double-oven GC system, Mondello isolated and quantified the relative enantiomeric ratios of monoterpene hydrocarbons, monoterpene alcohols (which include linalools), and linalyl acetate components for each of five differently produced oils. These results made possible, over the course of a full production cycle, the measurement of variations in the oils’ flavor components and the correlation between those alterations and the different processing technologies.

Hyphenated Chromatography

When standard chromatographic methods are combined with other analytical techniques, the result is commonly known as a "hyphenated method." The use of this term seems to have originated in 1980 (Hirschfeld, 1980), although the idea itself began with the coupling of GC and MS (see sidebar, "Techniques to the Right of the Hyphen") in the early 1970s.

Hyphenated methods, like their tandem counterparts, may be conducted either on- or off-line. Just as off-line methods originally predominated in coupled systems, they were also the original option of choice for hyphenated techniques. However, the regular need for cold trapping the fractional eluates before analysis proved to be a significant limitation. As a result, on-line processing of sample analytes became the norm, and numerous instrument companies now produce machines in which the coupled technologies are packaged, interfaced, and sold as a single unit.

GC is one of the most widely used quantitative analytical methods because of its ease of use, discriminating power, and ability to use a wide range of stationary phases. It is not surprising, therefore, that the first hyphenated method was GC-MS. GC has limitations, however (compounds may have similar retention times, only volatile substances can be analyzed, thermolabile compounds may not be separated, etc.), and GC-MS necessarily suffers from the same inherent deficiencies. As a result, the introduction of the LC-MS in 1972 was an obvious next step; the advent of the LC-MS not only overcame the GC-MS’s limitations but also avoided the problems of analyte derivitization while allowing for the direct determination of peak purity (Talroze et al., 1972).

The "barn door" to hyphenated method invention was then thrown open. One of the more potent technologies to develop involved the coupling of an LC column and an NMR (see sidebar, "Techniques to the Right of the Hyphen"). This novel combination of instruments allowed the ready separation and analysis of stereoisomers, pharmaceutical products, and environmental contaminants that were previously inseparable and unidentifiable. Further, when used on-line, light sensitive and pyrophoric compounds could also be processed.

In fact, it is more appropriate to discuss HPLC-NMR because it is most commonly used. The method is complicated, however, because NMR is relatively insensitive, and thus sample concentrations—in order to achieve an optimal signal-to-noise ratio after relatively few frequency pulses—must be maximized. The danger, however, is that in doing so, the HPLC column may become overloaded. Therefore, a careful balance in sample concentration is critical.

"Stopped flow" and "continuous flow" methods are now routine in HPLC-NMR systems. In a continuous-flow process, the LC effluent is funneled directly into the NMR for analysis. Thus, with careful control of the mobile phase’s flow rate, it is possible to obtain an NMR spectrum that corresponds to each chromatographic peak. Further, two-dimensional NMR plots can provide substantially more quantitative and structural information. Unfortunately, continuous-flow technology necessitates a short NMR acquisition time and thus requires an especially high analyte concentration. Stopped-flow processes avoid this problem by first collecting an analyte fraction and then halting the mobile phase’s flow until a suitable NMR spectrum is obtained. The stopped- flow instrument then cycles back, collects another fraction, and repeats the analysis process.

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Techniques to the Right of the Hyphen

Instrumental analysis techniques most commonly used in conjunction with chromatographic methods include the following:

Infrared Spectroscopy (IR, or Fourier Transform Infrared Spectroscopy, FTIR). Molecules exhibit vibrational motions (e.g., bending, stretching) that absorb infrared radiation at characteristic wavelengths. As a result, each molecule’s absorption spectrum is unique. In general, then, compounds are characterized using IR by recognizing these characteristic absorptions and attributing them to the presence of specific functional groups. So, for example, molecules that contain hydroxyl (–OH) groups will generally exhibit an absorption band around 3400 cm–1 (wave numbers).

Mass Spectrometry (MS). The sample of interest is first ionized under high-vacuum conditions. The resulting ionized, molecular fragments are then propelled through a magnetic mass analyzer that sorts the detected fragments based on their mass:charge ratio. The resulting spectrum thus provides quantitative information that can be used to characterize, and assist with the elucidation of, an unknown material’s chemical structure.

