Focus
Supercritical Fluids Can Be Nice Beginning chemistry students are taught that there are only three states of matter: solid, liquid, and gas. But there are those who feel that there is indeed a fourth state of matter, the supercritical fluid state. Supercritical fluids, which are currently finding wider application in separation science, are unique in their properties, differing from liquids and gases but having characteristics of both. In Figure 1, a phase diagram for water, the supercritical fluid state ex ists everywhere to the right of the ver tical line intersecting the χ axis at 374.1 °C, the critical temperature of water. Above the critical temperature, the molecules cannot stick together (condense) but continue to fly around like independent gas molecules, no matter how much pressure is applied. Dennis R. Gere of the HewlettPackard Company likes to remind people that they need not be afraid of supercritical fluids. "The prefix super sometimes throws people off and tends to frighten or intimidate them if they haven't thought about it," said Gere in a recent talk on supercritical fluid chromatography (SFC). "Some people relate supercritical fluids to su persaturated solutions, which are thermodynamically unstable. Super critical fluids are actually very stable." Gere emphasized that the mobile phase he has been using for most of his SFC experiments, supercritical CO2, is an especially friendly sub stance. "It's not toxic, it's not flamma ble, it has no odor, and it can be in haled or even ingested. It's readily available in pure form, and it's inex pensive, incredibly inexpensive." Gere spoke at the Eighth Food and Drug Administration (FDA) Science Symposium, "Recent Advances in An alytical Methodology in the Life Sci ences," held Aug. 16-18 in Bethesda, Md. Gere presented the paper on be half of himself and coauthors Robert Board, Henk Lauer, Douglass McManigill, Harry Weaver, and Douglas Smith. The theory and appli cations of SFC were also recently dis 0003-2700/82/A351-1249$01.00/0 © 1982 American Chemical Society
cussed in a detailed REPORT in ANA LYTICAL C H E M I S T R Y (Novotny,
Milos, et al. Anal. Chem. 1982, 53, 407-14 A). COVs critical temperature of just 31 °C and its critical pressure of 72.9 atm permit a wide latitude in selection of operating parameters before the temperature and pressure limitations of current liquid chromatography (LC) hardware are exceeded. Pioneers in SFC must modify high-perfor mance liquid chromatographic (HPLC) instrumentation to perform
the technique, since commercial SFC instrumentation is not available. CCVs good UV transparency makes UV detection possible even into the vacuum region, down to about 185 nm for a relatively pure grade of CO2. This is an advantage over the solvents used in liquid chromatography, many of which have UV cutoffs above 200 nm. Preparative collection is especially convenient with supercritical CO2 as the mobile phase. As the effluent exits from the SFC detector and the pres-
Temperature ('C)
Figure 1. Phase diagram for H20. Tb is the boiling point at 1 atm. Tc is the critical temperature. Note that the y axis is nonlinear ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982 · 1249 A
Focus sure drops to ambient, there is a strong cooling effect. If an autosampling vial is placed over the detector outlet as a peak registers on the^chromatogram, part or some of the eluting component will condense out, along with dry ice formed from the cooling of the mobile phase. The dry ice sublimes quickly at room temperature, leaving the pure component in the vial. When polar organic modifiers, such as methanol, are used in small concentrations (usually about 1%) in the SFC mobile phase, the collected component will be entrained in a small amount of the modifier. This is not always undesirable, but even this small amount of modifier can be easily evaporated away if desired. Diffusivity and viscosity have important effects on chromatographic behavior, explained Gere. In chromatography, the higher the diffusivity (the rate of diffusion of solutes in the mobile phase), the narrower the peaks tend to be, and thus the higher the efficiency of the technique. Since diffusion coefficients in supercritical fluids are intermediate in value between GC (gas chromatography) and LC, the efficiency, or separation power, of SFC is also intermediate. Thus, solutes can generally be separated and detected in one-fifth to one-tenth the time, and with the same efficiency, compared to a corresponding LC separation with the same relative retention (a) value. An order of magnitude viscosity advantage for SFC over LC (the viscosity is lower in supercritical fluids than in liquids) also contributes to this higher efficiency.
