Gas chromatography (GC) systems are similar to HPLC systems in that they are used to identify, separate and quantify compounds of interest. However, GC systems use an inert, gaseous mobile phase, as opposed to a liquid phase, to bring about the separation of molecules. The basic components of a GC system include an injection system, an oven, a column, a detector and a computer system to analyze results.
There have been significant changes in GC in the past decade, and most of them have focused on maximizing throughput and decreasing run time. One of the factors limiting throughput has been the rate at which proper oven temperatures can be reached. Recent efforts have led to the development of ultrafast GC systems that incorporate advanced column heating devices and controls that can rapidly heat and cool columns. Improving speed of analysis also has been the driving force leading to changes in column technology. The use of small, nanobore capillary columns has improved throughput without sacrificing efficiency or precision. “There is a market out there for ultrafast GC columns, although they need specialized instrumentation to run it,” says Rob Bunn, product manager for GC columns & consumables at Thermo Fisher Scientific. The GC columns also have undergone improvements in sensitivity that offer lower detection limits and ultralow column bleeds. “The deactivation process has improved significantly in the past few years and has led to low activity on the column,” says Bunn.
Another development in the GC field has focused on the use of multidimensional systems that incorporate different columns and detection systems to improve sample resolution and throughput. The strategy involves using multiple columns to facilitate the separation of co-eluting peaks, such as enantiomers, or of samples that contain complex mixtures or a large number of components. A switching valve is used to route portions of effluent from one column to another column, and under certain conditions, the columns can be operated independently to increase throughput. The ability to incorporate a variety of different detectors within the system also is a huge benefit. “Mass spectrometry is fabulous as a universal detector, but we are seeing resurgence in the use of selective detectors for very specific types of applications,” says Laura Chambers, senior product specialist for chromatography products at OI Analytical Corp., who works with customers to help them reconfigure their GC systems. “In some systems you can now have an MS and three other detectors that can work in tandem.” However, to take advantage of these improved technologies, customers must first understand what it is that they need. “Customers really need to know what they want to do with the system or they are going to waste a lot of money buying things they don’t need,” says Chambers. Since most methods for GC analysis are well standardized and documented, the application and protocol often determine the types of columns, detectors and other accessories to be used. Chambers therefore advises lab managers to think carefully about what the GC system is going to be used for, the skill level of the personnel using it and where it is going to be used. “That will help them make costeffective decisions as they go through their configuration processes,” she says. Taking the type of sample, the sample load and the sample preparation into consideration also is important. Thinking through these issues will determine if any special equipment is needed and will ensure that the samples don’t overwhelm certain components of the GC system. There also are other accessories like syringes, filters and septa that play important roles in sample analysis. “When people are involved in new method development, they try a series of different columns and sometimes find that they are not getting the results they are looking for,” says Bunn. “Often, when things don’t work people blame it on the GC column, but the choice of liner and the septa are equally as important.”
Planning ahead and consulting with the vendor are important, as technologies and applications continue to evolve. “Talk to your vendor, because they have experts who know those instruments and applications inside and out, and they can be an extraordinarily valuable resource,” says Chambers. Bunn also advises GC users to regularly scan resources on vendor websites. “There is not just product information, but there is detailed information on specific applications [as well as] work flow solutions—from sample collection to analysis. A lot of companies have resources on their Web pages that help users make informed decisions,” he says.
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.
An appropriate HPLC detector has the ability to sense the presence of a compound and send its corresponding electrical signal to a computer data station. The choice of a detector depends upon the characteristics and concentrations of the compounds that need to be separated and analyzed.
“Diode array detectors on the market today vary considerably in performance specifications and pricing. Researchers need to consider what they truly need - as it can dramatically impact the price they will pay,” says Moroni Mills, senior marketing specialist, Gilson, Inc. “A customer should be aware of the advantages a photo diode array detector brings to their analysis,” says John Pollard, vice president of sales, JASCO. “For example, some customers require a spectrum of the sample. A PDA detector offers spectra in addition to single wavelength data. The PDA detector also offers the added benefit of a generated library that is compiled as samples are run to help ensure sample purity and identification,” adds Pollard.
“Regarding technological advances, PDA detectors have been redesigned in recent years to allow faster data acquisition rates to ensure compatibility with UHPLC systems,” says Pollard.
According to Mills, “The researcher today really needs to determine what their true needs are in order to make the best decision on which diode array detector to choose.”
Many diode array detectors on the market today improve resolution up to 1024 diodes. Does the researcher require this amount of resolution? With the movement toward ultra fast HPLC, many diode array detectors have much faster acquisition speeds for detecting accurately peaks with widths as low as one second. Wavelength range is also something that differs greatly between different diode array detectors on the market. Some detectors approach NIR ranges (Near Infrared), while others may only go up to 600 nm. Some diode array detectors feature fiber optic technology that allows flow cells to be positioned outside of the detector and connect directly to the column output. All of these functions have a purpose.
