A number of samples which were part of collaborative studies were also analyzed using this method. One of these programs is ongoing, instituted by the Inspection Branch four years ago. Approximately 20 laboratories from Canada and the United States using a variety of methods participate in this study. Results are shown in Table I and good agreement is evident. Sample Types. Although the method has been primarily used for the analysis of fish tissue, other types of samples such as fish meal, blood, hair, and sediments have been analyzed with no special manipulations. This method is particularly suitable for routine work where a simple, rapid method is required. I t is appealing to the small laboratory since it allows for the digestion of a large number of samples in a small working area (in this case 140 in an area 18 X 14 inches.). Very little attention is required during the digestion process, thereby freeing the analyst. Moreover, the digestion process is simple in comparison to others ( I , 2 ) . Chemicals such as potassium permanganate and hydrogen peroxide are no longer required, resulting in a savings of money and time. The use of digestion tubes permits voluming the sample directly in the sample container, thereby eliminating the possibility of loss or contamination from transfers, together with the
time required for such manipulations. In addition, the cost of a graduated digestion tube is considerably less than that of a volumetric flask. The modified sampler permits sampling directly from the sample container and eliminates the need to transfer the sample a t any point throughout the entire procedure. Thus, in addition to the benefits mentioned above, errors due to misnumbering a sample are reduced to a minimum.
ACKNOWLEDGMENT We thank J. Harding and B. Berger for technical assistance and F. A. J. Armstrong for his advice in the writing of this paper. LITERATURE CITED (1) F. A. J. Armstrong and F. J. Uthe, At. Absorp. News/., 10, 101 (1971). (2) M. P. Stainton. Anal. Chem., 43, 625 (1971). (3) R. K. Munns and D. C. Holland, J. Assoc. Off. Anal. Chem., 54, 202 (1971). (4) R. Tkachuk and F. D. Kuzina, J. Sci. Fd. Agric., 23, 1183 (1972). (5) F. D. Deitz, J. L. Sell, and D. Bristol, J. Assoc. OM.Anal. Chem.,56, 378 (1973). (6) F. M. Teeny, J. Agric. Food Chem., 23, 668 (1975).
RECEIVEDfor review September 11, 1975. Accepted December 23,1975.
Heart Cutting Technique in High Resolution Gas Chromatography Applied to Sulfur Compounds in Cigarette Smoke W. Bertsch" University of Alabama, University, Alabama 35486
F. Hsu and A. Zlatkis University of Houston, Houston, Texas 77004
Complete resolution of trace organic compounds in complex mixtures has been a challenge since the invention of gas chromatography. The current trend in high resolution gas chromatography goes towards refinement of open tubular column technology. In spite of the impressive resolving power of such columns, the technique, unfortunately, has not found its way into the average laboratory. In many instances, inadequate packed columns are still employed for separations which demand the use of high resolution columns. The application of such columns is still considered a specialty by many potential users. The reluctance to switch from packed columns to capillary columns has to be attributed partially to difficulties in their preparation. On the other hand, even good capillaries often give disappointing results unless the instrument is properly adapted to such columns. Options to Effect Ultimate Resolution. In cases where a detailed characterization of all compounds in a mixture is required, the use of capillary columns is clearly advantageous. As complexity increases, a point will be reached a t which even the best capillary column with an optimally chosen stationary phase will not be capable of completely resolving all components and overlap will occur. Products from hydrocracking operations in the petroleum industry and tobacco smoke are examples of samples with extreme complexity. The resolving power of a column is a function of phase selectivity, capacity ratio, and efficiency. Keeping the first two parameters within reasonable limits, resolution can be 928
ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, M A Y 1976
