Anal. Chem. 1991. 63, 255-261
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limits for resolution, both chromatographic and mass spectral, are yet to be determined. Finally, we hope to examine the use of suspended-ion trapping procedures, recently developed by Laude and co-workers (13),to see if what we view as the major limitation of the technique, namely its limited mass spectral dynamic range, can be alleviated.
ACKNOWLEDGMENT We thank Mr. Eugene Ethridge and Mr. Frank Forgit of the University of California-Riverside, Department of Chemistry machine shop, for their contributions to both the design and construction of the probe interface.
LITERATURE CITED (1) Chester, T. L.; Pinkston, J. D. Anal. Chem. 1990, 62, 394R-402R. 12) Smith, R. D.; Wright, B. W. I n Microbore Column Chromatography; Yang, F. J., Ed.; Chromatographic Science 45; Marcel Dekker: New York, 1989; pp 307-368. (3) Sheeley, D. M.; Reinhold, V. N. J . Chromatogr. 1989, 474, 83-96. (4) Owens, G. D.; Burkes, L. J.; Pinkston, J. D.; Keough, T.; Simms. J. R.; Lacey, M. P. In Supercrltlcal Nuid Extraction and Chromatography; Charpentier, B. A., Sevenants, M. R., Eds.; ACS Symposium Series 366; American Chemical Society: Washington, DC, 1988; pp 191-207. (5) Smith, R. D.; Kalinoski, H. T.; Udseth, H. R. Mess Spectrom. Rev. 1987, 6 , 445-496. (6) Wright, B. W.; Kalinoski, H. T.; Udseth. H. R.; Smith, R. D. HRCB CC, J . High Resolot. Chromatogr Chromatogr Commun 1986, 9 , 145-153. (7) Smith, R. D.; Fjeidsted, J. C.; Lee, M. L. J . Chromatogr. 1982, 247, 231-243. (8) Smith, R. D.; Udseth, H. R.; Kalinoski, H. T. Anal. Chem. 1984, 56, 2971-2973. (9) Kalinoski. H. T.; Hargiss, L. 0. J . Chromatogr. 1989, 474, 69-82. (10) Huang, E. C.; Jackson, 8 . J.; Markides, K. E.;Lee, M. L. Anal. Chem. 1988, 60, 2715-2719. (11) Lee, E. D.; Henion, J. D.; Cody, R. B.; Kinsinger, J. A. Anal. Chem. 1987, 5 9 , 1309-1312. (12) Laude, D. A.; Pentoney, S. L.; Griffiths, P. R.; Wilkins, C. L. Anal. Chem. 1987, 59, 2283-2288. (13) Hogan, J. D.; Laude, D. A. Anal. Chem. 1990, 62, 530-535. (14) Hawthorne, S. B.; Miller, D. J. J . Chromatogr. 1989, 468, 115-125. (15) Sack, T. M.; McCrery, D. A,; Gross, M. L. Anal. Chem. 1985, 57, 1290-1295.
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.
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RECEIVED for review July 23,1990. Accepted October 29,1990. Partial support of this research by the National Science Foundation (Grant CHE-11685) is gratefully acknowledged.
Techniques for Postcolumn Derivatization in Gas ChromatographyIMass Spectrometry Woodfin V. Ligon, Jr.* and Hans Grade General Electric Company, Corporate Research and Development, Schenectady, New York 12301
The connection of the output of a conventional split-type ca-
INTRODUCTION
ceed at or near the ambient GC oven temperature and at atmospheric pressure. I n addition the reactions must be complete in less than 1 8. Bromlnatlon, deuterium exchange, and a variety of acylation reactions have been demonstrated.
