Capillary chromatography is in the mainstream of the technical evolution of chromatography, driven primarily by improvements in column technology that generally precede the development of the supporting instrumentation. At the 37th International Symposium on Capillary Chromatography (May 12–16, 2013, Palm Springs, CA), improved column technology was clearly leading, particularly in HPLC. Prof. Mary Wirth of Purdue University (West Lafayette, IN) presented compelling examples of improved separation speed arising from slip flow capillary columns packed with sub-1-μm particles. In GC, the emphasis was mostly on multicolumn separations (GC×GC or comprehensive GC). However, Prof. Dan Armstrong (University of Texas at Arlington) and Supelco (www.supelco.com) described significant advances in the use of ionic liquids (ILs) for stationary phases. ILs offer significant improvements in temperature range, which extends GC to larger molecules, and polarity options that can be tuned to provide higher chromatographic resolution. Significant applications for LC and GC include lipids, food contamination, flavors and odors, as well as fuels and biofuels.
Automated, multidimensional separations—particularly in GC×GC—dominated the first half of the meeting. This is reflected in the complete title, “37th International Symposium on Capillary Chromatography and 10th GC×GC Symposium.”
Adding more dimensions to the separation space greatly improves resolution, and if done carefully, can even reduce run time. Detection, particularly MS and MS×MS, also increases the dimensionality.
One interesting benchmark: A decade ago, chromatographs were so slow that Waters (www.waters.com) introduced the MUX for interfacing up to four HPLCs to one MS, since the mass specs were so much faster than the chromatographic peaks. Today, with ultrahigh-performance liquid chromatography (UHPLC) column technology, LC/MS throughput is often limited by the MS speed. Only time-of-flight (TOF)/MS really has the speed to be compatible with peaks that are less than a second wide. Each peak needs at least three samplings, and ideally 10, for quantitative analysis; data quality improves with more, up to about a dozen slices. So a 1-sec peak needs a minimum sample rate of 3 Hz, and ideally 10 Hz.
GC×GC: technique improvements
JEOL (www.jeol.com) markets GC×GC instruments equipped with TOF detectors, which are very fast. The AccuTOF™ JMS-T100 GCv 4G is a case in point. By reducing the diameter of the first column from 250 μm to 100 μm and the second from 100 to 50 μm, they achieved a 67% reduction in run time, with a corresponding 3× improvement in productivity.
The laboratory of Prof. Tadeusz Gorecki at the University of Waterloo (Ontario, Canada) developed a passively cooled consumable free thermal modulator that uses a capacitor discharge for a heat pulse. The modulator was evaluated for assay of volatiles from tea prepared from honeybush. The modulator provided improved run-to-run precision for repeat injections.
Another poster from the Gorecki lab showed tailing peaks, especially polar peaks, as are often observed in GC×GC assays. The top two suspected causes were incomplete desorption from the thermal modulator or partial adsorption in the injection port. In particular, when assaying pesticide residue, the organophosphates tailed badly. A valve-to-isolate and backflush of the injector 6 sec after injection reduced tailing of the organophosphates.
Applications of GC×GC
After more than a decade, it was encouraging to see applications of multidimensional separations expand beyond petroleum and biofuels. Reports focused on foods, beverages, the environment, and lipidomics.
Lipidomics is finally receiving attention. Despite the fact that lipids hold our cells together and are involved in their function, biochemists have largely ignored them while focusing on genomics and proteomics. The lack of tools such as suitable separation technology is one contributing factor, but this is changing. In GC, Prof. Dan Armstrong showed several ionic liquid phases that have greatly improved selectivity for lipids, plus the thermal stability to be practical. ILs are being commercialized by Supelco, which also presented detailed reports on separations for lipidomics.
