2578
Anal. Chem. 1986, 58, 2578-2581
A
= -0.92) with DM but to a lesser extent than methanol. This was the last peak discarded during the regression analysis. The major source of formaldehyde likely is the unesterified carboxyl groups. Figure 3 depicts a pyrogram of carrot pectin together with one of carrot fiber. The similarities of the two profiles and to the profile of citrus pectin are evident. Nevertheless, only 35% DM was calculated from the area percent of methanol and linear model given earlier, whereas the value was known to be greater than 75%. When the three-parameter model was used, 50% DM was found. The discrepancy is likely related to matrix effects and demonstrates that models derived for pectins from one fruit source cannot be generalized to pectins from other plant sources. This observation does not detract from the method presented here but supports the concept that calibrations must be derived from similar substrates for acceptable quantatitive information to be generated.
FiBER
I
I I
ACKNOWLEDGMENT The authors thank Edwin Piotrowski for performing the mass spectrometry experiments and Peter Hoagland for supplying carrot fiber and pectin. Registry No. Pectin, 9000-69-5;low-methoxyl pectin, 904934-7. 1
5
IO
I
I
15 MiNUTES
20
25
Flgure 3. Pyrograms of carrot fiber and pectin derived therefrom.
PY-GC. Statistically similar linear relationships between area of the methanol peak and DM were observed whether or not the mixtures were included in the data analysis. Apparently, even though the chemical environment around methoxy groups for a given DM is different within a polymer molecule than in a mixture of polymers prepared to simulate that DM, the chemistry of methoxy fragmentation is influenced minimally. For conditions described in the Experimental Section, a linear model % DM = 2.34 Area (MeOH) + 1.22 was derived with coefficient of determination of 95% and coefficient of variation (CV) of 11.71. Thii CV is comparable to CV reported in the literature for other methods used to determine DM (7). The amount of methanol measured in these pyrolysis experiments was calculated to represent 40-50% of known methylated galacturonate units. This diminished value could result from incomplete pyrolysis or from secondary reaction of methanol. The latter is more probable. Another peak eluting a t 4.2 min, identified tentatively as formaldehyde, correlated negatively (correlation coefficient
LITERATURE CITED (1) Fishman, M. L.; Pfeffer, P. E.; Barford, R. A,; Doner, L. W. J. Agric. FoodChem. 1984,32, 372-378. (2) Rees, D. A.: Wight, A. W. J . Chem. Soc. B 1971, 1366-1372. (3) Fishman, M. L.; Pepper, L.; Pfeffer, P. E. Polym. Mater. Sci. Eng. 1984,51. 561-565. (4) Kertesz, 2. I. The Pectic Substances: Interscience: New York. 1951; Chapter 111. (5) Schuitz, T. H. Methods in Carbohydrate Chemistry;Whistler, R. L., Ed., Academic Press: New York, 1954; Voi. 5, pp 189-194. (6) Waiter, R. H.; Sherrnen, R. M.: Lee, C. Y. J. Food Sci. 1983, 48, 1006-1007. (7) McFeeters, R. F.; Armstrong. S. A. Anal. Biochem. 1984, 139, 2 12-2 17. (8) Irwin, W. J. Analytical Pyrolysis; Marcel Dekker: New York, 1982; p 343. (9) Shuiten, H. R.; Bahr, V.; Giirtz. W. J. Anal. Appl. Pyrolysis l9Sll 1982.3. 229-241. (10) Zamorani. A.; Roda, G.: Lanzarini, G. Ind. Agrar. 1971, 9 , 35-40. (11) McReady, R. M. I n Methods in Carbohydrate Chemistry: Whistler, R. L., Ed.: Academic Press: New York, 1965: pp 166-179. (12) Wood, P. J.; Siddiqui, I. R. Anal. Blochem. 1971,39, 418-428. (13) Afifi, A. A.; Azen, S. P. Statistical Analysis: A Computer Oriented Approach; Academic Press: New York, 1972: pp 252-259. (14) Draper, N. R. and Smith, H. I n Applied Regression Analysis, 2nd ed.; Why: New York, 1981; pp 294-352.
RECEIVED for review January 7, 1986. Resubmitted June 4, 1986. Accepted June 9, 1986.
