Focus
Carleton Fred
Howard
Lytle
due to high spectral brightness of the laser used, collecting Raman-shifted radiation from a patch of sky only a few arc seconds in diameter, good laser monochromatization, a narrow time window on the detector, and subtraction of the background.
Flow Cytometry Another technique Hirschfeld discussed was flow cytometry, in which particulates are analyzed by flowing t h e m single file past a laser beam. " T h e r e are two books out on it," Hirschfeld remarked, "there have been several informational meetings, there are 700 instruments installed, and no analytical journal has ever mentioned it. Why? Because it's used for biochemical analysis, and t h a t is somebody else's work." One application of flow cytometry mentioned by Hirschfeld is the analysis of cells for DNA content. Cancer cells will have a high DNA content, since they are undergoing fast and uncontrolled subdivision. But in a million cells, Hirschfeld explained, only 10 may have abnormal DNA levels.
Fluorescence spectroscopy would be too slow. B u t if you p u t the cells in suspension and flow them single file past a laser beam, collect the fluorescence with a lens, and use a photomultiplier tube to detect the signal, you can generate a histogram of the DNA concentration distribution, Hirschfeld explained, and you can run this measurement at about 30 000 cells per second. Particulates are important not only in biochemistry, b u t in pollution, emulsion polymerization, and micelle studies as well. "Yet this technique hasn't made any inroads," complains Hirschfeld. "So perhaps it's time we became a little more aware of it." P. J. Hargis and J. P . Hohimer of Sandia Labs presented a paper on laser-induced fluorescence for trace element analysis. T h e y reported determination of thallium by laser fluorescence with a detection limit of 10 ppb. Since they were actually analyzing 50 ML of solution, they were detecting half a picogram of Tl. T h e investigators reported t h a t these results are two orders of magnitude better t h a n the best atomic absorption results. A further application of laser fluorescence investigated by the Sandia group was isotope detection. This can not be done with conventional light sources. B u t Hargis and Hohimer realized t h a t the monochromaticity of laser light would enable them to detect t h e small fluorescence shifts in elemental isotopes, and they have used the technique to quantitate isotopes of uranium, though they have yet to determine detection limits. T h e Sandia group also suggested applications to multielement analysis. A series of tunable dye lasers, each of which can be tuned over a certain range of frequencies, is used. Each laser is set to excite a particular atomic species. T h e laser outputs are timemultiplexed using different lengths of optical fiber cables, each laser's pulse being delayed to a different extent. T h e atomic fluorescence produced is then detected with a series of photomultipliers. Indeed, as Hirschfeld and Lytle pointed out, the laser has instigated a redefinition of the term "nonfluorescent". We are normally told a lot of nature is nonfluorescent. B u t the Raman spectroscopists will tell you, " T h a t isn't so." When a substance is
reported in the literature to be nonfluorescent, said Hirschfeld, "it is shorthand for saying, 'It doesn't fluoresce with a q u a n t u m efficiency exceeding 0.1% in the ultraviolet or blue region of the spectrum.' " There are arguments for placing a lower bound on the q u a n t u m efficiency defining the limits of fluorescence, now t h a t the laser is on the scene. Lytle of P u r d u e added t h a t one of his graduate students has recently written a thesis on the fluorescent properties of classically nonfluorescent molecules.