To attain the very highest chromatographic efficiencies, it is still necessary to use capillary GC. But large nonvolatile molecules cannot be handled by GC, because they cannot be volatilized without decomposition. These nonvolatile substances can be easily dissolved in a supercritical mobile phase, then separated and detected. Therefore, SFC seems to be finding its niche as a technique that can handle larger molecules than GC at higher efficiencies than LC. Gere reported on a number of applications of SFC he and his colleagues have been working on. Among these are separation of ubiquinones in extracts from bacteria cultures, including bacteria from the genus Legionella ; the separation of progesterone and estrone, two thermally labile biomolecules, in 12 min at a mild 34 °C; and the detection of caffeine in beverages. The group found that a liter of CocaCola had 82 mg caffeine, Pepsi-Cola had 77, Twining's Earl Grey tea had 68, and Dennis' Espresso Coffee had 731. "For industrial people, time is money," said Gere, "and being able to routinely assay caffeine in beverages every 36 s is very meaningful. In fact, the f R of caffeine can be reduced to 18 s under optimized conditions." "SFC has, I believe, gotten to a point now where it's a lot more practical," Gere continued. "The evolution of HPLC pumps, detectors, and packing materials have set the stage for another evolutionary discontinuity, a jump forward in the practical use of supercritical fluid chromatography."
SFC-MS With SFC attracting increasing interest, can hyphenated SFC be far behind? Development of a new supercritical fluid chromatograph-mass spectrometer (SFC-MS) has, indeed, recently been announced. The new SFC-MS is the product of a joint development effort by the Department of Energy's Pacific Northwest Laboratories (operated by Battelle Memorial Institute) and Brigham Young University (BYU). According to Battelle's Richard D. Smith, the instrument can handle molecules of high molecular weight and low volatility that are not capable of being analyzed by gas chromatography-mass spectrometry. Smith says the new system will be especially valuable in biological studies, since biomolecules are unusually sensitive to heat. In addition, the SFC-MS will be applicable to studies of the more polar and higher molecular weight fractions of synthetic fuels. The hyphenated instrument is based on an interface that permits the two techniques to work in tandem (Anal. Chem. 1982, 54, 1883-85). The interface, developed by Smith and John Fjeldsted, a graduate student at BYU, provides for direct fluid injection of the total SFC effluent into an MS chemical ionization source. The earliest SFC-MS instruments were developed in the late 1970s by Randall and Wahrhaftig and Gouw et al. and were often referred to as "dense gas chromatographmass spectrometers" at that time. Smith claims to have improved somewhat on these earlier designs.
1250 A · ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
SFC: Pro/Con There are those who would not agree completely with Gere's assessment of SFC's potential. The following are some points made by two other chromatographers interviewed by A N A L Y T I C A L C H E M I S T R Y .
• The higher efficiency of SFC may only be needed for complex samples containing 150-500 components. • One of the greatest strengths in LC is the ability to change the mobile phase to get a specific chemical interaction for a difficult separation. In SFC, one is much more constrained in the selection of mobile phase • The efficiency of LC columns is already so high that any improvement is going to be marginal in practical advantage. "It's trivial to say you can do a separation in IV2 rnin vs. 3 min. Unless you're doing a process analysis, you can't even make samples up that fast." "I don't think a factor of five in separation speed is marginal," replied Gere. • Both chromatographers also emphasized positive points about SFC. One stated that "SFC is interesting, worth pursuing, and it may have potential." The second explained that he though SFC would be useful for some applications: "If a good system is developed, for instance, whereby high molecular weight proteins and peptides could be separated by SFC, then I think that would be quite important."
NIR Litigation A small company that manufactures near-infrared instruments for the analysis of grain recently filed a $72million lawsuit against a competitor. In its suit, Trebor Industries, Inc., charges Pacific Scientific Company, Pacific's Gardner/Neotec division, and Grain Dryers Service, Inc., of conspiracy in the alleged theft of a Trebor instrument designed to measure protein and moisture levels in whole grain. Simultaneously, Pacific Scientific has filed a smaller countersuit, for $1 million, against Trebor. Pacific is charging that Robert D. Rosenthal, president of Trebor, prevented Neotec from pursuing development of a whole grain analyzer. Rosenthal was founder and former president of Neotec Corporation (forerunner of Gardner/Neotec) before he left to found Trebor.
