Issue Date: May 18, 2009
THIN-LAYER CHROMATOGRAPHY (TLC) has long been a staple of the organic laboratory, and its silica-on-glass plates are familiar even to undergraduate chemistry majors. But for advanced applications, TLC has lagged in popularity behind column chromatography, which offers better reproducibility and the ability to interface with identification techniques like mass spectrometry (MS).
Now, new planar chromatography instrumentation and stationary phases and improvements in MS are opening up TLC to applications that go far beyond a mere quick check on the progress of a chemical reaction.
To separate a mixture by TLC, a small amount of sample is spotted onto a thin layer of adsorbent, called the stationary phase, that's coated over a sheet of glass, plastic, or other material. The plate is placed into a solvent reservoir, and a liquid, called the mobile phase, ascends the plate by capillary action.
Key factors that have changed conventional TLC into high-performance TLC (HPTLC) include better plates, instrumentation, and standardized methodology, says Eike Reich, who heads the research laboratories for instrument manufacturer Camag. All three lead to better, more reproducible performance and an improved ability to validate methods.
These are critical characteristics for advanced TLC applications, such as determining when Artemisia annua plants should be harvested for artemisinin, used to treat malaria. The plants are grown in large plots in Asia, and local labs need to be able to determine quickly when the artemisinin content reaches a maximum, Reich says.
Analyzing for the molecule requires separating it from a complex plant matrix and finding a reliable way to identify and quantify it, even though it doesn't have a chromophore that would enable convenient spectroscopic detection. Camag, which develops TLC methods and instrumentation, developed an HPTLC procedure to separate the mixtures and then stain the bands with anisaldehyde to selectively detect and quantitate artemisinin (J. Liq. Chromatogr. Relat. Technol. 2007, 30, 2209).
Camag also has been working with the Official Food Control Authority of the Canton of Zurich to look for azo dyes that might be illegally added to spices, such as curry powder or paprika, to enhance their color. "These toxic dyes have actually entered food-processing plants and ended up in sausage or meat products and have to be detected there," Reich says.
The complex nature of either the spice or the food sample makes detection a challenge to which HPTLC is well suited, Reich says, and TLC also makes it convenient to separate, or run, a number of samples quickly in parallel. Methods developed by Camag and Zurich Food Control scientists can quantitatively detect the dyes down to 2 ppm (J. Liq. Chromatogr. Relat. Technol. 2009, 32, 1273).
An advantage of TLC for such applications is that it makes it easy to analyze samples side-by-side rather than sequentially, as with column chromatography, Reich says. This means greater flexibility and higher throughput with relatively little expense, he notes.
With TLC one can also see compounds that weren't separated because they ran with the solvent front or perhaps didn't migrate at all—something that's typically difficult to do with column chromatography, Reich adds. And after a run, samples remain on the plate—so a scientist can reevaluate the results or even do additional manipulations, such as chemical derivatization, rather than just revisiting a scanned image or chromatogram.
Instrumentation improvements have played a major role in turning conventional TLC into HPTLC. TLC plates are run in enclosures called plate development chambers. Using automated chambers to control conditions such as humidity, chamber saturation with mobile-phase vapor, and extent of mobile-phase migration can make a big difference in reproducibility of results, Reich says.
Silica, a common TLC adsorbent, is always in equilibrium with the humidity of the laboratory atmosphere. Because humidity affects the activity of silica, plates run in different locations worldwide, or in a cold, dry winter versus a hot, humid summer, can give strikingly different results. Controlling the humidity of the development chamber can go a long way toward mitigating those differences, Reich says.
Controlling the degree of saturation of the plate development chamber with mobile-phase vapor is also a critical factor in getting reproducible results. Simply opening the chamber to insert a plate disturbs the equilibrium of the chamber's atmosphere, potentially leading to irregular zones on the plate or a solvent front that's banana-shaped, instead of straight, the way it should be. "When you have the chamber saturation under control, you have straight zones across the plate," Reich says. Automated chambers allow reequilibration before placing a plate in contact with the mobile phase to start a run, and they may also include sensors to detect the advance of a solvent front so a plate can be automatically withdrawn and dried at a specific point.
ANOTHER BUDDING instrumentation trend involves taking TLC down to the nanoscale, which requires smaller plates, smaller spotting devices, and imaging or identification capability that can cope with nanoscale spots. Gertrud Morlock, a professor of food and analytical chemistry at the University of Hohenheim, in Germany, is collaborating with Michael Brett, a professor of electrical and computer engineering at the University of Alberta, in Edmonton, to produce plates in sizes from 1 × 1 cm to 4 × 4 cm, with stationary phases from 100 to 1,000 nm thick. Although the researchers are currently using standard stationary-phase materials such as silica, they could in principle use nearly any material, Brett says.
Nanoscale TLC would use smaller amounts of solvent, allow faster analysis times, and enable higher throughput on smaller instruments, Morlock says. Nanoscale TLC is in its infancy, she notes, but early results look promising.
In addition to developing instrumentation to enhance TLC performance, researchers are also working on new stationary phases for planar applications. Silica, the most common stationary phase, separates molecules by polarity—polar compounds interact more strongly with silica and thus migrate more slowly than nonpolar compounds. Use of so-called reverse stationary phases such as C18- functionalized silica has long extended TLC to separation of nonpolar molecules. More recent work is expanding the range of TLC to biomolecules.
