X-ray fluorescence analysis, matrix enhancement, and adsorption effects. High analytical sensitivity is obtained by using a target which has its characteristic K a X-ray energy just above the absorption edge of the element to be determined. Matrix enhancement is usually eliminated by this procedure and matrix absorption is compensated by normalizing to the backscatter peak of the target’s K a X-rays. The source-target assembly in Figure 1 has been used t o analyze glass and ceramic archaeological artifacts where the destruction of these artifacts was not permitted. A detailed description of the equipment used along with a multichannel pulse-height analyzer has been given elsewhere ( I ) . When present in low concentrations (1% or less), several elements with comparable X-ray energies often may be determined with the same target. Detection limits of 100 ppm and less for many elements can usually be obtained. Typically, analysis of a specimen for more than a dozen elements (Mn and higher atomic number elements) could be accomplished in less than one hour. Figure 2 shows a Mosley plot of a glass specimen containing 1.0% Cu, 0.15% Co, 0.8% Fe, and 0.Y:; Mn excited with ASK X-rays excited from an As203 target by an 241Amsource and detected with a lithium-drifted
silicon detector. It will be noted that the peaks from manganese, iron and cobalt are not resolved. With the data still in the memory of the pulse-height analyzer, the specimen is replaced by a piece of iron and the analyzer is run in the subtract mode until the iron X-ray peak, as seen on the oscilloscope screen, is reduced to the background level. The operation takes only a few seconds and the results are shown in Figure 3. The accuracy of many of these analyses was often good only t o 10%. An accuracy of around 1% should be obtainable for many analyses where the composition of the samples does not vary over a wide range, when particular care is taken in the sample preparation, and where sufficient care is taken in standardization. ACKNOWLEDGMENT
The author thanks the Lawrence Radiation Laboratory Berkeley Safety Services Department for designing and making the annular container for these sources. RECEIVED for review July 8, 1968. Accepted August 12, 1968. Work performed under the auspices of the U. S. Atomic Energy Commission Contract No. W-7405-eng-48.
A Micro-Preparative Gas Chromatograph and a Modified Carbon Skeleton Determinator Robert G. Brownlee and Robert M. Silverstein Stunford Reseurcli Institute, Menlo Park, Calif. 94025 WITHthe ability to identify submilligram amounts of organic molecules by spectrometric methods ( I ) has come the need for improved methods of handling small samples obtained by gas chromatography. Our integrated system has been built upon a modified Varian Aerograph 204 (Figure 1). This basic instrument is near ideal because of the large ovens and the interchangeable injectors. The devices we have added are a capillary variableratio effluent splitter, a thermal gradient collector, a glass capillary-breaking device, and a modified Beroza Carbon Skeleton Determinator (CSD), (2) which is an extremely useful adjunct to spectrometry. Capillary Variable-Ratio Effluent Splitter. None of the commercially available splitters had the requirements of variable ratio, low dead volume, and invariability of split ratio with flow rate. The splitter we designed is variable from 1 :10 t o 1 :>lo0 and is of annular design, with sampling from the center of the column flow. The split ratio is constant for any flow rate because of the capillary flow restriction; a total capillary volume of 3.78 pl ensures short residence times. The addition of an annular nitrogen make-up gas fitting between the end of the capillary and the detector prevents peak broadening and increases the sensitivity of the hydrogen flame detector (3).
(1) R. M. Silverstein and G. C. Bassler, “Spectrometric Identification of Organic Compounds,” 2nd ed., Wiley. New York, N. Y . , 1967. (2) Morton Beroza and Fred Acree, Jr., J. Ass. Oflc. Agr. Chem., 47, 1 (1964). (3) R . L. Hoffman and C . D. Evans, Science, 153, 172 (1966).