improved most dramatically by an increase of the column efficiency. Total efficiency, in turn, can be improved by a decrease of column diameter or an increase in column length, the maximum tolerable pressure drop being the principal limitation. A t the same time, however, other problems are generated arising from a decrease of loading capacity and need for longer analysis times. In many cases, complete characterization of complex mixtures may not be necessary and only a few compounds within a complex mixture may be of interest. The use of heart cutting techniques and multidimensional chromatography can, in principle, effect the resolution of even the most complex mixtures. In-line valves have been used for this purpose (1-3), but the valveless system proposed by Deans ( 4 ) has significant advantages. Several semiautomated versions have been described (5, 6), but no commercial instrument is available. Heart cutting also has advantages if ultimate sensitivity is required for only a few selected compounds, i.e., detection of pesticides (7) or electron capturing derivatives of biologically active materials (8) by ECD. In heart cutting, a small fraction of partially resolved compounds is isolated and diverted into another column of different selectivity where further separation takes place and overlap with coeluting substances is minimized. In principle, the process can be repeated making use of different phase selectivities until complete resolution is obtained. Practical arrangements usually require intermediate trapping to counteract spreading effects during the first
step of the separation. I t also provides a sharp injection for t h e following chromatography in the second column and several cuts can, if necessary, be collected during one run. Combinations with backflushing techniques can save analysis time for multiple runs. Both conventional packed columns and open tubular columns can be combined, but the most common approach employs a packed column in the first separation step and a capillary column for the final resolution. This arrangement is preferred if the detector sensitivity is the limiting factor. If substances are present in sufficient amounts, two capillary columns can also be combined. A simple and inexpensive system is described which can be installed in any gas chromatograph requiring only minimal modifications. I t is especially useful for a nonroutine situation in which complete resolution of a few selected components is required for identification by GC/MS. The purpose of our application was the complete resolution of trace sulfur compounds in cigarette smoke condensate. The system employed a sulfur specific flame photometric detector simultaneously with a flame ionization detector. The combination of a specific and a universal detector was chosen to facilitate component selection, but other detector combinations are equally applicable.
EXPERIMENTAL Apparatus. A Shimadzu GC 5AP5 gas chromatograph with flame ionization detector FID and flame photometric detector FPD was used with an inlet modification (9). Figure 1 shows the experimental arrangement of the components. Nickel capillary columns (IO) 300 f t X 0.02 in. were coated with Emulphor ON 870 or OV 17 silicone fluid (Supelco, Bellefonte, Pa.). Inlet and outlet traps were also made of short pieces of coated nickel tubing. The outlet trap was located outside the oven and connected to a vacuum line via an on/off ball valve (Whitey, Oakland, Calif.). Dividers for column effluent and makeup gas were lh-in. stainless steel Tees, bought locally. Two short pieces of stainless steel tubing, ?$ x 0.05 in. served as restrictors between outlet transfer line and detectors. All metal parts were silanized, using standard procedures. Helium served as carrier gas and nitrogen, regulated by fine metering valves (Nupro, Cleveland, Ohio) was the makeup gas. The flow rates for the detectors were individually optimized. Both detectors were fitted with low dead-volume tubing leading directly into the jets. Sampling and Analysis. A small apparatus for collection of cigarette smoke condensate was built. I t consisted of a vacuum source, a cigarette holder, and a valving system, capable of producing a standard puff of 35-ml volume and 2-s duration. Standard cigarettes were obtained from the University of Kentucky. The total smoke condensate was drawn into a glass tube filled with Tenax GC, 60-80 mesh (Applied Science Labs, Inc., State College, Pa.), and transferred into the cryogenically cooled precolumn with helium as purge gas. The system was flow-controlled during this process. The precolumn was then connected to the capillary column a t the end of the sample transfer process, and the inlet pressure previously set a t 1 2 psi was then the controlling factor. The outlet trap was also immersed in liquid nitrogen. T o monitor eluting components, the valve was kept closed, causing flow through the two detectors. Chromatographic peaks were diverted into the outlet trap by opening the outlet valve for the duration of the intended trapping period. At the end of the run, the capillary column was exchanged for another column of different selectivity and the outlet trap with the collected cuts was placed directly in front of it. T o save time, several inlet systems and columns were used simultaneously to allow backflushing of heavy ends and conditioning.
