AC Research
Anal. Chem. 1998, 70, 4223-4227
Articles
Effect of Column Flow Rate and Sample Injection Mode on Compound-Independent Calibration Using Gas Chromatography with Atomic Emission Detection Nanette A. Stevens*,† and Michael F. Borgerding†
Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061
Compound-independent calibration (CIC) is a theoretical concept in which a single compound is used to quantitate other compounds within a sample. Gas chromatography with atomic emission detection (AED) is one technique in which CIC can theoretically be applied. Reports are present in the literature, however, that both support CIC and indicate dependence of the AED response on compound structure. In this investigation, the effects of two gas chromatographic parameters (injection mode and column flow rate) on the atomic emission detector elemental response factor (ERF, peak area per nanogram of element injected) were studied. Seven compounds containing various nitrogen functional groups and three detection wavelengths (C at 179 nm, H at 486 nm, and N at 174 nm) were used in this work to explore CIC with GC-AED. When on-column injection was used, the calibration plots for the seven compounds fell on the same line (R ) 0.9995), indicating that CIC was possible. However, when splitless injection was employed, an apparent compound dependence of the AED response was observed due to discrimination at the injector. The flow rate dependence of AED response was demonstrated through studies performed with on-column injection and constant flow rates of 1.0, 2.8, and 5.0 mL/min. All three conditions produce compound-independent response, yet the average ERFs varied with the different flow rates. When a constant head pressure of 30 psi was used, the ERFs for the seven compounds became more variable due to the change in flow rate during a temperature-programmed chromatographic experiment. Thus, the experimental parameters of injection mode and column flow S0003-2700(98)00438-7 CCC: $15.00 Published on Web 09/12/1998
© 1998 American Chemical Society
rate must be considered in order to prevent apparent compound dependence of AED response. Gas chromatography (GC) is widely used for the separation of mixtures.1 Many information-rich GC detectors, e.g., mass selective, infrared, ultraviolet, have been developed which are useful for qualitative identification of sample components.1 Selective and sensitive detectors, e.g., flame ionization, thermal conductivity, nitrogen-phosphorus,1 have been applied for quantitative determinations of GC eluents as well. Quantification of individual sample components requires determination of the detector response factor for each compound of interest. Typically this is achieved by producing calibration curves using standard solutions having known concentrations of each component.2 Practically, however, this is not always a viable approach, e.g., when the sample is a complex mixture containing many compounds of interest, when the analytes are toxic, carcinogenic, mutagenic, or environmentally hazardous (causing disposal problems), or when standards for the compounds of interest simply are not available. Compound-independent calibration (CIC), the use of a single compound to quantify other components present in the sample, is an attractive alternative which could potentially eliminate these problems, as well as speed up analyses by reducing standard preparation and analysis time. One technique to which CIC can theoretically be applied is gas chromatography with microwave-induced plasma atomic emission detection (GC-AED). Following separation, compounds eluting from the GC column enter the AED plasma, where they † Current address: Bowman Gray Technical Center, R. J. Reynolds Tobacco Co., P.O. Box 1487, Winston-Salem, NC 27102. (1) McNair, H. M. LC-GC 1993, 11, 794-800. (2) Fundamentals of Analytical Chemistry; Skoog, D. A., West, D. M., Holler, F. J., Eds.; Saunders College Publishing: New York, 1988.