The column effluent is split (Figure 2) between a 152-mm X 1.6-mm o.d., 0.178 mm i.d. (0.007’’ i.d.) stainless steel (ss) capillary tube ( A ) and a 150-mm X 1.6-mm 0.d. ss tube (E)
that leads t o the collector. One end of the capillary tube is silver soldered into the Swagelok fitting that receives the column in the column oven. The other end is silver soldered into the nitrogen make-up gas fitting, which leads to the flame ionization detector through a 70-nim X 1.6-mm 0.d. ss tube. The minimum split ratio is set by a flow restrictor consisting of 5 mm length of 0.178-mm i.d. (0.007” i d . ) capillary tubing silver soldered to the end of the tube conducting the sample to the collector exit. The split can be varied by inserting lengths of stainless steel wire (Malin Music Wire Co., Cleveland, Ohio) into the capillary tube splitter at A . A 127-mm length of 0.127-mm (0.005”) wire gives a split ratio of 1 :54, and up to 0.152 mm (0.006”) wire can be used. The nitrogen make up gas, which enters at C, is preheated in a 1.5-m coil of 1.6 mm o.d., 1.19 mm i.d. ss tubing mounted in the detector oven. The nitrogen flow rate is controlled by a needle valve. Thermal Gradient Collector. Jennings and Sevenants ( 4 , 5) have demonstrated the efficiency of a thin-wall glass capillary for collecting microgram amounts of gas chromatography effluents. Schlenk and Sand used a heated section of a collector as a fog breaker (6). We have designed a collection device that facilitates rapid handling of the long, (4) W. G. Jennings, Berichte der Wissenschaftlich-Technischen Kommission Internationale Fruchtsaft-Union, Band VI, Luzerner Bericht, 1965, p 227. (5) M. R. Sevenants and W. G. Jennings, J . FoodSci., 31,81(1966). (6) Herman Schlenk and D. M. Sand, ANAL. CHEM..34, 1676 (1962). VOL. 40, NO. 13, NOVEMBER 1968
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Figure 2. Capillary variable-ratio effluent splitter with nitrogen make-up gas fitting
Figure 1. Varian Aerograph 204 modified for micropreparative gas-chromatography
fragile capillaries and establishes a reproducible, linear thermal gradient. The device also accommodates a 3-mm 0.d. glass tube with an attached reservoir for efficient collection of larger samples. The device consists of two parallel 215-mm long aluminum (T6) tubes, one 1.7 mm i.d., 3.17 mm 0.d. t o accept the thinwall glass capillary (30 cm x 1.6 mm 0.d. Matheson Scientific Co.), and the other, 3.4 mm i.d., 6.34 mm 0.d. to accept a 25-cm length of 3-mm 0.d. glass tubing. At one end, the aluminum tubes pass through a 38 mm cube aluminum block containing a 100-watt cartridge heater. At the other end, the tubes pass through a 38-mm X 38-mm X 50-mm aluminum block that has a 178-mm X 19-mm aluminum rod extending down into a cooling bath. Both blofks are mounted on, but insulated from, a length of aluminum angle, which is supported rigidly in a horizontal position bya bracket fastened with two screws t o the side of the gas chromatograph, Provision is made for vertical and lateral alignment of the aluminum tubes with the exit port of the gas chromatograph (GC). Final alignment is made at the desired operating temperature. The exit port (Figure 3) is fitted with a Swagelok fitting containing a Teflon (DuPont) seal (E). One-half turn of the Swagelok nut ensures a gas tight joint with the inserted glass collector tube. A short length of Teflon (DuPont) rod with a 1.6-mm hole, mounted on the distal end of the aluminum tubing, prevents frost accumulation, which would otherwise contaminate the collector tubes on introduction. Very efficient recoveries (>90%) of microgram samples in the 1.6-mm capillary have been made at flow rates between 25 and 50 cm3/minute. Obviously, the choice of coolant and flow rate will depend on the boiling or melting point and the viscosity of the sample. A 152-mm section of the thin wall tube containing the sample can be flame sealed and subsequently rechromatographed, or broken directly in the inlet system of a mass spectrometer or into the Beroza CSD as described herein. Glass Capillary Breaker. The commercial version (National Instrument Laboratories, Inc.) of the Beroza CSD is designed to accept an injected solution of a sample. This poses the problem of selecting solvents to avoid interference of solvent peaks with the peaks of the hydrogenolysis products. The use of solvents introduces complications of handling, transfer, and recovery of samples. In order to introduce neat samples directly from a sealed capillary collector tube, we designed a device (Figure 4) that
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ANALYTICAL CHEMISTRY
Figure 3. Low volume exit port fitting for use with glasscapillary tubes Teflon (DuPont) cone (6) is machined mth a 15” angle from 6.34mm ma