Capillary Gas Chromatography/Fourler Transform Infrared Spectroscopy Using an I njector/Trap Allen J. Fehl* and Curtis Marcott T h e Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, Ohio 45247 Capillary gas chromatography/Fourier transform infrared (GC/FT-IR) spectroscopy can be a very useful tool in molecular analysis, especially regarding isomer identification ( I ) . The technique has, however, suffered classically from a lack of sensitivity. Recent attempts have been directed a t increasing overall sensitivity by reducing system noise through the use of small-area detectors and optical revisions that
preclude lightpipe emission from reaching the detector (2). Another option for bettering sensitivity is to increase signal by injecting more sample. This is, however, not easily done because of chromatographic restrictions. Samples concentrated in solvent or collected from headspace over materials of interest are not readily introduced to a capillary GC because of the large volumes of solvent (or air) present with the sample.
0003-2700/86/0358-2578$01.50/00 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
VenI
t
V I Figure 1. Experimental arrangement of the caplllary GC/FT-IR in-
terface and injectorltrap.
To eliminate large volumes of unwanted solvent (or air), and to reconcentrate the sample, we incorporated an injector/trap (3) with a capillary GC/FT-IR interface of our design. With this system we have generated, for example, a good infrared spectrum of 29 ng of p-dioxane injected in 1.1 KL of dichloromethane and have identified minor components concentrated in 1OO-pL injections of Freon resulting from the steam distillation-extraction (SDE)of chocolate. EXPERIMENTAL SECTION Work on our GC/FT-IR interface was begun before capillary GC/IR accessories were commerically available. Basic design considerations were that the column flow rate would be 1mL/min and that minimum GC peak width at half peak height would be 6 s. This converts to a lightpipe volume of 100 pL, which will just contain a 6 s wide peak for optimum detection (4). Smooth-bore Pyrex tubing, 6.3 mm (1/4 in.) 0.d. and 1.0 mm i.d., was gold-coated in 30.5 cm lengths by the method of Azarraga (5), and a flawless 12.7-cm section cut out with a diamond saw. A V-shaped groove was cut in one end of this gold-coated tube, from outside circumferenceto center hole, with a diamond-tipped stylus. The groove is deep enough to accommodate a piece of 0.25 mm o.d., fused silica capillary. A cleaved piece of KBr crystal, 5 X 5 X 1 mm, was cemented over the end of the tube, with fused silica capillary in place, using Dow Corning 732 RTV sealant. There is no salt flat on the other end of the lightpipe. This assembly "floats" in a heated aluminum block with no applied pressure on either end of the tube. The assembly is kept at 250 "C continually. Lightpipe optics are 6X ellipsoids stripped from Perkin-Elmer 6X beam condensers used for microsampling. The accessory is contained within a purged chamber of approximately 75 L capacity and is purged continuously with nitrogen at 18 L/min. We have not experienced any difficulties resulting from condensation of lightpipe effluent on accessory optics within this chamber. The original optical design was essentially that of Figure 1 in Yang and Griffiths (2) but has since been modified somewhat as in their Figure 2. We have added a water-cooled aperture at the end of the lightpipe. Our optical design is shown in Figure 1. Collecting a smaller solid angle of light from the exit of the lightpipe leads to less detector saturation originating from lightpipe emission (unmodulated background). This permits the use of more sensitive, smaller focal chip detectors (2). The aperture is a 2.5 mm diameter hole in a plate of aluminum 12.7 X 17.8 X 0.3 cm (5 in. X 7 in. X 1/8 in.) mounted 0.95 mm (3/8 in.) from the end of the lightpipe. When this modification was put in place we observed a 30% increase in the amplitude of the interferogram signal collected using a mercury-cadmium-telluride (MCT) detector having a 1 mm X 1 mm focal chip. This detector has since been replaced with one having a 0.25 mm X 0.25 mm focal chip and biased to operate with a D* of 6.9 x 10'' cm HZ'/~/Wat 10 kHz (Infrared Associates, Inc., New Brunswick, NJ). The interferogram signal completely fills the analogto-digital (A/D) converter at minimum amplifier gain with no beam attenuation other than the lightpipe. When the beam is blocked, a single interferogram collection shows two to three bits of noise. This detector was in place for the experiments described. The GC/FT-IR interface is mounted permanently on a platform attached to the side of a Digilab FTS-15E interferometer spectrometer. A "collimated" beam exits the right side of the spec-