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are atomized.3 Next, the atoms undergo thermal excitation, followed by atomic emission.3 With the assumption that atomization is efficient, the detector response for each element should be independent of the structure of the original compound and should be proportional only to the number of atoms of each element in the plasma. Unfortunately, there are numerous factors that can affect the AED response at any particular wavelength. Among these are the nature and concentration of reagent gases in the plasma,4,5 the flow rate of makeup gas into the detector (which affects the residence time of emitting species in the plasma),5 and the presence of very large concentrations of carboncontaining species in the plasma, e.g., a solvent peak (which can cool the plasma and/or coat the discharge tube with elemental carbon, reducing the response of any compound eluting within several minutes of the large peak).5 Due to the complexity of the detector, it is not surprising that the literature contains conflicting reports concerning the compound independence of AED response. Some studies support the concept of CIC with the GC-AED,6-8 while others indicate an apparent dependence of AED response on compound structure.9,10 While the effect of various AED parameters on the ERF is understood, the role that GC parameters play in determining AED response has not been well established. In this paper, GC parameters, viz., injection mode and column flow rate, that potentially affect AED response are examined. Results from these studies demonstrate that CIC is possible using the GC-AED with careful attention to the chromatographic analysis parameters employed during separation. EXPERIMENTAL SECTION A Hewlett-Packard (HP) 5921A atomic emission detector (AED)4,11 coupled to a HP 5890 series II gas chromatograph was used for these studies. The gas chromatograph was equipped with a HP 7673 autosampler, a split/splitless injector with a deactivated splitless liner (HP part 5181-8818), and an on-column injector with electronically programmable pressure. The instrument was operated using the HP G2630AA ChemStation software. Separations were performed with a 30-m, 0.32-mm-i.d. DB-5 capillary column of 1-µm film thickness (J&W Scientific) coupled via fused-silica press fit connectors (J&W Scientific) to a 1-m deactivated fused-silica precolumn (0.53 mm i.d.) and a 2-m deactivated fused-silica transfer line (0.32 mm i.d.). All injections were 1 µL in volume. The on-column injector was operated with oven-track operational. The splitless injector was maintained at 200 °C with the purge valve off for 30 s. The GC oven temperature was held at 37 °C for 7.5 min and then was taken to 250 °C at 20 °C/min, where the temperature was held for 6 min. (3) McLean, W. R.; Stanton, D. L.; Penketh, G. E. Analyst 1973, 98, 432-442. (4) Quimby, B. D.; Sullivan, J. J. Anal. Chem. 1990, 62, 1027-1034. (5) van Dalen, J. P. J.; de Lezenne Coulander, P. A.; de Galan, L. Anal. Chim. Acta 1977, 94, 1-19. (6) Sullivan, J. J.; Quimby, B. D. J. High Resolut. Chromatogr. 1989, 12, 282286. (7) Kovacic, N.; Ramus, R. L. J. Anal. At. Spectrom. 1992, 7, 999-1005. (8) Szelewski, M. J. Empirical Formula Determinations and CompoundIndependent Calibration Using a GC-AED System; Hewlett-Packard Application Note 228-382. (9) Pedersen-Bjergaard, S.; Asp, T. N.; Greibrokk, T. Anal. Chim. Acta 1992, 265, 87-92. (10) Webster, C.; Cooke, M. J. High Resolut. Chromatogr. 1995, 18, 319-322. (11) Sullivan, J. J.; Quimby, B. D. Anal. Chem. 1990, 62, 1034-1043.
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Gas flows for the AED were set such that the nitrogen spectrometer purge was 2 mL/min, cavity pressure was 1.5 psi, and the cavity vent was 30.0 mL/min with the spectrometer window purge, all reagent gases, and the column flow turned off. Reagent gas cylinder pressures were set by comparing the areas of spectral peaks to those of helium. With only oxygen flowing, the ratio of O at 725 nm to He at 728 nm was 0.13. With only the hydrogen flowing, the ratio of H at 486 nm to He at 492 nm was 10. Data were collected for carbon at 179 nm, hydrogen at 486 nm, and nitrogen at 174 nm. This required two injections per sample. All chemicals were used as received from Aldrich. The seven nitrogen-containing compounds used were 2-nitrobenzene, 1-methylpyrrole, 2-methylpyrazine, 2-ethyl-3-methylpyrazine, nitrobenzene, 3-phenylpyridine, 