Nuclear Magnetic Resonance Spectroscopy (NMR). NMR works by placing a sample (usually a liquid held in a specially designed tube) directly into a magnetic field, thus causing certain spinning nuclei within the sample to align. When a second, weaker field is then applied perpendicularly, the aligned nuclei absorb characteristic energies and undergo transitions from one "spin state" to another. The energy required to cause this transition is based on numerous factors such as intermolecular interactions, chemical environments, and field strengths. This characteristic energy is ultimately used to generate spectra that can, both quantitatively and qualitatively, be used to confirm the chemical structure of an unknown compound.

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The hyphenated methods just introduced (GC/LC-MS and LC/HPLC-NMR) represent merely the tip of the iceberg. Techniques abound that combine, among other methods, IR (see sidebar above, "Techniques to the Right of the Hyphen"), absorbance/fluorescence spectroscopy, AES, and field ionization mass spectrometry (FIMS) with HPLC, GC, supercritical fluid, and even TLC. In addition, multidimensional hyphenated systems (see figure, "Schematic of LC-LC-UV-MS System") are extensively used for research and quality monitoring applications.

The Diverse World of Hyphenation

Given the wide range and high degree of specificity of hyphenated technologies, it is not possible to list all of their applications. The following examples illustrate some important uses in research today (see table).

Organic chemistry. Many of the applications for hyphenated chromatographic techniques fall within the confines of pure research science in which the ultimate goal is an understanding of specific chemical processes. A case in point is the use of GC coupled with FTIR to examine the photochemical degradations of phenoxyalkanoic acid derivatives (Climent and Miranda, 1997). (These compounds are used extensively as herbicides, and understanding their photolytic transformations is key to managing their environmental impact.) The combined results of separating and analyzing the photoproducts of two such derivatives, in fact, led a team of Spanish scientists to discover never-before-recognized degradation pathways in pesticide photochemistry that eventually could help scientists control the spread of these derivatives.

In the previous example, researchers used chromatographic techniques to advance scientific understanding. But a significant amount of research effort is routinely placed on developing and refining methods to separate and identify specific compounds within various mixtures. Such techniques have wide applicability in areas such as drug development, where new drug products typically consist of chiral molecules that are isolated as racemic mixtures. As a specific example, scientists at the Development Chemistry Department of GlaxoWellcome (now Glaxo SmithKline) in the U.K. studied ways to improve the separation and identification of enantiomeric molecules (Mistry et al., 1999). Their approach involved the direct coupling of chiral HPLC-NMR and HPLC-CD (circular dichroism, a method for determining the absolute configuration of a molecule). In their work, they successfully applied the chromatographic systems to the separation and structural identification of the organic drug compound, atracurium besylate.

In a similar vein, researchers at the University of Udine have reported the use of a combination of GC-FTIR and GC-MS techniques for the separation and identification of mononitro- and dinitro- isomers of diphenylmethane. In this case, the sophisticated elegance of the methodologies is also illustrated (Giumanini et al., 2001).

Inorganic chemistry. The design of host materials that act as ion extraction agents and ion sensors relies as much on the development of synthetic processes as it does on the ability to properly characterize them. In general, potentiometry, spectrophotometry, and NMR titrimetry are used to determine the metal-binding selectivities in host compounds that often consist of mixtures of macrocyclic compounds. Unfortunately, each of these analytical techniques is problematic due to combinations of low instrument sensitivities, limited solvent compatibilities, and so forth. Recently, however, Esther Kempen and her colleagues at the University of Texas at Austin developed a hyphenated chromatographic technique involving HPLC-MS to separate and characterize the metal-binding sites in a series of lariat ethers and substituted macrocycles (Kempen et al., 2001). For this particular application, an on-line, "HPLC-postcolumn complexation reaction-MS system" was designed; the purpose of the postcolumn reaction was to increase the sensitivity of the MS. The results of this chromatographic development helped the researchers identify host ion-selective reagents for Na+ and K+, as well as the alkali metal-binding site preferences within a mixture of calixarene compounds.

Environmental chemistry. Italian scientists have recently demonstrated that multiple pesticides can be recovered and analyzed using an instrumental process involving ion-interaction, micro HPLC-MS (Cappiello et al., 1999). Specifically, the separation methodology involves the use of an MS coupled to a reversed-phase HPLC column containing an ion-interaction reagent (IIR) in the mobile phase. (The IIR allows the simultaneous separation of hydrophilic and hydrophobic pesticides.) The advantage of this system is that chemically different water contaminants present in ultra-trace concentrations can readily be detected, identified, and quantified using a single chromatographic column.