allows introduction of the sample after the system is in equilibrium. A 160-mm long sealed glass capillary is held axially in a ss cylinder 152 mm X 50 mm o.d., 6.34 mm id., by means of fixtures (A) on either end, The center of the capillary tube is sheathed in a 100-mm length of 3.17 mm o.d., 1.75 mm i.d., ss tubing (E). The stem and bonnet from a #5 Whitey valve (F)are mounted in a threaded hole in the cylinder. When the valve handle is turned, the stem pushes against the center of the 3.17-mm ss tube and causes the glass capillary to break at both ends. A 1.8-m x 1.6-mm o.d., 1.19 mm i.d., ss tube, coiled around one end of the cylinder, serves as a carrier gas preheater. The cylinder and carrier gas are heated by asbestos insulated, nichrome resistance wire coated with liquid porcelain. The capillary breaker is mounted vertically so that the fixtures can be easily removed after breaking a sample. Figure 1 shows the box containing the capillary breaker, gas manifold system, and silicon control rectifier heater controls mounted on the side of the GC. Figure 5 shows the gas flow paths. A heated 1.6-mm o.d., 1.19-mm id., ss tube runs from the top Swagelok filling on the capillary breaker to a low volume “T”and then to a ss #O Whitey valve on a heated support on the injector shelf of the GC. The other branch of the “T” leads to a toggle valve for bleeding purge gas. The Whitey valve serves as the interface that isolates the capillary breaker from the rest of the GC system during sample loading or purging. Thus, the G C can always be maintained in a steadystate operating condition. The gas supply manifold consists of three toggle valves: the CSD valve, the capillary breaker valve, and the purge valve. The maximum displacement of the glass capillary io the design used is only 1.6 mm; therefore, to ensure breaking at both ends, it is necessary to scribe the glass capillary ends where the tube emerges from the end fixtures. Berovl Carbon Skeleton Determinator. Beroza has devised a novel method for determining the carbon skeleton of microgram quantities of organic compounds (2) by hydrogenolysis. The sample in a stream of hydrogen is passed
I F
5 cm
c
Figure 4. Glass capillary breaker C is a spring spacer and E is a Teflon (DuPont) washer seal
through a heated tube packed with a catalyst. The hydrogenolysis products are swept into a gas chromatograph and separated. Identification of the products can be made by matching retention time with known hydrocarbons or by collecting samples for mass spectrometry. We modified Beroza's CSD for use with the capillary breaker and for direct connection of the GC column to the outlet of the CSD. A Swagelok fitting was silver soldered to one end of a 230-mm x 9.5-mm i.d. ss tube, and a ss plug, through which pass three tubes, to the other end. One length of 1.6-mni 0.d. ss tubing conducts the sample from the Whitey interface valve; another length of 1.6 mm0.d. ss tubing, which makes several passes along the length of the catalyst tube, serves as a carrier gas preheater. A 50-mm length of 3.17 mm 0.d. ss tubing sealed at one end serves as a thermocouple well for measuring the catalyst temperature. The tube was insulated with a layer of asbestos paper, wrapped with nichrome wire heater and sealed with liquid porcelain. Several layers of glass wool insulate the catalyst tube from the injector shelf of the gas chromatograph. To begin operation,. all valves are closed except the CSD valve; this isolates the gas chromatograph system from the capillary breaker. The catalyst is maintained at the proper operating temperature and is supplied with preheated hydrogen. The glass capillary tube containing the neat sample is loaded into the capillary breaker from the bottom, and the Swagelok nut is tightened. The capillary breaker, sample line, and interface valve are all heated to whatever temperature is needed to maintain the sample as a vapor. The hydrogen purge gas valve and the bleed valve are opened. Preheated hydrogen gas purges the sample chamber of volatile contamination; the vented hydrogen is burned as a safety precaution. After purging, the purge bleed valve is closed; in about 20 seconds the purge valve is closed. The delay allows the pressure in the capillary breaker to attain the pres-
Figure 5. Schematic diagram showing gas supply manifold and sample path for use with glass capillary breaker and Beroza CSD sure of the rest of the system. The interface valve is opened, and hydrogen carrier gas is diverted from the CSD to the capillary breaker by closing the CSD valve and opening the capillary breaker valve. The glass capillary is then broken, The carrier flow can be diverted by opening the CSD valve, closing the interface valve, and closing the capillary breaker valve. The next sample can be loaded while the preceding sample is being chromatographed. The utility of the apparatus was demonstrated during our work on the isolation and identification of the sex attractant (brevicomin) from the frass of the Western pine beetle (7). Spectrometric evidence pointed to a bicyclic ketal with two substituents. Following trial runs on 2-pgram samples to establish conditions, a 50-lgram sample was hydrogenolyzed, and the major product was identified as n-nonane by mass spectrometry. This information eliminated a number of possibilities and finally led to the structure of brevicomin.
RECEIVED for review May 6, 1968. Accepted July 15, 1968.
(7) R. M. Silverstein, R. G. Brownlee, T.E. Bellas, D. L. Wood, and L. E. Browne, Science, 159, 889 (1968).
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