3-methyl-2-phenylpyridine. Six standard solutions containing all seven of the above compounds were prepared in chloroform with concentrations of each compound ranging from 5 × 10-5 to 2 × 10-3 M. To accurately subtract carbon background from the nitrogen signal, at least one component of the solution had to be a carbon-containing compound that did not contain nitrogen. Thus, each standard solution also contained 2-hexanone at a concentration comparable to the seven nitrogen-containing compounds. The nitrogen background amount was set by suppressing the 2-hexanone peak in each chromatogram. Each data point on the calibration plots represents the average peak area from five replicate injections. RESULTS AND DISCUSSION Injection Mode. In order for compound-independent response of the AED to be assessed, the amount of each compound reaching the detector must be accurately known. In the absence of leaks and adsorption onto the chromatographic column, it is assumed that compounds introduced onto the capillary column via the injection process are quantitatively transferred to the detector. The difficulty, however, is ensuring that the amount of each component that is delivered to the column is known accurately. Thus, the mode of injection must be critically examined before drawing conclusions about the detector response. It is generally recognized that the best way to ensure transfer of a known amount of each compound in a sample onto the GC column is to use cool on-column injection.12 Several earlier CIC studies, however, have used either split6,8 or splitless9,10 injection, presumably due to the “dirty” samples (i.e., samples containing many nonvolatile or sparingly volatile components which would contaminate the beginning of the column) evaluated in these investigations. In the present work, the effect of injection mode on the compound independence of the AED response for seven compounds containing various nitrogen functionalities was determined. Figure 1A shows the response curves for the analytes using on-column injection (head pressure 8 psi) and detection of carbon at 179 nm. When the peak area is plotted versus the amount of carbon injected (ng), all the compounds fall on a line (R ) 0.9995). This clearly indicates that under these conditions CIC is possible. Table 1 lists ranges, averages, and relative standard deviations (RSDs) of elemental response factors (ERFs, peak area per nanogram of element injected) for the seven (12) Sandra, P. In High-Resolution Gas Chromatography; Hyver, K. J., Ed.; HewlettPackard: Wilmington, DE, 1989.
Figure 1. Peak area vs amount of carbon injected (ng) for data collected with a constant head pressure of 8 psi using (A) on-column injection and (B) splitless injection. Six standards containing (+) 2-nitropropane, ([) 1-methylpyrrole, (2) 2-methylpyrazine, (]) 2-ethyl3-methylpyrazine, (O) nitrobenzene, (b) 3-phenylpyridine, and (3) 3-methyl-2-phenylpyridine were evaluated. Each data point is the average of five replicate measurements. Error bars representing one standard deviation are smaller than the plotting symbol for all points. The line in panel A is the linear regression of all the points (R ) 0.9995). (C) Elemental response factor vs compound for data collected with a constant head pressure of 8 psi using (+) on-column and (b) splitless injection. Compounds are, in order of elution, (1) 2-nitropropane, (2) 1-methylpyrrole, (3) 2-methylpyrazine, (4) 2-ethyl3-methylpyrazine, (5) nitrobenzene, (6) 3-phenylpyridine, and (7) 3-methyl-2-phenylpyridine.
compounds at C (179 nm), H (486 nm), and N (174 nm). For all three elements detected, the relative standard deviation for each ERF is less than 3.5%. This demonstrates compound independence of the AED response at all three wavelengths when oncolumn injection is used. Figure 1B shows comparable data for the same compounds with splitless injection. Clearly the compounds do not fall on a single line in this case. As seen in Table 1, the ranges of the ERFs (and the RSDs) for all three elements when splitless injection is used are significantly greater than those when on-