The complete analysis of industrial wastewater is of critical concern for environmental protection agencies around the world. In general, 20% of industrial effluent streams are composed of volatile organic components that can readily be separated and identified using traditional GC-MS techniques. The remaining 80% of the components, however, consist of nonvolatile, often polar compounds. To detect and characterize these contaminants requires a system with high separation efficiencies that can generate a maximum of structural information. LC-MS and stopped-flow LC-NMR have both been used for this purpose, but not until last year were the methods combined for environmental analyses. Through direct application within a German textile plant, numerous, previously undetected contaminants in the plant’s effluent stream were identified (Preiss et al., 2000).

The majority of MS analyses use electron impact ionization processes. However, FIMS is a technique that substantially reduces molecular ion fragmentation while yielding higher measured intensities. The result is that peak assignment is relatively straightforward and multicomponent mixtures can readily be identified. When FIMS is combined with traditional GC separation techniques, the result is a sophisticated method for identifying complex hydrocarbon mixtures. Researchers at SRI International in Menlo Park, CA, recently reported using such a hyphenated method to profile the components of diesel fuels (Briker et al., 2001). Typically, these fuels consist of complex mixtures that do not readily lend themselves to separation and analysis, but such tasks are doable with the GC-FIMS coupling approach.

One further example of the complexity of separating multiple components is found in the case of toxaphene, a commercial pesticide now banned in the United States but still widely produced elsewhere. The difficulty in separating and identifying this particularly hazardous material is that it is formed from a complex mixture of up to 33,000 differently substituted chlorine congeners. In 1995, however, scientists working on this problem introduced a successful hyphenated chromatographic approach using single-column GC-ECD (electron capture detection) (Alder et al., 1995). Even more recently, a different group of researchers showed that the components could be separated to a greater extent using sequential heartcutting techniques and multiple GC columns (de Boer et al., 1997). The result of these efforts is that proper monitoring of this pesticide, particularly in aquatic environments, is now possible.

Biotechnology (Non medical). Some coupled technologies lead scientists to bring their own physical senses into the analytical process. For example, GC-olfactometry (GC-O) is a technique in which fragrant mixtures are fractioned and then analyzed by smell. This technique is far from new, but the Citrus Research and Education Center at the University of Florida recently showcased it in the literature. Specifically, scientist Kevin Goodner and his colleagues investigated the presence of vanillin (4-hydroxy-3-methoxybenzaldehyde) in oranges, grapefruit, tangerines, lemons, and limes; they found it in all those fruits, albeit in varying concentrations (Goodner et al., 2000).

The combinations of HPLC-NMR and HPLC-MS are both well known; however, results obtained using these methods are often difficult to correlate with one another. The HPLC-NMR-MS triad, however, overcomes this problem. One application for such a system comes from the Imperial College of Science, Technology, and Medicine in London, where researchers have used it to investigate the metabolism of agrochemicals in hydroponically grown crops. Specifically, the scientists were able to characterize two metabolites of 5-trifluoromethylpyridone after its introduction into maize plants (Bailey et al., 2000).

Biotechnology (Medical). One of the more popular commercial uses of poly(dimethylsiloxane) is as the gel in silicone breast implants. The gel contains both high and low molecular weight (LMW) silicones, but only the LMW components are known to leak from the implants into organs and tissues.

Silicon has been identified in the serum and plasma of patients with implants, but whether the measured silicon levels correlate with the LMW silicones leached from the implants had never been made clear. Researchers at the University of Essen in Germany recently developed a one-step preparation for the detection of LMW silicones in blood samples (Flassbeck et al., 2001). Their method, which uses a traditional GC-MS instrument, requires a low solvent volume and is highly sensitive and reproducible; the experimental results conclusively show that women with silicone gel implants have increased concentrations of LMW cyclic siloxanes in their bloodstreams.

Similarly, researchers at Baylor University in Houston have used GC-MS along with GC coupled atomic emission detection (AED) methods to identify and quantify linear and cyclic siloxanes in biological tissues (Kala et al., 1997). Specifically, using GC-AED, the researchers were able to detect 12 silicon-containing peaks in an ultra-trace-level concentration sample of poly(dimethylsiloxane) and then identify each of those relative peaks using GC-MS.