Panel Discussions Symposium speakers and attendees had a chance to speak their minds at a series of panel discussions in the course of the symposium. Here is how some of the conversations went: • Laser T e c h n o l o g y Attendee: Fred Lytle mentioned t h a t his lasers are operational only 5 to 10% of the time. T h a t is a very critical area for laser manufacturers to be concerned about. E. L. Wehry, University of Tennessee (Knoxuille): I would second the motion there. When I left Knoxville, both of our major laser systems were down, and we have a X e - H g lamp t h a t has 11 000 hours on it. If you can't get the data, it doesn't matter what the theoretical possibilities are. Steve Harris: If you buy a nitrogen laser it may run for weeks or months without major problems. B u t nitrogen lasers are out of date now. I think one trouble is t h a t everyone wants a state of the art YAG or excimer laser, and the reason it's down all the time is because it was invented only a year ago. If you're willing to settle for a fiveyear-old laser, it will be more reliable. Hirschfeld: One of the problems with lasers is t h a t the specifications don't describe t h e m properly. For example, how many of us have learned about sideways beam wander the hard way? How many of us have found out t h a t the laser was 99% mode purity, which means t h a t 1% of the time it's totally in the wrong mode. We have nice lasers with all kinds of power at 4880 Â. In about 5% of the lasers I've seen, t h a t means you're lasing at 4889 Â at the same time. This can give you the most beautiful data, which you interpret and publish. • T h e Ideal Laser Hirschfeld: As to the specifications
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of the ideal laser, one comes u p with the following: It should cost a hundred bucks. It should be less than a cubic centimeter. It should have at least a few tens of milliwatts of power. It should be reliable, so that anybody with an IQ of 70 can operate it'. Its power supply should cost a hundred bucks or less. And it should be tunable over the range of your choice. With the exception of the last requirement, the laser exists and has already existed for 10 years. It's called a galliumarsenide diode laser. Researchers in t h e field expect t o be able t o improve the tunability by an order of magnitude. Of course, it's all in t h e near infrared. B u t since the gallium-arsenide cathodes have come out in t h e near infrared, that's no longer a region where photomultipliers don't work well. We still have this mental outlook t h a t if it's not visible you can't do it. We now have detectors t h a t have 10% quantum efficiency a t 8500 Â. So perhaps we ought to revise our prejudices a little. So we have a near ideal laser out there that's being sold commercially over t h e counter. • Take-off Point Hirschfeld: There are three techniques in laser analytical spectroscopy t h a t have just reached t h e take-off point. One is analytical cytology. There are now four manufacturers, there are about 700 instruments out, and t h e yearly increment in t h e number of people in t h e field exceeds 15%, in flow cytometry and slide scanning cytometry using lasers. Second take-off field: probes, sampling where you are not. It's now becoming established t h a t if you have a combustion research facility you must have some R a m a n equipment to probe t h e interior of the flame. There are, of course, all kinds of industrial processes where you would like to know what happens inside t h e reactor, and it's being extended t o this. We have been doing beautiful atomic analysis on micron-size samples. Now the R a m a n microprobe can do molecular analysis on micron-size samples. And last, b u t not least, fluorescence. I think we are about to see the last of the Hg arcs in t h e next few years. T h e simpler lasers are already competing very favorably in power and general signal levels with those you can obtain with a Hg lamp. T h e red lasers are already reliable. It's only a matter of time before we attain t h a t kind of reliability with the cheaper blue lasers. Right now t h e cost of lasers vs. the Hg lamp a n d its accessory optics comes out nearly equal. A Hg lamp requires more expensive optics and electronics, as well as much better filters. All this is part of the cost equation. In t h e next few years I think we are going to see a vast increase in the use of lasers in standard fluorimetry. These are t h e
short-term take-off fields I can see for lasers. Swofford, Standard Oil: If you want to come u p with a technique t h a t will catch a lot of interest, not only in this country b u t abroad, try to think of a laser technique t h a t will locate petroleum. Heckler: It's located in t h e storage tanks at Standard Oil. • Hyphenated Techniques Hirschfeld: If you make a matrix of all the techniques t h a t are good for
analyzing things and all t h e techniques t h a t are good a t separating things, you will find there are about 64 combinations, 30 of which make sense technically. Two or three of these combinations have ever been tried. If we are going to pool techniques, one instrument to analyze, one instrument t o separate, and a computer to make them work together, then why not go systematically t h e whole hog instead of accidentally as one or another technique gets popularized?