DISCUSSION The sulfur specific FPD was an ideal candidate for selection of a group of components in a large pool of interfering substances. Cigarette sulfur profiles are very sensitive to minor variations in sampling conditions, Le., larger puff volumes, longer sampling intervals, and shorter cigarette butts generate more complex profiles, especially of higher molecular weight sulfur compounds which otherwise may
ON/OFF VAI VF
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I I I
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Figure 1. Schematic diagram of GC flow system
be completely absent. A puff of 35-ml volume, drawn over a period of 10 s, will generate only 3-5 sulfur-containing substances which elute early in the chromatogram. The same puff volume, taken in a period of 1 s or less will produce in excess of 100 sulfur compounds which are clearly distinguishable over the entire range of the chromatogram (111. Sampling conditions therefore must be closely controlled. A comparison with smoke condensate from a standardized cigarette, obtained by conventional smoking machines, shows a much simpler pattern for the latter and increasing losses of sulfur compounds are observed under prolonged storage. No major changes in the chromatographic profiles were observed when the smoke condensate was adsorbed on Tenax and stored for periods u p t o several hours. Longer storage times were not investigated since samples were easy to generate, but previous experience with biologically important substances indicates excellent stability even under such conditions (12). In spite of some problems with reproducibility, it was therefore decided to use a sampling method capable of generating fresh cigarette smoke condensate. The gas chromatographic behavior of sulfur compounds is another important consideration in analysis. Some reactive and labile substances are difficult to elute from gas chromatographic columns because of absorption on active surfaces and secondary reactions. Some sulfur compounds are considered to fall into this category and special precautions must be taken to minimize losses. Appropriate deactivation of contact materials and avoidance of high temperatures are necessary prerequisites. Properly deactivated glass has previously been the most common column material for the gas chromatography of sulfur compounds in cigarette smoke (13-26). On the other hand, sulfur-containing substances of various functionality have been eluted from stainless steel capillary columns as well (17, 18). The proper choice of column material is related to the polarity of the stationary phase and the absolute amounts of sulfur compounds present in the sample. Even carbonized glass capillary columns can exhibit absorption effects and need to be preconditioned by injection with similar substances prior to analysis to cover exposed active sites (19). With polar phases, the problem seems to be less serious, since the substrate itself acts as a deactivating agent. For trace amounts, special precautions are necessary, but this does not represent a problem in tobacco smoke analysis since the quantities involved are relatively large. ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, M A Y 1976
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0
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Figure 3. Partial chromatogram of isolated fractions on phase of different selectivity. Upper recording FPD; lower recording FID; Sam-
pling conditions as above; Chromatographic conditions: Nickel capillary column 245 ft X 0.02 in. OV 17; carrier gas H e 8 psi; temp. prog. 30 'C (20 min) 30-180 'C, 2'/min
BO
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Figure 2. Top: Chromatograms of total cigarette smoke condensate; upper recording FPD; lower recording FID. Sampling conditions, see text. Chromatographic conditions: Nickel capillary 300 ft X 0.02 in.; Emulphor ON 870; carrier gas He 12 psi; temp. progr. 80 'C (10 min) 80-180 O C , 2'/min. Middle: Heart cutting of selected sulfur compounds. Conditions as above. Bottom: Reconstructed chromatograms of selected fractions. Conditions as above
Surprisingly, nickel capillary columns seem to have relatively inert surfaces. Biologically active materials which are difficult to chromatograph, such as derivatives of steroids or tetrahydrocannabinol, have been eluted easily from such columns (20). For quantitative analysis, the behavior of individual sulfur compounds, however, should be checked upon. I t was known from previous experiments that variations in profiles were due to the actual product distribution and not to the sampling method itself. If the exact reproduction of patterns is required, the adsorbent containing the smoke condensate can be subdivided into several portions. FID patterns show only minor variations for the bulk of the compounds with changing sampling conditions. In running two detectors simultaneously, each device can be separately optimized. I t is also possible to regulate the relative amount of effluent going into each by proper selection of the restrictions between the transfer line and each detector. Sample response was no problem for either detector, and both restrictions were chosen to be equal, providing for a 5050 split ratio as judged by the response obtained by interchanging the restrictions. The role of the makeup gas is primarily for quickly restabilizing the detector baseline which is temporarily upset by the application of a vacuum to the trap. The tendency of the vacuum to extinguish the flames is counteracted. 930