Oils and lipids were frequently mentioned as target analytes. For example, the composition of vegetable oils depends on the processing, according to Dr. Olga Vyviurska of the Institute of Analytical Chemistry (Bratislava, Slovakia). She used GC×GC MS to characterize the content of various vegetable oils as fatty acid methyl esters. She reports that virgin olive oil is enriched in hexadecanoic acid (16:0) and octadecanoic acid (18:0) compared to olive pomace oil. In general, chromatographic retention increases within any carbon number with more olefinic substitution. Also, within a homologous unsaturated group, analytes with the lowest ω number have the strongest retention.
Dr. Nikoleta Janoskova (Slovak University of Technology, Bratislava, Slovakia) used GC×GC TOF/MS to identify volatile organic analytes in acacia honey. She reports that 3-ethyl-3-heptanol, (E)-2-hexen-1-ol, and (E,E)-2-4-decadienal are detected in honey and flowers and are markers of botanic origin.
While GC×GC stole much of the limelight, good old single-dimensional capillary GC is the mainstay of our industrial society.
The uncertain supply of helium was a common concern, especially for legacy methods. Most see that hydrogen from hydrogen generators is probably the path of choice, unless detection involves MS. New, non-MS methods will almost certainly use hydrogen for both carrier gas and flame ionization detection (FID), which is a significant fraction of the market. When MS is involved, the fear is that hydrogen could build up inside the instrument in the event of a power failure. This raises the potential of reaching the explosive limit for hydrogen in some pocket of the instrument.
Restek (www.restek.com) presented an application note that described a web-based application to aid in changing carrier gas. It acknowledges a general advantage for replacing helium with hydrogen, but also describes nitrogen as an alternative. Nitrogen is slower, but in cases where all peaks are well resolved, it should be quite viable. Plus, the risk of reduction of olefins is a nonissue with nitrogen as the carrier gas.
PerkinElmer discusses the conversion issue here: http://www.perkinelmer.com/CMSResources/Images/44-136072TCH_010288_01-HydrogenCarrierGasGC.pdf.
Also, see this applications note from Agilent:
An inert flow path is critical for the assay of many reactive analytes such as pesticides, epoxides, and halogenated hydrocarbons, especially iodides. Agilent passed out a poster listing the “Top 5 Tips for GC Flow Path Inertness.” These are:
1. Maintain the inlet. Look for and replace worn septa, syringe needles, fittings, etc.
2. Prevent sample loss at injection. Sample liners collect contaminants with repeated use. Replacing with a new deactivated liner can improve recovery.
3. Select an inert column. Look for columns tested with test mixes containing labile probe analytes.
4. Examine your detector. All accessible metal surfaces should be inert and clean, especially with MS detection.
5. Use an indicating gas purifier to remove oxygen, oils, and particulates.
Injection artifact with amines dissolved in methanol
A poster by Dr. Che Leong Kee of the Health Sciences Authority in Singapore showed that amines dissolved in methanol produce several peaks from a condensation reaction. He attributes the condensation to the thermal decomposition of methanol formaldehyde at the hot surface of the injector.
Dr. Jack Cochran of Restek carried standard maintenance protocol to an extreme to see how short a GC column could be used. The particular protocol was assay of polybromodiphenyl ethers. The standard method (U.S. EPA 1614) requires a minimum resolution of 0.4 between two particular peaks. Commonly, the samples are dirty, which slowly degrades the column segment nearest the injector.
Agilent’s Method Translator software and GC Pressure/Flow Calculator is a useful guide to setting operating parameters (oven temperature, flow rate, and pressure) to compensate for the loss in column length. Often the new settings will be sufficient to avoid major changes in the data processing.
Assay for residual solvents
Society demands safety and efficacy. The FDA is concerned about residual solvents from the manufacturing process in the finished product. Jana Stavova and colleagues at Bristol Meyers Squibb (www.bms.com) reported a validated assay for residual petroleum ether in active pharmaceutical ingredients (APIs). The method detection limit was 0.003%.