One of the principal benefits of a tandem LC-LC system is its ability to separate individual proteins from complex mixtures that overwhelm simple, one-dimensional methods. The chief problem with this approach, however, is that all analytes must be separated equally in both dimensions. As a result, the separation is rarely complete. In 1997, James Jorgenson and his colleagues at the University of North Carolina and GlaxoWellcome proposed the use of a comprehensive, chromatographic, UV spectrometer and MS technique to separate and analyze large biomolecule mixtures (Opiteck et al., 1997). For this application, a cation-exchange column was followed with a reversed-phase HPLC column; the effluent from the separation was then passed, on-line, to a UV detector and finally to an MS. The result of these scientists’ efforts is a method that allows the molecular weight determination of component proteins from complex mixtures in less than two hours; it also appears to be the first example of an on-line LC-LC-MS coupling found in the literature.

Two brief examples of novel chromatographic applications illustrate the increasing complexity of the technology and the range of its utility: Stereostructure determination of new metabolites in plant extracts is now possible through a combined HPLC-CD technique, as is the use of SFE-SFC-MS systems to assess the presence of pesticides in animal tissue (Bringmann et al., 1999; Voorhees et al., 1998).

Researchers have long used the selective exchange of deuterium for hydrogen atoms in organic molecules to assist with their characterizations. One chromatographic application of this includes the use of deuterium oxide as a mobile phase for the analysis of pharmaceutical products. When researchers include deuterium oxide in standard HPLC and hyphenate it with UV-ESI (electrospray ionization)-MS, they dramatically improve their attempts to determine the structural identification of impurities and to elucidate the degradation products found in pharmaceutical compounds (Olsen et al., 2000). A further approach to this problem involves the use of superheated deuterium oxide as the eluent in HPLC-NMR and HPLC-NMR-MS systems (Smith et al.,1999).

Vitamin A is of obvious importance to humans, and several of its derivatives serve as the basis for various therapeutic products. However, because of the number of double bonds present in the vitamin A molecule, several stereochemical isomers exist, each of which exhibits a different biological activity. To differentiate the isomers, researchers have used several hyphenated methods. Most recently, Matthias Pursch and co-workers from the Institute of Organic Chemistry in Germany used a solid-state NMR and LC-NMR combination to study the temperature-dependent conformations of vitamin A acetate isomers. The results of their work are essentially academic; however, they do lead to a better understanding of the interphases between the various conformations (Pursch et al., 1996).

While combinations of hyphenated methods are increasing in number, they are becoming exponentially more sophisticated. For example, scientists at CalTech and SmithKline Beecham have recently reported using LC-electrospray MS-nanospray MS for phosphopeptide mapping (Annan et al., 2001). Their approach carefully considers sensitivity and selectivity issues, and it eliminates the need to radiolabel proteins.

In one final example of the complexity of "combinatorial chromatography," researchers at Hewlett-Packard have compared various on-line LC-ES-MS, offline LC-MALDI TOFMS, and CE-MALDI TOFMS techniques for the separation and characterization of posttranslational modifications in glycoproteins (Udiavar et al., 1998). This work is valuable because it addresses complications of microheterogeneity that are inherent in the characterization of the proteome. It is also of particular interest, though, as it effectively showcases the diverse future for chromatographic growth technologies.

Futures in Chromatography

The ability to separate completely and identify the components in a mixture of chemical compounds is key to chemistry and central to the field of chromatography. Since their conception in the 19th century, chromatographic techniques have improved, advanced, and been reincarnated; the result is an array of useful instruments that have made possible a wide range of products and discoveries.

By general agreement, however, single-column chromatographic techniques, and even multidimensional methods, have reached their theoretical limits of utility; no substantive advances in these fields have occurred within the past 15 years or so. As a result, it is increasingly rare to find examples in the literature that focus on the analytical techniques themselves rather than on their use as part of a routine chemical characterization.

Hyphenated methods, on the other hand, continue to develop and are regularly featured for their novelty and utility. In fact, original combinations of chromatographic systems are developed regularly and are featured as prominently in scientific journals as are the applications themselves. Increasingly, the design of combined systems is pushed beyond the limits of MS, IR, and even NMR; many of the latest innovations include coupling chromatographic systems with electrochemical methods (amperometry, polarography, coulometry, etc.) and various other instruments that measure optical activities, refractive indices, and chemical conductivities. Therefore, the number and complexity of chromatographic systems are staggering and imply that the world of separation and analysis technology remains in its infancy and will continue, therefore, to help chemistry remain the central science.