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Makeup gas is also beneficial in cutting down on dead volumes in the flow diverter-trap system and in preventing unwanted time lags between the detectors. The latter conditions are especially important if very narrow cuts are to be taken. Flow rates which are too fast will result in a drop of trapping efficiency and sample loss will occur. Optimization of flow rates must be done empirically, but only a few measurements are usually required. The application of a vacuum a t the trap improves the sharpness of the cuts significantly. Only a weak vacuum is required, but a quick shutoff valve should be installed if narrow cuts are to be taken. The nature of the analytical separation described requires a preliminary run to establish the exact location of the cutpoints. The selected peaks are then collected in the following run in time windows as narrow as possible to cut down on interferences. If quantitative regeneration is not required, cuts of only a few seconds can be taken close to the peak maxima. This will result in a favorable ratio of the quantity of desired substance to interferences. A third run on a phase of different selectivity will then further resolve the components of interest. The chances for overlap with interfering substances which were simultaneously collected are minimized if narrow cuts are well spaced from each other. If the selectivity of the secondary stationary phase is chosen to differ widely from the phase in the first column, additional chromatography is more of a theoretical possibility than a practical requirement. Figures 2 and 3 show chromatograms of fresh tobacco smoke in the universal and sulfur sensitive modes. The top chromatograms in Figure 2 represent the sulfur profile and FID profile of a standard cigarette puff. The FPD chromatogram is relatively simple, but considerable overlap is indicated by the FID. Differences in sensitivity and linearit y between the two detectors further complicate the situation, and a large FPD signal may hardly be visible on the FID a t the attenuations used. The chromatograms in the middle demonstrate the sharpness of cuts which can be taken, averaging from 2 to 10 s in this particular run. Detector baselines are only slightly disrupted. Baseline drops also constitute a convenient marker for the exact location of the cutting window. The bottom chromatograms represent reconstituted sulfur compounds which were cut in the previous run. All sulfur compounds selected for cutting are well recovered. The flame ionization detector demonstrates that the majority of interferences, in this case compounds which do not contain sulfur. have been removed at this
point. Figure 3 represents the first part of the final separation on a phase of different selectivity. Chromatographic conditions are optimized for the phase used. Components which were overlapping in the previous chromatographic runs are now far better resolved and spread out over a large part of the chromatogram.
ACKNOWLEDGMENT A gift of standard cigarettes and tobacco smoke condensate from John Benner, University of Kentucky, Lexington, Ky., is appreciated.
LITERATURE CITED D. J. McEwen, Anal. Chem., 36, 279 (1974). P. A. Schenck and C. H. Hall, Anal. Chim. Acta, 38, 65 (1967). J. R. Conder, J. H. Purnell, and R . Walsh, Talanta, 15, 145 (1968). D. C. Deans, Chromatographia, 1, 18 (1968). G. Schomburg and F. Weeke, in Gas Chromatography, 1972, Montreaux. s. G. Perry, Editor. (6) J. A. Rijks, J. H. M. Van Den Berg, and J. P. Diependaal, J. Chromatog., 91, 603 (1974). (7) P. R. McCullough and W. A. Aue, J. Chromatog., 82, 269 (1973).
(1) (2) (3) (4) (5)
(8) D. C. Fenimore, R. R. Freeman, and P. R . Loy, Anal. Chem., 45, 2331 (1973). (9) A. Zlatkis, H. A. Lichtenstein, and A. Tishbee, Chromatographia,6, 67 (1973). (10) W. Bertsch, F. Shunbo, R. C. Chang, and A. Zlatkis, Chromatographia, 7, 128 (1974). (11) W. Bertsch. unpublished results. (12) A. Zlatkis. H. A. Lichtenstein, and A. Tishbee, Chromatographia,6, 67 (1973). (13) P. J. Groenen and L. J. Van Gemert. J. Chromatog., 57, 239 (1971). (14) M. R . Guerin. Anal. Letters, 4, 751 (1971). (15) M. R . Guerin, G. Olerich, and A. D. Horton, J. Chromatog. Sci., 12, 385 (1974). (16) A. D. Horton. and M. R. Guerin. J. Chromatog.,90, 63 (1974). (17) G. A. F. Harrison, and C. M. Coyne. J. Chromatog., 41,453 (1969). (18) B. B. Agrawal, K. Tesarik, and J. Janak, J. Chromatog., 65, 207 (1972). (19) E. Leppin. K . Gollnick, and G. Schomburg, Chromatographia, 2, 535 (1969). (20) D. C. Fenimore. Texas Institute of Mental Sciences, Houston, Texas, private communication.