Capillary liquid chromatography
Traditionally, the Capillary Chromatography Meeting series has focused on gas chromatography, and this was true again in 2013, but the balance is shifting as capillary columns become more popular in liquid chromatography. Indeed, Prof. Mary Wirth of Purdue University updated the audience on the amazing increases in efficiency of capillary liquid chromatography (cLC) columns that arises from slip flow regime.
For several years, her research lab has been developing reversed-phase liquid chromatography (RPLC) capillary columns for top-down proteomics. She was intrigued with the observation that reducing the particle size to about 0.5 μm increased the efficiency for a protein separation by 100× for a nonporous packing. Sensing an opportunity, the lab started working with submicron-sized particles. Currently, the focus is on 470-nm nonporous silica RPLC particles. Photomicrographs show that these pack in a regular face centered crystal that show opalescence. This facilitates selecting well-packed columns by visual inspection.
The cLC columns are very efficient; for example, the HETP for bovine serum albumin (BSA) is only 15 nm, compared to the expected 1 μm. The efficiency is due to the idiosyncrasies of flow in narrow capillaries, where the flow at the wall is not zero, as it is in fat columns. The flow profile resembles a fat disk moving down the capillary. Wirth reports that the phenomenon has been known since the 1820s but has only recently been applied to chromatography. In a packed capillary, the only slow spot arises when a flow channel is blocked by a particle. However, the depth of this stagnant layer is only a few nm. Proteins are larger than the stagnant region, so they get swept along by the liquid rushing into the main channel.
Applications include studying the microheterogeneity of proteins and impurity profiles of proteins. For LC/MS, the capillaries are pulled to a point, but the column packing is retained without a frit.
If slip flow capillary technology is to be developed commercially, the size and exceptionally low band spreading will probably require a completely new instrument design. It will be a bigger transition than we saw for HPLC to UHPLC.
Multidimensional liquid chromatography
On the LC front, Prof. Guowang Xu (Dalian Institute of Chemical Physics, China) compared multidimensional LC×LC MS and GC×GC MS for lipidomics. Initial results with LC were disappointing since peak count was only about 500. Resolution was low due to the design of the valve interface between the two columns. He reasoned that the difference compared to GC×GC is that the modulators act as a trap and concentrate the analytes prior to a narrow plug injection on the second column. So he added a trap column to the LC valve interface. When the trap column was desorbed in the backflush mode, the resolution in the second column increased significantly. However, the peak counts are still less than 1000 of the 6500 lipids expected. Stay tuned for further improvements.
A report from the Mediterranean Separation Science Foundation Research and Training Center (Messina, Italy) described the use of LC/GC×GC MS to differentiate between mineral oil and vegetable oil in process foods such as baby food. They found that meat, vegetable oil, cornstarch, and sugar were all contaminated with mineral oil. The total found was significant and close to the regulatory admissible daily intake.
Conclusion and credits
The technical program was organized as two separate meetings—The 37th International Symposium on Capillary Chromatography and 10th GC×GC Symposium—co-located at one hotel. The emphasis on day 1 was on multidimensional separations, while days 3 and 4 focused on capillary columns. Day 2 was a transition between the two with a focus on hot applications. This seemed to work well.
- The meeting was organized by CASSS (www.CASSS.org). Ms. Renee Olsen led the CASSS team that provided logistical and meeting support. All deserve special thanks for a job well done.
- Professors Frank Svec (Lawrence Berkeley National Laboratory, Berkeley, CA) and Robert E. Synovec (University of Washington, Seattle) deserve special credit for organizing the meeting.
- Professors Jean-Marie Dimandja (Spellman College, Atlanta, GA) and Philip Marriott (Monash University, Berwick, Australia) organized the 10th GC×GC Symposium.
For related information, see:http://www.americanlaboratory.com/Blog/140014-Evolution-of-Petroleum-Assays-During-the-Last-50-Years/.
For more meeting reviews by Robert L. Stevenson, see:
Robert L. Stevenson, Ph.D., is a Consultant and Editor of Separation Science for American Laboratory/Labcompare; e-mail: firstname.lastname@example.org.