that can be filled with various catalysts to accomplish conversions such as hydrogenations and oxidations. Teeter et al. (7) have also described postcolumn hydrogenation. Chaffee et al. (8) have described a catalyst-filled microreactor for
0003-2700/91/0363-0255$02.50/00 1991 American Chemical Society
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capillary column effluents. None of the previous workers attempted to carry out their derivatizations on a peak by peak basis and in many cases the "reactors" resulted in losses of GC resolution. Ligon and Grade (9) have described preliminary experiments wherein the effluents of packed columns could be exposed to various reagents on a peak by peak basis with minimal loss of resolution. The goals of postcolumn derivatization are usually substantially different from those of precolumn derivatization. Precolumn methods have occasionally been used to improve mass spectral behavior but most often have been used to improve chromatographic behavior. By contrast, postcolumn methods are best directed toward functional group identification and toward improvements in mass spectral behavior. Assuming that a simple methodology could be found, there are a variety of practical reasons why relatively routine POstcolumn derivatization in GC/MS might be attractive. Consider, for example, a situation in which there exists a well-resolved unknown component in a complex mixture. A mass spectrum of this component can be readily obtained but the fragmentation pattern does not allow the analyst to decide between alternative structures which might include either alcohols or ethers. As a further complication, consider that several structural isomers of the substance may occur in the mixture at comparable levels. In such a case, precolumn derivatization by a method such as silylation can be useful only if the peak of interest does not react. In that case, the peak will occur unchanged at the same retention time and the analyst can thus infer that the substance is probably an ether. Note that it must be assumed that this "unknown" is still resolved chromatographically in the "new" mixture generated by the derivatization procedure. If, however, the peak does derivatize, then we are at a loss to know which of the many structural isomers in the mixture produced which of the products, since all have the same mass and since retention times and elution orders are not conserved after derivatization. In such a case, it will not be possible to unambiguously relate the derivative mass spectrum to the precursor mass spectrum. The analyst will, therefore, not be able to determine if the unknown peak had more than one hydroxyl and will not be able to draw any conclusions from comparisons of the fragmentations obtained for the derivatized and underivatized substances. An equally difficult analytical challenge occurs when a molecular ion and/or useful fragments cannot be obtained for an unknown in a complex mixture. Exactly the same limitations and ambiguities discussed above apply when precolumn derivatization is attempted as a solution to this problem. Situations like these are not at all unusual, especially in natural products chemistry. Furthermore, when the substance of interest is present in small quantities in a very complex mixture, GC/MS (and possibly GC/IR) may be virtually the only practical means of characterization. Under such circumstances postcolumn derivatization can be far more attractive than a lengthy isolation process which might otherwise be required to obtain the same information. Other situations which suggest postcolumn derivatization include mixtures obtained by thermal desorption, application of reagents which are incompatible with a GC column, and processes which are reversible on a GC column such as deuterium exchange. It should also be remembered that in natural products chemistry, precolumn derivatization can actually make a bad situation worse. This occurs when the derivatization procedure converts a mixture which has 50 volatile components and 150 nonvolatile components into a mixture with 200 voltaile components-many of which constitute potential interferences for the substance of interest. Consider further that biologically active materials (e.g. pheromones)
suppLy\I HELIUM
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may actually have been located in the chromatogram on the basis of their biological activity. Precolumn derivatization may bury the substance in a sea of interferences and at the same time destroy its biological activity. Chemical ionization mass spectrometry (CI) using a variety of reagent gases might be considered a competitor for POstcolumn derivatization, but only a very narrow range of chemical processes are observed under CI conditions. For a review of this technique see ref 10. In this paper we will describe the results of our efforts to develop methods and instrumentation which will allow simple and routine postcolumn introduction of reagents in combination with high-resolution capillary chromatography and high-performance mass spectrometry.
EXPERIMENTAL SECTION The apparatus which has been developed consists of specific modificationsto an existing GC/MS instrument. The instrument used was a Varian-MAT 311A employing a single-stagejet separator interface. (Note: A t t h e time that this paper was being prepared for publication, the 311A instrument was no longer available. Accordingly, some of the figures were obtained by reproducing the experiments on a similarly modified JEOL S X 1 0 2 mass spectrometer.) All of the mass spectra presented in this paper are of the standard 70-eV electron ionization type and have been acquired with an ion source temperature of about 250 "C and an ion source (housing) pressure of about 6 X lo4 Torr. The jet separator was interfaced simultaneously via a custom manifold to two GC columns. For materials that could be injected in solution, analyses were carried out by using a J&W Scientific DB-1 fused-silica capillary having a length of 30 m, an i.d. of 0.32 mm, and a film thickness of 1.0 Fm. For materials that required thermal desorption from a substrate, analyses were carried out on a Quadrex Corp., 5% phenyl/95% methyl silicone fused-silica capillary column having a length of 15 m, an i.d. of 0.53 mm, and a coating thickness of 5 Fm. The carrier gas was helium in all cases. The linear velocity of the carrier gas could be independently set to an optimum value for each column. The jet separator requires a certain minimum flow (about 20 mL/min) to operate correctly. In those cases where the optimum flow of the column in use fell below the jet separator minimum, the flow of the column not in use was adjusted to bring the total combined flow up to the value required by the jet separator. A diagram of the manifold that couples both GC columns to a common jet separator and allows the introduction of reagents is provided as Figure 1. The manifold was built to our specifications from 0.5 mm i.d. glass-lined stainless steel tubing by SGE, Austin, TX. As shown in the figure, part of the manifold is contained in the GC oven and part is contained in an interface oven between the gas chromatograph and the mass spectrometer. The portion in the interface oven is held at 290 "C. The overall length from the point at which reagents are added to the inlet
ANALYTICAL CHEMISTRY, VOL.