2579
trometer and is focused first by an off-axis paraboloid identical with those within the spectrometer. The lightpipe is scanned continuously at a mirror velocity of 1.28 cm/s and at a nominal resolution of 8 cm-l. A machined aluminum extension is bolted at a right angle to the heated lightpipe housing. This extends through a hole cut in the GC oven chamber at the rear of a Perkin-Elmer 900 GC unit located on the opposite side of the interface. A passage drilled through this aluminum extension carries the fused silica capillary, which is cemented to the lightpipe as previously described, into the GC oven where it is connected to the end of the GC column through a low dead-volume fitting (no makeup gas is used). At the front of the GC unit, the injector/trap is positioned on a platform such that the column from the GC oven passes into the injector/trap oven enclosure through a heated transfer line. The injector/trap, made in-house, consists of a Carle Model 4300 valve oven containing two Teflon trap housings as shown in Figure 1. The injector/trap oven is kept at 200 "C and traps 1and 2 can be electrically heated to 200 "C or cooled to a desired temperature under computer control. Cooling is accomplished by the controlled addition of liquid nitrogen to the traps, via solenoid valves. The GC column passes through a nichrome-wire-wrappedstainless steel tube within trap 2 to the valve (X). A tube within trap 1 consists of a piece of nichrome-wire-wrapped 3.2 mm in.) 0.d. glass-lined stainless steel pipe of 1mm i.d. and serves as the injector. Syringe needles were modified with ferrule and nut so that they make a tight fit with this injector pipe. Helium from a constant flow regulator is fed into the injector pipe in trap 1. A 60 m, 0.32 mm i.d., DB-1 fused silica column, with a film thickness of 1wm and helium carrier gas at 1.5 mL/min was used in the experimental work. Data were collected via Digilab's GCS software and stored continuously on a Control Data 300 M byte disk. The injector/trap cooling solenoids, trap heaters, and valving sequences are controlled by an Apple IIe computer. A typical loading sequence might be as follows: Cool trap 1 to -25 "C and inject a sample in 50 pL of CHpC1,with the valve (X) open to vent for 10 min. Close the vent, cool trap 2 to -125 "C and heat trap 1to 200 OC for 5 min. (The sample is transferred to the head of the column in trap 2.) Finally, heat trap 2 to 200 "C and begin the GC run simultaneously. The overall system sensitivity was evaluated by injecting microliter-size solutions of dichloromethane containing 21-156 ng quantities of p-dioxane using the loading sequence indicated above. The GC oven was held 2 min at 27 "C and programmed at 24 "C/min to 160 "C. The absorbances of the resulting infrared spectra were plotted vs. concentration to yield a linear calibration curve whose correlation coefficient was 0.99 with an intercept at zero absorbance. Commercially available chocolate chips (10.0 g) were steam distilled in 100 mL of distilled water and the distillate was simultaneously extracted with 50 mL of predistilled Freon-ll for 90 min. The 50 mL of Freon containing the chocolate volatiles after this steam distillation-extraction (SDE) (6) was reduced in volume to 0.25 mL by a gentle sweep of nitrogen. A 1WpL sample of this Freon solution was injected into trap 1set at +25" C; other conditions were the same as those listed above for a typical loading sequence. The GC oven was programmed at 4 OC/min to 250 "C after an initial holding time of 6 min at 40 OC.
RESULTS AND DISCUSSION The injector/trap provides a means of injecting analytes that have been concentrated in solvents by techniques such as SDE. Large volumes are required to get sufficient mass for detection. A 1WpL injection of solvent-containing analyte is typical. Injecting this much solvent on a capillary column without some means for solvent elimination could present several problems. With nonbonded columns, repeated large-volume injections will destroy the film stability of the column. Moreover, the use of bonded columns is not without problems: a large solvent peak would obscure important components and modify the chromatography, rendering retention index comparisons meaningless. Split injector techniques are not optimum for GC-IR because too little of the solvent is removed and some analyte loss must inevitably be
2580
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986 6 3377-
I
8X z e
1 1
3 0329 ..