Focus According to an article by Rudolph A. Pyatt, Jr., in the Washington Post (July 27, 1982, pp. D7-8), Pacific Scientific claims that "Rosenthal wanted to develop the analyzer for himself and that after resigning he obtained financing for research and development from a Swedish firm with which Neotec had had discussions." The near-infrared technique is not a new one. For instance, an I N S T R U -
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Petroleum in the Marine Environment
Advances in Chemistry Series No. 185 Leonidas Petrakis, Editor Gulf Research & Development Fred T. Weiss Editor Shell Development Company A symposium jointly sponsored by the Divisions of Petroleum and Analytical Chemistry of the American Chemical Society.
A carefully balanced presentation of analytical methodology and application that will be of major interest to environmentalists and energy conservationists. In this volume prominent chemists, marine ecologists, microbiologists, and toxicologists bring together for the first time the most important and most pertinent analytical techniques for the complex issues involved in this field. CONTENTS The Biogeochemistry of Fossil Fuel Hydrocarbons · Analytical Chemistry of Petroleum · Oil Spill Identification and Remote Sensing · Determination of Aromatic Hydrocarbons in Zooplankton and Macrofauna • High Resolution Gas Chromatography · Polynuclear Aromatic Hydrocarbons in Environmental Samples · Solubility Behavior of Polycyclic Aromatic Hydrocarbons · Multiple Gas-Phase Equilibration Method · Nonhydrocarbons in the Analysis of Virgin and Biodegraded Petroleum · Application of Trace Analytical Techniques · Analysis of Oily Water • Hydrocarbons in the Sediments of the Bermuda Region · Polycyclic Aromatic Hydrocarbons in Marine/Aquatic Sediments · Ambient-Temperature Extraction of Hydrocarbons from Marine Sediment · Distribution of Aromatic Hydrocarbons in Sediments from Selected Areas · Comparison of Methods for Analysis of Hydrocarbons in Marine Sediments 371 pages (1980) Clothbound $42.00 LC 79-25524 ISBN 0-8412-0475-6 Order from: SIS Dept. Box 07 American Chemical Society 1155 Sixteenth St., N.W. Washington, D.C. 20036 or CALL TOLL FREE 800-424-6747 and use your credit card.
1252 A · ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
M E N T A T I O N article on "Near Infrared Reflectance Spectrophotometric Analysis of Agricultural Products" by C. A. Watson appeared in the August 1977 issue of ANALYTICAL C H E M I S T R Y
(pp. 835-40 A). Rosenthal's Trebor90 Grain Tester differed somewhat from earlier designs, however. With earlier instruments, the grain was first ground and infrared radiation was reflected off the ground sample; whereas radiation is transmitted directly through a sample of whole grain with the Trebor-90, and grinding of the sample is no longer necessary. According to Trebor's suit, the three defendant firms conspired to steal a Trebor-90 unit to learn the trade secrets involved in its manufacture. An instrument such as this is normally protected by both patents and trade secrets. "When youtnke'out patents, all you've done is provide a track for other people to go around," said Rosenthal in a recent interview. "In other words, you've exposed your poker hand. A clever guy looks at it, and is perhaps able to come up with another way of doing the same job without infringing on the patent." Perhaps a better way of protecting yourself, explained Rosenthal, is the trade secret route, where you simply don't divulge publicly how you do something. This is the route that Coca-Cola has taken with its recipe, for instance. "Yes, we were covered by three patents on the Trebor-90," said Rosenthal, "but in addition to the patents there were a number of areas we considered proprietary. "It's not unusual for one company to buy another company's product," Rosenthal continued, "either directly or surreptitiously, and take that product apart. What's unusual in this case is that was not how it was done. A property owned by my company was, we allege, stolen and dismantled. We've taken great pains in our selling approach to try to avoid having someone purchase a unit and surreptitiously turn it over to a competitor. But in this case, we claim that an instrument owned by us and in the possession of an intermediate party was taken without permission and dismantled." Stuart A. Borman