of the jet separator is about 40 cm. Under normal operating conditions, the pressure at the inlet end of the manifold is at or slightly above atmospheric pressure, as is evidenced by the fact that removing the injector septum cap from either column results in back-flushing of that column due to the high downstream pressure. The 'reagent injector" was constructed from a standard Varian Model 2700 'f4-in. packed-column injector. The injector was modified in exactly the same fashion as can be used to provide a "split" type injector for a capillary column. The injector was in. 0.d. glass liner to which was attached a provided with a 'I4 1/4-1f16-in.compression fitting union. The union was modified by the addition of a in. 0.d. side tube that extended out of the union to a needle valve which could be used to set the split ratio. Helium (pressure regulated at 8-15 psi) was supplied to the injector by using the standard connection and served to transport the reagent through the injector and into the reaction zone. The connection between the injector and the manifold consisted of a 100 pm i.d. by 60 cm long section of fused-silica tubing that extended from a point inside the glass liner of the injector to a point where it was connected by a 'f16-in. fitting to the manifold. All connections to the fused-silica tubing were made with graphite ferrules. Heating was provided for this fused-silica line from the injector to the interior of the GC oven. The temperature of the reagent injector and associated transfer line was normally set to about 180 "C but was increased to 250 "C for work with high molecular weight reagents. Reagents. Deuterium oxide (99.8 atom % D),acetyl chloride, benzoyl chloride, trifluoroacetic anhydride, bromine, 2-(aminowere methyl)pyridine, and 2-chloro-1,3,2-benzodioxaphosphole obtained from Aldrich Chemical Co., Milwaukee WI. Bis(trimethylsily1)trifluoroacetamidewas obtained from Supelco, Inc., Bellefonte, PA.
RESULTS AND DISCUSSION It has been found that postcolumn derivatization can be readily accomplished by using an extremely simple instrument design. The use of a conventional split-type GC injector allows the manual injection of manageable quantities of reagent (milligrams) while delivering only small (nanogram to microgram) quantities of reagent to the reaction zone. A flow of helium gas through the injector is used to transport the reagent. As will be demonstrated later, the temporal distribution of reagents delivered in this way is comparable to the peak widths observed in analyses with modern capillary columns. The reaction zone itself consists of nothing more than a length of glass-lined stainless steel tubing to which is connected both the GC column and the reagent delivery line. At its downstream end, the reaction zone is connected to the inlet of the mass spectrometer's jet separator. (In a version now under development, an open split coupling has been successfully employed). The flow restriction of the glass-lined tubing is sufficient to produce pressures near atmospheric at the inlet end of the reactor. The use of glass, fused-silica, or glass-lined stainless steel ensures that even the most aggressive reagents (except hydrogen fluoride) can be delivered intact to the reaction zone. In the current design, part of the reaction zone lies in the GC oven and part in an interface oven, which is independently heated to about 290 "C. We have found that for those cases where the derivative is much less voltaile than the substrate, the derivative may partially condense in the reaction zone, thereby causing peak broadening. It would be advantageous, therefore, to enclose the entire reaction zone in an independently heated oven. This modification is currently being incorporated in a later version of the apparatus. Selection of Reagents. In order for derivatization to be successful, any reagents chosen must meet two criteria. They must be stable and voltaile a t temperatures and pressures typically available in gas chromatography experiments, and they must be capable of reacting with a reasonable fraction of the substrate in a very short period of time. It should also be noted that reactions which require solvent assistance (e.g.