column injection is employed. For example, the ranges of ERFs for C (179 nm) are 8.17-8.37 (a difference of 0.20) for on-column injection and 5.61-9.13 (a difference of 3.52) for splitless injection. Figure 1C makes this even more apparent. When the compounds are considered in order of elution, it is obvious that discrimination based on compound volatility is occurring. The later eluting compounds (e.g., compounds 5-7), which are least volatile, show a pronounced decrease in ERF. Also of interest is the enhanced response of 2-nitropropane (compound 1). Since the ERF for this compound is comparable to those for the other six compounds when on-column injection is used, a characteristic of the splitless injection mode must be causing more 2-nitropropane to enter the chromatographic column than is expected for the solution volume injected. This is likely due to the heated injection port (200 °C) causing selective vaporization of the compounds with highest vapor pressures (2-nitropropane and the solvent) from residual sample solution which normally remains in the needle. The splitless injection parameters used for this work are commonly used. Optimization of these parameters for the samples and gas flow rates used probably would reduce the discrepancies between the splitless and on-column injection results.13 However, the inherent problems associated with a heated injection port would remain to some degree. This further emphasizes the need to consider GC parameters when use of CIC with GC-AED is considered. Therefore, on-column injection is strongly recommended if consistent ERFs are desired from compounds having different vapor pressures. This injection mode is necessary when CIC is practiced with GC-AED analysis. Column Flow Conditions. Constant Flow. It has been reported by several authors5 that the flow rate of makeup gas into the AED plasma significantly effects the elemental response factors. Thus, the relationship between elemental response and column flow rate was studied in this work. Constant column flow rates of 1.0, 2.8, and 5.0 mL/min were investigated with on-column injection. Table 1 shows the results from these experiments for three detection wavelengths. For each flow rate and detection wavelength studied, the ERFs for the seven compounds were generally consistent, with RSDs of 3% or less. This indicates that the AED response is independent of the compound structure under these conditions. ERF values do, however, depend on the flow rate used. The lowest average ERF is observed with a flow rate of 2.8 mL/min for all three elements considered. Decreasing the flow rate to 1.0 mL/min causes an increase in the average ERF for each element. This is likely due to a small increase in the residence time of each element in the plasma. However, when the flow rate is increased to 5.0 mL/min, the average is even greater. This effect is probably due to both a decrease in peak width (i.e., more response per unit time) and a change in the compound interactions within the plasma.14 Therefore it is extremely important when data from different experiments are compared to ensure that all are collected at the same column flow rate. While observed differences in ERF for the various compounds at the same flow rate are small, it is important to actually test compound-independent calibration. Specifically, it must be verified (13) Klee, M. S. In GC-Inlets: An Introduction; Hewlett-Packard: Wilmington, DE, 1990; pp 47-58. (14) Andersson, J. T.; Schmid, B. Fresenius J. Anal. Chem. 1993, 346, 403409.
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Table 1. Comparison of AED Response Using Various GC Parameters ERFa GC conditions injection mode on-column
splitless
C (179 nm)
flow conditions constant flow, mL/min 5.0 2.8 1.0 constant pressure, psi 30 8 constant pressure, psi 8
H (486 nm)
rangeb
avc
RSD,d
9.08-9.72 7.01-7.20 7.41-7.54
9.51 7.14 7.42
7.30-7.90 8.17-8.37 5.61-9.13
%
N (174 nm)
range
av
RSD, %
range
av
RSD, %
2.7 0.9 0.2
99.1-102.4 83.6-85.4 92.4-96.8
100.8 84.8 95.4
1.8 1.4 1.6
25.6-26.6 15.1-16.1 19.2-20.7
26.0 15.9 19.8
1.6 2.0 3.0
7.58 8.26
3.6 1.0
85.7-96.4 104.3-112.2
91.3 106.9
4.3 3.4
17.2-19.7 20.7-22.8
18.3 21.6
4.8 3.2
7.71
17.0
52.2-115.0
77.6
20.0
13.5-23.4
18.3
18.6
a Elemental response factor, peak area per nanogram of element injected. b Minimum and maximum values of the ERFs for the seven compounds (2-nitropropane, 1-methylpyrrole, 2-methylpyrazine, 2-ethyl-3-methylpyrazine, nitrobenzene, 3-phenylpyridine, 3-methyl-2-phenylpyridine). c Average of the ERFs for the seven compounds. d Relative standard deviation of the ERFs for the seven compounds.
Table 2. CIC Calculations for Nitrobenzenea % differencec calibration compdb
C
H
N
2-nitropropane 1-methylpyrrole 2-methylpyrazine 2-ethyl-3-methylpyrazine nitrobenzene 3-phenylpyridine 3-methyl-2-phenylpyridine
0.1 0.4 0.9 0.4