Let's Get Small W h a t is carried around in a shirt pocket and beeps when there's too much vinyl chloride or other chemical hazard in t h e air? A pocket-sized gas chromatographic air analyzer does, and a team of electrical engineers a t Stanford University is working to make it a reality. Steve Terry has been working since 1971 on t h e development of the miniature gas chromatograph, the idea having originated at NASA in response to t h e need for a small instrument for the Viking flight to Mars. T h e instrument was not ready in time, and NASA had to use a small conventional GC on Viking, and on t h e Pioneer flight to Venus as well. Currently, work on t h e miniature GC is being funded by two agencies: NASA, which wants a flight instrument for future space missions, and the National Institute for Occupational Safety and Health (NIOSH), which is interested in a pocket-sized air contaminant monitor.
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Although Terry is an electrical engineer with a P h D from Stanford (1975), he has had to become intimately familiar with chromatography. His NASA technical report and t h e feasibility study he wrote for N I O S H are replete with fundamental chromatographic theory. Terry is no stranger to A N A L Y T I C A L C H E M I S T R Y either.
" T h e project I'm on has a little overlap with chemistry," Terry says, "so I've had to do a fair amount of digging through A N A L Y T I C A L C H E M I S T R Y . "
Terry also has plans to submit papers to t h e J O U R N A L in t h e near future. T h e reason for the marriage of electrical engineering and analytical chemistry in Terry's work is simple to explain: t h e chromatographic column is etched into a silicon wafer utilizing integrated circuit processing technology. T h e proposed instrument is a completely self-contained gas chromatograph, including a carrier gas supply and a microcomputer system for automatic control and data analysis. It will
Silicon wafer is compared to dime.
be small enough to be carried around by an industrial worker, and will con tinuously monitor the presence of up to 10 contaminant vapors in the work er's breathing zone. T h e chromatograph consists of an open tubular capillary column, a sam ple injection valve, and a thermal con ductivity detector, all fabricated on a single 5-cm diameter silicon wafer. T h e column is a Va-meter long groove etched into the silicon wafer and capped with a glass plate. T h e sample valve is a solenoid-actuated dia phragm valve with a nickel diaphragm and etched silicon valve seat. And the detector is a microbead thermistor suspended in an etched cavity in the o u t p u t gas stream. A state of the art integrated circuit (IC) microcomputer is included in the instrument to handle control and data processing functions. T h e instrument will be capable of sampling the atmo sphere once every two minutes for eight hours, measuring the concentra tions of up to 10 different vapors si multaneously to within 10% accuracy, calculating and storing the timeweighted average and peak concentra tions for each of the gases, measuring concentrations at the 10-ppm level, displaying any of the stored concen trations on demand, and sounding an alarm when any concentration exceeds the acute exposure limit for t h a t sub stance.
stationary phases used in gas chroma tography. To promote adherence of the lining liquid to the glass and sili con walls of the capillary channel, the column is treated with an organosilane compound. T h e stationary phase, dis solved in a volatile solvent, is then in troduced into the column and is left in a thin layer on the column walls as the solvent is blown out of the capil lary. "We are currently receiving tech nical help from NASA," Terry ex plains. "Several chemists from the Viking and Pioneer projects at Ames Research Center are helping us with different column linings for the GC." T h e sample injection system of the miniature GC is designed to collect at mospheric gas samples and inject pre cisely controlled amounts of the sam ple as short pulses into the carrier gas stream at the inlet of the column. It consists of a single normally-closed on/off valve integrated with the capil lary column and a servo-driven piston p u m p to draw in and pressurize the atmospheric sample. When a sample is to be injected, the pressure of the sample gas on the input side of the valve is raised above t h a t of the carrier gas and the valve is opened briefly. A plug of sample thus flows through the valve and forces its way into the capil lary. Terry has proved t h a t this injec tion configuration is capable of inject ing reproducible sample pulses as small as 1 nL in volume.