RECEIVEDfor review November 17, 1975. Accepted January 29, 1976. Work supported by the National Aeronautics and Space Administration, Life Sciences Directorate, Johnson Space Center, Houston, Texas (Contract NAS914534).
Spectrum Subtraction Techniques in Ion Scattering Spectrometry William L. Baun” Air Force Materials Laboratory (MBM), Wright-Patterson AFB, Ohio 45433
James S. Solomon University of Dayton Research Institute, Dayton, Ohio 45409
A new and powerful method of surface characterization and depth profiling is ion scattering spectrometry (ISS) ( 1 , 2). ISS is accomplished by measurement of the energy spectra of scattered noble gas ions a t a known scattering angle. Binary scattering is one of the many phenomena which take place when an ion beam is accelerated to a surface ( 3 ) .The technique is a simple one with the energy of each peak uniquely related to each element present in the first layer on the surface. However, in practice, limitations sometimes arise in the commercial instrument (3M Co., St. Paul, Minn.) because the spectrometer is small, and does not provide sufficient resolution to completely separate lines from each element. The recent introduction of a cylindrical mirror analyzer (CMA) will probably improve both sensitivity and resolution. Even when actual peaks are separated, often occurring asymmetrical backgrounds make absolute intensity measurements difficult. In theory when sufficient resolution or sensitivity is not obtained one changes to a scattering gas which provides optimum results ( 4 ) . Unfortunately, however, the choices of noble gas ions are limited in practice to He, Ne, and Ar which for certain combinations of elements do not provide complete separation of lines. In this note we describe how spectrum subtraction methods improve quantitative analysis, determination of background shape, and detection limits of minor constituents. EXPERIMENTAL All measurements were made here using the standard 3M Co. Model 520 ion scattering spectrometer. Ion beams of 4He or *ONe gas were accelerated a t 2500 V and focussed on the sample in a spot of -2-mm diameter. The scattered signal was routed through conventional pulse counting electronics to a commercial (TracorNorthern Scientific, Middleton, Wis.) signal averaging computer (SAC) Model 560.
RESULTS AND DISCUSSION The spectrum subtraction method is illustrated in Figure 1 where spectrum A is from a contaminated low-carbon steel. Carbon, oxygen, and sodium are seen in addition to iron. Spectrum B is from a “clean” sample of the same steel. In the curve shown labeled A-B, the “clean” spectrum is subtracted from the “contaminated” spectrum. The inset oscilloscope traces show one such run in which subtraction was performed by the SAC. The instrument is placed in positive mode and a given number of spectra are accumulated in memory. The instrument is then placed in negative mode and the spectrum to be subtracted is recorded until a predetermined background point reaches zero. Similarly, the SAC may be used to signal average and provide a convenient buffer storage prior to computer processing of data. Such a use is illustrated by the curves shown in Figure 1. These curves originate from the same samples and are marked the same, but represent a different run. In this mode, data for each spectrum are dumped to paper tape and computer processed. The curves are normalized to make the strongest feature in each spectrum equal and then subtracted giving the resultant curve labeled A-B. The extra “spike” on the high-energy side of the iron line appears to be real, resulting from a small difference in line width between spectra from “clean” and “dirty” surfaces. The origin of such a shift is not clear but could result from slight charging a t the surface. This SAC technique is a very sensitive method of measuring small spectral shifts. Many elements give ISS spectra which exhibit tailing to low EIEo values. This background tailing, not to be confused with high background near zero E/Eo due to sputtered species, has not yet been adequately explained but must represent inelastic collision phenomena. An element ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
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