There is no dearth of options—both in terms of variety of columns and vendors—for analyzing samples using column chromatography. The challenge is to be able to pick the right analytical column to analyze the right sample correctly. The decision is based on several factors: column specifications, dimensions, media particle and pore sizes, and chemistry of the bonded phase, all of which can affect separation efficiency, inertness, durability, pH range, batch-to-batch reproducibility, resolution, solvent usage, and more. There is also the complexity and quantity of the sample available and the desired cost and accuracy of analysis to be considered.
“What is really important to the consumer is lot-to-lot and column-tocolumn reproducibility,” says Dafydd Milton, product manager, LC and LC/MS columns, at Thermo Fisher Scientific “They have to have the confidence that the column will elute the analyte peaks at the same time, every time.” Along with elution times, the ability to get good peak shapes—sharp, narrow, symmetrical peaks— is also important, especially for applications such as method development. Conventional liquid chromatography uses plastic or glass columns that can range in size from a few centimeters to several meters in length. Commonly used lengths vary from 10 to 100 cm, with longer columns being used for preparative scale separations. High-performance liquid chromatography (HPLC) columns are made of stainless steel and are typically shorter, approximately 10 to 30 cm in length. Short, highly efficient HPLC columns allow shorter analysis times, better peak shapes and better quality data while also reducing cost per analysis. Narrower columns also offer better mass sensitivity and significantly reduce solvent use.
Milton mentions that in recent months there has been a shortage of acetonitrile, a solvent routinely used for HPLC analysis. Acetonitrile is a by-product of the automobile industry and, since there are no dedicated plants to manufacture acetonitrile, the recent slowdown in the production of cars has caused scarcity of the solvent. “We find many customers moving to smaller columns, packed with smaller particles (sub2-micron) because they use less solvent,” says Milton. Even before the solvent shortage occurred, the trend had been toward increasing the use of columns packed with smaller particles because of advantages associated with costs and efficiency, although slower, longer columns that offer better resolution are sometimes preferred to separate sample components in extremely complex samples. “It’s been a couple of years since the sub2- or 2.5-micron columns were introduced into the marketplace, and the smaller particle-size columns are now proving to be very important,” says Maureen Joseph, product manager in the Columns and Supplies division at Agilent Technologies Inc. These columns have proved very efficient in terms of cost and performance, and they cover a wide range of applications in industries that span food, environmental, pharmaceuticals, biofuels, and others. There is also an increased demand for the analysis of polar analytes for applications in both drug discovery and development, such as the identification of metabolites. Hence, many companies have now introduced hydrophilic interaction chromatography (HILIC) columns for analysis of such polar analytes. “More and more drugs that are being developed seem to have a polar component, which the traditional C18-type columns don’t seem to retain very well,” says Milton. The HILIC columns use hydrophilic interactions to facilitate the transfer of polar analytes to the stationary phase for increased retention and better sensitivity.
There are a lot of changes also taking place in the area of biomolecule analysis. “Along with preparative and process analysis, there are also analytical columns being introduced for the fast and accurate detection of biomolecules,” says Taegen Clary, who is also a product manager in the Columns and Supplies division at Agilent but is involved more with biomolecule analysis. People involved in antibody sizing, analysis of protein isoforms, and clinical samples are becoming increasingly concerned about time and cost of analysis. Reducing the time needed to analyze samples, increasing efficiency, and improving data quality can result in significant savings for laboratories that run hundreds of samples per day. Such laboratories are also continually evaluating alternative methodologies that can overcome some of the limitations associated with column chromatography. Microfluidics, for instance, is beginning to play a role in analyzing samples that are rare and available in small quantities, such as for proteomics. However, it has yet to play a role in mainstream analytical applications. While there is definitely a trend toward miniaturization, microfluidics is unlikely to completely replace column technology. “There will always be a place for traditional column technology,” says Joseph.
In recent years there has been a growing trend to work with live cells, for instance, in high-throughput screening for drug discovery, for stem cell research or for such applications as in vitro fertilization. “Certainly there is a lot more interest in live-cell imaging for looking at dynamic events, and microscopes are being built to achieve that,” says Joseph LoBiondo, product planning manager for Nikon Instruments Inc. “There is a lot of optics design going into achieving live-cell imaging.”
Self-contained, fully integrated live-cell imaging systems now come equipped with a built-in cell incubation chamber and a microscopy unit. Such companies as PerkinElmer, Carl Zeiss, Molecular Devices and Nikon all offer imaging systems with environmental controls for temperature, CO2 levels and humidity in order to ensure that cells can grow and survive for an extended period of time. “Cells stay alive for days in these incubation systems,” says LoBiondo. And in some instruments the cells don’t ever have to be taken out of the system for observation since the microscope is a part of the controlled environment. Units also come equipped with a full-sized incubator that can hold a variety of chamber slides and well plates. “Nikon’s BioStation CT has a motorized arm that pulls out the specified dish or plate, with minimum vibration or disruption to the cells,” says LoBiondo. “The unit is very methodical and slow and very carefully picks up the dish and takes it to the microscope.” The units also have some level of built-in security provided. “Certain program locks can be put in place so that if [researchers] are doing different experiments, each researcher will have access to only [his or her] cells.”