T h e miniature capillary column is formed by etching a long spiral groove into the surface of a silicon wafer and then hermetically sealing the wafer to a Pyrex glass cover plate. T h e cross section of the resulting gas channel is roughly rectangular, with a width of 200 μπι and a depth of 20 μηι. Fabrication of the column proceeds in a sequence of photolithography and etching steps very similar to those used in standard IC device processing. A layer of S1O2 approximately 1 μηι thick is thermally grown on a 5-cm di ameter silicon wafer to serve as an etch mask. T h e spiral pattern is then defined in the S1O2 using a standard photoresist and photolithography pro cess. T h e oxide is removed where the groove is to be formed, exposing the silicon surface. T h e wafer is then placed in a silicon etching solution where the spiral pattern, defined by the S1O2, is etched into the silicon. After the capillary groove and gas exit holes have been etched, Pyrex glass is bonded to the silicon wafer by an anodic bonding process which uses no bonding agent or filler material. T h e resulting seal between the glass and the silicon is irreversible and her metic, thus transforming the spiral groove into an enclosed capillary tube. T h e completed capillary column is next lined with one of the standard
Thermal conductivity detectors have been used in the miniature GC systems constructed to date. This type of detector consists of a bead thermis tor sealed in a cavity between the glass and silicon wafers of the integrated capillary column with its leads emerg ing through holes etched through the silicon wafer. T h e 200-nL volume and 20-ms time constant of this detector meet the requirements t h a t the smallvolume short-duration o u t p u t peaks impose on it. Utilizing this type of de tector, Terry estimates t h a t the minia ture GC will have a detection limit of 10 to 50 ppm. Laurence Doemeny, Chief of the Measurements Systems Section of NIOSH's Division of Physical Sci ences and Engineering, is interested in having automated pre-enrichment and desorption incorporated into fu ture versions of the instrument to im prove the limits of detection. Dr. Doe meny, who is coordinating with Terry on the project, explains: "We want to see how to make the instruments more versatile and to improve the sensitivi ty. If Dr. Terry could take 10 samples and enrich them, he'd already be down to less than a part per million." T h e main component of the GC's electronics system is presently a Zilog Z-80 microcomputer, although Terry explains t h a t this particular unit,
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though well suited for initial proto type development, is not adequate for the final hand-held units, mainly due to power considerations. But Terry ex pects t h a t a lower power alternative to the Z-80 will soon become available. T h e microcomputer will be pro grammed to automatically and contin uously step through the sequence of events necessary to sample, separate, and analyze the air for the several gases which the pocket-sized instru ment is set to monitor. First, the car rier gas pressure and column tempera ture are monitored, the detector am plifier is zeroed, and the base-line drift calculated. T h e n the air sample is drawn in, pressurized, and injected into the GC column. As the o u t p u t peaks are detected, their areas and concentrations are calculated, the peaks are identified by retention time, and the concentration values are stored in the computer's memory. This process is completed in less than one minute, and it is automatically re peated every two minutes throughout the day. At the end of the day, timeweighted average concentrations and peak exposures can be read from the gas analyzer's memory and recorded automatically. Terry envisions a wide variety of applications for the miniature gas chromatograph. T h e small diameter of the integrated column makes it more efficient than a conventional col umn at separating vapors, making the miniature GC approximately an order of magnitude faster than laboratory instruments at significantly reduced cost. T h e GC's high speed operation is potentially useful for process con trol or for patient monitoring in a medical situation, where rapid analy sis is necessary. In the toxic gas analy sis field, the miniature gas chromato graph is useful either as an individual exposure meter or as a portable field survey instrument. T h e initial project ed retail price is about $2000 per in strument, depending upon several data analysis and readout options. Terry is now at the point where he is getting some analytical results t h a t he can talk about. Soon he will deliver six prototypes to Doemeny at N I O S H for evaluation. Terry's group will then have another opportunity to improve the instrument before NIOSH sends then out for independent evaluation. Doemeny hopes the miniature GC will eventually be a more versatile in strument: " T h e columns operate at ambient temperature now. We could vastly improve the instrument by breaking it up into two packages—a battery pack on the belt and the moni tor up in the shirt pocket. This way we could have enough power to have a heated column, pre-enrichment, and thermal desorption."