Typical TLC layers can separate small peptides or oligonucleotides with molecular weights below about 2 to 3 kilodaltons, but such layers aren't suitable for larger biomolecules. An alternative approach uses porous polymers, or monoliths, which are also used in column chromatography (C&EN, Dec. 11, 2006, page 14).
Rania Bakry, a professor of analytical and radiochemistry at the University of Innsbruck, in Austria, and Frantisek Svec, director of the Molecular Foundry at Lawrence Berkeley National Laboratory, are using photopolymerization to generate new stationary phases for planar separations of biomolecules. By carefully controlling conditions, they can generate stable layers 20 to 200 μm thick with a well-defined porous structure. In their early development work, they used a layer made of poly(butyl methacrylate-co-ethylene dimethacrylate) to separate and identify the components of a mixture of large biomolecules—insulin, cytochrome c, lysozyme, and myoglobin (Anal. Chem. 2007, 79, 486).
Monoliths can also be used for two-dimensional separations to get better resolution of mixture components. Svec is experimenting with grafting hydrophilic, sulfonic acid-lined channels onto superhydrophobic monoliths. Aqueous mixtures of peptides can be run in the channels and separated through an ion-exchange mechanism. Once that separation is achieved, the researchers can distinguish the peptides further by turning the plate 90 degrees, switching to an organic mobile phase, and running the peptides through the monolith itself to separate them by polarity.
ADVANCE IN instrumentation and stationary phases notwithstanding, one fundamental drawback of planar chromatography is that the rate that the mobile phase advances diminishes with time, thereby slowing down the analysis. "Typically, the mobile phase moves rapidly the first few millimeters but by the end of a separation it may be moving very slowly," says David Nurok, a chemistry professor at Indiana University-Purdue University Indianapolis.
One way to speed up TLC analyses is to put the TLC plate on a wheel, with the mobile phase at the center, and spin the plate so that centrifugal force drives the solvent outward. But even here, the movement of the solvent front still continues to slow as it migrates.
Another approach, called overpressured-layer chromatography (OPLC), is to cover the surface of the plate with a membrane that is under pressure, so the mobile phase can be pumped through the stationary phase. OPLC keeps the mobile phase moving at a constant rate and improves analysis time over traditional TLC, but scientists would still like to achieve even faster turnover, Nurok says.
A third technique, called planar electrochromatography, uses an electric field across the stationary phase to induce electroosmotic flow of the mobile phase. This speeds up separation, but the electroosmotic force also drives the mobile phase to the surface of the plate, where liquid can accumulate and cause spot smearing. The movement of ions in the electric field also generates heat, and if the plate gets hot enough it will dry.
Nurok, in collaboration with Robert E. Santini, a senior scientist at the Jonathan Amy Facility for Chemical Instrumentation at Purdue University, has developed a technique called pressurized planar electrochromatography (PPEC) that addresses these drawbacks of electrochromatography and enables faster analysis than with OPLC. With PPEC, electrochromatography is carried out in a temperature-controlled, pressurized environment. The pressure prevents liquid from accumulating on the plate surface, and the pressurizing medium acts as a heat sink to prevent temperature buildup.
The end result is that separations are quick and efficient. In their proof-of-concept studies, Nurok, Santini, and colleagues separated all compounds of a nine-component mixture in a mere two minutes (Anal. Chem. 2006, 78, 2823) and an array of nine five-component samples in just one minute (Anal. Chem. 2004, 76, 1690). Recently, the two have formed a company, InChromatics, to commercialize the technology.
Once a mixture has been separated by TLC, it becomes critical to identify unknown components, by using a technique like MS. Developments in open-air ionization methods (C&EN, Oct. 8, 2007, page 13) have enabled significantly better coupling of TLC with MS than was previously possible. Whereas MS of TLC plates used to involve cutting the plate and putting the pieces in a vacuum chamber, various techniques can now be used to extract material from intact TLC plates on the benchtop. These include laser desorption, liquid or gas jet desorption, and extraction by a liquid stream. Ionization can be done after desorption or can be coupled with it, such as with desorption electrospray ionization (DESI) or matrix-assisted laser desorption ionization (MALDI).
Gary J. Van Berkel, head of the organic and biological mass spectrometry group at Oak Ridge National Laboratory, is working with collaborators to develop TLC stationary phases that are more amenable to MS analysis. He points to protein separation as an area where TLC-MS might be helpful.
"MS analysis of materials from gels can be a time-consuming and costly process," he says. "If you could replace a normal 2-D gel electrophoresis separation of proteins with something that's done on a TLC-like medium" and then use MS to identify proteins directly "without having to punch spots and do extensive sample clean-up, that would have an immediate application."
IN DEVELOPING MS-friendly plates, it's important to ensure that binders and additives on the TLC surface don't interfere with MS performance, Van Berkel says. Surfaces must also be robust. "With DESI, you have a miniature power washer," Van Berkel says. "If you destroy the surface you may still be able to do the analysis, but you'll blow stuff all over," compromising the methods and instrumentation.
As the qualities of HPTLC become better known, Camag's Reich expects to see it become popular in areas ranging from environmental analysis, wastewater treatment, dietary supplements, and cosmetics. For rapid screening of unknown contaminants, "I think it's a very important tool for the future because it's simple and very sensitive," Reich says. Perhaps HPTLC will soon be just as essential to the analytical lab as simple silica TLC plates have been to organic labs and chemistry majors.
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