8 n a
1
1 II I
llu!p u 'kc, ;I
1
1
1 1
i l l
n
1 1
-027201
0
10
20
30
40
50
60
70
Minutes
Flgure 3. Infrareddetected caplllary gas chromatogram of a 100-pL injection of Freon-1 1 containing chocolate volatiles.
tolerated. The injector/trap technique overcomes these objections by allowing all of the analyte to be placed on the column with elimination of almost all of the solvent. The small amount of residual solvent has not been a problem in any of our work. Water vapor is normally subtracted from all spectra. Shown in Figure 2 is the vapor-phase spectrum of 29 ng of p-dioxane constituting one point on the calibration curve discussed above. The spectrum has a signal-to-noise ratio (S/N)of approximately 13:l and a peak absorbance of 0.004 for the strongest bands. This spectral quality is believed to be typical of that achievable with about 30 ng of many organic compounds, considering that p-dioxane is only a moderate infrared absorber. Figure 3 is the Gram-Schmidt (7) chromatogram of a 1OO-pLinjection of a Freon extract containing chocolate aroma. The amount of residual Freon is equal to or less than the material in one of the detected GC peaks (i.e., the peak at 46 min). No attempt was made to dry the Freon after the simultaneous distillation with steam, hence the large GC water peak at 6 min. After water vapor subtraction, we obtained 85 interpretable infrared spectra beyond 25 min into the GC run. Strong to weak GC peaks associated with three of these
I
-01138
4000
3000
2000 Wavenumber
loo0 810
Figure 6. Trimethylpyrazine, vapor-phase infrared spectrum of peak C of Figure 3. Estimated at 300 ng by comparison with a sDectrum of a known injected amount of trimethylpyrazine.
spectra are shown in Figure 3 (arrows). The corresponding infrared spectra are shown in Figures (4-6),respectively, and have been positively identified as phenethyl acetate (peak A), tetramethylpyrazine (peak B), and trimethylpyrazine (peak C), all known to be constituents of chocolate (8). Trimethylpyrazine has an absorptivity about 3 times less than that of p-dioxane. In considering Figures 2 and 6 one would then estimate that the spectrum of Figure 6 with a S I N of
Anal. Chem. 1988, 58, 2581-2583
54 represents about 300 ng of trimethylpyrazine. Indeed a comparison of the spectrum of Figure 6 with a spectrum of 59 ng of injected trimethylpyrazine showed this to be the case. Thus, very weak absorbers such as trimethylpyrazine can be quite nicely concentrated by the SDE technique to give excellent infrared spectra when the solvent is eliminated by the injector/trap. Still weaker GC peaks presently in the noise in the chromatogram of Figure 3 can be intensified for identification by injecting more Freon solution or carrying out the SDE for longer periods of time. There is no indication of column degradation or alteration to date. ACKNOWLEDGMENT We are grateful to J. Anast and T. H. Eichhold for constructing the injector/trap and writing the software for its control. We thank L.'V. Haynes, G. P. Rizzi, and P. A. Ro~
2581
driguez for many helpful discussions and for supplying some of the materials in this work.
LITERATURE CITED (1) Griffiths, P. R.; de Haseth, J. A.; Azarraga, L. V. Anal. Chem. 1983, 55, 1361A-1387A. (2) Yang, P. W. J.; Grlffiths, P. R. Appl. Spectrosc. 1984, 38, 816-821. Rodriguez, P. A.; Eddy, C. L.; RkMer, G. M.; Culbertson, C. R. J . Chm(3) matogr. 1982. 236, 39-49. (4) Griffiths, P. R. Appl. Spectrosc. 1977, 3 1 , 284-288. (5) Azarraga, L. V. Appl. Spectrosc. 1980, 3 4 , 224-225. (6) RiJks, J.; Curvers, J.; Noy, T.; Cramers, C. J . Chromatogr. 1983, 279, 395-407. (7) de Haseth, J. A.; Isenhour, T. L. Anal. C b m . 1977, 4 9 , 1977-1981. (8) Shankaranarayana, M. L.; Abraham, K. 0.; Raghavan, B.; Natarajan, C. P. CRC Crlt. Rev. Food Sci. Nub. 1975, 6 , 271-315.
RECEIVEDfor review March 3,1986. Accepted May 22,1986.