03, NO. 3, FEBRUARY 1, 1991
257
solvated transition states) are unlikely to proceed under these gas-phase conditions. Since temperatures of a t least 300 "C are routinely attainable, many reagents are able to meet the volatility criterion. In practice, therefore, the reactivity criterion is the major limitation. Although the high temperatures available tend to enhance reactivity, the very short reaction times can be insufficient. On the basis of the reactor volume and the flow rate, the residence time in the reactor can be estimated to be slightly less than 1 s. Some mass spectrometric considerations make certain reagents more advantageous than others. For example, reagents which have a high mass and abundant fragments can provide a large "background" or interference signal when used for derivatization. If the same chemical objective can be obtained with another reagent, then such materials should be avoided, although their impact can be diminished through the use of background subtraction. We have not encountered any problems with jet separator plugging, mass spectrometer contamination, reduced filament lifetime, or pump degradation with any of the reagents examined. This is presumably due to the very small quantities of reagent which actually reach the spectrometer. Techniques for Derivatization. When a potentially useful reagent has been identified, a typical derivatization experiment proceeds as follows. A GC/MS acquisition is initiated at the mass spectrometry data system. Then a series of injections of the reagent alone are made and the mass spectra are acquired. In general, about 1 p L of the neat reagent is injected. If necessary, the reagent is dissolved in a compatible solvent. The spectra obtained in this way provide an evaluation of the background or interfering ions that will be contributed by the reagent itself. The reconstructed ion chromatogram (RIC) obtained for these injections allows an evaluation of the quantities of reagent reaching the reaction zone after being reduced by the split injector. This quantity can be optimized by either changing the amount injected or changing the split ratio of the reagent injector. In general, the quantity of reagent delivered is selected to give an RIC peak height roughly comparable to the anticipated peak height of the substrate. The peak shapes obtained from the RIC allow an evaluation of the efficiency with which the reagent is being delivered to and scavenged from the reaction zone. If tailing is observed, correction can usually be achieved by increasing the temperature of the reagent injector and associated inlet line. After it has been established that the reagent can be delivered efficiently and in an appropriate quantity, a new acquisition is initiated a t the data system and the sample of interest is injected into the gas chromatograph. As the components of the mixture elute from the gas chromatograph, they are monitored in real time utilizing the RIC capability of the mass spectrometer data system. As peaks of interest appear on the RIC, injections of reagent are made. The time between injection and the reagent reaching the reaction zone is substantially less than 1 s. Accordingly, it is possible to successfully derivatize GC peaks having peak widths a t halfheight as small as 5 s. This operation provides mass spectrometry data which is underivatized for the "front" portion of a GC peak and derivatized for the later portions of the GC peak. Because the "spikes" of reagent are very narrow in time, the reagent generally disappears from the reaction zone before the elution of subsequent peaks. This means that both derivatized and underivatized mass spectra can be obtained for sequentially eluting GC peaks. Finally, it is usually advantageous to make an injection of reagent in the part of the chromatogram which contains no eluting GC peaks so that the resulting mass spectrum of the
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
Table I. Summary of Successful Derivatization Reactions reagent deuterium oxide
acetyl chloride
benzoyl chloride
trifluoroacetic anhydride
2-chloro-1,3,2-benzodioxaphosphole (CyP)
bis( trimethylsily1)trifluoroacetamide
bromine
2-(aminomethy1)pyridine
product
substrate phenol 2,6-di-tert-butyl-4-methylphenol 2-propanol butanol pentanol hexanol 1-adamantanol N-ethylaniline dibutylamine 4-methylacetanilide silanol-terminated silicones 2,2-bis(4-hydroxypheny1)propane aniline aniline benzylamine dibutylamine n-octylamine n-hexylamine dioctylamine aniline benzylamine dibutylamine n-hexylamine dioctylamine n-octadecylamine aniline benzylamine dibutylamine n-octylamine n-hexylamine dioctylamine 4,4’-diaminobiphenyl benzyl alcohol n-pentanol 2-octanol 2-ethyl-1-hexanol n-decanol octadecanol benzyl alcohol 1-adamantanol hexylamine dibutylamine benzyl alcohol 2-octanol phenol 2,2-bis(4-hydroxyphenyl)propane octene decene allyl phenyl ether 2-allyl-6-methylphenol ethyl phenyl ether toluene m-xylene mesitylene aniline naphthalene hexadecanoyl chloride