Manufacturers started offering integrated units when interest in livecell imaging began to grow. Initially researchers, themselves, were integrating such individual components as motorized stages, filter wheels, shutters, cameras and software packages from a number of different manufacturers to create a system that would meet their needs. “Depending on how it is done, there are a lot of components that are needed to work together in order to make an integrated system,” says LoBiondo. “Integrated systems also tend to be less expensive. It could be about half to one-third of the costs of buying and putting together individual components.” The disadvantage is that an integrated system is less customizable and may compromise certain features, such as speed when switching between wavelengths, or flexibility in terms of number of objectives or software that can be used (the latter being crucial for certain applications). However, integrated systems work well for those laboratories in need of multiple units that can perform in a routine and reliable fashion but lack the time or expertise to build them.
However, with microscopes now being designed to cater to several different applications and supported by image analysis software and high-end computing hardware systems, matching products to a specific application may soon be a thing of the past. “Microscopes now have multimode capabilities to be used in multiuse facilities,” says LoBiondo. “They can now switch between total internal reflection fluorescence, confocal and live-cell imaging, with software control and motorization to do all those modalities.” Soon we may have a system that can do it all.
Mass spectrometry (MS) has been a widely used research and analytical tool for routine as well as specialized analysis of simple and complex samples. A truly versatile and powerful analytical technique, MS has continually evolved to meet the ever-changing demand for its expanding applications. The need for increased speed, sensitivity and resolution always has driven innovations in MS, but researchers now are asking for enhancements that go beyond performance-related attributes. “A theme that has been growing in intensity for the past year or so is the demand for MS systems to be as versatile as possible,” says Allan Millar, senior product manager for time-of-flight (TOF) MS at Waters Corp. “Versatility is becoming an increasingly important driver for new purchases in most laboratories. Particularly in a service lab environment or in a lab that faces a multitude of analytical challenges, having versatility in the instrumentation is a very attractive proposition.” Customers also are demanding increased accessibility and ease of use in MS systems. “The feedback that we get across many applications is that there is an increase in demand for instruments to be more accessible, as they need to be operated by people whose skills and experiences often are outside analytical chemistry and mass spectrometry, in particular,” says Millar. Hence, in newer systems, a lot of effort is put into matching the analytical performance of the mass spectrometer with its integration, data handling and automation capabilities. Newer systems are equipped with software programs that not only guide the user through the initial calibration and experimental design but also help streamline the workflow and simplify processes in the long run.
Informatics packages combining chemometrics with mass spectral de-convolution are being built specifically around the demands of certain applications. Applications such as proteomics, metabolomics and others demand mass analysis that probes deeper into the samples and offers a lot more detail, whereas other applications like forensic analysis and food safety require resolution of unknown and trace-level contaminants in complex and sometimes archived samples. This has led to the creation of chemically intelligent informatics to rapidly sift through very complex data sets for specific applications. “These informatics packages are the result of the embodiment of nearly a decade’s worth of experience and exposure to the challenges in that application area,” says Millar.
While instrumentation and bioinformatics are working to simplify the analysis, the samples that are being analyzed are getting more complex. “The trend is toward near realtime analysis and providing mass spectral confirmation of target compounds buried in complex matrices with great certainty,” says Nick Bukowski, product manager at ALMSCO International, a manufacturer of TOF MS and related software products. This trend toward analyzing increasingly complex samples, in real time, is in turn driving the need for turning high-quality data into high-quality information. The basic quantitative techniques measuring the integrated peaks and comparing them with calibration standards has not changed, says Bukowski. “What has changed however is the use of mathematical algorithms for pulling out information from trace signals buried in complex matrices and getting meaningful qualitative and quantitative information from them.” “In broad terms, MS is striving for better analytical performance, greater efficiency and productivity, ease of use and expanding its use beyond its core markets which have been proteomics, metabolism and drug discovery into environmental and clinical toxicology and food safety applications,” says Lester Taylor, strategic marketing director of Life Sciences Mass Spectrometry at Thermo Fisher Scientific Inc. Similarly, customers also are looking for more efficiency in terms of speed, time for sample prep and analysis, ease of use and streamlining workflow. “Customers always are looking for techniques that provide them more sensitivity, greater selectivity or specificity, and the ability to analyze complex samples,” says Taylor.
While in the past a single mass spectrometer had many interfaces, these days most labs have dedicated units for hyphenated applications like GC-MS or LC-MS. Hence, the choice of MS system, such as TOF, quadrupole or ion trap instrument, the nature of the sample, its complexity, the level of sensitivity required for analysis, the rate of data acquisition, the ease of use, and the skill set of the users are all important criteria that drive the decisions to buy or upgrade a system. The other attribute that customers have come to value is service. They tend to prefer solution providers that have dedicated fieldbased application scientists who can offer extensive in-house training and continued technical assistance. “There are challenges associated with adopting any technology,” says Millar. “There is the initial familiarization, and then there are other questions that come up as the user gets more proficient in operating the system. Hence, training is a critical step in the adoption of these types of techniques and is something that often is overlooked.”