Characterization of Mixtures of Organic Acids by Ion-Exclusion Partition Chromatography-Mass Spectrometry Frank Pacholec,' David R. Eaton,* and David T. Rossi2
Monsanto Co., 800 North Lindbergh Boulevard, St. Louis, Missouri 63167 With the advent of thermospray as a means for mass spectrometric sample introduction (I), liquid chromatography-mass spectrometry (LC-MS) has increased in popularity (2). Many applications of thermospray LC-MS have already been documented, and a review of some of these applications has recently been published (3). Most thermospray LC-MS applications involve the reversed-phase high-performance liquid chromatographic (HPLC) separation of neutral species, with subsequent ionization in the thermospray process, and monitoring by mass spectrometry. In the past several years, there has been a notable increase in the amount of literature pertaining to the chromatography of ionic species. For the determination of organic acids, ion-exclusion partition chromatography (IEPC) offers some advantages over other liquid chromatographic techniques with respect to sensitivity, capacity, and simplicity of the chromatographic system (4). In IEPC, the pH of the mobile phase is adjusted so that the acids of interest are partially protonated. Separation occurs by the Donnan exclusion principle, whereby neutral species can enter the pores of the column packing and be retained, while ionic species are excluded from the pores. Because acids with lower pK, values spend more time in an ionized form, they elute from the column earlier than acids with higher pK, values. This work demonstrates for the first time the combination of IEPC with mass spectrometry through a thermospray interface, facilitating the acquisition of mass spectral data of the organic acids. Four monoprotic acids were studied in this preliminary work, ranging in molecular weight from 60 to 88 m u . Limits of detection were evaluated for thermospray and discharge ionization, in the positive-ion mode.
EXPERIMENTAL SECTION Reagents. Mobile phase was prepared with Ultrex hydrochloric acid (J. T. Baker, Phillipsburg, NJ). Acetic and glycolic acids were reagent grade (Fisher Scientific, Fair Lawn, NJ). Propionic Present address: Suprex Corp., SFC Research Center, 125 William Pitt Way, Pittsburgh, PA 15238. Present address: Adria Laboratories,5000 Post Rd., Columbus, OH 43216.
0003-2700/86/0358-2581$01.50/0
and butyric acids were obtained from the PolyScience Corp. (Evanston,IL), as part of standard kit 65A. Deionized water used in mobile phase preparation and dilution of standards was obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA). Apparatus. The ion chromatography system used to perform the initial organic acid separation was a Model 16 ion chromatograph equipped with a conductivity detector (Dionex Corp., Sunnyvale, CA). The IEPC/MS system consisted of a Model 8700xR pump and Rheodyne 7125 injector (Spectra-Physics,San Jose, CA), a Model 5985B GC/MS (Hewlett-Packard, Palo Alto, CA), and a Vestec thermospray LC/MS interface (Vestec Corp., Houston, TX). For all separations, a Dionex HPICE-AS1 column was used. The column was connected to the Rheodyne valve and thermospray interface with Knurl-Lok adapters (Alltech Associates, Deerfield, IL). When the separation was monitored by conductivity detection, a Dionex packed bed HPICE suppressor column was placed in series with the separator column. The suppressor column served to remove protons and chloride ions from the mobile phase, thereby lowering the background conductivity. (This suppressor column was not used in the IEPC/MS work.) The thermospray ion source should be cleaned approximatelyonce a week to remove nonvolatile chloride salts resulting from the use of unsuppressed 1mM HC1 mobile phase. Also, water was flushed through the entire system at the end of each day. As long as these cleaning and flushing procedures were followed, the HC1 mobile phase appeared to have no detrimental effect on the thermospray or chromatographic equipment.
RESULTS AND DISCUSSION IEPC was chosen for preliminary experimentation due to its high sample capacity relative to other types of analytical ion chromatography (IC) and the high volatility of its mobile M HC1) relative to other types of IC (Na2C03, phase (1.0 X etc.). Ion chromatography columns on which compounds separate by an ion-exchange mechanism can be overloaded at solute concentrations as low as 100 ppm. In IEPC, retention is due to an exclusion-partition mechanism and good peak shape is maintained for solute concentrations up to at least 0.10%. Therefore, if initial thermospray experiments were performed under nonoptimal conditions resulting in poor ionization efficiencies, some mass spectral data might still be 0 1986 American Chemical Society