monodeuteration monodeuteration monodeuteration monodeuteration monodeuteration monodeuteration monodeuteration monodeuteration monodeuteration monodeuteration monodeuteration dideuteration dideuteration acetanilide N-benzylacetamide N,N-dibutylacetamide n-octylacetamide N-hexylacetamide N,N-dioctylacetamide benzanilide N-benzylbenzamide N,N-dibutylbenzamide N-hexylbenzamide N,N-dioctylbenzamide N-octadecylbenzamide trifluoroacetanilide N-benzyltrifluoroacetamide
N,N-dibutyltrifluoroacetamide N-octyltrifluoroacetamide N-hexyltrifluoroacetamide N,N-dioctyltrifluoroacetamide N,”-bis(trifluoroacetyl)-4,4’-diaminobiphenyl benzyltrifluoroacetate 2-(pentyloxy)-Xo 2-(2-octyloxy)-X 2-(2-ethyl-l-hexyloxy)-X 2-(decyloxy)-X 2-(octadecyloxy)-X 2-(benzyloxy)-X 2 4 1-adamanty1oxy)-X 2-(hexylamino)-X 2-(dibutylamino)-X 0-TMS 0-TMS 0-TMS bis-0-TMS + 2-Br + 2-Br + 2-Br + 1,2,3-Br + 1,2,(3)b-Br + 1-Br + (l)b-Br + I-Br + 1-Br + 1,2,3-Br + 1:Br N-(2-pyridylmethy1)hexadecanamide
X = 1.3.2-benzodioxauhosuhole. 0 = weak or very weak signal for derivative.
reagent alone can be utilized for background subtraction. Data manipulation of derivatized spectra involves subtracting the ions due to the reagent and the ions due to residual substrate. It is advantageous to use a data system that allows intensity scaling of the various background spectra. The removal of substrate ions is especially helpful when partial derivatization of the substrate has been accomplished. With these procedures, partial derivatization is a significant limitation only if unique ions for the derivative do not occur above the mass spectrometer’s detection threshold. Useful Derivatives. Table I provides a listing of the reagents that have been found useful, and whenever possible, the nature of the products obtained has been noted.
Deuterium oxide is a simple but powerful reagent. We have found that deuteron/proton exchange occurs rapidly and efficiently under our conditions. Figure 2 shows the effect of D 2 0 on a sample of 2,2-bis(I-hydroxyphenyl)propane. In the ,figure, we present the RIC for mass 20 (D,O), which demonstrates the very narrow and well-defined spike of reagent that is provided by the reagent inlet. The peak width at half-height for D 2 0 was 1.5 s. This value is typical of that obtained for all of the reagents studied. For the substrates reported, the yield of the deuteration process usually varies from about 30-50%, as the quantity of D 2 0 varies from 1:l to 5:l with respect to the substrate. D20 is removed cleanly from the interface and from the ion source. Experiments have
ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
259
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shown that introduction of a similar quantity of D20 into the ion source itself does not result in an analytically useful level of exchange. Acetyl chloride, benzoyl chloride, and trifluoroacetic anhydride react with a variety of amines to form the corresponding acyl-substituted derivatives. The conversions (product yields) observed fall in the 1-3070range. Especially for the case of benzoyl chloride, this reaction can substantially enhance the relative intensity of the molecular ion. All of the reactions can be used to verify the presence of primary or
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Result of the treatment of octadecylamine with the reagent benzoyl chloride. From the top, the boxes provide, respectively, the mass spectrum of octadecylamine, benzoyl chloride, and the crude reaction mixture and the product spectrum after subtraction of reagent and substrate. Neither the reagent nor the product showed evidence of tailing. Figure 3.
secondary amines. In Figure 3 we present an example of the reaction of a long-chain primary amine with benzoyl chloride as reagent. In Figure 4, we present the inverse reaction in which the substrate was the acid chloride of a long-chain fatty acid (hexadecanoyl chloride) and the reagent was an amine (2-(aminomethyl)pyridine). In this case the amide formed has an enhanced molecular ion and undergoes a stepwise fragmentation of the hydrocarbon chain (11). The small amount of free fatty acid evident at m/z 256 arises from traces of water in the reagent. With trifluoroacetic anhydride as reagent, a trace amount of conversion of benzyl alcohol to the trifluoroacetate can be observed, but in general alcohols are found to react very poorly, if a t all, with this reagent. Bis(trimethylsily1)tri-
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
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fluoroacetamide provides small (
